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Acetylcholine release in the rostral ventrolateral medulla of spontaneously hypertensive rats
Abstract
1. Central acetylcholine (ACh) has been implicated in the pathogenesis of experimental hypertension and the rostral ventrolateral medulla (RVLM) is an important area for cardiovascular regulation. The purpose of this study was to determine whether cholinergic neurotransmission in the RVLM is altered in spontaneously hypertensive rats (SHR).
2. Experiments were performed on male SHR (12–16 weeks) and age‐matched Wistar‐Kyoto rats (WKY). The rats anaesthetized with pentobarbital were placed in a stereotaxic apparatus. For determining the release of ACh in the RVLM, a dialysis probe was introduced into the RVLM.
3. An RVLM microinjection of cholinergic agents elicited an increase in blood pressure. The pressor response to physostigmine was greater in SHR than that of WKY whereas there was no difference in the pressor response to ACh or carbachol between SHR and WKY.
4. The release of ACh in the RVLM was greater in SHR than that of WKY. Physostigmine (0.5 mg/kg, i.p.) produced increases in medulla ACh contents. The increase in ACh content of the rostroventral medulla including the RVLM was greater in SHR than that of WKY whereas there was no difference in ACh contents of the other three parts of the medulla oblongata between SHR and WKY.
5. Depressor responses to scopolamine injected bilaterally into the RVLM were greater in SHR than those of WKY.
6. These results suggest that ACh release is enhanced specifically in the RVLM of SHR. It appears that this enhanced release of ACh in the RVLM of SHR contributes to the maintenance of hypertension in SHR.
... Although cardiovascular function in ChAT-/-mice has not been directly examined, several studies have shown ChAT upregulation in the rostral ventrolateral medulla (RVLM) in animal models of hypertension (Lin and Li, 1990) indicating that a compensatory upregulation of the cholinergic system can occur during hypertension. These studies also showed an increase in central ACh content in hypertensive rats and has been hypothesized to contribute to the maintenance of hypertension in these rodent models (Kubo et al., 1995). ...
... Although the mechanisms remain to be completely elucidated, alterations of neuronal membrane properties may underlie the exaggerated sympathetic outflow. Basal firing rates in central cardiovascular areas of SHR are elevated compared with normotensive rat strains (4,5,24), and activation of these areas by stress or direct stimulation elicits exaggerated pressor and sympathetic nerve responses in the SHR (4,16,21,42). Hyperexcitability has been observed in peripheral sympathetic neurons as a loss of spike accommodation (43) that is present in neonatal SHR neurons (22). Additionally, synaptic efficacy is enhanced in the sympathetic ganglion of SHR (27) via increased transmitter release from preganglionic nerve terminals (26), and catecholamine release is elevated at the SHR neuroeffector junction (41). ...
The A-type K+ current (IA) of superior cervical ganglion neurons acutely isolated from spontaneously hypertensive (SHR) and age-matched Wistar-Kyoto (WKY) rats was compared under whole cell voltage clamp. Activation parameters were similar in each strain. Steady-state inactivation was shifted approximately -6 mV in SHR, where one-half inactivation occurred at -81 mV vs. -75 mV in WKY rats. The shift was not present in prehypertensive SHR but remained in adult enalapril-treated SHR and, therefore, may represent a primary alteration of IA properties. IA amplitudes evoked from physiological potentials were similar, despite inactivation of a greater fraction of the current in the SHR. Comparing maximal IA densities revealed that current density is elevated in the SHR, which compensates for the inactivation shift. Current density decreased with age in WKY neurons but did not significantly decline in SHR neurons unless hypertension was prevented with enalapril. Thus adult SHR neurons may retain a high IA density as an adaptive response to offset potential hyperexcitability resulting from the hyperpolarized IA inactivation.
