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Rapid Decreases in Preoptic Aromatase Activity and Brain Monoamine Concentrations after Engaging in Male Sexual Behavior
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     Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Liege (C.A.C., M.B., C.D., J.B.), B-4020 Liege, Belgium

    Department of Experimental Pharmacology, Medical School, University of Athens (C.D., Z.P.-D.), 11527 Athens, Greece

    Department of Psychological and Brain Sciences, Johns Hopkins University (C.A.C., G.F.B.), Baltimore, Maryland 21218

    Abstract

    In Japanese quail, as in rats, the expression of male sexual behavior over relatively long time periods (days to weeks) is dependent on the local production of estradiol in the preoptic area via the aromatization of testosterone. On a short-term basis (minutes to hours), central actions of dopamine as well as locally produced estrogens modulate behavioral expression. In rats, a view of and sexual interaction with a female increase dopamine release in the preoptic area. In quail, in vitro brain aromatase activity (AA) is rapidly modulated by calcium-dependent phosphorylations that are likely to occur in vivo as a result of changes in neurotransmitter activity. Furthermore, an acute estradiol injection rapidly stimulates copulation in quail, whereas a single injection of the aromatase inhibitor vorozole rapidly inhibits this behavior. We hypothesized that brain aromatase and dopaminergic activities are regulated in quail in association with the expression of male sexual behavior. Visual access as well as sexual interactions with a female produced a significant decrease in brain AA, which was maximal after 5 min. This expression of sexual behavior also resulted in a significant decrease in dopaminergic as well as serotonergic activity after 1 min, which returned to basal levels after 5 min. These results demonstrate for the first time that AA is rapidly modulated in vivo in parallel with changes in dopamine activity. Sexual interactions with the female decreased aromatase and dopamine activities. These data challenge established views about the causal relationships among dopamine, estrogen action, and male sexual behavior.

    Introduction

    IN VERTEBRATES, TWO types of intracellular chemical communication systems are involved in the control of male sexual behavior: gonadal sex steroids (in particular, the androgen, testosterone) and neurotransmitters and neuropeptides of various types [in particular, monoaminergic transmitters such as the catecholamines, norepinephrine (NE), and dopamine (DA), and the indoleamine, serotonin (5-HT)]. Steroids hormones are thought to influence sexual behavior primarily via relatively slow (genomic) effects on neurotransmitter systems. More recently, converging evidence has accumulated indicating that there are also rapid effects of estrogens that can be discerned based on both cellular measures and behavioral measures.

    Many effects of testosterone in the brain (e.g. activation of male sexual behavior, sexual differentiation of the brain, and feedback on gonadotropic hormones) are mediated by its local conversion into estrogens by the enzyme aromatase or estrogen synthase (1, 2, 3). Avian species, such as the Japanese quail, are particularly useful in analysis of the control of aromatase activity (AA) because the concentration of the enzyme is relatively high in their brains, facilitating its detection (4). In the quail preoptic area, aromatase-immunoreactive neurons are almost exclusively located within the boundaries of the sexually dimorphic medial preoptic nucleus, a necessary and sufficient site for the activation by steroids of male copulatory behavior (5, 6). Furthermore, testosterone must be aromatized within this nucleus to exert its behavioral effects (1).

    In vertebrates, the regulation of brain AA is largely mediated by changes in enzyme concentration resulting from an increased transcription after exposure to androgens or estrogens (6, 7, 8). However, brain AA can also be rapidly (within minutes) changed by calcium-dependent phosphorylations (i.e. conformational changes without any change in the enzymatic concentration or by a nongenomic mechanism) (4, 7, 9). This rapid control of AA (and presumably of local estrogen bioavailability) appears necessary to sustain the rapid behavioral effects of estrogens that have been recently identified in quail (10) and rats (11). If rapid effects of estrogens really play a critical role in the activation of male sexual behavior, it might be expected that rapid changes in AA occur during the expression of the behavior.

    The involvement of monoamines in the control of male sexual behavior has been extensively documented over the last decades. Pharmacological studies indicate that dopamine (DA) stimulates male sexual behavior in mammals (12) as well as in birds (13). Inhibitory actions of norepinephrine (NE) have also been identified; similarly, 5-HT is generally considered to inhibit sexual behavior (12).

    Although their actions on sexual behavior have, by and large, been investigated independently, steroids and monoaminergic systems appear to interact extensively. Genomic actions of estrogens on monoaminergic systems have been identified in many studies (for recent reviews, see Refs.14, 15, 16). In addition, numerous studies have identified mechanisms by which estrogen can rapidly alter monoaminergic activity (17). It is also the case that experimental evidence suggests the existence of a reciprocal modulation of estrogen synthesis by monoaminergic systems on both short- and long-term bases (18, 19). In quail specifically, pharmacological experiments suggest that NE inhibits, whereas DA stimulates brain AA (6, 13). These changes in AA after pharmacological depletion of DA or NE are observed after several days of treatment, suggesting that they result from changes in the enzyme concentration. In addition, rapid changes in AA have been observed after exposure to DA and dopaminergic compounds in quail hypothalamic homogenates and explants (13, 20).

    Together, these data suggest that rapid changes in estrogen production and catecholaminergic activity modulate the expression of male sexual behavior, but the nature of these changes and the sequence in which they appear have never been investigated. The present study was designed to investigate whether AA and the brain concentration of monoamines vary rapidly during the expression of appetitive and consummatory aspects of male sexual behavior and whether these potential changes are temporally correlated.

    Materials and Methods

    Animals

    Ninety male Japanese quail (Coturnix japonica) served as subjects in these experiments. Birds were sexually naive before the experimental procedures. They were obtained from a local breeder at the age of 5 wk. Throughout their life at the breeding colony and in the laboratory, birds were exposed to a photoperiod simulating long days (16 h of light and 8 h of darkness/d) and had food and water available ad libitum. All experimental procedures were in agreement with the Belgian laws on Protection and Welfare of Animals and on the Protection of Experimental Animals and the International Guiding Principles for Biomedical Research Involving Animals published by the Council for International Organizations of Medical Sciences. The protocols were approved by the ethics committee for the use of animals at University of Liege.

