Spike broadening in magnocellular neuroendocrine cells of rat hypothalamic slices

Bursts of action potentials recorded from rat magnocellular neuroendocrine cells (MNCs) are known to be associated with enhanced release of oxytocin or vasopressin from their axon terminals in the neurohypophysis. Intracellular recordings from MNC somata in hypothalamic slices showed that spike broadening was characteristic of such bursts. Transitions from slow to fast firing caused spike broadening in all cells, whether they were silent, slow firing, phasic or fast-continuous. During phasic firing, broadening increased with the intraburst spike frequency. However, the parameters of maximal spike broadening recorded at the soma did not directly coincide with the previously described firing patterns evoking maximal hormone release from neurohypophysial terminals.     

Structural Reconstruction of the Perivascular Space in the Adult Mouse Neurohypophysis During an Osmotic Stimulation

Oxytocin (OXT) and arginine vasopressin (AVP) neuropeptides in the neurohypophysis (NH) control lactation and body fluid homeostasis, respectively. Hypothalamic neurosecretory neurones project their axons from the supraoptic and paraventricular nuclei to the NH to make contact with the vascular surface and release OXT and AVP. The neurohypophysial vascular structure is unique because it has a wide perivascular space between the inner and outer basement membranes. However, the significance of this unique vascular structure remains unclear; therefore, we aimed to determine the functional significance of the perivascular space and its activity‐dependent changes during salt loading in adult mice. The results obtained revealed that pericytes were the main resident cells and defined the profile of the perivascular space. Moreover, pericytes sometimes extended their cellular processes or ‘perivascular protrusions’ into neurohypophysial parenchyma between axonal terminals. The vascular permeability of low‐molecular‐weight (LMW) molecules was higher at perivascular protrusions than at the smooth vascular surface. Axonal terminals containing OXT and AVP were more likely to localise at perivascular protrusions than at the smooth vascular surface. Chronic salt loading with 2% NaCl significantly induced prominent changes in the shape of pericytes and also increased the number of perivascular protrusions and the surface area of the perivascular space together with elevations in the vascular permeability of LMW molecules. Collectively, these results indicate that the perivascular space of the NH acts as the main diffusion route for OXT and AVP and, in addition, changes in the shape of pericytes and perivascular reconstruction occur in response to an increased demand for neuropeptide release.     

Response of interleukin-1beta in the magnocellular system to salt-loading

Drinking 2% NaCl decreases interleukin (IL)-1beta in the neural lobe and enhances IL-1 Type 1 receptor expression in magnocellular neurones and pituicytes. To quantify cytokine depletion from the neural lobe during progressive salt loading and determine whether the changes are reversible and correspond with stores of vasopressin (VP) or oxytocin (OT), rats were given water on day 0 and then 2% NaCl to drink for 2, 5, 8 or 5 days followed by 5 days of water (rehydration). Control rats drinking only water were pair-fed amounts eaten by 5-day salt-loaded animals. Animals were decapitated on day 8, the neural lobe frozen and plasma hormones analysed by radioimmunoassay (OT, VP) or enzyme-linked immunosorbent assay (IL-1beta). IL-1beta, VP and OT in homogenates of the neural lobe were quantified by immunocapillary electrophoresis with laser-induced fluorescence detection. Differences were determined by ANOVA, Tukey's t-test, Dunnett's procedure, Fisher's least significant difference and linear regression analysis. In response to salt-loading, rats lost body weight similar to pair-fed controls, drank progressively more 2% NaCl and excreted greater urine volumes. Plasma VP increased at days 2 and 8 of salt-loading, whereas osmolality, OT and cytokine were enhanced after 8 days with IL-1beta remaining elevated after rehydration. In the neural lobe, all three peptides decreased progressively with increasing duration of salt-loading (IL-1beta, r2 = 0.98; OT, r2 = 0.94; VP, r2 = 0.93), beginning on day 2 (IL-1beta; VP) or 5 (OT), with only VP replenished by rehydration. IL-1beta declined more closely (P < 0.0001; ANOVA interaction analysis) with OT (r2 = 0.96) than VP (r2 = 0.86), indicative of corelease from the neural lobe during chronic dehydration. Local effects of IL-1beta on magnocellular terminals, pituicytes and microglia in the neural lobe with activation of forebrain osmoregulatory structures by circulating cytokine may sustain neurosecretion of OT and VP during prolonged salt-loading.     

