Marys Medicine

Plenary sessions:
Arvid Carlsson and Elias Eriksson
University of Gothenburg, Sweden ABERRATIONS IN BRAIN NEUROTRANSMITTER FUNCTION AS A POSSIBLE ROOT OF NEUROPSYCHIATRIC ILLNESS – WHICH CONCLUSIONS MAY BE DRAWN AFTER 60 YEARS OF RESEARCH? One important incentive for the many attempts to monitor brain molecules that have been undertaken during the past decades has been the wish to shed light on the possible importance of specific neurotransmitter abnormalities in psychiatric and neurological disease. But why did we ever come to believe that the pathophysiology of brain disorders may be partly explained in terms of transmitter aberrations? In this interview, psychopharmacologist Arvid Carlsson, who once pioneered this way of thinking by suggesting dopamine to be of importance for Parkinson's disease, and who later made important scientific contributions with respect to the involvement of brain neurotransmitters in disorders such as schizophrenia and depression, will comment on the history of the transmitter-centred perspective on neuropsychiatric disorders, and discuss both the virtues and limitations of this approach. Margaret E. Rice
NYU School of Medicine, SOMATODENDRITIC DOPAMINE RELEASE: THEN AND NOW Dopamine (DA) was one of the first substances to be studied by electrochemical methods, including fast-scan cyclic voltammetry. Factors that regulate axonal DA release in the striatum and other target regions are increasingly well understood, in large part because of these methods. Less well understood is the process of somatodendritic DA release from DA neurons in the midbrain. First identified four decades ago, somatodendritic release of DA appears to be exocytotic and action-potential dependent, albeit with a Ca2+ dependence that differs from that of axonal DA release. A novel set of exocytotic proteins and the involvement of Ca2+ release from intracellular stores in cell bodies and dendrites contribute to this difference. Nevertheless, many questions remain. These continue to be addressed using voltammetry, as well as by a novel biosensor, the DA neuron itself, which responds to local DA release during whole-cell recording with an inhibitory current produced by D2-autoreceptor activated G-protein-coupled inwardly rectifying K+ (GIRK) channels. Through this autoregulatory process, somatodendritic DA release modulates its neuronal activity and transmitter release. Thus, DA release from cell bodies and dendrites is a pivotal intrinsic feature of DA neurons that must be well defined in order to understand fully DA physiology and pathophysiology. Astrid C.E. Linthorst
University of Bristol, UK DYNAMIC REGULATION OF FREE GLUCOCORTICOID HORMONES AND GABA DURING STRESS AND EXERCISE Stress has become an increasingly important factor adversely affecting the health and wellbeing of millions of people. Inability to cope with stressful challenges is a key cause for the development of psychiatric disease. Using in vivo microdialysis, our research focuses on the effects of stress on the regulation of free (i.e. biologically active) glucocorticoid hormones and brain neurotransmitters. We recently discovered that corticosteroid-binding globulin (CBG) plays a much more dynamic role in the regulation of glucocorticoids than so far had been assumed based on its role in glucocorticoid hormone binding and transport. CBG is rapidly released from the liver into the bloodstream during moderate to strong forms of acute stress. This rise in circulating CBG levels results in an attenuated and delayed increase in free corticosterone in target organs including the brain. Furthermore, our recent studies have shown a significant role for astrocytic GABA transporters (GAT-3) in the regulation of extracellular GABA levels and tonic conductance in the hippocampus. Interestingly, voluntary exercise, which is known to promote stress resilience, increased hippocampal expression of GABA synthesising enzymes accompanied by a rise in extracellular GABA levels. This lecture will further explore the intricate interplay between stress, glucocorticoids and neurotransmitters. David Sulzer
Columbia University, USA UPDATE ON FLUORESCENT FALSE NEOROTRANSMITTERS TO VISUALISE CATECHOLAMINE FUNCTION IN BRAIN Optical techniques now provide means to examine neurotransmission at the level of individual synapses. These approaches include the use of endocytic tracers, most commonly the styryl dye FM1-43, and probes that measure calcium levels, primarily GCaMP. By developing fluorescent false neurotransmitters, derivatives of catecholamines that are accumulated by monoamine transporters and then released from synaptic vesicles during fusion, we can now record differences between synaptic properties in the acute brain slice and in the brain in vivo. We find a wide variety of synaptic properties, even between closely neighboring release sites of dopamine and norepinephrine axons. Current results suggest that even when they possess large presynaptic calcium responses, most boutons fil ed with synaptic vesicle are "silent": an important parameter in dopamine systems may be to regulate presynaptic silence or transport synaptic vesicles between silent and active sites. Anne M. Andrews
University of California, Los Angeles, USA THE FUTURE OF MONITORING SEROTONIN IN VIVO Great strides have been made in terms of understanding information content encoded in dopamine, acetylcholine, and recently, glutamate neurotransmitter signaling and the influence on behavior. Advances have been enabled by high spatial, temporal, and chemical resolution measurements associated with carbon-fiber microelectrode and biosensor technologies. Recently, improvements are also being made to enable similar high-resolution interrogation of serotonin signaling via fast microdialysis, chronoamperometry, fast-scan cyclic voltammetry, and serotonin-selective electronic biosensors. Serotonin is regarded as a neuromodulatory transmitter carrying out its actions through changes in basal homeostatic mechanisms. Nevertheless, the existence of 5-HT3 ligand-gated ion channels, which operate similarly to ion-channel glutamatergic, GABAergic, and cholinergic receptor subtypes portend fast-signaling mechanisms for serotonin as well. Maps of current and future landscapes regarding strategies for investigating the roles of serotonin signaling via in vivo measurements, including the development of electronic nanobiosensors, will be discussed. Kaj Blennow
The Sahlgrenska Academy at the University of Gothenburg, Sweden MASS SPECTROMETRY GUIDANCE TO MONITOR PROTEIN PROCESSING IN ALZHEIMER'S DISEASE Research advances in Alzheimer's disease (AD) molecular pathogenesis have given several drug candidates targeting β-amyloid (Aβ) that have been evaluated in clinical trials. However, the list of anti-Aβ trials that failed to show clinical benefits is growing, and there is now a consensus that trials need enrol patients before neurodegeneration is too advanced, to give drug candidates a chance to show clinical efficacy. To manage this, biomarkers will have a critical role. The CSF biomarkers total tau (T-tau), phospho-tau (P-tau) and Aβ (Aβ42 or Aβ42/40 ratio) have consistently been found to have high diagnostic accuracy to identify AD already in the early (prodromal) disease stage. Given that CSF Aβ42 and amyloid PET have been shown to have very similar diagnostic performance for AD and that the cost of CSF biomarker analyses is only a fraction of an amyloid PET scan, it is likely that CSF biomarkers will be important tools for AD diagnostics. However, standardization is needed since current ELISA methods have high between-lab and between-batch variability. The IFCC-WG for CSF proteins aims to develop a Certified Reference Material (CRM) for distribution to assay vendors and laboratories to harmonize assay readouts. A mass spectrometry-based Reference Measurement Procedure (RMP) has been developed for A42, and approved by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). Biotech companies have developed high-quality fully automated assays for the AD biomarkers, and the Cobas Elecsys method for CSF A42 was published in 2015, showing excellent analytical performance, and this method is also standardized against the RMP. Taken together, these efforts will allow uniform cut-off levels and enable the large-scale introduction of CSF biomarkers in diagnostic routine. New developments include assays to monitor synaptic dysfunction. Synaptic dysfunction is linked to clinical symptoms, and a very early pathogenic process in AD. We have used mass spectrometry to guide assay development, which has proved important since these synaptic proteins are cleaved into N- or C-terminal species before released from the neurons to the CSF. Recent studies show a marked increase in the CSF levels of the dendritic protein neurogranin in prodromal AD, with higher levels predicting a more rapid cognitive decline and with progression to AD dementia. This type of synaptic biomarkers may be valuable to select early AD cases for inclusion in trials. Importantly, in a large anti-A clinical trial, CSF neurogranin showed a dose-dependent decrease towards normalization with active treatment, indicating downstream effect on synaptic integrity by this class of compounds. Symposium: Monitoring neurotransmitters and related molecules in brain disorders
Chairperson: Jan Kehr Connie Sanchez
Lundbeck Research, Paramus, USA EMERGING MECHANISMS AND TREATMENTS FOR DEPRESSION BEYOND SSRIs AND SNRIs Whereas, the hypothesis that depression is ascribed to a reduced monoamine function has been prevailing since the 1960ies, recent research indicates that enhanced glutamate neurotransmission and downstream effects on neuronal plasticity may be the converging antidepressant mechanism. The SSRIs and SNRIs are currently first line treatments for depression. However, additional treatment options are needed since only 50% of patients go into clinical remission. Successful strategies have been to target 5-HT receptors and transporters in one molecule or to add an atypical antipsychotic to SSRI or SNRI treatment. Vortioxetine, a 5-HT reuptake inhibitor, 5-HT1A agonist, 5-HT1B partial agonist and 5-H1D, 5-HT3 and 5-HT7 antagonist, came out of the former strategy. Electrophysiology and microdialysis studies in rats show that vortioxetine modulates multiple neurotransmitter systems, i.e. 5-HT, NE, acetylcholine, histamine, glutamate and GABA. Of particular interest is that vortioxetine enhances glutamate neurotransmission via 5-HT3 receptor antagonism-mediated reduction of the GABAergic control of pyramidal cells. Preclinical and clinical studies indicate that vortioxetine in addition to its antidepressant effect has beneficial effects on cognitive dysfunction. Another recent strategy has been to target glutamate receptors directly, exemplified by the NMDA receptor antagonist ketamine. Interestingly, recent preclinical research indicates that ketamine's antidepressant activity depends on serotonergic tone. Craig Berridge
University of Wisconsin-Madison, USA THERAPEUTIC EFFICACY OF PSYCHOSTIMULANTS IN ADHD: PREFERENTIAL TARGETING OF PREFRONTAL CORTEX CATECHOLAMINE SIGNALING. At low doses, psychostimulants are highly effective in reversing the symptoms of attention deficit hyperactivity disorder (ADHD). The therapeutic efficacy of psychostimulants is linked to drug-induced improvements in prefrontal cortex (PFC)-dependent cognitive function, consistent with frontostriatal dysfunction in ADHD. Importantly, the cognition-enhancing effects of low-dose psychostimulants are not limited to ADHD patients, having been well-documented in healthy human and animal subjects. Microdialysis studies demonstrate that when administered in rats at cognition-enhancing doses that result in clinically-relevant plasma concentrations, methylphenidate (Ritalin) increases extracellular levels of catecholamines preferentially in the PFC, an action that involves mechanisms intrinsic to the PFC. These observations suggest the hypothesis that the cognition-enhancing and therapeutic effects of psychostimulants involve drug action directly within the PFC. Consistent with this, methylphenidate infusiong into the dorsomedial PFC, but not ventromedial PFC or dorsomedial or ventromedial striatum, improves both working memory and sustained attention in rats. Additional studies demonstrate the cognition-enhancing actions of low-dose methylphenidate are dependent on α2 and D1 receptor signaling in the PFC. Collectively, these observations demonstrate a critical role of PFC catecholamines in the therapeutic and cognition-enhancing actions of psychostimulants. These results have important clinical, policy and drug discovery implications. Pronexus Analytical AB, Karolinska Institute Science Park, Stockholm, Sweden EVALUATION OF NOVEL ANTIPSYCHOTIC DRUG CARIPRAZINE COMPARED TO ARIPIPRAZOLE BY MICRODIALYSIS IN RAT PCP MODEL OF SCHIZOFRENIA Currently used antipsychotics are effective in improving the positive symptoms, but have relatively little benefit on the negative symptoms and cognitive deficits. Cariprazine, a potent dopamine D3 /D2 receptor partial agonist with preferential binding to D3 receptors and partial agonism on 5-HT1A receptors (Kiss et al., 2010), has recently been approved for the treatment of schizophrenia and manic or mixed episodes in bipolar I disorder. Here, we compared cariprazine and aripiprazole in their ability to modulate the phencyclidine (PCP)-induced changes in extracellular levels of glutamate (Glu), GABA, dopamine (DA), noradrenaline (NA) and serotonin (5-HT), and the acidic metabolites DOPAC, HVA and 5-HIAA measured by microdialysis in the medial prefrontal cortex (mPFC) of rats while simultaneously recording their locomotor activity. Acute per oral treatment with cariprazine attenuated, in a dose-dependent manner, the PCP-induced increases in Glu, DA, NA and 5-HT levels, whereas the levels of DOPAC and HVA significantly increased. Cariprazine at all three doses tested attenuated the PCP-induced increase in locomotor activity. The intermediate dose (0.2 mg/kg) of cariprazine was comparable to, or even more efficient than aripiprazole (3 mg/kg) on attenuation of PCP-induced increases in Glu, DA and NA levels. The GABA levels were not significantly affected by PCP. John Cirrito
Washington University, USA USING MICRO-IMMUNOELECTRODES TO MEASURE RAPID SYNAPTIC-DEPENDENT AMYLOID-BETA GENERATION IN MOUSE MODELS OF ALZHEIMER'S DISEASE Alzheimer's disease (AD) is initiated by the progressive accumulation of amyloid-β (Aβ) peptide in the brain as toxic structures such as Aβ oligomers and plaques. Direct modulation of synaptic activity dynamically regulates brain Aβ levels in awake animals with increased synaptic activity increases brain interstitial fluid (ISF) Aβ levels and vice versa for suppressed activity (Cirrito et al., 2005). These findings strongly suggest a close temporal relationship between synaptic activity and Aβ generation. Aβ generation likely occurs on the order of seconds to minutes. We have recently adapted an electrochemical technique to study Aβ in brain every 60 seconds for several hours. We have covalently attached anti-Aβ antibodies to the electrode surface to provide specificity for Aβ detection to the exclusion of the other proteins and molecules present within the brain extracellular space. Using MIEs in vivo, we are able to detect a rapid increase in ISF Aβ following a rise in synaptic activity. Increases in synaptic activity raise Aβ levels within minutes in the APP/PS1 mouse brain, highlighting the close temporal relationship between synaptic activity and Aβ generation in the brain. Micro-immunoelectrodes provide a novel way to explore mechanisms of this relationship. Giovanna Paolone, Alberto Brugnoli, Ludovico Arcuri, Daniela Mercatelli, Michele Morari
University of Ferrara, Italy ELTOPRAZINE PREVENTS LEVODOPA-INDUCED DYSKINESIA BY REDUCING STRIATAL GLUTAMATE AND STRIATO-NIGRAL NEURON ACTIVITY Dyskinesia is a major complication of levodopa pharmacotherapy of Parkinson's disease. The serotonergic system plays a major role in levodopa-induced dyskinesia. Indeed, selective serotonin 5-HT1A or 5-HT1B receptor agonists, and, very recently, the mixed 5-HT1A/5-HT1B receptor agonist eltoprazine, proved effective in inhibiting levodopa-induced dyskinesia in experimental animals and parkinsonian patients. To investigate the mechanisms underlying the antidyskinetic effect of eltoprazine, microdialysis was employed in i) 6-hydroxydopamine-hemilesioned rats chronically treated with levodopa plus eltoprazine (priming protocol) ii) levodopa-primed dyskinetic rats acutely challenged with levodopa plus eltoprazine (expression protocol). Eltoprazine (0.3 mg/Kg) attenuated the development and expression of dyskinesia, preserving motor coordination on the rotarod. Eltoprazine prevented the rise of nigral amino acids and striatal glutamate levels, as well as the increase in striatal phosphorylated extracellular signal regulated kinase 1 and 2 (ERK1/2), associated with dyskinesia. However, eltoprazine did not affect the levodopa-induced increase of striatal dopamine. We conclude that eltoprazine inhibits the sensitization of striato-nigral GABA neurons (the direct pathway) to levodopa and their overactivation associated with dyskinesia appearance. Activation of 5-HT1A and 5-HT1B receptors regulating striatal glutamate transmission but not striatal ectopic dopamine release might underlie the symptomatic effect of this dose (0.3 mg/Kg) of eltoprazine. Symposium: Monitoring deep brain calcium signaling to illuminate temporal activity dynamics in
neuronal networks during behavior
Chairpersons: Louise Adermark and David M. Lovinger Rui M. Costa
Champalimaud Center for the Unknown, INVESTIGATING THE NEURAL CIRCUITS UNDERLYING ACTION INITIATION USING DEEP-BRAIN IMAGING The ability to decide when to perform and action and what action to perform is critical for survival. Many basal ganglia disorders affect movement initiation. We used deep brain imaging and optogenetics in behaving animals to understand how novel self-paced actions are initiated. Using endoscopic imaging and electrophysiology, we uncovered that transient activity in dopaminergic neurons precedes movement initiation. Using optogenetic manipulations of dopaminergic activity, we showed that dopaminergic activity gates movement initiation. Furthermore, we found using electrophysiology, endoscopic imaging and fiber photometry that basal ganglia direct and indirect pathways are both active before initiation. Finally, we show that both direct and indirect pathway are necessary for movement initiation, but have complementary roles. These data invite new models of how basal ganglia circuits modulate movement initiation. David M. Lovinger, David Kupferschmidt
National Institute on Alcohol Abuse and Alcoholism, USA PLASTICITY OF PRESYNAPTIC CALCIUM TRANSIENTS DURING SKILL LEARNING MEASURED WITH IN VIVO DEEP BRAIN PHOTOMETRY Different corticostriatal circuits are involved in early versus late stages of action learning. However, little is known about activity and plasticity of specific corticostriatal inputs during action learning and performance. We used in vivo fiber photometry to assess intracellular calcium in medial prefrontal cortex (mPFC) to dorsomedial striatum (DMS) and primary motor cortex (M1) to dorsolateral striatum (DLS) afferents. The genetically-encoded calcium indicator GCaMP6s was expressed in mPFC or M1 by viral injection. Optical fibers were later implanted into DMS (mPFC-injected mice) or DLS (M1-injected mice). Mice were then trained to run on an accelerating rotarod for 10 trials per day over 5 days. The M1-DLS afferents showed sustained calcium increases throughout running that rapidly returned to baseline after mice fell from the rod. These responses were robust on trial one, and steadily diminished in magnitude over each trial and each day of training. The mPFC-DMS afferents showed little change in calcium on initial trials, but increases were observed on later day 1 trials, followed by a rapid loss of response on days 2-5. Corticostriatal afferents show strong engagement early that diminishes with skill mastery, with the mPFC-DMS afferents having a very short time window of action-related activity. Dennis R. Sparta
University of Maryland, USA VISUALIZATION OF EXTENDED AMYGDALA CIRCUITS INVOLVED IN ANXIETY Anxiety disorders are the most common neuropsychiatric disorder, affecting over 18.1% of the population in the US. Although brain regions such as the prefrontal cortex, central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST) and hippocampus have been implicated in the development of anxiety disorders, current therapeutic strategies are limited by poor understanding of the specific cells and circuitry involved in mediating this disease. Here, we utilized in vivo calcium imaging in freely behaving mice along with optogenetics and in vivo electrophysiology to dissect genetically distinct populations of projection neurons within the CeA and BNST that modulate anxiety. Preliminary findings demonstrate that functionally opposing BNST to ventral tegmental area circuits regulate anxiety-like behavior, and may serve as a crucial circuit for bidrectionally normalizing maladaptive behaviors. Brian N. Mathur
University of Maryland, USA INHIBITORY MICROCIRCUIT DYNAMICS UNDERLYING ALCOHOL-INDUCED DISINHIBITION OF A HABIT CENTER Habit formation is mediated by the dorsolateral striatum and is expedited by exposure to alcohol. Across species, alcohol exposure disinhibits the dorsolateral striatum by dampening GABAergic transmission onto this structure's principal medium spiny projection neurons, providing a potential basis for habitual alcohol drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. Using a combination of whole-cell patch-clamp recordings and optogenetics, as well as in vivo calcium imaging, we identify the GABAergic striatal fast-spiking interneuron population as a key target of ethanol-induced dorsolateral striatum disinhibition. These findings provide new mechanistic insight to alcohol abuse and its comorbidity with other drug abuse disorders. Sam Golden
National Institute on Drug Abuse, USA THE ROLE OF THE VENTRAL STRIATUM IN ENCODING OPERANT BEHAVIOR FOR NATURAL REWARDS: FOOD AND AGGRESSION. Background and rationale:Inappropriate aggressive behavior, even in the face of severe negative consequences, is commonly co-morbid with a number of neuropsychiatric disorders and presents a major public health concern. The ventral striatum plays a critical role in modulating both adaptive and maladaptive reward behaviors. To identify, compare, and contrast the neural mechanisms governing such behavior we have adapted two tasks in mice for operant seeking of either highly palatable food reward or appetitive aggression reward. We have further paired these tasks with punishment-induced abstinence to reveal mechanisms governing resistance and susceptibility to the suppression of aggression reward or food seeking reward behavior. Experimental approach: In Experiment 1, we trained non-food-restricted outbred CD-1 mice to lever press for highly palatable 20-mg pellets (12.7% fat, 66.7% carbohydrate, 20.6% protein) for 10 consecutive daily 1-h sessions. We then tested the effect of ascending contingent or yoked non-contingent footshock (0.05 mA to 0.35 mA, 0.05 mA increments) on punishment-induced suppression of food-reinforced responding. In Experiment 2, we trained CD-1 mice under identical experimental conditions, but grouped them based on aggression reward phenotype, and then tested the effect of punishment-induced suppression on food-reinforced responding. In Experiment 3, we trained aggressive CD-1 mice to lever press for access to a subordinate social intruder using a block design and a second order conditioning schedule of reinforcement. Key results: Contingent, but not yoked non-contingent, footshock reliably resulted in punishment-induced abstinence from palatable food in mice. However, aggression reward phenotype did not predict susceptibility or resilience to punishment-induced abstinence to a natural food reward. Lastly, aggressive CD-1 mice will reliably lever-press for access to antagonistic aggressive social interactions with subordinate intruders. Conclusions and current studies: We have adapted a model of punishment-induced abstinence from palatable food to mice, and extended it for use with aggression reward. Future studies will reveal the efficacy of punishment-induced abstinence on aggression reward. We are currently using a custom in-house developed miniature fluorescent microscope (miniScope) for deep-brain calcium imaging in awake, behaving mice, that when used in conjunction with GRIN lens implantation and viral infection with genetically encoded calcium indicators, allows for in vivo recording of multicellular striatal activity that encodes aspects of self-administration of, and punishment-imposed abstinence from, natural rewards. Symposium: Hot topics in monitoring exocytosis
Chairperson: Ricardo Borges Kevin D. Gillis1,3 Timothy E. Glass2, Kenneth S. Hettie2, Le Zhang2, and Xin Liu1,3
1 Dept. of Bioengineering, University of Missouri, Columbia, USA 2 Dept. of Chemistry, University of Missouri, Columbia, USA 3 Dalton Cardiovascular Research Center, University of Missouri, Columbia, USA NEW FLUORESCENT SENSORS AND TRANSPARENT ELECTRODES FOR MONITORING EXOCYTOSIS OF CATECHOLAMINES Carbon-fiber microelectrodes have been used for many years to make significant contributions to our understanding of quantal exocytosis of catecholamines. More recently, electrochemical electrode arrays have been used to provide higher spatial resolution of exocytosis and to offer the possibility of higher throughput studies. Our group has developed transparent microelectrodes to allow fluorescent imaging of the site of exocytosis simultaneously with electrochemical measurements of the released transmitter. We have also developed new fluorescent sensors that (more or less) selectively bind norepinephrine and dopamine, but not secondary amines such as epinephrine. We will discuss the properties of these new sensors and how they can be combined with transparent electrodes to quantify the dynamics of catecholamine release. Manfred Lindau
Max Planck Institute, Germany COMBINING MULTI-ELECTRODE DEVICES WITH TIRF MICROSCOPY TO STUDY MOLECULAR MECHANISMS OF EXOCYTOSIS Carbon fiber amperometry is an invaluable technique to investigate properties of single vesicle fusion events and fusion pore dynamics. To investigate molecular events associated with fusion and transmitter release we developed ElectroChemical Detector arrays that consist of multiple amperometric Pt electrodes patterned on a glass coverslip. A cell can be placed in the space between the electrodes and its surface imaged with high resolution in Total Internal Reflection Fluorescence (TIRF) excitation mode. During imaging, the electrodes record fusion events amperometrically and analysis of the amperometric signals provides time and location of individual fusion pore openings with high temporal and spatial resolution. Expressing the SNAP-25 based FRET sensor SCORE in chromaffin cells indicates formation or a conformational change of SNARE complexes 90 ms before fusion and reversing within 5s after fusion. Whole cell patch clamp recordings show that SCORE rescues fusion completely in SNAP-25 knockout cells. Following pulse depolarization a transient FRET change is induced with a rise that parallels the increase of intracellular [Ca2+] and reverses with the same time course as the change associated with individual fusion events. A possible role of the vSNARE Synaptobrevin2 in this change is being investigated. Supported by ERC Grant ADG322699 NIH grant R01MH095046. Valentina Carabelli
University of Torino, Italy DIAMOND MULTIELECTRODE ARRAYS FOR REAL-TIME DETECTION OF OXIDIZABLE NEUROTRANSMITTER RELEASE AND CELL FIRING Taking advantage of diamond high-electrochemical activity, optical features and biocompatibility, we developed diamond-based multiarrays targeted to: i) monitoring catecholamine release with subcellular resolution (high-density arrays), ii) detecting exocytosis from several cells or adrenal slices (low-density arrays) iii) combining amperometric and potentiometric recordings from excitable cells. The diamond chips were constructed either by growing synthetic thin-films of boron-doped diamond on insulating substrates (NCD-MEA) or by drawing sub-superficial highly conductive graphitic micro-paths embedded in the insulating diamond matrix, using deep ion beam lithography (G-SCD-MEA). High-density electrode arrays of boron-doped nanocrystalline diamond electrodes (NCD) were designed with 9-12 electrodes, arranged into a circular hole (20 μm diameter). The secretory activity of single cel s was mapped with high-spatial resolution. Subcellular zones of secretory activity with different frequency of release but comparable spike parameters were identified. The low-density arrays (16-64 channels) effectively detected spontaneous and evoked-catecholamine release from isolated cells cultured on the device and freshly prepared adrenal slices. Sensitivity and time resolution of G-SCD-MEA allowed the detection of different secretory events, associated to "full fusion", "kiss and-run" and "kiss-and-stay" exocytosis. Concerning the last generation of NCD-MEAs (16 channels), different subsets of channels can be selected to specifically detect secretion or action potential firing. Ricardo Borges, Judith Estévez-Herrera, Ayoze G. Santana, Rebeca Baz and José D. Machado.
University of La Laguna. Spain HOW THE CHEMICAL COMPOSITION OF SECRETORY VESICLES AFFECTS EXOCYTOSIS The occurrence of large concentrations of neurotransmitters inside secretory vesicles (SVs) which usually exceed, by far, the theoretical tonicity of cytosol has captivated the attention of scientists along decades. For instance, chromaffin granules are able to accumulate, along with many other products, near molar concentration of catecholamines, hundreds of millimolar of ATP and notable concentrations of calcium, ascorbate, peptides, nucleotides and chromogranins thus forming a vesicular cocktail. We will expose our current view of the interactions of vesicular components emphasizing in their role in the accumulation and exocytosis of catecholamines. We will present data that directly implicate chromogranins (by altering endogenous expression), ATP (by acting on the expression of the vesicular nucleotide carrier) and pH (by pharmacological approach) in these crucial physiological functions. This work is partially paid by the grant BFU2013-45253-P from the MINECO (Spain) to RB and JDM. Raphaël Trouillon, Martin A.M. Gijs
Laboratory of Microsystems, EPFL, Lausanne, Switzerland CELLS-ON-PAPER AS AN OPPORTUNITY FOR EXOCYTOTIC TISSUE MEASUREMENT STUDIES Detecting and quantifying molecules secreted by cells are critical requirements for analyzing physiological phenomena, such as neurotransmission. If the molecule of interest is electroactive, electrochemistry provides powerful analytical tools, but often requires complicated techniques and equipment. A simple electroanalytical setup, allowing for the use of a wide range of samples, would therefore be beneficial. Here, a hybrid microfluidic/ electrochemical system is described for the detection of dopamine from a population of PC12 cells cultured onto the surface of filter paper. The system is fully characterized electrochemically. Furthermore, using fluorescence microscopy, it is demonstrated that the cells strongly adhere to the surface of the paper substrate. Dopamine release after stimulation with acetylcholine is observed, and the effects of the drug L-3,4-dihydroxyphenylalanine are also studied. These results are in good agreement with data previously published at the single cell level. However, dynamin inhibition, which is known to hinder exocytosis, is more potent on the paper sample than at the single cell level, thus hinting that a bottom-up collective cell approach can be required to translate individual cell mechanisms to tissue behavior. Overall, this study demonstrates the validity of our system for chemical analyses at cells or artificial cell constructs, such as organs-on-a-chip. Symposium: In vivo microdialysis and voltammetric techniques for studies of ethanol´s
mechanism of action along the mesolimbic dopamine reward system
Chairpersons: Bo Söderpalm and Rueben Gonzales Rueben Gonzales
The University of Texas at Austin, USA THE USE OF IN VIVO MICRODIALYSIS TO EXPLORE THE RELATIONSHIP BETWEEN BRAIN ETHANOL CONCENTRATIONS AND ACTIVATION OF MESOLIMBIC AND MESOCORTICAL DOPAMINE SYSTEMS Ethanol has long been known to stimulate dopamine release in the mesolimbic system, and this effect has been suggested to underlie some of the rewarding and motivational effects of ethanol. However, the dose-response for ethanol varies with respect to the dopamine release sites. To gain further insight into this variability in ethanol's actions, we performed microdialysis studies in various mesolimbic and mesocortical sites (subregions of the ventral and dorsal striatum and medial prefrontal cortex, mPFC). Microdialysis allows sampling of both dopamine and ethanol concentrations that reach the tissue. Male Long-Evans rats were used as subjects. Intravenous ethanol produced increases in dopamine in the shell that were significantly higher than those in the core. A significant dopamine response was also observed in the mPFC. The dorsal striatum was less sensitive to ethanol compared with the ventral striatum, but the dorsomedial striatum had a higher response than the dorsolateral striatum. The dopamine responses were dramatically different in the ventral striatum and medial prefrontal cortex after ethanol self-administration. Our results indicate that both contingent and noncontingent ethanol administration stimulates mesolimbic and mesocortical dopamine responses. Together, our findings support the idea that the stimulatory effects of ethanol on mesocortical and striatal dopamine signaling may contribute to the reinforcing properties of ethanol, albeit with differential behavioral roles. Scott Steffensen
Brigham Young University, USA DIFFERENTIAL EFFECTS OF ETHANOL ON BASAL AND PHASIC DOPAMINE RELEASE Dopamine (DA) neurons originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) have been implicated in alcohol reward. The prevailing dogma is that the rewarding properties of ethanol are mediated by enhancement of mesolimbic DA transmission to the NAc. Mainly, microdialysis studies have shown that spontaneuous "basal" DA release in the NAc is increased by ethanol and voltammetry studies have reported increases in DA transient frequency after ethanol. However, numerous ex vivo and in vivo voltammetry studies from our lab and others have reported decreases in high frequency electrically-evoked DA release by ethanol, a model of "phasic" release thought to emulate DA neuron bursting activity. We show that phasic DA release is inhibited by physiologically-relevant concentrations of ethanol and mediated by co-activation of VTA GABA neurons projecting to the NAc. Ethanol modulation of DA release at terminals in the NAc is mediated by atypical GABAARs on cholinergic interneurons (CINs) containing ρ1-subunits and terminals. Interestingly, ethanol enhancement of basal DA release in the NAc appears to be mediated by peripheral D2 receptors, which explains many of the discrepancies in the field regarding ethanol effects on DA neurons and DA transmission. Studies are ongoing to evaluate the role of peripheral mechanisms in mediating ethanol enhancement of basal DA release. Bo Söderpalm, Susanne Jonsson, Julia Morud, Mia Ericson
University of Gothenburg, Sweden SINGLE- AND DOUBLE-PROBE IN VIVO MICRODIALYSIS TECHNIQUES TO EXPLORE THE NEUROCIRCUITRY INVOLVED IN ETHANOL "REWARD" Activation of the mesolimbic dopamine system is a common mechanism involved in the reinforcing effects of drugs of abuse, including ethanol. Determining the mechanisms of action of ethanol involved along this system may open up for suggestions for treatments selectively preventing ethanol reinforcement, either by antagonism or by means of substitution with drugs less hazardous than ethanol. By applying one microdialysis probe in nucleus accumbens (nAc) and one in the ventral tegmental area (VTA) or in the lateral septum (LS) in the rat, and by administering ethanol locally, systemically or by voluntary intake, and simultaneously manipulate receptor populations via reversed perfusion, we have suggested that ethanol raises dopamine levels in nAc by engaging a nAc-(LS)-VTA-nAc neurocircuitry,,that involves glycine receptors in nAc and nicotinic acetylcholine receptors in VTA. Interestingly, manipulations of both these receptor populations can reduce ethanol intake in the rat and recent results from two randomized controlled trials in alcoholics (one in the U.S. and one in Sweden) indicate that the nicotinic receptor concept translates to the human situation. Mia Ericson, Rosita Stomberg, Bo Söderpalm
University of Gothenburg, Sweden COMBINED ANALYSIS OF BRAIN EXTRACELLULAR LEVELS OF AMINO ACIDS AND DOPAMINE FOR ELUCIDATING THE EFFECTS OF ETHANOL IN THE NUCLEUS ACCUMBENS Elucidating the mechanism by which ethanol activates the mesolimbic dopamine system is of great importance since dopamine elevation in the nucleus accumbens (nAc) is one of the few common denominators of all addictive drugs. When defining how ethanol increases nAc dopamine we previously found the glycine receptor (GlyR) to be of importance. Since others found ethanol to increase nAc taurine, an endogenous ligand of GlyRs, we started to utilize a paradigm where we were able to simultaneously monitor extracellular levels of dopamine and amino acids with affinity for the GlyR. Using in vivo microdialysis in Wistar rats, where we split the dialysate samples for analysis in two separate HPLC systems, we found that in order for ethanol to increase dopamine there needs to be an initial increase of extracellular levels of nAc taurine. We have also explored some pharmacological compounds known to decrease ethanol consumption in humans and found that several drugs indeed mediate their effects by modulating amino acids with affinity for GlyRs. Thus, combined monitoring of extracellular levels of dopamine and the amino acids glycine and taurine have provided new insights in the mechanism by which ethanol activates the mesolimbic dopamine system. Abigail G. Schindler1, Marta E. Soden1,2, Larry S. Zweifel1,2, Jeremy J. Clark1
1 Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA 2 Department of Pharmacology, University of Washington, Seattle, Washington, USA REVERSAL OF ADOLESCENT ALCOHOL-INDUCED DYSREGULATION IN DOPAMINE NETWORK DYNAMICS RESCUES MALADAPTIVE RISK TAKING Alcohol is the most commonly abused substance among adolescents, promoting the development of substance use disorders and compromised decision-making in adulthood. We have previously demonstrated that adolescent alcohol use results in adult risk-taking behavior that positively correlates with phasic dopamine transmission in the nucleus accumbens core (NAcc) in response to risky options, but the underlying mechanisms remain unknown. Here we utilize a multifaceted approach including fast-scan cyclic voltammetry (FSCV), slice electrophysiology, microdialysis coupled with UPLC-MS/MS and behavioral pharmacology to address these questions. Using these techniques we demonstrate that adolescent alcohol use produces maladaptive decision making through a disruption to dopamine network dynamics via increased GABAergic transmission within the VTA. This work supports a model where increased inhibitory tone on dopamine neurons leads to a persistent decrease in tonic dopamine levels and promotes a potentiation in stimulus-evoked phasic dopamine release that drives risky choice behavior. Based on these findings we demonstrate pharmacological reversal of risk-taking behavior in parallel with normalization of dopamine transmission using the GABA(A) allosteric agonist L-838,417. Together these results isolate the underlying circuitry involved in alcohol-induced maladaptive decision making and identify a potential therapeutic target. Wine and cheese session: Challenges for in vivo Biosensors
Chairperson: Ann-Sofie Cans Cátia M. Lourenço1, Ana Ledo1, João Laranjinha1, Greg A. Gerhardt2, Rui M. Barbosa1
1 Center for Neuroscience and Cell Biology and Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal 2 Department of Anatomy and Neurobiology, Center for Microelectrode Technology, University of Kentucky, Lexington, KY 40536, USA ENZYME-BASED MICROBIOSENSOR ARRAYS FOR HIGH-RESOLUTION MEASUREMENTS OF GLUCOSE AND OTHER METABOLITES IN THE BRAIN Enzyme-based microbiosensors associated with fast electrochemical techniques are attractive analytical tools to study brain energy metabolism, allowing monitoring metabolic substrates with high spatial and temporal resolution and minimal tissue damage. Ceramic-based platinum multisite microelectrode arrays (MEAs) fabricated by photolithographic techniques exhibit well-defined and reproducible geometrical design configurations and are of particularly interest for neurochemical measurements, allowing recordings from multiple brain areas or multiple neuroanalyte detection. Here, we explored such microelectrode platform for the development of enzyme-based microbiosensors for glucose and lactate measurements. Microbiosensors were prepared by immobilizing oxidase enzymes through cross-linking with glutaraldehyde and subsequent coating with m-phenylenediamine. Analytical performance was evaluated in terms selectivity, sensitivity, linear range, operational stability, response time, and oxygen dependency. Suitability for brain measurements was demonstrated by the ability to measure glucose and lactate with high temporal and spatial resolution in the hippocampus of anesthetized and awake rats. The use of a self-referenced coating scheme allowed us to estimate basal extracellular levels. Furthermore, the use of a microbiosensor to measure changes of metabolic markers together with local field potential-related currents in awake behaving rats is discussed. Acknowledgements: This work is funded by COMPETE and FCT under the projects PTDC/BBB-BQB/3217/2012, UID/NEU/04539/2013, and from Calouste Gulbenkian Foundation. Catherine Sweatman, Sarah Isherwood, Janet Nicholson, Kelly Allers
Boehringer Ingelheim, Germany QUANTIFICATION AND MODULATION OF GLUTAMATE RELEASE IN RESPONSE TO RESTRAINT STRESS IN RATS The advent of enzyme based biosensors holds great promise as a method of measuring changes in neurotransmitter release within discrete brain regions of conscious, freely moving animals, thus allowing physiology to be directly compared with behaviour. Biosensors allow second by second analysis of specific neurotransmitters via double-coating with the specific enzyme of interest, in this case Glutamate Oxidase, and Ascorbate Oxidase to protect against common electroactive interferents. The oxidation of glutamate via Glutamate Oxidase on the Pt-Ir electrode results in a nA signal and is recorded via Bluetooth by the Pinnacle Sirenia Acquisition program. Using glutamate specific biosensors, we investigated stress induced changes in glutamate level within the prefrontal cortex using a variety of different stressors and investigating differences between Wistar, Sprague Dawley and Lister Hooded rat strains. Using the restraint stress paradigm, we investigate the size and duration of the response, the effects of repeated exposure to restraint stress and mechanistic modulation of this stress response. Modulators of glutamate release (mGlu2 and mGlu5 positive allosteric modulators) are shown to effect restraint stress induced glutamate levels. Isabelle C. Samper, Sally A. N. Gowers, Michelle L. Rogers, Chi L. Leong, Sharon Jewell, Anthony J.
