Impaired Hypothalamic Regulation of Sympathetic Outflow in Primary Hypertension

The hypothalamic paraventricular nucleus(PVN) is a crucial region involved in maintaining homeostasis through the regulation of cardiovascular, neuroendocrine, and other functions. The PVN provides a dominant source of excitatory drive to the sympathetic outflow through innervation of the brainstem and spinal cord in hypertension. We discuss current findings on the role of the PVN in the regulation of sympathetic output in both normotensive and hypertensive conditions. The PVN seems to play a major role in generating the elevated sympathetic vasomotor activity that is characteristic of multiple forms of hypertension, including primary hypertension in humans. Recent studies in the spontaneously hypertensive rat model have revealed an imbalance of inhibitory and excitatory synaptic inputs to PVN presympathetic neurons as indicated by impaired inhibitory and enhanced excitatory synaptic inputs in hypertension.This imbalance of inhibitory and excitatory synaptic inputs in the PVN forms the basis for elevated sympathetic outflow in hypertension. In this review, we discuss the disruption of balance between glutamatergic and GABAergic inputs and the associated cellular and molecular alterations as mechanisms underlying the hyperactivity of PVN pre-sympathetic neurons in hypertension.     

Chronic stress‐induced neurotransmitter plasticity in the PVN

Chronic stress precipitates pronounced enhancement of central stress excitability, marked by sensitization of hypothalamic‐pituitary‐adrenocortical (HPA) axis responses and increased adrenocorticotropic hormone (ACTH) secretagogue biosynthesis in the paraventricular nucleus of the hypothalamus (PVN). Chronic stress‐induced enhancement of HPA axis excitability predicts increased excitatory and/or decreased inhibitory innervation of the parvocellular PVN. We tested this hypothesis by evaluating chronic variable stress (CVS)‐induced changes in total (synaptophysin), glutamatergic (VGluT2), GABAergic (GAD65), and noradrenergic (DBH) terminal immunoreactivity on PVN parvocellular neurons using immunofluorescence confocal microscopy. CVS increased the total PVN bouton immunoreactivity as well as the number of glutamatergic and noradrenergic immunoreactive boutons in apposition to both the corticotropin‐releasing hormone (CRH)‐immunoreactive cell bodies and dendrites within the parvocellular PVN. However, the number of GABAergic‐immunoreactive boutons in the PVN was unchanged. CVS did not alter CRH median eminence immunoreactivity, indicating that CVS does not enhance CRH storage within the median eminence. Taken together, the data are consistent with a role for both glutamate and norepinephrine in chronic stress enhancement of HPA axis excitability. These changes could lead to an enhanced capacity for excitation in these neurons, contributing to chronic stress‐induced hyperreactivity of stress effector systems in the brain. J. Comp. Neurol. 517:156–165, 2009. © 2009 Wiley‐Liss, Inc.     

Local gamma-aminobutyric acid and glutamate circuit control of hypophyseotrophic corticotropin-releasing factor neuron activity in the paraventricular nucleus of the hypothalamus

Paraventricular corticotropin-releasing factor (CRF) neurons play a pivotal role in regulating neuroendocrine responses to stress. The mechanisms by which synaptic inputs control the activity of these neurons are not well understood. The present study was undertaken to determine the role of the intrinsic gamma-aminobutyric acid (GABA)- and glutamatergic neural circuits of the hypothalamic paraventricular nucleus (PVN) in the control of CRF neural activity. We show that in organotypic cultures of the PVN, blockade of the intrinsic GABAergic neurotransmission by the GABAA receptor antagonist bicuculline resulted in a significant increase in CRF secretion. The bicuculline-induced CRF secretory activity was abolished by the coadministration of the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Electrical stimulation of the CRF cell division elicited glutamatergic extracellular field potentials that were dramatically enhanced by bicuculline and were suppressed by CNQX. These results show that the functional activity of CRF neurons in organotypic cultures of the PVN is under a tonic inhibitory influence of an intrinsic GABAergic circuit. Suppression of GABAergic transmission appears to have a permissive role for inducing an increased secretory activity of CRF neurons that is driven by an excitatory glutamatergic network via AMPA/kainate receptors.     

