Distribution of aromatase-immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): Implications for the neural action of steroids and nuclear definition in the avian hypothalamus

Cells immunoreactive for the enzyme aromatase were localized in the forebrain of male zebra finches with the use of an immunocytochemistry procedure. Two polyclonal antibodies, one directed against human placental aromatase and the other directed against quail recombinant aromatase, revealed a heterogeneous distribution of the enzyme in the telencephalon, diencephalon, and mesencephalon. Staining was enhanced in some birds by the administration of the nonsteroidal aromatase inhibitor, R76713 (racemic Vorozole) prior to the perfusion of the birds as previously described in Japanese quail. Large numbers of cells immunoreactive for aromatase were found in nuclei in the preoptic region and in the tuberal hypothalamus. A nucleus was identified in the preoptic region based on the high density of aromatase immunoreactive cells within its boundaries that appears to be homologous to the preoptic medial nucleus (POM) described previously in Japanese quail. In several birds alternate sections were stained for immunoreactive vasotocin, a marker of the paraventricular nucleus (PVN). This information facilitated the clear separation of the POM in zebra finches from nuclei that are adjacent to the POM in the preoptic area-hypothalamus, such as the PVN and the ventromedial nucleus of the hypothalamus. Positively staining cells were also detected widely throughout the telencephalon. Cells were discerned in the medial parts of the ventral hyperstriatum and neostriatum near the lateral ventricle and in dorsal and medial parts of the hippocampus. They were most abundant in the caudal neostriatum where they clustered in the dorsomedial neostriatum, and as a band of cells coursing along the dorsal edge of the lamina archistriatalis dorsalis. They were also present in high numbers in the ventrolateral aspect of the neostriatum and in the nucleus taeniae. None of the telencephalic vocal control nuclei had appreciable numbers of cells immunoreactive for aromatase within their boundaries, with the possible exception of a group of cells that may correspond to the medial part of the magnocellular nucleus of the neostriatum. The distribution of immunoreactive aromatase cells in the zebra finch brain is in excellent agreement with the distribution of cells expressing the mRNA for aromatase recently described in the finch telencephalon. This widespread telencephalic distribution of cells immunoreactive for aromatase has not been described in non-songbird species such as the Japanese quail, the ring dove, and the domestic fowl. © 1996 John Wiley & Sons, Inc.     

Neuroanatomical Distribution and Variations across the Reproductive Cycle of Aromatase Activity and Aromatase-Immunoreactive Cells in the Pied Flycatcher (Ficedula hypoleuca)

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

the present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.     

Distribution of aromatase-immunoreactive cells in the mouse forebrain

Summary The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.     

Aromatase immunoreactivity in the rat brain: Gonadectomy-sensitive hypothalamic neurons and an unresponsive “limbic ring” of the lateral septum-bed nucleus-amygdala complex

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.     

Effects of the nonsteroidal inhibitor R76713 on testosterone-induced sexual behavior in the Japanese quail (Coturnix coturnix japonica)

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid-metabolizing enzymes and without interacting with estrogen, progestin, or androgen receptors. This compound was tested for its capacity to intefere with the induction of copulatory behavior by testosterone (T) in castrated Japanese quail (Coturnix coturnix japonica). In a first experiment, R76713 inhibited (range 0.01 to 1 mg/kg) the activation of sexual behavior by T silastic implants and hypothalamic aromatase activity in castrated male quail in a dose-dependent manner. The 5 alpha- and 5 beta- reductases of T were not systematically affected. Stereotaxic implantation of R76713 in the medial preoptic area similarly blocked the behavior activated by systemic treatment with T, demonstrating that central aromatization of androgen is implicated in the activation of behavior. These inhibiting effects of R76713 on behavior were observed when implants were placed in the medial part of the nucleus preopticus medialis, confirming the implication of this brain area in the control of male copulatory behavior. Finally, the behavioral inhibition produced by R76713 could be reversed by simultaneous treatment with a dose of estradiol, which was not behaviorally effective by itself. This suggests that the behavioral deficit induced by the inhibitor was specifically due to the suppression of estrogen production. This also shows that the activation of copulatory behavior probably results from the interaction of androgens and estrogens at the brain level, as the two treatments separately providing these hormonal stimuli (T with the aromatase inhibitor on one hand and a low dose of estradiol on the other hand) had almost no behavioral effects but they synergized to activate copulation when given concurrently. These data confirm the critical role of preoptic aromatase in the activation of reproductive behavior and demonstrate that R76713 is a useful tool for the in vivo study of estrogen-dependent processes.     