Strong, Martyn G. Boutelle
Imperial College London, UK A NOVEL WIRELESS BIOSENSING SYSTEM FOR REAL-TIME DETECTION OF SPREADING DEPOLARISATIONS IN THE INJURED HUMAN BRAIN The recent consensus statement from the 2014 International Microdialysis Forum [1] identified the need for continuous real-time chemical monitoring of traumatic brain injury patients. Spreading depolarisations (SDs) are important secondary insults that place an extreme dynamic challenge on the energy supply to the tissue. Propagating through the cortex and around the injury, SDs are associated with poor patient outcome [2]. On-line neurochemical monitoring of energy availability in "at risk" tissue enables detection of the metabolic consequences of SDs giving a meassamanure of the expansion of the injury. Current wired systems prevent patient mobility and can obstruct routine clinical care. Therefore, there is a need to build a wearable device to extend the patient monitoring period. We have designed and built a device consisting of a 3D-printed microfluidic flow-cell that attaches to a clinical microdialysis probe. It holds glucose and lactate amperometric biosensors, connected to our newly engineered wireless potentiostat. Neurochemical changes in the injured brain were simulated using an automated microfluidic board and detected with this novel wireless system. These results will be presented together with preliminary patient data. 1. Hutchinson et al. Intensive care Med. DOI: 10.1007/s00134-015-3839-5 (2015) 2. Hartings et al. The Lancet Neurology, 10, 1058–64 (2011) Ricardo Santos
Ludwig Maximilian University of Munich, Germany PROBING CHOLINERGIC ACTIVITY AND CORTICO-HIPPOCAMPAL NETWORK DYNAMICS BY COMBINED ELECTROCHEMISTRY AND MULTICHANNEL ELECTROPHYSIOLOGY IN FREELY MOVING RATS Acetylcholine (ACh) is a key modulator of state-dependent activity and synaptic plasticity. Cholinergic signals span a wide time range, correlated to brain internal dynamics and sensory inputs and behavior. Here we simultaneously assessed cholinergic tone, cortico-hippocampal network dynamics and behavior, as crucial missing links to characterize ACh role in cognition. Cholinergic activity was monitored by amperometric measurement of H2O2 generated by choline oxidase (ChOx) in presence of Choline. The microelectrode was a Pt/Ir wire tetrode who's sites were differential y modified with gold and platinum plating, as well as m-phenylenediamine electropolymerization to optimize removal and separation of LFP-related currents and chemical interferents, while keeping response time and sampling volume minimal. Cholinergic and electrophysiological signals across cortical and hippocampal layers were recorded using a movable microbiosensor and a 32-channel linear probe, respectively. Rat behavior was 3D-tracked at fine spatio-temporal resolution in open-field arena. Cholinergic tone was higher during REM sleep and wakefulness than slow-wave sleep. Choline also changed at the seconds time-scale within states, associated with patterns of internal activity, including sharp-wave/ripples and with particular behavior correlated with arousal, novelty and locomotion. Results highlight ACh actions at multiple time-scales, providing new insights into its functional effects on network dynamics impacting cognition. Nako Nakatsuka
MONITORING NEUROTRANSMITTERS IN VIVO AT THE NANOSCALE TO CORRELATE CHEMICAL SIGNALS WITH BRAIN DISORDERS We are developing novel approaches to revolutionize our understanding of neural circuits involved in emotion-related behavior in healthy and disease-related states. Elucidating how alterations in complex neuronal networks contribute to brain disorders necessitates the use of chemically specific in vivo neurotransmitter sensors whose measurement parameters approach the length and time scales pertinent to intrinsically encoded information. To meet the need for ultra-fast, ultra-small, and chemically selective biosensors, we are selecting and investigating aptamers as artificial receptors. Aptamers are immobilized on field-effect transistors (FETs) produced by chemical lift-off and photolithography. Multiplexed-FETs function as neurotransmitter-recording elements. Devices functionalized with dopamine and serotonin-specific aptamers have been used to measure neurotransmitter levels in phosphate-buffered saline or cerebrospinal fluid to sub-picomolar levels in vitro. These devices can differentiate serotonin and dopamine in the presence of other closely structured neurotransmitters and metabolites. Ongoing work is focused on testing and transitioning these devices for use in vivo. Wine and cheese symposium: Novel electrochemical approaches to the study of cognition
Chairpersons: John P. Bruno and Mark Walton
Åsa Konradsson-Geuken, Thomas Viereckel and Åsa Mackenzie
Uppsala University and Karolinksa Institute, Sweden SHINING LIGHT ON THE ROLE OF GLUTMATE IN COGNITION In psychiatric disease, the level of cognitive function has been increasingly recognized as an important factor in determining treatment outcome. The prefrontal cortex plays a critical role in multiple cognitive functions, e.g. cognitive flexibility. However, our knowledge regarding the intricate cortical network regulating cognition as well as, the fast acting neurotransmitters involved in higher cognition is somewhat limited. In this study, we used optogenetics to activate distinct cell populations in transgenic animals to measure cortical glutamate release utilizing in vivo amperometry. Mice expressing Cre recombinase under regulatory control of the promoter for the Vglut2 gene in the ventral tegmental area were injected with Cre-dependent adeno-associated viruses carrying Channelrhodopsin-2. Optogenetics allowed us to control neuronal activities with millisecond precision in targeted neurons while the use of amperometry allowed real-time quantification of cortical glutamate with an outstanding spatial resolution in vivo. Our results will give a deeper insight in how and from what source, cortical glutamate regulates phasic release in cognitive control. Moreover, our data will provide new information on how new drug candidates can be modified to interact with the cortical glutamatergic system with better effect on cognitive dysfunctions. Mark Walton
University of Oxford, UK USING ELECTROCHEMISTRY TO TRACK REWARD PREDICTION IN ACTION The firing of many midbrain dopamine cells correlates with reward prediction errors derived from reinforcement learning models used to update value functions. However, pharmacological disruptions of dopamine transmission in terminal regions commonly cause changes in motivation and movement as well as in value updating. I will discuss some recent electrochemistry studies that attempt to shed light on the interactions between these factors during reward-guided behaviour. Kate M. Wassum, Melissa Malvaez, Harold Monbouquette
BASOLATERAL AMYGDALA GLUTAMATE DURING VALUE ENCODING AND DECISION MAKING Adaptive reward seeking requires the ability to extract from the environment and mentally represent information about specific available rewards. The basolateral amygdala (BLA) participates in this cognitive process, but precisely how is unknown. There is a particular lack of information regarding how BLA input signals relate to and influence reward-seeking behavior. The BLA receives dense glutamatergic innervation from both cortical and thalamic structures, so we focused on measuring this excitatory input signal during several tests of reward seeking. To achieve this, we used an electroenzymatic glutamate biosensor technology that affords near-real time, sensitive, and selective measurement of glutamate concentration changes in the brains of freely-behaving rodents. Transient elevations in glutamate concentration were detected in the BLA during reward-related behavior. Targeted behavioral tests revealed that these glutamate transients occurred when rats encoded the value of specific rewards and also when the mental representation of those rewards was called upon to guide reward-seeking decisions. Interference methods were used to provide a complementary causal analysis to the correlational recording approach. John P. Bruno, David Bortz, Valentina Valentini, and Jackson Schumacher
The Ohio State University, USA POSITIVE ALLOSTERIC MODULATORS OF ALPHA7 NICOTINIC RECEPTORS AND THEIR EFFECTS ON PREFRONTAL CHOLINE AND GLUTAMATE RELEASE: IMPLICATIONS FOR COGNITION-ENHANCING DRUGS Positive allosteric modulators (PAMs) of alpha7 nicotinic acetylcholine receptors (nAChRs) better preserve endogenous cholinergic transmission than direct agonists because agonists stimulate post-synaptic receptors independent of presynaptic afferent activity. In vitro data suggest that PAMs' effects depend upon nAChR activity, but in vivo demonstrations are lacking. Thus, we utilized a protocol of stimulating the nucleus accumbens shell (NacSh) with NMDA to dose-dependently evoke ACh/choline release in PFC (procedure also improved performance on an attention task) in order to test the hypotheses that a) this procedure increases cortical glutamate release, b) that alpha7 stimulation is necessary for glutamate release, and c) that PAM's would potentiate the levels of cortical glutamate as a function of type of PAM and local concentration of choline. Adult male Wistar rats received unilateral infusion cannulae into their NacSh and a choline- or glutamate-sensitive biosensor in their ipsilateral mPFC. Rats were infused with NMDA (aCSF, 0.05, or 0.30 µg/0.5 µL) 40 minutes after a systemic injection of either AVL3288 (type I PAM, 1-3 mg/kg) or PNU120596 (type II PAM, 3-9 mg/kg). Both PAMs displayed the capacity to potentiate glutamate release but only under conditions of moderate cholinergic activity. Thus, care must be exercised in selecting doses for studies testing cognition enhancement. Gary Gilmour
Eli Lilly & Co. Ltd., Erl Wood, UK IN VIVO OXYGEN AMPEROMETRY: A TRANSLATIONAL TOOL FOR MEASUREMENT OF BEHAVIOURAL CONTROL AND ITS UTILITY FOR NEUROPSYCHIATRIC DRUG DISCOVERY CNS drug discovery efforts have recently faced significant challenge from a series of failures. Often, these failures have happened in Phase III trials, where in many cases it was never unequivocally established that the drugs of interest got into the brain at the required levels and/or functionally engaged with the target of interest, in a manner that they would have been previously demonstrated in preclinical studies. Functional neuroimaging studies are increasingly being used in an attempt to bridge this translational gap, but these studies still happen relatively (and expensively) late in the discovery process. There is a clear need to be able to conduct the equivalent of functional neuroimaging studies in animals to help make decisions earlier, and ideally before moving to the clinical stage. In vivo oxygen amperometry is ideally placed to fill this gap, being a method of measuring brain tissue oxygen levels in real-time from several regions in awake, behaving animals. Amperometry in animals can effectively recapitulate the three main protocol variants of BOLD imaging, i.e. pharmacological MRI, task-based MRI, and MRI measures of functional connectivity, and each of these protocol variants can be illustrated by examples of current relevance to drug discovery. Some of the greatest advances in bridging the translational gap here lie in the field of behavioural control, where some striking homologies have been observed between studies of nucleus accumbens function in simple reward-motivated tasks between rodents and humans. This presentation will provide an overview of the state-of-the-art of this literature. Symposium: Probing, sniffing, lighting the brain in vivo: the multiple facets of biosensing
Chairpersons: Stephane Marinesco and Ann-Sofie Cans Yang Tian
East China Normal University, China ELECTROCHEMICAL DETERMINATION OF REACTIVE OXYGEN SPECIES AND RELATED MOLECULES IN THE LIVE RAT BRAINS Reactive oxygen species including superoxide anion, hydroxyl radical, and hydrogen peroxide, and so on has gained increasing attention in a variety of research fields, because they are considered as the mediators of the biochemistry of cellular pathology and may be involved in the etiology of aging and progressive neurodegenerative diseases, such as Parkinson's disease. However, one of the major obstacles to understand the roles of ROS play is the lack of suitable and efficient methods for in vivo detection of ROS due to their several special natures, such as low amount, short half-life, high reactivity, and easy conversion to each other, and so on. We are interested in development of new strategy for electrochemical detection of ROS and the related biological molecules in the live rat brains through rationally functionalizing electrode surface, aiming to the demand for selectivity, sensitivity, and accuracy. We developed a series of approaches for selective detection of ROS using dual recognition strategies by potential and specific enzyme to enhance the selectivity, and efficient construction of nanostructured surface. The ratiometric electrochemical surfaces were designed to greatly improve accuracy of in vivo detection in the live rat brains. Lanqun Mao
Chinese Academy of Sciences, China IN VIVO ELECTROCHEMISTRY TO UNDERSTAND PHYSIOLOGICAL ROLES OF ASCORBATE To understand the molecular basis of brain functions, researchers would like to be able to quantitatively monitor neurochemicals in vivo. However, the chemical and physiological complexity of the central nervous system (CNS) presents challenges for the development of these analytical methods. We used the redox nature of neurochemicals at the electrode/electrolyte interface to form a basis for selectively monitoring neurochemicals. In this presentation, I would introduce the recent process in our group on in vivo monitoring ascorbate in rat brain. Paul Slesinger1, Arnaud Muller2, Victory Joseph2 and David Kleinfeld2
1 Icahn School of Medicine at Mount Sinai, Manhattan, New York, USA 2 University of California, San Diego, USA A TOOL FOR OPTICALLY MEASURING IN VIVO THE CHANGES IN VOLUME TRANSMISSION OF DOPAMINE AND NOREPINEPHRINE IN MOUSE CORTEX DURING LEARNING We have developed implantable cell–based neurotransmitter fluorescent engineered reporters (CNiFERs) to study release of neuromodulators in vivo. A CNiFER is created by engineering a HEK293 cell to express a particular type of G protein-coupled receptor (GPCR) along with a FRET-based calcium detector. We have generated a CNiFER for detecting norepinephrine (NE), using the α1a adrenergic GPCR, and one for detecting dopamine (DA), using the D2 GPCR. To resolve the spatial and temporal dynamics of monoamine concentrations during conditioning, we used a two-photon microscope for in vivo imaging of α1a- and D2-CNiFERs implanted into the frontal cortex of mice. We monitored FRET over 5 days of conditioning trials, comprised of a 5-s tone followed 3-s later with a food reward. The onset of DA release correlated with that of licking and shifted from the time of the reward toward that of the cue upon conditioning. Release of NE did not correlate with licking or the cue. CNiFERs have a high spatial resolution (< 100 µm), a fast temporal resolution ( 2 s), and the ability to distinguish chemically similar transmitters. The molecular design of CNiFERs can be generalized to realize CNiFERs to optically detect any molecule that activates a GPCR. Martine Eilert-Olsen, Jarand Berg Hjukse, Rune Enger, Wannan Tang, Anna E. Thoren, Vidar Jensen,
Klas H. Pettersen, Erlend A. Nagelhus
University of Oslo, Norway A NEW LIGHT ON IONIC SHIFTS ELICITED BY ASTROCYTE SWELLING Being encased in the rigid skull the brain must possess mechanisms for effective volume control. Astrocytic endfoot processes form the outermost layer of nervous tissue and are strategically positioned to regulate the movement of ions and water between the parenchyma and extracerebral liquid compartments, e.g. the subarachnoid space and vasculature. Using the synthetic Ca2+ indicator Rhod2 we previously showed that brain swelling triggers Ca2+ signaling in astrocyte cell bodies and that deletion of the Aqp4 gene markedly interferes with these events (Thrane AS et al., PNAS 108:846-51, 2012). Limitations of the synthetic Ca2+ indicators prevented us from investigating the Ca2+ dynamics in delicate astrocytic processes and endfeet. The advent of ultrasensitive genetically encoded Ca2+ indicators have enabled investigation of Ca2+ signaling in all astrocytic microdomains, including subpial end perivascular endfeet. Here I will present and discuss our recent two-photon imaging data on the dynamics and mechanisms of swelling-induced Ca2+ signals within the entire astrocytic territory and of swelling-induced glutamate release into the extracellular space. The changes in [Ca2+]i and [glutamate]e were detected by GCaMP6f and iGluSnFR, respectively, delivered by recombinant adeno-associated virus 2-4 weeks prior to imaging. Our findings indicate that astrocytic endfeet display unique signaling responses during conditions of reduced extracellular osmolality. Emrah Eroglu, Markus Waldeck-Weiermair, Wolfgang F. Graier, Roland Malli
Medical University of Graz, Austria DEVELOPMENT OF NOVEL FP-BASED PROBES FOR LIVE-CELL IMAGING OF NITRIC OXIDE DYNAMICS Nitric oxide (NO•), a free radical with a wide range of biological effects, is practically impossible to visualize in single cells. Here we report the development of novel multicolored fluorescent quenching-based NO• probes by fusing a bacteria-derived NO•-binding domain close to distinct fluorescent protein variants. These genetical y encoded NO• probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular NO• dynamics and, hence, open a new era of NO• bio-imaging. The combination of geNOps with a Ca2+ sensor al owed us to visualize NO• and Ca2+ signals simultaneously in single cells. Moreover, targeting of the NO• probes was used to detect NO• signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of NO• signaling on the single (sub)cellular level. Symposium: CSF biological markers in psychiatric disorders
Chairperson: Mikael Landén and Erik Pålsson Kenji Hashimoto
Chiba University, Japan METABOLOMICS ANALYSIS OF CSF FROM PATIENTS WITH BIPOLAR DISORDER Although evidence for mitochondrial dysfunction in the pathogenesis of bipolar disorder (BD) has been reported, the precise biological basis remains unknown, hampering the search for novel biomarkers. In this study, we performed metabolomics of cerebrospinal fluid (CSF) from male BD patients and age-matched male healthy controls. Subsequently, postmortem brain analyses, gene analyses, metabolomics of CSF samples from rats treated with lithium or valproic acid were also performed. After multivariate logistic regression, isocitric acid (isocitrate) was significantly higher in the CSF from BD patients than healthy controls. Furthermore, gene expression of subtypes of isocitrate dehydrogenase (IDH) in the dorsolateral prefrontal cortex from BD patients was significantly lower than that of controls. Moreover, the expression of IDH protein in the postmortem brain from BD was lower than that of controls. In addition, chronic (4-weeks) treatment with lithium or valproic acid in rats did not alter CSF levels of isocitrate in the rat brain. These findings suggest that abnormality in the metabolism of isocitrate by IDH in the mitochondria plays a key role in the pathogenesis of BD, supporting the mitochondrial dysfunction hypothesis of BD. Ana Andreazza1, Gustavo Scola1, Joel Jakobson2 and Mikael Landen2
1 Departments of Pharmacology and Psychiatry , Centre for Addiction and Mental Health, University of Toronto, Canada 2 Department of Psychiatry and Neurochemistry, Sahlgrenska Academy of the University of Gothenburg, Sweden REDOX MODULATIONS IN PSYCHIATRIC ILLNESSES There is increasing evidence that energy dysfunction through mitochondrial dysfunction and consequent cellular redox modulation may play a role in the determination and expression of psychiatric illnesses. In recent years, sophisticated clinical measures are unveiling links between mitochondrial dysfunction, redox modulation and disease domains. Moreover, preclinical work is convergent in identifying redox modulations as a key element in neuronal connectivity and overall brain function. Thus, Dr. Andreazza will provide an overview of the role of mitochondrial bioenergetics and redox modulations in psychiatric disorders, including the presentation of unpublished data in cerebrospinal fluid that move the field forward in identifying these pathways as pathophysiological mechanisms in psychiatric disorders. She will first discuss clinical evidence of altered redox modulations and mitochondrial function in patients with mood disorders and schizophrenia, and then Dr. Andreazza will discuss the link among mitochondrial bioenergetics, redox modulations and inflammatory pathways in postmortem brain, cerebrospinal fluid and peripheral samples. Overall, Dr. Andreazza will highlight the above-mentioned mechanisms as functionally-linked cellular processes likely to be involved in the biological changes that underlie the expression of behavioral traits characteristic of psychiatric disorders. Sophie Erhardt
Karolinska Institute, Sweden DEFICIENT REGULATION OF KYNURENINE METABOLITES UNDERLIE VULNERABILITY TO PSYCHIATRIC ILLNESS The kynurenine pathway is activated by inflammation and might trigger psychiatric symptoms due to production of metabolites regulating glutamate neurotransmission. High levels of the pro-inflammatory cytokine interleukin (IL)-1b as well as of the N-methyl-D-aspartate (NMDA) receptor antagonist kynurenic acid (KYNA) are observed in the cerebrospinal fluid (CSF) of patients with psychosis. In suicide attempters, high levels of IL-6 and the NMDA receptor agonist quinolinic acid (QUIN) are observed. The enzyme aminocarboxymuconate-semialdehyde-decarboxylase (ACMSD) limits QUIN formation by competitive production of picolinic acid (PIC), and decreased activity of ACMSD therefore represents a plausible source of excess QUIN. In the present study, PIC and QUIN were analyzed in patients exhibiting suicidal behavior and healthy controls. Suicide attempters had reduced amounts of PIC and a decreased PIC/QUIN ratio in both CSF and blood. The minor C allele of the ACMSD SNP rs2121337 was associated with increased QUIN in CSF and was more prevalent in suicide attempters compared to healthy controls. Increased QUIN formation in patients exhibiting suicidal behavior may result from reduced activity of ACMSD. The plasma PIC/QUIN ratio could be a useful biomarker of suicide risk, and modulating the activity of ACMSD should be explored in order to develop novel anti-suicidal medications. Joel Jakobsson, Mikael Landén
Sahlgrenska Academy of the University of Gothenburg, Sweden LARGE-SCALE CEREBROSPINAL FLUID AND SERUM BIOMARKER PROFILING IN PATIENTS WITH BIPOLAR DISORDER Bipolar disorder is a severe psychiatric condition characterized by recurrent episodes of elevated and depressed mood. The biochemical abnormalities associated with the pathophysiology of bipolar disorder are unclear and we need a better understanding of disease mechanisms to develop new prevention and rehabilitation strategies. Previous studies on peripheral markers have been restricted to analyses of one or a few biomarkers at a time. Given the multifactorial etiology and heterogeneous expression of bipolar disorder it is unlikely that a single marker will have sufficient sensitivity and specificity for a certain diagnosis or clinical outcome and instead multi-marker profiles in combination with clinical assessments will be required. Here, we use a new multiplexed immunoassay technique that with high sensitivity and specificity can analyze 92 markers simultaneously in only one µl sample, Proseek Multiplex. We used three different panels (i.e., oncology, cardiovascular disease, and inflammation) to analyze cerebrospinal fluid and serum from two large independent bipolar disorder populations (St. Göran Bipolar Project), generating data on a total of 202 unique markers in CSF (N=209 patients and 144 controls) and serum (N=337 patients and 167 controls). Analytical data (sensitivity, specificity, intra- and inter-assay variation) and patient-control differences will be presented. Jessica Holmén Larsson1, Mikko Hölttä1, Carl Sellgren2, Carl-Johan Ekman3, Kaj Blennow1, Henrik
Zetterberg1,4, Johan Gobom1, Mikael Landén1
1 Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg,
2 The Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden
3 The Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
4 UCL Institute of Neurology, Queen Square, London, UK
The aim of this study was to explore pathophysiological mechanisms for Bipolar disorder (BD) and to
identify novel biological CSF markers for diagnostic purposes and for prediction/evaluation of treatment
responses. CSF contains large numbers of proteins and neuropeptides with great potential value as
We have analyzed CSF from 15 BD patients and 15 age- and sex-matched controls. The CSF proteins
were trypsin digested and labeled with isobaric Tandem Mass Tag (TMT) reagents that enable
multiplexed semi-quantification of hundreds of proteins from small
digested CSF proteins were subsequently analyzed with high-resolution liquid chromatography-mass
spectrometry (nano-LC-MS/MS).
In this exploratory study, 676 proteins were identified and semi-quantified. 36 proteins showed
significantly (p<0.05) altered levels in the BD patients as compared to controls. A majority of the altered
proteins are brain-specific and involved in e.g. cell growth/communication/adhesion, immune response
and protein metabolism. Some of them have previously been implicated in neuropsychiatric disorders on
genetic level, and some are novel findings and could be potential biomarker candidates. The results will
now be validated in two larger independent BD cohorts.
Symposium: Food for thought – monitoring the energetic costs of neurotransmission
Chairperson: Martyn G. Boutelle Eugene A. Kiyatkin
National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, USA DIRECT MONITORING OF GLUCOSE ENTRY INTO THE BRAIN: ENERGETIC AND INFORMATIONAL ASPECTS Glucose is the primary energetic substrate for the metabolic activity of brain cells and its proper delivery from the arterial blood is essential for normal brain functions. Here, I want to review our recent findings obtained with the use of enzyme-based glucose biosensors coupled with amperometry in freely moving rats. After a short introduction of the technical aspects of electrochemistry, I will consider normal physiological fluctuations in extracellular glucose levels in the nucleus accumbens (NAc) and demonstrate its relation to neural activity and activity-related changes in cerebral vascular tone. These changes in glucose consistently precede much slower changes in metabolic activity, suggesting "anticipatory" glucose entry into the brain to prevent any possible energetic deficit. In addition to rapid but relatively small increases in NAc glucose induced by arousing stimuli, glucose levels substantially increase after glucose-drinking behavior, correlating with the periods of inactivity between individual drinking bouts. This correlation suggests the role of glucose entry into the brain as a critical factor regulating glucose-drinking behavior and, possibly, any consummatory feeding behavior. Finally, I will present data on NAc glucose fluctuations induced by several drugs of abuse, suggesting important role of drug-induced changes in vascular tone in their mediation. J. Kealy, R. Bennett, John P. Lowry
Maynooth University, Ireland INVESTIGATING BRAIN ENERGY METABOLISM IN VIVO USING REAL-TIME MICROELECTROCHEMICAL SENSORS FOR GLUCOSE AND OXYGEN Neurodegenerative diseases represent a continually growing major unmet need for therapeutic intervention. Chronic deficits in energy metabolism, in particular, are known to occur with age and neurodegeneration, possibly contributing to both nerve cell loss and memory decline. Indeed, a reduction in cerebral glucose metabolism, as measured by FDG-PET, is commonly used in the diagnosis of Alzheimer's disease (AD). Also, glucose administered systemically to humans and rats significantly reverses age-related memory loss when given before or after information acquisition, and improves memory in patients with AD. Such observations have led to the speculation that understanding metabolism could better address the cause rather than symptoms of the disease. This is especially important given the increasing evidence linking diabetes with hippocampal lesions in AD. While there are many known effects of metabolic disruption (e.g. hypoglycaemia) in the periphery, its effects on the brain are less well studied. There is some evidence to suggest that along with long-term effects on brain function, short-term periods of hypoglycaemia may affect different regions of the brain. In order to understand this relationship metabolites were measured using implanted sensors in freely-moving animals to determine the effects of, for example, fasting, insulin administration, and reintroduction of food. Supported by Enterprise Ireland (PC/2009/ 523 and CP/2011/0103) and the European Regional Development Fund. Stephane Marinesco
INSERM, Lyon, France METABOLIC MONITORING AFTER TRAUMATIC BRAIN INJURY USING IMPLANTABLE MICROELECTRODE BIOSENSORS Local metabolic disturbances occurring after traumatic brain injury (TBI) provide important information about the ability of the nervous tissue to recover from the initial insult. Monitoring energy metabolites is therefore an important challenge for both pre-clinical and clinical research on TBI. Microelectrode biosensors are a promising technique to monitor the brain with a temporal resolution in the order of seconds. We have recently developed ultra-microelectrodes based on platinized carbon fibers that provide a basis for fabricating biosensors with less than 15 µm external diameter. Such biosensors avoid major mechanical injury to blood vessels and preserve the blood brain barrier at the site of implantation. Here, glucose and lactate microelectrode biosensors in addition to oxygen sensors and laser Doppler flowmetry were used to monitor the rat cortex during the first 5h following severe experimental TBI. These techniques revealed that local cerebral blood flow decreased and extracellular glucose concentrations remained low and stable throughout the monitoring period. Occasional cortical spreading depolarizations were detected and lowered oxygen levels and extracellular lactate concentrations, inducing a temporary metabolic crisis in the nervous tissue. Overall, implantable microelectrode biosensors revealed mild metabolic disturbances after TBI that can be significantly aggravated by the occurrence of cortical spreading depolarizations. Michelle L. Rogers1, Chi Leng Leong1, Sally A.N. Gowers1, Isabelle Samper1, Sharon Jewell2, Shumaila
Khan2, Anthony J. Strong2, Martyn G. Boutelle1
1 Imperial College, London, UK 2 Kings College London, UK MICROFLUIDIC ANALYSIS SYSTEM FOR MICRODIALYSIS MONITORING OF BRAIN INJURY PATIENTS In traumatic brain injury poor patient outcome is associated with Spreading depolarisations (SD). These are mass depolarisations of both neuronal and glial cells that originate from the initial injury site and radiate through surrounding tissues. This imposes a huge repolarisation task leading to a mismatch between supply and demand of key molecules in the local area [1]. SD's sometimes circle around the damaged core, leading to a clustering effect. Here, there is a high probability that the tissue will not be able to recover from the depolarisations [2]. Hence, the damaged tissue area grows in size. We are now monitoring this "at-risk" tissue using microdialysis. The dialysate is analysed in real-time using a microfluidic-based system. Potassium, detect the onset of the SD, glucose, lactate and pyruvate are monitored to determine the effects of the SD on the tissue metabolic state. Preliminary clinical results confirm the detrimental effect of SDs on the tissue [1] Rogers, ML. et al. ACS Chem Neurosci 4, 5, 799-807 (2013) [2] Feuerstein, D. et al. J Cereb Blood Flow Metab 30, 1343-1355 (2010) [3] Rogers ML. et al. Phys Chem Chem 13:5298-5303 (2011) Martin Eysberg, Lusi M. van Heerwaarden, Hendrik-Jan Brouwer, Nico J. Reinhoud
Antec, Netherlands METHOD DEVELOPMENT IN NEUROTRANSMITTER ANALYSIS TO IMPROVE SELECTIVITY, SENSITIVITY AND ROBUSTNESS In vivo microdialysis has become an invaluable tool that provides real-time information of neurotransmitter levels in living brain. Microdialysis samples are collected and stored, or analyzed immediately in an on-line configuration using UHPLC-ECD. Methods are presented for reproducible and accurate neurotransmitter analysis with detection limits in the low picomolar concentration range using only a few microliter of sample. To get as much as possible information from samples, methods have been optimized for selectivity, sensitivity and robustness. The ALEXYS Neurotransmitter analyzer has been applied for development of a number of integrated system solutions for trace analysis of neurotransmitters. Parallel and serial detection schemes using multiple flow cells have been used instead of running sequential trials for different neurotransmitters. Dual or triple loop injection valves are applied with minimum sample consumption on parallel UHPLC systems under completely different conditions. Getting more information out of fewer samples is not only saving time and money but - in the end - also rodents. Symposium: In vivo calcium imaging: a window into the behaving brain
Chairpersons: Erin S. Calipari and Rosemary C. Bagot
Erin S. Calipari
Icahn School of Medicine at Mount Sinai, Manhattan, New York, USA TEMPORALLY SPECIFIC D1 AND D2 MEDIUM SPINY NEURONAL SIGNALING MEDIATES ASSOCIATIVE LEARNING FOR DRUG REWARDS AND IS DYSREGULATED BY COCAINE TO DRIVE ADDICTIVE BEHAVIORS The reinforcing and rewarding properties of cocaine are attributed to its ability to increase dopaminergic transmission in nucleus accumbens (NAc). This action reinforces drug taking and seeking and leads to potent and long-lasting associations between the rewarding effects of the drug and the cues associated with its availability. The inability to extinguish these associations is a key factor contributing to relapse. Dopamine produces these effects by controlling the activity of two subpopulations of NAc medium spiny neurons that are defined by their predominant expression of either dopamine D1 or D2 receptors. Using fiber photometry calcium imaging we define D1 MSNs as the specific population of cells in NAc that encodes drug associations, and elucidate the temporal profile with which D1 activity is increased to drive drug seeking in response to contextual cues. Chronic cocaine exposure dysregulates these D1 signals to both prevent extinction and facilitate reinstatement of drug seeking to drive relapse. Directly manipulating these D1 signals using designer receptors exclusively activated by designer drugs (DREADDs) prevents contextual associations. Together, these data elucidate the responses of D1 and D2 type MSNs in NAc to acute cocaine and during the formation of context-reward associations and define how cocaine exposure dysregulates D1 signaling to drive relapse. Giovanni1,2, Bo Liang1, Lifeng Zhang1, Rong Chen3, Charles R. Gerfen4, Eugenio Culurciello2, Yun
Li1, Da-Ting Lin1,5,6
1 Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 333
Cassell Drive, Baltimore, MD 21224
2 Weldon School of Biomedical Engineering, Purdue University, 206 S. Martin Jischke Drive, West Lafayette, IN 47907 3 Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, 100 N Greene St, Baltimore, MD 21201 4 Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Building 49, Room 5A60, Bethesda, MD 20814 5 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 6 The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA. A NOVEL NEURAL ENCODING MODEL FOR DORSAL STRIATUM REVEALED BY IN VIVO CALCIUM IMAGING IN FREELY MOVING MICE An influential dogma of striatal function is that activity in the striatal direct and indirect output pathways promotes and inhibits movement, respectively. During normal behavior, activity occurs in both pathways, consistent with the model's notion that locomotion requires coordinated facilitation and inhibition of the behavior. However, more detailed neuronal mechanisms by which the direct and indirect pathways control locomotion remain elusive. Here, we developed a custom miniature fluorescent microscope system to concurrently record activity of hundreds of striatal neurons from freely moving mice. We demonstrated that striatal neurons in both direct and indirect pathways displayed an overall similar activity correlation with locomotion, with highest activity during ambulation and lowest activity during immobility. We further demonstrated that acute cocaine exposure enhanced mouse locomotor activity but exerted heterogeneous effects on neural activities in both direct and indirect pathways. Finally, using machine-learning algorithm, we were able to predict mouse ambulation state based on neural activity. The integration of in vivo imaging technique, neural clustering method, and machine-learning algorithm could be applied to many other brain regions and animal behavior models to unravel how activity of neural clusters controls behavior. Mazen Kheirbek
