Excitatory Amino Acid Neurotransmission at Sensory-Motor

JOURNALOF NEUROPHYSIOLOGY Vol. 70, No. 3, September 1993. Printed in U.S.A.

Excitatory Amino Acid Neurotransmission at Sensory-Motor and Interneuronal Synapses of Aplysia caZij?mica

LOUIS-ERIC TRUDEAU AND VINCENT F. CASTELLUCCI Laboratoire de Neurobiologie et Comportement, Institut de Recherches Cliniques de MontGal, Centre de Recherches en Sciences Neurologiques, Universite’ de Montrkal, Montreal, Quebec H2 W 1R 7, Canada

SUMMARY AND CONCLUSIONS

1. Although the gill and siphon withdrawal reflex of Aplysia has been used as a model system to study learning-associated changes in synaptic transmission, the identity of the neurotransmitter released by the sensory neurons and excitatory interneurons of the network mediating this behavior is still unknown. The identification of the putative neurotransmitter of these neurons should facilitate further studies of synaptic plasticity in Aplysia.

2. We report that sensory-motor transmission within this circuit is mediated through the activation of an excitatory amino acid receptor that is blocked by the non-N-methyl-D-aspartate excitatory amino acid receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX ) and 1- (4-chlorobenzoyl) -piperazine-2,3- dicarboxylic acid (CBPD). Compound postsynaptic potentials evoked in motor neurons by electrical stimulation of the siphon nerve were blocked by 92% with CNQX (75 PM) and 89% with CBPD (75 PM).

3. Simultaneous intracellular recordings were obtained from sensory neurons, excitatory interneurons, and motor neurons. Monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motor neurons by an action potential in a sensory neuron were blocked by 86% with CNQX (75 PM) and 7 1% with CBPD (75 PM). The two antagonists also blocked monosynaptic interneuronal EPSPs onto motor neurons by 65% and 67%, respectively.

4. Potential agonists of the synaptic receptors were puff-applied in the intact abdominal ganglion. Homocysteic acid (HCA) was found to mimic the action of the synaptically released transmitter because it strongly excites motor neurons. This effect was blocked by CNQX. Kainate and domoic acid were also effective agonists.

5. The actions of L- and D-glutamate as well as quisqualate were found to be mainly hyperpolarizing, whereas aspartate and ( +) -amino-3-hydroxy-5-methylisoxazole-4-proprionic acid had no effect.

6. Several reasons may be proposed to explain the inability of puff-applied glutamate to excite effectively the postsynaptic neurons in the intact ganglion. It is possible nonetheless that other endogenous amino acids such as HCA act as neurotransmitters at these synapses.

INTRODUCTION

Studies of synaptic transmission within the small neuronal networks mediating withdrawal reflexes in Aplysia have identified some cellular and molecular mechanisms that may take part in modifications of the CNS in relation to learning and memory (Braha et al. 1990; Byrne et al. 1993; Castellucci and Kandel 1976; Dash et al. 1990; Goldsmith and Abrams 199 1; Klein 1993; Klein et al. 1982; Mayford et al. 1992; Montarolo et al. 1986). The central component of the gill and siphon withdrawal ( GSW) reflex involves I )

excitatory synapses from sensory neurons to excitatory interneurons and to motor neurons, 2) excitatory synapses from excitatory interneurons to inhibitory interneurons and to motor neurons, and 3) inhibitory synapses that produce feedback inhibition onto excitatory interneurons and in some cases onto sensory neurons (Castellucci et al. 1970; Frost et al. 1988; Hawkins et al. 198 1; Trudeau and Castel- lucci 1992). Although it appears that acetylcholine ( ACh) activates transmitter-gated Cl- channels at many inhibitory synapses of the circuit ( Trudeau and Castellucci 1993 ), the identity of the transmitter( s) released at the synaptic terminals of sensory neurons and excitatory interneurons is still unknown.

In Aplysia CNS, the only fast excitatory synapses where the neurotransmitter is known are 1) the monosynaptic excitatory postsynaptic potential (EPSP) that is recorded in neuron R15 of the abdominal ganglion on threshold stimulation of an axon in the right pleuroabdominal connective (Frazier et al. 1967; Woodson et al. 1975) and 2) some of the synapses made by neuron LlO (Giller and Schwartz 197 1; Kandel et al. 1967; Segal and Koester 1980, 1982; Waziri and Kandel 1969). These EPSPs are produced by the activation of cationic cholinergic receptors with nico- tinic pharmacology. Previous work has shown, however, that sensory-motor EPSPs are not blocked by cholinergic antagonists such as d-tubocurarine and hexamethonium ( Segal and Koester 1982; Trudeau and Castellucci 1993 ). Together with the observation that most motor neurons of the GSW reflex are inhibited by ACh (Kandel et al. 1967), this makes it unlikely that ACh is the transmitter of sensory neurons.

