The bed nucleus of the stria terminalis is critical for sexual solicitation, but not for opposite-sex odor preference, in female Syrian hamsters

Successful reproduction in vertebrates depends critically upon a suite of precopulatory behaviors that occur prior to mating. In Syrian hamsters (Mesocricetus auratus), these behaviors include vaginal scent marking and preferential investigation of male odors. The neural regulation of vaginal marking and opposite-sex odor preference likely involves an interconnected set of steroid-sensitive nuclei that includes the medial amygdala (MA), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA). For example, lesions of MA eliminate opposite-sex odor preference and reduce overall levels of vaginal marking, whereas lesions of MPOA decrease vaginal marking in response to male odors. Although BNST is densely interconnected with both MA and MPOA, little is known about the role of BNST in female precopulatory behaviors. To address this question, females received either bilateral, excitotoxic lesions of BNST (BNST-X) or sham lesions (SHAM), and were tested for scent marking and for investigatory responses to male and female odors. Whereas SHAM females vaginal marked more to male odors than female odors on two days of the estrous cycle, BNST-X females marked at equivalent levels to both odors. This deficit is not due to alterations in social odor investigation, as both BNST-X and SHAM females investigated male odors more than female odors. Finally, BNST lesions did not generally disrupt the cyclic changes in reproductive behaviors that occur across the estrous cycle. Taken together, these results demonstrate that BNST is critical for the normal expression of solicitational behaviors by females in response to male odor stimuli.     

The bed nucleus of the stria terminalis is critical for sexual solicitation,but not for opposite-sex odor preference,in female Syrian hamsters

Successful reproduction in vertebrates depends critically upon a suite of precopulatory behaviors that occur prior to mating. In Syrian hamsters (Mesocricetus auratus), these behaviors include vaginal scent marking and preferential investigation of male odors. The neural regulation of vaginal marking and opposite-sex odor preference likely involves an interconnected set of steroid-sensitive nuclei that includes the medial amygdala (MA), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA). For example, lesions of MA eliminate opposite-sex odor preference and reduce overall levels of vaginal marking, whereas lesions of MPOA decrease vaginal marking in response to male odors. Although BNST is densely interconnected with both MA and MPOA, little is known about the role of BNST in female precopulatory behaviors. To address this question, females received either bilateral, excitotoxic lesions of BNST (BNST-X) or sham lesions (SHAM), and were tested for scent marking and for investigatory responses to male and female odors. Whereas SHAM females vaginal marked more to male odors than female odors on two days of the estrous cycle, BNST-X females marked at equivalent levels to both odors. This deficit is not due to alterations in social odor investigation, as both BNST-X and SHAM females investigated male odors more than female odors. Finally, BNST lesions did not generally disrupt the cyclic changes in reproductive behaviors that occur across the estrous cycle. Taken together, these results demonstrate that BNST is critical for the normal expression of solicitational behaviors by females in response to male odor stimuli.     

Male Syrian hamsters demonstrate a conditioned place preference for sexual behavior and female chemosensory stimuli

Sexual behavior is a natural reward for many rodent species, and it often includes chemosensory-directed components. Chemosensory stimuli themselves may also be rewarding. Conditioned place preference (CPP) is one paradigm frequently used to test the rewarding properties of a range of stimuli. Males and females of several rodent species show a CPP for sexual behavior; however, it is currently unknown whether sexual behavior can induce a CPP in male Syrian hamsters. As male Syrian hamsters are an animal model commonly used for investigation of the neurobiology of sexual behavior, understanding the rewarding components of sexual stimuli will better direct future research on brain regions and neurotransmitters involved in these behaviors. Experiment 1 tested the prediction that male hamsters show a CPP for sexual behavior. Female chemosensory stimuli are essential for the display of sexual behavior in male hamsters; however, the rewarding properties of female chemosensory stimuli contained in vaginal secretions (VS) are uncertain. Therefore, experiment 2 tested the prediction that male hamsters show a CPP for VS. This study is the first demonstration that both sexual behavior and VS induce a CPP in male hamsters. Thus, female chemosensory stimuli are a natural reward in a species that is dependent on these stimuli for reproductive fitness.     

