Clitoral stimulation induces conditioned place preference and Fos activation in the rat more

Hormones and Behavior 57 (2010) 112–118 Contents lists available at ScienceDirect Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y h b e h Clitoral stimulation induces conditioned place preference and Fos activation in the rat Mayte Parada a,⁎, Liliane Chamas a,b, Sabrina Censi a, Genaro Coria-Avila b, James G. Pfaus a a b Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 7141 Sherbrooke W., Montréal, QC Canada H4B 1R6 Instituto de Neuroetología, Universidad Veracruzana, CP 91190, Xalapa, VER, Mexico a r t i c l e i n f o a b s t r a c t The present study examined the ability of clitoral stimulation (CLS) to induce conditioned place preference (CPP) and Fos protein in the brain. Ovariectomized, hormone-primed Long–Evans rats were randomly assigned to receive either distributed CLS (1 stimulation every 5 s for 1 min prior to being placed in one distinctive side of a nonbiased CPP box for 2 min, after which the cycle of stimulation and CPP exposure were repeated for 4 more cycles, totaling 60 stimulations) or continuous CLS (1 stimulation per second for 1 min with 2 min in one side of the CPP box, repeated for 4 more cycles, totaling 300 stimulations). Two days later, females were placed into the other side of the CPP box without prior stimulation. CPP was tested after 5 sequential exposures each of CLS and no stimulation. Females given distributed stimulation developed a significant CPP whereas females given continuous stimulation did not. CLS induced Fos in hypothalamic and limbic structures, including the nucleus accumbens, piriform cortex, arcuate nucleus, and dorsomedial portion of the ventromedial hypothalamus, compared to no stimulation. However, distributed CLS induced more Fos in the medial preoptic area than continuous CLS or no stimulation. In contrast, continuous CLS induced more Fos in the posteroventral medial amygdala compared to no stimulation. These data indicate that CLS induces a reward state in the rat and a pattern of Fos activation in regions of the brain that process genitosensory input, incentive salience, and reward. © 2009 Elsevier Inc. All rights reserved. Article history: Received 20 April 2009 Revised 27 May 2009 Accepted 28 May 2009 Available online 9 June 2009 Keywords: Genitosensory stimulation Female rat Sexual reward Brain activation Introduction The ability of female rats to control or “pace” the initiation and rate of copulation supports the development of a positive hedonic state that results in a conditioned place preference (CPP; Oldenburger et al., 1992; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). Female rats will also selectively approach, solicit, and receive intromissions and ejaculations from males bearing an odor (e.g., almond) or strain cue (pigmented or albino) associated with paced copulation (Coria-Avila et al., 2005a,b, 2006). Both conditioned place and partner preferences are blocked by treatment with the opioid receptor antagonist naloxone during conditioning (Coria-Avila et al., 2008; Paredes and Martinez, 2001) whereas the dopamine antagonist flupenthixol blocks the development of conditioned partner preference for an odor, but not for strain cues (Coria-Avila et al., 2008) or conditioned place preference in female rats (García Horsman and Paredes, 2004). Naloxone infusions to regions of the hypothalamus (medial preoptic area (mPOA) or ventromedial hypothalamus (VMH)) or limbic system (medial amygdala (MEA) but not nucleus accumbens), block the development of pacing-induced CPP (García Horsman et al., 2008), indicating that opioid actions within those structures play an important role in pacing-induced sexual reward. ⁎ Corresponding author. Fax: +1 514 848 2817. E-mail address: may_para@live.concordia.ca (M. Parada). 