Many central excitatory synapses of vertebrates appear to utilize amino acids such as glutamate and possibly aspartate as their neurotransmitter (Brodin et al. 1988; Dryer 1988; Gasic and Hollmann 1992; Nicholls and Attwell 1990; Tsumoto 1990). This has been suggested to be the case for excitatory transmission from primary afferents in several vertebrate species (Brodin et al. 1987; Christenson and Grillner 199 1; Jahr and Yoshioka 1986; Takeuchi et al. 1983 ) . There is also some evidence that glutamate could be released at some excitatory synapses in the CNS of invertebrates (De Santis and Messenger 1988; Quinlan and Murphy 199 1). Other substances such as ATP (Edwards et al. 1992) and serotonin (Sugita et al. 1992) appear to be released at some excitatory synapses in the CNS of the rat. Homocysteic acid (HCA) has also been proposed as an endogenous neuroexcitant in the vertebrate CNS. It has been shown that in various brain regions HCA excites neurons

(X)22-3077/93 $2.00 Copyright 0 1993 The American Physiological Society 1221

1222 L.-E. TRUDEAU AND V. F. CASTELLUCCI

by activating excitatory amino acid receptors of the N- methyl-D-aspartate (NMDA) and non-NMDA types (Cuenod et al. 1990; Do et al. 1986a; Ito et al. 199 1; Neal and Cunningham 1989 ) . These latter observations raise the possibility that excitatory amino acid receptors may have as natural ligands amino acids other than glutamate or aspartate. A recent report has provided some indirect support for such a notion by demonstrating in the goldfish cerebellum the existence of high-affinity kainate binding sites that are glutamate insensitive (Davis et al. 1992). In the cockroach, Periplaneta americana, Wafford et al. ( 1992) have also demonstrated that an identified motoneuron could be strongly excited by kainate and domoate but inhibited by L-glutamate.

Here we report that the non-NMDA excitatory amino acid receptor antagonists 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX) (Honore et al. 1988) and I-( 4-chlorobenzoyl) -piperazine-2,3-dicarboxylic acid ( CBPD ) ( Davies et al. 1984; Ganong et al. 1986) are effective blockers of monosynaptic sensory-motor and interneuronal EPSPs of the GSW network. Additionally, HCA as well as kainic and domoic acid have a strong excitatory effect on neurons of the GSW reflex network. This effect is also blocked by CNQX. On the other hand, puff-applied glutamate, quisqualate, N-acetyl-aspartyl-glutamate, cysteic acid, and cysteic sulfinic acid have a predominantly hyperpolarizing effect, whereas aspartate, ( +) -amino-3-hydroxy+methylisoxazole-4-proprionic acid (AMPA) and NMDA have no effect. These results suggest that the neurotransmitter released at the synaptic terminals of sensory neurons of this circuit is an excitatory amino acid. HCA may be considered a candidate.

METHODS

Preparation

Aplysia calzfornica were obtained from Marinus (Venice, CA). Experiments were performed on the isolated and desheathed abdominal ganglion, which was dissected from the animal along with long sections of the siphon nerve, branchid nerve, and pleura- abdominal connectives. The nerves were aspirated into suction electrodes for extracellular stimulation. The ganglion was per- fused with artificial seawater ( ASW) that was composed of (in mM) 460 NaCl, 30 MgCI,, 25 MgSO,, 11 CaCl,, 10 KCl, and 10 N-2-hydroxyethylpiperazine-l\r-2-ethanesulfonic acid (HEPES) , pH 7.6. The high divalent cation ASW (2: 1 ASW), which contained twice the normal concentration of Mg2+ and 1.25 times the normal concentration of Ca2+, was (in mM) 368 NaCl, 10 1 MgCl,, 20 MgSO,, 13.8 CaCl,, 8 KCl, and 10 HEPES, pH 7.6 (Trudeau and Castellucci 1992).

Electroph ysiology

Intracellular recordings were made with glass microelectrodes filled with either 2 M KAc or 3 M KCl. Their resistance was between 2 and 20 M52. The signals were recorded with Axoclamp 2A and Axoprobe 1 A amplifiers (Axon Instruments). Data were stored in parallel on VHS tape (Vetter recorder, model 420E) and on the hard disk of a computer; they were analyzed with the SPIKE data analysis software (Hilal associates). The amplitudes of synaptic potentials were always measured at the first inflection whenever a polysynaptic component was recruited. For some experiments, traces were also directly recorded on a Hewlett-Pack- ard oscillograph paper recorder. Agonists were puff applied with

glass pipettes with a tip diameter of 5-6 pm. Pipettes were back- filled with light paraffin oil and tip-filled with the agonist. Brief pulses were applied with a pressure injection system (Medical Sys- tems). Two to five puffs of 20 ms in duration with a pressure of 35 kPa were usually used. Statistical analyses consisted of either analyses of variance (ANOVAs) (with post-hoc Tukey tests) or Stu- dent’s t test, where appropriate. Data are presented as means ~fi SE.

