Co-localization of midbrain projections, progestin receptors, and mating-induced fos in the hypothalamic ventromedial nucleus of the female rat

Hormones and Behavior 50 (2006) 52 – 60 www.elsevier.com/locate/yhbeh

Co-localization of midbrain projections, progestin receptors, and mating-induced fos in the hypothalamic ventromedial nucleus of the female rat Loretta M. Flanagan-Cato ⁎, Beney J. Lee, Lyngine H. Calizo Department of Psychology, Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA Received 9 May 2005; revised 19 January 2006; accepted 19 January 2006 Available online 20 March 2006

Abstract In female rats, sexual behavior requires the convergence of ovarian hormone signals, namely estradiol and progesterone, and sensory cues from the male on a motor output pathway. Estrogen and progestin receptors (ER and PR) are found in neurons in the hypothalamic ventromedial nucleus (VMH), a brain region necessary for lordosis, the stereotypic female copulatory posture. A subset of VMH neurons sends axonal projections to the periaqueductal gray (PAG) to initiate a motor output relay, and some of these projection neurons express PR. Previous studies showed that VMH neurons are activated during mating, based on the expression of the immediate early gene Fos. Many of the activated neurons expressed ER; however, it is not known if such activated neurons co-express PR. Fluorogold, a retrograde tracer, was injected into the PAG of ovariectomized rats to label neurons projecting from the VMH. Hormone-treated animals then were mated, and their brains were immunohistochemically stained for PR and Fos. Of the Fos-positive neurons, 33% were double-labeled for PR, 19% were double-labeled with Fluorogold, and 5% were triple-labeled for Fos, PR, and the retrograde tracer. The majority of triple-labeled neurons were found in the rostral, rather than caudal, portion of the VMH. These results show that PR-containing neurons are engaged during sexual behavior, which suggests that these neurons are the loci of hormonal–sensory convergence and hormonal–motor integration. © 2006 Elsevier Inc. All rights reserved. Keywords: Fos; Lordosis; Periaqueductal gray; Progesterone receptor; Sexual behavior; Ventromedial hypothalamus

Introduction Sexual behavior in female rodents includes lordosis, a stereotypic copulatory stance. The lordosis response depends on sequential exposure to the ovarian hormones, estradiol and progesterone, and is triggered by sensory cues originating from a male (Kow, 1976; Pfaff and Sakuma, 1979b; Pfaff et al., 1977; Pleim et al., 1989). During the estrous cycle, a gradual rise in estradiol levels is followed by a rapid increase in progesterone secretion. Although estradiol alone can promote sexual behavior, the subsequently elevated progesterone level is needed for the full expression of mating behavior (Barfield et al., 1984; Beach, 1942; Boling and Blandau, 1939; Rubin and Barfield, 1983). These hormones exert their behavioral effects ⁎ Corresponding author. Fax: 215 898 7301. E-mail address: [email protected] (L.M. Flanagan-Cato). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.01.012

in the ventrolateral region of the hypothalamic ventromedial nucleus (vlVMH) (Davis et al., 1982; Pleim et al., 1989; Rubin and Barfield, 1983), and receptors for both estradiol and progesterone are found in neurons in the vlVMH (Blaustein et al., 1988; DonCarlos et al., 1991; MacLusky and McEwen, 1980; Pfaff and Keiner, 1973; Simerly et al., 1990). The importance of progesterone receptor (PR) in the VMH for female reproductive behavior has been demonstrated by local infusion of either PR anti-sense oligonucleotides or a PR antagonist into the vlVMH, both of which reduced lordosis behavior (Etgen and Barfield, 1986; Mani et al., 1994; Ogawa et al., 1994). Our laboratory is interested in the configuration of neuronal connections within the vlVMH and how various neural elements may contribute to the control of reproductive behavior. An important type of vlVMH neuron sends descending projections to the periaqueductal gray (PAG)

