A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner.

One of the largest sex differences in brain neurochemistry is the expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate brain, with males having more AVP cells in the bed nucleus of the stria terminalis (BNST) than females. Despite the long-standing implication of AVP in social and anxiety-like behaviors, the circuitry underlying AVP's control of these behaviors is still not well defined. Using optogenetic approaches, we show that inhibiting AVP BNST cells reduces social investigation in males, but not in females, whereas stimulating these cells increases social investigation in both sexes, but more so in males. These cells may facilitate male social investigation through their projections to the lateral septum (LS), an area with the highest density of sexually differentiated AVP innervation in the brain, as optogenetic stimulation of BNST AVP → LS increased social investigation and anxiety-like behavior in males but not in females; the same stimulation also caused a biphasic response of LS cells ex vivo. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated all these responses. Together, these findings establish a sexually differentiated role for BNST AVP cells in the control of social investigation and anxiety-like behavior, likely mediated by their projections to the LS.

Endoplasmic reticulum and mitochondrial calcium handling dynamically shape slow afterhyperpolarizations in vasopressin magnocellular neurons.

Many neurons including vasopressin (VP) magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus (SON) generate afterhyperpolarizations (AHPs) during spiking to slow firing, a phenomenon known as spike frequency adaptation. The AHP is underlain by Ca2+-activated K+ currents, and while slow component (sAHP) features are well described, its mechanism remains poorly understood. Previous work demonstrated that Ca2+ influx through N-type Ca2+ channels is the primary source of sAHP activation in SON oxytocin neurons, but no obvious channel coupling was described for VP neurons. Given this, we tested the possibility of an intracellular source of sAHP activation, namely the Ca2+-handling organelles endoplasmic reticulum (ER) and mitochondria in male and female wistar rats. We demonstrate that ER Ca2+ depletion greatly inhibits sAHPs without a corresponding decrease in Ca2+ signal. Caffeine sensitized AHP activation by Ca2+ In contrast to ER, disabling mitochondria with CCCP or blocking mitochondria Ca2+ uniporter (MCU) enhanced sAHP amplitude and duration, implicating mitochondria as a vital buffer for sAHP-activating Ca2+ Block of mitochondria Na+-dependent Ca2+ release via triphenylphosphonium (TPP+) failed to affect sAHPs, indicating that mitochondria Ca2+ doesn't contribute to sAHP activation. Together, our results support that ER Ca2+-induced Ca2+ release activates sAHPs and mitochondria shape the spatiotemporal trajectory of the sAHP via Ca2+ buffering in VP neurons. Overall, this implicates organelle Ca2+, and specifically ER-mitochondria associated membrane contacts, as an important site of Ca2+ microdomain activity that regulates sAHP signaling pathways. Thus, this site plays a major role in influencing VP firing activity and systemic hormonal release.Significance Statement The slow afterhyperpolarization (sAHP) is mediated by a Ca2+-dependent K+ current. Despite its critical role in regulating neuronal spiking, the Ca2+-dependent mechanisms leading to its activation and spatiotemporal shape remains poorly understood. Here we show that in vasopressin (VP) neurons, dynamic interactions in Ca2+ handling between endoplasmic reticulum (ER) and mitochondria play a significant role in sAHP initiation (via ER Ca2+ release) and its spatiotemporal waveform (via mitochondrial Ca2+ uptake). Our results suggest that contact sites between ER and mitochondria represent Ca2+ microdomains critically involved in initiating the first steps of sAHP generation in VP neurons. Given that changes in the sAHP have been linked to abnormal firing activity in various diseases, our results have both wide-range physiological and pathological implications.

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+ blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonic INMDA in nondepolarized [Vh (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonic INMDA was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonic INMDA in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonic INMDA but not Mg2+-resistant tonic INMDA In the EU group VP neurons, an Mg2+-resistant tonic INMDA was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonic INMDA selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonic INMDA in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENT The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

Identification of Novel Cross-Talk between the Neuroendocrine and Autonomic Stress Axes Controlling Blood Pressure.

