Increased sociability and gene expression of oxytocin and its receptor in the brains of rats affected prenatally by valproic acid.

Autism is a neurodevelopmental disorder characterised by the disruption of social interactions. Autistic animal models play a crucial role in neurophysiologic research on this disorder. One of these models is based on rats that have been prenatally treated with valproic acid - VPA rats. The aim of our study performed with this model was to investigate changes in sociability and gene expression of neuropeptides and receptors involved in regulating social behaviour. We focused on gene expression in the hypothalamus, where the neuropeptides oxytocin (OT) and arginine-vasopressin (AVP) are produced, as well as oxytocin receptors (OTR) in certain neuronal structures involved in the creation of social abilities. Our research showed that VPA rats spent more time in the part with an unknown animal and less time in the central part of a three chamber sociability test apparatus than control animals. The latency period of VPA rats before initiating social contact was decreased. In addition, during weaning, VPA female rats spent more time in direct interaction with an unknown rat. We also found that adult VPA rats had an increased expression of OT in the hypothalamic supraoptic and paraventricular nuclei and of OTR in the medial prefrontal cortex, piriform cortex, cortex-amygdala transition zone and the region of the basolateral and basomedial amygdaloid nuclei compared with controls. To sum up, we observed that a single prenatal injection of VPA increased social behaviour and gene expression of OT and OTR in neurological structures connected with the social behaviour of rats. One unanticipated finding was the absence of one of the core symptoms of autism in VPA rats, suggesting a decreased ability to understand intraspecific communication signals.

Early weaning by maternal prolactin inhibition leads to higher neuropeptide Y and astrogliosis in the hypothalamus of the adult rat offspring.

The suppression of prolactin production with bromocriptine (BRO) in the last 3 d of lactation reduces milk yield (early weaning) and increases the transfer of leptin through the milk, causing hyperleptinaemia in pups. In adulthood, several changes occur in the offspring as a result of metabolic programming, including overweight, higher visceral fat mass, hypothyroidism, hyperglycaemia, insulin resistance, hyperleptinaemia and central leptin resistance. In the present study, we investigated whether overweight rats programmed by early weaning with maternal BRO treatment have hypothalamic alterations in adulthood. We analysed the expression of neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), pro-opiomelanocortin (POMC) and α-melanocyte-stimulating hormone (α-MSH) by immunohistochemistry in the following hypothalamic nuclei: medial and lateral arcuate nucleus (ARC); paraventricular nucleus (PVN); lateral hypothalamus (LH). Additionally, we sought to determine whether these programmed rats exhibited hypothalamic inflammation as indicated by astrogliosis. NPY immunostaining showed a denser NPY-positive fibre network in the ARC and PVN (+82% in both nuclei) of BRO offspring. Regarding the anorexigenic neuropeptides, no difference was found for CART, POMC and α-MSH. The number of astrocytes was higher in all the nuclei of BRO rats. The fibre density of glial fibrillary acidic protein was also increased in both medial and lateral ARC (6·06-fold increase and 9·13-fold increase, respectively), PVN (5·75-fold increase) and LH (2·68-fold increase) of BRO rats. We suggest that early weaning has a long-term effect on the expression of NPY as a consequence of developmental plasticity, and the presence of astrogliosis indicates hypothalamic inflammation that is closely related to overweight and hyperleptinaemia observed in our model.

Altered oxytocinergic hypothalamus systems in sepsis.

Sepsis is known to affect neuroendocrine circuits: injections of lipopolysaccaride are potent stimulators of oxytocin secretion from the posterior lobe, acute sepsis leads to uterus contractions and spontaneous abort. Here, we report changes in expression and distribution of hypothalamic oxytocin in rats that had been subjected to caecal ligation and puncture which led to acute sepsis. Septic animals showed loss of oxytocin immunostaining in perikarya of the supraoptic and paraventricular nuclei and an increase of oxytocin positive fibres, suggesting a shift of oxytocin pools into the axonal compartment. Immunostaining of the posterior lobe revealed reduction of oxytocin in septic rats. Magnocellular neurons in supraoptic- and to a lesser extent in paraventricular nuclei showed nuclear immunoreactivity for the protooncogene c-Fos, indicating stimulation of transcriptional activity upon sepsis. Contrary to magnocellular oxytocin immunoreactivity, we observed increased oxytocin immunoreactivity in cell bodies and processes of periventricular nucleus and in perivascular neurons. Oxytocin neurons in other regions of the hypothalamus and the preoptic region did not appear to be affected by acute sepsis. Our findings suggest a differential activation of neurohypophyseal and cerebrospinal fluid contacting oxytocin systems while centrally projecting oxytocin neurons may not be affected. Systemic oxytocin levels may serve as additional diagnostic marker for sepsis.

