Neurotransmitter and Peptide Receptors on Medial Vestibular Nucleus Neuronsa DAVID 0. CARPENTER AND NOBUAKI HORI Wadsworth Centerfor Laboratories and Research New York State Department of Health and School of Public Health Albany, New York 12201-0509 INTRODUCTION

The vestibular nuclei are the site of termination of most of the primary afferent inputs from the peripheral vestibular organs and play a central role in the processing of vestibular information. While a considerable amount is known about the inputoutput relations in these nuclei, less is known about the neurotransmitters and neuropeptides involved in integration in these structures. The goal of the present studies is to contribute to this body of information by study of the medial vestibular nucleus of the rat, using isolated brain slices in which one can record from the neurons using either extra- or intracellular techniques, iontophoretic application of substances suspected of having biologic activity, and bath perfusion of antagonist drugs. Several observations from this and previous studies are important in understanding the functioning of this nucleus, The neurons in the medial vestibular nucleus are all endogenous pacemakers, having an intrinsic rhythm which is modulated by excitatory or inhibitory synaptic inputs. It is relatively unusual for central nervous system (CNS) neurons to have such a regular pacemaker rhythm, but this property makes the neurons exquisitely sensitive to transmitter actions. The endogenous synaptic input from the eighth nerve is blocked by antagonists of the kainate/ quisqualate type of excitatory amino acid receptor, consistent with the conclusion that the transmitter is glutamate or a related excitatory amino acid. Finally, there are potent excitatory receptors for acetylcholine (muscarinic), opiate peptides (mu and delta), and the excitatory amino acids. There is an interaction between the opiate and muscarinic receptors on these neurons that has not been seen elsewhere in the nervous system, but the functional significance of this interaction remains to be elucidated. METHODS

Brain stem slices were prepared from male rats weighing 150-180 grams as shown diagrammatically in FIGURE 1. Animals were euthanized by cervical dislocation, and the brain rapidly removed and immediately placed in cold (4"C), oxygenated Krebs-Ringer solution containing (in mM) NaCI, 126; KCI, 5; MgSO,, 1.3; CaCI,, 2.4; 'Supported in part by a grant from the Aaron Diamond Foundation to the Capital District Center for the Study of Drug Abuse and Treatment. 668

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NaHC03,26; and glucose, 10. The brain stem was then blocked on wet filter paper, positioned on a vibratome, and 450-km sections cut containing the vestibular nuclei in the planes indicated in FIGURE 1A and B. The details of the preparation of brain slices from any area as developed in our laboratory have been previously published.' After the sections were cut they were incubated in oxygenated Krebs-Ringer at 37°C for at least two hours, after which a slice was positioned in the recording

A

C

Stim

FIGURE 1. Schematic representation of the rat brain with indication of the preparation of the rat medial vestibular nucleus brain slice. A and B show the preparation of the slice in the vertical and horizonal planes. Part C shows the details of the slice, with the placement of the stimulating (Stim) and recording (Rec) electrodes. VL = lateral vestibular nucleus. VM = medial vestibular nucleus.

chamber, covered with a nylon mesh, and submerged under oxygenated KrebsRinger, which flowed through the chamber at a rate of approximately 3 mL/minute. Recording electrodes for extra- or intracellular studies were positioned in the medial 1C. Extracellular electrodes were 3-5 vestibular nucleus as indicated in FIGURE megohm glass pipettes filled with Krebs-Ringer solution, while intracellular electrodes were pipettes of 100-150 megohm filled with 3 M potassium acetate. In some

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experiments synaptic activation of the medial vestibular neurons was achieved by activation of the eighth nerve from a point at the medial edge of the lateralvestibular nucleus, using a bipolar electrode, as indicated in FIGURE1C. Super maximal pulses (about 10 V) of 5O-p~duration were used. In other studies in which the responses of these neurons to iontophoretically applied transmitters and peptides were tested, a three-barreled iontophoretic electrode was prepared and positioned independently of the recording electrode in the dendritic tree (FIGURE2A). Drugs were applied using a Neurophore unit and a regular sequence where each substance was applied once per 90 seconds with 30-second intervals between applications of the various substances. The schematic of the neuron was drawn by Ms. Naomi Hori from sections prepared from the medial vestibular nucleus in which single neurons were injected with horseradish peroxidase, as previously described.2 All of the neurons (n = 11) injected with horseradish peroxidase that were examined had the general configuration shown in this drawing, with three or four major dendritic branches. The preparation and filling of all types of electrodes have been previously reported in detai1.I Part B of FIGURE 2 gives the concentrations and pHs used for the various substances applied by iontophoresis in these experiments, and the polarity of application. Note that the excitatory amino acids and enkephalins were applied by electroosmosis, as previously r e p ~ r t e d . ~ . ~ Recording procedures have also been described.' Signals were stored on tape, and hard records obtained by playing the tape at reduced speed onto a Gould pen recorder. Frequency histograms were obtained using a Princeton Applied Research Model 4203 signal avcrager in the time interval distribution mode. The bin width in all such recordings was 300 mseconds. In experiments in which active agents were applied by iontophoresis, extracellular recordings were obtained as illustrated in FIGURE 2C, and rate-meter records of the data obtained as previously de~cribed.~

