Neurons In Medulla Research Articles

A neurophysiological study of the medullary sympathetic pathway to the adrenal gland (1131.7)

Neurons in the rostral ventrolateral medulla (RVLM) control adrenaline secretion from the adrenal gland and participate in glucose homeostasis. Here, we examined sympathetic evoked responses recorded from the adrenal sympathetic nerve in response to intermittent stimulation of the RVLM. All experiments were performed using isoflurane/urethane‐anaesthetised, artificially‐ventilated, paralysed, male Sprague‐Dawley rats. The RVLM was first identified by extracellular single unit recording combined with field potential mapping of the facial nucleus. We compared the latencies of the evoked adrenal sympathetic nerve (ASNA) response to the lumbar sympathetic nerve (LSNA) response upon electrical stimulation of RVLM (0.5 Hz, 1 ms pulse width, 50‐300 μA) after conversion to urethane (1.4 g/kg, i.v.). RVLM stimulation elicited an evoked ASNA response that depended on whether the recording was primarily pre‐ or post‐ganglionic activity as judged by the effect of the ganglion blocker hexamethonium (Hex; 20‐40 mg/kg, i.v.). Pre‐ganglionic ASNA consisted of an early peak (latency ‐ 79±9 ms, n=4) and a late peak (latency ‐ 149±6 ms). Lumbar sympathetic nerve activity exhibited a prominent early peak (latency ‐ 87±7 ms; n = 5) that was eliminated by Hex. Pre‐ganglionic ASNA was reduced by 62 % after intrathecal kynurenate (0.5 μmole/10 μl; P<0.01, late peak, n=3). The early peak may represent sympathetic vasomotor activity in both the adrenal and lumbar sympathetic nerves. However, the late sympathetic response noted in the adrenal nerve is mediated by a glutamatergic input and may be involved in the control of catecholamine secretion from adrenal chromaffin cells.Grant Funding Source: National Health and Medical Research Council of Australia

Single unit recordings from putative presympathetic neurons in the rostral ventrolateral medulla of sedentary and physically active rats (686.35)

A sedentary lifestyle has been linked to the development of cardiovascular disease (CVD). The RVLM is believed to contribute to CVD through direct activation of sympathetic preganglionic neurons. Our previous work showed that sedentary rats exhibit enhanced sympathoexcitation after glutamate activation of the RVLM compared to active counterparts. We have also recently shown increased dendritic arborization in spinally‐projecting C1 RVLM neurons from sedentary rats. We hypothesized that the RVLM exhibits increased excitability due to structural and functional differences in sedentary versus active rats (14 weeks wheel running). To our knowledge, we are the first group to have performed in vivo single unit recordings from putative presympathetic RVLM neurons in sedentary and active rats to examine functional differences in presympathetic RVLM neurons. Preliminary results from Inactin‐anesthetized rats indicate that individual presympathetic RVLM neurons from sedentary rats appeared to have higher resting firing rates (14.0±5.1 Hz, n=4) compared to active rats (8.7±3.0 Hz, n=4). RVLM neurons also appeared to have greater responses to acute increases in blood pressure (∆‐11.4±5.3 Hz vs. ∆‐5.6±1.4 Hz, respectively), but decreases in blood pressure did not appear to elicit different responses (∆+3.1±1.4 Hz vs. ∆+2.9±0.3 Hz). Though our current sample size does not yet permit us to draw definitive conclusions, these preliminary data support our hypothesis and suggest that a sedentary lifestyle may increase sympathetic nerve activity by causing an increase in the activity of individual presympathetic RVLM neurons.Grant Funding Source: HL096787, WSU Rumble Fellowship

Different sub‐populations of pre‐sympathetic neurons are revelead by acute hypoxia in brainstem slices of adult rats (710.7)

