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Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus
Brain Research Bulletin, Vol. 53, No. 3, pp. 275–282, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
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Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus T. Kubo,* Y. Hagiwara, D. Sekiya, S. Chiba and R. Fukumori Department of Pharmacology, Showa Pharmaceutical University, Tokyo, Japan [Received 28 June 2000; Accepted 3 July 2000] ABSTRACT: The rostral ventrolateral medulla (RVLM) has cholinergic mechanisms responsible for pressor responses. Stimulation of the hypothalamic paraventricular nucleus (PVN) causes an increase of arterial pressure via activation of neurons in the RVLM. In this study, we examined whether PVN stimulation causes a pressor response via activation of cholinergic mechanisms in the RVLM. Male Wistar rats were used and they were anesthetized, paralyzed and artificially ventilated. Electrical stimulation of the PVN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine and the cholinesterase inhibitor physostigmine into the RVLM inhibited and potentiated, respectively, the pressor response induced by PVN stimulation. PVN stimulation also increased the firing rate of RVLM barosensitive neurons and the increase in the firing rate was inhibited and potentiated by scopolamine and physostigmine, respectively, iontophoretically applied on neurons. Microinjection of L-glutamate into the PVN produced a release of ACh in the RVLM. The inhibitory amino acid ␥-aminobutyric acid injected into the lateral parabrachial nucleus (LPBN) inhibited the pressor response induced by PVN stimulation. These results suggest that PVN stimulation causes an increase in arterial pressure via activation of cholinergic inputs in the RVLM. It appears that the pressor response is mediated, at least in part, via cholinergic inputs from the LPBN. © 2000 Elsevier Science Inc.
in the RVLM would thus improve our understanding of central mechanisms of hypertension. Electrical and chemical stimulation of the lateral parabrachial nucleus (LPBN) produces a pressor response via activation of RVLM sympathoexcitatory neurons [2,25,39]. Electrical stimulation of the central gray (CG) also increases blood pressure through activation of RVLM sympathoexcitatory neurons [6,38]. Previously we demonstrated that microinjections of the muscarinic receptor antagonist scopolamine into the RVLM inhibited the pressor response induced by LPBN or CG stimulation, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. LPBN or CG injection of glutamate produced a release of acetylcholine in the RVLM [17,18]. These findings suggest that pressor responses induced by LPBN or CG stimulation are mediated via activation of cholinergic mechanisms in the RVLM. The hypothalamus also plays an important role in blood pressure regulation [1,22,27,35,42]. Neurons in the paraventricular nucleus of the hypothalamus (PVN) also project to numerous structures involved in cardiovascular regulation including the RVLM [34]. Electrical and chemical stimulation of the PVN causes pressor responses and produces expression of c-fos protein in the RVLM of rats [15], suggesting that PVN stimulation leads to activation of neurons in the RVLM. Thus, it is possible that the pressor response elicited by PVN stimulation is also mediated via activation of cholinergic mechanisms in the RVLM. The purpose of the present study was to determine whether stimulation of the hypothalamic PVN causes an increase in arterial pressure via activation of cholinergic mechanisms in the RVLM.
KEY WORDS: Acetylcholine, Paraventricular nucleus, Rostral ventrolateral medulla, Pressor response, Scopolamine.
INTRODUCTION The rostral ventrolateral medulla (RVLM) is an area of crucial importance in regulation of blood pressure [9,20,28,40]. Neurons in the RVLM project to the intermediolateral cell column of the spinal cord, the main origin of the spinal sympathetic outflow [3,5,7]. The RVLM has an endogenous source of acetylcholine (ACh) responsible for pressor responses [4,11,12,21,23,26,31,32, 41]. The receptor involved is mainly the muscarinic receptor M2 subtype [4,16,21]. In addition, it has been demonstrated that RVLM cholinergic mechanisms are enhanced in spontaneously hypertensive rats (SHR), a rat model of genetic hypertension and that this enhanced RVLM cholinergic activity is related to the maintenance of hypertension in this strain [16,19]. Elucidation of cholinergic mechanisms responsible for blood pressure regulation
MATERIALS AND METHODS Male Wistar rats (300 –380 g) were used in this study. They were kept under alternative 12-h periods of dark and light and given standard rat chow and tap water ad libitum. Animals were anesthetized with urethane 1.2 g/kg, intraperitoneally. The femoral artery and vein were cannulated. The rats were paralyzed with D-tubocurarine (1 mg/kg, intramuscularly) and ventilated artificially with a respirator, as described [18]. All procedures were done in accordance with the guidelines outlined by the Institutional Animal Care and Use Committee of the Showa Pharmaceutical University.
* Address for correspondence: Dr. Takao Kubo, Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan. Fax: 042-721-1588.