Peripubertal exposure to male odors influences female puberty and adult expression of male-directed odor preference in mice

Testosterone-dependent olfactory signals emitted by male are well known to accelerate female puberty in mice (Vandenbergh effect). However, it remains unclear whether these chemosignals also influence adult expression of male-directed odor preference. Therefore, we exposed female mice to intact or castrated male bedding (vs clean bedding as control) during the peripubertal period (postnatal day (PD) 21–38) and measured male-directed odor preference in adulthood. At PD45 or PD60, females exposed to intact male odors, and thus showing puberty acceleration, preferred to investigate odors from intact males over females or castrated males. Females exposed to castrated male odors did not show puberty acceleration but preferred male (intact or castrated) over female odors. Finally, control females did not show any odor preference when tested at PD45, although a preference for male odors emerged later (PD60). In a second experiment, females that were exposed to intact male odors after pubertal transition (PD36–53) also preferred intact male over castrated male odors. In conclusion, our results indicate that peripubertal exposure to male odors induced early expression of male-directed odor preference regardless of puberty-accelerating effect and that induction of male-directed odor preference is not specific to the peripubertal period.     

PSA-NCAM in the posterodorsal medial amygdala is necessary for the pubertal emergence of attraction to female odors in male hamsters

During puberty, attention turns away from same-sex socialization to focus on the opposite sex. How the brain mediates this change in perception and motivation is unknown. Polysialylated neural cell adhesion molecule (PSA-NCAM) virtually disappears from most of the central nervous system after embryogenesis, but it remains elevated in discrete regions of the adult brain. One such brain area is the posterodorsal subnucleus of the medial amygdala (MePD). The MePD has been implicated in male sexual attraction, measured here as the preference to investigate female odors. We hypothesize that PSA-NCAM gates hormone-dependent plasticity necessary for the emergence of males' attraction to females. To evaluate this idea, we first measured PSA-NCAM levels across puberty in several brain regions, and identified when female odor preference normally emerges in male Syrian hamsters. We found that MePD PSA-NCAM staining peaks shortly before the surge of pubertal androgen and the emergence of preference. To test the necessity of PSA-NCAM for female odor preference, we infused endo-neuraminidase-N into the MePD to deplete it of PSAs before female odor preference normally appears. This blocked female odor preference, which suggests that PSA-NCAM facilitates behaviorally relevant, hormone-driven plasticity.     

Chemosensory and hormone information are relayed directly between the medial amygdala, posterior bed nucleus of the stria terminalis, and medial preoptic area in male Syrian hamsters

In many rodent species, including Syrian hamsters, the expression of appropriate social behavior depends critically on the perception and identification of conspecific odors. The behavioral response to these odors is mediated by a network of steroid-sensitive ventral forebrain nuclei including the medial amygdala (Me), posterior bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA). Although it is well-known that Me, BNST, and MPOA are densely interconnected and each uniquely modulates odor-guided social behaviors, the degree to which conspecific odor information and steroid hormone cues are directly relayed between these nuclei is unknown. To answer this question, we injected the retrograde tracer, cholera toxin B (CTB), into the BNST or MPOA of male subjects and identified whether retrogradely-labeled cells in Me and BNST 1) expressed immediate early genes (IEGs) following exposure to male and/or female odors or 2) expressed androgen receptor (AR). Although few retrogradely-labeled cells co-localized with IEGs, a higher percentage of BNST- and MPOA-projecting cells in the posterior Me (MeP) expressed IEGs in response to female odors than to male odors. The percentage of retrogradely-labeled cells that expressed IEGs did not, however, differ between and female and male odor-exposed groups in the anterior Me (MeA), posterointermediate BNST (BNSTpi), or posteromedial BNST (BNSTpm). Many retrogradely-labeled cells co-localized with AR, and a higher percentage of retrogradely-labeled MeP and BNSTpm cells expressed AR than retrogradely-labeled MeA and BNSTpi cells, respectively. Together, these data demonstrate that Me, BNST, and MPOA interact as a functional circuit to process sex-specific odor cues and hormone information in male Syrian hamsters.     

Blood supply of the bed nucleus of the stria terminalis in rat

Summary The topographical distribution of the blood vessels in the bed nucleus of the stria terminalis (NIST) has been mapped in rats. Arteries and veins were visualized in red and blue by using a double-ink perfusion technique. Arteries supplying the NIST arise from the anterior cerebral artery directly or through the anterior communicating and interhemispheric arteries. Only a few, dorsal branches derive from the medial cerebral artery through thalamostriatal arteries. According to their terminal branches, NIST arteries can be divided into five groups: medial, ventral, lateral, septal and dorsal, which have only a relatively small overlap in their territories. About 90% of veins from the NIST drain into the major basal veins. Medial branches run into the perioptic and interhemispheric veins, while the ventral branches and the large lateral vein drain directly into the anterior cerebral vein. A small proportion of NIST veins run dorsalward into the vena cerebri magna via thalamostriatal veins.     