The hypothalamic paraventricular nucleus (PVN) controls neuroendocrine axes and the autonomic nervous system to mount responses that cope with the energetic burdens of psychological or physiological stress. Neurons in the PVN that express the angiotensin Type 1a receptor (PVNAgtr1a) are implicated in neuroendocrine and autonomic stress responses; however, the mechanism by which these neurons coordinate activation of neuroendocrine axes with sympathetic outflow remains unknown. Here, we use a multidisciplinary approach to investigate intra-PVN signaling mechanisms that couple the activity of neurons synthesizing corticotropin-releasing-hormone (CRH) to blood pressure. We used the Cre-Lox system in male mice with in vivo optogenetics and cardiovascular recordings to demonstrate that excitation of PVNAgtr1a promotes elevated blood pressure that is dependent on the sympathetic nervous system. Next, neuroanatomical experiments found that PVNAgtr1a synthesize CRH, and intriguingly, fibers originating from PVNAgtr1a make appositions onto neighboring neurons that send projections to the rostral ventrolateral medulla and express CRH type 1 receptor (CRHR1) mRNA. We then used an ex vivo preparation that combined optogenetics, patch-clamp electrophysiology, and Ca2+ imaging to discover that excitation of PVNAgtr1a drives the local, intra-PVN release of CRH, which activates rostral ventrolateral medulla-projecting neurons via stimulation of CRHR1(s). Finally, we returned to our in vivo preparation and found that CRH receptor antagonism specifically within the PVN lowered blood pressure basally and during optogenetic activation of PVNAgtr1a Collectively, these results demonstrate that angiotensin II acts on PVNAgtr1a to conjoin hypothalamic-pituitary-adrenal axis activity with sympathetically mediated vasoconstriction in male mice.SIGNIFICANCE STATEMENT The survival of an organism is dependent on meeting the energetic demands imposed by stressors. This critical function is accomplished by the CNS's ability to orchestrate simultaneous activities of neurosecretory and autonomic axes. Here, we unveil a novel signaling mechanism within the paraventricular nucleus of the hypothalamus that links excitation of neurons producing corticotropin-releasing-hormone with excitation of neurons controlling sympathetic nervous system activity and blood pressure. The implication is that chronic stress exposure may promote cardiometabolic disease by dysregulating the interneuronal cross-talk revealed by our experiments.

Angiotensin II inhibits the A-type K+ current of hypothalamic paraventricular nucleus neurons in rats with heart failure: role of MAPK-ERK1/2 signaling.

Angiotensin II (ANG II)-mediated sympathohumoral activation constitutes a pathophysiological mechanism in heart failure (HF). Although the hypothalamic paraventricular nucleus (PVN) is a major site mediating ANG II effects in HF, the precise mechanisms by which ANG II influences sympathohumoral outflow from the PVN remain unknown. ANG II activates the ubiquitous intracellular MAPK signaling cascades, and recent studies revealed a key role for ERK1/2 MAPK signaling in ANG II-mediated sympathoexcitation in HF rats. Importantly, ERK1/2 was reported to inhibit the transient outward potassium current (IA) in hippocampal neurons. Given that IA is a critical determinant of the PVN neuronal excitability, and that downregulation of IA in the brain has been reported in cardiovascular disease states, including HF, we investigated here whether ANG II modulates IA in PVN neurons via the MAPK-ERK pathway, and, whether these effects are altered in HF rats. Patch-clamp recordings from identified magnocellular neurosecretory neurons (MNNs) and presympathetic (PS) PVN neurons revealed that ANG II inhibited IA in both PVN neuronal types, both in sham and HF rats. Importantly, ANG II effects were blocked by inhibiting MAPK-ERK signaling as well as by inhibiting epidermal growth factor receptor (EGFR), a gateway to MAPK-ERK signaling. Although no differences in basal IA magnitude were found between sham and HF rats under normal conditions, MAPK-ERK blockade resulted in significantly larger IA in both PVN neuronal types in HF rats. Taken together, our studies show that ANG II-induced ERK1/2 activity inhibits IA, an effect expected to increase the excitability of presympathetic and neuroendocrine PVN neurons, contributing in turn to the neurohumoral overactivity that promotes progression of the HF syndrome.