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FIG. 1. Effects of unilateral rostral ventrolateral medulla (RVLM) microinjection of scopolamine 0.088 nmol (A), 0.26 nmol (B), physostigmine 0.092 nmol (C) and 0.31 nmol (D) on the blood pressure (BP) response induced by electrical stimulation of the ipsilateral hypothalamic paraventriculat nucleus (PVN). (E) Effects of unilateral lateral parabrachial nucleus microinjection of ␥-aminobutyric acid (GABA) 3 nmol on the BP response induced by electrical stimulation of the ipsilateral PVN. ●, PVN electrical stimulation; Œ, RVLM or LPBN microinjection.
Microinjection Experiments The rats were placed in a stereotaxic apparatus with the head fixed at 15°. Microinjections were made using glass micropipettes as described [18]. All drugs were dissolved in phosphate-buffered saline (pH 7.4) and given in a vol. of 50 nl in 2 s. Physostigmine or scopolamine was microinjected into the RVLM as described [18]. The PVN was stimulated electrially or chemically by L-glutamate. Electrical stimulation or glutamate injection at the PVN was made at the coordinates: 2.0 –2.2 mm caudal to the bregma, 0.5 mm lateral to the midline and 7.2–7.5 mm below the dorsal surface of the brain. The PVN was electrically stimulated with a bipolar concentric electrode. Stimulation was performed with rectangular pulses (pulse duration of 300 s) at a frequency of 25 Hz and current intensities of 100 –250 A for 3 s. In some experiments, ␥-aminobutyric acid (GABA) was microinjected into the LPBN. Injections into the LPBN were made at the coordinates: 9.3 mm caudal to the bregma, 2.0 mm lateral to the midline and 5.0 mm below the dorsal surface of the cerebellum. At the end of experiments, the electrical stimulation site was
marked by the passage of 250 A DC current for 15 s. The injection site was marked by injecting 50 nl of a concentrated solution of Pontamine sky blue. The brains were cut and stained with Cresyl violet. The electrical stimulation sites and the injection sites were identified by visual inspection. Microiontophoretic Experiments Microiontophoretic experiments utilized four-barrel glass microelectrodes containing monofilament glass fibers [18]. One of the barrels was filled with 2 M sodium acetate and 5% Pontamine sky blue, and was used to record extracellular unit activity. Another barrel was filled with 2 M NaCl (pH 6) and used for automatic current balancing. Remaining barrels were filled with scopolamine hydrobromide (50 mM), physostigmine sulfate (20 mM) or L-glutamate monosodium (0.2 M). Extracellular recording was performed through the electrode, as described [18]. Briefly, electrical activity was displayed on a medical oscilloscope and filtered (band pass 0.1–10 kHz). A signal processor (Model 7T08; Nihondenki-San-ei Instrument, Tokyo, Japan) was used for compiling the data in the form of pulse density variation
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FIG. 2. Effects of unilateral rostral ventrolateral medulla microinjection of scopolamine (A) and physostigmine (B) on maximal changes in mean blood pressure (⌬BP) induced by electrical stimulation of the ipsilateral hypothalamic paraventricular nucleus (PVN). (C) Effects of unilateral lateral parabrachial nucleus microinjection of ␥-aminobutyric acid (GABA) on maximal changes in mean blood pressure induced by electrical stimulation of the ipsilateral PVN. Values are mean ⫾ SEM from six to seven animals. *p ⬍ 0.05, compared to before treatment. Open column, before treatment; dotted column, after treatment.
histograms. The site of unit recording was stained by expelling the Pontamine sky blue from the electrode by the passage of 20 –50 A DC current. The brain was removed and the injection sites were identified. Blood pressure was increased using an inflatable cuff wrapped around the aorta close to the bifurcation of the coeliac artery. Microdialysis Experiments Microdialysis experiments were made as described [18]. Briefly, a microdialysis probe (0.22 mm in diameter and 1 mm in length, Eicom A-I-12-01) was stereotaxically inserted into the sites identified with L-glutamate in the RVLM. Ringer solution (NaCl: 147 mM, KCl: 4 mM and CaCl2: 3.4 mM) containing physostigmine (100 M) was perfused into the probe at a rate of 1.2 l/min. Perfusates were successively collected every 20 min from 60 min
after the start of perfusion. ACh in perfusates was measured by a high-performance liquid chromatography with an electrochemical detector [17]. For chemical stimulation of the PVN, L-glutamate (1.6 nmol) was microinjected every 2 min into the PVN during the third fraction and during the sixth fraction. Animals were treated with hexamethonium as described [18], in order to avoid changes in ACh release due to increases in blood pressure induced by PVN glutamate microinjection. Physostigmine sulfate, scopolamine hydrobromide (Wako Pure Chemicals, Osaka, Japan) and L-glutamic acid monosodium salt (Nakarai Chemicals, Kyoto, Japan) were used. The results are expressed as mean ⫾ SEM. All results were analyzed by either paired t-test or analysis of variance combined with the Welch’s t-test or Student’s t-test to determine the significance of individual
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FIG. 3. Schematic diagrams of the transverse section of rat brainstem, showing the sites where microinjections of L-glutamate into the rostral ventrolateral medulla (A) and into the lateral parabrachial nucleus (C) and electrical stimulation of the hypothalamic paraventricular nucleus (B) produced hypertension. Abbreviations: IO, inferior olive; LPBN, lateral parabrachial nucleus; PVN, hypothalamic paraventricular nucleus; RF, retrofacial nucleus; 3V, third ventricle.