The bed nucleus of the stria terminalis has developmental and adult forms in mice,with the male bias in the developmental form being dependent on testicular AMH

Canonically, the sexual dimorphism in the brain develops perinatally, with adult sexuality emerging due to the activating effects of pubescent sexual hormones. This concept does not readily explain why children have a gender identity and exhibit sex-stereotypic behaviours. These phenomena could be explained if some aspects of the sexual brain networks have childhood forms, which are transformed at puberty to generate adult sexuality. The bed nucleus of stria terminalis (BNST) is a dimorphic nucleus that is sex-reversed in transsexuals but not homosexuals. We report here that the principal nucleus of the BNST (BNSTp) of mice has developmental and adult forms that are differentially regulated. In 20-day-old prepubescent mice, the male bias in the principal nucleus of the BNST (BNSTp) was moderate (360 ± 6 vs 288 ± 12 calbindin^+ ve neurons, p < 0.0001), and absent in mice that lacked a gonadal hormone, AMH. After 20 days, the number of BNSTp neurons increased in the male mice by 25% (p < 0.0001) and decreased in female mice by 15% (p = 0.0012), independent of AMH. Adult male AMH-deficient mice had a normal preference for sniffing female pheromones (soiled bedding), but exhibited a relative disinterest in both male and female pheromones. This suggests that male mice require AMH to undergo normal social development. The reported observations provide a rationale for examining AMH levels in children with gender identity disorders and disorders of socialization that involve a male bias.     

Vasotocin neurons in the bed nucleus of the stria terminalis preferentially process social information and exhibit properties that dichotomize courting and non-courting phenotypes

Neurons within the medial bed nucleus of the stria terminalis (BSTm) that produce arginine vasotocin (VT; in non-mammals) or arginine vasopressin (VP; in mammals) have been intensively studied with respect to their anatomy and neuroendocrine regulation. However, almost no studies have examined how these neurons process stimuli in the animals' immediate environment. We recently showed that in five estrildid finch species, VT-immunoreactive (-ir) neurons in the BSTm increase their Fos expression selectively in response to positively-valenced social stimuli (i.e., stimuli that should elicit affiliation). Using male zebra finches, a highly gregarious estrildid, we now extend those findings to show that VT-Fos coexpression is induced by a positive social stimulus (a female), but not by a positive non-social stimulus (a water bath in bath-deprived birds), although the female and bath stimuli induced Fos equally within a nearby control region, the medial preoptic nucleus. In concurrent experiments, we also show that the properties of BSTm VT-ir neurons strongly differentiate males that diverge in social phenotype. Males who reliably fail to court females (“non-courters”) have dramatically fewer VT-ir neurons in the BSTm than do reliable courters, and the VT-ir neurons of non-courters fail to exhibit Fos induction in response to a female stimulus.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Conversion of testosterone to estradiol may not be necessary for the expression of mating behavior in male Syrian hamsters (Mesocricetus auratus)

Male sexual behavior is mediated in part by androgens, but in several species, mating is also influenced by estradiol formed locally in the brain by the aromatization of testosterone. The role of testosterone aromatization in the copulatory behavior of male Syrian hamsters is unclear because prior studies are equivocal. Therefore, the present study tested whether blocking the conversion of testosterone to estradiol would inhibit male hamster sexual behavior. Chronic systemic administration of the nonsteroidal aromatase inhibitor Fadrozole (2.0 mg/kg/day) for 5 or 8 weeks did not significantly increase mount latency or reduce mount frequency, intromission frequency, ejaculation frequency, or anogenital investigation relative to levels shown by surgical controls. However, Fadrozole effectively inhibited aromatase activity, as evidenced by the suppression of estrogen-dependent progesterone receptor immunoreactivity in the male hamster brain. The JZB39 anti-progesterone receptor antibody labeled significantly more neurons in brains of sham-treated hamsters than in brains of Fadrozole-treated hamsters. These data suggest that aromatization of testosterone to estradiol is not necessary for normal mating behavior in Syrian hamsters.     

Patterns of neural activation associated with exposure to odors from a familiar winner in male golden hamsters

The neural mechanisms underlying recognition of familiar individuals and responses appropriate to them are not well known. Previous studies with male golden hamsters have shown that, after a series of brief aggressive encounters, a loser selectively avoids his own, familiar winner but does not avoid other males. Using this paradigm, we investigated activity in 20 areas of the brain using immunohistochemistry for c-Fos and Egr-1 during exposure to a familiar winner compared to control groups not exposed to another male. Behavioral data showed that 1 day after fights males that lost avoided the familiar winner, suggesting that they recognized this individual. The c-Fos and Egr-1 immunohistochemistry showed that the losers exposed to familiar winners had a greater density of stained cells in the basolateral amygdala, the CA1 region of anterior dorsal hippocampus and the dorsal subiculum than control groups had in these areas. These results suggest that these brain areas may be involved in the memory for other males, the learned fear of familiar winners, or related processes.     

