The effects of putative amino acid neurotransmitters on somata isolated from neurons of the locust central nervous system

Camp. B&hem.

Ph_vsiol. Vol. SOC, No. 2, pp. 231-236, 1985

Printed in Great Britain

0306-4492/85 S3.00 + 0.00 1985 Pergamon Press Ltd

c

THE EFFECTS

OF PUTATIVE

NEUROTRANSMITTERS NEURONS

OF THE D.

Department

ON SOMATA

LOCUST GILES*

of Zoology,

AMINO

CENTRAL

ACID

ISOLATED

FROM

NERVOUS

SYSTEM

and P. N. R. USHERWOOD

University of Nottingham, Nottingham Telephone: 0602-560101 13 June

(Received

NG7

2RD,

UK.

1984)

Abstract-l. Changes in spike frequency, membrane potential and input resistance of somata freshly isolated from neurons in the metathoracic ganglia of adult locusts (Schistocerca gregaria) during bath and ionophoretic application of putative amino acid transmitters and analogues were studied using intracellular techniques. 2. y-Aminobutyrate, glycine, taurine, cysteine and DL-ibotenate hyperpolarized the isolated soma, the response to kainic acid was depolarizing whereas L-glutamate and L-aspartate evoked a variety of potential changes. All of these compounds reduced the input resistance of the isolated soma. 3. Ionophoretic studies showed that the receptors for L-glutamate and y-aminobutyrate are diffusely distributed over the somal surface.

INTRODUCTION In previous publications technique for studying

from

our

laboratory

a new

the physiology and pharmacology of insect central neurons was described (Usherwood et al., 1980; Suter and Usherwood, 1985). This involved the exposure and dissociation of neuron somata of thoracic ganglia of the locust Schistocerca gregaria and their maintenance in vitro for periods of ca 12 hr post-excision. This technique was initially developed using locust neurons but is equally applicable to nervous tissue of other insects. The usefulness of this technique for studying the pharmacology of insect neurons has been underlined by preliminary studies of the influence of acetylcholine and other cholinometic agents on the isolated locust neuron soma (Usherwood et al., 1980; Suter and Usherwood, 1985) the results of which were qualitatively similar to those obtained from locust neurons (and other insect central neurons) in situ (Kerkut et al., 1969; David and Pitman, 1982; David and Sattelle, 1984). In this paper we describe the responses of neuron somata, isolated from the locust metathoracic ganglion, to a variety of putative amino acid neurotransmitters and analogues. We shall show that, despite the absence of synapses, the surface membrane of the locust neuron soma contains a rich array of extrajunctional receptors for amino acids. MATERIALS AND METHODS Dissociated neuron somata were obtained from adult Schistocerca gregaria (9) using the technique of Suter and Usherwood (1985). The isolated cell bodies were maintained in vitro for periods of 2-8 hr in saline of the following composition: NaCI, 214 mM; KCI 3.1 mM; CaCI,, 9.1 mM;

*Present address: Schering AG, Frohnau Research Postfach 650311, D-1000 Berlin 65, FRG.

Station,

231

Tris-HCI, 3.1 mM; pH adjusted to 7.0 with NaOH (modified after Kerkut et al., 1969). The electrophysiological techniques were identical to those used by Suter and Usherwood (1985), with intracellular recording and stimulatingelectrodes filled with 2 M potassium acetate. Two intracellular electrodes were used to determine input resistances. Drugs were applied either in the bath or by ionophoresis from micropipettes. The pipettes were tilled with 1 M amino acid

dissolved in water at appropriate undertaken at ca 20°C.

pH. Experiments were

RESULTS

The electrophysiological properties of the isolated soma were similar to those described by Usherwood et al. (1980) and Suter and Usherwood (1985). The resting potential was 49 + 11.9 mV (mean f SD; N = 16) and the input resistance 24.1 + 8.8 MR (N = 16). About 30% of the somata were spontaneously active, with action potentials occurring at a frequency of 0.1-5 Hz either at regular intervals (Fig. 1A) or in “bursts”. About 60% did not generate action potentials spontaneously but did so in response to current injection (Fig. 1B). The action potentials ranged between 10 and 100mV in amplitude (54.4 + 22.2mV; N = 16) often with negative afterpotentials of 10-50 mV (18.8 f 13 mV; N = 10). y-Aminobutyric

acid (GABA)

