Hypothalamic Magnocellular Neurons Research Articles

Translational and Posttranslational Dynamics in a Model Peptidergic System

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus, whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses to neuronal stimulation are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganization of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and are hyperphosphorylated following stimulation. By multiomic integration of transcriptome and proteomic data, we identify changes to proteins present in afferent inputs to this nucleus.

Transcription factor Creb3l1 maintains proteostasis in neuroendocrine cells

ObjectivesDynamic changes to neuropeptide hormone synthesis and secretion by hypothalamic neuroendocrine cells is essential to ensure metabolic homeostasis. The specialised molecular mechanisms that allow neuroendocrine cells to synthesise and secrete vast quantities of neuropeptides remain ill defined. The objective of this study was to identify novel genes and pathways controlled by transcription factor and endoplasmic reticulum stress sensor Creb3l1 which is robustly activated in hypothalamic magnocellular neurones in response to increased demand for protein synthesis. MethodsWe adopted a multiomic strategy to investigate specific roles of Creb3l1 in rat magnocellular neurones. We first performed chromatin immunoprecipitation followed by genome sequencing (ChIP-seq) to identify Creb3l1 genomic targets and then integrated this data with RNA sequencing data from physiologically stimulated and Creb3l1 knockdown magnocellular neurones. ResultsThe data converged on Creb3l1 targets that code for ribosomal proteins and endoplasmic reticulum proteins crucial for the maintenance of cellular proteostasis. We validated genes that compose the PERK arm of the unfolded protein response pathway including Eif2ak3, Eif2s1, Atf4 and Ddit3 as direct Creb3l1 targets. Importantly, knockdown of Creb3l1 in the hypothalamus led to a dramatic depletion in neuropeptide synthesis and secretion. The physiological outcomes from studies of paraventricular and supraoptic nuclei Creb3l1 knockdown animals were changes to food and water consumption. ConclusionCollectively, our data identify Creb3l1 as a comprehensive controller of the PERK signalling pathway in magnocellular neurones in response to physiological stimulation. The broad regulation of neuropeptide synthesis and secretion by Creb3l1 presents a new therapeutic strategy for metabolic diseases.

Plasticity in Intrinsic Excitability of Hypothalamic Magnocellular Neurosecretory Neurons in Late-Pregnant and Lactating Rats.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Purinergic P2 and glutamate NMDA receptor coupling contributes to osmotically driven excitability in hypothalamic magnocellular neurosecretory neurons.

Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+ -dependent manner extrasynaptic NMDAR function. The P2-NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs. These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs. The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine-tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole-cell patch-clamp recordings obtained from MNNs showed that ATP (100μM) induced an increase in firing rate, an effect that was blocked by either 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]2-pyridinyl]azo]1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10μM) or kynurenic acid (1mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP-potentiated glutamatergic receptor-mediated currents were evoked by focal application of L-glu (1mm) and NMDA (50μM), but not AMPA (50μM). ATP potentiation of NMDA-evoked currents was blocked by PPADS (10μM) and by chelation of intracellular Ca2+ with BAPTA (10mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55mOsm/kgH2 O) potentiated NMDA-evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

SummaryMisfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

Oestrogen inhibits salt-dependent hypertension by suppressing GABAergic excitation in magnocellular AVP neurons.

Abundant evidence indicates that oestrogen (E2) plays a protective role against hypertension. Yet, the mechanism underlying the antihypertensive effect of E2 is poorly understood. In this study, we sought to determine the mechanism through which E2 inhibits salt-dependent hypertension. To this end, we performed a series of in vivo and in vitro experiments employing a rat model of hypertension that is produced by deoxycorticosterone acetate (DOCA)-salt treatment after uninephrectomy. We found that E2 prevented DOCA-salt treatment from inducing hypertension, raising plasma arginine-vasopressin (AVP) level, enhancing the depressor effect of the V1a receptor antagonist (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-vasopressin, and converting GABAergic inhibition to excitation in hypothalamic magnocellular AVP neurons. Moreover, we obtained results indicating that the E2 modulation of the activity and/or expression of NKCC1 (Cl- importer) and KCC2 (Cl- extruder) underpins the effect of E2 on the transition of GABAergic transmission in AVP neurons. Lastly, we discovered that, in DOCA-salt-treated hypertensive ovariectomized rats, CLP290 (prodrug of the KCC2 activator CLP257, intraperitoneal injections) lowered blood pressure, and plasma AVP level and hyperpolarized GABA equilibrium potential to prevent GABAergic excitation from emerging in the AVP neurons of these animals. Based on these results, we conclude that E2 inhibits salt-dependent hypertension by suppressing GABAergic excitation to decrease the hormonal output of AVP neurons.