Early weaning is associated with higher neuropeptide Y (NPY) and lower cocaine- and amphetamine-regulated transcript (CART) expressions in the paraventricular nucleus (PVN) in adulthood.

The interruption of lactation for a short period, without the use of pharmacological substances or maternal separation, causes offspring malnutrition and hypoleptinaemia and programmes for metabolic disorders such as higher body weight and adiposity, hyperphagia, hyperleptinaemia and central leptin resistance in adulthood. Here, in order to clarify the mechanisms underlying the phenotype observed in adult early-weaned (EW) rats, we studied the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in different hypothalamic nuclei by immunohistochemistry and Western blot. In the EW group, the teats of lactating rats were blocked with a bandage to interrupt lactation during the last 3 d, while control pups had free access to milk throughout the entire lactation period. At age 180 d, EW offspring showed higher NPY staining in the paraventricular nucleus (PVN), as well as NPY protein content (+68 %) in total hypothalamus than control ones. AgRP showed no changes in staining or Western blot. POMC content was not affected; however, its distribution pattern was altered. CART-positive cells of EW offspring had lower immunoreactivity associated with reduced cell number in the PVN and lower protein content ( - 38 %) in total hypothalamus. The present data indicate that precocious weaning can imprint the neuronal circuitry, especially in the PVN, and cause a long-term effect on the expression of specific orexigenic and anorexigenic neuropeptides, such as NPY and CART, that can be caused by leptin resistance and are coherent with the hyperphagia observed in these animals.

[Effects of electroacupuncture at PC6 and BL15 on nerve electrical activity in spinal dorsal root and norepinephrine and dopamine contents in paraventricular nucleus of hypothalamus in rats with acute myocardial ischemia].

OBJECTIVE: To investigate the effects of electroacupuncture (EA) at Neiguan (PC6) and Xinshu (BL15) on the nerve electrical activity in spinal dorsal root and norepinephrine (NE) and dopamine (DA) concentrations in the paraventricular nucleus of the hypothalamus in rats with acute myocardial ischemia (AMI). METHODS: A total of 100 male Sprague-Dawley rats were randomly divided into sham-operation, model, EA at PC6, EA at BL15 and EA at both PC6 and BL15 groups with 20 rats in each group. The nerve electrical activity in spinal dorsal roots was recorded by bipolar electrodes. NE and DA concentrations in the paraventricular nucleus of the hypothalamus were detected by high-performance liquid chromatography. RESULTS: When compared with the sham-operation group, the nerve electrical activity in spinal dorsal roots was significantly increased while the NE and DA concentrations in the paraventricular nucleus of the hypothalamus were decreased in the model group (P<0.01). The nerve electrical activity in spinal dorsal roots was decreased and the NE and DA concentrations were increased in the paraventricular nucleus of the hypothalamus in the EA at PC6 group, the EA at BL15 group and the EA at both PC6 and BL15 group in comparison to those in the model group (P<0.01). The nerve electrical activity in spinal dorsal roots and the NE and DA concentrations in paraventricular nucleus of hypothalamus of the EA at both PC6 and BL15 group were significantly improved when compared to those of the EA at PC6 and EA at BL15 groups (P<0.05). CONCLUSION: EA at both PC6 and BL15 acupoints exhibits the synergistic protective effects against AMI. The possible mechanism is related to regulating the nerve electrical activity in spinal dorsal roots and the concentrations of NE and DA in paraventricular nucleus of the hypothalamus.

Direct visualization of retinoic acid in the rat hypothalamus: an immunohistochemical study.

In order to increase our knowledge about the distribution of vitamins in the mammalian brain, we have developed a highly specific antiserum directed against retinoic acid with good affinity (10(-8) M), as evaluated by ELISA tests. In the rat brain, no immunoreactive fibers containing retinoic acid were detected. Cell bodies containing retinoic acid were only found in the hypothalamus. This work reports the first visualization and the morphological characteristics of cell bodies containing retinoic acid in the mammalian paraventricular hypothalamic nucleus and in the dorsal perifornical region, using an indirect immunoperoxidase technique. The restricted distribution of retinoic acid in the rat brain suggests that this vitamin could be involved in very specific physiological mechanisms.

Localization of plasminogen in mouse hippocampus, cerebral cortex, and hypothalamus.

Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.

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.

The distribution of vasotocin and mesotocin immunoreactivity in the hypothalamic magnocellular neurosecretory nuclei of the Saharan herbivorous lizard, Uromastix acanthinurus Bell, 1825 (Sauria-Agamidae).

