Glutamate Spillover Dynamically Strengthens Gabaergic Synaptic Inhibition of the Hypothalamic Paraventricular Nucleus.

The hypothalamic paraventricular nucleus (PVN) is strongly inhibited by γ-aminobutyric acid (GABA) from the surrounding peri-nuclear zone (PNZ). Because glutamate mediates fast excitatory transmission and is substrate for GABA synthesis, we tested its capacity to dynamically strengthen GABA inhibition. In PVN slices from male mice, bath glutamate applied during ionotropic glutamate receptor blockade increased PNZ-evoked inhibitory postsynaptic currents (eIPSCs) without affecting GABA-A receptor agonist currents or single-channel conductance, implicating a presynaptic mechanism(s). Consistent with this interpretation, bath glutamate failed to strengthen IPSCs during pharmacological saturation of GABA-A receptors. Presynaptic analyses revealed that glutamate did not affect paired-pulse ratio, peak eIPSC variability, GABA vesicle recycling speed, or readily releasable pool (RRP) size. Notably, glutamate-GABA strengthening (GGS) was unaffected by metabotropic glutamate receptor blockade and graded external Ca2+ when normalized to baseline amplitude. GGS was prevented by pan- but not glial-specific inhibition of glutamate uptake and by inhibition of glutamic acid decarboxylase (GAD), indicating reliance on glutamate uptake by neuronal excitatory amino acid transporter 3 (EAAT3) and enzymatic conversion of glutamate to GABA. EAAT3 immunoreactivity was strongly localized to presumptive PVN GABA terminals. High bath K+ also induced GGS, which was prevented by glutamate vesicle depletion, indicating that synaptic glutamate release strengthens PVN GABA inhibition. GGS suppressed PVN cell firing, indicating its functional significance. In sum, PVN GGS buffers neuronal excitation by apparent "over-filling" of vesicles with GABA synthesized from synaptically released glutamate. We posit that GGS protects against sustained PVN excitation and excitotoxicity while potentially aiding stress adaptation and habituation.

Presence of a remote fear memory engram in the central amygdala.

Fear memory formation and recall are highly regulated processes, with the central amygdala (CeA) contributing to fear memory-related behaviors. We recently reported that a remote fear memory engram is resident in the anterior basolateral amygdala (aBLA). However, the extent to which downstream neurons in the CeA participate in this engram is unknown. We tested the hypothesis that CeA neurons activated during fear memory formation are reactivated during remote memory retrieval such that a CeA engram participates in remote fear memory recall and its associated behavior. Using contextual fear conditioning in TRAP2;Ai14 mice, we identified, by persistent Cre-dependent tdTomato expression (i.e., "TRAPing"), CeA neurons that were c-fos-activated during memory formation. Twenty-one days later, we quantified neurons activated during remote memory recall using Fos immunohistochemistry. Dual labeling was used to identify the subpopulation of CeA neurons that was both activated during memory formation and reactivated during recall. Compared with their context-conditioned (no shock) controls, fear-conditioned (electric shock) mice (n = 5/group) exhibited more robust fear memory-related behavior (freezing) as well as larger populations of activated (tdTomato+) and reactivated (dual-labeled) CeA neurons. Most neurons in both groups were mainly located in the capsular CeA subdivision (CeAC). Notably, however, only the size of the TRAPed population distributed throughout the CeA was significantly correlated with time spent freezing during remote fear memory recall. Our findings indicate that fear memory formation robustly activates CeA neurons and that a subset located mainly in the CeAC may contribute to both remote fear memory storage/retrieval and the resulting fear-like behavior.

Chronic intermittent hypoxia enhances glycinergic inhibition in nucleus tractus solitarius.

