cholinergic nerve terminals in the ventrolateral medullary pressor area: pharmacological evidence

Journal of the Autonomic Nervous System, 22 (1988) 221-228 221 Elsevier

JAN 00811

Cholinergic nerve terminals in the ventrolateral medullary pressor area: pharmacological evidence

Kalyana Sundaram 1 and Hreday Sapru 1,2

Section of Neurosurgery I and Department of Pharmacology 2, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103 (U.S.A.)

(Received 22 October 1987) (Revised version received 4 February 1988)

(Accepted 22 February 1988)

Key words: AFDX-116; Blood pressure; 3,4-Diaminopyridine; Heart rate; Hemicholinium; M2-muscarinic receptor; Scopolamine; Wistar rat

Abstract

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 rain. 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 M e 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 M 2 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 M 2 muscarinic receptors. The origin and physiological significance of these cholinergic terminals in the VLPA are not known.

Introduction

The pressor area in the ventrolateral medulla (VLPA) plays aft important role in the regulation of the cardiovascular system [4,6,13,22,29]. Excita- tion of neurons in the VLPA induced by microinjections of L-glutamate increases blood pressure (BP) and heart rate (HR) [29] while their inhibi- tion by muscimol decreases BP and HR [30,31].

Correspondence: H. Sapru, Neurosurgery, Medical Sciences Bldg., Room H592, New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, U.S.A.

Glutamate and muscimol act on neuronal cell bodies but not fibers of passage [15,35]. Electro- physiological and anatomic studies have shown that the neurons in the VLPA project to the intermediolateral column (IML) of the thoraco- lumbar cord [1,3,4,13,22,23]. Cardiovascular actions evoked from the depressor area of the ventrolateral medulla (VLDA) [19,33] and the hypothalamus [27,32] are also mediated via the VLPA. Lateral tegmental field neurons in the medulla may also exert their sympathoexcitatory actions via the VLPA [2,10]. VLPA has been shown to be the site of action of centrally acting hypertensive [20] and hypotensive [21] drugs. The role

222

of putative neurotransmitters in this area is just beginning to be investigated. We have recently reported that cholinergic agonists evoke a pressor response when injected into the VLPA and this action is mediated via the sympathetic nervous system [25,28,34]. Microinjections of the muscarinic receptor antagonists scopolamine hydrochloride or atropine sulfate or the specific M2 receptor blocker AFDX-116 [11,14] into the VLPA decrease BP and HR [19,20,25,28,34]. Based on these observations we hypothesized that this area may be under tonic excitatory cholinergic control [25,28,34]. This investigation was designed to demonstrate pharmacologically the presence of cholinergic nerve terminals in the VLPA and to study the effects of the release of endogenous acetylcholine in this area. 3,4-Diaminopyridine (DAP), which releases acetylcholine from cholinergic nerve terminals [12,17], was used for this purpose.

Materials and Methods

General procedures Male Wistar rats (Royal Hart, Middletown,

NY), weighing 300-350 g, were used. The rats were anesthetized with urethane (1.2-1.7 g/kg, i.p.) or pentobarbital sodium (45-50 mg/kg, i.p.). The trachea was cannulated with a polyethylene tubing and the rats were artificially ventilated using a rodent respirator (Harvard Apparatus, South Natick, MA; model 680). One of the femoral arteries was cannulated for monitoring pulsatile BP via a pressure transducer (Statham P23 Db). Mean arterial pressure (MAP) and HR were de- rived electronically from the BP waves. These tracings were recorded on a polygraph (Grass 7D). One of the femoral veins was cannulated for i.v. administration of various agents. The carotid sinus and aortic nerves were sectioned bilaterally in order to exclude the changes in baroreceptor input. Rectal temperature was monitored by a dig- ital thermometer (Sensortec, Clifton, N J; model BAT-8) and maintained at 3 7 _ 0.5 ° C. The re- suits obtained in anesthetized rats were confirmed in non-anesthetized mid-collicular decerebrate rats. The procedure for decerebration is described elsewhere [24].

Identification of the VLPA The rats were placed supine in a stereotaxic

instrument with the upper incisor bar at the level of the interaural line. The larynx, esophagus and underlying musculature were excised to expose the basal portion of the occipital bone. A window, approximately 6 mm wide and 7 mm long, was then created in this bone. The most caudal border of the occipital foramen served as a landmark. The VLPA was located 3.5-4.1 mm rostral to the landmark, 1.4-1.9 mm lateral to the midline and 0.5-1 mm deep from the ventral surface of the medulla. Microinjections of L-glutamate (1.77 nmol/si te) in this area evoked pressor responses. Details of this procedure are described elsewhere [29].