Drugs

L-HCA, L-aspartic acid, D-glutamic acid, N-acetyl-aspartyl-glutamate, and 2-amino-5-phosphonovaleric acid (AP5 ) were obtained from Sigma (St. Louis, MO). Ketamine hydrochloride, AMPA, NMDA, D-glutamylamino methanesulfonic acid (GAMS), kainic acid, and ( + )-MK-80 1 were from Research Bio- chemicals (Natick, MA). L-Glutamate was from Schwartz Biore- search (New York, NY). Tetrodotoxin (TTX) and L-cysteic acid were from Calbiochem (La Jolla, CA). L-Cysteic sulfinic acid was from Aldrich (Milwaukee, WI). CNQX, CBPD, philanthotoxin- 343 (PhTX), quisqualate, domoic acid, and HA-966 were from To& Neuramin (Bristol, UK).

RESULTS

Efect of CNQX and CBPD on compound EPSPs in motor neurons

We first verified the effect of various excitatory amino acid receptor blockers on EPSPs evoked in motor neurons of the GSW reflex by electrical stimulation of the siphon nerve. The interstimulus interval was 2 min. At this fre- quency of stimulation, the EPSPs are stable during the control period. A compound EPSP was recorded in a motor neuron of the LFS group or in motor neuron L7 polarized to -80 mV to prevent firing. This allowed us to screen si- multaneously for effects of the blockers on sensory-motor and interneuronal transmission because these responses are m 75% polysynaptic ( Trudeau and Castellucci 1992).

In preliminary experiments, excitatory amino acid receptor antagonists of the NMDA subtype (AP5, MK-80 1, ketamine, and HA-966) were found to have no effect on synaptic transmission (results not shown). On the other hand, bath application of the non-NMDA antagonist CNQX produced a powerful blockade of compound EPSPs. At a concentration of 75 PM, CNQX reduced the magnitude of compound EPSPs (as quantified by their surface area) by 91.9&2.8%(mean-tSE)(n=9)(t=5.3,iP<O.OOl)(Fig. 1 A). The effect was reversible on a 20-min wash. Another preferential non-NMDA excitatory amino acid receptor antagonist, CBPD, was found to have effects similar to those of CNQX. At 75 PM, compound EPSPs were reduced by 88.7 t 3.3% (n = 5) (t = 6.1, P < 0.001). Two additional preferential antagonists of the non-NMDA subtype were found to be ineffective at blocking compound EPSPs. At 100 PM, PhTX induced a nonsignificant decrease of 10.1 t 4.1% (n = 3)) whereas GAMS induced a nonsignificant decrease of 0.6 t 1.3% (n = 4).

Because these responses are partly polysynaptic, it was not clear whether transmission was blocked at sensory neuron synapses or at excitatory interneuron synapses. To clar- ify this point, the experiments with CNQX were repeated in an extracellular medium containing an elevated concentration of divalent cations to increase the threshold of action potential generation and to block polysynaptic transmis-

EXCITATORY AMINO ACID NEUROTRANSMISSION 1223

A ASW

FIG. 1. Effect of 6-cyano-7-nitroquinoxaline-2,3-

2:l ASW

I CNQX

dione (CNQX) on compound postsynaptic potentials in motor neurons of the gill and siphon withdrawal (GSW) reflex. A: compound postsynaptic potential evoked by siphon nerve stimulation and recorded in a motor neuron (MN) of the LFS group polarized to -80 mV. Appli- cation of 75 PM CNQX reduced these responses by 92%. Calibration: 10 mV, 40 ms. B: compound postsynaptic potential evoked by siphon nerve stimulation and recorded in an L7 motoneuron (MN) polarized with 2

- electrodes to -80 mV. This response was recorded in a medium containing a high concentration of divalent cations [ 2: 1 artificial seawater ( ASW )] and is mostly mono-

I CNQX

synaptic. Application of 75 PM CNQX reduced these responses by 79%. Cal ibratio n: 4 mV, 60 ms. C: mono-

C ASW

synaptic and cholinergic excitatory postsynaptic potential (EPSP) evoked in neuron RI 5 by stimulation of the right pleuroabdominal connective. The EPSP was unaf-

I fected by CNQX ( 75 PM ) . Calibration: 10 mV, 70 ms.

