Influence of putative neurotransmitters on brain electrical activity in Lithobius Forficatus L. (Myriapoda Chilopoda)

Camp.

Biochem.

0306~4492183 $3.00+ 0.00 Q 1983Pergamon Press Ltd

Vol. 76C, No. 2, pp. 237-240. 19X3

Physiol.

Printed in Great Britain

INFLUENCE OF PUTATIVE NEUROTRANSMITTERS ON BRAIN ELECTRICAL ACTIVITY IN ~IT~~~~US FU~FrCAT~S L. (MYRIAPODA CHILOPODA) MICIIEL DESCAMPS’ and BERNARD LASSALLE’ ‘LA au CNRS no. 148 (Endocrinologie comparhe des Invertibrds) and “Laboratoire de Morphogenese Animale, Service de Biologie Animale, Universitt de Lille I, 59655 Villeneuve d’Ascq Cedex, France. Telephone: (20)91-9222 (Rewived 28 February 1983) Abstract-l. Various putative neurotransmitters and related drugs were tested on the electrical activity of the brain of L. jii~ficatm, at various times during the year. 2. Indirect evidence exists for a depressant action from GABA. Ach, 5 HT, NA and DA have stimulatory effects. 3. However, DA effects were found to vary with the season, suggesting a role in the control of an annual cycle.

INTRODUCTION It has been shown in the centipede Lithobius forJicatus L. that the brain, mainly the endocrine areas of the protocerebrum, is involved in the control of various physiological processes such as the moulting cycle (Joly, 1966), spermatogenesis (Descamps, 1974, 1975) and oogenesis (Herbaut, 1975, 1976). Moreover, a steroid hormone, 20 OH-ecdysone, has a direct action on the brain? both on the electrical activity (Descamps and Lassalle, 1981) and on the synthetic activity of neurosecretory cells of the pars intercerebru~~~(Jamault-Navarro et af., 1983). The aim of the present experiments was to investigate the effects of various neurotransmitters and drugs on brain electrical responses in L. forfjcatus.

MATERIALS AND

METHODS

Experiments were carried out on adult male and female L. Jixjicatus L. (mean weight: 100 mg). The centipedes were anaesthetized with ethyl ether and the brain was exposed by surgical removal of the cephalic cuticle. &in electrical activity &as recorded by an extracellular SNEX 200 semi-micro bioolar electrode (Rhodes Medical Instruments, Inc.). This bipolar electrode‘ consists of two 0.1 mm insulated stainless steel wires separated by a 0.25 mm shaft. The entire electrode, except for the contacts, is insulated. The electrical activity was recorded with a Tektronix 5031 oscilloscope after amplification with a WPI differential preamplifier with a band width fixed between 100 Hz and 30 kHz. Electrical activity was also analysed with a discriminator (Neurolog System). We first recorded the basal electrical activity and then the activity resulting from the addition of various neurotransmitters and drugs into the haemolymph with a Hamilton micro-syringe. After several experiments, the amount of solution supplied to the animal was standardized at 0.5 ~1. The following drugs were used; unless otherwise indicated all products were of Sigma grade: Acetylcholine (Ach), Eserine (Fluka), Nicotine, ~-Tubocurarine, Tetra~thylammonium (TEA, Fluka), Gallamine, Muscarine, A&opine, y-amino butyric acid (GABA), y-hydroxybutyric

acid picrotoxin (Serva), L-glutamate, (GHB), 5-hydroxytryptamine (5 HT, serotonin), Imipramine, Reserpine (Serva), Nialamide, Methysergide (Sandoz), norepinephrine (noradrenalin, NA), Tetrabenazine (TBN) (Fluka), Propanolol, 3-Hydroxytyramine (Dopamine, DA) and Phenylhydrazine. These various products, generally in a 10m4 M Ringer solution (Ephrussi and Beadle, 1936), were tested at different times of the life cycle of the animals: during winter (physiological rest period, Joly and Descamps, 1969; Descamps, 1981), early spring (beginning of physiologica activation), late spring (June, period of maximum rate of moulting and spe~atocyte growth) and autumn (decrease of physiological activity). RESULTS

Five to ten animals were used in each experimental series. As previously described (Descamps and LasSalle, 1981), only basal electrical activity can be recorded from the brain of anaesthetized animals. As the basal electrical activity differs from one animal to the next, a control was recorded for each animal. No change in brain electrical activity was observed after the addition of Ringer’s kid.

