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Neuronal Colocalization of Androgen Receptor and Mating-Induced Fos: Hormone-sensitive Neurons Link Spinal Cord, Midbrain, and Forebrain Structures Regulating Copulation in Male Rats

Contact Person: David A Edwards (edwards@emory.edu)


 The most recent experiments that I write about here -- ones that document the colocalization of mating-induced Fos and androgen receptor (AR) -- were the result of a fortuitous collaboration between Béatrice Gréco, a recent graduate student now at the University of Massachusetts at Amherst, and colleagues Andrew Clancy and Doris Zumpe of the Emory University School of Medicine. Most of this work has been published, and I write only enough detail to provide the basis for some ideas about how hormone effects on sexual sensation and behavior could be represented in the organization of the brain and spinal cord.

 More than 15 years ago Hansen and Gummesson (15) showed that bilateral electrolytic lesions of the lateral tegmentum would eliminate mating in male rats, and pointed out that the importance of this region for mating seemed as fundamental as that of the medial preoptic anterior hypothalamic (MPAH) continuum. Two years later, Nancy Brackett and I (3) also reported that lesions of the lateral tegmentum would eliminate mating in male rats. Our midbrain lesions were placed dorsal to the lateral one-half of the substantia nigra at the lateral tip of the medial lemniscus. As Claire-Anne Maillard and I subsequently demonstrated (20,21), large axon-sparing excitoxin lesions of the lateral tegmentum would also eliminate mating, and there is no doubt that neuronal cell bodies intrinsic to the region are essential for copulation.

 The appearance of Fos protein in brain neurons is commonly used as an index of recent trans-synaptic activation (25). Baum and Everitt (2) were the first to note that mating induces c-fos expression in the lateral tegmentum of male rats. Following Simerly and Swanson (26), they referred to this region as the central tegmental field (CTF). Although we have used this designation too (9,10,11,12), the tegmental region of heightened Fos-immunoreactivity after mating actually includes only a small portion of the CTF. As noted in the work of Coolen (4,5) and in our own studies (9,10), mating-induced Fos-immunoreactive (Fos-ir) neurons in the lateral tegmentum are concentrated in the parvicellular subparafascicular nucleus of the thalamus (SPFp), and parts of the CTF, the caudal zona incerta, and the peripeduncular nucleus (PPN). By whatever name, however, this region is included within the lateral tegmental area whose destruction in male rats causes severe deficits in mating.

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The Research

Mating Induces c-fos Expression in Androgen Receptor Neurons in the Lateral Tegmentum and Other Brain Regions

 Mating in males of all mammalian species depends on testicular hormones, and androgens are believed to affect sexual performance by acting on brain neurons with androgen receptors (AR). Our first study of AR and Fos (9) was primarily intended to determine whether or not the lateral tegmentum contained neurons with AR, but was designed so that we could also compare the distribution of mating-induced Fos and AR in forebrain and midbrain structures of established sexual relevance. Like others (2,4,5), we found that mating to ejaculation was associated with the appearance of Fos-ir neurons within the lateral tegmentum, as well as the medial preoptic nucleus (MPN), the posterior part of the medial nucleus of the amygdala (MNA), and the posteromedial and posterolateral parts of the bed nucleus of the stria terminalis (BNST). Each of these regions also contained large numbers of AR-immunoreactive (AR-ir) neurons. Indeed, at least for these regions, the distribution of Fos-ir and AR-ir neurons was remarkably similar.

 Using male hamsters as subjects, Wood and Newman (27) had shown that many of the neurons in the MNA, BNST, and medial preoptic area that expressed c-fos after mating were also AR-ir. Following their lead, we used different fluorescent secondary antibodies in a way that permitted visualization of Fos-ir and AR-ir in the same neurons in recently-mated male rats. Over 80 percent of the Fos-ir neurons in the lateral tegmentum, MNA, BNST, and the MPN were also AR-immunoreactive! These percentages were generally higher than Wood and Newman had observed for male hamsters, but our conclusion was the same: in the male rat, as for the hamster, androgen-receptor neurons at multiple sites in the brain are activated during mating.

The involvement of the MNA, BNST, MPAH, and lateral tegmentum in the regulation of mating in male rats was first inferred from the fact that damage to each of these regions decreases sexual performance (22 for a review). That mating-induced Fos within these regions appears almost exclusively in AR neurons is consonant with the idea that androgens influence sexual performance by some action on androgen-sensitive MNA, BNST, MPAH and lateral tegmental neurons that are trans-synaptically activated during mating.