,155 A---l t

CNQX

sion (2: 1 ASW; see METHODS). Interstimulus interval was 2 sensory neurons, excitatory interneurons of the L34 and min. Under these conditions, the compound EPSPs pro- L29 groups, and LFS motor neurons. The LFS motor neu-

rons were used because it is easier to find synapses between duced by siphon nerve stimulation are mostly monosynaptic and stable in magnitude (Trudeau and Castellucci these neurons and excitatory interneurons. 1992). Figure 1 B illustrates that CNQX again produced a An LE sensory neuron was stimulated intracellularly clear reduction of the monosynaptic EPSPs. At a concen- every minute and the evoked EPSPs recorded in the moto- tration of 75 ,uM, a 79.1 t 4.1% ( YI = 8) decrease was ob- neuron and interneuron. The receptor blocker was bath ap- served (t = 2.4, P < 0.05). A dose-response curve for the plied after the second EPSP. In control experiments, no

drug was applied and the spontaneous decrement of the EPSP was monitored (n = 7). Figure 3, A and B show repre-

CNQX antagonism was obtained from four of these experiments. Figure 2 shows that 75 PM produced near maximal blockade and that the EC,, (concentration necessary to produce a 50% blockade) was ~50 FM. 120 -

The specificity of the antagonism produced by CNQX was tested by monitoring the effect of 75 ,uM CNQX on the cholinergic monosynaptic EPSPs evoked in neuron R15 of the abdominal ganglion by stimulation of the right pleuroabdominal connective (Frazier et al. 1967). The intestimu- lus interval was 2 min. The drug had no significant effect on this fast excitatory synapse (n = 3) (Fig. 1 C) . The peak amplitude of EPSPs was increased by 5.3 t 3.3% (t = 1.69, & not significant). b40 -

Sensory-motor EPSPs and excitatory interneuronal EPSPs 2o - are blocked by CNQX and CBPD

04 I I I I I To confirm the results obtained by studying compound 0 20 40 60 80 100

EPSPs, experiments with CNQX and CBPD were performed on identified monosynaptic EPSPs produced by sen-

CNQX (@A)

sory neurons of the LE cluster onto LFS motor neurons. FIG. 2. Dose-response curve of the effect of CNQX on compound

Simultaneous intracellular recordings were obtained in LE EPSPs evoked in motor neurons by siphon nerve stimulation. The EPSPs were recorded in 2: 1 ASW (n = 4).


t CNQX FIG. 3. Blockade by CNQX and I-(4-chlorobenzoyl)-

piperazine-2,3-dicarboxylic acid (CBPD) of monosynaptic junctions between identified sensory neurons and motor neurons of the GSW reflex. A-B: monosynaptic EPSPs evoked in an LFS motoneuron (MN) by an action potential in a sensory neuron (SN) of the LE cluster. Bath application

C

of CNQX ( 75 PM) (A ) or CBPD (75 PM) (B) significantly reduced the peak amplitude of the EPSPs. The traces were acquired successively with an interval of 1 min. Calibration for A: 20-35 mV, 50 ms. For B: 8-25 mV, 55 ms. C: sum- maryoftheeffectof75pMCNQX(n=7)orCBPD(n=5) on LE-LFS junctions. Interstimulus interval was 1 min. The

t

CBPD

antagonist was bath applied after the 2nd control EPSP (t ) . In the control experiments (n = 7) no drug was applied. The EPSPs are plotted relative to the initial control. Asterisks: significant difference between respective points on the control and CNQX (or CBPD) curves (P < 0.0 1).

Control

CBPD 75 phi CJ'WX 75 W

0123456789

TRIAL NO.

sentative pre- and postsynaptic traces from an experiment with CNQX and another one with CBPD. A clear reduction of synaptic transmission was obtained with both antagonists. Figure 3 C summarizes the results of seven experiments with CNQX and five experiments with CBPD. Two- way repeated-measures ANOVAs were used to analyze these data. The first factor was the group (control vs. CNQX or control vs. CBPD), whereas the repeated mea- surements acted as a second factor. For the CNQX data, the ANOVA confirmed that there was a significant overall effect of the treatment [ F( 1,ll) = 66.9, P < O.OOl] and a significant interaction between the treatment and the repeated measurement factor [ F( 7,77) = 13.3, P < O.OOl]. This last result indicated that the effect of CNQX depended on the trial number, i.e., there is no difference between the second EPSPs of the two curves. The overall effect of CBPD was also significant [ F( 1,9) = 26.3, P < 0.00 I], and a significant interaction was again obtained [F( 7,63) = 6.0, P < 0.00 I]. Average effects were calculated by comparing the last four points on the CNQX or CBPD curves (to allow for diffusion of the bath-applied antagonist) to the last four points on the control curve. With CNQX, the decreases for

the last four EPSPs compared with their respective untreated controls was 83.4%, 90.1%, 86.9%, and 85.2% for a mean decrease of 86.4 t 1.4%. With CBPD, the mean decrease for the last four EPSPs compared with the untreated controls was 70.7 t 1.8%.

