Copulation induces expression of the immediate early gene Arc in mating-relevant brain regions of the male rat

Abstract

The medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), and medial preoptic area (mPOA) are important for the regulation of male sexual behavior. Sexual experience facilitates sexual behaviors and influences activity in these regions. The goal of this study was to determine whether sexual experience or copulation induces plasticity in the MeA, BNST, or mPOA of male rats, as indicated by changes in levels of Arc, which is indicative of activity-dependent synaptic plasticity in the brain. To this end, sexually naïve or experienced males were placed in mating arenas either alone, with an inaccessible estrus female, or with an accessible estrus female. Arc protein levels were then quantified in these three regions using immunohistochemistry. As expected, sexual experience facilitated copulation, as evidenced by a reduction in latencies to mount, intromit, and ejaculate. Copulation also increased the number of Arc-positive cells in the MeA, anterior BNST, posterior BNST, and the posterior mPOA, but not in the central-rostral region of the mPOA. Surprisingly, prior sexual experience did not impact levels of Arc, suggesting that copulation-induced Arc occurs in both sexually naïve and experienced males.

1. Introduction

Sexual experience exerts a persistent influence over brain regions that are crucial for the expression of sexual behaviors [1–3]. In male rats, this system is integrated with the chemosensory system and includes the medial amygdala (MeA), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (mPOA). The main olfactory (MOB) and accessory olfactory (AOB) bulbs receive chemosensory stimulation from receptors in the nasal epithelium and vomeronasal organ (VNO) [4, 5], which then innervate the MeA, providing sex-relevant olfactory information. The MeA relays this information to both the BNST and mPOA. The BNST, which also receives direct afferents from the olfactory bulbs, sends efferents to the mPOA. The mPOA reciprocally innervates the BNST and the MeA [6, 7], which are also important for the patterning of sexual behaviors including copulation, genital reflexes, and sexual motivation [2, 3].

Experiments using targeted lesions of different brain regions support the crucial nature of these three structures in the regulation of copulation. For example, lesions of the MeA inhibit sexual behaviors in male rats [8, 9], hamsters [10], and gerbils [11]; they also negate the facilitation of sexual behaviors resulting from pre-exposure to an inaccessible estrus female in experienced male rats [12], suggesting that the MeA facilitates the response to and assimilation of sex-relevant stimuli. Lesions of the BNST also impair copulation in both sexually experienced and naïve male rats [13]. Specifically, BNST lesions increase the number of intromissions required for an ejaculation, increase the postejaculatory interval (PEI, a period of sexual quiescence after ejaculation), and decrease the number of ejaculations in a timed test [14]. Small lesions of the mPOA decrease sexual performance, while larger lesions abolish sexual behaviors in all studied species, including rats, monkeys, goats, dogs, cats, mice, guinea pigs, hamsters, ferrets, gerbils, snakes, birds, lizards, and fish [3]. In Syrian hamsters, the MeA and BNST act to channel sexually-relevant olfactory signals to the mPOA, illustrating that these three brain regions function in an integrated manner.

Experiments employing the protein byproduct of immediate-early genes like c-Fos, as a marker of neural activity, similarly support an important role for these regions in the regulation of copulation [15]. Both exposure to sex-relevant stimuli and increasing amounts of copulation induce increases in Fos-positive cells in the MeA, along with downstream regions including the BNST and mPOA [16–18]. In the BNST, for example, exposure to sex-relevant olfactory stimuli, non-contact erections, or copulation itself increased the number of Fos-positive cells in male rats [19] and hamsters [20]. Lastly, in the mPOA, like in the BNST and MeA, exposure to the odors of an estrus female and sexual activity increased the number of Fos-positive cells in male rats [16–18, 21–24], hamsters [25], and gerbils [26].

