Differential effects of gonadal steroids on dopamine metabolism in mesolimbic and nigro-striatal pathways of male rat brain

Thirty days after castration the concentration of dopamine (DA) was significantly reduced in the septum and n. accumbens septi, but not in the caudate-putamen, of male rat brain. The concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the principle metabolites of DA, also tended to be lower in septum and n. accumbens septi after castration. Chronic s.c. administration of testosterone (T), estradiol (E₂), 5α-dihydrotestosterone (DHT), or E₂ plus DHT in silastic capsules effectively reversed these effects of castration in septum and n. accumbens septi without affecting concentrations of DA, DOPAC, or HVA in caudate-putamen. The accumulation of DOPA after inhibition of aromatic amino acid decarboxylase activity, which was taken as an in vivo index of tyrosine hydroxylase activity, was not affected in these brain regions by long-term castration or by chronic administration of DHT to castrated males. Acute administration of haloperidol caused equivalent, significant increments in concentrations of DOPAC and HVA in all brain regions studied, regardless of whether castrated rats had been implanted with DHT capsules or no hormone. However, in the absence of haloperidol treatment the concentration of DOPAC in septum and n. accumbens septi, but not in caudate-putamen, was significantly higher in castrated rats implanted with DHT as opposed to no hormone. These results suggest that chronic exposure to T, or to its neural metabolites, E₂ and DHT, selectively enhances metabolic activity in mesolimbic DA neurons.     

Androgen and Estrogen Effects on Vasopressin Messenger RNA Expression in the Medial Amygdaloid Nucleus in Male and Female Rats

Vasopressin messenger RNA (AVP mRNA) expression in the medial amygdala and bed nucleus of the stria terminalis (BST) is almost completely dependent on gonadal steroids. In the BST, the effects of gonadal steroids on AVP mRNA expression are sexually dimorphic. Males have more cells that express AVP mRNA and more AVP mRNA per cell than females. Here we test whether this is also true for the MA. In gonadectomized rats that were treated with testosterone, males had more cells that were labeled for AVP mRNA than females. However, the labeling per cell did not differ between males and females. To assess contribution of testosterone metabolites to these differences, male and female rats were gonadectomized and implanted with empty tubing, or tubing filled with dihydrotestosterone (DHT), estradiol (E), or E plus DHT (E + DHT). The pattern of steroid effects on AVP mRNA expression in the MA was similar in both sexes. Hardly any labeled cells were found in rats with empty implants or rats treated with DHT. Significantly more labeled cells were found in rats treated with E, and even more cells in rats treated with E + DHT. The number of AVP mRNA-labeled cells was higher in males than in females for E as well as E + DHT treatment, but the labeling per cell did not differ between sexes. These data suggest that the number of MA cells that can express AVP mRNA is higher in males than in females, but the estrogen and androgen responsiveness of individual AVP mRNA-expressing cells in the MA does not differ between sexes.     

Oxytocin and vasopressin release in discrete brain areas after naloxone in morphine-tolerant and -dependent anesthetized rats: push-pull perfusion study.

The effects of naloxone on the release of oxytocin and vasopressin in discrete brain areas were investigated in control and morphine-tolerant/dependent female rats anesthetized with urethane. Two or three consecutive push-pull perfusates were collected for 30-40 min each and the peptide contents measured by radioimmunoassay; naloxone (5 mg/kg, i.v.) was given after the first perfusion. In control rats, naloxone did not increase oxytocin release from any of the regions studied: mediolateral septum, dorsal hippocampus, nucleus of tractus solitarius, or supraoptic nucleus. After naloxone, vasopressin release was approximately doubled in the nucleus of tractus solitarius (p less than 0.05), indicating endogenous opioid inhibition of vasopressin release. Naloxone increased oxytocin concentration in the circulation 3.7-fold (p less than 0.001) but did not affect vasopressin secretion. In rats made morphine tolerant/dependent by intracerebroventricular infusion of morphine for 5 d, oxytocin and vasopressin release in the perfused brain was initially similar to that in control rats, indicating tolerance to any initial morphine effects. In these rats, naloxone increased oxytocin release in the septum threefold relative to control rats (p less than 0.02) but did not alter oxytocin release in hippocampus or nucleus of tractus solitarius. Thus, the oxytocin neurons projecting to septum can develop morphine dependence and may be inhibited acutely by opioids acting via mu-receptors. The results indicate morphine acts selectively on oxytocin neurons projecting to mediolateral septum compared with other central projection areas and compared with centrally projecting vasopressin neurons. In the supraoptic nucleus, naloxone increased oxytocin release 2.3-fold (from 9.2 +/- 3.1 pg/30 min) and increased oxytocin release from axons of these neurons fivefold (from 7.8 +/- 3.2 pg/30 min). Naloxone had no significant effect on vasopressin release from any of the central sites, or on vasopressin secretion into blood, although oxytocin secretion was increased 36-fold (from 17.2 +/- 2.6 pg/ml; p less than 0.001), confirming dependence of magnocellular oxytocin neurons. The central processes of magnocellular supraoptic neurons may be a major source of central oxytocin released during morphine withdrawal.     

