A discrete neuronal circuit induces a hibernation-like state in rodents.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity1. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation4. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

Postsynaptic Depolarization Enhances GABA Drive to Dorsomedial Hypothalamic Neurons through Somatodendritic Cholecystokinin Release.

Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK.

Medial dorsal hypothalamus mediates the inhibition of reward seeking after extinction.

Extinction promotes abstinence from drug seeking. Extinction expression is an active process, dependent on infralimbic prefrontal cortex (ilPFC). However, the neurocircuitry mediating extinction expression is unknown. Here we studied the neural mechanisms for expression of extinction of alcoholic beer seeking in rats. We first examined the pattern of activation in prefrontal cortex projections to medial dorsal hypothalamus (MDH) (i.e., perifornical and dorsomedial nuclei) during extinction expression. Double labeling for retrograde tracer cholera toxin B subunit (CTb) and the neuronal activity marker c-Fos revealed significant recruitment of MDH projecting ilPFC neurons during extinction expression. We then studied the causal role of MDH in inhibiting alcoholic beer seeking during extinction expression. MDH infusion of the inhibitory neuropeptide cocaine- and amphetamine-regulated transcript prevented extinction expression, showing that MDH is necessary for extinction expression. Next we examined the pattern of activation in MDH projections to paraventricular thalamus (PVT) during extinction expression. Double labeling for CTb and c-Fos revealed significant recruitment of PVT projecting MDH neurons during extinction expression. We also showed, using triple-label immunofluorescence, that the majority of PVT projecting extinction neurons express prodynorphin, suggesting that actions at κ opioid receptors (KORs) in PVT may be critical for inhibiting alcoholic beer seeking. Consistent with this, infusions of a KOR agonist into PVT prevented reinstatement of alcoholic beer seeking showing that PVT KOR activation is sufficient to inhibit alcoholic beer seeking. Together, these findings identify a role for MDH and its ilPFC afferents and PVT efferents in inhibiting alcoholic beer seeking during extinction expression.

SIRT1 promotes the central adaptive response to diet restriction through activation of the dorsomedial and lateral nuclei of the hypothalamus.

Diet restriction retards aging and extends lifespan by triggering adaptive mechanisms that alter behavioral, physiological, and biochemical responses in mammals. Little is known about the molecular pathways evoking the corresponding central response. One factor that mediates the effects of diet restriction is the mammalian nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1. Here we demonstrate that diet restriction significantly increases SIRT1 protein levels and induces neural activation in the dorsomedial and lateral hypothalamic nuclei. Increasing SIRT1 in the brain of transgenic (BRASTO) mice enhances neural activity specifically in these hypothalamic nuclei, maintains a higher range of body temperature, and promotes physical activity in response to different diet-restricting paradigms. These responses are all abrogated in Sirt1-deficient mice. SIRT1 upregulates expression of the orexin type 2 receptor specifically in these hypothalamic nuclei in response to diet-restricting conditions, augmenting response to ghrelin, a gut hormone whose levels increase in these conditions. Our results suggest that in the hypothalamus, SIRT1 functions as a key mediator of the central response to low nutritional availability, providing insight into the role of the hypothalamus in the regulation of metabolism and aging in mammals.

Orexin-A directly depolarizes dorsomedial hypothalamic neurons, including those innervating the rostral ventrolateral medulla.

The dorsomedial hypothalamus (DMH) receives dense orexinergic innervation. Intra-DMH application of orexins increases arterial pressure and heart rate in rats. We studied the effects of orexin-A on DMH neurons, including those innervating the medullary cardiovascular center, the rostral ventrolateral medulla (RVLM), by using whole-cell recordings in brain slices. In the presence of tetrodotoxin, orexin-A (30-1000 nM) depolarized 56% of DMH neurons (EC50 82.4 ± 4.4 nM). Under voltage-clamp recording, orexin-A (300 nM) induced three types of responses characterized by different current-voltage relationships, namely unchanged, increased, and decreased slope conductance in 68%, 14%, and 18% of orexin-A-responsive neurons, respectively. The reversal potential of the decreased-conductance response was near the equilibrium potential of K+ and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is the underlying mechanism. In a low-Na+ solution, unchanged-, increased-, and decreased-conductance responses were observed in 56%, 11%, and 33% of orexin-A-responsive neurons, respectively, implying that a non-selective cation current (NSCC) underlies orexin-A-induced responses in a small population of DMH neurons. KBR-7943 (70 µM), an inhibitor of Na+-Ca2+ exchanger (NCX), suppressed orexin-A-induced depolarization in 7 of 10 neurons. In the presence of KBR-7943, the majority of orexin-A-responsive neurons exhibited decreased-conductance responses. These findings suggest that NCX activation may underlie orexin-A-induced depolarization in the majority of orexin-responsive DMH neurons. Of 19 RVLM-projecting DMH neurons identified by retrograde labeling, 17 (90%) were orexin-A responsive. In conclusion, orexin-A directly excited over half of DMH neurons, including those innervating the RVLM, through decreasing K+ conductance, activating NCX, and/or increasing NSCC.

