Hypothalamic cholinergic regulation of body temperature and water intake in rats.

Without disturbing the behavior of unanesthetized rats, the perfusion of neostigmine through microdialysis probe into the anterior hypothalamus (AH), paraventricular nucleus (PVN) and lateral ventricle (LV) decreased body temperature and increased water intake. On the other hand, the perfusion into the supraoptic nucleus (SON) increased the body temperature. The perfusion of neostigmine increased the extracellular concentration of acetylcholine in the perfusion sites except LV. Changes, both decrease and increase, in body temperature and increase in water intake were correlated with increases in c-fos-like immunoreactivity (Fos-IR) in the hypothalamus, pons and medulla. Distinct Fos-IR was found in the PVN, SON, median preoptic nucleus (MnPO), locus coeruleus (LC), area postrema and nucleus of the solitary tract (NTS). Co-administration of atropine with neostigmine completely suppressed the changes in the body temperature, water intake and Fos-IR, all of which were induced by the neostigmine perfusion into AH, PVN and SON. In the LV-perfused rats, on the other hand, co-administration of atropine and neostigmine only partially prevented body temperature reduction and still induced significant hypothermia. These results suggest that muscarinic receptor activation in specific regions of the hypothalamus and the activation of LC and NTS are implicated in the regulation of body temperature and water intake. Other receptor processes are involved in the LV-induced changes.     

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.     

Role of preoptic and anterior hypothalamic cholinergic input on water intake and body temperature

To elucidate the role played by cholinergic mechanism in the preoptic area (POA) and anterior hypothalamus (AH) in the control of body temperature and water intake of rats, we used microdialysis without disturbing the behavior of unanesthetized animals. After microdialysis, we also investigated immunoreactivity for c-Fos protein in the hypothalamus. Stimulation with neostigmine, an acetylcholine esterase inhibitor, through microdialysis probe increased extracellular concentration of acetylcholine (ACh) in the POA and AH, and was accompanied by a dose-dependent fall in body temperature and increased water intake. Addition of atropine, a muscarinic receptor antagonist, to the dialysis medium containing neostigmine suppressed the neostigmine-induced changes in rectal temperature and water intake. Neostignime markedly increased c-Fos-like immunoreactivity (Fos-IR) in certain hypothalamic areas, including the paraventricular nucleus, supraoptic nucleus and median preoptic nucleus. This increase was also attenuated by atropine. These results suggest that cholinergic inputs and activation of muscarinic processes in POA and AH induced a decline in body temperature and increased water intake.     

Colocalization of urocortin and neuronal nitric oxide synthase in the hypothalamus and Edinger-Westphal nucleus of the rat

Different lines of studies suggest that both the corticotropin-releasing hormone-related peptide Urocortin I (Ucn) and the neuromodulator nitric oxide (NO) are involved in the regulation of the complex mechanisms controlling feeding and anxiety-related behaviors. The aim of the present study was to investigate the possible interaction between Ucn and NO in the hypothalamic paraventricular nucleus (PVN), an area known to be involved in the modulation of these particular behaviors. Therefore, we mapped local mRNA and peptide/protein presence of both Ucn and the NO producing neuronal NO synthase (nNOS). This investigation was extended to include the hypothalamic supraoptic nucleus (SON) and the Edinger-Westphal nucleus area (EW), the latter being one of the major cellular Ucn-expressing sites. Furthermore, we compared the two predominantly used laboratory rat strains, Wistar and Sprague-Dawley. Ucn mRNA and immunoreactivity were detected in the SON and in the EW. A significant difference between Wistar and Sprague-Dawley rats was found in mRNA levels in the EW. nNOS was detected in all brain areas analyzed, showing a significantly lower immunoreactivity in the PVN and EW of Sprague-Dawley versus Wistar rats. Contrary to some previous reports, no Ucn mRNA and only a very low immunoreactivity were detectable in the PVN of either rat strain. Interestingly, double-labeling immunofluorescence revealed that in the SON approximately 75% of all cells immunoreactive for Ucn were colocalized with nNOS, whereas in the EW only approximately 2% of the Ucn neurons were found to contain nNOS. These findings suggest an interaction between Ucn and NO signaling within the SON, rather than the PVN, that may modulate the regulation of feeding, reproduction, and anxiety-related behaviors.     

