Neuronal nitric oxide synthase activity mediates Lycium barbarum polysaccharides-enhanced sexual performance without stimulating noncontact erection in rats.

RATIONALE: Lycium barbarum polysaccharide (LBP) is known to promote reproductive functions. However, its role in noncontact erection (NCE) of penis initiated by brain regions including medial preoptic area (MPOA) and paraventricular nucleus (PVN) regions responsible for sexual behavior has not been investigated. OBJECTIVES: Therefore, this study initially investigated the effects of LBP on male sexual function, and subsequently, the mechanistic insight was investigated through assessing the expression of neuronal nitric oxide synthase (nNOS) in the MPOA and PVN. METHODS: The adult male rats were treated with 100 mg/kg of LBP or vehicle by oral gavage. Before and after 14 days of treatment, copulatory behavior and noncontact erection (NCE) were recorded. After the last behavioral test, the brain was isolated to measure nNOS expression in the MPOA and PVN. RESULTS: Data showed that LBP treatment significantly increased both the frequencies of intromission as well as ejaculation, compared to the control group. Whereas, a reduced post-ejaculatory interval was observed compared to same group on day 0. Furthermore, the treatment led to an increased intromission ratio, inter-intromission interval, and the number of MPOA nNOS-immunoreactive cells (nNOS-ir). Additionally, a significantly positive correlation between ejaculation frequency and MPOA nNOS-ir cells was recorded. Of note, LBP treatment had no effects on NCE and PVN nNOS-ir expression. CONCLUSION: These findings suggest that LBP enhances sexual behavior through increased nNOS expression in the MPOA in male rats.

Ginkgo biloba treatment increases copulation but not nNOS activity in the medial preoptic area in male rats.

Nitric oxide (NO) is an important messenger in the central nervous system to mediate male copulatory behavior. EGb 761, a standardized extract of Gingko biloba, has been reported to facilitate male copulation in rats. The present study is to determine the effects of neuronal nitric oxide synthase (nNOS) in the medial preoptic area (MPOA) on copulation in male rats following EGb 761 treatment. Adult male rats were treated with 50mg/kg of EGb 761 or distilled water by oral gavage for 14 consecutive days. The animals were sacrificed approximately 14h after the last behavioral test and MPOA brain tissues were collected for nNOS immunohistochemistry. EGb 761 treatment for 14 days significantly increased the intromission frequency compared to the vehicle-treated controls on day 14. An increase in ejaculation frequency was also seen in the EGb 761-treated group compared to the vehicle-treated controls on day 14 and to the same group on day 0. However, EGb 761 treatment did not influence the number of nNOS-immunoreactive cells in the MPOA. These results suggest that enhanced male copulatory performance in sexually experienced rats administered EGb 761 may not be related to central nNOS activity in the MPOA.

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain.

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase-encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole-mount in situ hybridization protocols to detect gene expression in the anatomical three-dimensional context of whole juvenile brains. To confirm whether th2-expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2-expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish.

Experience modulates both aromatase activity and the sensitivity of agonistic behaviour to testosterone in black-headed gulls.

In young black-headed gulls (Larus ridibundus), exposure to testosterone increases the sensitivity of agonistic behaviour to a subsequent exposure to this hormone. The aim of this paper is twofold: to analyze whether social experience, gained during testosterone exposure, mediates this increase in hormonal sensitivity (priming), and whether this in turn is mediated by an increase in central aromatase activity. To this end, we performed three experiments. In the first juvenile gulls were exposed to two consecutive treatments with testosterone (T1 and T2), with more than a week interval in between. During T1, half of the birds were housed in social isolation (Iso) and the other half in groups (Soc). All birds were re-housed in a new social situation during the second treatment. The increase in social behaviour during T2 was significantly more rapid in Soc than Iso birds. In experiment 2 we show that 17beta-estradiol treatment facilitates the behaviour measured in experiment 1. In experiment 3 we used a set-up comparable with that of experiment 1, but birds were sacrificed early in the T2 period. Aromatase activity in the preoptic area and the hypothalamus was measured using the tritiated water releasing method. In some parts of the preoptic area and hypothalamus aromatase activity was higher in Soc birds relative to Iso birds. The results indicate that social experience can modulate the increase of social behaviour to testosterone via modulation of aromatase activity and independently of actual hormone levels.