Activation of neurons in the hypothalamic dorsomedial nucleus via hypothalamic projections of the nucleus of the solitary tract following refeeding of fasted rats.

We report that satiation evokes neuronal activity in the ventral subdivision of the hypothalamic dorsomedial nucleus (DMH) as indicated by increased c-fos expression in response to refeeding in fasted rats. The absence of significant Fos activation following food presentation without consumption suggests that satiation but not craving for food elicits the activation of ventral DMH neurons. The distribution pattern of the prolactin-releasing peptide (PrRP)-immunoreactive (ir) network showed remarkable correlations with the distribution of activated neurons within the DMH. The PrRP-ir fibers and terminals were immunolabeled with tyrosine hydroxylase, suggesting their origin in lower brainstem instead of local, hypothalamic PrRP cells. PrRP-ir fibers arising from neurons of the nucleus of the solitary tract could be followed to the hypothalamus. Unilateral transections of these fibers at pontine and caudal hypothalamic levels resulted in a disappearance of the dense PrRP-ir network in the ventral DMH while PrRP immunoreactivity was increased in transected fibers caudal to the knife cuts as well as in perikarya of the nucleus of the solitary tract ipsilateral to the transections. In accord with these changes, the number of Fos-expressing neurons following refeeding declined in the ipsilateral but remained high in the contralateral DMH. However, the Fos response in the ventral DMH was not attenuated following chemical lesion (neonatal monosodium glutamate treatment) of the hypothalamic arcuate nucleus, another possible source of DMH inputs. These findings suggest that PrRP projections from the nucleus of the solitary tract contribute to the activation of ventral DMH neurons during refeeding, possibly by transferring information on cholecystokinin-mediated satiation.

Temporal phase relation of circadian neural oscillations alters RFamide-related peptide-3 and testicular function in the mouse.

In order to study the effect of the temporal synergism of neural oscillations on reproductive regulation and the response of RFamide-related peptide-3 (RFRP-3; a mammalian ortholog of avian gonadotropin-inhibitory hormone), expression of immunoreactive RFRP-3 in the neurons of the dorsomedial nucleus of the hypothalamus was monitored in sexually immature and mature laboratory mice (study I). In study II, the effects of serotonin and dopamine precursors (5-hydroxytryptophan and L-dihydroxyphenylalanine; injected daily, 8 or 12 h apart, for 13 days in 3-week-old mice) on testicular activity and immunoreactive RFRP-3 neurons were studied until 24 days after treatment. Results indicate high levels of expression of immunoreactive RFRP-3 in the sexually immature and 8-hour mice (simulating gonadal suppression), while a low level was noted in mature and 12-hour mice (simulating gonadal stimulation). These findings not only suggest the modulation of gonadal development in mice (during the course of puberty attainment) by changing the temporal phase relation of serotonergic and dopaminergic oscillations (as in some seasonally breeding species), but also demonstrate an inverse correlation of RFRP-3 neurons and gonadal activity in both control and experimental conditions.

Cerebellar fastigial nuclear inputs and peripheral feeding signals converge on neurons in the dorsomedial hypothalamic nucleus.

Previous studies have indicated that neurons in the dorsomedial hypothalamic nucleus (DMN) receive feeding-related signals from the gastric vagal nerves, glycemia as well as leptin. On the other hand, it is intriguing that the cerebellum participates in regulating nonsomatic visceral activities including food intake via the direct cerebellohypothalamic projections. The present study was designed to examine, by using extracellular recordings in vivo in rats, whether the cerebellar fastigial nucleus (FN) could reach and converge with the feeding-associated gastric vagal, glycemia and leptin signals onto single DMN neurons. Of the 200 DMN neurons recorded, 104 (52%) responded to the cerebellar FN stimulation, in which 95 (91.3%) were also responsive to the gastric vagal stimulation, suggesting a convergence of cerebellar FN and gastric vagal inputs on the DMN neurons. Moreover, a summation of responses was observed (n = 10) when the cerebellar FN and gastric vagal nerve were simultaneously stimulated. Among the 18 DMN neurons receiving convergent inputs from the cerebellar FN and gastric vagal nerves, 16 (88.9%) cells also responded to the systemic administrations of glucose and leptin. These results demonstrated that the cerebellar FN-afferent inputs, together with the feeding signals from the gastric vagal nerves, blood glucose as well as leptin, converge onto single DMN neurons, suggesting that a somatic-visceral integration related to the feeding may occur in the DMN and the cerebellum may actively participate in the feeding regulation through the cerebellar FN-DMN projections.