Chronic intermittent hypoxia (CIH), an animal model of sleep apnea, has been shown to alter the activity of second-order chemoreceptor neurons in the caudal nucleus of the solitary tract (cNTS). Although numerous studies have focused on excitatory plasticity, few studies have explored CIH-induced plasticity impacting inhibitory inputs to NTS neurons, and the roles of GABAergic and glycinergic inputs on heightened cNTS excitability following CIH are unknown. In addition, changes in astrocyte function may play a role in cNTS plasticity responses to CIH. This study tested the effects of a 7-day CIH protocol on miniature inhibitory postsynaptic currents (mIPSCs) in cNTS neurons receiving chemoreceptor afferents. Normoxia-treated rats primarily displayed GABA mIPSCs, whereas CIH-treated rats exhibited a shift toward combined GABA/glycine-mediated mIPSCs. CIH increased glycinergic mIPSC amplitude and area. This shift was not observed in dorsal motor nucleus of the vagus neurons or cNTS cells from females. Immunohistochemistry showed that strengthened glycinergic mIPSCs were associated with increased glycine receptor protein and were dependent on receptor trafficking in CIH-treated rats. In addition, CIH altered astrocyte morphology in the cNTS, and inactivation of astrocytes following CIH reduced glycine receptor-mediated mIPSC frequency and overall mIPSC amplitude. In cNTS, CIH produced changes in glycine signaling that appear to reflect increased trafficking of glycine receptors to the cell membrane. Increased glycine signaling in cNTS associated with CIH also appears to be dependent on astrocytes. Additional studies will be needed to determine how CIH influences glycine receptor expression and astrocyte function in cNTS.NEW & NOTEWORTHY Chronic intermittent hypoxia (CIH) has been used to mimic the hypoxemia associated with sleep apnea and determine how these hypoxemias influence neural function. The nucleus of the solitary tract is the main site for chemoreceptor input to the CNS, but how CIH influences NTS inhibition has not been determined. These studies show that CIH increases glycine-mediated miniature IPSCs through mechanisms that depend on protein trafficking and astrocyte activation.

Local ionotropic glutamate receptors are required to trigger and sustain ramping of sympathetic nerve activity by hypothalamic paraventricular nucleus TNFα.

Tumor necrosis factor-α (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA) in cardiovascular disease models, but mechanisms are incompletely understood. As previously reported, bilateral PVN TNFα (0.6 pmol, 50 nL) induced acute ramping of splanchnic SNA (SSNA) that averaged +64 ± 7% after 60 min and +109 ± 17% after 120 min (P < 0.0001, n = 10). Given that TNFα can rapidly strengthen glutamatergic transmission, we hypothesized that progressive activation of ionotropic glutamate receptors is critically involved. When compared with that of vehicle (n = 5), prior blockade of PVN AMPA or NMDA receptors in anesthetized (urethane/α-chloralose) adult male Sprague-Dawley rats dose-dependently (ED50: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 2.48 nmol; D-(-)-2-amino-5-phosphonopentanoic acid (APV), 12.33 nmol), but incompletely (Emax: NBQX, 64%; APV, 41%), attenuated TNFα-induced SSNA ramping (n = 5/dose). By contrast, combined receptor blockade prevented ramping (1.3 ± 2.1%, P < 0.0001, n = 5). Whereas separate blockade of PVN AMPA or NMDA receptors (n = 5/group) had little effect on continued SSNA ramping when performed 60 min after TNFα injection, combined blockade (n = 5) or PVN inhibition with the GABA-A receptor agonist muscimol (n = 5) effectively stalled, without reversing, the SSNA ramp. Notably, PVN TNFα increased local TNFα immunofluorescence after 120, but not 60 min. Findings indicate that AMPA and NMDA receptors each contribute to SSNA ramping to PVN TNFα, and that their collective availability and ongoing activity are required to initiate and sustain the ramping response. We conclude that acute sympathetic activation by PVN TNFα involves progressive local glutamatergic excitation that recruits downstream neurons capable of maintaining heightened SSNA, but incapable of sustaining SSNA ramping.NEW & NOTEWORTHY The proinflammatory cytokine TNFα contributes to heightened SNA in cardiovascular disease models, but mechanisms remain obscure. Here, we demonstrate that TNFα injection into the hypothalamic PVN triggers SNA ramping by mechanisms dependent on local ionotropic glutamate receptor availability, but largely independent of TNFα autoinduction. Continued SNA ramping depends on ionotropic glutamate receptor and neuronal activity in PVN, indicating that strengthening and/or increased efficacy of glutamatergic transmission is necessary for acute sympathoexcitation by PVN TNFα.

Oxytocin Receptor Activation Rescues Opioid-Induced Respiratory Depression by Systemic Fentanyl in the Rat.

