RESUMEN

Glutamate and norepinephrine transmitter systems play critical roles in the synaptic control of hypothalamic magnocellular neurones. We recently reported on a norepinephrine-sensitive glutamate circuit within the paraventricular nucleus (PVN) that projects to magnocellular neurones. Here, we present evidence for norepinephrine regulation of glutamate release in the PVN and supraoptic nucleus (SON) via actions on presynaptic terminals. Whole-cell synaptic currents were recorded in magnocellular neurones of the SON and PVN in an acute slice preparation. Bath application of norepinephrine (100 microm) caused a robust, reversible increase in the frequency of spontaneous glutamatergic excitatory postsynaptic currents in 100% of SON neurones (246%) and in 88% of PVN magnocellular neurones (259%). The norepinephrine-induced increase in glutamate release was mediated by activation of both presynaptic alpha1 receptors and alpha2 receptors, but the alpha1-receptor component was the predominant component of the response. The presynaptic actions of norepinephrine were predominantly, although not completely, resistant to blockade of Na-dependent spikes, implicating a presynaptic terminal locus of action. Interestingly, the spike-dependent component of the response was greater in PVN than in SON magnocellular neurones. This robust presynaptic facilitation of glutamate release by norepinephrine, combined with the known excitatory postsynaptic actions of norepinephrine, activational effects on local glutamate circuits, and inhibitory effects on gamma-aminobutyric acid release, indicate a strong excitatory role of norepinephrine in the regulation of oxytocin and vasopressin release during physiological stimulation.

Asunto(s)

Ácido Glutámico/metabolismo , Hipotálamo Anterior/fisiología , Norepinefrina/farmacología , Núcleo Hipotalámico Paraventricular/fisiología , Terminales Presinápticos/metabolismo , Simpatomiméticos/farmacología , Anestésicos Locales/farmacología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipotálamo Anterior/citología , Masculino , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Tetrodotoxina/farmacología

RESUMEN

Parvocellular neurones of the hypothalamic paraventricular nucleus (PVN) comprise neurosecretory and non-neurosecretory subpopulations. We labelled neurosecretory neurones with intravenous injection of the retrograde tracer, fluoro-gold, and recorded from fluoro-gold-positive and negative PVN parvocellular neurones in hypothalamic slices. Non-neurosecretory parvocellular neurones generated a low-threshold spike (LTS) and robust T-type Ca2+ current, whereas neurosecretory neurones showed no LTS and a small T-current. LTS neurones were located in non-neurosecretory regions of the PVN, and non-LTS neurones were located in neurosecretory regions of the PVN. These findings indicate that neurosecretory and non-neurosecretory subtypes of parvocellular PVN neurones express distinct membrane electrical properties.

Asunto(s)

Núcleos Talámicos de la Línea Media/citología , Neuronas/fisiología , Potenciales de Acción , Animales , Canales de Calcio Tipo T/fisiología , Ratas

