Parallel analysis of c-Fos protein and interleukin-2 expression in hypothalamic cells under different influence

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.     

C-fos gene expression in the rat spinal cord and brain cells during stress and the use of different types of halothane anesthesia

C-fos gene expression was studied as a marker of nervous cells activation in the rat spinal cord and brain cells under different conditions (different kinds of narcosis). Using of 1.5% light halothane narcosis allowed the detection of c-Fos-like proteins expression in the spinal cord cells only. Under initial 1.5% halothane narcosis, c-Fos-like proteins expression in the rat spinal cord (lumbar segments) and the brain cells was observed after placing the rats into the hammock, noxious mechanical stimulation (NMS) or high frequency electromagnetic irradiation of the skin (EHF). The pattern of the brain structures reacting on the NMS by c-Fos proteins expression, was determined. It was shown that NMS increases the c-Fos positive cell quantity in the lateral hypothalamic area (LHA), ventro-medial (VMH), dorso-medial (DMH) hypothalamic nuclei and anterior hypothalamic area (AHN) by 116, 199, 101 and 157% resp., in comparison with the c-Fos immunoreactive cell quantity in intact animals. EHF irradiation of the skin decreased the intensity of c-Fos-like proteins synthesis induced by NMS in the most of the investigated structures (LHA, VMH, DMH and AHA by 32.8, 29, 15 and 33%, resp.). It was shown that only initial halothane narcosis allowed to determine the hypothalamic structures which realized the responses to the NMS, and modification effects of EHF skin irradiation on the intensity of these responses.     

Expression of c-Fos gene in the rat hypothalamus upon electric painful stimulation and EHF irradiation of the skin

Studies of the activation pattern of hypothalamic cells upon electric painful stimulation have revealed an increase in the counts of c-Fos-positive cells (72.02-98.95%) predominantly of a size ranging from 10 to 50 microm2. EHF irradiation of the skin activates hypothalamic cells of the size ranging from 10 to 70 microm2 in the paraventricular, dorsomedial nuclei, and in perifonical zone of the lateral hypothalamic area. The combined action of EHF skin irradiation and electric painful stimulation leads to a decrease in the counts of cells activated in result of the painful stimulation in the anterior hypothalamic area, ventromedial nucleus, and the basal part of the lateral area. Changes in size distributions of activated cells are found in the ventromedial nucleus and the basal part of the lateral area, occur mainly through decreased counts of smaller cells (10-30 microm2) and increased counts of lager cells. The modulatory effect of EHF irradiation of the skin was found to be expressed as a reduction in hypothalamic cell activation caused by electric painful stimulation.     

Brain activation following peripheral administration of the GLP-1 receptor agonist exendin-4

The aim of our study was to investigate the anorectic and brain stimulatory effects of various doses of exendin-4 (Ex-4) and to investigate the role of the vagus nerve in Ex-4-induced brain activation. A dose-related increase in c-fos mRNA expression was observed following Ex-4 administration (0.155-15.5 μg/kg). Doses of Ex-4 that caused anorexia without aversive effects (0.155, 0.775 μg/kg) induced c-fos expression in the hypothalamic arcuate and paraventricular (PVH; parvocellular) nuclei as well as in the limbic and brainstem structures. Doses of Ex-4 that caused aversion (1.55, 15.5 μg/kg) stimulated the same regions (in a more intense way) and additionally activated the magnocellular hypothalamic structures (supraoptic nucleus and PVH magnocellular). The brain c-fos pattern induced by Ex-4 showed both similarities and differences with that induced by refeeding. Subdiaphragmatic vagotomy significantly blunted the stimulation of c-fos mRNA expression induced by Ex-4 in the nodose ganglion, the medial part of nucleus of the solitary tract, and the parvocellular division of the PVH. Pretreatment with Ex-9-39 (330 μg/kg ip) impaired the neuronal activation evoked by Ex-4 in all brain regions and in the nodose ganglion. Effects of Ex-4 on hypothalamic-pituitary-adrenal axis activity were not altered by vagotomy. Results of this study demonstrate and relate the anorectic and brain stimulatory effects of aversive and nonaversive doses of Ex-4 and indicate that the activation of specific central regions induced by the peripheral administration of Ex-4 is, at least in part, dependent on the integrity of the vagus nerve.     