It is widely recognized that prior sexual experience alters sexual performance and protects against lesion-induced sexual deficits. However, the changes that occur in the brain as a result of experience, such as where in the brain sexual experience induces plasticity, are still not fully understood. The mPOA is a promising candidate in this regard, as it receives substantial input from both the MeA and BNST. Numerous studies suggest that changes in the mPOA underlie the behavioral changes associated with repeated sexual experience. For example, systemic and intra-mPOA injections of an NMDA receptor antagonist block the facilitation of copulation by pre-exposure to a female or by prior sexual activity, respectively [27, 28]. Sexual experience also impacts mating-induced activity in nitric oxide synthase-containing cells in the mPOA of male rats [22]. In males, sexual experience increases expression of numerous functionally important peptides within the mPOA, including oxytocin receptor [29]; phosphorylated DARPP-32, an integrator of cAMP and Ca²⁺ signaling [30]; androgen receptors in male mice [31]; and nitric oxide synthase in male rats [32]. More recently, Nutsch and colleagues [23] showed that sexual experience increases the number of cells in the mPOA containing D2-like dopamine receptors, and that sexually inexperienced animals copulating for the first time had a larger percentage of D2-positive cells coexpressing c-fos when compared to sexually experienced animals, and that, regardless of experience, animals that had sex prior to sacrifice had significantly more D2-positive cells coexpressing c-fos compared to animals that did not copulate. These results indicate that activity-dependent synaptic plasticity occurs in the brain’s sexual-behavior circuitry as a result of copulation. While the mPOA is clearly an important integrator, the exact location(s) of the plastic processes within these three sex-relevant regions involved in experience-induced changes have not been fully characterized.

As described above, successive increases in Fos immunoreactivity are observed in the MeA, BNST, and mPOA of males with copulation. Coolen and colleagues [33], for example, used Fos to characterize differences in cellular activation in the MeA, BNST, and mPOA depending on previous sexual experience and the nature of the sexual stimulus to which animals were exposed. To determine the degree to which these three regions undergo neuroplasticity following exposure to sex-relevant olfactory cues, sexual activity, or prior sexual experience, we exposed sexually naïve and sexually experienced male rats to an empty testing arena, an inaccessible estrus female, or an estrus female with which they could copulate. We used immunohistochemical staining for the protein product of the immediate early gene Arc, which is involved in neural processes underlying synaptic plasticity [34]. While c-fos is used as a marker of neural activity occurring in response to experimental stimuli, the Arc peptide may be more specifically indicative of neural plasticity. Therefore, the goal of this study was to determine whether sexual experience or copulation induces plasticity in the MeA, BNST, and mPOA of male rats, as indicated by changes in levels of Arc.

2. Materials and methods

All experimental procedures were approved by the Institutional Animal Care and Use Committee at The University of Texas at Austin and were in accordance with the National Institutes of Health Guidelines for the Use of Animals.

2.1. Subjects

Adult male Long-Evans rats (PN 58–64, 225–250g upon arrival, n=63, Harlan Laboratories, Indianapolis, IN) were singly housed in a temperature-controlled room (22°C, 30–70% humidity) with a reverse light/dark cycle (14 hours light/10 hours dark, lights off at 10 AM). Female conspecifics (PN 70–89, 200–225g, n=26, Harlan Laboratories) were double-housed in a separate room. Prior to all experiments and after approximately one week of acclimation to the animal colony, female rats underwent ovariectomies via ventral midline incisions under ketamine/xylazine hydrochloride anesthesia (50mg/kg and 4mg/kg, respectively; Animal Health International, Greeley, CO) using aseptic surgical procedures. They were injected with gentamicin antibiotic (5mg/kg, RXV Products, Westlake, TX), ketoprofen (5mg/kg, Fort Dodge Animal Health, Fort Dodge, IA), and 1–2 mL of sterile 0.9% saline (Hospira, Inc., Lake Forest, IL) to assist in recovery. At least one week after ovariectomy, females were given alternating subcutaneous injections of estradiol (0.02 mg/mL in sesame oil, 0.2 mL injection per animal) and progesterone (0.2 mg/mL in sesame oil, 0.2 mL injection per animal) every other day to restore sexual receptivity. After approximately two weeks of hormone replacement injections, they received progesterone injections 4 hours prior to experimental procedures, where they were used as stimulus females. Before each experiment, the females’ sexual receptivity was confirmed by placing them with stud males.