Reduced susceptibility of magnocellular neuroendocrine nuclei of the rat hypothalamus to transient focal ischemia produced by middle cerebral artery occlusion

Intraparenchymal injections of glutamate analogues into the diencephalon near the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus selectively spare magnocellular neuroendocrine cells. In this study we investigated for the first time the susceptibility of this neuronal population to ischemia. Temporary focal ischemia was produced using a three-vessel occlusion method involving unilateral middle cerebral artery and bilateral common carotid artery occlusion (MCAO/CCAO). Most of the 3-h ischemic period was maintained without anesthesia and reversed by microclip removal of the contralateral common carotid artery occlusion. In one subset of rats transcardial perfusion with India ink was used to estimate the degree of ischemia produced during MCAO/CCAO in the SON, lateral magnocellular nucleus of the PVN (PVL), caudoputamen (CP), and frontoparietal cortex (COR). Computer-assisted densitometry measurements of ink density indicated significant reductions in ink penetration in the territory of the occluded MCA within the SON (46%), PVL (45%), CP (53%), and COR (76%). In contrast, neither sham-operated rats nor rats subjected to occlusion of the MCA alone showed differences in ink optical densities between the sides ipsilateral and contralateral to MCAO. The other subset of rats were perfused 48-72 h after recovery and brain sections were examined for neurodegenerative changes. While the incidences of cerebral and caudoputamen infarction after MCAO/CCAO were 98.4 and 52%, respectively, the histological features of the SON or PVL in ischemic rats were similar to those of control rats. Reduced susceptibility of magnocellular neuroendocrine cells to ischemia may be due to a number of mechanisms including neuronal resilience, neuroprotection by glia and vascular/perivascular cells, and access to perivascular cerebrospinal fluid.     

Endothelin-mediated calcium responses in supraoptic nucleus astrocytes influence magnocellular neurosecretory firing activity

In addition to their peripheral vasoactive effects, accumulating evidence supports an important role for endothelins (ETs) in the regulation of the hypothalamic magnocellular neurosecretory system, which produces and releases the neurohormones vasopressin (VP) and oxytocin (OT). Still, the precise cellular substrates, loci and mechanisms underlying the actions of ETs on the magnocellular system are poorly understood. In the present study, we combined patch-clamp electrophysiology, confocal Ca(2+) imaging and immunohistochemistry to study the actions of ETs on supraoptic nucleus (SON) magnocellular neurosecretory neurones and astrocytes. Our studies show that ET-1 evoked rises in [Ca(2+) ](i) levels in SON astrocytes (but not neurones), an effect largely mediated by the activation of ET(B) receptors and mobilisation of thapsigargin-sensitive Ca(2+) stores. The presence of ET(B) receptors in SON astrocytes was also verified immunohistochemically. ET(B) receptor activation either increased (75%) or decreased (25%) SON firing activity, both in VP and putative OT neurones, and these effects were prevented when slices were preincubated in glutamate receptor blockers or nitric oxide synthase blockers, respectively. Moreover, ET(B) -mediated effects in SON neurones were also prevented by a gliotoxin compound, and when changes in [Ca(2+) ](i) were prevented with bath-applied BAPTA-AM or thapsigargin. Conversely, intracellular Ca(2+) chelation in the recorded SON neurones failed to block ET(B) -mediated effects. In summary, our results indicate that ET(B) receptor activation in SON astrocytes induces the mobilisation of [Ca(2+) ](i) , likely resulting in the activation of glutamate and nitric oxide signalling pathways, evoking in turn excitatory and inhibitory SON neuronal responses, respectively. Taken together, our study supports an important role for astrocytes in mediating the actions of ETs on the magnocellular neurosecretory system.     

