Brain Research 988 (2003) 139–145 www.elsevier.com / locate / brainres Research report Medial preoptic area dopaminergic responses to female pheromones develop during puberty in the male Syrian hamster Kalynn M. Schulz a , Heather N. Richardson a,c , Russell D. Romeo a,c , John A. Morris c , Keith J. Lookingland b,c , Cheryl L. Sisk a,c , * a b Department of Psychology, Michigan State University, East Lansing, MI 48824, USA Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA c Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA Accepted 15 July 2003 Abstract Chemosensory cues from receptive females do not elicit similar reactions before and after puberty in male hamsters. While pheromones facilitate a complex display of reproductive behavior in adults, prepubertal males do not engage in these same behaviors. Dopamine (DA) released from the medial preoptic area (MPOA) in response to a receptive female or her odors is an important component of the neural events underlying adult male rat sexual behavior. The current experiment investigated whether increased dopaminergic activity occurs in the adult male hamster MPOA in response to female pheromones, and if so, whether this response is absent in prepubertal males, which ¨ prepubertal and adult male hamsters were exposed to cotton swabs with or without pheromone from an do not mate. Sexually naıve estrous female for 0, 5, 15, or 25 min, after which brains were collected and frozen on dry ice. The MPOA was micropunched from frozen coronal sections (500 mm), and concentrations of DA and its primary metabolite DOPAC were determined by high-performance liquid chromatography–electrochemical detection. DOPAC was used as an index of dopaminergic activity. DOPAC levels significantly increased in adults after 15 min exposure to pheromone. In contrast, MPOA DOPAC concentrations did not increase in prepubertal males exposed to pheromone. These data demonstrate that the neural processing of sexually relevant chemosensory stimuli matures during puberty. The absence of a DA response to female pheromones prior to puberty may contribute to the inability of prepubertal males to display reproductive behavior.  2003 Elsevier B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Monoamines and behaviour Keywords: Dopamine; Pheromone; Reproductive behavior; Puberty; Medial preoptic area 1. Introduction Puberty is the developmental stage during which male rodent reproductive behaviors emerge. In the adult male Syrian hamster, full expression of reproductive behavior relies on both the presence of gonadal steroid hormones and exposure to female pheromones [10,26]. These pheromonal cues and steroidal signals are integrated within a forebrain neural circuit consisting of the medial *Corresponding author. Department of Psychology and Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, MI 48824, USA. Tel.: 11-517-355-5253; fax: 11-517-352-2744. E-mail address: sisk@msu.edu (C.L. Sisk). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03358-4 amygdala (Me), the bed nucleus of the stria terminalis (BNST) and nuclei within the medial preoptic area (MPOA) [26]. While the neural circuitry underlying chemosensory processing and the hormonal regulation of reproductive behavior are well studied in the adult male Syrian hamster, how these steroid-sensitive circuits develop during puberty to permit the expression of adult behavior is not clear. Unlike adults, prepubertal males do not mate with a receptive female, even when treated with adult levels of gonadal steroids [9,16,20]. Surprisingly, differences in neural responses to steroid hormones do not seem to account for these behavioral differences. For example, both testosterone and dihydrotestosterone increase brain an- 140 K.M. Schulz et al. / Brain Research 988 (2003) 139–145 drogen receptor immunoreactivity in prepubertal and adult castrates [9,16]. Testosterone is also capable of increasing hypothalamic aromatase activity both before and after puberty [19]. Estrogen receptor immunoreactivity is similar in pre- and postpubertal males, and estrogen treatment increases progesterone receptors before and after puberty [17,20]. Thus, many of the neural responses to steroid hormones that are important for the display of male reproductive behavior are in place before puberty, even though the behavior is not expressed until after puberty. Differences in the neural processing