Area postrema lesions attenuate LiCl-induced c-Fos expression correlated with conditioned taste aversion learning

Lesions of the area postrema (AP) block many of the behavioral and physiological effects of lithium chloride (LiCl) in rats, including formation of conditioned taste aversions (CTAs). Systemic administration of LiCl induces c-Fos immunoreactivity in several brain regions, including the AP, nucleus of the solitary tract (NTS), lateral parabrachial nucleus (latPBN), supraoptic nucleus (SON), paraventricular nucleus (PVN), and central nucleus of the amygdala (CeA). To determine which of these brain regions may be activated in parallel with the acquisition of LiCl-induced CTAs, we disrupted CTA learning in rats by ablating the AP and then quantified c-Fos-positive cells in these brain regions in sham- and AP-lesioned rats 1 h following LiCl or saline injection. Significant c-Fos induction after LiCl was observed in the CeA and SON of AP-lesioned rats, demonstrating activation independent of an intact AP. LiCl-induced c-Fos was significantly attenuated in the NTS, latPBN, PVN and CeA of AP-lesioned rats, suggesting that these regions are dependent on AP activation. Almost all of the lesioned rats showed some damage to the subpostremal NTS, and some rats also had damage to the dorsal motor nucleus of the vagus; this collateral damage in the brainstem may have contributed to the deficits in c-Fos response. Because c-Fos induction in several regions was correlated with magnitude of CTA acquisition, these regions are implicated in the central mediation of lithium effects during CTA learning.     

Cholinergic input to the supraoptic nucleus increases Fos expression and body temperature in rats

To examine the role played by cholinergic input and processes in the supraoptic nucleus (SON) in the control of body temperature and water intake in rats, we used microdialysis to stimulate and analyze SON without disturbing the behavior of unanesthetized rats. After microdialysis, we also investigated immunoreactivity for c-Fos protein in the brain as an index of neuronal activation. Stimulation with neostigmine, an acetylcholine esterase inhibitor, through the microdialysis probe increased the extracellular concentration of acetylcholine in the SON. This cholinergic stimulation dose-dependently increased body temperature but did not significantly change the water intake. The stimulation markedly increased c-Fos-like immunoreactivity (Fos-IR) in the SON and certain hypothalamic areas, including the paraventricular nucleus (PVN) and median preoptic nucleus (MnPO). Fos-IR was also evident in certain regions of the pons and brainstem, including the locus ceruleus (LC), area postrema (AP), and nucleus of the solitary tract (NTS). Addition of atropine, a muscarinic receptor antagonist, to the dialysis medium containing neostigmine attenuated the increase of Fos-IR and suppressed the neostigmine-induced responses in body temperature. These results suggest that cholinergic input and activation of the muscarinic cholinoceptive neurons in the SON contribute to the regulation of body temperature. Activation of noradrenergic pathways in the brainstem including LC and NTS may be involved in the thermoregulation mechanism.     

大鼠双侧海马伞离断对脂多糖诱导的下丘脑催产素能神经元功能活动的影响

为观察去海马传入对免疫应激时下丘脑室旁核、视上核催产素能神经元功能活动的影响 ,以探索海马调控外周免疫反应的脑内环路 ,本研究预先切断大鼠双侧海马伞 ,2 8d后腹腔内注射细菌内毒素脂多糖 (lipopolysaccharide,LPS,75 0μg/kg) ,用免疫组织化学双标记方法 ,以 F os蛋白作为神经元功能活动的标记物 ,观察下丘脑催产素能神经元中即刻早期基因的表达变化。结果显示 :双侧海马伞离断后 ,下丘脑室旁核中因腹腔脂多糖刺激而发生活化的催产素能神经元活化百分率显著下降 ;而视上核中催产素能神经元的活化未受明显影响。上述结果表明 ,海马对下丘脑 -垂体 -肾上腺轴以及免疫系统的抑制作用部分是通过其支配的下丘脑室旁核的催产素能神经元来完成的。     