Involvement of N-methyl- -aspartate receptor in the delayed transneuronal regression of substantia nigra neurons in rats

The substantia nigra pars reticulata (SNr) receives both inhibitory GABAergic and excitatory glutamatergic afferents from diverse origins. Ischemic injury to the striatum and/or the globus pallidus causes delayed transneuronal death of the SNr neurons, in the course of which neuronal disinhibition induced by loss of GABAergic inputs is supposed to trigger a lethal hypermetabolic process. In the in vivo experiment presented herein, we clarified the role of glutamatergic action via the N-methyl- -aspartate receptor in this cell death process. Continuous intraventricular infusion (0.5 μl/h) of the N-methyl- -aspartate receptor antagonist MK-801 (1000 μg/ml), or of saline (control group) was initiated 24 h after 2 h of transient middle cerebral artery (MCA) occlusion in rats, by which massive ischemic injury was produced in the striatopallidal regions. The measured rectal temperature was not significantly altered in the MK-801-infused and in the control rats throughout the time period examined. The rats were killed at 15 days after MCA occlusion. The volume of the focal ischemic infarction of the MK-801-infused group did not significantly differ from that of controls. Also, MK-801-infusion did not significantly ameliorate the nigral atrophy subsequent to MCA occlusion. In association with a marked depletion of GABAergic afferent fibers, neuronal cell number in the ipsilateral SNr was significantly decreased in the control group. In contrast, the neuronal cell loss in the nucleus was completely prevented in the MK-801-infusion group. The data suggested that withdrawal of GABAergic inputs may cause a severe imbalance between excitation and inhibition of the SNr neurons and may eventually result in neurotoxicity mediated by the N-methyl- -aspartate receptor. Suppression of glutamatergic excitatory effects by suitable drugs may be a reasonable therapy for the transneuronal death of the SNr neurons.     

Prenatal stress‐related alterations in synaptic transmission and 5‐HT7 receptor‐mediated effects in the rat dorsal raphe nucleus are ameliorated by the 5‐HT7 receptor antagonist SB 269970

Early life adversity exerts a detrimental influence on developing brain neuronal networks and its consequences may include mental health disorders. In rats, prenatal stress may lead to anxiety and depressive‐like behavior in the offspring. Several lines of evidence implicated an involvement of prenatal stress in alterations of the brain serotonergic system functions, but the effects of prenatal stress on its core, the dorsal raphe nucleus (DRN), still remain incompletely understood. The present study was aimed at finding whether prenatal stress induces modifications in the glutamatergic and GABAergic inputs to DRN projection cells and whether it affects DRN 5‐HT₇ receptors, which modulate activity of these synapses. Prenatal stress resulted in an increase in basal frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and in a decrease in basal frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from putative projection neurons in DRN slices ex vivo. While there were no changes in the excitability of DRN projection neurons, the 5‐HT₇ receptor‐mediated reduction in the sEPSC frequency and rise in the sIPSC frequency, seen in control rats, were largely absent in slices obtained from prenatally stressed rats. Repeated administration of SB 269970, a 5‐HT₇ receptor antagonist, resulted in a reversal of prenatal stress‐induced alterations in 5‐HT₇ receptor‐mediated effects on the sEPSC/sIPSC frequency. Moreover, the treatment reversed prenatal stress‐induced alterations in basal excitatory transmission and partially reversed the effect of stress on basal inhibitory transmission in the DRN.     