2.2. Sexual Behavior

All behavioral testing occurred under red light during the dark phase of the light-dark cycle in rectangular glass testing arenas (51cm long × 26cm wide × 32cm high) that contained a wire mesh basket (27cm long × 14cm wide × 15cm high) suspended from the back side 17 cm above the floor, and covered by a wire mesh lid. All male rats were placed alone in the arenas on four consecutive days for 30 minutes to habituate to the testing environment. Male rats were then divided into two groups: Experienced (n=30) and Naïve (n=33) groups. Over a two-week period, experienced males were placed in the testing arena with a receptive female on 5 separate occasions and were allowed to copulate with her for 30 minutes on the first four occasions and for 60 minutes on the final occasion. With the exception of being placed in the testing arena alone, naïve males were treated identically to experienced males. During the final sexual experience session, males were observed to confirm that they were able to resume intromission after ejaculation at least once within the 60-minute timeframe.

On the day of testing sexual behavior, both the experienced and naïve male groups were further divided into three subgroups: Alone, Female-exposed, or Mated. Animals in the Alone groups (Naïve Alone: n=8; Experienced Alone: n=8) were placed in the testing arena alone for 60 minutes. Animals in the Female-exposed groups (Naïve Female-exposed: n=10; Experienced Female-exposed: n=11) were placed in the testing arena for 60 minutes along with an inaccessible, sexually receptive female in the wire mesh basket within the testing arena. Finally, males in the mated groups (Naïve Mated: n=15; Experienced Mated: n=11) were placed in the testing arena with a receptive female with which they could copulate; they were removed after the first post-ejaculatory interval ended, which was marked by the first intromission following ejaculation. Two rats in the Naïve Mated group did not ejaculate after 60 minutes in the test arena and were removed from the experiment. During test sessions with Mated group males, blind observers scored behaviors to determine the latency to display mounts, intromissions, and ejaculation, and total number of mounts and intromissions, in addition to the post-ejaculatory interval (time between the ejaculation and the subsequent mount or intromission). Mount and intromission frequencies were also calculated for each animal by dividing the total number of behavioral displays by the time from the beginning of the trial to ejaculation in minutes.

2.3. Immunohistochemistry

Sixty minutes after either removal from the testing arena (Alone and Female-exposed groups) or their first ejaculation (Mated groups), males were intraperitoneally injected with a lethal dose of Euthasol (0.3 mL/animal, Virbac Animal Health, Inc., Fort Worth, TX). They were then perfused transcardially with 100 mL of 0.1M phosphate buffered saline (PBS) followed by 500 mL of 4% paraformaldehyde in 0.1M PB (filtered, pH 7.35). Brains were then removed, post-fixed in 4% PFA for 1 hour, transferred to 30% sucrose, and stored at 4°C for at least 48 hours prior to sectioning. Brains were cut into 35 μm coronal sections using a freezing microtome (Microm HM 450, ThermoFisher Scientific, Waltham, MA) and sections were stored in cryoprotectant solution (30% ethylene glycol, 30% sucrose, 0.00002% sodium azide in 0.1M PB) at −20°C until processing for immunohistochemical staining.

Free-floating sections of brain tissue containing the MeA, BNST, and mPOA were washed in 50mM Tris Buffer (TB) four times prior to and in between all incubations; TB also served as the diluent for incubation solutions, and all steps were done at room temperature. After the initial wash, antigen retrieval was performed by incubating sections in 10 mM sodium citrate buffer (pH 8.5) at 65°C for 15 minutes. Endogenous peroxidase activity was blocked by incubating the tissue in 1% H₂O₂ for 10 minutes. The tissue was then incubated in a blocking solution containing 0.4% Tween-20 and 5% BSA for 1 hour, after which it was transferred directly to primary rabbit anti-Arc antibody solution (Synaptic Systems, Gӧttingen, Germany, 1:5000 in blocking solution) and incubated overnight. The tissue was then incubated in biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, 1:1000 in blocking solution) for 1 hour. Signal amplification was achieved by incubation with avidin-biotin complex (ABC elite; Vector Laboratories, 1:1000) for one hour. Staining was visualized by incubation with 0.02% 3–3’diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO), 2% Ni2SO4 (ThermoFisher Scientific), and 0.01% H₂O₂ for 10 minutes. After the chromogen reaction was terminated by several TB washes, tissue was briefly transferred to 0.3% gelatin (Thermo-Fisher Scientific) solution, mounted on slides, counterstained with 0.5% methyl green (Sigma-Aldrich), dehydrated, cleared in xylene, and coverslipped with DPX (VWR Intl., Radnor, PA). To verify antibody specificity, control sections were treated identically except for the omission of the primary antibody from the incubation solution, which resulted in no staining.