Nonclassical mechanisms of progesterone action in the brain: II. Role of calmodulin-dependent protein kinase II in progesterone-mediated signaling in the hypothalamus of female rats.

In addition to the activation of classical progestin receptor-dependent genomic pathway, progesterone (P) can activate nonclassical, membrane-initiated signaling pathways in the brain. We recently demonstrated rapid P activation of second-messenger kinases, protein kinase A, and protein kinase C in the ventromedial nucleus (VMN) and preoptic area (POA) of rat brain. To determine whether P can activate yet another Ca+2 dependent kinase, we examined the rapid P modulation of calcium and calmodulin-dependent protein kinase II (CaMKII) in the VMN and POA in female rats. A rapid P-initiated activation of CaMKII basal activity was observed in the VMN but not the POA at 30 min. Estradiol benzoate (EB) priming enhanced this CaMKII basal activity in both the VMN and POA. CaMKII protein levels and phosphorylation of Thr-286 moiety on CaMKII, however, remained unchanged with EB and/or P treatments, suggesting that the changes in the CaMKII kinase activity are due to rapid P modulation of the kinase activity and not its synthesis or autoactivation. Furthermore, intracerebroventricular (icv) administration of a CaMKII-specific inhibitor, KN-93, 30 min prior to the P infusion, in EB-primed, ovariectomized female rats inhibited CaMKII activation but not protein kinase A and protein kinase C activities. Interestingly, icv administration of KN-93 30 min prior to P infusion (icv) resulted in a reduction but not total inhibition of P-facilitated lordosis response in EB-primed female rats. These observations suggest a redundancy or, alternately, a hierarchy in the P-regulated activation of kinase signaling cascades in female reproductive behavior.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Neuroanatomical specificity of sex differences in expression of aromatase mRNA in the quail brain.

In birds and mammals, aromatase activity in the preoptic-hypothalamic region (HPOA) is usually higher in males than in females. It is, however, not known whether the enzymatic sex difference reflects the differential activation of aromatase transcription or some other control mechanism. Although sex differences in aromatase activity are clearly documented in the HPOA of Japanese quail (Coturnix japonica), only minimal or even no differences at all were observed in the number of aromatase-immunoreactive (ARO-ir) cells in the medial preoptic nucleus (POM) and in the medial part of the bed nucleus striae terminalis (BSTM). We investigated by in situ hybridization the distribution and possible sex differences in aromatase mRNA expression in the brain of sexually active adult quail. The distribution of aromatase mRNA matched very closely the results of previous immunocytochemical studies with the densest signal being observed in the POM, BSTM and in the mediobasal hypothalamus (MBH). Additional weaker signals were detected in the rostral forebrain, arcopallium and mesencephalic regions. No sex difference in the optical density of the hybridization signal could be found in the POM and MBH but the area covered by mRNA was larger in males than in females, indicating a higher overall expression in males. In contrast, in the BSTM, similar areas were covered by the aromatase expression in both sexes but the density of the signal was higher in females than in males. The physiological control of aromatase is thus neuroanatomically specific and with regard to sex differences, these controls are at least partially different if one compares the level of transcription, translation and activity of the enzyme. These results also indirectly suggest that the sex difference in aromatase enzyme activity that is present in the quail HPOA largely results from differentiated controls of enzymatic activity rather than differences in enzyme concentration.

Targeting steroid receptor coactivator-1 expression with locked nucleic acids antisense reveals different thresholds for the hormonal regulation of male sexual behavior in relation to aromatase activity and protein expression.

