Paraventricular Nucleus Neuronal Activity Research Articles

Group III Metabotropic Glutamate Receptors Regulate Excitability of Hypothalamic Presympathetic Neurons and Sympathetic Output in Hypertension

Enhanced glutamatergic input to the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. Group III metabotropic glutamate receptors (mGluRs) are expressed in the PVN. In the present study, we determined the role of group III mGluRs in the control of the excitability of spinally projecting PVN neurons and sympathetic outflow in hypertension. We found that stimulation of group III mGluRs with L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) consistently inhibited the firing activity of PVN neurons in normotensive WKY rats. However, L‐AP4 inhibited 37%, but excited 48%, of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Both the inhibitory and excitatory effects of L‐AP4 on the firing activity were abolished by the group III mGluR antagonist CPPG. Inclusion of GDP‐β‐S in the intracellular recording solution eliminated the excitatory effect of L‐AP4 on the firing activity of PVN neurons in SHRs, but it did not significantly change the inhibitory effect of L‐AP4 on PVN neurons in WKY rats or SHRs. L‐AP4 significantly reduced the frequency of spontaneous excitatory postsynaptic currents of PVN neurons in both WKY rats and SHRs. Interestingly, pretreatment of brain slices with the group I mGluR agonist DHPG converted the L‐AP4 effect on PVN neurons from inhibitory to excitatory in WKY rats. Also, L‐AP4 consistently inhibited PVN neurons in SHRs after treatment with the group I mGluR antagonist MPEP. In addition, microinjection of L‐AP4 into the PVN dose‐dependently decreased arterial Bp and lumbar sympathetic nerve activity (LSNA) in WKY rats and SHRs. Microinjection of L‐AP4 in the presence of MPEP produced a more significant reduction in arterial BP and LSNA than did with microinjection of L‐AP4 alone in SHRs. These findings suggest that group III mGluRs in the PVN regulate the synaptic plasticity and excitability of PVN presympathetic neurons and sympathetic vasomotor activity in hypertension. The signaling of postsynaptic group III mGluRs is influenced by increased group I mGluR activity in PVN presympathetic neurons in hypertension. Supported by NIH grants HL131161 and HL142133.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased excitatory regulation of the hypothalamic paraventricular nucleus and circulating vasopressin results in the hypertension observed in polycystic kidney disease.

Hypertension and baroreflex dysfunction confer poorer outcomes in patients with polycystic kidney disease (PKD). We examined whether hypothalamic paraventricular nucleus (PVN) activation or circulating vasopressin contribute to hypertension and baroreflex dysfunction in the Lewis polycystic kidney (LPK) rat. Bilateral PVN inhibition with muscimol reduced SBP further in urethane-anaesthetized adult LPK rats than in control Lewis rats (-43 ± 4 vs. -18 ± 3 mmHg; P < 0.0001, n = 14), but was not associated with a greater reduction in sympathetic nerve activity (SNA) or improvement in HR or SNA baroreflex function. Blockade of ionotropic glutamatergic input to the PVN with kynurenic acid also reduced SBP (P < 0.001), but not SNA, further in both adult and juvenile LPK rats. No differences in AMPA or NMDA receptor mRNA expression were noted. Systemic V1A receptor antagonism using OPC-21268 reduced SBP in adult LPK rats only (P < 0.001) and had no effect on the depressor response to PVN inhibition (P = 0.39). Combined peripheral V1A receptor antagonism and PVN inhibition, however, normalized SBP in adult LPK rats (122 ± 11 vs. 115 ± 6 mmHg; LPK vs. Lewis, P > 0.05, n = 10). Our data show that in the LPK rat model of PKD, hypertension is contributed to by increased PVN neuronal activity and, through an independent mechanism, systemic V1A receptor activation. Treatments that reduce PVN neuronal activity and/or inhibit peripheral V1A receptors may provide novel treatment strategies to ameliorate hypertension in individuals with PKD and limit overall disease progression.

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.

α2δ-1 Is Essential for Sympathetic Output and NMDA Receptor Activity Potentiated by Angiotensin II in the Hypothalamus.