RESULTS The Origin of the Spontaneous Discharge of Medial Vestibular Neurons

All neurons recorded in the medial vestibular nucleus exhibited a regular, spontaneous discharge at a frequency varying from about 0.5 to 80 Hz, as was 2C in extracellular recordings. The majority of neurons in illustrated in FIGURE extracellular recordings, where there is less chance of injury from the electrode, showed discharge frequencies in the range of 10-30 seconds. Because of the very great regularity, this discharge might be a result of an endogenous paccmakcr, rather than due to synaptic drive from external sources. In order to establish that this is the case, several experiments were done. FIGURE3 shows intracellular recordings from a medial vestibular neuron, and the effects of application of hyperpolarizing current through the intracellular electrode using a bridge circuit for current application. At rest this neuron showed a spontaneous discharge of approximately 60/second. The regularity and the absence of indication of subthreshold synaptic potentials is suggestive of this being an endogenous rhythm. With hyperpolarizing current of increasing amplitude there is a progressive slowing of the discharge. However, the rhythm remains regular, and no synaptic potentials are seen. This is very strong evidence that the discharge is due to an endogenous FIGURE4 shows that the discharge of a vestibular neuron was not blocked by perfusion of a variety of drugs that antagonize various transmitter receptors. This figure shows extracellular action potentials. Perfusion of atropine, a muscarinic

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t

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50 rnM

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7.0

t

7.5

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.+

Enk

in 150 nM NaCl EAAs

10 rnM in 150 nM

NaCl GABA

0.5M

C ACh +5 nA

M +I5 nA

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FIGURE 2. Recording techniques used in medial vestibular slices. Part A shows a camera lucida drawing of a medial vestibular neuron that was injected intracellularly with horseradish peroxidase. Recording electrodes (RE) were positioned near or in the cell soma for extra- or intracellular recordings of electrical activity, respectively. In those experiments in which neuroactive substances were applied, a three-barreled iontophoretic electrode was used and positioned in the dendritic tree, where responses to effectively all agents tested were found to be largest. Part B shows the substances used, the concentrations prepared, pH, and polarity of application. ACh = acetylcholine; M = morphine; Enk = methionine enkephalin; EAAs = excitatory amino acids (N-methyl-D-aspartate, quisqualate, or glutamate); and GABA = y-aminobutyric acid. C shows extracellular recordings of a medial vestibular neuron and the response to acetylcholine, morphine, and N-methybaspartate (NMDA).

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1) Control

2) 100 pA

3) 600 pA

4) 1000 pA

20mV

I

40 msec

FIGURE 3. Pacemaker activity in medial vestibular neurons. All neurons recorded showed regular spontaneous activity the frequency of which was reduced by application of hyperpolarizing current. The records show the control (l), application of 100 pA of hyperpolarizing current (2), 600 pA (3), and 1000 pA (4). The fact that the discharge is smoothly slowed is strong indication that the discharge is endogenous and not due to synaptic input. acetylcholine antagonist, or curare, a nicotinic acetylcholine antagonist, did not significantly affect discharge. There was also no effect of amino phosphonobutyric acid (AF'B) or amino phosphonovaleric acid (APV), antagonists of two types of excitatory amino acid receptors. In studies not shown but done on other cells, there was also no effect of 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX), which is a selective antagonist of the quisqualate/kainate type of excitatory amino acid recep-