注射胸腺依赖和非胸腺依赖抗原雄性黑线毛足鼠的内分泌状态和气味吸引力

作者研究了胸腺依赖抗原 (SRBC) (羊红血细胞 ,sheepredbloodcells ,SRBC)和非胸腺依赖抗原 (细菌脂多糖 ,lipopolysaccharide ,LPS)的免疫活化对黑线毛足鼠气味信号和内分泌状态的影响。成年雄鼠注射SRBS5天后 ,其气味的性吸引力下降 ,这种结果伴随着粪便中睾酮含量的下降。SRBC处理后 ,雄性气味吸引力降低 ,这在体液免疫反应低的雄性个体中最明显。与SRBC的作用相反 ,注射LPS的雄性个体的气味吸引力增加 ,成熟雌性个体用于嗅闻嗅觉刺激上的时间差异与经LPS和盐处理过的雄鼠的粪便中睾酮浓度的差异呈正相关。作者讨论了这两种相反的嗅觉效应的适应性意义     

Immunoreactivity of hypothalamic orexin neurons and expression level of preproorexin gene in them after lipopolysaccharide injection

Hypothalamic orexin neurons are involved in the regulation of many physiological functions. The immunoreactivity of these neurons is shown to be altered after LPS injection. This phenomenon is characterized by definite time-space pattern and depends on dose of antigen applied. The expression level ofpreproorexin gene in rat hypothalamus was investigated in 2, 4 and 6 hours after injection of 25 and 500 mkg/kg b. w. LPS. Both injections of higher and lower doses resulted in the increase of expression level of preproorexin gene after 2 hours that could suggest an enhancement of orexin synthesis in neurons. There were no significant changes in 4 and 6 hours after injection. The comparative analysis of the data obtained earlier with immunohistochemistry, and the data shown in the present study suggest the mechanisms of orexin neurons reaction to injection of LPS in different doses, i. e. the more considerable prevalence of orexin utilization over its synthesis in hypothalamic cells after injection of subseptic (500 mkg/kg) dose of LPS.     

刺激和损毁下丘脑室旁核对针刺镇痛的影响

本文采用脑刺激和损毁法,探讨了大鼠下丘脑室旁核(PVH)在针刺镇痛中的作用。结果表明:电刺激PVH及PVH内微量注射谷氨酸钠均可增强“足三里”的针刺镇痛效果,呈量效关系;电解损毁PVH则削弱针刺镇痛效果;去除脑下垂体不能抑制PVH内注射谷氨酸钠的作用。     

Long-term effect of immunization with neural and nonneural antigens on the noradrenaline and serotonin levels of discrete brain areas in mice and the relationship between neurotransmitter levels and antibody production.

The effect of immunization with neural and nonneural antigens on hypothalamic and mesencephalic neurotransmission and antibody response have been investigated. Treatment of SJL/N mice with bovine myelin basic protein (BMBP), mouse spinal cord homogenate (MSCH) or bovine serum albumin (BSA) produced no significant changes in the average hypothalamic and mesencephalic noradrenaline (NA) or serotonin (5-HT) content. In Balb/c mice, however, treatment with BMBP caused a significant increase in mesencephalic NA, and treatment with MSCH caused a significant increase in hypothalamic 5-HT 3 weeks after the first antigenic challenge. The SJL/N strain showed a high antibody response to BMBP and BSA, and a moderate one to MSCH, while in Balb/c mice, there was a low response to BMBP, a moderate one to MSCH and a high response to BSA. Significant, positive correlations were found between the level of antigen-specific serum antibodies and the following neurotransmitters: hypothalamic NA in the BMBP and MSCH-treated groups, hypothalamic 5-HT in the MSCH-treated group, mesencephalic NA and 5-HT in the BMBP-treated group, and mesencephalic 5-HT in the MSCH-treated SJL/N animals. No significant correlations between the levels of antibodies and neurotransmitters have been found either in the BSA-immunized SJL/N animals, or in any of the groups of Balb/c mice treated with BMBP, MSCH or BSA.     