Induction of rapid,activity-dependent neuronal-glial remodelling in the adult rat hypothalamus in vitro

The hypothalamic oxytocinergic system offers a remarkable model of morphological plasticity in the adult because its neurons and astrocytes undergo mutual remodelling in relation to differing physiological conditions. Among various factors involved in such plasticity, oxytocin (OT) itself appears of primary importance as its central administration resulted in morphological changes similar to those brought on by physiological stimuli. In the present study, we applied OT on acute hypothalamic slices from adult rats that included the supraoptic nucleus. Using ultrastructural morphometric analyses, we found that it induced a significant reduction of astrocytic coverage of OT neurons, leaving their surfaces directly juxtaposed, to an extent similar to that detected in vivo under conditions like lactation. These neuronal-glial changes were rapid and reversible, occurring within a few hours, and specifically mediated via OT receptors. They were potentiated by oestrogen and depended on calcium mobilization and de novo protein synthesis. Moreover, they depended on concurrent neuronal activation brought on by hyperosmotic stimulation or blockade of inhibitory GABAergic neurotransmission; they were inhibited by blockade of glutamatergic receptors. Taken together, our observations show that intrahypothalamic release of OT affects not only neuronal activation of the OT system but its morphological plasticity as well. Moreover, the activity dependence of the OT-induced changes strongly suggests that astrocytes can sense the level of activity of adjacent neurons and/or afferent input and this can subsequently act as a signal to bring on the neuronal and glial conformational changes.     

Evidence for a tonic GABA-ergic inhibition of excitatory respiratory-related afferents to presympathetic neurons in the rostral ventrolateral medulla

The effect of blockade of ionotropic GABA and glutamate receptors in the rostral ventrolateral medulla (RVLM) on the relationship between phrenic nerve, splanchnic sympathetic nerve and lumbar sympathetic nerve activities was examined in urethane anesthetized, paralyzed and vagotomized Sprague-Dawley rats. Bilateral microinjection of the GABA-A receptor antagonist, bicuculline (4 mM, 100 nl), into the RVLM dramatically, and almost exclusively, increased the post-inspiratory related discharge in both splanchnic sympathetic nerve and lumbar sympathetic nerve activities and elicited hypertension with fluctuations of arterial pressure phase locked to the discharge of the phrenic nerve. Subsequent bilateral microinjection of kynurenate, a non-selective ionotropic excitatory amino acid receptor antagonist (50 mM, 100 nl), into the RVLM significantly attenuated the sympathoexcitation and hypertension evoked by injection of bicuculline. This was accompanied by an abolition of the post-inspiratory related burst discharge of splanchnic sympathetic nerve and lumbar sympathetic nerve activities. These data suggest that the GABAergic inputs to RVLM tonically inhibit glutamatergic inputs from central respiratory neurons that normally act to increase the firing of presympathetic neurons in the RVLM. Inputs from post-inspiratory neurons appear to be an especially potent excitatory synaptic drive to the presympathetic neurons in the absence of the GABAergic inhibition.     

Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus

The rostral ventrolateral medulla (RVLM) has cholinergic mechanisms responsible for pressor responses. Stimulation of the hypothalamic paraventricular nucleus (PVN) causes an increase of arterial pressure via activation of neurons in the RVLM. In this study, we examined whether PVN stimulation causes a pressor response via activation of cholinergic mechanisms in the RVLM. Male Wistar rats were used and they were anesthetized, paralyzed and artificially ventilated. Electrical stimulation of the PVN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine and the cholinesterase inhibitor physostigmine into the RVLM inhibited and potentiated, respectively, the pressor response induced by PVN stimulation. PVN stimulation also increased the firing rate of RVLM barosensitive neurons and the increase in the firing rate was inhibited and potentiated by scopolamine and physostigmine, respectively, iontophoretically applied on neurons. Microinjection of L-glutamate into the PVN produced a release of ACh in the RVLM. The inhibitory amino acid gamma-aminobutyric acid injected into the lateral parabrachial nucleus (LPBN) inhibited the pressor response induced by PVN stimulation. These results suggest that PVN stimulation causes an increase in arterial pressure via activation of cholinergic inputs in the RVLM. It appears that the pressor response is mediated, at least in part, via cholinergic inputs from the LPBN.     