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

KEY POINTS: 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. ABSTRACT: 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 (200 nM) 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 (10 mM) 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.

TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats.

Angiotensin II (AngII) is implicated in neuroinflammation, blood-brain barrier (BBB) disruption, and autonomic dysfunction in hypertension. We have previously shown that exogenous AngII stimulates Toll-like receptor 4 (TLR4) via AngII type 1 receptor (AT1R), inducing activation of hypothalamic microglia ex vivo, and that AngII-AT1R signaling is necessary for the loss of BBB integrity in spontaneously hypertensive rats (SHRs). Herein, we hypothesized that microglial TLR4 and AT1R signaling interactions represent a crucial mechanistic link between AngII-mediated neuroinflammation and BBB disruption, thereby contributing to sympathoexcitation in SHRs. Male SHRs were treated with TAK-242 (TLR4 inhibitor; 2 weeks), Losartan (AT1R inhibitor; 4 weeks), or vehicle, and age-matched to control Wistar Kyoto rats (WKYs). TLR4 and AT1R inhibitions normalized increased TLR4, interleukin-6, and tumor necrosis factor-α protein densities in SHR cardioregulatory nuclei (hypothalamic paraventricular nucleus [PVN], rostral ventrolateral medulla [RVLM], and nucleus tractus solitarius [NTS]), and abolished enhanced microglial activation. PVN, RVLM, and NTS BBB permeability analyses revealed complete restoration after TAK-242 treatment, whereas SHRs presented with elevated dye leakage. Mean arterial pressure was normalized in Losartan-treated SHRs, and attenuated with TLR4 inhibition. In conscious assessments, TLR4 blockade rescued SHR baroreflex sensitivity to vasoactive drugs, and reduced the SHR pressor response to ganglionic blockade to normal levels. These data suggest that TLR4 activation plays a substantial role in mediating a feed-forward pro-hypertensive cycle involving BBB disruption, neuroinflammation, and autonomic dysfunction, and that TLR4-specific therapeutic interventions may represent viable alternatives in the treatment of hypertension.

Tachycardia evoked from insular stroke in rats is dependent on glutamatergic neurotransmission in the dorsomedial hypothalamus.

BACKGROUND AND PURPOSE: Damage to the insula results in cardiovascular complications. In rats, activation of N-methyl-d-aspartate receptors (NMDARs) in the intermediate region of the posterior insular cortex (iIC) results in sympathoexcitation, tachycardia and arterial pressure increases. Similarly, focal experimental hemorrhage at the iIC results in a marked sympathetic-mediated increase in baseline heart rate. The dorsomedial hypothalamic region (DMH) is critical for the integration of sympathetic-mediated tachycardic responses. Here, whether responses evoked from the iIC are dependent on a synaptic relay in the DMH was evaluated. METHODS: Wistar rats were prepared for injections into the iIC and DMH. Anatomical (tracing combined with immunofluorescence) and functional experiments (cardiovascular and sympathetic recordings) were performed. RESULTS: The iIC sends dense projections to the DMH. Approximately 50% of iIC neurons projecting to the DMH express NMDARs, NR1 subunit. Blockade of glutamatergic receptors in the DMH abolishes the cardiovascular and autonomic responses evoked by the activation of NMDARs in the iIC (change in mean arterial pressure 7 ± 1 vs. 1 ± 1 mmHg after DMH blockade; change in heart rate 28 ± 3 vs. 0 ± 3 bpm after DMH blockade; change in renal sympathetic nerve activity 23% ± 1% vs. -1% ± 4% after DMH blockade). Experimental hemorrhage at the iIC resulted in a marked tachycardia (change 89 ± 14 bpm) that was attenuated by 65% ± 5% (p = 0.0009) after glutamatergic blockade at the DMH. CONCLUSIONS: The iIC-induced tachycardia is largely dependent upon a glutamatergic relay in the DMH. Our study reveals the presence of an excitatory glutamatergic pathway from the iIC to the DMH that may be involved in the cardiovascular alterations observed after insular stroke.