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(Canteras et al., 1994) as part of the motor output pathway critical for lordosis (Daniels et al., 1999; Hennessey et al., 1990; Sakuma and Akaishi, 1987; Sakuma and Pfaff, 1979, 1980, 1982). Previous research has found a modest colocalization of hormone receptor-containing neurons and PAG-projecting neurons (Akesson et al., 1994; Calizo and Flanagan-Cato, 2002, 2003; DonCarlos and Morrell, 1990; Morrell and Pfaff, 1982; Nielsen Ricciardi and Blaustein, 1994). VMH projection neurons that contain PR are of particular interest because this neural population represents the integration of a hormonal signal with a neural substrate directly linked to motor pathways. A second type of vlVMH neuron expresses estrogen receptors (ER). Estrogenic action up-regulates PR in the vlVMH in neurons that co-express ER (Blaustein and Turcotte, 1989; MacLusky and McEwen, 1978; Pleim et al., 1989; Romano et al., 1989; Warembourg et al., 1989). Thus, all neurons that express PR also contain ER, but there also may be ER-only neurons. Given that most ERcontaining neurons do not send axonal projections to the PAG, they may influence the output neurons through local synaptic connections. ER-containing neurons are among the neurons activated during mating behavior, as demonstrated by the co-localization of ER and Fos (Calizo and FlanaganCato, 2003). However, it remains uncertain whether these activated neurons co-express PR or represent the ER-only neurons. To distinguish between these two possibilities, this experiment used retrograde tracing and immunohistochemical staining for PR and mating-induced Fos. Mating behavior activates vlVMH neurons, as inferred by the induction of immediate early genes, including Fos and egr1 (Coolen et al., 1996; Flanagan-Cato and McEwen, 1995; Pfaus et al., 1993; Polston and Erskine, 1995; Tetel et al., 1993; Yang et al., 1999). If progesterone-sensitive ER-containing neurons were recruited during mating behavior, one would expect Fos to be co-localized with PR in proportions similar to the Fos-ER colocalization previously observed. Alternatively, if ER-only neurons were selectively activated during mating behavior, one would expect that Fos would not be found in PRcontaining neurons. Materials and methods Animals The brain sections used in this study came from animals used in a previous study (Calizo and Flanagan-Cato, 2003). Adult female Sprague–Dawley rats (n = 5) were housed in plastic tubs with standard bedding and with food and water continuously available. The temperature of the colony was maintained at 22°C with a 12:12 light:dark cycle. Animals were allowed at least 1 week to acclimate to the colony before any procedures were performed. The Institutional Animal Care and Use Committee of the University of Pennsylvania approved all procedures with animals.

Surgeries Ovariectomies were performed on all animals during anesthesia (33 mg/kg ketamine and 13 mg/kg xylazine, intraperitoneal) using aseptic surgical

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procedures. One week after ovariectomy, stereotaxic surgery was performed during anesthesia (50 mg/kg ketamine and 20 mg/kg xylazine, intraperitoneal). A solution of 2% Fluorogold in saline (0.3–0.5 μl) was unilaterally pressureinjected into the periaqueductal gray (PAG) over 15 min using a 28-gauge injector cannula (Plastics One; Roanoke, VA) connected with PE10 tubing to a 1-μl Hamilton syringe. Infusions were made at the following coordinates: 7.8 mm posterior and 0.08 mm lateral to Bregma and 5.7 mm ventral from the surface of the skull, targeting the caudal lateral/ventrolateral PAG. The cannula was left in place 5 min after the tracer infusion was completed and then removed. The incision then was closed with surgical staples, and each animal was returned to her home cage after recovery from anesthesia. The five animals used in this study all had injections that were accurate, discrete, without overlap with the aqueduct, and that allowed concomitant visualization of PR and Fos immunostaining.

Hormone treatment Hormone treatments began 1 day after tracer injection. Hormone treatments consisted of subcutaneous injections of estradiol benzoate (EB, 10 μg in 100 μl sesame oil, Sigma, St. Louis, MO) for two consecutive days. A subcutaneous injection of progesterone (P, 500 μg in 100 μl propylene glycol, Sigma, St. Louis, MO) was administered 48 h after the second EB injection. Four and a half hours after progesterone treatment, behavioral testing began.

