The effect of air puff stress on c‐Fos expression in rat hypothalamus and brainstem: central circuitry mediating sympathoexcitation and baroreflex resetting

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor‐sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c‐Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine‐synthesizing neurons, did express c‐Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS‐projecting neurons that were double‐labelled with c‐Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double‐labelled NTS‐projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor‐sympathetic reflex that is associated with psychological stress.     

Neurons in the caudal ventrolateral medulla mediate the somato-sympathetic inhibitory reflex response via GABA receptors in the rostral ventrolateral medulla.

In urethane-anesthetized rabbits, stimulation of the sural nerve, consisting of cutaneous afferents (A-fibers), evoked reflex responses consisting of an early small excitatory component followed by a prolonged inhibitory component in renal sympathetic nerve activity. Bilateral injections of GABA antagonist, bicuculline (4 nmol/site), into the rostral ventrolateral medulla (RVLM), where sympatho-excitatory reticulospinal neurons are located, attenuated the inhibitory component in a dose-dependent manner as well as the inhibition evoked by stimulation of the aortic nerve A-fibers (baroreceptor afferents). Bilateral injections of a neurotoxic agent, kainic acid (4 nmol/site, 3 sites/side), into the caudal ventrolateral medulla (CVLM), where sympatho-inhibitory neurons with axonal projection to the RVLM are located, diminished these sympatho-inhibitory responses. Therefore it is concluded that the sympatho-inhibition evoked by activation of somatic afferents was mediated by neurons in the CVLM and by GABA receptors in the RVLM, as was the sympatho-inhibition associated with the arterial baroreceptor reflex. Bilateral injections of kynurenic acid (4 nmol/site, 3 sites/side) into the CVLM did not affect the somato-sympathetic reflex response, but diminished the sympatho-inhibition produced by activation of the baroreceptor afferents. Sympatho-inhibitory neurons in the CVLM were activated by glutamate when baroreceptor afferents were activated, but another excitatory transmitter may participate in the somato-sympathetic reflex in the CVLM.     

AMPA/kainate receptors mediate sympathetic chemoreceptor reflex in the rostral ventrolateral medulla

Previous studies have reported that information from carotid chemoreceptors activates sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) exclusively viaN-methyl-d-aspartic acid (NMDA) receptors. In this study, we examined the possible involvement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in the RVLM on sympathetic chemoreceptor reflex in pentobarbitone anaesthetised, vagotomised and artificially ventilated rats. Carotid chemoreceptor stimulation with brief N₂ inhalation increased splanchnic sympathetic nerve activity and arterial pressure in animals that had received an intravenous injection of the non-competitive NMDA receptor blocker, MK-801 (2 mg/kg). RVLM sympathetic premotor neurons could also be activated by brief hypoxia in the presence of MK-801. However, microinjection of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective AMPA/kainate receptor antagonist, 2 mM, 100 nl) into the RVLM after intravenous MK-801 abolished the hypoxia evoked sympathoexcitatory response. These results demonstrate that AMPA/kainate receptors in the RVLM are involved in the chemoreceptor reflex pathway.     

Fos expression in spinally projecting neurons after hypotension in the conscious rabbit

Hypotension produces a reflex increase in the activity of sympathetic vasomotor nerves. Studies in anaesthetised animals have established that neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic vasomotor preganglionic neurons in the spinal cord are a critical component of the central pathways mediating this reflex response. There are also neurons in supramedullary regions (the A5 area in the pons and the paraventricular nucleus (PVN) in the hypothalamus), however, that project directly to the sympathetic vasomotor outflow. The aim of this study was to identify and map neurons within the A5 area and PVN, as well as in the RVLM, which may contribute to the reflex sympathoexcitatory response to a hypotensive challenge in conscious rabbits. In a preliminary operation, a retrogradely transported tracer was injected into a site centred on the intermediolateral cell column in the upper lumbar spinal cord. After a waiting period of at least 1 week, a moderate hypotension (decrease in arterial pressure of approximately 20 mm Hg) was induced in conscious rabbits for 60 min by continuous infusion of sodium nitroprusside. In confirmation of previous studies, hypotension resulted in the expression of Fos in the RVLM, the A5 area and PVN. There were also retrogradely labelled neurons in all these regions. In both the RVLM and A5 area, approximately 40% of the retrogradely labelled neurons were also immunoreactive for Fos. In contrast, in the PVN the proportion of retrogradely labelled neurons that were also Fos-positive was much less (approximately 6%). This study has demonstrated that, in the conscious rabbit, a significant proportion of spinally projecting neurons within discrete regions in the RVLM and A5 area are activated by hypotension (as indicated by Fos expression). In the PVN, only a very small proportion of spinally projecting neurons are activated by hypotension, and thus these neurons appear to be regulated primarily by inputs other than baroreceptor inputs.     