of female pheromones may account for prepubertal and adult differences in reproductive behavior. In adults, exposure to the pheromone-containing vaginal secretions of a female elicits an increase in plasma concentrations of testosterone 1 h later [18]. While this increase in testosterone is not required for the display of reproductive behavior, it is nevertheless a clear index of the activation of the hypothalamic–pituitary–gonadal axis by pheromonal exposure. Interestingly, prepubertal males do not exhibit an increase in testosterone after exposure to female pheromones, which suggests that the ability to detect and / or process female pheromones is immature prior to pubertal maturation [18]. It is apparent, however, that prepubertal males do detect female pheromones, since like adults, they display increased expression of the immediate-early gene product Fos within cell groups of the Me, BNST, and MPOA after exposure to pheromones [18]. This finding further suggests that differences between prepubertal and adult pheromonal processing are downstream of the initial pheromonal activation of cell groups that mediate mating behavior. DA release in the MPOA also occurs in response to female chemosensory cues, and this response is important for the expression of male rodent reproductive behavior [6]. For instance, microinjections of the DA receptor antagonist cis-flupenthixol into the MPOA impair ejaculatory behavior in rats, and decrease the percentage of trials in which males choose a female partner over a male partner [25]. Conversely, the DA receptor agonist apomorphine increases reproductive behavior in castrated rats [21]. Apomorphine also decreases both the latency to ejaculate and the length of time between ejaculations in male Syrian hamsters [3]. Microdialysis studies have confirmed that gonad-intact male rats release DA into the MPOA when they are exposed to the odors, sights, and sounds of an estrous female through a screen, and that copulation is accompanied by further increases in MPOA DA [6]. These data demonstrate that the integration of pheromonal and steroidal information in the mating circuitry results in DA release in the MPOA, which in turn stimulates reproductive behavior. We hypothesize that the inability of prepubertal males to engage in copulatory behavior is related to the immaturity of hypothalamic dopaminergic systems. Preliminary data suggest that MPOA dopaminergic activity increases in response to female pheromones in male hamsters, as it does in rats [12]. One objective of this study was to confirm and extend the observation that male hamster dopaminergic activity is increased within the MPOA in response to female chemosensory cues (experiment 1). The primary goal of this study was to determine whether DA responses to female pheromones occur prior to puberty (experiment 2). Prepubertal and adult hamsters were exposed to the vaginal secretions of estrous females, after which concentrations of DA and its primary metabolite DOPAC were measured in tissue extracts of the MPOA and the adjacent periventricular nucleus (PeVN). DOPAC is the primary metabolite of DA, and serves as an index of the activity of DA neurons [11]. We report here that female pheromones elicit increased dopaminergic activity in adult but not in prepubertal males, which may account for the inability of prepubertal males to engage in reproductive behavior. 2. Materials and methods 2.1. Animals Male Syrian hamsters were obtained from Charles River Laboratories (Kingston, NY, USA), singly housed in clear polycarbonate cages, and allowed at least 7 days to acclimate to their new environment before tests were conducted. The animals were maintained on a 14-h / 10-h light–dark schedule (lights out at 12.00 h) at 2162 8C. Food (Teklad Rodent Diet No. 8640; Harlan, Madison, WI, USA) and water were available ad libitum. All animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Michigan State University All University Committee for Animal Use and Care. 2.2. Experimental procedures 2.2.1. Experiment 1 Two groups of adults (66 days of age) were exposed to cotton swabs in their home cages starting 1 h into the dark phase of the light–dark cycle. The control group was exposed to a clean (blank) cotton swab, and the experimental group was exposed to a cotton swab containing the vaginal secretions of an estrous female. Animals were decapitated 15 min after introduction of the swab to the home cage. Brains were removed, frozen on aluminum foil placed on dry ice, and stored at 280 8C until sectioned. 