Effects of lipopolysaccharide on neurokinin A content and release in the hypothalamic-pituitary axis

The administration of bacterial lipopolysaccharide (LPS) markedly affects pituitary secretion, and its effects are probably mediated by cytokines produced by immune cells or by the hypothalamo-pituitary axis itself. Since neurokinin A (NKA) plays a role in inflammatory responses and is involved in the control of prolactin secretion, we examined the in vivo effect of LPS on the concentration of NKA in hypothalamus and pituitary (assessed by RIA) and serum prolactin levels in male rats. One hour after the intraperitoneal administration of LPS (250 microg/rat), NKA content was decreased in the posterior pituitary but not in the hypothalamus or anterior pituitary. Three hours after injection, LPS decreased NKA concentration in the hypothalamus and anterior and posterior pituitary. In all the conditions tested, LPS significantly decreased serum prolactin. We also examined the in vitro effects of LPS (10 microg/ml), interleukin-6 (IL-6, 10 ng/ml) and tumor necrosis factor alpha (TNF-alpha, 50 ng/ml) on hypothalamic NKA release. Interleukin-6 increased NKA release without modifying hypothalamic NKA concentration, whereas neither LPS nor TNF-alpha affected them. Our results suggest that IL-6 may be involved in the increase of hypothalamic NKA release induced by LPS. NKA could participate in neuroendocrine responses to endotoxin challenge.     

Immunosuppressive Effect of Bacterial Lipopolysaccharide on Antibody Response

Injection of endotoxins (bacterial lipopolysaccharide: LPS) several days prior to immunization causes the suppression of antibody response. The suppressive effects of several kinds of LPS preparations on the plaque-forming cell (PFC) antibody response in the spleen of mice were examined after immunization with sheep red blood cells (SRBC). Glycolipids obtained from heptoseless mutants (Re form) of salmonella or its lipid A preparation coupled artificially with bovine serum albumin (BSA) are capable, like LPS obtained from a wild type (S form) strain, of inducing suppression of the PFC response, while alkaline-detoxified LPS can not. The refractory periods of the PFC response induced by LPS injection last only a few days. However, the use of cyclophosphamide (CY) together with LPS can extend the refractory periods of antigenic stimulation for several weeks. Injections of LPS and CY can also induce unresponsive states of OH agglutinin antibody response to antigenic stimulation with formalin-killed organisms of Escherichia coli or Salmonella enteritidis (presumably both thymus-independent antigens). These unresponsive states induced by LPS and CY are easily terminated by a transfer of syngeneic bone marrow cells but not by thymocyte transfer.     

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.     

Hierarchy of T cell dependency in antibody response among different antigens.

T cell dependency of antibody response to polyvinylpyrrolidone (PVP), sheep red blood cells (SRBC), bovine gamma globulin (BGG), and bovine serum albumin (BSA) was examined. PVP and the other three are known as a T cell-independent antigen and T cell-dependent antigens respectively. Adult mice were thymectomized, X-irradiated, reconstituted with syngeneic bone marrow cells (TxXB mice), with bone marrow cells plus thymus cells (TxXBT mice), or with bone marrow cells treated with anti-Thy-1.2 serum and complement (TxXB-theta mice) and used as experimental animals. The anti-PVP response of TxXBT mice was significantly lower than that of TxXB mice, suggesting that T cells exerted a suppressive effect on the response to PVP. Both IgM and IgG responses to SRBC and BGG occurred even in TxXB-theta mice with the aid of bacterial lipopolysaccharide (LPS). However, a significant response to BSA was not observed in TxXB mice even in the presence of LPS or several other adjuvants. These results indicate that the T cell dependency of antigens is different among so called thymus-dependent antigens, that antibody response less dependent on the helper action of T cells can be supported by LPS in the absence of T cells, and that anti-BSA response seems to be extremely T cell dependent.     