Mitochondria Influence NMDA Receptor‐mediated Currents in Hypothalamic Magnocellular Neurons: Altered Mechanisms during Heart Failure

Hypothalamic magnocellular neurosecretory neurons (MNNs) located in the supraoptic (SON) and paraventricular nuclei (PVN) play a critical role in the pathophysiology of heart failure (HF). Mitochondria (MITO) plays an important role in the regulation of intracellular Ca2+ dynamics, and several studies observed that morphological and functional MITO changes contribute to the development and maintenance of various brain diseases. We recently demonstrated that NMDA receptors activation triggers a Ca2+−dependent stimulation of SK channel in MNNs from Sham rats, and that this coupling acts as a negative feedback mechanism restraining the magnitude of the NMDA receptor‐mediated current (INMDAR) and its excitatory effects. Importantly, we found that the NMDA‐SK channel coupling is blunted in MNNs from HF rats. Still, whether MITO regulation of intracellular Ca2+ dynamics influence INMDAR and whether this effect is altered during HF is unknown. To address this question, we obtained whole‐cell patch clamp recordings from MNNs (holding potential: −50 mV) in slices from Sham and HF rats. We found that focal application of NMDA (50 μM, 300 ms) evoked an INMDAR whose magnitude was similar in Sham and HF rats (p>0.05), as previously reported. Application of CCCP (10 μM, 5 s), a mitochondrial uncoupler, significantly increased the evoked INMDAR in MNNs from Sham rats (p<0.05), but had no effect in MNNs from HF rats (p>0.05). Intracellular dialysis of MNNs with the Ca2+ chelator BAPTA (10 mM) significantly augmented INMDAR in MNNs from Sham rats compared to the control condition (p<0.05), and occluded the CCCP‐induced INMDAR increase. Taken together, our data supports that mitochondria regulate, in a Ca2+‐dependent manner, the magnitude of INMDAR in MNNs from Sham rats, a phenomenon that is blunted during HF.Support or Funding InformationR01HL090948

Audiogenic kindling activates expression of vasopressin in the hypothalamus of Krushinsky-Molodkina rats genetically prone to reflex epilepsy.

The present study analysed the effects of audiogenic kindling on the functional state of the vasopressinergic system of Krushinsky-Molodkina (KM) rats. KM rats represent a genetic model of audiogenic reflex epilepsy. Multiple audiogenic seizures in KM rats lead to the involvement of the limbic structures and neocortex in the epileptic network. The phenomenon of epileptic activity that overspreads from the brain stem to the forebrain is called audiogenic kindling and represents a model of limbic epilepsy. In the present study, audiogenic kindling was induced by 25 repetitive audiogenic seizures (AGS) with 1 AGS per day. A proportion of KM rats did not express AGS to sound stimuli, and these rats were characterised as the AGS-resistant group. The data demonstrated that audiogenic kindling did not change activity of extracellular signal-regulated kinase 1/2 or cAMP response element-binding protein, although it led to an increase in vasopressin (VP) expression in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN). Additionally, we observed a decrease in GABAergic innervation of the hypothalamic neuroendocrine neurones after audiogenic kindling, whereas glutamatergic innervation of the SON and PVN was not altered. By contrast, analysis of AGS-resistant KM rats did not reveal any changes in the activity of the VP-ergic system, confirming that the activation of VP expression was caused by repetitive AGS expression, rather than by repetitive acoustic stress. Thus, we suggest that overspread of epileptiform activity in the brain is the main factor that affects VP expression in the hypothalamic magnocellular neurones.