FIG. 4. A coronal section of rat hypothalamic paraventricular nucleus (PVN) indicating the location of the stimulation site (arrow). Scale bar: 1 mm.
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FIG. 5. Rate histogram of rostral ventrolateral medulla neurons demonstrating the facilitatory effect of electrical stimulation (200 A, 25 Hz for 3 s) of the hypothalamic paraventricular nucleus (PVN) and their inhibition and potentiation during microiontophoretic application of scopolamine (30 nA) (A) and physostigmine (30 nA) (B), respectively, on neurons. Blood pressure (BP) responses to aortic constriction (AOc) and to PVN stimulation (●) were recorded.
paired comparisons. Differences between means were considered significant at p ⬍ 0.05. RESULTS Effects of RVLM Microinjection of Scopolamine and Physostigmine on Blood Pressure Responses Induced by Electrical Stimulation of the PVN The basal mean arterial pressure was 93 ⫾ 2 mmHg (n ⫽ 36). Unilateral electrical stimulation of the PVN produced a pressor response (Fig. 1). The pressor response was reproducible when the PVN was stimulated every 3–5 min. The pressor response was abolished by intravenous injection of hexamethonium (20 mg/kg) (n ⫽ 6), suggesting that the pressor response is mediated mainly by sympathetic excitation. The pressor response was dose-dependently inhibited by scopolamine (0.088 and 0.26 nmol) microinjected ipsilaterally into the RVLM (Figs. 1A,B and 2A). The pressor response was enhanced by physostigmine (0.092 and 0.31 nmol) injected ipsilaterally into the RVLM in a dose-dependent manner (Figs. 1C,D and 2B).
The dose 0.26 nmol of scopolamine injected into the RVLM was enough to block the pressor response to physostigmine (0.46 nmol) injected into the RVLM (data not shown). Postmortem histological examination confirmed that RVLM injection sites were located in an area containing the subretrofacial nucleus (Fig. 3A). PVN stimulation sites were located in an area of the PVN (Figs. 3B and 4). Effects of LPBN Microinjection of GABA on Blood Pressure Responses Induced by Electrical Stimulation of the PVN GABA (3 nmol) injected into the LPBN inhibited the pressor response induced by electrical stimulation of the PVN (Figs. 1E and 2C). GABA itself did not affect basal blood pressure. Effects of Iontophoretic Application of Scopolamine and Physostigmine on the Firing Response of RVLM Barosensitive Neurons Induced by Electrical Stimulation of the PVN Twelve neurons were inhibited during increases in blood pressure induced by aortic constriction (Fig. 5). Firing rates were
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KUBO ET AL. PVN every 2 min during the third fraction and during the sixth fraction. Blood pressure was not changed by glutamate injection in the hexamethonium-treated rats. DISCUSSION
FIG. 6. Effects of unilateral microinjections of L-glutamate into the hypothalamic paraventricular nucleus (PVN) on acetylcholine (ACh) release in the ipsilateral rostral ventrolateral medulla in hexamethonium (intravenous)-treated rats. L-Glutamate (1.6 nmol) or saline was microinjected into the PVN every 2 min during the third fraction and during the sixth fraction. The average of ACh release of the first and second fractions was considered as the basal release. Values are mean ⫾ SEM from seven animals. *p ⬍ 0.05, compared to saline.