The effects of bath-application of GABA (10m4 to lo-‘M) were studied on eight somata (Table 1). There was a lo-fold variation in sensitivity to this amino acid between cell bodies, but in all cases GABA elicited a hyperpolarization accompanied by a decrease in input resistance and a cessation of spike activity (Fig. 2). The hyperpolarizations ranged between 10 and 15 mV and the changes in input resistance between 60 and 90%. With prolonged (30-60 min) applications of GABA a slow but incomplete decline in responsiveness was recorded as the membrane potential and input resistance returned

D.

232

GILES and

P. N. R. USHERWOOD

A

A

l&M

GABA

U4M

GABA

-I ll-i

Fig. 1, Electrical activity of neuron somata freshly isolated from the metathoracic ganglion of Schistocerca gregaria. A. Intracellular recording (lower trace) from spontaneously active soma. Upper trace is zero membrane potential. Note large negative after potentials. Calibration; 10 mV, 200 msec. B. Action potential recorded intracellularly during passage of a depolarizing current (1 nA) through a second intracellular electrode. Resting potential of soma was 50mV. Calibration; IOmV, 200msec.

towards pretreatment levels. All of the effects of GABA were fully reversible when this drug was removed from the bathing medium. Although cell bodies responding to GABA ionophoresis were readily found, a few somata did not respond. We have no evidence that responsiveness to GABA ionophoresis was a property of specific soma types delineated, for example, by morphological and/or electrophysiological characteristics, but a systematic study may well reveal such relationships. Ionophoretic application of GABA evoked a transient hyperpolarization; the amplitude, time to peak and decay time of this response increased with increasing GABA dose (Fig. 3). This observation together with the fact that responses could be elicited over much of the soma .membrane indicates a diffusely distributed GABA receptor population. When GABA was ejected at pulse frequencies Table I. Summary

> 0.5 Hz the GABA potentials summated but there was no evidence of desensitization (Fig. 3B). For somata which were spontaneously electrically excitable, it was possible to inhibit action potential production with large (ca 5 nC) ionophoretic doses of GABA. Dose-response curves for GABA ionophoresis were more linear for low doses than for high doses (Fig. 3C,D). The maximum slopes of these relationships ranged between 5 and 10 mV/nC. Because the population of GABA receptors on the soma membrane was diffusely distributed and potential rather than current was the dependent variable, it is difficult to draw quantitative conclusions from the dose-response curves. L-GIuramate

Many cells (ca 60%) did not respond either with a potential change or with a change in input resistance

of responses to amino acids (bath-applied) of neuron somata freshly isolated from locust central nervous system

Amino acid (concentration) GABA (3 1O-4 M) L-Glutamate (>5 x 10-4M) L-Aspartate (>Sx IO-‘M) Kainic acid (1O-3 M) Ibotenic acid &5$;i4 M) (5 x IO-‘M) Taurine (5 x 10m3M) Cyst&e (5 x IO-‘M)

Fig. 2. Response of isolated somata to bath application of 10m4M GABA. A. Hyperpolarization of a spontaneously active soma with loss of spike activity. Note slow recovery of membrane potential in continued presence of the amino acid. Resting potential, -45 mV. Calibration; 20 mV, 10 sec. B. A second soma with a - 60 mV resting potential was hyperpolarized by GABA. This change in membrane potential was accompanied by a fall in input resistance (monitored by the change in amplitude of hyperpolarization to current pulse injections of 1 nA through a second intracellular electrode) with some evidence of “desensitization”. Calibration; 20 mV, 10 sec. Duration of drug application denoted by bars above traces. The effects of GABA were fully reversible.