Sexual differentiation of brain and behavior in quail and zebra finches: Studies with a new aromatase inhibitor, R76713

In many species of vertebrates, major sex differences affect reproductive behavior and endocrinology. Most of these differences do not result from a direct genomic action but develop following early exposure to a sexually differentiated endocrine milieu. In rodents, the female reproductive phenotype mostly develops in the absence of early steroid influence and male differentiation is imposed by the early action of testosterone, acting at least in part through its central conversion into estrogens or aromatization. This pattern of differentiation does not seem to be applicable to avian species. In Japanese quail (Coturnix japonica), injection of estrogens into male embryos causes a permanent loss of the capacity to display male-type copulatory behavior when exposed to testosterone in adulthood. Based on this experimental result, it was proposed that the male reproductive phenotype is “neutral” in birds (i.e. develops in the absence of endocrine influence) and that endogenous estradiol secreted by the ovary of the female embryo is responsible for the physiological demasculinization of females. This model could be recently confirmed. Females indeed display a higher level of circulating estrogens that males during the second part of their embryonic life. In addition, treatment of female embryos with the potent aromatase inhibitor, R76713 or racemic vorozole™ which suppresses the endogenous secretion of estrogens maintains in females the capacity to display the full range of male copulatory behaviors. The brain mechanisms that control this sexually differentiated behavior have not been identified so far but recent data suggest that they should primarily concern a sub-population of aromatase-immunoreactive neurons located in the lateral parts of the sexually dimorphic preoptic nucleus. The zebra finch (Taeniopygia guttata) exhibits a more complex, still partly unexplained, differentiation pattern. In this species, early treatment with exogenous estrogens produces a masculinization of singing behavior in females and a demasculinization of copulatory behavior in males. Since normal untreated males sing and copulate, while females never show these behaviors even when treated with testosterone, it is difficult to understand under which endocrine conditions these behaviors differentiate. In an attempt to resolve this paradox, we recently treated young zebra finches with R76713 in order to inhibit their endogenous estrogens secretion during ontogeny and we subsequently tested their behavior in adulthood. As expected, the aromatase inhibitor decreased the singing frequency in treated males but it did not affect the male-type copulatory behavior in females nor in males. In addition, the sexuality differentiated brain song control nuclei which are also masculinized in females by early treatment with estrogens, were not affected in either sex by the aromatase inhibitor. In conclusion, available data clearly show that sexual differentiation of reproductive behaviors in birds follows a pattern that is almost opposite to that of mammals. This difference may be related to the different mechanisms of sex determination in the two taxa. In quail, the ontogeny of behavioral differentiation is now well understood but we only have a very crude notion of the brain structures that are concerned. By contrast, in zebra finches, the brain mechanisms controlling the sexually differentiated singing behavior in adulthood have been well identified but we do not understand how these structures become sexually dimorphic during ontogeny.     

Comparative effects of the aromatase inhibitor R76713 and of its enantiomers R83839 and R83842 on steroid biosynthesis in vitro and in vivo

R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole) is a selective, non-steroidal aromatase inhibitor containing an asymmetric carbon atom. In this paper, we compare the effects of R76713 (racemate) with its enantiomers R83839 (the levo-isomer) and R83842 (the dextro-isomer) on steroid biosynthesis in rat cells in vitro and in the rat in vivo.