In this study we evaluated, in brainstem slices from adult rats, the effects of acute hypoxia (AH) on the firing rate of pre‐sympathetic neurons in rostral ventrolateral medulla sending projections to the intermediolateral column of spinal cord (RVLM‐IML neurons). Anesthetized male Wistar rats (PO 30) were submitted to microinjection of fluorescent retrograde tracer into the spinal cord (T3‐T5) to label the RVLM‐IML neurons. Five days later the rats were anesthetized to obtain brainstem coronal slices. The RVLM‐IML neurons were recorded using whole‐cell patch‐clamp and the firing rate was analyzed before and after perfusion of slice to AH for 2 minutes with hypoxic solution (95% N2 and 5% CO2) in the presence of synaptic blockers. After the AH protocol the viability of RVLM‐IML neurons were verified by the perfusion with normal solution (95% O2 and 5% CO2). All recorded RVLM‐IML neuronsof adult rats (n=17) presented intrinsic spontaneous firing activity. Three sub‐populations of RVLM‐IML neurons were identified after AH exposure: a) 3 neurons decreased their firing rate after AH(‐ 34,10%, p= 0,0225); b) 4 neurons increased their firing rate after AH (+ 40,86%, p= 0,0249) and c) 10 neurons were not affected by AH(± 3,54%, p= 0,5380. Hypoxia produced no changes in input resistance and resting membrane potential in these neurons. The data show that pre‐sympathetic neurons in the brainstem slices of adult rats present pacemaker activity and different sub‐populations of pre‐sympathetic neurons are revealed in response to AH.Grant Funding Source: Supported by FAPESP and CNPQ

Functional studies of rostral ventrolateral medulla neurons using manganese‐enhanced magnetic resonance imaging in sedentary versus physically active rats (686.36)

A sedentary lifestyle, a key risk factor in cardiovascular disease (CVD), is linked to increased sympathetic nerve activity (SNA). Control of SNA occurs via bulbospinal catecholaminergic (C1) and non‐C1 neurons primarily in the RVLM. We recently reported that sedentary, but not physically active rats, exhibit increased dendritic branching in bulbospinal C1 RVLM neurons in a specific caudal to rostral pattern. However, regionally specific increases in RVLM neuronal activation have not yet been associated with increased dendritic branching. In many brain regions, manganese (Mn2+), a paramagnetic ion, has been used as a contrast agent to detect neuronal activity via calcium channel uptake. The purpose of this study was to develop MeMRI for in vivo studies of the RVLM, with the hypothesis that sedentary rats would have increased RVLM neuronal activity and greater Mn2+ uptake. One sedentary and one active rat (12 wks wheel running) were administered Mn2+ chloride (66mg/kg, IP), and a control rat was given saline. After 24 hours, rats were imaged (7T, Bruker Avance) and saline values were subtracted from Mn2+ injected animals. Uptake of Mn2+ in the RVLM was greater in the sedentary versus active rat (e.g. 808 vs. 327 avg voxel intensity, respectively). Notably, only the sedentary rat had a pattern of increased uptake in the caudal to rostral RVLM, very similar to our structural studies. These preliminary studies suggest that increased dendritic branching in more rostral RVLM neurons may account for enhanced RVLM neuronal activity and the growing prevalence of CVD in sedentary individuals.Grant Funding Source: Supported by HL096787‐PJM; VA 1l01RX001095‐01‐AGH

Cholinergic involvement in control of REM sleep paralysis

REM sleep, also known as dreaming sleep, is marked by intense cortical activation and absence of skeletal muscle tone, so-called REM sleep paralysis (atonia). It is commonly believed that REM sleep paralysis functions to prevent movement during vivid dreams. Indeed, REM sleep behaviour disorder – a neurological condition marked by violent dream enactment – results from loss of REM sleep paralysis. For the last 50 years, biologists have focused on the identification of brain mechanisms responsible for REM sleep. A majority of evidence suggests that a brainstem region known as the sublaterodorsal nucleus (SLD), also called the subcoeruleus, is important for REM sleep generation (Jouvet 1962). However, there is uncertainty concerning the chemical mechanisms by which the SLD triggers REM sleep phenomena. For example, some data suggest that cholinergic modulation of SLD cells underlies REM sleep generation, whereas, other data suggest that GABAergic disinhibition and glutamatergic excitation of SLD cells are critical for REM sleep control (Boissard et al. 2002; Lu et al. 2006). The recent study by Weng et al. (2014) provides a potentially new framework for understanding REM sleep control by showing that both cholinergic and glutamatergic processes operating within the SLD could be important for triggering REM sleep paralysis.