2.2.2. Experiment 2 Prepubertal (28 days of age) and adult (66 days of age) males were exposed in their home cages to either blank or pheromone-containing swabs starting 1 h into the dark phase of the light–dark cycle. Animals were decapitated either 5, 15, or 25 min following the introduction of the swabs to the home cage (n58 each age / condition / time- K.M. Schulz et al. / Brain Research 988 (2003) 139–145 point). Groups of age-matched males that were not exposed to a cotton swab served as time-zero controls. Trunk blood was collected for determination of plasma testosterone concentrations, and brains were removed, frozen on dry ice, and stored at 280 8C until sectioned. 2.3. Plasma testosterone radioimmunoassay Plasma concentrations of testosterone were measured in 50 ml samples within a single assay using the Coat-ACount Total Testosterone Kit (Diagnostic Products, Los Angeles, CA, USA). This assay has been previously validated in our laboratory [14]. The intraassay C.V. was 9.2%, and the lower limit of detectability was 0.1 ng / ml. 2.4. Tissue microdissection The MPOA and PeVN were located within the same 500 mm coronal section that was cut on a cryostat and thawmounted onto a glass slide. The coronal section containing the MPOA and PeVN corresponds to Fig. 19 of Paxinos and Watson [15]. The PeVN served as a control brain region. All sections were stored on dry ice until tissue punches were dissected. The MPOA (experiments 1 & 2), and PeVN (experiment 2 only) were dissected using a modification of the technique of Palkovits [13]. Tissue samples were placed in 65 ml of 0.05 M sodium phosphate / 0.03 M citrate buffer (pH 2.5) containing 15% methanol and stored at 280 8C until extraction. 2.5. High-performance liquid chromatography ( HPLC) Concentrations of neurotransmitters and their metabolites in tissue samples of the MPOA (experiments 1 & 2) and PeVN (experiment 2) were determined by HPLC with electrochemical detection as previously described [4]. On the day of the assay, tissue samples were thawed, sonicated for 3 s (Sonicator Cell Disruptor; Heat Systems Ultrasonic, Plainview, NY, USA), and centrifuged for 30 s in a Beckman Microfuge B. The supernatant was then drawn off from each tissue sample and analyzed for DOPAC (experiments 1 & 2), DA (experiments 1 & 2), norepinephrine (NE) (experiment 2), serotonin (5-HT) (experiment 2), and the serotonin metabolite 5HIAA (experiment 2) by HPLC. NE, 5-HT, and 5HIAA were measured in the MPOA in order to determine whether the activity of other cell populations in the MPOA is altered after exposure to pheromones. The remaining tissue pellets were dissolved in 1.0 M NaOH and assayed for protein content [8]. A 50-ml volume of the supernatant was injected onto a C 18 reversed-phase analytical column (5 mm spheres; 25034.6 mm; Biophase ODS; Bioanalytical Systems, West Lafayette, IN, USA). The analytical column was protected by a precolumn cartridge filter (5 mm spheres; 3034.6 mm). The HPLC column was coupled to a single coulometric electrode-conditioning cell in series with dual 141 electrode analytical cells (ESA, Bedford, MA, USA). The conditioning cell electrode was set at 10.40 V, and the analytical electrodes were set at 10.12 V and 20.40 V, relative to internal silver reference electrodes. The HPLC mobile phase was composed of 0.1 M phosphate / citrate buffer (pH 2.5) containing 0.1 mM EDTA, 0.035% sodium octylsulfate, and 10–25% methanol. The quantities of neurotransmitters and their metabolites in each sample were determined by comparing peak heights (recorded from the 20.40 V analytical electrode by a HewlettPackard Integrator, Model 3393A) with those obtained from standards run on the same day. 2.6. Statistics Although each treatment group consisted of eight animals (experiments 1 & 2), actual sample sizes for each brain region analyzed varied due to occasional difficulties during sectioning or microdissection, which prevented tissue samples from a particular brain region or animal from being analyzed. In all cases the final sample sizes were $5. In experiment 1, a one-way analysis of variance (ANOVA) was used to compare DOPAC and DA concentrations in the MPOA of adults after a 15 min exposure to blank or pheromone containing swabs. Experiment 2 employed a three-way ANOVA to analyze the effects of age, swab type, and duration of swab exposure on concentrations of DOPAC, DA, NE, 5-HT, and 5HIAA in the MPOA and PeVN. Significant interactions were probed using Tukey HSD post-hoc tests. Differences were considered significant when P,0.05. 