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Seredynski et al. • Acute Effects of Neuroestrogens on Behavior J. Neurosci., January 2, 2013 • 33(1):164 –174 • 167
The two compartments are separated by a vertically sliding door that can Table 1. Details of experiments 1–3 conducted on the first group of males
be remotely controlled by strings and pulleys. A narrow vertical slit (1cm wide ⫻ 15 cm high) is located in the middle of this door and serves as a"window" that provides the male with a limited visual access to the female. A square area (30 ⫻ 30 cm) located on the floor of the main compartment in front of this door represents the test zone for the bird's position. The male present in the main compartment can only see the The experiments numbers (1–3) correspond to the order in which they were performed.
female located in the side compartment if he stands in front of the win- aE –BSA was administered at a dose equivalent to 50 ␮g of free estradiol following information provided by the dow in the test area. A male learns to stand continuously in front of the window providing visual access to the female after he has had a chance tocopulate with her in the experimental setup. Only one copulation isnecessary to establish this response.
Each behavioral test lasted a total of 10 min. The male was first placed in the main compartment, and the stimulus female was introduced in theadjacent smaller compartment. The amount of time that the male spentin the square test area in front of the window (Square) as well as the timethat he actually spent looking through the window (Look) was recordedfor 5 min. Looking behavior was defined as a stereotyped positioning ofthe male's head such that he could focus on the female through thenarrow slit in the door. These data provided a measure of the appetitivebehavior. Then, the door separating the two compartments was raised,allowing the two birds to physically interact for another 5 min, duringwhich the frequency and latency of first occurrence of copulatory behav-iors were directly recorded (see below). The animals were then moved Figure 2.
Chronic treatment with the aromatase inhibitor VOR almost completely eliminates back to their home cage until the next day.
CSB (group 1, preliminary phase). After the last pretest (Pre), CX males chronically treated with In the present experiment, eight acquisition tests were performed on 8 T were injected twice daily with VOR (1 mg/kg, i.m.; white bars, CX ⫹ T ⫹ VOR, n ⫽ 12) or its successive days that allowed all males to acquire a stable conditioned vehicle (PG; controls, black bars, CX ⫹ T, n ⫽ 4). Birds were then repeatedly tested every third response. Birds were then tested in extinction (female not released during day for the expression of CSB. CCM frequencies gradually decreased to almost zero within 9 d.
the second period of 5 min) in two additional tests to ensure that, as Data were analyzed by two-way ANOVA with treatment as independent factor (F previously described, the response remains stable even in the absence of p ⫽ 0.308) and successive tests as repeated factor (F ⫽ 17.532, p ⬍ 0.001; interaction, reinforcement The ⫽ 3.758, p ⫽ 0.016). *p ⬍ 0.05 versus controls (CX ⫹ T); †p ⬍ 0.05 and ††p ⬍ 0.01 effect of different endocrine treatments was then tested as detailed in the versus Pre same treatment, by Tukey's post hoc test.
specific section devoted to group 3. Importantly, the acquisition of thissocial proximity response occurs (1) only in gonadally intact males orcastrates chronically treated with T (2) n ⫽ 4) for the entire duration of the experiment (25 d). To monitor the only if males have been able to copulate in this experimental setup behavioral decline induced by this prolonged deprivation in estrogens and (3) in response to female but not male copulatory behavior was first evaluated before the stimuli Additionally, (4) the learned response is first VOR injection. Starting on the second day after the beginning of the markedly reduced after lesions of the preoptic area twice daily injection regimen, birds were then subjected to three addi- Together, these observations indicate that this learned response tional tests for copulatory behavior until a significant behavioral decline constitutes another good measure of ASB and reflects the propensity of was detected In a last test, all birds then received a vehicle intra- the male to engage in copulation (i.e., a measure of his underlying sexual cerebroventricular injection and were tested in sequence for RCSM and copulatory behavior in the aquarium in which all experimental tests wereperformed subsequently. This pretest confirmed the behavioral inhibi- Consummatory sexual behavior tion affecting both RCSM and CCM observed previously. All behavioral Consummatory sexual behavior (CSB) was assessed after presentation of tests took place 2 h after the morning injection and were given 3 d apart.
