Maternal dietary restriction alters offspring's sleep homeostasis.

Nutritional state in the gestation period influences fetal growth and development. We hypothesized that undernutrition during gestation would affect offspring sleep architecture and/or homeostasis. Pregnant female mice were assigned to either control (fed ad libitum; AD) or 50% dietary restriction (DR) groups from gestation day 12 to parturition. After parturition, dams were fed AD chow. After weaning, the pups were also fed AD into adulthood. At adulthood (aged 8-9 weeks), we carried out sleep recordings. Although offspring mice displayed a significantly reduced body weight at birth, their weights recovered three days after birth. Enhancement of electroencephalogram (EEG) slow wave activity (SWA) during non-rapid eye movement (NREM) sleep was observed in the DR mice over a 24-hour period without changing the diurnal pattern or amounts of wake, NREM, or rapid eye movement (REM) sleep. In addition, DR mice also displayed an enhancement of EEG-SWA rebound after a 6-hour sleep deprivation and a higher threshold for waking in the face of external stimuli. DR adult offspring mice exhibited small but significant increases in the expression of hypothalamic peroxisome proliferator-activated receptor α (Pparα) and brain-specific carnitine palmitoyltransferase 1 (Cpt1c) mRNA, two genes involved in lipid metabolism. Undernutrition during pregnancy may influence sleep homeostasis, with offspring exhibiting greater sleep pressure.

Water deprivation induces appetite and alters metabolic strategy in Notomys alexis: unique mechanisms for water production in the desert.

Like many desert animals, the spinifex hopping mouse, Notomys alexis, can maintain water balance without drinking water. The role of the kidney in producing a small volume of highly concentrated urine has been well-documented, but little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. In Notomys, we found that water deprivation (WD) induced a sustained high food intake that exceeded the pre-deprivation level, which was driven by parallel changes in plasma leptin and ghrelin and the expression of orexigenic and anorectic neuropeptide genes in the hypothalamus; these changed in a direction that would stimulate appetite. As the period of WD was prolonged, body fat disappeared but body mass increased gradually, which was attributed to hepatic glycogen storage. Switching metabolic strategy from lipids to carbohydrates would enhance metabolic water production per oxygen molecule, thus providing a mechanism to minimize respiratory water loss. The changes observed in appetite control and metabolic strategy in Notomys were absent or less prominent in laboratory mice. This study reveals novel mechanisms for appetite regulation and energy metabolism that could be essential for desert rodents to survive in xeric environments.

Upregulation of arginine vasopressin synthesis in the rat hypothalamus after kainic acid-induced seizures.

We examined the effects of kainic acid (KA)-induced seizures on arginine vasopressin (AVP) gene expression in the paraventricular (PVN) and the supraoptic nuclei (SON) of normal rats using in situ hybridization histochemistry. We also investigated the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene after KA-induced seizures in transgenic rats. AVP heteronuclear (hn) RNA levels in the PVN and the SON were significantly increased at 3h and 24h after subcutaneous (s.c.) administration of KA in normal rats. AVP mRNA levels in the PVN and the SON did not change significantly at 3h, 24h and 1 week after s.c. administration of KA in normal rats. In KA-administered transgenic rats, AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON were significantly stronger compared to vehicle-administered transgenic rats. By pretreatment with MK-801 (nonselective N-methyl-D-aspartate, NMDA, receptor antagonist), AVP-eGFP transgenic rats after administration of KA did not show preconvulsive symptoms or convulsions and AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON of these rats was significantly reduced. These results suggested that KA-induced increases in AVP transcripts and AVP were prevented by MK801 because seizure activity was prevented or reduced.

Angiotensin II type 1 receptor signaling regulates feeding behavior through anorexigenic corticotropin-releasing hormone in hypothalamus.