0018-506X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2009.05.008 Despite knowledge of the putative neurochemical mechanisms involved in linking place or partner cues to sexual reward in female rats, very little is known about the role of sensory stimulation received during copulation in this regard. Such stimulation could be olfactory or genitosensory in nature, and could arise from simple exposure to males or from the distributed stimulation received during each paced (or otherwise controlled) mount with pelvic thrusting and intromission from the male. Indeed, the development of significant CPP for copulation can occur in female rats in conditions where pacing is not optimized (Meerts and Clark, 2007), suggesting that pacing or control per se is not necessarily the rewarding element in copulation, but rather the distribution of stimulation at some preferred interval that is rewarding (Jenkins and Becker, 2003). Moreover, one recent study showed that 15 distributed artificial vaginocervical stimulations (VCSs) induced a reward state in female rats (Meerts and Clark, 2009), indicating that olfactory or visual cues provided by the presence of a male are not necessary for sexual reward. It is not known whether other types of genitosensory stimulation received by female rats during copulation could support the development of a sexual CPP, or whether electrical stimulation of the nerves that innervate the genitosensory tract (pelvic, pudendal, hypogastric, vagus), might stimulate a reward state. The clitoris is an important organ for genitosensory stimulation. In women, the clitoris is regarded as a gland that relays localized pleasurable sensory input (e.g., Giuliano et al., 2002; Masters et al., 1986; van Netten et al., 2008) and influences vaginal muscle function (Shafik M. Parada et al. / Hormones and Behavior 57 (2010) 112–118 113 et al., 2008). It has been studied in rats with regard to its innervation and vasculature during sexual arousal (Peters et al., 1987; Pacheco et al., 1989; Giuliano et al., 2001; Cruz et al., 2004) and its morphology in response to hormones (Hall, 1938; Munarriz et al., 2003; Pessina et al., 2006). However, its role in carrying sensory information during sexual activity in the female rat is currently unknown. However, the lordosis posture of the female facilitates the male's ability to mount with pelvic thrusting and intromission. This interaction stimulates the female's flanks, rump, tailbase, perineum, and perivaginal surfaces which include the clitoris (Pfaff et al., 1977), in addition to allowing the male to make penile intromission, which provides VCS. It is thus possible that clitoral stimulation in the rat could carry reward-related sensory inputs that are generated externally from the pelvic thrusting of the male during a mount and mounts with intromission. In addition to CPP, paced copulation with intromission, or artificial distributed VCS, induces a faster termination of estrus (Coopersmith et al., 1996a,b; Erskine and Baum, 1982; Lodder and Zeilmaker, 1976; Pfaus et al., 2000) and pseudopregnancy (Coopersmith and Erskine, 1994; Erskine et al., 2004; Lehmann and Erskine, 2004; Terkel, 1986). VCS activates the immediate-early gene product Fos in lumbar and sacral regions of the spinal cord, and in hypothalamic and limbic regions of the brain such as mPOA, posteriodorsal MEA (MEApd), bed nucleus of the stria terminalis (BNST), paraventricular nucleus (PVN), and VMH (Dudley et al., 1992; Erskine 1993; Pfaus et al., 1993, 1996; Pfaus and Heeb, 1997; Tetel et al., 1993; Wersinger et al., 1993). These regions also show differential activation in response to distributed VCS under different hormone priming regimens (Pfaus et al., 1996). Olfactory stimuli associated with paced copulation also induce Fos in those brain regions (Coria-Avila and Pfaus, 2007). One study using transneuronal tracing following pseudorabies virus injections to the clitoris has shown afferent input to lumbar and sacral spinal cord, and regions of the hypothalamus such as mPOA, PVN, and VMH (Marson, 1995), suggesting that clitoral stimulation (CLS) and VCS could carry a complex array of information relevant for reward and reproduction. Given the lack of information on the functional role of the rat clitoris in sexual behavior, the present study asked whether different types of CLS (continuous vs. distributed) could induce a reward state of sufficient intensity to induce CPP. We also examined where those two types of CLS induced Fos within hypothalamic and limbic structures previously shown to be activated by VCS or paced copulation. Materials and methods Animals and surgery Sexually