An immunohistochemical study of the magnocellular neurosecretory nuclei was performed in the hypothalamus of the desert lizard Uromastix acanthinurus using polyclonal antibodies against arginine vasotocin (AVT), mesotocin (MST) and neurophysins I and II (NpI, NpII). AVT- and MST-immunoreactivities were localized in individual neurons of the supraoptic, periventricular, and paraventricular nuclei and in scattered neurosecretory cells. The supraoptic nuclei (SONs) can be subdivided into rostral, medial and caudal portions. The rostral portion of the SONs was called the SON-ventral aggregation (V SON) because the neurosecretory neurons are present in the ventral part of the hypothalamus along the optic chiasma (OC). Their perikarya and fibres were only AVT-ir. The medial part of the SONs was constituted of two clusters of neurosecretory neurons located in the two lateral ends of the OC to form the SON-lateral aggregations (L SON). In the caudal end of the last one, some MST-ir perikarya appeared. The caudal part of the SONs was constituted of a dorso-lateral aggregation (D SON) of ir-neurons spreading over the lateral forebrain bundle (LFB). AVT- and MST- perikarya were observed in this caudal portion of the SONs, AVT-ir neurons being more numerous. AVTergic and MSTergic magnocellular neurons were present in the periventricular nuclei (PeVNs). Parvocellular and magnocellular AVT- and MST-ir were observed in the paraventricular nuclei (PVNs). The fibres emerging from the magnocellular neurons which belong to these nuclei and the scattered cells ran along the hypothalamic floor and entered the median eminence (ME) to end in the neural lobe of hypophysis. As a rule, immunoreactivity was also observed in all the regions of the forebrain with vasotocinergic and mesotocinergic perikarya and fibres. The immunoreactive distribution was similar to that described in other reptiles.

Molecular characterization and estrogen regulation of hypothalamic KISS1 gene in the pig.

Kisspeptin-GPR54 signaling plays an essential role in normal reproduction in mammals via stimulation of gonadotropin secretion. Here, we cloned the porcine KISS1 cDNA from the hypothalamic tissue and investigated the effect of estrogen on the distribution and numbers of KISS1 mRNA-expressing cells in the porcine hypothalamus. The full length of the cDNA was 857 bp encoding the kisspeptin of 54 amino acids, with the C-terminal active motif designated kisspeptin-10 being identical to that of mouse, rat, cattle, and sheep. In situ hybridization analysis revealed that KISS1-positive cell populations were mainly distributed in the hypothalamic periventricular nucleus (PeN) and arcuate nucleus (ARC). KISS1 expression in the PeN of ovariectomized (OVX) pigs was significantly upregulated by estradiol benzoate (EB) treatment. On the other hand, KISS1-expressing cells were abundantly distributed throughout the ARC in both OVX and OVX with EB animals. The number of KISS1-expressing neurons was significantly lowered by EB treatment only in the most caudal part of the ARC, but other ARC populations were not affected. The present study thus suggests that the PeN kisspeptin neurons could be responsible for the estrogen positive feedback regulation to induce gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) surge in the pig. In addition, the caudal ARC kisspeptin neurons could be involved in the estrogen negative feedback regulation of GnRH/LH release. This is the first report of identification of porcine KISS1 gene and of estrogen regulation of KISS1 expression in the porcine brain, which may be helpful for better understanding of the role of kisspeptin in reproduction of the pig.

Pituitary atrial natriuretic peptide of paraventricular nucleus origin.

Atrial natriuretic peptide-synthesizing neurons in the hypothalamic paraventricular nucleus constitute the major sources of ANP in the three lobes of the pituitary gland. Complete transection of the pituitary stalk eliminated 93% of ANP from the intermediate lobe, 47 and 77% from the anterior and the posterior lobes, respectively. Meantime, increased levels of immunoreactive ANP were measured in the median eminence, due to the accumulation of the peptide in the transected axons centrally to the transected stalk and in the paraventricular nucleus. It is likely that ANP neurons in the paraventricular nucleus innervate the pituitary, but those in the periventricular (median) preoptic nucleus and the organum vasculosum laminae terminalis may not contribute to the ANP innervation of the pituitary gland.

Ingestion of non-caloric liquid diet is sufficient to restore plasma corticosterone level, but not to induce the hypothalamic c-Fos expression in food-deprived rats.

Male Sprague-Dawley rats were subjected to four different conditions; free fed control (FC), 48 h of food deprivation (FD), 1 h of refeeding with chow (RF/CW) or with a non-caloric liquid diet following FD (RF/NC) and then sacrificed for c-Fos immunohistochemistry in the hypothalamic paraventricular nucleus (PVN) and the nucleus tractus of solitarius (NTS). Plasma corticosterone level and the postmortem weight of gastric contents were measured. Plasma level of corticosterone significantly increased during FD, and then decreased within 1 h after ad libitum access to chow or non-caloric liquid diet. c-Fos-ir in the brain regions was not changed by FD; however, significantly increased by chow refeeding, but not by non-caloric diet. Chow, but not the non-caloric, refeeding significantly increased gastric contents. Results suggest that caloric load and/or gastric distension may require for the postprandial activation of neurons in the PVN and NTS, but ingestion of non-caloric palatable mixture may be sufficient to normalize the fasting-induced increase of plasma corticosterone. In conclusion, feeding-related changes in the HPA axis activity may not be related with meal-induced c-Fos expression in the PVN and NTS.

