Aromatase, the enzyme that aromatizes androstenedione (A) to estrone and testosterone (T) to estradiol (E), affects androgen control of male sex behavior in many vertebrates. In male monkeys, rats and quail, E mimics the ability of T to promote mating, and aromatase inhibitors block mating induced by T but not E. Aromatase inhibitors include androgens with different A-rings than T and A, e.g., 1,4,6-androstatriene-3,17-dione (ATD), azoles, e.g., fadrozole, and androgens α-halogenated at carbon 6, e.g., 6α-bromoA, 6α-fluoroA and 6α-fluoroT. 6α-FluoroT is the only 6α-halogenated androgen studied in regard to mating. It promotes mating in male rats and quail and was studied, before it was known to inhibit aromatase, because it cannot be aromatized yet has the same A-ring as T. 6α-FluoroT might promote mating by binding estrogen receptors (ER) directly, i.e., unassisted, or by metabolism to an androgen that binds ER. Since neither process would require aromatase, this study tested both hypotheses by determining how mating induced in castrated male rats by 6α-fluoroT is affected by ATD and fadrozole. Both aromatase inhibitors inhibited the effects of 6α-fluoroT on mating. Thus, 6α-fluoroT does not promote mating by direct ER binding or metabolism to another androgen. Since aromatase underlies a process in which 6α-fluoroT, unlike most nonaromatizable androgens, mimics T effects on male sex behavior, the process must involve a feature that 6α-fluoroT shares with T but not other nonaromatizable androgens. A-ring structure is a candidate. A hypothesis is also offered for how aromatase may participate without aromatizing the androgen.

The posterodorsal preoptic nucleus (PdPN), lateral part of the posterodorsal medial amygdala (MeApd) and medial part of the medial preoptic nucleus (MPNm) are activated at ejaculation in male gerbils as assessed by Fos expression. We sought to immunocytochemically visualize substance P (SP), cholecystokinin (CCK), oxytocin, vasopressin and tyrosine hydroxylase (TH), a catecholaminergic marker, in the mating-activated cells, but the need for colchicine precluded behavioral testing. Instead, we detailed distributions of cells containing these molecules in the medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis (BST) and quantified their densities in the PdPN, MPNm and lateral MeApd for comparison to densities previously assessed for mating-activated efferents from these sites. TH cells were as dense in the PdPN and lateral MeApd as activated efferents to the anteroventral periventricular nucleus. In the lateral MeApd, TH cells were grouped where cells activated at ejaculation are clustered and where CCK cells form a ball. Lateral MeApd CCK cells and PdPN SP cells were as dense as activated efferents to the principal BST. Oxytocinergic PdPN cells and SP cells in the MPNm were as dense as mating-activated efferents to the lateral MeApd. If some oxytocin cells in the PdPN project to the neurohypophysis, as in rats, they could be a source of the oxytocin secreted at ejaculation. Since gerbils are monogamous and biparental, it was also interesting that, unlike monogamous prairie voles, they had few TH cells in the MeApd or dorsal BST, resembling promiscuous rats, hamsters and meadow voles.

Two clusters of forebrain neurons-one in the posterodorsal preoptic nucleus (PdPN) and one in the lateral part of the posterodorsal medial amygdala (MeApd)-are activated at ejaculation in male rats and gerbils as seen with Fos immunocytochemistry. To understand the functions of these cells and how they respond synchronously, it may be useful to identify their neurotransmitters. Nitric oxide (NO) was of interest because its levels in the preoptic area affect ejaculation, and it could synchronize clustered neurons through paracrine/volume transmission. Thus, we determined whether the ejaculation-related cells produce NO by assessing Fos co-localization with NO synthase (NOS) in recently mated male gerbils. We also studied NOS-Fos co-localization in the medial part of the medial preoptic nucleus (MPNm), where half of the neurons that express Fos after mating reflect ejaculation. We also quantified NOS co-localization with androgen receptor (AR) and NOS sensitivity to androgens at these sites. Without quantification, we extended these analyses throughout the hypothalamus and amygdala. Many mating-activated PdPN, lateral MeApd, and MPNm cells contained NOS (32-54%), and many NOS neurons at these sites expressed Fos (34-51%) or AR (25-69%). PdPN and MPNm NOS cells were sensitive to testosterone but not its androgenic metabolite dihydrotestosterone. The overall distribution of NOS and NOS-AR cells was similar to that in rats. These data suggest that NO may help to synchronize the activation of PdPN and lateral MeApd neurons at ejaculation and that NOS in PdPN and MPNm cells is regulated by testosterone acting via estradiol or without undergoing metabolism.