Correction to: Three-dimensional morphometric analysisreveals time-dependent structural changesin microglia and astrocytes in the centralamygdala and hypothalamicparaventricular nucleus of heart failure rats.

An amendment to this paper has been published and can be accessed via the original article.

Three-dimensional morphometric analysis reveals time-dependent structural changes in microglia and astrocytes in the central amygdala and hypothalamic paraventricular nucleus of heart failure rats.

BACKGROUND: Cardiovascular diseases, including heart failure, are the most common cause of death globally. Recent studies support a high degree of comorbidity between heart failure and cognitive and mood disorders resulting in memory loss, depression, and anxiety. While neuroinflammation in the hypothalamic paraventricular nucleus contributes to autonomic and cardiovascular dysregulation in heart failure, mechanisms underlying cognitive and mood disorders in this disease remain elusive. The goal of this study was to quantitatively assess markers of neuroinflammation (glial morphology, cytokines, and A1 astrocyte markers) in the central amygdala, a critical forebrain region involved in emotion and cognition, and to determine its time course and correlation to disease severity during the progression of heart failure. METHODS: We developed and implemented a comprehensive microglial/astrocyte profiler for precise three-dimensional morphometric analysis of individual microglia and astrocytes in specific brain nuclei at different time points during the progression of heart failure. To this end, we used a well-established ischemic heart failure rat model. Morphometric studies were complemented with quantification of various pro-inflammatory cytokines and A1/A2 astrocyte markers via qPCR. RESULTS: We report structural remodeling of central amygdala microglia and astrocytes during heart failure that affected cell volume, surface area, filament length, and glial branches, resulting overall in somatic swelling and deramification, indicative of a change in glial state. These changes occurred in a time-dependent manner, correlated with the severity of heart failure, and were delayed compared to changes in the hypothalamic paraventricular nucleus. Morphometric changes correlated with elevated mRNA levels of pro-inflammatory cytokines and markers of reactive A1-type astrocytes in the paraventricular nucleus and central amygdala during heart failure. CONCLUSION: We provide evidence that in addition to the previously described hypothalamic neuroinflammation implicated in sympathohumoral activation during heart failure, microglia, and astrocytes within the central amygdala also undergo structural remodeling indicative of glial shifts towards pro-inflammatory phenotypes. Thus, our studies suggest that neuroinflammation in the amygdala stands as a novel pathophysiological mechanism and potential therapeutic target that could be associated with emotional and cognitive deficits commonly observed at later stages during the course of heart failure.

NMDA receptors potentiate activity-dependent dendritic release of neuropeptides from hypothalamic neurons.