Synaptic mechanisms of adenosine A2A receptor‐mediated hyperexcitability in the hippocampus

Adenosine inhibits excitatory neurons widely in the brain through adenosine A₁ receptor, but activation of adenosine A_2A receptor (A_2AR) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A_2AR expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole‐cell recording from identified postsynaptic neurons we show that A_2AR facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A_2AR enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A_2AR has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A_2AR activation alters the synaptic excitation ‐ inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A_2AR promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high‐frequency electrical stimulation. Our results revealed selective synapse‐ and cell type specific adenosine A_2AR effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A_2AR's facilitatory effect on cortical network activity. © 2014 The Authors Hippocampus Published by Wiley Periodicals, Inc.     

Visualization of local afferent inputs to magnocellular oxytocin neurons in vitro.

We recently showed that oxytocin (OT) neurons in organotypic slice cultures obtained from postnatal rat hypothalamus display complex patterns of electrical activity, similar to those of adult magnocellular OT neurons in vivo. Here we used such cultures to investigate the identity and, in particular, the origin of afferent inputs responsible for this activity. Multiple immunostaining with light and confocal microscopy showed that the somata and dendrites of oxytocinergic neurons were contacted by numerous synapses, visualized by their reaction to the synaptic markers, synaptophysin or synapsin. Many were GABAergic, displaying immunoreactivities for glutamic acid decarboxylase or gamma-aminobutyric acid (GABA); others were enriched in glutamate immunoreactivity. Such afferents presumably arose from GABA- or glutamate-immunoreactive neurons, respectively, with distinct and characteristic morphologies and topographies. A few dopaminergic boutons (tyrosine hydroxylase- or dopamine-immunopositive) impinged on OT neurons; they arose from dopamine-positive neurons located along the third ventricle. No noradrenergic profiles were detected. Despite the presence of choline acetyl-transferase (ChAT)-immunoreactive neurons, there were no cholinergic contacts. Lastly, we found oxytocinergic synapses, identified by immunoreaction for OT-related neurophysin and synapsin, contacting OT somata and dendrites. Our observations thus demonstrate that inhibitory and excitatory inputs to OT neurons derive from local intrahypothalamic GABA and glutamate neurons, in close proximity to the neurons. They also reveal that OT neurons are innervated by hypothalamic dopaminergic neurons. Finally, they confirm the existence of homotypic OT synaptic contacts which derive from local OT neurons.     

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.     

Central nesfatin‐1 influences the excitability of ghrelin‐responsive gastric distension neurons in the arcuate nucleus and reduces gastric motility in rats

Although the novel satiety peptide nesfatin‐1 has been shown to regulate gastric motility, the underlying mechanisms have yet to be elucidated. The study aimed to explore the effects of nesfatin‐1 on ghrelin‐responsive gastric distension (GD) neurons in the arcuate nucleus (Arc), and potential regulation mechanisms of gastric motility by the paraventricular nucleus (PVN). Single‐unit discharges in the Arc were recorded extracellularly, and gastric motility in conscious rats was monitored during the administration of nesfatin‐1 to the Arc or electrical stimulation of the PVN. Retrograde tracing and fluo‐immunohistochemistry staining were used to determine NUCB2/nesfatin‐1 neuronal projections. Nesfatin‐1 inhibited most of the ghrelin‐responsive GD‐excitatory neurons, but excited ghrelin‐responsive GD‐inhibitory neurons in the Arc. Gastric motility was significantly reduced by nesfatin‐1 administration to the Arc in a dose‐dependent manner. The firing activity in the Arc and changes to gastric motility were partly reduced by SHU9119, an antagonist of melanocortin 3/4 receptors. Electrical stimulation of PVN excited most of the ghrelin‐responsive GD neurons in the Arc and promoted gastric motility. Nonetheless, pretreatment with an anti‐NUCB2/nesfatin‐1 antibody in the Arc further increased the firing rate of most of the ghrelin‐responsive GD‐excitatory neurons and decreased the ghrelin‐responsive GD‐inhibitory neurons following electrical stimulation of the PVN. Gastric motility was enhanced by pretreatment with an anti‐NUCB2/nesfatin‐1 antibody in the Arc following PVN stimulation. Furthermore, NUCB2/nesfatin‐1/fluorogold double‐labeled neurons were detected in the PVN. These results suggest that nesfatin‐1 could serve as an inhibitory factor in the Arc to regulate gastric motility via the melanocortin pathway. The PVN could be involved in the regulation of the Arc in gastric activity.     