DECONSTRUCTING VENTRAL HIPPOCAMPAL CONTROL OF LEARNED FEAR AND ANXIETY-RELATED BEHAVIOR Recent studies implicate the ventral hippocampus (vHPC) as an important node in the modulation of anxiety-related behavior. In this study we leveraged optical technologies to determine how the extended vHPC circuit can modulate innate and learned fear behavior. Using in vivo calcium imaging we tracked the activity patterns of vHPC neurons across multiple tests of anxiety-related behavior and contextual fear conditioning. Our imaging studies revealed that, at the population level, vHPC neurons exhibit a higher rate of calcium transients during exploration of innately anxiogenic environments, with a subpopulation of neurons recruited across multiple anxiety tests. In addition, we found that contextual fear learning increases correlated activity among vHPC neurons, with a small fraction of neurons recruited in both anxiety-related tests and contextual fear conditioning. Next, we dissected the relative contribution of vHPC outputs to the amygdala (BMA) and hypothalamus (LH) in the control of innate anxiety and learned fear. Acute optogenetic modulation of vHPC-BMA terminals impaired contextual fear conditioning while optical activation of vHPC-LH terminals increase anxiety-related behavior. These studies implicate a projection-specific functional dissociation of vHPC outputs in the control of innate anxiety-related behavior and learned contextual fear. Michael Halassa
New York University, USA FAST OPTICAL MEASUREMENTS OF INHIBITION IN THE AWAKE BRAIN How the brain selects appropriate sensory inputs and suppresses distractors is unknown. Given the well-established role of the prefrontal cortex (PFC) in executive function, its interactions with sensory cortical areas during attention have been hypothesized to control sensory selection. To test this idea and, more generally, dissect the circuits underlying sensory selection, we developed a cross-modal divided-attention task in mice that allowed genetic access to this cognitive process. By optogenetically perturbing PFC function in a temporally precise window, the ability of mice to select appropriately between conflicting visual and auditory stimuli was diminished. Equivalent sensory thalamocortical manipulations showed that behaviour was causally dependent on PFC interactions with the sensory thalamus, not sensory cortex. Consistent with this notion, we found neurons of the visual thalamic reticular nucleus (visTRN) to exhibit PFC-dependent changes in firing rate predictive of the modality selected. visTRN activity was causal to performance as confirmed by bidirectional optogenetic manipulations of this subnetwork. Using a combination of electrophysiology and intracellular chloride photometry, we demonstrated that visTRN dynamically controls visual thalamic gain through feedforward inhibition. Our experiments introduce a new subcortical model of sensory selection, in which the PFC biases thalamic reticular subnetworks to control thalamic sensory gain, selecting appropriate inputs for further processing. Rajendra D. Badgaiyan and Sampada Sinha
University of Minnesota, USA DEVELOPMENT OF THE SINGLE-SCAN DYNAMIC MOLECULAR IMAGING TECHNIQUE (SDMIT) AND ITS APPLICATION IN UNDERSTANDING PATHOPHYSIOLOGY OF PSYCHIATRIC CONDITIONS The ability of neuroimaging techniques to detect task-induced acute changes in the levels of neurotransmitters is extremely limited. As a result, we have poor understanding of the pathophysiology of psychiatric and neuropsychiatric conditions that are associated with dysregulated neurotransmission. To address this limitation, we developed the Single-scan Dynamic Molecular Imaging Technique (SDMIT). The technique allows detection, mapping and measurement of dopamine released acutely during performance of a cognitive or behavioral task in the live human brain. It uses positron emission tomography (PET) and exploits the competition between a neurotransmitter and its ligand for receptor occupancy. The PET data are analyzed using a specially developed receptor kinetic models (linear extension of simplified reference region model and extended simplified reference tissue model). These models measure the values of a number of receptor kinetic parameters including the binding potential (BP) before and after task initiation in the same scan session. By comparing these values, dopamine released acutely during task performance (phasic release) can be detected, mapped, and measured. The tonic release can be studied by measuring receptor kinetic parameters at rest. Development of this technique has made it possible to examine the validity of hypotheses that cannot be examined using other techniques. Recently, we used the technique to examine a hypothesis and resolve controversy concerning the status of dopamine neurotransmission in attention deficit hyperactivity disorder (ADHD). Earlier studies that have used indirect methods to measure dopamine neurotransmission have reported contradictory findings suggesting either increased, decreased, or unchanged levels of dopaminergic activity in ADHD. We hypothesized that in this condition the tonic release of dopamine is reduced and there is compensatory enhancement of the phasic release. Since indirect methods cannot differentiate between the tonic and phasic release, depending on the predominance of one or the other, earlier data indicated either increase, decrease, or no change in the activity. Because SDMIT allows direct measurement of the tonic and phasic release separately, we used this technique to examine the hypothesis. The experiment included 22 ADHD and 22 healthy control volunteers. The results indicated that at rest (tonic release) the BP of a dopamine receptor ligand 11Craclopride was significantly (p<0.003) higher (3.19±0.23) than that in healthy control volunteers (2.86±0.26) in the right caudate. It indicated reduced tonic release in ADHD. Further, the phasic release during performance of the Eriksen Flanker Task was significantly higher in ADHD volunteers in the same area. The BP in the right caudate of ADHD volunteers (2.17±0.55) was significantly lower (p<0.004) that that measured in healthy control volunteers (2.88±0.46). The results validated the hypothesis of reduced tonic and enhanced phasic release of dopamine in ADHD and help to resolve a long standing controversy concerning pathophysiology of ADHD. Further, this experiment demonstrated that the SDMIT can be used to study pathophysiology of other psychiatric and neuropsychiatric conditions that are associated with dysregulated neurotransmission. Wine and cheese symposium: Acute vs chronic voltammetry electrodes: benefits and limitations
Chairpersons: Sara R. Jones and Matthew Wanat R. Mark Wightman
University of North Carolina at Chapel Hill, USA PRINCIPAL COMPONENT REGRESSION AND VALIDATION OF IN VIVO VOLTAMMETRY SIGNALS Principal component regression is a multivariate calibration method that has been used to resolve overlapping voltammetric signals from recordings made in the brain with fast-scan cyclic voltammetry. The method also allows calibration of the concentration of the measured signals. However, to accomplish principal component regression, the technique requires information about current-concentration relationships across the potential window that is gained from analysis of training sets. The ability of the constructed models to resolve analytes critically depends on the quality of the training data. The training set should be recorded with identical noise characteristics and the species comprising the training set should be present at the same concentrations as they are in the brain. This study reveals the pitfalls that can occur when models are constructed with inappropriate data using both theoretical and experimental arguments. An outline of the theory of principal component regression we be presented lay the basis for this discussion. The findings demonstrate that the use of standard training sets leads to inappropriate assignment of the current-concentration relationships, which may result in inaccurate conclusions being drawn from experimental data. Thus, it is strongly advocated that training sets used for model construction be obtained under the experimental conditions to allow for accurate data analysis. J.G. Roberts, G.S. McCarty, Leslie Sombers
IN SITU ELECTRODE CALIBRATION FOR IN VIVO VOLTAMMETRIC MEASUREMENTS Advances in voltammetry have considerably expanded the scope of neurochemical studies by enabling selective quantification of fluctuating electroactive neurotransmitters, such as dopamine. However, difficulties associated with its practice have limited widespread use. One technical aspect that has not been sufficiently addressed is electrode calibration. Carbon-fiber microelectrodes are individually handmade, leading to significant variability in performance that renders calibration necessary. This is a drawback for many researchers, because experimental protocol makes post-calibration difficult, or in some cases impossible. Further, the relevance of ex vivo calibration can be questioned, as an ex vivo calibration can never fully recapitulate in vivo measurement conditions. We present a model that utilizes information contained in the background current to predict sensitivity to commonly encountered neurochemicals at any point in an in vivo electrochemical experiment. This method is particularly valuable in cases where calibration is difficult, such as when using chronically implanted electrodes. These enable investigations to be carried out over multiple days, but are very difficult or impossible to remove for calibration. Our approach to in situ electrode calibration simplifies electrochemical data collection protocols for in vivo applications, and promises to facilitate more widespread utilization of FSCV. Paul E. M. Phillips
University of Washington, USA STABLE LONG-TERM DOPAMINE DETECTION IN VIVO We have used fused-silica-insulated carbon-fiber microelectrodes for chronic implantation into the striatum of rodents for long-term tracking of extracellular dopamine concentrations with sub-second temporal resolution. Implantation of these electrodes produces minimal neuroinflammatory responses without glial encapsulation; and the recording sites appear unperturbed at the optical microscopy level, with normal levels of tyrosine-hydroxylase-positive staining around the implantation site. As assessed by both in-vitro calibration and the presentation of positive-control stimuli in vivo, electrode sensitivity is stable over months of implantation. Recordings from these electrodes faithfully reproduce the concentrations and kinetics observed with acutely implanted electrodes, but have the distinct advantage of being able to track within-in subject changes in neurotransmission over days, or even weeks, that result from psychological and/or pathological processes. The electrodes are sensitive to impulse-dependent dopamine neurotransmission as indicated by the detection of electrically stimulated changes in extracellular dopamine, by the attenuation of behaviorally evoked responses following the inactivation of midbrain dopamine neurons, and they have replicated many of the patterns of activity observed by electrophysiological recordings of dopamine neurons. Compared to glass-pulled electrodes, the epoxy-sealed fused-silica electrode design of the chronic electrode has lower and, importantly, less variable capacitance, making them more reproducible. Matthew Wanat
University of Texas at San Antonio, USA EXAMINING CONTEXT-DEPENDENT CHANGES IN DOPAMINE RELEASE The firing of dopamine neurons in response to conditioned stimuli (CS) is highly dependent upon the context in which the CSs are experienced. Within-session changes in dopamine transmission are commonly examined using electrophysiological recordings or voltammetry with acutely implanted electrodes. However, these methods are not well suited for examining between-session changes in dopamine transmission. Here, we utilized chronically-implanted voltammetry electrodes to probe dopamine release across sessions when we altered the training context in which Pavlovian CSs were experienced. In this Pavlovian task, two distinct audio CSs reflected different lengths of time since the previous reward (i.e. short wait CS and long wait CS). Rats first experienced these CSs in separate sessions, which revealed an elevated dopamine response to the short wait CS relative to the long wait CS. However, this CS-evoked dopamine release was asymmetrically updated upon experiencing the CSs together within the same training context for the first time. Specifically, dopamine release to the long wait CS was unchanged while dopamine release to the short wait CS was increased and accompanied by an elevation in conditioned responding. These results illustrate the power of utilizing chronically-implanted voltammetry electrodes to examine changes in dopamine transmission across training sessions. Symposium: State of the art Bioanalytical Appeoaches for Elucidating Pathomechanisms of
Alzheimers disease
Chairpersons: Henrik Zetterberg Frances A. Edwards
University College London, UK ANIMAL MODELS FOR ALZHEIMER'S DISEASE; WHAT ARE THEY GOOD FOR? In recent years positive results from mouse models for Alzheimer's disease, particularly for amyloid beta immunotherapies, were followed by a series of failed clinical trials. This has led to a general scepticism about the usefulness of mouse models. However all models have limitations and so the questions asked must be matched to the models. Available mouse models for Alzheimer's disease fall mostly into two categories. The first group are useful for understanding the earliest effects of rising amyloid beta and the laying down of amyloid plaques but these mice, with mutations for familial Alzheimer's disease, do not develop Tau tangles or substantial neurodegeneration. In contrast the second group, mice with mutations in Tau that cause various other forms of dementia, have neurofibrillary tangles and neurodegeneration and so are useful for studying these late stages of disease. There are however presently no models that elucidate the link between amyloid build up and late stage disease. Hence it is perhaps not surprising that treatments that decreased amyloid deposition in the early stage models were not effective in patients in which Tau tangles and neurodegeneration were well advanced. I will discuss the useful data that can be gained from present models, despite their limitations, and the ways in which we aim to develop improved models. Chihiro Sato, Nicolas Barthelemy, Kristopher Kirmess, Kevin Yarasheski, Tim Miller, Bruce Patterson,
Randall Bateman
Washington University, St.Louis, USA TAU KINETICS IN THE HUMAN CENTRAL NERVOUS SYSTEM We have previously pioneered a mass spectrometry-based method, Stable Isotope Labeling Kinetics (SILK), and measured the production and clearance of proteins such as Abeta, Apolipoprotein E (ApoE), Amyloid Precursor Protein (APP), and Superoxide dismutase 1 (SOD1) in the human central nervous system (CNS). We have recently developed the tau SILK method using two different clinical protocols and quantified tau half-life in the human CNS. Results indicate that tau has a long half-life (weeks) compared to other Alzheimer's Disease (AD) related CNS proteins, such as Abeta, ApoE and APP (hrs). We are currently testing the hypothesis that tau kinetics are altered in tauopathies such as in AD. Tau SILK will allow us to evaluate the efficacy of emerging tau-targeted therapies, which aim to decrease tau production or enhance tau clearance. Stephen N. Mitchell, N. Malik, G. Carter, M. Ligocki
Eli Lilly and Company, UK IN VIVO NEUROCHEMICAL CHARACTERISATION OF THE rTg4510 MOUSE MODEL OF HUMAN TAUOPATHY Alzheimer's disease is a complex disease associated with progressive cognitive and behavioural decline. Preclinical in vivo models capable of mimicking disease progression are important for understanding behavioural, neurochemical or neurophysiological sequelae associated with on-going pathology, as well as the development of disease modifying and symptomatic treatments. The rTg450 mouse is a robust and progressive model of tau-associated neurodegeneration, showing age-related tangle-like inclusions, brain atrophy, neuronal loss and cognitive impairment (Santacruz et al., 2005). We describe results of experiments aimed to investigate the in vivo neurochemical consequences of age-related tauopathy, by measuring potassium-evoked neurotransmitter efflux in of rTg4510 mice. Microdialysis probes were implanted into the hippocampus or caudate putamen. Experiments were conducted the day after surgery and the response to 50mM potassium measured after a suitable baseline period. Samples were analysed by LC-MS/MS following a derivatisation procedure, allowing detection of a number of neurotransmitters and metabolites. There was an age-dependent reduction in potassium-evoked GABA and glutamate efflux in the hippocampus, but not in the caudate putamen. In the hippocampus, there was no significant change in efflux of 5-HT, NA, DA, histamine and their metabolites, or ACh. The results show an age-dependent, transmitter-specific and regionally-selective effect despite widespread degenerative processes. Kina Höglund3, Ulf Andreasson1, Victor Lima1, Henrik Zetterberg1,2, Kaj Blennow1
1 Department of Psychiatry and Neurochemistry, University of Gothenburg, Sweden 2 UCL Institute of Neurology, Queen Square, London, UK 3 Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Disease Research, Karolinska Institutet, Sweden DIGITAL ELISA: BIOMARKER ANALYSIS OF LOW ABUNDANT CNS SPECIFIC PROTEINS IN BLOOD AND CSF IN ALZHEIMERS DISEASE A biomarker is an objective measure of a molecule that reflects an underlying biological event. In the quest for novel biomarkers reflecting disorders of the central nervous system (CNS), sensitivity of the analytical methods is a hurdle since many proteins originating from the brain are low abundant in the cerebrospinal fluid (CSF). For Alzheimer's disease (AD), there are three widely accepted CSF biomarkers, t-tau, p-tau and Aβ1-42, reflecting key pathological hallmarks of AD. Still, there is an overlap in terms of clinical symptoms and biomarker patterns in several neurodegenerative disorders which highlights the need for additional biomarkers reflecting concomitant pathology. In addition, having a blood based test, where the concentrations of brain specific proteins are even lower, would be very valuable in terms of feasibility and accessibility. We have successfully developed assays in an ultrasensitive digital ELISA format, yielding a 100- to 1000-fold improvement in sensitivity, enabling measurement of t-tau and Aβ42 in serum, protein aggregates (oligomers) in CSF and to monitor acute brain damage in serum (NFL). These assays allow us to evaluate the feasibility of blood derived biomarkers reflecting CNS disorders as well as studying protein aggregates hypothesized to be key players in several neurodegenerative disorders. Hideo Tsukada
Hamamatsu Photonics K.K., Japan EFFECTS OF AMYLOID-B DEPOSITION ON MITOCHONDRIAL COMPLEX-I ACTIVITY IN BRAIN: A PET STUDY IN AGED MONKEYS FDG-PET is a technique for quantitative imaging of regional cerebral metabolic rate of glucose (rCMRglc) in living brain. However, 18F-FDG was taken up into not only normal tissues but also inflammatory regions with microglial activation. To solve this problem, the translational research with 18F-BCPP-EF, a novel PET probe for mitochondrial complex 1 (MC-1) was conducted using an animal PET to assess the aging effects on MC-I activity in monkey brain. PET scans using 11C-PIB for A-beta, 11C-DPA-713 for inflammation, 18F-FDG for rCMRglc, and 18F-BCPP-EF for MC-1 were performed under conscious states in young and aged animals. When plotted VT of 18F-BCPP-EF against SUVR of 11C-PIB in the cerebral cortical regions, it showed a significant negative correlation. Plotting of SUV of 11C-DPA-713 against SUVR of 11C-PIB resulted in a significant positive correlation. In contrast, plotting of rCMRglc against SUVR of 11C-PIB did not reach statistically significant level. Furthermore, we recently reported in TauTg mice the negative correlation between the uptake of 11C-PBB3, a PET probe for imaging Tau deposition, and MC-I in hippocampus, and positive correlation between memory function and MC-I activity. These results suggested that 18F-BCPP-EF could discriminate the neuronal damaged areas with neuroinflammation, where 18F-FDG could not do. Symposium: Glycine receptors as new pharmacological targets
Chairpersons: Luis G. Aguayo and Joseph Lynch Hanns Ulrich Zeilhofer, Hendrik Wildner and Edmund Foster
University of Zurich & Swiss Federal Institute of Technology (ETH), Switzerland GLYCINERGIC NEURONS OF THE SPINAL DORSAL HORN IN PAIN AND ITCH Inhibitory neurons of the spinal dorsal horn take a center stage in the processing of nociceptive signals. These neurons fall into two large groups using either GABA, or GABA and glycine for fast synaptic inhibition. To specifically address the contribution of glycinergic neurons, which are concentrated in the deep dorsal horn, we made use of GlyT2::Cre transgenic mice suitable for virus-mediated retrograde tracing studies and for spatially precise ablation, silencing, and activation of glycinergic neurons. Local ablation or silencing glycinergic neurons in the lumbar spinal cord induced intense thermal and mechanical hyperalgesia, evoked signs of spontaneous discomfort including intense localized grooming, licking and biting of the affected innervation territory, and led to localized hair loss and skin lesions reminiscent of chronic itch. Conversely, activation of the same neurons through pharmacogenetics alleviated acute and chronic neuropathic pain, and reduced scratching responses to histamine and non-histamine-dependent pruritogens. These results indicate that the malfunctioning of glycinergic dorsal horn neurons induces signs of chronic pain and itch, whereas their pharmacogenetic activation alleviates acute and neuropathic pain and reduces histamine-dependent and histamine-independent itch. Joseph Lynch
Queensland Brain Institute, University of Queensland, NEW SMALL MOLECULE ANALGESTICS THAT WORK BY TARGETING THE ALPHA3 GLYCINE RECEPTOR CHLORIDE CHANNEL Novel pharmacophores for treating chronic pain are desperately needed. Glycine receptor (GlyR) Cl channels mediate inhibitory neurotransmission in the spinal cord and some brain regions. The vast majority of glycinergic synapses exclusively incorporate alpha1 GlyR subunits. However, glycinergic synapses on pain sensory neurons in the spinal cord are unique in that they incorporate alpha3 GlyR subunits. Inflammatory mediators (e.g., prostaglandin E2) induce chronic inflammatory pain by phosphorylating, and inhibiting, alpha3 GlyRs. This reduces the magnitude of glycinergic inhibitory synaptic currents in spinal pain sensory neurons, thereby ‘disinhibiting' these neurons and increasing the rate of transmission of pain impulses to the brain. This mechanism, which provides a paradigm for understanding chronic pain sensitisation, implies that drugs that selectively enhance (i.e., restore) alpha3 GlyR function should be analgesic. Here I describe the development and characterisation of novel small molecule, nanomolar-potent alpha3-specific potentiators that elicit potent analgesia in animal models of chronic pain. Robert J. Harvey
University College London, UK NEW BIOLOGICAL ROLES FOR GLYCINE RECEPTORS CONTAINING THE α2 AND α4 SUBUNITS Glycine receptors (GlyRs) are ligand-gated ion channels that mediate inhibitory synaptic transmission in the spinal cord, brainstem, cerebellum and retina. The major adult GlyR isoform, consisting of α1 and β subunits, has a major role the control of spinal motor reflex circuits. Mutations in the genes encoding this GlyR subtype cause startle disease/hyperekplexia, which affects newborn children and is characterised by noise or touch-induced seizures that result in muscle stiffness and neonatal apnoea episodes. However, it has recently emerged that other GlyR subtypes containing the α2, α3 and α4 subunits may play more diverse biological roles. For example, the GlyR α3 subunit acts as a molecular switch, modulating G-protein-coupled receptor mediated signaling pathways involved in inflammatory pain sensitization and rhythmic breathing. Although GlyR α2 subunit knockout mice were initially reported as lacking a behavioural or morphological phenotype, it was recently demonstrated that GlyR α2 is involved in cerebral cortical neurogenesis. Extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons activates voltage-gated Ca2+ channels that modulate actomyosin contractility to fine-tune neuronal migration in the developing cortical wall. Loss of GlyR α2 subunits leads to a selective depletion of cortical neurons and microcephaly in newborn Glra2 knockout mice. Lastly, although the GlyR α4 subunit gene is considered to be a pseudogene in humans, using structure-function and mutagenesis methods, we have found that GlyRs containing the α4 subunit are ful y functional in other species, including zebrafish, mice and apes. Gene knockdown, dominant-negative mutants and genetraps for glra4a in zebrafish suggest that GlyRs containing the α4 subunit underpin startle-evoked escape responses, leading to the intriguing hypothesis that humans respond to startle differently to other species. Supported by the Medical Research Council, Action Medical Research and UCL Impact studentships. Luis G. Aguayo
University of Concepcion, Chile ROLE OF GLYCERINE RECEPTORS ON ETHANOL TOXICITY AND CONSUMPTION Alcohol abuse affects several millions of people worldwide causing significant social, medical and economic burdens. Unfortunately, the current pharmacotherapeutic tools are limited, have low adherence and cause several side effects, which emphasizes the need for novel, mechanistically oriented therapies. In previous studies, we reported that basic amino acids (316-320 and 385/386) regulate the sensitivity of α1 glycine receptors (GlyR) to low concentrations of ethanol. The effects of the mutations are specific for ethanol, since the sensitivity to other positive and negative GlyR modulators were not affected. Recently, we generated and characterized a Knock In (KI) mouse for the α1 GlyR with mutations in residues 385/386 in the intracellular loop of the receptor. The KI mice had normal behavior and most importantly did not display a hyperexcitable phenotype. Examining spinal and brain stem neurons, we found that GlyRs were less affected by ethanol- and Gγβ− mediated modulations. Interestingly, the mice exhibited a reduced time in loss of righting reflex (LORR) when compared with wild type mice. Searching for inhibitory molecules, we found that a small peptide significantly reduced the ethanol-induced potentiation of GlyR activated currents and miniature inhibitory synaptic currents (mIPSCs). More recently, we found that a small molecule, M554 (an indol derivative), applied intracellularly was able to reduce ethanol potentiation of α1 GlyR. Additional y, i.p. injection of this compound was also able to reduce the LORR in mice. In conclusion, we identified important amino acids that participate in the modulation of GlyRs by ethanol. These findings could lead to the development of new therapy for alcohol related disorders. Supported by NIH RO1 and Fondecyt DPI 20140008 grants Bert Brône1, Giovanni Morelli1,2, Joris Comhair1,3, Laurent Nguyen2, Jean-Michel Rigo1
1BIOMED Research Institute, University Hasselt, Hasselt, Belgium 2GIGA, Université de Liège, Liège, Belgium 3Université Libre de Bruxelles, Brussels, Belgium THE GLYCERINE RECEPTOR ALPHA 2 CONTROLS CIRCUITRY FORMATION IN THE BRAIN The glycine receptor alpha 2 (GlyRa2) subunit is known to be expressed in the brain during early development whereas the expression level reduces gradually in favor of alpha1 and/or alpha3 subunits during brain maturation. We have previously shown that the embryonic alpha 2 receptor modulates neuronal proliferation and migration in mouse embryo's. Subsequent studies of our lab show that the embryonic GlyRa2 expression is necessary for normal network maturation in mice and that loss of the GlyRa2 results in increased excitability of the somatosensory cortex. A shift in the excitation/inhibition balance is a hallmark for epilepsy and GlyRa2 knockout animals showed an increased susceptibility for epilepsy. Interestingly, recently several loss of function mutations in the GlyRa2 were associated with autism spectrum disorder (ASD) and epilepsy is a known comorbidity in ASD. Although the expression level of the GlyRa2 subunit decreases during brain development, some evidence is present for its expression during later stages of life. However, the cell specific expression and the subsequent behavioral consequences largely remain elusive. Symposium: Miniature probes and non-mammalian systems
Chairpersons: Michael Johnson and Jill Venton Michael A. Johnson, Mimi Shin, Thomas M. Field and Mia N. Furgurson
University of Kansas, USA ELECTROCHEMICAL MEASUREMENT OF DOPAMINE RELEASE AND UPTAKE IN ZEBRAFISH Initially intended for the study of development, zebrafish are quickly becoming an important research model for the study of neuronal function due to their simplicity, low cost, and ease of genetic manipulation. However, the direct electrochemical measurement of neurotransmitter release and uptake in brain slices has only recently been described. Moreover, measurements obtained in the whole brain ex vivo and in vivo have not been reported. In this work, we describe methods for the measurement of dopamine release and uptake with fast-scan cyclic voltammetry at carbon-fiber microelectrodes (FSCV) in zebrafish whole brains, and compare these measurements with those obtained in brain slices. In addition to anatomical and electrochemical information, we have obtained pharmacological confirmation that the measured neurotransmitter was dopamine by treatment with inhibitors of dopamine uptake, synthesis, and D2-family autoreceptor function. We have further identified the optimal stimulation parameters for dopamine release in whole brain and brain slices. Our progress toward obtaining measurements in vivo will also be presented. Collectively, these results demonstrate the feasibility of measuring neurotransmitter release and uptake in zebrafish whole brain preparations and provide additional justification for the use of this model in studies of central nervous system function. Scott A. Shippy
University of Illinois at Chicago, USA DROSOPHILA HEMOLYMPH SAMPLING FOR PROTEINS AND SMALL MOLECULES Drosophila melanogaster has been used for over a century in a wide variety of biological studies. However, the small size has limited the types of chemical information that is available. Developments to sample the hemolymph or blood content from individual larvae or adult Drosophila provides an important window for understanding chemical content changes in hemolymph. In this work both large proteins and small molecules are assayed in separate studies. Protein analysis of nanoliter amounts of hemolymph is analyzed via a bottom-up approach utilizing chromatographic separation and peptide sequencing mass spectrometry. A fractionation approach was developed prior to liquid chromatographic-mass spectrometric analysis to improve protein coverage. Both reversed-phase and ion exchange based fractionation provided greater numbers of peptides identified, higher sequence coverage, and the discovery of proteins predicted at the transcription and gene level. Studies for smaller molecules explore the effects of anesthesia for characterizing hemolymph chemical content. Commonly used methods of anesthesia such as cold-shock, carbon dioxide and physical restriction were determined to provide different amino acid content profiles. The impact of the method of anesthesia appears to be related to the stress required and the state of the fly while hemolymph is collected. Michael L. Heien
University of Arizona, USA HIGH-THROUGHPUT ANALYSIS OF INSECT NEUROTRANSMITTERS Insects have been used extensively used as a model organism in genetics research and has significantly contributed to understanding molecular, cellular and evolutionary aspects of human behavior. However, chemical measurements of these mass-limited samples are still challenging. Research has focused on developing methods to obtain highly sensitive chemical quantification along with spatiotemporal information. The goal of chemical analysis is to relate neurochemical changes to either genetic mutations or to treatments that alter the state of the organism. Here, we develop methodology to rapidly quantify neurotransmitter and metabolite molecules. Chemical derivitization of biological molecules coupled with mass spectrometric detection will be used to measure neurotransmitter concentrations. The methods presented are broadly applicable and can be used with a wide range of analytes. Additionally, this scheme simplifies detection and makes rapid relative quantification possible, allowing comprehensive and rapid analysis of neurotransmitters. This enables the analysis of attomole amounts of neurotransmitters and metabolites. We investigated changes at individual cultured neurons from drosophila. Jill Venton, Eve Privman, Danielle Wolin, Leah Ostendorf
University of Virginia, USA DETECTING DOPAMINE CHANGES IN DROSOPHILA MODELS OF PARKINSON DISEASE Parkinson's disease (PD) is a disabling neurodegenerative movement disorder, caused by dopamine cel death. Drosophila models of PD, such as RNAi knockdown of Pink1 and Parkin genes, are used to study the progression of the disease. RING finger protein 11 (RNF11) is expressed by neurons in the substantia nigra of Parkinson's disease patients. We selectively knocked down RNF11, pink1, and parkin using RNAi in only dopaminergic cells and tested the extent to which dopamine neurotransmission is disrupted in 3rd instar larvae. Dopamine release was stimulated using pulsed red light activating CsChrimson and detected at a microelectrode with fast-scan cyclic voltammetry. The amount of dopamine released was significantly higher in RNF11 RNAi flies than in control animals. Vmax significantly increased in RNF11 knockdown flies, indicating a higher density of dopamine transporters in the neuropil but Km was unchanged from control flies. At this early developmental stage, pink1 and parkin knockdown did not change dopamine release, Vmax, or Km. Therefore, we have identified RNF11 as a protein that may be involved in early Parkinson's disease pathology and may be a target for timely intervention and pharmacological prevention of disease progression. Soodabeh MajdiI1, E. Carina Berglund2, Johan Dunevall1, Alexander I. Oleinick3, Christian Amatore3,
David Krantz4 and Andrew G. Ewing1,2
1 Department of Chemistry and Chemical Engineering Chalmers University of Technology Department, Kemivägen 10, 41296 Gothenburg, Sweden 2 Department of Chemistry and Molecular Biology University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden 3 Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités UPMC Univ Paris 06, CNRS UMR 8640 PASTEUR, 24, rue Lhomond, 75005 Paris, France 4 Department of Psychiatry and Biobehavioral Sciences, Gonda Center for Neuroscience and Genetics Research, David Geffen School of Medicine at University of California, Los Angeles, California,USA ELECTROCHEMICAL MEASUREMENTS OF OPTOGENETICALLY STIMULATED QUANTAL AMINE RELEASE FROM SINGLE NERVE CELL VARICOSITIES IN DROSOPHILA LARVAE This work faces the challenge to electrochemically measure neurotransmitter release from the smallest varicosities of a nerve cell. The nerve terminals found in the body wall of Drosophila larvae are
readily accessible to experimental manipulation. We used the light-activated ion channel
channelrhodopsin-2 (ChR2), which is expressed genetically in type II varicosities, to study octopamine
release in Drosophila larvae. Here we report the development of amperometry to measure release of the
neurotransmitter octopamine from individual varicosities in the Drosophila larval system by amperometry.
A carbon-fiber disk microelectrode was placed onto this cellular region of the muscle and held at a
potential above the formal oxidation potential of octopamine and the terminal stimulated by blue light. The
results suggest that optical stimulation can be used to evoke exocytosis release in the type II varicosities
of Drosophila larvae. When octopamine is released from the varicosities during stimulation, it is oxidized
at the electrode and a current spike results. We observe different types of release according to the
different shape of the spikes, and this we speculate is related to the mechanism of opening of the vesicle
to make the nanometer fusion pore. To determine the number of molecules released per vesicle, we
examined only single spikes rather than complex events. The single non-complex exocytosis events are
fast, with full width of 1.5±0.2 ms, and the mean number of molecules released per vesicle is
22700±4200.We use this method to quantify the amount of transmitter released and these subsecond
release events. This is a new approach to study millisecond release events in the nanoliter environment
of a neuromuscular junction.
