Action of gamma-aminobutyric acid derivatives on the bioelectrical activity of the metathoracic ganglion neurons in the cricket Gryllus domesticus

Comp. Gen. Pharmac., I974, Vol. 5~ PP. 127 to 134. Pergamon Press. Printed in Great Britain

127

A C T I O N OF G A M M A - A M I N O B U T Y R I C ACID D E R I V A T I V E S ON T H E B I O E L E C T R I C A L A C T I V I T Y OF T H E M E T A T H O R A C I C GANGLION N E U R O N S IN T H E C R I C K E T

GRYLLUS D O M E S T I C U S S. M. V E R E S H T C H A G I N , V. P. L A P I T S K Y AND I. A. SYTINSKY Laboratory of Comparative Physiology, Leningrad State University, U.S.S.R.

(Received 24 April I973) ABSTRACT I. Spontaneous impulses of the metathoracic ganglion neurons in the cricket Gryllus domesticus can be presented as unrhythmical and rhythmical discharges and the spikes in groups. 2. High concentrations (o"I M) of gamma-aminobutyric acid (GABA) and its derivatives (fl-alanine, gamma-amino-fl-hydroxybutyric acid (GABOB), gamma-hydroxybutyric acid (GHB), ~-phenyl-GABA, and homopantothenic acid) produced a pronounced inhibitory action on the bioelectrical activity of the cricket metathoracic ganglion which was restored by picrotoxin. 3. Low concentrations of these compounds (o.oi-o.ooi M) produced either a transitory activation of the bioelectrical activity preceding the following phase of depression, or showed no effect. THe. full identification of the nature of the transmitters of insects is still an elusive matter. GABA is the leading candidate for the inhibitory transmitter compound in the central nervous system (CNS) of insects (Kerkut & Walker, x966 ; Kerkut, 1969; Kerkut, Pitman & Walker, 1969a; Pitman & Kerkut, 197o ). The most frequent GABA effect in insect ganglia is depression of both spontaneous neuronal activity and the firing induced by some excitant compounds (picrotoxin, glutamic acid, etc.). There is some evidence for its role as a transmitter agent. Relatively high concentrations of GABA were detected in the insect CNS (Price, 196i ; Chen, I962 ; Frontali, I964; Ray, 1965; Jacobs, i 9 6 6 ; Jacobs, I968; Balogun, H a n i m a n n & C h e n , 1969; Fox & Larsen, i972 ; Hodgetts, 1972 ). The incubation of insect brain tissue homogenate with labelled glutamate shows its a-decarboxylation by conversion to GABA (Huggins, Rick & Kerkut, i967; Chen & Widmer, I968 ). Significant levels of the synthesizing

enzyme, glutamic acid decarboxylase (GAD; E.G. 4.1.1.15) were found in the brain of the honey-bee, Apis mellifera, and the housefly, Musca domestica (Frontali, 1964; Fox & Larsen, 1972 ). The new evidence that GABA is of importance in inhibitory synaptic transmission in insects was demonstrated by the presence of its degradative enzyme GABA: alpha (a) ketoglutaric acid aminotransferase (GABA-T; E.C. 2.6.1.19) in insect ganglia (Pasantes, Tapia, Ortega & Massiu, 1965). Howeyer, there is no evidence for the presence of GABOB and homopantothenic acids in the insect nervous system. Experimental data about the action of GABA and its derivatives on the bioelectrical activity of insect ganglia are not abundant. The inhibitory effect of GABA and fl-alanine was found at various stages of metamorphosis: caterpillar, pupa, butterfly (Vereshtchagin, Sytinsky & Tyshchenko, 1961 , a, b). Several reports in the literature indicate that the action of GABA on the nerve cord of grasshopper, locust & cockroach produced the inhibition of synaptic transmission (Suga &

198

VERESHTCI-I.AOINet al.