妊高征大鼠脑内室旁核、视上核及孤束核内血管紧张素Ⅱ1型受体的表达

目的观察妊娠高血压大鼠(PIH)与正常妊娠大鼠下丘脑室旁核(PVN)、视上核(SON)及孤束核(NTS)内血管紧张素Ⅱ1型受体(AT1)免疫阳性神经元的分布。方法用免疫荧光染色技术对PIH大鼠AT1分布的神经元进行了研究,并与正常大鼠进行比较。结果两组大鼠PVN、SON、NTS内AT1免疫阳性神经元的分布与形态基本类似。数据经统计学处理,结果表明,PIH组大鼠PVN和NTS内AT1阳性细胞数目较对照组多,而两组大鼠SON内AT1的表达无明显差异。结论实验结果提示AT1在下丘脑PVN和NTS内的血压神经内分泌调节活动中起着重要的介导作用,中枢AT1受体的异常增加可能与母体妊高征的发病有关。     

The role of catecholaminergic neurons in the hypothalamus and medullary visceral zone in response to restraint water-immersion stress in rats

The activity of catecholaminergic neurons in the hypothalamus and the medullary visceral zone (MVZ) in rats in response to restraint water-immersion stress (RWIS) was measured by use of dual Fos and tyrosine hydroxylase (TH) immunohistochemistry. In RWIS rats Fos immunoreactive (Fos-IR) nuclei dramatically increased in the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the dorsal motor nucleus of the vagus (DMV), the nucleus of the solitary tract (NTS), the area postrema (AP), and the ventrolateral medulla (VLM). A small number of TH-immunoreactive (TH-IR) and Fos/TH double-labeling neurons in the PVN, and their absence from the SON, were observed in both RWIS and nonstressed rats. More TH-IR neurons were observed in the MVZ of RWIS rats than in nonstressed rats. In RWIS and nonstressed rats, the percentage of Fos-IR nuclei in TH-IR neurons was 38.0 and 14.3% in the DMV, 34.4 and 9.7% in the NTS, 18.6 and 4.5% in the AP, and 45.7 and 18.9% in the VLM, respectively. In conclusion, catecholaminergic neurons in the MVZ are involved in the response to RWIS; although the PVN and SON also participate in the response to RWIS, the mechanism is not via catecholaminergic neurons.     

Exercise training differentially affects intrinsic excitability of autonomic and neuroendocrine neurons in the hypothalamic paraventricular nucleus

Oxytocinergic and vasopressinergic brain stem projections have been shown to play an important role in mediating cardiovascular adjustments during exercise training (ET). The aim of the present work was to determine whether the intrinsic excitability of hypothalamic neurons giving rise to brain stem peptidergic projections is altered as a consequence of ET. Whole cell patch-clamp recordings were obtained from nucleus of the solitarii tract (NTS)-projecting paraventricular nucleus of the hypothalamus (PVN) neurons and from supraoptic nucleus (SON) and PVN magnocellular cells (MNCs), in hypothalamic slices obtained from sedentary (S) and ET rats. Our results indicate that intrinsic excitability of PVN neurons that innervate the NTS (PVN-NTS) is enhanced by ET, resulting in a more efficient input-output function (increase number of evoked actions potentials, steeper frequency/current relationships and slower decaying frequency/time relationships). Changes in input-output function were accompanied by smaller hyperpolarizing afterpotentials (HAPs) and afterhyperpolarizing potentials (AHPs), during and after trains of spikes, respectively. On the other hand, a decreased efficacy in the input-output function was observed in SON/PVN MNCs during ET. Altogether, our results indicate that ET differentially affects the intrinsic excitability of autonomic and neurosecretory SON and PVN neurons. Increased excitability in PVN-NTS neurons may contribute to enhanced release of OT and VP peptides in the dorsal brain stem, and cardiovascular fine-tuning during exercise training.     