Microinjection technique Solutions of all agents, except AFDX-116, were

made in 0.9% saline. Glass micropipettes (Frederick Haer, Maine) with a tip diameter of approximately 50 /~m were filled with solutions. The micropipettes were connected, via PE-20 tubing, to a 1-/,1 microsyringe (Hamilton) mounted on an assembly for microinjections (Buxco Instru- ments, Sharon, CT, model STC-100). Microinjec- tions of various agents (50 nl volume) were de- livered in a 5-s interval. Control microinjections consisted of 50 nl of either artifical cerebrospinal fluid (CSF) or 0.9% saline. The pH of all microinjected solutions was 7.4.

Histology At the end of each experiment, the stereotaxic

coordinates of the sites were noted and an electrolytic lesion was made by passing a constant cur- rent of 5 mA for 30 s via a Tungsten microelec- trode (tip size approximately 1 btm). The brains were frozen in-situ by a spray of Histo-Freeze (Fisher Scientific, N J), removed and frozen again in liquid nitrogen. Sections (30/ tm) were cut in a cryostat (Hacker Instruments, N J), mounted and stained with Cresyl violet for histological identification of the injection sites using a standard atlas [18].

Statistical analysis Paired two-tailed t-test was used when animals

served as their own controls and an independent

t-test was used for testing the significance of differences between mean values from different groups of rats [5]. Differences were considered significant at P < 0.05. All values are expressed as mean + S.E.M.

Chemical agents used AFDX-116 (a specific antagonist for M2

muscarinic receptors [11,14]); Chlorisondamine chloride (a ganglion blocker); 3,4-diaminopyridine (releases acetylcholine from cholinergic nerve terminals [12,17]); hemicholinium bromide (impairs acetylcholine synthesis [16]); hexamethonium bromide (a nicotinic receptor blocker [25]); nicotine bitartrate; scopolamine HC1 (a muscarinic receptor blocker which does not distinguish between M1 and M2 receptors). The chemical agents were acquired from the following sources: AFDX- 116 (Boehringer Ingelheim, CT); 3,4-diaminopyridine (Aldrich Chemical, WI); L-glutamate monosodium, hexamethonium and nicotine were obtained from Sigma, MO. AFDX-116 was dis- solved in 0.05 N HC1. All other agents were dis- solved in 0.9% saline.

Results

Effect of microinjections of DAP into the FLPA Bilateral microinjections (50 nl) of 0.9% saline

into the VLPA evoked either no or a small increase (2 + 1.9 mm Hg) in BP. However, bilateral microinjections of L-glutamate (1.77 nmol /s i te ) evoked 35-50 mm Hg increase in MAP (Fig. 1A). Five minutes later, when the effects of L-glutamate had terminated, DAP was microinjected (1 n m o l / site) at the same sites. DAP evoked an increase in systolic and diastolic arterial pressure and HR (Fig. 1B). Dose-response relationships (n = 33) for the maximum hypertensive and tachycardic effects, which were obtained within 1-2 min of the microinjections, are shown in Fig. 1C and D, respectively. Control MAP and HR in these rats were 105 + 10 mm Hg and 380 + 30 bea ts /min (bpm), respectively. The doses of DAP varied between 0.1 and 2 nmol / s i te but the volume of microinjection and the pH of the solution were always 50 nl and 7.4, respectively. DAP induced an increase in MAP and HR within 2-5 s. The

223

A B

2o0 I

0 /

'°° r

eoor

(bpm)

2001- GLU ~ } DAP f

VLPA VLPA

1 lmin I

C D

~ 1 80

] ~ 60

~ 40 z 20

-z°20 - 0

0.1 0 .2 0 5 1 2 0.1 0.2 0 .5 1 2

DOSE OF DAP nmole/site DOSE OF DAP (nmolelsite)

Fig. 1. Legends for tracings in this and subsequent figures are as follows: top trace: mean arterial pressure (mrn Hg); middle trace: pulsatile BP (mm Hg); bottom trace: heart rate (beats/min). A: VLPA was identified bilaterally by microin- jecting L-glutamate monosodium 1.77 nmol/s i te (arrows). B: 5 min later, when the effects of glutamate had terminated, 3,4-diaminopyridine (DAP); 1 nmol/s i te) was microinjected bilaterally at the same sites; DAP induced an increase in BP and HR. The increase in BP (C) and HR (D) induced by DAP was dose-dependent (n = 33); the doses ranged between 0.1-2 nmol/si te . Each bar represents the peak response (expressed as mean + S.E.M.) which was obtained 1-2 min after the bilateral microinjections of DAP. The increase in BP and HR at 2 nmol /s i te was significantly smaller (P < 0.001) compared to that at 1 nmol/si te . Microinjections of artificial CSF or 0.9%

saline did not induce any significant effects.