naive female Long–Evans rats, weighing 200 to 250 g, were obtained from Charles River Canada, Inc., St. Constant, QC. Animals were housed in groups of two in shoebox cages in a colony room maintained on a reversed 12:12 h light/dark cycle (lights off at 08:00 h) at approximately 21 °C. Food and water were continuously available. Females were ovariectomized bilaterally through lumbar incisions un-der intraperitoneal (i.p.) injections (1 ml/kg of body weight) of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (4 mg/ml) anesthetic mixed in a ratio of 4:3 respectively. All females were given 1 week of post surgical recovery and maintained for the duration of the experiment on hormone replacement by subcutaneous injections of estradiol benzoate (EB; 10 μg in 0.1 ml of sesame oil) 48 h and progesterone (P; 500 μg in 0.1 ml of sesame oil) 4 h prior to testing. Long–Evans males from the same breeder were given 10 prior sexual experiences with sexually receptive females to generate maximal baseline rates of sexual responding (n = 20 per group). All animal procedures conformed to the guidelines of the Canadian Council on Animal Care, and were approved by the Concordia University Animal Research Ethics Committee. CPP apparatus Conditioning was conducted in 8 identical tri-colored PVC plastic rectangular boxes (21 × 21 × 68 cm), each containing three chambers separated by guillotine doors (Med-Associates Inc; St. Albans, VT). The two large end chambers were separated by a smaller center choice chamber, which was used on the pre-exposure and test days. One of the main chambers had black walls and a wire mesh floor; the other had a white wall located across from the guillotine door and a stainless steel rod floor with larger spaces. All floors were raised 5 cm to reduce the accumulation of urine and feces. Through the use of a computer interface, time spent in each chamber was recorded by means of infrared beam crossings. In each of the two end chambers, there were two beams separated by 8 cm. A rat was considered to be in an end chamber if the beam furthest from the door was broken. If only the beam closest to the door was broken, the rat was said to be in the center choice chamber. During conditioning and testing, the room was kept in semidarkness with only a single lamp reflecting light off one wall of the room. Conditioning procedure All conditioning and testing was conducted at 4-day intervals, 4 h after P injections, during the middle third of the rats' dark circadian cycle (as in Pfaus et al., 1999). The place conditioning procedure consisted of three phases: pre-exposure, conditioning, and a final CPP test. During the pre-exposure phase, each female was placed in the center choice chamber of the CPP box with the guillotine doors removed to allow access to the entire apparatus for 15 min. The amount of time spent in each chamber was monitored and used to assess unconditioned preferences. During the conditioning phase, females were randomly assigned to one of three stimulation conditions. The first group (n = 20) received continuous clitoral stimulation (CLS 1). CLS was applied once per second over the course of 1 min, after which females were placed into the previously non-preferred side of the CPP box for 2 min, after which they were removed and given CLS again. The cycle of CLS for 1 min and placement into the CPP apparatus for 2 min was repeated for a total of 5 cycles which totaled 300 stimulations. A second group (n = 20) received distributed CLS (CLS 5). This consisted of 1 CLS every 5 s for 1 min, after which females were placed in the previously non-preferred side of the apparatus for 2 min. The cycle of CLS and placement into the CPP apparatus was repeated for a total of 5 cycles which totaled 60 stimulations. A third group (n = 19) received sham (CNTL) clitoral stimulation prior to placement into the CPP apparatus. Each of the conditioning sessions lasted a total of 15 min per animal. On alternate days females received general handling stimulation, but no clitoral stimulation, and were placed into the previously preferred side of the CPP apparatus. Clitoral stimulations were given by the experimenter. The stimulations were made by lifting the base of the female's tail and