免疫抑制剂雷帕霉素对LPS免疫激发大鼠脑内免疫相关核团神经元Fos和nNOS表达的影响

观察免疫抑制剂雷帕霉素对大鼠中枢免疫核团Fos和nNOS表达的影响,并探讨雷帕霉素对中枢免疫调节的作用。将大鼠分为雷帕霉素(10mg/kg)实验组和生理盐水对照组。两组动物分别给药后,腹腔注射免疫激发剂细菌脂多糖(LPS,25mg/kg),用免疫组织化学方法显示并计数下丘脑室旁核、视上核和孤束核中Fos蛋白阳性、nNOS样免疫阳性神经元和Fos/nNOS双标神经元数量,并对数据进行统计学处理。结果显示:实验组动物的下丘脑室旁核、视上核和孤束核内Fos、Fos/nNOS神经元数目较对照组均显著减少(P<0.01)。这一结果表明免疫抑制剂雷帕霉素降低了中枢免疫相关核团内因LPS免疫应激所引起的nNOS的表达,提示雷帕霉素在抑制机体免疫机能的同时,也降低了神经-内分泌系统对免疫的抑制作用。     

Increased NADPH-diaphorase reactivity in the hypothalamic paraventricular nucleus and tanycytes following systemic administration of leptin in rats

Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.     

束缚应激大鼠下丘脑内一氧化氮合酶与c-fos蛋白的共存

为了探讨下丘脑内一氧化氮与应激之间的关系，本实验应用NADPH-d组化法和C-fos免疫组化技术相结合的方法，对束缚应激大鼠下丘脑内一氧化氮合酶（NOS）和c-fos蛋白的分布以及两者的共存关系进行了观察。结果表明，大鼠在束缚应激4h后，（1）室旁核大细胞部和视上核可见密集深染的NOS阳性大细胞；（2）室旁核小细胞部、背内侧核、穹窿周核、环状核、腹内侧核腹外侧部、结节内侧核、外侧区、室周区、乳头体前核和内侧核的外侧区等核团出现疏密不等和深浅不一的NOS阳性细胞；（3）C-fos蛋白以室旁核表达最为强烈，视前区、背内侧核、弓状核和外侧区亦有较强的表达；（4）在外侧区、室旁核小细胞部及其附近的室周区、背内侧核及乳头体前核腹侧部约有10％～15％的中、小型NOS阳性细胞同时表达C-fos蛋白，室旁核大细胞部则仅有较少的NOS阳性大细胞表达C－fos蛋白，在结节区、结节内侧核和视上核视交叉后部则偶见双标细胞。结果提示上述下丘脑核团内的部分一氧化氮能神经元与束缚应激反应有关。     

Cholecystokinin and hypothalamic corticotrophin-releasing factor participate in endotoxin-induced hypophagia

Cholecystokinin (CCK) provides a meal-related signal that activates brainstem neurons, which have reciprocal interconnections with the hypothalamic paraventricular nucleus. Neurons that express corticotrophin-releasing factor (CRF) in the hypothalamus possess anorexigenic effects and are activated during endotoxaemia. This study investigated the effects of CCK(1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia and hypothalamic CRF neuronal activation. Male Wistar rats were pretreated with a specific CCK(1) receptor antagonist (devazepide; 1 mg kg(-1); i.p.) or vehicle; 30 min later they received LPS (100 μg kg(-1); i.p.) or saline injection. Food intake, corticosterone responses and Fos-CRF and Fos-α-melanocyte-stimulating hormone (α-MSH) immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase immunoreactivity in the nucleus of the solitary tract (NTS) were evaluated. In comparison with saline treatment, LPS administration decreased food intake and increased plasma corticosterone levels, as well as the number of Fos-CRF and Fos- tyrosine hydroxylase double-labelled neurons in vehicle-pretreated rats; no change in Fos-α-MSH immunoreactivity was observed after LPS injection. In saline-treated animals, devazepide pretreatment increased food intake, but it did not modify other parameters compared with vehicle-pretreated rats. Devazepide pretreatment partly reversed LPS-induced hypophagia and Fos-CRF and brainstem neuronal activation. Devazepide did not modify the corticosterone and Fos-α-MSH responses in rats treated with LPS. In conclusion, the present data suggest that LPS-induced hypophagia is mediated at least in part by CCK effects, via CCK(1) receptor, on NTS and hypothalamic CRF neurons.     