a sexually mature female with which the male could freely interact during Experimental phase. VOR-treated birds were assigned to two experi- 5 min in three different test arenas: a small (60 ⫻ 40 ⫻ 50 cm; used during mental subgroups (n ⫽ 6 each) matched based on the mean behavioral all preliminary phases and for experimental tests in all groups) or large frequencies displayed during the last preliminary test. These birds were arena (90 ⫻ 90 ⫻ 50 cm; used for experimental tests in groups 2 and 3 involved in three experiments comparing the effect of the only) or the small aquarium used to quantify RCSM after removal of the control injection of the vehicle (PG) with either 50 ␮g of E (in 1 ␮l; glass partition between the two compartments immediately after the experiment 1), 100 ␮g of E (in 2 ␮l; experiment 2), or with its RCSM test (during the experimental phase in group 1). The frequency of membrane-impermeant analog E coupled with bovine serum albumin behavioral patterns that are part of the copulatory sequence, including (E –BSA; for more discussion on this compound, see below, Drugs; 50 neck grabs, mount attempts, mounts, and cloacal contact movements ␮g of E equivalent in 2 ␮l; experiment 3). Birds were tested every other (CCM) (for a detailed description of these behaviors, see day for 15 min after the intracerebroventricular injection of the experi- was recorded by an observer blind to the mental drug or the vehicle. As described in , treatments were treatments. Data relative to these four behavioral measures have been administered in a different order to the two subgroups, such that all birds analyzed and yielded the same statistical results and conclusions. As a served as their own control but half of them were first treated with the consequence, to avoid redundancy, only CCM results are shown here.
vehicle whereas the other half received the experimental injection.
Within the same experiment, animals were tested sequentially for ASB Specific experimental procedures
and CSB in the glass aquarium used for ASB quantification. As a conse- Effects of acute estrogen treatment in males chronically deprived of quence, the test lasted a total of 10 min: 2.5 min for RCSM in the absence estrogens (first group of birds) of the female, 2.5 min for RCSM in the presence of the female, and 5 min Preliminary phase. Two weeks after T implantation, 16 males were pre- for CCM. The control birds that were not treated with VOR (i.e., CX ⫹ T tested in small test chambers until they all displayed the full range of birds) were tested in parallel after a control intracerebroventricular in- copulatory behaviors. They were then injected twice daily in the pectoral jection of PG. These animals served as a reference point for fully active muscles with the potent aromatase inhibitor VOR (1 mg/kg; n ⫽ 12; birds black histogram bars) but were not included in the or its vehicle propylene glycol (PG)/saline (4:1; statistical analysis of the acute experiments because they were not tested 168 • J. Neurosci., January 2, 2013 • 33(1):164 –174
Seredynski et al. • Acute Effects of Neuroestrogens on Behavior Table 2. Details of experiments 4 –13 conducted on the second group of males
VOR and VOR ⫹ E VOR and VOR ⫹ E – biotin The experiment numbers follow the order in which the results are described, but the actual order in which the experiments were performed is provided in the second column. When E was administered along with VOR, the dose and timing of injection of both compounds are separated by the slash, with VOR being listed first.
Table 3. Details of experiments 14 –17 conducted on the third group of males
VOR versus VOR ⫹ E VOR versus VOR ⫹ E VOR versus VOR ⫹ E VOR versus VOR ⫹ E The experiment numbers follow the order in which the results are described, but the actual order in which the experiments were performed is provided in the second column. Because VOR and E were administered with different timings, the timing and doses of injections are both presented with VOR preceding E . LSPR, learned social proximity response.
with comparable experimental treatments. One bird lost its cannula be- and had thus to be excluded from the experiments, resulting in a tween the E and E –BSA tests and thus had to be excluded from exper- decrease in degrees of freedom in the ANOVAs.
Effects of blockade of local estrogen action or synthesis in sexually Effect of changes in brain-derived estrogens on the learned social active males (second group of birds) proximity response in sexually active males (third group of birds) Preliminary phase. Two weeks after T implantation, birds (n ⫽ 15) were Preliminary phase. Two weeks after T implantation, birds (n ⫽ 18) were offered several opportunities to copulate with a female to allow them to offered several opportunities to copulate with a female to acquire the full express the full repertoire of copulatory behavior and were tested once repertoire of copulatory behavior and tested once for RCSM to habituate for RCSM to habituate them to the test arena.
to the test arena. As described above (see Behavioral tests), they received Experimental phase. Based on the behavioral frequencies displayed eight acquisition tests given on 8 consecutive days until they had devel- during the preliminary phase, birds were assigned to one of three exper- oped a stable LSPR.
