Glucose-receptive neurones in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits.
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1. Patch-clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell-attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2. In whole-cell patch-clamp recordings, in the absence of ATP in the electrode solution, glucose-receptive neurones gradually hyperpolarized due to the induction of an outward current at -60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3. In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 microM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4. In cell-attached recordings diazoxide and the active fragment of leptin (leptin 22-56) reduced the firing rate of glucose-receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose-receptive neurones demonstrated the expression of the ATP-sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6. It is concluded that glucose-receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues. (+info)
Chronic hypoglycemia and diabetes impair counterregulation induced by localized 2-deoxy-glucose perfusion of the ventromedial hypothalamus in rats.
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Previous studies have demonstrated that the ventromedial hypothalamus (VMH) plays a critical role in sensing and responding to systemic hypoglycemia. To evaluate the mechanisms of defective counterregulation caused by iatrogenic hypoglycemia and diabetes per se, we delivered 2-deoxy-glucose (2-DG) via microdialysis into the VMH to produce localized cellular glucopenia in the absence of systemic hypoglycemia. Three groups of awake chronically catheterized rats were studied: 1) nondiabetic (with a mean daily glucose [MDG] of 6.9 mmol/l) BB control rats (n = 5); 2) chronically hypoglycemic nondiabetic (3-4 weeks, with an MDG of 2.7 mmol/l) BB rats (n = 5); and 3) moderately hyperglycemic insulin-treated diabetic (with an MDG of 12.4 mmol/l) BB rats (n = 8). In hypoglycemic rats, both glucagon and catecholamine responses to VMH glucopenia were markedly (77-93%) suppressed. In diabetic rats, VMH 2-DG perfusion was totally ineffective in stimulating glucagon release. The epinephrine response, but not the norepinephrine response, was also diminished by 38% in the diabetic group. We conclude that impaired counterregulation after chronic hypoglycemia may result from alterations of the VMH or its efferent pathways. In diabetes, the capacity of VMH glucopenia to activate the sympathoadrenal system is only modestly diminished; however, the communication between the VMH and the alpha-cell is totally interrupted. (+info)
Plasma leptin levels and triglyceride secretion rates in VMH-lesioned obese rats: a role of adiposity.
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To explore the role of adiposity on hypertriglyceridemia associated with obesity, we examined the relation between triglyceride secretion rate (TGSR) and plasma leptin, insulin, or insulin resistance in ventromedial hypothalamus (VMH)-lesioned rats in the dynamic and static phases (2 and 14 wk after lesions, respectively). VMH-lesioned rats gained body weight (BW) at fivefold higher rates in the dynamic phase compared with sham-operated control (sham) rats, and BW gain reached a plateau in the static phase. Parametrial fat pad mass was increased 2.5-fold in VMH-lesioned rats compared with sham rats in both phases. Leptin levels were sixfold higher in VMH-lesioned rats of the dynamic phase and even higher in the static phase. Insulin levels were twofold higher in VMH-lesioned rats than in sham rats in both phases. In the dynamic phase, VMH-lesioned rats had 2-fold higher plasma triglyceride (TG) levels and 2.6-fold higher TGSRs, whereas steady-state plasma glucose (SSPG) values, an indicator of insulin resistance, were lower. SSPG values became significantly higher in VMH-lesioned rats in the static phase, but TGSR was not further accelerated. TGSR was significantly associated with leptin, independent of insulin. Leptin was highly correlated with BW, fat mass, and nonesterified fatty acids (NEFA). These results suggest that adiposity itself plays a critical role in TGSR probably through increased NEFA flux from enlarged adipose tissues. Insulin resistance is not associated with the overproduction of TG in this animal model for obesity. (+info)
Pre- and postsynaptic actions of opioid and orphan opioid agonists in the rat arcuate nucleus and ventromedial hypothalamus in vitro.