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tor.' Bicuculline, an antagonist of y-aminobutyric acid (GABA) responses, had no effect, nor was there a significant alteration of discharge frequency by naloxone, an antagonist of opiate receptors. These observations imply not only that none of the above agonists is an excitatory transmitter driving the spontaneous discharge, but also that none of these agonists has a role in modulating the spontaneous discharge under the circumstances in which the experiment was done. This is particularly significant with regard to GABA, which is a common inhibitory transmitter, and implies that there is not a resting inhibitory background in the brain slices. If this rhythm is truly endogenous it should not be blocked by ionic manipulations of the perfusing solution that block synaptic transmission. FIGURE5 shows a frequency histogram of the discharge of a different neuron in the control KrebsRingers, and in low Caz+-high Mg2+Krebs-Ringer, in which essentially all synaptic transmission is blocked through blockade of transmitter release at the presynaptic terminal. The figure shows a plot of the interspike intervals, recorded over a period of 30 seconds. Note the little variability in the control solution. In the low Ca2+-high Mgz+solution there is a slight acceleration of the discharge, rather than the slowing

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ATRO P I NE (1 0%)

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APB

WASH

(10-5~)

BIC (10-%I)

APV

(5x 1 0 - 5 ~ )

(10-4~)

WASH

CURARE

WASH

NA LOXONE (10-5~)

0.3 mV 6 sec

FIGURE 4. Lack of effect of various transmitter receptor antagonists on spontaneous activity of medial vestibular neurons recorded extracellularly. Each drug was perfused for 10 minutes at the concentration indicated, followed by a 15-minute wash with control Krebs-Ringer. APB = amino phosphonobutyric acid; APV = amino phosphonovaleric acid; BIC = bicuculline.

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and cessation that would be expected if synaptic drive were the source of the discharge. The increase in frequency is almost certainly a reflection of the membrane instability and hyperexcitability induced by removal of calcium.8 With wash, the discharge frequency returns to control values. These observations constitute proof that the spontaneous activity represents an endogenous pacemaker discharge of medial vestibular neurons.

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FIGURE 5. Effects of perfusion of a high-Mg2+,no-added-Ca2+Krebs-Ringer solution on the spontaneous activity of a medial vestibular neuron, recorded extracellularly. Records show a histogram of spike interval following the last spike at time zero. The zero Ca*+, 6.3 mM Mg2+ solution, which should block all synaptic activity, resulted in a slight increase in frequency of spontaneous discharge as expected as a result of the effect of removing calcium on neuronal excitability. Each record represents accumulated spike intervals over a 30-second period.

The Endogenous Transminer Releasedfiom the Fibers of the Eighth Nerve Since there is considerable disagreement in the literature concerning what is the transmitter released from those afferent fibers projecting to the medial vestibular nucleus from the eighth nerve, we have performed a series of experiments in which synaptic excitation was evoked by electrical stimulation within the slice. The major problem in such studies is to find a site where excitatory drive is clearly not a result of

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direct current flow or the result of a relatively nonspecific activation of local interneurons. We have found that we could routinely drive these neurons by stimulation applied at the medial border of the lateral vestibular nucleus (shown diagrammatically in FIGURE 1C). FIGURE 6 shows results from one such study with extracellular recording. Following the stimulus artifact the cell is driven with a latency of about 3.5 mseconds. This is sufficiently long to indicate that the excitation is not due to current spread, and yet is sufficiently brief to be consistent with monosynaptic excitation.' The site of stimulation is consistent with what would be expected for the pathway of the eighth nerve. Therefore, we conclude that this stimulation is exciting eighth nerve fibers that are excitatory onto medial vestibular neurons. 4 were tested for efficacy in The various antagonists that were studied in FIGURE 6 illustrates one neuron in which CNQX, blocking the synaptic response. FIGURE APV, and atropine were studied. The synaptic drive was reversibly blocked by CNQX, but not by APV, an N-methy1-D-aspartate (NMDA) receptor antagonist, or by atropine, a muscarinic acetylcholine receptor antagonist. There also was no effect of curare or of naloxone. These results are consistent with the conclusion that the excitatory transmitter released by eighth nerve fibers onto medial vestibular neurons is an excitatory amino acid, presumably glutamate, which acts at a kainate or quisqualate receptor.