Rapid changes in the sensitivity of arcuate nucleus neurons to central ghrelin in relation to feeding status

Ghrelin, the endogenous ligand for the growth hormone secretagogue (GHS) receptor, stimulates feeding and increases body weight. Systemic ghrelin administration induces the immediate-early gene protein product, c-Fos, in the arcuate nucleus of the hypothalamus (ARC) of satiated rats and this increase is potentiated in fasted rats. The aim of this study was to determine whether potentiation was seen in fasted animals after intracerebroventricular (i.c.v) administration of ghrelin and to identify the hypothalamic nuclei activated by this peptide. In addition we investigated if allowing fasted animals to re-feed for 1 h prior to i.c.v. ghrelin injection affected the c-Fos response. Using c-Fos immunocytochemistry, we demonstrated that i.c.v. ghrelin activated several hypothalamic nuclei, including the ARC, paraventricular nucleus (PVH) and the lateral hypothalamus (LH). The c-Fos response was greater in fasted animals compared with satiated animals. Fasted rats allowed access to food for 1 h prior to central ghrelin administration showed an attenuated response in the ARC, similar to the response seen in fed animals. However, the response in the LH (including in the orexin neurons) was further potentiated. The latter may reflect a connection between the hypothalamus and regions of the brain responding to the reward value of the meal.     

加压素能和宫缩素能神经元在新生儿下丘脑的分布

本文用ABC法对加压素样和宫缩素样神经元在新生儿下丘脑的分布进行了观察。结果发现,两者的神经元集中分布于视上核、室旁核及副大细胞分泌核。根据两者的分布特点,又将视上核、室旁核和副大细胞分泌核再分为若干分区,各分区均以加压素样神经元为主,约占总数的80～90％。本文结果提示加压素样和宫缩素样神经元在下丘脑的分布具有动物种属和年龄特征。     

Descending pathways from the paraventricular nucleus contribute to the recruitment of brainstem nuclei following a systemic immune challenge

Hypothalamic nuclei, particularly the paraventricular nuclei (PVN), are important brain sites responsible for central nervous system responses during an immune challenge. The brainstem catecholamine cells of the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) have been shown to play critical roles in relaying systemic immune signals to the PVN. However, whilst it is well recognised that PVN divisions also innervate the NTS and VLM, it is not known whether descending PVN pathways can modulate the recruitment of brainstem cells during an immune challenge. Using systemic administration of the proinflammatory cytokine interleukin-1beta, in combination with Fos immunolabelling, we firstly investigated the effect of PVN lesions on NTS and VLM catecholamine and non-catecholamine cell responses. We found that ibotenic acid lesions of the PVN significantly reduced numbers of Fos-positive non-catecholamine, noradrenergic and adrenergic cells observable in the VLM and NTS after interleukin-1beta administration. We then investigated the origins of descending inputs to the VLM and NTS, activated by systemic interleukin-1beta, by mapping the distribution of Fos-positive retrogradely-labelled cells in divisions of the PVN after iontophoretically depositing choleratoxin-b subunit into the NTS or VLM one week prior to interleukin-1beta administration. We found that, after either NTS or VLM deposits, the majority of retrogradely-labelled Fos-positive cells activated by interleukin-1beta were localised in the medial and lateral parvocellular PVN divisions. Retrogradely-labelled Fos-positive cells were also observed in the NTS after VLM deposits, and in the VLM after NTS tracer deposits, suggesting reciprocal communication between these two nuclei after systemic interleukin-1beta. Thus the present study shows that the PVN has the capacity to modulate NTS and VLM responses after an immune challenge and that these may result from descending projections arising in the medial and lateral PVN divisions. These findings suggest that central nervous system responses to an immune challenge are likely to involve complex reciprocal connections between the PVN and the brainstem as well as between brainstem nuclei themselves.     