Steroid receptors such as the androgen and estrogen receptors require the presence of several proteins, known as coactivators, to enhance the transcription of target genes. The first goal of the present study was to define the role of SRC-1 on the steroid-dependent expression of the aromatase protein and its activity in male Japanese quail. The second goal was to analyze the rapid plasticity of the POM following antisense treatment interruption. We confirm here that the inhibition of SRC-1 expression by daily intracerebroventricular injections of locked nucleic acid antisense oligonucleotides in the third ventricle at the level of the preoptic area-hypothalamus (HPOA) significantly reduces testosterone-dependent male sexual behavior. In the first experiment, aromatase protein expression in HPOA was inhibited in SRC-1-depleted males but the enzymatic activity remained at the level measured in controls. We observed in the second experiment a recovery of the behavioral response to testosterone treatment after interruption of the antisense injection. However, several morphological characteristics of the POM were not different between the control group, the antisense-treated birds and antisense-treated birds in which treatment had been discontinued 3 days earlier. Antisense was also less effective in knocking-down SRC-1 in the present experiments as compared to our previous study. An analysis of this variation in the degree of knock-down of SRC-1 expression suggests dissociation among different aspects of steroid action on brain and behavior presumably resulting from the differential sensitivity of behavioral and neurochemical responses to the activation by testosterone and/or its estrogenic metabolites.

Rapid effects of aromatase inhibition on male reproductive behaviors in Japanese quail.

Non-genomic effects of steroid hormones on cell physiology have been reported in the brain. However, relatively little is known about the behavioral significance of these actions. Male sexual behavior is activated by testosterone partly through its conversion to estradiol via the enzyme aromatase in the preoptic area (POA). Brain aromatase activity (AA) changes rapidly which might in turn be important for the rapid regulation of behavior. Here, acute effects of Vorozole, an aromatase inhibitor, injected IP at different doses and times before testing (between 15 and 60 min), were assessed on male sexual behavior in quail. To limit the risk of committing both types of statistical errors (I and II), data of all experiments were entered into a meta-analysis. Vorozole significantly inhibited mount attempts (P < 0.05, size effect [g] = 0.527) and increased the latency to first copulation (P < 0.05, g = 0.251). The treatment had no effect on the other measures of copulatory behavior. Vorozole also inhibited appetitive sexual behavior measured by the social proximity response (P < 0.05, g = 0.534) or rhythmic cloacal sphincter movements (P < 0.001, g = 0.408). Behavioral inhibitions always reached a maximum at 30 min. Another aromatase inhibitor, androstatrienedione, induced a similar rapid inhibition of sphincter movements. Radioenzyme assays demonstrated that within 30 min Vorozole had reached the POA and completely blocked AA measured in homogenates. When added to the extracellular milieu, Vorozole also blocked within 5 min the AA in POA explants maintained in vitro. Together, these data demonstrate that aromatase inhibition rapidly decreases both consummatory and appetitive aspects of male sexual behavior.

Tyrosine hydroxylase expression is affected by sexual vigor and social environment in male Cnemidophorus inornatus.

Although the distribution of catecholamine-synthesizing cells has been described for a variety of taxa, less is known about the functional significance of particular populations in nonmammalian species, especially reptiles. To understand the role of these populations in the display of social behaviors in lizards, we studied the interactive effects of sexual vigor (sexually vigorous vs. sluggish) and social condition (housing in isolation vs. with females) on the number and somal areas of cells expressing tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine synthesis, in male whiptail lizards, Cnemidophorus inornatus. We found that, regardless of social condition, sexually vigorous males had more TH-immunoreactive (TH-ir) cells in the dorsal hypothalamus (DH) relative to sluggish males. Sexually vigorous males also had more TH-ir cells in the substantia nigra pars compacta (SNpc), but this difference was significant only among males housed with females. Sexually vigorous males that had been housed with females had smaller TH-ir cells in the preoptic area (POA) than vigorous males housed in isolation. On the other hand, no significant differences were found in the anterior hypothalamus. These results highlight the regional heterogeneity in the plasticity of TH expression and suggest that, just as in other species, the DH, SNpc, and POA might be involved in the expression of social behaviors and in behavioral plasticity following social experiences in lizards.