... The integrity of the AV3V region is essential for pressor responses by central cholinergic activation. In addition to the well-characterized roles of these forebrain structures, accumulating evidence showed that hindbrain sites (eg the nucleus tractus solitary (NTS) and the rostral ventrolateral medulla (RVLM)) are also involved in central cholinergically mediated cardiovascular responses (Gurtu et al, 1986;Kubo et al, 1995). Despite this information, it is noted that all those data are obtained from animals after birth. ...
Central cholinergic mechanisms play important roles in the control of cardiovascular responses. However, in utero development of brain cholinergic mechanism in regulation of arterial pressure before birth is largely unknown. This study investigated cardiovascular responses to central application of carbachol in fetuses and determined functional development of the central cholinergic systems controlling fetal pressor responses in utero. Chronically prepared near-term ovine fetuses (90% gestation) received an injection of carbachol intracerebroventricularly (i.c.v.). Fetal cardiovascular responses were measured, and the brains were used for c-fos mapping studies. In response to carbachol injection i.c.v., fetal systolic, diastolic, and mean arterial pressure (MAP) immediately increased, accompanied by a bradycardia. The maximum increase of MAP was at 30 min after the i.c.v. injection of carbachol and lasted 90 min. Associated with the pressor response, the neuronal activity marked with c-fos was enhanced significantly in the fetal anterior third ventricle (AV3V) region (including the median preoptic nucleus and organum vasculosum of the lamina terminalis) in the forebrain, and in the area postrema, lateral parabrachial nucleus, nucleus tractus solitary, and rostral ventrolateral medulla in the hindbrain. These results indicate that the central cholinergic mechanism is functional in the control of fetal blood pressure at the last third of gestation, and the central AV3V region and hindbrain have been intact relatively during in utero development in sheep at 90% gestational stage.
Moderate exercise reduces arterial pressure (AP) and heart rate (HR) in spontaneously hypertensive rats (SHR) and changes neurotransmission in medullary areas involved in cardiovascular regulation. We investigated if regularly swimming exercise (SW) affects the cardiovascular adjustments mediated by opioidergic neuromodulation in the RVLM in SHR and Wistar-Kyoto (WKY) rats. Rats were submitted to 6 wks of SW. The day after the last exercise bout, α-chloralose-anesthetized rats underwent a cannulation of the femoral artery for AP and HR recordings, and Doppler flow probes were placed around the lower abdominal aorta and superior mesenteric artery. Bilateral injection of endomorphin-2 (EM-2, 0.4 mmol/L, 60 nL) into the RVLM increased MAP in SW-SHR (20±4 mmHg, N=6), which was lower than in sedentary (SED)-SHR (35±4 mmHg, N=6). The increase in MAP in SW-SHR induced by EM-2 into the RVLM was similar in SED- and SW-WKY. Naloxone (0.5 mmol/L, 60 nL) injected into the RVLM evoked an enhanced hypotension in SW-SHR (-66±8 mmHg, N=6) compared to SED-SHR (-25±3 mmHg, N=6), which was similar in SED- and SW-WKY. No significant changes were observed in HR after EM-2 or naloxone injections into the RVLM. Changes in hindquarter and mesenteric conductances evoked by EM-2 or naloxone injections into the RVLM in SW- or SED-SHR were not different. Mu Opioid Receptor expression by Western blotting was reduced in SW-SHR than in SED-SHR and SW-WKY. Therefore, regularly SW alters the opioidergic neuromodulation in the RVLM in SHR and modifies the mu opioid receptor expression in this medullary area.
