Altered Cholinergic Mechanisms and Blood Pressure Regulation in the Rostral Ventrolateral Medulla of DOCA-Salt Hypertensive Rats

We examined whether cholinergic transmission in the rostral ventrolateral medulla (RVLM) of deoxycorticosterone acetate-salt hypertensive rats (DHR) is enhanced and the enhancement is involved in the maintenance of hypertension in DHR, and whether cholineacetyltransferase (ChAT) activities and ChAT mRNA expression are enhanced in neurons intrinsic to the RVLM of DHR. Rats were anesthetized, paralyzed, and artificially ventilated. Unilateral microinjection of cholinergic agents into the RVLM produced a pressor response. The pressor response to physostigmine was greater in DHR than in control rats, whereas the response to carbachol was the same in both sets of rats. Bilateral microinjection of scopolamine into the RVLM produced a decrease in blood pressure. The depressor response was greater in DHR than in control rats. The number of ChAT-activity-detected neurons in the RVLM was greater in DHR than in control rats. The number of ChAT mRNA-expressing neurons in the RVLM was also clearly greater in DHR than in control rats. These results demonstrate that cholinergic transmission in the RVLM is enhanced in DHR, and this enhancement may play a role in the maintenance of hypertension in DHR. It is probable that enhanced activity of cholinergic neurons intrinsic to the RVLM is at least in part, responsible for the enhanced cholinergic transmission in the RVLM of DHR.     

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.     

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.     

Neurons of the rostral para-ambiguual field have activity correlated to the 10-Hz rhythm in sympathetic nerve discharge of urethane-anesthetized cat

Using the techniques of time domain correlation (mid-signal spike-triggered averaging) and frequency domain correlation (neuron-to-nerve coherence), 24% (54) of a sample of 229 neurons of the rostral para-ambiguual field have shown to have activity correlated to the 8- to 13-Hz rhythm in the inferior cardiac sympathetic nerve discharge (SND) of urethane-anesthetized cat. This correlation existed in both baroreceptor-innervated and -denervated animals. Of the correlated neurons, 37% (20) were non-rhythmically firing and displayed flat autospectra, while 63% (34) fired rhythmically and contained well-defined peaks in their autospectra. The group firing rate of these neurons was 4.3±0.4 spikes/s, indicating that they are not pacemaker neurons for the 10-Hz SND rhythm. The group time of firing of these neurons to the next peak of the SND slow wave was 52±4 ms. Correlation of the activity of medullary neurons with the 8–13-Hz rhythm of the SND was previously claimed only for rostral ventrolateral medulla, caudal raphe, and rostral caudal ventrolateral medulla. This present finding suggests that this behavior may be more widely spread throughout the medulla.     

Morphology of rostral medullary neurons with intrinsic pacemaker activity in the rat

Neurons with regular ongoing activity attributable to intrinsic pacemaker properties were recorded in coronal tissue slices within the nucleus reticularis rostroventrolateralis of the rat medulla oblongata (RVL). The cells were injected with horseradish peroxidase or Lucifer yellow and their dendritic and proximal axonal characteristics were investigated (n = 15). These small-to-medium-sized neurons had a simple dendritic arborization (3-6 primary dendrites branching up to 3 times) apparently confined within the limits of nucleus RVL and with limited extension in the rostrocaudal direction. Their axons originated either from the cell body or from a primary dendrite and coursed in a dorsomedial direction without giving rise to local arborizations. It is concluded that RVL pacemaker neurons, presumed to represent a non-adrenergic class of sympathoexcitatory premotor neurons, exhibit characteristics reminiscent of the archetypal 'reticular core' neurons.     

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

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

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.     

Cholinergic mechanism and blood pressure regulation in the central nervous system

Cholinergic neurons in numerous brain regions have been implicated in blood pressure regulation. One of the most important brain regions where cholinergic neurons play a role in the pathogenesis of hypertension is the rostral ventrolateral medulla (RVL), an essential source of efferent sympathetic activity. Pharmacological and biochemical studies have revealed that acetylcholine release in the RVL is increased in experimental hypertension regardless of its etiology and that this enhanced release of acetylcholine leads to hypertension. The lateral parabrachial nucleus, another important hindbrain area involved in blood pressure regulation, is responsible for the enhanced release of acetylcholine in the RVL of hypertensive animals. Moreover, recent studies have demonstrated the involvement of the hypothalamic defence area, an area believed to be involved in the hypertension induced by chronic stress, in the release of acetylcholine in the RVL and also have demonstrated the existence of direct projections from the hypothalamic structures to the lateral parabrachial nucleus. More studies about mechanisms of the enhanced release of acetylcholine in the RVL of experimentally hypertensive animals will provide important information for central mechanisms of hypertension.     