imental subgroups (n ⫽ 5 each) matched based on the mean RCSM and Experimental phase. The effects of the experimental treatments (VOR CCM frequency produced during the preliminary phase. A total of 10 exper- vs VOR ⫹ E2 vs vehicle) were tested during the extinction phase of the iments were performed, each consisting of three repeated experimental tests learned social proximity response (that is when the female is no longer (one vehicle vs two drugs) surrounded by two "baseline" tests (one pretest released from her cage in the second step of this test) to prevent any and one posttest) to assess the effects of the blockade of estrogen synthesis or feedback from the experimentally induced decrease in copulatory behav- action on the expression of ASB measured by RCSM and on CSB investi- ior on the learned response (experiment 14) On the day after gated in small or large arenas. The experiments compared the following the last acquisition test, all birds received a first test in extinction. Two drugs with the vehicle: two estrogen receptor antagonists [ICI 182,780 days later, they all received a vehicle intracerebroventricular injection 30 min before a second test, which constituted the reference test for the basal triene-3,17-diol) (ICI), 100 ␮g; and tamoxifen (TMX), 100 ␮g]; two aroma- response (pretest). Birds were then assigned to one of three experimental tase inhibitors [VOR, 50 ␮g; and 1,4,6-androstatrien-3,17-dione (ATD), 50 subgroups (n ⫽ 6 each) matched based on the time spent in the square ␮g]; and VOR (50 ␮g) with or without E (50 ␮g) or E –biotin (50 ␮g of E and time spent looking at the female in the second extinction test of the equivalent; a membrane-impermeant E analog; for more discussion on this LSPR test. Then, three subgroups were repeatedly tested with the three compound, see below, Drugs). The effect of these drugs was tested after experimental treatments administered intracerebroventricularly, each latencies of 15 or 30 min depending on the experiment. The details of test given 3 d apart. Three days after the last experimental test, all sub- these experiments and the order in which they were actually conducted groups were tested again after a vehicle intracerebroventricular injection are represented in As illustrated in , each experiment serving as the posttest. A total of six tests were thus performed during the started with a pretest during which all birds were tested after a control extinction phase; the experimental phase was conducted on the last five intracerebroventricular injection of PG. Then, the three subgroups (one pretest, three experimental tests, and one posttest). Two birds in one were submitted to three independent tests assessing the effect of three subgroup lost their cannula during this experiment and were thus re- treatments administered in different orders on ASB or CSB measured moved from the statistical analysis.
15 or 30 min after injection. All birds were thus used as their own To replicate some of the data obtained with group 2, nine animals were control, with the different subgroups receiving the same exact treat- selected randomly among this group of birds and were reassigned to ments but in different orders to avoid any possible effects in final three different subgroups (n ⫽ 3) based on their mean CCM and RCSM results of long-term consequences of the acute treatments. Each series frequencies. They were then tested for the effects of VOR, VOR ⫹ E , or of three repeated tests was followed by a posttest after another control their vehicle (PG) on copulatory behavior measured in the small (exper- intracerebroventricular injection. All tests were conducted 3 d apart iment 15) and large (experiment 16) test arena and on RCSM frequencies throughout the entire experiment. All compounds tested were diluted (experiment 17; Table 3). As was done previously, these experiments in 1 ␮l of PG with the exception of the highest dose of E that was consisted of three experimental tests given 3 d apart surrounded by one diluted in 2 ␮l and administered intracerebroventricularly. Note that pretest and one posttest given 3 d before and after the first and last three birds (one in each subgroup) lost their cannula during the study experimental test, respectively (see and details of group 2).

Seredynski et al. • Acute Effects of Neuroestrogens on Behavior J. Neurosci., January 2, 2013 • 33(1):164 –174 • 169
RCSM frequencies did not reach the samelevel as in control birds that were notchronically treated with VOR. A higherdose of E2 (100 ␮g) increased RCSM fre-quency to the same extent (F 16.264, p ⫽ 0.002; vehicle, 26.5 ⫾ 8.7; E2,53.7 ⫾ 9.7; experiment 2). In contrast, E2(50 or 100 ␮g) had little or no effect onCCM frequency (50 ␮g: , F 2.500, p ⫽ 0.144, experiment 1; 100 ␮g: nobehavior was displayed by any subject re-gardless of the treatment; experiment 2).
To determine whether this effect of E2 was Figure 3.
E rapidly facilitates appetitive (ASB) but not consummatory sexual behavior (CSB) in males chronically deprived mediated by its action at the membrane or males of estrogens by its action at the membrane level (group 1, experimental phase). CX males were chronically treated with T and inside the cells, we tested the effect of E2– the aromatase inhibitor VOR (CX ⫹ T ⫹ VOR, white bars) or with its vehicle (CX ⫹ T; black bars, n ⫽ 4). The estrogen-deprived BSA, which does not cross the cell mem- males (CX ⫹ T ⫹ VOR) were then acutely injected with E (50 ␮g, n ⫽ 12, A, C), the membrane-impermeant analog E –BSA (50
analog mirrored the effect of the free es- respectively. Data from CX ⫹ T males (black bars) are shown as reference of fully stimulated behavior but were not integrated inthe final analyses. **p trogen on RCSM frequencies ; ⬍ 0.01 versus vehicle by Fisher's LSD post hoc test after identification of a significant treatment effect (repeated-measure) by two-way ANOVA.