The activation of renin-angiotensin system contributes to the development of metabolic syndrome and diabetes as well as hypertension. However, it remains undetermined how renin-angiotensin system is implicated in feeding behavior. Here, we show that angiotensin II type 1 (AT(1)) receptor signaling regulates the hypothalamic neurocircuit that is involved in the control of food intake. Compared with wild-type Agtr1a(+/+) mice, AT(1) receptor knock-out (Agtr1a(-/-)) mice were hyperphagic and obese with increased adiposity on an ad libitum diet, whereas Agtr1a(-/-) mice were lean with decreased adiposity on a pair-fed diet. In the hypothalamus, mRNA levels of anorexigenic neuropeptide corticotropin-releasing hormone (Crh) were lower in Agtr1a(-/-) mice than in Agtr1a(+/+) mice both on an ad libitum and pair-fed diet. Furthermore, intracerebroventricular administration of CRH suppressed food intake both in Agtr1a(+/+) and Agtr1a(-/-) mice. In addition, the Crh gene promoter was significantly transactivated via the cAMP-responsive element by angiotensin II stimulation. These results thus demonstrate that central AT(1) receptor signaling plays a homeostatic role in the regulation of food intake by maintaining gene expression of Crh in hypothalamus and suggest a therapeutic potential of central AT(1) receptor blockade in feeding disorders.

Hypothalamic vasopressin response to stress and various physiological stimuli: visualization in transgenic animal models.

Arginine vasopressin (AVP) is involved in the homeostatic responses numerous life-threatening conditions, for example, the promotion of water conservation during periods of dehydration, and the activation of the hypothalamo-pituitary adrenal axis by emotional stress. Recently, we generated new transgenic animals that faithfully express an AVP-enhanced green fluorescent protein (eGFP) fusion gene in the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the suprachiasmatic nucleus (SCN) of the hypothalamus. In these transgenic rats, marked increases in eGFP fluorescence and fusion gene expression were observed in the magnocellular division of the PVN and the SON, but not the SCN, after osmotic challenges, such as dehydration and salt loading, and both acute and chronic nociceptive stimuli. In the parvocellular division of the PVN, eGFP expression was increased after acute and chronic pain, bilateral adrenalectomy, endotoxin shock and restraint stress. In the extra-hypothalamic areas of the brain, eGFP expression was induced in the locus coeruleus after the intracerebroventricular administration of colchicine. Next, we generated another transgenic rat that expresses a fusion gene comprised of c-fos promoter-enhancer sequences driving the expression of monomeric red fluorescent protein 1 (mRFP1). In these transgenic rats, abundant nuclear fluorescence of mRFP1 was observed in the PVN, the SON and other osmosensitive areas after acute osmotic stimulation. Finally, we generated a double transgenic rat that expresses both the AVP-eGFP and c-fos-mRFP1 fusion genes. In this double transgenic rat, we have observed nuclear mRFP1 fluorescence in eGFP-positive neurons after acute osmotic stimulation. These unique transgenic rats provide an exciting new tool to examine neuroendocrine responses to physiological and stressful stimuli in both in vivo and in vitro preparations.

Similar changes of hypothalamic feeding-regulating peptides mRNAs and plasma leptin levels in PTHrP-, LIF-secreting tumors-induced cachectic rats and adjuvant arthritic rats.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in malignancy. However, it is difficult to explain the mechanism of anorexia/cachexia with PTHrP secretion in detail. Previously, we demonstrated that the expressions of orexigenic peptides increased and anorexigenic peptides decreased under cachectic conditions in rats carrying tumors secreting PTHrP. In this study, we investigated whether such changes in the expression of hypothalamic feeding-regulating peptides can be solely attributed to PTHrP or are a general response under cachectic conditions. Cachectic syndromes were induced in rats by: (i) inoculation of human lung cancer LC-6 cells that secreted PTHrP, (ii) inoculation of human melanoma SEKI cells that secrete not PTHrP but LIF1, (iii) injection of heat-killed Mycobacterium leading to arthritis (AA) and (iv) oral administration of a high dose of 1α,25(OH)(2)D(3) that resulted in hypercalcemia. The LC-6-bearing rats and AA rats were treated with or without anti-PTHrP antibody and indomethacin, respectively, and the expression of the hypothalamic feeding-regulating peptide mRNAs were examined by in situ hybridization histochemistry. The orexigenic peptide mRNAs, such as neuropeptide Y and agouti-related protein, were significantly increased, and that of anorexigenic peptide mRNAs, such as proopiomelanocortin, cocaine- and amphetamine-regulated transcript and corticotropin-releasing hormone were significantly decreased when they developed cachectic syndromes and AA. A high dose of 1α,25(OH)(2)D(3) caused hypercalcemia and body weight loss but did not affect the expression of hypothalamic feeding-regulating peptide mRNAs. The expressions of the hypothalamic feeding-regulating peptides change commonly in different chronic cachectic models without relating to serum calcium levels.