In these experiments, the excitatory interneuron was also stimulated every minute, - 15 s after stimulating the sensory neuron. Figure 4, A and B show that CNQX and CBPD also produced a significant decrease in the peak amplitude of these EPSPs. The blockade was somewhat less extensive than for sensory-motor EPSPs when a concentration of 75 FM CNQX (~1 = 5) or CBPD (n = 4) was used (Fig. 4C). The average decrease for the last four EPSPs compared with their respective controls was 64.6 t 2.1% with CNQX and 67.2 t 2.9% with CBPD. The effects were reversible on washout of the antagonist. For the CNQX data, the two-way repeated-measures ANOVA revealed that there was a significant overall effect of the treatment [ F( 1,11) = 7 1.7, P < 0.00 l] and a significant interaction between the treatment and the repeated measurement factor [ F( 7,77) = 10.0, P < 0.00 I]. The overall effect of CBPD was also significant [F( 1,lO) = 81.9, P < O.OOl], and a


MN

INT

B t

CNQX FIG. 4. Blockade by CNQX and CBPD of monosynaptic

junctions between identified excitatory interneurons and motor neurons of the GSW reflex. A-B: monosynaptic EPSPs evoked in an LFS motoneuron (MN) by an action

MNN --G potential in an excitatory interneuron (INT) . Bath application of CNQX (75 PM) (A) or CBPD (75 PM) (B) significantly reduced the peak amplitude of the EPSPs. The traces were acquired successively with an interval of 1 min. Calibra-

INT _/Iz /1_______ _?I?___-

tion for A: 9-25 mV, 50 ms. For B: 8-20 mV, 55 ms. C: summary of the effect of 75 PM CNQX ( y1 = 5 ) or 75 PM CBPD (~1 = 4) on INT-LFS EPSPs. Interstimulus interval

C t

was 1 min. The antagonist was bath applied after the 2nd control EPSP (t ). In the control experiments ( n = 8) no drug was applied. The EPSPs are plotted relative to the ini-

CBPD tial control. Asterisk: significant difference between respective points on the control and CNQX (or CBPD) curves

* (P < 0.01). * *

E 100 -

g 80- 2 2 60-

$ ti

40 -

8 20-

Control

CNQX 75 pM CBPD 75 pM

01’...‘.... 0123456789

TRIAL NO.

significant interaction was again obtained [ F( 7,70) = 10.3, P < 0.00 11. In two experiments, however, EPSPs produced by an excitatory interneuron onto a motoneuron were not blocked by CNQX. This lack of effect was obtained while a nearly complete blockade of the sensory-motor EPSP was being observed in parallel recordings. These data are not included in the graph of Fig. 4 C and in the statistical analyses. These results suggest that there is heterogeneity at excitatory interneuronal synapses, either in the type or subtype of receptors or in the neurotransmitter released by these neurons.

Action ofpotential agonists on motor neurons

The results presented above suggest that the neurotransmitter( s) released by sensory neurons and some excitatory interneurons interacts with a postsynaptic receptor sharing some of the pharmacological characteristics of the non- NMDA excitatory amino acid receptor of vertebrates. We therefore investigated the effect of various excitatory amino acids on the postsynaptic neurons. This was tested by apply- ing, through a pipette, small 20-ms puffs of concentrated agonist solutions (0.1 M) above the cell body of some identified motor neurons in the isolated. desheated ganglion. A

high concentration was used in an attempt to maximize the activation of receptors that were situated in the neuropil and that would not be likely to be recruited by the application of a small puff of diluted solution at the level of the cell body.

We first investigated whether L-glutamate could be the natural ligand at these synapses. If this was the case, application of exogenous L-glutamate to motor neurons of the GSW reflex should have evoked an excitatory response. The recording presented in Fig. 5 A illustrates the result of a representative experiment. L-glutamate (n = 7 preparations) produced a clear hyperpolarizing response in these neurons. This was followed at times by a small reboundlike excitatory response. These effects were also observed in the presence of (TTX) (30 PM) and CoC1, ( 1 PM) under conditions where synaptic transmission is blocked (n = 3). Aspartate, applied in the same way, completely failed to affect the motor neurons (n = 5 ). Because the usual ligand of non-NMDA receptors in vertebrates, glutamate, did not produce strong excitatory responses in motor neurons or interneurons of the GSW reflex, we looked for other known excitatory amino acids.