Sodium pumps, ouabain and aldosterone in the brain: A neuromodulatory pathway underlying salt-sensitive hypertension and heart failure

Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca2+ and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure.

Functional coupling between NMDA receptors and SK channels in rat hypothalamic magnocellular neurons: altered mechanisms during heart failure.

Glutamatergic NMDA receptors (NMDARs) and small conductance Ca2+ -activated K+ (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). In this work, we investigated whether NMDARs and SK channels in MNNs are functionally coupled, and whether an altered coupling may contribute to exacerbated neuronal activity in this condition. We report that NMDARs and SK channels form a functional Ca2+ -dependent negative feedback loop that restrains the excitatory effect on membrane potential and firing activity evoked by NMDAR activation. The negative feedback loop between NMDARs and SK channels was blunted or absent in MNNs of heart failure (HF) rats. These results help us better understand how synaptic and intrinsic mechanisms regulate hypothalamic neuronal activity, as well as how changes in the interaction among these disparate mechanisms contribute to altered neuronal activity during prevalent neurogenic cardiovascular diseases. Glutamatergic NMDA receptors (NMDARs) and small conductance Ca2+ -activated K+ (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs), both under physiological and pathological states, such as lactation and heart failure (HF). However, whether NMDARs and SK channels in MNNs are functionally coupled, and whether changes in this coupling contribute to exacerbated neuronal activity during HF is at present unknown. In the present study, we addressed these questions using patch-clamp electrophysiology and confocal Ca2+ imaging in a rat model of ischaemic HF. We found that in MNNs of sham rats, blockade of SK channels with apamin (200nM) significantly increased the magnitude of an NMDAR-evoked current (INMDA ). We also observed that blockade of SK channels potentiated NMDAR-evoked firing, and abolished spike frequency adaptation in MNNs from sham, but not HF rats. Importantly, a larger INMDA -ΔCa2+ response was observed under basal conditions in HF compared to sham rats. Finally, we found that dialysing recorded cells with the Ca2+ chelator BAPTA (10mM) increased the magnitude of INMDA in MNNs from both sham and HF rats, and occluded the effects of apamin in the former. Together our studies demonstrate that in MNNs, NMDARs and SK channels are functionally coupled, forming a local negative feedback loop that restrains the excitatory effect evoked by NMDAR activation. Moreover, our studies also support a blunted NMDAR-SK channel coupling in MNNs of HF rats, establishing it as a pathophysiological mechanism contributing to exacerbated hypothalamic neuronal activity during this prevalent neurogenic cardiovascular disease.

Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension.

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K+ channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca2+-dependent K+ channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K+ channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension.NEW & NOTEWORTHY Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.

SUN-472 Labile Ca-Permeable AMPA Receptors Comprise New Synapses Following Salt Loading-Induced Plasticity in Hypothalamic Magnocellular Neurons

Magnocellular neuroendocrine cells of the hypothalamus play an important role in the regulation of fluid and electrolyte homeostasis. They undergo a dramatic structural plasticity under sustained physiological activation, including an increase in glutamatergic synaptic innervation. We tested for plasticity in the glutamate AMPA receptor expression in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) induced by sustained activation by chronic salt loading-induced dehydration. We found that salt loading by with 2% saline drinking water for 5-7 days resulted in a significant selective increase in GluA1 protein expression in the rat SON, while no change in the mRNA expression or membrane localization of any of the AMPA receptor subunits was observed, suggesting a post-transcriptional up regulation of GluA1 at excitatory synapses on SON neurons. We performed whole-cell recordings of excitatory postsynaptic currents (EPSCs) to determine the contribution of Ca2+ -permeable AMPA receptors (CP-AMPARs) to synaptic transmission by the EPSC rectification index and the sensitivity of EPSCs to the selective antagonist of CP-AMPA receptors, 1-naphthyl-acetyl-spermine (NAS). We found that neurons from the salt-loaded group showed a significantly greater inward rectification and an increased sensitivity to NAS compared to the control group. To further determine the role of rapid protein turnover in the AMPAR plasticity, we preincubated slices for over 60 min in the translational inhibitor cycloheximide to block local protein synthesis, which. Blocking protein synthesis reverted the Ca permeability and reduced the EPSC frequency and EPSC rectification in SON neurons from in the salt-loaded group to the levels of controls. These findings together suggest that salt loading induces highly labile glutamate synapses in magnocellular neurons that are comprised almost exclusively of CP-AMPARs, and indicate an essential role for rapid protein synthesis in the maintenance of the new synapses. This glutamate receptor plasticity should result in an increase in glutamate-induced calcium influx, which could play a key role in the activity-dependent neuronal-glial remodeling of the magnocellular neurons that occurs during chronic dehydration. Supported by NIH R01 and NSF IOS grants.