Diurnal changes of arginine vasopressin-enhanced green fluorescent protein fusion transgene expression in the rat suprachiasmatic nucleus.

We have recently developed a new transgenic rat line expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The AVP-eGFP transgene is expressed in the paraventricular (PVN) and supraoptic (SON) nuclei and the suprachiasmatic nucleus (SCN) of the hypothalamus. Transgene expression in the PVN and SON showed an exaggerated response to salt loading and nociceptive stimulation. However, the expression of the AVP-eGFP transgene in the SCN did not change under these stressful conditions. Here, we examined daily profiles of the expression of the AVP-eGFP transgene in the SCN in comparison with the endogenous AVP and Period (Per1 and Per2) genes. While all of these genes elicited diurnal patterns of expression in the SCN, the rate of rhythmic change of transgene expression was significantly greater than that of the endogenous AVP gene. We also examined the effect of a light stimulus on the expression of the AVP-eGFP, AVP, Per1 and Per2 genes in the SCN of transgenic rats. Ninety minutes after a light stimulus, AVP-eGFP mRNA and AVP hnRNA levels in the SCN were significantly decreased, while Per2 mRNA levels were significantly increased. In addition, we observed the eGFP fluorescence in the SCN and recorded the electrophysiological properties of a dissociated SCN eGFP-positive neuron. The AVP-eGFP transgenic rat is a useful animal model to study the diurnal change and dynamics of the AVP system, and enables the facile identification of SCN AVP neurons both in vivo and in vitro.

Centrally administered relaxin-3 induces Fos expression in the osmosensitive areas in rat brain and facilitates water intake.

The expression of the relaxin-3 gene, detected as a new member of the insulin superfamily using human genomic databases, is abundantly present in the brain and testis. Intracerebroventricularly (icv) administered relaxin-3 stimulates food intake. Icv administered relaxin (identical to relaxin-2 in humans) affects the secretion of vasopressin and drinking behavior. Relaxin-3 partly binds relaxin family peptide receptor 1, which is a specific receptor to relaxin. Thus, we hypothesized that relaxin-3 would have physiological effects in the body fluid balance. However, the effects of relaxin-3 in the body fluid balance remain unknown. In the present study, we revealed that icv administered relaxin-3 induced dense Fos-like immunoreactivity (Fos-LI) in the rat hypothalamus and circumventricular organs including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, supraoptic nucleus (SON), the subfornical organ (SFO) and the paraventricular nucleus (PVN), that are related to the central regulation of body fluid balance. Icv administered relaxin-3 (54, 180 and 540 pmol/rat) also induced a significant increase in c-fos gene expression in a dose-dependent manner in the SON, SFO and PVN. Further, icv administered relaxin-3 (180 pmol/rat) significantly increased water intake, and the effect was as strong as that of relaxin-2 (180 pmol/rat). These results suggest that icv administered relaxin-3 activates osmosensitive areas in the brain and plays an important role in the regulation of body fluid balance.

Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary.

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt-eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt-eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

Robust up-regulation of nuclear red fluorescent-tagged fos marks neuronal activation in green fluorescent vasopressin neurons after osmotic stimulation in a double-transgenic rat.

The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomeric red fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected.

Centrally administered adrenomedullin 5 activates oxytocin-secreting neurons in the hypothalamus and elevates plasma oxytocin level in rats.

We examined the effects of i.c.v. administration of adrenomedullin 5 (AM5) on the brain of conscious rats. We used porcine AM5 in the present study because rat AM5 has not been detected. We observed Fos-like immunoreactivity (LI) in the hypothalamus and brainstem of conscious rats after i.c.v. administration of AM5 (2 nmol/rat). Fos-LI, measured at 90 min post-AM5 injection, was observed in various brain areas, including the supraoptic (SON) and the paraventricular nuclei (PVN). Dual immunostaining for Fos/oxytocin (OXT) and Fos/arginine vasopressin (AVP) revealed that OXT-LI neurones predominantly colocalized Fos-LI compared with AVP-LI neurones in the SON and the PVN. Plasma OXT levels were significantly increased 5 min after i.c.v. administration of AM5 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 15 and 30 min without changes in plasma AVP levels at any time. In situ hybridization histochemistry showed that i.c.v. administration of AM5 (0.2, 1 and 2 nmol/rat) caused a marked induction of the expression of the c-fos gene in the SON and the PVN. This induction was significantly but not completely reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37; 3 nmol/rat) and the AM receptor antagonist AM-(22-52; 27 nmol/rat). Although porcine AM5 has not been detected yet in the brain, these results suggest that centrally administered porcine AM5 may activate OXT-secreting neurosecretory cells in the hypothalamus partly through AM/CGRP receptors and elicit secretion of OXT into the systemic circulation in conscious rats.