Neuroactive Steroids Modulate GABAA Receptors in Peptidergic Nerve Terminals

Neuroactive steroid modulation of GABA_A receptors was investigated in the peptidergic nerve terminals of the posterior pituitary using patch clamp techniques. In common with GABA_A receptors in cell bodies, the nerve terminal GABA_A receptor was potentiated by the synthetic steroid alphaxalone and by physiological concentrations of the progesterone metabolite allopregnanolone. Both of these agents enhanced Cl^? currents elicited by GABA. Estradiol-17β had a weak inhibitory effect on GABA responses of nerve terminals, but only at high concentrations. The potentiating action was manifest as an increase in the probability of channel opening, with no effect on the rate of desensitization of the GABA_A receptor. Neuroactive steroids enhanced GABA-gated Cl～ channel activity in cell-free membrane patches, thus demonstrating a membrane delimited response. These results indicated that with regard to allosteric modulation by neuroactive steroids, the nerve terminal GABA_A receptor is similar to the GABA_A receptors of nerve cell bodies and endocrine cells. Neuroactive steroids are thus capable of altering the chemosensitivity of nerve terminal membranes by enhancing GABA inhibition at this location. The neuroactive steroid sensitivity of nerve terminal GABA_A receptors provides a pathway by which gonadal steroid derivatives could regulate peptide secretion from neurosecretory neurons. Such a pathway could participate in the coordination of neuropeptide secretion during complex neuroendocrine functions. With specific regard to the neurohypophysis, neuroactive steroid-induced changes in the sensitivity of nerve terminal GABA_A receptors could play a role in the initiation of oxytocin secretion during the transition between pregnancy and parturition.     

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague‐Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual‐label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin‐ or oxytocin‐labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post‐MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.     

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.     

Physiological Regulation of Magnocellular Neurosecretory Cell Activity: Integration of Intrinsic,Local and Afferent Mechanisms

The hypothalamic supraoptic and paraventricular nuclei contain magnocellular neurosecretory cells (MNCs) that project to the posterior pituitary gland where they secrete either oxytocin or vasopressin (the antidiuretic hormone) into the circulation. Oxytocin is important for delivery at birth and is essential for milk ejection during suckling. Vasopressin primarily promotes water reabsorption in the kidney to maintain body fluid balance, but also increases vasoconstriction. The profile of oxytocin and vasopressin secretion is principally determined by the pattern of action potentials initiated at the cell bodies. Although it has long been known that the activity of MNCs depends upon afferent inputs that relay information on reproductive, osmotic and cardiovascular status, it has recently become clear that activity depends critically on local regulation by glial cells, as well as intrinsic regulation by the MNCs themselves. Here, we provide an overview of recent advances in our understanding of how intrinsic and local extrinsic mechanisms integrate with afferent inputs to generate appropriate physiological regulation of oxytocin and vasopressin MNC activity.     

Effects of intrajugular hypertonic saline, vaginal distension and vulvar massage on activity of supraoptic neuroendocrine cells

Activity of 40 single antidromically identified supraoptic neurons was recorded and evaluated in response to a combination of tactile, vulvar massage, vaginal distension, and slow intrajugular 1.2 M sodium chloride infusion in unanesthetized, randomly hydrated ewes. Estradiol-implanted Southdown ewes were prepared according to techniques described by Jennings et al.⁵. Only 4 spontaneous firing patterns were observed in the supraoptic nuclei. Analysis of evoked activity indicated that each stimulus evoked alterations in mean firing rates or increased numbers of short interspike intervals in some cells. The resultant activity of units to sequential vulvar massage and 1.2 M sodium chloride infusion suggests a possibility of separate mechanisms of release of oxytocin and vasopressin.     