RESUMEN

Noradrenergic projections to the hypothalamic paraventricular nucleus have been implicated in the secretory regulation of several anterior pituitary hormones, including adrenocorticotropin, thyroid-stimulating hormone, growth hormone and prolactin. In an attempt to elucidate the effects of norepinephrine on the central control of pituitary hormone secretion, we looked at the actions of norepinephrine on the electrical properties of putative parvocellular neurons of the paraventricular nucleus using whole-cell current-clamp recordings in hypothalamic slices. About half (51%) of the putative parvocellular neurons recorded responded to norepinephrine with either a synaptic excitation or a direct inhibition. Norepinephrine (30-300microM) caused a marked increase in the frequency of excitatory postsynaptic potentials in about 36% of the parvocellular neurons recorded. The increase in excitatory postsynaptic potentials was blocked by prazosin (10microM), but not by propranolol (10microM) or timolol (20microM), indicating that it was mediated by alpha(1)-adrenoreceptor activation. It was also blocked by ionotropic glutamate receptor antagonists, suggesting that the excitatory postsynaptic potentials were caused by glutamate release. The increase in excitatory postsynaptic potentials was completely abolished by tetrodotoxin, indicating the spike dependence of the norepinephrine-induced glutamate release. In a separate group comprising 14% of the parvocellular neurons recorded, norepinephrine elicited a hyperpolarization (6.2+/-0.69mV) that was blocked by the beta-adrenoreceptor antagonists, propranolol (10microM) and timolol (20microM), but not by the alpha(1)-receptor antagonist, prazosin (10microM). This response was not blocked by tetrodotoxin (1.5-3microM), suggesting that it was caused by a direct postsynaptic action of norepinephrine. The topographic distribution within the paraventricular nucleus of the norepinephrine-responsive and non-responsive parvocellular neurons was mapped based on intracellular biocytin labeling and neurophysin immunohistochemistry. These data indicate that one parvocellular subpopulation, consisting of about 36% of the paraventricular parvocellular neurons, receives an excitatory input from norepinephrine-sensitive local glutamatergic interneurons, while a second, separate subpopulation, representing about 14% of the parvocellular neurons in the paraventricular nucleus, responds directly to norepinephrine with a beta-adrenoreceptor-mediated inhibition. This suggests that excitatory inputs to parvocellular neurons of the paraventricular nucleus are mediated mainly by an intrahypothalamic glutamatergic relay, and that only a relatively small subset of paraventricular parvocellular neurons receives direct noradrenergic inputs, which are primarily inhibitory.

Asunto(s)

Agonistas alfa-Adrenérgicos/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Norepinefrina/metabolismo , Norepinefrina/farmacología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Animales , Mapeo Encefálico , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Técnicas In Vitro , Lisina/análogos & derivados , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/citología , Núcleo Hipotalámico Paraventricular/citología , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/efectos de los fármacos , Receptores Adrenérgicos alfa/fisiología , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/fisiología

RESUMEN

Noradrenergic projections to the hypothalamus play a critical role in the afferent control of oxytocin and vasopressin release. Recent evidence for intrahypothalamic glutamatergic circuits prompted us to test the hypothesis that the excitatory effect of noradrenergic inputs on oxytocin and vasopressin release is mediated in part by local glutamatergic interneurons. The voltage response to norepinephrine (30-300 microM) was tested with whole-cell recordings in putative magnocellular neurons of the paraventricular nucleus (PVN) in hypothalamic slices (400 micrometers). Norepinephrine elicited an alpha1 receptor-mediated direct depolarization in 23% of the magnocellular neurons tested; however, the most prominent response, seen in 42% of the magnocellular neurons, was an alpha1 receptor-mediated increase in the frequency of EPSPs. The norepinephrine-induced increase in EPSPs was blocked by tetrodotoxin and by ionotropic glutamate receptor antagonists, suggesting that norepinephrine excited presynaptic glutamate neurons to cause an increase in spike-mediated transmitter release. The increase in EPSPs also was observed in a surgically isolated PVN preparation (64% of cells) and with microdrop applications of norepinephrine (1 mM, 33% of cells) and glutamate (0.5-1 mM, 28%) in the PVN, indicating that the norepinephrine-sensitive presynaptic glutamate neurons are located within the PVN. Biocytin injection and subsequent immunohistochemical labeling revealed that both oxytocin and vasopressin neurons responded to norepinephrine. Our data indicate that magnocellular neurons of the PVN receive excitatory inputs from intranuclear glutamatergic neurons that express alpha1-adrenoreceptors. These glutamatergic interneurons may serve as an excitatory relay in the afferent noradrenergic control of oxytocin and vasopressin release under certain physiological conditions.

Asunto(s)

Ácido Glutámico/fisiología , Interneuronas/fisiología , Norepinefrina/farmacología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Vías Nerviosas/efectos de los fármacos , Neuronas/química , Neuronas/efectos de los fármacos , Especificidad de Órganos , Oxitocina/análisis , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Vasopresinas/análisis

Asunto(s)

Ácido Glutámico/fisiología , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Núcleo Supraóptico/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Modelos Neurológicos , Neuronas/clasificación , Técnicas de Placa-Clamp , Ratas , Receptores de Glutamato Metabotrópico/efectos de los fármacos

RESUMEN

We conducted whole cell voltage-clamp and current-clamp recordings in slices of rat hypothalamus to test for local excitatory synaptic circuits. Local excitatory inputs to neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were studied with the use of electrical and chemical stimulation. Extracellular electrical stimulation provided indirect evidence of local excitatory circuits. Single stimuli evoked multiple excitatory postsynaptic potentials (EPSPs) or excitatory postsynaptic currents (EPSCs) in some PVN and SON cells, invoking polysynaptic excitatory inputs. Repetitive stimulation (10-20 Hz, 2-10 s) elicited long afterdischarges of EPSPs/EPSCs, suggesting a potentiation of upstream synapses in a polysynaptic circuit. Bath application of metabotropic glutamate receptor agonists provided more conclusive evidence for local excitatory circuits. Metabotropic receptor activation caused an increase in the frequency of EPSPs/EPSCs that was blocked by tetrodotoxin, suggesting that it was mediated by activation of local presynaptic excitatory neurons. The local excitatory inputs to SON and PVN neurons were mediated by glutamate release, because the EPSPs/EPSCs elicited with electrical stimulation and metabotropic receptor activation were blocked by ionotropic glutamate receptor antagonists. Finally, glutamate microstimulation furnished the most direct demonstration of local excitatory synaptic circuits. Glutamate microstimulation of perinuclear sites elicited an increase in the frequency of EPSPs/EPSCs in 13% of the PVN and SON neurons tested. Two sites provided most of the local excitatory synaptic inputs to PVN neurons, the dorsomedial hypothalamus and the perifornical region. These experiments provide converging physiological evidence for local excitatory synaptic inputs to hypothalamic neurons, inputs that may play a role in pulsatile hormone release.

Asunto(s)

Hipotálamo/fisiología , Red Nerviosa/fisiología , Transmisión Sináptica/fisiología , Animales , Técnicas de Cultivo , Potenciales Evocados/fisiología , Ácido Glutámico/fisiología , Masculino , Inhibición Neural/fisiología , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/fisiología , Núcleo Supraóptico/fisiología

RESUMEN

Local inhibitory synaptic inputs to neurons of the rat hypothalamic paraventricular nucleus (PVN) were studied by using glutamate microstimulation and conventional intracellular and whole-cell patch-clamp recording in coronal, horizontal, and parasagittal slices of rat hypothalamus. PVN cells were classified as magnocellular or parvocellular neurons on the basis of electrophysiological and post hoc immunohistochemical analyses; GABA-producing neurons were localized with in situ hybridization. Glutamate microstimulation of different sites around the PVN evoked volleys of postsynaptic potentials in 43% of the PVN neurons tested. Some responses to stimulation at each site were blocked by bicuculline, suggesting that they were mediated by the activation of presynaptic GABA neurons. In the coronal plane, presynaptic inhibitory sites were located lateral to the PVN and ventral to the fornix, corresponding to the lateral hypothalamic area and the posterior bed nucleus of the stria terminalis (BNST). In the horizontal plane, presynaptic inhibitory sites were found rostral, lateral, and caudal to the nucleus, corresponding to parts of the anterior hypothalamic area, the posterior BNST, the medial preoptic area, and the dorsomedial hypothalamus. In the parasagittal plane, presynaptic inhibitory neurons were revealed at sites rostral and caudal to the nucleus, corresponding to the medial preoptic area and the dorsomedial hypothalamus, and in a site dorsal to the optic chiasm that included the suprachiasmatic nucleus. These presynaptic sites each contained GABA-producing neurons based on in situ hybridization with a glutamic acid decarboxylase riboprobe and together formed a three-dimensional ring around the PVN. Unexpectedly, both magnocellular and parvocellular neurons received inhibitory synaptic inputs from common sites.

Asunto(s)

Mapeo Encefálico , Inhibición Neural/fisiología , Núcleo Hipotalámico Paraventricular/citología , Animales , Ácido Glutámico/farmacología , Inmunohistoquímica , Masculino , Vías Nerviosas , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

The medial septal area has been implicated in the control of the magnocellular neurosecretory cells of the hypothalamus, and in particular, in the regulation of neurons secreting oxytocin. The present study investigated the hypothesis that this medial septal pathway originates in the subfornical organ. Brief electrical stimulation of the subfornical organ or of the medial septum both evoked a transient rise in intramammary pressure equivalent to that caused by an i.v. injection of 1 mU oxytocin. The optimal frequency was 5-20 Hz for 5-10 s. Prolonged stimulation also elicited at its onset a single transient response, similar to that evoked by brief stimulation. Extracellular recordings were made from neurosecretory cells of the supraoptic nucleus identified by antidromic stimulation of the neural stalk and further classified as vasopressinergic and oxytocinergic by their reaction at the time of reflex milk ejection induced by suckling. Single-pulse stimulation of the subfornical organ rarely produced excitation, but short trains of stimuli evoked a large excitation in most oxytocinergic and vasopressinergic neurons. To delineate further the pathway from the subfornical organ to the magnocellular neurons, stimulations were combined with various lesions of the medial forebrain. The effects of stimulation of the subfornical organ were abolished after a section immediately rostral to the organ, and in most cases after lesion of the medial septum. Stimulation of the medial septum no longer had an effect after the subfornical organ had been lesioned a week prior to experiments, a period sufficient to allow degeneration of subfornical efferents. This study shows that the excitatory afferent input to the oxytocin and vasopressin-secreting neurons of the hypothalamus from the medial septal area originates in the subfornical organ. This input is not involved in the main afferent control of the milk ejection reflex since lesions of the subfornical organ and of the medial septum had no effect on the reflex. It is suggested, therefore, that the subfornical input to both oxytocin and vasopressin cells intervenes to facilitate synergistic action of both hormones in non-reproductive functions.

Asunto(s)

Lactancia/fisiología , Oxitocina/fisiología , Órgano Subfornical/fisiología , Vasopresinas/fisiología , Animales , Desnervación , Estimulación Eléctrica , Electrofisiología , Femenino , Vías Nerviosas/fisiología , Neuronas/fisiología , Ratas , Ratas Wistar , Reflejo/fisiología , Tabique Pelúcido/fisiología , Núcleo Supraóptico/citología , Núcleo Supraóptico/fisiología

RESUMEN

Abstract The possible role of the medial septum in the control of oxytocin release and of the milk ejection reflex induced by suckling was investigated in lactating rats by using electrical stimulations and lesions. In anaesthetized animals, brief electrical stimulation of the medial septum at 5 to 50 Hz elicited a single brief milk ejection similar to natural reflex milk ejections, whereas prolonged low frequency stimulations (5 to 10 Hz) induced a prolonged inhibition of the reflex. In acute experiments under anaesthesia, lesions of the medial septum did not impair the amplitude and pattern of reflex milk ejections. In chronic experiments, lesions of the medial septum resulted first in a loss of body weight of the mothers and a parallel reduction in growth of the litters. After a few days, the litters gained weight normally, and the pattern of milk ejections was normal. Thus, the pathways which pass through or originate from the medial septum and which are excitatory for oxytocin release appear not to be involved in the regulation of the milk ejection reflex. In view of these results and those from our previous study on the lateral septum, we conclude that the whole septum is not essential to the milk ejection reflex. However, the effects of septal stimulation suggest that the medial and lateral septum may be involved in a secondary neural circuitry that can inhibit the reflex.