12.41, p ⫽ 0.006; experiment 3), indicating that this effect is initiated at thecell membrane. In contrast, no copulatory behavior was displayed by any subject regardless of the treatment, VOR was graciously provided by Dr. R. DeCoster (Janssen Research suggesting the lack of a membrane-initiated effect on copulatory Foundation, Beerse, Belgium). PG, 17␤-E , E –BSA (membrane- impermeant analog of E ), ICI (Fulvestran), TMX, and T were obtained from Sigma. ATD and E – biotin (another membrane- No effect of the order of treatments (F ⱕ 0.659, p ⱖ 0.437) or impermeant analog of E ) were obtained from Steraloids.
interaction between the treatments and their order (F ⱕ 2.979, Two membrane-impermeant E analogs were used here. E –BSA is p ⱖ 0.118) was detected with the exception of an interaction that most commonly used in the literature, but there have been doubts re- was detected for E 2 at 50 ␮g ; F(1,10) 5.98, p ⫽ 0.034).
garding its stability and the purity of the commercially available product However, this interaction is associated with a more pronounced E – biotin was thus also used based on its recent use effect of E2 in the subgroup that received E2 in the first test com- for a similar purpose pared with the second test, suggesting that it cannot be attributed and the demonstration of a somewhat higher stability to a long-term treatment effect. Overall, this absence of treatment effects and interaction between treatments and orders suggeststhat there is no carryover effect indicative of long-term effects of In each of the 17 separate experiments, results obtained in pretests and Together, these data clearly indicate that a single injection of posttests were compared with each other with paired-samples t tests.
Data comparing the effects of experimental treatments in each of the 17 E2 or E2–BSA can acutely restore the appetitive component of independent experiments were analyzed by two-way ANOVAs with male sexual behavior in estrogen-deprived males. This suggests treatments as the repeated factor and their order (two or three sub- that the neural circuits underlying this aspect of the behavior are groups) as the independent factor to exclude the possibility of long-term significantly modulated by nongenomic actions of estrogens. The effects of treatments that would interfere with the short-term effects fact that E2 did not restore RCSM frequencies to the level dis- under investigation. When significant, these ANOVAs were followed by played by control animals suggests that some genomic priming post hoc Fisher's least significant difference (LSD) tests, Newman–Keuls by estrogens might additionally be necessary or alternatively that tests, or Tukey's honestly significant difference tests (as appropriate de- the acute treatment provided here was suboptimal. In contrast, pending on the number of comparisons) comparing all conditions to these data provide no information on whether estrogens acutely influence copulatory behavior. Males chronically depleted of es- All statistical analyses were performed with Statistica version 9 (StatSoft). Differences were considered significant for p ⬍ 0.05. All data trogens showed no or very little copulatory behavior. Neural cir- are expressed as mean ⫾ SEM.
cuits controlling this behavioral component may require a partialgenomic priming by estrogens before these steroids can exert any acute action. This idea was tested in a second group of experi- Effects of acute estrogen treatment in chronically estrogen-
ments by acutely blocking estrogen action or synthesis in males deprived males (group 1)
exposed to the androgenic and estrogenic metabolites of T.
To assess behavioral effects of a rapid increase in estrogen bio-availability, CX males were chronically treated with SILASTIC T Effects of blockade of local estrogen action or synthesis on
implants and received twice daily injections of the aromatase sexually active males (group 2)
inhibitor VOR. They were thus exposed to the full range of an- We next tested whether behavioral effects of endogenous estro- drogenic effects of T, whereas genomic effects of estrogens were gens produced by T aromatization in the brain would be rapidly dramatically reduced. As predicted this blocked by the estrogen receptor antagonists ICI and TMX. In treatment nearly eliminated copulation CX ⫹ T males displaying the full spectrum of male-typical sexual The acute intracerebroventricular injection of 17␤-E2 (50 ␮g) behaviors, both antagonists significantly reduced RCSM fre- to these animals resulted within 15 min in a doubling of RCSM quencies within 15 min ; F 23.69, p ⬍ 0.001; ex- frequency ; F 13.854, p ⫽ 0.004; experiment 1), yet periment 4). This inhibition was even more pronounced when

170 • J. Neurosci., January 2, 2013 • 33(1):164 –174
Seredynski et al. • Acute Effects of Neuroestrogens on Behavior Figure 4.
Effects of acute blockade of estrogen receptors on the ASB and CSB of fully active males (group 2). The blockade of estrogen action by the estrogen receptor antagonists ICI (50
␮g)andTMX(50␮g)acutelyinhibitsRCSMfrequency(A,B,n⫽15)butnotCCMfrequency(C,
D, n ⫽ 13 and 15, respectively) within 15 or 30 min compared with vehicle (Veh) injection
Figure 5.