    General procedures

    From their arrival in the laboratory and until the end of the experiment, the birds were housed in individual cages. At the age of 8–10 wk, they were submitted to three or four pretest trials for sexual behavior to let them acquire the copulatory pattern and ensure that all of them were able to copulate. Depending on the experiment they belonged to, subjects were then assigned to two, three, or four sub groups matched according to the mean frequency of behaviors produced during the pretest trials. These subgroups were confirmed to be statistically indistinguishable. During the same period, the two groups of birds that were later tested for appetitive sexual behavior tests (experiments 2 and 3) were habituated during five trials to being placed in the test aquarium for 5 min (see below). Approximately 1 or 2 wk after completion of the pretests, the feathers surrounding the cloacal gland of all these birds were removed to facilitate the observation of cloacal gland contractions. A few days later, the final test before brain collection was performed. The birds described below were studied in two to four conditions, assessing different aspects of the appetitive or consummatory sexual behavior depending on the experiment considered (see below). These tests during which experimental subjects could interact with conspecifics lasted for a duration of 1, 5, or 15 min. The frequencies and latencies of each behavior (see below) were recorded for the entire duration of these interactions.

    Immediately after the final behavioral test, birds were killed by decapitation, and their brains were rapidly (within 2–3 min) dissected out of the skull. The left and right parts of each brain were dissected separately (except in experiment 1, where only the whole preoptic-hypothalamic block was dissected). One half was used to measure monoamines and their metabolites by HPLC-electrochemical detection, and the other half was used to measure AA in the preoptic-hypothalamic block. The fresh preoptic-hypothalamic block (HPOA) was dissected by two coronal cuts at the level of the tractus septopallio-mesencephalicus (rostral edge of the preoptic area) and of the oculomotor nerves (caudal edge of hypothalamus), one parasagittal cut placed approximately 2 mm lateral to the brain midline and one horizontal cut approximately 2 mm above the floor of the brain. The telencephalon (the part rostral to the tractus septomesencephalicus and dorsal to the HPOA), the hindbrain (the part caudal to the oculomotor nerves), and the cerebellum were also isolated. All brain parts were frozen on dry ice immediately after their dissection. The entire dissection of the brain took approximately 5 min. The frozen brain samples were weighed and kept at –80 C until used for assays.

    Body mass and cloacal gland area (greatest length multiplied by greatest width in square microns) of each subject were also recorded on several occasions during the experiments. The cloacal gland is an androgen-sensitive structure whose size is highly correlated with plasma androgen levels (21). Only birds with a cloacal gland area larger than 200 μm2 were used in the experiments. Testicular weight was also measured after death.

    Behavioral tests

    Appetitive sexual behavior.

    The appetitive aspect of male sexual behavior was assessed by the measure of the rhythmic cloacal sphincter movements (RCSM). The cloacal gland is a large sexually dimorphic, androgen-sensitive, external protuberance of the caudal lip of the cloaca (21) that produces a stiff meringue-like foam, which is transferred into the female’s cloaca during copulation and enhances the fertilization success (22). It has been shown previously that sexually active (but not castrated) males rapidly increase the rate of these movements when they are provided with visual access to a female (22), whereas lesions of the preoptic area inhibit the female-induced movements of the gland (23). The movements thus provide a measure of appetitive male sexual behavior in quail (22). The tests for RCSM took place in an aquarium (20 x 40 cm) located on a raised platform. A mirror was placed under the aquarium at a 45° angle and provided the observer with an unobstructed view of the male’s cloacal area (1). The aquarium was divided into two chambers by a glass partition. One experimental male was placed in one of the chambers, and a stimulus egg-laying female (or a male in experiment 3) was placed in the other chamber. The male and the stimulus were only separated by the glass barrier; the male had visual access to the stimulus female or male, but they could not physically interact. RCSM were directly counted for the duration of the test. The RCSM were quantified by direct observation. A single stimulus female or male was used to test all males in a single experiment.

    Consummatory sexual behavior.

    The experimental bird was introduced into a test arena (60 x 40 x 50 cm) that contained a sexually mature female with which the male could freely interact during the duration of the test (5 min for pretest trials; 1, 5, or 15 min for final tests). The following behavior patterns were systematically noted: strut, neck grab (NG), mount attempt (MA), mount (M), and cloacal contact movement (CCM) (see Ref.24 for a detailed description). The latency and frequency of the male sexual behaviors (NG, MA, M, and CCM) providing a measure of the consummatory behavior of the birds were recorded during the 1–15 min of interactions. Two subjects were tested at a time in adjacent arenas; the order of testing was randomly varied each day as well as the stimulus female, so that even if the behavior of females varied during and between the days, and this had an effect on the male, the effect would be randomized in the different experimental groups. In the results, only scores of MA and CCM frequencies as well as CCM latencies are reported, because values for NG and M usually bring similar information.

    Specific procedures

    Experiment 1.

    Twenty-three intact males were used in this experiment. After three pretest trials, subjects were assigned to two subgroups that did not differ statistically in the mean frequencies of sexual behaviors produced during the last pretest [MA: F1.21 = 0.001; P = 0.9747; CCM: F1.21 = 1.607; P = 0.2188; CCM latency (CCM lat): F1.21 = 0.406; P = 0.5309]. During the final test, 1 wk later, birds were introduced for 15 min into the test arena, which was left empty (CTRL; n = 11) or contained a receptive female (SEX; n = 12) with which the male could freely interact for the duration of the test. After animals were killed, the brains were dissected and used to measure AA in the HPOA blocks (pooled left and right sides).

    Experiment 2.

    Twenty-nine intact males were subjected to an experiment investigating the effect of the production of RCSM in response to the view of a female and of copulation with a female for a period of 5 min on AA and on brain levels of monoamines. After four pretest trials, subjects were assigned to three groups that did not differ statistically in the mean frequencies of sexual behaviors displayed during the last pretest [MA: F2.26 = 0.185; P = 0.8319; CCM: F2.26 = 0.087; P = 0.9171; CCM lat: F2.26 = 0.353; P = 0.7060]. During the final test, 2 wk later, birds were introduced for 5 min into the test arena, which was either the aquarium containing a female behind the glass barrier (VIEW; n = 11) or the sex arena containing a receptive female with which male could freely interact (SEX; n = 10). The CTRL animals (n = 8) were handled like other subjects: they were taken out of their home cage and put back in for the duration of the test. All birds were then immediately killed, and brains were dissected and used to measure AA in the HPOA area of one half of the brain (left side) and monoamine levels in the four brain areas described above (right HPOA, telencephalon, hindbrain, and cerebellum) in the other half of the brain. AA was also measured in the left hindbrain to provide a specificity control for effects observed in the HPOA.