Symposium: The role of sigma receptors in brain pathologies
Chairperson: Michael Fritz Maurice Tangui
University of Montpellier, France TARGETING THE SIGMA 1 CHAPERONE PROTEIN FOR EFFECTIVE NEUROPROTECTION IN ALZHEIMER'S DISEASE The sigma-1 receptor (S1R) is a ligand-operated molecular chaperone localized in the endoplasmic reticulum (ER), mitochondria and plasma membranes. Its activation modulates IP3 receptor-dependent Ca2+ mobilizations, facilitates the activation of ER stress sensor proteins and kinase pathways. Under chronic activation, it also recomposes within membranes the highly functionalized lipid raft microdomains. Interestingly, the chaperone can be directly activated (or inactivated) by several classes of ligands. These S1R agonists are currently studied for their cytoprotective action in different neurodegenerative pathologies including Alzheimer's disease (AD). In AD, the S1R invalidation, in S1R KO mice, increased AD pathology. Two AD mouse models were used: a nontransgenic model by injection of amyloid-ß (Aß) protein [25-35] (Aß25-35) and transgenic lines overexpressing APPm. AD toxicity and behavioral deficits appeared significantly amplified in S1R KO mice injected with Aß25-35 and in S1R KO x APPm lines. On the contrary, administration of S1R agonists in Aß25-35-treated mice or APPSwe mice attenuated AD pathology. Current data suggest that S1R activity induces a wide-range cytoprotection by controlling cellular oxidative stress and mitochondrial integrity, mitigating Aß-induced apoptosis, regulating ATP/IP3-gated Ca2+ balance, modulating NMDA receptor activity and trophic factor effects, regulating neuroinflammation and microglial response, preventing Aß generation and decreasing Tau hyperphosphorylation. This pleiotropic action appears effective per se but can also be pertinent to define new drug combination therapies. Indeed, donepezil action appeared synergistically potentiated by S1R agonists. The S1R therefore appears as an effective endogenous neuroprotection system in neurodegenerative pathologies and S1R agonists could lead to potent neuroprotective and disease-modifying agents. Peter McCormick
University of East Anglia, THE ROLE OF SIGMA-1 RECEPTORS IN MODULATING G-PROTEIN COUPLED RECEPTOR FUNCTION AND ITS ROLE IN DRUG ADDICTION Sigma-1 receptor has been shown to modulate the signalling of several G-protein coupled receptors in the brain. We have found that this is accomplished via direct protein-protein interaction and has profound effects on both cell signalling and brain function. This talk will highlight some of the key findings we have made in this area, focusing on the biochemistry, cell biology, and in vivo experiments we have performed to examine these sigma-1-G-protein coupled receptor complexes. Anna M. Klawonn
Linköping University, Sweden TARGETING THE SIGMA-2 RECEPTOR SUBTYPE FOR TREATMENT OF PSYCHOSTIMULANT ADDICTION Neurosteroid sigma-receptors have been suggested to be involved in drug addiction. Two subtypes of the sigma-receptor have been identified based on drug selectivity and molecular mass: sigma-1R and sigma-2R. Whereas the role of sigma-1Rs in addiction has been studied, very little is known about the role of the sigma-2Rs. We found that the specific sigma-2R ligand Siramesine attenuated cocaine acquisition, expression and reinstatement in mice in conditioned place preference more effectively than the specific sigma-1R antagonist BD1063. Yet, Siramesine had no acute effect on cocaine induced locomotion, as BD1063 did. In vivo microdialysis revealed that Siramesine attenuates cocaine induced dopamine release in the striatum. Whole cell voltage clamp recordings of medium spiny neurons in nucleus accumbens (NAc) showed that Siramesine decreases presynaptic glutamate release probability. Consistent with this, fluorescent immunohistochemistry clarified that the sigma-2R/PGRMC1 is localized on glutamatergic end-terminals in the NAc. In conclusion, sigma-2Rs are located on neurocircuitry relevant for addiction and Siramesine, in a manner similar to the BD1063, attenuates cocaine induced behaviors. The neuromodulatory role of the sigma-2Rs in psychostimulant addiction is likely to occur through a cumulative effect on dopaminergic and glutamatergic circuitry. Jonathan L. Katz1, Weimin C. Hong2
1 National Institute on Drug Abuse 2 Butler University, USA A ROLE FOR SIGMA RECEPTORS IN STIMULANT ABUSE AND ADDICTION Stimulant self-administration induces self-administration of previously ineffective sigma-receptor (sR) agonists. Dual inhibition of sRs and the dopamine transporter (DAT) selectively blocks stimulant self-administration. Potential underlying mechanisms involve modulation of DAT function via sR activity. Linda Nguyen
Touro University California, USA MITIGATING PSYCHOSTIMULANT TOXICITY BY TARGETING SIGMA RECEPTORS Sigma receptors are compelling drug development targets for an array of neurological and psychiatric conditions. Psychostimulants such as cocaine and methamphetamine have significant affinity for sigma receptors, in addition to their actions on monoaminergic systems. The localization of sigma receptors in motor and limbic areas of the brain, as well as in monoaminergic neurons, provides a rational therapeutic target for mitigating the actions of psychostimulants. Over the years, novel, selective ligands for sigma receptors have been developed and shown to attenuate numerous toxicities caused by cocaine and methamphetamine in preclinical models. Cocaine-induced convulsions and lethality, in addition to acute stimulant effects, can be attenuated with sigma receptor antagonism. Methamphetamine-induced neurotoxicity and associated cognitive deficits can also be mitigated by sigma ligands. The neuroprotective effects of sigma ligands involve modulation of both neuronal and glial mechanisms that are common to many neurodegenerative conditions, suggesting additional therapeutic applications for these classes of compounds beyond drug abuse. Symposium: Advanced Molecular Imaging in Neuroscience
Chairporson: Jörg Hanrieder Stina Syvänen1, Xiaotian T Fang1, Linda M Cato1, Greta Hultqvist1, Johanna Fälting2, Gunnar Antoni3,4,
Lars Lannfelt1, Dag Sehlin1
1 Department of Public Health /Geriatrics, Uppsala University, Sweden 2 BioArctic Neuroscience AB, Sweden 3 Dept. of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, Sweden 4 Uppsala University Hospital PET-centre, Uppsala, Sweden AMYLOID-BETA PROTOFIBRILS CAN BE VISUALISED IN VIVO WITH PET USING A BISPECIFIC ANTIBODY-BASED RADIOLIGAND PET radioligand [11C]PIB binds to insoluble amyloid-beta (Aβ) plaques which are characteristic for Alzheimer's disease. However, PET images obtained with [11C]PIB are fairly static and do not reflect disease severity wel . Evidence today points to soluble Aβ protofibrils as being a neurotoxic agent in Alzheimer's disease (AD) and that levels of Aβ protofibrils correlate better than plaques with disease severity. This study therefore aimed at developing a PET ligand visualizing Aβ protofibrils in vivo. We have previously developed an antibody, mAb158, which selectively binds to Aβ protofibrils. To increase the brain distribution, a F(ab')2 fragment of the humanized variant of mAb158, F(ab')2-h158, was chemically fused with a transferrin receptor (TfR) antibody (8D3) to enable TfR mediated transcytosis of mAb158 into the brain. The fusion protein displayed almost 15-fold increased brain uptake, compared to mAb158. Ex vivo experiments demonstrated that the brain retention of 125I radiolabeled fusion protein increased with age and correlated closely with brain levels of Aβ protofibrils in AβPP transgenic mice (tg-ArcSwe), whereas it was low in wt mice. PET imaging with 124I labelled fusion protein confirmed the ex vivo results, showing a high signal in >12 months old tg-ArcSwe mice, which increased further with age, while it remained low in wild type mice of all ages. The PET-signal correlated with brain levels of Aβ protofibrils but not with total brain levels of Aβ40 and Aβ42, suggesting that the PET signal reflects Aβ protofibril pathology in vivo. To our knowledge this is the first time an antibody has been successfully used as a PET radioligand for a target within the CNS. Malin Andersson
Uppsala University, Sweden REGION- AND ENZYME-SPECIFIC BIOCONVERSION OF DYNORPHIN NEUROPEPTIDE DETECTED BY IN SITU HISTOCHEMISTRY AND MALDI IMAGING MASS SPECTROMETRY Expression of proteolytic enzymes in different regions modulates the biological activity of neuropeptides. Rapid and sensitive analytical methods to profile such activities are important for finding effective inhibitors with high therapeutic value. On-tissue histochemistry is a classic method for localizing enzyme activity directly on thin tissue sections, and by combining histochemistry with MALDI imaging mass spectrometry a highly sensitive method for un-biased analysis of neuropeptide conversion has been developed. General enzyme inhibitors, such as N-ethylmaleimide, acetic acid, and proteinase inhibitor cocktail almost completely, >90%, blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Specific inhibitors, such as phosphoramidon (a specific inhibitor of neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), whereas only opiorphin blocked striatal bioconversion to dynorphin B(2-13). These results may have direct impact on the development of novel drug therapies of L-DOPA-induced dyskinesia in Parkinson's disease, but wil also have implications for other areas, such as targeting inhibition of specific enzymes to alleviate pain. Combining histochemistry and MALDI imaging MS is here shown to be a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections. Nicola R. Sibson
University of Oxford, UK IMAGING THE INFLAMMATORY RESPONSE TO DETECT NEUROLOGICAL DISEASE The primary goal of what is now termed molecular imaging is the visualisation pathological processes at the cellular level, often long before disease symptoms become clinically apparent. The ability to do this confers two major benefits: earlier diagnosis of disease and targeting of individual therapy. Both PET and SPECT have been used in this way for many years, but with relatively limited spatial resolution. In recent years the idea of molecular MRI has evolved, with the advantage of considerably greater spatial resolution than either of the radioisotope-based methods. Typically, diagnostic MRI has relied on the effects of pathology on the water molecules in the tissue, and as a consequence has provided indirect and, frequently, rather non-specific information on the underlying processes. With the advent of molecular MRI we now have the ability to gain information on the expression, upregulation or downregulation, of specific molecules associated with pathology. Using this approach we have shown that we can detect specific adhesion molecules expressed on the vascular endothelium, as a component of the brain's inflammatory response, using ligand-targeted MRI contrast agents. We have shown that we can detect disease early in its pathogenesis and at a time when the presence of pathology is undetectable by either existing imaging methods or clinical scoring. In this talk I will discuss the various novel targeted contrast agents that we have been developing in Oxford and our in vivo work in experimental models of neurological disease, primarily multiple sclerosis and brain cancer. I hope, thereby, to demonstrate the potential of molecular MRI for the sensitive detection of neurological disease. Michael E. Kurczy
AstraZeneca, Sweden MEASURING DRUGS IN SINGLE CELLS Ideally, a drug molecule will concentrate preferentially at the therapeutic target leaving adjacent regions unaffected. Many of these targets are intracellular thus, the candidate molecule is required to transit the plasma membrane and in some cases the blood brain barrier. From a bioanalysis perspective these barriers mean that drug measurements from plasma or tissue may say little about distribution with the cell. Information concerning the intracellular concentration of drugs at target molecules is greatly needed in drug discovery yet difficult to measure. Furthermore, as we move toward new modalities we are finding that target engagement is of critical importance. ULTRA HIGH RESOUTION ION microprobe (NanoSIMS) imaging provides the spatial resolution required to answer the question of molecule localization within single cells. We are now beginning to integrate NanoSIMS imaging into the suite of technologies available for bioanalysis of cells and tissues. Symposium: Spotlight on brain noradrenaline, adenosine and histamine: we do not forget that
they are still neuromodulators
Chairpersons: Zoe McElligott and Sandrine Parrot Sandrine Parrot1, Barbara Ferry1, Marie-Jeanne Perrot-Minnot2, and Luc Denoroy1
1 Lyon Neuroscience Research Center, INSERM U1028, CNRS UMR5292, University of Lyon, Lyon, France. 2 Biogeosciences, Evolutionary Ecology Team, UMR CNRS UMR 6282, University of Burgundy and Franche-Comté (UBFC), Dijon, France CURRENT TOOLS TO MONITOR NOREPINEPHRINE, ADENOSINE, AND HISTAMINE IN ANIMAL BRAIN Brain norepinephrine, adenosine, and histamine play major roles in many neurobiological functions such as learning mechanisms, movement control, pain, neuroprotection, and/or sleep-waking cycle. However, the latest literature in monitoring molecules is less abundant for these three compounds than for dopamine and glutamate that are the most extensively studied molecules. This talk will briefly review both past and current works on norepinephrine, adenosine and histamine monitoring. Stress will be done on typical methodological issues which can be encountered for each compound including a review of the analytical methods applied from small invertebrates to mammals. Examples excerpted from literature and from experiments in our lab will illustrate the importance of the main steps in monitoring those biogenic molecules such as extraction/sampling, compound preservation, separation, detection, and/or experimental set-up. Besides, we will present our recent methodological and research data that are focused on norepinephrine and histamine. Natale R. Sciolino1, Nicholas W. Plummer1, Yu-wei Chen1, Sabrina D. Robertson2, Zoe A. McElligott3,4,
Patricia Jensen1
1 Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA. 2 Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA. 3 Bowles Center for Alcohol Studies, 4 Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, NC, USA. DUAL RECOMBINASE-DEPENDENT MOUSE LINE FOR CHEMOGENETIC ACTIVATION OF GENETICALLY DEFINED CELL TYPES Dissecting the function of heterogeneous cell populations, like the neurons of the central noradrenergic system, requires tools to visualize and manipulate their activity throughout development. Towards this aim, we generated a Flp/Cre recombinase-dependent Gq-coupled hM3 receptor (hM3Dq) DREADD mouse line for selective induction of cellular activity following administration of clozapine N-oxide (CNO). This new line uniquely permits non-invasive somatodendritic targeting of hM3Dq in genetically defined cells following Flp/Cre-mediated recombination, granting the unprecedented ability to control activity of previously inaccessible cell types to study their function and dysfunction. I will present data demonstrating the use of this new hM3Dq mouse line to uncover the unique role of two subsets of noradrenergic neurons in anxiety-like behavior. Zoe McElligott1, Jessica Vandenberg1, Anze Testen2, Viren Makhijani2
1 Bowles Center for Alcohol Studies, Department of Psychiatry, UNC Chapel Hill, USA 2 Neurobiology Program, UNC Chapel Hill NORADRENERGIC ADAPTATION IN THE MORPHINE DEPENDENT BRAIN The central noradrenergic system plays an extensive role in modulating behaviors associated with addiction and anxiety. There is significant evidence to suggest that norepinephrine (NE) plays a role in both the rewarding properties and the manifestation of withdrawal from opiate use. In particular, the ventral noradrenergic bundle, arising mainly from the A2/A1 cell groups and projecting to the bed nucleus of the stria terminalis (BNST), contributes significantly in the manifestation of both withdrawal and reward associated behaviors. My previous studies have demonstrated that morphine dependence can alter plasticity at noradrenergic synapses in the BNST, and that stress and drugs of abuse modulate glutamatergic synaptic plasticity within the same region. Here I present data exploring how drugs of abuse and dependence alter noradrenergic function on the level of synaptic activity within noradrenergic nuclei and in regions like the BNST that receive dense noradrenergic input using electrochemical and electrophysiological methods. Patrizio Blandina
University of Florence, Italy THE ENDOGENOUS LIPID MESSENGER OLEOYLETHANOLAMIDE FACILITATES FEAR MEMORY AND RELEASES HISTAMINE FROM BASOLATERAL AMYGDALA Neuronal histamine participates to oleoylethanolamide hypophagic effects. Since histamine is involved in mnemonic processes, we investigated if brain histamine is also required for oleoylethanolamide-induced promnesic effects. Wistar rats (280-300 g) were submitted to contextual fear conditioning, and immediately after received an oleoylethanolamide injection (10 mg/kg, ip). Memory was assessed 72 hours after injections. Oleoylethanolamide-treated animals showed a longer freezing time (255.3±48.5 s; n=11) as compared to saline-treated animals (176.6±34.4 s; n=8). Pretreatment with the histamine biosynthesis inhibitor alpha-fluoromethylhistidine (5 µg, i.c.v.) prevented such effect (150.7±61.3 s, n=9). A reduction of freezing time was observed in oleoylethanolamide-treated rats after intra-BLA infusions with the H1R antagonist pyrilamine (0.9 µM 142.4±35.4 s, n=7) or H2R antagonist zolantidine (0.1 µM 132.5±51.7 s, n=10) as compared with controls (247.4±55.2 s, n=10). Using microdilysis technique we observed an increase of HA release (up to 120%, basal HA release, 66.8±23.1 fmol/15min, n=5) from the BLA of freely moving rats after oleoylethanolamide i.p. injection at the same dosage used in the behavioural paradigm. But not from the hypothalamic ventromedial and paraventricular nuclei, whereas it decreased histamine release from the prefrontal cortex and nucleus accumbens. The involvement of histamine in the BLA, in oleoylethanolamid-induced promnesic is suggested. Salvatore Magara, Maria Lindskog
Karolinska Institute, Sweden HISTAMINE RECEPTORS MODULATE GLUTAMATE TRANSMISSION Histamine is a modulatory neurotransmitter that regulates neuronal activity in many brain areas. Modulatory neurotransmitters are the primary targets of antidepressant drugs that can exert their effects by modulating glutamatergic transmission and plasticity. Histamine receptors affect both cognition and mood, however, little is known regarding the underlying mechanism and the role of glutamate transmission modulation. We measured the effects of the H3R antagonist clobenpropit in the Flinders Sensitive Line (FSL), a rat model of depression with impaired glutamatergic transmission. Clobenpropit reduced immobility time in the forced swim test, enhanced long-term synaptic potentiation (LTP) in hippocampal slices, but did not affect the frequency or amplitude of excitatory inputs into CA1 pyramidal neurons. Interestingly, the clobenpropit-induced increase in LTP and the antidepressant effect were completely prevented by H1R and H2R antagonists. Thus, clobenpropit's antidepressant effects and the enhanced synaptic plasticity require hippocampal H1R and H2R activation. One proposed mechanism of fast-acting antidepressant drugs is to modulate the spontaneous glutamate transmission (the miniature EPSC, mEPSC). Notably, H1, but not H2, receptor activation reduces mEPSC frequency. Taken together our results show the effect of histamine receptors on glutamate transmission and plasticity, and propose a mechanism for the antidepressant effect of the histamine H3 receptor. ADDICTION AND BEHAVIOR
University of Gothenburg, The Sahlgrenska Academy, Sweden CENTRAL ADMINISTRATION OF THE ANOREXIGENIC PEPTIDE NEUROMEDIN U DECREASES ALCOHOL INTAKE AND ATTENUATES ALCOHOL-INDUCED REWARD IN RODENTS By investigating the neurochemical mechanisms through which alcohol activates the brain reward systems novel treatment strategies for alcohol use disorder (AUD) can be developed. In contrast to the common view of the function of gut-brain peptides, such as neuromedin U (NMU), to regulate food intake and appetite a novel role in reinforcement mediation has been implied. The anorexigenic effects of NMU are mediated via NMU2 receptors (NMUR2), preferably in the arcuate nucleus and paraventricular nucleus. The expression of NMUR2 is also expressed in several reward related areas in the brain, suggesting a role in reward regulation. The present experiments were therefore established to investigate the effect of intracerebroventricular administration of NMU on alcohol-mediated behaviors in rodents. We found that central administration of NMU attenuated alcohol-induced locomotor stimulation, accumbal dopamine release and the expression of conditioned place preference in mice. In addition, NMU dose dependently decreased alcohol intake in high, but not in low, alcohol-consuming rats. Central NMU administration did not alter blood alcohol concentrations nor change corticosterone levels in rodents. Given that AUD is a major healthcare challenge, where novel treatment strategies are warranted, our data suggest that NMU analogues deserves to be evaluated as novel treatment of AUD. Vinay Parikh, Robert D. Cole, Leonardo A. Ortega, Thomas J. Gould Department of Psychology and Neuroscience Program, Temple University, USA NICOTINE DEPENDENCE AND DEFICITS IN COGNITIVE FLEXIBILITY: NEUROCHEMICAL CIRCUIT MECHANISMS Behavioral and cellular mechanisms underlying nicotine addiction-related behaviors are not fully understood. Here, we investigated the effects of chronic nicotine and nicotine withdrawal on cognitive flexibility in mice. Because brain-derived neurotrophic factor (BDNF) modulates corticostriatal plasticity and striatal glutamate dynamics, and frontostriatal circuits are critical for cognitive flexibility, we also explored the effects of chronic nicotine on these neurochemical substrates. Chronic nicotine did not alter strategy switching but impaired reversal learning. Moreover, these deficits were associated with higher perseverance to the previously rewarded stimulus. BDNF levels and glutamate transmission was suppressed in the dorsal striatum of nicotine-exposed mice and striatal BDNF levels positively correlated with perseverative errors. On the other hand, nicotine withdrawal disrupted strategy switching by impairing the ability to execute a new decision strategy but not due to higher perseverance to the old strategy. Additionally, the ratio of striatal to prefrontal BDNF robustly increased during withdrawal reflecting overactive striatal BDNF signaling and glutamate transmission. Together, these data indicate that the effects of chronic nicotine on decision processes are linked to maladaptive changes in frontostriatal BDNF and glutamate dynamics. As cognitive deficits are hypothesized to predict relapse, normalizing BDNF signaling may restore executive function and facilitate smoking cessation. Lauren M. Burgeno, Nicole L. Murray, Ryan D. Farero, Ingo Willuhn, Paul E.M. Phillips University of Washington, USA DIAMETRIC CHANGES IN CUE-ELICITED PHASIC DOPAMINE RELEASE MEDIATE DRUG-TAKING AND DRUG-SEEKING Altered dopamine transmission is implicated in most contemporary theories of drug abuse, however the timing and directionality of these changes remain debated. Many studies, including recent work from our lab, demonstrate that dopamine release in the nucleus accumbens core (NAcc) serves as a satiety-like signal, thereby regulating drug intake. However, other work suggests NAcc dopamine promotes craving and drives cue-driven drug-seeking. How might cue-elicited dopamine transmission within the NAcc both decrease drug-taking and also increase drug-seeking? Since drug cues serve distinct roles during drug-taking and drug-seeking we hypothesized that there would be a divergence in cue-elicited dopamine responses in these differing contexts. We used fast-scan cyclic voltammetry at chronically implantable microelectrodes to test the prediction that dopamine responses elicited by unexpected presentation of drug- cues in a drug-seeking context would increase over the course of several weeks of drug-taking. As predicted, we find that non-contingent cue elicited phasic dopamine transmission increases with drug history and signals are larger in animals given long-access (6-hour/day) than those given short-access (1-hour/day). The directionality of changes in dopamine responses observed during drug-seeking directly oppose those previously observed during drug-taking, suggesting that NAcc dopamine transmission may contribute to multiple core symptoms of substance use disorders. Addiction Biology Unit, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Sweden DEEP BRAIN STIMULATION OF THE VTA - ACUTE BEHAVIORAL EFFECTS AND ACCUMBAL DOPAMINE Alcohol addiction is a major contributor to the global burden of disease, making the limited available options for treating alcohol addiction problematic. The dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens (nAc) is considered crucial for developing addiction. Deep Brain Stimulation (DBS) - electrical high frequency stimulation (HFS) delivered via implanted electrodes to specific parts of the brain, has emerged as a potential new treatment for addiction. Recent studies suggest that DBS of the nAc may alleviate cravings and alcohol consumption in patients. Interestingly, DBS appears to have an unknown mechanism of action and it is unclear if changes in dopaminergic signaling are involved. The aim of this project is to study how accumbal dopamine (DA) release and behavior are impacted by different stimulation settings. Using in vivo microdialysis in freely moving rats in combination with DBS, we found different settings produces differential acute behavioral effects as well as location-dependent influence on nAc DA release. Perhaps surprising, continuous HFS did not appear to induce a functional lesion of the VTA. We conclude that DBS in the brain reward system may produce interesting neurochemical and behavioral reactions that may be beneficial in relation to drug-induced adaptations. Jesper Vestlund, Daniel Vallöf, Jörgen A.Engel, Elisabet Jerlhag Institute of Neuroscience and Physiology, Department of Pharmacology, The Sahlgrenska Academy at the University of Gothenburg, Sweden THE ANOREXIGENIC PEPTIDE NEUROMEDIN U (NMU) ATTENUATES AMPHETAMINE-INDUCED LOCOMOTOR ATIMULATION, ACCUMBAL DOPAMINE RELEASE AND EXPRESSION OF CONDITIONED PLACE PREFERENCE IN MICE Amphetamine addiction, besides its substantial economical consequence, is a serious cause of mortality and morbidity. Addictive drugs activate the mesolimbic dopamine system a dopamine projection from the ventral tegmental area to the nucleus accumbens (NAc). The physiological function of gut-brain peptides are to regulate food intake but increasing evidence also suggest a role in drug reinforcement. A novel candidate for reward regulation is the anorexigenic peptide neuromedin U (NMU). The ability of intracerebroventricular (icv) administration of NMU to influence on amphetamine's effects on locomotor stimulation, accumbal dopamine release and conditioned place preference (CPP) was examined in mice. In addition the ability of accumbal administration of NMU to influence on amphetamine's effects on locomotor activity and CPP was examined in mice. Icv administration of NMU attenuates amphetamine-induced locomotor stimulation, accumbal dopamine release and expression of CPP in mice. NMU administration into the NAc blocks the ability of amphetamine to induce a locomotor stimulation but did not attenuate the amphetamine-induced expression of CPP in mice. Our novel data suggest that pharmacological agents targeting central NMUR2 represent a promising therapeutic strategy for treating drug addiction. Daniel Vallöf, Jörgen A Engel, Elisabet Jerlhag Department of Pharmacology, University of Gothenburg, Sweden GUT-BRAIN AXIS: INVOLVEMENT OF GASTROINTESTINAL HORMONES IN ALCOHOL USE DISORDER Alcohol use disorder (AUD), a chronic relapsing brain disease, is a serious health problem worldwide. The mesolimbic dopamine system, consists of dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), mediates the rewarding properties of addictive drugs including alcohol. By identifying the complex neurobiological mechanisms involved in this activation, novel targets for treatment of AUD can be identified. The findings that reward induced by food and drugs of abuse involve common mechanisms raises the possibility that gut-brain hormones traditionally known to control appetite, such as ghrelin, glucagon-like peptide 1 (GLP-1), neuromedin U (NMU) regulate reward. We have shown that a ghrelin receptor antagonist, GLP-1 receptor agonist and NMU receptor agonist attenuates the ability of alcohol, as well as other addictive drugs, to increase accumbal dopamine release. Specifically, we have shown that ventral tegmental ghreandreas receptors as well as accumbal NMU and GLP-1 receptors regulate alcohol-induced reward as measured by conditioned place preference and locomotor stimulation. In addition, these gut-brain peptides regulate the intake as well as motivation to consume alcohol. Collectively, these data contributes to understanding the neuronal mechanisms underlying the dependence-producing properties of alcohol and could tentatively contribute to new treatment strategies for AUD. Julia Morud Lekholm Addiction Biology Unit, Sahlgrenska Academy, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden PROLONGED NICOTINE ABSTINENCE PRODUCES DISINHIBITORY BEHAVIOR AND ALTERS ACCUMBAL GABAERGIC NEUROTRANSMISSION Nicotine is one of the most commonly abused substances worldwide, with a low cessation success rate and high risk of relapse. Nicotine has previously been considered as a cholinergic and dopaminergic drug, but studies also report nicotine-induced alterations in GABAergic neurotransmission. We therefore aimed to investigate tentative changes in GABAergic signaling in the nucleus accumbens (nAc) caused by repeated nicotine administration and subsequent abstinence. Adult male Wistar rats received daily nicotine administration during three weeks (0.36 mg/kg/day) after which accumbal GABAergic neurotransmission and disinhibitory behavior were evaluated following three months of nicotine abstinence. Our data indicate that protracted nicotine abstinence causes an increase in spontaneous disinhibitory behavior that parallels an altered electrophysiological response to GABAergic acting drugs and suppressed inhibitory activity in the nAc. To validate the involvement of accumbal GABAergic function in the disinhibitory behavior, rats were treated with local injections of diazepam (20 µg) prior testing on the elevated-plus maze. Diazepam increased disinhibition in this model, illustrating that increased GABAergic tone in the nAc produces disinhibitory and risk-taking behavior in the rat. The data presented here suggest neurotransmission, which might influence risk-taking behavior after extended abstinence. Drexel University, Philadelphia, USA HYPOCRETIN RECEPTOR 1 KNOCKDOWN DISRUPTS DOPAMINE AND BEHAIVIORAL RESPONSES TO COCAINE The hypocretin / orexin (HCRT) system has been recognized to modulate motivated behavior via actions on the mesolimbic dopamine (DA) system. This generally neuroexcitatory peptide system sends extensive projections to numerous reward-related regions including the ventral tegmental area (VTA) where both the HCRT receptor 1 (HCRTr1) and HCRT receptor 2 subtypes are found. Although, the existing literature points to the HCRTr1 as important for regulating reward and reinforcement, the majority of studies have employed acute and peripheral pharmacological manipulations of HCRT signaling. Consequently, little is known about the long-term, modulatory role of HCRTr1 manipulations or the specificity of actions at this receptor within the VTA. To address these issues, we knocked down HCRTr1 in the VTA and used fast scan cyclic voltammetry to measure changes to DA signaling in the NAc. Further, we evaluated the effects of HCRTr1 knockdown on cocaine self-administration behavior. Results indicate that knockdown disrupts DA neurotransmission in the NAc under baseline and cocaine conditions, and reduces cocaine self-administration. These observations provide further support for the involvement of HCRTr1 in regulating reward and reinforcement, and suggest that HCRTr1 may be a potential target for pharmacotherapies to treat cocaine abuse. Institute of Neuroscience and Physiology, University of Gothenburg, Sweden ANDROGEN RECEPTORS MODULATE SOCIAL RECOGNITION BUT NOT SOCIAL INVESTIGATION The role of sex and androgen receptors (ARs) for social preference and social memory is rather unknown. In this study we compared male and female mice and males lacking ARs specifically in the brain, using the mouse line ARNesDel, with respect to social preference, assessed with the three-chambered apparatus test, and social recognition, assessed with the social discrimination procedure. In the social discrimination test we also evaluated the tentative importance of the sex of the stimulus animal. Novel object recognition and olfaction were investigated to complement the results from the social tests. Gene expression analysis was performed to reveal molecules involved in the effects of sex and androgens on social behaviors. All three test groups showed social preference in the three-chambered apparatus test. In both social tests an AR-independent sexual dimorphism was seen in the persistence of social investigation of female conspecifics, whereas the social interest towards male stimuli mice was similar in all groups. Male and female controls recognized conspecifics independent of their sex, whereas ARNesDel males recognized female but not male stimuli mice. Moreover, the non-social behaviors were not affected by AR deficiency. The gene expression analyses indicated that Oxtr, Cd38, Esr1, Cyp19a1, Ucn3, Crh and Gtf2i were differentially expressed between the three groups. In conclusion, our results suggest that ARs are required for recognition of male but not female conspecifics, while being dispensable for social investigation towards both sexes. In addition, the AR seems to regulate genes related to oxytocin, estrogen and Wil iam's syndrome. Department of Pharmacology and Toxicology, Institute of Pharmacy and Center for Chemistry and Biomedicine (CCB), University of Innsbruck, Innsbruck, Austria THE MODULATORY ROLE OF SEPTAL RELAXIN-3/RXFP3 RECEPTOR SYSTEM IN NEUROENDOCRINE AND BEHAVIORAL STRESS FUNCTION IN RATS Previous studies suggest that the lateral septum (LS) plays an important role in stress mechanisms. However, the underlying neurochemical mechanisms of how LS neurons modulate neuroendocrine and behavioral stress responses are rather unknown. Several lines of evidence suggest a robust contribution of septal neuropeptides such as relaxin-3 (RLX3) system and its preferred receptor the RFP3 receptor in mediating these effects. Therefore, the aim of the present study was to examine the role of the septal RLX3/RXFP3 receptor system in neuroendocrine and behavioral stress responses. We found that RLX3 infusions into the LS attenuated stress-induced secretion of ACTH and corticosterone (CS) in the adult male rat but had no effect on basal stress hormone levels. Conversely, the administration of the selective RXFP3 receptor antagonist R3B1-22R induced the opposite effect, an enhancement in stress-induced increase of plasma ACTH levels. Moreover, our data show that intraseptal RLX3 infusions increased active coping and reduced immobility during the forced swim exposure as RLX3 treated animals showed increased struggling behavior and less floating behavior than controls. Taken together, our data suggest that the RLX3/RXFP3 receptor system in the LS is critically involved in the regulation of neuroendocrine and behavioral stress function. Funded by the Austrian-Science-Foundation (FWF). 11. Jenny Landin, Petronella Kettunen, Daniel Hovey, Anna Zettergren, Lars Westberg Department of Pharmacology, Department of Psychiatry and Neurochemistry, University of Gothenburg, Sweden AN OXYTOCIN RECEPTOR ANTAGONIST INHIBITS SOCIAL PREFERENCE IN ZEBRAFISH The zebrafish is a fairly novel model system that is particularly suitable for psychiatric research due to the developed behavioral tests, ease of pharmacological treatment and the available mutated lines of genes involved in human syndromes and disorders. Due to their strong tendency to socialize in shoals, zebrafish serves as a good model to study social behavior. Although the neuropeptide oxytocin is well-known to be crucial for social behaviors in mammals the role of isotocin, the fish homolog of oxytocin, for shoaling behavior in zebrafish is as yet not clarified. Aim: To investigate if an oxytocin receptor antagonist inhibits sociability in adult zebrafish. Methods: Adult male and female zebrafish were intraperitoneally injected with either the non-peptidergic oxytocin receptor antagonist L-368,899 or vehicle, and subsequently tested in a social preference paradigm. Results: Zebrafish treated with the oxytocin receptor antagonist displayed decreased social preference compared to fish injected with vehicle. Conclusions: These results indicate that endogenous isotocin is involved in social preference in zebrafish, and show promise for future explorations of the network underlying social behavior in the zebrafish. BIOMARKERS
12. S. Waters, P. Svensson, C. Sonesson, E. Ljung, B. Svanberg, N. Waters Integrative Research Laboratories, Gothenburg, Sweden ISP - AN IN VIVO BASED SYSTEMS PHARMACOLOGY PLATFORM FOR PHENOTYPIC CHARACTERIZATION OF CNS TREATMENTS, TRANSLATIONAL MODELLING AND DRUG DISCOVERY The CNS is a complex biological system, which limits the efficiency of highly reductionist approaches to understand physiological and pharmacological mechanisms in healthy or disease states, and to find new treatments. In drug discovery, integrative approaches, ie phenotypic screening, have proven more successful than target-based screening in generating first-in-class drugs. We developed a standardized process for CNS drug discovery, the Integrative Screening Process (ISP), based on in vivo systems-response profiling, using a comprehensive array of biomarkers to obtain sensitive fingerprints of the functional, network level effects of CNS-acting compounds. Biomarkers include monoaminergic neurochemistry, IEGs, and behavioural patterns. These fingerprints provide a basis for comparison/classification of compounds and predicting clinical properties, beyond what can be achieved using single-endpoint methodologies. ISP can be described as an efficient, in vivo based systems pharmacology platform for phenotypic characterization of CNS treatments and translational modelling. ISP has been systematically applied for more than a decade, resulting in an extensive database of in vivo systems response profiles of in-house compounds and >300 reference compounds with documented CNS effects in humans. ISP has generated 8 candidate drugs, four in phase I, one in late stage development for HD. Experimental/computational methodology and translational modelling strategies will be discussed. BIOSENSORS