duration of these actions was 10-30 min for doses of 0.1-0.2 nmol / s i t e and 20-50 min for doses of 0.5-2 nmol / s i t e . Maximal hypertensive and tachycardic responses were observed at 1 n m o l / site. At 2 nmol /s i te , the pressor and tachycardic responses were significantly ( P < 0 . 0 0 1 ) smaller than those observed at 1 nmol/s i te .

Microinjections of either L-glutamate (1.77 nmol /s i te ) or DAP (1 nmol /s i te ) into adjacent brain tissue (1-2 mm rostral to the VLPA) did not evoke any response. Anesthesia did not alter the

224

A B 200 r- ~ - - - - - ' -

M A P / ~ _ ~ ~ - ~ - ~ - (mmHg) /

0 L

200 I"

Ok.

6oor HR / - ~

(bpm)

200=- GLU ~. ~, DAP

VLPA VLPA

C D

200 r" M A P /

0 L

200 F

(mmHg) I I I I

o L

600 I"

(bpm) I 1rain I

200 L ' ib t CSD PE ~ DAP t

iV IV VLPA

Fig. 2. A: VLPA was identified bilaterally by microinjections of L-glutamate (1.77 nmol/s i te) . B: DAP (0.5 nmol /s i te) microinjected at the same sites produced an increase in BP and HR. C: chlorisondamine hydrochloride (CSD; 3 mg/kg , i.v.) decreased BP due to the blockade of the sympathetic ganglia. The BP was restored to control level by i.v. infusion (10 lag/min) of phenylephrine (large arrow). D: bilateral microinjections of DAP (0.5 nmol / s i te ) into the VLPA failed to evoke the usual increase in BP and HR indicating that these re-

sponses were mediated via the sympathetic nervous system.

responses observed because the responses were qualitatively identical in mild-collicular decerebrate and anesthetized rats.

Sympathetic nervous system mediated the effects of DAP

The pressor and tachycardic responses to the microinjections (0.5 nmol/s i te) of DAP into the VLPA (Fig. 2B) were blocked (Fig. 2D) by the

pretreatment of the rats (n = 4) with a ganglion blocker (chlorisondamine, 3 mg/kg, i,v.). The lack of responses to microinjections of DAP into the VLPA was not related to the decrease in BP induced by chlorisondamine (Fig. 2C) because the BP was restored to control level by i.v. infusions of phenylephrine (10/~g/min). Intravenous injections of the above-mentioned doses of DAP did not evoke any response.

Muscarinic receptors mediate the responses to DAP In these experiments (n = 5), the VLPA was

identified bilaterally with microinjections of L- glutamate (Fig. 3A) and the usual pressor and tachycardic responses to DAP (1 nmol/si te) were elicited (Fig. 3B). Microinjections of scopolamine (18 nmol /s i te ) into the VLPA produced a decrease in BP (Fig. 3C). An i.v. infusion of phenyl-

A 200 r

MAP / _ J \ ~ J ~ - (mmHg) L

0

(mniHg)

6oo r H.| (bpm) - -

200 t.. GLU

B C

~DAP~ sco

D E 2oor

MAP / - (rnmHg) I ~ '~ - " ~ ' ~ ~

/ 0~-

200 r (mmH

.00[ HR

(bprn) ~ ~

200 GLU~ ~ DAP

Fig. 3. A: VLPA was identified bilaterally by microinjections of L-glutamate (1.77 nmol/s i te) . B: bilateral microinjection of DAP (1 nmol /s i te ) increased BP and HR. C: scopolamine (SCO) (18 nmol / s i t e ) at the same sites decreased in BP. D: 25 min after the microinjections of scopolamine, L-glutamate was again microinjected at the same sites to ensure that the neurons in the VLPA were responsive. E: pressor response to subsequent microinjections of DAP at the same sites was abolished.