lightly brushing the clitoris with one stroke of a small, soft-bristle number 4 paintbrush dabbed with K-Y® jelly. As with manually applied stimuli, the intensity was likely not exactly the same every time; however care was taken to avoid brushing too strongly and to avoid stimulating the vagina. Sham stimulations were done by lifting the base of the tail but not touching the clitoris. Stimulation parameters were chosen specifically to examine distributed vs. continuous sensory stimulation of the clitoris. The 5-s interval was chosen on the basis of unpublished observations that fully primed female rats in bilevel chambers receive mounts with pelvic thrusting in bouts with approximately 5 s between successive mounts before an intromission is achieved. The 1-s interval was therefore chosen to provide continuous stimulation relative to the more distributed stimulation that occurs under normal circumstances. After 10 conditioning sessions, 5 reinforced and 5 non-reinforced, a final 15-minute preference test was conducted 24-hours after the last conditioning trial in the same manner as the initial preference test. 114 M. Parada et al. / Hormones and Behavior 57 (2010) 112–118 effects of conditioning on the activation of Fos by CLS). Females remained in the cage and were sacrificed 1-hour following the initiation of CLS and perfused to prepare tissue for immunocytochemistry. Histology and immunocytochemistry All rats were sacrificed by overdose of sodium pentobarbital (120 mg/kg, i.p.) and perfused intracardially with phosphate buffered saline (250 ml) and followed by 4% paraformaldehyde in 0.1 M phosphate buffer (250 ml). Brains were extracted and placed in fresh 4% paraformaldehyde for 4 hours and then overnight in 30% sucrose at 4 °C. Frozen coronal brain sections (40 μm) were cut from each brain through the medial prefrontal cortext and to the level of the ventral tegmental area on a cryostat. The sections were washed in cold Trisbuffered saline (TBS) and incubated sequentially with 30% hydrogen peroxide (H2O2) in TBS for 30 min at room temperature with 3% Normal goat serum (NGS) in 0.05% Triton-TBS for 90 min at 4 °C, with rabbit polyclonal anti-Fos (Fos ab5, Calbiochem, Mississauga, ON; diluted 1:40,000) in 0.05% Triton-TBS with 3% NGS for 72 h at 4 °C, with biotinylated goat anti-rabbit IgG (Vector Laboratories Canada, Burlington, ON; 1:200) in 0.05% Trition-TBS with 3% NGS for 1 h at 4 °C, and avidin–biotinylated–peroxidase complex (Vectastain ELITE ABC KIT, Vector Laboratories Canada; diluted 1:55) for 2 h at 4 °C. Sections were washed in TBS (3 × 5 min) between each incubation. Immunoreactions were stained by sequential treatments at room temperature with 50 mM Tris for 10 min, 3,3′-diaminobenzidine (DAB) in 50 mM Tris (0.1 ml of DAB/Tris buffer, pH 7.8) for 10 min, and 8% nickel chloride (400 μl per 100 ml of DAB/Tris buffer + H2O2). Sections were then mounted on gel-coated slides and allowed to dry, then dehydrated in alcohols (70%, 90% and 100%, 10 min each, respectively), cleared in Xyelines (2 h), coverslipped, and examined under a Leitz light microscope. Histological and statistical analyses Statistical analysis for CPP Pre-exposure and CPP test outcomes were determined by the total time spent in each chamber. To ensure that there was no initial bias of the chambers, a within-subjects repeated measures ANOVA was used to assess the effect of Chamber. Based on Paredes and Alonso (1997) there were two criteria used to determine whether place preference conditioning was successful. A preference score (time in the reinforced compartment / (time in the reinforced compartment + time in the non-reinforced compartment) was expected to increase significantly between pre-test and post-test. To account for the possibility that the rats are spending most of their time in the grey (neutral) chamber, a difference score was also calculated (difference between the time spent in the non-reinforced cage and time spent in the reinforced cage) and was expected to decrease significantly after conditioning. Preference scores and difference scores were analyzed for each stimulation type across pre-test and post-test using a paired-samples t-test to examine the change in scores prior to, and