    Experiment 3.

    Thirty-six intact males were subjected to an experiment investigating the effect of the view of a female or a male and of the copulation with a receptive female for a period of 1 min on AA and on brain levels of monoamines. After four pretest trials, subjects were assigned to four groups, which did not differ statistically in the mean frequencies of sexual behaviors produced during the last pretest (MA: F3.32 = 0.262; P = 0.8525; CCM: F3.32 = 0.544; P = 0.6560; CCM lat: F3.32 = 0.660; P = 0.5827). During the final test preceding death, 2 wk later, birds were introduced for 1 min into the test arena, which was either the aquarium containing a female (VIEW FEMALE; n = 9) or a male (VIEW MALE; n = 10) behind the glass barrier or the sex arena containing a receptive female with which the male could freely interact (SEX; n = 9). The CTRL birds (n = 8) were only taken out of their home cage and put back in for the duration of the test. All birds were then immediately killed, and brains were dissected and used to measure AA in the HPOA of one half of the brain (left) and monoamine levels in the four brain areas described above (right HPOA, telencephalon, hindbrain, and cerebellum) in the other half of the brain.

    Aromatase assay in HPOA homogenates

    AA was quantified by measuring the tritiated water production from [1-3H]androstenedione (25) adapted and validated for the quail brain (26). Samples were homogenized with an all-glass Potter homogenizer in ice-cold buffer containing 150 mM KCl, 10 mM Tris-HEPES, pH 7.2. On an ice bath, triplicate aliquots (50 μl) of homogenate containing approximately 1 mg wet weight were added to 50 μl 100 nM 1-[3H]androstenedione and 50 μl buffer. To initiate the assay, 50 μl reduced nicotinamide adenine dinucleotide phosphate were added to reach a final concentration of 1.2 mM. All of these steps were conducted at 4 C in 1.5-ml Eppendorf tubes, which were then quickly capped and incubated for 15 min at 37 C. The reaction was stopped by cooling the samples in an ice bath and adding 0.4 ml ice-cold 10% trichloroacetic acid containing 2% activated charcoal. After centrifugation at 1200 x g for 15 min, supernatants were applied to small columns made of Pasteur pipettes plugged with glass beads and filled (3 cm high) with a Dowex cation exchange resin (AG 50W-X4, 100–200 mesh; Bio-Rad Laboratories, Richmond, CA). The columns were then eluted three times with 0.6 ml distilled water. Effluents were collected in scintillation vials, and 10 ml Ecoscint A (National Diagnostics, Atlanta, GA) were finally added. Vials were counted for 3 min on a Packard Tri-Carb 1600 TR liquid scintillation analyzer (Downers Grove, IL).

    Within each experiment, blanks were obtained by processing brain samples in the presence of an excess (final concentration, 40 μM) of the potent and specific aromatase inhibitor, R76713 (racemic vorozole, Janssen Pharmaceutica, Beerse, Belgium). The blank values never exceeded 210 dpm, whereas active control samples had radioactivities ranging between 2,900 and 5,800 dpm. A recovery of 93 ± 2% was usually obtained from samples of 10,000 dpm tritiated water conducted throughout the entire purification procedure (incubation, centrifugation, and Dowex column). Enzyme activity was expressed as picomoles per hour or as picomoles per hour per milligram fresh weight after correction of the counts for quenching, recovery, blank values, and percentage of tritium in the -position in the substrate. Data are presented below as picomoles per hour without correction for fresh weight, because all aromatase-expressing neurons are in the center of the tissue block, and variations in dissection affect the total mass of the tissue, but not the AA.

    Neurochemical analyses

    The dissected tissues were homogenized and deproteinized in 500 μl 0.2 N perchloric acid solution (Merck KgaA, Darmstadt, Germany) containing 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA (both purchased from Riedel-de Han AG, Seelze, Germany). The homogenates were centrifuged at 14,000 rpm for 30 min at 4 C, and the supernatants were again stored at –80 C until analysis, which was performed by HPLC coupled to an electrochemical detector, as previously described with minor modifications (27, 28). Reverse phase ion pair chromatography was used to assay all samples for NE, DA, and its metabolites [3,4-dihydroxyphenylacetate (DOPAC) and homovanillic acid (HVA)], and 5-HT and its metabolite (5-HIAA). The mobile phase consisted of acetonitrile (Merck & Co., Darmstadt, Germany)-50 mM phosphate buffer (10.5:89.5), pH 3.0, containing 300 mg/liter 5-octylsulfate sodium salt as the ion pair reagent and 20 mg/liter Na2EDTA (Riedel-de Han AG). Reference standards were prepared in 0.2 N perchloric acid solution containing 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA (both from Riedel de Han AG). The sensitivity of the assay was tested for each series of samples using external standards. Assays were performed on a BAS-LC4C HPLC system with an amperometric detector (Bioanalytical Systems Inc., West Lafayette, IN). The working electrode was glassy carbon; the columns were Thermo Hypersil-Keystone, 150 x 2.1-mm 5μ Hypersil, Elite C18 (Thermo Electron, Cheshire, UK). The HPLC system was connected to a computer that was used to quantify with the help of specific HPLC software (Chromatography Station for Windows) all compounds by comparison of the area under the peaks with the areas of reference standards. The limit of detection was 1 pg/27 μl (injection volume). Additionally, the ratios of DOPAC/DA, HVA/DA, and 5-HIAA/5-HT were calculated as indexes of DA and 5-HT turnover rate (29, 30).

    The tissue concentrations in general correspond to inactive monoamines stored in vesicles in nerve terminals and, in a lower percentage, free monoamines released in the synaptic cleft or the extracellular space. After release, monoamines are rapidly metabolized. In particular, DA is inactivated, either by reuptake and subsequent intracellular metabolism to DOPAC or by extracellular metabolism to HVA. Thus, DOPAC and HVA are mainly derived from released dopamine, and as a consequence, increased or decreased levels of these metabolites are indicative of increased or decreased dopaminergic activity (31). Similarly, the HVA/DA, DOPAC/DA, and 5-HIAA/5-HT ratios represent indices of the activity of the cells that integrate the synthesis, release, reuptake, and/or metabolism of monoamines (32).