A switch from GABA inhibition to excitation of vasopressin neurons exacerbates the development angiotensin II-dependent hypertension.

Hypothalamic magnocellular neurons secrete vasopressin into the systemic circulation to maintain blood pressure by increasing renal water reabsorption and by vasoconstriction. When blood pressure rises, baroreflex activation normally inhibits vasopressin neurons via activation of GABAergic inputs. However, plasma vasopressin levels are paradoxically elevated in several models of hypertension and in some patients with essential hypertension, despite increased blood pressure. We have previously shown that vasopressin neuron activity is increased early in the development of moderate angiotensin II-dependent hypertension via blunted baroreflex inhibition of vasopressin neurons. Here, we show that antagonism of vasopressin-induced vasoconstriction slows the development of hypertension and that local administration of a GABAA receptor antagonist inhibits vasopressin neurons during, but not before, the onset of hypertension. Taken together, our data suggest that vasopressin exacerbates the increase in blood pressure evident early in the development hypertension and that blunted baroreflex inhibition of vasopressin neurons is underpinned by an excitatory shift in their response to endogenous GABA signalling. This article is protected by copyright. All rights reserved.

Functional Interaction Between NMDA Receptors and SK Channels in Hypothalamic Magnocellular Neurons: Role in Heart Failure

Neurohumoral activation, which includes elevated plasma levels of vasopressin, is a hallmark of heart failure (HF). However, the precise mechanisms that regulate hypothalamic magnocellular neurosecretory cells (MNCs) neuronal excitability during HF remains incompletely understood. Here, we evaluated whether an increase in intracellular Ca2+ induced by NMDA receptor activation would evoke a compensatory feedback inhibitory mechanism via activation of Ca2+‐dependent K+ channels, and whether this mechanism would contribute to the regulation of MNCs excitability in HF rats. Whole‐cell patch clamp recordings obtained from MNCs (holding potential: −50 mV) in slices from Sham and HF rats showed that focal application of NMDA (50 mM, 300 ms) evoked an inward NMDAR‐mediated current (INMDAR) whose magnitude was similar in Sham and HF rats (amplitude: Sham 0.16±0.02 vs HF 0.20±0.02 nA, p>0.05; area: Sham 94±24 vs HF 77±15 nA.ms, p>0.05). INMDAR was immediately followed by an outward current, whose magnitude was larger in MNCs from HF than Sham rats (amplitude: Sham 0.9±0.1 vs HF 1.6±0.3 nA, p<0.05; area: Sham 215±19 vs HF 410±76 nA.ms, p<0.01). Blockade of K+ currents with Cs+ revealed that the INMDAR was in fact significantly larger in MNCs from HF than Sham rats (amplitude: Sham 0.18±0.04 vs HF 0.86±0.02 nA, p<0.05; area: Sham 111±24 vs HF 263±54 nA.ms, p<0.05). Intracellular dialysis of MNCs with the Ca2+ chelator BAPTA significantly reduced the outward current triggered by NMDAR activation in both groups to the same extent (amplitude: Sham 0.17±0.05 vs HF 0.22±0.07 nA, p>0.05; area: Sham 18±9 vs HF 42±19 nA.ms, p>0.05). Bath application of the SK channel blocker apamin significantly decreased the outward current triggered by INMDAR in MNCs from HF rats (p<0.05), but this effect was minimal in Sham rats (p>0.05). Finally, current clamp recording showed that focal application of NMDA after SK channel blockade increased the number of evoked spikes both in Sham and HF rats compared to control conditions (Sham 168±25%, p<0.001 vs control; HF 311±71%, p<0.01 vs control), but this effect was exacerbated in HF rats (p=0.06 vs Sham). Taken together, our data supports the presence of a functional coupling between excitatory NMDAR and inhibitory SK channels in MNCs. This interaction is enhanced in HF rats contributing to dampening the NMDAR‐mediated hyperexcitability observed in MNCs during this condition.Support or Funding InformationR01HL090948This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects of osmotic stress on synaptic vs. paracrine signaling mechanisms in oxytocinergic magnocellular neurons.