Adrenomedullin 2/intermedin-like immunoreactivity in the hypothalamus and brainstem of rats.

Adrenomedullin 2 (AM2) (identical to intermedin)-like immunoreactivity (LI) was examined in the rat brain by immunohistochemistry after intracerebroventricular administration of colchicine (100 microg/rat) and chronic salt loading (2% saline to drink) for 5 days. In both vehicle-treated and euhydrated rats, AM2-LI neurons were observed in the hypothalamus and brainstem, including in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the ventromedial hypothalamic nucleus, the arcuate nucleus, the locus coeruleus, the nucleus of the tractus solitarius and the nucleus ambiguus. In colchicine-treated and salt loaded rats, AM2-LI neurons were visualized more strongly in the SON and the magnocellular part of the PVN than in those in each control. Some AM2-LI neurons appeared in the parvocellular part of the PVN in the colchicine-treated but not salt loaded rats. AM2-LI in the other areas of the hypothalamus and brainstem did not change after colchicine-treatment and chronic salt loading. These results suggest that AM2/intermedin in the hypothalamus and brainstem may play roles on neuroendocrine and autonomic functions, such as water/salt balance, in rats.

Reduced carnitine level causes death from hypoglycemia: possible involvement of suppression of hypothalamic orexin expression during weaning period.

The mechanism of onset of hypoglycemia in patients with carnitine deficiency has yet to be determined. Using mice with systemic carnitine deficiency (JVS mice), we examined this mechanism, focusing on the weaning period (days 14-28 postpartum). For normal mice, the survival rate was 100%, and no hypoglycemia was observed at all. Gastric lactose began to decrease on day 17, and cellulose increased sharply in amount thereafter. For JVS mice, the survival rate was 77% on day 14 and 28% on day 28. From day 21 on, hypoglycemia was noted. Gastric lactose had disappeared almost completely by day 17, and cellulose was almost undetectable from days 14 to 28. Expression of orexin mRNA in the hypothalamus did not differ between JVS and normal mice on day 14, but was suppressed in JVS mice on days 21 and 28. When JVS mice were fed a carnitine-rich diet, suppression of expression of orexin mRNA in hypothalamus was eliminated, and on day 28 lactose and cellulose were detected in the stomach without hypoglycemia. In conclusion, the suppression of the expression of orexin in the hypothalamus during the weaning period may be involved in the marked anorexia in JVS mice, which eventually leads to death from hypoglycemia.

Downregulation of prolactin-releasing peptide gene expression in the hypothalamus and brainstem of diabetic rats.

We investigated the prolactin-releasing peptide (PrRP) mRNA levels in the hypothalamus and brainstem of streptozotocin (STZ)-induced diabetic rats and fa/fa Zucker diabetic rats, using in situ hybridization histochemistry. PrRP mRNA levels in the hypothalamus and brainstem of STZ-induced diabetic rats were significantly reduced in comparison with those of control rats. PrRP mRNA levels in the diabetic rats were reversed by both insulin and leptin. PrRP mRNA levels in the fa/fa diabetic rats were significantly reduced in comparison with those of Fa/? rats. PrRP mRNA levels in the fa/fa diabetic rats were significantly increased by insulin-treatment, but did not reach control levels in the Fa/? rats. We also investigated the effect of restraint stress on PrRP mRNA levels in STZ-induced diabetic rats. The PrRP mRNA levels in the control and the STZ-induced diabetic rats increased significantly after restraint stress. The diabetic condition and insulin-treatment may affect the regulation of PrRP gene expression via leptin and other factors, such as plasma glucose level. The diabetic condition may not impair the role of PrRP as a stress mediator.

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats.

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM(22-52) and calcitonin gene-related peptide (CGRP) antagonist, CGRP(8-37) on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM(22-52) (27 nmol/rat) and CGRP(8-37) (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM(22-52)+CGRP(8-37) could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.