    Data analysis

    Unless otherwise mentioned, all data are expressed as the mean ± SEM. All results were analyzed by one-way ANOVA, with treatments and latencies as independent factors. The ratios of metabolites to the parent amine (DOPAC/DA, HVA/DA, and 5-HIAA/5-HT) were analyzed by the same methods without previous transformations, because these data met the condition of homogeneity of variance (homoscedasticity), and these results represent the ratios of two continuous variables that can vary from zero to the infinite and are therefore not bound by fixed limits like true percentages. Furthermore, all of these data were reanalyzed by the nonparametric Kruskal-Wallis ANOVA that identified exactly the same significant and nonsignificant effects (data not shown). ANOVAs were followed, when appropriate, by Tukey’s compromise honestly significant difference post hoc tests (Tukey HSD), which provide an optimal compromise between the risks of type I and type II errors. Effects were considered significant for P 0.05. All analyses were carried out with the Macintosh version of StatView, version 5.01 (Abacus Concepts, Inc., Berkeley, CA).

    Results

    Experiment 1: effect of copulation (15-min tests) on preoptic AA

    All males that were allowed to interact freely with a sexually mature female exhibited a substantial number of copulatory sequences during the 15-min test period (frequency of MA, 9.3 ± 2.1; frequency of CCM, 4.3 ± 0.8). As illustrated in Fig. 1A, no significant difference could be detected between the preoptic AA of birds that were allowed to copulate for 15 min and birds that had only been handled and returned to their cage (t21 = –1.061; P = 0.3009). In addition, no correlation between the enzymatic activity and sexual performance was detected in birds that were allowed to interact with a female (MA: r = 0.011; n = 12; P = 0.9718; CCM: r = 0.194; n = 12; P = 0.5459; CCM lat: r = 0.057; n = 12; P = 0.8615). The possible effect of visual access to a female alone was not tested at this latency (i.e. 15 min), but given that free access to a mature female that involved extensive sexual interactions did not modify AA, it is unlikely that the view of the female alone would have affected enzymatic activity.

    Experiment 2: effect of view of a female or of copulation (5-min tests) on preoptic AA and brain monoamine concentrations

    This experiment tested the effect of the expression of appetitive or consummatory sexual behavior (view of the female and production of RCSM or copulation) for 5 min on preoptic AA and brain levels of monoamines. All birds involved in this experiment were sexually active during the tests and expressed high frequencies of RCSM (186.8 ± 1.5) or copulatory behaviors (MA, 9.4 ± 2.4; CCM, 5.2 ± 1.5).

    AA

    A marked decrease in AA was observed in birds that had interacted with a female either visually (VIEW group) or physically (SEX group; see Fig. 1B). One-way ANOVA confirmed that sexual interactions for 5 min led to a significant change in AA (F2.26 = 4.522; P = 0.0206; Fig. 1B). Tukey’s HSD tests indicated that the mean enzymatic activity of control birds was significantly different from the activity in subjects exposed to the view of the female or of birds allowed to copulate with her (P < 0.05 in both cases). AA was also measured in the left hindbrain of these birds to evaluate the anatomical specificity of these effects. Absolute enzymatic activities were much lower than in the HPOA, and no significant difference between groups was detected (F2,27 = 3.345; P = 0.0503). Although not significant, the same pattern of effects was present for the AA in the two groups exposed to the female and was lower than in controls (CTRL: 0.487 ± 0.058 pmol/h; n = 9; VIEW: 0.345 ± 0.044; n = 11; SEX: 0.314 ± 0.045; n = 10; mean ± SEM).

    Postmortem analyses indicated that the testes weights were different in the three subgroups of males (F2.26 = 4.377; P = 0.0226; data not shown) and post hoc Tukey’s HSD tests identified the origin of this effect: the mean testes weight was lower in birds of the SEX groups compared with the control group (P < 0.05, by Tukey’s test). However, cloacal gland size, an accurate indicator of circulating testosterone levels (21, 33), was similar in all groups (F2.26 = 0.3560; P = 0.7038), indicating that the difference in AA between groups could not reflect a preexisting difference in circulating levels of testosterone.

    No correlation between AA and sexual performance was detected in birds that were allowed to copulate (MA: r = 0.048; n = 10; P = 0.8942; CCM: r = 0.177; P = 0.6243; CCM lat: r = 0.146; P = 0.6879) and in males provided with the view of the female (RCSM: r = 0.264; n = 11; P = 0.4337).

    Monoamine concentrations

    Mean concentrations of monoamines (NE, DA, and 5-HT), some of their metabolites (DOPAC, HVA, and 5-HIAA), and their ratio to the parent amine that had been measured in four brain regions (right HPOA, telencephalon, hindbrain, and cerebellum) were analyzed by one-way ANOVA, with the three experimental groups as independent factor. These ANOVA identified a significant difference between groups in the concentration of DA in the HPOA (F2.26 = 4.438; P = 0.0220; Fig. 2A); the mean DA concentration was significantly lower than in controls in subjects exposed to the view of the female or allowed to copulate with her (P < 0.05, in both cases by Tukey’s tests). A similar trend toward a decrease in the level of DOPAC was observed after sexual interactions, but this difference did not reach significance (F2.26 = 1.717; P = 0.1994; Fig. 2B). As a consequence, the DOPAC/DA ratio was similar in the three groups. Other amines or metabolites, including HVA, the other metabolite of DA (see Fig. 2C), were not affected by the treatments in the HPOA (not shown).

    No correlation between the DA concentrations and sexual performance was detected in birds that were allowed to copulate, although the relationship with CCM lat almost reached significance (MA: r = 0.145; n = 10; P = 0.6892; CCM: r = 0.188; n = 10; P = 0.6026; CCM lat: r = 0.646; n = 10; P = 0.0543). Similarly, males provided with the view of the female did not show a correlation between their DA levels and their production of RCSM (r = 0.081; n = 10; P = 0.8248). In contrast, a strong positive correlation between preoptic AA and DA levels was identified in the entire population of birds used for this experiment (r = 0.472; n = 29; P = 0.0097; see Fig. 2D). This correlation appears to result mostly from the treatment effects and not from individual differences present in each population, because no correlation was detected in each experimental group considered separately (CTRL: r = 0.375; n = 8; P = 0.3606; VIEW: r = 0.388; n = 10; P = 0.2684; SEX: r = 0.254; n = 11; P = 0.4505). No correlation was detected between AA and the concentration or ratios of other monoamines and their metabolites.

