Hypothalamic Magnocellular Neurosecretory Cells Research Articles

Somato-dendritic vasopressin and oxytocin secretion in endocrine and autonomic regulation.

Somato-dendritic secretion was first demonstrated over 30years ago. However, although its existence has become widely accepted, the function of somato-dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato-dendritic secretion was demonstrated and are among the neurones for which the functions of somato-dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato-dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra- and inter-population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato-dendritic vasopressin and oxytocin have also been proposed to act as hormone-like signals in the brain. There is some evidence that somato-dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin- or oxytocin-containing axons but, to date, there is no conclusive evidence for, or against, hormone-like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.

Lactation induces increased IPSC bursting in oxytocinergic neurons.

Hypothalamic magnocellular neurosecretory cells (MNCs) undergo dramatic structural reorganization during lactation in female rats that is thought to contribute to the pulsatile secretion of oxytocin critical for milk ejection. MNCs from male rats generate robust bursts of GABAergic synaptic currents, a subset of which are onset‐synchronized between MNC pairs, but the functional role of the IPSC bursts is not known. To determine the physiological relevance of IPSC bursts, we compared MNCs from lactating and non–lactating female rats using whole–cell recordings in brain slices. We recorded a sixfold increase in the incidence of IPSC bursts in oxytocin (OT)‐MNCs from lactating rats compared to non–lactating rats, whereas there was no change in IPSC bursts in vasopressin (VP)‐MNCs. Synchronized bursts of IPSCs were observed in pairs of MNCs in slices from lactating rats. Our data indicate, therefore, that IPSC bursts are upregulated specifically in OT‐MNCs during lactation, and may, therefore, contribute via rebound depolarization to the spike trains in OT neurons that lead to reflex milk ejection.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

Altered NMDA receptor-evoked intracellular Ca2+ dynamics in magnocellular neurosecretory neurons of hypertensive rats.

NMDA receptor (NMDAR)-mediated Ca2+ signalling plays a critical role in modulating hypothalamic neurosecretory function. However, whether an altered NMDAR-evoked changes in Ca2+ (NMDAR-ΔCa2+ ) signalling in magnocellular neurosecretory cells (MNCs) may contribute to neurohumoral activation during disease states is unknown. We show that activation of NMDARs evoked similar inward currents in MNCs of sham and renovascular hypertensive (RVH) rats. Despite this, a prolonged and larger NMDAR-ΔCa2+ response was observed in the latter. The exacerbated NMDAR-ΔCa2+ responses in MNCs of RVH rats affected both somatic and dendritic compartments. Inhibition of the endoplasmic reticulum sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump prolonged NMDAR-ΔCa2+ responses in sham rats, but not in RVH rats. Our study supports an altered spatiotemporal dynamic of NMDAR-ΔCa2+ signalling in MNCs from RVH rats, partly due to blunted endoplasmic reticulum Ca2+ buffering capacity. A growing body of evidence supports an elevated NMDA receptor (NMDAR)-mediated glutamate excitatory function in the supraoptic nucleus and paraventricular nucleus of hypertensive rats that contributes to neurohumoral activation in this disease. However, the precise mechanisms underlying altered NMDAR signalling in hypertension remain to be elucidated. In this study, we performed simultaneous electrophysiology and fast confocal Ca2+ imaging to determine whether altered NMDAR-mediated changes in intracellular Ca2+ levels (NMDAR-ΔCa2+ ) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in renovascular hypertensive (RVH) rats. We found that despite evoking a similar excitatory inward current, activation of NMDARs resulted in a larger and prolonged ΔCa2+ in MNCs from RVH rats. Changes in NMDAR-ΔCa2+ dynamics were observed both in somatic and dendritic compartments. Inhibition of the sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump activity with thapsigargin prolonged NMDAR-ΔCa2+ responses in MNCs of sham rats, but this effect was occluded in RVH rats, thus equalizing the magnitude and time course of the NMDA-ΔCa2+ responses between the two experimental groups. Taken together, our results support (1) an exacerbated NMDAR-ΔCa2+ response in somatodendritic compartments of MNCs of RVH rats, and (2) that a blunted ER Ca2+ buffering capacity contributes to the altered NMDAR-ΔCa2+ dynamics in this condition. Thus, altered spatiotemporal dynamics of the NMDAR-ΔCa2+ response stands as an underlying mechanism contributing to neurohumoral activation in neurogenic hypertension.

Effects of Salt Loading on the Regulation of Rat Hypothalamic Magnocellular Neurosecretory Cells by Ionotropic GABA and Glycine Receptors.

Synaptic and extrasynaptic transmission mediated by ionotropic GABA and glycine receptors plays a critical role in shaping the action potential firing (spiking) activity of hypothalamic magnocellular neurosecretory cells and therefore determines the rate at which vasopressin and oxytocin are released from the neurohypophysis. The inhibitory effect of these transmitters relies on the maintenance of a low concentration of intracellular chloride ions such that, when activated by GABA or glycine, a hyperpolarisation of the neuronal membrane potential results. In this review, we highlight the various ways by which the two types of inhibitory receptors contribute to homeostasis by fine-tuning the spiking rate of vasopressin-releasing magnocellular neurosecretory cells in a manner dependent on the hydration state of the animal. In addition, we review the currently available evidence on how the strength of these inhibitory pathways can be regulated during chronic hypernatraemia via a form of activity-dependent depolarisation of the chloride reversal potential, leading to an abolition of these inhibitory pathways potentially causing sodium-dependent elevations in blood pressure.

Induction of hypertension blunts baroreflex inhibition of vasopressin neurons in the rat.

Vasopressin secretion from the posterior pituitary gland is determined by action potential discharge of hypothalamic magnocellular neurosecretory cells. Vasopressin is a potent vasoconstrictor, but vasopressin levels are paradoxically elevated in some patients with established hypertension. To determine whether vasopressin neurons are excited in hypertension, extracellular single-unit recordings of vasopressin neurons from urethane-anaesthetized Cyp1a1-Ren2 rats with inducible angiotensin-dependent hypertension were made. The basal firing rate of vasopressin neurons was higher in hypertensive Cyp1a1-Ren2 rats than in non-hypertensive Cyp1a1-Ren2 rats. The increase in firing rate was specific to vasopressin neurons because oxytocin neuron firing rate was unaffected by the induction of hypertension. Intravenous injection of the α1-adrenoreceptor agonist, phenylephrine (2.5 μg/kg), transiently increased mean arterial blood pressure to cause a baroreflex-induced inhibition of heart rate and vasopressin neuron firing rate (by 52 ± 9%) in non-hypertensive rats. By contrast, intravenous phenylephrine did not inhibit vasopressin neurons in hypertensive rats, despite a similar increase in mean arterial blood pressure and inhibition of heart rate. Circulating angiotensin II can excite vasopressin neurons via activation of afferent inputs from the subfornical organ. However, the increase in vasopressin neuron firing rate and the loss of inhibition by intravenous phenylephrine were not blocked by intra-subfornical organ infusion of the angiotensin AT1 receptor antagonist, losartan. It can be concluded that increased vasopressin neuron activity at the onset of hypertension is driven, at least in part, by reduced baroreflex inhibition of vasopressin neurons and that this might exacerbate the increase in blood pressure at the onset of hypertension.

Fluorescent Visualisation of Oxytocin in the Hypothalamo-neurohypophysial/-spinal Pathways After Chronic Inflammation in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rats.

Oxytocin (OXT) is a well-known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT-mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT-mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up-regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN-spinal pathway may be involved in sensory modulation. OXT-mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in invitro preparations.

GABAergic inhibition is weakened or converted into excitation in the oxytocin and vasopressin neurons of the lactating rat.

BackgroundIncreased secretion of oxytocin and arginine vasopressin (AVP) from hypothalamic magnocellular neurosecretory cells (MNCs) is a key physiological response to lactation. In the current study, we sought to test the hypothesis that the GABAA receptor-mediated inhibition of MNCs is altered in lactating rats.ResultsGramicidin-perforated recordings in the rat supraoptic nucleus (SON) slices revealed that the reversal potential of GABAA receptor-mediated response (EGABA) of MNCs was significantly depolarized in the lactating rats as compared to virgin animals. The depolarizing EGABA shift was much larger in rats in third, than first, lactation such that GABA exerted an excitatory, instead of inhibitory, effect in most of the MNCs of these multiparous rats. Immunohistochemical analyses confirmed that GABAergic excitation was found in both AVP and oxytocin neurons within the MNC population. Pharmacological experiments indicated that the up-regulation of the Cl− importer Na+-K+-2Cl− cotransporter isotype 1 and the down-regulation of the Cl− extruder K+-Cl− cotransporter isotype 2 were responsible for the depolarizing shift of EGABA and the resultant emergence of GABAergic excitation in the MNCs of the multiparous rats.ConclusionWe conclude that, in primiparous rats, the GABAergic inhibition of MNCs is weakened during the period of lactation while, in multiparous females, GABA becomes excitatory in a majority of the cells. This reproductive experience-dependent alteration of GABAergic transmission may help to increase the secretion of oxytocin and AVP during the period of lactation.

Purinergic signaling pathways in endocrine system.

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling.

A functional coupling between extrasynaptic NMDA receptors and A-type K+ channels under astrocyte control regulates hypothalamic neurosecretory neuronal activity.

Neuronal activity is controlled by a fine-tuned balance between intrinsic properties and extrinsic synaptic inputs. Moreover, neighbouring astrocytes are now recognized to influence a wide spectrum of neuronal functions. Yet, how these three key factors act in concert to modulate and fine-tune neuronal output is not well understood. Here, we show that in rat hypothalamic magnocellular neurosecretory cells (MNCs), glutamate NMDA receptors (NMDARs) are negatively coupled to the transient, voltage-gated A-type K(+) current (IA). We found that activation of NMDARs by extracellular glutamate levels influenced by astrocyte glutamate transporters resulted in a significant inhibition of IA. The NMDAR-IA functional coupling resulted from activation of extrasynaptic NMDARs, was calcium- and protein kinase C-dependent, and involved enhanced steady-state, voltage-dependent inactivation of IA. The NMDAR-IA coupling diminished the latency to the first evoked spike in response to membrane depolarization and increased the total number of evoked action potentials, thus strengthening the neuronal input/output function. Finally, we found a blunted NMDA-mediated inhibition of IA in dehydrated rats. Together, our findings support a novel signalling mechanism that involves a functional coupling between extrasynaptic NMDARs and A-type K(+) channels, which is influenced by local astrocytes. We show this signalling complex to play an important role in modulating hypothalamic neuronal excitability, which may contribute to adaptive responses during a sustained osmotic challenge such as dehydration.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

Chronic Hyperosmotic Stress Converts GABAergic Inhibition into Excitation in Vasopressin and Oxytocin Neurons in the Rat

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.

Metaplasticity of Hypothalamic Synapses following In Vivo Challenge

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Cerebral Complications of Diabetes Mellitus

According to WHO estimates, more than 180 million people suffer from diabetes mellitus worldwide and this number could double within 15 years. Normal function of the brain is dependent on continuous supply of glucose. In hypoglycemia, production of counterregulatory hormones (glucagon, epinephrine, growth hormone, and cortisol) increases, the sympathetic system becomes stimulated, and features of neuroglycopenia appear in order to save the homeostasis. Hypoglycemia is an alarming, actually life-threatening condition, but the exposures to chronic hyperglycemia have a more detrimental effect on the brain than recurrent exposure to severe hypoglycemia. The active neural response to hyperglycemia induces changes in gene expression and function. The first steps against hyperosmolality are initially adaptive, but later hyperactivation of the hypothalamic magnocellular neurosecretory cells leads to their structural damage. Changes in hippocampal gene transcription are partially implicated in the deterioration of declarative memory. Neurologically passive shunting of excess glucose through alternative cellular metabolic pathways induces atherogenic, vascular lesions, free radicals, leukoencephalopathy and atrophy of the brain, thus leading to cognitive deficits. In physiological conditions insulin has a neuroprotective effect. However, insulin resistance in the central nervous system correlates with insulin resistance in the periphery. Loss of responsiveness to insulin could render neurons more susceptible to neurotoxic insults, the protective effect of insulin diminishes, and apoptosis, neurodegeneration and the resultant cognitive decline are all increased in insulin-resistant patients. Some unclear relations appear between diabetes mellitus and Alzheimer’s disease. Diabetic patients with APOE-4 gene have an increased risk for Alzheimer’s disease. Prevalence of depression is higher in patients with diabetes mellitus and in turn, depression is a risk factor for diabetes mellitus. Simultaneous presence of depression and diabetes mellitus tends to worsen the course of both.

Cerebral Complications of Diabetes Mellitus

According to WHO data more than 180 million people suffer from diabetes mellitus worldwide and this number could double within 15 years. Normal function of the brain is dependent on continuous supply of glucose. In hypoglycemia, production of counterregulatory hormones (glucagon, epinephrine, growth hormone, and cortisol) increases, the sympathetic system becomes stimulated, and features of neuroglycopenia appear in order to save the homeostasis. Hypoglycemia is an alarming, actually life threatening condition, but the exposure to chronic hyperglycemia has a more detrimental effect on the brain than recurrent exposure to severe hypoglycemia. The active neural response to hyperglycemia induces changes in gene expression and function. The first steps against hyperosmolality are initially adaptive, but later hyperactivation of the hypothalamic magnocellular neurosecretory cells leads to their structural damage. Changes in hippocampal gene transcription are partially implicated in the deterioration of declarative memory. Neurologically passive shunting of excess glucose through alternative cellular metabolic pathways induces atherogenic, vascular lesions, free radicals, leukoencephalopathy and atrophy of the brain and thus leading to cognitive deficits. In physiological conditions insulin has neuroprotective effect. However, insulin resistance in the central nervous system correlates with insulin resistance in the periphery. Loss of responsiveness to insulin could render neurons more susceptible to neurotoxic insults, the protective effect of insulin diminishes, and apoptosis, neurodegeneration and the resultant cognitive decline are all increased in insulin-resistant patients. Some unclear relations appear between diabetes mellitus and Alzheimer's disease. Diabetic patients with APOE-4 gene have an increased risk for Alzheimer's disease. Prevalence of depression is higher in patients with diabetes mellitus and in turn, depression is a risk factor for diabetes mellitus. Simultaneous presence of depression and diabetes mellitus tends to worsen the course of both.

Dopamine Modulates Use-Dependent Plasticity of Inhibitory Synapses

The release of the hormones oxytocin (OT) and vasopressin (VP) into the circulation is dictated by the electrical activity of hypothalamic magnocellular neurosecretory cells (MNCs). In the paraventricular nucleus of the hypothalamus (PVN), MNC neuronal activity is exquisitely sensitive to changes in input from inhibitory GABAergic synapses. To explore the hypothesis that efficacy at these synapses is dictated by the rate at which a given synapse is activated, we obtained whole-cell recordings from MNCs in postnatal day 21-27 male Sprague Dawley rat brain slices. IPSCs were elicited by electrically stimulating GABAergic projections from either the suprachiasmatic nucleus or putative interneuron populations immediately ventral to the fornix at 5, 10, 20, and 50 Hz. Short-term plasticity was observed at 88% of the synapses tested. Of this group, synaptic depression was observed in 58%, and synaptic facilitation was observed in 41%. Identification of cells using a combined electrophysiological and immunohistochemical approach revealed a strong correlation between cell phenotype and the nature of the plasticity. Short-term facilitation was observed preferentially in OT cells (86%), whereas short-term depression was predominant in VP neurons (69%). We next examined the effects of dopamine, which increases MNC excitability, on short-term plasticity. Activation of presynaptic D(4) receptors decreased the frequency of miniature IPSCs and prevented the development of synaptic depression at higher rates of activity. Synaptic facilitation, however, was unaffected by dopamine. These findings demonstrate that, by lowering GABA release probability, dopamine confers high-pass filtering properties to the majority of inhibitory synapses onto MNCs in PVN.

Local Morphine Withdrawal Increases c-fosGene, Fos Protein, and Oxytocin Gene Expression in Hypothalamic Magnocellular Neurosecretory Cells

We measured stimulation of c-fos and oxytocin gene expression during excitation of oxytocin cells induced by systemic or local morphine withdrawal. Female rats were made morphine-dependent by intracerebroventricular morphine infusion over 5 d. Morphine withdrawal, induced by systemic injection of the opioid antagonist naloxone (5 mg/kg) in conscious or anesthetized rats, increased the density of c-fos messenger RNA and of oxytocin heterogeneous nuclear RNA in supraoptic nucleus cells compared with those of nonwithdrawn rats; c-fos messenger RNA was also increased in the magnocellular and parvocellular paraventricular nuclei of withdrawn rats. Morphine withdrawal increased the number of Fos-immunoreactive cells in the supraoptic and magnocellular paraventricular nuclei of conscious or pentobarbitone-anesthetized rats. Morphine withdrawal also increased Fos-immunoreactive cell numbers in the parvocellular paraventricular nucleus of conscious but not anesthetized rats. Central administration of the alpha(1)-adrenoreceptor antagonist benoxathian (5 microg/min) did not prevent morphine withdrawal-induced increases in the numbers of Fos-immunoreactive neurons in the supraoptic or magnocellular paraventricular nucleus. Unilateral microdialysis administration of naloxone (10(-5) M) into the supraoptic nucleus of anesthetized morphine-dependent rats increased Fos-immunoreactive cell numbers compared with the contralateral nucleus. Finally, we investigated whether dependence could be induced by chronic unilateral infusion of morphine into a supraoptic nucleus; systemic naloxone (5 mg/kg) increased Fos-immunoreactive cell numbers in the morphine-infused nucleus compared with the contralateral nucleus. Thus, morphine withdrawal excitation increases c-fos and oxytocin gene expression in supraoptic nucleus neurons. This occurs independently from excitation of their ascending noradrenergic inputs, and both dependence and withdrawal can be induced within the supraoptic nucleus.

Galanin Inhibits Continuous and Phasic Firing in Rat Hypothalamic Magnocellular Neurosecretory Cells

The effects of galanin (GAL) on magnocellular neurosecretory cells (MNCs) were examined during microelectrode recordings from supraoptic neurons in superfused hypothalamic explants. Application of the full-length peptide (GAL1-29) or of the N-terminal fragment GAL1-16 produced reversible membrane hyperpolarization with an IC50 near 10 nM. These effects were associated with an increase of membrane conductance, with a reversal potential near -70 mV, and were not blocked by tetrodotoxin, indicating that the receptors mediating these effects are located postsynaptically. Hyperpolarizing responses were also observed in response to the GAL-like chimeric ligands M35 and M40, suggesting that these behave as partial agonists at galanin receptors. The reversal potential of the GAL-mediated effect was unaffected by reducing extracellular chloride or by intracellular chloride injection, indicating that the effects of galanin are not mediated by modulation of chloride conductances. In contrast, reducing the external concentration of potassium ions from 3 to 1 mM shifted the reversal potential of the responses to -85 mV, suggesting the involvement of a potassium conductance. When tested on spontaneously active MNCs, the hyperpolarizing effects of galanin were associated with a suppression of firing in both continuously active and phasically active neurons. Inhibition of phasic bursts was mediated both through the inhibitory effects of the hyperpolarization and through a GAL-mediated inhibition of the depolarizing afterpotential that is responsible for the production of individual bursts. These results suggest that galanin may be a potent endogenous modulator of firing pattern in hypothalamic neuroendocrine cells.

Chapter 24 Synaptic and neurotransmitter regulation of activity in mammalian hypothalamic magnocellular neurosecretory cells

Publisher Summary This chapter describes the synthesis of recent observations on synaptic and transmitter regulation of activity in neurohypophysial neurons on the basis of data mainly obtained in the rat supra-optic nucleus and emphasizes on vasopressin (VP)-synthesizing neurons and an elaboration of a portion of known inputs and associated transmitters regulating their excitability, particularly those of cardiovascular origin. The magnocellular neurons of the neurohypophysial system synthesize VP or oxytoxin (OXY) for release into the systemic circulation in the neurohypophysis. Oxytocin and vasopressin have been shown to influence the excitability of central neurons and to have a transmitter-like role, part of which is directed toward the magnocellular neurons themselves. Magnocellular neurons also contain several co-existing peptides, of which the opioid peptides have been most intensely studied. Opioid peptides are also present in afferent pathways to neurosecretory neurons and appear to have a predominantly depressant action on their excitability while other peptides located in pathways to magnocellular neurons include inhibin-related peptides and substance P.

Functional N‐Methyl‐D‐Aspartate and Non‐N‐Methyl‐D‐Aspartate Receptors are Expressed by Rat Supraoptic Neurosecretory Cells in vitro

Abstract A considerable amount of evidence has accumulated to support a role for excitatory glutamatergic transmission in the regulation of the hypothalamo-neurohypophysial system. Glutamate immunoreactivity has been found in axon terminals forming asymmetric synapses on to magnocellular neurosecretory cells and kynurenic acid, a broad spectrum glutamate receptor antagonist inhibits 1) spontaneous electrical activity in vivo, 2) excitatory postsynaptic potentials in hypothalamic slices, and 3) osmotically-evoked vasopressin release from hypothalamic explants. While this provides strong evidence for glutamatergic regulation of hypothalamic magnocellular neurosecretory cells, the subtypes of glutamate receptors expressed by these cells have not been defined. We have, therefore, obtained current and voltage clamp recordings from supraoptic magnocellular neurosecretory cells in vitro to investigate the functional and pharmacological properties of their glutamate receptors. Application of micromolar concentrations of L-glutamate, or of the agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA), produced reversible and dose-dependent depolarizations in all cells tested. These responses were mediated by postsynaptic receptors since they persisted during chemical synaptic blockade with Ca(2 +) -free or tetrodotoxin-containing solutions. The inward current induced by NMDA showed a marked Mg(2+)-sensitive voltage dependence, and was blocked by D, L-2-amino-5-phosphonovalerate. In contrast, currents induced by kainate and quisqualate showed linear current-voltage properties and were antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione. We conclude that both NMDA and non-NMDA receptors are expressed by magnocellular neurosecretory cells of the rat supraoptic nucleus.