18.5 ⫾ 3.1 spikes/s. Electrical PVN stimulation increased the firing rate in 11 of 12 barosensitive RVLM neurons (Fig. 5). The excitation of unit activity was inhibited by iontophoretic application of scopolamine (30 nA) onto neurons in all of 6 RVLM neurons tested (Fig. 5A). Scopolamine (30 nA) itself did not affect the basal neuronal activity in five neurons but slightly inhibited it in one neuron. The excitation of unit activity induced by PVN stimulation was potentiated by iontophoretic application of physostigmine (30 nA) onto neurons in all of five RVLM neurons tested (Fig. 5B). Physostigmine (30 nA) slightly increased the unit activity of RVLM neurons. On the other hand, microiontophoretic application of the noncholinergic agent L-glutamate (60 nA) onto RVLM neurons increased the firing rate of neurons, but scopolamine (30 nA) applied similarly did not affect the glutamate-induced excitation of unit activity in all cases tested (n ⫽ 6). ACh Release in the RVLM in Response to PVN Injection of LGlutamate Microinjection of L-glutamate into the PVN also produced an increase in blood pressure. We determined whether PVN injection of L-glutamate causes an increase of ACh release in the RVLM. The average ACh content of two consecutive samples beginning 60 min after the start of RVLM perfusion was 5.3 ⫾ 0.4 pmoles/20 min (n ⫽ 14) in anesthetized rats treated with hexamethonium. Release of ACh was gradually decreased over 140 min after the first fraction (Fig. 6). The release of ACh in the RVLM was increased by ipsilateral microinjection of L-glutamate into the
In the present study, electrical stimulation of the PVN of the rat caused a pressor response. The pressor response induced by PVN stimulation was inhibited by the muscarinic receptor antagonist scopolamine and potentiated by the cholinesterase inhibitor physostigmine, microinjected ipsilaterally into the RVLM. Furthermore, chemical stimulation of the PVN with L-glutamate caused an increase of ACh release in the RVLM. It has been demonstrated that ACh microinjection into the rat RVLM causes a pressor response via activation of muscarinic receptors [19,26,41]. Thus, the results of the present study suggest that PVN stimulation activates cholinergic mechanisms in the RVLM and that this cholinergic activation results in an increase in blood pressure. In the present study, we stimulated the PVN electrically or chemically by L-glutamate. Electrical stimulation excites both cell bodies and axons of passage, whereas glutamate excites only cell bodies but not axons of passage on the central nervous system [13]. Thus, the results of the present study suggest that neurons in the PVN may be responsible for activation of cholinergic mechanisms in the RVLM. PVN stimulation activated barosensitive neurons in the RVLM. The activation effect on RVLM neurons was inhibited and potentiated by iontophoretic application of scopolamine and physostigmine, respectively. The application of scopolamine (30 nA) did not affect the excitation of unit activity induced by iontophoretic application of L-glutamate, suggesting that scopolamine (30 nA) has no local anesthetic-like action. Thus, the results of the present study suggest that the PVN is, at least in part, involved in mediation of cholinergic inputs to RVLM barosensitive neurons. Choline acetyltransferase activity, an index of impulse flow in cholinergic neurons [10], and release of ACh in the RVLM area are increased in SHR, and that the enhanced ACh release in the RVLM is involved in the development and maintenance of hypertension in this strain [19]. Therefore, we can speculate that the PVN area may be involved in mediation of the enhanced cholinergic activity in the RVLM of SHR and thus involved in mechanisms of hypertension in this model. Indeed, it has been demonstrated that lesioning of the PVN attenuates the development and maintenance of hypertension in SHR [8]. In the present study, scopolamine (0.26 nmol) injected into the RVLM inhibited but did not abolish the pressor response induced by PVN stimulation, although the dose 0.26 nmol of scopolamine was enough to block the pressor response to physostigmine (0.46 nmol) injected into the RVLM. Corticotropin-releasing factor and angiotensins are also reported to be involved in excitatory synaptic inputs to pressor neurons in the RVLM arising from activation of the PVN [24,36]. In addition, it has been suggested that a portion of some pressor responses induced by PVN stimulation might be mediated by neural pathways that do not include a component within the RVLM [14]. Immunocytochemical evidence suggests that neurons in the PVN that contain oxytocin and vasopressin project to and may influence the activity of preganglionic neurons in the intermediolateral cell column of the spinal cord [29,30,33]. Previously, we have demonstrated that neurons in the LPBN are essential for mediation of cholinergic inputs responsible for pressor responses in the RVLM [17]. Anatomical studies have demonstrated that neurons in the PVN project to the LPBN [37]. Electrical and chemical stimulation of the PVN led to increases in numbers of neurons with c-fos protein in the LPBN, suggesting that PVN stimulation leads to activation of neurons in the LPBN
HYPOTHALAMIC BLOOD PRESSURE CONTROL [15]. Thus, it could be speculated that PVN stimulation leads to activation of neurons in the LPBN, which next leads to activation of cholinergic inputs to neurons in the RVLM. In the present study, indeed, the inhibitory amino acid GABA injected into the LPBN inhibited the pressor response induced by PVN stimulation. If similar alterations of central cholinergic function occurred in human essential hypertension, selectively acting anticholinergic medication might be expected to be useful for its treatment. In contrast, generalized increases in cholinergic activity with acetylcholinesterase inhibitors such as those used in Alzheimer’s disease might accelerate hypertension. In conclusion, the present study demonstrated that PVN stimulation causes pressor responses via activation of cholinergic mechanisms in the RVLM. Since cholinergic inputs to the RVLM are enhanced in SHR and this enhanced activity is involved in maintenance of hypertension in this strain, the results of the present study would provide an important information for our understanding of central mechanisms of hypertension.