The origin of the vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum

The origin of the vasopressin-containing fibers in the rat lateral septum was studied by means of lesioning specific areas, in which vasopressin-containing cells are found, or by surgically separating the septum from the underlying structures. Following these procedures sections of the brain were stained immunocytochemically for the presence of vasopressin. In addition, retrograde labelling tracers were injected in the lateral septum.Lesioning of the paraventricular nucleus did not result in the disappearance of vasopressin fibers from the lateral septum, nor from the various other areas studied. It did, however, cause the disappearance of fibers from the nucleus of the solitary tract and the nucleus ambiguus. By contrast, after the same lesion practically the whole oxytocinergic innervation of the brain disappeared. Injection of tracers into the lateral septum revealed retrograde labeled cells, e.g. in the bed nucleus of the stria terminalis, but not in the paraventricular and supraoptic nucleus.Horizontal cuts under the lateral septum, intersecting the diagonal band of Broca, resulted in a dramatic decrease of the vasopressin fibers in the lateral septum, suggesting that the fibers enter the septum via this structure. Moreover, since the vasopressin fiber density was found to decrease drastically in the lateral septum after lesioning the bed nucleus of the stria terminalis, the vasopressin cells found in this area are probably the source of these fibers. Other areas where fibers were seen to decrease after lesions of the bed nucleus are the diagonal band of Broca, the area of the anterior amygdala, the lateral habenular nucleus, the periventricular gray, and the locus coeruleus.     

The vasopressinergic innervation of the lateral septum of the rat after chronic alcohol consumption and withdrawal

We have recently reported that ethanol ingestion induces morphological changes in the vasopressinergic neurons of the supraoptic nucleus and that withdrawal from alcohol partially reverses these alterations. Since the production of vasopressin is not restricted to the magnocellular neurons of the hypothalamus, we investigated the effects of long-term ethanol intake and withdrawal on the lateral septum, an area heavily innervated by vasopressinergic fibers. Besides, as ethanol leads to a decrease of the plasma levels of testosterone, a hormone which plays a pivotal role in the development and maintenance of the vasopressinergic innervation of the lateral septum, we included groups of alcohol-fed animals submitted to testosterone replacement both in physiological and supraphysiological doses. In ethanol-treated rats there was a marked reduction in the number of vasopressin-immunoreactive fibers in the lateral septum. Following ethanol withdrawal a partial recovery in the number of vasopressin-immunoreactive fibers was observed. In both groups of ethanol + testosterone-treated animals the vasopressinergic innervation was increased when compared to the alcohol-fed group, although a complete reversal was not achieved. Therefore, two mechanisms might be regarded as underlying the impoverishment of the vasopressinergic innervation in the lateral septum after prolonged alcohol consumption: alcohol-induced cell death in the bed nucleus of the stria terminalis, from where these fibers arise, and/or alcohol-induced decrease in testosterone plasma levels.     

Testosterone supplementation restores vasopressin innervation in the senescent rat brain

The vasopressin (AVP) innervation in the male rat brain is decreased in senescence. This decrease is particularly pronounced in brain regions where AVP fiber density is dependent on plasma levels of sex steroids. Since plasma testosterone levels decrease progressively with age in the rat, the possibility of restoring central AVP innervation by peripheral testosterone supplementation was investigated by giving senescent (33 months) Brown-Norway rats subcutaneous implants of either empty or testosterone-filled silastic tubes for the period of 1 month. Plasma testosterone levels of testosterone-treated animals were restored to values which did not differ from those of young animals. The results show that the age-related decline in AVP fiber density can indeed be reversed by testosterone supplementation. In contrast, oxytocin innervation, which was previously shown not to be testosterone-dependent, was not restored. These results show for the first time restoration of a specific innervation pattern in the senescent rat brain mediated by peripheral hormones and indicate that a considerable plasticity is retained in the aging central nervous system.     