13. Melissa Malvaez University of California, USA GLUTAMATE RELEASED INTO THE BASOLATERAL AMYGDALA TRACKS REWARD VALUE ENCODING AND THE USE OF THIS INFORMATION TO GUIDE REWARD SEEKING Reward-seeking decisions are heavily controlled by the value of the specific reward they attain. This value is acquired and updated through the incentive learning process that occurs when the reward is experienced in a relevant motivational state (e.g., experience with a specific food in a novel hungry state). The basolateral amygdala (BLA) participates in this process, but precisely how is unknown. Because the BLA is densely innervated by both cortical and thalamic glutamatergic projections, we hypothesized that glutamate released into the BLA would track changes in reward value important for value-guided reward seeking. To test this, we used electroenzymatic biosensors to make near-real time measurements of BLA glutamate concentration changes during an incentive learning experience and during a subsequent reward-seeking test. We detected transient elevations in BLA glutamate concentration that tracked the incentive learning process. Interestingly, we also detected transient glutamate elevations immediately preceding bouts of reward-seeking activity following incentive learning. These data suggest that transient BLA glutamate release tracks the reward evaluation process and use of this value information for guiding reward-seeking decisions. Ongoing experiments are evaluating the source of these signals to provide information on how the BLA functions within a broader circuit to coordinate reward-seeking actions. University of Leicester, UK DOPAMINE D4 AGONIST ATTENUATES TONIC GLUTAMATE RELEASE IN THE PREFRONTAL CORTEX Cognitive and attentional processing deficits are seen in multiple pathological conditions including schizophrenia and Alzheimer's disease. The frontal cortex, and in particular the glutamate system, is crucially involved in both attention and cognition. Cognitive tasks in rats have been used to assess potential cognitive enhancing drugs which may reverse these deficits. Cognitive and attentional behaviours in the rat are attenuated by the dopamine D4 receptor agonist, PD168077. Thus there is a link between cognition, and the frontal cortex glutamate and dopamine systems. We therefore examined the effects of PD168077 on glutamate release within the prefrontal cortex in vitro. We used enzyme based glutamate sensors to record tonic levels in vitro within three frontal subregions. There were differences in glutamate levels between the three subregions. In addition, PD168077 produced a dose dependent decrease in the tonic levels of glutamate within the prelimbic cortex, showing that activation of dopamine D4 receptors in the prefrontal cortex reduces tonic levels of glutamate. There is, therefore, a mechanistic link between dopamine D4 receptors and the prefrontal glutamate system. This interaction may play an important role in cognitive functions in the normal and pathological brain. 15. Saidhbhe L. O'Riordan1, J.P. Lowry1 1 Neurochemistry Research Unit, BioAnalytics Laboratory, Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland REAL-TIME ELECTROCHEMICAL MONITORING OF PHYSIOLOGICAL BIOMARKERS; METHODS TO INVESTIGATE DISORDERS OF THE BRAIN AND EVALUATE MUSCLE TISSUE VIABILITY. The association of oxidative stress as an initiating factor or merely as a downstream event, in the pathophysiology of various neuronal dysfunctions, remains inconclusive. It is possible to measure the onset and progression of oxidative stress in the brain via the real-time detection of hydrogen peroxide. We have previously successfully demonstrated the in-vitro characterisation of a catalase-based hydrogen peroxide sensor. We now demonstrate the real-time detection of brain hydrogen peroxide in the freely-moving animal using our catalase-based electrochemical sensor. We have demonstrated the ability of our sensors and associated methodology to examine normal brain function and monitor neurological disease related biomarkers in rodents. With a view to potential clinical implementation, we have modified the application area of a platinum based electrochemical microsensor for the detection of brain tissue oxygen. We now seek to provide evidence, of the suitability of this oxygen sensor, to measure the onset and progression of ischemia in the peripheral muscle tissue. This research was funded by the Health Research Board [RP/2004/44 and HRA-POR-2014-694]. 16. Isabel Álvarez-Martos and Elena E. Ferapontova Interdisciplinary Nanoscience Center (iNANO) and Danish National Research Foundation: Center for DNA Nanotechnology (CDNA), Aarhus University, Denmark SPECIFIC ANALYSIS OF DOPAMINE IN SERUM AND IN THE PRESENCE OF STRUCTURALLY RELATED NEUROTRANSMITTERS BY ELECTROCHEMICAL LABEL-FREE APTASENSOR Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and thus in vivo analysis of dopamine in the presence of structurally related neurotransmitters represents a Holy Grail of Neuroscience. Interference from those neurotransmitters generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer, tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The aptasensor allowed continuous 0.5 h amperometric analysis of dopamine in 10% serum, within the physiological 100 nM—1 µM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and L-DOPA. In a flow-injection mode, the aptasensor response to dopamine was ∼1s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 nA μM-1 cm-2 and the LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT, and thus may be further applicable in biomedical research. 17. Yuanmo Wang and Ann-Sofie Cans Chalmers University of Technology, Gothenburg, Sweden DEVELOPMENT OF ULTRA-FAST, NANO-STRUCTURED BIOSENSORS FOR PROBING NEURONAL COMMUNICATION Neuronal activities are very fast and occur in the time scale of milliseconds. In order to capture and monitor the rapid transients of metabolites and non-electroactive molecules involved in neuronal activities in the brain, a novel method for construction and design of an enzyme based electrochemical sensor has been developed in our lab. The sensor is constructed by adsorbing GOx onto a gold nanoparticle coated carbon fiber microelectrode. The AuNp are modified onto the surface of the carbon fiber to increase the surface area, surface curvature and to maximize the retained enzymatic activity upon adsorption. With this new approach we developed a sensor that can detect acetylcholine (i.e. neurotransmitter) with a time resolution in millisecond. Currently the glucose sensor and glutamate sensor are under development. 18. Jenny Bergman University of Gothenburg, Gothenburg, Sweden ENZYME COVERAGE AND IN SITU CHARACTERIZATION OF NANOPARTICLES ON ELECTRODE SURFACES –KEY PARAMETERS FOR A FAST BIOSENSOR We have focused on new strategies for enzyme based amperometric biosensor design for electrochemical detection of non-electroactive molecules important for brain function such as acetylcholine, glutamate and glucose. These sensors are aimed for recording of transients of these analytes at single cells as well as brain tissue. To be able to monitor the fast alternations of neurotransmitters released from single vesicle exocytotic events or brain tissue a sensor with high temporal resolution is required. By careful characterization and design of an enzyme-based gold nanoparticle coated carbon fiber electrode and limiting the enzyme coverage to close to monolayer, we recently showed that an enzymatic biosensor can reach response times fast enough to resolve single vesicle release events(1). Here we present a facile method to directly quantify the amount of glucose oxidase bound to the surface of different nanoparticle materials coating a carbon electrode surface. This is performed by combining an electrochemical stripping technique(2) to dissolve the bioconjugates from the electrode surface together with a fluorimetry assay to quantify the surface bound fluorescently labeled enzymes. This new method approach is a means to directly control the enzyme thickness at the electrode surface, which is important for optimizing the time resolution for the sensors. (1) J.D. Keighron ACS Chem. Neurosci., 2015, 6 (1), pp 181–188 (2) Ying Wang Analyst, 2012, 137, 4693. 19. Carlos Cordeiro University of Groningen; Brains On-Line BV, Netherlands IN VIVO REAL-TIME BIOMONITORING WITH GOLD COATED TUNGSTEN (W-Au) BASED MICROBIOSENSORS Amperometric enzyme-based biosensors are increasingly employed in continuous in vivo biomonitoring. Initially confined to continuous glucose measurements, its applications have reached other biomedical areas, including experimental neuroscience. Nowadays, implantable biosensors are mostly based on Pt or Pt/Ir needle-type microelectrodes, functionalized with matrices containing enzymes specific for the target analyte(s). However, when implanted, biosensors often bend, damaging its surface, degrading its bioanalytical properties. Additionally, Platinum and Pt/Ir alloys are highly malleable, hindering biosensor downscale. The use of low malleability wires (W), coated with a highly conductive layer (Au) in microelectrode construction, will enable biosensor downscale, reduce tissue damage, allowing longer implantation periods. We developed implantable needle-type gold coated tungsten (W-Au) microelectrodes for in vivo biomonitoring. We functionalized the surface of W-Au microelectrodes with permselective membrane(s) followed by the assembly of an enzymatic hydrogel. All biosensors were characterized in vitro (at 37ºC), at constant potential (600-800 mV vs Ag/AgCl). For in vivo evaluation, biosensors were implanted in the brain of anesthetized animals, that were submitted to relevant pharmacological challenges. In vitro calibration revealed that the biosensors were selective and sensitive for the target analyte(s). Preliminary data showed that W-Au needle-type microelectrodes were able to continuously monitor, in real time, changes in the target analyte, induced by pharmacological modulation. CIRCUITS / TOXICITY
20. Youmna Nasser Ayad Alexandria University, Egypt THE PROTECTIVE EFFECT OF TURMERIC AGAINST NEUROAFLATOXICOSIS Aflatoxin B1 is one of the most prevalent known mycotoxins. It affects human and animals by eating Aflatoxin contaminated foods or by inhaling dust particles of Aflatoxin B1. Turmeric has been used for 4,000 years to treat a variety of conditions. It may help in fighting infections and some cancers, reducing inflammation. Moreover, it has gotten a lot of press regarding its anti-oxidizing effect. In this study, adult albino rats were divided into 4 groups (Group 1: Negative control, Group 2: Positive control (DMSO-treated group), Group 3: Aflatoxin-treated group, Group 4: Aflatoxin and Turmeric-treated group). Our results show that the total serum level of NSE (Neuron Specific Enolase) in Aflatoxin-treated animals was significantly (p < 0.05) elevated as compared to the controls. This result indicates a possible increased level of inflammation in the brain of Aflatoxin-treated rats. Despite the slight increase (non-significant) in the NSE level after oral administration of turmeric together with Aflatoxin were as compared to controls, its level was interestingly lower than that of the Aflatoxin-treated animals. This indicates a possible anti-Aflatoxin protective effect resulting from administration of turmeric. Our histological examination revealed neuronal disorganization and neuropil deterioration in the Aflatoxin injected animal group. 21. B. Laughlin, S. Rice, I. Bailey, K. Drew Institute of Arctic Biology, University of Alaska Fairbanks, USA ADENOSINE 1AR TARGETED TEMPERATURE MANAGEMENT IN RATS AND RESULTANT PHYSIOLOGICAL EFFECTS OF A PHARMACOLOGICAL INDUCED HYPOMETABOLIC STATE. Hype and hope has been generated by promises of inducing suspended animation. Biased reporting of positive results underestimates complications that ultimately limit translation. In this study we apply the Adenosine 1A receptor (A1AR) agonist N6-Cycolhexyladenosine (CHA) in a manner similar to prior work in this lab found to induce a hibernation-like state in rats. Temperatures, oxygen consumption, and cardiovascular parameters are monitored throughout the experiment. Core body temperature is maintained between 30-34°C for 24-48hrs by adjusting the dose of CHA and/or adjusting the surface temperature of the cage via a thermoelectric cooling device. Side effects of bradycardia and hypotension are managed with administration of an adenosine receptor antagonist, 8-SPT. Results indicate individual variation in response to the CHA. Low doses of CHA resulted in bradycardia but had little to no effect on the inhibition of thermogenesis. Starting administration at a low concentration while doubling the concentration every 4 hours resulted in the development of tolerance to the drug's effect on thermogenesis but not bradycardia. Brain temperature follows core body temperature closely during the cooling and rewarming phases. Oxygen consumption requirements decreased after CHA administration. CHA is a potent inhibitor of thermogenesis and metabolism at doses greater than 0.4mg/kg. 22. Sebastian Illes University of Gothenburg, Institute for Neuroscience and Physiology, Gothenburg, Sweden MONITORING THE FUNCTIONAL CONSEQUENCES OF HUMAN CEREBROSPINAL FLUID-DERIVED MOLECULES ON NEURONAL CIRCUITS For decades it has been hypothesized that molecules within the cerebrospinal fluid (CSF) diffuse into the brain parenchyma and influence the function of neurons. However, the functional consequences of CSF on neuronal circuits are largely unexplored and unknown. A major reason for this is the absence of appropriate neuronal in vitro model systems, and it is uncertain if neurons cultured in pure CSF survive and preserve electrophysiological functionality in vitro. We present an approach to address how human CSF (hCSF) influences neuronal circuits in vitro. We validate our approach by comparing the morphology, viability, and electrophysiological function of single neurons and at the network level in rat organotypic slice and primary neurons cultivated either in hCSF or in defined standard culture media. Our data indicate that hCSF represents a physiological environment for neurons in vitro and a superior culture condition compared to the defined standard media. Moreover, this experimental approach paves the way to monitor the functional consequences of CSF-derived molecules on neuronal circuits. DISORDERS
23. S. Barde1, J. Ruegg2, T. Ekström2, Miklos Palkovits3, G. Turecki4, J. Prud'homme5, N. Mechawar4 and T. Hökfelt1 1 Department of Neuroscience, Karolinska Institutet, Sweden; 2 Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; The Center for Molecular Medicine, Sweden; 3 Neuromorphological and Neuroendocrine Research Laboratory, Department of Anatomy, Histology and Embryology, Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary and Human Brain Tissue Bank and Laboratory, Semmelweis University, Hungary, 4 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Canada; 5 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Canada; Integrated Program in Neuroscience, McGill University, Montréal, QC, Canada; Department of Psychiatry, McGill University, Canada ALTERED EXPRESSION OF GALANIN AND ITS RECEPTOR TRANSCRIPTS IN POSTMORTEM BRAIN TISSUE OF SUICIDES WITH MAJOR DEPRESSIVE DISORDER Major depressive disorder (MDD) is a serious illness affecting around 10-20% of people in the Western world. Current pharmacological treatments mainly target the monoamine transporters, but the results are not satisfactory. Galanin is a 29 (30 in humans) amino acid neuropeptide that is expressed in many regions of the rodent brain. Here, we used qPCR to study transcript expression of galanin and its receptors in five different regions of post-mortem human brains, including Broadmann area (BA) 8/9, BA 24, locus coeruleus (LC), dorsal raphe nucleus (DRN) and medullary raphe nucleus (MRN). In total, 212 samples from age- and gender-matched controls and suicides (with MDD) were processed. Galanin mRNA was upregulated in BA8/9 of females in addition to LC, DRN and MRN of males and females. For the receptors, GalR1 was upregulated in BA8/9 and DRN of males and females and LC of female brain samples. GalR3 was upregulated in the LC, DRN and MRN of male and female brain samples versus downregulated in the male BA8/9. These findings suggest involvement of the galanin system (galanin, GalR1 and R3) in control of mood and behaviour and that some galanin receptors may represent a novel target for development of antidepressant therapeutics. 24. Rong Ma, Bengt Gustafsson Departement of Physiology, Institute of Neuroscience and Physiology at Sahlgrenska Academy, University of Gothenburg, Sweden LOW FREQUENCY SYNAPTIC DEPRESSION AT PERFORANT-PATH SYNAPSES ONTO CA1 PYRAMIDAL CELLS IN THE DEVELOPING HIPPOCAMPUS IS NOT EXPLAINED BY VESICLE DEPLETION In the developing hippocampus (2nd postnatal week) the transmission at Schaffer collateral synapses onto CA1 pyramidal cells (SC-CA1 synapses) is labile in that the synaptic response declines even at very low frequency activation (0,013-1Hz), this depression explained by AMPA silencing. Such very low frequency depression can also be observed at the perforant path synapses onto these neurones (PP-CA1 synapses), but in contrast to the SC-CA1 synapse, the depression at PP-CA1 synapses increases substantially with increasing frequency within this frequency range. 25. Mark M. Rasenick University of Illinois College of Medicine/ Pax Neuroscience. USA A BIOMARKER FOR DEPRESSION, ANTIDEPRESSANT RESPONSE AND THERAPEUTIC SELECTION Although approximately 1 in 6 will suffer from Major-Depressive-Disorder (MDD) during their lifetime, many patients with MDD go undiagnosed in primary care settings, and 20% are incorrectly diagnosed with MDD. Given the substantial medical, economic and social costs, there is tremendous need for a simple objective biomarker test to aid clinicians in accurately identifying MDD. No test currently exists that can accurately diagnose MDD. Further, clinical response to antidepressant therapy requires as much as two months, necessitating a test that can predict response within a few days of the inception of treatment. Providers can use this for diagnosis and as an early predictor of response, while Phama can use this to stratify subjects for clinical trials. The Pax Neuroscience diagnostic biomarker Gαs-Sequestration-Assay (GSA) indicates that MDD patients have a greater proportion of Gαs captured in lipid rafts compared to non-depressed controls and can identify accurately, MDD. Pax used the same principles that underpin the GSA to develop a C6 glioma (Gαs -GFP) FRAP assay which can reliably predict antidepressant efficacy and that can be used to identify compounds with potential antidepressant activity (including rapid-acting molecules like ketamine). A FRAP-based high-content screen is being designed as an ex-vivo test to predict individual therapy. This technology offers an underlying technology to improve diagnosis. develop new drugs and personalize therapy. 26. Monica M. Marcus, Carl Björkholm, Anna Malmerfelt, Annie Möller, Ninni Påhlsson, Vladimir Ivanov, Åsa Konradsson-Geuken, Kristin Feltmann, Kent Jardemark, Björn Schilström, Torgny H. Svensson Department of Physiology and Pharmacology, Section of Neuropsychopharmacology, Karolinska Institute, Stockholm, Sweden ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR AGONISTS AND PAMS AS ADJUNCTIVE TREATMENT IN SCHIZOPHRENIA. AN EXPERIMENTAL STUDY Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Here we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive al osteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release and NS1738 enhanced burst firing and frequency of dopamine cells in the ventral tegmental area. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Taken together, our data support the utility of drugs targeting the α7 nAChRs to potentiate the effect of atypical antipsychotic drugs and suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia. 27. Torgny H. Svensson, Carl Björkholm, Åsa Konradsson-Geuken, Kent Jardemark, Monica M. Department of Physiology and Pharmacology, Section of Neuropsychopharmacology, Karolinska Institute, Stockholm, Sweden THE NOVEL ATYPICAL ANTIPSYCHOTIC DRUG BREXPIPRAZOLE, ALONE AND IN COMBINATION WITH ESCITALOPRAM, FACILITATES PREFRONTAL GLUTAMATERGIC TRANSMISSION VIA A DOPAMINE D1 RECEPTOR-DEPENDENT MECHANISM Brexpiprazole, a novel D2 receptor (R) partial agonist, was recently approved as monotherapy for schizophrenia, improving both positive and negative symptoms, and as adjunctive therapy to antidepressants for the treatment of major depressive disorder. To explore whether these clinical effects might involve modulation of prefrontal glutamatergic transmission we investigated the effect of brexpiprazole, alone and in combination with escitalopram, using in vitro electrophysiological intracellular recordings in deep layer pyramidal cells of the rat medial prefrontal cortex. Clinically relevant concentrations of brexpiprazole potentiated NMDAR-induced currents and electrically evoked EPSPs via activation of dopamine D1Rs, similar to the effects of clozapine. The corresponding effect of a sub-effective concentration of brexpiprazole was significantly potentiated by the addition of escitalopram. The combination of brexpiprazole and escitalopram, but not either drug alone, also potentiated AMPAR-mediated neurotransmission, similar to the potent and fast acting antidepressant drug ketamine. The effect on the AMPAR-mediated currents was also D1R dependent. Our results propose that brexpiprazole exerts a clozapine-like potentiation of prefrontal NMDAR-mediated currents, which may explain its efficacy in schizophrenia, including improved cognition. Add-on treatment with brexpiprazole to escitalopram also potentiated AMPAR-mediated transmission, which may provide a neurobiological explanation to the potent antidepressant effect of adjunctive therapy with brexpiprazole. 28. Linda Nguyen West Virginia University, Morgantown, WV PRECLINICAL BEHAVIORAL AND BIOCHEMICAL ANALYSES OF THE FAST-ACTING ANTIDEPRESSANT-LIKE POTENTIAL OF DEXTROMETHORPHAN Ketamine can exert rapid antidepressant effects, but its use is limited by abuse liability and adverse events. Herein, the over-the-counter antitussive dextromethorphan (DM) was investigated as a potential alternative to ketamine due to overlapping pharmacology. Mice were injected with DM, and behavioral (antidepressant-like effects in forced swim test (FST) and tail suspension test (TST)) and biochemical (pro- and mature brain-derived neurotrophic factor (BDNF) expression using western blot) tests were performed. Also, the role of AMPA and sigma-1 receptors in DM's antidepressant-like actions was examined, as these mechanisms may facilitate a rapid onset of antidepressant efficacy and are relevant to ketamine's effects. Our results revealed DM produces antidepressant-like actions in the FST and TST, similar to ketamine. In the FST, pretreatment with NBQX (AMPA antagonist) or BD1063 (sigma-1 antagonist) blocked DM's effects, indicating that AMPA and sigma-1 receptors play pivotal roles in mediating its antidepressant-like behaviors. DM however did not alter hippocampal pro-BDNF or BDNF levels, while ketamine increased pro-BDNF. These data suggest DM shares some, but not all, antidepressant-like effects with ketamine. Together with recent clinical evidence of DM's efficacy for bipolar depression and our preclinical findings, further studies are needed to examine DM's potential as a safe, fast-acting antidepressant. EXOCYTOSIS
29. Adam R. Meier University of Arizona, USA A NOVEL APPROACH TO SINGLE-CELL AMPEROMETRY USING AN ALL-POLYMER MICROFLUIDIC CHIP Single-cell amperometry is a powerful technique for measuring neurochemical release from cells and elucidating the mechanisms of these release events. Existing methodologies for single-cell amperometry rely on manual fabrication and positioning of carbon-fiber disk electrodes. In this work, we detail the fabrication, characterization, and implementation of a microfluidic chip that is capable of capturing single cells on a polymer microelectrode and measuring chemical release from that cell amperometrically. The device is fabricated in two parts: (i) PEDOT electrodes are patterned using standard photolithography techniques on one part of the chip and (ii) the fluidics, including the cell-capture channel, are made by injection molding on a second part of the chip. The two halves are then permanently bonded yielding a device that can be used to measure chemical release from single cells. This design eliminates the need for manual fabrication and positioning of electrodes for this type of measurement. A common model system for dopamine vesicle release, PC-12 cells, were measured using this new device. Cells were successfully captured at the electrode surface using software-controlled suction and exocytosis events were measured. This novel chip design provides a more efficient and controlled method to perform single-cell amperometry. 30. Takuya Hikima. Christian R. Lee, Edwin J. Vazquez-Cintron, Konstantin Ichtchenko and Margaret Department of Neurosurgery, NYU School of Medicine, USA DETECTION OF SOMATODENDRITIC DOPAMINE RELEASE USING WHOLE-CELL RECORDING: A ROLE FOR EXOCYTOSIS? A key feature of midbrain dopamine (DA) neurons is that they release DA from their cell bodies and dendrites (somatodendritic DA release). This release activates D2 dopamine receptors (D2Rs) on DA neurons, which inhibit spontaneous activity via G-protein-coupled inwardly-rectifying potassium channels (GIRKs). However, the basic question of whether somatodendritic DA release is exocytotic remains unresolved. We have begun to unravel this by testing the effect of derivatives of botulinum neurotoxin-A (BoNT-A), which cleaves SNAP-25, a SNARE-complex protein, on evoked D2R-mediated inhibitory currents (D2ICs) in voltage-clamped DA neurons as an index of somatodendritic DA release in midbrain slices. Initial recordings of evoked EPSCs in cortical cultures were used as a model system to confirm toxin efficacy. Recordings in substantia nigra pars compacta DA neurons were made in the presence of ionotropic glutamate and GABA receptor antagonists to isolate D2ICs. We found that endogenous application of the D2 agonist quinpirole or local electrical stimulation induced sulpiride-sensitive D2ICs. Importantly, intracellular BoNT-A analogue application abolished D2ICs. By contrast, no attenuation was seen with an in-activatable BoNT-A analogue. These data support a role for exocytosis in somatodendritic DA release, and pave the way for examination of neurotoxins that target other SNARE-complex components. 31. Neda Najafinobar, Andrew Ewing Chalmers University of Technology, Sweden THE EFFECT OF EXCITED FLUOROPHORE ON VESICLE FUSION AT THE SURFACE OF THE ELECTRODE Electrochemistry is a technique that can be used to detect the contents of neurotransmitter molecules in vesicles or liposomes by electrochemically oxidizing the content upon vesicle release. Previous work in quantifying the neurotransmitter content of single secretory vesicles has been performed using a technique called electrochemical cytometry. In our lab we have recently further developed this technique to become easier in performance and involves direct lysing of vesicles onto an amperometric electrode surface and without the need for a preceding separation step. Here we present how the electrochemical response from applying this new method to single adrenal chromaffin vesicles can be used to quantify vesicle content as well as study vesicle fusability as they impact a 33-um diameter disk-shaped carbon electrode. The frequency of the recorded amperometric spikes in each experiment has been used to probe the fusability of vesicle as a function of fluorofor concentration in the chromaffin vesicles membrane. Chromaffin granules were incubated with different concentration of fluorescent-labeled phospholipids before each experiment. In these experiments we used Fluorescent probe Rh-DOPE and or NBD-PS and probing vesicle fusion before and after subjection of samples to excitation wavelength of red (570nm) and blue (490nm) light respectively for each of the probes used. The data suggest a significant increase in the number and frequency of vesicles fusion onto the surface of the electrode when exciting the fluorophor of the granules incubated with fluorescently labeled phospholipid. Our finding show that the light itself or only labeled vesicle dose not change the frequency of vesicle fusion but the combination of theses two increase the chance of vesicle fusion. Therefore in this study we have shown that excited fluorescent-labeled phospholipids can change the membrane properties and facilitate vesicle fusability. 32. Xianchan Li1, Johan Dunevall2 and Andrew G. Ewing1,2 1 Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden 2 Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden SINGLE CELL AMPEROMETRY REVEALS CISPLATIN TREATMENT MODULATES THE RELEASE OF NEUROTRANSMITTERS DURING EXOCYTOSIS Chemotherapy with cis-diamminedichloroplatinum (cisplatin) induces mild cognitive impairment (commonly called chemo brain) including memory loss, difficulty for multi-tasking, difficulty focusing confusion etc. High doses usage of cisplatin can also causes peripheral neuropathy. So far, the cause of mild cognitive impairment or peripheral neuropathy by chemotherapy is unknown. So it is interesting to investigate the influence of cisplatin on the automomic nervous system. In the present study attempts have been made to determine the influence of cisplatin on catecholamine release from single cultured pheochromocytoma (PC12) cells. Pretreatment of PC12 cells with cisplatin influences cell exocytotic ability in a dose dependent manner. Low concentration of cisplatin stimulates catecholamine release while high concentration inhibits it. Single cell amperometry reflects that low concentration of cisplatin treatment increases exocytotic event frequency and event current with shorter duration, whereas high concentration of cisplatin treatment decreases exocytotic event frequency and event current with longer duration. In addition, the stability of the fusion pore formed during exocytosis and the late kinetic characteristics of the fusion pore are also regulated differentially by different concentration of cisplatin. This study suggests that cisplatin influences exocytosis by multiple mechanisms. 33. Lin Ren1, Soodabeh MajdiI1, Per Malmberg1, Andrew Ewing1,2 1 Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden. 2 Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden SYNAPTIC ZINC EFFCTS NEUROTRANSMITTER RELEASE DURING EXOCYTOSIS We have used electrochemistry at single cells to investigat the regulating functions of synaptic zinc in the release of neurotransmitters in pheochromocytoma (PC12) cells during exocytosis. In the last few years a number of studies have indicated the importance of zinc in regulating ion channels and intracellular pathways involved in neuroplasticity and also learning and memory.[1-3] The abundance of zinc in vesicles in the cortex, hippocampus and amygdala areas of the brain areas associated with memory formation, storage, and retrieval further support the neurophysiological importance of synaptic zinc. Furthermore, it is becoming more and more clear that the secreted zinc is not just some epiphenomenon that accompanies secretion of the "real" messengers from these cells. Synaptic vesicle zinc in these neurons is also thought to function as a neuromodulator upon its release into the synaptic cleft.[4-5] However, some reports have suggested that removing zinc from synaptic vesicles does not impair spatial learning, memory, or sensorimotor functions in the mouse. Whether synaptic vesicle zinc is involved in normal functioning of zinc-rich regions of the brain and spinal cord remains a big challenge in zinc-biology field. Therefore, it is of great significance to uncover the role of synaptic zinc in neuron activities. We induced zinc-rich and zinc-depleted models to study the efficacy of zinc on dopamine release in PC12 cells. We used single cell amperometry at carbon fiber electrodes to quantify the effects of zinc on exocytosis. We found that zinc inhibits the release of dopamine. And we also used fluorescence microscopy to localize vesiclar zinc to get more insight into the mechanism of how synaptic zinc affects exocytosis. REFERENCES 1. S. Martin, P. Grimwood, and R. Morris, Annu Rev Neurosci, 2000. 23: p. 649-711. 2. C. Sindreu, and D. Storm, Front Behav Neurosci, 2011. 5: p. 68. 3. R. Frederickson, C. Frederickson, and G. Danscher, Behav Brain Res, 1990. 38(1): p. 25-33. 4. K. Keller, Y. Chu, A. Grider, and J. Coffield, J Nutr, 2000. 130(6): p. 1633-40. 5. N. Whittle, M. Hauschild, G. Lubec, A. Holmes, and N. Singewald, J Neurosci, 2010. 30(41): p. 13586-96. 34. Daixin Ye1, Andrew Ewing1,2 1 Department of Chemistry, University of Gothenburg, Gothenburg, Sweden 2 Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden EFFECTS OF ANESTHETICS ON EXOCYTOTIC DOPAMINE RELEASE General anesthetics have potent effects on synaptic transmission, both on presynaptic terminals, by altering neurotransmitter release, and on postsynaptic neurons, by modulating their responses to neurotransmitters. Local anesthetics can block almost every nerve between the peripheral nerve endings and the central nervous system. Anesthetics are essential to modern medicine, and yet a detailed understanding of their mechanisms of action is lacking especially for the effects on the excytosis in single cell level. This work we mainly concentrate on the effects of the local anesthetics and general anesthetics on exocytosis in PC 12 cells and compare the differences on exocytosis between local anesthetics and general anesthetics. 35. Johan Dunevall1, Hoda Fathali1, Jelena Lovric1, Xianchan Li1, Soodabeh Majdi1 and Andrew G. 1 Department of Chemistry, Chalmers University of Technology, Sweden 2 Department of Chemistry and Molecular Biology, Gothenburg University, Sweden BEHVIOR OF MAMMALIAN VESICLES AT ELECTRODE SURFACES - ADSORPTION AND BURSTING We have developed an electrochemical method that can be used to quantify the content of individual vesicles. The biological system we have used is large-dense core vesicles isolated from bovine chromaffin cells. The vesicles adsorb onto a 33 µm diameter disk-shaped carbon electrode surface and subsequently rupture through a yet unknown mechanism to release its content in close vicinity to the electrode. These contents are then oxidized and a current (or amperometric) peak results from each vesicle that bursts. This method allows different aspects of vesicle loading, such as the effect of drugs, osmotic pressure, and pH to be studied in a cell-free environment. By collecting data under different conditions, like potential, temperature, and osmotic pressures, in combination with complementary techniques, such as quartz crystal microbalance (QCM-D) and total internal reflection (TIRF) microscopy. We are developing a theoretical model to understand and describe the key aspects of vesicle bursting at the electrode surface. J. Dunevall, H. Fathali, N. Najafinobar, J. Lovric, J. Wigström, A-S. Cans, A. G. Ewing, J. Am. Chem. Soc., 2015, 137 (13), pp 4344–4346 36. Ayoze González-Santana1, Judith Estévez-Herrera1; Michelle Juan-Bandini 1, José David Machado1 and Ricardo Borges1, 2 1 Pharmacology Unit, Medical School, Tenerife, Spain. 2 Instituto de Bio-Orgánica "Antonio González". University of La Laguna, Tenerife, Spain MODULATION OF CATECHOLAMINE EXOCYTOSIS THROUGH INCRETIN RECEPTORS Regulated exocytosis is highly regulated and determines the neuronal communication and secretory response of many neuroendocrine cells. The exocytosis is modulated by second messengers as Ca2+ or cAMP affecting the number of secreted vesicles and the amount and manner in which each vesicle releases its content. In this work, we have studied the regulation of catecholamine secretion by incretin receptor ‘glucagon-like-peptide-1' (GLP-1R). This receptor might be acting through two cAMP-dependent activation pathways, PKA and/or EPAC. To test which of them are involved in GLP-1R effects on secretion, we have combined single cell amperometry and whole cell patch-clamp in bovine chromaffin cells treated with the GLP-1R agonist exendine-4. Our finding show that GLP-1R agonists potentiate the cathecolamine secretion and increase the quantum size by activating the cAMP/PKA but not the EPAC route. 1. Drucker, DJ & Nauck, MA (2006) Lancet 368, 1696-1705
2. Machado, JD, et al (2001) Mol Pharmacol 60, 514-520
AGS has a FPI fellowship from MINECO (Spain). This work has been supported by the grant
BFU2013-45253-P from the MINECO to R. Borges and JD. Machado
37. Michelle Juan-Bandini1, Judith Estévez-Herrera1, Ayoze González-Santana1, José David Machado1, Ricardo Borges1,2 1 Pharmacology Unit, Medical School and 2 Instituto de Bio-Orgánica "Antonio González". Universidad de La Laguna, Tenerife, Spain WHO'S FIRST? THE STIMULUS-SECRETION COUPLING ACCORDING TO THE AGE OF SECRETORY GRANULES An adrenomedullary chromaffin cell contains 10,000-20,000 secretory vesicles (SV). However, as a response to strong stimuli only 2,000 – 6,000 vesicles will be released. The remained pool of SV usually never gets exocytosis and is called ‘non-releasable' or reserve pool. Although the ‘maturation theory' says that this reserve pool wil supply SV to the releasable pools Duncan et al., (2003) and Giner et al., (2005) got data indicating that young vesicles are the first in getting exocytosis "youths jumps the queue". However, this explanation has not been completely demonstrated. To elucidate whether are the youngest or the oldest the preferential SV in getting exocytosis we have monitored, by real time secretion catecholamines, chimeric neuropeptide Y (NPY-EGFP) and chromogranins by on-line electrochemistry and dot-blot respectively. Our results have found that NPY-EGFP (which must come from youngest SV) are present only in the initial releasing pulses elicited by nicotinic stimuli. This work is partially paid by the grant BFU2013-45253-P from the MINECO (Spain) to RB and JDM. Duncan RR, et al. (2003) Nature 422: 176-180. Giner D, et al. (2005) J Cell Sci 118: 2871-80. 38. Judith Estévez-Herrera1, Ayoze González-Santana1, Rebeca Baz Dávila1, Ricardo Borges1,2 and José David Machado1 1 Unidad de Farmacología, Facultad de Medicina; 2 Instituto Universitario de Bio-Orgánica ‘Antonio González', University of La Laguna, Tenerife, Spain ATP AS MORE THAN A CO-TRANSMITTER: UNDERSTANDING THE CAPACITY OF TRANSMITTERS ACCUMULATION INSIDE SECRETORY VESICLES. ATP is transported and co-stored along with other transmitters at high concentrations in almost all secretory vesicles described. In the chromaffin granule, ATP seems to promote the formation of intravesicular complexes between the major constituents: catecholamines (CA) and chromogranins to account for the huge amount of CA stored in these vesicles (ranging 0.8-1 M), that largely exceed the theoretical osmotic limits. Despite the existence of many in vitro contributions that demonstrated that ATP is essential for the accumulation of CA, the function in living cells still remains unknown. In this work, we used specific siRNAs against the vesicular nucleotide transporter (VNUT) as a gene silencing strategy and we explored its impact in the accumulation and the exocytosis of CAs in bovine chromaffin cells. The VNUT knock-down cells (VNUT-KD) exhibited a reduction of more than 50% of transporter expression, which resulted in a decrease of about 40% of ATP and adrenaline secreted by exocytosis without changes in total CAs levels or intracellular Ca2+. Moreover, the incubation of cells with L-DOPA yields a dopamine secretion higher in control than in VNUT-KD cells. Our data suggest that the mechanisms of accumulation of vesicular CAs in VNUT-KD cells are deficient. The amperometric recordings of VNUT-KD cells showed a reduction over a half of CAs secretion compared with control cells. This difference was not due only to a reduction of the quantal size (more tan 50%) but also by a reduction in the frequency of secretory events, indicating a lower degree of recruitment of granules affected by the lack of ATP. 39. Rajtarun Madangopal NIDA IRP / NIH, USA DEVELOPMENT OF IN VIVO CALCIUM IMAGING IN FREELY BEHAVING RATS TO ASSESS THE FORMATION AND MAINTENANCE OF FOS-EXPRESSING NEURONAL ENSEMBLES Since proposed by Hebb in 1949, evidence supporting the hypothesis that learned associations are encoded in sparsely distributed ‘cel assemblies' (neuronal ensembles) has primarily been correlational. Recently, we used our Daun02 inactivation procedure to determine that sparsely distributed Fos-expressing neurons mediate the formation and expression of learned behaviors. While this ensemble-targeted approach allows us to demonstrate a causal role for these neurons in learned behaviors, the neural circuit dynamics that lead to the formation of these sparse networks and the relationship between the most strongly activated Fos-expressing sub-population and the adjacent less-activated neurons in these brain areas is currently unexplored. We are currently using single cell resolution mapping of circuit-level calcium dynamics in freely behaving rats using GRIN endoscope-based in vivo epifluorescent microscopy. This approach allows us to monitor the Fos-positive and Fos-negative neuronal sub-populations over time to understand how they modulate network activity over different time scales, as well as understand the detailed relationships among neural activity, calcium dynamics and Fos expression. 40. Junyong Sun, Feng Gao