    No significant experimental effect was detected in the telencephalon, although the concentration of HVA tended to increase and the DA level tended to decrease after a visual or physical interaction with the female. As a result, the HVA/DA ratio increased almost significantly after these interactions (F2.26 = 3.025; P = 0.0659). The other amines did not differ between groups. A direct correlation was identified between the HVA/DA ratio and MA frequency (r = 0.661; n = 10; P = 0.0375). No other significant correlation was detected.

    In the hindbrain, the levels of dopaminergic metabolites (DOPAC and HVA) as well as the ratios of these metabolites to their parent amine increased after visual or sexual interactions with a female. However, this effect only reached statistical significance for DOPAC/DA ratio (F2.26 = 3.548; P = 0.0434). This effect resulted from a significant increase of this ratio in the SEX (P < 0.05), but not in the VIEW group compared with the control situation. DA, NE, 5-HT, and 5-HIAA concentrations did not differ between groups.

    Finally, in the cerebellum, a single significant experimental effect was observed: HVA concentrations were significantly affected by the treatments (F2.26 = 4.256; P = 0.0252) due to an increase in the SEX group (P < 0.05, by Tukey test), but not in the VIEW group by comparison with controls. The ratios of metabolite levels to their parent amine levels did not differ between groups, and no correlation was detected between HVA levels and frequencies of consummatory sexual behavior.

    Experiment 3: effect of view of a male or female conspecific or of copulation (1-min tests) on preoptic AA and brain monoamine concentrations

    This experiment tested the effect of short social interactions on the same neurochemical measures as experiment 2 (preoptic aromatase and brain levels of monoamines). The only difference in protocol was that the duration of interactions (view of a conspecific or copulation) was decreased from 5 to 1 min, and an additional group was included to compare the effects of visual exposure to a female and to a male. Once again, all subjects in this experiment were sexually active. During the 1-min test, birds in the SEX group performed active copulatory behavior (frequency of MA, 5.5 ± 1.3; frequency of CCM, 3.1 ± 0.5), whereas both groups of subjects exposed to the view of a conspecific expressed large numbers of RCSM. Interestingly, the numbers of RCSM produced in response to conspecifics of either sex were nearly identical (VIEW FEMALE, 53.4 ± 6.7; VIEW MALE, 52.4 ± 4.9; t17 = 0.094; P = 0.9259), in contrast with previous results showing that males provided with the view of a receptive female produce significantly more RCSM than males provided with the view of a male (22).

    AA

    Social interaction for a period as short as 1 min did not produce significant overall changes in AA if the four groups were considered together (F3.32 = 1.442; P = 0.2488). However, a numerical decrease was observed in all groups that had been exposed to a female (see Fig. 1C). Accordingly, the ANOVA performed on data corresponding to the groups CTRL, VIEW FEMALE, and SEX (i.e. after exclusion of the additional VIEW MALE group) indicated a clear trend toward a decrease (F2.23 = 2.704; P = 0.0882). Furthermore, if only the groups CTRL and SEX were compared, this decrease became significant (F1.15 = 8.040; P = 0.0125). In contrast, comparison of the CTRL and VIEW FEMALE groups was not significant (F1.15 = 8.862; P = 0.1113). These data thus suggest that after only 1 min, the full sexual interaction with a female copulation might decrease brain AA.

    No difference between groups was identified in the testes weights (F3.32 = 0.999; P = 0.4058) or cloacal gland sizes (F3.32 = 0.606; P = 0.6160), indicating that the difference observed at the enzymatic level does not result from a difference in circulating levels of testosterone.

    No correlation between AA and sexual performance was detected in birds that were allowed to copulate (MA: r = 0.549; n = 9; P = 0.1260; CCM: r = 215; n = 9; P = 0.5782; CCM lat: r = 0.046; n = 9; P = 0.9059) or between AA and the number of RCSM produced in response to the view of a female (r = 0.070; n = 9; P = 0.8571) or a male (r = 538; n = 10; P = 0.1087).

    Monoamine concentrations

    The analysis of the effects of the experimental manipulations on the levels of monoamines and their metabolites was carried out by the same methods as those used in experiment 2 (see above), and significant effects were observed during this experiment exclusively in the HPOA (see Fig. 3). No significant change was detected in the three other brain areas investigated (P 0.1396; data not shown).

    In the HPOA, the levels of parent amines (NE, DA, and 5-HT) were similar in the four experimental groups (see Fig. 3, A–C), but significant differences between groups were identified for two metabolites, DOPAC (F3.32 = 3.415; P = 0.0290; Fig. 3D) and 5-HIAA (F3.32 = 3.782; P = 0.0198; Fig. 3F), and their ratio to the parent amine (DOPAC/DA ratio: F3.32 = 4.356; P = 0.0111; 5-HIAA/5-HT ratio: F3.32 = 3.723; P = 0.0211; see Fig. 3, G and I, respectively). Nearly significant differences were also observed for HVA levels (F3.32 = 2.539; P = 0.0693; Fig. 3E) and the HVA/DA ratio (F3.32 = 2.787; P = 0.0565; Fig. 3H). As illustrated in Fig. 3, the same pattern of response was observed in each case. A reduction in the metabolite level and in the corresponding ratio to the parent amine was observed in birds allowed to see a female. A more pronounced reduction was observed in birds allowed to copulate than in birds that only saw the female. In contrast, the levels of these monoamines in birds exposed to the view of a stimulus male remained apparently unchanged, suggesting that although experimental subjects produced similar numbers of RCSM in response to a male or a female, they were nevertheless able to discriminate between the two types of stimuli. Post hoc Tukey tests indicated that the differences between results in the CTRL or VIEW-MALE groups and the SEX group were statistically significant in almost every case where the ANOVA had detected significant overall effects. Although average concentrations of metabolites or of ratios changed in the same direction in birds that had been allowed to see the female only, these effects did not reach statistical significance by Tukey’s post hoc tests (see detail of Tukey’s HSD tests in Fig. 3).