Transmitterand Peptide Receptors on Medial Vestibular Neurons

FIGURE 7 shows the responses of one of these neurons to iontophoretic application of morphine (M), quisqualate ( a ) , and acetylcholine (ACh). All three substances excited this neuron. In this neuron the response to quisqualate quickly recovered to baseline discharge, while for both acetylcholine and morphine there was a rapid initial response followed by a prolonged excitation, which may reflect a second component. This figure shows the effect of increasing the iontophoretic current on the response to morphine. The threshold was to currents of less than 5 nA. There was little increase in the early peak discharge with currents beyond 15 nA, but the duration of discharge was prolonged when higher currents were used. Of over 57 neurons recorded in the medial vestibular nucleus, all of those tested were excited by quisqualate and acetylcholine. The great majority of neurons were excited by morphine. Morphine is known to be primarily a mu receptor agonist, but does also act at delta receptors. We tested the effect of methionine enkephalin (which acts primarily at delta receptors) iontophoresis in five neurons, and found it to be excitatory in all cases. In two neurons excited by morphine, we tested the effects of the specific mu agonist DAGO and the specific delta agonist DPDPE and found that both were excitatory. These observations, although preliminary, suggest that the medial vestibular neurons have excitatory receptors for both mu and delta agonists. Occasionally (3 of 45 cells), pure inhibitory responses were seen to morphine. Biphasic (excitatory-inhibitory) responses were common for acetylcholine, and these 2C. Previous studies by are illustrated in the raw data records shown in FIGURE Gallagher et aL1"suggest that the biphasic response to acetylcholine may be due to activation of inhibitory interneurons, releasing GABA, but we have not tested this possibility directly. The response of one of the neurons that showed pure inhibition 8. Both the morphine inhibition and acetylcholine by morphine is shown in FIGURE excitation were depressed by naloxone. We have not yet characterized the inhibitory responses in regard to receptor type.

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CONTROL

CNOX (lo%, 6 min) (20 min)

WASH

WASH

I

I

NEW CONTROL

(10min) ,

ATROPINE (10

%, 1 o min)

WASH (10 min)

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FIGURE 6. Blockade of synaptic activation of a medial vestibular neuron by stimulation of the eighth nerve by CNQX,an antagonist of excitatory amino acid receptors of the quisqualate/ kainate type. Records are extracellular recordings of responses following stimulation as 2. The stimulation was through a bipolar electrode, using an interval of 4 illustrated in FIGURE seconds and pulses of 50 pseconds duration and approximately 10 V intensity. The left peak in each recording is the stimulus artifact, while the biphasic response on the right is the neuronal spike. Note the response was blocked by CNQX but not by APV or atropine.

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FIGURE 9 shows another neuron and the effects of naloxone on responses to acetylcholine, morphine, and quisqualate. All responses in this cell are pure excitation. Naloxone caused a depression of both the morphine and acetylcholine responses, although there was no effect on the response to quisqualate. Naloxone has long been used as a relatively specific antagonist of opiate receptors, and therefore its effect on acetylcholine responses is surprising. However, 10 shows the this action was a consistent one on all neurons tested (n = 14). FIGURE effects of naloxone on NMDA, acetylcholine, and morphine responses. The NMDA response was unaffected, while the acetylcholine and morphine responses were depressed to effectively equal degrees. When a higher concentration of naloxone was used to give effectively total blockade of the morphine response, there was also effectively total blockade of the acetylcholine response (FIGURE11).

10 sec

FIGURE 7. Rate-meter recordings of responses of a medial vestibular neuron to morphine (M), quisqualate and acetylcholine (ACh). The effects of dose on the response to morphine are also shown. The rate meter reset every 300 mseconds.

(a),

FIGURE12 shows results obtained from one neuron in which it was possible to test the interactions of several agonists and antagonists on the acetylcholine and morphine responses. In this cell we iontophoresed acetylcholine, morphine, and quisqualate. The quisqualate response was selectively blocked by CNQX. Naloxone depressed the acetylcholine and morphine responses to an equal degree without effect on the response to quisqualate. When morphine was perfused in the bath there was clear and reversible potentiation of the acetylcholine response. Finally, when atropine, an antagonist of muscarinic acetylcholine responses, was applied there was an almost total blockade of both the acetylcholine and morphine responses without any effect on the response to quisqualate.

CONTROL

NALOXONE (5 x I O - ~ M 5, min) II

ACh (15 nA)

M (30 nA)

10 sec

FIGURE 8. An unusual hyperpolarizing response to morphine in a medial vestibular neuron. Both the depolarizing acetylcholine and the hyperpolarizing morphine responses were partially but not completely blocked by naloxone. The rate meter reset every 300 mseconds.