following, clitoral stimulation (Dominguez-Salazar et al., 2005; Meerts and Clark, 2007; Meerts and Clark, 2009). As per Paredes and Alonso (1997) a significant increase in the preference score and a significant decrease in the difference score are the main criteria when a conditioned place preference is developed. Fos activation by CLS One week following the CPP experiment the same females were primed with EB and P as above, placed in a Plexiglas cage identical to their home cages (36 cm × 26 cm × 19 cm), and allowed to acclimate for 20 min, after which females were randomly re-assigned to one of the three CLS groups described above (to eliminate any previous Tissue sections were examined at 40×, and the number of Fospositive cells was counted bilaterally for each region from 3 sections per area per rat using Scion Image 1.63. The regions were defined by using the atlas of Paxinos and Watson (1986): mPOA (medial preoptic area), VMH (ventromedial hypothalamus), MeA (medial amygdala), BLA (basolateral amygdala), NAcc (nucleus accumbens), VTA (ventral tegmental area), PirCtx (piriform cortex), LH (lateral habenula), BNST (basal nucleus of the stria terminalis), and ArcN (Arcuate Nucleus). A mean was calculated for each area in each rat from the 3 bilateral sections per area, and statistical analyses was conducted for 5 rats in each stimulation group (15 sections per group for each brain area, as Table 1 Average numbers of Fos-positive cells in different hypothalamic and limbic structures as a function of sexual stimulation (mean ± SEM). Area Nacc shell core mPOA LS BNST VMH Arc MEApv LH PirCtx VTA Stimulation condition CNTL 45.9 ± 9.7 33.2 ± 9.5 16.8 ± 1.8 17.7 ± 5.9 6.7 ± 4.5 32.3 ± 6.3 37.0 ± 6.6 39.4 ± 6.5 20.8 ± 5.4 91.0 ± 10.8 57.0 ± 11.0 CLS 1 38.1 ± 8.9 56.6 ± 18.9 17.05 ± 1.9 28.7 ± 12.3 12.8 ± 5.6 42.7 ± 14.6 40.4 ± 7.5 123.2 ± 27.2⁎ 28.5 ± 7.7 140.6 ± 26.5 47.0 ± 9.2 CLS 5 64.9 ± 17.5 53.4 ± 14.6 59.7 ± 8.2⁎ 37.9 ± 7.3 19.7 ± 4.3 29.1 ± 2.6 50.7 ± 4.5 82.6 ± 15.6 27.7 ± 5.8 83.0 ± 13.1 56.4 ± 20.2 Fig. 1. Mean + SEM preference scores (top) and difference scores (bottom) on the pretest (pre-conditioning, white bars) and test (post-conditioning, black bars) for female rats that received sham stimulation (CNTL), continuous stimulation (CLS 1), or distributed stimulation (CLS 5) (n = 20 per group). Rats receiving distributed stimulation displayed CPP for the compartment associated with that treatment. This coincides with a significant decrease in difference score, ⁎p b 0.05. CNTL, sham stimulation; CLS 1, continuous clitoral stimulation; CLS 5, distributed clitoral stimulation (n = 5 per group). ⁎ p b 0.05 from CNTL. M. Parada et al. / Hormones and Behavior 57 (2010) 112–118 115 we have done previously for Fos induction by VCS; Pfaus et al., 1993, 1996). A between-subjects analysis of variance (ANOVA) was performed to assess differences in Fos induction between females that received CLS 1, CLS 5, and sham stimulation. For each significant ANOVA, post hoc analysis of mean differences was made using the least significant difference (LSD) method, p b 0.05. Results CPP associated with manual clitoral stimulation Rats that received CLS 5 (distributed CLS) showed both a significant increase in preference scores at the post-test following the conditioning trails (t (19) = − 3.394, p b 0.05) and a significant decrease in difference scores (t (19) = 3.213, p b 0.05) (Fig. 1). Rats that received CLS 1 (continuous CLS) or sham stimulation did not show any significant change in preference or difference scores. Fos induction by CLS Examination of brain sections from the medial prefrontal cortex back to the level of the ventral tegmental area of the midbrain revealed clusters of Fos in areas associated with tactile genitosensory stimulation. Specifically Fos was found in and around the mPOA, VMH, MEA, BLA, NAcc, VTA, PirCtx, LH, BNST, and ArcN (see Table 1). Although clusters of Fos were present in these areas only the mPOA Fig. 2. Representative digitized images of the mPOA (left side) and MEApv (right side) taken at 40×. Areas that were analyzed for Fos IR are outlined in black. Fos activation for the mPOA as a function of stimulation type (CLS 5: distributed; CLS 1: continuous, CTRL: sham stimulation) are displayed along the left side and Fos activation for the MEApv is displayed along the right side. 3 V, third ventricle; opt, optic tract. 