Alpha-adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro

Extracellular recordings from 141 rat supraoptic nucleus neurons maintained in vitro in a perfused hypothalamic explant indicated that the excitability of 85% of cells was enhanced by the addition of 10-200 microM norepinephrine (NE) and the alpha-agonist methoxamine (MOXY), but not by isoproterenol, to the perfusion medium or by pressure application. Similar responses were observed from 7 cells maintained in synaptic isolation in medium containing 15 mM Mg2+. At the lowest effective concentration (10 microM), NE and MOXY induced bursting activity without an overall increase in firing frequency; at higher concentrations an initial increase in firing frequency was followed by the appearance of phasic activity patterns. The actions of NE and MOXY were reversibly blocked by administration of the alpha-adrenergic antagonists phenoxybenzamine (1-10 microM), yohimbine (greater than 10 microM) and irreversibly blocked by prazosin (10 nM-1 microM). These observations suggest that NE has a predominantly facilitatory role to enhance the excitability and promote bursting activity in supraoptic neurosecretory neurons through an alpha-1 adrenoreceptor mechanism.     

Glucoresponsive neurons in rat ventromedial hypothalamic tissue slices in vitro

Extracellular action potentials were recorded from rat ventromedial hypothalamic nucleus (VMH) tissue slices in vitro. The identified glucoresponsive and non-glucoresponsive neurons were then visually located and observed by intracellular horseradish peroxidase staining. Glucoresponsive neurons, which were predominantly multipolar, were found near the center of the VMH, while non-glucoresponsive neurons, half of which were bipolar and half multipolar, were found randomly throughout the VMH.     

Independent glucose effects on rat hypothalamic neurons: an in vitro study

The effects of changes in glucose concentration were studied on 256 ventromedial (VMH), 212 dorsomedial (DMH) and 59 lateral (LHA) neurons recorded from coronally-oriented rat hypothalamic slices. When glucose concentration of the medium was increased (5.5-20 mM), these neurons exhibited 3 response patterns: excitation, excitation followed by inhibition, and inhibition. Twenty percent of neurons in VMH, 33% in DMH, and 41% in LHA responded to increases in glucose concentration. The majority in VMH and DMH were excited, and in LHA, inhibited. Only minor modifications of these ratios were obtained by isolating VMH from the other areas. In isolated DMH, equal numbers were excited and inhibited. Both glucose-responsive and non-glucose-responsive neurons in VMH and DMH were identified by intracellular horseradish peroxidase staining. The dendritic arborizations of glucose-responsive neurons were richer than in non-glucose-responsive neurons. These results suggest that the different populations of glucose-responsive neurons in the VMH, DMH and LHA might have different functions in the regulation of glucose.     

Increased secretion of somatostatin-28 from hypothalamic neurons of aged rats in vitro

The purpose of this experiment was to determine whether increased secretion of somatostatin or alterations in its molecular form contribute to the age-related decline in growth hormone secretion. Median eminences were removed from male Sprague-Dawley rats (3–4 and 21–24 months of age) and superfused with Krebs-Ringer bicarbonate buffer with 0.5 mg/ml bacitracin. After 40 min, tissues were stimulated with 55 mM K⁺ for 5 min and fractions collected for 60 min. Tissue and superfusate were analyzed for somatostatin using a highly specific antiserum which cross-reacts with somatostatin-14 and -28. Somatostatin release was expressed as a fractional efflux of somatostatin tissue content. In young rats, somatostatin increased from basal levels of22 ± 5×10⁻⁴ to57 ± 3×10⁻⁴ in response to 55 mM K⁺ and returned to basal levels. Old rats exhibited similar basal levels of somatostatin efflux(25 ± 5×10⁻⁴) but increased to91 ± 19×10⁻⁴ in response to K⁺. This representsan 89% greater increase in somatostatin fractional efflux in old as compared to young rats(P<0.05). The molecular form of somatostatin released during K⁺ stimulation was determined by fractionating samples on Sephadex G-25. In young animals, both somatostatin-14 (31% of total immunoreactivity) and somatostatin-28 (69% of total immunoreactivity) were released by hypothalamic neurons. In old rats, similar absolute levels of somatostatin were released in response to K⁺ but greater quantities of somatostatin-28 (87% of total immunoreactivity) were secreted. We conclude that (1) immunoreactive somatostatin fractional efflux in response to 55 mM K⁺ is greater in old than in young rats, and (2) this increase in somatostatin is due to somatostatin-28, known to exhibit greater biological activity than somatostatin-14 in inhibition of growth hormone release. The increased release of somatostatin-28 in old rats may be partly responsible for the decline in secretion of growth hormone and somatomedin-C, and the reduction in protein synthesis in aging rats.     