1

CONTROL

NALOXONE (5 x

ACh (25 nA)

i o - s ~5, rnin)

M (60 nA)

Ill Q (25 nA)

10 sec

FIGURE 9. Naloxone reduces both morphine and acetylcholine responses without effect on responses to quisqualate. The rate meter reset every 300 mseconds.

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DISCUSSION Medial Vestibular Neurons Exhibit Endogenous Pacemaker Activity

Endogenous pacemaker discharge in neurons was first definitively documented by Alving, who physically isolated neurons of Aplysiu and demonstrated that spontaCONTROL

I

NALOXONE

(5 x 1 0 - 5 ~ 1,0 min)

NMDA (30 nA)

ACh

(5 nA)

M (15 nA)

FIGURE 10. Naloxone reduces both morphine and acetylcholine responses without effect on responses to NMDA.

neous, patterned activity did not depend upon synaptic input." Such pacemaker activity is commonly observed in invertebrate preparations6 but definitive identification of pacemaker activity in the mammalian CNS has not previously been possible. It is not possible to prove that spontaneous activity is a result of endogenous activity in in vivo preparations where a total blockade of synaptic activity is incompatible with

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life, but the advent of use of isolated brain slices has allowed investigators new freedom to alter the environment without being dependent upon heart beat and respiration. Using brain slices, several authors have reported spontaneous activity, which they have ascribed to an endogenous pacemaker a~tivity.I*-~~ Previous studies using the medial vestibular nucleus have commented on the regular spontaneous activity and have presented the possibility that the discharge was e n d o g e n o u ~ . ~ J ~ l ~ The present studies demonstrate that the regular spontaneous activity of medial vestibular neurons depends upon membrane potential, is independent of individual

FIGURE 11. Concentrations of naloxone that give effectively total blockade of the morphine response also give almost total blockade of the acetylcholine response.

transmitter systems, and is present when all synaptic activity is blocked by low Caz+-high Mg2+medium. Therefore, there can be no other conclusion than that these are true pacemaker neurons, having an endogenous rhythm resembling that of heart muscle. Thus, these observations represent the most convincing evidence to date that central neurons may exhibit endogenous pacemaker discharges similar to those characteristic of invertebrate neurons. The presence of pacemaker activity, especially activity at a relatively high

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FIGURE 12. Effects of CNQX, naloxone, and atropine on responses to acetylcholine (ACh), morphine (M), and quisqualate (Q). While the quisqualate responses were selectively blocked by CNQX, both naloxone and atropine blocked both the acetylcholineand morphine responses. The rate meter resets every 300 mseconds.

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discharge rate observed in these neurons (0.5 to 80 Hz), has considerable implications for understanding information processing in this nucleus. Clearly the simplistic expectation that all is quiet until a stimulus comes in, cannot apply here. Those neurons postsynaptic to this nucleus are constantly receiving input at a high rate of activity. Thus the information transfer must be in the patterning, not just in the presence or absence, of activity. A second feature of pacemaker neurons is that they are exquisitely sensitive to small inputs. One characteristic of pacemaker neurons in invertebrates is that they tend to have high input resistances secondary to a relatively low resting potassium cond~ctance.~ The high input resistance and the fact that the discharge can be accelerated or decelerated by depolarizing or hyperpolarizing currents, respectively, make the discharge very sensitive to small synaptic currents, which would usually be totally subthreshold in a nonpacemaker neuron. There is, however, little or no understanding how the nervous system can decipher information encoded in these relatively subtle changes in discharge frequency. This problem poses a very interesting challenge to physiologists interested in the vestibular system.