College of Chemistry and Materials Science, Anhui Normal University, China A TWO-PHOTON EXCITED SEMICONDUCTING POLYMER DOTS FOR RATIOMETRIC FLUORESCENCE DETECTION AND IMAGING OF TYROSINASE ACTIVITY Semiconducting polymer dots (Pdots) are fluorescent materials which offer many advantages such as extraordinary fluorescence brightness, high flexibility, excellent photostability, high emission rate and nontoxic characteristics for bioanalysis and bioimaging, and are complementary to revolutionary advances in fluorescence technology. Two-photon (2P) imaging holds great promise for in vivo microscopic physiological studies in areas such as neurobiology, immunology, and tissue engineering. Herein, a one, two-photo excited Pdots, PFO/CN-PPV, were designed and prepared with nanoprecipitation method. The Pdots were further functionalized with air-stable tyrosine methyl ester(TYR-OME) through electrostatic assembly for highly selective detection of tyrosinase (TR) activity. Based on the selective quenching of orange emission of Pdots with electron-transfer process by the catalytic oxidation product of tyrosine methyl ester, a two-photon ratiometric fluorescence detection of tyrosinase activity have been established. The proposed fluorescent probe has also been explored to image the tyrosinase activity of melanoma cells B16-F1 with two-color confocal microscopy. 41. Jelena Lovric, Andrew Ewing Chalmers University of Technology, Sweden HIGH-RESOLUTION MASS SPECTROMETRY IMAGING OF THE NEUROCHEMISTRY AT SINGLE NEUROTRANSMITTER VESICLES LEVEL Imaging mass spectrometry was used to study the neurochemistry in small environments such as large dense core vesicles (LDCVs). The major goals are to investigate the impact of pharmaceuticals like L-3,4-dihydroxyphenylalanine (L-DOPA) and reserpine onto metabolic pathways of neurotransmitter dopamine and to measure the dopamine molecule distribution between sub-vesicular compartments in PC12 cells. Cells were treated with isotopically labeled L-DOPA and chemically fixed, resin embedded and sectioned for transmission electron microsocopy (TEM). Imaging inside vesicles was done with a Cameca NanoSIMS 50L ion microprobe using Cs+ primary ions with approx. 50 nm spatial resolution. High spatial resolution negative secondary ion images of isotopic ratios were acquired from micrometer-sized cell regions. The overlays of isotopic ratio images and TEM images were used to localize the transmitters in the cells and to reveal the possible transmitter vesicular regions. The use of NanoSIMS opens the possibilities for the study of neurochemistry in single transmitter vesicles and allows us to identify vesicle compartments. The major goals of this research are to understand new mechanisms of exocytosis and transmitter storage in vesicles as well as to investigate the impact of pharmacology on regulation of neurotransmitter release. 42. E. Hernández-Martín1, F. Marcano1, O. Casanova1, C. Modroño1, J. Plata1, J.L. González-Mora1 1 Faculty of Medicine. University of La Laguna, Spain INCREASED STATISTICAL POWER IN DIFFUSE OPTICAL TOMOGRAPHY IMAGES USING BAYESIAN FILTERING. Functional near infrared spectroscopy (fNIRS) is an optical tool used to estimate changes in cerebral oxygenated (HbO2) and deoxygenated hemoglobin (HbR) concentration due to local cortical activation. Three-dimensional images of hemoglobin concentration in human cerebral cortex can be obtained using Diffuse Optical Tomography (DOT), a modality of fNIRS that calculates absorption changes of near infrared light over a finite element model of the brain. To achieve an increase of statistical power in DOT image corresponding to design of a cognitive paradigm for this experiment, we applied DRIFTER (Dynamic Retrospective Filtering of Physiological Noise) algorithm, which models and remove physiological noise, allowing accurate dynamical tracking of the variations in the cardiac and respiratory frequencies. This filter was applied over raw DOT data during preprocessing. The same paradigm was repeated six times with a participant using an MRI scanner and Nirx Medical System equipment. Images collected from both techniques were co-registered to the structural images and analyzed using SPM8. We generated a contrast image comparing blocks of tasks vs rest for each probe (p-value < 0.001). A conjunction spatial analysis (p-value<0.05) revealed a large number of voxels in the common areas between DOT and fMRI images using DRIFTER other than conventional filters. 43. Masoumeh Dowlatshahi Pour1,2 , Eva Jennische3, Stefan Lange3, Per Malmberg1,2, Andrew FOOD-INDUCED CHANGES OF LIPIDS AND VITAMINS IN RAT NEURONAL AND INTESTINAL TISSUE VISUALIZED BY IMAGING TOF-SIMS 1 Chalmers University of Technology, Sweden; 2 National Center of Mass Spectrometry, Sweden; 3 Institute of Biomedicine, Sweden; 4 University of Gothenburg, Sweden Antisecretory Factor (AF) is a 41 kDa endogenous protein which is commonly secreted in plasma and other tissue fluids in mammals with proven regulatory function on inflammation and fluid secretion. In general, most of the AF is in an antisecretory inactive state in the plasma of a healthy person however, some bacterial toxins or intake of certain food components have been found to activate AF (aAF). Specially processed cereals (SPC-flakes) is one of these food components that can increase aAF-level in plasma. Intake of such cereals has been shown to decrease symptoms in diseases where inflammation and/or secretory disturbances are involved such as inflammatory bowel disease (IBD), endocrine diarrhoea and Menieres disease. The exact mechanism for the activation process of AF at the cellular level remains unclear. Here, in order to gain more clear insights into the possible mechanisms involved and to examine changes in lipid distribution, Time of Flight Secondary Ion mass spectrometry (ToF-SIMS) was employed to investigate the lipid localization in brain and intestine tissue sections from rats fed specially processed cereals and control rats fed ordinary feed. A TOF-SIMS 5 instrument equipped with a Bi cluster ion gun was used to analyze the tissue sections. Data from 15 brain and 15 intestine samples from control and activated AF rats were recorded using the stage scan macro raster with a lateral resolution of 5μm. Data obtained from brain and intestine samples respectively were then exported to MatLab and pretreated by mean centering and Poisson scaling before subjecting to principal components analysis (PCA). The preliminary data clearly indicated the involvement of certain lipids in the aAF working mechanism. In the brain tissue samples, PCA score plots showed a good separation in lipid distribution between the control and the aAF group. The loadings plot revealed that changes in cholesterol and c18:2, c16:0 fatty acid distribution as well as some short chain monocarboxylic fatty acid compositions caused the main separation between two groups. PCA score plots from intestine sections also showed a well-defined separation between the control and the aAF group. Here, the c18:2, c18:1, c16:0 as well as the distribution of several other short chain monocarboxylic fatty acids were changed between the two groups. Hence ToF-SIMS has provided important insight into the aAF working mechanism and provided clues for a subsequent targeted quantitative analysis of the lipid content in brain and intestine. 44. Amir Saedi Mohammadi, Nhu Phan, John Fletcher, Andrew Ewing
Chalmers University of Technology, Sweden LABLE FREE MASS SPECTRAL IMAGING OF LIPID SPECIES IN BRAIN SAMPLES: TECHNICAL ADVANCES Identification and localization of specific biomolecules such as peptides and lipids within cellular membranes is currently a major challenge in metabolomics and biological studies. With recent technological and methodological improvements imaging mass spectrometry methods including secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption ionization (MALDI) are now promising techniques in the field of molecular imaging of biological samples such as tissue sections or cells. In biological investigations SIMS and MALDI often provide complimentary information. SIMS provides detection of small molecules at high spatial resolution, whereas MALDI is capable of ionizing larger molecules sample, albeit at reduced spatial resolution. Although these MS techniques have associated abilities there are some limitations with each method. The chemistry of the sample is normally significantly altered in MALDI due to the addition of the matrix. This can be eliminated by using Au nanoparticles as a matrix (Nanoparticle Assisted-LDI) on tissue allowing lipid molecular ion species and relevant mass fragments to be imaged with enhanced spatial resolution. To extend the useful mass range of SIMS imaging we have applied gas cluster ion beams (GCIBs) that have been developed to analyze and image organic compounds. The high energy Ar GCIB shows potential for ionizing molecular ion lipids and intact neuropeptides as well as high mass fragments of large proteins produced by on surface tryptic digestion. 45. Wojciech Michno, Jörg Hanrieder University of Gothenburg, Sweden HYPERSPECTRAL IMAGING AND MASS SPECTROMETRIC PROBING OF AMYLOID BETA PLAGUE PATHOLOGY IN ALZHEIMER'S DISEASE Alzheimer's disease (AD) is a chronic, neurodegenerative disorder. It is characterized by accumulation of amyloid-β (Aβ) peptides into different extracel ular plaques. While this amyloid pathology has been suggested to be a critical inducer of AD pathogenesis, the correlation of plaque burden and AD progression has been questioned. For instance, cognitively normal pathological aging (PA) patients exhibit amyloid pathology. However, Aβ plaques present in PA brains are mostly diffuse in nature, while plaques in AD brain tissue are mostly mature/compact. Diffuse plaques can be a consequence of an alternative, neuroprotective aggregation mechanism. Alternatively, they can represent an immature non-toxic state of mature compact amyloid plaques. Therefore, discrimination between structural and molecular plaque architecture are of essential interest to resolve Aβ plaque pathology in AD. Luminescent conjugated oligothiophenes (LCO), suggested to exhibit differential binding to the various amyloid aggregation states, offer a way to approach this problem. We use a hyperspectral imaging paradigm employing LCO staining and fluorescence immunolabelling to detect and discriminate Aβ plaque heterogeneity in AD and PA based on chemical and structural features. Further, the approach is applicable to laser microdissection and offline mass spectrometric analysis, which validate the presence of various C-terminal Aβ in the plaques. 46. McGinnity C.J.1,2,3, Riaño Barros D.A. 2,3, Myers J.F.2, Stokes P.R.2, Lingford-Hughes A.R.2, Koepp M.J.4, Hammers A.1,2,3 1 Division of Imaging Sciences & Biomedical Engineering, King's College London; 2 Division of Brain Sciences, Faculty of Medicine, Imperial College London; 3 Medical Research Council Clinical Sciences Centre; 4 Department of Clinical and Experimental Epilepsy, University College London Institute of Neurology QUANTIFICATION OF GABA-A α5 SUBUNIT AVAILABILITY IN MRI-NORMAL TEMPORAL LOBE EPILEPSY USING [11C]RO15 4515 PURPOSE: Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. In animal models of epilepsy, expression of the α5 subunit of the GABA-A receptor is decreased in the hippocampus. We quantified the availability of the α5 subunits in patients with temporal lobe epilepsy and normal magnetic resonance imaging (MRI; nTLE) using the positron emission tomography (PET) tracer [11C]Ro15 4513. METHODS: Twenty-three controls and 12 patients with nTLE had a 90-minute [11C]Ro15 4513 PET scan with arterial blood sampling and T1-weighted MRI. We calculated volumes-of-distribution (VT) images using exponential spectral analysis. VT was compared between groups using statistical parametric mapping (SPM12). RESULTS: The patients with TLE showed a trend towards higher global [11C]Ro15 4513 VT (p = 0.08). The TLE group had non-significant bilateral decreases in VT in the medial temporal lobes (ipsilateral > contralateral, z score 3), as well as non-significant widespread bilateral increases mainly in the inferior parietal lobules (z scores 4). CONCLUSIONS: These findings suggest there is decreased α5 subunit availability in the temporal lobe in MRI-normal TLE. They should be corroborated with partial volume effect correction to exclude subtle volume defects. Further study is required to investigate the capacity of [11C]Ro15 4513 PET to lateralise nTLE. 47. Csaba Adori1, Nicolas Renier2, Zsofia Magloczky3, Gabor G. Kovacs4, Andrea Kliewer5, Marc Tessier-Lavigne2, Tomas Hökfelt1 1 Department of Neuroscience, Karolinska Institute, Sweden 2 The Rockefeller University, Laboratory of Brain Development and Repair, New York, USA 3 Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary 4 Medical University of Vienna, Institute of Neurology, Austria 5 Institute for Pharmacology and Toxicology Jena University Hospital, Germany THREE-DIMENSIONAL IMMUNO-IMAGING OF RODENT AND HUMAN BRAINS IN NORMAL AND PATHOLOGICAL CONDITIONS WITH THE NEWLY DEVELOPED VISUALIZATION TECHNIQUE iDISCO iDISCO volume imaging (immunolabelling-enabled three dimensional imaging of solvent-cleared organs) is a powerful method enabling three dimensional (volume) imaging of cleared and (double/triple) immunolabelled structures in complex tissues (e.g. entire smaller human nuclei; or pieces of neocortex, including all layers). The short time required for clearing makes iDISCO especially suitable for immunostaining procedures. It allows both qualitative and quantitative approaches, in combination with light-sheet and two-photon microscopy and the ImarisTM analysis software. Light sheet microscopy will enable us to quickly image very large fields of views (over a cm in size). In the present work we use iDISCO volume imaging and show (i) ‘neuropeptide S' immunoreactive neuronal populations in the mouse brainstem mice, (ii) the detailed 3D distribution of degenerative noradrenergic fibers in the forebrain of somatostain receptor 2 knockout mice as well as (iii) the 3D visualization of the human nucleus locus coeruleus from controls and subjects having suffered from schizophrenia and Alzheimer's disease. Advanced molecular imaging using the iDISCO method allows direct exploration of the connectome and large scale imaging of neurotransmitter systems of the brain in health and disease and may help to develop novel early biomarkers and/or therapeutic interventions. University of Gothenburg, Gothenburg, Sweden LIPID IMAGING OF DROSOPHILA BRAIN BY SECONDARY ION MASS SPECTROMETRY Drosophila melanogaster is a common biological model system, which has relatively simple anatomy and behaviors but possesses highly conserved molecular and cellular processes compared to humans. We have applied time of flight secondary ion mass spectrometry (ToF-SIMS) to study lipid structural effects of stimulant drugs (methylphenidate, cocaine) on the fly brain. Different distributions of various biomolecules, particularly fatty acids, eye pigment, diacylglycerides, phospholipids have been found across the fly brain. The lipid structures dramatically altered following the administration of the drugs.1 The results have an important implication that high curvatured and low curvatured lipids are affects differently by the drugs and therefore the structure of lipids might exert an important role in learning and memory. In addition, the MS/MS imaging with a high energy 40 keV Ar4000+ gas cluster primary ion beam (GCIB) have been successfully applied on the fly brain in order to elucidate the distribution of specific MS/MS fragments of the brain biomolecules. ToF-SIMS imaging shows great potential to study chemical distributions in the fly brain in relation to endogenous and exogenous effects. IN VIVO ELECTROCHEMISTRY / APPLICATIONS
49. Nathan Rodeberg University of North Carolina at Chapel Hill, USA NEUROBIOLOGICAL AND BEHAVIORAL FACTORS GOVERNING PHASIC DOPAMINE RELEASE IN INTRACRANIAL SELF-STIMULATION Intracranial self-stimulation (ICSS) of the ventral tegmental area is a frequently used behavioral paradigm to determine the rewarding or aversive impact of a variety of pharmacological manipulations. Dopamine has been determined as a key mediator of this task through pharmacological and histological studies, though its exact role remains controversial. While experiments using microdialysis have investigated changes in tonic dopamine levels during this task, the role of phasic dopamine release remains less clear. A previous study using fast-scan cyclic voltammetry (FSCV) during continuous ICSS (Garris et al. 1999) revealed a dissociation of phasic dopamine release from ICSS behavior. However, the waveform employed by this study (anodic limit of +1.0 V) is less sensitive than modern waveforms, making it unclear whether dopamine release was abolished or simply diminished to previously undetectable levels. Ongoing studies utilizing FSCV with an extended waveform (anodic limit of 1.3 V) reveal a progressive attenuation, but not abolishment, of dopamine release during continuous ICSS. This study investigates the source of this attenuation through ICSS procedure modifications (fixed-ratio vs. fixed-interval ICSS), changes in reward expectancy (ICSS vs. yoked stimulation), and pharmacological manipulation. 50. Lindsay Walton University of North Carolina at Chapel Hill, USA GLUTAMATE RECEPTOR INFLUENCE ON LOCALIZED OXYGEN METABOLISM Homeostasis in the brain is achieved through mediating local cerebral blood flow (CBF) supply based on neuronal activity. CBF increases renew locally depleted glucose and oxygen following neurotransmission. This system is dysregulated in disease states like Alzheimer's and stroke, as is neurotransmitter supply, making the relationship between CBF and neurotransmission important to understand. The largest percentage of neurons in the brain release excitatory glutamate, but its behavior is not well understood outside of the cortex or in slices. We aim to understand glutamatergic neurovascular influence deeper within intact brains. This research explores the postsynaptic neuronal firing and oxygen responses to exogenous glutamate. Iontophoresis, a local drug delivery technique, ejects precisely glutamate and/or drugs in close proximity to a neuron. Cell firing is detected using single unit elecrophysiology and oxygen responses are recorded with fast-scan cyclic voltammetry at the same microelectrode. Our method of glutamate delivery is gentle enough to not provoke CBF changes, and instead probes the intensity and duration of oxygen utilization that follows action potentials. We present glutamate-elicited neurotransmission and subsequent oxygen changes under exposure to glutamatergic drugs in both the nucleus accumbens and cortex. University of Colorado, Denver, USA IN VIVO ASSESSMENT OF ACCUMBAL DOPAMINE RELEASE EVENTS IN A NOVEL BEHAVIORAL ECONOMICS BASED FOOD-SEEKING TASK The mesolimbic dopamine system is strongly implicated in motivational processes. Currently accepted theories suggest that transient mesolimbic dopamine release events are involved in assessing the value of reward predictive stimuli and/or in generating motivated action sequences. Here, we investigate the role of cue- and reward-evoked dopamine release on cue-motivated food seeking. To address this research question we developed a novel behavioral economics food-seeking task. In this task, food is provided to rats across 10 different unit-prices (i.e., response requirement/reward magnitude). Using fast-scan cyclic voltammetry we first determined that the concentration of accumbal dopamine time-locked to cue presentation decreases as a function of unit-price. We next sought to assess the effect of optically augmenting release both at reward delivery and cue presentation. Our data reveal that optically facilitating dopamine release at the cue decreases motivation for food; whereas, facilitating release at reward delivery increases motivation for food. Augmenting release at both cue presentation and reward delivery decreased response latency, consistent with an invigoration of responding that might be dissociable from value-based changes in motivation. It is possible that augmenting cue-evoked dopamine release decreases motivation in our task because we are violating the animal's expectation. 52. Anne Collins VENTRAL STRIATAL CHOLINERGIC MODULATION OF DOPAMINE SIGNALING AND CUE-INDUCED INENTIVE MOTIVATION Striatal acetylcholine signaling has been demonstrated to modulate the release of dopamine, but whether this occurs in behaving animals with natural sensory input is unknown, preventing understanding of the functional significance of such modulation. Cholinergic signaling through nicotinic receptors on dopamine terminals may work to blunt the phasic release of dopamine that should occur when the neurons burst fire, with muscarinic autoreceptors opposing this function. In the nucleus accumbens core (NAc), such phasic dopamine signaling mediates cue-motivated behavior. Therefore, we tested the hypothesis that NAc cholinergic signaling has a primarily suppressive effect on cue-motivated behavior via modulation of dopamine release. We first found that blockade of NAc nicotinic receptors enhanced the invigorating influence of a reward-predictive cue over reward-seeking actions, while blockade of muscarinic receptors selectively attenuated this Pavlovian-to-instrumental transfer (PIT) effect. We next monitored NAc dopamine with fast-scan cyclic voltammetry in rats behaving in the PIT task and found that unilateral blockade of NAc nicotinic receptors in the recording zone augmented, while unilateral blockade of NAc muscarinic receptors attenuated both cue- and reward-evoked dopamine responses. NAc acetylcholine might, therefore, gate a cue's motivating influence through modulatory action at dopamine terminals, perhaps signaling the current adaptive utility of cue-motivated action. 53. Katherine Brimblecombe University of Oxford, UK SUBSTANCE P WEIGHTS STRIATAL DOPAMINE TRANSMISSION DIFFERENTLY WITHIN THE STRIOSOME-MATRIX AXIS The striatum is organised not only topographically, but also biochemically, into striosomes and matrix compartments. Striosomes form patchy, labyrinth-like structures that are enriched in μ-opioid receptors (MORs) and Substance P (SP) relative to the surrounding matrix, which is enriched in calbindin-D28K and acetylcholinesterase. Evidence for a third "peristriosomal" region at the striosome-matrix interface, has been proposed from high met-Enkephalin expression. The functional implications of this organisation are poorly understood. Dopamine transmission occurs throughout striosomes and matrix, and is reported to be modulated by SP. However reported effects are conflicting, ranging from facilitation to inhibition. We addressed whether dopamine transmission is modulated differently in striosome-matrix compartments by SP. We paired detection of electrically evoked dopamine release at carbon-fibre microelectrodes using fast-scan cyclic voltammetry in mouse striatal slices with post-hoc immunolabelling for MOR-immunoreactivity to define striosomes. SP modulated dopamine release via NK1-receptors, however the effect of SP on extracellular dopamine ranged from a 70% enhancement to 50% reduction. The direction of modulation was determined by location within the striosomal-matrix axis: Dopamine release was boosted in striosome centres, diminished in striosomal-matrix borders and unaffected in the matrix. In summary, dopamine signals are differentially weighted along the striosome-matrix axis by SP. 54. Dan P. Covey University of Maryland, USA NUCLEUS ACCUMBENS DOPAMINE RELEASE AND MOTIVATED BEHAVIOUR ARE COMPROMISED IN THE Q175 MOUSE MODEL OF HUNGTINGTON'S DISEASE Huntington's disease (HD) is a fatal, neurodegenerative disease characterized by profound motor abnormalities. Additionally, cognitive and emotional deficits affecting memory, attention, executive function and mood manifest early in disease progression, prior to motor dysfunction. Clinical and preclinical work suggests compromised striatal dopamine function represents a common neuropathological feature of HD that may precede motor deficits. However, a direct association between dopamine function and behavior in HD is lacking. Motivational deficits represent a primary non-motor symptom in HD and motivation is tightly regulated by dopamine in the nucleus accumbens (NAc). We therefore assessed motivated responding using a progressive ratio (PR) food-seeking task while monitoring NAc dopamine release with fast-scan cyclic voltammetry in the Q175 knock-in mouse HD model. Q175 mice displayed significantly lower breakpoints on the PR task relative to wild-type controls. Impaired motivation was associated with compromised dopaminergic encoding of reward receipt. Genotypic differences in dopamine release increased as the PR session progressed and were dramatically different during the final trials as animals reached their breakpoint. This work indicates that attenuated dopamine release accompanying reward pursuit contributes to an apathetic state characteristic of HD. 55. Meining Zhang Renmin University of China, China IN VIVO ELECTROCHEMISTRY WITH CARBON NANOTUBE-MODIFIED CARBON FIBER MICROELECTRODES Development of highly selective and sensitive microelectrodes with excellent analytical properties is of great importance for in vivo monitoring of neurochemicals in real time nature. The distinct electronic and structural properties and ready functionalization of carbon nanotubes (CNTs) make them quite useful for the development of electrochemical sensors and biosensors to probe brain chemistry. By using carbon nanotubes as the electrode materials, we found that the use of carbon nanotubes (CNTs) could greatly facilitate the oxidation of ascorbate at low potential (ca. −50 mV), opening a new avenue for selective ascorbate detection. The uses of CNTs could offer a support to confine redox mediators and electrocatalysts employed in the biosensing. Therefore, we functionalized CNTs with platinum for in vivo amperometric monitoring of O2 in rat brain, and Prussian blue (PB) for monitoring H2O2 and glucose in rat brain. 56. Megan E. Fox University of North Carolina at Chapel Hill, USA RECIPROCAL CATECHOLAMINE RELEASE DURING ACUTE OPIATE EXPOSURE AND PRECIPITATED WITHDRAWAL The transition from drug-use to drug-abuse is thought to occur following a change in an individual's motivation to take drugs. Both dopamine and norepinephrine are involved in reinforcing drug-use and the switch from impulsive to compulsive drug administration is accompanied by adaptations in catecholamine-containing circuits. Since dopamine and norepinephrine respond reciprocally to appetitive and aversive stimuli, we hypothesized they would signal differently during the development of addiction. We used fast-scan cyclic voltammetry and single-unit electrophysiology in awake animals to measure catecholamine signaling during acute morphine exposure and naloxone-precipitated withdrawal. We found spontaneous dopamine release in the nucleus accumbens shell (NAcsh) under morphine that was absent during precipitated withdrawal. Conversely, we measured spontaneous norepinephrine overflow in the ventral bed nucleus of the stria terminalis (vBNST) only during the withdrawal period. Norepinephrine release tracked somatic indices of withdrawal and was enhanced following α2 antagonism. Electrically evoked dopamine release was unchanged after the morphine-naloxone treatment, but norepinephrine was attenuated as compared with control rats. Our data demonstrate contrasting roles for catecholamine signaling during acute drug exposure/ withdrawal that may contribute to plasticity and be important for developing drug dependence. 57. Heramb Chadchankar, James E. McCutcheon Department of Neuroscience, Psychology & Behaviour, University of Leicester, UK CALORIE-PAIRED FLAVOURS EVOKE GREATER NEURONAL ACTIVITY IN NUCLEUS ACCUMBENS THAN NUTRITIONALLY-DEVOID FLAVOURS Dopamine plays an important role in appetitive behaviours and is sensitive to the nutritional value of food. Caloric foods, and cues that predict them, evoke greater dopamine release in nucleus accumbens (NAc) core than non-caloric foods. However, how this dopamine release affects the encoding of nutritional value by NAc neurons is not known. We used in vivo fibre photometry to measure neuronal activity in NAc core during a Pavlovian calorie-seeking task. Rats were trained to drink from a retractable sipper containing either non-caloric (0.2% saccharin) or caloric (0.2% saccharin + 8% polycose) solution paired with an auditory cue. Each solution was flavoured with cherry or grape. After six days, rats were presented with sippers containing either grape or cherry-flavoured saccharin solution in a pseudorandom order. In contrast to studies on dopamine release, no difference in neuronal activity was observed between cues that predicted caloric and non-caloric rewards. However, neuronal activity during presentation of the calorie-paired flavour was significantly elevated relative to the nutritionally devoid flavour. This difference in neuronal activity was observed even in the absence of a behavioural difference in sipper approach or amount of solution consumed suggesting a specific role for NAc neurons in encoding nutritional value of food. 58. T. Pouvreau, E. Tagliabue, S. Eybrard, F. Meyer, Alain Louilot DOPAMINERGIC VARIATIONS IN THE SHELL AND CORE PARTS OF THE NUCLEUS ACCUMBENS TO MK-801 ARE DIFFERENTIALLY DISRUPTED IN ADULT RATS AFTER POSTNATAL FUNCTIONAL BLOCKADE OF THE PREFRONTAL CORTEX A striatal dopaminergic (DA) disturbance in schizophrenia is generally accepted and may be dependent of a prefronto-striatal disconnection involving glutamatergic NMDA receptors. The present study was designed to investigate the effects of the non-competitive NMDA receptor antagonist, MK801, in adult rats on DA responses in the dorsomedial shell and core parts of the nucleus accumbens (Nacc), following a postnatal inactivation of the left PFC (infralimbic/prelimbic region). Transient functional inactivation of the left PFC was carried out by local tetrodotoxin (TTX) microinjection in 8-day-old rats, i.e a critical time of the neurodevelopmental period. DA variations were recorded in the two parts of Nacc using in vivo voltammetry in freely moving adult rats (11 weeks). The obtained results were the following : 1) A clear dose-dependent decrease in dorsomedial shell DA levels was observed in PBS-microinjected animals whereas a dose-dependent DA increase was observed with the two MK801 doses (0.1 mg/kg or 0.2 mg/kg s.c.) in TTX- microinjected animals; 2) A clear dose-dependent increase in core DA levels was observed for the two conditions (PBS and TTX microinjected at PND8). In conclusion, these findings may help to understand the involvement of NMDA glutamatergic receptors in the pathophysiology of schizophrenia. University of Leicester, UK NMDA RECEPTOR MODULATION OF ELECTRICALLY STIMULATED DOPAMINE RELEASE IN NUCLEUS ACCUMBENS SLICED IN VITRO Chronic pre-treatment with phencyclidine produces behavioural deficits in rats, which model aspects of schizophrenia in people. This effect is, at least partially, mediated through glutamate-dopamine interactions in nucleus accumbens (NAc): glutamate dysregulation may result in changes in NAc dopamine function associated with schizophrenia. We used fast cyclic voltammetry in rat brain slices in vitro, to measure the effects of local activation of NMDA-type glutamate receptors on electrically-stimulated release of dopamine from NAc shell. Further, we investigated whether the changes were affected by sub-chronic pre-treatment with phencyclidine. NMDA cause dose-dependent decrease in electrically-stimulated dopamine release. The decrease caused by 30µM NMDA was similar in phencyclidine pre-treated animals, and saline pre-treated controls, and was not affected by the GABA-A antagonist, picrotoxin. However, DHbE, a nicotinic (α2β4 subunit) antagonist reduced the effect of NMDA in saline pre-treated animals, but not phencyclidine pre-treated animals. These results indicate that NMDA receptor activation reduces stimulated dopamine release in NAc, through cholinergic mediated mechanisms in normal, but not phencyclidine pre-treated animals. Hence, we believe that cross-talk involving NMDAR and nicotinic receptors on dopaminergic terminals in NAc may modulate dopamine release. This interaction may be disrupted by phencyclidine pre-treatment, which may give insights into abnormal mechanisms in schizophrenia. 60. Mallikarjunarao Ganesana Department of Chemistry, University of Virginia, USA TRANSIENT ADENOSINE RELEASE FREQUENCY INCREASED DURING ISCHEMIC BRAIN INJURY Adenosine is an important neuromodulator in the central nervous system, which plays a vital role in a wide variety of physiological and pathophysiological processes. Previous studies have shown that tissue adenosine levels increase during ischemic events and attenuate the excitotoxic neuronal injury. Recently, our lab developed an electrochemical fast-scan cyclic voltammetry (FSCV) method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale. We found that adenosine can be transiently released and cleared in about 3 s. In this study, transient adenosine release was studied in the caudate-putamen of anesthetized rats during the progression of ischemia-reperfusion (I-R) for the first time. Transient adenosine measurements were carried out continuously for a two hour period of normoxia, followed by the induction of 30 min ischemia through bilateral common carotid artery occlusion and 90 min of reperfusion. There was a significant increase in the frequency of transients detected and the inter event time decreased during ischemia and reperfusion. The cumulative adenosine concentration increased fivefold during ischemia and reperfusion periods combined compared to normoxia. These findings provide us an initial understanding on the time course release of transient adenosine during normoxia and I-R periods. 61. Catarina Owesson-White Department of Chemistry, The University of North Carolina at Chapel Hill, USA CUE-EVOKED DOPAMINE RELEASE RAPIDLY MODULATES D2 NEURONES IN THE NUCLEUS ACCUMBENS DURING BEHAVIOUR Dopaminergic neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) fire in response to unpredicted rewards or to cues that predict reward delivery. In the NAc, the dopaminergic projections form boutons on medium spiny neurons (MSNs), GABAergic cells that also receive glutamatergic input from the prefrontal cortex, hippocampus, and basolateral amygdala. While electrophysiological studies show that presentation of a cue or unpredicted reward triggers an increase in firing rate of dopaminergic neurons, and voltammetric studies show subsequent dopamine release, the functional consequences of these findings are not clear. Here, we uncover dopamine's actions during a reward-based behavioral task using a multimodal sensor that simultaneously probes 3 aspects of neuronal communication: neurotransmitter release, cell firing, and identification of dopamine receptor subtype. The cellular resolution of the sensor, coupled to its subsecond temporal resolution, reveals discrete dopamine release events modulating sequential activation of different populations of MSNs during reward seeking. These results support the view that dopaminergic neurons respond quickly to reward based signals and that dopamine-mediated responses following cues are linked to actions at a subset of MSNs containing D2 receptors. 62. Caddy N. Hobbs University of North Carolina at Chapel Hill, USA SPREADING DEPRESSION ELICITS CHANGES IN OXYGEN AND DOPAMINE: STUDIES IN THE CORTEX AND NUCLEUS ACCUMBENS Spreading depression (SD) is a pathophysiological event that occurs in brain tissue following localized trauma. Waves of SD propagate from the site of injury causing a massive cell depolarization, followed by a quiescent period. The passing wave breaks down ion gradients across the cell membrane and provokes extensive neurotransmitter release. These passing waves increase probability of neuronal death, enlarging the necrotic core. Here, we simultaneously record electrochemical and electrophysiological signals during SD using carbon fiber microelectrodes. We investigated changes during SD with minimal tissue damage from the electrode in the cortex and nucleus accumbens. Single-unit recordings showed a burst of cell firing at onset of the SD wave, followed by diminished activity. Using fast-scan cyclic voltammetry, chemically specific data on oxygen and dopamine fluctuations were obtained. In both cortex and nucleus accumbens, oxygen increased during the SD wave and then fell below baseline at its termination. In the dopamine-rich nucleus accumbens, the oxygen response was accompanied by a massive release of dopamine. We show that carbon fiber microelectrodes allow for a novel method capable of simultaneous detection of oxygen, electroactive transmitters, and cell firing in discrete brain regions previously inaccessible due to probe size. 63. Kayla A. Siletti, Jyoti C. Patel, Ariana Rabinowitsch, Soledad Cabeza de Vaca, Kenneth D. Carr, Margaret E. Rice Department of Neurosurgery, New York University School of Medicine, USA DIET-DEPENDENT CHANGES IN INSULIN SENSITIVITY OF STRIATAL DOPAMINE RELEASE Previous studies suggest that insulin may communicate caloric value and contribute to post-ingestive reward processes. Activation of insulin receptors (InsRs) on cholinergic interneurons (ChIs) in ex vivo striatal slices has been shown to elevate ChI excitability and acetylcholine (ACh) signaling, which amplifies single-pulse-evoked extracellular dopamine via nicotinic ACh receptors. This effect of insulin on dopamine release can be modulated by diet. A food-restricted diet, during which circulating insulin is relatively low, augments sensitivity to insulin. An obesogenic diet, during which circulating insulin is relatively high, diminishes sensitivity to insulin. Diet, therefore, modulates the concentration of insulin required to yield a potentially rewarding increase in dopamine release. As this outcome may alter feeding behavior, the present investigation explores the reversibility of these diet-dependent changes. Adult male Sprague-Dawley rats were placed on a food-restricted diet regimen. One subset was then exposed to ad libitum food access for two days; a second subset was exposed for two weeks. Evoked dopamine release was monitored using fast-scan cyclic voltammetry in striatal slices from each group. Sensitivity to 10 nM insulin increased with food-restriction compared to control; on-going experiments test the hypothesis that decreased insulin responsiveness may accompany refeeding, as seen with an obesogenic diet. 64. Jyoti C. Patel, Melissa A. Stouffer, Kenneth D. Carr and Margaret E. Rice Department of Neurosurgery, NYU School of Medicine, USA DIET AND INSULIN DEPENDENT ALTERATIONS IN STRIATAL DAT ACTIVITY FOLLOWING EVOKED DOPAMINE RELEASE Evoked extracellular dopamine [DA]o in the striatum is decreased with both food-restricted (FR) and obeseogenic (OB) diets versus an ad-libitum (AL) fed diet. We examined whether these diets influence DA uptake via the dopamine transporter (DAT) in adult rats. FR rats received titrated amounts of chow to maintain body weight at 80% control. OB rats consumed a moderately high fat chocolate liquid. Diets lasted 21-30 days after which single-pulse evoked [DA]o was assessed using fast-scan cyclic voltammetry in striatal slices. To evaluate changes in DAT kinetics, the initial falling phase of evoked [DA]o curves was fitted to the Michaelis-Menten equation to extract Vmax (maximal uptake rate) using a fixed Km (0.2 µM). In AL rats Vmax was higher in the caudate putamen (CPu) versus nucleus accumbens core (NAc) reflecting the greater density of DAT in this sub-region. However, while there was a trend for lower Vmax in OB striatum, Vmax was significantly lower in FR versus AL in both CPu and NAc, preumably a homeostatic response to low DA release. Insulin (30 nM) increased Vmax in CPu and NAc of AL rats. Whether insulin's effect on Vmax is PI3 kinase-dependent or altered with FR or OB diets will be established. 65. Aimilia Lydia Kalafateli1, Daniel Vallöf1, Markus Heilig2, Jörgen A. Engel1, Elisabet Jerlhag1 1 Institute of Neuroscience and Physiology, Department of Pharmacology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden 2 Center for Social and Affective Neuroscience, Division of Neuro and Inflammation Sciences, Linköping University, Sweden A CANNABINOID RECEPTOR ANTAGONIST ATTENUATES GHRELIN-INDUCED ACTIVATION OF THE MESOLIMBIC DOPAMINE SYSTEM IN MICE The orexigenic peptide ghrelin increases appetite as well as activates the mesolimbic dopamine system, i.e. the dopamine projection from the ventral tegmental area (VTA) to nucleus accumbens (NAc). Preclinical studies report that ghrelin receptor (GHS-R1A) antagonism decreases drug reinforcement, suggesting that the gut-brain peptide increases the incentive salience of motivated behaviours. The present experiments were designed to explore the involvement of cannabinoid receptors type 1 (CB1), specifically in the VTA, for the ability of ghrelin to stimulate the mesolimbic dopamine system. Indeed, we showed that peripheral (intraperitoneal, ip) administration of Rimonabant (SR141716A), a CB1 antagonist, attenuates the ability of central (intracerebroventricular, icv) ghrelin administration to increase locomotor activity as well as accumbal dopamine release in mice. Ghrelin (icv)-induced food intake was not attenuated by the CB1 antagonist. Finally, we showed that ventral tegmental administration of Rimonabant attenuates ghrelin- into VTA, induced locomotor stimulation, suggesting that ventral tegmental CB1 regulates ghrelin-induced activation of the mesolimbic dopamine system. Since elevated plasma levels of ghrelin are associated with craving in patients with alcohol dependence our present data highlights a potential therapeutic strategy involving CB1 control of ghrelin action at the level of the mesolimbic dopamine system for treatment of alcohol use disorder. 66. Ana Ledo1,2, Cátia M. Lourenço 2, João Laranjinha 2,3, Greg A. Gerhardt 4 and Rui M. Barbosa 2,3 1 BrainSense, Biocant Park, Cantanhede, Portugal, 2 Center for Neuroscience and Cell Biology, University of Coimbra, Portugal 3 Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, 4 Department of Anatomy and Neurobiology, Center for Microelectrode Technology, University of Kentucky, Lexington, USA MONITORING NEUROMETABOLIC CHANGES IN THE CENTRAL NERVOUS SYSTEM OF AWAKE-BEHAVING RODENT MODELS OF EPILEPSY Temporal lobe epilepsy (TLE) is a form of acquired epilepsy displaying recurrent seizures arising from one or both lobes of the brain. Extreme changes in neuronal excitability perturb energetic demand and produce a vascular response. The spatiotemporal dynamics of neurovascular and neurometabolic coupling during the evolution of an ictal event are poorly understood. The measurement of changes in interstitial [O2] is attractive as it reflects both neurometabolic and vascular function. In vivo electrochemistry combining fast electrochemical techniques (amperometry) with neuromorphic multisite platinum microelectrode arrays allows us to directly measure interstitial [O2] in the brain with high spatiotemporal resolution. Here we investigate changes in O2 in the hippocampus of awake-behaving pilocarpine-treated rats to induce TLE. We show that seizure activity is accompanied by drastic changes in O2. Seizure onset is accompanied by increased O2 consumption followed by increase in interstitial [O2] resulting from typical increase in CBF at the epileptic focus. Furthermore, the use of a microelectrode to measure changes in local field potential related currents in awake behaving rats allowed us to confirm seizure activity through the evolution of frequency power. Acknowledgements: This work is funded by COMPETE (UID/NEU/04539/2013) and Calouste Gulbenkian Foundation. 67. Zachary Brodnik Drexel University College of Medicine, USA INDIVIDUAL DIFFERENCES IN THE RESPONSE TO STRESS PREDICT ELEVATIONS IN COCAINE SELF-ADMINISTRATION AND DISRUPTIONS IN TERMINAL REGULATION OF DOPAMINE Patients with post-traumatic stress disorder (PTSD) display a heightened vulnerability to developing substance use disorders (SUD). Despite this evidence, preclinical studies of prior stress experience on self-administration behavior have been inconsistent, with reports that stress increases, does not affect, or decreases, the propensity of an animal to self-administer cocaine. In these studies we used predator scent stress paired with segregation based on elevated plus maze and context avoidance behavior. We found that subjects classified as susceptible display an increased propensity to self-administer cocaine, but that resilient subjects do not. We then used ex vivo fast scan cyclic voltammetry to query mechanisms of terminal dopamine release and reuptake, as well as terminal cocaine sensitivity across susceptible, resilient, and control subjects. Overall, our findings indicate that susceptible and resilient subjects are differentially sensitive to the reinforcing effects of cocaine, and these behavioral changes correspond with distinct adaptations to terminal regulation of dopamine release and uptake, as well as changes in terminal sensitivity to cocaine. This variance in the behavioral and physiological adaptations that occur following stress may explain some of the inconsistencies between clinical and preclinical studies focused on PTSD and SUD comorbidity. 