    Interestingly, DOPAC levels and DOPAC/DA ratios were significantly correlated with several measures of copulatory behavior (DOPAC with MA: r = 0.827; n = 9; P = 0.0060; with CCM: r = 0.933; n = 9; P = 0.0002; DOPAC/DA with CCM: r = 0.707; n = 9; P = 0.0331; with CCM lat: r = 0.746; n = 9; P = 0.0211). These correlations are illustrated in the case of CCM frequencies (see Fig. 4); there was an obvious inverse relationship between the frequency of consummatory behavior and the level of DOPAC or the DOPAC/DA ratio measured in the HPOA. In contrast, no association was found between the HPOA concentrations of monoamines and the production of RCSM by males exposed to the view of a female. There was also no association between the levels of monoamines or their ratios to the amines in the HPOA and the preoptic AA.

    Discussion

    During the last few years, evidence has accumulated indicating that AA can be regulated at two different time domains that are associated with different cellular mechanisms of action: the slow genomic and the fast nongenomic. To date, the transcriptional control of aromatase concentration is the only mode of control that has been clearly demonstrated to be involved in the control of male sexual behavior (1). The present study constitutes the first demonstration of a rapid modulation in vivo of AA, presumably through a nongenomic mechanism associated with the expression of sexual behavior. The preoptic AA significantly decreased by about 20% 1 or 5 min after expression of male sexual behavior, but had almost returned to baseline after 15 min. Interestingly, just being provided with visual access to a female for 5 min along with the associated production of RCSM decreased preoptic AA. Rapid changes in preoptic monoaminergic activity were also observed in these conditions, and they seemed to occur more rapidly than the changes in AA, which raises the possibility that rapid changes in monoamines may modulate brain estrogen production. Several of these provocative findings deserve detailed discussion.

    Changes in brain monoamine concentrations: behavioral implications

    Previous studies in mammals have demonstrated that during sexual interactions, DA and 5-HT are released in various brain regions, including the preoptic area (12, 34, 35, 36, 37). This rise of extracellular DA seems to be required for the initiation of copulation, because males that do not exhibit this enhanced release of DA do not copulate (34). Changes in 5-HT activity have also been related to ejaculation and satiety processes. The elevation of preoptic 5-HT levels is delayed compared with the rise of extracellular DA and is observed in postejaculatory samples only (36, 37, 38), supporting its role in the control of sexual satiety (12).

    The present data demonstrate that concentrations of monoamines and their metabolites also change in the quail HPOA after the expression of both appetitive and consummatory sexual behaviors. Although a few changes in DA activity were identified in the three other areas after 5 min of interaction, most changes were detected in the HPOA after either 1 or 5 min of interaction, supporting the potential involvement of these changes in the control of reproductive function.

    However, in contrast to the data derived from rodent studies, dopaminergic and serotonergic activities appear to decrease after these interactions (decrease in metabolites and their ratio to the parent amine, suggesting diminished release). This apparent species difference between the responses of monoaminergic systems to sexual interactions might arise from differences in the temporal organization of the behavioral pattern or from technical differences between experiments.

    Studies of dopaminergic activity during copulation in rats and quail have to date employed different measurement protocols. Many studies in rats assessed the release of DA in the extracellular medium by either voltammetry or in vivo dialysis coupled with the assay of amines by HPLC (34, 36, 37). In the present work on quail, we measured the tissue concentrations of the amines and their major metabolites. Because we found a decrease in DOPAC and, to a lesser extent, HVA levels after sexual interactions, we assumed that dopaminergic activity had decreased. This conclusion must be considered cautiously and is not established with the same degree of confidence as the increase in extracellular DA identified by in vivo dialysis in rat. However, one study of rats employing the same methodology as the present one (involving the measurement of total concentrations of amines and their metabolites) reached a conclusion consistent with all literature on rodents, i.e. that sexual interactions of a male with a female lead to an increase in dopamine release (38). We are therefore inclined to believe that the difference observed between rats and quail indeed reflects a true species difference.

    This difference may relate to differences in the copulatory patterns in these two species. In rats, the achievement of a full copulatory sequence can last as long as 30–40 min and is paralleled by progressive increases in DA and 5-HT that take several minutes to reach a plateau after progression of the interaction from the first contact to the final ejaculation (34, 36). Conversely, in quail, ejaculation is often observed after as little as 5 sec of interaction between partners. One hypothesis that provides some clarification for the species difference is that an optimal degree of DA activity is maintained throughout the reproductive season in quail, so that immediate mating could be achieved as soon as suitable stimuli become available. A rise in DA activity consequently might not be required for the completion of a successful cloacal contact and ejaculation in quail. These differences in the pattern of DA release might also be related to the fact that quail do not possess an intromittent organ, and many of the effects of preoptic DA in rats seem causally related to the activation of penile movements and reflexes (12).

    If a rise in DA is not necessary to trigger copulatory behavior in quail, one might expect that monoamine concentrations in the quail HPOA should be higher than those in rats, and this appears to be the case to the extent that different studies can be directly compared [in quail (39), present study, and in rodents (38, 40, 41)]. This neurochemical difference between species could thus explain why no rise in dopaminergic activity is detected during copulation in quail. Future work should investigate whether this surprising response of monoaminergic systems to sexual interactions constitutes an adaptation of a species to copulate extremely rapidly or a particular feature of all avian species that have no intromittent organ. Interestingly, DA concentrations in the preoptic area of zebra finches are also quite high and in the same range as those in quail (42), but this species has not been submitted to an intense selection for breeding (correlated with a hyperactive sexual behavior) and displays extensive courtship displays before copulation. The high concentration of DA in the preoptic area could thus be a general feature of birds as opposed to mammals. This idea is also supported by the observation of a similarly high concentration of DA in the chicken brain (43).

    It should be noted that a recent study in quail demonstrated that, contrary to what had been expected based on studies using specific dopaminergic agonists and antagonists, the injection of DA into the third ventricle inhibits both appetitive and consummatory aspects of male sexual behavior (44). In this context, a reduction of DA activity in the medial preoptic area could alternatively be viewed as the removal of a dopaminergic inhibition that results in the stimulation of copulatory behavior. More experiments employing behavioral pharmacology tools need to be performed to assess the functional significance of these results.

    Rapid in vivo changes in AA: control mechanisms

    Previous experiments conducted in our laboratory demonstrated that AA is rapidly (within 5 min) and reversibly modulated in vitro by Ca2+-dependent phosphorylations (7, 9). The present study shows for the first time that very rapid changes in AA do also occur in vivo in a physiologically relevant context. A significant decrease in AA was observed in our study after only 1 or 5 min of sexual interaction with a female, but was no longer present after 15 min. The mechanism(s) mediating this enzymatic change is unidentified to date, but two obvious options can be considered.