The Transmitter in VestibularAfferent Fibers Until very recently it was believed that the transmitter of vestibular afferents was acetylcholine.1g23These studies, using in vivo preparations, resulted in conclusions that are not supported by more recent experiments using isolated brain slices, however. There has been suggestive evidence for some time that excitatory amino ~.~~ acids played a role in vestibular afferent fibers, based on b i o ~ h e m i c a l ~and immunocytochemica126evidence. In the frog there has been direct evidence that the excitatory postsynaptic potential (EPSP) evoked by stimulation of vestibular afferents is blocked by non-NMDA receptor antagonist^.^^ Two recent studies have utilized brain slice preparations similar to ours, and both have concluded that the transmitter receptor was a non-NMDA excitatory amino a ~ i d .The ~ . present ~~ results confirm this conclusion, even though all three studies used somewhat different methods for stimulation of vestibular afferents. Lewis et al. utilized local stimulation,w and obtained very short latency responses which were blocked by kynurenic acid, a relatively nonspecific excitatory amino acid antagonist, and amino phosphonobutyric acid, a substance closely related to other excitatory amino acid antagonists, which probably acts pre~ynaptically.~~ Doi et al. attempted to include the root of the eighth nerve in their slices of the medial vestibular nucleus, but they had to use very long stimulation pulses to excite the n e ~ r o n sThey . ~ were, however, able to distinguish mono- and polysynaptic excitation and to determine a synaptic delay to be of the ordcr of 2 mseconds. Our method differs in that we could consistently obtain drive of medial neurons by placing the stimulating electrode at the medial margin of the lateral vestibular nucleus. While we have no direct proof that this is the pathway of the eighth nerve, the potent drive and the consistent brief latency are consistent with the conclusion that we are monosynaptically exciting the neurons by stimulation of the afferent fibers of the vestibular system. The consistency of conclusion among the three studies is perhaps the best evidence that the endogenous transmitter is glutamate or a related excitatory amino acid. The lack of any effect of perfusion of the various transmitter antagonists on the endogcnous discharge is of interest in light of reports that APV, microinjected into intact animals, blocks a major component of the resting discharge of vestibular neurons.30 This observation is understandable in light of the fact that in the slice, the great majority of synaptic inputs are cut and therefore silenced.

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Opiate Receptors in the VestibularNucleus, and the Interaction of Opiate and Muscannic Receptors

Opiate receptors are widely distributed in the central nervous system, and both enkephalin-containing cell bodies and terminals are found in the vestibular nuclei.3'-33 Beitz et al. report that the medial vestibular nucleus contains the greatest numbers of enkephalin-labeled neurons among all brain stem structure^.^^ The opiates have a number of important functions, but it is relatively unusual for there to be clear excitatory responses on the great majority of neurons in any specific population. In the first place, most responses to opiate peptides are i n h i b i t ~ r y . In ~~.~~ addition, many if not most opiate receptors are on the presynaptic terminals and not the soma on neurons.36The fact that most neurons of the medial vestibular nucleus who have receptors for opiate peptides was reported by Yasnetsov and Pravdi~tsev,~' recorded in anesthetized cats. Our observations confirm their report that a very high percentage of vestibular neurons have both mu and delta opiate receptors, although the frequency of observing inhibitory responses was considerably less in our studies than theirs. These authors also reported that naloxone, a specific antagonist of opiate receptors, blocked both excitatory and inhibitory effects of morphine and enkephalin, an observation that our experiments confirm. The interaction between morphine and acetylcholine responses that we have observed was unexpected. Early in vivo studies with iontophoresis of morphine and enkephalins showed that morphine could block some responses to excitatory amino acids and a c e t y l ~ h o l i n eby ~ ~unknown *~~ mechanisms. In contrast, Bradley and Dray reported that morphine blocked excitation of unidentified brain stem neurons in the rat by acetylcholine, norepinephrine, and serotonin, but did not affect excitation by .~~ glutamate or homocysteic acid or inhibition by any of the above s ~ b s t a n c e sThey did note that occasionally they saw a potentiation of the excitatory responses by morphine, but provided no further details in a preliminary report. While this reference could be to a process similar to the one that we observe, the interaction we see is both opposite in direction and more specific to one transmitter system than the common effect reported in all of these studies. Moreover, the interaction is demonstrated by the cross effectiveness of both naloxone, the specific opiate antagonist, and atropine, the specific muscarinic antagonist, and by the total lack of interaction with either the NMDA or the quisqualate receptors. A very similar interaction between acetylcholine and opiate responses has been reported on Renshaw cells of the cat spinal ~ o r d . ~These ' . ~ ~neurons are excited by acetylcholine, morphine, and leu-enkephalin as well as by the excitatory amino acids. The response to the opiate peptides and acetylcholine but not the excitatory amino acids is blocked by naloxone. Furthermore, the response to acetylcholine is enhanced by prolonged application of morphine. This action of morphine on acetylcholine responses of Renshaw cells had been previously reported by Lodge et al.43Atropine was tested by Duggan et al. on four neurons and found to reduce both morphine and acetylcholine excitation!l Therefore, in all regards this appears to be the same interaction as we observe. Nabatame et al. report that lateral vestibular neurons are excited by phencyclidine, and that this excitation is blocked by atropine.23This may reflect a similar effect to that observed here. The molecular mechanisms responsible for this interaction are unknown and will be the subject of future investigations. Clearly this interaction is important for understanding vestibular function, particularly so since cholinergic mechanisms are so important in such vestibular-dependent events as motion sickness." Further investigations will be required to elucidate the details of these interactions and their physiological implications.

