Neurons In Paraventricular Nucleus Research Articles

A novel mechanism strengthens GABAergic inhibition in the hypothalamic PVN in proportion to the level of synaptic L‐glutamate spillover

Perhaps the most robust property of pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) in unstressed animals is their state of near quiescence. Although discharge quiescence has been strongly linked to the dominance of GABAergic synaptic inhibition, mechanisms that govern the strength of local GABAergic inhibition remain to be fully elucidated. Here, whole cell patch‐clamp recordings were made from identified parvocellular PVN neurons in brain slices and GABAergic inhibitory postsynaptic currents (IPSCs) were evoked by electrical stimuli delivered to the surrounding peri‐nuclear zone. To investigate the impact of GABA synthesis and vesicular packaging on GABAergic IPSC amplitude, we compared eIPSC amplitude before and after bath application of the GABA synthetic substrate L‐glutamate (L‐Glu; 100 μM, 10 min). Within 3–5 minutes of L‐Glu washout, eIPSC amplitude suddenly and significantly increased (+23 ± 6%; P<0.002). This response was unaffected by selective inhibition of glial L‐Glu uptake with dihydrokainate (DHK; 250 μM), but was nearly abolished (−77 ± 8.5%) by DL‐threo‐β‐benzyloxyaspartic acid (TBOA; 100 μM), a pan‐specific inhibitor of L‐glutamate transporters, suggesting that strengthening of GABAergic inhibition by L‐Glu depends on uptake by the neuronal L‐Glu transporter EAAT3. Similar results were obtained when local synaptic activity was increased by bath perfusion with high K+ (10 mM) aCSF. In current‐clamp recordings of spontaneous firing, high bath K+ caused a delayed reduction of discharge (1.58 ± 0.59Hz to 1.18 ± 0.51Hz; P<0.03) that was not observed when depolarizing current was injected to mimic the spiking response to high K+. These findings suggest that GABAergic IPSC strengthening is sufficient to reduce PVN neuronal discharge. High frequency stimulation‐induced GABAergic synaptic depression/vesicular depletion revealed that exposure to bath L‐Glu did not increase quantal content (i.e., the number of GABA vesicles released during each IPSC), but enhance recovery of IPSC amplitude by +36 pA within 10 s. These data indicate that bath L‐Glu selectively increases the number of GABA molecules contained within each GABA vesicle. In anesthetized rats (n=6), acute blockade of GABA‐A receptors in the PVN with picrotoxin (PTX) increased renal sympathetic nerve activity (+23%). Prior PVN injection of L‐Glu greatly amplified the PTX response (+72%), which is consistent with increased functional inhibitory tonus. Collectively, our findings indicate that GABA synaptic vesicles are “over‐filled” when GABA is synthesized from L‐Glu under conditions of heightened L‐Glu availability. This process depends on EAAT3‐mediated L‐Glu uptake, likely into GABA terminals. As over‐filled vesicles transition to the readily releasable pool, the amplitude of IPSCs increases, allowing synaptic inhibitory tonus to increase in proportion to the level of synaptic L‐Glu spillover. This local mechanism is postulated to help prevent neuronal hyper‐activation under conditions of heightened glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxytocinergic Neurons in the Hypothalamic Paraventricular Nucleus Stimulate Brown Adipose Tissue Thermogenesis through Rostral Medullary Raphe