Behavioral experience All behavioral testing occurred during the dark phase under dim red illumination. Animals were placed in a test cage (1.2 × 0.9 × 0.4 m, length × width × height) with one of four sexually experienced male rats. Females were mated for 50 min. Individual males were replaced either after ejaculation or 5 min of inactivity to ensure that each female received a minimum of 35 intromissions during the testing period, including at least 10 intromissions within the first 15 min of testing. The numbers of mounts, intromissions, and ejaculations by the male and lordosis responses performed by the female were recorded. The lordosis quotient (LQ = # lordosis/# mounts received × 100) also was determined for each female.

Perfusion and sectioning After behavioral testing, animals were anesthetized (50 mg/kg ketamine and 20 mg/kg xylazine, intraperitoneal). They were perfused 60 to 75 min after the beginning of behavioral testing transcardially with 100 ml saline followed by 200 ml 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA). The brains were isolated, post-fixed in paraformaldehyde overnight at 4°C, then submerged in 20% sucrose in 0.1 M phosphate buffer. Coronal sections encompassing the rostrocaudal extent of the VMH were cut on a freezing microtome into four serial sets of 30-μm-thick sections. One set (7–9 sections) from each animal underwent immunocytochemical staining and analysis. Coronal sections of the PAG also were cut into four serial sets of 30-μmthick sections on a freezing microtome. PAG sections were mounted on slides, air-dried, and coverslipped in DPX mounting media (Electron Microscopy Sciences; Fort Washington, PA). The location of each injection site within the PAG was documented with fluorescence microscopy.

Immunocytochemistry Sections were washed in Tris-buffered saline (TBS; pH 7.4) then incubated with a progesterone receptor antibody (1:1000, MAB462, mouse, Chemicon, Temecula, CA) and a Fos antibody (1:20,000, sc-52, rabbit; Santa Cruz Biotechnology, Santa Cruz, CA) in TBS with 0.2% TritonX-100 and 3% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) overnight at 4°C. After several washes, sections were incubated in cy3-conjugated donkey antimouse antiserum (1:100, Jackson ImmunoResearch) and cy2-conjugated donkey anti-rabbit antiserum (1:50, Jackson ImmunoResearch) in TBS with 0.2% TritonX-100 and 3% normal donkey serum (Jackson ImmunoResearch) for 2 h at room temperature. After several washes, sections were mounted on

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slides, air-dried, and coverslipped with DPX mounting media (Electron Microscopy Sciences). The rat PR is expressed in two isoforms: a full-length form (PR-B) and an Nterminally truncated form (PR-A). Both PR isoforms are derived from the same gene and are generated from either alternative transcriptional or translational start sites (Conneely et al., 1989; Kastner et al., 1990). In the hypothalamus, estradiol up-regulates both forms, and progesterone treatment down-regulates both (Guerra-Araiza et al., 2003). Therefore, an antibody was employed that detected a peptide sequence present in both forms of PR. To determine possible antibody cross-reactivity, brain sections from a randomly chosen animal were incubated in both of the secondary antibodies using the same staining conditions described above with either the Fos primary antibody or the PR primary antibody removed from the staining procedure. Labeling for the excluded antibody was eliminated, thus confirming the lack of cross-reactivity.