The number of Arc-positive cells was quantified in the following brain areas, as delineated by Paxinos and Watson [35]: lateral and medial anterior BNST (Bregma −0.24 mm), central mPOA (Bregma −0.24 mm), lateral and medial posterior BNST (Bregma −0.96mm), posterior mPOA (Bregma −0.96 mm), and dorsal and ventral posterior MeA (Bregma −2.76 mm). The area counted for each brain area is depicted in Figure 1. Each brain area was quantified on both the left and the right side for each animal; the averages of these two numbers were used for analyses. Figure 2 contains representative micrographs from animals that either had or did not have sex before being sacrificed.

Figure 1.

Brain diagrams depicting areas that were examined. In panel A, the following demarcations indicate the following regions: (1) central mPOA; (2) amBNST; (3) alBNST. In panel B: (4) posterior mPOA; (5) pmBNST; (6) plBNST. In panel C: (7) pdMeA and (8) pvMeA. All brain atlas figures adapted from Paxinos and Watson (2007).

Figure 2.

Representative micrographs showing Arc staining in sex-relevant brain regions (each row) of animals that either had (left column) or did not have (right column) sex on the day of testing. Bar length is 50 μm.

2.4. Statistical Analyses

All data were analyzed using R statistical computing software (version 3.1.0). Measurements of sexual behavior from sexually experienced and sexually naïve animals were compared using Welch’s independent sample t-tests. Differences in the number of Arc-positive cells depending on prior sexual experience and test day stimulus were identified using two-way ANOVAs independently for each brain area. When appropriate, Tukey HSD post-hoc tests were used to evaluate differences driving significant main effects. Pearson’s correlation coefficient tests were used in analyses of all correlations.

3. Results

Main effects of test stimulus on the number of Arc-positive cells were found in all but one of the brain areas examined (Figure 3). In both the alBNST and amBNST, the stimulus main effect (F_(2,53)=3.38, p = 0.042 and F_(2,58)=5.39, p = 0.0074, respectively) was driven by greater Arc-positive cell counts in males that had sex as compared to those exposed to females (p = 0.045, d=0.74 and p = 0.0083, d=0.93, respectively). In both posterior BNST subdivisions, the stimulus main effect (plBNST: F_(2,47)=6.48, p = 0.0035; pmBNST: F_(2,57)=5.52, p = 0.0067) resulted from elevated Arc-positive cell counts in sex groups as compared to both the female groups (plBNST, p = 0.0049, d=0.99; pmBNST, p = 0.014, d=0.87) and the groups placed in the testing arena alone (plBNST, p = 0.020, d=0.84; pmBNST, p = 0.029, d=0.77). Because the pattern of differences in Arc counts was identical for both the lateral and medial BNST subdivisions, these were combined to give one average Arc-positive cell count each for the anterior and posterior BNST. In the anterior BNST, a stimulus main effect (F_(2,56)=4.32, p = 0.019) resulted from increased Arc levels in sex groups compared to female groups (p = 0.029, d=0.76). A significant main effect of stimulus was also found in the posterior BNST (F_(2,56)=6.85, p = 0.0023), but there sex groups had more Arc-positive cells than both Female (p = 0.0059, d=0.98) and Alone (p = 0.013, d=0.89) groups. The stimulus main effect in the posterior dorsal MeA (F_(2,56)=10.3, p < 0.001) reflected greater Arc-positive cell counts in sex groups as compared to both Female (p = 0.0030, d=1.22) and Alone (p < 0.001, d=1.48) groups. In the posterior ventral MeA, however, the stimulus main effect (F_(2,57)=6.24, p = 0.0037) was driven by increased Arc-positive cell counts in sex groups as compared only to Alone groups (p = 0.0028, d=1.20). Interestingly, there was a significant main effect of stimulus in the posterior mPOA (F_(2,60)=5.20, p = 0.0085) but not in the central mPOA. Sex groups had increased Arc counts as compared to both Female (p = 0.022, d=0.79) and Alone (p = 0.024, d=0.86) groups in the posterior mPOA. No significant main effects of prior sexual experience or experience by stimulus interactions were found in any of the brain areas examined.