Projection neurons of the vestibulo‐sympathetic reflex pathway

Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo‐sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c‐Fos protein. The retrograde tracer Fluoro‐Gold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c‐Fos protein and Fluoro‐Gold concomitantly. We observed activated projection neurons of the vestibulo‐sympathetic reflex pathway in the caudal half of the spinal, medial, and parvocellular medial vestibular nuclei. Approximately two‐thirds of the cells were ipsilateral to Fluoro‐Gold injection sites in both the RVLM and CVLM, and the remainder were contralateral. As a group, cells projecting to the RVLM were located slightly rostral to those with terminals in the CVLM. Individual activated projection neurons were multipolar, globular, or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo‐sympathetic reflex. J. Comp. Neurol. 522:2053–2074, 2014. © 2013 Wiley Periodicals, Inc.     

Role of the rostral ventrolateral medulla in the generation of synchronized sympathetic rhythmicities in the rat.

In artificially ventilated, paralyzed rats anesthetized with Nembutal or urethane, power density spectral analysis (PDS), using direct FFT algorithm, was used to quantify rhythmicities in the sympathetic cervical and lumbar nerves after bilateral microinjections into rostral ventrolateral medulla (RVLM) of CoCl2 and MgCl2--unspecific synaptic transmission blockers. Later overall sympathetic activity, phrenic nerve discharge, heart rate and arterial blood pressure were recorded. Block of synaptic transmission in RVLM was tested by elimination of sympathoinhibitory or sympathoexcitatory reflex responses to aortic nerve and vagal afferents stimulation respectively. In animals vagotomized with bilateral section of carotid sinus nerve the power in all frequency bands was very significantly reduced to a level not different from that which remained after spinal cord transsection. If carotid baroreceptors were intact, a small peak corresponding to cardiac frequency band persisted. Overall, non-synchronized sympathetic activity and arterial blood pressure increased. All effects were transient, lasted up to 15 min, and could be reproduced several times in one experiment. Respiratory rhythmic activity was unchanged yet respiratory-sympathetic synchronization was abolished. It is concluded that RVLM reticulospinal sympathoexcitatory neurons are responsible for non-synchronized tonic sympathetic activity but are not able to generate synchronized sympathetic rhythms. Synaptic input, presumably inhibitory, either from local neuronal circuits within ventral medulla and/or from other brain stem neuronal circuitries is needed to shape out the flexible pattern of sympathetic oscillations.     

Differential effects on sympathetic nerve activities elicited by activation of neurons in the pressor areas of dorsal and rostral ventrolateral medulla in cats.

Changes of the nerve activity of the sympathetic renal and vertebral nerves were elicited by microinjection of sodium glutamate (50 nmol/100 nl) into the pressor areas of the dorsal (DM) and rostral ventrolateral medulla (RVLM) in cats under urethane-chloralose anesthesia. Animals were bilaterally vagotomized, artificially ventilated, and paralyzed with gallamine triethiodide. The vertebral nerve activity always increased when pressor responses were induced by DM or RVLM stimulation. However, the effects of medullary stimulation on the renal nerve activity were variable. Three types of renal nerve responses concomitant with the pressor responses were observed in either baroreceptor-intact or baroreceptor-denervated cats. They were: (1) augmentation (type I); (2) attenuation (type II); and (3) insignificant change (type III). Type I responses were often elicited by RVLM stimulation whereas type II responses were often elicited by DM stimulation. Findings suggested that neurons integrating these sympathetic nerve activities were not equally distributed in the pressor areas of DM and RVLM. This result supports the notion that neurons located in different pressor areas of the brainstem exert differential effects over different sympathetic nerve activities.     