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ANNALS NEW YORK ACADEMY OF SCIENCES

The principal conclusions from this study are as follows: 1. Medial vestibular neurons, studied in rat brain slices, are endogenous pacemak-

ers, and therefore an understanding of their physiological function necessitates study of the patterning of their output, not just discharge activity. 2. The neurotransmitter utilized by eighth nerve terminals is an excitatory amino acid, probably glutamate, acting at CNQX-sensitive receptors of the kainate/ quisqualate type. 3. Opiate peptides are potent excitants of medial vestibular neurons, acting at both mu and delta receptors. There is an interaction between opiate and cholinergic receptors such that they both are blocked by the opiate antagonist naloxone and the muscarinic antagonist atropine. REFERENCES 1. HORI,N., N. AKAIKE& D. 0. CARPENTER. 1988. Piriform cortex brain slices: techniques for isolation of synaptic inputs. J. Neurosci. Methods 25: 197-208. 2. ~~RENCH-MULLEN, J. M. H., N. HORI,H. NAKANISHI, N. T. SLATER& D. 0. CARPENTER. 1983. Asymmetric distribution of acetylcholine receptors and M channels on prepyriform neurons. Cell. Mol. Neurobiol. 2: 153-182. 3. CARPENTER, 1988. Excitation of area D. O., D. B. BRIGGS,A. P. KNOX& N. STROMINGER. postrema neurons by transmitters, peptides and cyclic nucleotides. J. Neurophysiol. 59: 358-369. J. M. H., N. HORI & D. 0. CARPENTER.1986. Receptors for the 4. ~~RENCH-MULLEN, excitatory amino acids on neurons in rat pyriform cortex. J. Neurophysiol. 55: 12831294. D. 0.1973. Ionic mechanisms and models of endogenous discharge ofAplysiu 5. CARPENTER, neurons. I n Proceedings of the Symposium on Neurobiology of Invertebrates: Mechanism of Rhythm Regulation: 35-58. Hungarian Academy of Sciences. Tihany, Hungary. 6. CARPENTER, D. O., Ed. 1982. Cellular Pacemakers, 1 and 2. John Wiley and Sons. New York, N.Y. 7. HONORE,T., S. N. DAVIES,J. DRFJER, E. J. FLETCHER, P. JACOBSEN, D. LODGE& F. E. NIELSEN.1988. Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science 241: 701-703. D. 0.& R. GUNN.1970. The dependence of pacemaker discharge ofAplysiu 8. CARPENTER, neurons upon Na+ and Ca2+.J. Cell. Physiol. 75: 121-127. 9. Dot, K., T. TSUMOTO & T. MATSUNAGA. 1990. Actions of excitatory amino acid antagonists on synaptic inputs to the rat medial vestibular nucleus: and electrophysiological study in vifro. Exp. Brain Res. 82: 254-262. 1992. Modulation of J. P., K. D. PHELAN& P. SHINNICK-GALIAGHER. 10. GALLAGHER, excitatory transmission at the rat medial vestibular nucleus synapse in vitro. Ann. N.Y. Acad. Sci. (This volume.) B. 0. 1968. Spontaneous activity in isolated somata of Aplysiu pacemaker 11. ALVING, neurons. J. Gen. Physiol. 51: 29-45. D. 1987. Endogenous bursting by rat supraoptic neuroendocrine cells is calcium 12. ANDREW, dependent. J. Physiol. London 384: 451-465. 13. HAITON,G. I. 1982. Phasic bursting activity of rat paraventricular neurones in the absence of synaptic transmission. J. Physiol. London 327: 273-284. 14. INOUYE, S . T. & H. HAWAMURA. 1979. Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus. Proc. Natl. Acad. Sci. USA 76 5962-5966. 15. LEWIS,M. R., J. P. GALLAGHER & P. SHINNICK-GALLAGHER. 1987. An in vitro brain slice preparation to study the pharmacology of central vestibular neurons. J. Pharmacol. Methods 1 8 267-273.

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