The ventral bed nuclei of the stria terminalis (BST) and medial preoptic nucleus (MPN) of gerbils contain cells that regulate male sex behavior via a largely uncrossed pathway to the retrorubral field (RRF). Our goal was to learn more about cells at the pathway source and target. To determine if the pathway uses GABA as its transmitter, we used immunocytochemistry (ICC) to study glutamic acid decarboxlyase67 (GAD67) colocalization with fluoro-gold (FG) in the ventral BST and MPN after applying FG to the RRF. To determine if the pathway is activated with mating, we studied FG-Fos colocalization in the ventral BST of recently mated males. The ventral BST expresses Fos with mating and is the major pathway source. To determine to what extent other GABAergic cells in the ventral BST are activated with mating, we studied Fos colocalization with GAD67 mRNA visualized by in situ hybridization (ISH). We also looked for GAD67 mRNA in RRF cells. Almost all ventral BST and MPNm cells projecting to the RRF (95–97%) and most ventral BST cells activated with mating (89%), were GABAergic. GABAergic cells were also seen in the RRF. RRF-projecting cells represented 37% of ventral BST cells activated with mating. Their activation may reflect arousal and anticipation of sexual reward. Among ventral BST cells that project to the RRF, 14% were activated with mating, consistent with how much of this pathway is needed for mating. The activated GABAergic cells that do not project to the RRF may release GABA locally and inhibit ejaculation.

The medial preoptic nucleus (MPN) and ventral bed nuclei of the stria terminalis (BST) are needed to maintain mating in sexually experienced male gerbils and rats. The gerbil ventral BST is also activated with mating, as assessed by Fos expression, as is the medial MPN (MPNm) of both species. In gerbils, many of those mating-activated cells contain glutamic acid decarboxylase (GAD), the enzyme that synthesizes γ-aminobutyric acid (GABA). Some of those cells are projection neurons, but others may release GABA locally. Through actions in the medial preoptic area, GABA inhibits and testosterone (T) promotes male sex behavior. Thus, T may promote mating, in part, by decreasing GAD in MPNm or ventral BST cells. In rats, T increases GAD mRNA in the central MPN (MPNc), where MPN GABAergic cells are densest, but mating behavior does not change in sexually experienced males when the MPNc is ablated. Therefore, this study focused on the MPNm and ventral BST to ask whether their GABAergic cells respond to T or are sexually dimorphic. This was done by visualizing cells immunoreactive (IR) for GAD67, an isoform found primarily in cell bodies, in male and female gerbils and in castrated males with and without T. At both sites, males had more GAD67-IR cells than females, and T decreased GAD67-IR cell numbers in males. Thus, the MPNm and ventral BST have GABAergic cells that are sexually dimorphic and in which T decreases GAD, consistent with local effects of T and GABA on mating.

This research identified the rat counterpart of the lateral cell group of the sexually dimorphic area (SDA) found in medial preoptic area (MPOA) gerbil of gerbils. The lateral SDA (lSDA) is critical for mating in male gerbils and contains most of the SDA cells projecting to the retrorubral field (RRF), a projection that is also important for mating. Therefore, to locate the counterpart of the lateral SDA, we traced the inputs to the rat RRF, which were dense in the ventral part of the bed nucleus of the stria terminalis (BST). To determine if the ventral BST or its projection to the RRF affects mating in male rats, we disrupted them bilaterally by placing cell-body lesions bilaterally in the ventral BST or unilaterally there and in the contralateral RRF. We also studied the effects of RRF lesions in both rats and gerbils. Bilateral ventral BST lesions, which left the medial preoptic nucleus intact, produced persistent and severe mating deficits. Disconnecting the ventral BST from the RRF also had long-lasting, but less severe, consequences. RRF lesions produced only temporary mating deficits in rats, but virtually eliminated mating in gerbils. The recovery of mating in rats after RRF, but not ventral BST, lesions, and the intermediate effects of disconnecting these areas from each other suggest that the ventral BST may contain mating-related projection neurons other than those projecting to the RRF or that its RRF-projecting cells send collaterals to another site. In either case, the pedunculopontine tegmental nucleus or raphe nuclei may be involved.