116 M. Parada et al. / Hormones and Behavior 57 (2010) 112–118 Fig. 3. Mean number of Fos cells/side (+SEM) as a function of different types of CLS are shown for the mPOA (top) and MEApv (bottom) for females receiving no stimulation (CNTL), continuous stimulation (CLS 1), and distributed stimulation (CLS 5) (n = 5/ stimulation condition). Rats receiving distributed stimulation showed a significantly greater number of Fos cells in the mPOA than rats receiving continuous stimulation and no stimulation. Rats receiving continuous stimulation showed a significantly greater number of Fos cells in the MEApv compared to no stimulation, ⁎p b 0.05. and the MEApv had significantly different levels of Fos in response to the type of CLS received. Fos in the mPOA In the mPOA (Fig. 2), distributed CLS produced a clustered pattern of activation located around the medial, central, and most ventral aspect of the medial preoptic nucleus, with a scattering of induction in the medial, dorsal aspect of this nucleus. This pattern of activation was not induced by continuous or sham CLS. For the induction in the mPOA, the ANOVA detected a significant main effect of CLS, F (2,15) = 19.388, p b 0.0001. Post-hoc comparisons revealed that only distributed CLS induced Fos over that observed in the continuous CLS and control groups (Fig. 3). Fos in the MEA In the MEA (Fig. 2), both distributed and continuous CLS induced a cluster of Fos activation located within the ventromedial portion of the MEApv only, with scattered Fos cells radiating laterally away from this cluster. Little to no Fos cells were observed in other regions of the MEA including the MEApd. For the induction in the MEApv, the ANOVA detected a significant main effect of CLS, F (2, 15) = 5.104, p b 0.05. Post-hoc comparisons revealed that continuous CLS produced a significantly greater amount of Fos activation in the MEApv compared to the control group (Fig. 3). Discussion The present study demonstrates both a behavioral and neural response to clitoral stimulation in the rat. This study identified manual distributed CLS as a strong inducer of sexual CPP and also that CLS induces Fos protein within regions of the brain generally associated with tactile genitosensory stimulation when females are in a receptive state. Further, the ability of CLS to induce Fos-immunoreactivity in the mPOA and the MEApv is dependant on the type of CLS that is received by the female, with distributed CLS resulting in a significantly larger number of cells with Fos in the mPOA, and continuous CLS resulting in a significantly larger number of cells with Fos in the MEApv. We suggest that the rat clitoris, like that of the human, carries reward-related tactile stimulation during sexual interaction to the brain. In the rat, however, a distributed form of stimulation is necessary to induce reward of sufficient intensity to support CPP, similar to recent studies using VCS as the tactile cue (Meerts and Clark, 2009). We speculate that the clitoris may well be stimulated by pelvic thrusting during mounts, intromissions and ejaculations during copulation. Thus, together with VCS, CLS may form an important component of sexual reward. Distributed CLS induced a significantly greater number of Fos cells in the mPOA compared to continuous CLS and controls. It has been shown previously that mechanical stimulation of the cervix excites neurons in the mPOA (Haskins and Moss, 1983). Further, expression of Fos immunoreactivity has also been shown in this region in response to vaginocervical stimulation both from a male partner during copulation and from artificial manual stimulation (Pfaus et al., 1993, 1996; Tetel et al., 1993, 1994a; Wersinger et al., 1993). It is interesting that different subregions of the mPOA appear to be activated by CLS and VCS. Whereas VCS activates a central and medial region (Pfaus et al., 1996), CLS activates a more ventral region of the mPOA. This suggests a specific area of activation for clitoral stimulation. This also suggests that clitoral stimulation during copulation may be independent of other forms of tactile