Effects of acute deprivation in brain-derived estrogens on the ASB and CSB of fully (white bars). The "Pre" and "Post" black bars provide reference behavior frequencies after active males (group 2). Blockade of local estrogen synthesis by the aromatase inhibitors ATD (50 vehicle intracerebroventricular injections performed before and after the acute treatments, but these data are not included in the statistical analyses. ***p ⬍ 0.001 versus vehicle; †p ⬍ 0.05 E (50 ␮g; C) or its membrane-impermeant analog E – biotin (E -BIO, 50 ␮g E equivalent; D)
versus ICI by Newman–Keuls post hoc tests after identification of a significant treatment effect injected 15 min after VOR counteracts its effect on RCSM. The "Pre" and "Post" black bars provide (repeated-measure) by two-way ANOVA.
reference behavior frequencies after vehicle intracerebroventricular injections performed be-fore and after the acute treatments, but these data are not included in the statistical analyses.
**p ⬍ 0.01 and ***p ⬍ 0.001 versus vehicle (Veh); †††p ⬍ 0.001 vs VOR by Newman–Keuls tests were performed 30 min after the intracerebroventricular post hoc tests after identification of a significant treatment effect (repeated measure, n ⫽ 12 in injection ; F 24.705, p ⬍ 0.001; experiment 5). In each case) by two-way ANOVA.
contrast, regardless of timing, these antagonists did not affect copu-latory behavior ,D; 15 min: F 1.122, p ⫽ 0.345, exper- iment 6; 30 min: F 0.101, p ⫽ 0.903, experiment 7). The acute iment 8), thus demonstrating that the suppression of estrogen blockade of estrogen action thus significantly affected a measure of production has the same effect as the blockade of its action and, ASB but not its consummatory aspect, just as did the acute stimula- notably, that the relevant estrogens are produced locally in the tion by E2 in the first group of experiments.
brain. However, CSB was still unchanged by aromatase inhibitors No order effect of the treatment was detected (F ⱕ 1.769, p ⱖ 0.161, p ⫽ 0.851; experiment 9).
0.212) except for RCSM tested after 30 min (F 11.02, p ⫽ No order effect (F ⱕ 0.438, p ⱖ 0.657) nor interaction was 0.010; baseline behavior was slightly different between sub- detected in any of these experiments (F ⱕ 1.146, p ⱖ 0.366). In groups). However, no interaction was detected in any of these addition, none of the comparisons of the pretests and posttests experiments (F ⱕ 1.695, p ⱖ 0.183). In addition, all comparisons was significant (t ⱕ 0.168, p ⱖ 0.869). These results again indicate of the pretests and posttests was not significant (t ⱕ 1.389, p ⱖ that these effects are not associated with long-term actions of 0.190). Thus, these behavioral responses do not seem to be af- fected by long-term effects of estrogens.
Importantly, the VOR-induced inhibition of ASB was almost Nongenomic effects of estrogens are potentially mediated by completely prevented by an E2 intracerebroventricular injection different membrane receptors, including nuclear estrogen recep- (50 or 100 ␮g) administered 15 min after VOR ; 50 ␮g: tors of the ␣ or ␤ subtype associated with the neuronal membrane 50.152, p ⬍ 0.001; experiment 10). Thus, the behavioral but also G-protein-coupled inhibition does not result from nonspecific adverse effects of the receptors, such as GPR30 aromatase inhibitor but specifically from the depletion of endog- and Gq-mER ICI and TMX block enous estrogens. This rescue of VOR treatment effects by E2 was some, but definitely not all, of these receptors. ASB and CSB mimicked by another membrane-impermeable estrogen analog could thus be acutely regulated by estrogens through different 2– biotin (50 ␮g; ; F(2,18) 31.01, p ⬍ 0.001; experiment receptors, thus explaining why these two aspects of behavior were 11), further confirming that rapid effects of E2 are initiated at the differentially affected by these antagonists. To test this possibility, membrane. Again, no order effect (F ⱕ 0.231, p ⱖ 0.797) was birds were tested again but this time 30 min after an acute block- detected in these experiments. No interaction was found after E2 ade of estrogen synthesis. A single injection of both the steroidal injection (F 2.686, p ⫽ 0.064), but a significant interaction (ATD) and nonsteroidal (VOR) aromatase inhibitors signifi- was detected with E 2– biotin (F(4,18) 4.093, p ⫽ 0.015). This cantly decreased ASB ; F 15.31, p ⬍ 0.001; exper- interaction results from a decreased stimulation by E2– biotin in

Seredynski et al. • Acute Effects of Neuroestrogens on Behavior J. Neurosci., January 2, 2013 • 33(1):164 –174 • 171
tion but not sexual performance. To assess this possibilityfurther, another group of CX ⫹ T males was used to test the acuteeffects of brain-derived estrogens on another measure of ASB, thelearned social proximity response. These animals were also usedto demonstrate the reproducibility of some of the results pre-sented previously.