Effects of hyperosmotic stimulation and adrenalectomy on vasopressin mRNA levels in the paraventricular and supraoptic nuclei of the hypothalamus: in situ hybridization histochemical analysis using a synthetic oligonucleotide probe

The effects of salt loading and adrenalectomy on arginine vasopressin (AVP) mRNA levels in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus were studied by semiquantitative in situ hybridization histochemistry, using a synthetic oligonucleotide probe and a computer-assisted image analysis system. Salt loading (2% NaCl) for 7 days produced marked increases in AVP mRNA levels in the magnocellular neurons of the PVN, SON, and accessory nuclei. Adrenalectomy caused an increase in AVP mRNA expression in the magnocellular part of the PVN and the expansion of hybridization signals into its medial parvocellular region, where the cell bodies of corticotropin-releasing hormone (CRH) neurons are located. No apparent alteration of AVP mRNA levels was observed in the SON following adrenalectomy. These results indicate that hyperosmotic stimulation and the loss of circulating glucocorticoids had differential effects on AVP gene expression in the PVN and SON, and that the magnocellular PVN and SON neurons responded in different manners to the loss of feedback signals.     

Lipopolysaccharide suppresses activation of the tuberomammillary histaminergic system concomitant with behavior: a novel target of immune-sensory pathways

Infection and inflammation strongly inhibit a variety of behaviors, including exploration, social interaction, and food intake. The mechanisms that underlie sickness behavior remain elusive, but appear to involve fatigue and a state of hypo-arousal. Because histaminergic neurons in the ventral tuberomammillary nucleus of the hypothalamus (VTM) play a crucial role in the mediation of alertness and behavioral arousal, we investigated whether the histaminergic system represents a target for immune activation and, if so, whether modulation by ascending medullary immune-sensitive projections represents a possible mechanism. Rats were injected intraperitoneally with either the pro-inflammatory stimulus lipopolysaccharide (LPS) or saline, and exposed to one of various behavioral tests that would induce motivated behavior (exploration, play behavior, social interaction, sweetened milk consumption). Upon kill, brains were processed for c-Fos and histidine decarboxylase immunoreactivity. LPS treatment reduced behavioral activity and blocked behavioral test-associated c-Fos induction in histaminergic neurons of the VTM. These effects of LPS were prevented by prior inactivation of the caudal medullary dorsal vagal complex (DVC) with a local anesthetic. To determine whether LPS-responsive brainstem projection neurons might provide a link from the DVC to the VTM, the tracer Fluorogold was iontophoresed into the VTM a week prior to experiment. Retrogradely labeled neurons that expressed c-Fos in response to LPS treatment included catecholaminergic neurons within the nucleus of the solitary tract and ventrolateral medulla. These findings support the hypothesis that the histaminergic system represents an important component in the neurocircuitry relevant for sickness behavior that is linked to ascending pathways originating in the lower brainstem.     

大鼠下丘脑弓状核神经元的神经支配

用ＨＲＰ逆行追踪和免疫细胞化学与电镜结合法研究了下丘脑弓状核神经元的神经支配。结果表明，室旁核向弓状核投射的神经元中，有一部分为催产素免疫反应阳性，一部分为后叶加压素免疫反应阳性，视上核也有部分ＯＴ免疫反应阳性神经元发出纤维投射至弓状核。将零乱毒素Ｂ亚单位结合ＨＲＰ注入第三脑室后，可见弓状核内有逆行标记细胞，电镜观察发现弓状核内的脑啡肽免疫反应阳性的树突接受ＨＲＰ反应阳性的触液神经元的轴突形成突触     

