Rat Rostral Ventrolateral Medulla Neurons Research Articles

PSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension

Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Fourthly, we find that there is basal level of activation of TH neurons in the lateral, medial and central regions (~70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (~80%) and medial (~90%) C1 neurons in the RVLM. Support or Funding Information This work was supported by the Australian Research Council (Discovery Early Career Researcher Award DE120100992), the National Health and Medical Research Council of Australia (Grants 1065485 and 1082215 and Fellowship 1024489), Sydney University, Macquarie University and the Heart Research Institute. TH staining, pTH staining and merged staining in rat brainstem.

PSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension

Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Finally, we find that there is basal level of activation of TH neurons in the lateral, central and medial regions (∼70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (∼80%) and medial (∼90%) C1 neurons in the RVLM.

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia by D'Agostino D, Mazza E, and Neubauer JA

the effect of hypoxia to stimulate breathing and the cardiovascular system has been well known for more than a century (for a review, see Ref. [11][1]). In 1938, Heymans was awarded the Nobel Prize for Physiology or Medicine for demonstrating the importance of carotid body chemoreceptors. However,

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia.

Heme oxygenase has been linked to the oxygen-sensing function of the carotid body, pulmonary vasculature, cerebral vasculature, and airway smooth muscle. We have shown previously that the cardiorespiratory regions of the rostral ventrolateral medulla are excited by local hypoxia and that heme oxygenase-2 (HO-2) is expressed in the hypoxia-chemosensitive regions of the rostral ventrolateral medulla (RVLM), the respiratory pre-Bötzinger complex, and C1 sympathoexcitatory region. To determine whether heme oxygenase is necessary for the hypoxic-excitation of dissociated RVLM neurons (P1) cultured on confluent medullary astrocytes (P5), we examined their electrophysiological responses to hypoxia (NaCN and low Po(2)) using the whole-cell perforated patch clamp technique before and after blocking heme oxygenase with tin protoporphyrin-IX (SnPP-IX). Following the electrophysiological recording, immunocytochemistry was performed on the recorded neuron to correlate the electrophysiological response to hypoxia with the expression of HO-2. We found that the responses to NaCN and hypoxia were similar. RVLM neurons responded to NaCN and low Po(2) with either depolarization or hyperpolarization and SnPP-IX blocked the depolarization response of hypoxia-excited neurons to both NaCN and low Po(2) but had no effect on the hyperpolarization response of hypoxia-depressed neurons. Consistent with this observation, HO-2 expression was present only in the hypoxia-excited neurons. We conclude that RVLM neurons are excited by hypoxia via a heme oxygenase-dependent mechanism.

Acute tolerance to ethanol inhibition of NMDA-induced responses in rat rostral ventrolateral medulla neurons.

The present study was performed to examine the effects of acute ethanol exposure on N-methyl-D-aspartate (NMDA)-induced responses and the development of acute tolerance in rat rostral ventrolateral medulla (RVLM) in vivo and in vitro. Repeated microinjections of NMDA (0.14 nmol) into the RVLM every 30 min caused reproducible increases in mean arterial pressure in urethane-anesthetized rats weighing 325-350 g. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA-induced pressor effects in a blood-concentration-dependent and reversible manner. The inhibitory effect of ethanol was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol (0.32 g). A high dose of ethanol (0.32 g) had no significant effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, gamma-aminobutyric acid and glycine-induced changes in blood pressure. In vitro studies showed that ethanol (10- 100 mM) dose-dependently inhibited inward currents elicited by pressure ejection of NMDA (10 mM) in RVLM neurons of neonatal brainstem slice preparations. When the superfusion time of ethanol (100 mM) was increased to 50 min, its inhibitory effect decreased gradually after 30-40 min in 60% of RVLM neurons examined. These data suggested that ethanol inhibition and subsequent tolerance development is associated with changed sensitivity to NMDA in the RVLM, which may play important roles in the ethanol regulation of cardiovascular function.

CGMP/protein kinase G-dependent potentiation of glutamatergic transmission induced by nitric oxide in immature rat rostral ventrolateral medulla neurons in vitro.