Image analysis Images were acquired with a digital camera (Diagnostic Instruments, Sterling Heights, MI, model RTKE) from hemisections ipsilateral to the Fluorogold injection site. For each animal, digital micrographs for the individual single labels, PR (cy3), Fos (cy2), and Fluorogold, were imported into Adobe Photoshop 4.0 for nine to twelve immunocytochemically labeled sections per animal using the 20× objective. To ensure that images of individual labels would be accurately aligned for the determination of double or triple labeling, care was taken to prevent changes in the location of each section for all sets of digital micrographs. Images for an individual animal were taken on the same day, maintaining the same camera settings for a given label. However, different animals were photographed on different days and the camera settings were adjusted for each animal so that the digital image matched that seen in the microscope. The cluster of PR-IR neurons is located along the ventrolateral edge of the vlVMH. Mating-induced Fos is expressed in the vlVMH, but not the dorsomedial VMH (Flanagan et al., 1993; Pfaus et al., 1993; Polston and Erskine, 1995; Yang et al., 1999). The VMH PR cluster was delimited by one 400 × 400 μm square box centered on the cluster of PR-stained cells (Fig. 1). A threshold optical density was established to standardize quantification of labeled objects. In particular, a minimum level of average pixel intensity (optical density) was set and only objects with an average pixel intensity above this level were counted as labeled cells. Pixel intensity was measured using the public domain program NIH Image 1.62 (http://rsb.info.nih.gov/nih-image/). For each label (PR, Fos, and Fluorogold), the average pixel intensity value from at least five cells and three corresponding background regions of the same size and area were taken from one hemisection from each animal. Only labeled objects with an average pixel intensity value two standard deviations above the mean background pixel intensity were counted as labeled cells. This minimum pixel intensity was determined for each label from each animal to account for any individual differences during digital photography. Labeled cells within the VMH PR cluster were counted manually in triplicate from the digitized images. To determine whether any specific cell was double- or triple-labeled, digital images of sections containing single labels, cy3 (PR), cy2 (Fos), and Fluorogold, were placed into individual image layers that were then overlaid using Adobe Photoshop 4.0. A change in the color of a cell was not always an accurate indicator of double labeling because background labeling in one layer could at times alter the appearance of an individual cell on the overlaid image such that a single-labeled cell could appear double-labeled. Alternatively, a brightly labeled cell in one layer could obscure the appearance of a second label on the overlaid image. Thus, double or triple labeling was determined by comparing the overlaid image with the individual layers making up the overlay. A cell was considered to be double- or triple-labeled only if a matching cell was found labeled in another layer.

Statistical analysis The average numbers of labeled cells per hemisection and behavioral data are expressed as the mean ± SEM. Two statistical comparisons were performed. First, the percentage of Fos-PR double labeling compared with the percentage of

Fig. 1. Illustration of the Fluorogold injection sites in the PAG. The injection sites were composed of a necrotic center (dark gray circles) surrounded by a bright halo of dye (light gray circles) that covered approximately half of the PAG hemisection ipsilateral to the injection. The sizes of the necrotic centers were proportional to the surrounding halos. Drawings are based on Paxinos and Watson (1986). The numbers to the right indicate the coordinates posterior to bregma. The black area indicates the aqueduct. DR refers to the dorsal raphe.

Fos-Fluorogold double labeling was analyzed within these five animals. The paired χ2 test was performed to assess possible differences in these relative distributions. Second, the number of labeled cells per section in the rostral versus caudal aspects of the vlVMH was compared using Student's t test. Significance was set at P < 0.05.

Results Fluorogold injections were localized to the caudal lateral/ ventrolateral PAG, as described previously (Calizo and Flanagan-Cato, 2003). The injection site consisted of a necrotic center (area of necrotic center = 0.058 ± 0.012 mm2) surrounded by a bright halo of dye (area of necrotic center and halo = 0.622 ± 0.075 mm2) that covered approximately half of the PAG hemisection, ipsilateral to the injection site (Fig. 1). Although some dye was seen beyond the PAG, most of the injected Fluorogold was found within the boundaries of the PAG. The Fluorogold injection into the PAG did not interfere with behavioral performance. These females received 112 ± 9 mounts and 61 ± 7 intromissions, and their lordosis quotient was 89 ± 2. Retrogradely labeled projection neurons were located throughout the VMH. As in other studies, PR-containing neurons were clustered along the ventrolateral border of the ventrolateral subdivision of the VMH, and neurons that expressed mating-induced Fos were located within the cluster of PR-containing neurons (Fig. 2). Single-, double-, and triplelabeled neurons were clearly discernable. Although somewhat scattered, these neurons were within the confines of the cluster

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Fig. 3. Bar graph illustrating the distribution of vlVMH neurons that project to the PAG. Numbers above each bar indicate the percentage of projection neurons with that particular labeling pattern. Abbreviations: FG, Fluorogold; PR, progesterone receptor. Values are expressed as mean ± SEM.