HCA has been previouslv described as one of the most


A J ASW

4 L-GLU

B J

ASW

4 L-HCA

C --J ASW

chx

4 L-HCA

D

4*-

+ L-HCA

E --I

4 L-HCA

potent amino acids with excitatory actions in the CNS (Curtis and Watkins 1963 ) . Puff application of HCA (0.1 M) to LFS motor neurons produced a powerful excitation, usually preceded by a small hyperpolarization ( YL = 10 preparations) (Fig. 5 B) . Similarly, the excitatory interneurons, which are synaptic targets for sensory neurons, were also powerfully excited by HCA ( YI = 3; results not shown). Cysteic acid has also been reported to have excitatory actions in the vertebrate CNS (Curtis and Watkins 1963; Cur- tis et al. 196 1). We found that this molecule, similar to HCA but with a shorter side chain, strongly hyperpolarized motor neurons ( YI = 2). Cysteic sulfinic acid produced a similar effect (~2 = 2 ) . The dipeptide N-acetyl-aspartyl-glutamate, another molecule suspected to be an endogenous neurotransmitter in some regions of the vertebrate CNS (Jones and Sillito 1992), also produced mostly hyperpolarizing responses in motor neurons (n = 5 ).

If HCA activates the same type of receptors as those present at the synapses of sensory neurons and excitatory interneurons onto motor neurons, then its effect on motor neurons should be antagonized by CNQX because the antagonist blocks the synaptic responses. We found that in the presence of 75 PM CNQX, puff application of HCA to motor neurons produced effects that were considerably diminished relative to control conditions. The trace presented in

ASW T;X

ch2

ASW T;X

+ coc12

ci;ax

FIG. 5. Effects of homocysteic acid ( HCA ) and glutamate on motor neurons. A : puff application (see METH- ODS) of L-glutamate ( L-GLU) (0.1 M) to an LFS motoneuron produced a hyperpolarizing response that caused a pause in the spontaneous firing of the neuron. Resting potential: -45 mV. Calibration: 45 mV, 10 s. B: in a different LFS motoneuron, puff application of HCA (L- HCA) (0.1 M) produced a marked increase in the firing rate and a depolarization. Note the brief initial hyperpolarizing response. Resting potential: -47 mV. Calibra- tion: 45 mV, 10 s. C: in the presence of 75 PM CNQX, the excitatory effect of puff-applied HCA was considerably reduced. Note that the initial hyperpolarization was still evoked. Same neuron as in B. Calibration: 45 mV, 10 s. D: in the presence of 30 PM tetrodotoxin (TTX) and 1 mM CoCl, to block action potentials and synaptic transmission, puff application of HCA evoked a depolar- izing response in an LFS motoneuron. Note that small TTX-resistant action potentials were still produced. Resting potential: - 50 mV. Calibration: 22.5 mV, 10 s. E: in the presence of 75 ,uM CNQX as well as 30 PM TTX and 1 mM CoCl,, puff application of HCA had a greatly diminished effect. Same neuron as in D. Calibra- tion: 22.5 mV, 10 s. In all cases, the intracellular electrodes contained 2 M KAc.

Fig. 5C illustrates the response to HCA of an LFS motoneuron in the presence of CNQX. The same neuron responded very powerfully in control conditions (Fig. 5 B). These observations were replicated in five other experiments.

To confirm that the motor neurons were directly depolarized by HCA and to provide quantitative data on the blockade by CNQX, the experiments were repeated in the presence of TTX (30 PM) and also of CoCl, ( 1 mM) to block synaptic transmission and indirect effects produced by activating nearby interneurons. Figure 5 D illustrates that HCA can directly depolarize LFS motor neurons. This effect is again blocked by CNQX (75 PM) (Fig. 5 E; same neuron). This observation was confirmed in three other experiments. The surface area of these responses was decreased by 75.7 t 6.2% (t = 4.4, P < 0.01, y2 = 4).