No change in the electrical activity was recorded after addition of 10e4M Ach. A concentration of lo-* M was necessary for an electrical response (Fig. 1). The addition in the haemolymph of eserine (lo-* M), known as anti-cholinesterase, led to an increase in the brain electrical activity (Fig. 2). Effects of Ach can be potentiated by simultaneous injection of eserine. In this case only 10e3 M solutions were necessary (Fig. 3). Nicotine, in lo-’ or lo-“M solutions, also increased the electrical activity (Fig. 4). TEA antagonized the effect of Ach (Fig. 5) but d-Tubocurarine and gallamine had no effect. Muscarine in 1W4 or lo-‘M solutions led to a weak stimulation (Fig. 6). Atropine (lo-’ M) had an inhibitory effect on brain electrical responses (Fig. 7).

MICHEL DFSCAMPSand BERNAKDLASSALLE As.tyklmnnaW’M

80‘P,. t

Figs. 1-7. Effects of various drugs on the electrical activity of the brain of Lifhobius. Figures 2 and 4 represent the recordings of spike discharge before (upper fine) and after (lower line) addition of eserine (IO-’ M) (Fig. 2) and,nicotine 10s4 M (Fig. 4). Vertical bar: 100FV, horizontal bar: 400 msec. Figures

1, 3. 5, 6 and

7

represent the graphic recordings of discharge frequency before and after addition of the drugs indicated in the figures.

GABA

GABA and GHB (structural analogue of GABA, but more effective) had no effect. Nevertheless, picrotoxin, an antagonist of GABA, had a typical stimulatory effect (Fig. 8).

No physiological response was obtained with this compound. 5HT

Serotonin had a st~mulatory effect on brain electrical activity (Figs 9 and 10). This effect, obtained after a latency period of 15-30 set disappeared after washing with Ringer or after adding methysergide (Fig. 10). It was possible to moderate or stimulate the electrical activity alternately by successive injections of methysergide and 5 HT. Nialamide, another putative antagonist of 5 HT, was without effect. Of the agonistic compounds tested (imipramine and reserpine) only imipramine had a weak effect. NA

Injection of NA had Iittie effect on the electrical activity. It was only after discriminator analysis (Fig. I I) that it was possible to observe a stjmulatory effect of NA. TBN did not antagonize NA action whereas propanolol did. DA

Results were very interesting: the use of all other compounds led to the same response throughout the

year, but for DA responses were related to the period of experiment. Electrical activity was stimulated by DA in winter (essentially with 10e3 M solutions), but results were not constant in early spring and no effect could be recorded in June. Concerning this last case, it must be pointed out that the centipedes which were put at 5°C for at least 3 or 4 hr showed a typical increase in electricai activity after injection of DA (Fig. 12). Shorter exposures (30 min and I hr) had no effect. While this cold shock of 4 hr was sufficient for 100% ‘*unlocking” of the animals in June, only about 60% were unlocked in autumn experiments. In late November the responses obtained were comparable to those of early spring. The effect of DA injections was antagonized by addition of phenylhydrazine. It must be pointed out that this effect was sometimes obtained after a period of increased activity (Fig. 12). DISCUSSION

As shown by our results, a wide range of neurotransmitters can act on brain electrical activity in L. .forjicarus. In short, with the exception of r&ttamate, all the putative neurotransmitters tested have an electrical action on brain activity. Ach, 5 HT, NA and DA have stimuIatory effects and GABA seems to be the depressant neurotransmitter of the central nervous system. Concerning Ach, it may be postulated that the action of this neurotransmitter, only revealed at high doses, is related, as in insects, to large amounts of cholinesterases; this finding is in agreement with the eserine (anticholinesterase) action

Neurotransmitters on centipede brain

9

8

Control

239

Sarotonin

Methyssrgide

10m4M

Noradrenalin

Control

10m4M

I

IO-” M

80 sp/s t 20s 11 Figs. 8-l 1. Effects of various drugs on the electrical activity of the brain of ~~~~~~~~~. Figures 8 and 9 represent the recordings of spike discharge before (upper line) and after (lower line) addition of picrotoxin (10m4M) (Fig. 9). Vertical bar: 100 pV; horizontal bar: 400 msec. Figures 10 and 11 represent the graphic recordings of discharge frequency before and after addition of serotonin and methysergide (Fig. 10) and noradrenalin (Fig. 1I).

and with the potentiation of the effects of Ach and eserine. There is only indirect evidence of the inhibitory effect of GABA, since picrotoxin has a potent stimulating action on the brain electrical activity. Why is there no response when GABA is added in the haemolymph? It may be due either to a brain barrier or to the Lack of receptors open to exogenous GABA. After NA injection only weak increasing responses can be recorded: it seems to be related to the number