Behavior‐dependent activity patterns of GABAergic long‐range projecting neurons in the rat hippocampus

Long‐range glutamatergic and GABAergic projections participate in temporal coordination of neuronal activity in distributed cortical areas. In the hippocampus, GABAergic neurons project to the medial septum and retrohippocampal areas. Many GABAergic projection cells express somatostatin (SOM+) and, together with locally terminating SOM+ bistratified and O‐LM cells, contribute to dendritic inhibition of pyramidal cells. We tested the hypothesis that diversity in SOM+ cells reflects temporal specialization during behavior using extracellular single cell recording and juxtacellular neurobiotin‐labeling in freely moving rats. We have demonstrated that rare GABAergic projection neurons discharge rhythmically and are remarkably diverse. During sharp wave‐ripples, most projection cells, including a novel SOM+ GABAergic back‐projecting cell, increased their activity similar to bistratified cells, but unlike O‐LM cells. During movement, most projection cells discharged along the descending slope of theta cycles, but some fired at the trough jointly with bistratified and O‐LM cells. The specialization of hippocampal SOM+ projection neurons complements the action of local interneurons in differentially phasing inputs from the CA3 area to CA1 pyramidal cell dendrites during sleep and wakefulness. Our observations suggest that GABAergic projection cells mediate the behavior‐ and network state‐dependent binding of neuronal assemblies amongst functionally‐related brain regions by transmitting local rhythmic entrainment of neurons in CA1 to neuronal populations in other areas. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.     

Somatic afferent modulation of thoracic (T9‐T10) spinal neurons receiving gastric mechanical input in rats

Objectives: The aim was to determine whether somatic afferent fiber stimulation influences thoracic spinal neuronal activity responding to gastric distensions. Materials and Methods: Extracellular potentials of single T9‐T10 spinal neurons were recorded in anesthetized male rats. Ipsilateral median and peroneal nerve afferent stimulation (MNAS, PNAS) was delivered by electrodes. Inflation of a latex balloon was used to produce gastric distension. Results: MNAS and PNAS (1.5 mA, 50 Hz, 10 sec) altered activity of 63% and 66% of the spinal neurons excited or inhibited by gastric distension, respectively. MNAS more frequently reduced spinal neuronal activity with excitatory responses to gastric distension than did PNAS (p < 0.05). PNAS more likely increased neuronal activity with low‐threshold excitatory responses to gastric distension than MNAS (p < 0.05). Conclusions: Peripheral somatic afferent information utilizes central pathways to modulate gastric afferent processing in T9‐T10 spinal neurons. Thus, somatic afferent stimulation might be used to treat gastric pain and/or hypersensitivity.     

The rostral parvicellular reticular formation neurons mediate lingual nerve input to the rostral ventrolateral medulla

In rats that had been anesthetized by urethane-chloralose, we investigated whether neurons in the rostral part of the parvicellular reticular formation (rRFp) mediate lingual nerve input to the rostral ventrolateral medulla (RVLM), which is involved in somato-visceral sensory integration and in controlling the cardiovascular system. We determined the effect of the lingual nerve stimulation on activity of the rRFp neurons that were activated antidromically by stimulation of the RVLM. Stimulation of the lingual trigeminal afferent gave rise to excitatory effects (10/26, 39%), inhibitory effects (6/26, 22%) and no effect (10/26, 39%) on the RVLM-projecting rRFp neurons. About two-thirds of RVLM-projecting rRFp neurons exhibited spontaneous activity; the remaining one-third did not. A half (13/26) of RVLM-projecting rRFp neurons exhibited a pulse-related activity, suggesting that they receive a variety of peripheral and CNS inputs involved in cardiovascular function. We conclude that the lingual trigeminal input exerts excitatory and/or inhibitory effects on a majority (61%) of the RVLM-projecting rRFp neurons, and their neuronal activity may be involved in the cardiovascular responses accompanied by the defense reaction.     