Both the sympathetic nervous system and the renin-angiotensin system are critically involved in hypertension development. Although angiotensin II (Ang II) stimulates hypothalamic paraventricular nucleus (PVN) neurons to increase sympathetic vasomotor tone, the molecular mechanism mediating this action remains unclear. The glutamate NMDAR in the PVN controls sympathetic outflow in hypertension. In this study, we determined the interaction between α2δ-1 (encoded by Cacna2d1), commonly known as a Ca2+ channel subunit, and NMDARs in the hypothalamus and its role in Ang II-induced synaptic NMDAR activity in PVN presympathetic neurons. Coimmunoprecipitation assays showed that α2δ-1 interacted with the NMDAR in the hypothalamus of male rats and humans (both sexes). Ang II increased the prevalence of synaptic α2δ-1-NMDAR complexes in the hypothalamus. Also, Ang II increased presynaptic and postsynaptic NMDAR activity via AT1 receptors, and such effects were abolished either by treatment with pregabalin, an inhibitory α2δ-1 ligand, or by interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide. In Cacna2d1 knock-out mice (both sexes), Ang II failed to affect the presynaptic and postsynaptic NMDAR activity of PVN neurons. In addition, the α2δ-1 C terminus-interfering peptide blocked the sympathoexcitatory response to microinjection of Ang II into the PVN. Our findings indicate that Ang II augments sympathetic vasomotor tone and excitatory glutamatergic input to PVN presympathetic neurons by stimulating α2δ-1-bound NMDARs at synapses. This information extends our understanding of the molecular basis for the interaction between the sympathetic nervous and renin-angiotensin systems and suggests new strategies for treating neurogenic hypertension.SIGNIFICANCE STATEMENT Although both the sympathetic nervous system and renin-angiotensin system are closely involved in hypertension development, the molecular mechanisms mediating this involvement remain unclear. We showed that α2δ-1, previously known as a calcium channel subunit, interacts with NMDARs in the hypothalamus of rodents and humans. Angiotensin II (Ang II) increases the synaptic expression level of α2δ-1-NMDAR complexes. Furthermore, inhibiting α2δ-1, interrupting the α2δ-1-NMDAR interaction, or deleting α2δ-1 abolishes the potentiating effects of Ang II on presynaptic and postsynaptic NMDAR activity in the hypothalamus. In addition, the sympathoexcitatory response to Ang II depends on α2δ-1-bound NMDARs. Thus, α2δ-1-NMDAR complexes in the hypothalamus serve as an important molecular substrate for the interaction between the sympathetic nervous system and the renin-angiotensin system. This evidence suggests that α2δ-1 may be a useful target for the treatment neurogenic hypertension.

High salt intake enhances swim stress-induced PVN vasopressin cell activation and active stress coping

PurposeStress contributes to many psychiatric disorders; however, responsivity to stressors can vary depending on previous or current stress exposure. Relatively innocuous heterotypic (differing in type) stressors can summate to result in exaggerated neuronal and behavioral responses. Here we investigated the ability of prior high dietary sodium chloride (salt) intake, a dehydrating osmotic stressor, to enhance neuronal and behavioral responses of mice to an acute psychogenic swim stress (SS). Further, we evaluated the contribution of the osmo-regulatory stress-related neuropeptide arginine vasopressin (VP) in the hypothalamic paraventricular nucleus (PVN), one of only a few brain regions that synthesize VP. The purpose of this study was to determine the impact of high dietary salt intake on responsivity to heterotypic stress and the potential contribution of VPergic-mediated neuronal activity on high salt-induced stress modulation, thereby providing insight into how dietary (homeostatic) and environmental (psychogenic) stressors might interact to facilitate psychiatric disorder vulnerability. ApproachSalt loading (SL) with 4% saline for 7 days was used to dehydrate and osmotically stress mice prior to exposure to an acute SS. Fluid intake and hematological measurements were taken to quantify osmotic dehydration, and serum corticosterone levels were measured to index stress axis activation. Immunohistochemistry (IHC) was used to stain for the immediate early gene product c-Fos to quantify effects of SL on SS-induced activation of neurons in the PVN and extended amygdala – brain regions that are synaptically connected and implicated in responding to osmotic stress and in modulation of SS behavior, respectively. Lastly, the role of VPergic PVN neurons and VP type 1 receptor (V1R) activity in the amygdala in mediating effects of SL on SS behavior was evaluated by quantifying c-Fos activation of VPergic PVN neurons and, in functional experiments, by nano-injecting the V1R selective antagonist dGly[Phaa1,d-tyr(et), Lys, Arg]-VP bilaterally into the amygdala prior to the SS. FindingsSL increased serum osmolality (P < 0.01), which positively correlated with time spent mobile during, and time spent grooming after a SS (P < 0.01, P < 0.01), and SL increased serum corticosterone levels (P < 0.01). SL alone increased c-Fos immunoreactivity among PVN neurons (P = .02), including VP positive neurons (P < 0.01). SL increased SS-induced c-Fos activation of PVN neurons as well (P < 0.01). In addition, SL and SS each increased the total number of PVN neurons that were immunoreactive for VP (P < 0.01). An enhancing effect of SL and SS was observed on c-Fos positive cell counts in the central (P = .02) and basolateral (P < 0.01) nuclei of the amygdala and bilateral nano-injections of V1R antagonist into the amygdala reduced time spent mobile both in salt loaded and control mice during SS (P < 0.05, P < 0.05). SummaryTaken together, these data indicate that neuronal and behavioral responsivity to an acute psychogenic stressor is potentiated by prior exposure to high salt intake. This synergistic effect was associated with activation of PVN VP neurons and depended, in part, on activity of V1 receptors in the amygdala. Findings provide novel insight into neural mechanisms whereby prior exposure to a homeostatic stressor such as osmotic dehydration by excessive salt intake increases responsivity to a perceived stress. These experiments show that high dietary salt can influence stress responsivity and raise the possibility that excessive salt intake could be a contributing factor in the development of stress-related psychiatric disorders.