As shown previously the animals pro- gressively learned to stand in front of the window (from ⬃60 sduring the first test to 250 s of 300 s at the plateau) and spent anincreasing amount of time looking at the female (from ⬃45 sduring the first test to 200 s at the plateau). Thus, there was asignificant change in these behaviors across the eight acquisition Figure 6.
Effects of acute blockade of estrogen action or synthesis on copulatory be- tests (repetition effect: Square, F 12.530, p ⬍ 0.0001; Look, havior of behaviorally active males tested in large test chambers (group 2). The acute 13.936, p ⬍ 0.0001). This response was then stable over blockade of estrogen action by general estrogen antagonists (A, n ⫽ 14) or synthesis by
time even when tested in extinction as revealed by the com- aromatase inhibitors (B, n ⫽ 12) profoundly inhibits CSB when measured in a large test
arena (90 ⫻ 90 ⫻ 50 cm) 30 min after injection compared with control vehicle (Veh)
parison of the time spent in the square and looking between injection. The "Pre" and "Post" black bars provide reference behavior frequencies after the last acquisition test and the first two extinction tests (rep- vehicle intracerebroventricular injections performed before and after the acute treat- etition effect: Square, F 2.213, p ⫽ 0.129; Look, F(2,26) ments, but these data are not included in the statistical analyses. **p ⬍ 0.01 and ***p ⬍ 2.328, p ⫽ 0.117).
0.001 versus vehicle (Veh) by Newman–Keuls post hoc tests after identification of a sig- The learned social proximity response, as assessed by the time nificant treatment effect (repeated measure) by two-way ANOVA.
spent near the window (square) and looking at the female, wasacutely reduced by aromatase inhibition, and this effect was com-pletely prevented by E 2 , B; Square, F(2,26) 4.867, p ⫽ the subgroup that received this treatment last (order in this sub- 0.016; Look, F 9.284, p ⬍ 0.001; experiment 14). No effect group PG/VOR/VOR ⫹ E2– biotin). This decreased reaction can- of the order of the treatments (Square, F 0.009, p ⫽ 0.991; not be ascribed to lasting effects of the previous injection of VOR 0.114, p ⫽ 0.893) or their interaction with these because a marked stimulation by E2– biotin was observed in the treatments (Square, F 0.094, p ⫽ 0.983; Look, F(4,26) subgroup exposed in sequence to VOR/VOR ⫹ E2– biotin/PG. In 0.284, p ⫽ 0.885) was noticed. In addition, the pretests and post- addition, none of the comparisons of the pretests and posttests tests did not differ either (t ⱕ 0.509, p ⱖ 0.618).
were found to be statistically significant (t ⱕ 0.219, p ⱖ 0.830).
The effects of these treatments (VOR and VOR ⫹ E2 com- Overall, these findings support the independence of these effects pared with PG) were then tested on the RCSM frequency, and from long-term actions of estrogens.
copulatory behavior was assessed in the small and large test One explanation for the striking discrepancy between rapid chambers. These experiments fully confirmed the effects ob- effects of estrogens on ASB and CSB could reside in the fact that served in the second set of animals in the current study: changes copulatory behavior was tested in a small arena in which motiva- in brain-derived estrogens induced parallel changes in RCSM tional decreases might not affect performance because both part- frequency ; F ⫽ 13.965, p ⬍ 0.001; experiment 15), as ners are in such close proximity that males reflexively perform the well as in sexual performance when measured in a large arena in copulatory sequence in the absence of sexual motivation; the which the female can easily escape the male ; F simple contact with the mate would trigger the performance of the motor copulatory sequence. To test this hypothesis, effects of 7.429, p ⫽ 0.008; experiment 17) but not in the small arena in estrogen receptor antagonists and aromatase inhibitors on CSB which both partners are in very close proximity ; F(2,12) were tested again but this time in a larger arena in which females 0.888, p ⫽ 0.437; experiment 16). Again, no order effect (F ⱕ could more easily escape so that males had to actively pursue 0.864, p ⱖ 0.468) nor interaction (F ⱕ 2.279, p ⱖ 0.120) was them to copulate successfully. In this new context, TMX, but not detected in any of these experiments, and no difference was found ICI, significantly inhibited copulation ; F between the behavioral frequencies displayed during the pretests p ⬍ 0.001; experiment 12). Furthermore, both aromatase inhib- and posttests (t ⱕ 0.577, p ⱖ 0.579), with the exception of the itors profoundly inhibited CSB ; F RCSM test (t ⫽ 6.271, p ⬍ 0.0002; overall minor decrease in all 10.943, p ⬍ 0.001; experiment 13). Thus, the acute blockade of estrogen ac- subgroups over the course of the experiment).
tion affects CSB in contexts in which males must be sexually Together, these results further demonstrate that acute changes motivated and actively chase the female to mate.