Sex differences and hormone influences on tyrosine hydroxylase immunoreactive cells in the leopard frog.

We examined sex differences in tyrosine hydroxylase immunoreactive (TH-ir) cell populations in the preoptic area (POA), suprachiasmatic nucleus (SCN), posterior tuberculum (TP), and caudal hypothalamus (Hy) in the leopard frog (Rana pipiens), in addition to the effects of natural variation in sex steroid hormones on these same populations in both sexes. All four of these populations have been shown to be dopaminergic. Gonadal sex, androgens, and estrogen all influenced TH-ir cell numbers, but in a complicated pattern of interactions. After factoring out the effects of sex steroids by multiple regression, TH-ir cell numbers in all four areas differed between the sexes, with males having a greater number of TH-ir cells. The influence of androgens and estrogen differed by region and sex of the animals. Androgens were the main influence on TH-ir cell numbers in the POA and SCN. Plasma androgen concentrations were positively correlated with TH-ir cell numbers in both areas in males. In females, androgen concentration was negatively correlated with TH-ir cell numbers in the POA; there was no significant relationship in the SCN in females. In the more caudal populations, estrogen (E2) levels were positively correlated with TH-ir cell numbers in the TP of both males and females. In the caudal hypothalamus, E2 levels were positively correlated with TH-ir cell numbers in females, but there was no significant correlation in males. The results indicate that gonadal sex imposes a baseline sex difference in the four TH-ir (dopamine) populations, resulting in a higher number of such cells in males. Individual and sex-linked differences in gonadal steroid hormones lead to variation around this baseline condition, with androgens having a greater influence on rostral populations and estrogen on caudal populations. Last, an individual's gonadal sex determines the effect that androgens and estrogen have on each population.

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus.

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.

Maternal and perinatal brain aromatase: effects of dietary soy phytoestrogens.

Phytoestrogens are extensively investigated for their potential to prevent many hormone-dependent cancers and age-related diseases, however little is known about their effects in brain. Brain aromatase and plasma phytoestrogen levels were determined in Sprague-Dawley rats fed a phytoestrogen-rich diet during pregnancy/lactation. Ingested phytoestrogens cross the placenta and become concentrated in maternal milk as evident from high infantile plasma concentrations. Dietary phytoestrogens, however, do not alter brain aromatase during pregnancy/lactation or perinatal development.

Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats.

Phytoestrogen [plant estrogenic-like molecule(s)] research has grown rapidly in recent years due to their potential health benefits. However, little is known about phytoestrogen's effects on the CNS. Androgen metabolizing enzymes are known to regulate neuroendocrine functions and reproductive behaviors, while calcium-binding proteins are associated with protecting against neurodegenerative diseases. Therefore, we examined aromatase and 5alpha-reductase enzyme activities in the medial basal hypothalamic and preoptic area (mbh-poa) and characterized mbh-poa and amygdala (amy) calbindin and calretinin levels (via Western analysis) from animals fed a phytoestrogen-free (P-free) vs. a phytoestrogen-containing diet [(P-600); that had 600 microg/g of phytoestrogens]. After approximately 5 weeks on the diets, the male rats were killed at 105 days. P-600 plasma phytoestrogen levels were 78-fold higher than the P-free values and the mbh-poa phytoestrogen content was 8-fold higher than the P-free group, demonstrating the passage of phytoestrogens into brain. In general, brain aromatase or 5alpha-reductase activity levels were not significantly altered by the experimental diets. However, independent of brain site (i.e., mbh-poa or amy) the abundance of calbindin from male P-600 rats was significantly lower than P-free animals. Conversely, for calretinin there were no significant alterations in the mbh-poa tissue site, while in the amy a similar pattern of expression was seen to that of the calbindin results. These data suggest that consumption of phytoestrogens via a soy diet for a relatively short interval can significantly: (1) elevate plasma and brain phytoestrogens levels and (2) decrease brain calcium-binding proteins without altering brain androgen metabolizing enzymes.

