Identification of central nervous system sites involved in the water diuresis response elicited by central microinjection of nociceptin/ Orphanin FQ in conscious rats via c-Fos and inducible cAMP early repressor immunocytochemistry

Intracerebroventricular (i.c.v.) administration of the opioid-like peptide, nociceptin/Orphanin (nociceptin), in conscious rats produces diuretic and antinatriuretic effects. The present study utilised changes in Fos and inducible cAMP early repressor (ICER) immunocytochemistry expression to examine the central nervous (CNS) sites activated or inhibited, respectively, by central administration of nociceptin. Urine samples were collected during control (15 min) and after i.c.v. vehicle (5 microl, n = 12) or nociceptin (10 microg/5 microl; n = 12). Four additional urine samples (15-min) were collected after the i.c.v. injection. The brain was processed for Fos using a commercially available antibody (Oncogene AB-5) and for ICER using a polyclonal anti-ICER antibody raised in rabbits. In vehicle-injected conscious rats, renal excretion of water or sodium was not altered. However, nociceptin produced a rapid and marked increase in urine flow (V) and a decrease in urinary sodium excretion rate. In addition, i.c.v. nociceptin produced a significant increase in Fos staining in the dorsomedial nucleus of the hypothalamus, the perinuclear zone of the supraoptic nucleus, the organum vasculosum of the lamina terminalis (OVLT), the lateral preoptic area and the lateral hypothalamic area compared to control. By contrast, Fos expression decreased in the area postrema and locus coeruleus compared to controls. Furthermore, ICER staining was significantly increased in the perinuclear zone of the supraoptic nucleus, supraoptic nucleus, median preoptic nucleus, OVLT, medial preoptic area, central nucleus of the amygdala, and medial nucleus of the solitary tract. Together, central opioid receptor-like type 1 activation in these CNS regions may participate in the neural pathways involved in the diuretic and antinatriuretic effects of nociceptin.     

Glucocorticoid modulation of neuronal activity and hormone secretion induced by blood volume expansion

The present study evaluated the involvement of glucocorticoid in the activation of vasopressinergic and oxytocinergic neurons of hypothalamic nuclei and plasma levels of vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP) and corticosterone (CORT) in response to both isotonic and hypertonic blood volume expansion (BVE). Rats were subjected to isotonic (0.15 M NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 M NaCl, 2 ml/100 g b.w., i.v.) BVE with or without pre-treatment with dexamethasone (1 mg/kg, i.p.). Results showed that isotonic BVE increased OT, ANP and CORT, and decreased AVP plasma levels. On the other hand, hypertonic BVE enhanced AVP, ANP, OT, and CORT plasma concentrations. Both hypertonic and isotonic BVE induced an increase in the number of Fos-OT double-labeled magnocellular neurons in the PVN and SON. Pre-treatment with dexamethasone reduced OT secretion, as well as Fos-OT immunoreactive neurons in response to both isotonic and hypertonic BVE. We also observed that dexamethasone pre-treatment had no effect on AVP secretion in response to hypertonic BVE, although this effect was associated with a blockade of Fos expression in the vasopressinergic magnocellular neurons in the PVN and SON. In conclusion, these data suggest that, not only the rapid OT release from storages, but also the oxytocinergic cellular activation induced by BVE are modulated by glucocorticoids. However, this pattern of response was not observed for AVP cells, suggesting that dexamethasone is not likely to influence rapid release of AVP but seems to modulate the activation of these neurons in response to hypertonic BVE.     

Expression of the arginine vasopressin gene in response to salt loading in oxytocin gene knockout mice

