Repeated peripheral administration of lipidized prolactin-releasing peptide analog induces c-fos and FosB expression in neurons of dorsomedial hypothalamic nucleus in male C57 mice.

Previous studies indicate that hypothalamic prolactin-releasing peptide (PrRP), signaling via GPR10 and neuropeptide FF2 receptor, is involved in energy homeostasis, stress responses, and cardiovascular regulation. Energy homeostasis depends on the balance between food intake regulation and energy expenditure, in which the hypothalamus plays a key role. The lipidization of PrRP31 with palmitoyl acid allows it to produce its anorexigenic effect after repeated peripheral administration and to reduce body weight and improve metabolic parameters in diet-induced obese (DIO) mice. The aim of this study was to reveal the transient and long-lasting changes in neuronal activity via c-Fos and FosB immunohistochemistry in brain nuclei related to food intake regulation and energy homeostasis during the first days of treatment with a newly designed lipidized analog of PrRP31 (palm11-PrRP31) with promising antiobesity effects. The data revealed that the anorexigenic effect of repeated application of palm11-PrRP31 was associated with delayed but gradually significantly reduced cumulative food intake in mice as well as with a significant reduction in their body weight. Moreover, while the repeated application of palm11-PrRP31 was associated with a significant reduction in acute cell activity in the paraventricular hypothalamic nucleus (PVN) and nucleus of the solitary tract (NTS) compare to its acute treatment, both acute and long-lasting cell activity in the dorsomedial hypothalamic nucleus (DMN) were increased. The data indicate that DMN neurons might be tonically activated after repeated administration of lipidized PrRP analogs that may be associated with the process of long-term adaptation to modified energy homeostasis.

Blockade of the dorsomedial hypothalamus and the perifornical area inhibits respiratory responses to arousing and stressful stimuli.

The dorsomedial hypothalamus (DMH) and the perifornical area (DMH/PeF) is one of the key regions of central autonomic processing. Previous studies have established that this region contains neurons that may be involved in respiratory processing; however, this has never been tested in conscious animals. The aim of our study was to investigate the involvement of the DMH/PeF area in mediating respiratory responses to stressors of various intensities and duration. Adult male Wistar rats (n = 8) received microinjections of GABAA agonist muscimol or saline into the DMH/PeF bilaterally and were subjected to a respiratory recording using whole body plethysmography. Presentation of acoustic stimuli (500-ms white noise) evoked transient responses in respiratory rate, proportional to the stimulus intensity, ranging from +44 ± 27 to +329 ± 31 cycles/min (cpm). Blockade of the DMH/PeF almost completely abolished respiratory rate and tidal volume responses to the 40- to 70-dB stimuli and also significantly attenuated responses to the 80- to 90-dB stimuli. Also, it significantly attenuated respiratory rate during the acclimatization period (novel environment stress). The light stimulus (30-s 2,000 lux) as well as 15-min restraint stress significantly elevated respiratory rate from 95 ± 4.0 to 236 ± 29 cpm and from 117 ± 5.2 to 189 ± 13 cpm, respectively; this response was abolished after the DMH/PeF blockade. We conclude that integrity of the DMH/PeF area is essential for generation of respiratory responses to both stressful and alerting stimuli.

Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats.

Anxiety disorders in humans reduce both the heart rate variability (HRV) and the sensitivity of the cardiac baroreflex (BRS). Both may contribute to sudden death. To elucidate the mechanisms underlying these alterations, male rats were subjected to social defeat sessions on four consecutive days. Five days later, the rats were found to be in an anxiety-like state. At this time point, we analysed HRV and BRS in the defeated rats, with or without treatment with the anxiolytic chlordiazepoxide (CDZ). HRV was reduced after social defeat, due to changes in the autonomic balance favouring the sympathetic over the parasympathetic component. Spontaneous and pharmacological baroreflex gains were also reduced. CDZ abolished anxiety-like symptoms as well as HRV and BRS alterations. Inhibition of the dorsomedial hypothalamus (DMH) with muscimol reversed all cardiovascular alterations, whereas blockade of the nucleus tractus solitarii (NTS) 5-HT3 receptor by the local or systemic administration of granisetron restored only baroreflex gains and the parasympathetic component of HRV. In conclusion, repeated social defeat in the rat lead to an anxiety-like state that was associated with lasting reduction in HRV and baroreflex gains. The DMH and the NTS were responsible for these chronic cardiovascular alterations. These regions may therefore constitute new therapeutic targets for reducing cardiac dysfunction and fibrillation in anxiety disorders.