Acute hypernatremia promotes natriuresis in part by increasing activity of oxytocinergic magnocellular neurons in the hypothalamus that promote synaptic release of oxytocin (OT) from axon terminals in the neurohypophysis. Acute hypernatremia also blunts stress‐induced activation of the hypothalamic‐pituitary‐adrenal axis via a mechanism that we believe is likely to depend on central paracrine (rather than synaptic) release of OT from magnocellular dendrites. Interestingly, prior work from other groups indicates that acute hypernatremia produces an increase in the concentration of circulating OT significantly more rapidly than it creates an increase in the concentration of central OT. The current project was designed to develop a better understanding of the underlying cellular mechanisms through which osmotic stressors may plausibly promote both synaptic and paracrine release of OT from the same population of osmosensitive hypothalamic neurons, but with distinctly different temporal dynamics. Towards that end, we used the Cre‐LoxP system to generate OT‐reporter mice that express tdTomato in neurons that synthesize OT. Initial studies indicated a strong colocalization of tdTomato with OT/neurophysin1 throughout the hypothalamus. Whole‐cell patch clamp recordings from tdTomato positive magnocellular neurons in the paraventricular nucleus of the hypothalamus revealed expected intrinsic properties, including transient outward rectification produced by a prominent A‐type K+ current (IA). Initial imaging studies revealed that the dendrites of OT positive magnocellular neurons are passive conductors, with robust activity dependent dendritic calcium influx that diminishes substantially with increasing distance from the soma. Bath application of an external osmolyte (15 mM mannitol) produced a clear increase in the frequency of spontaneous action potentials observed in OT positive magnocellular neurons, and yet intriguingly, also produced a clear decrease in activity dependent dendritic calcium influx. This effect was also much more prominent in distal dendrites than in proximal dendrites, consistent with a mechanism that depends on decreased dendritic input resistance. Also consistent with that interpretation, we found that acute application of 15 mM mannitol significantly reduces the amplitude of evoked glutamatergic EPSCs recorded at the soma, as produced by a small theta glass stimulator placed near the distal dendrites. Interestingly, preliminary data suggest that parvocellular OT neurons lack similar osmosensitivty. Collectively, these results provide a partial mechanistic explanation for why acute osmotic stress is likely to initially favor synaptic over paracrine release of OT from osmosensitive hypothalamic magnocellular neurons.Support or Funding InformationSupported by NIMH R0104641This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A reduction in SK channels contributes to increased activity of hypothalamic magnocellular neurons during heart failure.