68. Ladislav Mrzljak1, Holden Brown Janssens2, Arash Rassoulpour2, Ignacio Munoz-Sanjuan1 and 1 CHDI Foundation/CHDI Management, Los Angeles, CA, USA. 2 Brains On-Line LL, San Francisco, CA, USA. ALTERED DOPAMINE RECEPTOR 2 SIGNALING IN THE ZQ175 MOUSE MODEL OF HUNTINGTON'S DISEASE Huntington's disease (HD) is a genetically inherited neurodegenerative disorder characterized by severe motor dysfunction, cognitive decline and psychiatric disturbances, associated with profound striatal and cortical loss. Altered dopaminergic transmission is thought to play a key role in HD pathophysiology, and has been reported in HD patients as well as in rapidly-progressing rodent models of HD. To enhance our understanding of such differences, we aimed to characterize dopamine dynamics in the slower-progressing knock-in zQ175 mouse model of HD. Electrically-evoked striatal dopamine release from anesthetized 7 month old heterozygous zQ175 and WT mice was detected by fast-scan cyclic voltammetry. Oxidative currents were measured in the ventral striatum while increasing intensities of electrical stimulation were delivered to the VTA/SNc after vehicle and after raclopride (D2 antagonist; 1 mg/kg) i.p. injection. Dopamine levels detected from WT and Q175 mice after vehicle treatment were not significantly different. However, raclopride-evoked enhancement of phasic dopamine release was attenuated in Q175 animals as compared with WT. There was no difference in the rate of reuptake between genotypes as measured by Tau75. These results indicate presynaptic D2 receptor dysfunction in Q175 mice, and suggest that similar functional alterations may exist at the postsynaptic level, impairing striato-pallidal indirect pathway transmission. 69. S.Smith, C.Lee, C.Doster, J.Baird, D.Rao, G.McCarty, L.Sombers Department of Chemistry, North Carolina State University, USA MONITORING SUBSECOND GLUCOSE DYNAMICS IN RESPONSE TO INTRAVENOUS GLUCOSE AND COCAINE REVEALS SPATIALLY HETEROGENEOUS MICRO ENVIRONMENTS IN THE RAT DORSAL STRIATUM Brain cells utilize glucose to fuel metabolic processes, and glucose consumption is increased upon neuronal activation. Indeed, brain energy demands are high and this accounts for at least 20% of the entire body's glucose consumption. As such, real-time molecular detection of glucose dynamics is imperative to understanding the regulation of brain energy utilization and its involvement in neuropathological disorders, as well as the adaptations that occur upon exposure to substances of abuse. Extracellular glucose dynamics are dependent on two opposing forces: glucose availability (through cerebral blood flow) and glucose utilization. The location of intraparenchymal microvessels is heterogeneous across brain locations, and brain regions are differentially activated in response to stimuli. Therefore, it is likely that there is a great deal of variation in glucose dynamics both between brain regions, and within a single brain nucleus. In the caudate putamen (CPu), dopamine-related neurochemical adaptations occur in in habit-formation, goal-directed behaviors, and motor control. However, far less is known regarding glucose signaling in this brain region. Direct attempts to assess glucose heterogeneity within discrete brain locations have been hindered due to lack of technology available for these measurements with sufficient spatiotemporal resolution. This work employs fast-scan cyclic voltammetry (FSCV) in conjunction with glucose-oxidase (GOx) modified carbon fiber microelectrodes to monitor glucose dynamics with sub-second temporal resolution in the CPu. We have assessed heterogeneity in glucose signaling that occurs in response to intravenous administration of saline, glucose, and a cocktail comprised of cocaine and raclopride. These investigations will advance our understanding of the neuroenergetics at work in normal brain functions, as well adaptations associated with substance abuse. 70. Christie Lee, Lingjiao Qi, Kristen Blanton, Leslie Sombers North Carolina State University, USA VENTRAL TEGMENTAL AREA MU OPIOID RECEPTOR MODULATION OF DOPAMINE DYNAMICS IN THE VENTRAL STRIATUM The actions of endogenous opioids in the ventral tegmental area (VTA) are critical to normal dopamine (DA) function, and pathology within this region likely leads to aberrant DA signaling in drug addiction. However, it is becoming increasing clear that we only partially understand the mechanisms by which mu opioid receptors (MORs) control VTA activity. This project uses fast-scan cyclic voltammetry to directly assess the effects of intra-VTA infusion of MOR-selective drugs on sub-second DA release in the nucleus accumbens (NAc) of awake rats, and to correlate these measurements with conditioned place preference to improve our understanding of how this circuitry underlies reinforcement. We have found that VTA-MOR activation does not always elicit striatal DA release via a straightforward mechanism, and striatal DA elicited by these manipulations is not always sufficient to elicit place preference. Specifically, intra-VTA infusion of the MOR agonist DAMGO (1.5 μg) significantly increased the frequency and amplitude of DA transients recorded in NAc, and elicited a place preference. Interestingly, intra-VTA infusion of the antagonist CTOP (160 ng) also produced robust DA transients, despite eliciting a place aversion. These results challenge accepted ideas and demonstrate that there are complex mechanisms underlying MOR actions in the VTA. 71. Leslie R. Wilson North Carolina State University, USA REAL-TIME STRIATAL MEASUREMENTS OF OXIDATIVE STRESS AND DOPAMINE IN THE DYSKINETIC RAT DURING CHRONIC L-DOPA TREATMENT FOR PARKINSON'S DISEASE Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the preferential loss of nigrostriatal dopaminergic neurons. The substantial decreases in striatal dopamine (DA) result in devastating hypokinetic movements and motor disturbances. Increased generation of reactive oxygen species, such as hydrogen peroxide (H2O2), is also thought to contribute to Parkinsonian symptoms. However, the precise role of H2O2 in the initiation, progression, and maintenance of the disease remains unclear, as reactive oxygen species are difficult to monitor in brain tissue. Further, several lines of evidence suggest that the standard treatment strategy of Levodopa (L-DOPA; L-3,4 dihydroxyphenylalanine) may serve to increase oxidative stress and even potentiate cell death. We aim to investigate how striatal H2O2 and DA dynamics underlie behavioral changes that result from chronic L-DOPA administration in unilateral 6-OHDA lesioned rats using fast-scan cyclic voltammetry with carbon-fiber microelectrodes in the dorsal striatum to simultaneously quantify rapid H2O2 and DA fluctuations over several weeks. Chemical fluctuations are correlated with behavioral abnormalities that develop over the course of treatment. These studies will aid in our understanding of how oxidative stress modulates nigrostriatal DA signaling, and will demonstrate how these signals correspond with the development of dyskinetic movements in the treatment of PD. IN VIVO ELECTROCHEMISTRY / METHODS
Department of Chemistry, Capital Normal University, Beijing, China FACILE AND FAST DEVELOPMENT OF AU-RING MICROELECTRODES AND ARRAYS FOR IN VIVO AND SINGLE CELL ANALYSIS USING NON-TOXIC POLYDOPAMINE AS MULTIFUNCTIONAL MATERIAL We develop a facile and fast wet deposition technique to bottom-up fabricate Au-ring microelectrodes(Au-RMEs) using non-toxic polydopamine as multifunctional grafting material instead of commonly used (3-aminopropyl)-trimethoxysilane (APTMS). The Au-RMEs are fabricated by growing Au film uniformly inside of a pulled glass capillary. Au-RMEs with tip apex diameter ranging from 15 to 50 μm were fabricated involving four consequent steps, i.e. hydroxylating the inside wall of a pulled glass capillaries, grafting adhesive polydopamine (PDA) film to hydroxyl group surface, seeding gold nano-particles (AuNPs) onto PDA surface and finally growing thickness-tunable gold layer on top of gold nanoparticles. After 3-mercaptopropionic acid (MPA) self-assembled monolayers (SAMs) modification, the Au-RMEs obtain improved specificity and sensitivity for monitoring of dopamine (DA) with respect to ascorbic acid (AA) interference. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, Au-RMEs were used for electrochemical monitoring of DA release stimulated by K+ in the striatum of rats. The whole fabrication process is simple and economic, which can be carried out in ordinary laboratories. The further modified methods can be extended to fabricate Au ring-ring microelectrode and corresponding Au ring arrays for multiple species sensing and single cell manipulation and analysis. University of Virginia, USA LASER TREATED CARBON NANOTUBE YARN MICROELECTRODES FOR RAPID AND SENSITIVE DETECTION OF DOPAMINE IN VIVO Carbon nanotube yarn microelectrodes (CNTYMEs) exhibit rapid and selective detection of dopamine with fast-scan cyclic voltammetry (FSCV), however, the sensitivity limits their application in vivo. In this study, we introduce laser treatment as a simple, reliable, and efficient approach to improve the sensitivity of CNTYMEs by three fold while maintaining high temporal resolution. Laser treatment increases surface area and oxygen containing functional groups on the surface, which provides more adsorption sites for dopamine than at unmodified CNTYMEs. Moreover, similar to unmodified CNTYMEs, dopamine signal at laser treated CNTYMEs barely dropped with increasing repetition frequency compared to a significant decrease at carbon fiber microelectrodes (CFMEs). This is presumably caused by the significantly larger surface roughness which would trap dopamine-o-quinone within each scan and amplify dopamine signal without influencing temporal resolution. In addition, CNTYMEs were applied as an in vivo sensor with FSCV for the first time and laser treated CNTYMEs maintained high dopamine sensitivity compared to CFMEs with an increased scan frequency of 50 Hz. CNTYMEs with laser treatment are advantageous of their easy fabrication, high reproducibility, and rapid in vivo measurement of dopamine with high sensitivity and fast electron transfer, which might be expected to be a potential alternative of CFMEs. 74. Thitaphat Ngernsutivorakul, Woonghee Lee, Erik Guetschow, Jenny Wong, Robert Kennedy University of Michigan, USA MICROFABRICATION OF SAMPLING PROBES FOR IN VIVO NEUROCHEMICAL MONITORING WITH HIGH SPATIO-TEMPORAL RESOLUTION Sampling probes, i.e. push-pull sampling and microdialysis, are essential tools for in vivo neurochemical monitoring. However, conventional probes are typically handmade and have several limitations, such as large in size (> 200 µm in diameter) and limited flexibility in their designs. We have therefore used a microfabrication approach to overcome these disadvantages. The probes were microfabricated from a 525 µm thick silicon wafer coated with SiO2 mask. Microchannels were created using lithographic patterning and plasma etching tools. The channels were then sealed with poly Si using low pressure chemical vapor deposition. Lithography and plasma etching were used to assign and etch probe shapes and sampling areas with desired dimensions. For microdialysis probes, nanoporous anodic aluminum oxide was adapted for use as embedded membranes. We have successfully fabricated small sampling areas and three microchannels within a probe, which has overall dimension of < 100 µm. Over a hundred probes can also be fabricated within a wafer. Furthermore, the probes can feasibly be coupled to other microfluidic devices for droplet formation and sample derivatization as well as miniaturized analytical methods for improved temporal resolution. Preliminary studies have shown suitability for in vivo neurochemical monitoring with 10 s temporal resolution. 75. Douglas Kirkpatrick University of North Carolina at Chapel Hill, USA ADVANCES IN QUANTITATIVE MICROIONTOPHORESIS Microiontophoresis is a neurochemical tool describing the ejection of a solution from a micropipette using a controlled current. This technique allows for highly localized drug delivery on a short time scale and is advantageous for studies of individual cellular behavior and characterization. Although much effort has been devoted to determining precise ejection amounts, no robust method exists. This prevents its incorporation into concentration based studies and limits the technique to qualitative use. To address this, ejection rate characteristics were investigated to further development as a quantitative tool. Ejection amounts of various chemical species are determined using electrochemical and chromatographic methods. Factors such as molecular charge, ejection current magnitude, and the ionic composition of the drug solution are examined for their effects on the delivered quantity. Particular attention is paid to the ejection duration, as shorter ejections are more susceptible to error due to the initial time dependent nature of ejection rates. From these conclusions, quantitative neurochemical studies are performed which demonstrate the abilities and limitations of microiontophoresis in quantitative applications. 76. Justin Allen Johnson The University of North Carolina at Chapel Hill, USA METHOD FOR REMOVAL OF NON-FARADAIC CONTRIBUTIONS TO FAST-SCAN CYCLIC VOLTAMMETRY RECORDINGS Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes has proven an essential tool for monitoring subsecond fluctuations in electroactive neurotransmitters (e.g. dopamine and norepinephrine) in awake, freely moving animals. The background current that arises at the high voltammetric scan rates employed for FSCV necessitates the use of digital background subtraction to resolve the signal of interest. Success of such an approach depends critically on the background's stability during the neurobiological phenomenon under study. However, ionic fluctuations, which occur both naturally (e.g. potassium waves in spreading depression) and artificially (e.g. iontophoretic ejections), alter the electrode capacitance and, consequently, background current, complicating identification of the catecholamine signal. Here, we describe and characterize a procedure for the removal of these ionic contributions to the signal. This method relies on use of a modified waveform to probe the electrode's capacitive response prior to the triangular FSCV sweep, which is used for subtraction of the non-Faradaic component of the voltammetric response. We demonstrate the application of this methodology for removal of such signals from in vitro (e.g. flow-cell analysis of dopamine-potassium and glutamate mixtures) and in vitro (e.g. iontophoretic ejections in anesthetized animals) FSCV recordings. 77. Anna Larsson, Andrew Ewing University of Gothenburg, Sweden IN VIVO ELECTROCHEMICAL MEASUREMENTS OF THE EFFECTS OF DROSOPHILA VESICULAR MONOAMINE TRANSPORTER (DVMAT) TRAFFICKING IN D. MELANOGASTER LARVA Drosophila vesicular monoamine transporter (DVMAT) is responsible for loading monoamine neurotransmitters such as octopamine and dopamine into both synaptic vesicles (SV) and large dense core vesicles (LDCV). Mutations disrupting the trafficking of DVMAT have previously been shown to increase DVMAT localization in LDCV versus SV and also affect behaviors such as larval locomotion. But the quantitative information regarding to content of SV and LDCV is still unknown. This work faces the challenge to electrochemically measure effects of DVMAT trafficking on neurotransmitter release in D. Melanogaster larva. Recently, we developed a method to measure octopamine release from the neuromuscular junction (NMJ) in D. Melanogaster larvae by using amperometry. In this method, the light-activated ion channel channelrhodopsin-2 (ChR2) is expressed genetically in type II varicosities, and then blue light could be used to activate octopamine release. Upon illumination, octopamine released from varicosities detected with a carbon-fiber microelectrode, which was placed onto this cellular region of the muscle. This is a new approach to study millisecond release events in the nanoliter environment of the NMJ, and it can be used to understand how changes in monoamine release regulate synaptic transmission and behavior in the fly. 78. Anne Meiller NeuroChem Platform, Claude Bernard University Lyon1, France IN VIVO DETECTION OF BRAIN NITRIC OXIDE USING SILANE-COATED CARBON FIBER ELECTRODES Nitric oxide (NO) is an important component of the excitotoxic damage induced by N-methyl-D-aspartate (NMDA) receptor activation in neurons. Direct in vivo detection of NO released by NMDA application is therefore an important challenge to understand the mechanisms of oxidative stress and excitotoxic insult. Electrochemical in situ analysis using carbon fiber or platinum wire microelectrodes is a promising strategy to circumvent the high lability of the NO molecule. Here, we used carbon fiber microelectrodes coated with Nickel Porphyrin and (heptadecafluoro-1,1,2,2,-tetrahydrodecyl)trimethoxysilane. Such sensors had a detection limit of about 20nM and a linear range extending up to 2µM. They showed improved selectivity against nitrite, serotonin and ascorbate compared with conventional Nafion-coated electrodes. They were stable when stored at room temperature for a week and lost only 34% sensitivity after three hours of in vivo monitoring. In vivo, these electrodes could detect NO released by the local administration of NMDA (30 mM, 250nL). NMDA administration induced a rise in oxidation current of about 25 pA that related to 430nM of NO, and the signal disappeared when the NO synthase inhibitor, L-NNA, was applied 2 hours before NMDA. Overall, silane-coated electrodes are an advantageous method to detect endogenous NO release in vivo. 79. Anna Belle, A.C. Tooker, J.A. Pebbles, K.Y. Lee, A.S. Sperry, V.M. Tolosa Lawrence Livermore National Laboratory, USA IMPLANTABLE, FLEXIBLE PROBES FOR ELECTRICAL STIMULATION AND RECORDING OF UNIT ACTIVITY AND CHEMICAL RELEASE Electrical stimulation of brain tissue to induce or suppress neuronal activity is an important therapeutic application of implantable sensor technologies. Understanding why a specific stimulation is successful and what causes changes in the brain's response to the same electrical stimulation over time is a key step to improving brain stimulation based therapies. We are developing a flexible probe that allows for monitoring of local tissue responses in an awake, freely moving rodent in response to acute and chronic electrical or environmental stimuli. Iridium oxide electrodes on this probe can be used to electrically stimulate tissue, record unit activity or record pH changes. Integration of amperometric electrochemical sensors onto this same probe creates the opportunity to monitor changes in chemical messengers like glutamate and dopamine that may serve as predictive markers for the success or failure of an electrical stimulation. These probes also include integrated reference and ground electrodes to minimize noise and further tissue alteration during experimentation. Here we present data from one probe design, capable of recording single-unit activity from eight sites and chemical release from eight discrete sensors to better examine the dynamics of glutamatergic and dopaminergic neurotransmission during cell activity. 80. Gregory S. McCarty1, Christie A. Lee1, Sarah E. Calhoun1, Carl J. Meunier1 and Leslie A. 1 Department of Chemistry, North Carolina State University, USA ELECTROANALYTICAL MEASUREMENTS OF TYROSINE-CONTAINING NEUROPEPTIDES: CHASING THE ENKEPHALINS Opioid neuropeptides regulate a broad spectrum of biological functions and are heavily implicated in pain management and in hedonic behaviors associated with addiction. Opioid neuropeptides modulate the mesolimbic and nigrostriatal dopamine (DA) circuits and intact opioid signaling pathways are required for key aspects of cocaine abuse. However, the precise mechanisms that underlie opioid modulation of DA systems remain ambiguous. Although several methods exist for monitoring DA fluctuations, few tools are available for selectively monitoring dynamic fluctuations of endogenous opioid neuropeptides. This work addresses the issues involved with monitoring endogenous opioid neuropeptides by optimizing and employing an innovative electrochemical approach to monitor sub-second fluctuations of the tyrosine-containing opioid neuropeptides methionine-enkephalin (M-ENK). By combining multiple scan rate voltammetry with constant-potential amperometry, we have optimized an electroanalytical technique for measuring M-ENK fluctuations. The waveform development described in this work has leveraged the knowledge gained from years of utilizing electrochemistry to monitor dopamine, and adapted it to M-ENK detection. Given opioid signaling has been implicated in mechanisms accompanying robust increases in extracellular DA elicited by L-DOPA or cocaine, our study investigates cocaine-induced effects on putative M-ENK fluctuations in the striatum of an L-DOPA treated rat. Importantly, our electrochemical approach enables selective detection of chemical species, in this case DA and M-ENK release. As such, on-going work is evaluating simultaneous M-ENK and DA dynamics in the striatum following acute administration of cocaine. Biomedical Engineering Department, Hanyang University, South Korea IN VIVO MONITORING OF THE EXTRACELLULAR DOPAMINE EFFLUX BY CHARGE-BALANCED MULTIPLE WAVEFORM FAST-SCAN CYCLIC VOLTAMMETRY The background current of fast-scan cyclic voltammetry should be subtracted to monitor small change of dopamine in limited time. The subtraction time point has to be renewed every 30 60 seconds to monitor phasic activity because the background signal drifts over a minute. Herein, we developed charge-balanced multiple fast-scan cyclic voltammetry (CBM-FSCV) to monitor extracellular dopamine efflux for prolonged time (> 1hour) with fixed background subtraction time point. By using CBM-FSCV, we confirmed the background from CBM-FSCV doesn't changed for 2 days in vitro. In pharmacological in vivo test, we were able to detect the final concentration of dopamine efflux by nomifensine (reuptake inhibitor, +235±60nM, SEM) and α-Methyl-DL-tyrosine (synthesis inhibitor, -72.5±4.8nM, SEM). In this research, we successfully measure the long term change of dopamine in vivo both positive and negative direction and we show that CBM-FSCV is an essential technique to monitor dopamine efflux for prolonged time. 82. Diana Campos-Beltrán1, Sonja Binder1, Ping Chai Koo1, Åsa Konradsson-Geuken2, George E. Quintero3, Lisa Marshall1,4 AMPEROMETRIC RECORDINGS OF D-SERINE USING MICROELECTRODE ARRAYS 1 Institute of experimental Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany 2 Karolinska Institutet, the Department of Neuroscience, Stockholm, Sweden and Uppsala University, the Department of Comperative Physiology, Uppsala, Sweden 3 CenMeT, University of Kentucky, Lexington, USA and Quanteon LLC, Kentucky, USA 4 Graduate School for Computing in Medicine and Life Sciences, University of Lübeck, Lübeck, Germany D-serine is the major D-amino acid in the mammal central nervous system. As a dominant endogenous NMDAR co-agonist, D-serine plays a role in learning, memory and plasticity, with alterations of D-serine linked to mental disorders like schizophrenia. Given this, recording D-serine concentration in vivo is of major interest. Based on the method by Pernot et al., 2008 for cylindrical platinum microelectrodes, we designed a procedure for detecting D-serine with ceramic-based microelectrode arrays (MEAs) using the FAST-16 amperometric system from Quanteon, LLC. Applying D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) we demonstrate for in vitro MEA D-serine biosensor recordings with a sensitivity of -0.0058 ± 0.0005 nA/µM D-serine and a LOD of 0.33 ± 0.06 µM (MEAN ± SEM; n=6). D-serine is catalyzed by RgDAAO to produce H2O2 which is oxidized on the electrode surface to generate a current (nA), under constant potential amperometry (+0.7V vs Ag/AgCl), that indicates the D-serine concentration (µM). Two non-RgDAAO coated channels act to self-reference and afford the possibility of measuring basal D-serine levels. Selectivity over other neurochemicals is achieved by polymerizing m-Phenylenediamine (mPD) onto the recording sites. In conclusion, we find that RgDAAO coated MEAs can be used to record D-serine in vivo. 83. Tazima Nur, Shree Hari Gautam, Julie A. Stenken, Woodrow L. Shew University of Arkansas, USA MEASURING FUNCTIONAL IMPLICATIONS OF INHOMOGENEOUS ACETYLCHOLINE DISTRIBUTION IN CEREBRAL CORTEX Neuromodulators are critical for sensory information processing in cortical microcircuits. Acetylcholine (ACh) is a neuromodulator associated with attention, arousal, learning, and memory. Dysfunctional ACh modulation is associated with various neural disorders including Alzheimer's disease. Previous studies do not address the possibility that ACh concentrations are not spatially uniform. The effect of nonuniform ACh concentration gradients across the spatial extent of a microcircuit on the function of the microcircuit is not well understood yet. To better elucidate the effects of ACh on a neural circuit, we have combined an implanted microdialysis probe with a microelectrode array in anesthetized rat somatosensory cortex. Acetylcholine is administered via the microdialysis probe and the concomitant neural activity is recorded with the microelectrode array. Here we present preliminary results demonstrating that neural response to somatosensory (whisker) stimulation depends on the spatial gradient of ACh. Our initial results constitute the first steps towards better understanding the effect of inhomogeneous distribution of ACh on sensory information processing. 84. Irene Mollinedo-Gajate1,2, Javier González-Maeso3, J. Javier Meana1,2 1 Department of Pharmacology, University of the Basque Country UPV/EHU, 2 Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and BioCruces Health Research Institute, Spain 3 Departments of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, USA METABOTROPIC GLUTAMATE 2 RECEPTOR HAS AN ESSENTIAL ROLE FOR THE APPROPRIATE DOPAMINE RELEASE MEDIATED BY SEROTONIN 2A RECEPTOR IN MOUSE FRONTAL CORTEX The presence of metabotropic glutamate mGlu2 receptors (mGluR2) is necessary for the cell signaling mediated by hallucinogenic serotonin 5-HT2A receptor (5-HT2AR) agonists. Besides, the activation of mGluR2 by selective agonists is able to block psychosis-like behaviors. This study focused on the interaction of 5-HT2AR and mGluR2 by using in vivo microdialysis to evaluate extracellular dopamine (DA) release in the frontal cortex (FC) of mGluR2 and 5-HT2A knock-out mice. Reversal-dialysis application of the 5-HT2AR agonist (±)DOI (300 µM) increased DA in mGluR2+/+ (Emax=163±15%) and in 5-HT2AR+/+ (Emax=225±21%) mice. The stimulation was blocked by local presence of the 5-HT2A antagonist M100907 (300 µM) (Emax=127±6%). In mGluR2-/- (Finteraction[13,325]=3.267;p<0.001) and was insensitive to M100907 (Emax=132±15%). (±)DOI displayed a lower release of DA in 5-HT2AR-/- (Emax=155±16%) than in HT2AR+/+ mice (Finteraction[13,169]=7.089;p<0.001). Overexpression of mGluR2 in mGluR2-/- mice, by local injection in the FC of herpes simplex virus-mediated transgene with mGluR2 (mGluR2-HSV) led to recover DA release induced by (±)DOI (Emax=172±18%), as compared to mGluR2-/- animals infected with herpes simplex virus-mediated control transgene (GFP-HSV) (Emax=137±10%) (Finteraction[13,260]=3.223;p<0.001). These findings indicate that the existence of mGluR2 is necessary for the appropriate regulation of DA release by hallucinogenic 5-HT2AR agonist drugs. 85. Jason S. Katner, A. Rassoulpour, X. Chai, E. Chernet, J.E. Cooper, K. Huinink, T.A. Day, M. Hayashi, K.A. Svensson Eli Lilly & Company, USA DEVELOPMENT OF AN IN-VIVO MICRODIALYSIS METHOD TO ANALYZE FULL-LENGTH, C-TERMINAL AND N-TERMINAL TAU PROTEIN IN THE HIPPOCAMPUS OF TG4510 MICE. Alzheimer's disease (AD) is a debilitating neurological disorder that is characterized by elevated levels of Tau protein, which manifests as neurofibrillary tangles and is thought to initiate neuronal cell death. The aim of this study was to develop a push/pull in-vivo microdialysis technique to measure extracellular Tau levels of TG4510 mice, a transgenic strain of mice that over-expresses the tau mutant P301L gene. Our microdialysis method was optimized for artificial CSF composition, flow rate, and probe construction. We collected extracellular dialysates from the hippocampus in 18-19 week-old male and female TG4510 mice and compared these levels using a Tau-specific ELISA. Along with full-length Tau, we also utilized ELISA methods that measured truncated N-Terminal and C-Terminal Tau. We were able to consistently measure extracellular levels of Tau in the hippocampus of both male and female TG4510 mice at regular intervals. This may prove useful in further characterizing tau pathology and support discovery of novel AD therapies. 86. Chi Leng Leong Imperial College London, UK DEVELOPMENT OF MICROFLUIDIC DROPLET SYSTEM FOR IONIC ANALYSIS OF OFFLINE MICRODIALYSIS SAMPLES FROM TRAUMATIC BRAIN INJURY PATIENTS Spreading depolarization, a spontaneous phenomenon linked to the development of secondary brain injury, is characterized by a temporary disruption of the ionic homeostasis in the brain, with a transient reversal of the cellular ionic gradients of potassium, sodium, calcium and magnesium. In order to recover, the ionic balance must be restored; failure to do so results in tissue death and hence resulting in secondary brain damage. Whilst we have concentrated on online analysis, many clinical centers employ offline microdialysis and collect samples that are frozen for later analysis. One of the challenges with offline microdialysis is the small sample volume that imposes a stringent restriction on conventional analysis. We have developed a microfluidic system that aspirates a small precise volume from the dialysate collection vial, segmented it with immiscible oil to eliminate mixing and dilution of the sample. The sample segment is then moved to the sensing region of the system where its ionic content will be measured by an array of miniaturized ion-selective electrodes. Once analyzed, the sample segment can then be returned to its sampling vial with minimal volume loss, meaning the sample is preserved and can then be passed on for further analysis. 87. Martin Eysberg Antec, Netherlands ANALYSIS OF DOPAMINE AND SEROTONIN UNDER 2 MIN TO FURTHER IMPROVE TIME RESOLUTION IN ON-LINE MICRODIALYSIS Microdialysis of neurotransmitters in vivo has become an invaluable tool to study neurotransmission in the living brain. Extracellular fluid of the brain is sampled via a microdialysis probe and fractions are collected for further analysis. Typical flow rates in microdialysis are 1 - 2 µL/min, decreasing the fraction size to a few microliters enables a temporal resolution of a few minutes. However, it also requires an analytical system that has the sensitivity for reliable quantification of neurotransmitters and the capability to handle samples of only a few microliters. In case of on-line analysis, the sample fractions are collected in a sample loop and analyzed immediately. For uninterrupted analysis in such a setup, the UHPLC analysis time should ‘match' the time required to collect a sample. We developed a fast and reliable method for analysis of dopamine (DA) and serotonin (5-HT). Small samples of less than 2 µL were analyzed on a UHPLC system with a new electrochemical detector, the DECADE Elite and the new Sencell. This flow cell has a proprietary Adjustable Spacer Technology (AST) to improve the detection limit. DA and 5-HT were quantified in less than 2 min analysis time. An increased data rate was used to analyze the fast chromatographic peaks and an elevated column temperature further facilitated the speed of separation. The principle and feasibility of this set-up is shown with the analysis of dopamine (DA) and serotonin (5-HT), which showed a detection limit of about 0.2 nmol/L (1 µL injection so about 0.2 fmol on column), with a temporal resolution of about 1 min. 88. Lisa Ulenius1, Louise Adermark1, Bo Söderpalm1,2, Mia Ericson1
1 Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, The
Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden
2 Beroendekliniken, Sahlgrenska University Hospital, Gothenburg, Sweden VOLUME REGULATED ANION CHANNELS CONTROLS ETHANOL-INDUCED DOPAMINE AND TAURINE RELEASE IN THE RAT NUCELUS ACCUMBENS Alcohol activates the mesolimbic dopamine system and increases dopamine levels in the nucleus accumbens (nAc), an effect associated with positive reinforcement. When trying to understand the underlying mechanism of action we have focused on taurine, an endogenous amino acid with a wide range of tasks in the brain including osmoregulatory properties. We have demonstrated that in order for ethanol to increase dopamine in the nAc an initial enhancement of extracellular levels of taurine is required. We have also shown that administrating ethanol in a hypertonic saline solution prevents ethanol-induced taurine release in the same region. Taurine can be released into the extracellular space via volume regulated anion channels (VRACs) which is why we hypothesized that VRAC antagonism will prevent ethanol-induced dopamine elevation. Thus, in vivo microdialysis in rats was used to measure the effects of ethanol and the VRAC inhibitor DCPIB on accumbal dopamine and taurine levels. We found that local perfusion with DCPIB blocked the ethanol-induced dopamine release and partially blockade the taurine release. In conclusion, ethanol-induced elevation of extracellular taurine is mainly mediated via VRACs. These data also support previous findings that the elevations of dopamine and taurine in the nAc after ethanol are closely related. 89. Amir Lotfi, Julia Morud, Valentina Licheri, Bo Söderpalm, Mia Ericson, Louise Adermark University of Gothenburg, Sweden AGE-DEPENDENT MODULATIONS IN NEUROTRANSMISSION AND DOPAMINE LEVELS BY NICOTINE IN SUBREGIONS OF STRIATUM Acute exposure to nicotine exerts profound neurophysiological effects that cause an array of cognitive and behavioral properties, many of which are attributed to increased dopamine levels. There appears to be an age-dependent component to these effects, and both clinical and preclinical studies indicate that rewarding and stimulatory properties of nicotine are greater in adolescents. By means of in vivo microdialysis and ex vivo electrophysiological recordings, the aim of this study was to explore the role of age on nicotine-induced changes in dopamine output and neurotransmission in defined subregions of the striatum, a key brain structure with respect to drug-reinforcement and reward-guided behavior. Our data shows that nicotine increases dopamine output in a subregion selective manner in nicotine naïve rats. In addition, the increase in dopamine sustains for a longer time period in older rats (>35 weeks). Electrophysiological recordings support a role of age in the neuromodulatory properties of nicotine, where nicotine depresses striatal output in juvenile rats but not in older rats. The data presented here suggests that nicotine affects neurotransmission in a subregion specific, and age-dependent manner. These age-dependent effects might be important neurophysiological underpinnings contributing to the high risk of developing nicotine addiction in adolescents. University of Texas at Austin, USA ETHANOL STIMULATES EXTRACELLULAR NOREPINEPHRINE IN DIALYSATES COLLECTED FROM THE PREFRONTAL CORTEX OF LONG EVANS RATS Ethanol is known to enhance brain dopamine signaling, but little is known about effects of ethanol on norepinephrine (NE). We examined this using microdialysis and an HPLC-electrochemical detection method modified from Reinhoud et al. (2013); pump flow rate = 0.09 mL/min, mobile phase pH = 5.6, and methanol (5%) was the organic modifier. NE standards were stabilized in 10 mM phosphate (pH about 3), and the retention time of NE was 4.1 minutes. The signal-to-noise ratio of the lowest tested standard (78 pM) was 3.9. In vivo microdialysis was performed in the prefrontal cortex (PFC) of male, adult Long Evans rats (n=3). The basal NE concentration was 0.38±0.07 nM. Ethanol infusion (1 g/kg, intravenous) increased dialysate NE to about 75±4% over basal levels within ten minutes, whereas saline infusion did not increase PFC NE concentrations relative to baseline. Deletion of calcium from the probe perfusate reduced NE by 77% indicating that extracellular NE was largely exocytotic. Overall, we show that NE in the PFC is stimulated by ethanol suggesting that NE may also contribute to its intoxicating properties. 91. Shannon L. Zandy The University of Texas at Austin, USA HIGH SENSITIVITY METHOD FOR ANALYSIS OF IN VIVO EXTRACELLULAR GABA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION USING O-PHTHALDIALDEHYDE (OPA)/SULFITE DERIVATIZATION Reversed-phase HPLC with pre-column derivatization with ortho-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysates. However, OPA/sulfite derivatives were reported to be less sensitive with fluorescence. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of OPA/sulfite in the derivatizing solution. Optimal λex=220 nm, and λem=400 nm. GABA fluorescence was unaffected by reaction time (0, 2.5, 5, 10 min), but a pH of 10.4 was better than 9.4 or 8.4. Dilution of the OPA/sulfite solution by 30-fold did not alter the GABA signal, but did reduce blank peaks, whereas 100-fold dilution decreased the GABA signal. Varying the molar ratio of OPA:sulfite (1:1, 1:3, 1:10) did not affect GABA, but increasing sulfite reduced blank peaks. Microdialysis was performed in adult Long Evans rats from dorsal striatum (DS) and ventral tegmental area (VTA), and GABA was determined using external standards and standard additions. Basal GABA concentrations in the DS (n=4) were 12.9±2.2 and 14.5±2.2 nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=4) were 4.0±1.0 and 4.2±0.9 nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative. 92. V. Valentini1,2, D.M. Bortz1, J. Schumacher1, G.P. Serra2, D. Phenis1 and J.P. Bruno1 1 Departments of Psychology and Neuroscience, The Ohio State University, Columbus, Ohio 2 Department of Biomedical Sciences, University of Cagliari, Italy PREFRONTAL-ACCUMBENS BIDIRECTIONAL REGULATION OF TRANSMITTER RELEASE: IMPLICATIONS FOR SCHIZOPHRENIA AND THE DISCOVERY OF COGNITION-ENHANCING THERAPEUTICS Executive functions (attention, working memory), contribute to cognitive control via top-down regulation of goal-directed behavior and are mediated by a distributed neural network including the nucleus accumbens (NAC), basal forebrain, and prefrontal cortex (PFC). Disruptions in these interactions produce cognitive deficits that are core symptoms in several neuropsychiatric disorders (schizophrenia, ADD, drug addiction). Previously we reported, using both traditional microdialysis and a more novel enzyme-based biosensor, that intra-NAC stimulation with NMDA increases prefrontal ACh levels and facilitates cognition, increasing the resistance to distraction in a sustained attention task. Here we demonstrate, using both techniques, reciprocal interactions between NAC and PFC. Intra-NAC NMDA (0.05 – 0.3 μg) evoked ACh release (75-150% above baseline); leading to elevated prefrontal glutamate (400-900%) and dopamine levels (100%). This effect required stimulation of local α7nACh receptors, as α7nACh antagonists (MLA or α-BGT) abolish glutamate/dopamine release. Cortical levels of ACh, glutamate, and DA are also regulated by an endogenous negative modulator, kynurenic acid (KYNA). We enhanced prefrontal activity (mimicking top-down control) by inhibiting KYNA synthesis with local perfusion of S-ESBA (5.0 or 10 mM). Glutamate/dopamine release was assessed in NAC. Collectively, these bidirectional transmitter interactions justify the focus on these receptor targets for the discovery of cognition-enhancing drugs. 93. Roger Cachope1, Jane Sutcliffe3, Arash Rassoulpour4, Mariette S. Heins2, Leticia Toledo Sherman1, Cristina Schlumbohm3, Kerstin Hoffman3, Joost H.A. Folgering2,3, Thomas I.F.H. Cremers2,3, Marcel M. Van Galen3, Vinod Khetarpal1 , Celia Dominguez1, Ignacio Munoz SanJuan1 and Ladislav Mrzljak1 1 CHDI Management Inc., Princeton and Los Angeles, USA 2 Brains On-Line, Netherlands 3 Encepharm, Goettingen, Germany 4 Brains On-Line, USA KMO INHIBITION IN THE CONSCIOUS NON-HUMAN PRIMATE: KYNURENINE PATHWAY METABOLITE AND NEUROTRANSMITTER RELEASE IN THE CAUDATE NUCLEUS AND PREFRONTAL CORTEX Metabolites of kynurenine pathway (KP) are implicated in the pathophysiology of neurodegenerative disorders including Huntington's disease (HD). Kynurenine 3- monooxygenase (KMO) inhibitors may have potential to treat HD, given that KMO inhibition should shift the metabolism of kynurenine to increase the formation of the neuroprotective metabolite kynurenic acid (KYNA) and reduce the neurotoxic metabolites 3-hydoxykynurenine (3-OH-KYN) and quinolinic acid (QA) in the brain. This has been confirmed in rodent microdialysis studies using the potent KMO inhibitor CHDI-00340246. Here, we studied the effects of CHDI-00340246 on level of KP metabolites, free compound and neurotransmitter release in the dorsal caudate nucleus and the dorsolateral prefrontal cortex (PFC - area 9) in conscious young adult male cynomolgus macaques using microdialysis. Oral administration of 10-30 mg/kg of CHDI-00340246 increased concentrations of KYNA and AA in both brain areas. Importantly, CHDI-00340246 reduced concentrations of the neurotoxic metabolites 3-OH-KYN and QA in both brain areas. The compound did not affect the neurotransmitters dopamine, serotonin, glutamate, glycine and GABA, except that dopamine concentrations were reduced in the PFC (highest dose only). These findings demonstrate that oral dosing of CHDI-00340246 modulates the pathway centrally in primates and may have neuroprotective potential in HD. 94. Ashish Asthana1, S. de Groot1, T. Phisonkunkasem2, S.H. van Heiningen2, E.A. Tolner2,3, A.M.J.M. van den Maagdenberg2,3, M. Odijk1 1 BIOS − Lab on a Chip group, MESA+ Institute for Nanotechnology and MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands 2 Department of Human Genetics, Leiden University Medical Centre, The Netherlands 3 Department of Neurology, Leiden University Medical Centre, The Netherlands