    First, radioenzyme assays performed on synaptosomal preparations and immunocytochemistry at the light and electron microscopic levels have demonstrated that aromatase is located in the presynaptic terminals (45, 46, 47). In the quail and rat brain, aromatase-immunoreactive material is observed at the surface of synaptic vesicles located in presynaptic boutons (47). This raises the possibility that estrogens produced at the synapse could be released in a manner similar to neurotransmitters by the extrusion of vesicles. The identified sequence of the aromatase gene indicates that the protein contains a hydrophobic segment assumed to be a membrane-spanning domain (3), and one report mentions the presence of aromatase on the surface of cells (48). Aromatase could thus be released together with estrogen at the synaptic cleft, or the enzyme could be exposed to the extracellular milieu after the exocytosis of estradiol when the membrane of the vesicle fuses with the presynaptic membrane, as observed for dopamine--hydroxylase, the synthesis enzyme of NE (49). The aromatase enzyme exposed to the extracellular milieu could then be transiently inactivated (a substantial recovery is observed after 15 min).

    Alternatively, copulation could trigger neurotransmitter changes (e.g. changes in glutamatergic or dopaminergic action) that would transitorily inactivate the enzyme (via conformational changes), as suggested by in vitro work demonstrating that the stimulation of glutamate or DA receptors inhibits AA (4, 13, 20, 50). Although various types of glutamate receptors are known to be present in brain areas expressing aromatase-immunoreactive cells groups in quail (51), potential changes in brain glutamatergic activity during copulation have not been documented in this species. In contrast, the present study strongly suggests that preoptic dopaminergic activity markedly decreases during or immediately after the expression of appetitive or consummatory sexual behavior in quail, and previous work indicates that DA modulates AA in multiple ways. In vivo pharmacological experiments suggest that DA action might, after a few days, enhance aromatase transcription and thus activity in quail (20, 52), and there is evidence that catecholamines (NE or DA) or their second messenger cAMP could have similar effects in mammalian tissues (18, 19, 53, 54). In contrast, on a short-term basis, DA as well as dopaminergic compounds inhibit in vitro AA within minutes in adult quail HPOA homogenates and explants (13, 20, 26).

    In the present studies we observed within 1–5 min after copulation parallel changes in aromatase and dopaminergic activities. This raises the possibility that these two neurochemical changes could be causally related. Because changes in dopaminergic activity were already statistically significant 1 min after testing, whereas the maximal decrease in AA occurred after a latency of 5 min, one could hypothesize that changes in DA caused the changes in AA. This idea is also supported by the fact that in experiment 2, which investigated these neurochemical changes after 5 min of sexual interactions, a positive correlation was identified between individual variations in AA and DA concentration in the preoptic area.

    The cellular mechanisms that could mediate the control of AA by DA, however, are poorly understood at present. High densities of tyrosine hydroxylase-immunoreactive fibers are known to make close contacts with aromatase-immunoreactive neurons in the quail brain (55). Aromatase and low levels of dopaminergic receptor-like binding are coexpressed in the same brain areas (20, 55, 56), but, due to the lack of suitable antibodies (antibodies to dopaminergic receptors do not cross-react with these receptors in birds in which aromatase immunocytochemistry is the most successful), it has been impossible to assess whether dopaminergic receptors are specifically expressed by aromatase-positive neurons. The absence of DARPP-32 immunoreactivity on aromatase-immunoreactive cells suggests, however, that dopaminergic receptors of the D1 subtype are not expressed by aromatase neurons (57). It is therefore difficult to model at the cellular level how the effects of DA on AA are controlled and whether these effects involve the aromatase-containing neurons only or concern multineuronal circuits in which DA would act on one type of neuron that would then transsynaptically control a distinct population of other neurons expressing aromatase.

    Independent of these questions related to cellular mechanisms that remain unanswered, there is another issue to consider. The decrease in AA observed in the present experiments cannot simply reflect an effect of variation in dopaminergic activity that is limited to the in vitro situation. One might argue that after homogenization of brain tissue (by allowing an interaction between DA and aromatase that could not take place in vivo), regulations of AA by DA are observed that are not physiological and have no relevance for the in vivo situation. However, it has been clearly established that the addition of DA to HPOA homogenates (with disrupted cells) markedly inhibits AA (26). With this logic, the decreased dopaminergic activity observed after the expression of sexual behavior should have resulted in an increased AA, whereas the opposite was detected here. If the decrease in dopaminergic tone is involved in the changes in AA that were detected, it must therefore be via an unidentified mechanism operating in vivo rather than through an artifact related to homogenization.

    It is, however, also possible that these two groups of neurochemical changes are not directly related. Although it is reasonable to hypothesize that there are direct causal links between the changes in AA and in dopaminergic activity, this is not necessarily the case, and the two types of changes could be modulated independently by the expression of behavior without having any direct relationship to each other. Previous work on explants indeed indicates that DA inhibits AA when added to the incubation medium (13, 20). It is therefore difficult to reconcile this effect with the parallel decrease in both dopaminergic activity and AAs that was observed here. It should also be noted that after 5 min of sexual activity, AA and dopaminergic activity both decreased in the HPOA, but in the hindbrain these two measures tended to vary in opposite directions (not a fully significant decrease in AA, but an increase in the DOPAC/DA ratio). This makes it even less likely that changes in DA activity control the short-term changes in aromatase. Additional work should be carried out to research whether the changes in AA observed in the HPOA are anatomically specific or concern other brain regions innervated, or not, by dopaminergic inputs. Potential relationships with changes in glutamatergic activity should also be considered.

    Rapid in vivo changes in AA: behavioral significance

    A substantial amount of literature indicates that estrogens derived from the local aromatization of testosterone in the brain play a critical role in the activation of male sexual behavior in quail, rodents, and many other species (1). Most of the work demonstrating a role for aromatase in the control of male sexual behavior, however, relates to slow effects mediated by changes in the transcription of the enzyme and to genomic effects of the locally produced estrogens. Evidence has also recently accumulated indicating that estradiol affects the expression of sexual behavior in the short-term domain. Acute ip injections of estradiol are able to stimulate sexual behavior within 15 min, and conversely, the inhibition of aromatization by a single injection of an aromatase inhibitor results in a rapid (within 15–30 min) reduction in the expression of appetitive and consummatory aspects of male sexual behavior (10, 58).