Figure 3.

Copulation increases Arc-positive cells independent of sexual experience. Black bars indicate the mean number of immunopositive cells in naïve males and white bars experienced males. In the (A) alBNST, (B) amBNST, and (C) aBNST, mated groups had more Arc-positive cells than female-exposed groups. In the (D) plBNST, (E) pmBNST, (F) pBNST, (G) pdMeA, and (J) mPOAp, mated groups had higher number of Arc-positive cells than Female-groups or Alone-groups. In the (H) pvMeA, mated groups had higher number of Arc-positive cells than Alone-groups. Finally, in the (K) mPOAc, there were no significant differences in Arc counts among groups. All values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Sexually experienced males displayed shorter latencies to mount, intromit, and ejaculate, when compared to sexually naïve males (mount latency: t_10.6=3.81, p = 0.0031, d=1.48; intromission latency: t_11.3=2.77, p = 0.018, d=1.15; ejaculation latency: t_15.4=2.87, p = 0.0011, d=1.16) (Figure 4); these results are similar to those originally reported by Dewsbury [36], and several others since [2, 3].

Figure 4.

Sexual experience improved measures of copulation. Naïve males displayed higher (A) mount, (B) intromission, and (C) ejaculation latencies than experienced males. Experienced males, however, displayed higher (H) intromission frequencies during copulation than naïve males. Naïve and experienced males did not differ in the number of (D) mounts, number of (E) intromissions, duration of the (F) post-ejaculatory interval, or (G) mount frequency. All values are expressed as mean ± SEM. *p<0.05, **p<0.01.

Correlations between the number of Arc-positive cells in different brain areas and between the numbers of Arc-positive cells and behaviors for all copulating males were also calculated. Correlations in Arc counts between different brain areas after separation by test day stimulus are shown in Figure 5 and listed in Table 1. Correlations between Arc counts and behaviors for all copulating males are shown in Figure 6 and listed in Table 2. This was an exploratory analysis and, as such, significance demarcations presented in heat maps do not reflect a correction for multiple comparisons.

Figure 5.

Pearson’s r values are shown for Arc counts in all pairs of brain areas with males separated by test stimulus. Data from naïve and experienced males were combined. Darker red colors indicate stronger positive r-values while darker blue colors indicate stronger negative r-values. On top, the cladogram is shown depicting functional groupings derived from the correlations. Solid light blue lines in the heat map represent the r value for each correlation relative to r=0, which is represented by the dashed blue line. The color key and histogram inset shows the distribution of all calculated r-values. Statistically significant correlations are labeled as follows: *p<0.05, **p<0.01, ***p<0.001.

Table 1.

Correlations between the number of immunopositive cells are shown for males exposed to different stimuli on the day of testing, independent of prior experience. Pearson’s r and associated p-values are reported.