High fat diet impact on Fos expression in ovariectomized female C57BL/6 mice: effect of colchicine and response of different neuronal phenotypes.

OBJECTIVES: Activity of neuropeptide Y (NPY), tyrosine hydroxylase (TH), corticoliberine (CRH), and oxytocin (OXY) producing cells was investigated in the ovariectomized (OVX) female C57BL/6 mice kept on the high fat diet for 16 weeks and their response to colchicine stress in selected brain areas, including the hypothalamic paraventricular (PVN), dorsomedial (DMN) and arcuate (ARC) nuclei, A1/C1 (in the ventrolateral medulla), and A2/C2 (in the nucleus of the solitarii tract, NTS) catecholaminergic cell groups. METHODS: The OVX female C57BL/6 mice kept on high fat diet were sacrificed by transcardial perfusion with fixative 48 h after intracerebroventricular injection of colchicine (18 µg mice). Dual Fos/neuropeptide immunohistochemistry was employed to investigate Fos/neuropeptide colocalizations. RESULTS: In the OVX saline-treated mice (sham control) with standard diet (St diet), no immunopositive CRH and NPY neurons were identified in the PVN and weak Fos immunostainig was visible in TH neurons in the DMN and ARC nuclei. Colchicine treatment in the OVX mice with St diet increased the number of CRH and OXY immunopositive neurons in the PVN as well as the number of NPY and TH neurons in DMN and ARC nuclei and NPY neurons in the middle NTS (mNTS) and A1/C1 cell group. Prolonged HF diet in OVX sham control mice moderately increased the number of Fos/TH neurons in the mNTS and commissural NTS (cNTS) in comparison with St diet mice. However, prolonged HF diet in OVX colchicines-treated mice reduced the number of Fos/NPY neurons in the anterior NTS (aNTS) and A1/C1 cell group in comparison with colchicines-treated animals with St diet as well as Fos-TH neurons in the mNTS and cNTS in comparison with saline-treated animals with HF diet. CONCLUSION: The data of this pilot study indicate that prolonged high fat diet might: 1) represent itself a light/moderate stimulus for activation of TH neurons in the NTS and A1/C1 cell group as well as NPY neurons in the A1/C1 cell group and 2) interfere with colchicines-induced and time-delayed Fos activation in the NPY and TH neurons in both the above mentioned brain nuclei.

Activation of orexin neurons in dorsomedial/perifornical hypothalamus and antidepressant reversal in a rodent model of depression.

Chronic stressful life events are risk factors for depression often accompanied by homeostatic disturbances. Hypothalamic neuropeptides, such as orexins (OXs) and melanin-concentrating hormone (MCH), are involved in regulation of several autonomic functions that are altered in depression. However, little is known about the link between orexinergic or MCH-ergic systems and depression. Using double immunohistochemical labeling for OX- or MCH-containing neurons and Fos protein, we studied the effects of a chronic selective serotonin reuptake inhibitor antidepressant treatment (fluoxetine) on the OX and MCH neuronal activation in mice exposed to unpredictable chronic mild stress (UCMS), a rodent model of depression. Western blot was also performed to assess OX and MCH receptor expression in various brain areas. Finally, almorexant, a dual OX receptor antagonist, was assessed in the tail suspension test. UCMS induced physical and behavioral disturbances in mice reversed by 6-week fluoxetine treatment. Orexinergic neurons were more activated in the dorsomedial and perifornical hypothalamic area (DMH-PFA) of UCMS-subjected mice compared to the lateral hypothalamus (LH), and this increase was reversed by 6-week fluoxetine treatment. UCMS also reduced expression of OX-receptor 2 in the thalamus and hypothalamus, but not in animals chronically treated with fluoxetine. MCH neurons were neither affected by UCMS nor by antidepressant treatment, while UCMS modulated MCH receptor 1 expression in thalamus and hippocampus. Finally, chronic but not acute administration of almorexant, induced antidepressant-like effect in the tail suspension test. These data suggest that OX neurons in the DMH-PFA and MCH-ergic system may contribute to the pathophysiology of depressive disorders.