Potentiation by angiotensin II of spontaneous excitatory postsynaptic currents in rat supraoptic magnocellular neurones

The physiological actions of angiotensin II in the supraoptic (SON) and paraventricular nuclei have been widely demonstrated, including the modulation of firing rate and release of arginine vasopressin and oxytocin. Here, we investigated whether angiotensin II modulates synaptic inputs into the SON. To do this, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat SON neurones in thin slice preparations using the whole-cell patch-clamp technique. Angiotensin II reversibly increased the frequency of spontaneous EPSCs in a dose-related manner without affecting the amplitude, indicating that angiotensin II potentiated EPSCs via a presynaptic mechanism. Angiotensin II-induced potentiation of EPSCs was unaffected in the presence of tetrodotoxin. On the other hand, angiotensin II did not cause significant effects on IPSCs. The potentiation of EPSCs by angiotensin II was potently suppressed by previous exposure to the angiotensin type 1 (AT1) receptor antagonist, losartan. Our results suggest that angiotensin II potentiates the excitatory synaptic inputs into SON neurones, via the AT1 receptors.     

Membrane properties of rat magnocellular neuroendocrine cells in vivo

Membrane properties of rat magnocellular neuroendocrine cells (MNCs) were examined during intracellular recordings in vivo. Current-clamp experiments revealed sustained outward rectification positive to −55 mV and transient outward rectification of depolarizing responses elicited from negative holding potentials. Trains of impulses were associated with a progressive increase of spike duration. Such features, which were not observed in neighboring non-neuroendocrine cells, are similar to those of MNCs in slices or explants of rat hypothalalus. In contrast, however, cells recorded in vivo were characterized by intense synaptic inhibition and a lower specific impedance than commonly observed from MNCs impaled in vitro.     

Assessment of spike activity in the supraoptic nucleus

Novel approaches to the characterization of coding carried by spike trains are discussed. Measuring firing frequency alone may only partially reflect spike patterning, and can only quantify changes of the most obvious kind. We have devised a method that combines probabilistic and information approaches to quantify the variability of the interspike intervals in a way that is independent of spike frequency. To illustrate the technique, the firing of an oxytocin cell and a vasopressin cell were compared before and after osmotic stimulation. A bimodal lognormal function was fitted to the interspike interval histograms. The entropy of the log interval histogram was used to measure the variability of intervals and to reflect the coding capacity of the cell per spike. A perfect metronome shows no variability in interval and thus has no greater coding capacity than is conveyed by its frequency, whereas the variability of intervals of magnocellular neurones means that their irregular activity has greater potential for coding. While the mean spike frequency increased in both the oxytocin and vasopressin cells in response to osmotic stimulation, the changes in their irregularity showed differences. Osmotic stimulation reduced the entropy of the oxytocin cell, reflecting an increase in the regularity of its spike activity. Conversely, osmotic stimulation had little effect on the entropy of the vasopressin cell. Such differences are not evident from a simple inspection of ratemeter activity. The comparison highlights the limitations of mean spike frequency as a measure of spike coding. Parameters based on the interspike intervals constitute informative measures of spike activity that allow objective comparisons to be made between the activity under different physiological conditions.     

Oxytocin in Brattleboro Rats: Increased Synthesis is Contrasted by Blunted Intrahypothalamic Release From Supraoptic Nucleus Neurones

Adult male Brattleboro rats were used to investigate the impact of the congenital absence of vasopressin on the release pattern of oxytocin (OXT) within the hypothalamic supraoptic nucleus (SON) in response to a 10-min forced swimming session and osmotic stimulation. Both immunohistochemical and in situ hybridisation data suggest that vasopressin-deficient animals have more oxytocin-synthesising neurones in the SON than homozygous wild-type controls. Unexpectedly, both forced swimming and peripheral osmotic stimulation resulted in a blunted release profile of oxytocin within the SON of vasopressin-deficient rats compared to controls. A similar intranuclear OXT response to direct osmotic stimulation of the SON by retrodialysis with hypertonic Ringer's solution in both genotypes confirmed the capability of SON neurones to locally release oxytocin in vasopressin-deficient rats, indicating an altered processing of information originating from multisynaptic inputs rather than a deficit in release capacity. Taken together with data obtained in previous studies, the present findings provide evidence suggesting that autocrine and paracrine signalling of magnocellular neurones differs within the paraventricular nucleus and the SON. Thus, significant alterations in intra-SON oxytocin mRNA levels cannot easily be extrapolated to intranuclear release profiles and the local signal intensity of this neuropeptide after physiological stimulation.     

Phasic bursts in rat magnocellular neurosecretory cells are not intrinsically regenerative in vivo

Vasopressinergic hypothalamic magnocellular neurosecretory cells fire in phasic bursts. Burst initiation involves summation of postsynaptic potentials to generate action potentials. Action potentials are each followed by a nonsynaptic depolarizing after-potential that summates temporally to generate a plateau potential and so sustain activity throughout the burst. It is unknown whether this plateau potential exceeds spike threshold in vivo to cause intrinsic regenerative firing or simply approaches threshold to increase the probability that excitatory postsynaptic potentials will trigger further action potentials. Here we show that pharmacological blockade of ionotropic glutamatergic transmission by microdialysis application of kynurenic acid into the supraoptic nucleus of anaesthetized rats prevents spontaneous bursts and bursts (after-discharge) evoked by short trains of antidromically stimulated action potentials in magnocellular neurosecretory cells. Even during prolonged depolarization induced by 1 m NaCl infusion, kynurenic acid microdialysis application still blocked after-discharge. The ability of kynurenic acid to block after-discharge during osmotic stimulation was not caused by an unmasking of inhibitory postsynaptic potentials as kynurenic acid was equally effective in the presence of the ionotropic gamma-aminobutyric acid receptor antagonist, bicuculline, nor did it result from inhibition of plateau potential amplitude as this was unaffected by kynurenic acid and bicuculline in vitro, as was after-discharge evoked in vitro. We conclude that phasic bursts are nonregenerative in vivo but rather require continued excitatory synaptic input activity superimposed upon a subthreshold plateau potential to sustain burst activity.     

Synaptic inputs and electrical coupling among magnocellular neuroendocrine cells

This paper first briefly reviews the evidence for synaptic and nonsynaptic plasticity among the neurons and glia of the magnocellular hypothalamo-neurohypophysial system. Emphasis is placed upon the importance of the roles played by astrocytes in the remodeling of the magnocellular nuclei under various conditions of increased hormone demand. Evidence is then reviewed from more recent studies showing that there is electrical coupling among magnocellular neurons, and that this coupling shows plasticity similar to that shown for other characteristics of the system (e.g., chemical synapses, dendritic bundling etc.). Further, evidence is presented that extent of electrical coupling can be modified not only by manipulating the physiological state of the animal (such as lactation), but also by electrical stimulation of newly described olfactory afferent inputs to the cells of the supraoptic nucleus. The possible functional significance of these findings is discussed in relation to the behavior of nursing rats.     

Changes in the active membrane properties of rat supraoptic neurones during pregnancy and lactation

To better understand the plasticity of intrinsic membrane properties of supraoptic magnocellular neuroendocrine cells associated with reproductive function, intracellular recordings were performed in oxytocin (OT) and vasopressin (VP) neurones from virgin, late pregnant (E19-22), and lactating (8-12 days of lactation) rats in vitro, using hypothalamic explants. OT neurones from virgin rats displayed a narrower spike width than neurones from pregnant and lactating rats, characterized by faster rise and decay times. Spike width changes in VP neurones were not as prominent as those observed in OT neurones. In OT neurones, the amplitude and the decay of the afterhyperpolarization following spike trains was significantly larger and faster, respectively, in pregnant and lactating rats compared to virgin rats. These properties did not change during pregnancy and lactation in VP neurones. The incidence of the depolarizing afterpotential following spikes significantly increased from approximately 20% in virgin rats to 40-50% during pregnancy and lactation in OT neurones, but was stable (80-90%) across states in VP neurones. Repetitive firing properties (frequency adaptation, the first interspike interval frequency and frequency-current (F-I) relationship) were altered during pregnancy and lactation in OT neurones, but not VP neurones. The increased incidence of depolarizing afterpotentials in OT neurones enhances excitability, while the increased afterhyperpolarization results in suppression of firing rate. Thus, the changes may favour the short bursting activity seen in OT neurones during lactation. These results confirmed reproductive state-dependent changes in intrinsic membrane properties of OT neurones during lactation, and suggest these changes are in place during late pregnancy. This argues that the plasticity in the electrical properties in OT neurones associated with lactation is not instigated by suckling.