In rat granulosa cells, aromatase activity was inhibited by 50% at concentrations of 0.93 nM of R76713, 240 nM of R83839 and 0.44 nM of R83842, revealing a 545-fold difference in activity between both enantiomers.

Up to 1 μM, none of the compounds had any effect on steroid production in primary cultures of rat testicular cells. Above this concentration all three compounds showed a similar slight inhibition of androgen synthesis with a concomitant increase in the precursor progestins, indicative for some effect on the 17-hydroxylase/17,20-lyase enzyme. In rat adrenal cells none of the compounds showed any effect on corticosterone synthesis. At concentrations above 1 μM there was an increase in the levels of 11-deoxycorticosterone pointing towards an inhibition of the 11-hydroxylase enzyme. This increase was more pronounced for R83839 than for R76713 and R83842.

In vivo, in PMSG-primed rats, R83842 reduced plasma estradiol by 50%, 2 h after oral administration of 0.0034 mg/kg, whereas 0.011 mg/kg of R76713 and 0.25 mg/kg of R83839 were needed to obtain the same result.

Oral administration of up to 20 mg/kg of the compounds did not significantly affect plasma levels of adrenal steroids in LHRH/ACTH-injected rats. Plasma testosterone was lowered at 10 and 20 mg/kg of R83842 and at the highest dose (20 mg/kg) of R76713 and R83839.

In conclusion, the present study shows that the aromatase inhibitory activity of R76713 resides almost exclusively in its dextro-isomer R83842. R83842 exhibits a specificity for aromatase as compared to other enzymes involved in steroid biosynthesis of at least a 1000-fold in vitro as well as in vivo. This confirms the extreme selectivity previously found for the racemate.     

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

Quantitative Analysis of Long-Form Aromatase mRNA in the Male and Female Rat Brain

In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.     

Capsaicin-induced changes in LTP in the lateral amygdala are mediated by TRPV1

The transient receptor potential vanilloid type 1 (TRPV1) channel is a well recognized polymodal signal detector that is activated by painful stimuli such as capsaicin. Here, we show that TRPV1 is expressed in the lateral nucleus of the amygdala (LA). Despite the fact that the central amygdala displays the highest neuronal density, the highest density of TRPV1 labeled neurons was found within the nuclei of the basolateral complex of the amygdala. Capsaicin specifically changed the magnitude of long-term potentiation (LTP) in the LA in brain slices of mice depending on the anesthetic (ether, isoflurane) used before euthanasia. After ether anesthesia, capsaicin had a suppressive effect on LA-LTP both in patch clamp and in extracellular recordings. The capsaicin-induced reduction of LTP was completely blocked by the nitric oxide synthase (NOS) inhibitor L-NAME and was absent in neuronal NOS as well as in TRPV1 deficient mice. The specific antagonist of cannabinoid receptor type 1 (CB1), AM 251, was also able to reduce the inhibitory effect of capsaicin on LA-LTP, suggesting that stimulation of TRPV1 provokes the generation of anandamide in the brain which seems to inhibit NO synthesis. After isoflurane anesthesia before euthanasia capsaicin caused a TRPV1-mediated increase in the magnitude of LA-LTP. Therefore, our results also indicate that the appropriate choice of the anesthetics used is an important consideration when brain plasticity and the action of endovanilloids will be evaluated. In summary, our results demonstrate that TRPV1 may be involved in the amygdala control of learning mechanisms.     