Gonadal steroids regulate oxytocin receptors but not vasopressin receptors in the brain of male and female rats. An autoradiographical study

The distribution and the amount of [3H]oxytocin binding were studied in the brain of adult rats of either sex, as well as in male and female castrates, some of which received injections of estradiol or testosterone. Intact males were treated with an aromatase inhibitor. Castration and inhibition of aromatase activity reduced, whereas estradiol and testosterone increased oxytocin binding, particularly in regions of the brain assumed to be involved in reproductive functions, such as the ventrolateral part of the hypothalamic ventromedial nucleus and the islands of Calleja and neighbouring cell groups. Binding of oxytocin to the uterus was also estrogen-dependent. In the same animals, we also studied the distribution of [3H]vasopressin binding sites present in the brain. It was similar in males and females, and was not affected by experimentally manipulating gonadal hormone levels. In immunocytochemical studies we noticed, as others had previously, that the vasopressin content of certain areas of the rat brain was affected by castration, whereas the oxytocin innervation was not. These results are discussed in relation to the possible functions of oxytocin in the brain and of the lack of correspondence between the immunocytochemical and the autoradiographic data.     

Chronic morphine treatment inhibits oxytocin synthesis in rats

The changes of oxytocin content and mRNA expression in some nuclei were investigated in morphine-dependent rats using radioimmunoassay (RIA) and in situ hybridization (ISH). After chronic administration of morphine, the oxytocin content in supraoptic nucleus (SON) and nucleus accumbens (NAc) decreased, and increased in the ventral tegment area (VTA) and locus coeruleus (LC), but did not change in other nuclei including the paraventricular nucleus (PVN), lateral septum (SEPTUM), raphe magnus nucleus (NRM) and periaquaductal gray (PAG). In morphine-L dependent rats, naloxone increased the levels of oxytocin in SON and PVN, but decreased that in LC. ISH first showed that chronic morphine treatment inhibited the oxytocin synthesis in SON but not in PVN. The present study demonstrates that chronic morphine treatment alters the brain oxytocin system, suggesting that oxytocin might contribute to the behavioral and neuroendocrine responses to morphine.     

Effects of lesions in the hypothalamic paraventricular, supraoptic and suprachiasmatic nuclei on vasopressin and oxytocin in rat brain and spinal cord

The content of arginine vasopressin and oxytocin in various extrahypothalamic sites of the rat brain and spinal cord was determined by specific radioimmunoassays after lesions had been made in either the paraventricular (PVN), supraoptic (SON) or suprachiasmatic nuclei (SCN). In some animals all 3 nuclei were destroyed together. The PVN provided a considerable amount of the vasopressin innervation of the solitary tract nucleus, and most of that in the spinal cord. Oxytocin was removed from some areas after lesions of the PVN and, again, most of this peptide was lost from the spinal cord. Lesions of the SCN did not appear to be followed by significant quantitative changes in either hormone in any of the areas studied. Lesions of the SON resulted in loss of oxytocin, particularly in the periventricular grey and some other areas, suggesting that extrahypothalamic projections from this nucleus may be more important than was previously assumed. Lesions of all 3 nuclei which included destruction of accessory hypothalamic nuclei resulted in a much more widespread loss of vasopressin and oxytocin, but there was preservation of both peptides in the dorsal raphe nucleus and much of those present in the locus coeruleus. It is concluded that the contribution of the classical hypothalamic nuclei to the extrahypothalamic content of vasopressin and oxytocin in rat brain is less than was originally believed, and that there are areas of the brain such as the locus coeruleus and dorsal raphe nucleus in which the source of these peptides may be outside the hypothalamus.     