Although both nitric oxide (NO) and glutamate within the rostral ventrolateral medulla (RVLM) are important mediators of the central cardiovascular regulation, little is known about the functional interactions between these two mediators. Herein, we investigated the possible role of NO on the glutamatergic transmission of RVLM neurons. Whole-cell patch-clamp recordings were performed on visualized RVLM neurons in the brainstem slice preparation of rats. We found that bath application of l-arginine, the substrate for NO production, significantly increased the amplitude of excitatory postsynaptic currents (EPSCs). This enhancement was completely abolished by coadministration of the NO synthase inhibitor 7-nitroindazole and mimicked by the NO donors 3-morpholinylsydnoneimine and spermine NONOate. Bath application of a NO-sensitive guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or a protein kinase G (PKG) inhibitor, Rp-8-bromo-guanosine 3',5'-cyclic monophosphorothioate, fully prevented the l-arginine-, 3-morpholinylsydnoneimine-, and N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl]-1,3-propanediamin (spermine NONOate)-induced synaptic potentiation. Direct activation of PKG with 8-(4-chlorophenylthio)-cGMP mimicked the action of NO donors. Furthermore, the augmentation by spermine NONOate of EPSC was accompanied by a reduction of the paired-pulse facilitation and synaptic failure rate of EPSCs. Spermine NONOate also significantly increased the frequency of both spontaneous and miniature EPSCs without altering their amplitude distribution. Pretreatment with the N-type Ca2+ channel blocker omega-conotoxin GVIA selectively blocked the spermine NONOate-induced synaptic potentiation. These results suggest that NO acts presynaptically to elicit a synaptic potentiation on the RVLM neurons through an enhancement of presynaptic N-type Ca2+ channel activity leading to facilitating glutamate release. The presynaptic action of NO is mediated by a cGMP/PKG-coupled signaling pathway.

Pressor response to microinjection of orexin/hypocretin into rostral ventrolateral medulla of awake rats

Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 μM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. l-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.

Endomorphin-1 and endomorphin-2, endogenous ligands for the μ-opioid receptor, inhibit electrical activity of rat rostral ventrolateral medulla neurons in vitro

The classic opioid peptide, enkephalin, and the novel member of the opioid family, nociceptin/orphanin FQ, inhibit the spontaneous electrical activity of neurons recorded from the rostral ventrolateral medulla, presumably cardiovascular neurons. In this study, the putative effects of endomorphin-1 and endomorphin-2, the newly discovered endogenous ligands for the μ-opioid receptor, on the electrical activity of rostral ventrolateral medulla neurons were investigated in rat brain slices in vitro. Like enkephalin and nociceptin, perfusion of endomorphin-1 or endomorphin-2 profoundly inhibited spontaneous discharges of 43% and 38% of the medullary neurons, respectively. No excitatory response to perfusion of either endomorphin was found in all neurons surveyed. Both endomorphins produced concentration-dependent inhibition. However, endomorphin-1 was more potent than endomorphin-2 for production of the inhibition, as demonstrated by the greater and longer suppression induced by endomorphin-1 than that induced by endomorphin-2 at the same concentration. Among the four opioid agonists tested, ec 50 values (in nM) were 3.17 (endomorphin-1), 3.02 (nociceptin), 10.1 (endomorphin-2) and 150.0 (enkephalin). The non-selective opioid receptor antagonist, naloxone, blocked the inhibitory responses of the neurons to endomorphin-1, endomorphin-2 and enkephalin, but not to nociceptin. The selective μ antagonist, β-funaltrexamine, prevented the neuronal inhibition induced by endomorphins, but not by enkephalin and nociceptin. Neither naloxone nor β-funaltrexamine alone had a significant effect on the firing rate of the neurons. These results demonstrate that endomorphin-1 and, to a lesser extent, endomorphin-2 exert an inhibitory modulation of the electrical activity of rostral ventrolateral medulla neurons, which is mediated through the stimulation of μ-opioid receptors.

Catechol activation in rat rostral ventrolateral medulla after systemic isocapnic metabolic acidosis.

The catechol signal recorded using in vivo voltammetry within the rat rostral ventrolateral medulla (RVLM) can be interpreted as a catechol-specific index of the integrated activity of RVLM adrenergic barosensitive bulbospinal and nonbulbospinal neurons. To test the hypothesis that systemic acidosis leads to the activation of RVLM adrenergic neurons, the RVLM catechol signal was observed in rats after mild systemic acidosis (pH 7.20-7.25 for 30 min) induced by 1 M HCl under halothane anesthesia, controlled mechanical ventilation, and continuous infusion of Ringer lactate. Particular attention was paid to ensure that changes in mean arterial pressure (MAP) were <15 mmHg during HCl challenge. Saline administration was not associated with any significant change in all considered variables (n = 5). Mild isocapnic systemic acidosis was associated with an increase in catechol signal (n = 5), irrespective of carotid sinus nerve section (n = 5). In keeping with the aim of the study, there were minor (<15 mmHg) but significant changes in MAP among saline, intact, and deafferented groups. Changes in heart rate were not significant. In conclusion, a catechol activation is observed in the RVLM when arterial pressure is maintained during isocapnic systemic metabolic acidosis. This catechol activation appears primarily centrally mediated. Therefore, adrenergic RVLM neurons may relay inputs from the central respiratory generator to the sympathetic system and/or act as chemosensors for H+ next to the surface of the ventrolateral medulla.

Post- and presynaptic GABA B receptor activation in neonatal rat rostral ventrolateral medulla neurons in vitro

Whole-cell patch recordings were made from immature (six- to 12-day-old) rat rostral ventrolateral medulla neurons in brainstem slices. GABA or the specific GABA B receptor agonist (−)baclofen (10–50 μM) by superfusion or by pressure ejection induced an outward current or a hyperpolarization, which persisted in a tetrodotoxin (0.3 μM)-containing Krebs' solution in nearly every cell tested. The GABA B receptor antagonists 2-hydroxy saclofen (50–200 μM) and CGP 35348 (50–200 μM) dose-dependently suppressed baclofen-currents. Baclofen-currents were suppressed by barium (1 mM) but not by tetraethylammonium (20 mM), low Ca 2+ (0.24 mM) solution or in a solution containing the Ca 2+ chelator BAPTA-AM (10 μM). The outward current had an estimated reversal potential of −98, −77 and −52 mV in 3.1, 7 and 15 mM [K +] o. Pre-incubation of slices with pertussis toxin (500 μg/ml for 5–7 h) or intracellular dialysis with GDP- β-S (500 μM) markedly reduced baclofen-currents. Baclofen in low concentrations (1–3 μM) that caused slight or no change of holding currents and of inward or outward currents induced by exogenously applied glutamate or glycine/GABA, decreased excitatory and inhibitory postsynaptic currents by an average of 86.5±4.3% and 78.4±2.7%. The GABA B antagonist CGP 35348 (100 μM) increased the excitatory postsynaptic currents by an average of 64%, without causing a significant change in holding currents in 10/18 cells tested. Our results indicate the presence of post- and presynaptic GABA B receptors in the rostral ventrolateral medulla neurons. Activation of postsynaptic GABA B receptors induces an outward K + current which is barium-sensitive, Ca 2+-independent and may be coupled to a pertussis-sensitive G-protein. Activation of presynaptic GABA B receptors attenuates excitatory or inhibitory synaptic transmission. More importantly, the observation that CGP 35348 enhanced excitatory synaptic currents implies a removal of tonic activation of presynaptic GABA B receptors by endogenously released GABA (disinhibition), supporting the hypothesis that these receptors may have a physiological role in regulating the input and output ratio in a subset of rostral ventrolateral medulla neurons in vivo.

GABA- and glycine-mediated inhibitory postsynaptic potentials in neonatal rat rostral ventrolateral medulla neurons in vitro

Whole-cell patch recordings were made from rostral ventrolateral medulla neurons of two in vitro preparations: (i) brainstem–spinal cords of two- to five-day-old rats, and (ii) coronal brainstem slices of eight- to 12-day-old rats, and the inhibitory synaptic activities in these neurons have been studied. In brainstem–spinal cord preparations, Lucifer Yellow was diffused into the recording neurons at the end of experiments. Medullary neurons were characterized as: (i) spinally projecting by the appearance of an antidromic spike following electrical stimulation of the spinal tract between T2 and T3 segments, and (ii) adrenergic by the detection of phenylethanolamine- N-methyltransferase immunoreactivity in Lucifer Yellow-filled neurons. Of the 13 spinally projecting and phenylethanolamine- N-methyltransferase-positive medullary neurons, focal stimulation elicited in the presence of glutamate receptor antagonists an inhibitory postsynaptic potential in nine neurons. Inhibitory synaptic potentials were reversibly eliminated by the GABA A receptor antagonist bicuculline (10–20 μM) in six of nine neurons, by the glycine receptor antagonist strychnine (0.1–1 μM) in two and by a combination of bicuculline and strychnine in one neuron. In brainstem slice preparations, focal stimulation elicited three types of synaptic potential: (i) an excitatory postsynaptic potential, (ii) an inhibitory postsynaptic potential and (iii) a biphasic synaptic potential consisting of an excitatory synaptic potential followed by an inhibitory synaptic potential. Inhibitory synaptic potentials had a reversal potential between −70 and −80 mV, reversed their polarity in a low (6.7 mM) Cl −–Krebs' solution, and suppressed or blocked by either bicuculline or strychnine or both. Elimination of inhibitory synaptic potentials unmasked in some cells an excitatory synaptic potential or enhanced the excitatory synaptic potential component in medullary neurons with a biphasic response, indicating a marked convergence of excitatory and inhibitory inputs onto a single neuron. A population of medullary neurons appeared to be pacemaker neurons whereby they discharged spontaneously. When discharges were suppressed by membrane hyperpolarization, focal stimulation elicited inhibitory synaptic potentials in 8/23 neurons tested. Our results suggest that inhibitory synaptic potentials in medullary neurons are mediated by either GABA and/or glycine which open primarily Cl − channels. The prevalence of inhibitory synaptic potentials in medullary neurons indicates an essential role of inhibitory transmission in controlling the input and output ratio of these neurons.

Catechol changes in the rat rostral ventrolateral medulla following changes in systemic CO2.

A catechol signal recorded with in vivo voltammetry within the rat rostral ventrolateral medulla (RVLM) was taken as an index of the activity of RVLM adrenergic neurons and related to the level of arterial PCO2, under halothane anesthesia. Reversible increases in catechol signal were observed during reversible increases in arterial partial CO2 pressure (PaCO2) from 20 to 60 mmHg after alteration of tidal volume (n = 5 intact rats, n = 5 after carotid sinus deafferentation). A reversible increase in inspiratory CO2 combined with constant tidal volume led to changes in PaCO2 from 40 mmHg to 50 or 60 or 70 mmHg for 60 min (n = 5 in each group) and to a reversible increase in catechol signal (r = 0.76). These changes were also observed after carotid sinus deafferentation (PaCO2 = 40 to 60 to 40 mmHg, n = 5). Lowering the PaCO2 from 40 to 20 mmHg led to a minor, nonsignificant reduction in catechol signal (n = 5). Changes in arterial pressure were minimal, although they reached statistical significance in some groups of experiments. The level of catechol metabolism in the RVLM 1) is continuously related to the level of arterial CO2, 2) functions close to its resting level under baseline nonstimulated condition with respect to CO2, and 3) is reversibly modified on changes in capnia. Sensitivity of the catechol signal, recorded in the rostral ventrolateral medulla, to CO2 appears primarily to be centrally mediated. Thus adrenergic RVLM neurons may relay inputs from the central respiratory generator to the sympathetic chemoreflex or act as chemosensors for CO2, next to the ventrolateral medulla surface.

Neuronal inhibition by a GABAB receptor agonist in the rostral ventrolateral medulla of the rat

We recorded the effects of the gamma-aminobutyric acid class B (GABAB) receptor agonist baclofen on neuronal activity in the rat rostral ventrolateral medulla (RVLM) in tissue slices and in vivo. In vitro, baclofen (3 microM) produced hyperpolarization (13 of 17), decrease in input resistance (12 of 16), and reduction of spontaneous synaptic activity (7 of 14). Baclofen inhibited 84 of 87 spontaneously active neurons recorded extracellularly in vitro. Inhibition was concentration dependent (0.1-3 microM, maximum inhibition: 94 +/- 4%, n = 16) and persisted in low-Ca2+/high-Mg2+ medium (n = 19). The GABAB receptor antagonists CGP-54626A (1 microM, n = 19), CGP-55845A (1 microM, n = 15), and 2-hydroxysaclofen (0.5 mM, n = 3) attenuated inhibition by baclofen (1-3 microM) but not by muscimol or GABA. In vivo, iontophoresis of baclofen inhibited 31 of 32 RVLM neurons, including bulbospinal barosensitive (15 of 16) and respiratory ones (7 of 7). CGP-55845A attenuated baclofen inhibition (6 of 9). Bicuculline attenuated the effect of GABA but not that of baclofen (4 of 4). In summary, RVLM presympathetic neurons have somatodendritic GABAB receptors that may contribute to baclofen-induced hypotension in humans.

Neuronal excitation by angiotensin II in the rostral ventrolateral medulla of the rat in vitro.

We examined the effects of angiotensin II (ANG II) on spontaneous unit activity in slices of the rat rostral ventrolateral medulla (RVLM), ANG II (1-3 microM) excited 61% of a population of slowly and irregularly firing RVLM neurons (predrug, 1.2 +/- 0.1 spikes/s; postdrug, 4.6 +/- 0.3 spikes/s; n = 52). ANG II had no effect on pacemaker-like rapidly firing neurons (predrug, 8.6 +/- 0.4 spikes/s; n = 33). The effect of ANG II on slowly firing cells was repeatable and was reduced 75% by 3 microM losartan (baseline, 1.7 +/- 0.4 spikes/s; ANG II, 5.3 +/- 0.7 spikes/s; ANG II+losartan, 2.4 +/- 0.6 spikes/s; n = 12). The ongoing activity of slowly firing neurons was unaffected by 0.5-1 mM kynurenic acid (an ionotropic excitatory amino acid receptor antagonist). Most ANG II-responsive neurons (10 of 11) were inhibited by the alpha 2-adrenergic receptor agonist UK-14,304, but pacemaker-like neurons were not. In conclusion, the RVLM contains neurons excited by AT1 receptor agonists. These neurons are distinct from the previously described pacemaker nonadrenergic presympathetic cells. They may be responsible for the pressor effects produced by injecting ANG II into the RVLM in vivo.

PNMT-containing neurons of the C1 cell group express c-fos in response to changes in baroreceptor input.

The immunocytochemical detection of Fos, the protein product of the immediate-early gene c-fos, was used as a marker for activated neurons to examine whether the C1 neurons in the rat rostral ventrolateral medulla (RVLM) respond to changes in baroreceptor afferent activity. After hydralazine-induced hypotension or sinoaortic denervation, two treatments that reduce baroreceptor afferent activity, numerous Fos-positive neurons were observed in the RVLM. The number of Fos-positive neurons in the RVLM was counted in brain stem sections from hydralazine-treated rats that had been previously injected with Fluorogold into the upper thoracic spinal cord to label spinally projecting RVLM neurons as well as stained for phenylethanolamine-N-methyltransferase (PNMT) as a marker of C1 neurons. The results indicate that approximately 80% of the C1 neurons expressed Fos in response to hydralazine injection; this was true of spinally projecting C1 neurons as well as those C1 neurons that were not labeled with Fluorogold. Furthermore, in hydralazine-treated rats, the majority of Fluorogold-labeled Fos-positive neurons contained PNMT. These results suggest that C1 neurons are sensitive to baroreceptor afferent input and support a role of these neurons in cardiovascular regulation.

Chemoreflexes from the heart and lungs.

Chemoreflexes from the heart and lungs.