Accumulating evidence suggests that both oxytocin and arginine vasopressin (AVP) are vital components in the regulation of body fluid balance. However, the physiological role of oxytocin and possible cooperative interactions between oxytocin and AVP in sodium balance remain obscure, even though recent studies using oxytocin knockout (OTKO) mice suggested that oxytocin may contribute to the regulation of salt appetite. In the present study, we examined the effects of salt loading (drinking 2% NaCl for 5 days) on the expression of the AVP gene in the paraventricular (PVN) and supraoptic nuclei (SON) of wild-type, OTKO and heterozygous littermates using in situ hybridization histochemistry. In addition, the effects of salt loading on the expression of the oxytocin gene were also examined in wild-type and heterozygous mice. Under the non salt-loaded condition, the levels of AVP mRNA in the PVN and SON of OTKO mice were significantly decreased compared to those in wild-type mice. Nevertheless, the up-regulation of the expression of the AVP gene in response to salt loading was preserved in OTKO mice. The degree of the up-regulation in OTKO mice tended to be greater compared to those in wild-type mice, suggesting compensatory up-regulation of the expression of the AVP gene in OTKO mice after salt loading. The basal levels of oxytocin mRNA in the PVN and SON of heterozygous mice were significantly lower than those in wild-type mice. Salt loading caused an increase of oxytocin mRNA levels in the PVN and SON of both wild-type and heterozygous mice. The ratios of increase of oxytocin mRNA levels were very similar between wild-type and heterozygous mice, suggesting that the single remaining oxytocin gene in heterozygous mice responds normally to an osmotic cue. Finally, salt loading tended to increase the serum concentration of sodium regardless of genotype, and there were no genotype differences in both the control and salt-loaded groups. These results suggest ways in which oxytocin may play a cooperative role together with AVP in the regulation of sodium balance.     

Regulation of rat APJ receptor messenger ribonucleic acid expression in magnocellular neurones of the paraventricular and supraopric nuclei by osmotic stimuli

The novel apelin receptor (APJ receptor, APJR) has a restricted expression in the central nervous system suggestive of an involvement in the regulation of body fluid homeostasis. The endogenous ligand for APJR, apelin, is also highly concentrated in regions that are involved in the control of drinking behaviour. While the physiological roles of APJR and apelin are not fully known, apelin has been shown to stimulate drinking behaviour in rats and to have a regulatory effect on vasopressin release from magnocellular neurones of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. To determine the role of APJR in the regulation of water balance, this study examined the effects of osmotic stimulation on the expression of APJR mRNA in the magnocellular PVN (mPVN) and SON of salt-loaded and water-deprived rats. Intake of 2% NaCl and water deprivation for 48 h induced expression of APJR mRNA in the mPVN and SON. Using dual-label in situ hybridization histochemistry, we also investigated whether APJR is colocalized within vasopressin neurones in control, salt-loaded and water-deprived rats. APJR mRNA was found to colocalize with a small population of vasopressin-containing magnocellular neurones in control and water-deprived rats. Salt-loading resulted in an increased colocalization of APJR and vasopressin mRNAs in the SON. These data verify a role for APJ receptors in body fluid regulation and suggest a role for apelin in the regulation of vasopressin-containing neurones via a local autocrine/paracrine action of the peptide.     

Monoamine Content of the Stalk-Median Eminence and Hypothalamus in Adult Female Sheep as Affected by Daylength

In the ewe, plasma luteinizing hormone and prolactin concentrations exhibit seasonal variations. During long days, inhibition of pulsatile luteinizing hormone secretion is mediated by monoamines. In a model of ovariectomized ewes bearing a subcutaneous oestradiol implant, we previously showed that the steroid-dependent inhibition of luteinizing hormone involves the A15 dopaminergic nucleus of the retrochiasmatic area. In the present work, we compared the aminergic activities of tele-diencephalic structures in groups of ovariectomized ewes under artificial illumination for short versus long days (8 versus 16 h/day of light, respectively). Half the animals in each group were bearing a subcutaneous oestradiol implant. Using high-performance liquid chromatography and electrochemical detection, we measured the levels of amines and amine metabolites in ‘punches’ of tissues from regions containing luteinizing hormone-releasing hormone axon terminals or cell bodies and catecholaminergic structures. Concurrently, we checked the pulsatile luteinizing hormone release and plasma prolactin concentration to assess the ability of our model to mimic seasonal changes in the hormonal status. As expected, ovariectomized ewes with a subcutaneous oestradiol implant showed an inhibition of the pulsatile luteinizing hormone release under long days. A higher concentration of plasma prolactin was also observed under long days, without any effect of the steroid treatment. Under this light regimen, statistically significant higher contents of dopamine than under short days were found in the stalk-median eminence. Larger contents of homovanillic acid, a dopamine metabolite, and 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), a noradrenaline metabolite were observed in the infundibular nucleus, while the catechola-mines themselves remained unchanged. Furthermore, oestradiol also significantly increased the content of MHPG in the latter structure. During long days, animals without oestradiol treatment exhibited a significant lower content of noradrenaline in the A15 nucleus, without any alteration of the dopamine content. Daylength or oestradiol treatment had no significant effects on the levels of amines or amine metabolites in the preoptic or septal areas. Thus, our results in the ewe underline the role played by the medial basal hypothalamus in the catecholaminergic regulation of seasonal changes in hormone release and suggest modifications in the turnover of the neurotransmitters in some structures.     