Baroreflex dependent and independent roles of the caudal ventrolateral medulla in cardiovascular regulation

The caudal ventrolateral medulla (CVLM) plays a critical role in cardiovascular regulation. Convincing data now support the hypothesis that inhibition of sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM) by CVLM neurons constitutes the necessary inhibitory link in baroreceptor reflex mediated control of sympathetic vasomotor outflow. Inhibition or destruction of the CVLM produces severe acute hypertension, consistent with blockade of baroreceptor reflexes and withdrawal of inhibition of RVLM sympathoexcitatory neurons. However, other data indicate that the CVLM also tonically inhibits RVLM sympathoexcitatory neurons in a manner not driven by baroreceptor input. In some studies, inhibition of the CVLM results in an increase in arterial pressure (AP) without inhibiting baroreceptor reflexes, possibly reflecting baroreceptor-independent and baroreceptor-dependent sub-regions of the CVLM. Furthermore, in baroreceptor-denervated rats, inhibition of the CVLM still leads to large increases in AP. In addition, in spontaneously hypertensive rats (SHR) central processing of baroreceptor reflexes appears normal but CVLM-mediated inhibition of the RVLM seems to be attenuated, suggesting that it is specifically a baroreceptor-independent mechanism of cardiovascular regulation in SHR that is altered. Taken together, these findings support an important, tonic, baroreceptor-independent inhibition of RVLM sympathoexcitatory neurons exerted by the CVLM.     

Adrenal epinephrine secretion is not regulated by sympathoinhibitory neurons in the caudal ventrolateral medulla.

By providing the principal inhibitory regulation of the discharge of sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), neurons in the caudal ventrolateral medulla (CVLM) play a major role in regulating the level of sympathetic nerve activity (SNA) to cardiovascular targets. To determine whether adrenal medullary secretion of epinephrine (EPI) is also regulated by sympathoinhibitory inputs from the CVLM to the RVLM, we compared levels of plasma EPI obtained after disinhibition of RVLM neurons with levels obtained after inhibition of CVLM neurons, both of which result in sustained elevations in arterial blood pressure (AP), SNA, and heart rate (HR). Plasma norepinephrine (NE) concentrations were significantly elevated following bilateral microinjection either of bicuculline (BIC) into the RVLM or of muscimol into the CVLM of urethane/chloralose-anesthetized, artificially-ventilated rats. In sharp contrast, although plasma EPI concentrations were significantly elevated following disinhibition of neurons in the RVLM, they were unchanged by inhibition of neurons in the CVLM. These results demonstrate that the discharge of sympathetic premotor neurons in the RVLM regulating adrenal secretion of EPI is modulated by a tonic, GABA-ergic inhibition that arises from a source that is different from the sympathoinhibitory neurons in the CVLM that project to RVLM sympathetic premotor neurons controlling vasoconstrictor and cardiac targets.     

Reticulospinal neurons inactivated by warming of the preoptic area and anterior hypothalamus of rabbits

To identify the premotor neurons for vasoconstrictors of the skin, activities of reticulospinal neurons in the rostroventral medulla, the ear sympathetic nerve (ESNA) and the renal sympathetic nerve (RSNA) were recorded in anesthetized and immobilized Japanese White or New Zealand White rabbits. Two groups of neurons were identified according to their responses to thermal stimulation of the preoptic area and the anterior hypothalamus (POAH) and to electrical stimulation of baroreceptor afferents, the aortic nerve (AN). Neurons (Type I neurons, n = 21) whose activity was inhibited by warm stimulation of the POAH but not inhibited by the AN stimulation were located in sites medial to the rostral ventrolateral medulla (RVLM). The other neurons (Type II neurons, n = 20) whose activity was not inhibited by warm stimulation of the POAH but inhibited by the AN stimulation were located in the RVLM. Because the time course of the inhibitory response of Type I neurons to warm stimulation of the POAH was very similar to that of the inhibitory response of the ESNA and activities of these neurons and the ESNA were not inhibited by the stimulation of the AN, it was suggested the Type I neurons might participate in regulation of activity of the vasoconstrictors of the ear skin. The Type II neurons are considered to be the barosensitive RVLM neurons that regulate systemic arterial pressure by controlling the activity of visceral or muscular sympathetic vasoconstrictors or cardiac sympathetic fibers.     