3. Results 3.1. Experiment 1 3.1.1. DOPAC and DA in the MPOA Adults exposed to a pheromone swab for 15 min had significantly greater concentrations of MPOA DOPAC than adults exposed to a blank swab for 15 min [F(1,12)57.45, P,0.02]. No significant differences in MPOA DA concentrations were detected between males exposed to blank and pheromone-containing swabs (Fig. 1). 3.2. Experiment 2 3.2.1. Plasma testosterone There were no main effects of swab type or swab duration on plasma testosterone, and these variables were therefore collapsed to compare testosterone levels of prepubertal and adult males. Adults had significantly greater plasma concentrations of testosterone than did prepubertal males [2.7360.215 vs. 0.4060.115 ng / ml, respectively; F(1,123)591.18, P,0.0001]. 142 K.M. Schulz et al. / Brain Research 988 (2003) 139–145 Fig. 1. Pheromone exposure increases dopaminergic activity in the MPOA of adult males. Mean concentrations (ng / mg protein) of DOPAC and dopamine (DA) in medial preoptic area (MPOA) tissue punches of adults exposed to blank or pheromone-containing swabs for 15 min. All values are means6S.E.M. Asterisk indicates P,0.05. The number of animals in each group is indicated. 3.2.2. DOPAC in the MPOA The three-way ANOVA revealed a significant three-way interaction between age, swab type, and swab duration [F(3,81)54.66, P,0.01]. When exposed to pheromonecontaining swabs, adults exhibited significantly greater DOPAC levels than prepubertal males [F(1,81)513.01, P,0.001] (Fig. 2A and B). In contrast, no differences between prepubertal and adult males were observed when exposed to blank swabs [F(1,81)53.706, P.0.05] (Fig. 2C and D). Furthermore, when the DOPAC responses of prepubertal and adult males were compared across the Fig. 2. MPOA DOPAC increases in adult but not prepubertal males after pheromone exposure. Mean concentrations (ng / mg protein) of DOPAC in medial preoptic area (MPOA) tissue punches of prepubertal and adult males exposed to blank or pheromone-containing swabs for 0, 5, 15, or 25 min. The asterisk indicates that adult males display significantly increased DOPAC concentrations 15 min after pheromone exposure while prepubertal males do not. All values are means6S.E.M. The number of animals in each group is indicated. Fig. 3. MPOA DA levels in prepubertal and adult males. Mean concentrations (ng / mg protein) of dopamine (DA) in medial preoptic area (MPOA) tissue punches of prepubertal and adult males exposed to blank or pheromone-containing swabs for 0, 5, 15, or 25 min. Main effect bar graph illustrates that adults exhibit overall higher concentrations of DA. All values are means6S.E.M. Asterisk indicates P,0.05. The number of animals in each group is indicated. different pheromone-swab exposure durations, Tukey tests revealed that adults had significantly greater concentrations of DOPAC than prepubertal males after 15 min of exposure to a pheromone-swab [F(1,81)524.00, P,0.01], but not after 0, 5, or 25 min exposure (Fig. 2A and B). 3.2.3. DA in the MPOA Adults exhibited significantly greater overall concentrations of MPOA DA than prepubertal males [F(1,815 20.90), P,0.001] (main effect of age, Fig. 3). DA concentrations were not significantly affected by the type of swab presented, or the duration of swab exposure. 3.2.4. DA and DOPAC in the PeVN Adults exhibited significantly greater overall concentrations of PeVN DA than prepubertal males [F(1,75)5 5.53, P,0.03] (main effect of age, Fig. 4). No changes in Fig. 4. Prepubertal and adult male PeVN DA levels. Mean concentrations (ng / mg protein) of dopamine (DA) in periventricular nucleus (PeVN) tissue punches of prepubertal and adult males exposed to blank or pheromone-containing swabs for 0, 5, 15, or 25 min. Main effect bar graph illustrates the higher overall concentration of PeVN DA in adult males. All values are means6S.E.M. Asterisk indicates P,0.05. The number of animals in each group is indicated. K.M. Schulz et al. / Brain Research 988 (2003) 139–145 Fig. 5. Prepubertal and adult NE levels in the MPOA. Mean concentrations (ng / mg protein) of norepinephrine (NE) in medial preoptic area (MPOA) tissue punches of prepubertal and adult males exposed to blank or pheromone-containing swabs for 0, 5, 15, or 25 min. Main effect bar graph illustrates that adult males exhibit overall greater concentrations of norepinephrine (NE). All values are means6S.E.M. Asterisk indicates P,0.05. The number of animals in each group is indicated. DA concentration were observed with exposure to blank or pheromone-containing swabs. No significant differences were observed between prepubertal and adult PeVN concentrations of DOPAC, nor were there any significant changes in DOPAC concentrations with exposure to blank or pheromone containing swabs (data not shown). 3.2.5. NE in the MPOA Analysis of MPOA NE concentrations revealed a main effect of age, such that adults exhibited significantly greater concentrations of NE than prepubertal males [F(1,81510.97, P,0.01] (main effect of age, Fig. 5). NE levels did not significantly vary with the type of swab or with the duration of swab exposure. Fig. 6. Prepubertal and adult 5-HT levels. Mean concentrations (ng / mg protein) of serotonin (5-HT) in medial preoptic area (MPOA) tissue punches of prepubertal and adult males exposed to blank or pheromonecontaining swabs for 0, 5, 15, or 25 min. No significant effects of age, swab type, or time were observed for 5-HT levels in the MPOA. All values are means6S.E.M. The number of animals in each group is indicated. 143 Fig. 7. Prepubertal and adult levels of the serotonin metabolite 5HIAA. Mean concentrations (ng / mg protein) of 5HIAA in medial preoptic area (MPOA) tissue punches of prepubertal and adult males exposed to blank or pheromone-containing swabs for 0, 5, 15, or 25 min. No significant effects of age, swab type, or time were observed for 5HIAA levels in the MPOA. All values are means6S.E.M. The number of animals in each group is indicated. 3.2.6. 5 -HT and 5 HIAA in the MPOA No differences were observed between prepubertal and adult levels of 5-HT (Fig. 6) or 5HIAA (Fig. 7) in the MPOA. Further, no significant changes in 5-HT or 5HIAA concentrations were observed after exposure to a blank or pheromone-containing swab. 4. Discussion This study demonstrates that increased MPOA dopaminergic activity in response to female pheromones occurs in males only after pubertal maturation. The failure of prepubertal males to display increases in DOPAC in response to female pheromones is the first direct demonstration of differences in the neural processing of sexually relevant information between prepubertal and adult males, and this difference may contribute to the inability of prepubertal males to engage in mating behavior. Thus, expression of reproductive behavior in adulthood likely involves maturation of the hypothalamic DA system during puberty. Furthermore, we have confirmed that increased dopaminergic activity in the MPOA occurs in adult male hamsters when presented with female odors [12]. Thus, female sensory stimuli elicit MPOA DA responses in male hamsters as well as in male rats [6]. Two important aspects of neural maturation during puberty were elucidated in this study. First, the pubertal development of increased dopaminergic activity in response to female chemosensory stimuli was demonstrated, as indexed by increased DOPAC concentrations in adults, but not prepubertal males, after exposure to pheromones. Second, DA responses to chemosensory cues seem to be 144 K.M. Schulz et al. / Brain Research 988 (2003) 139–145 specific to the MPOA, and MPOA responses to pheromones seem to be specific to dopaminergic activity. The nearby PeVN did not show increased DOPAC concentrations in response to female sensory cues, and within the MPOA, NE, 5HIAA, and 5-HT levels did not change with exposure to pheromone. Interestingly, overall levels of DA in the MPOA and PeVN were greater in adults, which suggests that the hypothalamic DA system undergoes general developmental changes during puberty. MPOA norepinephrine levels were also significantly higher in adults than prepubertal males. MPOA concentrations of serotonin and its metabolite 5HIAA, however, did not change with pubertal maturation. Thus, while both DA and NE-producing neurons of the hypothalamus undergo maturational changes during puberty, only the DA neurons terminating in the MPOA acquire responsiveness to pheromones. In both hamster and rat, the MPOA has a greater concentration