KEY POINTS: Using 'sniffer' cell biosensors, we evaluated the effects of specific firing patterns and frequencies on activity-dependent somatodendritic release of vasopressin from paraventricular nucleus neurones. Somatodendritic release of vasopressin was rarely observed during continuous firing but was strengthened by clustered activity. Moreover, release evoked at any given frequency was robustly potentiated by NMDA receptor (NMDAR)-mediated firing. Differently from axonal release, NMDAR activation was necessary for somatodendritic release to occur at physiological firing frequencies, acting thus as a gating mechanism by which activity-dependent release from these two neuronal compartments could be independently regulated. The NMDAR-mediated potentiation was independent of a specific firing pattern and was not accompanied by increased spike broadening, but correlated with higher dendritic Ca2+ levels. Our studies provide fundamental novel information regarding stimulus-secretion coupling at somatodendritic compartments, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites could be regulated in a spatially compartmentalized manner. ABSTRACT: Dendrites are now recognized to be active transmitting neuronal compartments subserving complex brain functions, including motor behaviours and homeostatic neurohumoral responses. Still, the precise mechanisms underlying activity-dependent release of dendritic signals, and how dendritic release is regulated independently from axonal release, remains largely unknown. We used 'sniffer' biosensor cells to enable the measurement and study of activity-dependent dendritic release of vasopressin (VP) from hypothalamic neurones in brain slices. SnifferVP responses were dose-dependent, with a threshold detection level of 0.5 nM for VP, being thus a highly sensitive tool to detect endogenous physiological levels of the neuropeptide. Somatodendritic release of VP was rarely observed in response to a burst of action potentials fired in continuous mode, but was strengthened by clustered firing activity. Moreover, release evoked at any given frequency was robustly potentiated when firing was triggered by NMDA receptor (NMDAR) activation. Differently from axonal release, NMDAR activation was necessary for dendritic release to occur at physiological firing frequencies. Thus, we propose that NMDARs may act as a gating mechanism by which activity-dependent release from these two neuronal compartments can be independently regulated. The NMDAR-mediated potentiation of dendritic release was independent of a particular action potential waveform, firing pattern evoked, or a more pronounced spiked broadening, but correlated with higher dendritic Ca2+ levels. Overall, our studies provide fundamental novel information regarding stimulus-secretion coupling at neuronal dendrites, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites can be regulated in a spatially compartmentalized manner.

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.

A Unique "Angiotensin-Sensitive" Neuronal Population Coordinates Neuroendocrine, Cardiovascular, and Behavioral Responses to Stress.

Stress elicits neuroendocrine, autonomic, and behavioral responses that mitigate homeostatic imbalance and ensure survival. However, chronic engagement of such responses promotes psychological, cardiovascular, and metabolic impairments. In recent years, the renin-angiotensin system has emerged as a key mediator of stress responding and its related pathologies, but the neuronal circuits that orchestrate these interactions are not known. These studies combine the use of the Cre-recombinase/loxP system in mice with optogenetics to structurally and functionally characterize angiotensin type-1a receptor-containing neurons of the paraventricular nucleus of the hypothalamus, the goal being to determine the extent of their involvement in the regulation of stress responses. Initial studies use neuroanatomical techniques to reveal that angiotensin type-1a receptors are localized predominantly to the parvocellular neurosecretory neurons of the paraventricular nucleus of the hypothalamus. These neurons are almost exclusively glutamatergic and send dense projections to the exterior portion of the median eminence. Furthermore, these neurons largely express corticotrophin-releasing hormone or thyrotropin-releasing hormone and do not express arginine vasopressin or oxytocin. Functionally, optogenetic stimulation of these neurons promotes the activation of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, as well as a rise in systolic blood pressure. When these neurons are optogenetically inhibited, the activity of these neuroendocrine axes are suppressed and anxiety-like behavior in the elevated plus maze is dampened. Collectively, these studies implicate this neuronal population in the integration and coordination of the physiological responses to stress and may therefore serve as a potential target for therapeutic intervention for stress-related pathology.SIGNIFICANCE STATEMENT Chronic stress leads to an array of physiological responses that ultimately rouse psychological, cardiovascular, and metabolic impairments. As a consequence, there is an urgent need for the development of novel therapeutic approaches to prevent or dampen deleterious aspects of "stress." While the renin-angiotensin system has received some attention in this regard, the neural mechanisms by which this endocrine system may impact stress-related pathologies and consequently serve as targets for therapeutic intervention are not clear. The present studies provide substantial insight in this regard. That is, they reveal that a distinct population of angiotensin-sensitive neurons is integral to the coordination of stress responses. The implication is that this neuronal phenotype may serve as a target for stress-related disease.

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

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

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

KEY POINTS: 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. ABSTRACT: 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 (200 nm) 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.

KEY POINTS: 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. ABSTRACT: 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.

Vasopressin casts light on the suprachiasmatic nucleus.