CHANGES IN SEROTONIN LEVEL AND TURNOVER IN DISCRETE HYPOTHALAMIC NUCLEI AFTER PINEALECTOMY AND MELATONIN ADMINISTRATION TO RATS

The influence of the pineal gland on the hypothalamic serotonergic function was examined by studying the effects of long-term pinealectomy (1 month) and melatonin replacement (500 μg/kg; 10 days) on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content as well as on the in vivo 5-HT synthesis rate in discrete hypothalamic nuclei. Pinealectomy was followed by a significant decrease of 5-HT content in the anterior hypothalamic nuclei (AHN) and the ventromedial hypothalamic nuclei (VMHN), and also in 5-HIAA content in lateral (LPON) and medial preoptic nuclei (MPON). The 5-HT synthesis rate, estimated from the accumulation of 5-hydroxytryptophan after blockade of the 1-amino acid decarboxylase activity, were also decreased in the AHN and the paraventricular hypothalamic nuclei (PVHN) of pinealectomized rats. In contrast, an enhanced 5-HT synthesis rate and basal 5-HIAA content were found in the suprachiasmatic nuclei (SCN) after pinealectomy. Daily treatment with melatonin for 10 days reversed most of the effects induced by pinealectomy. Thus, melatonin increased the levels of 5-HT in the AHN and VMHN, and slightly increased the 5-HIAA content in preoptic nuclei. In addition, melatonin increased the 5-HT synthesis rate in the AHN and VMHN, but also in the MPON, VMHN and dorsomedial hypothalamic nuclei (DMHN) where pinealectomy had no effect. By contrast, melatonin treatment did not affect SCN 5-HT synthesis rate, although it decreased 5-HIAA levels. The results demonstrate that melatonin is able to stimulate 5-HT metabolism in most of the hypothalamic areas, but inhibits SCN 5-HT function. Some of the effects of melatonin seems to be exerted by modulating the synthesis of the amine, although melatonin likely also interacts with other regulatory processes of 5-HT function (i.e. release/uptake). The well defined presence of melatonin receptors in the rat SCN, and its absence in other hypothalamic structures, suggest that this may be the mechanism mediating the differential response to endogenous melatonin. Moreover, the larger effect of exogenous melatonin in relation to pinealectomy suggests the presence of melatonin unespecific effects possibly owing to supraphysiological doses. The present findings may be relevant for the mode of action of melatonin and its implication in several endocrine and behavioral functions mediated by serotonergic neurons. Copyright © 1996 Elsevier Science Ltd     

Endotoxin administration increases hypothalamic somatostatin mRNA through nitric oxide release

Acute inflammation induced by endotoxin (LPS) administration inhibits insulin-like growth factor (IGF-I) and growth hormone (GH) secretion. The aim of this study was to elucidate the role of glucocorticoids and nitric oxide (NO) in the effect of LPS on hypothalamic somatostatin gene expression. Adult male Wistar rats were injected with different doses of LPS (5, 10 and 100 microg/kg). Rats received two i.p. injections of LPS (at 17:30 and 8:30 h the following day) and were killed 4 h after the second injection. LPS administration at the dose of 100 microg/kg increased the hypothalamic somatostatin mRNA content, as well as the serum concentrations of corticosterone. Glucocorticoids do not seem to be involved in LPS-induced increase in hypothalamic somatostatin mRNA since adrenalectomy did not prevent this effect. In order to analyze the possible effect of NO, aminoguanidine, an inducible nitric oxide synthase inhibitor, was injected (100 mg/kg s.c.) simultaneously with LPS injection. Aminoguanidine administration did not modify somatostatin mRNA in saline injected rats, but it prevented LPS-induced increase in hypothalamic somatostatin mRNA. These data suggest that the stimulatory effect of endotoxin on hypothalamic somatostatin gene expression is not mediated by glucocorticoids, but instead by the increase in NO release.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.     