225

A B

200 MAP r

(mmHg) / ~ /

O L

2001"

(mrnHg)

O'- eOOl" H" /

(bpm)

200~- ~, GLU ~' ~' AFDX ~'

C D 200 I" MAPI

(mmHg) / ~ /

0 L

200 r

OL

(bpml / - - 2001,- 11min I

t GLU t t DAP t

Fig. 4. A: VLPA was identified bilaterally by microinjections of L-glutamate (1.77 nmol/s i te) . B: bilateral microinjection of AFDX-116 (1.6 nmol /s i te ) at the same sites induced a decrease in BP. C: 45 rain after the microinjections of AFDX-116, L-glutamate was again microinjected at the same sites to ensure that the neurons in the VLPA were responsive. D: pressor response to subsequent microinjections of DAP at the same

sites was abolished.

ephrine (10 /tg/min) was used to restore BP to control level. Scopolamine (18 nmol/site) caused depression of neurons initially because responses to subsequent microinjections of L-glutamate were attenuated. However, the response to L-glutamate was restored within 20-25 min (Fig. 3D). Subse- quent injections of DAP into the VLPA failed to evoke the usual pressor response (Fig. 3E). This effect lasted for 1-2 h.

In another group of rats (n = 4), M 2 receptor blocker, AFDX-116 (1.6 nmol/site) was microin-

jected into the VLPA (Fig. 4B) which was previ- ously identified with microinjections of L-glutamate (Fig. 4A). Microinjections of AFDX-116 into the VLPA produced a decrease in BP. Initially AFDX-116 rendered the neurons in the VLPA unresponsive to L-glutamate. When the response to L-glutamate recovered (40-50 min; Fig. 4C), subsequent microinjections of DAP at the same sites failed to evoke the usual pressor response (Fig. 4D).

Nicotinic receptors in the VLPA did not mediate the effects of DAP as shown by the following experiments (n = 4). Microinjections of nicotine (0.5 nmol/site) into the VLPA (Fig. 5A) increased BP (50 + 10 mm Hg) and HR (30 + 20 beats/min) which lasted for 20-35 rain. Microin- jection of hexamethonium (5.5 nmol/site) into the

2OOr MAPI

(mmHg 1

Ok

A B

(bPm) / 2 0 0 L

#Mco ~ t c6

D E

(mmHg) I J ~ / OL.

2OOr

OL.

(barn) I ~ - -

2001- t GLU t tDAP~' t NICO t

Fig. 5. A: VLPA was identified bilaterally by microinjections of L-glutamate (not shown). Microinjeetions of nicotine (NICO) (0.5 nmol / s i t e ) at the same sites increased BP. B: microinjec- dons of hexamethonium ((:6) (5.5 nmol / s i te ) into the same sites decreased BP. C: 15 min later, the responses to L-glutamate were again tested. D: at this time microinjections of DAP (0.5 nrnol/si te) continued to increase BP even though the

responses to the same dose of nicotine were blocked (E).

226

VLPA caused a decrease in BP (25 _+ 10 m m Hg) and H R (30 _+ 10 bea t s / r a in ) (Fig. 5B). However, the response to microinjections of L-glutamate re- mained unaltered (Fig. 5C). Subsequent microinjections of D A P (0.5 nmol / s i t e ) continued to elicit pressor and tachycardic responses (Fig. 5D) while the same doses of nicotine failed to do so (Fig. 5E).

Acetylcholine synthesis blocker attenuated the responses to D A P

In another group of rats (n = 8), control pressor and tachycardic responses to D A P (1 n m o l / site) were obtained (Fig. 6B) after identifying the VLPA bilaterally (Fig. 6A). Microinjections of hemicholinium (3 nmol / s i t e ) into the same sites

A B C

2oor _ _ _ - MAP / ,. .... i'~_ ~

200 r

(mmHg)

O L

600 r ""| (bpm) ""

2OO / t GLU4' DAp ~' ~tHC 3

D E

20o r MAP /

(mmHg) / - f ..... " /" -

I OL

2oo r

OL

6001'- ""| (bpm) I 1rain I

2001-

Fig. 6. A: VLPA was identified bilaterally. B: microinjections of DAP (1 nmoi /s i te) produced the usual increase in BP. C: microinjections of hemicholinium (HC3) produced a decrease in BP. D: 1 h later, the responses to L-glutamate were tested. E: subsequent microinjections of DAP (1 nmol /s i te ) at the s ame sites produced significantly (P < 0.001) attenuated re-

sponses.

Fig. 7. This section was cut 2.2 mm rostra] to the calamus scriptorius. The site of microinjection in the VLPA, marked by an electrolytic lesion, included the PGi, ventrolateral border of nA and ventromedial border of Gi. The agents were microinjected bilaterally unless indicated otherwise. However, in this section the site is marked on the left side so that various nuclei could be labelled on the right side. dmnX, dorsal motor nucleus of vagus; Gi, nucleus reticularis gigantocellularis; nA, nucleus ambiguus; nTS, nucleus tractus solitarius; py, pyra-

mids; PGi, paragigantocellular reticular nucleus [19].