Neuronal expression of Fos protein in the hypothalamus of rats with heart failure

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.     

Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus)

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P 相似文献   

Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light

Per1 and Per2, two clock genes rhythmically expressed in the suprachiasmatic nucleus (SCN), are implicated in the molecular mechanism of the circadian pacemaker and play a major role in its entrainment by light. To date, it is not known if every cell of the SCN, a heterogeneous structure in respect of neuropeptide content, expresses clock genes equally. The aim of this study was to identify, by single and double non-radioactive and/or radioactive hybridizations, the cell types (AVP, VIP and GRP) expressing Per1 or Per2 in the SCN of rats, (1) when Per are highly expressed during the daytime, and (2) after induction of Per expression by a light pulse at night. Our results indicate that, during the daytime, Per1 and Per2 genes are both mainly expressed in the AVP cells of the dorso-median part of the SCN, whereas only a few VIP cells in the ventral part of the SCN exhibit Per gene expression. In contrast, following a light pulse at night, there is differential induction of the two Per genes. Per1 expression essentially occurs in the ventro-lateral GRP cells, while Per2 expression is not restricted to the retinorecipient part of the SCN as it also occurs in AVP cells. Altogether, our results suggest that Per1 and Per2 are mainly expressed in AVP cells during the daytime and suggest that GRP cells play an important role in resetting of the clock by light.     

Oxytocin receptor gene loss influences expression of the oxytocin gene in C57BL/6J mice in a sex‐ and age‐dependent manner

Parental care and sensory stimulation are critical environmental factors that influence oxytocin (OXT) and its receptor (OXTR). Because developmental Oxt mRNA expression is enhanced by sensory‐rich early life experience and reduced by sensory deprivation, we predicted that compared to wild‐type (WT) littermates, mice with congenital loss of OXTR (OXTR KO), as a genetically induced deprivation, would show impaired Oxt mRNA expression in the offspring hypothalamus during development. Oxt mRNA levels of male and female OXTR KO mice were not different from WT littermates from postnatal day (P)0 to P6, although, by P8, OXTR KO showed significantly decreased Oxt mRNA expression in the hypothalamus compared to WT littermates. At P14, male and female OXTR KO mice had significantly decreased Oxt mRNA expression specifically in the paraventricular nucleus (PVN), but not the supraoptic nucleus (SON), compared to WT littermates. We investigated whether this effect persisted in adulthood (P90) and found a significant genotype by sex interaction where male OXTR KO mice displayed a reduction in Oxt expression specific to the PVN compared to male WT littermates. By contrast, male and female OXTR KO adults had increased Oxt mRNA levels in the SON. These findings suggest that OXTR plays a role in developmental Oxt mRNA expression with sex by genotype interactions apparent at adulthood. We then measured OXT and neural activation in the PVN and SON at P14. We observed more OXT‐immunoreactive cells in the PVN of OXTR KO mice but significantly fewer c‐Fos immunoreactive cells. There were no genotype differences in immunoreactivity for OXT and no c‐Fos activity in the SON at P14. Combined, these data suggest that OXTR WT P14 mice have more PVN activity and are more likely to release OXT than OXTR KO mice. Future experiments are warranted to understand which OXTR‐expressing neural circuits modulate the development of the PVN oxytocin system.     