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Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus T. Kubo,* Y. Hagiwara, D. Sekiya, S. Chiba and R. Fukumori Department of Pharmacology, Showa Pharmaceutical University, Tokyo, Japan [Received 28 June 2000; Accepted 3 July 2000] ABSTRACT: The rostral ventrolateral medulla (RVLM) has cholinergic mechanisms responsible for pressor responses. Stimulation of the hypothalamic paraventricular nucleus (PVN) causes an increase of arterial pressure via activation of neurons in the RVLM. In this study, we examined whether PVN stimulation causes a pressor response via activation of cholinergic mechanisms in the RVLM. Male Wistar rats were used and they were anesthetized, paralyzed and artificially ventilated. Electrical stimulation of the PVN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine and the cholinesterase inhibitor physostigmine into the RVLM inhibited and potentiated, respectively, the pressor response induced by PVN stimulation. PVN stimulation also increased the firing rate of RVLM barosensitive neurons and the increase in the firing rate was inhibited and potentiated by scopolamine and physostigmine, respectively, iontophoretically applied on neurons. Microinjection of L-glutamate into the PVN produced a release of ACh in the RVLM. The inhibitory amino acid ␥-aminobutyric acid injected into the lateral parabrachial nucleus (LPBN) inhibited the pressor response induced by PVN stimulation. These results suggest that PVN stimulation causes an increase in arterial pressure via activation of cholinergic inputs in the RVLM. It appears that the pressor response is mediated, at least in part, via cholinergic inputs from the LPBN. © 2000 Elsevier Science Inc.
in the RVLM would thus improve our understanding of central mechanisms of hypertension. Electrical and chemical stimulation of the lateral parabrachial nucleus (LPBN) produces a pressor response via activation of RVLM sympathoexcitatory neurons [2,25,39]. Electrical stimulation of the central gray (CG) also increases blood pressure through activation of RVLM sympathoexcitatory neurons [6,38]. Previously we demonstrated that microinjections of the muscarinic receptor antagonist scopolamine into the RVLM inhibited the pressor response induced by LPBN or CG stimulation, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. LPBN or CG injection of glutamate produced a release of acetylcholine in the RVLM [17,18]. These findings suggest that pressor responses induced by LPBN or CG stimulation are mediated via activation of cholinergic mechanisms in the RVLM. The hypothalamus also plays an important role in blood pressure regulation [1,22,27,35,42]. Neurons in the paraventricular nucleus of the hypothalamus (PVN) also project to numerous structures involved in cardiovascular regulation including the RVLM [34]. Electrical and chemical stimulation of the PVN causes pressor responses and produces expression of c-fos protein in the RVLM of rats [15], suggesting that PVN stimulation leads to activation of neurons in the RVLM. Thus, it is possible that the pressor response elicited by PVN stimulation is also mediated via activation of cholinergic mechanisms in the RVLM. The purpose of the present study was to determine whether stimulation of the hypothalamic PVN causes an increase in arterial pressure via activation of cholinergic mechanisms in the RVLM.
KEY WORDS: Acetylcholine, Paraventricular nucleus, Rostral ventrolateral medulla, Pressor response, Scopolamine.
INTRODUCTION The rostral ventrolateral medulla (RVLM) is an area of crucial importance in regulation of blood pressure [9,20,28,40]. Neurons in the RVLM project to the intermediolateral cell column of the spinal cord, the main origin of the spinal sympathetic outflow [3,5,7]. The RVLM has an endogenous source of acetylcholine (ACh) responsible for pressor responses [4,11,12,21,23,26,31,32, 41]. The receptor involved is mainly the muscarinic receptor M2 subtype [4,16,21]. In addition, it has been demonstrated that RVLM cholinergic mechanisms are enhanced in spontaneously hypertensive rats (SHR), a rat model of genetic hypertension and that this enhanced RVLM cholinergic activity is related to the maintenance of hypertension in this strain [16,19]. Elucidation of cholinergic mechanisms responsible for blood pressure regulation
MATERIALS AND METHODS Male Wistar rats (300 –380 g) were used in this study. They were kept under alternative 12-h periods of dark and light and given standard rat chow and tap water ad libitum. Animals were anesthetized with urethane 1.2 g/kg, intraperitoneally. The femoral artery and vein were cannulated. The rats were paralyzed with D-tubocurarine (1 mg/kg, intramuscularly) and ventilated artificially with a respirator, as described [18]. All procedures were done in accordance with the guidelines outlined by the Institutional Animal Care and Use Committee of the Showa Pharmaceutical University.
* Address for correspondence: Dr. Takao Kubo, Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan. Fax: 042-721-1588.
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FIG. 1. Effects of unilateral rostral ventrolateral medulla (RVLM) microinjection of scopolamine 0.088 nmol (A), 0.26 nmol (B), physostigmine 0.092 nmol (C) and 0.31 nmol (D) on the blood pressure (BP) response induced by electrical stimulation of the ipsilateral hypothalamic paraventriculat nucleus (PVN). (E) Effects of unilateral lateral parabrachial nucleus microinjection of ␥-aminobutyric acid (GABA) 3 nmol on the BP response induced by electrical stimulation of the ipsilateral PVN. ●, PVN electrical stimulation; Œ, RVLM or LPBN microinjection.
Microinjection Experiments The rats were placed in a stereotaxic apparatus with the head fixed at 15°. Microinjections were made using glass micropipettes as described [18]. All drugs were dissolved in phosphate-buffered saline (pH 7.4) and given in a vol. of 50 nl in 2 s. Physostigmine or scopolamine was microinjected into the RVLM as described [18]. The PVN was stimulated electrially or chemically by L-glutamate. Electrical stimulation or glutamate injection at the PVN was made at the coordinates: 2.0 –2.2 mm caudal to the bregma, 0.5 mm lateral to the midline and 7.2–7.5 mm below the dorsal surface of the brain. The PVN was electrically stimulated with a bipolar concentric electrode. Stimulation was performed with rectangular pulses (pulse duration of 300 s) at a frequency of 25 Hz and current intensities of 100 –250 A for 3 s. In some experiments, ␥-aminobutyric acid (GABA) was microinjected into the LPBN. Injections into the LPBN were made at the coordinates: 9.3 mm caudal to the bregma, 2.0 mm lateral to the midline and 5.0 mm below the dorsal surface of the cerebellum. At the end of experiments, the electrical stimulation site was
marked by the passage of 250 A DC current for 15 s. The injection site was marked by injecting 50 nl of a concentrated solution of Pontamine sky blue. The brains were cut and stained with Cresyl violet. The electrical stimulation sites and the injection sites were identified by visual inspection. Microiontophoretic Experiments Microiontophoretic experiments utilized four-barrel glass microelectrodes containing monofilament glass fibers [18]. One of the barrels was filled with 2 M sodium acetate and 5% Pontamine sky blue, and was used to record extracellular unit activity. Another barrel was filled with 2 M NaCl (pH 6) and used for automatic current balancing. Remaining barrels were filled with scopolamine hydrobromide (50 mM), physostigmine sulfate (20 mM) or L-glutamate monosodium (0.2 M). Extracellular recording was performed through the electrode, as described [18]. Briefly, electrical activity was displayed on a medical oscilloscope and filtered (band pass 0.1–10 kHz). A signal processor (Model 7T08; Nihondenki-San-ei Instrument, Tokyo, Japan) was used for compiling the data in the form of pulse density variation
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FIG. 2. Effects of unilateral rostral ventrolateral medulla microinjection of scopolamine (A) and physostigmine (B) on maximal changes in mean blood pressure (⌬BP) induced by electrical stimulation of the ipsilateral hypothalamic paraventricular nucleus (PVN). (C) Effects of unilateral lateral parabrachial nucleus microinjection of ␥-aminobutyric acid (GABA) on maximal changes in mean blood pressure induced by electrical stimulation of the ipsilateral PVN. Values are mean ⫾ SEM from six to seven animals. *p ⬍ 0.05, compared to before treatment. Open column, before treatment; dotted column, after treatment.
histograms. The site of unit recording was stained by expelling the Pontamine sky blue from the electrode by the passage of 20 –50 A DC current. The brain was removed and the injection sites were identified. Blood pressure was increased using an inflatable cuff wrapped around the aorta close to the bifurcation of the coeliac artery. Microdialysis Experiments Microdialysis experiments were made as described [18]. Briefly, a microdialysis probe (0.22 mm in diameter and 1 mm in length, Eicom A-I-12-01) was stereotaxically inserted into the sites identified with L-glutamate in the RVLM. Ringer solution (NaCl: 147 mM, KCl: 4 mM and CaCl2: 3.4 mM) containing physostigmine (100 M) was perfused into the probe at a rate of 1.2 l/min. Perfusates were successively collected every 20 min from 60 min
after the start of perfusion. ACh in perfusates was measured by a high-performance liquid chromatography with an electrochemical detector [17]. For chemical stimulation of the PVN, L-glutamate (1.6 nmol) was microinjected every 2 min into the PVN during the third fraction and during the sixth fraction. Animals were treated with hexamethonium as described [18], in order to avoid changes in ACh release due to increases in blood pressure induced by PVN glutamate microinjection. Physostigmine sulfate, scopolamine hydrobromide (Wako Pure Chemicals, Osaka, Japan) and L-glutamic acid monosodium salt (Nakarai Chemicals, Kyoto, Japan) were used. The results are expressed as mean ⫾ SEM. All results were analyzed by either paired t-test or analysis of variance combined with the Welch’s t-test or Student’s t-test to determine the significance of individual
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FIG. 3. Schematic diagrams of the transverse section of rat brainstem, showing the sites where microinjections of L-glutamate into the rostral ventrolateral medulla (A) and into the lateral parabrachial nucleus (C) and electrical stimulation of the hypothalamic paraventricular nucleus (B) produced hypertension. Abbreviations: IO, inferior olive; LPBN, lateral parabrachial nucleus; PVN, hypothalamic paraventricular nucleus; RF, retrofacial nucleus; 3V, third ventricle.