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1. Using whole-cell patch clamp recording from neurones in an in vitro slice preparation, we have examined opioid- and orphanin FQ (OFQ)-mediated modulation of synaptic transmission in the rat arcuate nucleus and ventromedial hypothalamus (VMH). 2. Application of OFQ activated a Ba2+-sensitive and inwardly rectifying K+ conductance in approximately 50 % of arcuate nucleus neurones and approximately 95 % of VMH neurones. The OFQ-activated current was blocked by the nociceptin antagonist [Phe1Psi(CH2NH)Gly2]-nociceptin(1-13) NH2 (NCA), a peptide that on its own exhibited only weak agonist activity at high concentrations (> 1 microM). Similar current activation was observed with the mu agonist DAMGO but not delta (DPDPE) or kappa (U69593) agonists. 3. In arcuate nucleus neurones, DAMGO (1 microM), U69593 (1 microM) and OFQ (100 nM to 1 microM) but not DPDPE (1 microM) were found to depress the amplitude of electrically evoked glutamatergic postsynaptic currents (EPSCs) and decrease the magnitude of paired-pulse depression, indicating that opioid receptors were located presynaptically. 4. In VMH neurones, DAMGO strongly depressed the EPSC amplitude in all cells examined. DAMGO decreased the magnitude of paired-pulse depression, indicating that mu receptors were located presynaptically. U69593 weakly depressed the EPSC while OFQ and DPDPE had no effect. 5. In VMH neurones, DAMGO depressed the frequency of miniature EPSCs (-58 %) in the presence of tetrodotoxin and Cd2+ (100 microM), suggesting that the actions of mu receptors could be mediated by an inhibition of the synaptic vesicle release process downstream of Ca2+ entry. 6. The data presented show that presynaptic modulation of excitatory neurotransmission in the arcuate nucleus occurs through mu, kappa and the orphan opioid ORL-1 receptors while in the VMH presynaptic modulation only occurs through mu opioid receptors. Additionally, postsynaptic mu and ORL-1 receptors in both the arcuate nucleus and VMH modulate neuronal excitability through activation of a K+ conductance. (+info)
Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats.
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We studied the effects of microinjection of leptin into the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) on glucose uptake in peripheral tissues in unanesthetized rats. The rate of glucose uptake was assessed in vivo by 2-[3H]deoxyglucose incorporation. Single injection of leptin into VMH increased glucose uptake in brown adipose tissue (BAT), heart, skeletal muscles, and spleen but not in white adipose tissue or skin. On the other hand, microinjection of leptin into LH had little effect on glucose uptake in those tissues. The plasma concentrations of glucose and insulin were unaltered by intrahypothalamic injection of leptin into either VMH or LH. Among skeletal muscles, the increase in glucose uptake induced by intrahypothalamic injection of leptin was greater in the soleus than in the extensor digitorum longus. Likewise, the increased glucose uptake in the gastrocnemius in response to leptin was more prominent in the red part than in the white part of the tissue. When surgical sympathetic denervation of the interscapular BAT was performed, the enhanced glucose uptake by BAT in response to intrahypothalamic leptin was completely suppressed. These findings suggest that intrahypothalamic injection of leptin preferentially increases glucose uptake by some peripheral tissues through activation of the VMH-sympathetic (or its neighboring medial hypothalamus-sympathetic) nervous system, thereby contributing to the maintenance of energy balance. (+info)
Hypothalamopontine projections in the rat: anterograde axonal transport studies utilizing light and electron microscopy.