α2δ‐1 Is Essential for Angiotensin II‐Induced Sympathoexcitation and NMDA Receptor Hyperactivity in the Hypothalamus

Both the sympathetic nervous system and renin‐angiotensin system are critically involved in hypertension development. Although angiotensin II (Ang II) stimulates presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) to increase sympathetic vasomotor tone, the molecular mechanism responsible for this action remains unclear. The glutamate N‐methyl‐D‐aspartate receptor (NMDAR) in the PVN controls sympathetic outflow in hypertension. In this study, we determined how Ang II affects synaptic NMDARs in spinally projecting PVN neurons. Coimmunoprecipitation assays showed that α2δ‐1, commonly known as a voltage‐gated calcium channel subunit, interacted with the NMDAR in the hypothalamus of rats and humans. Ang II increased the synaptic expression level of α2δ‐1–NMDAR complexes. Also, Ang II increased pre‐ and postsynaptic NMDAR activity via AT1 receptors, and such effects were abolished either by treatment with pregabalin, an inhibitory α2δ‐1 ligand, or by interrupting the α2δ‐1–NMDAR interaction with α2δ‐1Tat peptide. In α2δ‐1 knockout mice, Ang II failed to affect the pre‐ and postsynaptic NMDAR activity of PVN neurons. In addition, the α2δ‐1Tat peptide blocked the sympathoexcitatory response to the microinjection of Ang II into the PVN. Our findings indicate that by promoting the synaptic trafficking of α2δ‐1–bound NMDARs, Ang II augments sympathetic vasomotor tone and excitatory glutamatergic input to PVN presympathetic neurons.Support or Funding InformationThis work was supported by the National Institutes of Health (Grant R01 HL131161) and the N.G. and Helen T. Hawkins Endowment (to H.‐L.P.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regional distribution of hypothalamic paraventricular nucleus neurons activated by acute intermittent hypoxia

Upon returning to normoxic ventilation following exposure to acute intermittent hypoxia (AIH), sympathetic nerve activity (SNA) is observed to progressively increase – a response termed sympathetic long term facilitation (sLTF). We recently reported that inhibition of the hypothalamic paraventricular nucleus (PVN) prior to AIH attenuated post‐AIH sLTF in anesthetized rats. Although this study implicated PVN neuronal activity in post‐AIH sLTF, the specific PVN cell groups involved and their neurochemical phenotypes were not determined. Here we hypothesized that AIH activates parvocellular subregions of the PVN enriched with sympathetic regulatory neurons, and tested this using c‐Fos IHC to identify neurons activated by 10 cycles of AIH in awake rats. Separate groups were exposed to AIH in which FIO2 was reduced every 6 minutes to nadirs of 10%, 8%, or 6%, with each nadir lasting ~75 s. Compared to control rats exposed to sham AIH (n=6), the number of Fos+ nuclei in the medial parvocellular (MP) region of the PVN was progressively greater in rats exposed to AIH to 10% (367%, P&lt;0.01, n=4), 8% (566%, P&lt;0.01, n=5) and 6% (1054%, P&lt;0.01, n=4) FIO2. Fos+ counts increased in the dorsal parvocellular and central magnocellular regions of the PVN as well, but only in response to AIH to 6% FIO2 (P&lt;0.05, n=4–5). No level of AIH increased Fos+ nuclei in the caudally located lateral parvocellular area (P&gt;0.05, n=4–5). Neurochemical phenotypes of neurons activated by AIH to 8% FIO2 were examined by co‐immunostaining for corticotropin‐releasing hormone (CRH) or arginine vasopressin (AVP), which are stress‐responsive neurons enriched in the MP region. AIH to 8% FIO2 was selected for this analysis because it induced robust Fos expression without eliciting escape behavior as was observed in rats exposed to AIH to 6% FIO2. Whereas CRH+ neurons (~772) outnumbered AVP+ neurons (~50), the population size of each was similar between sham treated and AIH exposed rats. A significantly greater fraction of CRH+ neurons (~14%, P&lt;0.05) and AVP+ neurons (~13%, P&lt;0.05) was Fos+ in AIH exposed rats (n=4) compared to sham treated controls (CRH+, ~3%; AVP+, ~0%; n=4). Further analysis revealed that ~30% of AIH‐activated PVN neurons were CRH+ (110/371) while only ~2% (7/371) were AVP+. Thus a sizable fraction of AIH‐activated PVN neurons were CRH‐containing, while the neurochemical phenotype(s) of the majority of activated neurons (~68%) remains to be identified. These data suggest that responses to AIH are unlikely to be mediated by PVN‐AVP neurons, and raise the possibility that CRH neurons could contribute to endocrine and/or autonomic responses to AIH, including post‐AIH sLTF. Studies are needed to test the latter possibility and to define the role of AIH‐activated PVN neurons whose chemical phenotypes remain unknown. Determining intrinsic and synaptic mechanisms leading to AIH activation of PVN neurons could provide insight into mechanisms of persistent sympathetic activation in cardiovascular and metabolic diseases.Support or Funding InformationSupport: NIH HL088052 &amp; AHA 25710176 (GMT)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Proinflammatory Cytokines Is Upregulated in the Hypothalamic Paraventricular Nucleus of Dahl Salt-Sensitive Hypertensive Rats.