Smelling of odorous sex hormone-like compounds causes sex-differentiated hypothalamic activations in humans.

The anatomical pathways for processing of odorous stimuli include the olfactory nerve projection to the olfactory bulb, the trigeminal nerve projection to somatosensory and insular cortex, and the projection from the accessory olfactory bulb to the hypothalamus. In the majority of tetrapods, the sex-specific effects of pheromones on reproductive behavior is mediated via the hypothalamic projection. However, the existence of this projection in humans has been regarded as improbable because humans lack a discernable accessory olfactory bulb. Here, we show that women smelling an androgen-like compound activate the hypothalamus, with the center of gravity in the preoptic and ventromedial nuclei. Men, in contrast, activate the hypothalamus (center of gravity in paraventricular and dorsomedial nuclei) when smelling an estrogen-like substance. This sex-dissociated hypothalamic activation suggests a potential physiological substrate for a sex-differentiated behavioral response in humans.

Alterations in RFamide-related peptide expression are coordinated with the preovulatory luteinizing hormone surge.

The preovulatory LH surge is triggered when the circadian pacemaker, the bilateral suprachiasmatic nucleus (SCN), stimulates the GnRH system in the presence of high estrogen concentrations (positive feedback). Importantly, during the remainder of the estrous cycle, estradiol inhibits LH release via negative feedback. We have recently documented the presence of a novel mammalian RFamide-related peptide (RFRP), a putative gonadotropin-inhibitory hormone (GnIH), that presumably acts upstream of GnRH to modulate the negative feedback effects of estrogen. The present series of studies used female Syrian hamsters to examine the possibility that, in addition to driving the LH surge positively, the SCN concomitantly coordinates the removal of steroid-mediated RFRP inhibition of the gonadotropic axis to permit the surge. We found that the SCN forms close appositions with RFRP cells, suggesting the possibility for direct temporal control of RFRP activity. During the time of the LH surge, immediate-early gene expression is reduced in RFRP cells, and this temporal regulation is estrogen dependent. To determine whether projections from the SCN regulate the timed reduction in activation of the RFRP system, we exploited the phenomenon of splitting. In split animals in which the SCN are active in antiphase, activation of the RFRP system is asymmetrical. Importantly, this asymmetry is opposite to the state of the GnRH system. Together, these findings point to novel circadian control of the RFRP system and potential participation in the circuitry controlling ovulatory function.

The dynamics of neuronal activation during food anticipation and feeding in the brain of food-entrained rats.

Rats can anticipate a daily mealtime when they are maintained on restricted feeding schedules (RFS). Neural substrates of the food-entrainable oscillator (FEO) are not yet fully understood. The numerous lesions of a single brain region failed to abolish the behavioral anticipation of a daily meal, suggesting that the FEO may be represented by a distributed neuronal network. The present study was designed to detect the dynamics of neuronal activation, using as a marker the expression of c-fos mRNA in the brain of rats subjected to 2-hour daily RFS, 3, 2 and 1 h before the expected meal, at the time of the usual feeding, and 1 h after feeding. We also aimed to clarify whether the increase in plasma corticosterone in food-anticipating rats coincides with the increase in expression of corticotropin releasing factor (CRF) mRNA in the paraventricular hypothalamic nucleus (PVH). The obtained results revealed that the neuronal activation occurring 3 h before the expected meal was not confined to one brain structure, but was evident in the anterior hippocampal continuation and septohippocampal nucleus (AH/SHi), the anterior part of the paraventricular thalamic nucleus (PVTa), and the dorsomedial hypothalamic nucleus (DMH), thus representing distributed septohippocampal-thalamo-hypothalamic circuitry that may act as the FEO. The pattern of neuronal activation after feeding was different from that detected during food anticipation for some specific nucleus or subregions. The increase in plasma corticosterone during food anticipation was not accompanied by an increase in CRF mRNA levels, suggesting that factors other than CRF are involved in the control of adrenocortical secretion under RFS.

