Amygdala enterostatin induces c-Fos expression in regions of hypothalamus that innervate the PVN

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus (PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 microl) into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala, lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in the arcuate, LH and VMH, which innervate the PVN.     

Hypothalamic mechanisms associated with corticotropin-releasing factor-induced anorexia in chicks

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with potent anorexigenic effects in rodents and chickens. However, the mechanism underlying this effect remains unclear. Hence, the objective of the current study was to elucidate the hypothalamic mechanisms that mediate CRF-induced anorexia in 4 day-old Cobb-500 chicks. After intracerebroventricular (ICV) injection of 0.02 nmol of CRF, CRF-injected chicks ate less than vehicle chicks while no effect on water intake was observed at 30 min post-injection. In subsequent experiments, the hypothalamus samples were processed at 60 min post-injection. The CRF-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial hypothalamus (VMH), and paraventricular nucleus (PVN) of the hypothalamus than vehicle-treated chicks. CRF injection was associated with decreased whole hypothalamic mRNA abundance of neuropeptide Y receptor sub-type 1 (NPYR1). In the ARC, CRF-injected chicks expressed more CRF and CRF receptor sub-type 2 (CRFR2) mRNA but less agouti-related peptide (AgRP), NPY, and NPYR1 mRNA than vehicle-injected chicks. CRF-treated chicks expressed greater amounts of CRFR2 and mesotocin mRNA than vehicle chicks in the PVN and VMH, respectively. In the DMN, CRF injection was associated with reduced NPYR1 mRNA. In conclusion, the results provide insights into understanding CRF-induced hypothalamic actions and suggest that the anorexigenic effect of CRF involves increased CRFR2-mediated signaling in the ARC and PVN that overrides the effects of NPY and other orexigenic factors.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Involvement of nuclei in the hypothalamus in cardiac sympathoexcitatory reflexes in cats

The hypothalamus is considered to be an important area in the central regulation of cardiovascular function. However, its role in processing excitatory cardiovascular reflexes induced by stimulation of cardiac afferents has not been established. In the present study, using c-Fos immunoreactivity, we located neurons in the hypothalamus activated by inputs from cardiac sympathetic afferents. Following bilateral barodenervation and cervical vagotomy in anesthetized cats, bradykinin (BK, 1-10 microg, in 0.1 ml; n=7) was applied repetitively (6x, every 20 min) to the anterior epicardial surface of the left ventricle. This chemical stimulation caused consistent excitatory cardiovascular reflexes characterized by increases in blood pressure (BP) and heart rate (HR), while the vehicle for BK (0.9% saline, n=6) produced no such responses. Compared to control cats, c-Fos immunoreactive cells were significantly increased (P<0.05) in the arcuate nucleus (ARC), dorsal hypothalamic area (HDA), dorsomedial nucleus, paraventricular hypothalamic nucleus (PVN) and periventricular nucleus in the BK-treated animals. More neurons double-labeled with c-Fos and nitric oxide synthase (NOS) were observed in the PVN following epicardial application of BK (P<0.05). There was no significant increase in co-localization of these two labelings in the other nuclei. These results suggest that several nuclei in the hypothalamus respond to activation of cardiac sympathetic afferents, leading to sympathoexcitatory reflexes. Nitric oxide (NO) may function as a neurotransmitter or as a neuromodulator in the PVN during these cardiac-cardiovascular responses.     

Neonatal Nicotine Exposure Leads to Hypothalamic Gliosis in Adult Overweight Rats

Astrocytes and microglia, the immune competent cells of central nercous system, can be activated in response to metabolic signals such as obesity and hyperleptinaemia. In rats, maternal exposure to nicotine during lactation leads to central obesity, hyperleptinaemia, leptin resistance and alterations in hypothalamic neuropeptides in the offspring during adulthood. In the present study, we studied the activation of astrocytes and microglia, as well as the pattern of inflammatory mediators, in adult offspring of this experimental model. On postnatal day 2 (P2), osmotic minipumps releasing nicotine (NIC) (–6 mg/kg/day) or saline for 14 days were s.c . implanted in dams. Male offspring were killed on P180 and hypothalamic immunohistochemistry, retroperitoneal white adipose tissue (WAT) polymerase chain reaction analysis and multiplex analysis for plasma inflammatory mediators were carried out. At P180, NIC astrocyte cell number was higher in the arcuate nucleus (ARC) (medial: +82%; lateral: +110%), in the paraventricular nucleus (PVN) (+144%) and in the lateral hypothalamus (+121%). NIC glial fibrillary acidic protein fibre density was higher in the lateral ARC (+178%) and in the PVN (+183%). Interleukin‐6 was not affected in the hypothalamus. NIC monocyte chemotactic protein 1 was only higher in the periventricular nucleus (+287%). NIC microglia (iba‐1‐positive) cell number was higher (+68%) only in the PVN, as was the chemokine (C‐X3‐C motif) receptor 1 density (+93%). NIC interleukin‐10 was lower in the WAT (?58%) and plasma (?50%). Thus, offspring of mothers exposed to nicotine during lactation present hypothalamic astrogliosis at adulthood and microgliosis in the PVN.     

Regulation of Neuronal Activation by Alpha2A Adrenergic Receptor Agonist

Stress factors induce neuronal activation in brain areas that are related to anxiety and fear. High doses of caffeine induce neuronal activation with Ca2+ influx followed by expression of the immediate early gene c-fos. In the present study, we investigated c-Fos protein expression in stress-responsive brain areas induced by caffeine, as well as the role of alpha2A receptor in the regulation of neuronal activation. Immunohistochemical analysis showed that an acute effect of caffeine induced c-Fos protein expression in the hippocampus, the bed nucleus of stria terminalis (BNST), the lateral septum, the basolateral and central amygdala, the paraventricular hypothalamic nucleus (PVN), the locus coeruleus, and the lateral parabrachial nucleus (LPBN). However, c-Fos expression was attenuated after repeated treatment of caffeine, spaced 24 h apart, compared to a single acute effect. Alpha2A receptor activation with the agonist guanfacine attenuated the acute effect of caffeine in terms of c-Fos expression in neurons in the CA1-CA3 areas of hippocampus, the locus coeruleus and the LPBN as compared with effect of caffeine alone, whereas the number of c-Fos expressing neurons increased in the lateral septum, the dorsal BNST, the central amygdala, and the PVN, areas that are densely innervated by noradrenergic neurons. Guanfacine alone induced c-Fos protein expression in neurons in the central amygdala, the dorsal BNST, the PVN, the LPBN, and the caudal nucleus of the solitary tract. Guanfacine alone also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in neurons expressing c-Fos in the dorsal BNST, the central amygdala, and the LPBN. These results suggest that alpha2A receptor activation modulates synaptic transmission in neuronal circuits that are correlated with stress in vivo.     

FosB expression in the central nervous system following isotonic volume expansion in unanesthetized rats

We examined c-Fos and FosB staining in the central nervous system 8 and 24 h following acute volume expansion in unanesthetized rats. Male rats were instrumented with a femoral artery catheter for measurement of blood pressure and heart rate (HR), a jugular venous catheter for measurement of central venous pressure (CVP), and a femoral vein catheter for i.v. infusion. After 48 h, rats were volume expanded with isotonic saline (10% of body weight for 10 min i.v.) or given a control infusion (0.01 ml/min for 10 min i.v.). After a period of 8 or 24 h, the rats were deeply anesthetized and perfused transcardially with 4% paraformaldehyde. Separate sets of serial sections of the hypothalamus were processed for either FosB (Santa Cruz) or c-Fos (Oncogene AB-5) immunocytochemistry. The volume expansion protocol significantly increased central venous pressure but did not affect blood pressure or heart rate. Volume expansion produced a significant increase in FosB-positive cells in the paraventricular nucleus (PVN) of the hypothalamus, the supraoptic nucleus (SON), the perinuclear zone (PNZ) of the supraoptic nucleus, the nucleus of the solitary tract (NST), and the caudal ventrolateral medulla (CVL) in both the 8- and 24-h groups. In the area postrema (AP), the number of FosB-positive cells was significantly increased only at 8 h post-infusion. However, c-Fos was not significantly increased above control levels at either time point. The results demonstrate that FosB activation is maintained for at least 24 h following an acute increase in central venous pressure.     

The alteration of glucocorticoid receptor-immunoreactivity in the rat forebrain following short-term and long-term adrenalectomy

To examine the effect of short-term and long-term adrenalectomy (ADX) on the glucocorticoid receptor (GR) expression, we performed an immunohistochemical study on the rat forebrain. One day after ADX, the GR-immunoreactivity significantly decreased or disappeared in most forebrain structures, while relatively strong GR-immunoreactivity was still found within the hypothalamus especially in the arcuate nucleus (ARC) and the parvocellular paraventricular nucleus (PVN). Two weeks following ADX, GR-immunoreactive cells disappeared in many structures of the forebrain including most parts of hypothalamus while moderate GR-immunoreactivity was still observable in the ARC and PVN. More than 3 months after ADX, the rats still survived when they received replacement of corticosterone during the first 2 weeks following the operation. Moderate GR-immunoreactivity in the ARC and PVN of the hypothalamus was exhibited whereas no immunoreactive cells remained in the cerebral cortex, thalamus and other forebrain structures when these animals showed obvious cell death in the granule cells of the dentate gyrus, identified with the silver impregnation method for degenerating cells. Massive cell loss in this hippocampal region is an indicator of a complete ADX, in addition to the blood corticosterone level. These results demonstrate topographic differences of GR expression in the rat forebrain after ADX with only continuous immunoreactivity in the ARC and PVN of the hypothalamus, suggesting that some neurons in the ARC and PVN could keep active GR probably in order to maintain their survival after removing the adrenal gland.     

Effect of calcitonin gene-related peptide (CGRP) on avian appetite-related processes

Calcitonin gene-related peptide (CGRP) is released from the gastrointestinal tract following ingestion and causes satiety in mammals. Its effects on appetite in non-mammalian vertebrates are unreported. In Experiment 1, fasted chicks reduced food and water intake after central injection of CGRP. These effects were not associated with increased plasma corticosterone concentration. In Experiment 2, we showed that the effect on water intake was independent of food intake. In Experiment 3, central CGRP caused increased c-Fos immunoreactivity in the arcuate (ARC) nucleus, paraventricular nucleus (PVN), periventricular (PHN) and ventromedial (VMH) hypothalamic nuclei. The results of Experiment 4 demonstrate that intraperitoneal injection of CGRP also causes reduced food and water intake. c-Fos immunoreactivity was increased in the ARC, PHN, PVN and VMH in Experiment 5 after intraperitoneal injection of CGRP. Lastly in Experiment 6, we showed that central CGRP changes the type of pecks from feeding to exploratory, and reduces the number of escape attempts. The effect of CGRP appears to be primary on appetite in chicks. In conclusion, the mechanisms of CGRP induced satiety have some similarities and differences between avian and rodent models. The results presented here provide new insight into the evolution of vertebrate satiety regulatory mechanisms.     

The central anorexigenic mechanism of adrenocorticotropic hormone involves the caudal hypothalamus in chicks

Adrenocorticotropic hormone (ACTH), consisting of 39 amino acids, is most well-known for its involvement in an organism's response to stress. It also participates in satiety, as exogenous ACTH causes decreased food intake in rats. However, its anorexigenic mechanism is not well understood in any species and its effect on appetite is not reported in the avian class. Thus, the present study was designed to evaluate central ACTH's effect on food intake and to elucidate the mechanism mediating this response using broiler chicks. Chicks that received intracerebroventricular (ICV) injection of 1, 2, or 4 nmol of ACTH reduced food intake, under both ad libitum and 180 min fasted conditions. Water intake was also reduced in ACTH-injected chicks under both feeding conditions, but when measured without access to feed it was not affected. Blood glucose was not affected in either feeding condition. Following ACTH injection, c-Fos immunoreactivity was quantified in key appetite-associated hypothalamic nuclei including the ventromedial hypothalamus (VMH), dorsomedial hypothalamus, lateral hypothalamus (LH), arcuate nucleus (ARC) and the parvo- and magno-cellular portions of the paraventricular nucleus. ACTH-injected chicks had increased c-Fos immunoreactivity in the VMH, LH, and ARC. Hypothalamus was collected at 1 h post-injection, and real-time PCR performed to measure mRNA abundance of some appetite-associated factors. Neuropeptide Y, pro-opiomelanocortin, glutamate decarboxylase 1, melanocortin receptors 2–5, and urocortin 3 mRNA abundance was not affected by ACTH treatment. However, expression of corticotropin releasing factor (CRF), urotensin 2 (UT), agouti-related peptide (AgRP), and orexin (ORX), and melanocortin receptor 1 (MC1R) mRNA decreased in the hypothalamus of ACTH-injected chicks. In conclusion, ICV ACTH causes decreased food intake in chicks, and is associated with VMH, LH, and ARC activation, and a decrease in hypothalamic mRNA abundance of CRF, UT, AgRP, ORX and MC1R.     

Differential sensitivity of specific neuronal populations of the rat hypothalamus to prolactin action

Prolactin stimulates dopamine release from neuroendocrine dopaminergic (NEDA) neurons in the hypothalamic arcuate nucleus (ARC) to maintain low levels of serum prolactin. Elevated prolactin levels during pregnancy and lactation may mediate actions in other hypothalamic regions such as the paraventricular nucleus (PVN) and rostral preoptic area (rPOA). We predicted that NEDA neurons would be more sensitive prolactin targets than neurons in other regions because they are required to regulate basal prolactin secretion. Moreover, differences in the accessibility of the ARC to prolactin in blood may influence the responsiveness of this population. Therefore, we compared prolactin-induced signaling in different hypothalamic neuronal populations following either systemic or intracerebroventricular (icv) prolactin administration. Phosphorylation of the signal transduction factor, STAT5 (pSTAT5), was used to identify prolactin-responsive neurons. In response to systemic prolactin, pSTAT5-labeled cells were widely observed in the ARC but absent from the rPOA and PVN. Many of these responsive cells in the ARC were identified as NEDA neurons. The lowest icv prolactin dose (10 ng) induced pSTAT5 in the ARC, but with higher doses (>500 ng) pSTAT5 was detected in numerous regions, including the rPOA and PVN. NEDA neurons were maximally labeled with nuclear pSTAT5 in response to 500 ng prolactin and appeared to be more sensitive than dopaminergic neurons in the rPOA. Subpopulations of oxytocin neurons in the hypothalamus were also found to be differentially sensitive to prolactin. These data suggest that differences in the accessibility of the arcuate nucleus to prolactin, together with intrinsic differences in the NEDA neurons, may facilitate homeostatic feedback regulation of prolactin release.     

Relaxin-3/INSL7 Regulates the Stress-response System in the Rat Hypothalamus

Relaxin-3 (RLN3) is a neuropeptide belonging to the insulin–relaxin superfamily. RLN3-expressing neurons are predominantly located in the dorsal pons known as the nucleus incertus, and project their axons to the forebrain including the hypothalamus. RLN3 has been suggested to be involved in the stress response. In the present study, we investigated the hypothalamic action of RLN3 in the stress-response system by intracerebroventricular (icv) administration of RLN3. Compared with saline icv injection, 1 nmol icv RLN3 injection induced c-Fos expression in the paraventricular nucleus of the hypothalamus (PVN) at 1 h after administration. Some RLN3-induced c-Fos-positive cells in the PVN were also corticotropin-releasing factor (CRF)-expressing neurons. CRF and c-fos mRNA levels in the PVN were increased at 2 h after RLN3 administration. Plasma adrenocorticotropic hormone (ACTH) levels were also increased after RLN3 administration. These results suggest that RLN3 is able to stimulate the hypothalamopituitary CRF–ACTH system during the acute response.     

Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action

Orexin A and B, a recently identified pair of neuropeptides, are produced in perikarya located in the lateral and perifornical hypothalamus (LH and PFH). Immunoreactive fibers from these neurons innervate several nuclei in the hypothalamus. Orexin A and orexin B stimulate feeding when administered intracerebroventricularly to rats. To identify the specific sites of orexin action, orexin A and B were microinjected into a number of hypothalamic and extrahypothalamic sites in rats. Orexin A was found to enhance food intake when injected into four hypothalamic sites, the paraventricular nucleus (PVN), the dorsomedial nucleus (DMN), LH and the perifornical area, but was ineffective in the arcuate nucleus (ARC), the ventromedial nucleus (VMN), and the preoptic area (POA) as well as the central nucleus of the amygdala (CeA) and nucleus of the tractus solitarius (NTS). Orexin B was not effective at any site tested. These findings demonstrate that orexin A receptive sites for stimulation of food intake exist primarily in a narrow band of neural tissue within the hypothalamus that is known to be involved in control of energy homeostasis.     

Differential Expression of RFamide‐Related Peptide,a Mammalian Gonadotrophin‐Inhibitory Hormone Orthologue,and Kisspeptin in the Hypothalamus of Abadeh Ecotype Does During Breeding and Anoestrous Seasons

Gonadotrophin‐inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that was discovered in birds as an inhibitory factor for gonadotrophin release. RFamide‐related peptide (RFRP) is a mammalian GnIH orthologue that inhibits gonadotrophin synthesis and release in mammals through actions on gonadotrophin‐releasing hormone (GnRH) neurones and gonadotrophs, mediated via the GnIH receptor (GnIH‐R), GPR147. On the other hand, hypothalamic kisspeptin provokes the release of GnRH from the hypothalamus. The present study aimed to compare the expression of RFRP in the dorsomedial hypothalamus and paraventricular nucleus (DMH/PVN) and that of kisspeptin in the arcuate nucleus (ARC) of the female goat hypothalamus during anoestrous and breeding seasons. Mature female Abadeh does were used during anoestrus, as well as the follicular and luteal phases of the cycle. The number of RFRP‐immunoreactive (‐IR) neurones in the follicular phase was lower than in the luteal and anoestrous stages. Irrespective of the ovarian stage, the number of RFRP‐IR neurones in the rostral and middle regions of the DMH/PVN was higher than in the caudal region. By contrast, the number of kisspeptin‐IR neurones in the follicular stage was greater than in the luteal stage and during the anoestrous stage. Irrespective of the stage of the ovarian cycle, the number of kisspeptin‐IR neurones in the caudal region of the ARC was greater than in the middle and rostral regions. In conclusion, RFRP‐IR cells were more abundant in the rostral region of the DMH/PVN nuclei of the hypothalamus, with a greater number being found during the luteal and anoestrous stages compared to the follicular stage. On the other hand, kisspeptin‐IR neurones were more abundant in the caudal part of the ARC, with a greater number recorded in the follicular stage compared to the luteal and anoestrous stages.     

Disruption of arcuate/paraventricular nucleus connections changes body energy balance and response to acute stress.

The mediobasal hypothalamus regulates functions necessary for survival, including body energy balance and adaptation to stress. The purpose of this experiment was to determine the contribution of the arcuate nucleus (ARC) in controlling these two functions by the paraventricular nucleus (PVN). Circular, horizontal cuts (1.0 mm radius) were placed immediately above the anterior ARC to sever afferents to the PVN. In shams the knife was lowered to the same coordinates but was not rotated. Food intake and body weight were monitored twice daily, at the beginning and end of the light cycle, for 1 week. On the final day the animals were restrained for 30 min. Lesioned animals had increased food intake in light and dark periods, higher weight gain per day, and more body fat as compared with shams. There was no difference in caloric efficiency. Unlike shams, lesioned rats had no predictable relationship between plasma insulin and leptin. Plasma ACTH was increased at 0 min in lesioned rats but was decreased 15 and 30 min after restraint as compared with shams. There was no difference in plasma corticosterone. Immunostaining revealed that alpha-melanocortin (alphaMSH) and neuropeptide Y (NPY) accumulated below the cuts, and both were decreased in PVN. Food intake and body weight were correlated negatively to alphaMSH, but not NPY in PVN. There was no difference in proopiomelanocortin (POMC) mRNA, but NPY mRNA was reduced in the ARC of lesioned animals. We conclude that ARC controls body energy balance in unstressed rats, possibly by alphaMSH input to PVN, and that ARC also is necessary for PVN regulation of ACTH.