A combined electron microscopic HRP and immunocytochemical study of the limbic projections to rat hypothalamic nuclei containing vasopressin and oxytocin neurons

Light microscopic studies in our laboratory have indicated that the lateral septum, amygdala, and ventral subiculum project in a perinuclear fashion to the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei (Oldfield et al., '82; Silverman and Oldfield, '84). In the present paper a combined anterograde HRP and immunocytochemical procedure has been used to determine the connectivity between these limbic efferents and peptide-containing processes emanating from the above mentioned hypothalamic nuclei. Synaptic associations were found to exist between efferents from (1) the septum and both vasopressin (VP)- and oxytocin (OX)-positive dendrites derived from cells in the PVN and SON, (2) the septum and VP dendrites dorsal to the SCN, (3) the ventral subiculum and both VP and OX dendrites arising from the PVN and SON, and (iv) the amygdala and VP dendrites from the PVN. These observations help clarify an apparent discrepancy between electrophysiological data, in which limbic efferents have been shown to influence the activity of VP and OX neurons in the PVN and SON, and anatomical evidence which indicates only a perinuclear innervation from these sites not encroaching on the hypothalamic nuclei themselves. In each case the synaptic connections are made on dendrites external to the nucleus: those lateral and ventrolateral to the PVN, dorsal to the SON, and dorsal or dorsolateral to the SCN.     

Androgens alter corticotropin releasing hormone and arginine vasopressin mRNA within forebrain sites known to regulate activity in the hypothalamic-pituitary-adrenal axis

To reveal direct effects of androgens, independent of glucocorticoids, we studied the effects of gonadectomy (GDX) in adrenalectomized (ADX) rats with or without androgen replacement on corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression within various forebrain sites known to regulate the hypothalamic-pituitary-adrenal axis. These included the medial parvocellular portion of the paraventricular nucleus of the hypothalamus (mp PVN), the central and medial nuclei of the amygdala and bed nuclei of the stria terminalis (BNST). In the mp PVN, ADX stimulated both CRH and AVP mRNA expression. Combined ADX + GDX inhibited only AVP, and testosterone and dihydrotestosterone (DHT) restored AVP mRNA. In the central nucleus of the amygdala, ADX decreased CRH mRNA expression, and this response was unaffected by GDX +/- testosterone or DHT replacement. In the medial amygdala, AVP mRNA expression was decreased by ADX, abolished by ADX + GDX, and restored by androgen replacement. ADX had no effect on CRH and AVP mRNA expression in the BNST. GDX + ADX, however, reduced CRH mRNA expression only within the fusiform nuclei of the BNST and reduced the number of AVP-expressing neurones in the posterior BNST. Androgen replacement reversed both responses. In summary, in ADX rats, AVP, but not CRH mRNA expression in the amygdala and mp PVN, is sensitive to GDX +/- androgen replacement. Both CRH- and AVP-expressing neurones in the BNST respond to GDX and androgen replacement, but not to ADX alone. Because androgen receptors are not expressed by hypophysiotropic PVN neurones, we conclude that glucocorticoid-independent, androgenic influences on medial parvocellular AVP mRNA expression are mediated upstream from the PVN, and may involve AVP-related pathways in the medial amygdala, relayed to and through CRH- and AVP-expressing neurones of the BNST.     

Regional differences in testosterone effects on vasopressin receptors and on vasopressin immunoreactivity in intact and castrated Siberian hamsters

Vasopressin binding sites were detected in the brain of the Siberian hamster, using [³H]vasopressin and a ¹²⁵I-labelled linear vasopressin antagonist specific for V₁ vasopressin receptors. In the ventromedial and remammillary nuclei, the density of the binding was lower in the females than in the males. The effect of castration and of testosterone replacement was assessed in males. Two distinct effects were observed. Orchidectomy diminished significantly the vasopressin binding in the ventromedial nucleus, an effect which was prevented by implantation of a mini-pump releasing testosterone. On the contrary, in the premammillary nucleus no significant differences were noticed following castration and testosterone treatment. In addition, vasopressin immunoreactivity was examined in males, in females and in castrated males. No sex differences were evident. However, in the bed nucleus of the stria terminalis and the lateral septal nucleus, castration decreased vasopressin immunoreactivity in either sex. This effect of castration was prevented by testosterone. Vasopressin immunoreactivity was detected neither in the ventromedial nor in the premammillary hypothalamic nuclei. Our observations suggest that, in adult Siberian hamster premammillary nucleus, the expression of vasopressin receptors is not controlled by gonadal steroids but is sex related and could be induced during fetal or early postnatal life.     