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin, which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that oxytocin can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v.) of oxytocin to counter fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. Oxytocin dose-dependently rescued fentanyl OIRD by almost immediately reversing PNA burst arrest (P = 0.0057) and restoring baseline burst frequency (P = 0.0016) and amplitude (P = 0.0025) at low but not high doses, resulting in inverted bell-shaped dose-response curves. Oxytocin receptor antagonism (40 nmol/kg, i.v.) prevented oxytocin reversal of OIRD (arrest: P = 0.0066, frequency: P = 0.0207, amplitude: P = 0.0022). Vasopressin 1A receptor (V1aR) antagonism restored high-dose oxytocin efficacy to rescue OIRD (P = 0.0170 to P < 0.0001), resulting in classic sigmoidal dose-response curves, and prevented (P = 0.0135) transient hypertension from V1aR cross-activation (P = 0.0275). Alone, vasopressin (5 nmol/kg, i.v.) failed to reverse fentanyl respiratory arrest (P = 0.6184). The nonpeptide oxytocin receptor agonist WAY-267464 (75 nmol/kg, i.v.), which has V1aR antagonist properties, quickly reversed fentanyl OIRD (P < 0.0001), with rapid recovery of PNA frequency (P = 0.0011) and amplitude (P = 0.0044) without adverse hemodynamic consequences (P = 0.9991). Findings indicate that peptide and nonpeptide agonist activation of oxytocin receptors without V1aR cross-activation rescues fentanyl OIRD. Oxytocin receptor agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia. SIGNIFICANCE STATEMENT: Oxytocin receptor activation produces analgesia. Here, we demonstrate that activation by the US Food and Drug Administration-approved agonist oxytocin and the nonpeptide partial agonist WAY-267464 can each reverse fentanyl cardiorespiratory depression. Selective targeting of oxytocin receptors for resuscitation from opioid overdose, alone or in combination with an opioid antagonist, could eliminate or attenuate negative side effects associated with traditional opioid receptor antagonism.

Intermittent hypercapnic hypoxia induces respiratory hypersensitivity to fentanyl accompanied by tonic respiratory depression by endogenous opioids.

KEY POINTS: Sleep apnoea increases susceptibility to opioid-induced respiratory depression (OIRD). Endogenous opioids are implicated as a contributing factor in sleep apnoea. Rats exposed to sleep-phase chronic intermittent hypercapnic hypoxia (CIHH) for 7 days exhibited exaggerated OIRD to systemic fentanyl both while anaesthetized and artificially ventilated and while conscious and breathing spontaneously, implicating heightened CNS inhibitory efficacy of fentanyl. CIHH also induced tonic endogenous opioid suppression of neural inspiration. Sleep-related episodes of hypercapnic hypoxia, as in sleep apnoea, promote hypersensitivity to OIRD, with tonic respiratory depression by endogenous opioids implicated as a potential underlying cause. ABSTRACT: Sleep apnoea (SA) increases opioid-induced respiratory depression (OIRD) and lethality. To test the hypothesis that this results from chronic intermittent bouts of hypercapnic hypoxia (CIHH) accompanying SA, we compared OIRD across continuously normoxic control rats and rats exposed to sleep-phase (8 h/day) CIHH for 1 week. OIRD sensitivity was first assessed in anaesthetized (urethane/α-chloralose), vagotomized and artificially ventilated rats by recording phrenic nerve activity (PNA) to index neural inspiration and quantify PNA burst inhibition to graded doses (0, 2, 20, 50 µg kg-1 , i.v.) of the synthetic opioid fentanyl. Fentanyl dose-dependently reduced PNA burst frequency (P = 0.0098-0.0001), while increasing the duration of burst quiescence at 50 µg kg-1 (P < 0.0001, n = 5-6/group/dose). CIHH shifted the fentanyl dose-phrenic burst frequency response curve to the left (P = 0.0163) and increased the duration of burst quiescence (P < 0.0001). During fentanyl recovery, PNA burst width was increased relative to baseline in normoxic and CIHH rats. Systemic naloxone (1 mg kg-1 , i.v.) reversed fentanyl-induced PNA arrest in both groups (P = 0.0002), and increased phrenic burst amplitude above baseline (P = 0.0113) in CIHH rats only. Differential sensitivity to anaesthesia as a cause of CIHH-related OIRD hypersensitivity was excluded by observing in conscious spontaneously breathing rats that fentanyl at 20 µg kg-1 (i.v.), which silenced PNA in anaesthetized rats, differentially increased breathing variability in normoxic versus CIHH rats (P = 0.0427), while significantly reducing breathing frequency (P < 0.0001) and periodicity (P = 0.0003) in CIHH rats only. Findings indicate that CIHH increased OIRD sensitivity, with tonic inspiratory depression by endogenous opioids as a likely contributing cause.