Immunotargeted Lesions of Paraventricular CRF and AVP Neurons in Developing Rats Reveal the Pattern of Maturation of these Systems and their Functional Importance

Pituitary ACTH secretion in the rat is controlled by a number of hypothalamic secretagogues, like CRF and AVP and by inhibitory feedback provided by glucocorticoids. During development, little is known about the precise regulation of ACTH release by hypothalamic neuropeptides and glucocorticoids. We used immunotargeted chemical PVN lesions to investigate the role of CRF and AVP neurons of the hypothalamic paraventricular nucleus (PVN) in the control of ACTH secretion in neonatal rats under basal conditions and 5 days after adrenalectomy (ADX). Neonates aged day (d) 4 or d14 were injected over the PVN with ricin A toxin associated with either non-specific antibodies (IgG/Tx), or monoclonal antibodies directed towards CRF (CRF/Tx) or AVP (AVP/Tx). Rats from each group received either sham surgery (SHAM) or were adrenalectomized (ADX). Pups were sacrificed 5 days after PVN treatment and adrenal surgery (d9 or 19). Plasma ACTH and corticosterone (B) levels were measured by RIAs. Changes in CRF and AVP expression in the PVN and other brain regions were determined by immunohistochemistry (ICC) and in situ hybridization. Injection of the toxin associated with IgGs did not have non specific effects on body weight gain, neuropeptide expression or plasma ACTH and B secretion compared to intact, uninjected rats. Lesions of CRF or AVP neurons greatly reduced peptide expression and mRNA levels in the PVN and median eminence at both ages. However, the specificity of the lesion was greater in older than in young pups. At both ages, we observed a dissociation between the morphological effects of the lesions and hormonal responses. In d14–19 pups, CRF and AVP lesions prevented ADX-induced changes in mRNA levels and peptide expression but did not reduce ACTH secretion under basal or stimulated (post ADX) conditions. However, CRF and AVP lesions increased the expression of CRF in the central amygdala and the bed nucleus of the stria terminalis. Lesions with AVP also stimulated CRF expression in the PVN. Thus, these compensatory changes could take over some of the hypophysiotropic actions of the damaged PVN neurons. In young pups (d4–9), we did not observe the typical increase in CRF and AVP mRNA levels and peptide expression found after ADX in older pups or adults. Lesions of the CRF neurons also affected the AVP system and reciprocally. We suggest that this could be explained by a high degree of colocalization of CRF and AVP observed in parvocellular and small, immature magnocellular neurons in young pups. The lesions did not affect basal or ADX-induced ACTH secretion, suggesting that during the early neonatal period, the pituitary is the major site of glucocorticoid inhibitory feedback on ACTH secretion and that the hypothalamus does not exert a tonic control over basal pituitary secretion. These results unravel ontogenetical differences in the regulation of ACTH secretion by hypothalamic CRF and AVP. During the first 10 days of life, within the adrenal stress hyporesponsive period, hypothalamic CRF and AVP neurons are not sensitive to glucocorticoid feedback and basal ACTH secretion appears to be relatively independent from hypothalamic input. After the second week of life, maturation of glucocorticoid receptors, neuronal phenotype and connections of the PVN to other brain structures (bed nucleus of the stria terminalis, central amygdala) allows for the full expression of corticosterone effect on hypothalamic neurons and for compensatory changes to occur following lesions. These results emphasize the extraordinary capacity of the developing central nervous system to adapt to changes in functionning of some neuronal areas critical for homeostatic balance and the important potential role of intra-hypothalamic and extrahypothalamic relationships in maintaining control over ACTH and glucocorticoid production during development.     

Direct cellular peptidomics of hypothalamic neurons

The chemical complexity of cell-to-cell communication has emerged as a fundamental challenge to understanding brain systems. This is certainly true for the hypothalamus, where neuropeptide signals are heterogeneous, localized and dynamic. Thus far, most hypothalamic peptidomic studies have centered on the entire structure; however, recent advances in collection strategies and analytical technologies have enabled direct, high-resolution peptidomic profiles focused on two regions of interest, the suprachiasmatic and supraoptic nuclei, including their sub-regions and individual cells. Suites of peptides now can be identified and probed for function. High spatial and analytical sensitivities reveal that discrete hypothalamic nuclei have distinct peptidomic signatures. Peptidomic discovery not only reveals unanticipated complexity, but also peptides previously unknown that act as key circuit components. Analysis of tissue releasates identifies peptides secreted into the extracellular environment and available for transmitting intercellular signals. Direct sampling techniques define peptide-releasate profiles in spatial, temporal and event-dependent patterns. These approaches are providing remarkable new insights into the complexity of neuropeptidergic cell-to-cell signaling central to neuroendocrine physiology.     