Accumulating evidence indicates that inflammation is implicated in hypertension. However, the role of brain proinflammatory cytokines (PICs) in salt sensitive hypertension remains to be determined. Thus, the objective of this study was to test the hypothesis that high salt (HS) diet increases PICs expression in the paraventricular nucleus (PVN) and leads to PVN neuronal activation. Eight-week-old male Dahl salt sensitive (Dahl S) rats, and age and sex matched normal Sprague Dawley (SD) rats were divided into two groups and fed with either a HS (4% NaCl) or normal salt (NS, 0.4% NaCl) diet for 5 consecutive weeks. HS diet induced hypertension and significantly increased cerebrospinal fluid (CSF) sodium concentration ([Na+]) in Dahl S rats, but not in normal SD rats. In addition, HS diet intake triggered increases in mRNA levels and immunoreactivities of PVN PICs including TNF-α, IL-6, and IL-1β, as well as Fra1, a chronic marker of neuronal activation, in Dahl S rats, but not in SD rats. Next, we investigated whether this increase in the expression of PVN PICs and Fra1 was induced by increased CSF [Na+]. Adult male SD rats were intracerebroventricular (ICV) infused with 8 μl of either hypertonic salt (4 μmol NaCl), mannitol (8 μmol, as osmolarity control), or isotonic salt (0.9% NaCl as vehicle control). Three hours following the ICV infusion, rats were euthanized and their PVN PICs expression was measured. The results showed that central administration of hypertonic saline in SD rats significantly increased the expression of PICs including TNF-α, IL-6, and IL-1β, as well as neuronal activation marker Fra1, compared to isotonic NaCl controls and osmolarity controls. Finally, we tested whether the increase in PICs expression occurred in neurons. Incubation of hypothalamic neurons with 10 mM NaCl in a culture medium for 6 h elicited significant increases in TNF-α, IL-6, and Fra1 mRNA levels. These observations, coupled with the important role of PICs in modulating neuronal activity and stimulating vasopressin release, suggest that HS intake induces an inflammatory state in the PVN, which, may in turn, augments sympathetic nerve activity and vasopressin secretion, contributing to the development of salt sensitive hypertension.

Korean red ginseng excitation of paraventricular nucleus neurons via non-N-methyl-D-aspartate glutamate receptor activation in mice.

It has been reported that Korean red ginseng (KRG), a valuable and important traditional medicine, has varied effects on the central nervous system, suggesting its activities are complicated. The paraventricular nucleus (PVN) neurons of the hypothalamus has a critical role in stress responses and hormone secretions. Although the action mechanisms of KRG on various cells and systems have been reported, the direct membrane effects of KRG on PVN neurons have not been fully described. In this study, the direct membrane effects of KRG on PVN neuronal activity were investigated by using a perforated patch-clamp in ICR mice. In gramicidin perforated patch-clamp mode, KRG extract (KRGE) induced repeatable depolarization followed by hyperpolarization of PVN neurons. The KRGE-induced responses were concentration-dependent and persisted in the presence of tetrodotoxin, a voltage sensitive Na+ channel blocker. The KRGE-induced responses were suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM), a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, but not by picrotoxin, a type A gamma-aminobutyric acid receptor antagonist. The results indicate that KRG activates non-NMDA glutamate receptors of PVN neurons in mice, suggesting that KRG may be a candidate for use in regulation of stress responses by controlling autonomic nervous system and hormone secretion.