FIG. 4. A coronal section of rat hypothalamic paraventricular nucleus (PVN) indicating the location of the stimulation site (arrow). Scale bar: 1 mm.
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FIG. 5. Rate histogram of rostral ventrolateral medulla neurons demonstrating the facilitatory effect of electrical stimulation (200 A, 25 Hz for 3 s) of the hypothalamic paraventricular nucleus (PVN) and their inhibition and potentiation during microiontophoretic application of scopolamine (30 nA) (A) and physostigmine (30 nA) (B), respectively, on neurons. Blood pressure (BP) responses to aortic constriction (AOc) and to PVN stimulation (●) were recorded.
paired comparisons. Differences between means were considered significant at p ⬍ 0.05. RESULTS Effects of RVLM Microinjection of Scopolamine and Physostigmine on Blood Pressure Responses Induced by Electrical Stimulation of the PVN The basal mean arterial pressure was 93 ⫾ 2 mmHg (n ⫽ 36). Unilateral electrical stimulation of the PVN produced a pressor response (Fig. 1). The pressor response was reproducible when the PVN was stimulated every 3–5 min. The pressor response was abolished by intravenous injection of hexamethonium (20 mg/kg) (n ⫽ 6), suggesting that the pressor response is mediated mainly by sympathetic excitation. The pressor response was dose-dependently inhibited by scopolamine (0.088 and 0.26 nmol) microinjected ipsilaterally into the RVLM (Figs. 1A,B and 2A). The pressor response was enhanced by physostigmine (0.092 and 0.31 nmol) injected ipsilaterally into the RVLM in a dose-dependent manner (Figs. 1C,D and 2B).
The dose 0.26 nmol of scopolamine injected into the RVLM was enough to block the pressor response to physostigmine (0.46 nmol) injected into the RVLM (data not shown). Postmortem histological examination confirmed that RVLM injection sites were located in an area containing the subretrofacial nucleus (Fig. 3A). PVN stimulation sites were located in an area of the PVN (Figs. 3B and 4). Effects of LPBN Microinjection of GABA on Blood Pressure Responses Induced by Electrical Stimulation of the PVN GABA (3 nmol) injected into the LPBN inhibited the pressor response induced by electrical stimulation of the PVN (Figs. 1E and 2C). GABA itself did not affect basal blood pressure. Effects of Iontophoretic Application of Scopolamine and Physostigmine on the Firing Response of RVLM Barosensitive Neurons Induced by Electrical Stimulation of the PVN Twelve neurons were inhibited during increases in blood pressure induced by aortic constriction (Fig. 5). Firing rates were
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KUBO ET AL. PVN every 2 min during the third fraction and during the sixth fraction. Blood pressure was not changed by glutamate injection in the hexamethonium-treated rats. DISCUSSION
FIG. 6. Effects of unilateral microinjections of L-glutamate into the hypothalamic paraventricular nucleus (PVN) on acetylcholine (ACh) release in the ipsilateral rostral ventrolateral medulla in hexamethonium (intravenous)-treated rats. L-Glutamate (1.6 nmol) or saline was microinjected into the PVN every 2 min during the third fraction and during the sixth fraction. The average of ACh release of the first and second fractions was considered as the basal release. Values are mean ⫾ SEM from seven animals. *p ⬍ 0.05, compared to saline.