Age- and Sex-Specific Plasticity in Dopamine Transporter Function Revealed by Food Restriction and Exercise in a Rat Activity-Based Anorexia Paradigm.

Eating disorders such as anorexia typically emerge during adolescence, are characterized by engagement in compulsive and detrimental behaviors, and are often comorbid with neuropsychiatric disorders and drug abuse. No effective treatments exist. Moreover, anorexia lacks adolescent animal models, contributing to a poor understanding of underlying age-specific neurophysiological disruptions. To evaluate the contribution of dopaminergic signaling to the emergence of anorexia-related behaviors during the vulnerable adolescent period, we applied an established adult activity-based anorexia (ABA) paradigm (food restriction plus unlimited exercise access for 4 to 5 days) to adult and adolescent rats of both sexes. At the end of the paradigm, measures of plasma volume, blood hormone levels, dopamine transporter (DAT) expression and function, acute cocaine-induced locomotion, and brain water weight were taken. Adolescents were dramatically more affected by the ABA paradigm than adults in all measures. In vivo chronoamperometry and cocaine locomotor responses revealed sex-specific changes in adolescent DAT function after ABA that were independent of DAT expression differences. Hematocrit, insulin, ghrelin, and corticosterone levels did not resemble shifts typically observed in patients with anorexia, though decreases in leptin levels aligned with human reports. These findings are the first to suggest that food restriction in conjunction with excessive exercise sex-dependently and age-specifically modulate DAT functional plasticity during adolescence. The adolescent vulnerability to this relatively short manipulation, combined with blood measures, evidence need for an optimized age-appropriate ABA paradigm with greater face and predictive validity for the study of the pathophysiology and treatment of anorexia. SIGNIFICANCE STATEMENT: Adolescent rats exhibit a distinctive, sex-specific plasticity in dopamine transporter function and cocaine response after food restriction and exercise access; this plasticity is both absent in adults and not attributable to changes in dopamine transporter expression levels. These novel findings may help explain sex differences in vulnerability to eating disorders and drug abuse during adolescence.

Neuroinflammation Contributes to High Salt Intake-Augmented Neuronal Activation and Active Coping Responses to Acute Stress.

High dietary salt intake increases risk of stress-related neuropsychiatric disorders. Here, we explored the contribution of high dietary salt intake-induced neuroinflammation in key stress-responsive brain regions, the hypothalamic paraventricular nucleus and basolateral amygdala, in promoting exaggerated neuronal activation and coping behaviors in response to acute psychogenic stress. Mice that underwent high dietary salt intake exhibited increased active stress coping behaviors during and after an acute swim stress, and these were reduced by concurrent administration of minocycline, an inhibitor of microglial activation, without affecting body fluid hyperosmolality caused by high dietary salt intake. Moreover, minocycline attenuated high dietary salt intake-induced increases of paraventricular nucleus tumor necrosis factor-α, activated microglia (ionized calcium-binding adaptor molecule 1), and acute swim stress-induced neuronal activation (c-Fos). In the basolateral amygdala, similar effects were observed on ionized calcium-binding adaptor molecule 1+ and c-Fos+ counts, but not tumor necrosis factor-α levels. These data indicate that high dietary salt intake promotes neuroinflammation, increasing recruitment of neurons in key stress-associated brain regions and augmenting behavioral hyper-responsivity to acute psychological stress.

Burst patterning of hypothalamic paraventricular nucleus-driven sympathetic nerve activity in ANG II-salt hypertension.