Brain Area	Brain Area	Alone		Female		Sex
		R	p	R	p	R	p
pdMeA	mPOAc	−0.70	0.0053**	0.04	0.8727	0.57	0.0042**
pdMeA	mPOAp	−0.45	0.1030	0.11	0.6679	0.53	0.0083**
pdMeA	amBNST	−0.13	0.6629	0.53	0.0209*	0.27	0.2135
pdMeA	pmBNST	0.25	0.3864	−0.01	0.9830	0.53	0.0076**
pdMeA	pvMeA	0.15	0.6294	0.36	0.1454	0.44	0.0313*
pvMeA	mPOAp	−0.02	0.9407	0.15	0.5211	0.46	0.0254*
pvMeA	alBNST	−0.11	0.7156	0.58	0.0137*	0.09	0.6837
pvMeA	amBNST	−0.20	0.4937	0.61	0.0047**	0.62	0.0015**
pvMeA	pmBNST	0.01	0.9642	0.01	0.9680	0.44	0.0312*
alBNST	mPOAc	0.52	0.0453*	−0.17	0.5029	0.05	0.8158
alBNST	mPOAp	0.68	0.0050**	0.25	0.3106	0.44	0.0435*
amBNST	plBNST	0.14	0.6549	0.48	0.0391*	0.68	0.0052**
amBNST	pmBNST	0.71	0.0032**	0.28	0.2634	0.39	0.0667
amBNST	mPOAp	0.35	0.2062	0.44	0.0462*	0.42	0.0481*
plBNST	pmBNST	0.79	0.0012**	0.55	0.0185*	0.81	0.0002***
plBNST	mPOAp	0.17	0.5765	0.58	0.0097**	0.34	0.1997
pmBNST	mPOAp	0.27	0.3094	0.39	0.1051	0.43	0.0380*
mPOAc	mPOAp	0.75	0.0012	0.70	0.0005***	0.08	0.7070

Statistically significant correlations are labeled as follows:

^*p<0.05

^**p<0.01

^***p<0.001

Figure 6.

Correlations between Arc-induction and sexual behaviors. Pearson’s r-values are shown for all brain area, Arc count-behavior pairs in all males that copulated on the day of the test (data from naïve and experienced males is combined). Darker red colors indicate stronger positive r-values while darker blue colors indicate stronger negative r-values. The cladograms shown on top, indicate functional groupings derived from the correlations; the more similar the pattern of correlations for two given behaviors or brain areas, the closer together they are located in the cladogram. Solid light blue lines in the heat map represent the r value for each correlation relative to r=0, which is represented by the dashed blue line. The color key and histogram inset shows the distribution of all calculated r-values. NM = total number of mounts, IL = intromission latency, EL = ejaculation latency, MF = mount frequency, ML = mount latency, PEI = post-ejaculatory interval, NI = total number of intromissions, IF = intromission frequency. Statistically significant correlations are labeled as follows: *p<0.05, **p<0.01, ***p<0.001.

Table 2.

Correlation between Arc and sexual behaviors. Correlations between the number of immunopositive cells and behavior, when all copulating males are combined independent of experience. Pearson’s r and associated p-values are reported. IL= intromission latency, NM = total number of mounts, IF = intromission frequency.

Brain Area	Behavior	R	p
pvMeA	IL	0.51	0.0134*
plBNST	NM	0.73	0.0018**
pmBNST	NM	0.81	0.0000***
mPOAc	IF	−0.44	0.0385*
mPOAp	IL	0.53	0.0087**

Statistically significant correlations are labeled as follows:

^*p<0.05

^**p<0.01

^***p<0.001

4. Discussion

The present study was conducted to assess whether exposure to a sexually receptive female, copulation, or sexual experience differentially influenced levels of Arc in sex-relevant brain regions of male rats. Copulation increased Arc-positive cell counts in all BNST subregions, pMeA subregions, and in the posterior mPOA. The only brain region in which Arc expression did not increase after mating was the central mPOA. This is surprising given the importance of cellular activity in both central and posterior regions of the mPOA for consummatory sexual behaviors [37]. Considering the different roles in appetitive and consummatory sexual behaviors played by different parts of the mPOA [38, 39], and the differences in neural projections to the mesolimbic reward system as one moves from the anterior to the posterior mPOA [40], plastic changes following sexual experience might also differ depending on exact location within the mPOA.

Interestingly, throughout the BNST and pMeA, Arc expression in response to copulation was more widely distributed than Fos expression as observed in previous studies. Specifically, sex-induced Fos expression is observed only in the pmBNST and pdMeA, and is occasionally seen in the amBNST as well [41–43]. This may indicate differences in the roles played by these different substructures in mediating mating-induced Arc versus Fos.

Exposure to an inaccessible estrus female did not induce Arc expression in any of the brain areas examined. Given the enhancement in sexual performance such exposure typically confers [27, 28, 30], we expected to find more Arc in at least some of these sub-regions in response to an inaccessible estrus female. It is possible, however, that Arc induction requires both cellular activity due to ascending input generated by sex-relevant stimuli and descending input from integrative brain areas resulting from copulation itself. This appears to be the case in the AOB, where Arc expression is much higher after copulation in an estrus odor-rich environment compared to exposure to the estrus odor exposure without copulation [44]. This may also represent an important difference between Arc induction and c-fos induction, which is increased to the same degree in the AOB after either exposure to estrus odors or copulation [19].

There was no significant effect of prior experience on copulation-induced Arc expression in any of the brain areas examined. We predicted that experienced animals would show elevated Arc expression in sex-relevant brain areas compared to naïve animals, as an up-regulation of plastic processes could help explain improvements in performance that result from prior sexual experience. Instead, the results suggest that continuous modification of neural circuitry underlying sexual behavior might be advantageous to both naïve and experienced males. Other studies have examined Arc as an indicator of sex-induced plastic changes in the context of female sexual behavior. Christensen and colleagues [45], for example, monitored the effects of mating and sexual experience on the number of neurons expressing Arc in the mediobasal hypothalamus in females. They showed that that the behavioral induction of Arc in the arcuate nucleus was in fact associated with reduced sexual receptivity. In addition, estradiol-treated experienced females had significantly reduced sexual receptivity when compared to naïve rats that received similar estradiol treatments. They also discovered that blocking Arc expression prevented the blunting of lordosis behavior in experienced estradiol-only-treated rats [45]. In a different set of experiments, Flanagan-Cato and colleagues [46] also examined the link between Arc and mating in females. They found that paced mating increased Arc levels in the ventrolateral hypothalamic ventromedial nucleus (vlVMH) in both sexually naïve and sexually experienced females one hour after copulation. In the same study, a separate group of females had reduced spine densities on one of the three dendritic compartments examined in the vlVMH five days after mating compared to females that never copulated. This reduction in spine density does not necessarily indicate what role Arc protein plays in the affected neurons, however; Arc might help stabilize any expansion that occurred in the remaining spines as a result of LTP, but it could also be involved in endocytosis of AMPARs at spines that were eventually eliminated [47, 48]. Arc may also contribute to LTP and LTD processes differently in sexually experienced versus sexually naïve animals. Future studies examining the consequences of Arc expression within specific dendritic compartments of both naïve and experienced males will help illustrate how plastic changes in sex-relevant brain areas contribute to experience-dependent improvement in sexual behavior. The dependence of copulatory improvement on protein synthesis [49] and NMDA receptor activation [27, 28], and direct observations of short-term synaptic plasticity in the mPOA [50], all suggest that plasticity does occur in sex-relevant brain circuits, and the presence of Arc may indicate the degree to which this happens in different brain areas. In the context of female sexual behavior, Yang and colleagues [51] characterized and compared the mating-induced neural activation patterns within the amygdalar and hippocampal regions of rats using expression of Arc and other immediate early genes. They discovered that paced mating induces changes in Arc in the CA1 region of the hippocampus and the basolateral, central, and cortical regions of the amygdala, areas of the brain traditionally associated with learning [51]. Therefore, it is possible that when future studies examine effects of sexual learning in males, analyses of these other regions may yield interesting and different results than the ones reported here.

With regards to correlations between Arc expression in the examined brain areas, in the Alone group, significant positive correlations in Arc-positive cells were found both within and between the BNST and mPOA (see Figure 5). This suggests that Arc induction, and perhaps related plastic processes, are observed in both the BNST and the mPOA when they occur in the absence of exposure to a female or copulation. The only other significant correlation in Alone males was a negative correlation between Arc counts in the pdMeA and mPOAc, suggesting that plastic changes tend to occur in only one or the other of these two areas in the absence of sexually-relevant stimuli.

While significant correlations in Arc counts were found in six brain-area-to-brain-area comparisons in males placed in the arena alone, eight such correlations were found in males exposed to inaccessible females. These correlations in males exposed to females were also more widespread among brain regions examined, and were found both within the mPOA and BNST and between the mPOA, BNST, and MeA. Positive correlations between the pmBNST and plBNST and between the mPOAp and mPOAc were similar to significant correlations seen in males in the Alone group; the remaining six correlations in Female group males were not observed in those in the Alone group. The data suggest, then, that exposure to a female increases the co-occurrence of plastic changes, as indicated by Arc induction, between specific subdivisions of the mPOA, BNST, and MeA.