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use γ‐aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron‐specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67‐kDa form of glutamic acid decarboxylase (GAD 67 ) and in situ hybridization for GAD 67 messenger RNA (mRNA). Colocalization of Fos and GAD 67 mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described. J. Comp. Neurol. 459:290–300, 2003. © 2003 Wiley‐Liss, Inc.

The posterodorsal preoptic nucleus (PdPN) and the lateral part of the posterodorsal medial amygdala (MeApd) express Fos with ejaculation in male gerbils. Ejaculation‐activated cells participate in the PdPN and MeApd projections to each other and to the sexually dimorphic preoptic area (SDA), but those projections involve less than 20% of the activated PdPN cells and less than 50% of the activated MeApd cells. To identify other potential targets of ejaculation‐activated cells, we traced PdPN and lateral MeApd outputs using biotinylated dextran amine. The principal part of the bed nucleus of the stria terminalis (BSTpr) and the anteroventral periventricular nucleus (AVPv) were labeled from both sites and were injected with Fluoro‐Gold to determine whether PdPN and lateral MeApd cells that express Fos with ejaculation would be retrogradely labeled. Fluoro‐Gold was also applied to the dorsomedial hypothalamus (DMH) and retrorubral field (RRF) because such injections label PdPN cells in rats. The PdPN‐DMH projection is minimal in gerbils, involving few, if any, ejaculation‐related cells. Ejaculation‐activated PdPN cells project to the AVPv (43%), dorsal BSTpr (30%), and RRF (12%). Those in the lateral MeApd project to the dorsal BSTpr (43%) and AVPv (18%). When these percentages are combined with those for ejaculation‐activated cells involved in the PdPN and lateral MeApd projections to each other and to the medial SDA, the totals reach 100%. Thus, every PdPN and MeApd cell activated with ejaculation may participate in one of these projections. Similar projections may contribute to the similar behavioral effects of the PdPN and MeApd. J. Comp. Neurol. 444:75–94, 2002. © 2002 Wiley‐Liss, Inc.

Based on Fos expression, four areas of the gerbil brain are activated with ejaculation, i.e., the posterodorsal preoptic nucleus (PdPN), the lateral part of the posterodorsal medial amygdala (MeApd), the medial cell group of the sexually dimorphic preoptic area (medial SDA), and the parvicellular part of the subparafascicular thalamus (SPFp). The SPFp and medial SDA also express Fos earlier in the context of mating. To study connections among these areas, we injected one with FluoroGold and assessed the colocalization of FluoroGold and mating‐induced Fos in the others. To determine if any of these areas activates the others, we lesioned one unilaterally and measured mating‐induced Fos ipsilaterally and contralaterally in the others. Half of the SPFp cells projecting to the medial SDA, PdPN, and MeApd were activated with mating. SPFp lesions also decreased Fos expression in those areas. However, those areas do not project to the SPFp or affect its Fos expression with mating. Projections from the lateral MeApd to the medial SDA and PdPN, and from the medial SDA to the lateral MeApd, were also activated with mating, but lesions in these areas did not affect Fos expression in the others. Because 32–50% of the mating‐activated cells in the SPFp participated in each SPFp projection identified, projections may have been identified for all of the mating‐activated cells in the SPFp. In contrast, most of the mating‐activated cells in the lateral MeApd, PdPN, and medial SDA do not participate in any projection studied, suggesting that they are either interneurons or project elsewhere. J. Comp. Neurol. 439:248–258, 2001. © 2001 Wiley‐Liss, Inc.