Hypothalamic neurons in the paraventricular nucleus (PVN) produce several neuropeptides, including oxytocin and vasopressin, and send their axons to various brain regions. These PVN neurons have been considered to serve neuroendocrine and autonomic functions, and many studies have demonstrated the involvement of oxytocin in social and emotional behaviors (e.g. maternal and sexual behaviors, and stress responses). Although these behaviors are accompanied with autonomic responses, it is unclear how oxytocin modulates the activities of the autonomic nervous system. To investigate the roles of PVN oxytocinergic neurons in the central regulation of autonomic function, in this study, we first performed neural tract tracing in rats from PVN oxytocinergic neurons to the rostral medullary raphe region (rMR), which contains sympathetic premotor neurons controlling brown adipose tissue (BAT) thermogenesis. In the rMR, oxytocin‐immunoreactive fibers, but not vasopressin‐immunoreactive fibers, were distributed, and oxytocin‐containing terminals were apposed to tryptophan hydroxylase‐expressing (i.e., serotonergic) neurons, potentially BAT sympathetic premotor neurons. Retrograde tracing from the rMR with cholera toxin b‐subunit (CTb) labeled a portion of oxytocinergic neurons in the caudal PVN, but not in the supraoptic nucleus, suggesting that the PVN is a major source of oxytocinergic fibers in the rMR. Then, we performed in vivo BAT sympathetic nerve recording combining optogenetic stimulation of the PVN‐derived oxytocinergic axon terminals in the rMR. Recombinant adeno‐associated viral vectors were injected into the PVN to express the engineered channel rhodopsin variant, ChIEF, under the oxytocin promoter. We confirmed that most of PVN neurons expressing ChIEF‐mCherry contained oxytocin and that their axon fibers were densely distributed in the rMR. Optogenetic stimulation of these fibers evoked BAT sympathetic nerve activities as well as increased expired CO2 and heart rate. Similar thermogenic and cardiac responses were elicited by nanoinjection of oxytocin into the rMR, and these responses were blocked by prior injection of the oxytocin receptor antagonist, L‐368,899 into the rMR. In addition, oxytocin injection into the rMR also potentiated NMDA‐evoked BAT sympathetic nerve activities. These findings indicate that PVN oxytocinergic neurons stimulate thermogenic sympathetic outflow to BAT through activation of rMR neurons. This oxytocinergic pathway may play an important role in emotion‐related autonomic responses.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Numbers JP16H05128, JP15H05932, JP26118508 (KN) and by AMED under Grant Number JP18gm5010002 (KN)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(Pro)renin receptor knockdown in the paraventricular nucleus of the hypothalamus attenuates hypertension development and AT1 receptor-mediated calcium events.

Activation of the brain renin-angiotensin system (RAS) is a pivotal step in the pathogenesis of hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a critical part of the angiotensinergic sympatho-excitatory neuronal network involved in neural control of blood pressure and hypertension. However, the importance of the PVN (pro)renin receptor (PVN-PRR)-a key component of the brain RAS-in hypertension development has not been examined. In this study, we investigated the involvement and mechanisms of the PVN-PRR in DOCA-salt-induced hypertension, a mouse model of hypertension. Using nanoinjection of adeno-associated virus-mediated Cre recombinase expression to knock down the PRR specifically in the PVN, we report here that PVN-PRR knockdown attenuated the enhanced blood pressure and sympathetic tone associated with hypertension. Mechanistically, we found that PVN-PRR knockdown was associated with reduced activation of ERK (extracellular signal-regulated kinase)-1/2 in the PVN and rostral ventrolateral medulla during hypertension. In addition, using the genetically encoded Ca2+ biosensor GCaMP6 to monitor Ca2+-signaling events in the neurons of PVN brain slices, we identified a reduction in angiotensin II type 1 receptor-mediated Ca2+ activity as part of the mechanism by which PVN-PRR knockdown attenuates hypertension. Our study demonstrates an essential role of the PRR in PVN neurons in hypertension through regulation of ERK1/2 activation and angiotensin II type 1 receptor-mediated Ca2+ activity. NEW & NOTEWORTHY PRR knockdown in PVN neurons attenuates the development of DOCA-salt hypertension and autonomic dysfunction through a decrease in ERK1/2 activation in the PVN and RVLM during hypertension. In addition, PRR knockdown reduced AT1aR expression and AT1R-mediated calcium activity during hypertension. Furthermore, we characterized the neuronal targeting specificity of AAV serotype 2 in the mouse PVN and validated the advantages of the genetically encoded calcium biosensor GCaMP6 in visualizing neuronal calcium activity in the PVN.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