Histoautoradiographic detection of oxytocin- and vasopressin-binding sites in the telencephalon of the rat

Localization of oxytocin- and vasopressin-binding sites has so far been studied in the rat brain by means of film autoradiographs. The disposal of iodinated ligands with high specificity has allowed us to develop histoautoradiography on emulsion-coated sections and to reinvestigate on a microscopic scale the distribution of these sites in the telencephalon (septum, striatopallidal system, amygdala and hippocampus). This technique showed that oxytocin and vasopressin labelling presented distinct distributions and coincided with delimited zones, corresponding to anatomical subdivisions defined on cytoarchitectural and immunocytochemical bases. Vasopressin sites were seen in the dorsal and intermediate parts of the lateral septum and the juxtacapsular nucleus of the bed nucleus of the stria terminalis. Oxytocin sites were located in the ventral and intermediate parts of the lateral septum, the oval and the principal nuclei of the bed nucleus of the stria terminalis and the septofimbrial nucleus. In the striatopallidal system, vasopressin sites were found in the accumbens nucleus and the fundus striati, whereas oxytocin sites were in the accumbens nucleus, the head, and the posterolateral parts of the caudate-putamen, the striatal cell bridges, and the olfactory tubercle. In the amygdala, vasopressin sites were not found, but oxytocin sites were located in the central, medial, and basomedial nuclei. In the hippocampus, vasopressin sites were located in the dentate gyrus (polymorph and molecular layers), and oxytocin sites, in the subiculum (molecular and pyramidal layers) and in the field CA1 of Ammon's horn (lacunosum moleculare and pyramidal layers). The localization of the binding sites at the microscopic level permitted us to reinvestigate whether or not correlation existed in a same area between innervation, electrophysiological effects, and presence of binding sites. © 1993 Wiley-Liss, Inc.     

Septal and Hippocampal Release of Oxytocin, but not Vasopressin, in the Conscious Lactating Rat During Suckling

The central release of both oxytocin and vasopressin within the septum and dorsal hippocampus in response to suckling was studied in conscious, freely-behaving lactating rats. Three consecutive 30-min push-pull perfusions were carried out before, during and after suckling (suckled group) or without suckling (control group). As compared to control levels, suckling resulted in a significantly increased oxytocin release within both limbic brain areas (septum: to 140%, dorsal hippocampus: to 1,600%). After removal of the suckling pups, the oxytocin concentration in the final perfusates remained at the stimulation level (septum) or tended to return to control values (dorsal hippocampus). In contrast to oxytocin, the vasopressin perfusate levels did not differ significantly between unsuckled and suckled rats.     

Release of Oxytocin into Blood and into Cerebrospinal Fluid Induced by Naloxone in Anaesthetized Morphine-Dependent Rats: The Role of the Paraventricular Nucleus

Opioid actions on oxytocin secretion into blood and cerebrospinal fluid (CSF) were investigated in urethane-anaesthetized female rats after intracerebroventricular (icv) infusion of morphine sulphate or vehicle for 5 days. Serial femoral arterial blood samples and cisterna magna CSF samples were collected for radioimmunoassay. Naloxone was given to assess endogenous opioid tone in icv vehicle-infused rats and to precipitate withdrawal in morphine-dependent animals. Initial plasma oxytocin concentration was not affected by icv morphine infusion. In control rats receiving icv vehicle, naloxone increased plasma oxytocin 11-fold within 5 min, and in icv morphine-infused rats, naloxone increased plasma oxytocin 80-fold within 5 min. In both groups, 90 min after naloxone plasma oxytocin was still 5 and 10 times, respectively, the initial concentration. Without naloxone, neither plasma nor CSF oxytocin concentration changed significantly with time (up to 90 min) in either icv treatment group. In the icv vehicle group, there was a 2-fold increase in CSF oxytocin 90 min after naloxone. In the icv morphine-infused group, CSF oxytocin was increased 5-fold 40 min after naloxone. In another group of icv morphine-infused rats, intravenous infusion of oxytocin to achieve plasma levels similar to those seen after naloxone, did not significantly increase CSF oxytocin. In a further group of icv morphine-infused rats, [³H]oxytocin was infused intravenously immediately after naloxone was given; in these rats oxytocin transfer from blood to CSF could account at most for only 20% of the increase in CSF oxytocin after naloxone. A further group of rats underwent bilateral microknife ablation of the paraventricular nuclei (PVN) 9 days before icv vehicle or morphine infusions were started; blood and CSF samples were collected under urethane anaesthesia. Initial concentrations of oxytocin in CSF and in plasma were similar in both groups with PVN ablation. In all PVN-lesioned rats initial plasma concentrations of oxytocin were undetectable (<5 pg/ml) and thus less than in intact rats. In contrast, initial levels of oxytocin in CSF were 8-fold greater in PVN-lesioned rats than in intact animals. Naloxone increased plasma oxytocin concentration in the icv vehicle group at least 10-fold within 30 min and in the icv morphine group at least 100-fold within 5 min. CSF oxytocin in the icv vehicle group was not altered by naloxone, but in the icv morphine group CSF oxytocin was increased 5-fold 40 min after naloxone. There were no consistent differences between the icv vehicle- and icv morphine-treated groups in the initial plasma levels of vasopressin, growth hormone and adrenocorticotrophin; PVN ablation did not affect adrenocorticotrophin levels. After naloxone growth hormone levels did not change, vasopressin concentration rose moderately only after 90 min and only in the icv vehicle-treated group, and adrenocorticotrophin concentrations decreased with time whether or not naloxone was given. The results imply an endogenous opioid tone on neurons releasing oxytocin into CSF, and morphine-dependence of these neurons. Furthermore, in PVN-lesioned rats, magnocellular supraoptic neurons could be a source of oxytocin release into CSF.     