Physiological significance of 2-buten-4-olide (2-B4O), an endogenous satiety substance increased in the fasted state

A sugar acid, 2-B4O, has been found to increase from 3.5 to 13 microM in rat serum at 36 h after food deprivation. Injections of 2-B4O (2.5 microM) into the rat III cerebral ventricle (III ICV) suppress food intake and single neuronal activity in the lateral hypothalamic area (LHA). 2-B4O is effective even in 72 h food-deprived rats. 2-B4O hyperpolarizes glucose-sensitive neurons in the LHA via Na+-K+ pump activation, but depolarizes glucoreceptor neurons in the ventromedial nucleus (VMH) via closure of ATP-sensitive K channels. The plasma levels of glucose, corticosterone, and catecholamines, and the firing rate in both parvocellular neurons in the paraventricular nucleus (PVN) and sympathetic efferent nerves, all increase 2-B4O intravenous (iv) injection, indicating activation of the hypothalamo-pituitary-adrenal axis. A 2-B4O iv injection facilitates emotional and spatial learning and memory, and pretreatment with anti-acidic fibroblast growth factor (aFGF) antibody ICV eliminates these effects. aFGF is released from ependymal cells in the III cerebral ventricle in response to the glucose increase in CSF induced by 2-B4O iv injection. 2-B4O also suppresses the clinical symptoms of experimental allergic encephalomyelitis (EAE) in Lewis rats [induced by immunization with a myelin basic protein (MBP)], a model for human multiple sclerosis. After immunization with MBP, the delayed-type hypersensitivity response to MBP is also reduced in 2-B4O-treated rats. 2-B4O thus suppresses autoimmune responses. These results indicate that 2-B4O is not only a powerful satiety substance, but also effective as an activator of the hypothalamo-pituitary-adrenal axis and sympathetic efferent outflow, and as a memory facilitation and a modulator of immune functions.     

Testosterone 5 alpha-reductase in discrete hypothalamic nuclear areas in the rat: effect of castration

The conversion of testosterone into 5 alpha-dihydrotestosterone (DHT) has been studied in different hypothalamic nuclear areas and in the superficial layers of the cerebral cortex of normal and castrated male rats. The tissue fragments utilized in each incubation have been punched from frozen brain sections utilizing calibrated needles. Castration has been performed 12 (short term) and 180 (long term) days before sacrifice. The nuclear areas studied include: the medial preoptic nucleus (MPN), the lateral preoptic nucleus (LPN), the anterior hypothalamic nucleus (AHN), the lateral hypothalamic nucleus (LHN), the posterior hypothalamic nucleus (PHN), the nucleus ventromedialis (HVM), the arcuate nucleus (AR), the median eminence (ME), the nucleus paraventricularis (HPV), the supraoptic nucleus (SO) and the suprachiasmatic nucleus (SC). The possible effect of castration on the 5 alpha-reductase, were assessed in the MPN,LPN,AHN,LHN,PHN and in the cerebral cortex. The results indicate that, in the male rat: 1) the lateral preoptic(LPN) and the lateral hypothalamic nuclei(LHN) possess a 5 alpha-reductase activity higher than that present in the cerebral cortex and in the other hypothalamic nuclei considered; 2)the suprachiasmatic nucleus (SC) apparently possesses a testosterone metabolizing activity lower than that found in any other nervous structures studied so far; 3) castration does not seem to influence the 5 alpha-reductase activity either in the hypothalamic nuclear structures considered or in the cerebral cortex.