In gerbils, the posterodorsal preoptic nucleus (PdPN) and the lateral part of the posterodorsal medial amygdala (MeApd) express Fos with ejaculation. In contrast, the medial/central part of the MeApd expresses Fos when a sexually experienced male reenters the environment associated with mating. The parvicellular part of the subparafascicular thalamic nucleus (SPFp) of gerbils expresses Fos under both conditions. To study the role of the PdPN and MeApd in male sex behavior, male gerbils were tested for mating before and after these areas were bilaterally lesioned by infusions of N-methyl-D-aspartate (NMDA). Controls received the vehicle or inactive isomer, NMLA. Lesions in either area reduced mounting, but MeApd lesions, which were more complete than PdPN lesions, also delayed ejaculation when males intromitted. To determine if the MeApd and PdPN affect mating via a common pathway, they were bilaterally disconnected by lesioning them unilaterally, contralateral to each other. Other groups received ipsilateral lesions, NMLA, or bilateral lesions of the PdPN or MeApd. In addition, the SPFp was studied using bilateral lesions. MeApd and PdPN lesions again decreased mounting, and this time both lesions, which were quite complete, delayed ejaculation when males intromitted. Contralateral lesions that bilaterally disconnected these cell groups from each other mimicked both effects. Thus, the MeApd and PdPN affect mounting and ejaculation, at least in part, via their connections with each other. In contrast, SPFp lesions did not affect mating. Thus, SPFp cells activated at ejaculation may react to ejaculation rather than trigger it, possibly initiating preparations for paternity.

The sexually dimorphic area of the gerbil hypothalamus is essential for male sex behavior. To determine which aspects of mating activate its cells, or cells near or connected to it, we visualized c-Fos in the brains of male gerbils that had been exposed to various types of sex-related stimuli or that had displayed various aspects of sex behavior. Five groups of males were placed in familiar arenas containing sex-related odors. All subjects had previously mated in these arenas. For four groups, a female was introduced and remained with the male until he ejaculated, intromitted, mounted or sniffed her. Males in the fifth group remained in the arena alone. Males in a sixth group were placed in a clean arena in another room. These males were also familiar with this arena but had never encountered a female there. The seventh group remained in their home cages. The posterodorsal preoptic nucleus, the lateral part of the posterodorsal medial amygdala, the medial part of the sexually dimorphic area and the parvicellular part of the subparafascicular nucleus of the thalamus expressed c-Fos after ejaculation. Whether these cells triggered ejaculation or responded to it is not clear. The latter two areas also expressed c-Fos whenever males were exposed to the sex arena, but the sexually dimorphic area pars compacta did not express c-Fos under any condition. The medial core of the nucleus accumbens, the ventrolateral septum, the caudomedial bed nucleus of the stria terminalis, the medial/central part of the posterodorsal medial amygdala and the lateral part of the sexually dimorphic area also expressed c-Fos when males entered the sex arena. The ventrolateral part of the ventromedial nucleus of the hypothalamus expressed c-Fos whenever males were with females. None of the 31 areas studied responded to mounting or intromission, but the zona incerta, the amygdalohippocampal area, the lateral part of the sexually dimorphic area and the area lateral to the medial part of the sexually dimorphic area showed progressive increases in c-Fos expression as mating progressed. The area dorsal to the medial part of the sexually dimorphic area, the paraventricular nucleus of the hypothalamus, the ventral premammillary nucleus and the retrorubral field showed the same level of c-Fos expression when males were exposed to the non-sexual context as when they were exposed to the sexual one. While a projection to the retrorubral field from the sexually dimorphic area is critical for male sex behavior, the retrorubral field did not show a sex-related c-Fos response. The data suggest that brain regions involved in male sex behavior are involved in different aspects of it and that this can also apply to different subsets of cells in each area. The data also indicate that cells involved in mating do not necessarily show mating-related patterns of c-Fos expression. Thus, while c-Fos is useful for identifying areas involved in mating, or other behaviors, its characteristics could cause relevant areas to be overlooked.