Altered balance between excitatory and inhibitory inputs onto CA1 pyramidal neurons from SV2A‐deficient but not SV2B‐deficient mice

Synaptic vesicle protein 2 (SV2) is a glycoprotein that exists in three isoforms, SV2A, SV2B, and SV2C. SV2A knockout (KO) mice and SV2A/SV2B double KO (DKO) mice, but not SV2B KO animals, start to experience severe seizures and weight loss 7 days after birth and die at about postnatal day (P)14–P23. Because excitatory and inhibitory inputs play a major role in controlling neuronal excitability in the hippocampus, we examined the effects of SV2A and/or SV2B deletions on glutamatergic and GABA_A neurotransmission in hippocampal CA1 pyramidal neurons. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, mEPSCs, sIPSCs, and mIPSCs, respectively) were recorded using the whole‐cell patch‐clamp technique in slices from P6–P14 mice. The frequency of sEPSCs was increased in SV2A KO and SV2A/SV2B DKO mice, but their amplitude was unchanged. Such changes were not observed in SV2B KOs. On the contrary, the frequency and amplitude of sIPSCs were decreased in SV2A KO and SV2A/SV2B DKO mice but not in SV2B KO animals, as reported previously for the CA3 region. Kinetic parameters of sIPSCs and sEPSCs were unchanged. Importantly, no changes were observed in any genotype when examining mEPSCs and mIPSCs. We conclude that action potential‐ and Ca²⁺‐dependent glutamatergic and GABAergic synaptic transmission are differentially altered in the hippocampus of SV2A‐deficient mice, whereas the mechanism of exocytosis itself is not changed. The altered balance between these major excitatory and inhibitory inputs is probably a contributing factor to seizures in SV2A KO and SV2A/SV2B DKO mice. © 2012 Wiley Periodicals, Inc.     

Sympathoinhibition after angiotensin receptor blockade in the rostral ventrolateral medulla is independent of glutamate and gamma-aminobutyric acid receptors.

Bilateral blockade of angiotensin (Ang) receptors in the rostral ventrolateral medulla (RVLM) causes a profound fall in arterial pressure. In this study, we tested whether this effect is due to an interaction between Ang receptors and either glutamatergic or gamma-aminobutyric acidergic (GABAergic) synaptic inputs to RVLM sympathoexcitatory neurons. In urethane-anaesthetised rats, bilateral microinjections of the Ang receptor antagonists [Sar1,Thr8]Ang II or [Sar1,Ile8]Ang II into the RVLM pressor region caused large decreases in arterial pressure, heart rate and renal sympathetic nerve activity (RSNA). These responses were not significantly altered following bilateral microinjections into the RVLM of the glutamate receptor antagonist kynurenic acid (4.5 nmol). Furthermore, bilateral injections of kynurenic acid plus the GABA(A) receptor antagonist bicuculline (200 pmol) into the RVLM increased the baseline arterial pressure and RSNA, but did not alter the percentage decreases in these variables evoked by bilateral microinjections of [Sar1,Ile8]Ang II. However, the level of arterial pressure and RSNA following bilateral injections of kynurenic acid, bicuculline and [Sar1,Ile8]Ang II were similar to the levels before injection of any of these compounds. The effectiveness of the microinjections of kynurenic acid and bicuculline into the RVLM was demonstrated by the observation that they virtually abolished the somato-sympathoexcitatory and baroreceptor-sympathoinhibitory reflexes, which are mediated by glutamatergic and GABAergic synapses, respectively, in the RVLM. These results indicate that (1) blockade of Ang receptors greatly reduces the firing rate of RVLM sympathoexcitatory neurons via a mechanism that is independent of glutamatergic or GABAergic neurotransmission, and (2) in the absence of inputs mediated by ionotropic glutamate, GABA(A) and Ang receptors, there are other mechanisms which generate a level of tonic activity in RVLM sympathoexcitatory neurons sufficient to maintain a normal level of sympathetic vasomotor activity.     