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.

Blunted sympathoinhibitory responses in obesity‐related hypertension are due to aberrant central but not peripheral signalling mechanisms

The gut hormone cholecystokinin (CCK) acts at subdiaphragmatic vagal afferents to induce renal and splanchnic sympathoinhibition and vasodilatation, via reflex inhibition of a subclass of cardiovascular-controlling neurons in the rostroventrolateral medulla (RVLM). These sympathoinhibitory and vasodilator responses are blunted in obese, hypertensive rats and our aim in the present study was to determine whether this is attributable to (i) altered sensitivity of presympathetic vasomotor RVLM neurons, and (ii) aberrant peripheral or central signalling mechanisms. Using a diet-induced obesity model, male Sprague-Dawley rats exhibited either an obesity-prone (OP) or obesity-resistant (OR) phenotype when placed on a medium high fat diet for 13-15weeks; control animals were placed on a low fat diet. OP animals had elevated resting arterial pressure compared to OR/control animals (P<0.05). Barosensitivity of RVLM neurons was significantly attenuated in OP animals (P<0.05), suggesting altered baroreflex gain. CCK induced inhibitory responses in RVLM neurons of OR/control animals but not OP animals. Subdiaphragmatic vagal nerve responsiveness to CCK and CCK1 receptor mRNA expression in nodose ganglia did not differ between the groups, but CCK induced significantly less Fos-like immunoreactivity in both the nucleus of the solitary tract and the caudal ventrolateral medulla of OP animals compared to controls (P<0.05). These results suggest that blunted sympathoinhibitory and vasodilator responses in obesity-related hypertension are due to alterations in RVLM neuronal responses, resulting from aberrant central but not peripheral signalling mechanisms. In obesity, blunted sympathoinhibitory mechanisms may lead to increased regional vascular resistance and contribute to the development of hypertension.

ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla

This study investigated the effects of ANG II on slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rostral ventrolateral medulla (RVLM) in urethane-anesthetized rabbits to determine whether the sympathetic baroreflex modulation induced by application of ANG II into the RVLM can be explained by the total action of ANG II on individual RVLM neurons. In response to pharmacologically induced slow ramp changes in mean arterial pressure (MAP), individual RVLM neurons exhibited a unit activity-MAP relationship that was fitted by a straight line with upper and lower plateaus. Iontophoretically applied ANG II raised the upper plateau without changing the slope, and, thereby, increased the working range of the baroreflex response. An asymmetric sigmoid curve that was determined by averaging individual unit activity-MAP relationship lines became more symmetric with ANG II application. The characteristics of the average curves, both before and during ANG II application, were consistent with the renal sympathetic nerve activity-MAP relationship curves obtained under the same experimental conditions. ANG II also affected rapid baroreflex responses of RVLM neurons that were induced by cardiac beats, as application of ANG II predominantly raised the average unit activities in the downstroke phase of arterial pulse waves. The present study provides a possible explanation for the ANG II-induced sympathetic baroreflex modulation based on the action of ANG II on barosensitive bulbospinal RVLM neurons. Our results also suggest that ANG II changes both static and dynamic characteristics of baroreflex responses of RVLM neurons.