Copulation further increased the number of significant correlations in Arc levels between brain areas to twelve. Specifically, Arc was positively correlated both within the MeA and BNST and between the mPOA, MeA, and BNST. Six of the correlations were unique to males that copulated, suggesting that sexual behavior also coordinates the induction of Arc, and perhaps synaptic plasticity, between specific pairs of mPOA, MeA, and BNST subregions. Three correlations in copulating males that all involved the amBNST were very similar to correlations also observed in female-exposed, but not alone, males. However, this result is difficult to interpret given that the amBNST is not a subdivision typically associated with copulation. Interestingly, the only significant result observed in all three groups of males was the positive correlation between the pmBNST and the plBNST; perhaps, then plastic changes occur together in these subdivisions independent of sex-related stimuli in male rats. Finally, the negative correlation observed between pdMeA and mPOAc Arc levels in Alone group males was replaced by a significant positive correlation in copulating males, suggesting that the co-induction of plastic changes in these two brain areas is particularly important for sexual behavior.

Regarding correlations between Arc expression and sexual behavior, significant positive correlations were found between intromission latency, which was shorter in experienced males, and Arc levels in the pvMeA and mPOAp, which both showed copulation-dependent Arc increases. Taken together, these results seem to indicate that longer intromission latencies, which were associated with naïve males, might increase plastic neural changes in the pvMeA and mPOAp. It is also possible that greater exposure on testing day for animals in the naïve group may have provided sufficient sensory stimulation to match levels of Arc in the experienced males, providing a possible explanation as to why experience did not influence levels of Arc in these regions. Additionally, higher Arc counts in both posterior BNST subdivisions were strongly associated with increased mounting behavior. Lesions of the pmBNST increase the number of mounts during copulation [13]; increased Arc levels might contribute to the reduction in number of mounts prior to ejaculation by altering neural connections in this brain area. Interestingly, this potential role of the pBNST in sexual behavior does not appear to be related to sexual experience, as neither we nor Claro and colleagues [13] found a difference in mount frequency between experienced and naïve males. Alternatively, as per the classical Rescorla-Wagner model of learning [52], an animal with poor behavioral performance has a lot more room for improvement than an animal that starts out doing well. As such, poorly performing animals would be expected to undergo more rapid initial plasticity than animals that performed well (e.g. those that had short initial latencies). Finally, a significant negative correlation was found between intromission frequency, which was higher in experienced males, and Arc counts in the mPOAc.

In conclusion, levels of the immediate early gene Arc, which is indicative of synaptic plasticity in response to an experimental stimulus, increased throughout the BNST, MeA, and posterior mPOA of male rats in response to copulation. Interestingly, there was no effect of prior sexual experience on copulation-induced Arc expression, suggesting that plastic changes occur throughout sex-relevant brain regions in both naïve and experienced males. Finally, olfactory cues from an estrus female, which increased Fos expression in the central mPOA, did not induce Arc expression in any of the brain areas examined, perhaps indicating that co-occurrence of olfactory cues and successful copulation is required for plastic changes to occur, as measured by levels of Arc.

Correlations between Arc levels in five brain areas and three different measures of sexual behavior were identified for all copulating males, regardless of prior experience. Stronger Arc induction in males that take longer to initiate intromissions, intromit less frequently, and mount more may trigger plastic changes to alter future sexual behavior similarly in both naïve and experienced males. Future studies identifying the type of plastic changes indicated by increased Arc levels after sexual activity would help in the development of experiments examining causal relationships between prior sexual experience, Arc levels, and measures of copulation.

Highlights.

• Levels of the immediate early gene Arc increased throughout the BNST, MeA, and posterior mPOA of male rats in response to copulation.

• There was no effect of prior sexual experience on copulation-induced Arc expression.

• Correlations exist between Arc levels in five brain areas and three different measures of sexual behavior were identified for all copulating males, regardless of prior experience.

• Males that required more time to initiate intromissions, intromitted less frequently, and displayed more mounting behaviors also experienced stronger Arc induction.