stimulation (e.g., flank stimulation), and may contribute information to the mPOA about the timing of pleasurable anogenital sensations. A similar effect was observed within the MEA. Previous work has shown that VCS activates Fos in cells of the MEApd in a linear fashion, with more activation in response to increasing amounts of stimulation (Erskine, 1993; Pfaus et al., 1993, 1996; Polston and Erskine, 1995; Tetel et al., 1993). However the present study did not find Fos activation in the MEApd, but rather in the posteroventral MEA (MEApv) in response to continuous CLS. This suggests that specific regions of the MEA respond to different forms of genitosensory stimulation with VCS activating the MEApd and CLS activating the MEApv. The idea that separate regions of the amygdala contribute differentially to female sexual behavior is not new. Lesion studies of the corticomedial amygdala have reported a reduction of the lordosis response whereas lesions of the basolateral and lateral nuclei of the amygdala facilitate lordosis (Masco and Carrer, 1983; McGinnis et al., 1978; Nance et al., 1974). Neural implants of estrogen to the MEApd have been shown to facilitate lordosis (Lisk and Barfield, 1975) and VCS in females primed with estrogen results in an increase in Fos-immunoreactivity in this same region but only to threshold levels in the MEApv (Pfaus et al., 1996). Lidocaine infusions to the medial amygdala, which include regions around the MEApv and MEApd, block the induction of pseudopregnancy when administered both before and after receiving intromissions, which has been suggested to provide evidence that the MEApd may modulate neuroendocrine and behavioral functions that are activated by VCS (Coopersmith et al., 1996a,b; Pfaus et al., 1996). It is possible that activation of the MEApv by a large amount of CLS may also contribute to the modulation of neuroendocrine and behavioral functions. Like distributed CLS, distributed VCS induces significant CPP (Meerts and Clark, 2009). There is considerable overlap in the sensory innervation of both clitoris and vagina, including the proximal perineal branch of the sacral plexus that innervates the skin between the clitoris and vagina, the viscerocutaneous branch of the pelvic nerve, and the distal perineal branch of the pudendal nerve that M. Parada et al. / Hormones and Behavior 57 (2010) 112–118 117 innervates the skin between the vagina and anus, and the distal perineal branch of the pudendal nerve and the proximal perineal branch of the sacral plexus that innervates the skin lateral to the vaginal opening (Cruz et al., 2004). Cruz et al. (2004) suggested that such overlap might help to ensure that somatosensory information necessary to trigger reproductive and non-reproductive reflexes actually reaches the spinal cord and brain. It is therefore possible that a mount with intromission and pelvic thrusting from a male rat activates the clitoris, vagina, and cervix of the female, thus activating reward- and reproductive-related circuits of the spinal cord and brain simultaneously, including the mPOA and VMH. It is important to note that although CLS and VCS are strong inducers of CPP and thus sexual reward during copulation, it is possible that pacing copulation or distributing stimulation to induce pseudopregnancy drives reward. Females may pace the behavior to ensure that enough intromissions occur to induce ejaculation and pregnancy, and that this may be “more” rewarding than the behavior itself. It is also possible that the reward value of the somatosensory stimulation drives the reproductive consequences, or that the two occur by simultaneous activation of reward- and reproductive-related brain systems. It would be interesting to examine pseudorabies virus traces at different times after injection, given that the virus travels approximately 1 synapse per day. It would also be instructive to perform selective unilateral lesions of reward-(e.g., VTA, NAcc, mPOA, VMH) and reproductive-(VMH, mPOA, MEApd)related brain regions prior to the pseudorabies virus injection, to examine the actual synaptic flow of afferent information. It is also important to note that the stimulations given in this experiment differed not only in terms of distribution but