Fig. 2. Micrographs of labeling for PR, Fluorogold, and Fos. PR (red), Fos (green), and Fluorogold (blue/purple). Potentially double- and triple-labeled neurons appear as yellow (PR/Fos), teal (Fos/Fluorogold), pink (PR/Fluorogold), or white (PR/Fos/Fluorogold), although as detailed in Materials and methods, color was not the sole indicator of multiple labeling. Images at high magnification of single, double, and triple labeling. Double or triple labeling was determined by comparing the overlaid image with the individual layers making up the overlay. A cell was counted as double- or triple-labeled only if a corresponding labeled cell was found in another layer. Scale bar = 25 μm.

of PR-containing neurons. On average, there were 73.6 ± 22.0 Fluorogold-labeled neurons/hemisection, 64.5 ± 9.1 PR-labeled neurons/hemisection, and 22.7 ± 3.2 Fos-labeled neurons/ hemisection, all ipsilateral to the Fluorogold injection. PR was somewhat co-localized with the projection neurons. In particular, 12% of the projection neurons were double-labeled with PR, and 13% of the PRimmunoreactive (PR-IR) neurons were double-labeled with Fluorogold. Most Fluorogold-labeled neurons were singly labeled (77%), whereas 6% were double-labeled with mating-induced Fos, and 5% were triple-labeled (Fig. 3). Similarly, of the PR-IR neurons, 70% were singly labeled, 11% co-expressed mating-induced Fos, and only 5% were triple-labeled (Fig. 4). Of the three labels, neurons that expressed mating-induced Fos were the least abundant, yet showed the greatest incidence

of double and triple labeling (Fig. 5). Only 33% were singly labeled, while 33% were double-labeled with PR, 19% were double-labeled with Fluorogold, and 15% were triple-labeled. There was a significantly higher percentage of Fos-PR double labeling compared with Fos-Fluorogold co-labeling (χ2 test, P < 0.02). The distribution of labeling was analyzed in the coronal plane and along the rostral–caudal axis of the vlVMH. In the coronal plane, no segregation of the double- or triple-labeled neurons was discerned, but rather they appeared randomly dispersed among the singly labeled neurons in the dorsal– ventral and medial–lateral axes. To assess the rostrocaudal distribution of labeled neurons, the sections were categorized as rostral (−2.12 to −2.80 mm from Bregma) and caudal (−3.14 to −3.6 mm from Bregma), based on Paxinos and Watson (1986). For each animal, four to six sections were averaged in both rostral and caudal zones. The density of all PR-labeled neurons was 48% higher in the caudal sections (t = −4.13, P < 0.02), and the density of all Fluorogoldlabeled neurons was 79% higher in the rostral sections (t = 5.43, P < 0.01). The number of Fos-labeled neurons per hemisection was not significantly different between the rostral and caudal sections. Nevertheless, there were rostrocaudal differences in the cell-types that co-expressed Fos. In the rostral sections, 23% of the Fos-labeled neurons were triplelabeled. However, in the caudal sections, only 6% of the Fos-

Fig. 4. Bar graph illustrating the distribution of PR-IR neurons. Numbers above each bar indicate the percentage of PR-IR neurons with that particular labeling pattern. Abbreviations: FG, Fluorogold; PR, progesterone receptor. Values are expressed as mean ± SEM.