Hypothalamic PVN contributes to acute intermittent hypoxia-induced sympathetic but not phrenic long-term facilitation.

Blackburn MB, Andrade MA, Toney GM. Hypothalamic PVN contributes to acute intermittent hypoxia-induced sympathetic but not phrenic long-term facilitation. J Appl Physiol 124: 1233-1243, 2018. First published December 19, 2017; doi: 10.1152/japplphysiol.00743.2017 .- Acute intermittent hypoxia (AIH) repetitively activates the arterial chemoreflex and triggers a progressive increase of sympathetic nerve activity (SNA) and phrenic nerve activity (PNA) referred to as sympathetic and phrenic long-term facilitation (S-LTF and P-LTF), respectively. Neurons of the hypothalamic paraventricular nucleus (PVN) participate in the arterial chemoreflex, but their contribution to AIH-induced LTF is unknown. To determine this, anesthetized rats were vagotomized and exposed to 10 cycles of AIH, each consisting of ventilation for 3 min with 100% O2 followed by 3 min with 15% O2. Before AIH, rats received bilateral PVN injections of artificial cerebrospinal fluid (aCSF; vehicle) or the GABA-A receptor agonist muscimol (100 pmol in 50 nl) to inhibit neuronal activity. Thirty minutes after completing the AIH protocol, during which rats were continuously ventilated with 100% O2, S-LTF and P-LTF were quantified from recordings of integrated splanchnic SNA and PNA, respectively. PVN muscimol attenuated increases of SNA during hypoxic episodes occurring in later cycles (6-10) of AIH ( P < 0.03) and attenuated post-AIH S-LTF ( P < 0.001). Muscimol, however, did not consistently affect peak PNA responses during hypoxic episodes and did not alter AIH-induced P-LTF. These findings indicate that PVN neuronal activity contributes to sympathetic responses during AIH and to subsequent generation of S-LTF. NEW & NOTEWORTHY Neural circuits mediating acute intermittent hypoxia (AIH)-induced sympathetic and phrenic long-term facilitation (LTF) have not been fully elucidated. We found that paraventricular nucleus (PVN) inhibition attenuated sympathetic activation during episodes of AIH and reduced post-AIH sympathetic LTF. Neither phrenic burst patterning nor the magnitude of AIH-induced phrenic LTF was affected. Findings indicate that PVN neurons contribute to AIH-induced sympathetic LTF. Defining mechanisms of sympathetic LTF could improve strategies to reduce sympathetic activity in cardiovascular and metabolic diseases.

Overactivity of Liver-Related Neurons in the Paraventricular Nucleus of the Hypothalamus: Electrophysiological Findings in db/db Mice.

Preautonomic neurons in the paraventricular nucleus (PVN) of the hypothalamus play a large role in the regulation of hepatic functions via the autonomic nervous system. Activation of hepatic sympathetic nerves increases glucose and lipid metabolism and contributes to the elevated hepatic glucose production observed in the type 2 diabetic condition. This augmented sympathetic output could originate from altered activity of liver-related PVN neurons. Remarkably, despite the importance of the brain-liver pathway, the cellular properties of liver-related neurons are not known. In this study, we provide the first evidence of overall activity of liver-related PVN neurons. Liver-related PVN neurons were identified with a retrograde, trans-synaptic, viral tracer in male lean and db/db mice and whole-cell patch-clamp recordings were conducted. In db/db mice, the majority of liver-related PVN neurons fired spontaneously; whereas, in lean mice the majority of liver-related PVN neurons were silent, indicating that liver-related PVN neurons are more active in db/db mice. Persistent, tonic inhibition was identified in liver-related PVN neurons; although, the magnitude of tonic inhibitory control was not different between lean and db/db mice. In addition, our study revealed that the transient receptor potential vanilloid type 1-dependent increase of excitatory neurotransmission was reduced in liver-related PVN neurons of db/db mice. These findings demonstrate plasticity of liver-related PVN neurons and a shift toward excitation in a diabetic mouse model. Our study suggests altered autonomic circuits at the level of the PVN, which can contribute to autonomic dysfunction and dysregulation of neural control of hepatic functions including glucose metabolism.SIGNIFICANCE STATEMENT A growing body of evidence suggests the importance of the autonomic control in the regulation of hepatic metabolism, which plays a major role in the development and progression of type 2 diabetes mellitus. Despite the importance of the brain-liver pathway, the overall activity of liver-related neurons in control and diabetic conditions is not known. This is a significant gap in knowledge, which prevents developing strategies to improve glucose homeostasis via altering the brain-liver pathway. One of the key findings of our study is the overall shift toward excitation in liver-related hypothalamic neurons in the diabetic condition. This overactivity may be one of the underlying mechanisms of elevated sympathetic activity known in metabolically compromised patients and animal models.

Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin-3-RXFP3 signalling.

Relaxin-3 is a stress-responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3-dependent, inhibitory action of relaxin-3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin-3. We observed a Gαi/o -protein-dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium-sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin-3-immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin-3-containing fibres/terminals within the PVN. The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra-PVN injections of the neuropeptide relaxin-3 or selective relaxin-3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin-3, acting through its cognate G-protein-coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin-3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3-A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3-A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri-PVN and sparse intra-PVN relaxin-3-immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin-3 neurons, identify a strong inhibitory influence of relaxin-3-RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

Src Kinases Regulate Glutamatergic Input to Hypothalamic Presympathetic Neurons and Sympathetic Outflow in Hypertension.

The elevated sympathetic outflow associated with hypertension is maintained by increased N-methyl-d-aspartate receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus. Synaptic NMDAR activity is tightly regulated by protein kinases, including the Src family of tyrosine kinases. We determined whether Src kinases play a role in increased NMDAR activity of PVN neurons projecting to the rostral ventrolateral medulla and in elevated sympathetic vasomotor tone in spontaneously hypertensive rats (SHRs). The Src protein level in the PVN was significantly greater in SHRs than in normotensive Wistar-Kyoto (WKY) rats and was not significantly altered by lowering blood pressure with celiac ganglionectomy in SHRs. Inhibition of Src kinase activity with 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) completely normalized the higher amplitudes of evoked NMDAR-mediated excitatory postsynaptic currents and puff NMDA-elicited currents of rostral ventrolateral medulla-projecting PVN neurons in SHRs. PP2 treatment also attenuated the higher frequency of NMDAR-mediated miniature excitatory postsynaptic currents of these neurons in SHRs. However, PP2 had no effect on NMDAR-excitatory postsynaptic currents or miniature excitatory postsynaptic currents of rostral ventrolateral medulla-projecting PVN neurons in WKY rats. NMDAR activity increased by an Src-activating peptide was blocked by PP2 but not by inhibition of casein kinase 2. In addition, microinjection of PP2 into the PVN not only decreased lumbar sympathetic nerve discharges and blood pressure but also eliminated the inhibitory effect of the NMDAR antagonist on sympathetic nerve activity and blood pressure in SHRs. Collectively, our findings suggest that increased Src kinase activity potentiates presynaptic and postsynaptic NMDAR activity in the PVN and sympathetic vasomotor tone in hypertension.

Astrocytes Contribute to Angiotensin II Stimulation of Hypothalamic Neuronal Activity and Sympathetic Outflow.

Angiotensin II (AngII) is a key neuropeptide that acting within the brain hypothalamic paraventricular nucleus regulates neurohumoral outflow to the circulation. Moreover, an exacerbated AngII action within the paraventricular nucleus contributes to neurohumoral activation in hypertension. Although AngII effects involve changes in paraventricular nucleus neuronal activity, the precise underlying mechanisms, cellular targets, and distribution of AngII receptors within the paraventricular nucleus remain largely unknown. Thus, whether AngII effects involve direct actions on paraventricular neurons, or whether it acts via intermediary cells, such as astrocytes, is still controversial. To address this important gap in our knowledge, we used a multidisciplinary approach combining patch-clamp electrophysiology in presympathetic paraventricular neurons and astrocytes, along with in vivo sympathetic nerve recordings and astrocyte-targeted gene manipulations. We present evidence for a novel mechanism underlying central AngII actions, which involves astrocytes as major intermediary cellular targets. We found that AngII type 1 receptor mRNA is expressed in paraventricular astrocytes. Moreover, we report that AngII inhibited glutamate transporter function, increasing in turn extracellular glutamate levels. This resulted in the activation of neuronal extrasynaptic NMDA (N-methyl-d-aspartate) receptors, increased presympathetic neuronal activity, enhanced sympathoexcitatory outflow, and increased blood pressure. Together, our studies support astrocytes as critical intermediary cell types mediating brain AngII regulation of the circulation and indicate that AngII-mediated neuronal and sympathoexcitatory effects are dependent on a unique neuroglial signaling modality involving nonsynaptic glutamate transmission.