 Testosterone is metabolized into estradiol by aromatase and into dihydrotestosterone by 5 alpha-reductase, and there is abundant evidence (briefly summarized in 11) that androgens and estrogens interact to activate mating in male rats. In sections from the MPN through the lateral tegmentum we (11) looked for the presence of AR and estrogen receptor (ER) immunoreactivity in males recently mated to ejaculation. In the MNA, BNST, and MPN 80-90 percent of the ER-ir neurons were also AR-ir, representing a subset of approximately 30 percent of the AR-ir neurons present. In these regions about 30 percent of the Fos-ir neurons were also ER-ir. No ER-ir neurons were found in the lateral tegmentum. These results are consonant with the view that both androgens and estrogens work together to regulate some aspects of mating, perhaps by acting on forebrain neurons containing both types of receptors.

 The MPN Receives Projections from Amygdala and Lateral Tegmental AR Neurons Activated During Mating

 Bilateral destruction of the MPAH that includes substantial damage to the MPN virtually eliminates copulation in male rats and males of all other species studied (22 for a review). So, this region is of fundamental importance for mating. Baum and Everitt (2) provided convincing evidence that afferent inputs from the lateral tegmentum and medial amygdala contribute to the activation of BNST and MPAH neurons during mating. We wanted to know if the contributions from the lateral tegmentum and amygdala were made by AR neurons. To determine this we (10) injected the retrograde tracer Fluorogold (FG) into the MPN on one side of the brain of male rats. Four days later the males were mated to several ejaculations and their brains were subsequently examined for FG transport, Fos-ir, and AR-ir. We found FG in the cytoplasm of neurons in a wide variety of regions known to project to the MPN. These regions included the BNST, the MNA, and the lateral tegmentum. In the MNA and lateral tegmentum (we did not quantify for the BNST), nuclear colocalization of AR and Fos was present in 20 and 43 percent respectively of the FG-labeled neurons. This result confirms the lateral tegmentum and MNA as sources of sexually-relevant input to the MPN, and shows that at least some of the information is carried by androgen-sensitive neurons.

 AR Neurons in the Spinal Cord Are Activated During Mating

 The penis is innervated by the pelvic and pudendal nerves which emerge from the lumbosacral trunk of the spinal cord, and somatosensory information relayed from the penis to the spinal cord via the pudendal nerve appears to be crucial for intromissive and ejaculatory components of mating in the male rat (24). Are spinal neurons activated during mating also sensitive to androgens as appears to be the case for the brain? Following our usual protocol, male rats were killed after ejaculating several times and we (12) used dual immunocytochemical techniques to identify Fos-ir and AR-ir neurons in the lumbosacral spinal cord. AR-ir neurons were present in a number of regions of the lumbosacral cord, with particularly dense concentrations appearing in the L5, L6 and S1 segments. In these segments, concentrations of AR-ir neurons were highest in the dorsal part (above and laterally surrounding the central canal) of Lamina X. In the spinal cord, as for the brain, mating-induced Fos was predominantly localized in AR neurons. For example, in the dorsal part of Lamina X of the mated males, virtually all of the Fos-ir neurons were AR-ir, representing, depending on spinal level, 21 to 35 percent of the AR-ir neurons present. Thus, lumbosacral spinal neurons activated during mating are androgen-sensitive.

 The Lateral Tegmentum Receives Projections from AR Neurons in Spinal Cord and Forebrain, Many of Which Are Activated During Mating

 Do androgen-sensitive spinal neurons activated during mating project to the lateral tegmentum? To answer this question we (13) injected FG into the lateral tegmentum of male rats. Ten days later we killed the males after they had mated to several ejaculations. Both spinal cord and brain were examined for FG transport, and AR and Fos immunoreactivity. In the spinal cord, we found FG-labeled neurons in the dorsal and ventral parts of Lamina X at the L5, L6, and S1 segments, many of which contained both AR-ir and Fos-ir. For example, summing the dorsal and ventral parts of Lamina X across the three segments, 51 percent of the FG neurons contained both AR-ir and Fos-ir. In brain, we found FG-labeled neurons in many regions known to project to the lateral tegmentum including the BNST and MPAH. In the BNST, depending on region, about 41-68 percent of the FG neurons were AR-ir, and between 7-15 percent of the FG neurons contained both AR-ir and Fos-ir. In the MPAH, FG neurons were present in a region immediately dorsolateral to the MPN, and about 40 percent of these contained both AR-ir and Fos-ir. Thus, the lateral tegmentum appears to receive input from androgen-sensitive spinal cord, BNST, and MPAH neurons activated during mating. 

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The Hormonal Activation of Mating in Male Rats

For the spinal cord and brain, only about 20-40 percent of the AR-neurons in the regions we studied express c-fos in association with mating. Diverse populations of hormone-sensitive neurons probably coexist within regions critical for reproduction. Different populations may influence different physiological and/or behavioral phenomena. However, the absence of Fos after copulation should not be used to infer an absence of mating-related trans-synaptic activation. Fos is only one of the proteins produced by immediate-early genes, and not all neurons produce Fos in response to stimulation (25, and see 12,16).