Photoreceptor-Derived Activin Promotes Dendritic Termination and Restricts the Receptive Fields of First-Order Interneurons in Drosophila

How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. Here, we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ∼16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

The Novel Endocannabinoid Receptor GPR18 Is Expressed in the Rostral Ventrolateral Medulla and Exerts Tonic Restraining Influence on Blood Pressure

Systemic administration of the G-protein-coupled receptor 18 (GPR18) agonist abnormal cannabidiol (Abn CBD) lowers blood pressure (BP). Whether GPR18 is expressed in the central nervous system (CNS) and plays a role in BP control is not known despite the abundance of the GPR18 ligand N-arachidonoyl glycine (NAGly) in the CNS. Therefore, we first determined whether GPR18 is expressed in the presympathetic tyrosine hydroxylase (TH) immunoreactive (ir) neurons of the brainstem cardiovascular regulatory nuclei. Second, we investigated the impact of GPR18 activation and blockade on BP and heart rate (HR) and neurochemical modulators of sympathetic activity and BP. Immunofluorescence findings revealed GPR18 expression in TH-ir neurons in the rostral ventrolateral medulla (RVLM). Intra-RVLM GPR18 activation (Abn CBD) and blockade (O-1918, 1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-,cyclohexen-1-yl]benzene) elicited dose-dependent reductions and elevations in BP, respectively, along with respective increases and decreases in HR in conscious male Sprague-Dawley rats. RVLM GPR18 activation increased neuronal adiponectin (ADN) and NO and reduced reactive oxygen species (ROS) levels, and GPR18 blockade reduced neuronal ADN and increased oxidative stress (i.e., ROS) in the RVLM. Finally, we hypothesized that the negligible hypotensive effect caused by the endogenous GPR18 ligand NAGly could be due to concurrent activation of CB(1)R in the RVLM. Our findings support this hypothesis because NAGly-evoked hypotension was doubled after RVLM CB(1)R blockade (SR141716, rimonabant). These findings are the first to demonstrate GPR18 expression in the RVLM and to suggest a sympathoinhibitory role for this receptor. The findings yield new insight into the role of a novel cannabinoid receptor (GPR18) in central BP control.

Chapter 2 - Coupling of respiratory and sympathetic activities in rats submitted to chronic intermittent hypoxia

The respiratory modulation of sympathetic activity relies on the balance between excitatory and inhibitory inputs from the brainstem respiratory network to presympathetic neurons of the rostral ventrolateral medulla. This central respiratory-sympathetic contributes for the generation of respiratory-related rhythmical oscillations in heart rate and arterial pressure levels, whose functional effects on the blood gas exchange/perfusion and cardiac work remain to be elucidated. Herein, we discuss the experimental evidence describing the potential neural mechanisms underlying the entrainment between respiratory and sympathetic activities at baseline conditions as well as under conditions of metabolic challenges. We also discuss the possible implications of changes in the strength or pattern of the central respiratory-sympathetic coupling in the genesis of sympathetic overactivity and neurogenic hypertension, including that associated with the exposure to chronic intermittent hypoxia.

Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair.

Peripheral nerve injuries are common in clinical practice because of traumas such as crushing and sectioning. Lesions of the nerve structure result in lost or diminished sensitivity and/or motor activity in the innervated territory. The degree of lesion depends on the specific nerve involved, the magnitude and type of pressure exerted, and the duration of the compression. The results of such injuries commonly include axonal degeneration and retrograde degeneration of the corresponding neurons in the spinal medulla, followed by very slow regeneration (Rochkind et al., 2001). The adverse effects on the daily activities of patients with a peripheral nerve injury are a determining factor in establishing the goals of early recovery (Rodriguez et al., 2004). The most severe form of nerve damage involves complete transection of the nerve, which results in the loss of sensory and motor function at the site of injury. Although a degree of recovery can be expected in most untreated nerve injuries, the process is slow and often incomplete. Moreover, despite considerable advances in microsurgical techniques, the functional results of peripheral nerve repair remain largely unsatisfactory. The regrowth of nerves across large gaps is particularly challenging, usually requiring a nerve graft to correctly bridge the proximal and distal nerve stumps. At present, nerve autografting is the most common treatment used to repair peripheral nerve defects. However, this recognized “gold standard” technique has a number of inherent disadvantages, such as limited availability of donor tissue (IJkema-Paassen et al., 2004), secondary deformities, potential differences in tissue structure and size (Nichols et al., 2004), and numbness at donor sites (Bini et al., 2004). Although xenografts and allografts have been proposed as alternatives to autografts, the success rate of these techniques remains poor, often resulting in immune rejection. Thus, researchers have invested considerable effort in developing synthetic nerve conduits for the repair of peripheral nerve defects.

Activation of Neurons in the Rat Medulla Following a Gag Reflex Stimulus

Despite the fact that a number of individuals present with a hypersensitive gag reflex, research in this area has been limited to clinical applications. An animal model that describes the neurologic underpinnings of the gag reflex has not been reported. Thus, the objective of the current study was to localize activated neurons in the rat medulla and to investigate their neurochemistry following mechanostimulation to the posterior pharyngeal wall eliciting a gag reflex response. Activated neurons, determined using c-fos immunohistochemistry, were observed in subnuclei of the nucleus tractus solitarius (NTS) and motor regions such as dorsal motor vagus, hypoglossal, and nucleus ambiguus. The lateral and dorsal paragigantocellular nuclei and the medial rostral ventrolateral nucleus involved in bitter taste processing, also showed robust activation. Tyrosine hydroxylase and/or choline acetyltransferase immunoreactivity was localized in many activated neurons. The results of the present study provide the first documentation of activated neurons and preliminary neurochemistry in brainstem nuclei that are unique to mechanostimulation to the posterior pharyngeal wall. Our results support postulated medullary structures involved with the gag reflex and propose new regions to be added to the circuitry model. Further, our results reveal an activation pattern within the NTS unique to the gag reflex. Keywords: Brain stem, Nucleus of the solitary tract, neuroanatomy, animal model, pediatric feeding. Read more →

Activity of respiratory neurons in the rostral medulla during vocalization, swallowing, and coughing in guinea pigs

To examine the relationship between the neuronal networks underlying respiration and non-respiratory behaviors such as vocalization and airway defensive reflexes, we compared the activity of respiratory neurons in the ventrolateral medulla during breathing with that during non-respiratory behaviors including vocalization, swallowing, and coughing in guinea pigs. During fictive vocalization the activity of augmenting expiratory neurons ceased, whereas the other types of expiratory neurons did not show a consistent tendency of increasing or decreasing activity. All inspiratory neurons discharged in synchrony with the phrenic nerve activity. Most of the phase-spanning neurons were activated throughout the vocal phase. During fictive swallowing, many expiratory and inspiratory neurons were silent, whereas many phase-spanning neurons were activated. During fictive coughing, many expiratory neurons were activated during the expiratory phase of coughing. Most inspiratory neurons discharged in parallel with the phrenic nerve activity during coughing. Many phase-spanning neurons were activated during the expiratory phase of coughing. These findings indicate that the medullary respiratory neurons help shape respiratory muscle nerve activity not only during breathing but also during these non-respiratory behaviors, and thus suggest that at least some of the respiratory neurons are shared among the neuronal circuits underlying the generation of breathing and non-respiratory behaviors.

Physical (in)activity-dependent structural plasticity in bulbospinal catecholaminergic neurons of rat rostral ventrolateral medulla.

Increased activity of the sympathetic nervous system is thought to play a role in the development and progression of cardiovascular disease. Recent work has shown that physical inactivity versus activity alters neuronal structure in brain regions associated with cardiovascular regulation. Our physiological studies suggest that neurons in the rostral ventrolateral medulla (RVLM) are more responsive to excitation in sedentary versus physically active animals. We hypothesized that enhanced functional responses in the RVLM may be due, in part, to changes in the structure of RVLM neurons that control sympathetic activity. We used retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) to identify bulbospinal catecholaminergic (C1) neurons in sedentary and active rats after chronic voluntary wheel-running exercise. We then digitally reconstructed their cell bodies and dendrites at different rostrocaudal levels. The dendritic arbors of spinally projecting TH neurons from sedentary rats were more branched than those of physically active rats (P < 0.05). In sedentary rats, dendritic branching was greater in more rostral versus more caudal bulbospinal C1 neurons, whereas, in physically active rats, dendritic branching was consistent throughout the RVLM. In contrast, cell body size and the number of primary dendrites did not differ between active and inactive animals. We suggest that these structural changes provide an anatomical underpinning for the functional differences observed in our in vivo studies. These inactivity-related structural and functional changes may enhance the overall sensitivity of RVLM neurons to excitatory stimuli and contribute to an increased risk of cardiovascular disease in sedentary individuals.

Physical (in)activity‐dependent structural plasticity in bulbospinal catecholaminergic neurons of rat rostral ventrolateral medulla

ABSTRACTIncreased activity of the sympathetic nervous system is thought to play a role in the development and progression of cardiovascular disease. Recent work has shown that physical inactivity versus activity alters neuronal structure in brain regions associated with cardiovascular regulation. Our physiological studies suggest that neurons in the rostral ventrolateral medulla (RVLM) are more responsive to excitation in sedentary versus physically active animals. We hypothesized that enhanced functional responses in the RVLM may be due, in part, to changes in the structure of RVLM neurons that control sympathetic activity. We used retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) to identify bulbospinal catecholaminergic (C1) neurons in sedentary and active rats after chronic voluntary wheel‐running exercise. We then digitally reconstructed their cell bodies and dendrites at different rostrocaudal levels. The dendritic arbors of spinally projecting TH neurons from sedentary rats were more branched than those of physically active rats (P &lt; 0.05). In sedentary rats, dendritic branching was greater in more rostral versus more caudal bulbospinal C1 neurons, whereas, in physically active rats, dendritic branching was consistent throughout the RVLM. In contrast, cell body size and the number of primary dendrites did not differ between active and inactive animals. We suggest that these structural changes provide an anatomical underpinning for the functional differences observed in our in vivo studies. These inactivity‐related structural and functional changes may enhance the overall sensitivity of RVLM neurons to excitatory stimuli and contribute to an increased risk of cardiovascular disease in sedentary individuals. J. Comp. Neurol. 522:499–513, 2014. © 2013 Wiley Periodicals, Inc.

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

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.

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.

Distribution and neurochemical characterization of neurons in the rat ventrolateral medulla activated by glucoprivation

Hypoglycemia elicits physiological and behavioral responses which are mediated in part by neurons within the ventrolateral medulla (VLM). The present study describes the neurochemistry of neurons activated by glucoprivation (2-deoxy-D-glucose, 2DG), specifically those within regions containing the A1, caudal C1 (cC1) and rostral C1 (rC1) cell groups. 2DG induced c-Fos immunoreactivity throughout the VLM. Activated neurons expressing prepro-cocaine and amphetamine-regulated transcript (PPCART), neuropeptide Y (NPY), glutamic acid decarboxylase (GAD67) or prepro-enkephalin (PPE) mRNA and/or immunoreactivity (-ir) for tyrosine hydroxylase (TH) were identified. TH(+) neurons were recruited in a dose-dependent manner. At high doses of 2DG [400 mg/kg, (n = 6)], 76 ± 1.2 % of activated neurons were TH(+) representing 52 ± 1.3 % of the total TH population. Virtually all activated neurons in the A1 and cC1 regions but only 60 % in the rC1 region were TH(+). Within the A1 region, TH(+), TH(+)NPY(+) and TH(+)NPY(+)PPE(+) subpopulations were activated and likely regulate vasopressin, oxytocin, and corticotrophin releasing hormone (CRH) from the hypothalamus. Within the cC1 region, non-TH neurons, TH(+)NPY(+), TH(+)NPY(+)PPCART(+), and TH(+)NPY(+)PPE(+) subpopulations were activated, likely regulating autonomic hypothalamic neurons or CRH and thyrotropin releasing hormone secretion. Within the rC1 region, non-TH neurons (40 % of those activated) were predominantly PPE(+) and were recruited by higher 2DG doses. Of the TH(+) activated neurons in the rC1 region, many expressed PPCART and half expressed NPY. The activated spinally projecting population was almost entirely TH(+)PPCART(+) and is likely to regulate adrenaline and glucagon release. These data indicate that glucoprivation activates at least nine phenotypically distinct populations of neurons in the VLM.