Renal Denervation Improves Exaggerated Sympathoexcitation in Rats With Heart Failure: A Role for Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus.

Renal denervation (RDN) has been postulated to reduce sympathetic drive during heart failure (HF), but the central mechanisms are not completely understood. The purpose of the present study was to assess the contribution of neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN) in modulating sympathetic outflow in rats with HF that underwent RDN. HF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, bilateral RDN was performed. Rats with HF had an increase in FosB-positive cells in the PVN with a concomitant increase in urinary excretion of norepinephrine, and both of these parameters were ameliorated after RDN. nNOS-positive cells immunostaining, diaphorase staining, and nNOS protein expression were significantly decreased in the PVN of HF rats, findings that were ameliorated by RDN. Microinjection of nNOS inhibitor N(G)-monomethyl l-arginine into the PVN resulted in a blunted increase in lumbar sympathetic nerve activity (11±2% versus 24±2%) in HF than in sham group. This response was normalized after RDN. Stimulation of afferent renal nerves produced a greater activation of PVN neurons in rats with HF. Afferent renal nerve stimulation elicited a greater increase in lumbar sympathetic nerve activity in rats with HF than in sham rats (45±5% versus 22±2%). These results suggest that intact renal nerves contribute to the reduction of nNOS in the PVN, resulting in the activation of the neurons in the PVN of rats with HF. RDN restores nNOS and thus attenuates the sympathoexcitation commonly observed in HF.

Paraventricular Nucleus Modulates Excitatory Cardiovascular Reflexes during Electroacupuncture.

The paraventricular nucleus (PVN) regulates sympathetic outflow and blood pressure. Somatic afferent stimulation activates neurons in the hypothalamic PVN. Parvocellular PVN neurons project to sympathoexcitatory cardiovascular regions of the rostral ventrolateral medulla (rVLM). Electroacupuncture (EA) stimulates the median nerve (P5-P6) to modulate sympathoexcitatory responses. We hypothesized that the PVN and its projections to the rVLM participate in the EA-modulation of sympathoexcitatory cardiovascular responses. Cats were anesthetized and ventilated. Heart rate and mean blood pressure were monitored. Application of bradykinin every 10-min on the gallbladder induced consistent pressor reflex responses. Thirty-min of bilateral EA stimulation at acupoints P5-P6 reduced the pressor responses for at least 60-min. Inhibition of the PVN with naloxone reversed the EA-inhibition. Responses of cardiovascular barosensitive rVLM neurons evoked by splanchnic nerve stimulation were reduced by EA and then restored with opioid receptor blockade in the PVN. EA at P5-P6 decreased splanchnic evoked activity of cardiovascular barosensitive PVN neurons that also project directly to the rVLM. PVN neurons labeled with retrograde tracer from rVLM were co-labeled with μ-opioid receptors and juxtaposed to endorphinergic fibers. Thus, the PVN and its projection to rVLM are important in processing acupuncture modulation of elevated blood pressure responses through a PVN opioid mechanism.

ATP stimulates rat hypothalamic sympathetic neurons by enhancing AMPA receptor-mediated currents.

We have previously shown that ATP within the paraventricular nucleus (PVN) induces an increase in sympathetic activity, an effect attenuated by the antagonism of P2 and/or glutamatergic receptors. Here, we evaluated precise cellular mechanisms underlying the ATP-glutamate interaction in the PVN and assessed whether this receptor coupling contributed to osmotically driven sympathetic PVN neuronal activity. Whole-cell patch-clamp recordings obtained from PVN-rostral ventrolateral medulla neurons showed that ATP (100 μM, 1 min, bath applied) induced an increase in firing rate (89%), an effect blocked by kynurenic acid (1 mM) or 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10 μM). Whereas ATP did not affect glutamate synaptic function, α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor-mediated currents evoked by focal application of AMPA (50 μM, n = 13) were increased in magnitude by ATP (AMPA amplitude: 33%, AMPA area: 52%). ATP potentiation of AMPA currents was blocked by PPADS (n = 12) and by chelation of intracellular Ca(2+) (BAPTA, n = 10). Finally, a hyperosmotic stimulus (mannitol 1%, +55 mosM, n = 8) potentiated evoked AMPA currents (53%), an effect blocked by PPADS (n = 6). Taken together, our data support a functional stimulatory coupling between P2 and AMPA receptors (likely of extrasynaptic location) in PVN sympathetic neurons, which is engaged in response to an acute hyperosmotic stimulus, which might contribute in turn to osmotically driven sympathoexcitatory responses by the PVN.