Neuropeptide Y in the rostral ventrolateral medulla blocks somatosympathetic reflexes in anesthetized rats

The rostral ventrolateral medulla (RVLM) is a major integrative center of cardiovascular reflexes that modulate vasomotor tone. The functions of Neuropeptide Y (NPY) in the RVLM on cardiorespiratory responses remain unknown. Arterial blood pressure (AP), heart rate (HR), splanchnic sympathetic (sSNA) and phrenic nerve activities, and responsiveness to baro-, somatosympathetic, and chemoreflex stimulation were recorded before and after bilateral NPY injection (100 pmol, 200 nl/side) in the RVLM of vagotomized urethane-anesthetized rats (n=7). Responses were characterized by an initial increase in AP followed by prolonged hypotension (P<0.01). A similar biphasic effect was exerted on HR (P<0.01). NPY caused a large increase of sSNA (P<0.01), that gradually recovered towards baseline. Somatosympathetic responses evoked by tibial nerve stimulation were largely abolished following NPY microinjection (P<0.01), but sympathoexcitatory responses evoked by acute hypoxia or sympathoinhibition evoked by aortic depressor nerve stimulation were unchanged following NPY. There was no effect of NPY on phrenic nerve amplitude or frequency. We conclude that NPY exerts excitatory effects on sympathetic tone, but inhibits responses evoked by somatic inputs. We speculate that this apparent contradiction may be due to differential expression of NPY receptor subtypes on the soma of sympathetic premotor neurons in the RVLM and on the presynaptic terminals of neurons that comprise excitatory afferent pathways.     

AV3V lesions reduce the pressor response to L-glutamate into the RVLM

Neurons from the rostral ventrolateral medulla (RVLM) directly activate sympathetic pre-ganglionic neurons in the spinal cord. Hypertensive responses and sympathetic activation produced by different stimuli are strongly affected by lesions of the preoptic periventricular tissue surrounding the anteroventral third ventricle (AV3V region). Therefore, in the present study, we investigated the effects of acute (1 day) and chronic (15 days) electrolytic lesions of the AV3V region on the pressor responses produced by injections of the excitatory amino acid L-glutamate into the RVLM of unanesthetized rats. Male Holtzman rats with sham or electrolytic AV3V lesions and a stainless steel cannula implanted into the RVLM were used. The pressor responses produced by injections of L-glutamate (1, 5 and 10 nmol/100 nl) into the RVLM were reduced 1 day (9 +/- 4, 39 +/- 6 and 37 +/- 4 mm Hg, respectively) and 15 days after AV3V lesions (13 +/- 6, 39 +/- 4 and 43 +/- 4 mm Hg, respectively, vs. sham lesions: 29 +/- 3, 50 +/- 2 and 58 +/- 3 mm Hg, respectively). Injections of L-glutamate into the RVLM in sham or AV3V-lesioned rats produced no significant change in the heart rate (HR). Baroreflex bradycardia and tachycardia produced by iv phenylephrine or sodium nitroprusside, respectively, and the pressor and bradycardic responses to chemoreflex activation with iv potassium cyanide were not modified by AV3V lesions. The results suggest that signals from the AV3V region are important for sympathetic activation induced by L-glutamate into the RVLM.     

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.     

Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are activated by haemorrhage

The retrogradely-transported tracer, rhodamine-tagged microspheres, was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to identify paraventricular neurons in the hypothalamus that project to the RVLM. The protein, Fos, was detected immunohistochemically and used to highlight neurons that were activated by a hypotensive haemorrhage. Compared to controls, Fos production was increased by approximately 3-fold in the paraventricular nucleus (P<0.009) and there was a significant increase in the number of retrogradely-labelled cells that expressed Fos. These represented 5% of the retrogradely-labelled cell population. The results suggest that a small subpopulation of PVN neurons projecting to the RVLM are activated by haemorrhage and may be involved in the reflex responses initiated by that stimulus.     