Role of orexin input in the diurnal rhythm of locus coeruleus impulse activity.

Activation of noradrenergic locus coeruleus (LC) neurons promotes wakefulness and behavioral arousal. In rats, LC neurons receive circadian inputs via a circuit that originates in the suprachiasmatic nucleus (SCN) and relays through the dorsomedial hypothalamus (DMH) to LC; this circuit input increases LC activity during the active period. DMH neurons expressing the peptide neurotransmitter orexin/hypocretin are ideally situated to act as a relay between SCN and LC due to their synaptic inputs from SCN and innervation of LC. Here, we examined the hypothesis that orexin is involved in transmitting circadian signals to LC using single-unit recordings of LC neurons in anesthetized rats maintained in 12:12 light-dark housing. We replicated earlier findings from this lab that LC neurons fire significantly faster on average during the active compared to rest periods. Local microinjection of an orexin antagonist, SB-334867-A attenuated the impulse activities of the fastest firing population of LC neurons during the active period. We also found that DMH orexin neurons project preferentially to LC and express a diurnal rhythm of activation that correlates with LC neuronal firing frequency. Therefore, we propose that DMH orexin neurons play a role in modulating the day-night differences of LC impulse activity.

The dorsomedial hypothalamic nucleus is not necessary for the expression of circadian food-anticipatory activity in rats.

Restricted daytime feeding generates food-anticipatory activity (FAA) by entrainment of a circadian pacemaker separate from the light-entrainable pacemaker located in the SCN. The dorsomedial hypothalamic nucleus (DMH) has been proposed as the site of food-entrainable oscillators critical for the expression of FAA, but another study found no effects of complete DMH ablation on FAA. To account for these different results, the authors examined methodological factors, including (1) cage configuration and feeding method and (2) use of social cues. Intact and DMH-ablated rats were maintained on one 4-h daily meal in the middle of the light period, using caging and feeding methods matching those of Gooley et al. (2006). Rats with partial or complete DMH ablation were less nocturnal during ad lib food access but exhibited normal FAA during restricted feeding, as quantified by FAA magnitude, ratios, latency to appearance, duration, and precision. To evaluate the use of social cues, intact rats naive to restricted-feeding schedules were food deprived for 72 h on 4 tests. Daytime activity increased during food deprivation, but the magnitude and waveform of this activity was not influenced by the presence of food-entrained rats exhibiting robust FAA in adjacent cages. Thus, hungry intact rats do not use social cues to anticipate a daily mealtime, suggesting that DMH-ablated rats do not anticipate meals by reacting to sounds from food-entrained intact rats in adjacent cabinets. These results confirm our previous finding that the DMH is not critical for normal expression of FAA in rats, and this observation is extended to food restriction methodologies used by other labs. The methodological differences that do underlie discrepant results remain unresolved, as does the location of food-entrainable oscillators, input pathways, and output pathways critical for FAA.

Importance of melanocortin signaling in refeeding-induced neuronal activation and satiety.

To identify regions in the hypothalamus involved in refeeding and their regulation by alpha-MSH, adult rats were subjected to a 3-d fast, and 2 h after refeeding, the distribution of c-Fos-immunoreactive neurons was elucidated. Compared with fed and fasted animals, a significant increase (P < 0.001) in the number of c-Fos-immunoreactive cells was identified in refed animals in the supraoptic nucleus, magnocellular and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus (PVNv), and the dorsal and ventral subdivisions of the dorsomedial nucleus (DMNd and DMNv, respectively). Refeeding shifted the location of c-Fos-labeled neurons from the medial to lateral arcuate where c-Fos was induced in 88.7 +/- 2.2% of alpha-MSH-containing neurons. alpha-MSH-containing axons densely innervated the PVNv, DMNd, and DMNv and organized in close apposition to the majority of refeeding-activated c-Fos-positive neurons. To test whether the melanocortin system is involved in induction of c-Fos in these regions, the melanocortin 3/4 receptor antagonist, agouti-related protein (AGRP 83-132), was administered to fasting animals just before refeeding. Compared with artificial cerebrospinal fluid, a single intracerebroventricular bolus of agouti-related protein (5 microg/5 microl) not only significantly increased the total amount of food consumed within 2 h but also nearly abolished refeeding-induced c-Fos expression in the PVNv and DMNd and partially reduced c-Fos immunoreactivity in the DMNv. We conclude that refeeding activates a subset of neurons in the PVN and DMN as a result of increased melanocortin signaling and propose that one or more of these neuronal populations mediate the potent anorexic actions of alpha-MSH.

Short-term cold exposure may cause a local decrease of neuropeptide Y in the rat hypothalamus.

Neuropeptide Y (NPY) is an orexigenic and hypothermic peptide. To understand its role in hypothermic conditions, male rats were placed in a 24 degrees C or 4 degrees C air chamber for 1.5 h. The expression of c-Fos protein, and NPY mRNA and protein, was analyzed in the hypothalamus 1 h-2 h later. The cold treatment increased the number of c-Fos-immunoreactive cells in the paraventricular hypothalamic nucleus (PVN) and arcuate nucleus (ARC). At the same time it decreased the density of NPY-immunoreactive components in the PVN, dorsomedial hypothalamic nucleus and ARC, as well as of NPY transcripts in the PVN and ARC. No colocalization of c-Fos with NPY was detected. These results suggest that short-term cold exposure should reduce indirectly NPY production in some hypothalamic nuclei to facilitate thermogenesis without inducing feeding behavior.

Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei.

In the brain preproglucagon expression is limited to a cluster of neurons in the caudal part of the nucleus of the solitary tract (NTS) as well as a smaller number of neurons that extend laterally from the NTS through the dorsal reticular area into the A1 area. These neurons process preproglucagon to glucagon-like peptide-1 (GLP-1), GLP-2, oxyntomodulin and glicentin. The neurons project mainly to the hypothalamus, where especially two nuclei involved in appetite regulation--the paraventricular (PVN) and dorsomedial (DMH) hypothalamic nuclei--are heavily endowed with GLP-immunoreactive nerve fibres. To gain further insight into this neurocircuitry, we injected the retrograde tracers cholera toxin, subunit B (ChB) and Fluorogold (FG) into the PVN and the DMH, respectively. Of thirty-five injected rats, six had successful injections that predominantly restricted within the boundaries of the PVN and DMH. Hindbrain sections from these rats were triple labelled for ChB, FG and GLP-2. A total of 24+/-1% of the PVN-projecting NTS-neurons contained GLP-2-ir whereas 67+/-4% of the DMH-projecting neurons were also stained for GLP-2, suggesting that the NTS-projections to the DMH arise mainly from preproglucagon neurons. Approximately 20% of backfilled cells in the NTS contained both retrograde tracers, therefore presumably representing neurons projecting to both the PVN and the DMH. The results of the present study demonstrate that the majority of the preproglucagon-expressing neurons in the NTS project in a target-specific manner to the hypothalamus. It is therefore possible that individual subgroups of GLP-containing neurons can mediate different physiological responses.

Obesity and endocrine dysfunction in mice with deletions of both neuropeptide Y and galanin.

Neuropeptide Y (NPY) and galanin have both been implicated in the regulation of body weight, yet mice bearing deletions of either of these molecules have unremarkable metabolic phenotypes. To investigate whether galanin and NPY might compensate for one another, we produced mutants lacking both neuropeptides (GAL(-/-)/NPY(-/-)). We found that male GAL(-/-)/NPY(-/-) mice ate significantly more and were much heavier (30%) than wild-type (WT) controls. GAL(-/-)/NPY(-/-) mice responded to a high-fat diet by gaining more weight than WT mice gain, and they were unable to regulate their weight normally after a change in diet. GAL(-/-)/NPY(-/-) mice had elevated levels of leptin, insulin, and glucose, and they lost more weight than WT mice during chronic leptin treatment. Galanin mRNA was increased in the hypothalamus of NPY(-/-) mice, providing evidence of compensatory regulation in single mutants. The disruption of energy balance observed in GAL(-/-)/NPY(-/-) double knockouts is not found in the phenotype of single knockouts of either molecule. The unexpected obesity phenotype may result from the dysregulation of the leptin and insulin systems that normally keep body weight within the homeostatic range.

Rostral ventromedial periaqueductal gray: a source of inhibition of the sympathetic outflow to brown adipose tissue.

Central inhibitory pathways play a significant role in determining the level of sympathetic outflow to the cold defense efferents in mammals. We tested the hypothesis that neurons in the rostral ventromedial periaqueductal gray (rvmPAG) are a source of inhibitory regulation of the sympathetic nerve activity (SNA) to brown adipose tissue (BAT). In urethane/chloralose-anesthetized, paralyzed, artificially ventilated rats, microinjection of PGE2 (200 pmol in 70 nl) into the medial preoptic area (POA) or microinjection of the GABAA antagonists, bicuculline or SR95531 (60 pmol in 60 nl), into the dorsomedial hypothalamic area (DMH) increased BAT SNA by +853 +/- 176 and +898 +/- 249% of control, respectively. These evoked increases in BAT SNA were reversed by microinjection of bicuculline (60 pmol in 60 nl) into the rvmPAG at the level of the posterior commissure. Microinjection of muscimol (160 pmol in 80 nl) into the rvmPAG increased BAT SNA by an amount (+191 +/- 92% of control) that was significantly (P < 0.05) smaller than the peak increase observed after bicuculline microinjection into the rostral raphe pallidus (+1340 +/- 547% of control), but not different from that observed after transaction of the midbrain posterior to the rvmPAG (+423 +/- 123% of control). We conclude that the rvmPAG contains neurons that exert an inhibitory influence on the sympathetic outflow to BAT. These BAT sympathoinhibitory neurons are, themselves, under a tonic GABAergic inhibition. Blockade of this tonic inhibition reveals an inhibitory influence of rvmPAG neurons that is capable of reversing BAT SNA activations from POA or from DMH. Augmenting the tonic inhibition of rvmPAG neurons elicits a modest increase in BAT SNA. Neurons in rvmPAG provide some, but not all, of the tonic inhibition regulating the discharge of BAT sympathetic premotor neurons in RPa and ultimately the level of thermogenesis in BAT.

Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice.

The arcuate nucleus of the hypothalamus (ARH) is a critical component of forebrain pathways that regulate a variety of neuroendocrine functions, including an important role in relaying leptin signals to other parts of the hypothalamus. However, neonatal rodents do not lose weight in response to leptin treatment in the same way as do adults, suggesting that certain aspects of leptin signaling pathways in the hypothalamus may not be mature. We tested this possibility by using DiI axonal labeling to examine the development of projections from the ARH to other parts of the hypothalamus in neonatal mice, paying particular attention to the innervation of the paraventricular nucleus (PVH), the dorsomedial nucleus (DMH), and the lateral hypothalamic area (LHA), each of which have been implicated in the regulation of feeding. The results indicate that ARH projections are quite immature at birth and appear to innervate the DMH, PVH, and LHA in succession, within distinct temporal domains. The projections from the ARH to the DMH develop rapidly and are established by the sixth postnatal day (P6), whereas those to the PVH develop significantly later, with the mature pattern of innervation first apparent between postnatal day 8 (P8)-P10. Furthermore, the ability of leptin to activate Fos in the PVH, DMH, and LHA appears to be age-dependent and correlates with the arrival of ARH projections to each nucleus. Taken together, these findings provide new insight into development of hypothalamic circuits and suggest an anatomical basis for the delayed postnatal regulation of food intake and body weight by leptin.

KiSS-1 expression and metastin-like immunoreactivity in the rat brain.

Metastin, the gene product of metastasis suppressor gene KiSS-1, is the endogenous ligand for the G-protein-coupled receptor GPR54 (or AXOR12, or OT7T175). The expression of KiSS-1 gene and peptide and the distribution of metastin were studied in the rat central nervous system by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical methods. KiSS-1 gene and peptide expression was higher in the hypothalamus than in the brainstem and spinal cord. In the brain, metastin-like immunoreactivity (irMT) was found mainly in three groups of cells: dorsomedial hypothalamic nucleus, nucleus of the solitary tract, and caudal ventrolateral medulla. Immunoreactive fibers of varying density were noted in bed nucleus of stria terminalis, septal nuclei, nucleus accumbens, caudate putamen, diagonal band, amygdala, hypothalamus, zona incerta, thalamus, periaqueductal gray, raphe nuclei, lateral parabrachial nucleus, locus coeruleus, spinal trigeminal tract, rostral ventrolateral medulla, and medullary reticular nucleus. Preabsorption of the antiserum with metastin peptide fragment (45-54)-NH2 (1 microg/ml) resulted in no staining in any of the sections. The biological activity of metastin was assessed by monitoring intracellular calcium [Ca2+]i in cultured hippocampal neurons, which are known to express GPR54. Metastin increased [Ca2+]i in a population of cultured hippocampal neurons. The results show that metastin is biologically active in rat central neurons, and its anatomical distribution suggests a possible role in nociception and autonomic and neuroendocrine functions.