    These recent experiments on short-term effects of estrogens leave open a number of questions concerning the specific relationships between short-term changes in AA and modulations of sexual behavior. In particular, if a blockage of estrogen production results in the inhibition of an estrogen-dependent behavior, additional mechanisms must exist to terminate the action of estrogens already present in brain tissue. This can theoretically be achieved by a dilution of the steroid around the site of production and action or by enzymatic degradation, so that in both cases, local concentrations become subthreshold. Enzymes that catabolize estrogens, such as 2- and 4-hydroxylase, glucuronidase, sulfonase, and O-methylase, are known to be present in the brain (58, 59). It has been shown that in the placenta, aromatase also catalyzes estrogen 2-hydroxylation (60), so that the same enzymatic protein could produce and degrade estrogens in an anatomically specialized manner. It is therefore conceivable that aromatase establishes high local concentrations of estrogens at the presynaptic level, and this steroid acts nongenomically on the postsynaptic membrane, but that dilution or catabolism rapidly inactivates estrogenic neural modulation.

    The half-life of estradiol in brain tissue is not known and may be difficult to establish experimentally given the very low concentrations that are present. A recent study reported brain concentrations of estrogens in neonatal rats (that express very high levels of aromatase) ranging from 10–20 pg/mg protein, i.e. 0.5–1 pg/mg tissue, assuming a protein content of about 5% (61). Our preliminary attempts to measure estradiol in the quail HPOA by gas chromatography coupled with mass spectrometry (62) suggest even lower concentrations (Balthazart, J., P. Liere, and M. Schumacher, unpublished observations). It will thus be very difficult, if not impossible, to assess the rate of decline of such a low concentration after the interruption of estrogen synthesis. Old pharmacokinetic studies estimated the half-life of estradiol in the blood to be in the range of 2–4 min in humans (63) and 1.1–27.5 min (fast and slow components of a two-compartment model) in chickens (64). Similarly, transformation of the published estradiol clearance rates from the blood in humans or pigs [t1/2 = (estimated distribution volume x ln2)/clearance] leads to estimates of half-life for estradiol ranging from 5–15 min (65, 66). It is therefore plausible that enzymatic degradation combined with dilution around the site of synthesis and action could within minutes interrupt estrogen-dependent signaling in the brain. This would explain the inhibition of male sexual behavior observed within 15–30 min after an acute injection of the aromatase inhibitor vorozole (10, 58) and the even faster changes (within 1 min) in response to painful stimuli of quail that had received an intrathecal injection of vorozole, which blocked AA localized in the dorsal (sensitive) horn of the spinal cord (67).

    The available experimental data are thus consistent with the idea that estradiol stimulates male sexual behavior in the short term as well as in the long term, and it was somewhat unexpected to observe in this study that the expression of sexual behavior results in a decrease in preoptic AA. It must be noted, however, that the behavioral effects of estradiol or aromatase inhibitors were observed after latencies of 15–30 min, whereas the changes in AA were identified after only 1–5 min of interaction with a female and had almost completely vanished after 15 min. These different time scales could contribute to the apparent discrepancies. It is, on the one hand, possible that a change in estradiol action in the brain could result in a behavioral change more rapidly than 15–30 min, but that systemic pharmacological treatments are not able to affect so rapidly the local estrogen concentration at the level of the critical brain targets. On the other hand, it should also be pointed out that the changes in AA observed in our study do not necessarily reflect what happens in the brain during the expression of sexual behavior and could, rather, be associated with its termination. When exposed to a sexually mature female, experienced male quail usually copulate within seconds, and in most cases, males used in the present experiments copulated several times before being killed (even in the 1-min tests). Accordingly, the decrease in AA could potentially reflect mechanisms associated with sexual satiation, rather than with the activation of behavior. Male sexual behavior in quail is organized into temporal bouts of short duration lasting 1 to 3–4 min, separated by periods of sexual inactivity (68). This relatively rapid cycling of AA and therefore, presumably, of estrogen availability could be one way that these bouts of sexual behavior are regulated. This interpretation, however, is partly contradicted by the fact that a decrease in AA was also detected in males that expressed RCSM in response to visual interaction with a female. Physical interactions with the female and the associated sexual satiation thus do not appear to be required to induce this enzymatic change. Together, these data support the idea that the changes in AA relate to variations in sexual motivation; whether they are the cause or the consequence of these variations, however, cannot be established from the available data.

    In conclusion, the present study provides the first demonstration of rapid changes in AA occurring in vivo. Curiously, sexual interactions decreased enzymatic activity in the preoptic area, and the functional significance of these neuroendocrine changes is not fully understood at present. Concomitant changes were observed in catecholaminergic transmission; thus, these data also raise the question of whether changes in dopaminergic activity rapidly regulate AA in the preoptic area. Additional studies are needed to determine whether changes in AA and catecholaminergic activity are directly related in a causal manner. These data do suggest that there is a rapid cycling of AA activity and catecholamine concentrations relating to temporal changes in copulatory behavior in quail. Because quail have a qualitatively different temporal patterning of sexual behavior and use a completely different effector organ system (a cloaca rather than a penis), the temporal dynamics of AA and catecholamine associated with male sexual performance per se seem quite distinct in some ways from what has been described in mammals. These differences should be exploited to sharpen our hypotheses concerning the functional significance of steroid-catecholamine interactions in relation to different aspects of sexual functioning among vertebrate species.

    Footnotes

    This work was supported by grants from the National Institutes of Health (R01-NIH/MH-50388; to G.F.B.), the Belgian Fonds de la Recherche Fondamentale collective (2.4562.05; to J.B.), and the French Community of Belgium (ARC99/04-241; to J.B.). C.A.C. was a Belgian National Fund for Scientific Research Fellow and is currently a Belgian American Educatonal Foundation postdoctoral fellow.

    Abbreviations: AA, Aromatase activity; CCM, cloacal contact movement; CTRL, control; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetate; HPOA, preoptic-hypothalamic block; HSD, honestly significant difference post hoc test; 5-HT, serotonin; HVA, homovanillic acid; lat, latency; M, mount; MA, mount attempt; NE, norepinephrine; NG, neck grab; RCSM, rhythmic cloacal sphincter movement; SEX, cage contained receptive female; VIEW, aquarium containing a female behind the glass barrier.

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