Katsuki, i96r ; Vereshtchagin, Sytinsky & Tyshchenko, I963, I964; Gahery & Boistel, i965; Kerkut, Leake, Shapira, Cowan & Walker, i965; Kerkut & Walker, I966, I967; Boistel, I968; Kerkut, 0liver, Rick & Walker, I97o; Pitman & Kerkut, I97o ). Kerkut, Pitman & Walker (I969b) found that iontophoretically applied GABA in small quantities caused hyperpolarization of cells in the metathoracic ganglion of Periplaneta americana. The initial excitatory phase was shown to precede the inhibitory action of GABA in isolated insect ganglia (Sittler & De Rerner, I967). Our previous work has shown that the GABA derivative, GABOB, inhibited the bioelectrical activity of butterflies, Dasychira pudibunda and Gastropacha quercifolia (Vereshtchagin, Sytinsky & Tyshchenko, I96I c). The action of GIIB led to a significant decrease of spontaneous locomotor activity of Drosophila melanogaster (Connolly, Tunnicliff & Rick, i97i). There are no data concerning the action of fl-phenyl-GABA (' phenyboot ') and of homopantothenic acid on the insect ganglia. The present paper describes the data of comparative investigation of six various derivatives of GABA on the metathoracic ganglion in the cricket Gryllus domesticus. The different concentrations of solutions tested were used for the investigation of the inhibitory action of these drugs. We hoped to detect various phases of depression which could not be found using the high concentrations. MATERIALS AND METHODS All the experiments were performed on adult house crickets Gryllus domesticus L. during three months (April-June). The animals were kept at room temperature (approximately 2o-25°C). The extracellular recordings of the bioelectrical activity of the metathoracic (third thoracic) ganglion neurons in the zone of the motor neuropile were obtained (FIo. I). The glass microelectrodes were filled with 2"5 M KC1 and had a resistance of io-2o M~. These electrodes were introduced into the ganglion by means of micromanipulator MM-I. Action potentials were amplified (using an amplifier UBP-I-o2), displayed on an oscilloscope (a bifilar oscillograph MPO-2) and photographed. In some cases for electrophoretic GABA (IM) administration the micro-

Comp. Gen. Pharmac.

FiG. i.--Diagram of the cricket metathoracic ganglion. The activity of single neurons was recorded from the hatched zones.

pipette was used with constant currents of 100-200 nA of appropriate polarity. The compounds tested were dissolved in a physiological solution for insects, pH 7.2 (Kerkut et al., I969 a, b). The following substances were used: GABA and fl-alanine (Reanal-BudapestHungary), GABOB (' Gamibetal ', Ono pharmaceutical Co, Ltd, Japan), GHB (production of USSR; Arendaruk, Serebraykov & Skaldinov, x963), homopantothenic acid (production of USSK; Kopelevich, Evdokimova, Marieva & Shmuylovich, I97I), fl-phenyl-GABA (' phenyboot', production of USSR; Perekalin & Zobacheva, 1959). The following concentrations of these drugs were applied: o.oox M; o.oi M; o-I M; 0. 5 M; x M. Picrotoxin ('Sigma' U.S.A.) was used as o'o25~o or o'I ~o. In most experiments the drug solution was introduced into the bath and left in contact with the preparation. RESULTS T H E SPONTANEOUS BIOELECTRICAL ACTIVITY

The bioelectrical activity of the central nervous system of insects is the result of the activity of many nervous cells. There are various cells which are capable of giving unrhythmical and rhythmical discharges, and spikes in groups (Vereshtchagin & Lapitsky, I971 ). Unrhythmical neurons have an asynchronous activity (FIG. 2a). T h e character of distribution of interimpulsive intervals has no obvious maximum on the histogram. The bioelectrical activity of rhythmical neurons is presented by the following action potentials on FIG. 2b. The distribution of inter-impulsive intervals of these neurons has an evident maximum.

I974, 5

aA~A ON

CRICKETGANGLION NEURONS

There is also a group of neurons giving the grouped discharges which consist of two, three or a greater number of the action potentials (Fla. 2c). The neurons with these types of bioelectrical activity were discovered in metathoracic ganglion of the cricket Gryllus domesticus L. This distribution of neurons into such groups is not absolute. There are ceils which change the rhythm of their activity with a definite temporal interval. The activity of these neurons is presented on FIo. 2 d-f. The rhythm of the action potentials appears to be defined by the functional condition of the neuron and the level of its membrane polarization. The stability of the frequency of the bioelectrical activity during short intervals (about 5 minutes) was of great importance for the experiments with drugs. ACTION OF HIOH CONCENTRATIONS OF G A B A A N D ITS D E R I V A T I V E S (0" I M )