To characterize further the pharmacological profile of the excitatory amino acid receptors that were activated by HCA, other agonists were tested in the same manner. We found that kainate powerfully excited all motor neurons tested in a fashion analogous to HCA ( n = 5 ) ( Fig. 6A ) . As for HCA, the effects of kainate were strongly reduced by 75 PM CNQX (n = 3) (Fig. 6 B) . Domoic acid, known to act preferentially at non-NMDA receptors of the kainate subtype in vertebrate systems (Watkins et al. 1990), was also found to excite motor neurons (~1 = 3), an effect that was


k KA

k KA

-I FIG. 6. Effects of excitatory amino acid receptor ago- . - nists on motor neurons. A: puff application (see MEW- ASW

&ax

J

ODS) of kainate (KA) (0.1 -M) to an LFS motoneuron produced a marked increase in the firing rate and a depolarization. Note the brief initial hyperpolarizing response. Resting potential: -50 mV. Calibration: 45 mV, 10 s. B: in the presence of 75 PM CNQX, the excitatory effect of puff-applied kainate was considerably reduced. Note that the initial hyperpolarization was unaffected. Same neuron as in A. Calibration: 45 mV, 10 s. C: in a different LFS motoneuron, puff application of quisqualate (QUIS) (0.1 M) produced a hyperpolarizing response that caused a pause in the spontaneous firing of the neuron. Resting potential: -47 mV. Calibration: 45

--I

k AMPA

mV, 10 s. D: puff application of ( &)-amino-3-hydroxy-5- methylisoxazole-4-proprionic acid (AMPA) (0.1 M) had no marked effect on the firing rate of LFS motor neurons. The brief hyperpolarizing response was of spontaneous origin. Resting potential: -47 mV. Same neuron as in C. Calibration: 45 mV, 10 s. E: puff application of

ASW D-glutamate (D-GLU) (0.1 M) hyperpolarized LFS motor neurons and caused a pause in their spontaneous firing. Resting potential: -50 mV. Calibration: 45 mV, 10 s. In all cases, the intracellular electrodes contained 2 M KAc.

k D-GLU

CNQX sensitive (~1 = 3). On the other hand, AMPA (~1 = blocked by the antagonists (Fig. 3, A and B). Monosynaptic 3) (Fig. 6C) and NMDA ( n = 3) had no effect, whereas EPSPs evoked in motor neurons by an action potential in quisqualate ( n = 3) (Fig. 6 D) and D-glutamate ( n = 3 ) (Fig. 6 E) produced mostly hyperpolarizing responses.

DISCUSSION

NMDA excitatory amino acid receptors of the kainate sub-

Our results provide direct evidence that excitatory synaptic action produced by sensory neurons and some excit-

type found in vertebrates because they are effectively

atory interneurons of the GSW reflex network ofAplysia is mediated through the activation of excitatory amino acid

blocked by CNQX and CBPD and activated by kainate and

receptors. These receptors share some similarities with non-

domoic acid (Watkins et al. 1990). We found that compound postsynaptic potentials

evoked in motor neurons by stimulation of the siphon nerve were antagonized by -90% by 75 PM CNQX or CBPD (Fig. 1). This suggested that most sensory neurons of the reflex release the same neurotransmitter. More direct

excitatory interneurons were also significantly decreased by CNQX at five of the seven synapses tested and by CBPD at four of four synapses tested (Fig. 4, A and B).

tors, depending for example on the location of synaptic contacts on a given postsynaptic neuron or on the identity

The finding that in two preparations excitatory interneuronal transmission was unaffected by CNQX may be explained by either or both of two possibilities. First, it is

of the postsynaptic neuron. Testing these possibilities

possible that excitatory interneurons of the GSW circuit

would require simultaneous intracellular recordings of

release different neurotransmitters. Second, all excitatory interneurons may release the same neurotransmitter, but they may activate different subtypes of postsynaptic recep-

more than one excitatory interneuron and a common fol- lower and/or simultaneous recordings of an excitatory interneuron and different postsynaptic targets.

Glutamate produced mostly hyperpolarizing responses in motor neurons of the GSW reflex, similar to what has been previously reported in the CNS of other invertebrates and for some Aplysia neuro ns (Bolshakov et al. 199 1; Has-

experiments performed at identified monosynaptic sensory-motor synapses con firmed that th ese connection s are


soni et al. 1992; Katz and Levitan 1993; Kehoe 1978; King and Carpenter 1989; Parmentier and Case 1972; Quinlan and Murphy 199 1; Yarowsky and Carpenter 1976). We observed in some experiments that a small and slow depolarization followed the prominent hyperpolarization induced by glutamate. This small rebound excitation was not blocked by CNQX (results not shown). These same neurons were, however, strongly depolarized by HCA, an effect that was antagonized by CNQX at the same concentration that blocks the synaptic responses (Fig. 5 ) . Our preliminary data indicate, furthermore, that the current produced by HCA in motor neurons has a reversal potential similar to that of the synaptic currents (between 0 and +20 mV).

The possibility that glutamate may nonetheless be the endogenous transmitter at these synapses may, however, not be rejected for the moment because of the three follow- ing reasons. First, it is possible that highly effective uptake systems prevent sufficient amounts of glutamate from reaching the synaptic receptors in the neuropil. An indirect argument against this possibility is that D-glutamate pro- duces the same effect as L-glutamate. It has been previously shown that the former is less effectively captured by high affinity uptake systems (Balcar 1992; Rosenberg et al. 1992; Steffgen et al. 199 1). Second, it is possible that the excitatory currents produced by glutamate desensitize ex- tremely rapidly (Huettner 1990) such that only inhibitory responses are detected. Because we have not used a very rapid agonist application system, this is certainly a possibility. It is possible that HCA, kainate, and domoate fail to induce a similar rapid desensitization of the receptors (Huettner 1990), so that their overall effect remains excitatory despite the simultaneous activation of inhibitory responses. Previous studies have, however, reported clearly detectable excitatory responses to glutamate applied by slow perfusion in some central neurons of Helisoma (Quin- lan and Murphy 199 1) and in the leech Hirudo medicinalis (Mat Jais et al. 1984). A third and complementary argument is that excitatory and inhibitory receptors may have a different topographical distribution, as is the case for locust muscle fibres (Cull-Candy 1976; Cull-Candy and Usher- wood 1973; Gration et al. 1979). The extrajunctional receptors could be inhibitory and possibly more numerous than junctional excitatory receptors; glutamate would therefore produce mostly hyperpolarizing responses when applied near the soma. Additionally, one would have to postu- late that HCA as well as kainate and domoic acid are more effective than glutamate at activating the excitatory receptors, perhaps simply because they produce less desensitization, as discussed above.

Evidence has accumulated in the last few years in favor of the hypothesis that HCA could be an endogenous ligand for excitatory amino acid receptors in the rat CNS (Cox et al. 1977; Curtis and Watkins 1963; Curtis et al. 196 1; Do et al. 1986a,b; Herrling et al. 1989; Ito et al. 199 1; Jones and Sillito 1992; Lee et al. 1988; Neal and Cunningham 1989; Ortega et al. 1990; Vyklicky and Vlachova 1992). How- ever, there has been no definitive evidence for an HCA-re- leasing synapse to this date in the vertebrate nervous system. Such synapses have been difficult to identify, partly because every region of the CNS that demonstrates excitatory responses to HCA is also excited by glutamate. It is

possible that the neurotransmitter released by sensory neurons and some excitatory interneurons of Aplysia is HCA or a substance closely related to it. However, we cannot exclude glutamate for the moment, and it will be necessary to directly detect which amino acid is present in these neurons and released in a Ca2+-dependent manner.

Our initial characterization of the synaptic receptors activated by the neurotransmitter of sensory neurons and excitatory interneurons should contribute to a better characterization of the learning-related plastic properties of synaptic transmission in Aplysia. Because of the availability of agonists and antagonists, it could now be possible to verify the involvement of receptor desensitization or downregulation in the process of short- and long-term habituation of the GSW or tail withdrawal reflexes. Also, the possible involvement of postsynaptic mechanisms in short- or long-term sensitization, besides the presynaptic processes that have already been described, may now be tested more easily. Fi- nally, because the transmitter of the facilitator/excitor L29 interneurons of the GSW reflex is still unknown (Kistler et al. 1985), it would be interesting to verify whether these neurons release an excitatory amino acid as suggested by our finding that most excitatory interneuronal synapses can be affected by CNQX and CBPD. The possibility that the activation of presynaptic receptors to excitatory amino acids leads to facilitation or inhibition of sensory-motor synapses should also be investigated. In conclusion, our results show that most fast excitatory synaptic transmission within the GSW reflex is mediated through the activation of excitatory amino acid receptors that are blocked by CNQX and CBPD but not by NMDA receptor antagonists. The endogenous ligand for these receptors is as yet unidentified but may be HCA or a closely related substance.

We thank Drs R. Dubuc, M. Klein, and J.-C. Lacaille as well as T. Ouimet for comments on the manuscript, Dr. G. Massicotte for helpful discussions, I. Morin and C. Charbonneau for the illustrations, and B. Emond for typing the manuscript.

This work was funded by Grant MT-12092 from the Medical Research Council of Canada and by the Richard and Edith Strauss Canada Founda- tion. L.-E. Trudeau is the recipient of a 1967 scholarship from the Conseil de Recherches en Sciences Naturelles et en Genie du Canada.

Address for reprint requests: V. F. Castellucci, Institut de Recherches Cliniques de Montreal 110, Avenue des Pins Ouest, Montreal, Quebec H2W lR7, Canada.

Received 16 February 1993; accepted in final form 5 May 1993.

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Excitatory Amino Acid Neurotransmission at Sensory-Motor