It is well established that normal aging is associated with progressive increases in efferent sympathetic nerve discharge (SND). Type II diabetes, obesity, heart failure, and hypertension are pathologies that have been attributed to both the processes of aging and sympathetic dysfunction, exemplifying the importance of understanding central regulation of SND during aging. However, the central mechanisms mediating altered SND with advancing age remain unclear. The rostral ventral lateral medulla (RVLM) is a brainstem region critically involved in setting the basal level of sympathetic outflow and cardiovascular function. Indeed, the RVLM is the only presympathetic region that when bilaterally inactivated results in profound reductions in both SND and arterial pressure. Glutamatergic influences in RVLM activity are powerfully inhibited by tonic GABAergic neural inputs originating from the caudal ventral lateral medulla (CVLM); effects that are mediated by GABAA receptors located on presympathetic neuronal cell bodies within the RVLM. In the present study we proposed that reductions in GABA[subscript A] receptor subunit gene expression may reflect withdrawal of GABAergic tone in the RVLM thereby contributing to the basal sympathetic activation that occurs with advancing age. Therefore, the objective of the current study was to identify age-related changes in the constitutive expression of genes related to GABAergic and muscarinic, nicotinic and dopaminergic receptor systems due to their reported involvement in modulating GABA[subscript A] receptor function, in the RVLM of adult young (3-5 mo. old), middle-aged (12 mo. old), weight stable presenescent (24-25 mo. old) and senescent (>24 mo. old) Fischer 344 (F344) rats using a commercially available real-time PCR array. Real-time analysis revealed nonuniform and age-associated changes in the RVLM GABA, muscarinic, nicotinic and dopaminergic neurotransmitter gene expression profile between young and middle-aged F344 rats. Heterogeneous expression of genes related to these neurotransmitters was also observed between presenescent and senescent F344 rats. Our results suggest that potential changes in neurotransmitter synthesis and degradation, uptake, transport, signaling and receptor subunit composition may account for the sympathoexcitatory state that is commonly observed in the aged. Doctor of Philosophy Doctoral Department of Anatomy and Physiology Michael J. Kenney
We demonstrated previously that central muscarinic cholinergic receptor (mAChR) activation increased splanchnic sympathetic nerve activity and sympathetic baroreflex function via activation of mAChR in the rostral ventrolateral medulla (RVLM), and we found that some RVLM bulbospinal neurons contain muscarinic M2R mRNA. Here, we examined the gene expression, cellular distribution and functional role of muscarinic receptors in the RVLM in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats.
Using the sensitive technique of quantitative real time reverse transcriptase-PCR, M2R mRNA level was elevated two-fold (P<0.05) and M4R mRNA was downregulated two-fold (P<0.001), with all other receptors expressed at similar levels, in the rostral ventral medulla of SHR compared with WKY. Bulbospinal, but not catecholaminergic neurons, in the RVLM expressed M2R mRNA (M2RR), and similar numbers were found in the RVLM of SHR and WKY. Could elevated M2R within individual neurons or enhanced presynaptic activity reflects enhanced cholinergic effects in the RVLM? Activation of central mAChR using oxotremorine evoked a larger increase in mean arterial pressure in SHR compared with WKY (P<0.01); however, oxotremorine-induced increases in splanchnic sympathetic nerve activity, and sympathetic baroreflex function were similar in SHR and WKY.
These data indicate that enhanced pressor responses in SHR, following centrally mediated mAChR activation, are not associated with RVLM-mediated constriction of the splanchnic circulation or effects on the sympathetic baroreflex, but could reflect modified mAChR gene expression elsewhere. RVLM-dependent splanchnic sympathetic nerve activity effects, evoked by mAChR activation, are not mediated by the differential M2/M4 receptor mRNA levels identified in SHR compared with WKY.
The application of in vivo microdialysis to the study of acetylcholine (ACh) release has contributed greatly to our understanding of cholinergic brain systems. This article reviews standard experimental procedures for dialysis probe selection and implantation, perfusion parameters, neurochemical detection, and data analysis as they relate to microdialysis assessments of cholinergic function. Particular attention is focused on the unique methodological considerations that arise when in vivo microdialysis is dedicated expressly to the recovery and measurement of ACh as opposed to other neurotransmitters. Limitations of the microdialysis technique are discussed, as well as methodological adaptations that may prove useful in overcoming these limitations. This is followed by an overview of recent studies in which the application of in vivo microdialysis has been used to characterize the basic pharmacology and physiology of cholinergic neurons. Finally, the usefulness of the microdialysis approach for testing hypotheses regarding the cholinergic systems' involvement in cognitive processes is examined. It can be concluded that, in addition to being a versatile and practical method for studying the neurochemistry of cholinergic brain systems, in vivo microdialysis represents a valuable tool in our efforts to better comprehend ACh's underlying role in a variety of behavioral processes.
We have studied the responses to electrical and chemical stimulation of the ventrolateral medulla in the chloralose-anesthetized, paralyzed, artificially ventilated rat. Locations of most active pressor responses were compared to regions containing neurons labeled immunocytochemically for phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing the synthesis of adrenaline. Elevations of arterial pressure (+81.6 +/- 2.5 mm Hg) and cardioacceleration (+73 +/- 13.6 bpm) were elicited with low current (5 times threshold of 9.5 +/- 1.1 microA) electrical stimulation in a region of rostral ventrolateral medullary reticular formation we have termed the nucleus reticularis rostroventrolateralis (RVL). Electrical stimulation of the RVL increased plasma catecholamines (16.8-fold for adrenaline, 5.3-fold for noradrenaline, and 1.9-fold for dopamine) and vasopressin (1.7-fold before spinal transection, 4.7-fold after). The location of the most active pressor region in the ventrolateral medulla corresponded closely with the location of C1 adrenaline-synthesizing (PNMT-containing) neurons. In addition, the location of the most active pressor region in the dorsomedial medulla corresponded with the location of a bundle of PNMT-containing axons. Unilateral injections into the RVL of the excitatory amino acid monosodium L-glutamate (50 pmol to 10 nmol), but not saline, caused transient dose-dependent and topographically specific elevations (maximum +71.6 +/- 4.9 mm Hg) of arterial blood pressure and tachycardia. Injections of the rigid structural analogue of glutamate, kainic acid, caused large, prolonged (at least 15 min) pressor responses and tachycardia. Unilateral injections of the inhibitory amino acid gamma-aminobutyric acid (GABA) into the RVL caused transient dose-dependent hypotension (maximum -40.8 +/- 6.6 mm Hg) and bradycardia, whereas the specific GABA antagonist bicuculline caused prolonged (10 to 20 min) elevations (+64.2 +/- 6.8 mm Hg) of arterial pressure and tachycardia. By contrast, injections of the glycine antagonist strychnine had no significant effect. Bilateral injections of the neurotoxin, tetrodotoxin, dropped arterial pressure to low levels (51.7 +/- 4.7) not changed by subsequent spinal cord transection at the first cervical segment (52.5 +/- 6.2). We propose the following. (1) Neurons within the RVL, most probably C1 adrenaline-synthesizing neurons, exert an excitatory influence on sympathetic vasomotor fibers, the adrenal medulla, and the posterior pituitary. (2) These neurons are tonically active and under tonic inhibitory control, in part via GABAergic mechanisms--perhaps via the nucleus of the solitary tract (NTS).(ABSTRACT TRUNCATED AT 400 WORDS)
Intravenous injection of physostigmine evoked a pressor response in unanaesthetized rats. Spontaneously hypertensive rats (SHR) showed increased pressor responses, but the responses were within normal limits in renal hypertensive and DOCA-saline hypertensive rats. The pressor effect in SHR was abolished by the i.v. injection of atropine sulphate but not by the i.v. injection of atropine methylbromide. After inhibition of the peripheral muscarinic receptors by atropine methyl bromide, oxotremorine also produced a pressor response in unanaesthetized rats. In contrast to the pressor effect of physostigmine, there was no difference between the oxotremorine-induced pressor response of SHR and that of normotensive Wistar-Kyoto rats. The pressor effect of oxotremorine in SHR was blocked by the i.v. injection of atropine sulphate.
This study aimed to demonstrate the role of acetylcholine receptors in the rostral ventrolateral medulla (RVL) in the central regulation of the cardiovascular system in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The effects of cholinergic drugs, microinjected into the rostral ventrolateral medullary vasopressor area, on blood pressure and heart rate in anesthetized and artificially ventilated rats were investigated. Unilateral microinjection of carbachol (1 nmol/site), physostigmine (300 pmol/site) or 3,4-diaminopyridine (500 pmol/site) into the RVL elicited a pressor and tachycardiac response, of which only the pressor response was significantly greater in SHR than in WKY. Bilateral microinjection of atropine (1 nmol/site) caused a depressor and bradycardiac response. The depressor response produced by atropine injected in the RVL was also significantly greater in SHR than in WKY. These results suggest that there are tonic cholinergic mechanisms in the RVL of the rats, which exert an excitatory cardiovascular action, and that the enhanced responsiveness to acetylcholine receptor stimulation in the RVL may contribute to the sustained elevation of blood pressure in the SHR.
In several models of essential hypertension in the rat, the pressor response to central cholinergic stimulation is enhanced with respect to age-matched normotensive controls. Neurochemical evidence is available from previous studies to suggest that both pre- (transmitter synthesis and release) and post-synaptic (muscarinic receptors) components of cholinergic transmission may be enhanced in hypertensive rats and that such alterations might be responsible for the exaggerated pressor response to centrally-acting cholinergic agonists. The present study, employing pharmacological approaches, was designed to determine whether pre- or post-synaptic components of central cholinergic transmission were more important in this regard. The pressor response to intravenous injection of the indirect-acting agonist, physostigmine, but not to that of the direct-acting agonist, arecoline, was significantly reduced by pretreatment with hemicholinium-3 (to deplete acetylcholine in brain). The pressor response to physostigmine, but not to arecoline, was enhanced in adult, spontaneously-hypertensive rats, with respect to their normotensive controls. The pressor response to oxotremorine was partially inhibited by pretreatment with hemicholinium-3, but was only partially effective at inducing an exaggerated pressor response in spontaneously hypertensive rats. These results are consistent with the hypothesis that heightened cholinergic activity in spontaneously hypertensive rats is derived primarily through altered pre-synaptic mechanisms, and that the actions of oxotremorine may involve a multi-synaptic cholinergic pathway.
We studied the responses to chemical stimulation of the ventrolateral medulla in pentobarbital-anaesthetized, paralyzed, normotensive and spontaneously hypertensive (SHR) rats. When unilaterally injected into a circumscribed region of the rostral ventrolateral medulla, L-glutamate (0.16-1.6 nmol) elicited a dose-dependent increase in arterial pressure and heart rate. Bilateral microinjections of L-glutamate diethylester (63 nmol), an antagonist of excitatory amino acids, into the glutamate-sensitive sites markedly reduced arterial pressure and heart rate. gamma-Aminobutyric acid (GABA) (0.3-3 nmol) injected into the glutamate-sensitive sites caused a dose-dependent decrease in arterial pressure and heart rate. The depressor response to GABA was smaller in SHR than that in normotensive Wistar Kyoto rats (WKY), while there were no differences between WKY and SHR in the pressor response to L-glutamate. Thus, a depressor function involving the ventrolateral medulla appears to be diminished in SHR.
This investigation was designed to demonstrate the presence of cholinergic nerve terminals in the pressor area of the ventrolateral medulla (VLPA) and to study the effects of the release of endogenous acetylcholine in this area. Bilateral microinjections (0.1-2 nmol)/site) of 3,4-diaminopyridine (DAP), which releases acetylcholine from cholinergic nerve terminals, into the VLPA in anesthetized rats evoked an increase in blood pressure and heart rate which lasted for 20-40 min. Intravenous injections of the same doses of this agent failed to evoke a response. The ganglion blocker, chlorisondamine (3 mg/kg, i.v.) abolished the responses to microinjections of DAP indicating that the responses were mediated by the sympathetic nervous system. Microinjections of scopolamine or a specific M2 muscarinic receptor antagonist (AFDX-116) into the VLPA prevented the pressor and tachycardic responses to subsequent microinjections of DAP at the same sites indicating that the responses were mediated via M2 receptors. Microinjections of hemicholinium (3 nmol/site; which impairs acetylcholine synthesis) attenuated the responses to the subsequent microinjections of DAP at the same sites. These results indicate that the substance released from the terminals in the VLPA may be predominantly acetylcholine which evokes pressor and tachycardic responses via M2 muscarinic receptors. The origin and physiological significance of these cholinergic terminals in the VLPA are not known.
The cardiovascular effects associated with the microinjection (100 nl) of carbachol, physostigmine and atropine into the pressor area in the ventrolateral medulla (VLPA) were studied. In urethane anesthetized rats, VLPAs were functionally identified bilaterally by microinjection of the neuroexcitatory amino acid L-glutamate (300 ng/site). L-Glutamate microinjections into the VLPA cause a transient rise in blood pressure (BP) and heart rate (HR). The bilateral microinjection of carbachol into the VLPA caused a prolonged dose-related increase in BP and HR in the dose range of 0.8 to 400.0 ng/site. At higher doses (1-10 micrograms), carbachol caused a decrease in BP and HR; indicative of depolarization blockade. Acetylcholine esterase inhibition by physostigmine microinjections in the VLPA caused cardiovascular effects similar to those observed with carbachol. The hypertensive responses evoked by muscarinic receptor stimulation in the VLPA were mediated by increasing sympathetic outflow. The pathway by which cholinergic receptor stimulation in the VLPA activates vasomotor outflow appears to be entirely descending as coronal knife cuts at the level of the trapezoid body failed to alter the hypertensive responses. Pressor responses elicited by both physostigmine and carbachol were reversed completely by the i.v. administration of atropine sulfate (0.5-3 mg/kg i.v.). Muscarinic receptor blockade in the VLPA after atropine sulfate microinjection caused a dose-related (0.4-15.0 micrograms/site) fall in the BP and HR suggesting that cholinergic mechanisms in the VLPA are tonically active.
Intravenous injection of the cholinesterase inhibitor physostigimine evoked a hypertensive response in the unanesthetized 12- to 14-week-old spontaneously hypertensive rat (SHR). These responses were greatly enhanced in magnitude when compared to similar injections in normotensive Wistar-Kyoto (WKY) control rats. Stimulation of autonomic ganglia with dimethylphenylpiperazinium (DMPP) also evoked a pressor response in SHR; however, the magnitude of these responses was not different from those obtained in WKY. In unanesthetized, freely moving SHR, the intracerebroventricular (icv) injection of hemicholinium-3 (HC-3) to block the synthesis of brain acetylcholine (ACh) produced a marked hypotensive response. No reduction in arterial pressure was observed following similar injections of HC-3 in WKY. The decline in arterial pressure following icv HC-3 was correlated with the initial decline in brain ACh levels, particularly in the hypothalamus. These results indicate that enhanced sympathetic nerve activity and the resultant hypertensive state present in young SHR is dependent on increased central cholinergic activity.
Intracerebroventricular (icv) injection of hemicholinium-3 (HC-3) in doses of 10–20 μg causes a dose-related decrease in the blood pressure of conscious spontaneous hypertensive (SH) rats but not of normotensive rats. HC-3 also reduces heart rate (HR) in both SH and normotensive rats. The bradycardia was blocked by intravenous injection of methylatropine, implicating increased vagal activity as a cause of the response. The decrease in HR also was blocked by icv injection of atropine but not by icv injection of mecamylamine, suggesting that the bradycardia is mediated via central muscarinic receptors. In contrast, the fall in blood pressure in SH rats was not influenced by intravenous administration of methylatropine or by the icv injection of either atropine or mecamylamine.