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Projections to the basilar pontine nuclei (BPN) from a variety of hypothalamic nuclei were traced in the rat utilizing the anterograde transport of biotinylated dextran amine. Light microscopy revealed that the lateral hypothalamic area (LH), the posterior hypothalamic area (PH), and the medial and lateral mammillary nuclei (MMN and LMN) are the four major hypothalamic nuclei that give rise to labeled fibers and terminals reaching the rostral medial and dorsomedial BPN subdivisions. Hypothalamopontine fibers extended caudally through the pontine tegmentum dorsal to the nucleus reticularis tegmenti pontis and then coursed ventrally from the main descending bundle toward the ipsilateral basilar pontine gray. Some hypothalamopontine fibers crossed the midline in the tegmental area just dorsal to the pontine gray to terminate in the contralateral BPN. Electron microscopy revealed that the ultrastructural features of synaptic boutons formed by axons arising in the LH, PH, MMN, and LMN are similar to one another. All labeled hypothalamopontine axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions with dendritic shafts as well as dendritic appendages, and occasionally with neuronal somata. Some labeled boutons formed the central axon terminal in a glomerular synaptic complex. In summary, the present findings indicate that the hypothalamus projects predominantly to the rostral medial and dorsomedial portions of the BPN which, in turn, provide input to the paraflocculus and vermis of the cerebellum. Since the hypothalamic projection zones in the BPN also receive cerebral cortical input, including limbic-related cortex, the hypothalamopontine system might serve to integrate autonomic or limbic-related functions with movement or somatic motor-related activity. Alternatively, since the cerebellum also receives direct input from the hypothalamus, the BPN may function to provide additional somatic and visceral inputs that are used by the cerebellum to perform the integrative function. (+info)
Comparing the hypothalamic and extrahypothalamic actions of endogenous hyperleptinemia.
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To determine whether the depletion of body fat caused by adenovirus-induced hyperleptinemia is mediated via the hypothalamus, we used as a "bioassay" for hypothalamic leptin activity the hypothalamic expression of a leptin-regulated peptide, cocaine- and amphetamine-regulated transcript (CART). The validation of this strategy was supported by the demonstration that CART mRNA was profoundly reduced in obese rats with impaired leptin action, whether because of ablation of the ventromedial hypothalamus (VMH) or a loss-of-function mutation in the leptin receptor, as in Zucker diabetic fatty rats. We compared leptin activity in normal rats made hyperleptinemic by adenovirus-leptin treatment (43 +/- 9 ng/ml, cerebrospinal fluid leptin 100 pg/ml) with normal rats made hyperleptinemic by a 60% fat intake (19 +/- 4 ng/ml, cerebrospinal fluid leptin 69 +/- 22 pg/ml). CART was increased 5-fold in the former and 2-fold in the latter, yet in adenovirus-induced hyperleptinemia, body fat had disappeared, whereas in high-fat-fed rats, body fat was abundant. Treatment of the high-fat-fed rats with adenovirus-leptin further increased their hyperleptinemia to 56 +/- 6 ng/ml without changing CART mRNA or food intake, indicating that leptin action on hypothalamus had not been increased. Nevertheless, their body fat declined 36%, suggesting that an extrahypothalamic mechanism was responsible. We conclude that in diet-induced obesity body-fat depletion by leptin requires supraphysiologic plasma concentrations that exceed the leptin-transport capacity across the blood-brain barrier. (+info)
Estrogen selectively regulates spine density within the dendritic arbor of rat ventromedial hypothalamic neurons.
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Estrogen acts in the hypothalamic ventromedial nucleus (VMH) to promote female sexual behavior. One potential mechanism through which estrogen may facilitate this behavior is by reconfiguring synaptic connections within the VMH. Estrogen treatment increases the number of synapses and dendritic spines in the VMH, but how this remodeling occurs within the context of the local, behaviorally relevant microcircuitry is unknown. The goal of this study was to localize estrogen-induced changes in spine density within the VMH and relate these to dendritic morphology and the presence of nuclear estrogen receptor. The hypothalami from ovariectomized rats, treated with either vehicle or estradiol, were lightly fixed, and VMH neurons were iontophoretically filled with Lucifer yellow. Confocal microscopy was used to examine neuronal morphology. Estrogen treatment increased dendritic spine density by 48% in the ventrolateral VMH but had no effect on spine density in the dorsal VMH. The primary dendrites of VMH neurons were differentially affected by estrogen. Estrogen treatment increased spine density twofold on the short primary dendrites but did not affect spine density on long primary dendrites. Immunocytochemical staining showed that none of the filled neurons expressed estrogen receptor-alpha. Thus, although the effect of estrogen on spine density is localized to a VMH subdivision where estrogen receptor is expressed, estrogen treatment induces spines on neurons that lack estrogen receptor. Taken together, our results suggest that the effect of estrogen on ventrolateral VMH spines is selective within the dendritic arbor of a neuron and may be mediated by an indirect, possibly transynaptic, mechanism. (+info)