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Fig. 5. Bar graph illustrating Fos expression in neuronal populations in the vlVMH. Numbers above each bar indicate the percentage of cells in a given population that express mating-induced Fos. Abbreviations: PR, progesterone receptor; FG, Fluorogold; PR−/FG−, neurons that neither contain PR nor project to the PAG; PR+/FG−, PR neurons that do not project to the PAG; PR−/FG+, projection neurons that do not contain PR; PR+/FG+, PR neurons that project to the PAG. Values are expressed as mean ± SEM.

labeled neurons were triple-labeled. Thus, the triple-labeled neurons were found in four-fold higher density in the rostral zone of the vlVMH compared with the caudal zone (t = 3.9, P < 0.02; Fig. 6). Discussion The present study was undertaken to better characterize the network of neurons activated in the vlVMH during mating behavior. A striking proportion (67%) of the Fos-labeled neurons could be classified as PR-containing, PAG-projecting, or both. In addition, rostrocaudal differences in the distribution of PR-, Fluorogold-, and triple-labeled neurons in the vlVMH were documented. The modest overlap of the projection neurons with PR-labeled neurons was consistent with our previous estimates of hormone-sensitive projection neurons (Calizo and Flanagan-Cato, 2002, 2003; Daniels et al., 1999), as well as studies from other laboratories (DonCarlos and Morrell, 1990; Dufourny and Warembourg, 2001; Nielsen Ricciardi and Blaustein, 1994). The implications of these results will be discussed in terms of VMH cell types, mating-induced Fos, and VMH topography. The neurochemical phenotypes of PR-containing and PAGprojecting neurons in the vlVMH have not been well defined. Both glutamate and gamma-aminobutyric acid (GABA), and their respective receptors, have been found in the VMH (Commons et al., 1999; Gratten and Selmanoff, 1997; O'Connor et al., 1988; Schumacher et al., 1989). Moreover, there is electrophysiological and behavioral evidence that both glutamate and GABA are important neurotransmitters within the VMH (Diano et al., 1997; Frankfurt et al., 1984; Jang et al., 2001; Kow and Pfaff, 1985; Luine et al., 1997, 1998; McCarthy et al., 1990, 1991; Meeker et al., 1994; Roy et al., 1985). Immunocytochemical studies have detected GABAergic neurons within the primate VMH; in fact, all of the progestinreceptor-containing VMH neurons expressed the biosynthetic enzyme for GABA, glutamic acid decarboxylase (GAD) (Leranth et al., 1991). Nevertheless, neither glutamate nor GABA has been linked to a particular cell type, such as PRcontaining or PAG-projecting, in the rat VMH.

Several peptides have been localized to neurons in the vlVMH, most notably enkephalin, substance P, and prolactin (Akesson and Micevych, 1988; Blaustein et al., 1988; De Kloet et al., 1986; Harlan et al., 1983; Pfaff and Keiner, 1973; Yamano et al., 1986). Moreover, each of these peptides has been shown in fibers projecting from the VMH to the PAG (Dornan et al., 1990; Nielsen Ricciardi and Blaustein, 1994; Nishizuka et al., 1990; Yamano et al., 1986). Although not reported for enkephalin and prolactin, quantitative analysis suggests that these substance P-labeled fibers represent a small proportion (8%) of the PAG-projecting neurons. Substance P also has been detected in ER- and PR-containing neurons (Akesson and Micevych, 1988; Blaustein et al., 1991). Enkephalin has been co-localized with GABA terminals in the VMH, although it is unknown if these are intrinsic or extrinsic connections (Commons et al., 1999). Additional studies are needed to reveal whether these peptides are co-localized with glutamate, GABA, and/or each other. The interpretation of Fos expression is limited by the uncertain status of Fos-negative neurons, the time dynamics of Fos expression, and the unknown target genes. The level of Fos expression may not accurately represent the level of neural activity for a number of reasons (Hoffman and Murphy, 2000). Measures of neural activity in awake behaving animals would allow more informative conclusions in this regard. Nevertheless, the analysis of Fos expression can provide information about neurons undergoing behavior-induced genomic activation. It is also important to consider the timing of the behavioral test and brain collection with respect to the dynamics of Fos translation and degradation. Fos protein becomes apparent 30 min after the onset of a stimulus and begins to decline approximately 1 h after the cessation of the stimulus (Demmer et al., 1993). Therefore, the Fos labeling observed in the present study was likely based on mating stimulation during the first

Fig. 6. Bar graph illustrating the rostrocaudal distribution of labeled neurons. White bars represent rostral sections, and black bars represent caudal sections. Values are expressed as mean ± SEM. Abbreviations: PR, progesterone receptor; FG, Fluorogold. Asterisk indicates significant difference between the rostral and caudal regions (*P < 0.05).