 As noted earlier, AR-ir neurons of the lateral tegmentum are contained within a region that includes the SPFp, and parts of the CTF, the caudal zona incerta, and PPN. Because of the odd shape of this distribution it has been difficult for us to make lateral tegmental excitotoxin lesions restricted to the steroid-sensitive parts of this region. However, considering both published (20,21) and unpublished work, I believe that for excitoxin lesions of the lateral tegmentum to eliminate mating the damage must not only include but also extend beyond the areas of highest concentration of AR-ir and Fos-ir neurons. Immunocytochemical assays for the protein products of other immediate early genes could be helpful in identifying neighboring populations of neurons trans-synaptically activated during mating.

 Although testicular hormones probably activate mating by actions on AR at multiple sites in the brain (22 for a review), the colocalization of mating induced Fos and AR in spinal neurons suggests that hormone action on steroid-sensitive spinal neurons could make an important contribution to this process -- an idea first suggested by Hart and Haugen 20 years ago (14). The MNA, BNST, and lateral tegmentum are important for mating. These regions are linked by androgen-sensitive neurons, many of which are activated during mating, and these links are almost certainly essential for normal male sexual performance.

 In reviewing his studies of the effects of testosterone replacement on sexual function in hypogonadal men, Julian Davidson (6) advanced the hypothesis that androgen acts to promote sensitivity to the pleasurable awareness of sexual thoughts and activity, and in his view sexual appetite has much to do with pleasure in sexual sensation. Androgen effects on sexual sensation could be mediated by steroid-sensitive neurons acting on neurons relaying sexually-relevant sensation. In their particularly elegant analysis, Wood and Newman (28) make the case that mating in male hamsters is regulated by two parallel but interconnected subcircuits -- one carrying chemosensory information about females, the other comprised of steroid receptor containing neurons -- linking the MNA, BNST, and medial preoptic area. In the male rat, although neither is essential for mating, olfactory and genital somatosensory sensations are clearly important for normal copulation (e.g.,1,7,8,24). The MNA and BNST appear to be important parts of the central olfactory pathway through which volatile odors from estrous females elicit sexual arousal (18,19), and lateral tegmental neurons appear to relay genital somatosensory information from the spinal cord to the forebrain (2). Our research, which documents the presence of mating-induced Fos in a subsets of AR neurons in MNA, BNST, the lateral tegmentum, and the lumbosacral spinal cord, suggests that androgens might modify olfactory and somatosensory sexual sensation by a direct effect on neurons carrying sexually-relevant chemosensory or somatosensory information.

 The importance of the lateral tegmentum as a target site for the hormonal activation of mating has not been systematically studied. However, as part of a large study of the effects of intracerebrally-implanted testosterone on the reactivation of mating in castrated male rats, Kierniesky and Gerall (17) included four males with implants in what we identify as the androgen-sensitive lateral tegmentum -- all four males mated to ejaculation after hormone implantation. A more detailed study, one that uses immunocytochemical techniques to evaluate intracerebral spread away from the implantation site (e.g.,28), is obviously needed.

 As Nancy Brackett and I showed some years ago (3), a unilateral lateral tegmental lesion combined with a lesion of the MPAH on the other side of the brain eliminates mating in male rats. In other words, asymmetric brain damage that bilaterally destroys the connections between the lateral tegmentum and MPAH eliminates mating. We concluded that the connections between these two regions were essential for copulation. Part of this contribution may be made via hormone-sensitive lateral tegmental projections to the MPAH. However, as our recent studies show, the lateral tegmentum receives afferents from androgen-sensitive preoptic and BNST neurons activated during mating. These connections too, are likely to contribute something to sexual performance. Although effects of MPAH lesions on mating are permanent, transplantation of fetal hypothalamic tissue into the region of the preoptic damage can restore copulation, and this effect is associated with reestablished reafferentation of the lateral tegmentum by preoptically located neurons (23). Taken together, these results suggests an integrative role in mating for the lateral tegmentum that goes far beyond the simple relay of sexually-relevant somatosensory information to the forebrain. 

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 This research was supported by NSF Grant IBN-9421208 to David Edwards, Whitehall Foundation Grant J96-24 to Andrew N. Clancy, and USPHS Grant MH-19506 to Richard P. Michael, who graciously made the resources of his laboratory available for the immunocytochemical studies of Fos, AR, and ER. We thank Dr. Gail Prins for her generous donation of the anti-AR antibody used in the research.

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Edwards, DA.; (1998). Neuronal Colocalization of Androgen Receptor and Mating-Induced Fos: Hormone-sensitive Neurons Link Spinal Cord, Midbrain, and Forebrain Structures Regulating Copulation in Male Rats. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/baum/edwards0440/index.html
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