Comparison of brain activity between dopamine D2 receptor-knockout and wild mice in response to dopamine agonist and antagonist assessed by fMRI

It has been reported that antipsychotic dopamine-D2-receptor (D2R) antagonists affected other neurotransmitter systems. In the present study, the effects of a D2R agonist, bromocriptine, and a D2R antagonist, spiperone, on brain activity were investigated using wild-type mice (WT) with intact D2Rs, and D2R-knockout mice (D2R-KO) lacking D2Rs by functional magnetic resonance imaging. In the WT, flow-weighted signal intensity significantly increased after administration of bromocriptine in the hippocampal formation. In contrast, signal intensity significantly decreased after administration of spiperone in the somatosensory-motor cortices, thalamus, anterior cingulate cortex, caudate-putamen, nucleus accumbens, hippocampal formation, and amygdala. In the D2R-KO, however, no significant changes were observed after administration of either bromocriptine or spiperone. The present results indicated that the D2R-KO lacked sensitivity to D2R agonist and antagonist in agreement with its genetic defects, which confirmed that the changes in brain activity in the WT after administration of either drug were mediated through D2Rs. These results suggest that antipsychotic D2R antagonists affect activity of the same brain regions of human patients through D2Rs, as observed in the present study. These changes in brain activity might be related to therapeutic efficacy as well as side effects of antipsychotic drugs on schizophrenic patients.     

R 76713, a new specific non-steroidal aromatase inhibitor

The effects of R 76713, a new triazole derivative, on rat ovarian, testicular and adrenal steroidogenesis were investigated both in vitro and in vivo. In vitro R 76713 is a very potent inhibitor of the aromatase enzyme in rat granulosa cells, showing an IC50-value of 3.0 +/- 0.2 nM. The compound is about 1000 times more active than aminoglutethimide which shows an IC50-value of 3900 +/- 2800 nM in the same system. R 76713 is also a highly selective aromatase inhibitor. In cultures of ovarian, testicular and adrenal cells, formation of progesterone, androgens and glucocorticoids was only affected by drug concentrations higher than 1 microM. In vivo, single oral drug doses of 0.05 mg/kg lowered plasma estradiol levels of PMSG-primed female rats by more than 90%. An ED50-value of 0.005 mg/kg could be calculated. A single oral dose of 1 mg/kg suppressed plasma estradiol levels almost completely for 24 h. A dose of 0.1 mg/kg lowered plasma estradiol by more than 90% for 8 h. In vivo, R 76713 also showed a highly selective profile. In LHRH/ACTH-injected rats, plasma levels of testicular and adrenal steroids remained unchanged after administration of a drug dose of 20 mg/kg. R 76713 at drug concentrations of 10 microM, showed no interaction in vitro with estrogen-, progestin-, androgen- and glucocorticoid-receptors. Given orally at 20 mg/kg for 3 days the compound also showed no estrogen or androgen agonistic or antagonistic effects.     

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled complementary (c)RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III-VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular, supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.     

Organizational effects of estrogens on brain vasotocin and sexual behavior in quail

Reproductive behavior is sexually differentiated in quail: The male-typical copulatory behavior is never observed in females even after treatment with high doses of testosterone (T). This sex difference in behavioral responsiveness to T is organized during the embryonic period by the exposure of female embryo to estrogens. We showed recently that the sexually dimorphic medial preoptic nucleus (POM), a structure that plays a key role in the activation of male copulatory behavior, is innervated by a dense steroid-sensitive network of vasotocin-immunoreactive (VT-ir) fibers in male quail. This innervation is almost completely absent in the female POM and is not induced by a chronic treatment with T, suggesting that this neurochemical difference could be organizational in nature. This idea was tested by injecting fertilized quail eggs of both sexes on day 9 of incubation with either estradiol benzoate (EB) (25 μg, a treatment that suppresses the capacity to show copulatory behavior in adulthood) or the aromatase inhibitor R76713 (10 μg, a treatment that makes adult females behaviorally responsive to T), or with the solvents as a control (C). At 3 weeks posthatch, all subjects were gonadectomized and later implanted with Silastic capsules filled with T. Two weeks later, all birds were perfused and brain sections were processed for VT immunocytochemistry. Despite the similarity of the adult endocrine conditions of the subjects (all were gonadectomized and treated with T Silastic implants providing the same plasma level of steroid to all subjects), major qualitative differences were observed in the density of VT-ir structures in the POM of the different groups. Dense immunoreactive structures (fibers and a few cells) were observed in the POM of C males but not females; EB males had completely lost this immunoreactivity (and lost the capacity to display copulatory behavior); and, conversely, R76713 females displayed a male-typical VT-ir system in the nucleus (and also high levels of copulatory behavior). Similar changes in immunoreactivity were seen in the nucleus of the stria terminalis and in the lateral septum (VT-ir fibers only in this case) but not in the magnocellular vasotocinergic system. These neurochemical changes closely parallel the effects of the embryonic treatments on male copulatory behavior. The vasotocinergic system of the POM can therefore be considered an accurate marker of the sexual differentiation of brain circuits mediating this behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 684–699, 1998     

Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.     

The Hypothalamic–Pituitary–Adrenal Axis and Serotonin Metabolism in Individual Brain Nuclei of Mice with Genetic Disruption of the NK1 Receptor Exposed to Acute Stress

Mice lacking the substance P (SP) neurokinin-1 (NK1) receptor (NK1R?/?mice) were used to investigate whether SP affects serotonin (5-HT) function in the brain and to assess the effects of acute immobilisation stress on the hypothalamic–pituitary–adrenocortical (HPA) axis and 5-HT turnover in individual brain nuclei. Basal HPA activity and the expression of hypothalamic corticotropin-releasing hormone (CRH) in wild-type (WT)- and NK1R?/? mice were identical. Stress-induced increases in plasma ACTH concentration were considerably higher in NK1R?/? mice than in WT mice while corticosterone concentrations were equally elevated in both mouse lines. Acute stress did not alter the expression of CRH. In the dorsal raphe nucleus (DRN), basal 5-HT turnover was increased in NK1R?/? mice and a 15 min stress further magnified 5-HT utilisation in this region. In the frontoparietal cortex, medial prefrontal cortex, central nucleus of amygdala, and the hippocampal CA1 region, stress increased 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA) concentrations to a similar extent in WT and NK1R?/? mice. 5-HT turnover in the hypothalamic paraventricular nucleus was not affected by stress, but stress induced similar increases in 5-HT and 5-HIAA in the ventromedial and dorsomedial hypothalamic nuclei in WT and NK1R?/? mice. Our findings indicate that NK1 receptor activation suppresses ACTH release during acute stress but does not exert sustained inhibition of the HPA axis. Genetic deletion of the NK1 receptor accelerates 5-HT turnover in DRN under basal and stress conditions. No differences between the responses of serotonergic system to acute stress in WT and NK1R?/? mice occur in forebrain nuclei linked to the regulation of anxiety and neuroendocrine stress responses.     

Distribution of corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay: comparison and characterization of ovine and rat/human CRF antisera

The distribution of corticotropin releasing factor (CRF)-like immunoreactivity in the rat brain has been demonstrated by immunohistochemistry and radioimmunoassay using 4 different antisera. Two antisera were directed against synthetic ovine CRF, two antisera were directed against synthetic rat/human CRF. Immunohistochemistry revealed that there are discrete regions where CRF immunoreactive cell bodies are seen with all 4 antisera (e.g., the paraventricular nucleus, the dorsolateral tegmental nucleus) whereas there are cells observed only with one rat CRF antiserum (e.g., in the cortex) or terminal fields observed only with ovine CRF antisera (e.g., the spinal trigeminal tract, the substantia gelatinosa, the spinal cord). Radioimmunoassay showed different cross reactivity of the antisera with synthetic ovine or rat/human CRF and sauvagine, however, there was no cross reactivity with a variety of other peptides. Tissue values of CRF obtained by RIA of micropunched brain nuclei with the 4 antisera were frequently dissimilar suggesting that different antisera recognize different substances. High performance liquid chromatography and radioimmunoassay of brain tissue samples, revealed that there is more than one form of CRF-like immunoreactivity present. There is indirect evidence that there exists at least one peptide in the rat brain, prominent in the medulla and the spinal cord, which cross reacts with antisera directed to ovine CRF only.