Fos-like immunoreactivity and thirst following hyperosmotic loading in rats with subdiaphragmatic vagotomy

If receptors in the gut relay information about increases in local osmolality to the brain via the vagus nerve, then vagotomy should diminish this signaling and reduce both thirst and brain Fos-like immunoreactivity (Fos-ir). Water intake in response to hypertonic saline (i.p. or i.g., 1 M NaCl, 1% BW; i.g., 0.6 M NaCl, 0.5% BW) was reduced during 120 min in rats with subdiaphragmatic vagotomy (VGX) compared to sham-VGX rats. Brain Fos-ir was examined in response to both i.g. loads. After the smaller load, VGX greatly reduced Fos-ir in the supraoptic nucleus (SON) and the magnocellular and parvocellular areas of the paraventricular nucleus (PVN). Fos-ir in the subfornical organ (SFO) and nucleus of the solitary tract (NTS) was not affected. After the larger load, VGX significantly reduced Fos-ir in the parvocellular PVN and in the NTS, but not in the other regions. Thus, decreased water intake by VGX rats was accompanied by decreased Fos-ir in the parvocellular PVN after the same treatments, indicating a role for the abdominal vagus in thirst in response to signaling from gut osmoreceptors. The decreased water intake in the VGX group was not reflected as a decrease in Fos-ir in the SFO. Absorption of the larger i.g. load may have activated Fos-ir through more rapidly increasing systemic osmolality, thereby obscuring a role for the vagus at this dose in the SON and magnocellular PVN.     

Mapping of cholinergic neurons associated with rat supraoptic nucleus: Combined immunocytochemical and histochemical identification

Recent electrophysiological experiments have suggested that electrical stimulation of an area dorsolateral to the rat supraoptic nucleus (SON) activates a cholinergic pathway to the vasopressin neurons of the SON. As no detailed information is available concerning the distribution and projections of the cholinergic neurons in this area, we have sought to provide this using a combination of choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. In some cases, these techniques were applied to the same neurons. Almost all neurons just outside of the SON that showed ChAT-like immunoreactivity also stained densely for AChE. These cells were distributed in a region dorsolateral to the SON. Light, punctate AChE staining around SON neurons was observed predominantly in the more ventral and posterior parts of the nucleus and were suggestive of synaptic terminals. Cholinergic fibres were found to enter the SON mainly from a lateral direction, turning in an anterior or posterior direction inside the nucleus. These results support the conclusion of earlier studies that the major cholinergic input to the SON arises in its immediate vicinity. We hypothesize that these ChAT/AChE-positive neurons are those responsible for cholinergically mediated, osmotically-stimulated release of vasopressin.     

Regional suppression by water intake of c-fos expression induced by intraventricular infusions of angiotensin II

Intracerebroventricular (i.c.v.) infusions of angiotensin II (AII) reliably induced c-fos expression in the supraoptic (SON) and paraventricular (PVN) nuclei, as well as other areas of the basal forebrain including the OVLT, subfornical organ (SFO), and bed nucleus (BNST). Double-labelling showed that AII-induced c-fos was observed in both vasopressin (AVP-) and oxytocin (OXY)-containing neurons of the SON and PVN in male rats. Allowing rats to drink water after AII infusions suppressed c-fos expression both AVP- and OXY-stained magnocellular neurons. Intragastric infusions of water were also effective, showing that oro-pharyngeal stimuli were not critical. Maximal suppression occurred in rats in whom water had been infused intragastrically about 5 min before i.c.v. AII infusions, suggesting that changes in osmolarity were responsible. i.c.v. AII also induced c-fos expression in a number of brainstem structures, including the solitary nucleus (NTS), lateral parabrachial nucleus (LPBN), locus coeruleus (LC), and the area postrema (AP). These results indicate that AVP and OXY-containing neurons in the magnocellular parts of the SON and PVN alter their immediate-early gene response to AII after water intake, and that this does not depend upon oro-pharyngeal factors. Furthermore, AII can induce c-fos expression in a number of brainstem nuclei associated with autonomic function, and these do not respond to water intake.     

Expression of Fos in rat brain in relation to sodium appetite: furosemide and cerebroventricular renin

Two experiments were performed to investigate the relationship between the expression of sodium appetite and the appearance of Fos-like immunoreactivity (Fos-IR) in the brain of rats. In the first experiment, rats were depleted of sodium by treatment with furosemide 24 h prior to sacrifice and without access to either food or sodium solution. Some rats had access to distilled water, and others had no fluids available during the 24 h. All of the furosemide-treated rats showed Fos-IR in both the subfornical organ (SFO) and around the organum vasculosum laminae terminalis (OVLT). Rats with access to distilled water during the depletion period showed no Fos-IR in the supraoptic (SON) or paraventricular hypothalamic nuclei (PVN) and, in parallel behavioral studies, comparably-treated rats consumed only 0.3 M NaCl solution at the end of the 24 h. In rats that had no fluids during the deprivation period, only about one half showed Fos-IR in SON and PVN and, in parallel behavioral studies, comparably treated rats consumed both water and 0.3 M NaCI solution at the end of 24 h. In a second experiment, cerebroventricular administration of renin stimulated short latency intake of 0.3 M NaCI and water. The relative intakes of water and NaCl were comparable at a low dose of renin, but intake of water exceeded that of NaCl after higher doses. Renin induced Fos-IR in SFO, MnPO, peri-OVLT region, SON and PVN. Both Fos-IR and fluid intake were antagonized by administration of losartan, an angiotensin 11 type 1 receptor antagonist. Thus, only the circumventricular organs of the lamina terminalis showed Fos-IR during each natriorexigenic regimen in these studies. These data support the view that Ang 11 of both central and peripheral origin activates the SFO and/or peri-OVLT region and contributes to sodium appetite.     

The role of cardiovascular and muscle afferent systems in control of body water balance

Influences of afferent inputs from cardiovascular and muscle receptors on the activities of neurosecretory neurons in the hypothalamus, which secrete vasopressin (ADH) were studied. Recordings were made from identified neurosecretory neurons in the supraoptic (SON) and paraventricular nuclei (PVN) of cats and rats. Activation of baroreceptors in the carotid sinus and aortic arch and atrial receptors inhibited SON and PVN neuron activities, while activation of chemoreceptors in the carotid sinus excited them. Repetitive electrical stimulation of the carotid sinus and aortic nerves showed that weak stimulation produced excitation and stronger stimulation produced inhibition of SON and PVN neurons. Electrical stimulation of these nerves and the nucleus tractus solitarius (NTS) by a single or short train of pulses showed that 'fast' and 'slow' pathways between the NTS and the SON existed, while these two types of pathways were not observed between the NTS and the PVN. Evidence of direct connections from the NTS to the PVN was found by means of antidromic stimulation of the PVN. Electrical stimulations of group I afferent fibers from the gastrocnemius muscle did not change SON neuron discharges, while activation of group III and IV afferent fibers excited them. Injection of chemicals (NaCl, KCl, bradykinin) into arteries supplying the muscle excited SON neurons. The excitation disappeared after section of the muscle nerves. The results indicated that activation of small afferents from the muscle excites the SON neurons, leading to an increase in vasopressin secretion. All these studies show that afferent inputs from receptors in the cardiovascular system and in the muscle have modulatory effects on neurosecretory neurons, and participate in control of body water balance by regulating vasopressin secretion from the neurohypophysis.     

Functional and anatomic relationship between cholinergic neurons in the median preoptic nucleus and the supraoptic cells

The median preoptic nucleus (MePO) has been suggested to be an important area in the brain for the regulation of vasopressin (VP) release under the condition of osmotic stimulation. Fos immunoreactivity (Fos-ir), choline acetyltransferase (ChAT) immunoreactivity and retrograde labeling with fluoro-gold were used in this study to determine whether cholinergic neurons in the MePO can be activated by hypertonic NaCl, and to characterize the specific MePO cells that have anatomic projections to the supraoptic nuclei (SON). The results showed that c-fos expression specifically induced by hypertonic NaCl was found in the ChAT cells of the MePO. A retrograde tracing experiment demonstrated that the MePO neurons projecting to the SON were cholinergic. In addition, hypertonic saline-induced Fos-ir was colocalized with the MePO neurons back labeled with fluoro-gold from the SON. Together, these data provide evidence that the MePO cholinergic neurons are activated by osmotic stimulation, and suggest that cholinergic cells in the MePO are functionally important in the control of the SON neurons under the condition of hypertonic stimulation.     

Olfactory and visceral projections to the paraventricular nucleus

Electrophysiological studies were performed to determine if neurons of the paraventricular nucleus (PVN) which receive inputs from the stomach via vagal afferents also respond to nucleus of the solitary tract (NTS) and olfactory bulb (OB) stimulation. We found that the NTS, OB stimulation, and gastric distension depress the firing frequency of PVN neurons. The pathway from the NTS to the PVN contains larger fibers than the projection from the PVN to the NTS.     

Connections of neurons in the region of the nucleus tractus solitarius with the hypothalamic paraventricular nucleus: Their possible involvement in neural control of the cardiovascular system in rats

Extracellular recordings were made from 607 spontaneously firing neurons within the nucleus tractus solitarius (NTS) and its vicinity in urethane-anesthetized male rats. Following electrical stimulation of the hypothalamic paraventricular nucleus (PVN) area, 21% of the neurons were orthodromically excited, 6% were inhibited and 2.5% were antidromically activated. The antidromic spike latencies were 22-64 ms. Among those orthodromically responding neurons, 81 neurons were tested by pressure pulse stimulation of the isolated carotid sinus. The pressure stimulation produced excitation in 7 and inhibition in 13 neurons. Of the 8 tested neurons which were antidromically activated, one neuron was excited and another neuron inhibited by the pressure pulse stimulation. These results provide electrophysiological evidence for reciprocal connections between neurons in the NTS region and the PVN, and give support to the hypothesis that the PVN is involved in the neural control of the cardiovascular system.     

Identification of central sites involved in butorphanol-induced feeding in rats

Butorphanol (BT), a mixed kappa- and mu-opioid receptor agonist, induces vigorous food intake in rats. Peripheral injection of BT seems to increase food intake more effectively than intracerebroventricular administration. To further elucidate the nature of BT's influence on consummatory behavior, we examined which feeding-related brain areas exhibit increased c-Fos immunoreactivity (IR) following subcutaneous injection of 4 mg/kg body weight BT, a dose known to induce a maximal orexigenic response. We also evaluated whether direct administration of BT into the forebrain regions activated by peripheral BT injection affects food intake. Peripheral BT administration induced c-Fos-IR in the hypothalamic paraventricular nucleus (PVN), central nucleus of the amygdala (CeA), and nucleus of the solitary tract (NTS). However, 0.1-30 microg BT infused into the CeA, failed to increase food intake 1, 2, and 4 h after injection. Only the highest dose of BT (30 microg) injected into the PVN increased feeding. These results suggest that the PVN, CeA, and NTS mediate the effects of peripherally-injected BT. The PVN or CeA are probably not the main target sites of immediate BT action.     

Functional relationship between subfornical organ cholinergic stimulation and cellular activation in the hypothalamus and AV3V region

The subfornical organ (SFO) has been suggested to be important for water intake and secretion of vasopressin (AVP). However, the role of the SFO cholinergic mechanism in the control of body fluid regulation is not clear. This study determined the effects of local cholinergic stimulation in the SFO produced by administration of physostigmine on drinking and cellular excitation in the anterior third ventricle (AV3V) region and in the supraoptic and paraventricular nuclei (SON and PVN). The results showed that injection of physostigmine into the SFO induced water intake and c-fos expression in the AV3V area as well as in the AVP containing neurons in the hypothalamus. Pretreatment of the SFO with mecamylamine, a nicotinic receptor antagonist, had no effect on physostigmine induced behavioral and c-fos responses. The muscarinic receptor blocker atropine, however, abolished both drinking and cellular activation after injection of physostigmine into the SFO. Immunostaining experiments demonstrated positive acetyltransferase (ChAT) in the SFO. Intensive ChAT immunoreactivity was located in the cholinergic fibers in the SFO. Together, the results indicate that SFO cholinergic mechanisms are important in co-operation with the AV3V and hypothalamic neurons in the control of thirst and AVP-mediated body fluid homeostasis.     

Hypotension preferentially induces c-fos immunoreactivity in supraoptic vasopressin neurons.

Immunoreactivity to Fos protein (Fos-IR) was detected in rat hypothalamic neurons within 1 h of onset of hemorrhage by withdrawing 4-5 ml of blood, which lowered the arterial blood pressure to 50-70 mm Hg. About 70% of vasopressin (AVP)-containing neurons in the supraoptic nucleus (SON) and 20% in the paraventricular nucleus (PVN) expressed Fos-IR. In contrast, 5% of oxytocin (OXY)-containing neurons in the SON and < 1% in PVN were Fos-IR. Intravenous infusion of the vasodilating agent, nitroprusside, which lowered the blood pressure to levels comparable to that attained by hemorrhage, induced Fos-IR in greater than 65% of AVP-containing neurons in the SON, while relatively few AVP neurons in the PVN were Fos positive. These results suggest that hemorrhage or hypotension preferentially induces c-fos expression in supraoptic AVP-containing neurons.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Sodium deprivation and salt intake activate separate neuronal subpopulations in the nucleus of the solitary tract and the parabrachial complex

Salt intake is an established response to sodium deficiency, but the brain circuits that regulate this behavior remain poorly understood. We studied the activation of neurons in the nucleus of the solitary tract (NTS) and their efferent target nuclei in the pontine parabrachial complex (PB) in rats during sodium deprivation and after salt intake. After 8-day dietary sodium deprivation, immunoreactivity for c-Fos (a neuronal activity marker) increased markedly within the aldosterone-sensitive neurons of the NTS, which express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). In the PB, c-Fos labeling increased specifically within two sites that relay signals from the HSD2 neurons to the forebrain--the pre-locus coeruleus and the innermost region of the external lateral parabrachial nucleus. Then, 1-2 hours after sodium-deprived rats ingested salt (a hypertonic 3% solution of NaCl), c-Fos immunoreactivity within the HSD2 neurons was virtually eliminated, despite a large increase in c-Fos activation in the surrounding NTS (including the A2 noradrenergic neurons) and area postrema. Also after salt intake, c-Fos activation increased within pontine nuclei that relay gustatory (caudal medial PB) and viscerosensory (rostral lateral PB) information from the NTS to the forebrain. Thus, sodium deficiency and salt intake stimulate separate subpopulations of neurons in the NTS, which then transmit this information to the forebrain via largely separate relay nuclei in the PB complex. These findings offer new perspectives on the roles of sensory information from the brainstem in the regulation of sodium appetite.     

Water deprivation induces regional expression of c-fos protein in the brain of inbred polydipsic mice

We studied the effects of water deprivation on the expression of c-fos protein (Fos) in the brain of inbred polydipsic mice, STR/N strain, that show extreme polydipsia without a lack of vasopressin in the body. Non-polydipsic mice, ICR strain, were used as controls. All male animals were deprived of water for 24 and 48 h. Fos-like immunoreactivity (Fos-LI) in the brain was studied by immunohistochemical techniques. In both groups of mice water deprivation induced a remarkable increase in Fos-LI in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the median preoptic nucleus (MnPO), the organum vasculosum laminae terminalis (OVLT) and the subfornical organ (SFO). A far more increase, however, was seen in the MnPO, the SFO and the area postrema (AP) of the polydipsic mice compared to those of the non-polydipsic control mice. In the nucleus of the tractus solitarius (NTS) and in the anteroventral part of the PVN (avPVN), water deprivation caused a clear increase in Fos-LI in the polydipsic mice, while in the non-polydipsic mice the same treatment induced no Fos-LI in the NTS and no change in the avPVN. These results indicate that neurons in the circumventricular organs and the NTS are strongly activated by water deprivation in the polydipsic mice, suggesting that these brain structures play an important role in the polydipsia.     

迷走神经参与胃伤害性信息向下丘脑的传递

目的　 研究迷走神经是否参与胃伤害性信息向下丘脑室旁核的传递。 方法　 检测下列条件下c Fos蛋白在孤束核及下丘脑室旁核的表达 :①胃内注入福尔马林引起伤害性刺激 ;②福尔马林刺激结合双侧膈下迷走神经切断术。结果　胃内注入福尔马林引起的伤害性刺激可以诱导c Fos蛋白在孤束核和下丘脑室旁核等脑区的表达 ,但在胸段脊髓的I,V ,VII和X层无明显表达。胃内注入生理盐水的对照组则仅有极少量的表达 ,双侧膈下迷走神经切断术可以减少c Fos蛋白在这些部位的表达。 结论　 该研究结果表明 ,迷走神经参与了胃内脏伤害性信息向孤束核及下丘脑室旁核的传递