in local estrogen bioavailability do not alter the ability of male Again, the analyses did not reveal any order effect (F ⱕ2.367, quail to produce a highly coordinated sexual motor response.
p ⱖ 0.149) nor interaction between treatments and their order Indeed, the lack of effect in the small arena demonstrates that the (F ⱕ 1.317, p ⱖ 0.294). In addition, none of the comparisons absence of estrogens does not impair the ability of the male to between the pretests and posttests was significant (t ⱕ 1.000, p ⱖ mount and perform the highly stereotypical CCM. However, 0.338). Overall, this absence of order effects and of interaction copulatory behavior can only be observed after the male has between treatments and orders suggests that there is no carryover searched for and approached a female. When examined in the indicative of long-term effects of estrogens.
large arena, an absence of sexual performance thus indirectlyreflects a lack of motivation. That sexual performance is only Effect of changes in brain-derived estrogens on the learned
affected by estrogens in the large arena clearly suggests that brain- social proximity response in sexually active males (group 3)
derived estrogens rapidly affect sexual motivation but not perfor- The results described so far suggested that the nongenomic ac- mance as supported by the results obtained from two tions of estrogens might preferentially control the sexual motiva- independent measures of ASB.

172 • J. Neurosci., January 2, 2013 • 33(1):164 –174
Seredynski et al. • Acute Effects of Neuroestrogens on Behavior Discussion
The present experiments show that
17␤-E2 significantly activates and estro-
gen receptor antagonists inhibit, with la-
tencies in the order of minutes, the
expression of behaviors reflecting sexual
motivation in male quail. Mechanistically,
these effects appear to be initiated at the
cell membrane level because these effects
aremimickedbytheinjectionofmembrane-
impermeable estrogens, such as E2–BSA and
E2–biotin. Finally, the observation that ASB
is blocked also within minutes by a single
intracerebroventricular injection of aroma-
tase inhibitors indicates that endogenous es-
trogens locally produced in the brain are
actually exerting these rapid controls of sex-
ual motivation.
The activation of appetitive and con- summatory male sexual behavior by thegenomic action of brain-derived estro-gens has been extensively documentedacross Figure 7.
Effects of acute blockade of brain estrogen synthesis associated or not with an acute E injection on appetitive and consummatory aspects of male sexual behavior in sexually active males (group 3). Blockade of local estrogen synthesis by the aromatase inhibitor VOR (50 ␮g) acutely reduces whereas E (50 ␮g) restores the time spent near the window (A) and looking at
the female (B) in the learned social proximity test (n ⫽ 16). Similar effects are observed on RCSM frequency (C, n ⫽ 9) and
specifically, the activation of copulatory copulatory behavior assessed by the frequency of CCM measured in the large arena (D, n ⫽ 9). However, no such effects were
behavior and appetitive responses, such as detected on copulatory behavior measured in the small arena (E, n ⫽ 9). The "Pre" and "Post" black bars provide reference
the female-induced increase in RCSM fre- behavior frequencies after vehicle intracerebroventricular injections performed before and after the acute treatments. **p ⬍ 0.01 quency and the learned social proximity and ***p ⬍ 0.001 versus vehicle (Veh); (†)p ⬍ 0.10, †p ⬍ 0.05, ††p ⬍ 0.01 versus VOR by Newman–Keuls post hoc tests after response, requires local T aromatization identification of a significant treatment effect (repeated measure) by two-way ANOVA.
in the preoptic area Recently, it had been suggested Together, these data demonstrate that centrally administered that estrogens also acutely control both behavioral components 17␤-E2 rapidly stimulates sexual motivation but not perfor- with a more pronounced effect on the appetitive aspect of male- mance (unless performance depends on motivation, as in the typical sexual behavior in birds at least tests performed in the large arena). Importantly, the basal (con- trol) behavioral activity was remarkably stable across tests despite these effects were highly variable. This might in part be explained repeated treatments with various drugs. No effect of the order in by the fact that all compounds were administered peripherally which treatments were administered to different subgroups of but also by the relative lack of previous genomic priming by the birds could be generally detected, and there were usually no dif- androgenic and/or estrogenic metabolites of T. Previous studies ferences in behavioral frequencies between pretests and posttests, have indeed tested whether acute administration of E indicating that there is no long-term (presumably genomic) store sexual behavior in CX rats or in effect coupled to these acute treatments. Moreover, these CX quail treated with a suboptimal dose of T whose effects membrane-initiated effects are faster than the typical hours/days were quite difficult to titrate The present required for genomic actions. These data imply that the effects design circumvented these problems by administering all are independent of new transcription and rely on nongenomic acute treatments directly in the brain (third ventricle) and testing the effects of E It was proposed recently that 17␤-E 2 either (1) in males chronically supplied 2 satisfies most, if not all, with physiological T concentrations but chronically depleted criteria needed to classify the steroid in the brain as a neuro- in estrogens (group 1 of birds) or (2) in males chronically modulator rather than a hormone treated with T but acutely deprived of estrogens (groups 2 and Among these criteria, the ability to exert 3). The present data demonstrate that brain-derived estrogens rapid actions and the existence of rapid regulations of local syn- can acutely facilitate a measure of ASB in the absence of thesis were described in vitro, but clear evidence that E2 acts as a genomic activation by estrogens (experiments 1 and 3), although neuromodulator to alter behavioral responses was primarily restoration seems to be more complete when genomic priming by lacking. The remarkable reduction of sexual motivation observed both androgenic and estrogenic metabolites is present and here after central blockade of either estrogen action or synthesis local aromatization is only inhibited acutely (experiments 10, provides a clear example of rapid estrogenic effects on a complex 11, and 15). In contrast, the absence of effect of E behavioral response. More importantly, it provides direct evi- inhibited copulatory behavior of males chronically deprived dence that the estrogens relevant for this rapid control are pro- of estrogens (experiment 2) indicates that the neural circuits duced locally. Because membrane receptors underlying these underlying the control of the copulatory sequence critically effects are probably available continuously, local E2 concentra- depend on the genomic activation by estrogens.