Reward Inhibits Paraventricular CRH Neurons to Relieve Stress

Chronic, uncontrollable stress can lead to various pathologies [1-6]. Adaptive behaviors, such as reward consumption, control excessive stress responses and promote positive health outcomes [3, 7-10]. Corticotrophin-releasing hormone (CRH) neurons in paraventricular nucleus (PVN) represent a key neural population organizing endocrine, autonomic, and behavioral responses to stress by initiating hormonal cascades along the hypothalamic-pituitary-adrenal (HPA) axis and orchestrating stress-related behaviors through direct projections to limbic and autonomic brain centers [11-18]. Although stress and reward have been reported to induce changes of c-Fos and CRH expression in PVN CRH neurons [19-23], it has remained unclear how these neurons respond dynamically to rewarding stimuli to mediate the stress-buffering effects of reward. Using fiber photometry of Ca2+ signals within genetically identified PVN CRH neurons in freely behaving mice [24-26], we find that PVN CRH neurons are rapidly and strongly inhibited by reward consumption. Reward decreases anxiety-like behavior and stress-hormone surge induced by direct acute activation ofPVN CRHneurons or repeated stress challenge.Repeated stress upregulates glutamatergic transmission andinduces an N-methyl-D-aspartate receptor (NMDAR)-dependent burst-firing pattern in these neurons, whereas reward consumption rebalances the synaptic homeostasis and abolishes theburst firing. Anatomically, PVN CRH neurons integrate widespread information from both stress- and reward-related brain areas in the forebrain andmidbrain, including multiple direct long-range GABAergic afferents. Together, these findings reveal a hypothalamic circuit that organizes adaptive stress response by complementarily integrating reward and stress signals and suggest that intervention in this circuit could provide novel methods to treat stress-related disorders.

Rapid, biphasic CRF neuronal responses encode positive and negative valence.

SUMMARYCorticotropin-releasing factor (CRF) that is released from the paraventricular nucleus (PVN) of the hypothalamus is essential for mediating stress response by activating the hypothalamic-pituitary-adrenal (HPA) axis. CRF-releasing PVN neurons receive inputs from multiple brain regions that convey stressful events, but their neuronal dynamics on the timescale of behavior remain unknown. Here, our recordings of PVN CRF neuronal activity in freely behaving mice revealed that CRF neurons are activated immediately by a range of aversive stimuli. By contrast, CRF neuronal activity starts to drop within a second of exposure to appetitive stimuli. Optogenetic activation or inhibition of PVN CRF neurons was sufficient to induce a conditioned place aversion (CPA) or preference (CPP), respectively. Furthermore, CPA or CPP induced by natural stimuli was significantly decreased by manipulating PVN CRF neuronal activity. Together, these findings suggest that the rapid, biphasic responses of PVN CRF neurons encode the positive and negative valences of stimuli.

Programming of Stress-Sensitive Neurons and Circuits by Early-Life Experiences.

Early-life experiences influence brain structure and function long-term, contributing to resilience or vulnerability to stress and stress-related disorders. Therefore, understanding the mechanisms by which early-life experiences program specific brain cells and circuits to shape life-long cognitive and emotional functions is crucial. We identify the population of corticotropin-releasing hormone (CRH)-expressing neurons in the hypothalamic paraventricular nucleus (PVN) as a key, early target of early-life experiences. Adverse experiences increase excitatory neurotransmission onto PVN CRH cells, whereas optimal experiences, such as augmented and predictable maternal care, reduce the number and function of glutamatergic inputs onto this cell population. Altered synaptic neurotransmission is sufficient to initiate large-scale, enduring epigenetic re-programming within CRH-expressing neurons, associated with stress resilience and additional cognitive and emotional outcomes. Thus, the mechanisms by which early-life experiences influence the brain provide tractable targets for intervention.

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3.

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation. Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.

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

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. 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 0.5nM 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.

The sympathetic role of glutamatergic paraventricular nucleus neurons in blood pressure regulation.

The sympathetic role of glutamatergic paraventricular nucleus neurons in blood pressure regulation.

Late onset obesity in mice with targeted deletion of potassium inward rectifier Kir7.1 from cells expressing the melanocortin-4 receptor.

Energy stores in fat tissue are determined in part by the activity of hypothalamic neurones expressing the melanocortin-4 receptor (MC4R). Even a partial reduction in MC4R expression levels in mice, rats or humans produces hyperphagia and morbid obesity. Thus, it is of great interest to understand the molecular basis of neuromodulation by the MC4R. The MC4R is a G protein-coupled receptor that signals efficiently through GαS , and this signalling pathway is essential for normal MC4R function in vivo. However, previous data from hypothalamic slice preparations indicated that activation of the MC4R depolarised neurones via G protein-independent regulation of the ion channel Kir7.1. In the present study, we show that deletion of Kcnj13 (ie, the gene encoding Kir7.1) specifically from MC4R neurones produced resistance to melanocortin peptide-induced depolarisation of MC4R paraventricular nucleus neurones in brain slices, resistance to the sustained anorexic effect of exogenously administered melanocortin peptides, late onset obesity, increased linear growth and glucose intolerance. Some MC4R-mediated phenotypes appeared intact, including Agouti-related peptide-induced stimulation of food intake and MC4R-mediated induction of peptide YY release from intestinal L cells. Thus, a subset of the consequences of MC4R signalling in vivo appears to be dependent on expression of the Kir7.1 channel in MC4R cells.