α-Adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro

Extracellular recordings from 141 rat supraoptic nucleus neurons maintained in vitro in a perfused hypothalamic explant indicated that the excitability of 85% of cells was enhanced by the addition of 10–200 μM norepinephrine (NE) and the α-agonist methoxamine (MOXY), but not by isoproterenol, to the perfusion medium or by pressure application. Similar responses were observed from 7 cells maintained in synaptic isolation in medium containgng 15 mM Mg²⁺. At the lowest effective concentration (10 μM), NE and MOXY induced bursting activity without an overall increase in firing frequency; at higher concentrations an initial increase in firing frequency was followed by the appearance of phasic activity patterns. The actions of NE and MOXY were reversibly blocked by administration of the α-adrenergic antagonists phenoxybenzamine (1–10 μM), yohimbine (> 10 μM) and irreversibly blocked by prazosin (10 nM−1 μM). These observations suggest that NE has a predominatly facilitatory role to enhance the excitability and promote bursting activity in supraoptic neurosecretory neurons through an α-1 adrenoreceptor mechanism.     

Differentiation of rat hypothalamic dopaminergic neurons is stimulated in vitro by target cells: the melanotrophs

We have investigated in vitro the influence of pituitary intermediate lobe melanotrophs on the differentiation of their afferent hypothalamic dopaminergic neurons. The presence of melanotrophs in primary cultures of foetal hypothalamic neurons induces an increase of the number of dopaminergic neurons (while the total neuronal population remains unchanged) and induces a stimulation of their neuritic outgrowth. These effects are mediated by diffusible factors since they are reproduced by application of conditioned medium issued from co-cultures with intermediate lobe cells from newborn rats. Moreover, by immunoneutralization of α-melanocyte-stimulating hormone (αMSH) in the co-culture or conditioned medium, or by application of the peptide itself, we demonstrate that the neuritotrophic effect on dopaminergic neurons is mediated by αMSH, the main secretory product of melanotrophs, whereas the inductive effect on the number of dopaminergic neurons is attributable to another diffusible neurotrophic factor(s) present in foetal, but not adult, adenohypophysis. Similar effects are observed on cultures of newborn hypothalamic neurons. However, at this stage of neuronal development, αMSH also increases the number of dopaminergic neurons, which could be due to a change of neuronal receptivity. We show that the neuritotrophic influence of αMSH is restricted to the dopaminergic neurons connected to the melanotrophs, and that in addition, these neurons systematically co-express the tyrosine hydroxylase and glutamate decarboxylase as the neurons innervating the melanotrophs in situ. These findings indicate that the differentiation of dopaminergic hypothalamic neurons is influenced by the target cells, melanotrophs, and that this trophic influence implicates αMSH.     

Expression of peptides derived from the melanin-concentrating hormone precursor in serum-free culture of rat fetal hypothalamic neurons: role of attachment factors.

A serum-free medium culture was developed in order to study the secretory behavior of neurons producing the melanin-concentrating hormone (MCH) precursor. The present results show that our culture conditions (supplemented RPMI 1640, poly-D-lysine substrate) are efficient in promoting attachment and growth of MCH neurons dissociated from rat fetal hypothalamus. These neurons acquire a differentiation stage in which neuropeptides of interest to us are expressed in a pattern similar to that observed on tissue sections: (1) coexpression of salmon MCH, growth-hormone-releasing factor (GRF37), alpha-melanocyte-stimulating hormone and acetylcholinesterase immunoreactivities, and (2) different intracellular distribution of salmon MCH and 1-37 sequence of GRF37 staining. Neurite growth was rapid and interneuronal connections were observed early. These observations suggest that our model of defined medium culture is suitable for functional investigations on MCH neurons.     

Effects of mazindol on rat lateral hypothalamic neurons

In order to elucidate the mechanism of action of the anorectic drug, mazindol, effects of electrophoretically applied mazindol were examined on glucose-sensitive and non glucose-sensitive neurons in the rat lateral hypothalamic area (LHA), which is functionally important in food intake control. Mazindol was found to significantly suppress the firing rate of glucose-sensitive neurons. Ouabain a Na-K pump inhibitor, attenuated mazindol induced suppression of neuronal firing rate. Intracellular recordings revealed hyperpolarization of the membrane with no change in membrane conductance by perfusion of brain slice with 0.1 mM mazindol in bath. This was similar to the effect of 30 mM glucose. Results suggest that the inhibitory action of mazindol is mediated by activation of the Na-K pump. Spiroperidol, a dopamine antagonist, did not affect the inhibitory response to mazindol, suggesting direct action of mazindol on LHA neurons, independent of dopamine.     