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30–45 min of behavioral testing, whereas the last 10 min of our behavioral test probably contributed little to the observed Fos expression. The function of mating-induced Fos in the vlVMH remains unclear. The same mating cues that induce Fos expression in the vlVMH (Pfaus et al., 1996; Polston and Erskine, 1995) also enhance sexual receptivity (Bennett et al., 2002; Rodriquez-Sierra et al., 1975), alter female pacing of sexual behavior (Coopersmith et al., 1996; Yang and Clemens, 1997), and promote estrous termination (Lodder and Zeilmaker, 1976; Reading and Blaustein, 1984). Thus, it seems plausible that Fos is part of a genomic effector pathway that modifies subsequent mating behavior in the VMH. When the target genes of Fos are better understood, future studies may explore the molecular links between Fos induction and subsequent behavioral changes. It is well established that sexual stimulation induces Fos expression in the female vlVMH. Many studies focused on specific sensory channels that contribute to mating-induced Fos expression in the female vlVMH, including odors from the male (Bennet et al., 2002), flank stimulation (Coolen et al., 1996; Pfaus et al., 1993; Yang et al., 1999), certain temporal aspects of stimulus administration (Tetel et al., 1994b), and vaginal–cervical stimulation (VCS) (Coolen et al., 1996; Pfaus et al., 1996; Polston and Erskine, 1995; Wersinger et al., 1993). Electrophysiological studies in anesthetized rats have verified that vlVMH neurons are responsive to olfactory, tactile, and VCS cues (Bueno and Pfaff, 1976; Chan et al., 1984). All of these sensory cues were presented to our females and therefore contributed to the observed Fos induction. However, in addition to activation that directly reflects sensory afferents, some of the Fos induction may represent the activation of an efferent pathway. Presently, very little is understood about sensory– motor transformation in the VMH. The presence of matinginduced Fos in a subset of the projection neurons suggests that the VMH does not simply transmit a hormonedependent priming signal to the PAG or passively receive mating cues. Several previous studies examined Fos co-localization with ER or PR in the VMH. When making comparisons between studies, any differences in the incidence of colocalization may merely reflect methodological differences in staining and/or quantification. While keeping this caveat in mind, it is worth considering possible interpretations if these differences reflect true differential activation. One set of studies examined the co-localization of VCS-induced Fos in the VMH with ER- and PR-containing neurons (Tetel et al., 1994a). Fos was induced in the VMH by exposing animals to 13 min of VCS. Approximately 24% of the Fos-containing neurons co-expressed ER, whereas 30–40% co-expressed PR. In the ER study, with the lower incidence of double labeling, the animals were not hormone-treated; however, in the PR study, animals were primed with estradiol and progesterone. This difference in hormone treatment may explain the differential level of co-localization. Thus, VCS and the full array of sexual stimuli may induce Fos in a similar

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proportion of hormone-sensitive neurons provided that hormonal conditions are similar. Another interesting finding was that progesterone treatment induced Fos expression in the vlVMH, but only 20 to 30% of the Fos-labeled neurons were double-labeled for PR (Auger and Blaustein, 1997). One interpretation of this result is that direct effects of progesterone on PR-labeled neurons transynaptically induce Fos in other VMH neurons. Likewise, in our study, some Fos-labeled neurons may have been activated directly by extrinsic sensory inputs while others were activated secondarily by local synapses. The higher percent of Fos-PR colocalization in the present study suggests that PR-containing neurons are more genomically activated by sexual stimuli than by progesterone. Previously, our laboratory reported a triple labeling study performed on the same animals but in an adjacent set of brain sections that were immunostained for ER rather than PR (Calizo and Flanagan-Cato, 2003). Others have shown that, in the vlVMH, PR is expressed only in ER-containing neurons, although not all ER-containing neurons express PR (Blaustein and Turcotte, 1989; Warembourg et al., 1989). This led to the question of whether the ER-containing neurons doublelabeled with mating-induced Fos co-expressed PR or were “ER-only.” The pattern of Fos activation and retrograde labeling in the PR-labeled neurons was strikingly similar to that previously found in ER-labeled neurons (Table 1). Most notably, the proportion of Fos-labeled neurons that coexpressed ER was nearly identical to the proportion that coexpressed PR (35% versus 33%). Similarly, the percentage of Fos-labeled neurons with triple labeling was nearly identical in both studies (12% versus 15%). If a much smaller percent of Fos-labeled neurons double-labeled for PR, it might be inferred that ER-only neurons are mainly activated during Table 1 Percentages of Fluorogold (FG; top), Fos (middle), and steroid-receptor-labeled (bottom) neurons in our previous study (Calizo and Flanagan-Cato, 2003) (left) and the present study (right) Percent of total for each single label Previous study (ER)