Activation of the hypothalamic paraventricular nucleus by acute intermittent hypoxia: Implications for sympathetic long-term facilitation neuroplasticity

Exposure to acute intermittent hypoxia (AIH) induces a progressive increase of sympathetic nerve activity (SNA) that reflects a form of neuroplasticity known as sympathetic long-term facilitation (sLTF). Our recent findings indicate that activity of neurons in the hypothalamic paraventricular nucleus (PVN) contributes to AIH-induced sLTF, but neither the intra-PVN distribution nor the neurochemical identity of AIH responsive neurons has been determined. Here, awake rats were exposed to 10 cycles of AIH and c-Fos immunohistochemistry was performed to identify transcriptionally activated neurons in rostral, middle and caudal planes of the PVN. Effects of graded intensities of AIH were investigated in separate groups of rats (n = 6/group) in which inspired oxygen (O2) was reduced every 6 min from 21% to nadirs of 10%, 8% or 6%. All intensities of AIH failed to increase c-Fos counts in the caudally located lateral parvocellular region of the PVN. c-Fos counts increased in the dorsal parvocellular and central magnocellular regions, but significance was achieved only with AIH to 6% O2 (P < 0.002). By contrast, graded intensities of AIH induced graded c-Fos activation in the stress-related medial parvocellular (MP) region. Focusing on AIH exposure to 8% O2, experiments next investigated the stress-regulatory neuropeptide content of AIH-activated MP neurons. Tissue sections immunostained for corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) revealed a significantly greater number of neurons stained for CRH than AVP (P < 0.0001), though AIH induced expression of c-Fos in a similar fraction (~14%) of each neurochemical class. Amongst AIH-activated MP neurons, ~30% stained for CRH while only ~2% stained for AVP. Most AIH-activated CRH neurons (~82%) were distributed in the rostral one-half of the PVN. Results indicate that AIH recruits CRH, but not AVP, neurons in rostral to middle levels of the MP region of PVN, and raise the possibility that these CRH neurons may be a substrate for AIH-induced sLTF neuroplasticity.

Synaptic adaptations in the central amygdala and hypothalamic paraventricular nucleus associated with protracted ethanol abstinence in male rhesus monkeys.

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

Exercise training normalizes elevated firing rate of hypothalamic presympathetic neurons in heart failure rats.

Exercise training (ExT) normalizes elevated sympathetic nerve activity in heart failure (HF), but the underlying mechanisms are not well understood. In this study, we examined the effects of 3 wk of ExT on the electrical activity of the hypothalamic presympathetic neurons in the brain slice of HF rats. HF rats were prepared by ligating the left descending coronary artery. The electrophysiological properties of paraventricular nucleus neurons projecting to the rostral ventrolateral medulla (PVN-RVLM) were examined using the slice patch-clamp technique. The neuronal firing rate was elevated in HF rats, and ExT induced a reduction in the firing rate ( P < 0.01). This ExT-induced decrease in the firing rate was associated with an increased frequency of spontaneous and miniature inhibitory postsynaptic current (IPSCs; P < 0.05). There was no significant change in excitatory postsynaptic current. Replacing Ca2+ with Mg2+ in the recording solution reduced the elevated IPSC frequency in HF rats with ExT ( P < 0.01) but not in those without ExT, indicating an increase in the probability of GABA release. In contrast, ExT did not restore the reduced GABAA receptor-mediated tonic inhibitory current in HF rats. A GABAA receptor blocker (bicuculline, 20 μM) increased the firing rate in HF rats with ExT ( P < 0.01) but not in those without ExT. Collectively, these results show that ExT normalized the elevated firing activity by increasing synaptic GABA release in PVN-RVLM neurons in HF rats. Our findings provide a brain mechanism underlying the beneficial effects of ExT in HF, which may shed light on the pathophysiology of other diseases accompanied by sympathetic hyperactivation.