Mating, marking and the sexually dimorphic area (SDA) were studied in male gerbils that were castrated or sham-operated on the day of birth and in females given testosterone (T) propionate or oil 1 day later. Other females received dihydrotestosterone (DHT), R1881, estradiol benzoate (EB), R2858, DHT and EB, or DHT and R2858. Females and control males were castrated as adults. Then half of each group was given T and tested for marking and male sex behavior. They were still exposed to T at perfusion. The rest were given EB and progesterone and were tested for marking and lordosis. They were perfused 1 month after hormone treatment ended. Neonatal castration eliminated mounting, decreased T-induced marking and increased lordosis in males. Neonatal exposure to TP decreased lordosis and increased mounting, intromission patterns and T-induced marking in females. DHT and R1881 did not affect marking or mating when given alone. EB and R2858 decreased lordosis and increased mounting. DHT enhanced the former effect but not the latter. Only combined treatments increased T-induced marking. Total SDA volume was not affected by neonatal treatments, but "dark volume" was largest in control males. The SDA pars compacta (SDApc) was seldom seen in females unless they received steroid neonatally but was present in males that were castrated neonatally. SDApcs of neonatal castrates and steroid-treated females differed, though, from SDApcs of control males in that they were smaller in the absence of T and did not enlarge when exposed to T. Another cell group, the cmSDApc, was affected by T neonatally but was not seen consistently in either sex. SDApc volume correlated positively with T-induced marking and an index of male sex behavior. Both SDApc and cmSDApc volumes correlated negatively with lordosis. The correlations with marking and male sex behavior probably reflect constraints on sexual differentiation rather than causal relationships since these behaviors persist when the SDApc is gone. The effects of direct manipulations of the SDApc on lordosis have not been studied.

The research summarized here shows that the two major cell groups of the sexually dimorphic area (SDA) of the gerbil hypothalamus are essential for male sex behavior. Bilateral cell-body lesions of either the medial or lateral SDA virtually eliminate mating in sexually experienced male gerbils given exogenous testosterone. Similar deficits occur when the SDA is bilaterally disconnected from the retrorubral field (RRF) as a result of unilateral cell-body lesions in the SDA and contralateral RRF. The A8 cells of the RRF do not account for this effect. Bilaterally disconnecting the SDA from the caudomedial part of the bed nucleus of the stria terminalis (caudal BSTm) also eliminates sexual activity. Disconnecting the SDA from the medial amygdala does not mimic this effect. Neither does disconnecting the medial amygdala from the caudal BSTm. Thus, caudal BSTm neurons that are essential for mating via connections with the SDA do not simply relay information from the medial amygdala.

The sexually dimorphic area (SDA) of the gerbil hypothalamus is essential for male sexual behavior. To determine (a) if the SDA can affect mating via laterally projecting axons and (b) which SDA afferents might affect mating, male gerbils were given bilateral, parasagittal knife cuts lateral to the medial or lateral SDA. Others were given cuts with a knife coated with horseradish peroxidase to label cells of which axons were cut. Medial cuts eliminated mating and consistently labeled cells in the medial SDA. Lateral cuts did neither. Medial cuts also labeled more cells in the ventral part of the lateral septal nucleus, the encapsulated part of the bed nucleus of the stria terminalis, the medial nucleus of the amygdala, the amygdalohippocampal area, and the ventral premammillary nucleus than lateral cuts did. Thus, medial cuts may disrupt mating by severing SDA efferents or by severing SDA afferents from 1 or more of these 5 sites.