Regional distribution and control of tyrosine hydroxylase activity in the quail brain.

Tyrosine hydroxylase (TH) activity, the rate-limiting step in the synthesis of catecholamines, was quantified in the preoptic area-hypothalamus of adult male Japanese quail by a new assay measuring the tritiated water production from 3,5-[3H]-L-tyrosine. Maximal levels of activity were observed at a 20-25 microM concentration of substrate, with more than 50% inhibition of the activity being recorded at a 100 microM concentration. TH activity was linear as a function of the incubation time during the first 20 min and maximal at a pH of 6.0. TH was heterogeneously distributed in the quail brain with highest levels of activity being found (in decreasing order) in the mesencephalon, diencephalon, and telencephalon. Given the large size of the telencephalon, this is the brain area that contains, as a whole, the highest level of enzyme activity. TH inhibitors that have been well-characterized in mammals, such as 3-iodo-L-tyrosine and L-alpha-methyl-p-tyrosine (AMPT) completely inhibited the enzyme activity at a 100 microM concentration. In mammals, the accumulation of catecholamines exerts a negative feedback control on TH activity. Similar controls were observed in the quail brain. Two inhibitors of the DOPA decarboxylase that should lead to accumulation of DOPA depressed TH activity by 60% or more, and the inhibitor of the dopamine beta-hydroxylase, fusaric acid that should cause an accumulation of dopamine, suppressed 90% of the TH activity. The addition of exogenous DOPA, dopamine, or norepinephrine to the brain homogenates also strongly inhibited TH activity, independently confirming the feedback effects of the enzyme products on the enzyme activity. These data demonstrate that TH activity in the quail brain is heterogeneously distributed and acutely regulated, as it is in mammals, by the accumulation of its products and of the derived catecholamines.

Site-specific induction of Fos immunoreactivity in preoptic and hypothalamic NADPH-positive neurons during glucoprivation.

Neuronal nitric oxide synthase, e.g. NADPH diaphorase (NADPH-d), catalyzes formation of the free radical, nitric oxide (NO), and occurs within brain structures that have functional significance for energy fuel homeostasis. The following studies examined whether populations of NADPH-d-positive neurons in the hypothalamus and nearby preoptic area express immunoreactivity for the nuclear transcription factor, Fos, in response to glucose substrate imbalance. Eight days after bilateral ovariectomy (OVX) and subcutaneous implantation of silastic capsules containing 30 microgram estradiol benzoate/ml, female rats were injected i.p. with the glucose antimetabolite, 2-deoxy-D-glucose (2DG; 400 mg/kg), or the vehicle, saline. The animals were sacrificed by transcardial perfusion 2 h after these treatments. Sections at 150-micrometer intervals throughout preoptic area and anterior and tuberal regions of the hypothalamus were processed for dual cytoplasmic NADPH-d enzyme activity and nuclear Fos-immunoreactivity (-ir). The glucose antimetabolite elicited expression of nuclear Fos-ir by NADPH-d-positive neurons in several neural structures, including the medial preoptic area, median preoptic nucleus, anterior commissural, periventricular magnocellular supraoptic nucleus, paraventricular nucleus, and medial part of the bed nucleus of the stria terminalis. In contrast, the extensive populations of NADPH-d-positive neurons in the ventromedial hypothalamic nucleus and lateral hypothalamic area showed very little immunolabeling for Fos in response to glucoprivation. This demonstration of nuclear immunoreactivity for Fos suggests that cellular glucopenia elicits the transcriptional activation, via AP-1 regulatory sites, of multiple populations of hypothalamic neurons characterized by the functional capacity to generate NO, and thus that this gaseous neurotransmitter may fulfill a role(s) in central neural mechanisms governing regulation of compensatory motor responses to metabolic imbalance.

NADPH-diaphorase expression in the hypothalamo-hypophysial system of different catfish.