Regulation of Neuropeptides in Adult Rat Forebrain by the Neurotrophins BDNF and NGF

The expression of neuropeptides and neurotrophic factors is altered in the hippocampus after seizure induction in rats. Because the increase in brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) mRNAs precede changes in neuropeptide expression after seizure, it is possible that BDNF and NGF mediate subsequent alterations in peptide expression. To test this hypothesis directly, BDNF or NGF was infused into the hippocampus and cortex of adult rats. To ascertain the regional specificity of any observed effects of neurotrophin administration on neuropeptide expression, infusions into the striatum were also studied. To control for specificity, vehicle was also infused into the same sites. Peptide and mRNA alterations were assessed by Northern analysis, immunohistochemistry and radioimmunoassay. BDNF produced elevations of peptide and mRNA for neuropeptide Y and cholecystokinin in hippocampus and cortex, and somatostatin in cortex. BDNF increased mRNAs for neuropeptide Y, cholecystokinin, substance P and dynorphin in striatum. In contrast, BDNF decreased dynorphin peptide and mRNA in hippocampus. NGF's effects were limited to small mRNA increases, without corresponding changes in peptide levels, for neuropeptide Y in hippocampus and striatum, substance P in cortex and cholecystokinin in striatum. The distinct and limited effects of NGF infusion on neuropeptide expression demonstrate that BDNF's effects are not non-specific results of protein infusion into the brain. These findings indicate that BDNF may play a regionally specific role in modulating neuropeptide expression in the normal brain as well as in various pathophysiological states.     

迷走神经切断对胃肠道给予大鼠伤寒杆菌诱发的下丘脑室旁核和视上核Fos表达的影响

为研究迷走神经在自然感染状态下向脑传递免疫信息的作用。应用免疫组织化学方法,观察了切断隔下迷走神经对大鼠消化道内给予鼠伤寒杆菌刺激诱发的下丘脑室旁核和视上核的Fos表达变化的影响。结果发现,接受细菌刺激的动物与仅给予生理盐水的动物相比,回肠和肠系膜淋巴结有明显炎症存在,室旁结果发现,接受细菌刺激的动物与仅给予生理盐水的动物相比,回肠和肠系膜淋巴结有明显炎症存在,室旁核外侧部和视上核背侧部的Fos阳性细胞数增加;膈下迷走神经切断后,手术 细菌组与假手术 细菌组相比,室旁核的外侧部和视上核背部Fos表达减少。因此迷走神经途径在自然感染性免疫应答过程中,特别是在其早期阶段可能是传递腹腔免疫信息的重要途径之一。     

A quantitative and morphometric evaluation of I-alpha-bungarotoxin binding in the rat hypothalamus

The distribution of 125I-alpha-bungarotoxin (alpha-BTX), a putative nicotinic cholinergic receptor ligand was studied both in vitro and in vivo in the suprachiasmatic nucleus (SCN), anterior hypothalamic area (AHA), and supraoptic nucleus (SON) of the hypothalamus. For in vitro studies 20 micron frozen frontal sections containing SCN were incubated with either radioligand or, unlabeled alpha-BTX plus 125I alpha-BTX and tissues were processed for light microscopic autoradiography. Areas of cresyl violet stained SCN sections were measured using a Bioquant Analysis System and grain counts and distributions were determined. For in vivo investigations third ventricular infusion of either 125I alpha-BTX, or unlabeled alpha-BTX with 125I alpha-BTX was performed, and 24 hours later animals were perfused pericardially and 1 micron serial plastic sections of the SCN were processed for light microscopic autoradiography. Localization of silver grains in 1 micron serial sections was evaluated in a double blind study. In vitro and in vivo labeling patterns in the hypothalamus were the same and compared well with previously examined paraffin-processed tissues from animals which had received third ventricular infusions of the neurotoxin. We observed a distinctive and specific labeling pattern of the SCN. Grains tended to localize diffusely and uniformly in more rostral regions, but clustered densely in the dorsal and lateral mid-SCN, and dorsally in the mid-caudal SCN. Grains were localized in the SCN where larger neurons were found. In the most caudal regions of the SCN no labeling was observed. Tissues from unlabeled alpha-BTX plus 125I alpha-BTX in vitro or in vivo studies did not demonstrate grain counts above background levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immunohistochemistry. Zolpidem was administered intraperitoneally in dose 10 mg/kg of BW and 60 min later the animals were sacrificed by transcardial perfusion with fixative. The Fos/OXY co-labelings were analyzed on 40 microm thick serial coronal sections using computerized light microscopy. Zolpidem elicited a concordant Fos/OXY staining in all four PVN sub-areas investigated, including the anterior (15.71+/-2.35%), middle (14.52+/-2.53%), dorsal (13.34+/-2.61%), and periventricular (18.21+/-4.75%) ones, however, had no significant stimulatory effect on OXY cells in the SON. In response to Zolpidem, statistically significant activations were also seen in certain groups of accessory structures including the circular nucleus (13.99+/-3.43%), small clusters of accessory neurons (10.55+/-1.94%), and the lateral hypothalamic perivascular nucleus (9.42+/-2.74%). Between the naive and vehicle controls, the dual Fos/OXY labelings did not elicit any significant differences. Our data provide insight into the topographic patterns of brain activity within the clusters of magnocellular OXY cells in the hypothalamus associated with stimulation of GABA(A) benzodiazepine receptors and for the first time illustrate the triggering contemporaneousness within the cells of the principal and accessory magnocellular nuclei in response to Zolpidem treatment. The present study provides a comparative background that may help in the further understanding of a possible extend of Zolpidem effect on the brain.     