GABA The first experiments were made on the perfused metathoracic ganglion with a partly removed covering. Fie. 3 a shows the rhythmical activity of single neuron. The frequency of the potentials was decreased three-fold within 2 minutes of the injection of GABA (o.oi M) in the perfusate. One minute later a decrease of the electrical activity was observed with the total disappearance of potentials (FIo. 3 b & c). This ganglion bioelectrical activity could be restored after 3 mlnutes washing by a physiological solution, but its frequency was decreased 7-fold. The preservation of the ganglioncovering did not affect the inhibitory 'action of GABA (o.x M). The decrease of the bioelectrical activity caused by GABA was observed within one minute. This recorded depression could be blocked under the influence of picrotoxin (o.o5%). Iontophoretic injection of GABA (I M, IOO-2OO nA) led to the decreased frequency of potentials which later were depressed. After switching off the current the activity was restored to the initial level and even increased in frequency (Fro. 4, a-d). The microelectrodes filled with I M NaCI

129

were used in control experiments. In these cases the iontophoretic current did not show any change during 6 minutes.

~-alanine Approximately the same effects were recorded while fl-alanine (o- I M) was apphed. The inhibitory effect disappeared under the influence of picrotoxin (FIG. 5).

GABOB A direct effect of GABOB was a decrease in frequency of the potentials, but the unrhythmical character of spontaneous activity was retained. Later on, a depression of electrical activity was developed till its complete cessation which was reversed under the influence of picrotoxin. In this case it was possible to obtain some increase in the frequency of the action potentials (FIo. 6).

GHB Injection of GHB (o.I M) in the perfusate led to a pronounced inhibitory effect (FIo. 7). This drug showed .that depression occurred during the first lO-i 5 seconds and had reached its maximum by the third minute. This inhibitory influence of GHB was observed on the effect caused by the previous use of picrotoxin (o.o25--o.i%) on the ganglion. In its turn the initial level of this activity with some bursts of potentials could be observed under the repeated application of picrotoxin.

Homopantothenic acid This drug (o. 5 M) has a specific action on the action potentials of the ganglion. At first an increase in the bioelectrical activity was observed, and the frequency of potentials doubled (FIG. 8). After this reaction, a 5-6-fold drop in activity level occurred. This decrease of the potentials could be restored afterwards to the initial level under the influende of picrotoxin (o.o25%).

~-phenyl-GABA This compound has a comparatively quick depressive reaction on the activity of nervous ganglia. Fie. 9 shows an example of the complete cessation of spikes in groups which occurred in 3 minutes and continued for 23 minutes (FIG. 9b). The effect of

Corot:. Gen. Pharmac.

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picrotoxin led to the appearance of the bursts of potentials which were intermittent with many discharges (frequency I6O impulses]second) (FIo. 9c). O F LOW C O N C E N T R A T I O N S O F GABA ITS D E R I V A T I V E S (O'OI--O'OOI M)

ACTION AND

o.oi M concentration For this concentration the usual response was a decrease in the activity of the metathoracic ganglion but not a complete cessation. In some cases the pronounced inhibitory effect of this drug was not seen. And in 3o-5o% of the cases, the injections of GHB or fl-phenyl-GABA were not followed by a depression of the electrical activity (Table I). o.ooi M concentration The effect of this concentration was rather variable. Sometimes it was possible to observe both the inhibitory and activatory effects. In 2o-25% of the cases the ganglion would seem to be unaffected by this low concentration of GHB, fl-phenyl-GABA or fl-alanine. It should be noted that the action of these compounds at this concentration (o.ooi M) is a cyclic change in the frequency of the action potentials. As a rule, the response most frequently observed began with a decrease of the frequency of the action potentials. In 4 ° seconds an increase in the

activity took place, followed by a secondary drop in 2 minutes, a rise in 5 minutes followed by a subsequent drop) (FIo. IO). Spontaneous changes of bioelectrical activity which were recorded during a more considerable period of time in comparison with the effect of these drugs confirm the specific character of their action. DISCUSSION At high concentrations of GABA and its derivatives the usual response was the pronounced depression of the bloelectrical activity which was observed earlier (Vereshtchagin et al., I96Ia , b, 1954; Gahery & Boistel, I965; Kerkut & Walker, I966 , 1967). Apparently, the effect of these drugs does not depend on the existence or absence of a covering. However, the period of the development of depression has a delay of from 3 up to 8 minutes. Perhaps the observed effect comes from the chemical structure of these compounds and, specifically, from the length of their carbon chain. Table I shows the variability in the sensitivity of the ganglion to GABA and its derivatives. In the concentration range of the compound tested we can establish that the effects of fl-phenyl-GABA and GHB were revealed most rapidly. It is necessary to indicate that fl-phenyl-GABA is synthetic compound which

i32

VERESHTCHAOINet al.