腹腔注射2-脱氧-D-葡萄糖可诱发大鼠视上核和室旁核神经元表达Fos

目的 探讨腹腔注射2-脱氧-D-葡萄糖（2-DG）能否激活大鼠下丘脑视上核（SON）和室旁核（PVN）神经元而表达Fos。方法 健康雄性SD大鼠12只,随机分为腹腔注射2-DG组（6只）、生理盐水对照组（3只）及正常对照组（3只）。各自处理后,应用免疫组织化学方法,观察各组下丘脑SON和PVN内Fos表达及其与催产素（OT）和加压素（VP）的双标情况,同时采用ELISA方法对血清中OT和VP的含量进行检测。 结果与生理盐水对照组和正常对照组相比,2-DG引发的特异性Fos免疫阳性产物主要集中分布于下丘脑外侧区和穹隆周区,在SON、PVN也有密集表达。SON和PVN内的Fos表达与该区的特异性神经活性物质OT和VP有共存。OT/Fos双标细胞率（双标细胞占OT阳性细胞的百分率）在SON和PVN分别为87.10%、90.57%,明显高于VP/Fos在这两个核团的双标率（双标细胞占VP阳性细胞的百分率,68.42%、76.92%）,两者比较差异有统计学意义（P<0.05）。ELISA检测结果显示,2-DG组动物血清中OT和VP水平与对照组相比无明显变化。 结论 腹腔注射2-DG可激活大鼠下丘脑SON和PVN内OT和VP神经元表达Fos,SON和PVN可能参与2-DG诱导的急性应激反应。     

Hypobaric hypoxia induces fos and neuronal nitric oxide synthase expression in the paraventricular and supraoptic nucleus in rats

This study examined the effects of high altitude exposure on neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus in adult and neonatal rats. In adult control rats, occasional Fos-like immunoreactive neurons were localized in both the hypothalamic nuclei. A marked increase in Fos positive cells was induced at 1-4 h following altitude exposure but it was reduced to levels comparable to the controls at 24 h. The expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the PVN and SON followed a similar temporal pattern. The nNOS immunoreactivity, which was constitutively expressed in the hypothalamic neurons in the control rats, was noticeably augmented at 1-4 h, but it was comparable to the controls at 24 h following altitude exposure. In postnatal rats, Fos expression was not detected in the hypothalamic neurons of the controls. Induction of Fos expression was observed in some neurons at 1-4 h following altitude exposure but it was diminished at 24 h. There was no noticeable change in nNOS expression in both the control and altitude exposed postnatal rats; in both instances, it was barely detectable. It is concluded that both the PVN and SON of the adult rats are activated at high altitude exposure and that they may be involved in the regulation of neuroendocrine, cardiovascular and respiratory functions in hypobaric hypoxia. This study has also shown the differential response of the hypothalamus neurons between the two age groups to the hypoxic insult. Our results suggest that the adult neurons are probably more sensitive to the reduced oxygen levels in hypobaric hypoxia, as reflected by the upregulated NOS expression in this age group but not in the postnatal rats.     

New central projections of neuropeptide FF: colateral branching pathways in the brainstem and hypothalamus in the rat

Neuropeptide FF (NPFF), a morphine modulatory peptide, has been identified within discrete autonomic regions in the brainstem and hypothalamus. Triple fluorescence labelling was employed to identify collateral branching projections of NPFF neurons located within the nucleus tractus solitarius (NTS) and in the region of the hypothalamus between the dorsomedial and ventromedial hypothalamus. Injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres into the pontine parabrachial nucleus (PBN) and the ventrolateral medulla resulted in labelling of NPFF neurons in the NTS that contained one (double-labelled) or both (triple-labelled) tracers. Within the NTS, most double- and triple-labelled NPFF neurons were localized at the level of the area postrema or just rostral to it and within the medial and dorsomedial subdivisions of the nucleus. Injections of tracers into the PBN and hypothalamic paraventricular nucleus revealed double- and triple-labelled NPFF neurons, a majority of which were located in a zone between the dorsomedial and ventromedial hypothalamus. These results indicate that NPFF neurons in the brainstem and hypothalamus may simultaneously transmit signals to their target nuclei in the brainstem and forebrain. This coordinated signalling may lead to synchronized responses of NPFF target sites and provide insights into the role of this peptide in cardiovascular and nociceptive responses.