Rostroventrolateral medulla neurons with commissural projections provide input to sympathetic premotor neurons: anatomical and functional evidence

The activity of neurons in the rostral ventrolateral medulla (RVLM) is critical for the generation of vasomotor sympathetic tone. Multiple pre‐sympathetic pathways converge on spinally projecting RVLM neurons, but the origin and circumstances in which such inputs are active are poorly understood. We have previously shown that input from the contralateral brainstem contributes to the baseline activity of this population: in the current study we investigate the distribution, phenotype and functional properties of RVLM neurons with commissural projections in the rat. We firstly used retrograde transport of fluorescent microspheres to identify neurons that project to the contralateral RVLM. Labelled neurons were prominent in a longitudinal column that extended over 1 mm caudal from the facial nucleus and contained hybridisation products indicating enkephalin (27%), GABA (15%) and adrenaline (3%) synthesis and included 6% of bulbospinal neurons identified by transport of cholera toxin B. Anterograde transport of fluorescent dextran‐conjugate from the contralateral RVLM revealed extensive inputs throughout the RVLM that frequently terminated in close apposition with catecholaminergic and bulbospinal neurons. In urethane‐anaesthetised rats we verified that 28/37 neurons antidromically activated by electrical stimulation of the contralateral pressor region were spontaneously active, of which 13 had activity locked to central respiratory drive and 15 displayed ongoing tonic discharge. In six tonically active neurons sympathoexcitatory roles were indicated by spike‐triggered averages of splanchnic sympathetic nerve activity. We conclude that neurons in the RVLM project to the contralateral brainstem, form synapses with sympathetic premotor neurons, and have functional properties consistent with sympthoexcitatory function.     

Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin

Systemic administration of the gastrointestinal hormone cholecystokinin (CCK) selectively inhibits splanchnic sympathetic vasomotor discharge and differentially affects presympathetic vasomotor neurons of the rostroventrolateral medulla (RVLM). Stimulation of the sympathoexcitatory region of the periaqueductal grey (PAG) produces profound mesenteric vasoconstriction. In this study, our aim was to identify phenotypically different populations of RVLM presympathetic vasomotor neurons using juxtacellular neuronal labelling and immunohistochemical detection of the adrenergic neuronal marker phenylethanolamine-N-methyl transferase (PNMT) and to determine whether the PAG provides functional excitatory input to these neurons. Fifty-eight percent (36/62) of RVLM presympathetic neurons were inhibited by systemic administration of CCK. These cells had conduction velocities (3.6 +/- 0.2 m/sec) in the non-C-fiber range consistent with neurons possessing lightly myelinated spinal axons. Of these, 79% (22/28) were excited by PAG stimulation, and 59% (10/17) were not immunoreactive for PNMT. Conversely, 42% (26/62) of RVLM presympathetic neurons were either unaffected or activated by CCK administration and had slower conduction velocities (1.4 +/- 0.3 m/sec) than cells inhibited by CCK. Fifty percent (11/22) of these cells were driven by PAG stimulation, and most (11/14 or 79%) were PNMT-positive. These results suggest that cardiovascular responses elicited by PAG stimulation occur via activation of non-C1 and C1 RVLM presympathetic neurons. RVLM neurons inhibited by CCK were more likely to be driven by PAG stimulation and may be a subset of neurons responsible for driving gastrointestinal sympathetic vasomotor tone. CCK-induced inhibition of a subpopulation of RVLM presympathetic neurons may be implicated in postprandial hyperemia and postprandial hypotension.     

Evidence for tonic disinhibition of RVLM sympathoexcitatory neurons from the caudal pressor area

Previous studies in the rat have shown that a significant proportion of the tonic activity of presympathetic neurons in the rostral ventrolateral medulla (RVLM) is dependent on the tonic activity of neurons within the caudal pressor area (CPA), located in the most caudal part of the caudal ventrolateral medulla (CVLM). In this study, we determined the extent to which tonically active neurons in the CPA contribute to sympathetic vasomotor tone, and we also investigated the pharmacological mechanisms by which these neurons affect the tonic activity of RVLM presympathetic neurons. In anaesthetised rabbits, bilateral injections of the neuroinhibitory compound muscimol into the CVLM at the level of the most caudal part of the lateral reticular nucleus, which corresponds to the anatomical location of the CPA as mapped in the rat, resulted in an immediate profound hypotension and almost complete abolition of renal sympathetic nerve activity (rSNA). In contrast, microinjections into surrounding regions had little or no effect or else evoked a delayed hypotensive response. The hypotensive and sympathoinhibitory response evoked by inhibition of the CPA was greatly delayed by prior injections of the GABA receptor antagonist bicuculline into the RVLM. In contrast, injections of the glutamate receptor antagonist kynurenic acid into the RVLM did not alter the hypotensive and sympathoinhibitory response. The results indicate that neurons within the CPA tonically inhibit other neurons, which, in turn, inhibit RVLM sympathoexcitatory neurons, via a GABAergic synapse. This disinhibition of RVLM neurons by CPA neurons is essential for maintaining resting sympathetic vasomotor tone.     

Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM‐CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM‐CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine‐β‐hydroxylase‐expressing neurons in mice [DβH^Cre/0;VGLUT2^flox/flox mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM‐CA neurons in cKO vs. control mice (DβH^Cre/0), using channelrhodopsin‐2 (ChR2–mCherry) optogenetics. ChR2–mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90–99% colocalisation with tyrosine hydroxylase). RVLM‐CA neurons had similar morphology and axonal projections in DβH^Cre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2‐positive RVLM‐CA neurons in DβH^Cre/0 and cKO mice. Photostimulation in conscious mice produced frequency‐dependent respiratory activation in DβH^Cre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH^Cre/0 mice only. Vagal responses were unaffected by α₁‐adrenoreceptor blockade. In conclusion, two responses evoked by RVLM‐CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM‐CA neurons are mediated by glutamate.     

Medial prefrontal depressor response: involvement of the rostral and caudal ventrolateral medulla in the rat

The importance of neurones of the caudal and rostral ventrolateral medulla (CVLM and RVLM, respectively) in mediation of the medial prefrontal cortex depressor response was studied in halothane-anaesthetised rats. Blockade of GABA(A) receptors in the RVLM produced by microinjection of bicuculline (50 nl, 2 mM, n = 6) resulted in reversal of the depressor (-9.5 +/- 1.2 mm Hg) and lumbar sympathetic (-6.5 +/- 5.7 units) responses to pressor (+7.8 +/- 3.5 mm Hg) and sympathoexcitatory (+19.3 +/- 12.5 units) responses and simultaneous blockade of baroreceptor reflex-mediated sympathoinhibition. Baroreflex blockade was reflected by a significant reduction in the gain (slope of the blood pressure vs. lumbar sympathetic nerve discharge regression line) of the reflex. Microinjection of the excitatory amino acid antagonist kynurenic acid (100 nl, 50 mM, n = 6) into the CVLM blocked the baroreflex and significantly reduced the depressor (-9.6 +/- 0.4 to -6.9 +/- 0.6 mm Hg) and lumbar sympathetic (-4.0 +/- 2.1 to 2.9 +/- 1.9 units) responses to medial prefrontal cortex stimulation. These results support the hypothesis that the medial prefrontal cortex depressor response is mediated by a pathway which converges at the level of the RVLM and which is only partly dependent on an excitatory input to caudal ventrolateral medullary neurones.     

Pancreatic vasoconstrictor responses are regulated by neurons in the rostral ventrolateral medulla

The pancreas receives sympathetic input which arises from several premotor cell groups in the CNS including the rostral ventrolateral medulla (RVLM). In this study, we examined the influence of electrical stimulation of the RVLM on pancreatic blood flow measured by laser Doppler flowmetry and gastric blood flow measured by ultrasonic Doppler flowmetry in halothane-anesthetized rats. The laser Doppler flow measurement technique was validated by demonstration that pancreatic conductance was reduced by systemic administration of the vasoconstrictor phenylephrine and increased by the vasodilator sodium nitroprusside. Sympathetic vasomotor withdrawal induced by either administration of phenylbiguanide (2 and 10 microg/kg, i.v.) or electrical stimulation of the central end of the cervical vagal trunk (5 Hz, 2 ms, 50-150 microA) produced depressor responses and increases in pancreatic and gastric vascular conductance. Electrical stimulation of the RVLM (50 Hz, 0.5 ms, 25-75 microA) produced pressor and tachycardic responses accompanied by decreases in pancreatic and gastric vascular conductance. All responses to RVLM stimulation were abolished by blockade of ganglionic neurotransmission (hexamethonium bromide, 20 mg/kg, i.v.). These data suggest that RVLM presympathetic vasomotor neurons are a primary source of tonic sympathetic vasomotor drive to the pancreatic and gastric vasculature.