(Fig. 6C) caused a decrease in MAP (20 _+ 5 mm Hg) and H R (20 _+ 10 bea t s /min ) which lasted for 1 h. At this time the neurons in the VLPA were responsive to L-glutamate (Fig. 6D). The responses to subsequent microinjections of D A P were significantly ( P < 0.001) at tenuated (Fig. 6E); the increase in M A P induced by microinjections of D A P (1 n m o l / s i t e ) into the V L P A was 100 + 10 mm Hg before and 25 + 5 m m Hg after the microinjections of hemicholinium.

Fig. 7 shows the site of microinjection (marked by an electrolytic lesion) in the VLPA in one of the experiments. The V L P A site included paragi- gantocel lu lar ret icular nucleus, ventrola tera l border of nucleus ambiguus and ventromedial border of gigantocellular reticular nucleus [19].

Discussion

The V L P A seems to be the final station in the brainstem through which cardiovascular function is regulated [4,6,13,22,29]. Microinjections of cholinergic agonists into this area evoke pressor responses [25,28,34] which may be the result of excitation of sympathoexci ta tory neurons [27]. Possible excitation of respiratory neurons located in the vicinity of this area cannot be responsible

for these BP changes because such excitation has been reported to produce hyperventilation [7] which, in turn, is expected to cause depressor responses. Although, hypotension has been elicited by the application of cholinergic agents to the ventral surface of the medulla using plexiglass rings [7,8], this effect may be due to the diffusion of the drugs to the caudal depressor area [29] where microinjections of cholinergic agents evoke hypotension [25,26]. In this investigation we used DAP, which has been shown to facilitate the release of neurotransmitters from nerve terminals [12,17] to demonstrate the presence of cholinergic nerve terminals in the VLPA. Electrophysiological experiments have shown that aminopyridines decrease outward potassium ion movement, leading to prolongation of action potential duration and potentiation of calcium currents, thereby increas- ing the quantity of acetylcholine released from cholinergic nerve terminals [171. However, these agents do not increase the sensitivity to acetylcholine on cholinergic receptors or alter the kinetics of reaction of acetylcholine with receptors and are devoid of anticholinesterase activity [12].

The pressor and tachycardic responses to the microinjections of DAP into the VLPA were mediated by the sympathetic nervous system because i.v. administered ganglion blocker (chlorisondamine) prevented them. The lack of responses to the i.v. injection of the same doses of DAP further supported the conclusion that the responses were central in origin. Microinjections of scopolamine prevented the effects of DAP indicating that the substance released from the nerve terminals in the VLPA was acting via muscarinic receptors. Prevention of the actions of DAP by AFDX-116 indicated that the muscarinic receptors involved were of M2 type [11,14,28]. On the other hand, microinjections of hexamethonium in doses sufficient to block the effects of nicotine failed to alter the responses to the same doses of DAP suggesting that nicotinic receptors were not involved in mediating these effects. Initially, scopolamine and AFDX-116 attenuated the responses to L-glutamate but these responses recovered within 40-50 rain. The effect of these antagonists on the actions of DAP were tested when the response to L-glutamate was restored.

227

Hemichol inium impairs the synthesis of acetylcholine because it blocks the high-affinity uptake of choline by cholinergic nerve terminals [16]. Microinjections of this agent into the VLPA significantly decreased BP and HR. Significant reduction in acetylcholine synthesis has been reported to occur within 1 h of microinjections of hemicholinium into the brain tissue [9]. In our study, the responses to microinjections of DAP were significantly ( P < 0.001) attenuated by prior microinjection of hemicholinium.

These results indicate that the substance released in the VLPA by DAP was probably acetylcholine. Although DAP may release other transmitters from non-cholinergic terminals [12], complete blockade of the effects of DAP by prior microinjections of scopolamine or AFDX-116 indicate that acetylcholine m a y be the predomi- nant transmitter released.

In summary, these results suggest that cholinergic terminals are present in the VLPA and acetylcholine released from these terminals acts via M2 muscarinic receptors. The origin of these cholinergic terminals and their physiological role remains to be established.

Acknowledgements

This work was supported by the following grants awarded to H.S.: N.I.H. (HL24347) and American Heart Association (N J). A generous gift of AFDX-116 from Boehringer Ingelheim Phar- maceuticals Inc., CT, U.S.A., is gratefully acknowledged.

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cholinergic nerve terminals in the ventrolateral medullary pressor area: pharmacological evidence

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