Circadian modulation of calcium levels in cells in the suprachiasmatic nucleus

There is reason to believe that resting free calcium concentration [Ca2+]i in neurons in the suprachiasmatic nucleus (SCN) may vary with the circadian cycle. In order to start to examine this hypothesis, optical techniques were utilized to estimate resting Ca2+ levels in SCN cells in a rat brain slice preparation. [Ca2+]i measured from the soma was significantly higher in the day than in the night. Animals from a reversed light-dark cycle were used to confirm that the phase of the rhythm was determined by the prior light-dark cycle. The rhythm in Ca2+ levels continued to be expressed in tissue collected from animals maintained in constant darkness, thus confirming the endogenous nature of this variation. Interestingly, the rhythm in Ca2+ levels was not observed when animals were housed in constant light. Finally, the rhythm in Ca2+ levels was prevented when slices were exposed to tetrodotoxin (TTX), a blocker of voltage-sensitive sodium channels. Similar results were obtained with the voltage-sensitive Ca2+ channel blocker methoxyverapamil. These observations suggest a critical role for membrane events in driving the observed rhythm in Ca2+. Conceptually, this rhythm can be thought of as an output of the circadian oscillator. Because [Ca2+]i is known to play a critical role in many cellular processes, the presence of this rhythm is likely to have many implications for the cell biology of SCN neurons.     

Expression of Fos immunoreactivity in rat brain during dehydration: effect of duration and timing of water deprivation

Water deprivation induces expression of the immediate early gene c-fos in specific brain regions, most likely as a result of the activation of cells that are responsive to changes in osmolality and/or blood volume. We hypothesized that the magnitude of c-fos expression would be a function of both the duration of water deprivation and the time of day at which the deprivation started. This study was designed to examine the pattern of Fos-like immunoreactivity (FLI) following water deprivation in rats under normal light/dark conditions (nLD) and reverse light/dark conditions (rLD). Rats were deprived of water but not food either for 0, 5, 16, 24 or 48 h. As expected, hematocrit ratio (HCT), osmolality (OSM), plasma renin activity (PRA) and weight loss increased as a function of duration of water deprivation. In non-deprived rats (0 h), very little FLI was observed in most brain regions. The number of cells showing FLI increased with duration of water deprivation in the supraoptic nucleus (SON), paraventricular nucleus (PVN), organum vasculosum laminae terminalis (OVLT), median preoptic nucleus (MnPO) and subfornical organ (SFO) in both nLD and rLD conditions. However, the pattern of FLI differed between nLD and rLD conditions. Compared to corresponding nLD groups after 5 or 24-h water deprivation, rLD groups had significantly more FLI in SON and PVN, and higher PRA and HCT. Also, weight loss and FLI in the MnPO were greater after 5 h, and FLI in the SFO was greater after 24 h under rLD compared to nLD conditions. Our findings indicate that the magnitude of c-fos expression, and change in weight and plasma parameters were a function of both the duration of water deprivation and the time of day at which the deprivation started. This may result from ingestion of food early in the deprivation periods during the rLD tests, thus producing greater change in osmolality and blood volume.