Sympathoexcitatory neurons of rostral ventrolateral medulla exhibit pacemaker properties in the presence of a glutamate-receptor antagonist

Intracisternal (i.c.) administration of the glutamate-receptor antagonist kynurenate to halothane-anesthetized rats (paralyzed, ventilated) produced an initial hypertension associated with an increase in lumbar sympathetic nerve discharge. Kynurenate (i.c.) blocked or greatly reduced all sympathetic reflexes investigated (somatosympathetic 70% reduction; vagal pressor and depressor responses, 100%; hypothalamic mixed responses, 90%; baroreflex, 100%) and increased the firing rate of reticulospinal sympathoexcitatory cells of the rostral ventrolateral medulla (PGCL-SE neurons) by 33%. After i.c. kynurenate, these cells exhibited a rhythmic, non-bursting firing pattern which could be reset by spinal cord stimulation only when antidromic spikes were elicited. Cells with similar characteristics were recorded in the nucleus paragigantocellularis lateralis (PGCL) in an in vitro rat bulb preparation perfused through the basilar artery. Their 'pacemaker-like' discharge pattern was observed even in the absence of kynurenate and was reset by orthodromic activation. Cells with similar characteristics were also recorded within the PGCL in 500-microns coronal slices in vitro. At 37 degrees C their discharge rate was similar to that of PGCL-SE neurons recorded in vivo after i.c. kynurenate; it was also pacemaker-like and was insensitive to glutamate receptor blockade. It is suggested that the tonic discharge of PGCL-SE neurons is normally due to an intrinsic pacemaker activity which is modulated in vivo by a variety of synaptic inputs.     

Central Amino Acids Mediate Cardiovascular Response to Angiotensin II in the Rat

To elucidate the role of the rostral ventrolateral medulla (RVLM) in cardiovascular control through the release of central amino acid neurotransmitters, experiments were performed in Sprague–Dawley (normotensive) rats and spontaneously hypertensive rats (SHR) anesthetized with urethane by using microdialysis sampling from the RVLM for determination of amino acid neurotransmitters. The baseline release of the excitatory amino acid neurotransmitter, glutamate (GLU) from the RVLM in SHR was higher and those of the inhibitory amino acid neurotransmitters, glycine (GLY), taurine (TAU), and γ-aminobutyric acid (GABA), were lower than in normotensive rats. Microinjection of angiotensin II (ANG II) into the RVLM caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR), accompanied by increased release of GLU in the RVLM. In contrast, microinjection of the ANG II type 1 receptor (AT₁) antagonist CV 11974 into the RVLM reduced MAP and HR, accompanied by increased release of GLY, TAU and GABA. These changes in MAP and HR after administration of ANG II or AT₁ antagonist were partially blocked by the use of the corresponding antagonist of each amino acid neurotransmitter. Furthermore, these effects were more prominently seen in SHR than in normotensive rats. These results suggest that the release of amino acid neurotransmitters mediate the cardiovascular effects of the angiotensin system in the RVLM, which may be involved in the generation of hypertension in SHR.     

Electrical stimulation of the rostral ventrolateral medulla promotes wakefulness in rats

Objectives

Previous studies suggest that central sympathetic activity might carry information on wakefulness, so we tested the hypothesis that direct activation of the rostral ventrolateral medulla (RVLM), a well-studied sympathetic vasomotor center, promotes wakefulness.

Methods

A bipolar stimulating electrode was implanted in the right RVLM of Wistar-Kyoto rats or in a brainstem control site. Bioelectrical signals were recorded using a telemetry system. The experiment comprised a baseline session and a 6-h electrical stimulation session (50 μA, 50 Hz for 3 min every 20 min). Sleep–wake stages were defined by the electroencephalogram (EEG) and electromyogram (EMG) as active waking (AW), nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Autonomic function was assessed using cardiovascular variability analysis.

Results

During the RVLM stimulation session, AW time increased from 38.48 ± 5.82 to 99.91 ± 8.23 min compared with baseline (P < .001), while REM sleep was decreased from 110.10 ± 4.91 to 50.74 ± 13.01 min (P = .004). Analysis of the RVLM stimulation bouts delivered during NREM sleep showed a significantly higher probability of awakening; it also showed that the latency to arousal was significantly shorter than the latency for 10% blood pressure (BP) increase (1.50 ± 0.30 vs 7.42 ± 1.83 s; P = .009).

Conclusions

Our findings show that direct stimulation of the RVLM promotes wakefulness, suggesting that sleep disturbance may result from central sympathetic activation.     

Difference in distribution of glutamate-immunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: a double-labeling study

Glutamate immunoreactivity was found in 19% and 21% of the neurons of the central autonomic nuclei projecting into the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), respectively, using a double-labeling technique in combination with glutamate immunocytochemistry. Double-labeled neurons were distributed in 22 nuclei or subnuclei in the limbic system, hypothalamus, midbrain, pons and medulla. The average number of glutamate-immunoreactive neurons per thousand in SHR was significantly higher in the ipsilateral lateral parabrachial nucleus (P < 0.05) and Koelliker-Fuse nucleus (P < 0.01) than in WKY, while it was significantly lower in the ipsilateral medial subnucleus (P < 0.05) and the commissure subnucleus (P < 0.05) of the nucleus tractus solitarii in SHR than in WKY. The results indicate that: (1) glutamate-immunoreactive neurons (possibly glutamatergic) in many central autonomic nuclei project into the sympathetic vasomotor control neurons in the SRF; (2) the large population of glutamate-immunoreactive neurons in the lateral parabrachial nucleus and the Koelliker-Fuse nucleus of SHR is likely to increase excitatory inputs to the SRF vasomotor control neurons, while the smaller population of glutamate-immunoreactive neurons in the medial and commissure subnuclei of the nucleus tractus solitarii is likely to decrease excitatory inputs to the GABAergic neurons intrinsic to the SRF.     

Evidence of disynaptic projections from the rostral ventrolateral medulla to the thoracic spinal cord

Sympathetic outflow is regulated by a direct pathway of the rostral ventrolateral reticular formation (rvlm) to the thoracic spinal cord. For the first time, a dual retrograde/anterograde transport technique was used to demonstrate by light microscopy, potential disynaptic pathways from the rvlm to the thoracic spinal cord in the rat. An anterograde tracer, biotinylated dextran amine (BDA) was injected into the rvlm and a retrograde tracer, FluoroGold (FG) deposited into the upper thoracic spinal cord in the same animal. Rostral ventrolateral medullary efferents labeled with BDA were apposed to thoracic reticulospinal neurons labeled with FG in the ventrolateral tegmentum, ipsilateral and contralateral to the injection site in the rvlm. Suggestive evidence was obtained of synaptic interactions with neuronal somata and proximal dendrites. The results support the idea that the rvlm projects to the thoracic cord via disynaptic, intrareticular pathways paralleling the well established monosynaptic projection.     

Anatomical evidence that hypertension associated with the defence reaction in the cat is mediated by a direct projection from a restricted portion of the midbrain periaqueductal grey to the subretrofacial nucleus of the medulla

Wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injections were made at sites within a restricted portion of the midbrain periaqueductal grey region (PAG) of the cat at which microinjection of the excitant amino acid, d,l-homocysteic acid, elicits the strongest form of a defence reaction, including a hypertensive response. Among the revealed projections, significant anterograde labelling was found in a discrete region of the rostral ventrolateral medulla, the subretrofacial nucleus (SRF). In the cat, the SRF contains pressor neurones which project to the spinal preganglionic sympathetic outflow. The labelling was most marked ipsilaterally, although substantial contralateral labelling was also observed. To verify that the projection to the SRF originated from the restricted ‘defence region’ of the PAG, WGA-HRP or rhodamine-labelled microspheres were injected into physiologically-identified sites in the SRF. In all experiments, labelled neurones were found in the same restricted region of the PAG at which DLH injection evokes hypertension and behavioural signs of the defence reaction. The results are consistent with the hypothesis that a discrete cell group within the PAG mediates both somatic and autonomic components of the defence reaction and that the characteristic hypertensive response is mediated by a direct pathway from these PAG cells to pressor neurones in the SRF.     

Responses of cardiovascular neurons in the rostral ventrolateral medulla of the normotensive Wistar Kyoto and spontaneously hypertensive rats to iontophoretic application of angiotensin II

In female pentobarbital-anesthetized Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), changes in spontaneous discharges of cardiovascular neurons in the rostral ventrolateral medulla (RVL) in response to iontophoretic application of angiotensin II (Ang II) were studied and compared. It was found that iontophoretic application of Ang II to RVL increased the spontaneous neuronal activities of 30% of the cardiovascular neurons in both types of rats and that the increase was significantly greater in SHR than in WKY. In both types of rats, there was an increase in arterial blood pressure in response to iontophoretic release of Ang II to RVL. The pressor response was accompanied by tachycardia, which was significantly greater in SHR than in WKY. The present study provides evidence that Ang II acts directly on cardiovascular neurons in RVL, and in SHR, an enhanced sensitivity and responsiveness of the RVL cardiovascular neurons to Ang II may augment the sympathetic outflow from RVL and contribute to the genesis of hypertension.     

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.     

Obesity due to high fat diet decreases the sympathetic nervous and cardiovascular responses to intracerebroventricular leptin in rats

Obesity is associated with an increase in plasma leptin levels primarily derived from enhanced expression of the leptin gene in the adipose tissue. Leptin levels and expression are higher in females than males. The main functions of leptin are to decrease food intake and increase sympathetic nerve activity, especially in the brown adipose tissue. The high levels of leptin in obese, female rats suggest leptin resistance. In this article we describe experiments designed to investigate the effect of the intracerebroventricular (i.c.v.) administration of leptin on lumbar sympathetic nerve activity (LSNA) and cardiovascular parameters in female rats fed a low fat diet (control), a high fat diet (obese), or high fat diet followed by a period of food restrictions (reduced). The i.c.v. leptin administration increased LSNA in control rats, but decreased it in obese rats. In weight reduced animals the LSNA response to leptin returned to control levels. The i.c.v. leptin increased the mean arterial pressure in control and wt. reduced rats, but not in obese animals. The heart rate did not respond to leptin in any animal group. These results suggest that obesity decreases the central nervous system (CNS)-mediated lumbar sympathetic nervous and cardiovascular responses to leptin and that these responses recover following food restriction and wt. reduction. We conclude that obesity is associated with a decreased CNS response to leptin leading to a decrease in leptin effects to increase the activities of the autonomic nervous and cardiovascular systems.