Proportion of cells responsive

Spike generation (spontaneous)

S/S 12/30 l/30 3111 4117 3113 l/3

Inhibited Inhibited Enhanced Inhibited Enhanced Enhanced Not present

Hyperpolarization Hyperpolarization Depolarization Hyperpolarization Depolarization Depolarization Hyperpolarization

416

Inhibited

Hyperpolarization

215

Inhibited

Hyperpolarization

216

Not present

Hyperpolarization

A membrane

potential

Effect of amino A

acid transmitters

on locust

neurons

233

B

K 0 5I =-----’ “id---2‘

iMfv 4 ’

.

tili/vfl/L/

““i-l----

J A

I

Dose(nCi

lb

I

lb

Dose(nC)

Fig. 3. Hyperpolarizations to ionophoretic application of GABA to freshly dissociated somata. A. Increasing doses of GABA increased the amplitude of the hyperpolarizations, their rise times and the duration of their falling phases. Calibration; 10 mV, 100 msec. B. Repeated application of 3 nC pulses of GABA lead to summation of hyperpolarizations but there was no evidence for desensitization. Calibration; 10 mV, 200 msec. C. Log-log plot of amplitude of peak GABA response (hyperpolarization) vs ionophoretic dose of GABA. D. Lower graph; log-log plot of GABA dose vs response. Upper graph; relationship between time to peak GABA response vs ionophoretic dose.

to bath applied L-glutamate, even at concentrations as high as lo-‘M (Table 1). The rest, with 5 x 10m4 to lo-’ M L-glutamate either were hyperpolarized by 15-25mV (Fig. 4) or exhibited biphasic potential changes (a transient hyperpolarization of lCk20mV 5 x

of l-5 set duration followed by a sustained hyperpolarization) or were slightly (l-2 mV) depolarized. In all instances these changes in membrane potential were accompanied by reductions in input resistance of 50-90°/0 and loss of action potentials in spontane10m4 M

L-glutamate

A

5 x 10e4 M L -glutamate

Fig. 4. A. Response of isolated soma to bath applied L-glutamate (5 x 10m4 M). This soma had a resting potential of -45mV. Application of L-glutamate hyperpolarized the soma and inhibited spontaneous spiking, but the response waned within 1 min such that the membrane potential returned to its pretreatment value and spike activity was resumed. Calibration; 20mV, 20sec. B. Hyperpolarization, reduction in input resistance (reduction in amplitude of hyperpolarization to current injection) and loss of spike production during bath application of 5 x 10m4 M L-glutamate to an isolated soma (resting - 45 mV). Calibration; 20 mV, 25 sec. Duration of t-glutamate application denoted by bars potential, above traces. The effects of L-glutamate were fully reversible.

234

D. A

GILES

and P. N. R.

USHERWOOD

B

K 0 5-,-

’ -,Y

bT?-

5-

-l <

Dose

Dose

tnC)

(nC)

Fig. 5. A. Hyperpolarizations elicited by ionophoresis of L-glutamate to an isomated soma. The amplitude, time to peak and decay time of the response varied with ionophoretic dose. Calibration; 10 mV, 200 msec. B. Repeated application of L-glutamate (6 nC) caused desensitization. Calibration; 10 mV, 100 msec. C. Log-linear plot of response amplitude vs ionophoretic dose of L-glutamate. D. Lower graph; log-log plot of response amplitude vs ionophoretic dose. Upper graph; change in time to peak of ionophoretic glutamate potential with dose.

spiking neurons. The biphasic changes in membrane potential are reminiscent of those recorded during L-glutamate application to extrajunctional membrane of locust leg muscle (Lea and Usherwood, 1973; Cull-Candy and Usherwood, 1973) where two populations of receptors for this amino acid co-exist. Usually the membrane potential and input resistance returned to pre-treatment values (and in appropriate cases spiking activity resumed) after 3&90 set in L-glutamate (5 x lo--” M) (Fig. 4A). If the glutamate concentration in the bath was then increased (e.g. to 10m3M) further changes in membrane potential, input resistance and spike production did not arise. Ionophoretic application of glutamate evoked transient hyperpolarizations, 24 mV in amplitude with rise times of 40-500msec (Fig. 5A,B). ously

A

Neither depolarizations nor biphasic responses to t-glutamate ionophoresis were observed. Repetitive application of L-glutamate by ionophoresis induced desensitization (Fig. 5B). Doseeresponse curves for L-glutamate (Fig. SC,D) were similar to those obtained during GABA ionophoresis. The increasing rise and decay times of the glutamate depolarization with increasing ionophoretic dose suggests a diffuse distribution of glutamate receptors over the soma membrane. In fact glutamate potentials were readily obtained on any part of the surface of the isolated soma, but not on the axon stump. Interaction

between GABA

and glutamate

responses?

When GABA and L-glutamate were applied ionophoretically to the same area of somal membrane it f3

C

Fig. 6. Ionophoresis of 3 nC of L-glutamate onto a hyperpolarization (A), but a second application However, when a 3 nC dose of L-glutamate (D) hyperpolarization was recorded. This indicates that receptor sites. although they may gate the same

an isolated soma (resting potential, -45 mV) elicited of drug delivered 5 set later elicited no response (B). followed 5 set after a 0.6 nC dose of GABA (C) a GABA and L-glutamate do not compete for the same class of ionic channel. Calibration; 5 mV, 200 msec.

Effect of amino

acid transmitters

was found that for a given hyperpolarization or a given dose the time to peak of the glutamate response was longer than that for GABA, but there was no interaction between the responses (Fig. 6). Both responses were first converted to depolarizations and then abolished when chloride in the bathing medium was replaced isosmotically by the supposedly impermeant anion sulphate. (The depolarizing and hyperpolarizing Cl--transients which accompanied removal and restoration of Cl-, respectively, suggest that this anion is permeant in the resting soma and passively distributed across the somal membrane.) However, replacement of chloride by sulphate sometimes initially converted a GABA hyperpolarization to a biphasic response with a depolarization followed by a hyperpolarization. Pitman and Kerkut (1970) have previously shown that the GABA responses of cockroach cell neuron somata are Cl--dependent with perhaps some contribution from K+. This also seems to be true for the GABA and glutamate hyperpolarizations recorded extrajunctionally from the isolated locust neuron soma. L-Aspartate

This amino acid was applied to 17 somata (Table 1). Seven somata responded reversibly to L-aspartate but only when exposed to concentrations 3 5 x 10m3 M. Three somata were hyperpolarized by 5-15 mV (10.3 f 4.2) with 5 x 10m3 M L-aspartate, a change which was accompanied by a fall in input resistance of 3&70% and loss of spontaneous spiking. In two of these somata the response to t_-aspartate remained constant for 10 min but in the other cell body the response waned within 1 min. Four somata were depolarized by t_-aspartate and exhibited an increase in spontaneous spike frequency. Both changes were accompanied by a fall in input resistance of 30-50x. Attempts (> 100 somata tested) to obtain responses to L-aspartate ionophoresis were unsuccessful even with doses as high as 300 nA applied for > 1 sec. ix-Ibotenic cysteine

acid,

taurine,

A minority of somata acids (Table 1).

glycine,

responded

kainic

acid

and

to these amino

on locust

neurons

235

bearing here, the most likely reason for the difference is the loss of glial investments during soma isolation. The presence of glial cells in vivo may play a major role in controlling the amino acid environment of the neuron soma. A similar explanation has been invoked to explain the higher sensitivity of freshly isolated locust somata to acetylcholine (Usherwood et al., 1980; Suter and Usherwood, 1985) although the extracellular level of this putative transmitter is probably controlled in uivo by acetylchohnesterase on glia and somata rather than by sequestration into glia. Until more extensive pharmacological studies are undertaken on isolated locust neuron somata it would be unwise to generalize on their pharmacological properties. All that can be said at present is that they clearly contain a rich array of extrajunctional receptors which deserve further study. But perhaps this is not a surprising conclusion since Usherwood and Machih (1966, 1968), Dowson and Usherwood (1972) and Fulton and Usherwood (1977) have shown that the axon terminal membrane of locust motoneurons is also well-endowed with receptors for many different putative neurotransmitters including amino acids. Although the functions of these receptors are unclear it has been suggested that they reflect the expression by the motoneurons of postjunctional receptors with similar ligand binding properties (Usherwood, 1981). There is already evidence that acetylcholine and GABA synapses are present on cockroach central neurons (Pitman and Kerkut, 1970) and this may also be true for locust motoneurons. It remains to be established whether synapses for some of the other amino acids referred to herein are also present. Because of the large size of the neuron somata used in our studies we have broadly assumed that they all originated from motoneurons. However, more detailed studies will be required to substantiate this conclusion. What has become abundantly clear from these and associated studies (Usherwood et al., 1980; Suter and Usherwood, 1985) is that the isolated neuron soma provides a convenient and reliable pharmacological assay and that its high potential for voltage clamp and patch clamp studies of receptors for putative neurotransmitters in the insect central nervous system awaits exploitation.

DISCUSSION

of GABA on insect have indicated a low sensitivity to this amino acid. For example, high concentrations (lo-* M) of GABA were required to evoke a response from intact cockroach ganglia (Callec, 1974), ionophoretic application of GABA to neurons in the cockroach 6th abdominal ganglion indicated a low sensitivity of ca 0.05 mV/nC (calculated from Fig. 3.26, Callec, 1974) and Kerkut et al. (1969) obtained a sensitivity of only ca 0.1 mV/nC for GABA ionophoresis onto neurons in the cockroach metathoracic ganglion. According to our studies the sensitivity of somata freshly isolated from the locust central nervous system is l-2 orders of magnitude higher than the figure quoted by Kerkut et al. (1969). While ion redistribution across the membrane of the locust soma during its isolation may have some Previous

central

studies

nervous

of the action

systems

REFERENCES

Callec J. J. (1974) Synaptic transmission in the central nervous system of insects. In Insect Neurobiology (Edited by J. R. Treherne), pp. 119-186. North Holland, Amsterdam. Cull-Candy S. G. and Usherwood P. N. R. (1973) Two populations of L-glutamate receptors on locust muscle fibres. Nature New Biol. 246, 62-64. David J. A. and Pitman R. M. (1982) The effects of axotomy upon the extrasynaptic acetylcholine sensitivity of an identified motoneurone in the cockroach Periplanefu americana. J. exp. Biol. 98, 329-341. David J. A. and Satelle D. B. (1984) Actions of cholinergic pharmacological agents on the cell body membrane of the fast coxal depressor motoneurone of the cockroach (Periplaneta americana). J. exp. Biol. 108, 119-136. Dowson R. J. and Usherwood P. N. R. (1973) The effect of

236

D. GIL= and P. N. R. USHERW~~D

low concentrations of L-glutamate and L-aspartate on transmitter release at the locust excitatorv nerve-muscle synapse. J. Physiool.229, 13-14. Futton B. and Usherwood P. N. R. (1977) Presvnaotic \ acetylcholine action at the locust neuromuscular jut&on. I~e~&~ophurmaco~o~.~ 16, 877-880. Kerkut G. A., Pitman R. and Walker R. (1969) Ionophoretic application of acetyicholine and GABA onto insect central neurones. Coq. Biochem. Physiol. 31, r

<

61 l-633.

Lea T. J. and Usherwood P. N. R. (1973) The site of action of ibotenic acid and the identification of two populations of glutamate receptors on insect muscle-fibres. Comp. gen. Pharmuc. 4, 333-350.

Pitman R. M. and Kerkut G. A. (1970) Comparison of the actions of ionophoretically applied acetylcholine and

gamma aminobutyric acid with the EPSP and IRSP in cockroach central neurones. Camp. gen. Pharmac. 1, 221-230.

Suter C. and Usherwood P. N. R. (1985) Action of acetylcholine and antagonists on somata isolated from locust central neurones. Camp. &o&em. Physiol. SOC, 221-229. Usherwood P. N. R. (1981) Glutamate synapses and receptors on insect muscle. In Glutamate as a Neurotransmitter (Edited by Di Chiari G. and Gess G. L.), pp. 183-193. Raven Press, New York. Usherwood P. N. R. and Machili P. (1966) Chemical transmission at the insect excitatory neuromuscular synapse. Nature, Lond. 210, 634-636. Usherwood P. N. R. and Machili P. (1968) Pharmacological properties of excitatory neuromuscular synapses in the locust. J. exp. Biol. 49, 341-361.