Chronic morphine treatment and withdrawal increase extracellular levels of norepinephrine in the rat bed nucleus of the stria terminalis

Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an alpha(2)-adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal.     

Inactivation of the medial preoptic area or the bed nucleus of the stria terminalis differentially disrupts maternal behavior in sheep

The present study was designed to investigate the role of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST) in the onset and maintenance of maternal behavior in sheep. In a first experiment, the MPOA or BNST were transiently inactivated during the first 2 h postpartum in primiparous ewes with the use of the anaesthetic lidocaine. MPOA inactivation greatly impaired the display of maternal behavior whereas inactivation of BNST or of adjacent sites (septum or diagonal band of Broca) or infusion of cerebrospinal fluid did not. In a separation/reunion lamb test (S/R) performed at 2 h postpartum, ewes with MPOA inactivation exhibited little reaction after separation of their lambs and did not show any motivation to reunite with them. Ewes with BNST inactivation showed intermediate performances in the S/R test. Moreover, in control ewes that were maternal for the first 2 h postpartum, MPOA or BNST inactivation performed in the following 12 h induced deficits in the S/R test, indicating that the MPOA and to a lesser extent BNST are also involved in the maintenance of maternal behavior. A second experiment showed that, in multiparous ewes, MPOA inactivation at parturition induced less deficit in the display of maternal behavior and in the S/R test than in primiparous mothers. These findings indicate that the MPOA and, to some extent, the BNST are functionally involved in the initiation and in the maintenance of maternal behavior in sheep, but this involvement is influenced by maternal experience.     

Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: role of Bax-dependent cell death

Neuron number in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is greater in adult male mice than in females. Deletion of the proapoptotic gene, Bax, increases the number of BNSTp cells in adulthood and eliminates the sex difference in cell number. Here, we map the ontogeny of sex differences in nuclear volume and cell number in the BNSTp of neonatal mice, and evaluate the role of cell death in the development of these differences. We find that BNSTp volume and cell number do not differ between male and female wild-type mice on postnatal days P3, P5, or P7. Sex differences emerge after the first postnatal week and both measures are significantly greater in males than in females on P9 and P11. Cell death, assessed by TUNEL staining, was observed in the BNSTp of both sexes from P1-P8. Females had more TUNEL-positive cells than males from approximately P3-P6, with the maximum number of dying cells observed on P5/P6. To test whether the Bax gene is required for sexually dimorphic cell death in the BNSTp, TUNEL cells were counted on P6 in Bax -/- mice and their Bax +/+ siblings. Bax gene deletion nearly abolished TUNEL-positive cells in the BNSTp of both sexes. Together, these findings support the interpretation that the sex difference in BNSTp cell number seen in adulthood is due to Bax-dependent, sexually dimorphic cell death during the first week of life.     

Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat.

The central nucleus of the amygdala, bed nucleus of the stria terminalis, and central gray are important components of the neural circuitry responsible for autonomic and behavioral responses to threatening or stressful stimuli. Neurons of the amygdala and bed nucleus of the stria terminalis that project to the midbrain central gray were tested for the presence of peptide immunoreactivity. To accomplish this aim, a combined immunohistochemical and retrograde tracing technique was used. Maximal retrograde labeling was observed in the amygdala and bed nucleus of the stria terminalis after injections of retrograde tracer into the caudal ventrolateral midbrain central gray. The majority of the retrogradely labeled neurons in the amygdala were located in the medial central nucleus, although many neurons were also observed in the lateral subdivision of the central nucleus. Most of the retrogradely labeled neurons in the BST were located in the ventral and posterior lateral subdivisions, although cells were also observed in most other subdivisions. Retrogradely labeled neurotensin, corticotropin releasing factor (CRF), and somatostatin neurons were mainly observed in the lateral central nucleus and the dorsal lateral BST. Retrogradely labeled substance P-immunoreactive cells were found in the medial central nucleus and the posterior and ventral lateral BST. Enkephalin-immunoreactive retrogradely labeled cells were not observed in the amygdala or bed nucleus of the stria terminalis. A few cells in the hypothalamus (paraventricular and lateral hypothalamic nuclei) that project to the central gray also contained CRF and neurotensin immunoreactivity. The results suggest the amygdala and the bed nucleus of the stria terminalis are a major forebrain source of CRF, neurotensin, somatostatin, and substance P terminals in the midbrain central gray.