Central nervous regulatory mechanism of myocardial ischemia-reperfusion injury in rats: excitability of neurons in hypothalamic paraventricular nucleus

Objective To evaluate the relationship between excitability of neurons in hypothalamic paraventricular nucleus(PVN) and central nervous regulatory mechanism of myocardial ischemia-reperfusion(I/R) injury in rats. Methods Clean-grade healthy adult male Sprague-Dawley rats, in which PVN catheters were successfully implanted, aged 6-8 weeks, weighing 260-300 g, were divided into 4 groups(n=6 each) using a random number table method: sham operation group(S group), myocardial I/R group(I/R group), γ-aminobutyric acid group(GABA group) and L-glutamic acid group(L-Glu group). Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery for 30 min followed by 120-min reperfusion. Normal saline 2.4 μl/h was infused for 50 min via the PVN catheter starting from 10 min before ischemia in S and I/R groups. GABA 30 μmol/L and L-glutamic acid 30 μmol/L were infused for 50 min via the PVN catheter at a rate of 2.4 μl/h starting from 10 min before ischemia in GABA and L-Glu groups. The heart rate(HR) and mean arterial pressure(MAP) were recorded at 10 min before ischemia(T0), beginning of ischemia(T1), 30 min of ischemia(T2) and 120 min of reperfusion(T3). Rate-pressure product(RPP) was calculated. Blood samples were collected at T3 for determination of plasma cardiac troponin I(cTnI) concentrations by chemiluminescence assay. Rats were then sacrificed and myocardial specimens were obtained for measurement of myocardial infarct size(IS) and area at risk(AAS), and IS/AAR percentage was calculated. PVN tissues were taken to detect the expression of c-fos by Western blot. Results No myocardial infarction was found in group S, and myocardial infarction was marked in the other three groups. Compared with group S, the plasma cTnI concentrations were significantly increased, and the expression of c-fos in PVN was up-regulated in I/R, GABA and L-Glu groups(P<0.05), and MAP, HR and RPP were significantly decreased at T1-3 in I/R and GABA groups and at T3 in group L-Glu(P<0.05). Compared with group I/R, IS and IS/AAR percentage were significantly decreased, the plasma cTnI concentrations were decreased, the expression of c-fos in PVN was down-regulated, and HR, MAP and RPP were decreased at T1, 2 in group GABA, and IS and IS/AAR percentage were significantly increased, the plasma cTnI concentrations were increased, the expression of c-fos in PVN was up-regulated, and HR, MAP and RPP were increased at T1, 2 in group L-Glu(P<0.05). Conclusion Excitability of neurons in hypothalamic PVN is involved in central nervous regulatory mechanism of myocardial I/R injury in rats: decreased excitability can attenuate myocardial I/R injury and increased excitability aggravates myocardial I/R injury. Key words: Paraventricular hypothalamic nucleus; Neurons; Myocardial reperfusion injury

Overexpression of melanocortin 2 receptor accessory protein 2 (MRAP2) in adult paraventricular MC4R neurons regulates energy intake and expenditure

ObjectiveMelanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2. MethodsTo target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII. ResultsMc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females. ConclusionsOur data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure.

Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.

Recurrent periods of over-excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over-activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin-2 leads to an immediate frequency-dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)-salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. Neuro-cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over-excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin-2 using an adeno-associated virus (CamKII-ChR2-eYFP-AAV2) in wildtype mice (n= 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency-dependent (2, 10 and 20Hz) increase in BP from baseline by ∼9mmHg at 20Hz stimulation (P< 0.001). Additionally, in vGlut2-cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre-dependent caspase (AAV2-flex-taCASP3-TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA-salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P< 0.05) resulted in an attenuated rise in BP following DOCA-salt treatment (P< 0.05 at 7day time point, n= 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA-salt treatment in the lesioned animals (P< 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP.

Electrophysiological Effects of Ghrelin in the Hypothalamic Paraventricular Nucleus Neurons.