18.5 ⫾ 3.1 spikes/s. Electrical PVN stimulation increased the firing rate in 11 of 12 barosensitive RVLM neurons (Fig. 5). The excitation of unit activity was inhibited by iontophoretic application of scopolamine (30 nA) onto neurons in all of 6 RVLM neurons tested (Fig. 5A). Scopolamine (30 nA) itself did not affect the basal neuronal activity in five neurons but slightly inhibited it in one neuron. The excitation of unit activity induced by PVN stimulation was potentiated by iontophoretic application of physostigmine (30 nA) onto neurons in all of five RVLM neurons tested (Fig. 5B). Physostigmine (30 nA) slightly increased the unit activity of RVLM neurons. On the other hand, microiontophoretic application of the noncholinergic agent L-glutamate (60 nA) onto RVLM neurons increased the firing rate of neurons, but scopolamine (30 nA) applied similarly did not affect the glutamate-induced excitation of unit activity in all cases tested (n ⫽ 6). ACh Release in the RVLM in Response to PVN Injection of LGlutamate Microinjection of L-glutamate into the PVN also produced an increase in blood pressure. We determined whether PVN injection of L-glutamate causes an increase of ACh release in the RVLM. The average ACh content of two consecutive samples beginning 60 min after the start of RVLM perfusion was 5.3 ⫾ 0.4 pmoles/20 min (n ⫽ 14) in anesthetized rats treated with hexamethonium. Release of ACh was gradually decreased over 140 min after the first fraction (Fig. 6). The release of ACh in the RVLM was increased by ipsilateral microinjection of L-glutamate into the
In the present study, electrical stimulation of the PVN of the rat caused a pressor response. The pressor response induced by PVN stimulation was inhibited by the muscarinic receptor antagonist scopolamine and potentiated by the cholinesterase inhibitor physostigmine, microinjected ipsilaterally into the RVLM. Furthermore, chemical stimulation of the PVN with L-glutamate caused an increase of ACh release in the RVLM. It has been demonstrated that ACh microinjection into the rat RVLM causes a pressor response via activation of muscarinic receptors [19,26,41]. Thus, the results of the present study suggest that PVN stimulation activates cholinergic mechanisms in the RVLM and that this cholinergic activation results in an increase in blood pressure. In the present study, we stimulated the PVN electrically or chemically by L-glutamate. Electrical stimulation excites both cell bodies and axons of passage, whereas glutamate excites only cell bodies but not axons of passage on the central nervous system [13]. Thus, the results of the present study suggest that neurons in the PVN may be responsible for activation of cholinergic mechanisms in the RVLM. PVN stimulation activated barosensitive neurons in the RVLM. The activation effect on RVLM neurons was inhibited and potentiated by iontophoretic application of scopolamine and physostigmine, respectively. The application of scopolamine (30 nA) did not affect the excitation of unit activity induced by iontophoretic application of L-glutamate, suggesting that scopolamine (30 nA) has no local anesthetic-like action. Thus, the results of the present study suggest that the PVN is, at least in part, involved in mediation of cholinergic inputs to RVLM barosensitive neurons. Choline acetyltransferase activity, an index of impulse flow in cholinergic neurons [10], and release of ACh in the RVLM area are increased in SHR, and that the enhanced ACh release in the RVLM is involved in the development and maintenance of hypertension in this strain [19]. Therefore, we can speculate that the PVN area may be involved in mediation of the enhanced cholinergic activity in the RVLM of SHR and thus involved in mechanisms of hypertension in this model. Indeed, it has been demonstrated that lesioning of the PVN attenuates the development and maintenance of hypertension in SHR [8]. In the present study, scopolamine (0.26 nmol) injected into the RVLM inhibited but did not abolish the pressor response induced by PVN stimulation, although the dose 0.26 nmol of scopolamine was enough to block the pressor response to physostigmine (0.46 nmol) injected into the RVLM. Corticotropin-releasing factor and angiotensins are also reported to be involved in excitatory synaptic inputs to pressor neurons in the RVLM arising from activation of the PVN [24,36]. In addition, it has been suggested that a portion of some pressor responses induced by PVN stimulation might be mediated by neural pathways that do not include a component within the RVLM [14]. Immunocytochemical evidence suggests that neurons in the PVN that contain oxytocin and vasopressin project to and may influence the activity of preganglionic neurons in the intermediolateral cell column of the spinal cord [29,30,33]. Previously, we have demonstrated that neurons in the LPBN are essential for mediation of cholinergic inputs responsible for pressor responses in the RVLM [17]. Anatomical studies have demonstrated that neurons in the PVN project to the LPBN [37]. Electrical and chemical stimulation of the PVN led to increases in numbers of neurons with c-fos protein in the LPBN, suggesting that PVN stimulation leads to activation of neurons in the LPBN
HYPOTHALAMIC BLOOD PRESSURE CONTROL [15]. Thus, it could be speculated that PVN stimulation leads to activation of neurons in the LPBN, which next leads to activation of cholinergic inputs to neurons in the RVLM. In the present study, indeed, the inhibitory amino acid GABA injected into the LPBN inhibited the pressor response induced by PVN stimulation. If similar alterations of central cholinergic function occurred in human essential hypertension, selectively acting anticholinergic medication might be expected to be useful for its treatment. In contrast, generalized increases in cholinergic activity with acetylcholinesterase inhibitors such as those used in Alzheimer’s disease might accelerate hypertension. In conclusion, the present study demonstrated that PVN stimulation causes pressor responses via activation of cholinergic mechanisms in the RVLM. Since cholinergic inputs to the RVLM are enhanced in SHR and this enhanced activity is involved in maintenance of hypertension in this strain, the results of the present study would provide an important information for our understanding of central mechanisms of hypertension.
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