Co-expression of c-Fos with oestradiol receptor α or somatostatin in the arcuate nucleus, ventromedial nucleus and medial preoptic area in the follicular phase of intact ewes: alteration after insulin-induced hypoglycaemia.

The aim of this study was to investigate how acute insulin-induced hypoglycaemia (IIH) alters the activity of cells containing oestradiol receptor α (ERα) or somatostatin (SST) in the arcuate nucleus (ARC) and ventromedial nucleus (VMN), and ERα cells in the medial preoptic area (mPOA) of intact ewes. Follicular phases were synchronized with progesterone vaginal pessaries. Control animals were killed at 0 h or 31 h (n = 5 and 6, respectively) after progesterone withdrawal (PW; time zero). At 28 h, five other animals received insulin (INS; 4 iu/kg) and were subsequently killed at 31 h. Hypothalamic sections were immunostained for ERα or SST each with c-Fos, a marker of neuronal transcriptional activation. Insulin did not alter the percentage of activated ERα cells in the ARC; however, it appeared visually that two insulin-treated animals (INS responders, with no LH surge) had an increase in the VMN (from 32 to 78%) and a decrease in the mPOA (from 40 to 12%) compared to no increase in the two INS non-responders (with an LH surge). The percentage of activated SST cells in the ARC was greater in all four insulin-treated animals (from 10 to 60%), whereas it was visually estimated that activated SST cells in the VMN increased only in the two insulin responders (from 10 to 70%). From these results, we suggest that IIH stimulates SST activation in the ARC as part of the glucose-sensing mechanism but ERα activation is unaffected in this region. We present evidence to support a hypothesis that disruption of the GnRH/LH surge may occur in insulin responders via a mechanism that involves, at least in part, SST cell activation in the VMN along with decreased ERα cell activation in the mPOA.

Suppression of leptin-induced hypothalamic JAK/STAT signalling and feeding response during pregnancy in the mouse.

Hyperphagia during pregnancy, despite rising concentrations of the satiety hormone leptin, suggests that a state of leptin resistance develops. This study investigated the satiety response and hypothalamic responses to leptin during pregnancy in the mouse. Pregnant (day 13) and nonpregnant mice received an i.p. injection of either leptin or vehicle and then 24-h food intake was measured. Further groups of pregnant and nonpregnant mice were perfused 2 h after leptin or vehicle injections and brains were processed for pSTAT3 and pSTAT5 immunohistochemistry. Leptin treatment significantly decreased food intake in nonpregnant mice. In pregnant mice, however, leptin treatment did not suppress food intake, indicating a state of leptin resistance. In the arcuate nucleus, leptin treatment increased the number of cells positive for pSTAT3, a marker of leptin activity, to a similar degree in both nonpregnant and pregnant mice. In the ventromedial nucleus (VMN), the leptin-induced increase in pSTAT3-positive cell number was significantly reduced in pregnant mice compared to that in nonpregnant mice. In nonpregnant mice, leptin treatment had no effect on the number of pSTAT5-positive cells, suggesting that in this animal model, leptin does not act through STAT5. In pregnant mice, basal levels of pSTAT5 were higher than in nonpregnant mice, and leptin treatment led to a decrease in the number of pSTAT5-positive cells in the hypothalamus. Overall, these results demonstrate that during pregnancy in the mouse, a state of leptin resistance develops, and this is associated with a reduced sensitivity of the VMN to leptin.

The action of leptin on appetite-regulating cells in the ovine hypothalamus: demonstration of direct action in the absence of the arcuate nucleus.

It is widely accepted that leptin acts on first-order neurons in the arcuate nucleus (ARC) with information then relayed to other hypothalamic centers. However, the extent to which leptin mediates its central actions solely, or even primarily, via this route is unclear. We used a model of hypothalamo-pituitary disconnection (HPD) to determine whether leptin action on appetite-regulating systems requires the ARC. This surgical preparation eliminates the ARC. We measured effects of iv leptin to activate hypothalamic neurons (Fos labeling). In ARC-intact animals, leptin increased the percentage of Fos-positive melanocortin neurons and reduced percentages of Fos-positive neuropeptide Y neurons compared with saline-treated animals. HPD itself increased Fos labeling in the lateral hypothalamic area (LHA). Leptin influenced Fos labeling in the dorsomedial nucleus (DMH), ventromedial nucleus, and paraventricular nucleus (PVN) in HPD and normal animals, with effects on particular cell types varying. In the LHA and DMH, leptin decreased orexin cell activation in HPD and ARC-intact sheep. HPD abolished leptin-induced expression of Fos in melanin-concentrating hormone cells in the LHA and in CRH cells in the PVN. In contrast, HPD accentuated activation in oxytocin neurons. Our data from sheep with lesions encompassing the ARC do not suggest a primacy of action of leptin in this nucleus. We demonstrate that first order to second order signaling may not represent the predominant means by which leptin acts in the brain to generate integrated responses. We provide evidence that leptin exerts direct action on cells of the DMH, ventromedial nucleus, and PVN.