The role of N-methyl-D-aspartate receptor subunits in the rat thalamic mediodorsal nucleus during central sensitization.

We have identified tooth pulp-driven neurons (TPDNs) in the thalamic mediodorsal nucleus (MD) in rats and showed that the TPDNs' responsiveness in the MD is increased by chemical conditioning stimulation of allyl-isothiocyanate (mustard oil) to the molar tooth pulp. The aim of the present study was to address the role of N-methyl-d-aspartate receptors (NMDA receptors) in the sensitized central nervous system following the mustard oil application to the rat tooth pulp. Microinjection of MK-801, a noncompetitive NMDA receptor antagonist, to the thalamic MD nucleus reduced the TPDNs' responsiveness in the thalamic MD nucleus. Gene expression analysis showed that expression levels of NMDA receptor subunits NR2A and NR2D mRNAs in the thalamus were increased by the mustard oil application and that the increases were reduced by MK-801. When naloxone, an opioid receptor antagonist, was given systemically following the MK801 microinjection, the TPDNs' responsiveness was rekindled and expression levels of NR2D and NR2A mRNAs were increased. Moreover, lidocaine pretreatment abolished the mustard oil-induced upregulation of NR2D and NR2A mRNAs. These results suggest that, during central sensitization, interaction of NMDA receptors and endogeneous opioid-related inhibitory mechanisms plays critical role in the alteration of the TPDNs' responsiveness in the thalamic MD nucleus.

The action of leptin on appetite-regulating cells in the ovine hypothalamus: demonstration of direct action in the absence of the arcuate nucleus.

It is widely accepted that leptin acts on first-order neurons in the arcuate nucleus (ARC) with information then relayed to other hypothalamic centers. However, the extent to which leptin mediates its central actions solely, or even primarily, via this route is unclear. We used a model of hypothalamo-pituitary disconnection (HPD) to determine whether leptin action on appetite-regulating systems requires the ARC. This surgical preparation eliminates the ARC. We measured effects of iv leptin to activate hypothalamic neurons (Fos labeling). In ARC-intact animals, leptin increased the percentage of Fos-positive melanocortin neurons and reduced percentages of Fos-positive neuropeptide Y neurons compared with saline-treated animals. HPD itself increased Fos labeling in the lateral hypothalamic area (LHA). Leptin influenced Fos labeling in the dorsomedial nucleus (DMH), ventromedial nucleus, and paraventricular nucleus (PVN) in HPD and normal animals, with effects on particular cell types varying. In the LHA and DMH, leptin decreased orexin cell activation in HPD and ARC-intact sheep. HPD abolished leptin-induced expression of Fos in melanin-concentrating hormone cells in the LHA and in CRH cells in the PVN. In contrast, HPD accentuated activation in oxytocin neurons. Our data from sheep with lesions encompassing the ARC do not suggest a primacy of action of leptin in this nucleus. We demonstrate that first order to second order signaling may not represent the predominant means by which leptin acts in the brain to generate integrated responses. We provide evidence that leptin exerts direct action on cells of the DMH, ventromedial nucleus, and PVN.

Changes in central sodium and not osmolarity or lactate induce panic-like responses in a model of panic disorder.

Panic disorder is a severe anxiety disorder characterized by recurrent panic attacks that can be consistently provoked with intravenous (i.v.) infusions of hypertonic (0.5 M) sodium lactate (NaLac), yet the mechanism/CNS site by which this stimulus triggers panic attacks is unclear. Chronic inhibition of GABAergic synthesis in the dorsomedial hypothalamus/perifornical region (DMH/PeF) of rats induces a vulnerability to panic-like responses after i.v. infusion of 0.5 M NaLac, providing an animal model of panic disorder. Using this panic model, we previously showed that inhibiting the anterior third ventricle region (A3Vr; containing the organum vasculosum lamina terminalis, the median preoptic nucleus, and anteroventral periventricular nucleus) attenuates cardiorespiratory and behavioral responses elicited by i.v. infusions of NaLac. In this study, we show that i.v. infusions of 0.5 M NaLac or sodium chloride, but not iso-osmolar D-mannitol, increased 'anxiety' (decreased social interaction) behaviors, heart rate, and blood pressure responses. Using whole-cell patch-clamp preparations, we also show that bath applications of NaLac (positive control), but not lactic acid (lactate stimulus) or D-mannitol (osmolar stimulus), increases the firing rates of neurons in the A3Vr, which are retrogradely labeled from the DMH/PeF and which are most likely glutamatergic based on a separate study using retrograde tracing from the DMH/PeF in combination with in situ hybridization for vesicular glutamate transporter 2. These data show that hypertonic sodium, but not hyper-osmolarity or changes in lactate, is the key stimulus that provokes panic attacks in panic disorder, and is consistent with human studies.