KEY POINTS: A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light-induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo-entrainment of biological rhythms. ABSTRACT: In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light-evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c-fos in the SCN, which is involved in photic entrainment of circadian rhythms.

A-type K+ channels contribute to the prorenin increase of firing activity in hypothalamic vasopressin neurosecretory neurons.

Recent studies have supported an important contribution of prorenin (PR) and its receptor (PRR) to the regulation of hypothalamic, sympathetic, and neurosecretory outflows to the cardiovascular system, including systemic release of vasopressin (VP), both under physiological and cardiovascular disease conditions. Still, the identification of precise cellular mechanisms and neuronal/molecular targets remain unknown. We have recently shown that PRR is expressed in VP neurons and that their activation increases neuronal activity. However, the underlying ionic channel mechanisms are undefined. Here, we performed patch-clamp electrophysiology from identified VP neurons in acute hypothalamic slices obtained from enhanced green fluorescent protein-VP transgenic rats. Voltage-clamp recordings showed that PR inhibited the magnitude of A-type K+ current (IA; ~50% at -25 mV), a subthreshold voltage-dependent current that restrains VP firing activity. PR also increased the inactivation rate of IA and shifted the steady-state voltage-dependent inactivation function toward more hyperpolarized membrane potential (~7 mV shift), thus resulting in less channel availability to be activated at any given membrane potential. PR also inhibited a sustained component of IA ("window" current). PR-mediated changes in action potential waveform and increased firing activity were occluded when IA was blocked by 4-aminopyridine. Finally, PR failed to increase superoxide production within the supraoptic nucleus/paraventricular nucleus, and PR excitatory effects persisted in slices treated with the SOD mimetic tempol. Taken together, these experiments indicated that PR excitatory effects on vasopressin neurons involve inhibition of IA, due, in part, to increases in its voltage-dependent inactivation properties. Moreover, our results indicate that PR effects did not involve an increase in oxidative stress.NEW & NOTEWORTHY Here, we demonstrate that prorenin/the prorenin receptor is an important signaling unit for the regulation of vasopressin firing activity and, thus, systemic hormonal release. We identified A-type K+ channels as key molecular targets mediating prorenin stimulation of vasopressin neuronal activity, thus standing as a potential therapeutic target for neurohumoral activation in cardiovascular disease.

Neuronal-derived nitric oxide and somatodendritically released vasopressin regulate neurovascular coupling in the rat hypothalamic supraoptic nucleus.

The classical model of neurovascular coupling (NVC) implies that activity-dependent axonal glutamate release at synapses evokes the production and release of vasoactive signals from both neurons and astrocytes, which dilate arterioles, increasing in turn cerebral blood flow (CBF) to areas with increased metabolic needs. However, whether this model is applicable to brain areas that also use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the rat hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritic release of neuropeptides, including vasopressin (VP), constitutes a key signaling modality influencing neuronal and network activity. Using a multidisciplinary approach, we investigated vasopressin-mediated vascular responses in SON arterioles of hypothalamic brain slices of Wistar or VP-eGFP Wistar rats. Bath-applied VP significantly constricted SON arterioles (Δ-41 ± 7%) via activation of the V1a receptor subtype. Vasoconstrictions were also observed in response to single VP neuronal stimulation (Δ-18 ± 2%), an effect prevented by V1a receptor blockade (V2255), supporting local dendritic VP release as the key signal mediating activity-dependent vasoconstrictions. Conversely, osmotically driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide-mediated vasodilation (Δ19 ± 2%). Activity-dependent vasodilations were followed by a VP-mediated vasoconstriction, which acted to limit the magnitude of the vasodilation and served to reset vascular tone following activity-dependent vasodilation. Together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between nitric oxide and activity-dependent dendritic released VP, in the generation of proper NVC responses.

Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction.

Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca(2+) dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca(2+) imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca(2+). Astrocytic Ca(2+) responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca(2+). In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca(2+)) and enhanced when high Ca(2+) or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K(+) and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone.