Effects of nitric oxide and GABA interaction within ventrolateral medulla on cardiovascular responses during static muscle contraction

We hypothesized that nitric oxide (NO) has opposing roles in regulating cardiovascular responses within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla by modulating release of gamma-aminobutyric acid (GABA). We have measured GABA concentrations within the RVLM and CVLM during increases in mean arterial pressure (MAP) and heart rate (HR) following a 2-min tibial nerve stimulation-evoked static muscle contraction before and after microdialysis of the NO precursor, L-arginine (1.0 microM), for 30 min, and after the NO inhibitor, L-NMMA (1.0 microM), for 30 min. In eight anesthetized rats, muscle contraction significantly increased MAP, HR and GABA levels within the RVLM area (from 0.53+/-0.09 to 1.22+/-0.10 ng/10 microl). Following microdialysis of L-arginine, muscle contraction augmented GABA levels (from 0.45+/-0.07 to 2.18+/-0.09 ng/10 microl) and attenuated changes in MAP and HR. Subsequent application of L-NMMA significantly decreased GABA levels (from 0.47+/-0.08 to 0.22+/-0.07 ng/10 microl) but potentiated MAP and HR responses to a muscle contraction. In contrast, muscle contraction significantly increased MAP and HR but decreased GABA concentrations within the CVLM (from 1.20+/-0.20 to 0.78+/-0.17 ng/10 microl). Following microdialysis of L-arginine, muscle contraction significantly attenuated GABA levels (from 1.34+/-0.19 to 0.33+/-0.10 ng/10 microl) and augmented changes in MAP and HR in response to muscle contraction. A subsequent microdialysis of L-NMMA into the CVLM reversed the effects of L-arginine. These results demonstrate that NO within the RVLM and CVLM differentially modulates cardiovascular responses during static muscle contraction and that NO influences exercise-induced cardiovascular responses by modulating GABA release within the ventrolateral medulla.     

Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion

We hypothesized that static skeletal muscle contraction-induced systemic cardiovascular responses, and central glutamate/GABA release in rostral (RVLM) and caudal ventrolateral medulla (CVLM), would be modulated by cerebral ischemia. In sham-operated rats, a 2-min tibial nerve stimulation induced static contraction of the triceps surae, evoked pressor responses, increased glutamate in both the RVLM and CVLM, decreased GABA in the CVLM, and increased GABA in the RVLM. In rats with a temporary 90-min left middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, pressor responses during muscle contractions were attenuated, as were glutamate within the left RVLM and left CVLM. Glutamate within the right RVLM and right CVLM were unaltered and similar to those in sham rats. In contrast, GABA increases during muscle contractions were enhanced in the left RVLM and CVLM but changes within the right CVLM and RVLM were similar to those in sham rats. These results indicate that unilateral ischemia increases ipsilateral GABA/glutamate ratios during muscle contraction in the RVLM. In contrast, opposite changes in ipsilateral glutamate and GABA release within the RVLM and CVLM were observed following a 90-min right-sided MCAO followed by 24 h reperfusion. However, cardiovascular responses during muscle contraction were depressed following such an ischemic brain injury. These data suggest that transient ischemic brain injury attenuates cardiovascular responses to static exercise via modulating neurotransmission within the ventrolateral medulla.     

Simultaneous glutamate and γ-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats

The purpose of this study was to determine if baroreflex modulates cardiovascular responses and neurotransmitter release within rostral (RVLM) and caudal (CVLM) ventrolateral medulla during static contraction of skeletal muscle using anesthetized rats. We evoked cardiovascular responses by a static muscle contraction and measured simultaneous release of glutamate and γ-aminobutyric acid (GABA) in both the RVLM and CVLM using microdialysis probes, two inserted bilaterally into the RVLM and two into the CVLM. In intact anesthetized rats, a muscle contraction increased release of glutamate concomitantly in both the RVLM and CVLM along with significant increases in heart rate and arterial blood pressure. In contrast, concentrations of GABA increased within the RVLM, but decreased significantly within the CVLM during the pressor response. These changes were due to contraction-evoked activation of muscle afferents since tibial nerve stimulation following muscle paralysis failed to evoke glutamate, GABA, or any cardiovascular changes. On the other hand, static muscle contractions in baroreceptor denervated rats augmented the increases in heart rate and blood pressure. Furthermore, muscle contraction significantly enhanced the release of glutamate in the RVLM but attenuated its release in the CVLM. In addition, concentrations of GABA within the RVLM were attenuated following a muscle contraction in denervated rats without any changes in GABA within the CVLM. These results demonstrate that the baroreceptors influence cardiovascular responses to static muscle contraction associated with dynamic changes in glutamate and GABA release within the RVLM and CVLM.     

Cardiovascular responses to muscle contraction following microdialysis of nitric oxide precursor into ventrolateral medulla

We determined the effects of administering L-arginine, a precursor for the synthesis of nitric oxide, and L-NMMA (NG-monomethyl-L-arginine), a nitric oxide synthase blocker, into the rostral (RVLM) and caudal (CVLM) ventrolateral medulla on cardiovascular responses elicited during static contraction of the triceps surae muscle. Two microdialysis probes were inserted bilaterally into the RVLM or CVLM of anesthetized Sprague-Dawley rats using stereotaxic guides. For RVLM experiments, static muscle contraction evoked by stimulation of the tibial nerve increased mean arterial pressure (MAP) and heart rate (HR) by 29+/-3 mmHg and 44+/-7 bpm, respectively (n=8). Microdialysis of L-arginine (1.0 microM) for 30 min attenuated the contraction-evoked increases in MAP and HR. After discontinuing L-arginine, L-NMMA (1.0 microM) was microdialyzed into the RVLM for an additional 30 min followed by a muscle contraction. This contraction augmented the pressor response (37+/-4 mmHg) and HR (61+/-11 bpm) with respect to control values. For CVLM experiments, muscle contraction increased MAP and HR by 23+/-3 mmHg and 25+/-5 bpm, respectively (n=9). Microdialysis of L-arginine (1.0 microM) for 30 min potentiated the contraction-evoked increases in MAP and HR. Subsequent administration of L-NMMA (1.0 microM) into the CVLM for an additional 30 min blocked the augmented MAP and HR responses. Developed tensions did not alter during contractions throughout both RVLM and CVLM protocols. These results suggest that nitric oxide, within the RVLM and CVLM, plays an opposing role in modulating cardiovascular responses during static muscle contraction.     

Glutamate neurotransmission and nitric oxide interaction within the ventrolateral medulla during cardiovascular responses to muscle contraction

We previously reported that nitric oxide, within the RVLM and CVLM, plays an opposing role in modulating cardiovascular responses during static muscle contraction [B.J. Freda, R.S. Gaitonde, R. Lillaney, A. Ally, Cardiovascular responses to muscle contraction following microdialysis of nitric oxide precursor into ventrolateral medulla, Brain Res. 828 (1999) 60-67]. In this study, we determined whether the effects of administering L-arginine, a precursor for the synthesis of nitric oxide, and N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor, into the rostral (RVLM) and caudal (CVLM) ventrolateral medulla on cardiovascular responses elicited during static muscle contraction were mediated via an alteration of localized glutamate concentrations using microdialysis techniques. In experiments within the RVLM (n=8), muscle contraction increased MAP and HR by 21+/-2 mmHg and 22+/-3 bpm, respectively. Glutamate increased from 1.1+/-0.4 to 4.4+/- 0.6 ng/5 microl measured from bilateral RVLM areas. Microdialysis of L-arginine (1.0 microM) for 30 min attenuated the contraction-evoked increases in MAP, HR, and glutamate levels. After subsequent microdialysis of L-NMMA (1.0 microM) into the RVLM, contraction augmented the pressor and tachycardic responses and glutamate release. In experiments within CVLM (n=8), muscle contraction increased MAP and HR by 22+/-3 mmHg and 20+/-2 bpm, respectively. Glutamate increased from 0.8+/-0. 4 to 3.6+/-0.6 ng/5 microl measured from the CVLM. L-Arginine augmented the cardiovascular responses and glutamate release and L-NMMA attenuated all the effects. Results suggest that nitric oxide within the RVLM and CVLM plays opposing roles in modulating cardiovascular responses during static exercise via decreasing and increasing, respectively, extracellular glutamate levels.     

Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway

Experiments were done to test the hypothesis that caudal ventrolateral medulla (CVLM) neurons excited by activation of arterial baroreceptors and by stimulation of depressor sites in the nucleus tractus solitarii (NTS) project monosynaptically to the rostral ventrolateral medulla (RVLM). In urethan anaesthetized and artificially ventilated rats we recorded extracellular activity from 46 spontaneously firing units in the CVLM. Twenty of these units were excited by baroreceptor activation (1–3 μg phenylephrine i.v.) and of these 6 were excited (mean latency of9.8 ± 2.3ms) by single pulses (0.1 ms,30 ± 8.3 μA) delivered once per second to a depressor site in the ipsilateral NTS. These 6 units were also antidromically activated with a latency of4.1 ± 0.12ms by stimulation of a pressor region in the ipsilateral RVLM. These results provide evidence for the existence of an excitatory projection from the NTS to the CVLM which, in turn, projects monosynaptically to sympathoexcitatory neurons in the RVLM.     

Comparison of the pressor effects of angiotensin II and III in the rostral ventrolateral medulla

Microinjection of angiotensin II and III into the rostral ventrolateral medulla of anesthetized barodenervated rabbits elicited in both cases pressor responses, which were of similar magnitude and time course. The responses to angiotensin II and III were either unchanged or increased in the presence of compounds which inhibit their degradation to shorter length peptides. The results indicate that both angiotensin peptides are independently capable of eliciting pressor responses in the rostral ventrolteral medulla.     

Essential hypertension as a result of neurochemical changes at the rostral ventrolateral medulla

Acute ischemia of the brainstem has been known to produce hypertension. After an initial review of central nervous system mechanisms contributing to systemic hypertension and the impact of the rostral ventrolateral medulla (RVLM) on arterial pressure, the authors propose that essential hypertension involves neurochemical changes at the level of the RVLM which are triggered by cerebral ischemia. Experimental and clinical data are presented to show that there is a link between ischemia of the brainstem and chronic hypertension. Atherosclerosis of the cerebral circulation leads to ischemia of the RVLM and other regions with autonomic function. This ischemic process results in increased availability of angiotensin II in the RVLM, which maintains the chronic hypertensive state via either direct stimulation of the RVLM or exacerbation of brainstem ischemia due to increased vasoconstriction.     

Cardioacceleratory responses to hypocretin-1 injections into rostral ventromedial medulla

Intracisternal injections of hypocretin-1 (hcrt-1) have been shown to elicit sympathoexciatory responses. However, the location of central sites that may mediate these cardiovascular effects have not been clearly elucidated. This study was done in male Wistar rats to investigate the effects of microinjections of hcrt-1 into the rostral ventromedial medulla (RVMM) on mean arterial pressure (MAP), heart rate (HR) and the arterial baroreflex. An initial series of experiments was done to provide a detailed mapping of the location of hcrt-1- and hcrt-1 receptors (hcrtR-1)-like immunoreactivity (i.r.) in the RVMM region. Hcrt-1 and hcrtR-1 ir were found throughout the RVMM region, but primarily within the magnocellular reticular nucleus and the adjacent nucleus paragigantocellularis lateralis. In the second series, this region containing hcrt-1 and hcrtR-1 ir was explored for sites that elicited changes in MAP and HR in the anaesthetized rat. Microinjection of hcrt-1 (0.5-2.5 pmol) into the region of magnocellular reticular nucleus elicited a dose-dependent increase in HR, with little or no change in MAP. Administration (i.v.) of the muscarinic receptor antagonist atropine methyl bromide significantly attenuated ( approximately 62%) the HR response whereas, the total autonomic blockade abolished the HR response. Finally, unilateral or bilateral microinjection of hcrt-1 into the magnocellular reticular nucleus significantly attenuated the reflex bradycardia resulting from the activation of the baroreflex following the increase in MAP from an iv injection of phenylephrine. These data suggest that hcrt-1 in the RVMM region activates neuronal circuits that both inhibit vagal activity and increase sympathetic activity to the heart, and that it alters the excitability of central circuits that reflexly control the circulation.     

Activation of skeletal muscle afferents evokes release of glutamate in the subretrofacial nucleus (SRF) of cats

The subretrofacial nucleus (SRF) is a region of the rostral ventrolateral medulla known to play a crucial role in sympathoexcitation. SRF neurons send direct projections to the intermediolateral cell columns of the spinal cord where they form synaptic contact with preganglionic sympathetic motor neurons. Activation of this neural pathway increases sympathetic outflow to the heart and blood vessels affecting cardiac function and vasomotor tone. Previous studies utilizing electrophysiological recording techniques and c-Fos expression have established that the activity of SRF neurons is increased during skeletal muscle contraction. However, the excitatory neurotransmitter mediating this increased activity remains in question. In the present study, static contraction of the triceps surae was induced by electrical stimulation of L7 and S1 ventral roots in anesthetized cats (n=12). Endogenous release of glutamate (Glu) from the SRF was recovered by microdialysis and measured by HPLC. Static muscle contraction for 4 min increased mean arterial pressure (MAP) 38+/-4 mmHg from a control level of 102+/-12 mmHg (P< 0.05). During muscle contraction the extracellular concentration of Glu recovered from the SRF increased from 623+/-117 to 1078+/-187 nM (P<0.05). To determine the effect of muscle contraction on Glu release in the absence of synaptic input from other reflexogenic areas, contraction was repeated following acute sinoaortic denervation and vagotomy. Following this denervation, muscle contraction increased MAP 41+/- 4 mmHg (P < 0.05) and Glu concentration from 635+/-246 to 1106+/-389 nM (P < 0.05). Muscle paralysis prevented the increases in MAP and Glu concentration during ventral root stimulation. These results suggest that: (i) Glu is released in the SRF during activation of contraction-sensitive skeletal muscle afferent fibers in the cat; and (ii) synaptic input from other reflexogenic areas appears to be ineffective in modulating the release of Glu in the SRF during static muscle contraction.     

Differential responses of barosensitive neurons of rostral ventrolateral medulla to hypoxia in rats

We examined the responses to hypoxia of 48 spontaneously active barosensitive neurons of the rostral ventrolateral medulla (RVL) of anesthetized rats. Twenty-nine projected to the spinal cord while 19 did not. All spinal barosensitive neurons increased their discharges in advance of an elevation of arterial pressure in the presence or absence of arterial chemoreceptors. In contrast, 18/19 of the non-spinal barosensitive neurons were not excited by hypoxia. The results indicate that barosensitive RVL neurons consist of two populations differing in efferent pathway and responsivity to hypoxia and that the spinal barosensitive RVL neurons are functionally discrete and selectively sensitive to hypoxia.     

Depressor responses to L-proline microinjected into the rat ventrolateral medulla are mediated by ionotropic excitatory amino acid receptors

The essential amino acid L-proline produces a depressor response when microinjected into the caudal ventrolateral medulla (CVLM) of anesthetized rats. L-proline may activate some excitatory amino acid (EAA) receptors. The present study tested this hypothesis by investigating the effects of two ionotropic excitatory amino acid receptor antagonists on the depressor response to L-proline in the CVLM: the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the N-methyl-D-aspartate (NMDA) receptor-selective antagonist MK801. Urethane-anesthetized rats received arterial catheters and their ventrolateral medulla surface was exposed. Injections of the antagonists CNQX and MK801 (2 mM, 68 nl in each case) into the CVLM completely blocked depressor responses to subsequent administration of AMPA (2 pmol/34 nl) and NMDA (2 pmol/34 nl), respectively. The depressor response to L-proline (3.4 nmol/34 nl) was strongly inhibited by prior injection of CNQX (2 mM, 68 nl) and significantly attenuated by prior injection of a high dose (20 mM, 68 nl), but not a low dose (2 mM, 68 nl), of MK801. The results indicate that the depressor response to L-proline in the CVLM includes mechanisms of ionotropic excitatory amino acid receptors.     

The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus

We recently reported that a train of episodic neural discharges within the ventromedial nucleus of the hypothalamus (VMH) associated with suppression of the circulatory system had been determined by monitoring multiple unit activity (MUA). Abrupt increases in neural activity (MUA volleys; 1 to 4 min in duration) accompanied transient decreases in heart rate (HR) and blood pressure (BP), and showed circadian rhythm, occurring every 15 to 30 min in the light phase but seldom in the dark phase. The present study was aimed to determine if neurons in the vasomotor area of the rostral ventrolateral medulla (RVL) are involved in this VMH-induced cardiovascular suppression. MUAs of the VMH and RVL were monitored simultaneously with HR and BP in urethane-anesthetized rats. In synchrony with each MUA volley in the VMH, spontaneous activity of RVL neurons significantly decreased, as well as HR and BP. These RVL neurons are most likely vasomotor neurons because MUA of the RVL was attenuated by baroreceptor reflex activation, and electrical stimulation of these cells through the MUA recording electrodes produced pressor responses. These data suggest that VMH neurons that show a train of episodic discharges suppress the circulatory system at least in part by inhibiting the excitability of vasomotor neurons in the RVL.     

Anatomical substrates for baroreflex sympathoinhibition in the rat

The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS-->CVL-->RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks.     

Modulation of baroreflex function by angiotensin II endogenous to the caudal ventrolateral medulla

Neurons in the ventrolateral medulla (VLM) mainly determine the tonic sympathetic activity. The caudal VLM (CVLM) relays baroreflex signals to the rostral VLM. We have reported that endogenous angiotensin II (ANG II) contributes to the ongoing activity of the VLM neurons. In the present study, we examined if ANG II endogenous to the CVLM modulates the baroreflex function in anesthetized normotensive Sprague-Dawley rats. Changes in renal sympathetic nerve activity (RSNA) in response to changes in mean arterial pressure (MAP) induced by i.v. infusion of phenylephrine and nitroglycerin were recorded before and after bilateral microinjection of (Sar1, Thr8]-ANG II, an ANG II antagonist, into the CVLM. The ANG II antagonist injection into the CVLM significantly increased MAP and RSNA by 17.6 ± 8.0 mmHg (mean ± S.D.) and 36.3 ± 18.1%, respectively. It also significantly increased the baroreflex sensitivity (BS) from −0.49 ± 0.38 to −0.74 ± 0.37%/mmHg during nitroglycerin infusion. In contrast, the BS examined by phenylephrine infusion was not altered by the pretreatment with ANG II antagonist. Injection of artificial CSF affected neither the baseline values of MAP and RSNA nor the BS. These results suggest that ANG II endogenous to the CVLM exert a modulating role in baroreflex control of RSNA.     

Reciprocal connections between nucleus tractus solitarii and rostral ventrolateral medulla

Experiments were done to test the hypothesis that there are reciprocal connections between the nucleus of the tractus solitarius (NTS) and the rostral ventrolateral medulla (RVLM). Spontaneous activity was recorded from units in the right RVLM or NTS of urethan-anesthetized and artificially ventilated rats. Twenty-four of 42 RVLM and 12 of 21 NTS units were classified as cardiovascular because they were inhibited by baroreceptor activation and displayed a cardiac rhythm. Electrical stimulation of depressor sites in the NTS inhibited 14 and excited 10 RVLM units. Stimulation of pressor sites in the RVLM excited 10 and inhibited 2 NTS units. None of the units in the NTS or in the EVLM could be activated antidromically. These results provide evidence that there are reciprocal excitatory and inhibitory connections between NTS and RVLM and that these connections are not monosynaptic.     

Effects of clonidine on sympathoexcitatory neurons in the rostral ventrolateral medulla

The effects of intravenous and iontophoretic clonidine were determined on the firing rates of sympathoexcitatory neurons in the rostral ventrolateral medulla of the cat. As previously reported in the rat, we found that sympathoexcitatory neurons could be differentiated based on their sensitivity in clonidine. Approximately 50% of the neurons were inhibited by clonidine. There was only a weak correlation between the inhibition of unit activity and whole sympathetic nerve activity. The discharge rates of the remaining neurons were either not altered or were increased by clonidine. Unlike the rat, these two groups of neurons could not be further differentiated on the basis of axonal conduction velocity or discharge frequency. These data are discussed and the effects of clonidine and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on sympathoexcitatory neurons are compared.     

Spinal cord substance P mediates carbachol-induced cardiovascular responses from the rostral ventrolateral medulla

Intrathecal (i.t.) infusion of substance P (SP) antagonist, (d-Pro₂,d-Trp_7.9) P (20 μg), lowered blood pressure profoundly without any significant change of heart rate. The hypertensive and tachycardic responses elicited by microinjection of carbachol (25 ng/site) into the bilateral rostral ventrolateral medulla (rVLM) were blocked by i.t. infusion of SP antagonist. These data provided evidence that an excitatory cardiovascular effect induced by cholinergic system in rVLM may be mediated mainly by the SP receptors in spinal cord.     

Rapid effects of corticosterone on cardiovascular neurons in the rostral ventrolateral medulla of rats

The present study has explored possible fast actions of corticosteroid hormones on activity of cardiovascular neurons of the rostral ventrolateral medulla. Experiments were conducted in 60 urethane-anesthetized, artificially ventilated adult rats. Extracellular recordings of unitary firings were made from the RVLM with multi- or single-barreled microelectrodes. Barosensitive cardiovascular neurons were identified through activation of the baroreceptor reflex by electrical stimulation of the aortic nerve and by intravenous injection of phenylephrine. In 52 barosensitive cardiovascular neurons, iontophoretically applied corticosterone sulfate increased the ongoing activity of 30 (57.7%) neurons, the other 22 (42.3%) neurons being unaffected. In 16 bulbospinal pre-sympathetic neurons, iontophorized corticosterone increased the firing rate of 12 neurons. Intravenously applied corticosterone (0.2 mg) increased the firing rates of 5 out of 12 bulbospinal pre-sympathetic neurons. The average baseline activity of cardiovascular neurons sensitive to corticosterone was found to be significantly less than that of the cardiovascular neurons insensitive to corticosterone. In 64 non-cardiovascular neurons, the firing rate of 13 (20.3%) neurons increased, 23 (36.0%) decreased and 28 (43.7%) remained unchanged following local application of corticosterone. The changes in firing rates of RVLM neurons following application of corticosterone occurred rapidly and were dependent on the doses of the agent. RU-38486 was able to reduce or block the rapid effects of corticosterone on cardiovascular and non-cardiovascular neurons. The results demonstrated that corticosterone may fast, non-genomically, modulate the activity of central regulators of the cardiovascular system and suggested that fast non-genomic actions of corticosteroid hormones may be an important mechanism in the integration of the autonomic nervous and the cardiovascular systems during some conditions such as stress.