ANG II-salt hypertension selectively increases splanchnic sympathetic nerve activity (sSNA), but the extent to which this reflects increased respiratory versus cardiac rhythmic bursting is unknown. Here, integrated sSNA was elevated in ANG II-infused rats fed a high-salt (2% NaCl) diet (ANG II-HSD) compared with vehicle-infused rats fed a normal-salt (0.4% NaCl) diet (Veh-NSD; P < 0.01). Increased sSNA was not accompanied by increased inspiratory or expiratory bursting, consistent with no group difference in central inspiratory drive. Consistent with preserved inhibitory baroreflex entrainment of elevated sSNA in ANG II-HSD rats, the time integral ( P < 0.05) and amplitude ( P < 0.01) of cardiac rhythmic sSNA were increased. Consistent with activity of hypothalamic paraventricular nucleus (PVN) neurons supporting basal SNA in ANG II-salt hypertension, inhibition of PVN with the GABA-A receptor agonist muscimol reduced mean arterial pressure (MAP) and integrated sSNA only in the ANG II-HSD group ( P < 0.001). PVN inhibition had no effect on respiratory rhythmic sSNA bursting in either group but reduced cardiac rhythmic sSNA in ANG II-HSD rats only ( P < 0.01). The latter likely reflected reduced inhibitory baroreflex entrainment subsequent to the fall of MAP. Of note is that MAP as well as integrated and rhythmic burst patterns of sSNA were similar in vehicle-infused rats whether they were fed a normal or high-salt diet. Findings indicate that PVN neurons support elevated sSNA in ANG II-HSD rats by driving a tonic component of activity without altering respiratory or cardiac rhythmic bursting. Because sSNA was unchanged in Veh-HSD rats, activation of PVN-driven tonic sSNA appears to require central actions of ANG II. NEW & NOTEWORTHY ANG II-salt hypertension is strongly neurogenic and depends on hypothalamic paraventricular nucleus (PVN)-driven splanchnic sympathetic nerve activity (sSNA). Here, respiratory and cardiac bursts of sSNA were preserved in ANG II-salt rats and unaltered by PVN inhibition, suggesting that PVN neurons drive a tonic component of sSNA rather than modulating dominant patterns of burst discharge.

Activation of the hypothalamic paraventricular nucleus by forebrain hypertonicity selectively increases tonic vasomotor sympathetic nerve activity.

We recently reported that mean arterial pressure (MAP) is maintained in water-deprived rats by an irregular tonic component of vasomotor sympathetic nerve activity (SNA) that is driven by neuronal activity in the hypothalamic paraventricular nucleus (PVN). To establish whether generation of tonic SNA requires time-dependent (i.e., hours or days of dehydration) neuroadaptive responses or can be abruptly generated by even acute circuit activation, forebrain sympathoexcitatory osmosensory inputs to PVN were stimulated by infusion (0.1 ml/min, 10 min) of hypertonic saline (HTS; 1.5 M NaCl) through an internal carotid artery (ICA). Whereas isotonic saline (ITS; 0.15 M NaCl) had no effect (n = 5), HTS increased (P < 0.001; n = 6) splanchnic SNA (sSNA), phrenic nerve activity (PNA), and MAP. Bilateral PVN injections of muscimol (n = 6) prevented HTS-evoked increases of integrated sSNA and PNA (P < 0.001) and attenuated the accompanying pressor response (P < 0.01). Blockade of PVN NMDA receptors with d-(2R)-amino-5-phosphonovaleric acid (AP5; n = 6) had similar effects. Analysis of respiratory rhythmic bursting of sSNA revealed that ICA HTS increased mean voltage (P < 0.001) without affecting the amplitude of inspiratory or expiratory bursts. Analysis of cardiac rhythmic sSNA likewise revealed that ICA HTS increased mean voltage. Cardiac rhythmic sSNA oscillation amplitude was also increased, which is consistent with activation of arterial baroreceptor during the accompanying pressor response. Increased mean sSNA voltage by HTS was blocked by prior PVN inhibition (muscimol) and blockade of PVN NMDA receptors (AP5). We conclude that even acute glutamatergic activation of PVN (i.e., by hypertonicity) is sufficient to selectively increase a tonic component of vasomotor SNA.

Blood pressure is maintained during dehydration by hypothalamic paraventricular nucleus-driven tonic sympathetic nerve activity.

Resting sympathetic nerve activity (SNA) consists primarily of respiratory and cardiac rhythmic bursts of action potentials. During homeostatic challenges such as dehydration, the hypothalamic paraventricular nucleus (PVN) is activated and drives SNA in support of arterial pressure (AP). Given that PVN neurones project to brainstem cardio-respiratory regions that generate bursting patterns of SNA, we sought to determine the contribution of PVN to support of rhythmic bursting of SNA during dehydration and to elucidate which bursts dominantly contribute to maintenance of AP. Euhydrated (EH) and dehydrated (DH) (48 h water deprived) rats were anaesthetized, bilaterally vagotomized and underwent acute PVN inhibition by bilateral injection of the GABA-A receptor agonist muscimol (0.1 nmol in 50 nl). Consistent with previous studies, muscimol had no effect in EH rats (n = 6), but reduced mean AP (MAP; P < 0.001) and integrated splanchnic SNA (sSNA; P < 0.001) in DH rats (n = 6). Arterial pulse pressure was unaffected in both groups. Muscimol reduced burst frequency of phrenic nerve activity (P < 0.05) equally in both groups without affecting the burst amplitude-duration integral (i.e. area under the curve). PVN inhibition did not affect the amplitude of the inspiratory peak, expiratory trough or expiratory peak of sSNA in either group, but reduced cardiac rhythmic sSNA in DH rats only (P < 0.001). The latter was largely reversed by inflating an aortic cuff to restore MAP (n = 5), suggesting that the muscimol-induced reduction of cardiac rhythmic sSNA in DH rats was an indirect effect of reducing MAP and thus arterial baroreceptor input. We conclude that MAP is largely maintained in anaesthetized DH rats by a PVN-driven component of sSNA that is neither respiratory nor cardiac rhythmic.