Cardiovascular and thermal responses evoked from the periaqueductal grey require neuronal activity in the hypothalamus.

Stimulation of neurons in the lateral/dorsolateral periaqueductal grey (l/dlPAG) produces increases in heart rate (HR) and mean arterial pressure (MAP) that are, according to traditional views, mediated through projections to medullary autonomic centres and independent of forebrain mechanisms. Recent studies in rats suggest that neurons in the l/dlPAG are downstream effectors responsible for responses evoked from the dorsomedial hypothalamus (DMH) from which similar cardiovascular changes and increase in core body temperature (T(co)) can be elicited. We hypothesized that, instead, autonomic effects evoked from the l/dlPAG depend on neuronal activity in the DMH. Thus, we examined the effect of microinjection of the neuronal inhibitor muscimol into the DMH on increases in HR, MAP and T(co) produced by microinjection of N-methyl-D-aspartate (NMDA) into the l/dlPAG in conscious rats. Microinjection of muscimol alone modestly decreased baseline HR and MAP but failed to alter T(co). Microinjection of NMDA into the l/dlPAG caused marked increases in all three variables, and these were virtually abolished by prior injection of muscimol into the DMH. Similar microinjection of glutamate receptor antagonists into the DMH also suppressed increases in HR and abolished increases in T(co) evoked from the PAG. In contrast, microinjection of muscimol into the hypothalamic paraventricular nucleus failed to reduce changes evoked from the PAG and actually enhanced the increase in T(co). Thus, our data suggest that increases in HR, MAP and T(co) evoked from the l/dlPAG require neuronal activity in the DMH, challenging traditional views of the place of the PAG in central autonomic neural circuitry.

Gabaergic mechanisms of hypothalamic nuclei in the expression of conditioned fear.

The amygdala, the dorsal periaqueductal gray (dPAG), and the medial hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN), the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses.

Nicotine administration effects on feeding and cocaine-amphetamine-regulated transcript (CART) expression in the hypothalamus.

In previous studies food intake and meal size significantly decreased in rats two days after injecting 4 mg/kg/day nicotine tartrate. Food intake returned to normal after nine days of continued nicotine treatment, when reduced meal size is countered by an increase in meal number. Nicotine also reduced body weight after nicotine injection and body weight remained low after nine days. To begin characterizing the mechanism that modulates these changes in feeding behavior and/or body weight during nicotine exposure the transcript levels for agouti related protein (AGRP), cocaine-amphetamine-regulated transcript (CART), corticotropin releasing hormone receptor one (CRH-R1), melanocortin receptors three and four (MC3R/4R), neuropeptide Y (NPY), NPY Y1 and Y5 receptors and/or pro-opiomelanocortin (POMC) were analyzed in the arcuate (ARC), dorsomedial (DMN) and paraventricular (PVN)/periventricular (PE) hypothalamic nuclei on the second and ninth day of saline or nicotine treatment. Results show that the transcript levels of the anorexigenic molecule CART increased in the PVN and/or PE two days after nicotine treatment but after nine days CART levels equalize. In contrast, nine days of nicotine treatment reduced CART levels in the DMN as compared to saline controls. To investigate CART's role in regulating feeding, infusion of CART (55-102) into the third ventricle reduced food intake and meal size. These results are consistent with nicotine modulating feeding behavior and body weight, in part, by affecting CART transcript levels in the DMN, PVN and/or PE.

Neural substrate of cold-seeking behavior in endotoxin shock.

Systemic inflammation is a leading cause of hospital death. Mild systemic inflammation is accompanied by warmth-seeking behavior (and fever), whereas severe inflammation is associated with cold-seeking behavior (and hypothermia). Both behaviors are adaptive. Which brain structures mediate which behavior is unknown. The involvement of hypothalamic structures, namely, the preoptic area (POA), paraventricular nucleus (PVH), or dorsomedial nucleus (DMH), in thermoregulatory behaviors associated with endotoxin (lipopolysaccharide [LPS])-induced systemic inflammation was studied in rats. The rats were allowed to select their thermal environment by freely moving in a thermogradient apparatus. A low intravenous dose of Escherichia coli LPS (10 microg/kg) caused warmth-seeking behavior, whereas a high, shock-inducing dose (5,000 microg/kg) caused cold-seeking behavior. Bilateral electrocoagulation of the PVH or DMH, but not of the POA, prevented this cold-seeking response. Lesioning the DMH with ibotenic acid, an excitotoxin that destroys neuronal bodies but spares fibers of passage, also prevented LPS-induced cold-seeking behavior; lesioning the PVH with ibotenate did not affect it. Lesion of no structure affected cold-seeking behavior induced by heat exposure or by pharmacological stimulation of the transient receptor potential (TRP) vanilloid-1 channel ("warmth receptor"). Nor did any lesion affect warmth-seeking behavior induced by a low dose of LPS, cold exposure, or pharmacological stimulation of the TRP melastatin-8 ("cold receptor"). We conclude that LPS-induced cold-seeking response is mediated by neuronal bodies located in the DMH and neural fibers passing through the PVH. These are the first two landmarks on the map of the circuitry of cold-seeking behavior associated with endotoxin shock.

Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats.

Relatively little is known concerning the interaction of psychostimulants with hypothalamic neuropeptide systems or metabolic hormones implicated in regulation of energy balance. The present studies tested whether methamphetamine alters the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), two important orexigenic neuropeptides, or proopiomelanocortin (POMC), the precursor for the anorexigenic peptide alpha-melanocyte-stimulating hormone, or the secretion of leptin, insulin and ghrelin, concomitant with inhibition of food intake. Female rats were either fed ad libitum (AL) or placed on a scheduled feeding (SF) regimen, with access to food limited to 4 h/day. Administration of (+/-)-methamphetamine (7.5 mg/kg, i.p.) 2 h prior to food presentation significantly inhibited food intake in SF animals, but did not affect intake in AL animals. In a separate study, AL and SF animals were killed just prior to expected food presentation, and expression of NPY, AgRP and POMC mRNAs in hypothalamus was determined using in situ hybridisation; concentrations of leptin, insulin and ghrelin in serum were determined with radioimmunoassays. In saline-treated, SF controls, NPY and AgRP mRNA expression in arcuate nucleus and serum ghrelin were significantly elevated, and serum leptin and insulin were significantly reduced. Methamphetamine reversed the up-regulation of NPY mRNA expression observed in the SF condition, without affecting AgRP mRNA or the serum concentrations of metabolic hormones. However, in AL animals, NPY mRNA expression in arcuate and dorsomedial nuclei was significantly increased by methamphetamine, which also reduced serum leptin and insulin and increased serum ghrelin concentrations. These findings suggest that the inhibition of NPY expression in SF animals may be a mechanism underlying the anorexigenic effect of methamphetamine seen in this condition. The increase in NPY expression produced by methamphetamine in AL animals may be mediated by the ability of this drug to decrease secretion of leptin and insulin and increase secretion of ghrelin.

Disinhibition of maternal behavior following neurotoxic lesions of the hypothalamus in primigravid rats.

Virgin female rats do not respond maternally to foster pups due to an endogenous neural circuit that actively inhibits the display of maternal behavior. Once pregnant, primigravid rats will continue to avoid foster pups until just prior to or at parturition. Anosmia or lesions of the olfactory tract, medial amygdala, and areas of the hypothalamus will stimulate virgin females to display maternal behavior rapidly, but little is known of the effect of these lesions in primigravid rats. The objective of the present study was to determine if neurotoxic lesions of the dorsomedial (DMH) and ventromedial nuclei (VMH) of the hypothalamus will advance the onset of maternal behavior in primigravid rats. Nulliparous Sprague-Dawley female rats were mated and then on day 8 of gestation bilaterally infused with N-methyl-d-aspartic acid (NMDA; 8 microg/0.2 microl/side) or vehicle directed toward either the DMH or VMH. Beginning on day 15 of gestation until parturition, females were tested daily for maternal responsiveness. DMH and VMH lesions significantly advanced the onset of maternal behavior (5-6 days vs. 0-1 day before parturition) in first-time pregnant rats. These results indicate that the DMH and VMH are involved in the regulation of maternal behavior and may be part of an endogenous neural circuit that inhibits maternal behavior during pregnancy.

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.

Multiple hypothalamic sites control the frequency of hippocampal theta rhythm.

Stimulation of a neural pathway originating in the brainstem reticular formation, with synapses in the medial hypothalamus, activates the hippocampal theta rhythm. The frequency of reticular-elicited theta is determined in the medial supramammillary nucleus (mSuM) completely in anaesthetised rats, but only partially when the animal is awake. We tested other medial hypothalamic sites for their capacity to control theta frequency in awake rats. Blockade of sodium channels (1 microl fast infusion of the local anaesthetic procaine, experiment 1) or increased inhibition by GABA (Chlordiazepoxide [CDP], experiment 2) was found to reduce or increase the frequency of reticular-elicited theta, depending on the precise site of injection, in the region of the dorsomedial hypothalamic nucleus (DMH) and the posterior hypothalamic nucleus (PH). A band of null sites for CDP was located in the region of the ventral border of PH and dorsal border of mSuM. Using 0.5 and 1 microl CDP, and slow infusions (experiment 3), it was found that effective PH sites were also separate from mSuM in the rostrocaudal direction. In experiment 4, the DMH/PH region was mapped with unilateral and bilateral slow infusions of 0.5 microl CDP. CDP significantly reduced frequency in medial (periventricular) and dorsal PH, but not DMH. Bilateral injections appeared to generally sum the usual effects of unilateral injection, producing effects of intermediate size. However, the absolute frequency change in any given site, or with any pair of sites, did not exceed 1 Hz, which is similar to what is seen with single injections in mSuM. Overall, it appears that at, any one time, theta frequency may be determined by a complex interplay between distinct but interacting modulatory regions in the medial hypothalamus.

Hypothalamic bHLH transcription factors are novel candidates in the regulation of energy balance.

The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake.

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Dopaminergic agonists normalize elevated hypothalamic neuropeptide Y and corticotropin-releasing hormone, body weight gain, and hyperglycemia in ob/ob mice.

Hypothalamic neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) influence feeding and levels of plasma glucose, insulin, free fatty acids, and triglycerides. Treatment of genetically obese, ob/ob mice, with dopamine receptor D(1)/D(2) agonists normalizes hyperphagia, body weight gain, hyperglycemia, and hyperlipidemia. We therefore examined whether levels of NPY and CRH immunoreactivity in discrete hypothalamic nuclei are altered in ob/ob mice, and whether dopaminergic treatment reverses this alteration. Female ob/ob mice were treated daily at 1 h after light onset with the D(1)/D(2) agonists, SKF-38393 (20 mg/kg) and bromocriptine (15 mg/kg), respectively or vehicle for 2 weeks. Such treatment, while normalizing body weight gain and hyperglycemia, also significantly reduced elevated NPY immunoreactivity in the suprachiasmatic (by 39%), intergeniculate (by 43%), paraventricular (PVN; by 31%), and arcuate (by 41%) nuclei in obese mice to levels observed in lean mice. This treatment also caused a 45-50% decline in levels of CRH in the PVN and dorsomedial hypothalamus compared to obese controls to levels observed in lean mice. Taken together, these findings suggest that dopaminergic D(1)/D(2) receptor coactivation may improve hyperphagia, hyperglycemia, and obesity in the ob/ob mouse, in part, by normalizing elevated levels of both NPY and CRH.

Insulin selectively downregulates alpha2-adrenoceptors in the arcuate and dorsomedial nucleus.

Intracerebroventricular infusion of insulin (2 mU/ day) produced selective downregulation of 3H-paraminoclonidine binding to alpha2-adrenoceptors in the hypothalamic arcuate (14%) and dorsomedial (19%) nuclei out of 16 forebrain areas in Wistar rats. Binding of 3H-prazosin to alpha1-adrenoceptors was unaffected. This is in keeping with the known effect of insulin on catecholamine and neuropeptide Y metabolism in these brain regions that play an important role in energy homeostasis.