Understanding of neuro-chemical dynamics in the brain of a living animal is fundamental for developing cures for brain anomalies. Microdialysis of cerebrospinal fluid coupled with analytical tools has been well established by the Kennedy group for quantitative in-vivo monitoring. In the present work, a stand-alone, extremely low L.O.D. (less than 20 nM for fluorescent products) and portable set up has been developed for online and in-vivo monitoring of dialysate from the brain of a rodent. The dialysate is mixed with neurotransmitter-specific fluorogenic reagents and compartmentalized into nanoliter droplets within an oil stream to preserve temporal history of neurotransmitter concentrations. The resulting fluorescence is read downstream to correlate with neurotransmitter concentration. A case study of dynamics of excitatory neurotransmitter, glutamate has been chosen as a basis for investigating the pathophysiology of migraine. Initially commercial microdialysis probes are used in conjunction with DC-recordings to study disease-related changes in glutamate levels in anesthetized mice. This will be followed by use of miniaturized hybrid push-pull type neuroprobes with simultaneous ionselective recordings at spatial resolution of the order of 100 microns. 95. Michael Hogard, Nathan Oborny, Susan M. Lunte, Craig E. Lunte University of Kansas, Ralph N. Adams Institute for Bioanalytical Chemistry, USA DEVELOPMENT OF AN ONLINE MICROCHIP ELECTROPHORESIS WITH LED-INDUCED FLUORESCENCE SYSTEM FOR NEAR REAL-TIME MONITORING OF EXCITATORY AMINO ACID NEUROTRANSMITTERS INVOLVED IN TRAUMATIC BRAIN INJURY Excitatory amino acid neurotransmitters (EAAs) are associated with a wide number of neurodegenerative diseases. This includes traumatic brain injury (TBI), a leading cause of death and disability worldwide. The development of analytical techniques that can directly monitor EAAs in vivo could assist in medical treatments for TBI patients, as well as research on animal models of the disease. The objective of these studies was to develop a method for the separation and detection of EAAs using microchip electrophoresis (ME) with fluorescence detection that could be used on-site. The amino acids were derivatized with naphthalene-2,3-dicarbozaldehyde (NDA) for fluorescence detection. Glutamate and aspartate, as well as other neurologically relevant amino acids, were separated with this method and detected in rat brain microdialysis (MD) samples. Since traditional benchtop analytical equipment is too cumbersome for use in hospitals, a mobile and fully integrated light-emitting diode-induced fluorescence (LED-IF) detection system was designed and built in-house and used for these studies. The MD-ME-LED-IF system with integrated online derivatization will be used to monitor in vivo changes in EAA concentration in near real-time in a rat epilepsy model. In the future, the system will be used to study the role of EAAs in animal models of TBI. 96. Amanda M. Furness, Craig Lunte, Susan Lunte University of Kansas, Ralph N. Adams Institute for Bioanalytical Chemistry, USA INVESITGATION OF THE EFFECTS OF INDOMETHACIN ON THE NEUROCHEMICAL RESPONSE ASSOCIATED WITH SEIZURE EVENTS Epilepsy is a well-known neurological disease that results in seizures. In approximately 70% of epileptic patients, these seizures occur in specific localized regions of the brain. Since patients are only diagnosed with epilepsy after experiencing two or more unprovoked seizures, an animal model for local epilepsy was developed where multiple seizures are induced within one experiment to more accurately represent the disease in comparison to ¬in vitro or in silico studies. Additionally, the nonsteroidal anti-inflammatory drug indomethacin has been shown to reduce the negative effects of oxidative stress caused by epilepsy and shorten recovery time post-seizure. The objective of these studies was to monitor the neurochemical response of the rats after being dosed with indomethacin using the multiple seizure animal model. An intercranial microdialysis probe was used to locally administer the convulsant 3-mercaptopropionic acid (a glutamic acid decarboxylase inhibitor) to the hippocampus of a rat while simultaneously collecting the brain dialysate. Glutamate, GABA, norepinephrine, and dopamine concentrations were determined in the dialysate samples using LC with fluorescence and electrochemical detection. By monitoring both the relevant amino acid and catecholamine neurotransmitters, it was determined if and how the dosing of indomethacin altered the levels of these neurotransmitters associated with seizures. 97. J. Boulanger Bertolus, Sandrine Parrot, A.M. Mouly Lyon Neuroscience Research Center, University Lyon1, France ENCODING TIME INTERVALS IN ODOR FEAR CONDITIONING: ROLE OF THE DORSAL STRIATUM In pavlovian fear conditioning, an initially neutral stimulus (conditioned stimulus, CS) predicts the arrival of an aversive unconditioned stimulus (e.g. a foot-shock, US) at a fixed time interval. We previously showed that odor fear acquisition was associated with an increase in 2-deoxyglucose uptake in the dorsomedial striatum of paired rats suggesting that this structure might be involved in encoding the odor-shock association and/or the temporal link between the two stimuli. The aim of the present study was to investigate the role of the striatum in timing interval durations. A setup allowing the recording of respiration, ultrasonic vocalizations and behavior during training (10 Odor-Shock pairings, with a 20s CS-US interval) enabled us to detect the emergence of temporal patterns in the animal's fear response during acquisition, indicating that rats are able to encode CS/US interval duration after a few training trials. Besides, respiratory rate fluctuations observed during transient inactivation of the dorsomedial striatum suggest that a functional striatum favors the stability of a learned temporal pattern. We then measured striatal DA levels using intracerebral microdialysis during an acquisition session which included 7 odor-shock pairings, 6 with a 20s CS-US interval and the last one with a 30s interval. We observed a decrease in DA concentration at the onset of the pairings, which stabilized throughout the 6 trials with a 20s CS-US interval and a further decrease when the interval duration was shifted to 30s. Taken together, the present data suggest that the dorso-medial striatum is not involved in the formation of the odor-shock association per se but might be involved in the production of a stable temporal pattern of behavior related to the duration of the CS-US interval. 98. Barbara Ferry and Sandrine Parrot Lyon Neuroscience Research Center, University of Lyon, France WHEN MICRODIALYSIS TECHNIQUE UNVEILS THE REAL EFFECTS OF MICROINFUSION: A FOCUS ON α2-ADRENERGIC SYSTEM IN THE BASOLATERAL AMYGDALA Recent neuropharmacological experiments in the rat have shown that α2-adrenoceptor antagonist idazoxan enhanced, whereas the agonist UK 14,304 decreased inhibitory avoidance performance when locally infused into the BLA. In order to study the effects of these α2-adrenergic drugs on norepinephrine (NE) release in the BLA, anaesthetized male Sprague-Dawley rats unilaterally implanted with a microdialysis probe coupled to a microinfusion cannula in the BLA received acute dexefaroxan (0.3 µg/0.2 µl) or UK 14,304 (3 ng/0.2 µl) 15 minutes before the start of the experiment. Comparativel groups received either acute systemic injection of dexefaroxan or UK 14,304 (0.63 mg/kg) or were retrodialyzed 45 min with dexefaroxan (0.1 mM) or UK 14,304 (10 µM) in the BLA. Results showed that systemic and local retrodialysis of dexefaroxan induced an enhancement of NE release of about 200% whereas acute local microinfusion of dexefaroxan induced an increase corresponding to 500% of the baseline. In addition, systemic and local retrodialysis of the α2-adrenoceptor agonist UK 14,304 induced a reduction of NE release of about 45% whereas acute microinfusion of UK 14,304 induced a decrease corresponding to 60% of the baseline. These results points to a fundamental reflection when it is question about physiological interpretation of results obtained with pharmacological manipulation commonly used in behavioral studies. 99. Gennady Smagin PsychoGenics INC, USA L-KYNURENINE (KYN) METABOLISM INVESTIGATED USING IN VIVO MICRODIALYSIS COUPLED WITH A NOVEL HIGH CONTENT LC/MS/MS METHOD Degradation of tryptophan along the kynurenine pathway (KP) yields many neuroactive intermediates. The limited ability of KYN-derived metabolites to cross the BBB suggests that their CNS concentration is largely regulated by enzyme activity. KYN is actively transported into the brain but its fate is poorly understood and was investigated using a labeled analogue [13C6]L-KYN and in vivo microdialysis. A sensitive LC/MS/MS method was utilized to provide a time course for labeled metabolite formation. Mice and rats were implanted with guide cannula placed into the striatum and a microdialysis probe was inserted. Microdialysis samples were collected for 6 hrs after i.p. administration of 5 mg/kg of [13C6]L-KYN and analyzed for labeled and unlabeled L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA) and quinolinic acid (QUIN), using LC/MS/MS method. In mice, [13C6]L-KYN metabolism showed a stronger kynurenine 3-monooxygenase (KMO) branch response with increased incorporation of 13C label in 3-HK, 3-HAA, and QUIN. [13C6]L- KYN metabolism in rats progressed predominantly down the kynurenine aminotransferase (KAT) pathway. The results confirm the utility of LC/MS/MS analysis of stable isotope labeled KP metabolite from in vivo microdialysis samples. Further studies of brain-penetrable modulators of KP may be beneficial for the treatment of these diseases. 100. Jacqueline Keighron, Amy H. Newman, Gianluigi Tanda Medication Development Program, NIDA-IRP, NIH/DHHS, USA MONITORING EXTRACELLULAR DOPAMINE IN THE ACCUMBENS WITH MICRODIALYSIS AND FAST SCAN CYCLIC VOLTAMMETRY (FSCV) AFTER INHIBITION OF UPTAKE The dopamine transporter (DAT) is the main pharmacologic target involved in the psychostimulant actions of cocaine that can lead to abuse. However, the atypical uptake inhibitors, despite binding with relatively high affinity and selectivity to the DAT, do not produce cocaine-like behavioral/reinforcing effects. Here we show the effects of cocaine-like (cocaine and methylphenidate) and atypical uptake inhibitors (JHW 007 and R-Modafinil) on extracellular dopamine monitored by microdialysis or FSCV in the nucleus accumbens shell of Swiss-Webster mice. All drugs significantly increased extracellular dopamine with different potencies (rank order: Met> JHW > Coc >MOD), efficacies (Met> Coc > JHW >MOD), onset (Coc=Met>MOD>JHW) and offset (JHW>MOD>Coc>Met) of effects measured by microdialysis in freely moving mice. Using FSCV in anesthetized mice the drugs affect extracellular dopamine by increasing DAMax (maximum DA concentration detected) and dopamine clearance after stimulation of the medial forebrain bundle. Based on known DAT binding affinities, there is an apparent trend with dopamine clearance, but DAMax data suggests a more complex mechanism of action than simple DAT blockade for some drugs tested. Together these data provide insight into the underlying mechanisms involved in cocaine abuse and its possible treatment. Funded by NIH/DHHS 101. Meha Fatima University of Karachi, Pakistan RESTORATION OF INSULIN SIGNALING IN BRAIN CELLS AND INHIBITION OF MEMORY LOSS IN ALZHEIMER'S DISEASE BY A NOVEL SCHIFF BASE OF ISATIN Dementia is one of the hallmarks of Alzheimer's disease characterized by formation of amyloid plaques and neurofibril ary tangles (NFTs) that contain hyperphosphorylated tau. Alzheimer's disease is one of the complications of diabetes. Glycation frequently occurs in diabetes and glycated proteins have been found in plaques and NFTs. Insulin signaling regulates phosphorylation of tau. Disruption of insulin signaling as occurs in diabetes leads to hyperphosphorylation of tau which results in formation of NFTs and consequently neurodegeneration. Current therapy for Alzheimer's disease target neurotransmitter levels but does not provide neuroprotection. Isatins are endogenously produced metabolites of tribulin that serve as monoamine oxidase (MAO) inhibitor. Schiff base of Isatin has previously shown anticonvulsant activity. In this study, we have shown that a synthetic Schiff base of Isatin URM-II-81 possess potent antiglycation activity. We have defined the mechanism of action of URM-II-81 in inhibition of advanced Glycation End Products (AGEs) and restoration of insulin signaling in brain. URM-II-81 was able to restore insulin signaling and prevent axonal damage in Neuro2A cells. Also, URM-II-81 inhibited memory loss due to NFTs. Our findings suggest that URM-II-81 can develop as a new drug target for treatment of Alzheimer's disease. 102. Jane Cooper, S.M. Sossick, C. Kerridge, T.K. Murray, S.Bose, M.J. O'Neill Eli Lilly & Company, UK INVESTIGATING THE CONSEQUENCES OF TAU PROPAGATION ON TAU, GABA & GLUTAMATE; CLASSICAL AND PUSH-PULL MICRODIALYSIS IN P301S TRANSGENIC MICE We have previously described a novel in vivo model of tau propagation using human P301S tau transgenic mice infused unilaterally with brain extract containing tau aggregates. Infusion-related tau pathology was induced rapidly and robustly, and increased in a stereotypic manner; contralateral and anterior / posterior spread of tau pathology was also evident and was dependent on structural connectivity, not spatial proximity. We have since used the rapid and robust propagation of tau pathology in this model to test the efficacy of tau-based therapies and investigate the mechanisms of tau spread. Monitoring changes in proteins implicated in neurodegenerative diseases could play a key role in elucidating processes involved in neurodegeneration and facilitate the development of novel therapeutics. Microdialysis experiments were performed in the same P301S transgenic mouse model using classical and push-pull microdialysis to evaluate any functional consequences of tau propagation in the hippocampus of the mice; dialysates were analysed by LC-MS/MS or ELISA to determine basal and evoked release of GABA, glutamate and total tau 4 and 8 weeks following the induction of tau propagation. The application of microdialysis may provide information regarding the nature of tau release and mechanisms of spread in Alzheimer's disease and other tauopathies. 103. Alberto Brugnoli University of Ferrara, Italy BLOCKADE OF STRIATAL M1 AND M4 MUSCARINIC RECEPTORS ATTENUATES LEVODOPA-INDUCED DYSKINESIA ALONG WITH ITS NEUROCHEMICAL MARKERS It was previously reported that elevated cholinergic transmission in striatum contributes to motor complications arising from long-term levodopa therapy of Parkinson's disease. Since both M1 receptors (M1R) and M4 receptors (M4R) regulate the activity of medium-sized GABAergic striatofugal neurons, we sought to investigate whether M1R and M4R blockade could impact levodopa-induced In vivo microdialysis was performed in 6-OHDA-hemilesioned, levodopa-primed dyskinetic rats. The preferential M1R and M4R antagonists, telenzepine and PD-102807, respectively, were perfused through a probe implanted in the dopamine-depleted striatum at concentrations expected to generate extracellular levels just above affinity values for M1R and M4R, while levodopa was administered systemically. Glutamate and GABA release was monitored in the dopamine-depleted striatum and ipsilateral substantia nigra reticulata, and dyskinetic movements scored Both telenzepine and PD-102807 attenuated LID expression (by 40% and 60%, respectively) and prevented the associated rise of nigral GABA and striatal glutamate levels. PD-102807 also prevented the LID-associated increase of nigral glutamate. We conclude that endogenous acetylcholine contributes to the LID-associated overactivity of striato-nigral GABA neurons (i.e. the direct pathway) via striatal M1R and M4R. These data suggest that M1R and M4R antagonists may represent a novel therapeutic approach to LID. 104. Sarah Threlfell University of Oxford, UK DAT INHIBITORS REVERSE DA TRANSMISSION DEFICITS IN DORSAL STRIATUM OF ALPHA-SYNUCLEIN-OVEREXPRESSING PD MOUSE MODEL BAC-transgenic mice that overexpress human alpha-synuclein under the control of the whole wild-type SNCA genomic locus on a mouse alpha-synuclein-null (Snca-/-) background were generated (SNCA-OVX) to model Parkinson's-associated SNCA gene multiplications in humans. Evoked DA release in dorsal striatum in acute slices is 30% less in SNCA-OVX mice compared to Snca-/- littermate controls. Moreover, this deficit is region-specific: it is most marked in dorsal striatum, a region in humans particularly vulnerable to PD, and less pronounced in ventral striatal regions. Deficits in DA release are present throughout the SNCA-OVX lifespan (3-22 months). Deficits in evoked extracellular DA ([DA]o) were not attributable to any deficiency in DA content or any detectable increase in rate of DA uptake. However, inhibitors of DA uptake, cocaine and GBR 12935, increased [DA]o to a greater extent in dorsal striatum of SNCA-OVX mice than Snca-/- controls. This was not due to changes in DA transporter (DAT) levels, or to enhanced roles for serotonin or norepinephrine transporters. DAT inhibitors eliminate deficits in DA release in dorsal striatum between SNCA-OVX and Snca-/- mice, and could suggest vesicle mobilization deficits in this model of PD that are rescued by the reported mobilizing effects of DAT inhibitors. 105. Michelle Doran, N.J. Finnerty, J.P. Lowry Maynooth University, Department of Chemistry, Ireland THE ROLE OF OXIDATIVE STRESS MANIFESTATION IN PARKINSON'S DISEASE Parkinson's disease (PD) produces a range of symptoms, including tremor, rigidity, slowness of movement and problems with balance and coordination. Patients may also have memory problems, depression, and sleep complaints. Animal models have been extensively used to study neuronal and behavioural alterations caused by PD. The administration of reserpine to rodents has been suggested as a pharmacological model of PD based on the effects of this monoamine-depleting agent on motor activity. Reserpine interferes with the storage of monoamines in intracellular vesicles causing monoamine depletion in nerve terminals and transient hypolocomotion and muscular rigidity depending on the dose. It works by inhibiting the vesicular monoamine transporter 2 (VMAT-2). The blockage of dopamine vesicular uptake results in the accumulation of neurotoxic dopamine oxidation byproducts which potentially increases brain oxidative stress leading to neuronal damage. In order to understand this relationship, and to determine the effects of reserpine on the sleep –wake cycle, markers of oxidative stress (e.g. NO, H2O2 and O2) where monitored in real time using implanted sensors in freely-moving animals. Supported by the Irish Research Council (Project ID RS/2012/152) 106. Eyassu Chernet, J.S. Katner, S. Wu, T.A. Day, F.D. Tingley, X. Chai, M. Pogue, M. Hayashi, Eli Lilly & Company, USA ELISA QUANTITATION OF FULL-LENGTH, N-TERMINAL AND C-TERMINAL TAU LEVELS IN MALE VS FEMALE TRANSGENIC 4510 MICE EXTRACELLULAR, CEREBROSPINAL FLUID & BRAIN TISSUE SAMPLES COLLECTED BY IN VIVO MICRODIALYSIS Neurofibrillary tangles, composed of hyperphosphorylated aggregated tau, are a pathological hallmark of Alzheimer's disease (AD). Differentiating the disease stages of AD from controls may be dependent on the subset of extracellular fluid (ECF) & cerebrospinal fluid (CSF) Tau species measured. N-terminal Tau fragments may be a more sensitive AD biomarker than full-length Tau. To answer these questions, microdialysis probes were placed in the hippocampus of male and female (Tg) 4510 mice overexpressing Tau and ECF was collected. CSF was collected from these same mice followed by brain tissue collection. Full-length, C-terminal & N-terminal Tau in the ECF, CSF, hippocampus and frontal cortex of female Tg mice will be compared to similar age group of male Tg mice by ELISA. We will present results of these ongoing studies, evaluating differences in tau levels in the different compartments of male and female Tg mice. 107. Kate L. Parent University of Arizona, USA REAL-TIME MEASUREMENT OF KETAMINE-INDUCED TONIC DOPAMINE FLUCTUATIONS IN FREELY MOVING RATS Dopamine fluctuations occur on two timescales; rapid, sub-second (phasic) firing and slower, minute-to-minute (tonic) changes. Fast-scan cyclic voltammetry (FSCV) has long been used to study phasic dopamine transmission. However, this technique requires background subtraction. Thus baseline levels, and with them tonic changes, are inaccessible. Fast-scan controlled-adsorption voltammetry (FSCAV), a modification of FSCV, provides access to these tonic concentrations with sub-minute temporal resolution. Sub-anesthetic ketamine has recently been investigated as a therapeutic for depression and to reduce L-DOPA-induced dyskinesias resultant from long-term Parkinson's disease (PD) treatment. Here, we use FSCAV to monitor fluctuations in tonic dopamine at chronically implanted carbon-fiber microelectrodes in the dorsolateral striatum (terminal region of substantia nigra dopaminergic neurons highly degraded in PD) of freely moving rats at thirty-second intervals. Measurements were made across several weeks and in response to ketamine injection. Two three-hour no-injection control sessions were recorded and stable tonic levels were observed ([DA]tonic = 250 ± 20 nM ). In separate sessions, either vehicle or sub-anesthetic ketamine (20 mg/kg) was administered intraperitoneal after one hour of baseline measurement. Preliminary data suggests saline administration does not affect tonic dopamine while ketamine injection elicits a rapid (timescale) reduction in concentration which recovers within 30 min. 108. Maria Rosanna Bronzuoli Sapienza University of Rome, Italy EFFECT OF PALMYTOILETHANOLAMIDE IN A TRIPLE TRANSGENIC MODEL OF ALZHEIMER'S DISEASE Alzheimer's disease (AD) is a neurodegenerative disorder whose main features are β-amyloid (Aβ) plaques and neurofibrillary tangles, both responsible for neuronal loss and synapses reduction. Recently, the focus of research shifted from a neurocentric point of view to consider the role of non-neuronal cells in promoting disease progression; this is because a continuous astrocyte activation named "reactive gliosis" is seen in AD brain. Many models are developed to reproduce at best the hallmarks of AD and one of these is the 3xTg-AD. Aim of the study was to investigate any differences in the expression of the principal astrocyte activation markers (GFAP and S100β) in 3xTg-AD mice, in comparison with Non-Tg ones, and later evaluate the action of palmitoylethanolamide (PEA). PEA is an endogenous lipidic compound with well known anti-inflammatory properties, mainly produced by glial cells in the central nervous system. We found an increased 3xTg-AD-astrocytes activation compared to Non-Tg, imputable to an increased GFAP expression. PEA was able to control reactive gliosis, bringing cell's functionality to a physiological state. These results strongly suggest that PEA could be a promising tool in the therapeutic strategy against AD. 109. Evgeniya Efimova1,2, Damiana Leo3, Ilya Sukhanov3,4, Placido Illiano3, Liudmila Mus3,4, Stefano Espinoza4, Tatyana D. Sotnikova1,2,3, Marius C. Hoener5 and Raul R. Gainetdinov1,2,3 1 Institute of Translational Biomedicine, St. Petersburg State University, Russia 2 Skolkovo Institute of Science and Technology, Russia 3 Fondazione Istituto Italiano di Tecnologia, Neuroscience and Brain Technologies Department, Italy. 4 Department of Psychopharmacology, Institute of Pharmacology, Pavlov Medical University, Russia 5 Neuroscience Research, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Switzerland NEUROCHEMICAL CHARACTERIZATION OF DOPAMINE TRANSPORTER DEFICIENT RATS A variety of neuropsychiatric such as schizophrenia, Parkinson's disease, substance abuse and attention deficit hyperactivity disorder are widely accepted to have a basis in a dysfunction of the dopaminergic systems. The major function of Dopamine Transporter (DAT) is the control of dopamine (DA) dynamics by rapid uptake of neurotransmitter into presynaptic nerve terminals, making it an important regulatory element of both the synaptic action of DA and the intracellular stores of DA. We present a newly developed strain of rats (DAT-KO rats) in which the gene encoding the DAT has been disrupted by using Zinc Finger Nuclease technology. In our studies we showed that DAT-KO rats have pronounced changes in striatal DA system: DAT-KO rats have down to 20% of DA WT level, however extracellular level of DA is 7 fold higher, that could be explained by much longer (over 60 seconds) DA clearance time. DAT-KO rats demonstrate hyperlocomotion that can be normalized by amphetamine. However injection of amphetamine, as well as cocaine had no effect on extracellular level of DA in striatum. Pronounced changes in DA system of DAT-KO rats make them a novel valuable model for human diseases involving aberrant DA function and/or mutations affecting the DAT or DAT-related regulatory mechanisms. 110. Dimitri Brinet, Jörg Hanrieder Department of Psychiatry and Neurochemistry, University of Gothenburg, Sweden INSIGHT INTO Aβ OLIGOMERS IN ALZHEIMER'S DISEASE, TOWARDS NOVEL BIOMARKERS Alzheimer's disease is the most prevalent neurodegenerative disorder. The exact mechanisms underlying AD pathogenesis are still not fully understood, significantly hampering diagnostic strategies and the development of therapeutic treatment. AD is a progressive disorder characterized in part by amyloid deposition. This pathological hallmark is mostly composed of amyloid β-peptide (Aβ). Aβ is produced by a sequential cleavage of the amyloid protein precursor. Then Aβ can undergo a β-sheet transition and through self-association form higher-order assemblies. This dynamic process involves formation of growing species such as soluble oligomers, protofibrils or fibril. Evidence has emerged that soluble, oligomeric forms of Aβ have potent neurotoxicity. This deleterious phenomenon hasn't yet been elucidated, partially due to challenges associated with the monitoring of these metastable species. Here we have developed a method based on capillary electrophoresis and MALDI, with the aim to separate, identify, characterize and measure the different oligomer species, using both synthetic peptides and human samples. Development of techniques for monitoring oligomers is necessary to understand the self-association of Aβ and identify new biomarker. Thus, it might provide potential molecular targets for therapeutic intervention as well as molecular markers for diagnosis, prognosis and to follow treatment efficiency. 111. Giesbert R. Alken1, Torun Malmlöf2, Kristin Feltmann2, Åsa Konradsson-Geuken2, Frank Schneider1,Björn Schilström2, Torgny H. Svensson2 1 BDD GmbH, Fontanestr, Germany. 2 Karolinska Institute, Dept. of Physiology and Pharmacology, Section of Neuropsychopharmacology, Sweden LESS DYSKINESIA AT MOTOR-EQUIVALENT DOSES OF TRIPLE-DEUTERATED L-DOPA vs. L-DOPA AFTER CHRONIC ADMINISTRATION IN RATS Treatment with L-DOPA may via toxic metabolites contribute to motor complications, particularly dyskinesias, in Parkinson´s disease (PD). Our data show that replacement of hydrogen by deuterium in the α-,β-,β-positions (D3-L-DOPA) reduces the catabolism of dopamine (DA) via MAO and DA-β-hydroxylase, avoiding potential excess of e.g. toxic DOPAL levels. D3-L-DOPA exhibits similar plasma concentration kinetics in rats as L-DOPA but significantly elevated and sustained striatal DA concentrations, a decreased DOPAC/DA ratio and a reduced norepinephrine response compared to L-DOPA. In rats with unilateral striatal 6-OH-DA lesions and pretreated with a peripheral dopa-decarboxylase inhibitor the dose-response curve for the motor stimulation (during 3h) by D3-L-DOPA vs. L-DOPA was shifted to the left and the equipotent dose of D3-L-DOPA vs. L-DOPA (EC50) corresponded to ≈ 60% of a given L-DOPA dose. Moreover, D3-L-DOPA 5mg/kg caused significantly less (p<0.05) dyskinesias than L-DOPA 8mg/kg (the motor equivalent dose) during chronic treatment (3 weeks), assessed by the median cumulative AIMS scores using the cylinder test. In conclusion, D-3-L-DOPA allows vs. L-DOPA a significant dose reduction without loss of motor efficacy and reduced dyskinesias, i.e. offers a wider therapeutic window, and may represent the first drug which hence appears superior to L-DOPA for the treatment of PD. 112. N. Malik1, M. Conway1, G. Carter1, J. Huxter1, M. Ligocki1, K.L. Baker2, J.P. Lowry2, S.N. Mitchell1 1 In Vivo Pharmacology, Eli Lilly and Company Ltd., Erl Wood Manor, Windlesham, UK 2 Department of Chemistry, National University of Ireland, Ireland SIMULTANEOUS MEASUREMENT OF ACETYLCHOLINE RELEASE IN THE mPFC OF FREELY MOVING RATS USING AMPEROMETRIC SENSORS AND MICRODIALYSIS Memory decline is one of the main symptoms in the initial stages of AD, and one of the major recognisable neurochemical deficits is central cholinergic hypofunction, suggesting that measurement of brain levels of acetylcholine (ACh) and/or its precursor and metabolite, choline, may be useful for predicting efficacy of therapeutic compounds. Biosensors for the amperometric measurement of extracellular choline allow monitoring at a sub-second resolution compared to the multiple-minute resolution seen with microdialysis techniques, and therefore have yielded new insights in the regulation and function of ACh release in freely moving and task-performing animals. In these studies, a first generation Pt-based enzyme composite biosensor was used in order to measure real time increases in choline resulting from hydrolysis of newly released ACh by endogenous acetylcholinesterase (AChE). In the present study, we validated the choline biosensor by simultaneously measuring the levels of ACh and choline using microdialysis in the rat medial prefrontal cortex, and by assessing the impact of two compounds known to increase ACh efflux: donepezil and a thyrotropin releasing hormone (TRH) agonist. Locomotor activity and sleep were also measured in to assess the impact of these behavioural and physiological measures on choline and acetylcholine. 113. Dan P. Covey University of Maryland, USA CANNABINOID TYPE 1 RECEPTORS FACILITATE CONDITIONED REINFORCEMENT EVOKED OPTOGENETIC STIMULATION OF DOPAMINE RELEASE Cannabinoid type 1 receptors (CB1Rs) potently regulate conditioned reinforcement, such that increasing or decreasing CB1R signaling potentiates or curtails cue-directed reward seeking, respectively. This is thought to arise from CB1-mediated regulation of dopamine projections to the nucleus accumbens (NAc), but a direct and causal link has not been determined. In the current study, we used fast-scan cyclic voltammetry in the mouse NAc and observed consistent dopamine release time-locked to optogenetic self-stimulation of midbrain dopamine neurons. When a predictive cue was introduced to signal reward availability, latency to lever press decreased over consecutive trials while cue-elicited NAc dopamine release increased. Both measures were facilitated by the indirect CB1 receptor agonist JZL-184. Thus, dopamine neuronal firing sufficiently reinforces NAc dopamine release accompanying reward prediction and this is positively modulated by CB1Rs. Next, we confirmed that blocking CB1Rs with AM-251 inhibited cue-evoked NAc dopamine release and reward seeking in a food-reinforced task. Deficits were reversed by pairing cue presentation with optical stimulation of dopamine neurons, suggesting that decreased cue-directed reward seeking arise from suppressed CB1-dependent facilitation of cue-evoked dopamine release. Collectively, this work confirms and refines current understanding of how CB1R signaling regulates conditioned reinforcement. 114. Thomas Viereckel, Åsa Konradsson-Geuken, Åsa Wallen-Mackenzie Uppsala University, Sweden A NOVEL APPROACH FOR MEASURING LIGHT-EVOKED NEUROTRANSMITTER RELEASE Electrochemical measurements with amperometry allow in vivo quantification of specific neurotransmitters with an outstanding temporal- and spatial resolution. Most studies used today utilize electrical- or pharmacological stimulation with exogenous neurotransmitters to evoke neurotransmitter release. These approaches, however, lack spatial selectivity. The aim of this study was to utilize optogenetic activation of restricted cell populations in transgenic animals to measure glutamate release with in vivo amperometry. Mice expressing Cre recombinase in Pitx2-positive cells in the subthalamic nucleus as well as mice with Cre expression in the Trpv1-positive cells of the ventral tegmental area were injected with Cre-dependent adeno-associated viruses carrying Channelrhodopsin 2. Glutamate release was measured in both these Cre-systems at +0.7 V holding potential with Glutamate Oxidase (GluOx)-coated multi-channel electrodes. Possible laser artifacts were identified with sentinel channels lacking GluOx and the use of a holding potential of +0.25 V at which no glutamate activity is present. Enzyme activity was tested through local exogenous glutamate application. In summary, by combining optogenetics with amperometry in the anesthetized mouse, we have established a new protocol for the analysis of glutamate release from spatially restricted cell populations. 115. Brian O'Neill, Kayla A. Siletti, Margaret E. Rice Department of Neurosurgery, NYU School of Medicine, USA D2 RECEPTORS ON STRIATAL CHOLINERGIC INTERNEURONS HAVE LITTLE INFLUENCE ON DOPAMINE RELEASE REGULATION Previous studies suggest that dopamine-induced inhibition of dopamine (DA) release is mediated entirely by D2 autoreceptors on striatal DA axons. However, D2 receptors are also on cholinergic interneurons (ChIs), which play a critical role in facilitating DA release during local electrical stimulation, via nicotinic acetylcholine receptor (nAChR) activation. We tested whether there is a role for D2 receptors on ChIs in DA release autoinhibition in a new way, namely by using optogenetics for specific activation of DA axons in striatal slices and comparing this release (detected by fast-scan cyclic voltammetry) to that of electrical stimulation in the same preparation with a D2 antagonist, but in the presence or absence of nAChR blockade. These studies support the conclusion that D2 receptors on ChIs play a minimal role in local DA release regulation. Finally, data from a conditional choline acetyltransferase knock-out mouse also suggests a marginal role for D2 receptors on ChIs – similar to our pharmacological data. These studies suggest that D2 receptors on ChIs, although known to negatively regulate release of acetylcholine, must be positioned to be regulated only by supraphysiological levels of DA – or positioned to control release of acetylcholine from sites that do not in turn regulate DA release. 116. Yolanda Mateo, Brady Atwood, Joe Cheer and David Lovinger Laboratory for Integrative Neuroscience, NIAAA/NIH. USA CORTICAL AFFERENTS EXPRESSING CB1 RECEPTORS CONTROL PHASIC DOPAMINE RELEASE CAUSED BY CHOLINERGIC ACTIVATION Although dopamine release is strongly linked to firing of dopamine neurons, recent developments have revealed the importance of local presynaptic control at dopaminergic terminals within the striatum with the interplay between glutamatergic and cholinergic systems drawing most attention. The endocannabinoid system elicits also an important role regulating dopamine release in the nucleus accumbens independently of actions at the cell body. We observe that endocannabinoid signaling at CB1 receptors profoundly decreases subsecond dopamine release driven by optogenetic activation of cholinergic interneurons. We further report that selective optogenetic activation of excitatory prefrontocortical projections to the nucleus accumbens directly depolarize dopamine terminals via AMPA receptors expressed on nucleus accumbens dopamine terminals and that this response is robustly inhibited by a CB1 receptor agonist. These results demonstrate the plastic nature of endocannabinoid actions within different nodes of a common network and provide another crucial regulatory target for mesolimbic dopamine release of relevance to reward seeking. 117. Erik Naylor, Seth Gabbert, Daniel V. Aillon, Donna A. Johnson and David A. Johnson Pinnacle Technology, Inc., USA AN IN VIVO SYSTEM FOR OPTICAL STIMULATION WITH SIMULTANEOUS NEURONAL AND BIOSENSOR RECORDINGS Optogenetics is a broadly applicable research tool. To focus on the experiment and not system design, researchers require standard techniques and turn-key systems. Pinnacle's system includes LED fiber probes, precise timing, TTL inputs and outputs, various recording headstages (sleep, seizure, biosensors, etc.) and synchronized video. No optical commutator is required. In validation experiments, mice (hippocampus) were stimulated for 20s with blue (445 nm) or deep red (660 nm) light at 20 Hz, 10% duty cycle, (intensity > 100 mW/mm2) while measuring EEG via a depth electrode and 2 cortical electrodes. Control mice (C57BL6/J) showed no response to optical stimulation including no Becquerel effect. In response to blue light stimulation, transgenic mice (Thy1-COP4/EYFP)9Gfng - Jackson Laboratory) expressing channelrhodopsin had EEG frequency changes in the hippocampus corresponding to the timing of the stimulation pulse. In 70% of the trials, the response propagated to both hemispheres resulting in large-amplitude cortical seizure-like activity lasting 20-30s beyond end of stimulation. Glutamate in the frontal cortex consistently declined (-0.17 +/- 0.02 uM) during periods of blue light stimulation. As an additional control, stimulation at 660 nm (deep red) did not result in any measurable response. This research was supported by NIH grant #R44AG046030. 118. Mai Hoang1, Nhu Phan2, Per Malmberg1, Andrew Ewing1,2 1Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Sweden; 2Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden

Cocaine is a popular drug of abuse that has influences on the central nervous system, body temperature, the cardiovascular system, the sympathetic nervous system, and nerve conduction. Cocaine binds to transporters of dopamine and serotonin resulting in the inhibition of the re-uptake of these monoamines into presynaptic cells. Some studies have assessed the effect of cocaine on brain lipid metabolism in humans but the specific lipid species altered following the drug exposure are unclear. The lipids are involved in numerous functions, including recognition and processing of lipid head groups and side chains, membrane fusion during exo- and endocytosis. We focus on lipids in this work because of their participation in synaptic transmission and plasticity thus possibly forming short- and long-term memories. In order to investigate how cocaine exposure effects lipid distribution and structures in the nervous system, Drosophila melanogaster (fruit fly) is an ideal model owing to short life cycle, high fecundity, and simple genetic system (4 pairs of chromosomes). In this project, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to directly map the distribution of lipids and lipid-related, such as diacylglycerides, phosphatidylcholine, phosphatidylethanolamine, fatty acids and phosphatidylinositol, compounds in the fly brain. Two instrumental approaches have been used to image the chemical localization of lipids in the fly brain: the TOF-SIMS V equipped with a 25keV Bismuth liquid metal ion gun (LMIG) and the J105 ToF-SIMS equipped with a 40keV Ar gas cluster ion beam (GCIB). The LMIG has advantage in high lateral resolution allowed for lipid detection at the nanoscale, whereas higher mass species can be imaged with the GCIB as a primary ion source. Data analysis using principle component analysis shows changes in the lipid distribution and abundance in fly brain after cocaine use. The exposure mainly effected diacylglycerides, phosphatidylcholines, and fatty acids. Lipid structures in the brain are altered in the cocaine-treated flies suggesting that cocaine can induce alterations in brain function. Recent studies indicate that addictive drugs, such as cocaine, morphine, ethanol, etc induce long-term synaptic plasticity in the reward neurons of the brain consistent with our data.



Available online at Biochemistry & Physiology Pesticide Biochemistry and Physiology 91 (2008) 90–95 Warfarin resistance in Rattus losea in Guangdong Province, China Jianshe Wang a, Zhiyong Feng b, Dandan Yao b, Jingjing Sui b, Wenqin Zhong a, Ming Li a, Jiayin Dai a,* a Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101, PR China

Involvement of the dorsolateral prefrontal cortex and superior temporal sulcus in impaired social perception in schizophrenia

Contents lists available at Progress in Neuro-Psychopharmacology & Biological Involvement of the dorsolateral prefrontal cortex and superior temporalsulcus in impaired social perception in schizophrenia Jung Eun Shin ,, Soo-Hee Choi Hyeongrae Lee , Young Seok Shin , Dong-Pyo Jang Jae-Jin Kim ,a Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Koreab Institute of Behavioral Science in Medicine, Yonsei University College of Medicine, Seoul, Republic of Koreac Department of Psychiatry, Seoul National University College of Medicine and Institute of Human Behavioral Medicine, SNU-MRC, Seoul, Republic of Koread Magnetoencephalography center, Department of Neurosurgery, Seoul National University Hospital, Seoul, Republic of Koreae Department of Biomedical Engineering, Hanyang University, Seoul, Republic of Koreaf Department of Psychiatry, Yonsei University College of Medicine, Seoul, Republic of Korea