Small conductance Ca2+ -activated K+ (SK) channels play an important role in regulating the excitability of magnocellular neurosecretory cells (MNCs). Although an increased SK channel function contributes to adaptive physiological responses, it remains unknown whether changes in SK channel function/expression contribute to exacerbated MNC activity under disease conditions. We show that the input-output function of MNCs in heart failure (HF) rats is enhanced. Moreover, the SK channel blocker apamin enhanced the input-output function in sham, although not in HF rats. We found that both the after-hyperpolarizing potential magnitude and the underlying apamin-sensitive IAHP are blunted in MNCs from HF rats. The magnitude of spike-induced increases in intracellular Ca2+ levels was not affected in MNCs of HF rats. We found a diminished expression of SK2/SK3 channel subunit mRNA expression in the supraoptic nucleus of HF rats. Our studies suggest that a reduction in SK channel expression, but not changes in Ca2+ -mediated activation of SK channels, contributes to exacerbated MNC activity in HF rats. Small conductance Ca2+ -activated K+ channels (SK) play an important role in regulating the activity of magnocellular neurosecretory cells (MNCs) and hormone release from the posterior pituitary. Moreover, enhanced SK activity contributes to the adaptive responses of MNCs to physiological challenge, such as lactation. Nevertheless, whether changes in SK function/expression contribute to exacerbated MNC activity during diseases such as heart failure (HF) remains unknown. In the present study, we used a combination of patch clamp electrophysiology, confocal Ca2+ imaging and molecular biology in a rat model of ischaemic HF. We found that the input-output function of MNCs was enhanced in HF compared to sham rats. Moreover, although the SK blocker apamin (200nm) strengthened the input-output function in sham rats, it failed to have an effect in HF rats. The magnitude of the after-hyperpolarizing potential (AHP) following a train of spikes and the underlying apamin-sensitive IAHP were blunted in MNCs from HF rats. However, spike-induced increases in intracellular Ca2+ were not affected in the MNCs of HF rats. Real-time PCR measurements of SK channel subunits mRNA in supraoptic nucleus punches revealed a diminished expression of SK2/SK3 subunits in HF compared to sham rats. Together, our studies demonstrate that MNCs from HF rats exhibit increased membrane excitability and an enhanced input-output function, and also that a reduction in SK channel-mediated, apamin-sensitive AHP is a critical contributing mechanism. Moreover, our results suggest that the reduced AHP is related to a down-regulation of SK2/SK3 channel subunit expression but not the result of a blunted activity-dependent intracellular Ca2+ increase following a burst of action potentials.

An increased extrasynaptic NMDA tone inhibits A-type K+ current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.

A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR-IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA , contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats. We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR-IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch-clamp electrophysiology and real-time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1-NR2A-D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.

A Reduction in SK Channel Function Contributes to Increased Excitability of Hypothalamic Magnocellular Neurons During Heart Failure

Neurohumoral activation, which includes elevated plasma levels of vasopressin, is a hallmark of heart failure (HF) and contributes to morbidity and mortality in this disease. Here, we evaluated whether changes in Ca2+‐activated K+ (SK) channels contribute to enhanced hypothalamic magnocellular neurosecretory cell (MNCs) activity in HF rats. Whole‐cell patch clamp recordings obtained from MNCs in slices from Sham and HF rats showed that the spike frequency evoked by direct current injection was significantly higher in the HF rats compared to Sham rats (Sham 16±0.9 vs HF 24±1.2 spikes, p<0.001). Bath application of the SK channel blocker apamin significantly increased the evoked firing frequency in MNCs from Sham rats, but no effect was observed in HF rats (Sham 23±0.9, p< 0.05 vs control; HF 26±0.7 spikes, p>0.05 vs control). The medium afterhyperpolarization potential (mAHP) following a train of spikes was significantly smaller in MNCs from HF rats compared to Sham rats (Sham 9.7±1.1 vs HF 6.6±0.6 mV, p<0.05) and apamin reduced the mAHP in both groups (Sham 2.9±0.5 vs HF 1.6±1.1 mV). Voltage‐clamp recordings of SK tail currents revealed that the apamin‐sensitive current amplitude and area were significantly smaller in MNCs from HF rats (amplitude: Sham 0.2±0.03 vs HF 0.09±0.03 nA, p<0.05; area: Sham 15.4±2.5 vs HF 7.2±1.2 nA.ms, p<0.01) whereas intracellular dialysis of MNCs with BAPTA reduced the currents in both groups to the same extent. No significant differences were observed in cell capacitance (Sham 30±2 vs HF 26±3 pF, p>0.05) or input resistance (Sham 960±87 vs HF 945±167 MΩ, p>0.05) between groups. Taken together, our data indicates that a decrease in SK channel function/activity and/or expression contributes to the increased excitability of MNCs in HF rats.Support or Funding InformationNIH R01HL090948

Hypothalamic Vasopressinergic Projections Innervate Central Amygdala GABAergic Neurons: Implications for Anxiety and Stress Coping.

The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

Electrophysiology of Hypothalamic Magnocellular Neurons In vitro: A Rhythmic Drive in Organotypic Cultures and Acute Slices.

Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.