Neuroanatomical plasticity in the gonadotropin‐releasing hormone system of the ewe: Seasonal variation in glutamatergic and γ‐aminobutyric acidergic afferents

Temperate zone animals time the onset of reproductive events to coincide with specific portions of the sidereal year. Although the neural mechanisms involved remain poorly understood, a marked annual variation in the brain's sensitivity to estradiol negative feedback is thought to mediate many of the changes in neuroendocrine hormone secretion, especially that of the gonadotropin‐releasing hormone (GnRH) neurons, via neural afferents. The aim of the present study was to determine whether glutamatergic inputs to GnRH neurons in sheep vary seasonally and to expand our previous observations of seasonal changes in γ‐aminobutyric acid (GABA)‐ergic inputs. Brains from adult sheep were collected during the breeding season (N = 8) or the nonbreeding season (anestrus; N = 7). Confocal microscopy and optical sectioning were used to quantify the density of labeled VGLUT2 and VGAT immunoreactivity onto GnRH neurons. The results reveal a significantly greater number of VGLUT2‐ir inputs to GnRH dendrites during the breeding season vs. the nonbreeding season but no seasonal changes on GnRH cell somas. The number of VGAT‐ir terminals onto GnRH dendrites was reduced in the breeding season compared with the nonbreeding season. GnRH neurons were also found to receive dual‐phenotype (VGLUT + VGAT) inputs; these varied with season in a manner similar to VGAT inputs. Morphologically, the numbers of branches of proximal dendrites increased significantly in a subset of GnRH neurons located near the midline. Together these results reveal a dynamic seasonal reorganization of identified inputs onto GnRH neurons and lend additional support to the overall hypothesis that seasonal modulation of GnRH neurons involves glutamatergic and GABAergic neural plasticity. J. Comp. Neurol. 515:615–628, 2009. © 2009 Wiley‐Liss, Inc.     

Brain sources of inhibitory input to the rat rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM) contains neurons critical for cardiovascular, respiratory, metabolic, and motor control. The activity of these neurons is controlled by inputs from multiple identified brain regions; however, the neurochemistry of these inputs is largely unknown. Gamma‐aminobutyric acid (GABA) and enkephalin tonically inhibit neurons within the RVLM. The aim of this study was to identify all brain regions that provide GABAergic or enkephalinergic input to the rat RVLM. Neurons immunoreactive for cholera toxin B (CTB‐ir), retrogradely transported from the RVLM, were assessed for expression of glutamic acid decarboxylase (GAD67) or preproenkephalin (PPE) mRNA using in situ hybridization. GAD67 mRNA was expressed in CTB‐ir neurons in the following regions: the nucleus of the solitary tract (NTS, 6% of CTB‐ir neurons), area postrema (AP, 8%), caudal ventrolateral medulla (17%), midline raphe (40%), ventrolateral periaqueductal gray (VLPAG, 15%), lateral hypothalamic area (LHA, 25%), central nucleus of the amygdala (CeA, 77%), sublenticular extended amygdala (SLEA, 86%), interstitial nucleus of the posterior limb of the anterior commissure (IPAC, 56%), bed nucleus of the stria terminals (BNST, 59%), and medial preoptic area (MPA, 53%). PPE mRNA was expressed in CTB‐ir neurons in the following regions: the NTS (14% of CTB‐ir neurons), midline raphe (26%), LHA (22%), zona incerta (ZI, 15%), CeA (5%), paraventricular nucleus (PVN, 13%), SLEA (66%), and MPA (26%). Thus, limited brain regions contribute GABAergic and/or enkephalinergic input to the RVLM. Multiple neurochemically distinct pathways originate from these brain regions projecting to the RVLM. J. Comp. Neurol. 521:213–232, 2013. © 2012 Wiley Periodicals, Inc.