The distribution of NADPH-d activity was studied in the hypothalamus and in the pituitary gland of 15 species of catfish. Seven hypothalamic nuclei, four fiber bundles, as well as cells located in the adenohypophysis were labeled by NADPH-d histochemistry. Reactive somata were found in the nucleus praeopticus periventricularis, the paraventricular division of the nucleus praeopticus, the supraoptic division of the nucleus praeopticus, the nucleus lateralis tuberis, the paraventricular organ, the nucleus recessus lateralis, the nucleus recessus posterioris, and in the adenohypophysis. In some species, an inconsistent number of these structures lacked NADPH-d activity. These results are compatible with the notion that NADPH-d activity expressing cells in the hypothalamus and in the pituitary are involved in the control of hormone regulation.

Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area.

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.

Constitutive nitric oxide synthase in hypothalami of normal and hereditary diabetes insipidus rats and mice: role of nitric oxide in osmotic regulation and its mechanism.

Constitutive nitric oxide synthase (cNOS) was immunolocalized to study its role in osmotic regulation. Immunoreactivity was observed in all major hypothalamic osmoregulatory structures, the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, and supraoptic and paraventricular nuclei. These nuclei were compared in normal Long-Evans rats and homozygous Brattleboro rats with hereditary hypothalamic diabetes insipidus and in normal mice and mice with hereditary nephrogenic diabetes insipidus. About 50% of supraoptic neurons in Long-Evans rats and 90% in Brattleboro rats were cNOS immunopositive; a qualitatively similar difference occurred in the paraventricular nucleus. Mice with hereditary nephrogenic diabetes insipidus also showed a greater proportion of cNOS-positive supraoptic neurons (50%) than normal mice (20%). However, the number of cNOS-positive cells in the organum vasculosum laminae terminalis, subfornical organ, and median preoptic nucleus dis not differ significantly between diabetic and normal animals. The similar changes in cNOS in two mutant strains in which the only common feature is chronic osmotic stimulation shows that differences in vasopressin and oxytocin are not involved in the regulation of cNOS. The results suggest strongly that cNOS is involved in long term modulation of the hypothalamo-neurohypophysial system and, hence, body water and electrolyte homeostasis, and that cNOS is itself regulated by body osmotic status.

Progesterone receptor immunoreactivity in aromatic L-amino acid decarboxylase-containing neurons of the guinea pig hypothalamus and preoptic area.

A double-labeling immunofluorescence procedure was used to determine whether progesterone receptor (PR)-immunoreactive (IR) neurons in the preoptic area and hypothalamus of female guinea pigs also contained aromatic L-amino acid decarboxylase (AADC), an enzyme involved in the synthesis of both catecholamines and serotonin. Immunostaining was performed on cryostat sections prepared from ovariectomized guinea pigs primed by estradiol to induce PR. The nuclear presence of PR was visualized by a red fluorescence while the AADC-containing perikarya showed a yellow-green fluorescence. The topographic distribution of AADC-IR neurons was investigated by using a specific antiserum obtained by immunization of rabbits with a recombinant protein beta-galactosidase-AADC in the two regions known to contain the densest populations of estradiol-induced PR-IR cells: the preoptic area and the mediobasal hypothalamus. The localization of PR-IR and AADC-IR cell populations showed considerable overlap in these areas, mainly in the medial and periventricular preoptic nuclei and in the arcuate nucleus. A quantitative analysis of double-labeled cells estimated that about 15% to 23% of AADC-IR cells in the preoptic area and about 11% to 21% of AADC-IR cells in the arcuate nucleus possessed PR. This colocalization persisted throughout the rostrocaudal extent of these areas and represented 3% to 9% of the population of PR-IR cells. These findings provide neuroanatomical evidence that a subset of AADC neurons is directly regulated by progesterone. The exact physiological role of this enzyme in target cells for progesterone is not understood. AADC may be involved in functions other than that for the synthesis of the classical neurotransmitters.