Gonadal Steroids have Paradoxical Effects on Brain Oxytocin Receptors

Specific brain receptors for oxytocin have been described in several mammalian species. The distribution of these receptors differs greatly across species and in the rat, receptor binding in specific brain regions appears to depend upon gonadal steroids. This study used in vitro receptor autoradiography to examine the effects of testosterone on oxytocin receptor binding in the mouse forebrain. Three groups of male mice were compared: castrates treated with blank capsules, castrates treated with testosterone filled capsules, and intact males. Irrespective of steroid treatment, the distribution of oxytocin receptors in mouse forebrain differed markedly from patterns previously described in the rat. In addition to these species differences in receptor distribution, testosterone had effects in the mouse which differed from the induction of receptors previously reported in the rat. In the mouse ventromedial nucleus of the hypothalamus, binding in the untreated castrate males was approximately double that observed in either the intact or the testosterone-treated castrates. In other regions of the mouse brain, such as the intermediate zone of the lateral septum, binding to oxytocin receptors was increased with testosterone treatment. These results suggest that the brain oxytocin receptor varies across species not only in its distribution but also in its regional regulation by gonadal steroids. These apparently paradoxical changes in oxytocin receptor binding may result from either direct or indirect effects of gonadal steroids in mouse brain.     

Vasopressin innervation of sexually dimorphic structures of the gerbil forebrain under various hormonal conditions.

The distribution of vasopressin-immunoreactive fibers in the forebrain of male and female gerbils was studied, focusing on the lateral septum and the sexually dimorphic area (SDA) found at the border between the medial preoptic area and the anterior hypothalamus. To study hormonal influences on the densities of these fibers, some animals of each sex were gonadectomized or gonadectomized and given testosterone. Others were given sham operations. High densities of vasopressin-immunoreactive fibers were found in the lateral septum. In the SDA, the densities of these fibers varied considerably. Many were found in the medial half of the medial SDA, but few in the lateral SDA. Vasopressin-immunoreactive fibers were also sparse in the lateral half of the medial SDA, except for a dense cluster in the SDA pars compacta of males. Similar but smaller clusters were seen in the same location in females although the SDA pars compacta could not be detected in Nissl-stained sections from the female brains. Fiber densities in two areas, the lateral septum and the lateral SDA, were sensitive to gonadal steroids. In both cases, castration reduced fiber density and testosterone enhanced it. In addition, fiber densities in two areas, the lateral septum and the medial SDA, were sexually dimorphic. In each case, fiber density was greater in males. There was no hormonal effect, however, on the fiber densities in the medial SDA. The fact that the fiber plexuses in the lateral septum and the medial SDA respond differently to gonadal steroids suggests that they arise from different cells and possibly from different areas of the brain. The vasopressin-immunoreactive fibers in the lateral septum probably come from steroid-sensitive vasopressin neurons in the bed nucleus of the stria terminalis. Those in the medial SDA may originate in the dorsal aspect of the suprachiasmatic nucleus where vasopressin-immunoreactive cell bodies were seen.