Estrogen regulation of proteins in the rat ventromedial nucleus of the hypothalamus.

The effects of estradiol (E2) on the expression of proteins in the pars lateralis of the ventromedial nucleus of the hypothalamus (VMNpl) in ovariectomized rats was studied using 2-dimensional gel electrophoresis followed by RPLC-nanoESI-MS/MS. E2 treatment resulted in the up-regulation of 29 identified proteins. Many of these proteins are implicated in the promotion of neuronal plasticity and signaling.

Silencing of estrogen receptor alpha in the ventromedial nucleus of hypothalamus leads to metabolic syndrome.

Estrogen receptor alpha (ERalpha) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERalpha signaling in ERalpha knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERalpha in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERalpha expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERalpha levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERalpha in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.

Hypothalamic galanin and early state of hyperphagia in obese Zucker rats.

Galanin (GAL) stimulates food intake in normal rats when it is injected in different hypothalamic areas involved in feeding such as the paraventricular and ventromedial nuclei and the lateral hypothalamus. At adulthood, the hyperphagic obese Zucker rat is characterized by a general dysregulation of some important neuropeptides involved in the regulation of food intake including GAL. The aim of this study was to measure GAL in different microdissected brain areas in 2- and 4-week-old lean (FA/-) and obese (fa/fa) male Zucker rats in order to know if GAL actively participates in triggering abnormal feeding behavior in obese rats. There was a significant increase (40%-220%) in GAL concentration with age in the arcuate and dorsomedial nuclei and in the above areas except for the lateral hypothalamus. Genotype differences were observed in the arcuate and paraventricular nuclei only. GAL levels were globally lower in obese than in lean rats (-15% to -25%) and the difference was significant at 2 weeks of age in the paraventricular nucleus and at 4 weeks of age in the arcuate nucleus. In agreement with human observations, these data suggest that GAL is not an early player in the development of overeating.

Regulation of corticotropin-releasing factor and its types 1 and 2 receptors by leptin in rats subjected to treadmill running-induced stress.

The present study was conducted to investigate the long-term effects of subchronic elevation of central leptin levels on the expression of corticotropin-releasing factor (CRF) and its types 1 and 2 receptors in the brain of rats subjected to treadmill running-induced stress. PBS or recombinant murine leptin was infused continuously for a period of 5 days into the third ventricle of rats with the aid of osmotic minipumps at a delivery rate of 2 mug/day. On the fifth day of infusion, rats were killed under resting conditions or after a session of treadmill running, which is known to induce a stress response in rats. Leptin treatment significantly decreased food intake, body weight, white adipose tissue weight, glucose and insulin plasma contents, and blunted the treadmill running-induced elevation in plasma levels of corticosterone. Leptin infusion prevented stress-induced de novo synthesis of CRF in the paraventricular hypothalamic nucleus (PVN), which was measured using the intronic probe for CRF heteronuclear RNA. The induction of the type 1 CRF receptor (CRF(1)R) in the PVN and supraoptic nucleus in running rats was also significantly blunted by leptin. In contrast, leptin treatment strongly increased the expression of type 2 CRF receptor (CRF(2)R) in the ventromedial hypothalamic nucleus (VMH). The present results suggest that subchronic elevation of central levels of leptin blunts treadmill running-induced activation of the hypothalamic-pituitary-adrenal axis through the inhibition of activation of the CRFergic PVN neurons, and potentially enhances the anorectic CRF effects via the stimulation of expression of CRF(2)R in the VMH.

[Participation of progesterone receptors in plus-maze behavior in female mice].

