Impact of environmental thermal stimulation on activation of hypothalamic neuronal nitric oxide synthase during the prenatal ontogenesis in Muscovy ducks.

The aim of the study is to investigate the influence of prenatal temperature stimulation on neuronal NO synthase (nNOS) expression in the anterior hypothalamus of Muscovy duck embryos. Experiments were performed on embryonic day (E) E20, E23, E28, and E33 using histochemistry for identification of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as marker of NOS-containing neurons. Until the experiments, all duck embryos were incubated under standard temperature conditions (37.5°C). During 3 hours before the start of the experiments, one group was incubated at 37.5°C (control group), the second was warm-experienced at 39°C, and the third was cold-experienced at 34°C. In normal and warm-incubated duck embryos, nNOS activity could be first detected on E23. Particularly, after cold stimulation, a significant increase in nNOS activity was found in all embryos investigated even on day 20. Warm stimulation obviously induces the opposite effect, but at later embryonic age (E33). It can be concluded that probably in late-term bird embryos NO acts as a mediator of the neuronal cold pathway in the anterior hypothalamus, which might be improved by prenatal cold stimulation.

Membrane-type 4 matrix metalloproteinase (MT4-MMP) modulates water homeostasis in mice.

MT4-MMP is a membrane-type metalloproteinase (MMP) anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) motif. GPI-type MT-MMPs (MT4- and MT6-MMP) are related to other MT-MMPs, but their physiological substrates and functions in vivo have yet to be identified. In this manuscript we show that MT4-MMP is expressed early in kidney development, as well as in the adult kidney, where the highest levels of expression are found in the papilla. MT4-MMP null mice had minimal renal developmental abnormalities, with a minor branching morphogenesis defect in early embryonic kidney development and slightly dysmorphic collecting ducts in adult mice. Interestingly, MT4-MMP null mice had higher baseline urine osmolarities relative to wild type controls, but these animals were able to concentrate and dilute their urines normally. However, MT4-MMP-null mice had decreased daily water intake and daily urine output, consistent with primary hypodipsia. MT4-MMP was shown to be expressed in areas of the hypothalamus considered important for regulating thirst. Thus, our results show that although MT4-MMP is expressed in the kidney, this metalloproteinase does not play a major role in renal development or function; however it does appear to modify the neural stimuli that modulate thirst.

Long-term modulation of tyrosine hydroxylase activity and expression by endothelin-1 and -3 in the rat anterior and posterior hypothalamus.

Brain catecholamines are involved in the regulation of biological functions, including cardiovascular activity. The hypothalamus presents areas with high density of catecholaminergic neurons and the endothelin system. Two hypothalamic regions intimately related with the cardiovascular control are distinguished: the anterior (AHR) and posterior (PHR) hypothalamus, considered to be sympathoinhibitory and sympathoexcitatory regions, respectively. We previously reported that endothelins (ETs) are involved in the short-term tyrosine hydroxylase (TH) regulation in both the AHR and PHR. TH is crucial for catecholaminergic transmission and is tightly regulated by well-characterized mechanisms. In the present study, we sought to establish the effects and underlying mechanisms of ET-1 and ET-3 on TH long-term modulation. Results showed that in the AHR, ETs decreased TH activity through ET(B) receptor activation coupled to the nitric oxide, phosphoinositide, and CaMK-II pathways. They also reduced total TH level and TH phosphorylated forms (Ser 19 and 40). Conversely, in the PHR, ETs increased TH activity through a G protein-coupled receptor, likely an atypical ET receptor or the ET(C) receptor, which stimulated the phosphoinositide and adenylyl cyclase pathways, as well as CaMK-II. ETs also increased total TH level and the Ser 19, 31, and 40 phosphorylated sites of the enzyme. These findings support that ETs are involved in the long-term regulation of TH activity, leading to reduced sympathoinhibition in the AHR and increased sympathoexcitation in the PHR. Present and previous studies may partially explain the cardiovascular effects produced by ETs when applied to the brain.

Activation of pedunculopontine tegmental protein kinase A: a mechanism for rapid eye movement sleep generation in the freely moving rat.

Cells in the pedunculopontine tegmentum (PPT) play a key role in the generation of rapid eye movement (REM) sleep, but its intracellular signaling mechanisms remain unknown. In the current studies, the role of PPT intracellular protein kinase A (PKA) in the regulation of REM sleep was evaluated by comparing PKA subunit [catalytic (PKA(C alpha)) and regulatory (PKA(RI), PKA(RII alpha), and PKA(RII beta)) types] expression and activity in the PPT at normal, high, and low REM sleep conditions. To compare anatomical specificity, REM sleep-dependent expressions of these PKA subunits were also measured in the medial pontine reticular formation (mPRF), medial prefrontal cortex (mPFC), and anterior hypothalamus (AHTh). The results of these PKA subunit expression and activity studies demonstrated that the expression of PKA(C alpha) and PKA activity in the PPT increased and decreased during high and low REM sleep, respectively. Conversely, PKA(C alpha) expression and PKA activity decreased with high REM sleep in the mPRF. Expression of PKA(C alpha) also decreased in the mPFC and remained unchanged in the AHTh with high REM sleep. These subunit expression and PKA activity data reveal a positive relationship between REM sleep and increased PKA activity in the PPT. To test this molecular evidence, localized activation of cAMP-dependent PKA activity was blocked using a pharmacological technique. The results of this pharmacological study demonstrated that the localized inhibition of cAMP-dependent PKA activation in the PPT dose-dependently suppressed REM sleep. Together, these results provide the first evidence that the activation of the PPT intracellular PKA system is involved in the generation of REM sleep.

Changes in the metabolic activity of neurons in the anterior hypothalamic nuclei in rats during hyperthermia, fever, and hypothermia.

Histochemical methods were used to detect differently directed changes in the metabolic activity of neurons in the anterior hypothalamic nuclei in rats during hyperthermia, fever, and hypothermia. Hyperthermia induced by high temperatures was associated with increases in the activities of enzymes involved in energy metabolism, with increases in RNA contents in neurons in the supraoptic, paraventricular, and median preoptic nuclei of the anterior hypothalamus of the rat, which is evidence for increases in metabolic activity in the neurons of these nuclei. Endotoxin-induced fever was accompanied by decreases in the metabolic activity of neurons in the median preoptic nucleus, while activity in neurons of the supraoptic and paraventricular nuclei showed no significant change. The development of hypothermia induced by low temperatures was characterized by decreases in the metabolic activity of neurons in the supraoptic, paraventricular, and median preoptic nuclei of the anterior hypothalamus. It is suggested that the differently directed changes in metabolic activity in the neurons of the anterior hypothalamus in hyperthermia, fever, and hypothermia are associated with their roles in the central mechanisms of thermoregulation (median preoptic nucleus) and neurosecretory processes (supraoptic and paraventricular nuclei).

Deoxycorticosterone stimulates the activity of nicotinamide adenine dinucleotide phosphate-diaphorase/nitric oxide synthase immunoreactivity in hypothalamic nuclei of rats.

Mineralocorticoids (MC) play an important role in development of salt appetite. Part of this effect involves the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, in which MC treatment increases arginine vasopressin (AVP) synthesis and release. Since the AVP system is also modulated by nitric oxide (NO), we studied if deoxycorticosterone acetate (DOCA) treatment changed the number of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) active neurons and neuronal NO synthase (nNOS)-immunoreactive (IR) cells in the PVN and SON. After four injections of DOCA (10 mg/rat per day), rats developed a salt appetite and increased NADPH-d active and nNOS-IR neurons in both nuclei. A single DOCA injection did not change salt consumption or nNOS-IR cells, but increased the number of NADPH-d positive neurons in the PVN only. Therefore, while acute MC treatment stimulated the activity of pre-existing enzyme, chronic steroid treatment recruited additional neurons showing nNOS immunoreactivity/NADPH-d activity. These data suggest a role for NO produced in the PVN and SON in DOCA stimulatory effects on AVP mRNA and salt appetite.

Increased somatostatin mRNA expression in periventricular nucleus of rat hypothalamus during hypoxia.

We reported that hypoxia inhibited the growth hormone (GH) and induced somatostatin (SS) release from the hypothalamic median eminence (ME) of rats. This study is designed to examine the SS mRNA alterations in the periventricular nucleus (PeN) of the hypothalamus in rats and the possible involvement of glucocorticoid (GC) during hypoxia. Rats were exposed to hypoxia in a simulated hypobaric chamber. SS mRNA levels in the PeN were tested by in situ hybridization. Hypoxia of 5-km altitude (10.8% O(2)) for 2, 5 and 24 h increased the SS mRNA expression by 34.72%, 50.31% and 95.05% (p<0.05), respectively. Severe hypoxia of 7-km altitude (8.2% O(2)) enhanced the SS expression by 79.08% (p<0.01), 74.90% (p<0.01) and 71.40% (p<0.05), respectively. Prolonged hypoxia (5 km for 5 days) exposure augmented a 2.5-fold SS mRNA (p<0.001). One week post adrenalectomy (ADX), SS mRNA level was significantly increased. During hypoxia, 5 km for 5 h, SS mRNA in ADX rats was not further increased. An increased SS mRNA was showed by pretreatment with low dose of dexamethasone (DEX) (125 microg/kg, i.p.) to ADX animals but this increase was depressed by a high dose of DEX (500 microg/kg, i.p.). The data suggested that (1) hypoxia stimulated the expression of SS mRNA in the PeN of rat hypothalamus. (2) Increased circulating GC levels might play a role in upregulating the SS mRNA in the rat PeN during hypoxia.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-æm sections at 120-æm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 + or - 206, N = 6; 1020 + or - 150, N = 6; nonhandled and handled, respectively), paraventricular (584 + or - 85, N = 8; 682 + or - 62, N = 9) or periventricular (743 + or - 118, N = 7; 990 + or - 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats.

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-microm sections at 120-microm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 +/- 206, N = 6; 1020 +/- 150, N = 6; nonhandled and handled, respectively), paraventricular (584 +/- 85, N = 8; 682 +/- 62, N = 9) or periventricular (743 +/- 118, N = 7; 990 +/- 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Involvement of tyrosine kinase in the pyrogenic fever exerted by NOS pathways in organum vasculosum laminae terminalis.

Nitric oxide synthase (NOS) is an enzyme which has a distinct cytokine-inducible isoform (iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used two tyrosine kinase inhibitors (genistein and lavendustin A) to investigate the potential role of tyrosine kinase activation in the induction on both iNOS and fever caused by lipopolysaccharide (LPS) in rabbits. Direct administration of LPS into the organum vasculosum laminae terminalis (OVLT) increased iNOS expression. These increases paralleled the increase in deep body temperature in unanesthetized rabbits. Pretreatment with genistein or lavendustin A not only reduced the fever but also attenuated the iNOS expression in the OVLT following an intra-OVLT dose of LPS. These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates the induction of both iNOS and fever elicited by LPS in the OVLT of rabbit brain.

Partial co-existence of NADPH-diaphorase and acetylcholinesterase in the hypothalamic magnocellular secretory nuclei of the rat.

Co-localization of NADPH-diaphorase (ND) and acetylcholinesterase (AChE) activities were explored in the magnocellular secretory nuclei of the rat hypothalamus by means of a double histochemical staining of the same sections. Partial co-existence was found in all the nuclei studied (paraventricular, supraoptic, fornicals and circular nuclei). No particular location of the neurons expressing both markers was found, although in the paraventricular nucleus all of them (ND +, AChE + and neurons expressing both markers) were preferentially located in the magnocellular subdivisions whereas in the parvicellular ones only some neurons belonging to all three types were detected, mainly located in the periventricular and medial subdivisions. The lowest degree of co-existence was found at the level of the main magnocellular nuclei (supraoptic and paraventricular) when compared with the accessory magnocellular nuclei, especially the posterior fornical and the circular nuclei. These results extend previous data on the chemical nature of the neurons producing nitric oxide in the neurosecretory nuclei and the possible functional role of this atypical messenger in the hypothalamus.

Aromatic L-amino acid decarboxylase- and tyrosine hydroxylase-immunohistochemistry in the adult human hypothalamus.

The distribution of cell bodies immunoreactive for tyrosine hydroxylase and aromatic L-amino acid decarboxylase was studied in the adult human hypothalamus. Many neurons in the posterior (A11) and caudal dorsal hypothalamic areas (A13) as well as in the arcuate (A12) and periventricular (A14) zone were immunoreactive for the two enzymes, suggesting that they were dopaminergic. Numerous tyrosine hydroxylase-immunoreactive neurons, which were not immunoreactive for aromatic L-amino acid decarboxylase, could be seen in the paraventricular, supraoptic and accessory nuclei (A15) as well as in the rostral dorsal hypothalamic area. These were considered to be non-dopaminergic. Conversely, large numbers of small neurons immunoreactive for aromatic L-amino acid decarboxylase but not for tyrosine hydroxylase, were identified in the premammillary nucleus (D8), zona incerta (D10), lateral hypothalamic area (D11), anterior portion of the dorsomedial nucleus (D12), suprachiasmatic nucleus (D13), medial preoptic area and bed nucleus of the stria terminalis (D14). In the human hypothalamus, besides dopaminergic cell bodies, there exists a large number of tyrosine hydroxylase-only and aromatic L-amino acid decarboxylase-only neurons, whose physiological roles remain to be determined.

Sex differences in androgen responsiveness in the rat brain: regional differences in the induction of aromatase activity.

The transformation of testosterone (T) to estrogens in brain tissue by cytochrome P-450 aromatase is required for the expression of sexual behaviors in adult male rats. Androgens regulate aromatase activity in the medial preoptic nucleus (MPN), as well as in a reciprocally connected group of forebrain nuclei involved in the regulation of male sexual behaviors. The levels of aromatase in these nuclei are generally greater in males than in females due to sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic. The current experiments were undertaken: (1) to characterize and compare the kinetic properties of aromatase in male and female rats and (2) to study sex differences in the dose-response relationship between the administered doses of T and the induction of aromatase in microdissected brain regions. Saturation analysis of aromatase activity in the MPN, bed nucleus of the stria terminalis (BNST), periventricular preoptic area (PVPOA), anterior hypothalamus (AH), and ventromedial hypothalamic nucleus (VMN) indicates that the greater aromatase activity observed in intact males reflects a sex difference in the maximal enzyme velocity, and not a sex difference in the apparent affinity of enzyme for substrate (Michaelis constant). The dose-response study of aromatase induction in the BNST, PVPOA, and VMN indicated a sex difference in aromatase activity over a range of circulating T levels varying from 0.3 to 35 ng/ml. No sex difference in inducible aromatase activity in AH was observed at any dose of T. The results of this study clearly demonstrate a sexually dimorphic effect of androgen action in the rat brain. Since T both regulates and is the substrate for aromatase in the brain, this sexual dimorphism is potentially an important limitation to the action of T in females and may relate to the enhanced expression of T-stimulated copulatory behavior in males compared to females.

NADPH diaphorase activity around the suprachiasmatic nucleus in rat brain.

The 'nicotinamide adenine dinucleotide phosphate diaphorase' histochemical technique was used as a marker of neuronal nitric oxide synthase to assess the presence of the enzyme in the anterior hypothalamus of the rat. Particular attention was focused on the subparaventricular zone, periventricular area and suprachiasmatic nucleus. The results show that there is strong staining in the anterior hypothalamus particularly in the subparaventricular zone by the perinuclear regions of the suprachiasmatic nucleus, and in the periventricular nucleus. Some diaphorase activity was also seen within the suprachiasmatic nucleus, but this was much weaker than in the surrounding areas. These results, taken together with existing evidence, would further suggest the involvement of nitric oxide in the signal transduction pathway in the suprachiasmatic nucleus.

Nitric oxide synthase in the hypothalamo-pituitary pathways.

Several histochemical and physiological studies in the literature suggest that nitric oxide (NO) is involved in the posterior pituitary regulation. This study was set out to demonstrate the distribution of the pituitary projecting nitric oxide containing hypothalamic pathways. The fluorescent retrograde tracer fluorogold (FG) was injected into the pituitary gland in order to reveal the hypothalamic nuclei projecting to the pituitary gland. Nitric oxide synthase (NOS) was subsequently visualized from the same sections by NADPH-diaphorase histochemistry. More than 70% of the FG labelled neurons in the preoptic area, paraventricular, supraoptic, retrochiasmatic, anterior commissural and circular nuclei as well as in many other small neurosecretory centers in the hypothalamus contained also NADPH-diaphorase activity (NADPH-DA). In addition, some of the neurons in the periventricular nucleus as well as occasional neurons in the lateral hypothalamus projecting to the pituitary gland contained NADPH-DA. The present results give the first systematic neuroanatomical description of the hypothalamic NO synthase containing neurons projecting into the pituitary gland. They indicate that the pituitary receives a widely distributed NO innervation, which originates mostly in the magnocellular neurosecretory hypothalamo-pituitary system. In addition, the finding that some presumably non-neurosecretory pituitary-projecting neurons contain NO as well suggests that NO might be a more widely used regulator of the posterior pituitary secretion than previously expected.

Adenylyl cyclase mRNA expression does not reflect the predominant Ca2+/calmodulin-stimulated activity in the hypothalamus.

Only three (Types I, II, V) of the six currently-described subtypes of adenylyl cyclase are prominently expressed in the rat brain. These species are differently sensitive to Ca2+, beta gamma subunits of G-proteins and protein kinase C. A knowledge of the susceptibility of the cAMP-signalling system in particular brain regions to these diverse modes of regulation can shed light on the mechanism of action of the neurotransmitters that modify neuronal activity in such regions. Cyclic AMP is extensively involved in the physiological functions of the hypothalamus. We have used in situ hybridization histochemistry with synthetic oligonucleotides to examine the expression in the rat hypothalamus of the three major brain subtypes of adenylyl cyclase-Ca2+/calmodulin-stimulable (Type I), Ca(2+)-insensitive (Type II) and Ca(2+)-inhibitable (Type V). The hypothalamus expresses high levels only of Type II mRNA, particularly in the supraoptic and paraventricular nuclei. Curiously, the strong expression of the Ca(2+)-insensitive Type II mRNA and the lack of expression of the major brain specific Type I mRNA does not correlate with the adenylyl cyclase activity, which is largely Ca2+/calmodulin stimulable in plasma membranes prepared from the hypothalamus.

Immunocytochemical and histochemical analyses of gonadotrophin releasing hormone, tyrosine hydroxylase, and cytochrome oxidase reactivity within the hypothalamus of chicks showing early sexual maturation.

In order to determine the changes in gonadotrophin releasing hormone (GnRH) and dopaminergic activity within the brain during the onset of sexual precocity, a Halasz-like knife was developed to produce discrete parasagittal cuts in 2-week-old male broiler chicks. At 5 weeks of age, sexually precocious respondents were selected on the basis of advanced secondary sex characteristics and randomly paired with sham-operated controls. Each pair of birds was perfused with heparinized saline followed by 4% paraformaldehyde. Sections 40 microns thick, obtained throughout the hypothalamus, were immunostained with either anti-GnRH or anti-tyrosine hydroxylase (TH) to ascertain dopaminergic activity. Alternate sections from each pair of brains were also treated with cytochrome oxidase to determine metabolic activity levels or with Nissl stain to localize the knife cuts. Analysis revealed an increase in GnRH immunoreactivity within the bed nucleus of the pallial commissure (nCPa) and paraventricular nucleus (PVN), as well as the median eminence (ME). An increase in TH immunoreactivity was observed in the nucleus intramedialis (nI). Also an increase in metabolic activity was seen in the PVN as revealed by cytochrome oxidase reactivity. It is hypothesized that during the onset of puberty there is an increase in immunoreactive GnRH cell numbers as a result of a decrease in the inhibition of the GnRH system, possibly involving the nI and PVN. The source of the dopamine reported in the ME could be from the nI and other nearby nuclei. Dopamine from the tubero-infundibular area may be one of the putative neurotransmitters responsible for the increased activity of GnRH within the ME of chicks showing precocious puberty.

Recovery from soman-induced hypothermia is due to an increase in acetylcholinesterase activity but not new protein synthesis.

Organophosphate-induced hypothermia in rodents appears to be due to stimulation of muscarinic receptors in the anterior hypothalamus. The nerve agent soman (pinacolyl methylphosphonofluoridate) produced a transient hypothermia in mice. Concomitant with the recovery from soman-induced hypothermia, which was complete within 6 hr, there was a parallel recovery of the hypothalamic acetylcholinesterase activity. Pretreatment of mice with the protein synthesis inhibitor, cycloheximide, did not affect the recovery from soman-induced hypothermia nor the recovery of hypothalamic acetylcholinesterase activity. The results suggest that the recovery from soman-induced hypothermia may be due to the recovery of acetylcholinesterase, perhaps from the assembly of previously synthesized precursors.

Asymmetry of brain aromatase activity: region- and sex-specific developmental patterns.

Developmental patterns of aromatase activity (AA) were characterized in individual forebrain regions of the rat at gestational day (GD) 22 and postnatal days (PN) 6 and 15. Aromatase activity was measured separately in homogenates of left and right preoptic area, anterior amygdaloid area, medial amygdaloid nucleus, anterior hypothalamic area and posterior hypothalamic area, by the tritiated water method with [1 beta-3H]-androstenedione as a substrate. Region- and sex-dependent asymmetries of AA with either left-to-right or right-to-left gradients were found. They change between GD22 and PN6 and PN15 according to region-specific patterns. Thus, AA of the male medial amygdaloid nucleus of the left side is higher at GD22, lower at PN6 and equal to the right side at PN15; in females, AA of the left side is lower than AA of the right side at GD22 and higher at PN6 and PN15. In preoptic area, a side difference (left side higher) was only detected in males. Asymmetries may result from differences in the expression of the enzyme by individual cell groups, or from differences in the number of cells per area expressing the enzyme. In either case, the stage-dependent patterns of asymmetry in AA would be expected to influence sex steroid-dependent differentiation processes in individual forebrain areas.

Detection and distribution of aromatase in hypothalamus of the male rat.

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase. An in vitro assay for aromatase activity, which quantitates the production of 3H2O during the conversion of 1 beta-3H-androstenedione to estrone, was developed and validated in the present study. The distribution of aromatase activity in discrete hypothalamic nuclei was also explored. Individual hypothalamic nuclei were microdissected from 200-micron frozen sections. Tissues from 3 animals were pooled and assayed for aromatase activity. It was found that the rate of aromatase activity was linear with time up to 1 hour, and with protein concentrations up to 900 micrograms/ml tissue homogenate. A maximal rate was obtained with a substrate concentration of 0.3 microM. The aromatase activity was heterogeneously distributed within the rat brain. The greatest amount of aromatase activity was found in the preoptic area. The suprachiasmatic nucleus also contained a high level of aromatase activity. The aromatase activities in the anterior hypothalamus, ventromedial nucleus, arcuate nucleus and dorsomedial nucleus were less than half that found in the preoptic area.