tion must vary rapidly to control these on/off switches in moti- Seredynski et al. • Acute Effects of Neuroestrogens on Behavior J. Neurosci., January 2, 2013 • 33(1):164 –174 • 173
vation. This conclusion provides a functional significance to the changes are mediated by classical neurotransmitters, we demon- rapid fluctuations in aromatase activity described recently in strate here that the rapid on/off switches in sexual motivation are brain areas controlling sexual behavior controlled, at least in part, by brain-derived estrogens. These re- sults indicate that the same molecule has evolved mechanisms to It has been demonstrated that preoptic aromatase activity is control different aspects of behavior in distinct temporal do- very rapidly inhibited in the male quail brain after sexual inter- mains. Given the pleiotropic effects of estrogens, one may won- actions with a female Somewhat der whether other responses associated with reproduction (e.g., surprisingly, however, this enzymatic decrease was already ob- territoriality, parenting) also depend on a slow genomic regula- served after only visual exposure to the female without the male tion of the neuronal plasticity underlying behavioral activation having had an opportunity to copulate with her and an acute control of motivation to engage in behavior. Ex- This decrease is therefore tending this duality to non-reproductive responses, such as not the direct result of engaging in copulation per se. Based on changes in cognition or neuroprotection, might have profound this observation, it was hypothesized that the high aromatase activity and the resulting elevated local concentration of estro-gens might be critical for promoting sexual motivation and ap- petitive behavior through which the male searches for and courts Adkins EK, Adler NT (1972) Hormonal control of behavior in the Japanese the female. Copulatory performance sensu stricto would then no quail. J Comp Physiol Psychol 81:27–36. longer depend on these rapid nongenomic effects of estrogens Bakker J, Honda S, Harada N, Balthazart J (2004) Restoration of male sexual but rather on the action of other neurotransmitters, such as do- behavior by adult exogenous estrogens in male aromatase knockout mice.
pamine, that have been primed and/or enhanced by modifica- Horm Behav 46:1–10. tions of brain neurochemistry resulting from the transcriptional Ball GF, Balthazart J (2010) Japanese quail as a model system for studying the neuroendocrine control of reproductive and social behaviors. ILAR J effects of estrogens (for a detailed discussion, see This interpretation fits well with the present results Balthazart J, Ball GF (2006) Is brain estradiol a hormone or a neurotransmit- demonstrating that 17␤-E2 activates within minutes appetitive ter? Trends Neurosci 29:241–249. aspects of male sexual behavior in quail but that the activation of Balthazart J, Foidart A, Hendrick JC (1990a) The induction by testosterone copulatory performance sensu stricto requires a more sustained of aromatase activity in the preoptic area and activation of copulatory stimulation by estrogens and is not affected by these steroids on a behavior. Physiol Behav 47:83–94. short-term timescale. Evidence coming from radioenzyme assays Balthazart J, Evrard L, Surlemont C (1990b) Effects of the non-steroidal inhibitor R76713 on testosterone-induced sexual behavior in the Japanese of aromatase activity after sexual interactions quail (Coturnix coturnix japonica). Horm Behav 24:510 –531. and from behavioral effects of intracerebroventricular pharmacological treatments (present Balthazart J, Reid J, Absil P, Foidart A, Ball GF (1995) Appetitive as well as study) thus converges to indicate that estrogens independently consummatory aspects of male sexual behavior in quail are activated by modulate appetitive and consummatory aspects of male sexual androgens and estrogens. Behav Neurosci 109:485–501. behavior by nongenomic and genomic mechanisms, respectively.
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