Dopamine

Control I

1

Phenylhydrazint

of NA neurons in the protocerebrum (perhaps only 2, unpublished data). The difference of action of DA with the season is interesting. Moreover, the fact that DA receptors can be activated after a cold exposure to 5°C for at least 3 or 4 hr suggests that DA is probably involved in the control of an annud cycle. Results following phenylhydrazine injection can be explained by the antide~rboxylasic action of this drug. Indeed, based upon the metabolic pathways of neurotransmitters in Insecta, as described in the

10m3M

1

10m4M

Fig. 12. Graphic recording of the discharge frequency of the brain of Lirhobiusbefore and after addition of dopamine and phenylhydrazine.

240

MICHEL DESCAMPS and BERNARD LASSALLE

review of Lafon-Cazal (1978), the increased electrical activity would be the result of the inhibiting action of phenylhydrazine on glutamate decarboxylase, i.e. a decrease in the level of GABA leads to an increase in the electrical activity. But other decarboxylasic systems are also inhibited, for example DOPA and Shydroxytryptophane. This inhibition leads to a decrease in DA but also in NA and 5 HT, with a decrease in electrical activity as a result. The transient increase of activity seen first may be due to the fact that GABA represents the sole depressant system in the brain, other neurotransmitters constituting the stimulatory system. Our results are in agreement with general findings in Insecta and Crustacea (for reviews, see Gerschenfeld, 1973; Lafon-Cazal, 1978; Leake and Walker, 1980). However, some differences exist between L. forficatus and the scorpion Androctonus australis (Goyffon and Niaussat, 1975; Goyffon et al., 1980). In this last animal Ach has an excitatory effect whereas 5 HT as well as GABA (at high doses) have depressant actions. Results comparable to ours have been observed in another Chelicerate, Limulus polyphemus: GABA has an inhibitory effect and Ach a stimulatory action. Receptors for other neurotransmitters such as 5 HT, DA and NA are aiso present (Walker and James, 1978, 1980; James and Walker, 1979; James et al., 1982). Acknowledgements-The authors thank Miss A. Rousseau for her technical assistance, and Mr F. Carey for checking the English.

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Li?habi~ ,fo~~c~fus L. (Myriapoda: Chiiopoda). Reprod. Nutr. Develop. 21, 681-687. Ephrussi 8. and Beadle G. W. (1936) A technique of -transplantation for Drosophila. Am. Nat. 70, 218-225. Gerschenfeld H. M. (1973) Chemical transmission in ini vertebrate central nervous systems and neuromuscular junctions. Physiol. Rev. 53, I-119. Goyffon M. and Niaussat P. (1975) Interrelations entre mbcanismes cholinergiques et monoaminergiques dans le diterminisme de I’activ& ilectrique cCrebrale spontanire du Scorpion. Annls Endocr. 36. 101-102. Goyffon c., Drouet J. and Francaz J. M. (1980) Neurotransmitter amino acids and spontaneous electrical activity of the prosomian nervous system of the scorpion. Comp. Biachem. Phy.~iol. 6&T, 59-64. Herbaut C. (1975) Etude exp~~mentale de I’ovoge&se chez L~thobiusforjcat~ L. (Myriapode Chitopode). RBie de la pars intercerebralis. Gen. camp. Endocr. 27, 34-42. Herbaut C. (1976) Etude expCrimentale de la r&ulation endocrinienne de I’ovoge&se chez Lithabius,j@jcatus L. (Myriapode Chilopode). RBle du complexe “ceilules neurest-c&rices protoctr&brales-glandes c&brales”. G’en. romp. Endow. 28, 264-276. Jamault-Navarro C., Joly R. and Descamps M. (1983) Activation of neurosecretory protocerebral-cells by 20-hvdroxyecdysone in Lithabius farficatus L. (Mvriapoda Chilbpoda). Gen. camp. End&r. 50, 36-42.’ ’ James V. A.. Roberts C. J. and Walker R. J. (19821 Evidence for y-aminobutyric acid (GABA) as an inhibitory transmitter in the c&al nervous system of Limul& po&~hemus. Camp. Biochem. Phwiol. 71C. 229-238. James V. A. and Walker R. J. (1979) ‘The responses of acetylcholine, y-aminobutyric acid (GABA). dopamine, octopamine and other putative transmitters on Limulus polyphemus central neurons. Comp. