Hypothalamic Supraoptic Neurons Research Articles

CIRCADIAN DYNAMICS OF OPTIC DENSITY OF MELATONIN RECEPTORS IN THE NEURONS OF HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RATS UNDER ALTERED PHOTOPERIOD

Introduction. Melatonin production is considerably suppressed by light and affects the ability to transfer daily rhythm information from the hypothalamus to other neural target sites and thus alters the expression of some biological rhythms. The hormone controls the state of the hypothalamic-pituitary system and endocrine gland activity through melatonin receptors (membrane, cytosolic and nuclear ones). In addition, using a mechanism of the feedback, it interferes with the activity of supraoptic nucleus of the hypothalamus, which regulates water-salt metabolism and responses to stress. Objective: to provide quantitative circadian characteristics of melatonin receptors density in the neurons of the hypothalamic supraoptic nucleus of rats being under light stimulation as well as the correction of changes after injecting exogenous melatonin. Material and methods. The experiments were conducted on 60 white mongrel mature male rats weighing 150 – 180 g. The test animals were divided into 3 parts each with 2 groups, kept under the conditions of standard light regime, hyperilluminated and the injection of exogenous melatonin and day-round lighting within 7 days. To perform immunohistochemical methods, we used polyclonal antibodies to melatonin 1A receptors produced by Abcam and streptavidin biotin visualization system LSAB2 (peroxidase mark + diaminobenzidine) produced by Chemicon International Inc. We adhered to protocol standardization of methods for all sections. Additional staining of nuclei was performed with Mayer hematoxylin. Results. The indices of optical density of specific melatonin 1A receptors of supraoptic neurocytes staining obtained in the intact group (at 02.00 AM- 0,488 ± 0,0024, at 02.00 P.M. - 0,464 ± 0,0023, p = 0.002) and in animals subjected to light stress (at 02.00 AM-0,295 ± 0, 0019, at 02.00 P.M.- 0,286 ± 0,0018, p = 0,012) had a probable value and were characterized by a clear diurnal periodicity. In the group of animals with pineal gland hypofunction modulation (at 02.00 A.M.- 0,216 ± 0,0017, at 02.00 P.M. - 0,214 ± 0,0021, p> 0,05). Conclusions The density of 1A melatonin receptors in rat’s hypothalamic supraoptic neurons are normally characterized by an accurate circadian rhythm. The highest density of receptors is observed at 02.00 AM, and at 02.00 PM it is significantly lower (p = 0.002). Immunohistochemical studies revealed that under inhibition of pineal gland activity the circadian rhythm of melatonin receptors density in neurons of supraoptic nuclei of the hypothalamus gets disturbed, which is characterized by an incredible difference of indices in the tested periods of the day.

Variation in sodium current amplitude between vasopressin and oxytocin hypothalamic supraoptic neurons

Biophysical characteristics of tetrodotoxin-sensitive sodium (Na(+)) currents were studied in vasopressin (VP) and oxytocin (OT) supraoptic neurons acutely isolated from rat hypothalamus. Na(+) current density (pA/pF) was significantly greater in VP neurons than in OT neurons. No significant difference between VP and OT neurons was detected regarding the voltage dependence of activation and steady-state inactivation, or rate of recovery from inactivation of Na(+) currents. In both VP and OT neurons, the macroscopic inactivation of the Na(+) currents was best fitted with a double-exponential expression suggesting two rates of inactivation. Also in both types, the time course of recovery from inactivation proceeded with fast and slow time constants averaging around 8 and 350 ms, respectively, suggesting the presence of multiple pathways of recovery from inactivation. The slower time constant of recovery of inactivation may be involved in the decrease in action potential (AP) amplitude that occurs after the first spike during burst firing in both neuronal types. The larger amplitude of Na(+) currents in VP vs. OT neurons may explain the previous observations that VP neurons exhibit a lower AP threshold and greater AP amplitude than OT neurons, and may serve to differently tune the firing properties and responses to neuromodulators of the respective neuronal types.

High-threshold, Kv3-like potassium currents in magnocellular neurosecretory neurons and their role in spike repolarization.

We identified Kv3-like high-threshold K+ currents in hypothalamic supraoptic neurons using whole cell recordings in hypothalamic slices and in acutely dissociated neurons. Tetraethylammonium (TEA)-sensitive currents (< 1 mM TEA) evoked from -50 mV were characterized by a large component that inactivated in 10-30 ms, and a smaller, persistent component that inactivated in 1-2 s. I/V relations in dissociated neurons revealed TEA-subtracted currents with a slope and voltage dependency consistent with the presence of Kv3-like channels. In slices, tests with 0.01-0.7 mM TEA produced an IC50 of 200-300 nM for both fast and persistent currents. The fast transient current was similar to currents associated with the expression of Kv3.4 subunits, given that it was sensitive to BDS-I (100 nM). The persistent TEA-sensitive current appeared similar to those attributed to Kv3.1/3.2 subunits. Although qualitatively similar, oxytocin (OT) and vasopressin (VP) neurons in slices differed in the stronger presence of persistent current in VP neurons. In both cell types, the IC50 for TEA-induced spike broadening was similar to that observed for current suppression in voltage clamp. However, TEA had a greater effect on the spike width of VP neurons than of OT neurons. Immunochemical studies revealed a stronger expression of the Kv3.1b alpha-subunit in VP neurons, which may be related to the greater importance of this current type in VP spike repolarization. Because OT and VP neurons are not considered fast firing, but do exhibit frequency- and calcium-dependent spike broadening, Kv3-like currents may be important for maintaining spike width and calcium influx within acceptable limits during repetitive firing.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

Time course of Fos and Fras expression in the hypothalamic supraoptic neurons during chronic osmotic stimulation

The Fos family comprises Fos and several subtypes of Fos-related proteins (Fras) such as FosB, Fra-1, Fra-2, ΔFosB, and chronic Fras. Changes in the expression of Fos family proteins with time are not well elucidated, particularly during chronic stimulation. In the present experiments, we investigated quantitatively the time course changes in Fos, FosB and Fras immunoreactivity in the magnocellular neurons of the supraoptic nucleus (SON) during acute and chronic osmotic stimulation. A small number of Fos- and FosB-positive neurons were observed in the SON of control rats, while many Fras-positive neurons were seen in control animals. Significant increases in the numbers of Fos-, FosB-, and Fras-positive neurons were observed 2 h after acute osmotic stimulation by intraperitoneal (i.p.) injection of 3% NaCl solution. Although the number of Fos-positive neurons returned to the control level 4 h after i.p. injection, a significant number of FosB- and Fras-positive neurons were still observed 8 h after i.p. injection. During chronic osmotic stimulation by giving 2% NaCl solution for 2 and 5 days, a large number of Fos-positive neurons were observed, but the cessation of chronic osmotic stimulation by normal water drinking immediately decreased the number of Fos-positive neurons to the control level within 2 h. The number of FosB-positive neurons was increased with period of chronic osmotic stimulation, and a significant number were observed 2–8 h after the cessation of the stimulation. The number of Fras-positive neurons was also significantly higher during chronic osmotic stimulation, and this number was significantly high 2–8 h after the cessation of the stimulation. RT-PCR analysis demonstrated the persistent expression of c-fos mRNA in the SON during chronic osmotic stimulation. These results suggest that c-fos mRNA and Fos protein are constitutively elevated during chronic osmotic stimulation and the time course changes in Fos are different from those seen in FosB and Fras.

Inhibition of spontaneous inhibitory postsynaptic currents (IPSC) by noradrenaline in rat supraoptic neurons through presynaptic α 2-adrenoceptors

It has been shown that noradrenergic activation has great influence on the activities of hypothalamic supraoptic neurons. No direct evidence has been reported on the presynaptic effects of adrenoceptors in the actions of noradrenaline on supraoptic neurons, although postsynaptic mechanisms have been studied extensively. In the present study, we explored presynaptic effects of noradrenaline on the supraoptic neurons by measuring spontaneous inhibitory postsynaptic currents (IPSC) with the whole-cell patch-clamp technique. Noradrenaline reduced the frequency of IPSCs in a dose-dependent (10 −9 to 10 −3 M) and reversible manner. Noradrenaline did not affect the amplitude of IPSCs at concentrations of 10 −9 to 10 −5 M, but reduced the amplitude of IPSCs at high concentrations (10 −4 and 10 −3 M). The inhibitory effects of noradrenaline were mimicked by the α 2-agonist clonidine (10 −4 M), but not by the α 1-agonist methoxamine (10 −4 M) nor by the β-agonist isoproterenol (10 −4 M). Moreover, the inhibitory effects of noradrenaline on IPSCs were blocked by the non-selective α antagonist phentolamine (10 −4 M) or the selective α 2-antagonist yohimbine (10 −4 M), but not by the α 1-antagonist prazosin (10 −4 M). These results suggest that noradrenaline inhibits release of GABA from the presynaptic GABAergic terminals of the supraoptic neurons by activating presynaptic α 2-adrenoceptors and such presynaptic mechanisms may play a role in the excitatory control of SON neurons by noradrenergic neurons.

Morphological and electrophysiological classification of hypothalamic supraoptic neurons

In mammals, the magnocellular neurons of the supraoptic nucleus (SON) have been classified into vasopressin- (VP) and oxytocin- (OT) producing subtypes. The degree to which these neurons have distinguishable characteristics is considered in the present review. Most of the cytoarchitectonic diversity observed in some Golgi studies has yet to be attributed to differences between OT and VP neurons. The predominant SON cell type is a large bipolar neuron with relatively short and simply branching dendrites. Based on intracellular filling, large multipolar neurons probably represent a small subset of neurosecretory cells. Parvicellular multipolar and bipolar neurons may represent interneurons or subsets of neurosecretory cells. Suggestive evidence that axonal origin and spine density may differ between OT and VP neurons remains to be confirmed in rat. Different fiber systems are thought to preferentially innervate VP or OT subgroups, but only rarely have inputs to OT and VP neurons been compared at the ultrastructural level. Potentially selective inputs to OT somata may derive from the raphe system and the nucleus of the solitary tract, whereas the apparent preferential innervation of VP neurons (e.g. from the A1 region of the ventrolateral medulla) is less certain because of the overlapping dendritic fields of OT and VP neurons. Electrophysiologically, OT and VP neurons are best distinguished in vivo by their reaction to gastric, cardiovascular and suckling stimuli. The firing patterns of activated OT and VP neurons often differ, but can transiently appear indistinguishable in vivo and especially in vitro. Classification in vitro without immunochemical labelling may be aided by the presence of phasic bursting (mostly in VP neurons) and by the differential response of these neurons to certain neurochemicals or to stimulation of certain inputs. The membrane properties of OT and VP neurons are generally similar in vitro, but the range of tests has not been extensive. The depolarizing afterpotential is more often exhibited by, but is not exclusive to, VP neurons.

N-methyl- d-aspartate receptor antagonist ketamine selectively attenuates spontaneous phasic activity of supraoptic vasopressin neurons in vivo

Supraoptic neurosecretory neurons express a prominent N-methyl- d-aspartate receptor system. Recent in vitro evidence reveals that N-methyl- d-aspartate receptor activation dramatically alters the spontaneous discharge patterns of supraoptic neurons. In this study we evaluate whether N-methyl- d-aspartate receptors in vivo contribute to the development of characteristic phasic discharge patterns displayed by vasopressin-secreting neurons. Intravenous administration of ketamine hydrochloride, a non-competitive N-methyl- d-aspartate receptor antagonist, was used to examine whether N-methyl- d-aspartate receptor blockade influences patterned spontaneous discharge observed in supraoptic neuro-secretory neurons. Extracellular recordings were obtained from identified hypothalamic supraoptic neurons in pentobarbital-anaesthetized Long-Evans rats. Systemic administration of ketamine (≤1.5 mg/kg) potently suppressed spontaneous phasic discharge in 16/19 putative vasopressin-secreting cells. The ketamine-induced blockade was dose dependent, fully reversible and was associated with the complete blockade of activity evoked by local pressure application of N-methyl- d-aspartate, but not the activity evoked by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor agonists (6/6 cells). Ketamine had no detectable effect on threshold or shape of antidromic action potentials. By comparison, the activity in 9/10 continuously active neurons (putative oxytocin-secreting) was unaffected by administration of identical doses of ketamine. These data suggest that N-methyl- d-aspartate receptors play an important role in regulating the onset and maintenance of spontaneous phasic activity patterns displayed by rat supraoptic vasopressin neurons in vivo.

Effects of interleukin-1 beta on membrane potentials of hypothalamic supraoptic neurons in rats in vitro.

Effects of interleukin-1 beta on membrane potentials of hypothalamic supraoptic neurons in rats in vitro.

Inhibitory response in neurosecretory cells of the hypothalamic supraoptic nucleus to subiculum stimulation in lactating rats

The effects of subiculum stimulation were investigated in 80 antidromically identified hypothalamic supraoptic neurons in lactating rats. Inhibition manifesting as suppression of antidromic action potentials (or of their somatodendritic component) was revealed in 26% of cells, induced by applying conditioned and test stimuli to the subiculum and neurohypophysial stalk. In some instances inhibition arose following a latency of 5–25 msec after each subicular stimulus and lasted only briefly; it set in gradually in other cases, leading to stable long-term changes in the excitability of neurosecretory cells. No activation was produced by this stimulation. It is deduced that subicular inhibitory inputs follow different patterns, thus reflecting morphological organizational aspects of synaptic inhibitory inputs to neurosecretory cells.

Influence of lateral septum and amygdala stimulation on the excitability of hypothalamic supraoptic neurons. An electrophysiological study in the rat

Extracellular recordings were obtained from 116 phasically-active (putative vasopressinergic) and 113 continuously-active (putative oxytocinergic) neurosecretory neurons in the hypothalamic supraoptic nucleus of urethane or pentobarbital anesthetized male Sprague-Dawley rats. Single 1 Hz pulse stimulation in most regions of the amygdala and the ipsilateral lateral septum was followed by a transient (20–140 ms) reduction in the excitability of more than 90% of responsive cells; one third displayed a reduction in excitability to both amygdala and lateral septum stimulation. Amygdala or lateral septum stimuli delivered in brief trains of 20–100 pulses at 5–20 Hz during ongoing phasic discharges could induce silent periods lasting to 30 or more seconds beyond the time of application. The same stimuli also reduced ongoing activity among continuously-firing SON cells but their response lasted only as long as the duration of the applied stimulus. These data suggest that neurons in both the ipsilateral lateral septum and the various amygdaloid nuclei exert a predominantly inhibitory influence on the excitability of both vasopressinergic and oxytocinergic SON neurons in the rat.

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 Mg 2+. 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.

Spontaneous "phasic-firing' in supraoptic neurons recorded from hypothalamo-neurohypophysial explants in vitro.

Single unit, extracellular electrical activity of 97 hypothalamic supraoptic neurons was recorded with micropipettes from rat hypothalamo-neurohypophysial explants maintained acutely in vitro. Spontaneously firing as well as quiescent neurons were encountered in roughly in roughly equal numbers. Of the spontaneously active neurons, 36.3% fired in a phasic mode quantitatively similar to that of "vasopressin-secreting' neurons recorded in vivo under conditions of enhanced hormone release. A small portion (approximately 14%) of the recorded supraoptic neurons had spikes which showed constant latency and high frequency following responses to stimulation of the neural stalk, responses characteristic of antidromically stimulated neurons. The mean latency and the range of latencies to these responses were similar to that recorded in vivo. Hypothalamo-neurohypophysial explants maintained in vitro thus provide a useful tool for investigating the electrical activity of supraoptic neurosecretory cells. Since the explant contains both the supraoptic neurons and their neurohypophysial projection path, this preparation offers an opportunity for characterizing further the relationship between electrical activity and hormone release.

Effects of chemoreceptor and baroreceptor stimulation on the discharge of hypothalamic supraoptic neurones in rats.

Experiments have been performed to examine the effects of activating the carotid body chemoreceptors and the arterial baroreceptors on the discharge of neurones within the hypothalamic supraoptic nucleus of the rat. Chemoreceptors were activated by intracarotid injection of 0.9% NaCl solution equilibrated with 100% CO2. The baroreceptors of the carotid sinus and aortic arch were activated by raising the blood pressure with an intravenous injection of phenylephrine. Chemoreceptor stimulation activated and baroreceptor stimulation inhibited the discharge of all the phasically discharging neurones tested. Neither stimulus had any consistent effect on non-phasically discharging neurones, although slight inhibition occasionally occurred. Anaesthesia of the carotid bifurcation abolished the effects of cardiovascular stimulation on the supraoptic neurones. Responses resumed when the anaesthesia wore off. However, the anaesthesia also seemed to alter the phasic pattern of discharge. The results are discussed with reference to the influence of the cardiovascular receptors upon the neurones in the supraoptic nucleus, and with reference to possible roles for the cardiovascular reflexes in control of vasopressin secretion.

Mammalian endocrine neurones: Control of phasic activity by antidromic action potentials

Hypothalamic supraoptic neurones were identified by antidromic invasion following stimulation of the pituitary stalk in urethane anaesthetized rats. These endocrine neurones either discharged continuously at low average rates (random) or were intermittently active (phasic). Phasic cells are thought to be vasopressinergic. With phasic neurones, antidromic activation with short trains of stimuli during the quiescent period triggered the 'on-phase' of spike discharge, and the probability of this response increased as the 'off-phase' progressed. Stimulation was ineffective in triggering high frequency firing when applied below the threshold for antidromic invasion, and caused no parallel response of random neurones. Arrest of firing during the 'on-phase' was achieved by stimulation with higher currents and longer pulse trains. These studies demonstrate that the switching process determining the firing pattern of phasic neurones depends on the cells' ongoing activity. Further, they reveal progressive changes in the cells' capacity to alter their electrophysiological state, a capacity that is not expressed in terms of the temporal pattern of spike discharges.

Excitation of phasically firing hypothalamic supraoptic neurones by carotid occlusion in rats.

1. The activity of supraoptic neurones has been recorded extracellularly during bilateral occlusion of the common carotid arteries in anaesthetized rats. 2. Experiments in lactating rats showed that occlusion liberated sufficient amounts of neurohypophysial hormones to cause a rise in intramammary pressure 15-25 s after the onset of occlusion. 3. Ninety-one percent of the phasic neurones (defined as those showing bursts of activity alternating with periods of silence) were activated by carotid occlusion less than 10 s after the onset of occlusion. Most randomly firing neurones were inhibited or were unaffected. 4. The activation of phasic neurones is unlikely to be just a nonspecific effect, because in the same animals, phasic neurones were excited whilst random neurones were not. 5. Moreover, in phasic neurones, statistical analysis shows (a) that the intervals during which an occlusion was performed were significantly shorter than the intervals between spontaneously occurring bursts, and (b) that this activation was followed by a period of reduced firing probability. 6. The results are discussed with reference to the correlation of supraoptic neuronal activity with hormone release. The possibility is considered of relating the tendency of some supraoptic neurones to fire in bursts with the secretion of vasopressin.

Hypothalamic supraoptic neurones: rates and patterns of action potential firing during water deprivation in the unanaesthetized monkey

Extracellulary recorded action potentials were obtained from hypothalamic supraoptic neurones in unanaesthetized rhesus monkeys. Rates and patterns of firing were studied during an initial control period, during 5 successive days of water deprivation and during 4 further days when drinking water was again available. During water deprivation, plasma osmolarity increased progressively from about 300 mOsmoles/kg to about 340 mOsmoles/kg; control values were again reached after 3 days of rehydration. Systematic changes in action potential firing accompanied the changes in plasma osmolarity. Under control conditions, the majority of cells fire slowly and irregularly (type i), whilst a few cells exhibited phases of alternating activity and silence (type p). As dehydration progresses, the frequency of neuronal firing increase and the pattern of firing changes. By the third day the majority of cells are type p with few type i cells being found. By the fourth day, the population consists of type p cells with some others showing a high continuous rate of firing (type c). By the fifth day, these two cell types are found in approximately equal proportions. Rehydration of the animal reverses the situation. We propose that type i cells contribute little, if at all, to hormone secretion, while type p and type c cells would be in a more actively secreting state. According to this view, the three firing patterns would represent different activity states of the same functional population stimulated by the unspecific stimulus of water deprivation rather than functionally different neurones. However, the use of stimuli which selectively release their oxytocin or vasopressin may be needed to answer this problem.

Firing patterns of hypothalamic supraoptic neurons during water deprivation in monkeys.

Water deprivation in monkeys caused an acceleration of action potential firing of supraoptic neurons, but not of neurons located 2 to 3 millimeters above the hypothalamic supraoptic nucleus. Whereas in the normally hydrated animal only 12 percent of the neuroendocrine cells discharged periodically, the proportion of these periodic bursters increased markedly with increasing plasma osmolarity. This finding suggests that such periodically firing supraoptic neurons are those engaged in active neurohypophyseal hormone secretion.