Parathyroid hormone-related protein induces cachectic syndromes without directly modulating the expression of hypothalamic feeding-regulating peptides.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. EXPERIMENTAL DESIGN: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating peptide genes and serum leptin levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. RESULTS AND CONCLUSION: The mRNA levels for the orexigenic peptides, such as the agouti-related protein and the neuropeptide Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the proopiomelanocortin in the Arc, the cocaine and amphetamine-regulated transcript in the Arc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for orexin. Thus, PTHrP induces HHM and concurrent cachectic syndromes by mechanisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.

The short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons but not arginine vasopressin-secreting neurons in the hypothalamus of rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-neurohypophyseal system were examined in rats. Plasma oxytocin (OXT) levels were significantly increased 15-60 min after i.p. administration of 2-B4O (100 mg/kg), whereas plasma arginine vasopressin (AVP) did not change. Dual immunostaining revealed that Fos-like immunoreactivity (LI) was predominantly observed in OXT-secreting neurons in the paraventricular (PVN) and the supraoptic nuclei (SON) 120 min after i.p. administration of 2-B4O. In addition, many Fos-LI neurons were observed in the nucleus of the tractus solitarius (NTS) after i.p. administration of 2-B4O. These results suggest that a peripherally administered high dose of 2-B4O activates OXT-secreting neurons in the hypothalamus through activation of NTS neurons, possibly as a result of a stress response.

Effects of cholecystokinin (CCK)-8 on hypothalamic oxytocin-secreting neurons in rats lacking CCK-A receptor.

Peripheral administration of cholecystokinin (CCK)-8 selectively activates oxytocin (OXT)-secreting neurons in the supraoptic (SON) and the paraventricular nuclei (PVN) with the elevation of plasma OXT level in rats. We examined the effects of intravenous (iv) administration of CCK-8 on the neuronal activity of hypothalamic OXT-secreting neurons and plasma OXT level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a congenital defect in the expression of the CCK-A receptor gene. In situ hybridization histochemistry (ISH) for c-fos mRNA revealed that the expression of the c-fos gene was not induced in the SON, the PVN, the nucleus of the tractus solitarius (NTS) and the area postrema (AP) 30 min after iv administration of CCK-8 (20 and 40 microg/kg) in OLETF rats. In Long-Evans Tokushima Otsuka (LETO) rats (controls), c-fos mRNA was detected abundantly in those nuclei 30 min after iv administration of CCK-8 (20 microg/kg). Immunohistochemistry for c-fos protein (Fos) showed that the distributions of Fos-like immunoreactivity (LI) were identical to the results obtained from ISH. Dual immunostaining for OXT and Fos revealed that Fos-LI was mainly observed in OXT-secreting neurons in the SON and the PVN of LETO rats 90 min after iv administration of CCK-8 (20 microg/kg). Radioimmunoassay for OXT and arginine vasopressin (AVP) showed that iv administration of CCK-8 did not cause significant change in the plasma OXT and AVP levels in OLETF rats, while iv administration of CCK-8 caused a significant elevation of plasma OXT level without changing the plasma AVP level in LETO rats. These results suggest that peripheral administration of CCK-8 may selectively activate the hypothalamic OXT-secreting neurons and brainstem neurons through CCK-A receptor in rats.

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway.

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.

Six-hour selective REM sleep deprivation increases the expression of the galanin gene in the hypothalamus of rats.

The effect of short-term selective REM sleep deprivation (RSD) on the gene expression of galanin in the rat hypothalamus was examined using in situ hybridization histochemistry. Monitoring an electroencephalogram (EEG) and electromyogram (EMG) on an on-line computer screen, as the RSD rats entered REM sleep, they were gently stroked on their backs using a brush to wake them during the RSD period. Galanin mRNA levels in the preoptic area (POA) were significantly increased by RSD for a period of 6 h. RSD had no significant effect on the mRNA levels of corticotrophin-releasing factor (CRF), arginine vasopressin (AVP), oxytocin (OXT) or orexins. These results suggest that 6-h selective RSD may not be sufficient to induce the activation of the hypothalamo-pituitary adrenal axis, and that the expression of the galanin gene in the hypothalamus reacts more readily against the loss of REM sleep in comparison to other hypothalamic neuropeptides such as arginine vasopressin, oxytocin and orexins.