the total number received during a given conditioning session. Females in the distributed group received a total of 60 clitoral stimulations during the 15-minute period in contrast to the 300 stimulations received by females in the continuous group. It is therefore possible that CPP developed as a result of the total number of stimulations rather than the pattern of distribution. However, in this case, the results of the current study would suggest that more is less in terms of the activation of CPP and Fos in the mPOA, a site that plays a critical role in linking sexual reward to appetitive sexual responses, such as solicitations in females (Hoshina et al., 1994; Paredes, 2009). This is in contrast to studies that have revealed a positive relationship between the amount or intensity of VCS and the induction of Fos in various brain regions (Erskine, 1993; Pfaus et al., 1993, 1996; Tetel et al., 1993; Wersinger et al., 1993), and strongly suggests that an optimal timing exists for the conduction of reward-related CLS. Finally, the parameters for the stimulation conditions used in the present study were chosen specifically to examine continuous vs. distributed CLS. They were based on unpublished observations from our laboratory that mounting stimulation often occurs in bouts that consist of several trains of pelvic thrusting prior to intromission and dismount. We do not yet know whether the stimulation parameters used here were in any way optimal for particular behavioral end-points (e.g., CPP, partner preference, estrus termination, etc.). It is important to acknowledge that there is inconsistency throughout the literature regarding artificial VCS and its method of application, the intensity of the stimulation, and the timing which makes it difficult to approximate what a female rat would experience in the real world. Attempts have been made to mimic the timing of intromissions to what is observed on average during a copulatory bout (Meerts and Clark, 2009) or to compare Fos activation from differing amounts of artificial VCS to what is seen following an hour of copulation (Pfaus et al., 1993). However, it is not known whether the same temporal parameters used for artificial VCS would induce a similar pattern of Fos induction with CLS. The present study found statistical trends toward activation of other regions (e.g., BNST, LS, PirCtx), and it may be the case that other patterns of CLS will result in significant increases (or decreases) in Fos induction in those regions. Further work will need to address the optimization of CLS, although it may be the case that individual differences will exist for female rats in the optimization of CLS for reward, as is acknowledged in women (Masters et al., 1986). In summary, the present study confirms that clitoral stimulation is a form of tactile genitosensory stimulation that has a reinforcing value strong enough to induce CPP. This type of conditioned preference occurs when the stimulation is distributed rather than continuous, and induces Fos-immunoreactivity in areas previously associated with the processing of genitosensory stimulation for reward, estrus termination, and pseudopregnancy. Throughout a copulatory bout females typically receive both CLS and VCS from mounts. CLS likely occurs when the male's testicles or pelvis come into contact with the clitoris during pelvic thrusting, whereas VCS occurs during intromissions and ejaculations. This would stimulate both the vagina and cervix and also the clitoris through internal contact with the nerves that feed into it. Because the innervation of the clitoris and vagina share common sensory pathways in humans and other animals (e.g., Foldes and Buisson, 2009), receiving both distributed CLS combined with VCS may be an important sensory experience that links reward with reproduction. Acknowledgments Supported by Discovery Grant OGP-138878 from the Natural Sciences and Engineering Research Council (NSERC) of Canada to JGP. References Coopersmith, C., Erskine, M.S., 1994. Influence of paced mating and number of intromissions on fertility in the laboratory rat. J. Reprod. Fertil. 102, 451–458. Coopersmith, C., Candurra, C., Erskine, M.S., 1996a. 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