To determine if the sexually dimorphic area (SDA) of the gerbil hypothalamus affects male sexual behavior through its projections to the retrorubral field (RRF) or ventrolateral periaqueductal gray (PAGvl), these pathways were lesioned asymmetrically. Unilateral radio frequency lesions of the lateral SDA (ISDA), the major source of the pathways, impaired mating when combined with contralateral RRF, but not PAGvl, lesions. N-methyl-D-aspartate (NMDA) lesions of the medial SDA, ISDA, and the area between them (the total pathway source) eliminated mating when combined with contralateral, but not ipsilateral, NMDA lesions of the RRF. To determine if A8 cells contributed to these effects, males received NMDA in the SDA and NMDA or 6-hydroxydopamine in the contralateral RRF. When combined with large SDA lesions, A8 lesions impaired but did not eliminate mating. Thus the SDA-RRF pathway, but not an SDA-A8 pathway, is essential for sexual behavior in male gerbils.

The sexually dimorphic area (SDA) of the gerbil hypothalamus is essential for mating in male gerbils. To determine if it affects mating through its connections with the ventral part of the lateral septal nucleus (LSv), the caudal part of the medial bed nucleus of the stria terminalis (caudal BSTm), or the medial amygdala-amygdalohippocampal area (MA-AHi), these connections were severed. Unilateral cell-body lesions were made in the SDA and in the contralateral LSv, caudal BSTm, or MA-AHi. Controls received the same lesions ipsilaterally. Other gerbils received lesions in the caudal BSTm and contra- or ipsilateral MA-AHi. Only contralateral lesions of the SDA and caudal BSTm severely impaired mating. Because contralateral lesions of the SDA and MA-AHi, or BSTm and MA-AHi, did not mimic this effect, the BSTm neurons that are needed for male sexual behavior through their connections with the SDA do not simply relay information to or from the MA-AHi.

Male reptiles, birds and mammals do not copulate if the medial preoptic area (MPOA) is destroyed but the MPOA cell groups necessary for male sexual behavior were not known. Here, two cell groups essential for copulation are identified in the sexually dimorphic area (SDA) of the gerbil (Meriones unguiculatus) MPOA. Bilateral cell-body lesions of either the medial or lateral SDA eliminated mating in sexually experienced male gerbils given testosterone. Nearby MPOA lesions did not. The medial and lateral SDA affect sex behavior via separate pathways since lesioning the medial SDA on one side of the brain and the lateral SDA on the other did not stop sexual behavior.

Outputs of the sexually dimorphic area (SDA) of the gerbil hypothalamus were identified by injecting Phaseolus vulgaris‐leucoagglutinin into the medial or lateral SDA (mSDA, ISDA) in males and females. They were verified by injecting Fluoro‐Gold or rhodamine‐labeled beads into over half the areas that contained labeled fibers. Both anterograde and retrograde tracing showed that the mSDA and ISDA project to many of the same sites but often to differing degrees. The mSDA projects more heavily than the ISDA to many of their forebrain targets including the ventral part of the lateral septal nucleus, the bed nucleus of the stria terminalis, the medial tuberal area, and the anteroventral periventricular, arcuate, ventromedial and ventral premammillary nuclei of the hypothalamus. The ISDA projects more heavily than the mSDA to many of their mid‐ and hindbrain targets including the caudal, ventrolateral part of the periaqueductal gray, the retrorubral field, the pedunculopontine tegmental nucleus, and the locus coeruleus. In many other areas of the brain, the projections of the mSDA and ISDA are similar in size. These areas include the substantia innominata, the vascular organ of the lamina terminalis, the anterior amygdela, the posterior hypothalamus, the reuniens and paraventricular nuclei of the thalamus, and the pontine periaqueductal gray lateral to the fourth ventricle. The SDA pars compacta (SDApc), a small cell group embedded in the mSDA of males, projects to many fewer areas than the surrounding mSDA. It was strongly labeled when retrograde tracers were injected into the encapsulated part of the bed nucleus of the stria terminalis, the anteroventral periventricular nucleus, or the mSDA. It was also labeled from the vascular organ of the lamina terminalis, the caudal part of the lateral bed nucleus of the stria terminalis, the ISDA, the area lateral to the mSDA, the arcuate nucleus, the ventral premammillary nucleus, and the ventrolateral part of the ventromedial nucleus of the hypothalamus. Nothing resembling an SDApc was identified during retrograde tracing in females. © 1993 Wiley‐Liss, Inc.