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.

Coping with dehydration: sympathetic activation and regulation of glutamatergic transmission in the hypothalamic PVN.

Autonomic and endocrine profiles of chronic hypertension and heart failure resemble those of acute dehydration. Importantly, all of these conditions are associated with exaggerated sympathetic nerve activity (SNA) driven by glutamatergic activation of the hypothalamic paraventricular nucleus (PVN). Here, studies sought to gain insight into mechanisms of disease by determining the role of PVN ionotropic glutamate receptors in supporting SNA and mean arterial pressure (MAP) during dehydration and by elucidating mechanisms regulating receptor activity. Blockade of PVN N-methyl-D-aspartate (NMDA) receptors reduced (P < 0.01) renal SNA and MAP in urethane-chloralose-anesthetized dehydrated (DH) (48 h water deprivation) rats, but had no effect in euhydrated (EH) controls. Blockade of PVN α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors had no effect in either group. NMDA in PVN caused dose-dependent increases of renal SNA and MAP in both groups, but the maximum agonist evoked response (Emax) of the renal SNA response was greater (P < 0.05) in DH rats. The latter was not explained by increased PVN expression of NMDA receptor NR1 subunit protein, increased PVN neuronal excitability, or decreased brain water content. Interestingly, PVN injection of the pan-specific excitatory amino acid transporter (EAAT) inhibitor DL-threo-ß-benzyloxyaspartic acid produced smaller sympathoexcitatory and pressor responses in DH rats, which was associated with reduced glial expression of EAAT2 in PVN. Like chronic hypertension and heart failure, dehydration increases excitatory NMDA receptor tone in PVN. Reduced glial-mediated glutamate uptake was identified as a key contributing factor. Defective glutamate uptake in PVN could therefore be an important, but as yet unexplored, mechanism driving sympathetic hyperactivity in chronic cardiovascular diseases.

Discharge of RVLM vasomotor neurons is not increased in anesthetized angiotensin II-salt hypertensive rats.

Neurons of the rostral ventrolateral medulla (RVLM) are critical for generating and regulating sympathetic nerve activity (SNA). Systemic administration of ANG II combined with a high-salt diet induces hypertension that is postulated to involve elevated SNA. However, a functional role for RVLM vasomotor neurons in ANG II-salt hypertension has not been established. Here we tested the hypothesis that RVLM vasomotor neurons have exaggerated resting discharge in rats with ANG II-salt hypertension. Rats in the hypertensive (HT) group consumed a high-salt (2% NaCl) diet and received an infusion of ANG II (150 ng·kg(-1)·min(-1) sc) for 14 days. Rats in the normotensive (NT) group consumed a normal salt (0.4% NaCl) diet and were infused with normal saline. Telemetric recordings in conscious rats revealed that mean arterial pressure (MAP) was significantly increased in HT compared with NT rats (P < 0.001). Under anesthesia (urethane/chloralose), MAP remained elevated in HT compared with NT rats (P < 0.01). Extracellular single unit recordings in HT (n = 28) and NT (n = 22) rats revealed that barosensitive RVLM neurons in both groups (HT, 23 cells; NT, 34 cells) had similar cardiac rhythmicity and resting discharge. However, a greater (P < 0.01) increase of MAP was needed to silence discharge of neurons in HT (17 cells, 44 ± 5 mmHg) than in NT (28 cells, 29 ± 3 mmHg) rats. Maximum firing rates during arterial baroreceptor unloading were similar across groups. We conclude that heightened resting discharge of sympathoexcitatory RVLM neurons is not required for maintenance of neurogenic ANG II-salt hypertension.

Rats selectively bred for differences in aerobic capacity have similar hypertensive responses to chronic intermittent hypoxia.