Intracellular recordings from rat nucleus accumbens neurons in vitro

Intracellular recordings were obtained from rat nucleus accumbens (NAC) neurons in brain slice preparations. Local stimulations evoked depolarizing postsynaptic potential (DPSP). Injections of low intensity depolarizing currents decreased the amplitude of the DPSP and reversed a later portion of the DPSP into a hyperpolarizing potential. Superfusion of pentobarbital facilitated the reversal of this later portion of DPSP and bicuculline abolished this polarity reversal. These data suggested that the DPSP evoked by local stimulation consisted of a combination of an excitatory and an inhibitory postsynaptic potential, and that the latter was probably mediated by gamma-aminobutyric acid.     

Postsynaptic actions of neurotensin on preoptic-anterior hypothalamic neurons in vitro

This study examines the effects of neurotensin (NT) on single neurons in explants of the preoptic-anterior hypothalamus (POAH) in vitro. Standard in vitro electrophysiological techniques were employed. Cultures were prepared from newborn rats and maintained in roller tubes for 3-4 weeks. NT was administered either through the superfusion fluid or via micropressure ejection (0.5-10 psi). Pressure ejection of NT produced a consistent, dose-related, excitatory effect on 71% of the cells studied. The remaining cells were either unresponsive to NT or inhibited by it. In addition, the excitatory effects of microiontophoretically applied glutamate (10-100 nA) were markedly enhanced by NT applied at concentrations that did not alter spontaneous rate. The effects of NT at higher concentrations were additive with glutamate. The effects of NT persisted in Ca2+-free medium when synaptic activity was suppressed. These data indicate that NT exerts a potent excitatory effect on single neurons in the POAH in vitro. Moreover, this effect proved to be additive with that of glutamate. The persistence of these effects in Ca2+-free medium suggests that the actions of NT are postsynaptic in nature.     

Colocalization of proenkephalin peptides in rat brain neurons

A serial, thin-section immunocytochemical study of the anatomical distribution of Leu-enkephalin and BAM-22P (an adrenal proenkephalin peptide) demonstrated that both immunoreactivities occur within the same neurons throughout brain. However, neither peptide immunoreactivity could be observed in neurons containing dynorphin A immunoreactivity. These results are consistent with the possibility that the enkephalin precursor in brain is similar to that sequenced in adrenal, but fail to support the hypothesis that the dynorphin precursor is a major source of Leu-enkephalin in brain.     

Dynamics of 5-hydroxytryptamine released from dopamine neurons in the caudate putamen of the rat

Fast-scan cyclic voltammetry has been used for the detection of 5-hydroxytryptamine (5-HT) in vivo. The applied potential waveform was previously optimized to maximize response times to concentration changes. This technique has been used to reinvestigate 5-HT release from striatal dopaminergic terminals after pharmacological pretreatment as reported by Stamford et al. [13]. Our results concur with those set forth by Stamford and further show that the dopamine transporter is responsible for 5-HT uptake in this experiment.     

Action of neurotropin on rat hypothalamic neurons in tissue slices

To clarify some of the actions of Neurotropin (NSP), intra- and extracellular measurements were made of 140 paraventricular (PVN) and 48 ventromedial nucleus (VMH) neurons in rat hypothalamic tissue slices in vitro while perfusing NSP, an extract from the inflamed skin of rabbits inoculated with vaccinia virus, through the recording chamber. NSP mostly decreased activity in PVN neurons (inhibition, 46; excitation, 27; excitation-inhibition, 5; no response, 62), and mostly increased it in VMH neurons (excitation, 12; inhibition, 3; no response, 33). Inhibition of PVN neurons by NSP was due to hyperpolarization with no change in membrane conductance. Since ouabain antagonized the NSP-induced inhibition of PVN neurons, the effect was probably due to activation of the sodium pump. Activity of some NSP-responsive PVN neurons was increased by increase of extracellular osmolarity, and activity of some NSP-responsive VMH neurons was increased by glucose application. The results suggest central modulation of autonomic or neuroendocrinological function by distinct NSP influence on PVN and VMH neural activity.     

Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.     

Cerebellar modulation of feeding-related neurons in rat dorsomedial hypothalamic nucleus

Cerebellum has newly been implicated in many more nonsomatic functions other than motor control. Previous studies indicate that the cerebellum is involved in feeding regulation and that the gastric vagal nerves transmit short-term meal-related visceral signals, including cholecystokinin (CCK), into the hypothalamus. Recently, the dorsomedial hypothalamic nucleus (DMN) has been thought to play an important role in feeding control. Here we investigate whether the inputs from cerebellar interpositus nucleus (IN) can reach and converge onto single DMN neurons with some feeding-related visceral signals, including gastric vagal inputs, CCK, and blood glucose, whose concentration is closely linked to food intake. Among the 259 DMN neurons recorded, 120 (46.3%) and 169 (65.3%) responded to the cerebellar IN and gastric vagal stimulations, respectively. Within the 120 DMN neurons responsive to the cerebellar IN stimulation, 98 (81.7%) also responded to the gastric vagal stimulus, and a summation of the responses was observed further (n = 20), suggesting a convergence and interaction of cerebellar and gastric vagal inputs on the cells. Moreover, among the 98 cells receiving convergent inputs from cerebellar IN and gastric vagal nerves, 69 (70.4%) were identified to be glycemia sensitive, and 22 (68.8%) of the 32 tested neurons were also sensitive to systemic CCK. These results demonstrate that the DMN integrates somatic information forwarded by the cerebellar IN and visceral signals related to food intake, including gastric vagal, CCK and glycemia, and electrophysiologically reveal a novel cerebellohypothalamic IN-DMN pathway through which the cerebellum may actively participate in short-term feeding regulation.     

Thermal responses of ventromedial hypothalamic neurons in vivo and in vitro

The ventromedial hypothalamic neurons were recorded in anesthetized rats and in slice preparations. Whereas almost all thermosensitive neurons (27 out of 28) in the slice preparations showed warm sensitivity, cold-responsive neurons were recorded more frequently than warm-responsive ones during preoptic thermal stimulation in anesthetized rats. These results suggest that most cold-responsive neurons excited by preoptic cooling in the anesthetized rats were influenced by synaptic inputs.     

Effects of serotonin on perifornical-lateral hypothalamic area neurons in rat

The hypocretin (HCRT) system of the perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the facilitation of behavioral arousal. HCRT neurons receive serotonergic afferents from the dorsal raphe nucleus. Although in-vitro pharmacological studies suggest that serotonin (5-HT) inhibits HCRT neurons, the in-vivo effects of 5-HT on HCRT neurons in the PF-LHA and associated behavioral changes have not been described. We examined the effects of 5-HT delivered locally into the PF-LHA using reverse microdialysis on its neuronal activity and the consequent sleep-wake changes in rats. First, we quantified Fos expression (Fos-IR) in HCRT and other PF-LHA neurons following unilateral 5-HT perfusion in awake rats. Second, we determined the transient effects of 5-HT perfusion on the extracellular activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe. Third, we examined the effects of 5-HT perfusion into the PF-LHA on the sleep-wake profiles of the rats during the lights-off period. Unilateral perfusion of 5-HT into the PF-LHA in awake rats dose-dependently decreased the number of HCRT neurons exhibiting Fos-IR. 5-HT also inhibited the discharge activity of four of five responsive wake-related, putative HCRT neurons. However, unilateral perfusion of 5-HT into the PF-LHA did not produce significant behavioral changes during the 2-h recording period. These results confirm the in-vitro findings that 5-HT exerts inhibitory influences on HCRT neurons but further suggest that the inactivation of a limited number of HCRT neurons by unilateral 5-HT microdialysis may not be sufficient to induce behavioral changes.     

Burst firing of oxytocin neurons in male rat hypothalamic slices

Burst firing and single spike activity play different roles in the modulation of local neuronal circuit activity and neurosecretion. In hypothalamic oxytocin (OT) neurons in vivo, burst firing is associated with pulsatile secretion of OT in the milk ejection reflex, and can be observed in slices from both immature and lactating rats in vitro. Whether OT neurons from male rats also possess burst firing capability is still an open question. To examine this possibility, whole-cell patch clamp recordings were made in supraoptic nucleus OT neurons in brain slices from male rats. In low Ca(2+) medium, the alpha(1)-adrenoceptor agonist, phenylephrine evoked bursts that were highly similar to those from lactating rats in vivo and in vitro: explosive onset, short-duration, quickly reaching peak firing rate and displaying an exponential decay in returning to the pre-burst rate. During bursts, spike durations increased, and spike amplitudes decreased, while riding on an arc of depolarization around peak rate. In comparison to those from lactating rats in vitro, the rising phase of male bursts was more rapid, the decay phase was slower, and the rising phase of the spike after hyperpolarization was faster. No significant differences, however, were seen in burst characteristics that are most important in determining the amount of peptide release: burst amplitudes (the number of spikes in a burst), firing frequency within bursts or peak firing rate. Thus, we conclude that OT neurons in males are capable of burst firing highly similar to that seen in lactating rats.