of NE than DA (current study, and Ref. [7]). Because DA is a precursor of NE, both DA and DOPAC may be produced by noradrenergic neurons. Therefore, it is possible that the DOPAC measured in tissue punches of the MPOA originated from noradrenergic neurons, and not DA neurons. However, we conclude that the changes in DOPAC concentration observed in the current study are the result of increased dopaminergic activity. The relative levels of DA, DOPAC, and NE in this study most closely resemble what is typical for DA neuron activation, and not noradrenergic activation. Specifically, when noradrenergic MPOA neurons are activated, DOPAC and DA concentrations increase significantly while NE decreases significantly [24]. This pattern is observed because when noradrenergic neurons are activated, the rate of DA synthesis exceeds the rate of DA uptake and conversion to NE by DA b-hydroxylase [24]. Therefore, precursor DA accumulates in the cytoplasm and is metabolized to DOPAC by monoamine oxidase in the mitochondria [1,5,22]. Given that no changes in concentrations of NE across experimental groups were observed in the current study, and only a slight (non-significant) increase in DA was observed after exposure to pheromone, we conclude that DOPAC concentrations measured in the MPOA reflect the activity of DA neurons and not NE neurons. While adults displayed greater plasma concentrations of testosterone than prepubertal males, neither age group displayed increased plasma concentrations of testosterone after exposure to pheromone swabs. This stands in contrast with previously published work in which adult but not prepubertal males displayed increased plasma testosterone concentrations 60 min after exposure to female pheromones [18]. Most likely, the different results of these two studies are attributable to differences in the time between exposure to the pheromone swab and euthanasia of animals. In the present study, 0–25 min passed between swab exposure and animal sacrifice, which may not have been sufficient time for increases in plasma testosterone concentrations to be observed. What are the relationships among testosterone, DA responses to pheromones, and expression of male reproductive behavior? One week after removal of endogenous testosterone via castration, some, but not all, male rats cease to copulate with a female [6]. Castrates that do not engage in sexual behavior also fail to release DA in the MPOA when exposed to an estrous female. However, castrates that do engage in sexual behavior also release DA in the MPOA in anticipation of and during copulation [6]. These results indicate that DA release is more closely tied to the expression of male reproductive behavior than are circulating testosterone levels, and that circulating testosterone is not required for the DA response to female sensory stimuli. Thus, it seems likely that the absence of a DA response to female pheromones in prepubertal males is not due to their low levels of circulating testosterone. Furthermore, if DA release is the critical neural response for the induction of reproductive behavior, then we predict that DA responses to pheromones would not occur in prepubertal males treated with testosterone, because testosterone treated prepubertal males do not display mating behavior. The factors responsible for the pubertal increase in DA responses to pheromones are presently unknown. One possibility is that gonadal hormones, acting specifically during the time of puberty, program or organize DA responses to female sensory stimuli. If this is the case, then depriving males of hormones during puberty via prepubertal gonadectomy should reduce dopaminergic activity and mating behavior in testosterone treated adult males. While we know that prepubertal gonadectomy of male hamsters compromises the activation of mating behavior by testosterone in adulthood [23], we do not know whether this attenuation of reproductive behavior is due to a reduction in the MPOA DA response to female pheromones. Alternatively, pubertal maturation of hypothalamic dopaminergic systems may not depend on the presence of testosterone during pubertal development. For instance, Andersen et al. [2] reported that the overproduction and subsequent pruning of striatal DA receptors that occurs during puberty in male rats does not depend on gonadal hormones. 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