Activation of corticotropin-releasing factor receptors in the rostral ventrolateral medulla is required for glucose-induced sympathoexcitation.

Energy expenditure is determined by metabolic rate and diet-induced thermogenesis. Normally, energy expenditure increases due to neural mechanisms that sense plasma levels of ingested nutrients/hormones and reflexively increase sympathetic nerve activity (SNA). Here, we investigated neural mechanisms of glucose-driven sympathetic activation by determining contributions of neuronal activity in the hypothalamic paraventricular nucleus (PVN) and activation of corticotropin-releasing factor (CRF) receptors in the rostral ventrolateral medulla (RVLM). Glucose was infused intravenously (150 mg/kg, 10 min) in male rats to raise plasma glucose concentration to a physiological postprandial level. In conscious rats, glucose infusion activated CRF-containing PVN neurons and TH-containing RVLM neurons, as indexed by c-Fos immunofluorescence. In α-chloralose/urethane-anesthetized rats, glucose infusion increased lumbar and splanchnic SNA, which was nearly prevented by prior RVLM injection of the CRF receptor antagonist astressin (10 pmol/50 nl). This cannot be attributed to a nonspecific effect, as sciatic afferent stimulation increased SNA and ABP equivalently in astressin- and aCSF-injected rats. Glucose-stimulated sympathoexcitation was largely reversed during inhibition of PVN neuronal activity with the GABA-A receptor agonist muscimol (100 pmol/50 nl). The effects of astressin to prevent glucose-stimulated sympathetic activation appear to be specific to interruption of PVN drive to RVLM because RVLM injection of astressin prior to glucose infusion effectively prevented SNA from rising and prevented any fall of SNA in response to acute PVN inhibition with muscimol. These findings suggest that activation of SNA, and thus energy expenditure, by glucose is initiated by activation of CRF receptors in RVLM by descending inputs from PVN.

An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

K(+)-dependent Na(+)/Ca(2+)-exchangers are broadly expressed in various tissues, and particularly enriched in neurons of the brain. The distinct physiological roles for the different members of this Ca(2+) transporter family are, however, not well described. Here we show that gene-targeted mice lacking the K(+)-dependent Na(+)/Ca(2+)-exchanger, NCKX4 (gene slc24a4 or Nckx4), display a remarkable anorexia with severe hypophagia and weight loss. Feeding and satiety are coordinated centrally by melanocortin-4 receptors (MC4R) in neurons of the hypothalamic paraventricular nucleus (PVN). The hypophagic response of Nckx4 knock-out mice is accompanied by hyperactivation of neurons in the PVN, evidenced by high levels of c-Fos expression. The activation of PVN neurons in both fasted Nckx4 knock-out and glucose-injected wild-type animals is blocked by Ca(2+) removal and MC4R antagonists. In cultured hypothalamic neurons, melanocyte stimulating hormone induces an MC4R-dependent and sustained Ca(2+) signal, which requires phospholipase C activity and plasma membrane Ca(2+) entry. The Ca(2+) signal is enhanced in hypothalamic neurons from Nckx4 knock-out animals, and is depressed in cells in which NCKX4 is overexpressed. Finally, MC4R-dependent oxytocin expression in the PVN, a key essential step in satiety, is prevented by blocking phospholipase C activation or Ca(2+) entry. These findings highlight an essential, and to our knowledge previously unknown, role for Ca(2+) signaling in the MC4R pathway that leads to satiety, and a novel non-redundant role for NCKX4-mediated Ca(2+) extrusion in controlling MC4R signaling and feeding behavior. Together, these findings highlight a novel pathway that potentially could be exploited to develop much needed new therapeutics to tackle eating disorders and obesity.

Protein kinase CK2 contributes to diminished small conductance Ca2+-activated K+ channel activity of hypothalamic pre-sympathetic neurons in hypertension.

Small conductance calcium-activated K(+) (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up-regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre-sympathetic neurons in SHRs. Perforated whole-cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar-Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin-sensitive SK currents and depolarization-induced medium after-hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after-depolarization potential of PVN neurons in SHRs. In addition, CK2-mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2-mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre-sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre-sympathetic neurons and increased sympathetic vasomotor tone in hypertension. Small conductance calcium-activated K(+) (SK) channels, calmodulin, and protein kinase CK2 form a molecular complex and regulate neuronal excitability. Our study suggests that augmented CK2 activity in hypertension can increase calmodulin (CaM) phosphorylation, which leads to diminished SK channel function in pre-sympathetic neurons. Diminished SK channel activity plays a role in hyperactivity of pre-sympathetic neurons in the hypothalamus in hypertension.