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.

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague-Dawley rats.

The research on components of the renin-angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT(1) receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT(1) receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT(1) receptors we performed IHC on AT(1) receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague-Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT(1) receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT(1)-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT(1)-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT(1) receptors.

Influence of dehydration on the expression of neuropeptide Y Y1 receptors in hypothalamic magnocellular neurons.

Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24-72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.

Involvement of brain-derived neurotrophic factor in the regulation of hypothalamic somatostatin in vivo.

Brain-derived neurotrophic factor (BDNF) has been extensively studied in the central nervous system as a survival and differentiation factor and in plasticity processes. In vitro, BDNF has been shown to stimulate cellular differentiation and neurohormones synthesis and release. We demonstrated that BDNF is a potent and specific stimulatory agent of somatostatin (SRIH) synthesis in primary cultures of hypothalamic neurons. However, less information is available about its function on SRIH neurons in vivo. In the present study, we examined the effect of in vivo intracerebroventricular BDNF administration in adult non-anesthetized male rats. Two distinct experimental approaches were used: acute intracerebroventricular injection and long-term (14 days) continuous infusion (Alzet micro-pumps). We demonstrate that single intracerebroventricular BDNF injections (5 microg/rat) induce an early (60 and 180 min) decrease in the SRIH mRNA signal in the hypothalamic periventricular nucleus (PeVN) accompanied by a decrease of the hypothalamic SRIH content. 48 h after the acute injection, SRIH mRNA levels and peptide content strongly and significantly increased. After continuous intracerebroventricular BDNF administration (12 microg/day for 14 days), a significant increase in the SRIH hypothalamic content was observed. Nevertheless, the increase in peptide content was not correlated with a similar increase in the PeVN messenger level. These findings show the involvement of BDNF in the in vivo regulation of somatostatinergic neurons in adult rats, which clearly differs according to the BDNF administration mode.

Effects of fluoxetine administration on regional galanin expression in obese Zucker rat hypothalamus.

The aim of the present work was to study the potential involvement of hypothalamic galanin system in the anorectic mechanism of fluoxetine in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. Significant decreases in food intake, final body weight and total body fat were observed after fluoxetine treatment. Although fluoxetine-treated rats showed a decrease in urine elimination, this effect was not enough to compensate decreased water intake, leading to dehydration, as showed by decreased body water content. Chronic fluoxetine administration increased the numbers of galanin positively immunostained neural cells in medial and lateral preoptic areas, lateral hypothalamic area and paraventricular nucleus (rostral and magnocellular regions), without changes in dorsomedial, ventromedial, supraoptic, suprachiasmatic and arcuate nuclei. Taken into account that galanin stimulates appetite, these results could represent rather a compensatory response against reduced food intake than a direct anorectic mechanism. Changes in the magnocellular region of the hypothalamic paraventricular nucleus suggest a role for galanin neural circuits at this level in fluoxetine-induced hydro-osmotic impairment.

Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo.

The regulation of TRH gene expression in the paraventricular nucleus of the hypothalamus (PVH) by leptin is critical for normal function of the thyroid axis in rodents and humans. The TRH neuron in the PVH expresses both leptin and melanocortin-4 receptors, suggesting that both signaling systems may regulate TRH gene expression in vivo. Indeed, the TRH promoter responds to both of these signaling pathways in cell culture through identified cis-acting elements, which include signal transducer and activator of transcription (STAT) 3 and cAMP-response element binding protein binding sites that mediate leptin and melanocortin responses, respectively. To determine whether leptin signaling can directly target the TRH promoter in vivo, we developed a chromatin immunoprecipitation assay to use on leptin-treated animals. After a single injection of leptin in fasting animals, we could detect a significant increase in TRH gene expression in the PVH that correlated well with the induction of phosphorylated-STAT3 in the hypothalamus. Furthermore, using a STAT3 antibody, we could immunoprecipitate the STAT-binding site containing regions of both the TRH promoter and the promoter of the suppressor of cytokine signaling-3 gene, another well-defined target of leptin action. In contrast, upstream regions of these promoters that lack STAT sites were not precipitated. Taken together these experiments demonstrate that STAT3 mediates transcriptional effects of leptin in vivo and that the TRH promoter is a likely direct site of leptin action. In addition, these experiments demonstrate that chromatin immunoprecipitation can be used to characterize leptin-signaling in vivo.

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.