is not a normal metabolite of the nervous system as are GABA and its other derivatives. The GHB structure has no amine group. GABA, CABOB and fl-alanine, possessing a structural relationship, had approximately the same latent period for the depressions The homopantothenic acid, with the larger length of the carbon chain, had the maximum latent period. At the same time we can observe the initial distinct phase of excitation which usually preceded the subsequent depression. The similar effect of the rise in activity at the start of GABA injection was shown by investigating the neurons of a cockroach Periplaneta americana (Sittler & De Remer, I967). For the full explanation of the possible mechanism of this phenomenon additional experiments are necessary. Picrotoxin, the GABA antagonist, blocked its depressant effects. The study of the interactions of picrotoxin and GABA or fl-alanine shows that GABA- (or fl-alanine)induced depression of the bioelectrical activity was abolished by picrotoxin during 3 minutes. Pierotoxin also altered the depressant action of GLIB, CABOB or flphenyl-GABA within 4-5 minutes. It should be emphasized, however, that for 4 minutes picrotoxin also antagonized the effect of the homopantothenic acid which had the maximum latent period for its depressant effect. Low concentrations of the tested compounds (o-oI-o-ooi M) showed various effects. Perhaps the absence of the depressant action or even in some cases the strengthening of bioelectrical activity are caused by functional shifts in the nervous ceils, connected with their membrane potential. It is concluded that fl-phenyl-GABA, an artificial compound, has the greatest variability in its effect. The interesting phenomenon connected with the effect of the low concentrations of all GABA derivatives (o.ooI M), which show cyclic recurrence of the action potentials frequency alterations, should be noted. These changes cannot b e explained by spontaneous alterations of the bioelectrical activity. This recurrence may be connected with compensatory reactions of nervous cells which attempt to restore the normal rhythm

disturbed by derivatives.

6'omp. Gen. Pharmac. the

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SUMMARY High concentrations (o.I M) of GABA and its derivatives produced a depressant action on the biopotentials of the metathoracic ganglion neurons in the cricket Gryllus domesticus L. The degree of the depression was dependent upon the concentration of these compounds. Picrotoxin (o.o25-o-i%) blocked this inhibitory action but its excitatory effect was depressed by GABA and its analogues. A low concentrations of these compounds (o.oi-o.ooi M) produced some initial activation of the bioelectrical activity which was changed by its depression or did not reveal any effect. T h e character of action of GABA and its derivatives was dependent upon its molecular structure, its concentration and the functional condition of the neurons.

REFERENCES ARENDARUK, A. P., SEREBRAYKOV, L. A., & SKALDINOV, A. P. (I96]) , ~ Synthesis and some

pharmacodynamic problems of y-aminobutyric and y-oxybutyric acids ', Med. Ind. USSR, 5, 6-8 (in Russian). BALOOUN, R . A., HANIMA.NN, F., & CI-rEN, P. S. (z969), 'Separation of free amino acids and derivatives in the tsetse fly Glossina palpalis (Diptera) by ion-exchange chromatography', Experientia, 25, 93-95. BOISTEL,J. (r968), 'The synaptic transmission and related phenomena in insects ', Adv. Insect Physiol., 5, 1--64" CHEN, P. S. (I962), ' Free amino acids in insects ', in Amino Acids Pools; Distribution, Formation and Function of Free Amino Acids (ed. Holden), pp. 1 15-I 19. New York: Elsevier. CrmN, P. S., & Wm~mR, B. (x968), ' Content and synthesis of y-amlnobutyric acid in the larval brain of Drosophila melanogaster ", Experientia, 24, 516-517 . CONNOLLY, K., TtmmeLIFF, G., & RICK, J. T. (i971), ' T h e effect of hydroxybutyric acid on spontaneous locomotor activity and dopamine level in a selected strain of Drosophila melanogaster ',Comp. Biochem. Physiol., 4oB, 321-326. FROWrAU, N. (I964), 'Brain glutamic acid decarboxylase and synthesis of y-aminobutyric acid in vertebrate and invertebrate species ', in Comparative aVeuroehemist~y(ed. Richter), pp. x85I92. Oxford: Pergamon Press.

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133

miniature end-plate potentials and contractures Fox, P. M., & LARSEN,G. R. (I972), ' Glutamic of the coxal muscles of the cockroach Periplaneta acid decarboxylase and the GABA shunt in the supraoesophageal ganglion of the honey-bee, americana L. ', Comp. Biochem. Physiol., 28, 999xoo3. Apis mellifera ', 07. Insect Physiol., xS, 439-457. GA~mRY, J., & BOISTEL,J. (x965) , ' Study of some KOPELEVICH, V. M., EVDOKIMOVA, T. S.,MARIEVA, O. D., & SHmUYLOWCH, L. IVL (197x), 'Synpharmacological substances which modify the thesis of D-homopantothenic acid ', Chemicoelectrical activity of the sixth abdominal pharmac. 07.,9, 21-22 (in Russian). ganglion of the cockroach Periplaneta americana ', in The Physiology of the Insect Central .Nervous OLIVER, G. W. O., TABERNER, P. V., RICK, J. r., & KERKUT, G. A. (i97o), ~ Changes in G A B A System (eds. Treherne & Beament), pp. 73-78. level, G.A.D. and ChE activity in the CNS of London & New York: Academic Press. an insect during learning ', Comp. Biochem. HODOEa'rs, R. B. (I972), ' Biochemical characterPhysiol., 3813, 529-535 . ization of mutants affecting the metabolism of fl-alanine in Drosophila ', 07. Insect Physiol., I8, P m ~ r ~ L m , V. V., & ZOBACHEVA, M. M. (I959) , ' Synthesis of y-amino acids and pyrrolidones ', 937-947. 07. Gen. Chem. U.S.S.R., 29, 29o5-29Io. HUOGINS, A. K., RICK, . J . T . , & KERKUT,G. A. (I967), ' A comparative study of the inter- PITMAN, L. M., & KERKUT, G. A. (I97O), ' Comparison of the actions of iontophoretically mediary metabolism of L-glutamate in muscle applied acetylcholine and gamma aminobutyric and nervous tissue '. Comp. Biochem. Physiol., '~x, acid with the EPSP and IPSP in cockroach 23-29 . central neurons ', Comp. Gen. Pharmac., x, 221JAcoBs, H. E. (x966), ' Deposition of labelled beta230. alanine in ebony and non-ebony Drosophila melanogaster with notes on other amino acids ', PRmE, G. IVL (x9.6I), ' Some aspects of amino acid metabolism m the adult housefly Musca Genetics, 53, 777-784 • domestica ', Biochem. 07., 80, 42o-428. JACOBS, M. E. (I968), ' fl-alanine use by ebony and normal Drosophila melanogaster with notes on PASANTES, H., TAPIA, R., ORTEGA,B., & MAssm, G. (I965), ' F r e e amino acids and activity of glucose, uracil, dopa and dopamine ', Biochem. some pyridoxal phosphate-dependent enzymes Genet., i , 267-275. in the nervous system of ~ahree arthropoda KERKUT, G. A. (I969) , 'Neurochemistry of species', Comp. Biochem. Physiol., x6, 523 Invertebrates '. In Handbook of .Neurochemistry 528. (ed. LAJTHA) Vol. 2, pp. 539-562" New York: RAy, S. W. (x965), ' The free amino-acid pool of Plenum Press. cockroach (Periplaneta americana: Dictyoptera) K~RKUT, G. A., & WALKER, R. J. (i966), ' The central nervous system ', in The Physiology of the effect of L-glutamate, acetylcholine and Insect Central .Nervous System, (eds. Treherne & gamma-aminobutyric acid on the miniature Beament), pp. 31-37. London & New York: end-place potentials and contractures of the Academic Press. coxal muscles of the cockroach, Periplaneta SUOA, N., & KATSUra, J. (x96x), ' Pharmacological americana ', Comp. Biochem. Physiol., I7~ 435studies on the auditory synapses in a grass454. hopper ', 07. Exp. Biol., 38, 759-77o. KERKUT,G. A., PITMAN,R. M., & WALKER,R.J. SITTLBR, O. D., & DE I ~ m R , R. J. (i967), (1969a), ' Sensitivity of neurones of the insect ' G a m m a aminobutyric acid-induced changes central nervous system to iontophoretically in the spontaneous firing rates of insect neurons', applied acetylcholine or GABA', .Nature, Lond., Experientia, 23, 284-288. 222, Io75-xo76. VEI~ESHTCHAGIN, S. M., SYTINSKY, I. A., & KERKUT, G. A., PITMAN,R. M., & WALKER,R. J. TYSHCI-mNXO, V. P. (I96Ia), ' T h e effect of 7(1969b), ' Iontophoretic application of acetylaminobutyric a c i d ' and fl-alanine on biocholine and GABA onto insect central electric activity of nerve ganglia of the pine neurones ', Comp. Biochem. Physiol., 3x, 6 I I moth caterpillar, (Dendrvlimus pini) ', 07. Insect. 633. Physiol., 6, 21-25. KERKUT, G. A., OLrVER, G. W. O., RICK, J. T., & VERESHTCHAGIN, S. iV[., SYTrNSKY, I. A., & WALKER, R.J. (I97O), ¢ The effectsof drugs on TYSCHm'~O, V. P. (I96Ib), ' T h e effect of learning in a simple preparation ', Comp. Gen. 7-aminobutyric acid and fl-alanine on motor Pharmac., I, 437-483 . effects and bioelectrical activity in Annelids and KERKUT, G. A., LEAKE, L. D., SHAPIRA, A., Arthropods ', 07. Gen. Biol., 2 2 , 467-47 x (in CowAN, S., & WALKER, R. J. (I965), ' T h e Russian). presence of glutamate in nerve-muscle perfusates VER.ESHTCFIAGIN, S. M., SYTINSKY, I. A., & of Helix, Carcinu~ and Periplaneta ', Comp. TYSHCH~NKO, V. e. (196IC), 'Action of Biochem. Physiol., i5, 485-5o2. 13-oxy-y-aminobutyric acid on the bio-electrical K~RKUT, G. A., & WALKER, R. J. (i967) , ' T h e activity of ganglia of isolated nerve cord of effect of iontophoretic injection of L-glutamic Lepidoptera ', Dokl. Akad..Nauk SSSR, x38 , acid and 7-amino-.N-butyric acid on the 722-724 (in Russian).

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VERESHTCItAGIN, S. IV[., S'~TmSKY, I. A., & TYSHCHENKO, V. P. (I963), ' T h e inhibitory action of 7-aminobutyric acid upon the central synapses of insects ', .Nervous System Leningrad University, 4, xoS-i lo (in Russian). VERESHTCHAGIN, S, M,, SYTINSKY, I. A., & TvsHcrmm~o, V. P. (x964), '7-Aminobutyric acid and the inhibitory problem of the nervous system of Annelida and Arthropoda ', in The Role of Gamma-Aminobutyric Acid in Activity of .NervousSystem. (Ed. Leningrad University), pp. 95-Ioo (in Russian).

Comp. Gen. Pharmac.

Vza~srrrcaAoxr~, S. M., & LAPITSKY,V. P. (x97x), ¢The bioelectrical activity of the insect neurons ', Vestnik of Leningrad University, x5~ 146--x48 (in Russian).

Key Word Index: GABA, gamma-amino-flhydroxybutyric acid, gamma-hydrox~/butyric acid, fl-alanine, homopantothenic acid, insect CNS, Gr~llus domesticus.

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FIc. 4.--Effect of iontophoretically applied GABA (IM) on tbe spontaneous activity of the ganglion: (a) before GABA, (b) 3o seconds after the iontophoretic GABA (Ioo nA), (c) response to the same effect in 3 minutes. (d) activity restored 5 minutes after cessation of current.

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FIc. & - - T h e effect of GABOB (o.I M) on the spontaneous activity of the metathoracic ganglion neuron: (a) normal, (b) IO secouds after GABOB, (c) 8 minutes after GABOB, (d) activity restored after the action of o.o-o5 % picrotoxin in 3 minutes. Calibration: -o5o tzV, 2o mseconds.

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FIo. 7 . - - T h e effect of GHB (o. I M) on bioelectricai activity of the neuron: (a) normal, (b) 3 minutes after GHB, (c) after washing with a physiological solution in 4minutes. Calibration: 25ot~V , 2o mseconds

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Fro. 8 . - - T h e effect of the bomopantc.henic acid (0. 5 M) on the spontaneous neuron activity: (a) normal, (b) 4 minutes after action of tb~. homopantothenic acid, (c) 15 minutes after its action, (d) response after the action of picrotoxin to.o25 ~o) in 4 minutes. Calibration: 05o p.\z, 2o mseconds.

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Fro. 9 . - - T h e effect of fi-phenyI-GABA (o.z M) on the action potentials of neuron: (a) normal, (b) 3 minutes after action of ~-phenyl-GABA, (c) activity restored after the action of picrotoxin (o'I ~/o) in 5 minutes. Calilar,a tipn: 250/xV, 20 mseconds.