Current study (PR)

Fluorogold (FG) FG only FG + Fos FG + ER/PR Triple-labeled

78 5 12 5

78 6 12 5

Fos Fos only Fos + FG Fos + ER/PR Triple-labeled

41 12 35 12

33 19 33 15

ER/PR ER/PR only ER/PR + FG ER/PR + Fos Triple-labeled

70 12 13 5

70 13 11 5

Values for single, double, and triple labeling are listed separately and as percentages of each of the three individual labels.

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sexual behavior. Instead, these results suggest that matinginduced Fos expression occurs in ER-PR neurons rather than ER-only neurons. A more definitive experiment to demonstrate ER, PR, Fos, and Fluorogold co-localization would require quadruple labeling, which is methodologically problematic due to the overlapping wavelengths of available fluorescent labels. Thus, an alternative strategy was employed, comparing different combinations of triple labeling in adjacent sections to make inferences about the likely incidence of four-way co-localization. The triple labeling strategy is often used to identify cellular co-localization of all three labels. In addition, it can be applied to specify a segregation of markers or define various permutations of double labeling. An interpretation is straightforward when either the co-localization or the segregation is nearly complete. Partial co-localization creates an ambiguity for interpreting the relative importance of various subsets of labeling. In the present case, for example, the low absolute number of triple-labeled neurons may suggest that they play a minor role. However, these same neurons represent 50% of the PR/Fluorogold double-labeled neurons, which may indicate that this cell type has an important function. Until this ambiguity can be resolved with a better model of the VMH neural circuitry, it seems reasonable to focus on the more general finding that multiple neuronal types are induced to express Fos during sexual behavior. Rostrocaudal differences have occasionally been noted in studies of the vlVMH. Electrophysiological recordings of projection neurons were more readily found in the rostral, rather than caudal, VMH (Akaishi and Sakuma, 1986), which is consistent with our finding more PAG-projecting neurons in the rostral zone. Similarly, our laboratory previously found that transneuronal transport of PRV from lumbar epaxial muscle to the vlVMH showed rostrocaudal differences; however, given that different landmarks were used, direct comparisons are difficult (Daniels and FlanaganCato, 2000). Finally, consistent with our finding that PRlabeled neurons are more abundant in the caudal VMH, the hormonal induction of oxytocin receptors was more pronounced in the caudal, rather than rostral, VMH, using the same atlas demarcation (Johnson et al., 1989). Thus, although the VMH has striking dorsoventral differences in neurochemistry and function, there also are rostrocaudal gradients in neuronal cell types. In summary, mating behavior is associated with genomic activation of neurons in the vlVMH. Although the neurotransmitter phenotypes of these neurons are not well characterized, many of the activated neurons can be functionally defined as either hormonally responsive or projecting to the midbrain. A rostrocaudal gradient was observed in the vlVMH, with more projection neurons in the rostral pole and more PR-containing neurons in the caudal pole. The triple-labeled neurons were overwhelmingly found in the rostral, rather than caudal, vlVMH. These results refine our model of the components of the neural network in the vlVMH that controls female sexual behavior.

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