Exposure to chronic intermittent hypoxia (CIH) is an animal model that mimics the repetitive bouts of hypoxemia experienced by humans with sleep apnea. Rats exposed to CIH develop hypertension that depends on the activation of sympathetic nerve activity (SNA). Since obesity and metabolic syndrome have been linked to neurogenic hypertension and sleep apnea, and because sleep apnea can adversely affect aerobic exercise capacity, we tested the hypothesis that rats bred for selection of low aerobic capacity running (LCR) would have a greater hypertensive response to CIH than rats bred for high aerobic capacity running (HCR). Blockade of ganglionic transmission was performed to compare the contribution of SNA to the maintenance of resting mean arterial pressure (MAP). Next, hypertensive responses to 7 days of CIH were compared across LCR and HCR rats (14-16 mo old). Finally, the contribution of the hypothalamic paraventricular nucleus (PVN) to the maintenance of SNA and hypertension after CIH was determined and compared across groups. Although LCR rats were less active and had greater body weights than HCR rats, resting MAP, the contribution of ongoing SNA to the maintenance of MAP, and hypertensive responses to CIH were similar between groups. Contrary to our hypothesis, chemical inhibition of the PVN with muscimol (1 mmol/100 nl) caused a larger fall of MAP in HCR rats than in LCR rats. We conclude that LCR rats do not have resting hypertension or an exaggerated hypertensive response to CIH. Interestingly, the maintenance of CIH hypertension in LCR rats compared with HCR rats appears less reliant on ongoing PVN neuronal activity.

Chronic intermittent hypoxia increases sympathetic control of blood pressure: role of neuronal activity in the hypothalamic paraventricular nucleus.

Like humans with sleep apnea, rats exposed to chronic intermittent hypoxia (CIH) experience arterial hypoxemias and develop hypertension characterized by exaggerated sympathetic nerve activity (SNA). To gain insights into the poorly understood mechanisms that initiate sleep apnea/CIH-associated hypertension, experiments were performed in rats exposed to CIH for only 7 days. Compared with sham-treated normoxic control rats, CIH-exposed rats (n = 8 rats/group) had significantly increased hematocrit (P < 0.001) and mean arterial pressure (MAP; P < 0.05). Blockade of ganglionic transmission caused a significantly (P < 0.05) greater reduction of MAP in rats exposed to CIH than control rats (n = 8 rats/group), indicating a greater contribution of SNA in the support of MAP even at this early stage of CIH hypertension. Chemical inhibition of neuronal discharge in the hypothalamic paraventricular nucleus (PVN) (100 pmol muscimol) had no effect on renal SNA but reduced lumbar SNA (P < 0.005) and MAP (P < 0.05) more in CIH-exposed rats (n = 8) than control rats (n = 7), indicating that CIH increased the contribution of PVN neuronal activity in the support of lumbar SNA and MAP. Because CIH activates brain regions controlling body fluid homeostasis, the effects of internal carotid artery injection of hypertonic saline were tested and determined to increase lumbar SNA more (P < 0.05) in CIH-exposed rats than in control rats (n = 9 rats/group). We conclude that neurogenic mechanisms are activated early in the development of CIH hypertension such that elevated MAP relies on increased sympathetic tonus and ongoing PVN neuronal activity. The increased sensitivity of Na(+)/osmosensitive circuitry in CIH-exposed rats suggests that early neuroadaptive responses among body fluid regulatory neurons could contribute to the initiation of CIH hypertension.

Zinc, copper, and blood pressure: Human population studies.

Copper and zinc are essential trace biometals that regulate cardiovascular homeostasis, and dysregulation of these metals has been linked to vascular diseases, including hypertension. In this article, we review recent human population studies concerning this topic, focusing on: 1) the relationship between blood pressure and levels of zinc and copper; 2) correlations between trace metals, the renin-angiotensin system, obesity, and hypertension; 3) the relationship between environmental metal pollution and the development of hypertension; and 4) methods commonly employed to assay zinc and copper in human specimens. Moreover, based on the findings of these studies, we suggest the following topics as the basis for future investigations: 1) the potential role of environmental metal pollution as a causal factor for hypertension; 2) metal profiles within specific pathogenic subsets of patients with hypertension; 3) standardizing the experimental design so that the results between different studies are more comparable; and 4) the requirement for animal experiments as complementary approaches to address mechanistic insight that cannot be studied in human populations.

Anterior basolateral amygdala neurons comprise a remote fear memory engram.

Introduction: Threatening environmental cues often generate enduring fear memories, but how these are formed and stored remains actively investigated. Recall of a recent fear memory is thought to reflect reactivation of neurons, in multiple brain regions, activated during memory formation, indicating that anatomically distributed and interconnected neuronal ensembles comprise fear memory engrams. The extent to which anatomically specific activation-reactivation engrams persist during long-term fear memory recall, however, remains largely unexplored. We hypothesized that principal neurons in the anterior basolateral amygdala (aBLA), which encode negative valence, acutely reactivate during remote fear memory recall to drive fear behavior. Methods: Using adult offspring of TRAP2 and Ai14 mice, persistent tdTomato expression was used to "TRAP" aBLA neurons that underwent Fos-activation during contextual fear conditioning (electric shocks) or context only conditioning (no shocks) (n = 5/group). Three weeks later, mice were re-exposed to the same context cues for remote memory recall, then sacrificed for Fos immunohistochemistry. Results: TRAPed (tdTomato +), Fos +, and reactivated (double-labeled) neuronal ensembles were larger in fear- than context-conditioned mice, with the middle sub-region and middle/caudal dorsomedial quadrants of aBLA displaying the greatest densities of all three ensemble populations. Whereas tdTomato + ensembles were dominantly glutamatergic in context and fear groups, freezing behavior during remote memory recall was not correlated with ensemble sizes in either group. Discussion: We conclude that although an aBLA-inclusive fear memory engram forms and persists at a remote time point, plasticity impacting electrophysiological responses of engram neurons, not their population size, encodes fear memory and drives behavioral manifestations of long-term fear memory recall.

Early central cardiovagal dysfunction after high fat diet in a murine model.

High fat diet (HFD) promotes cardiovascular disease and blunted cardiac vagal regulation. Temporal onset of loss of cardiac vagal control and its underlying mechanism are presently unclear. We tested our hypothesis that reduced central vagal regulation occurs early after HFD and contributes to poor cardiac regulation using cardiovascular testing paired with pharmacology in mice, molecular biology, and a novel bi-transgenic mouse line. Results show HFD, compared to normal fat diet (NFD), significantly blunted cardio/pulmonary chemoreflex bradycardic responses after 15 days, extending as far as tested (> 30 days). HFD produced resting tachycardia by day 3, reflected significant loss of parasympathetic tone. No differences in bradycardic responses to graded electrical stimulation of the distal cut end of the cervical vagus indicated diet-induced differences in vagal activity were centrally mediated. In nucleus ambiguus (NA), surface expression of δ-subunit containing type A gamma-aminobutyric acid receptors (GABAA(δ)R) increased at day 15 of HFD. Novel mice lacking δ-subunit expression in vagal motor neurons (ChAT-δnull) failed to exhibit blunted reflex bradycardia or resting tachycardia after two weeks of HFD. Thus, reduced parasympathetic output contributes to early HFD-induced HR dysregulation, likely through increased GABAA(δ)Rs. Results underscore need for research on mechanisms of early onset increases in GABAA(δ)R expression and parasympathetic dysfunction after HFD.

Intrinsic control of sodium excretion in the distal nephron by inhibitory purinergic regulation of the epithelial Na(+) channel.

PURPOSE OF REVIEW: This review summarizes the new evidence for an intrinsic control system in the aldosterone-sensitive distal nephron in which purinergic signaling regulates sodium transport and governs renal sodium excretion. RECENT FINDINGS: Electrophysiological studies identify epithelial Na(+) channels (ENaC) as final effectors of purinergic signaling via P2Y(2) receptors in the distal nephron. Inhibition of ENaC by autocrine/paracrine purinergic signaling reduces sodium reabsorption allowing an appropriately graded pressure-natriuresis response when delivery of sodium to the distal nephron is high. Disruption of this intrinsic control mechanism decreases sodium excretion and therefore has a prohypertensive effect. Because purinergic inhibition of ENaC is tonic yet submaximal, its enhancement increases sodium excretion and therefore has an antihypertensive action. SUMMARY: Purinergic inhibitory regulation of ENaC is a key component of an intrinsic control system that enables the distal nephron to respond appropriately to the delivered load of sodium. This control system is physiologically important and functions in parallel with extrinsic control by the renin-angiotensin-aldosterone system, enabling sodium excretion to keep pace with sodium intake, especially when intake is high, and thereby maintaining arterial blood pressure. Disruption of intrinsic control of sodium transport by the distal nephron likely contributes to diseases such as arterial hypertension.