The paraventricular nucleus (PVN) is involved in the control of sympathetic tone and the secretion of hormones, both functions known to be influenced by ghrelin, suggesting direct effect of ghrelin in this nucleus. However, the effects of ghrelin on the excitability of different PVN neuronal populations have not been demonstrated. This study assessed the effects of ghrelin on the activity of PVN neurons, correlating the responses to subpopulations of PVN neurons. We used a 64 multielectrode array to examine the effects of ghrelin administration on extracellular spike frequency in PVN neurons recorded in brain slices obtained from male Sprague-Dawley rats. Bath administration of 10 nM ghrelin increased (29/97, 30%) or decreased (37/97, 38%) spike frequency in PVN neurons. The GABAA and glutamate receptors antagonists abolish the decrease in spike frequency, without changes in the proportion of increases in spike frequency (23/53, 43%) induced by ghrelin. The results indicate a direct effect of ghrelin increasing PVN neurons activity and a synaptic dependent effect decreasing PVN neurons activity. The patch clamp recordings showed similar proportions of PVN neurons influenced by 10 nM ghrelin (33/95, 35% depolarized; 29/95, 30% hyperpolarized). Using electrophysiological fingerprints to identify specific subpopulations of PVN neurons we observed that the majority of pre-autonomic neurons (11/18 -61%) were depolarized by ghrelin, while both neuroendocrine (29% depolarizations, 40% hyperpolarizations), and magnocellular neurons (29% depolarizations, 21% hyperpolarizations) showed mixed responses. Finally, to correlate the electrophysiological response and the neurochemical phenotype of PVN neurons, cell cytoplasm was collected after recordings and RT-PCR performed to assess the presence of mRNA for vasopressin, oxytocin, thyrotropin (TRH) and corticotropin (CRH) releasing hormones. The single-cell RT-PCR showed that most TRH-expressing (4/5) and CRH-expressing (3/4) neurons are hyperpolarized in response to ghrelin. In conclusion, ghrelin either directly increases or indirectly decreases the activity of PVN neurons, this suggests that ghrelin acts on inhibitory PVN neurons that, in turn, decrease the activity of TRH-expressing and CRH-expressing neurons in the PVN.

Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN-RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN-RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN-RVLM neurons. Male rats received microinjections into the PVN with an adeno-associated virus (AAV) vector that encoded a hybrid of channelrhodopsin-2/1 with the reporter tdTomato (ChIEF-tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2-GFPretro ). Under anaesthesia with urethane and α-chloralose, photostimulation (473nm wavelength) of PVN-RVLM neurons, achieved by laser illumination of either RVLM of ChIEF-tdTomato rats (n= 8) or PVN of ChR2-GFPretro rats (n= 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF-tdTomato rats were closely associated with tdTomato-labelled, PVN-derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF-tdTomato rats (n= 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN-RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons.

Stimulation of brain nicotinic acetylcholine receptors activates adrenomedullary outflow via brain inducible NO synthase-mediated S-nitrosylation.

We have demonstrated that i.c.v.-administered (±)-epibatidine, a nicotinic ACh receptor (nAChR) agonist, induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla with dihydro-β-erythroidin (an α4β2 nAChR antagonist)-sensitive brain mechanisms. Here, we examined central mechanisms for the (±)-epibatidine-induced responses, focusing on brain NOS and NO-mediated mechanisms, soluble GC (sGC) and protein S-nitrosylation (a posttranslational modification of protein cysteine thiol groups), in urethane-anaesthetized (1.0g·kg-1 , i.p.) male Wistar rats. (±)-Epibatidine was i.c.v. treated after i.c.v. pretreatment with each inhibitor described below. Then, plasma catecholamines were measured electrochemically after HPLC. Immunoreactivity of S-nitrosylated cysteine (SNO-Cys) in α4 nAChR subunit (α4)-positive spinally projecting neurones in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of adrenomedullary outflow) after i.c.v. (±)-epibatidine administration was also investigated. (±)-Epibatidine-induced elevation of plasma catecholamines was significantly attenuated by L-NAME (non-selective NOS inhibitor), carboxy-PTIO (NO scavenger), BYK191023 [selective inducible NOS (iNOS) inhibitor] and dithiothreitol (thiol-reducing reagent), but not by 3-bromo-7-nitroindazole (selective neuronal NOS inhibitor) or ODQ (sGC inhibitor). (±)-Epibatidine increased the number of spinally projecting PVN neurones with α4- and SNO-Cys-immunoreactivities, and this increment was reduced by BYK191023. Stimulation of brain nAChRs can induce elevation of plasma catecholamines through brain iNOS-derived NO-mediated protein S-nitrosylation in rats. Therefore, brain nAChRs (at least α4β2 subtype) and NO might be useful targets for alleviation of catecholamines overflow induced by smoking.