The current study tested delayed effect pf progesterone on the anxiety level of female mice. The elevated plus maze (EPM) behavior was assessed in ovariectomized mice injected for 7 days with estradiol benzoate and progesterone or progesterone alone after 6 hrs of the last treatment. One group of ovariectomized mice was injected with progesterone receptor blocker Mifepristone before 2 hrs of the last treatment. The immunocytochemistry method was used to visualize cells in different brain areas having immunoreactivity (ir) for progesterone receptors. In the EPM, progesterone administration significantly increased the anxiety levels of ovariectomized mice as compared with estradiol benzoate and progesterone administration. The participation of nuclear progesterone receptors in anxiety levels regulation is confirmed by high correlation of the change of progesterone receptor-ir cell number in some brain areas and anxiety levels. Mifepristone decreased anxiety levels and progesterone receptor-ir cell number in both groups of mice that suggests involvement of genomic mechanisms in anxiety regulation in female mice.

PAC1 receptors mediate pituitary adenylate cyclase-activating polypeptide- and progesterone-facilitated receptivity in female rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a feed-forward, paracrine/autocrine factor in the hypothalamic ventromedial nucleus (VMN) for receptivity and sensitizes pituitary hormone release for ovulation. The present study examined receptor(s) and signaling pathway by which PACAP enhances rodent lordosis. PACAP binds to PACAP (PAC1)- and vasoactive intestinal peptide-preferring receptors (VPAC1, VPAC2). Ovariectomized rodents primed with estradiol (EB) were given PACAP or vasoactive intestinal peptide directly onto VMN cells. Only PACAP facilitated receptivity. Pretreatment with VPAC1 and VPAC2 inhibitors blocked both PACAP- and progesterone (P)-induced receptivity. Antisense (AS) oligonucleotides to PAC1 (not VPAC1 or VPAC2) inhibited the behavioral effect of PACAP and P. By real-time RT-PCR, EB, P and EB+P enhanced VMN mRNA expression of PAC1. Within the total PAC1 population, EB and EB+P induced expression of short form PAC1 and PAC1hop2 splice variants. Finally, blocking cAMP/protein kinase A signaling cascade by antagonists to cAMP activity and protein kinase A or by antisense to dopamine- and cAMP-regulated phosphoprotein of 32 kDa blocked the PACAP effect on behavior. Collectively, these findings provide evidence that progesterone receptor-dependent receptivity is, in part, dependent on PAC1 receptors for intracellular VMN signaling and delineate a novel, steroid-dependent mechanism for a feed-forward reinforcement of steroid receptor-dependent reproductive receptivity.

In vitro increase in intracellular calcium concentrations induced by low or high extracellular glucose levels in ependymocytes and serotonergic neurons of the rat lower brainstem.

Pancreatic glucokinase (GK)-like immunoreactivities are located in ependymocytes and serotonergic neurons of the rat brain. The present study investigated in vitro changes in intracellular calcium concentrations ([Ca(2+)](i)) in response to low (2 mm) or high (20 mm) extracellular glucose concentrations in isolated cells from the wall of the central canal (CC), raphe obscurus nucleus (ROb), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LHA) in male rats. An increase in [Ca(2+)](i) was found in cells from the CC (21.1% or 9.8% of ependymocytes), ROb (10.9% or 14.5% of serotonergic neurons), VMH (7.8% and 25.2% of neurons), and LHA (20% or 15.7% of neurons), when extracellular glucose levels were changed from 10 to either 2 or 20 mm, respectively. Most of the ependymocytes and serotonergic neurons responding to the glucose changes were immunoreactive to the anti-GK in the CC (96.8% for low glucose and 100% for high glucose) and ROb (100% for low and high glucose). The [Ca(2+)](i) increase was blocked with calcium-free medium or L-type calcium channel blocker. Cells with an increase in [Ca(2+)](i) in response to low glucose did not respond to high glucose and vice versa. Inhibition of GK activity with acute alloxan treatment blocked low or high glucose-induced [Ca(2+)](i) increases in most GK-immunoreactive cells from the CC or ROb. The glucose-sensitive [Ca(2+)](i) increase in neurons of the VMH and LHA was also alloxan-sensitive, but no cells taken from the VMH and LHA were immunoreactive to the antibody used. The present study further indicates that ependymocytes of the CC and serotonergic neurons in the ROb are also sensitive to the changes in extracellular glucose in a GK-dependent manner, but that the subtype of GK in these cells could be different from that in the VMH and LHA.

Caloric intake and hypothalamic neurotransmitters in Zucker rats made acutely diabetic with streptozocin.

Zucker rats, lean and obese, treated with low dose intraperitoneal injections of streptozocin become hyperglycemic within 24h. Insulin levels fall, although the obese animal remains hyperinsulinemic. Associated with these changes in glucose and insulin there are transient decreases in caloric intake. Macronutrient selection studies show that protein consumption decreases. There is a trend for fat intake to decrease. The levels of hypothalamic neurotransmitters in the lean animals are not altered by streptozocin. The levels of 5-hydroxyindoleacetic acid increases in the streptozocin-treated obese animal in the paraventricular region, ventromedial region and the raphe. Serotonin is also significantly increased in the paraventricular region of the obese rat. These results suggest that acutely, treatment with streptozocin injures pancreatic islets, causing, in turn, decreases in insulin levels so that hyperglycemia ensues in both phenotypes. Associated with these perturbations are decreases in caloric intake. The magnitude of change in insulin levels is much greater in the obese rat. It is hypothesized that in the obese Zucker rat decrements in food intake are mediated by increase in serotonin turnover in the hypothalamus and these changes are related to changes of insulin levels. These data support the concept that circulating insulin affects hypothalamic neurotransmitters.

Hormonal-neural integration in the female rat ventromedial hypothalamus: triple labeling for estrogen receptor-alpha, retrograde tract tracing from the periaqueductal gray, and mating-induced Fos expression.

The lordosis reflex, a stereotypic posture adopted by female rats during sexual behavior, requires the convergence of a hormonal signal, estrogen, with a descending neural pathway from the ventromedial hypothalamic nucleus (VMH). The VMH contains at least three lordosis-relevant neural populations: estrogen receptor-alpha immunoreactive (ERalpha-IR) neurons, VMH neurons that project to the periaqueductal gray (PAG), and neurons that are ERalpha-IR and project to the PAG. Expression of Fos, a marker for neuronal activation, is increased in the VMH after mating. However, it is unknown which, if any, of these lordosis-relevant populations is activated. The majority of ERalpha-IR and projection neurons were not colocalized. Of the Fos-positive neurons, 41% neither contained ERalpha nor projected to the PAG, and 35% contained ERalpha but did not project to the PAG. Only 25% of Fos-positive neurons projected to the PAG, including projection neurons that expressed ERalpha. Our results suggest that mating activates several distinct VMH neuron types. However, ERalpha-IR neurons are activated to a greater extent compared with the PAG-projecting neurons.

Abnormalities of leptin and ghrelin regulation in obesity-prone juvenile rats.

Rats selectively bred to develop diet-induced obesity (DIO) spontaneously gain more body weight between 5 and 7 wk of age than do those bred to be diet resistant (DR). Here, chow-fed DIO rats ate 9% more and gained 19% more body weight from 5 to 6 wk of age than did DR rats but had comparable leptin and insulin levels. However, 6-wk-old DIO rats had 29% lower plasma ghrelin levels at dark onset but equivalent levels 6 h later compared with DR rats. When subsequently fed a high-energy (HE; 31% fat) diet for 10 days, DIO rats ate 70% more, gained more body and adipose depot weight, had higher leptin and insulin levels, and had 22% lower feed efficiency than DR rats fed HE diet. In DIO rats on HE diet, leptin levels increased significantly at 3 days followed by increased insulin levels at 7 days. These altered DIO leptin and ghrelin responses were associated with 10% lower leptin receptor mRNA expression in the arcuate (ARC), dorsomedial (DMN), and ventromedial hypothalamic nuclei and 13 and 15% lower ghrelin receptor (GHS-R) mRNA expression in the ARC and DMN than in the DR rats. These data suggest that increased ghrelin signaling is not a proximate cause of DIO, whereas reduced leptin sensitivity might play a causal role.

Neurochemical characteristics of the ventromedial hypothalamus in mediating the antiaversive effects of anxiolytics in different models of anxiety.

In experiments on rats using an "illuminated area" avoidance test and a "threatening situation" avoidance test, preliminary i.p. administration and subsequent microinjection into the ventromedial hypothalamus of various combinations of monoamines, transmitter amino acids, and their agonists and antagonists demonstrated differences in the functional importance of the neurochemical profile of this limbic formation in mediating anxiety states of different origins. The neurochemical analysis with local intrahypothalamic administration of anxiosedative and anxioselective substances showed that the antiaversive actions of Campirone are obtained only in conditions in which the dominant motivation is fear, while chlordiazepoxide, Phenibut, and Indoter are also active in anxiety induced by negatively stressful zoosocial influences; these actions are mediated respectively by serotoninergic and GABAergic types of synaptic switching in the ventromedial hypothalamus.

Chemical coding of GABA(B) receptor-immunoreactive neurones in hypothalamic regions regulating body weight.

Gamma-aminobutyric acid (GABA) interacts with hypothalamic neuronal pathways regulating feeding behaviour. GABA has been reported to stimulate feeding via both ionotropic GABA(A) and metabotropic GABA(B) receptors. The functional form of the GABA(B) receptor is a heterodimer consisting of GABA(B) receptor-1 (GABA(B)R1) and GABA(B) receptor-2 (GABA(B)R2) proteins. Within the heterodimer, the GABA-binding site is localized to GABA(B)R1. In the present study, we used an antiserum to the GABA(B)R1 protein in order to investigate the cellular localization of GABA(B)R1-immunoreactive neurones in discrete hypothalamic regions implicated in the control of body weight. The colocalization of GABA(B)R1 immunoreactivity with different chemical messengers that regulate food intake was analysed. GABA(B)R1-immunoreactive cell bodies were found in the periventricular, paraventricular (PVN), supraoptic, arcuate, ventromedial hypothalamic, dorsomedial hypothalamic, tuberomammillary nuclei and lateral hypothalamic area (LHA). Direct double-labelling showed that glutamic acid decarboxylase (GAD)-positive terminals were in close contact with GABA(B)R1-containing cell bodies located in all these regions. In the ventromedial part of the arcuate nucleus, GABA(B)R1-immunoreactive cell bodies were found to contain neuropeptide Y, agouti-related peptide (AGRP) and GAD. In the ventrolateral part of the arcuate nucleus, GABA(B)R1-immunoreactive cell bodies were shown to contain pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript. In the LHA, GABA(B)R1 immunoreactivity was present in both melanin-concentrating hormone- and orexin-containing cell populations. In the tuberomammillary nucleus, GABA(B)R1-immunoreactive cell bodies expressed histidine decarboxylase, a marker for histamine-containing neurones. In addition, GAD and AGRP were found to be colocalized in some nerve terminals surrounding GABA(B)R1-immunoreactive cell bodies in the parvocellular part of the PVN. The results may provide a morphological basis for the understanding of how GABA regulates the hypothalamic control of food intake and body weight via GABA(B) receptors.

Ventrolateral preoptic nucleus contains sleep-active, galaninergic neurons in multiple mammalian species.

The ventrolateral preoptic nucleus (VLPO) is a group of sleep-active neurons that has been identified in the hypothalamus of rats and is thought to inhibit the major ascending monoaminergic arousal systems during sleep; lesions of the VLPO cause insomnia. Identification of the VLPO in other species has been complicated by the lack of a marker for this cell population, other than the expression of Fos during sleep. We now report that a high percentage of the sleep-active (Fos-expressing) VLPO neurons express mRNA for the inhibitory neuropeptide, galanin, in nocturnal rodents (mice and rats), diurnal rodents (degus), and cats. A homologous (i.e. galanin mRNA-containing cell group) is clearly distinguishable in the ventrolateral region of the preoptic area in diurnal and nocturnal monkeys, as well as in humans. Galanin expression may serve to identify sleep-active neurons in the ventrolateral preoptic area of the mammalian brain. The VLPO appears to be a critical component of sleep circuitry across multiple species, and we hypothesize that shrinkage of the VLPO with advancing age may explain sleep deficits in elderly humans.

Expression of cAMP response element binding protein-binding protein in the song control system and hypothalamus of adult European starlings (Sturnus vulgaris).

In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP-ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors.

A progestin antagonist blocks vaginocervical stimulation-induced fos expression in neurones containing progestin receptors in the rostral medial preoptic area.

Vaginocervical stimulation (VCS) has a variety of effects on the brain, physiology and behaviour. Previous work demonstrated that a progestin antagonist blocked neuronal response to VCS (i.e. Fos expression) in the absence of progesterone in some neurones, and suggested that some of the effects of VCS on the brain are mediated by ligand-independent activation of progestin receptors (PRs). Although it had been reported previously that some of the cells in which VCS induces Fos expression also contain PRs, it had not been determined if a progestin antagonist blocked Fos expression in these particular neurones. The purpose of this experiment was to determine if a progestin antagonist decreases Fos expression specifically in cells that also express PRs in the preoptic area and ventromedial hypothalamus. As has been shown previously, VCS increased Fos-immunoreactive (ir) expression in the particular areas studied. In the rostral medial preoptic area, VCS increased Fos expression in cells that coexpressed PRs, as well as in cells that do not. However, in the caudal medial preoptic area, VCS only increased Fos expression in cells that did not coexpress PRs. Injection of the progestin antagonist, RU 486, decreased Fos expression in the rostral, but not caudal medial preoptic area, and it decreased Fos expression only in cells that coexpressed PR-ir. In contrast to a previous report, in the present study, the progestin antagonist did not inhibit VCS-induced Fos expression in the ventromedial hypothalamic area. The results of this experiment suggest that the progestin antagonist inhibits VCS-induced Fos expression in some neurones by blocking PRs, and they provide further support for the idea that VCS influences neuronal response in some cells by ligand-independent activation of PRs in those cells.

Immunohistochemical analyses of thyroid-specific enhancer-binding protein in the fetal and adult rat hypothalami and pituitary glands.

Thyroid-specific enhancer-binding protein (T/EBP), also known as NKX2.1 or TTF-1, regulates the expression of thyroid- and lung-specific genes. The t/ebp/Nkx2.1-null mutant mouse was stillborn but lacked the thyroid gland, pituitary gland, ventral region of the forebrain and normal lungs. These data demonstrated that T/EBP/NKX2.1 plays an important role not only in tissue-specific gene expressions in adults but also in genesis of these organs during development. Although the expression of t/ebp/Nkx2.1 in the brain has been reported, its function in the brain remains unknown. The present study was designed to determine the localization of T/EBP/NKX2.1 in the hypothalamus and pituitary gland of fetal and adult rats by immunohistochemistry as the first step toward understanding the function of T/EBP/NKX2.1 in the rat brain. In the fetal rat hypothalamus, T/EBP/NKX2.1 was localized widely in the ventral hypothalamic areas. In the adult rat brain, T/EBP/NKX2.1 was localized in the ventromedial hypothalamic nucleus, medial tuberal nucleus, arcuate nucleus and mammillary body. No T/EBP/NKX2.1 immunoreactivity was observed in the anterior or intermediate lobe of the pituitary gland in either fetal or adult rats. On the other hand, immunoreactive T/EBP/NKX2.1 was found in the posterior lobe of the pituitary gland. This paper presents results of detailed analyses of the distributions of T/EBP/NKX2.1 protein in the fetal and adult rat hypothalami and pituitary glands, and these results should provide important information for understanding the function of T/EBP/NKX2.1 in the brain.

Development of obesity and neurochemical backing in aurothioglucose-treated mice.

To clarify the neurochemical backing of aurothioglucose (ATG)-induced obesity in mice, we investigated lesion sites, hypothalamic neurotransmitters and c-Fos-like immunoreactivity (Fos-IR). At day 2 after ATG, tissue loss or cells death was observed in several parts of the ventral area of the ventromedial hypothalamic nucleus (VMH), and the dorsal area of arcuate nucleus and in the nucleus of the solitary tract (NTS). However, the greater part of the VMH was retained. Body weight began to increase in week 1. Hypothalamic serotonin (5-HT) and the metabolites were increased at day 2. The contents of acetylcholine, norepinephrine and dopamine in the hypothalamus showed no significant change. In week 1, the area shown tissue loss was compacted and plugged up. In the control group, most obvious c-Fos-like immunoreactive region was paraventricular nucleus (PVN). At day 2, Fos-IR was observed around destroyed regions in the hypothalamus and NTS, but few Fos-IR was found in the other regions including PVN. The Fos-IR around destroyed regions diminished after week 1. In week 3, Fos-IR in the PVN increased. These results suggest that the development of ATG-induced obesity cannot be attributed to solely VMH destruction. The restoration processes of the neuronal dysfunction involving PVN seem to play an important role in the development of obesity. NTS lesion and 5-HT system might contribute to decrease in food intake for several days after ATG.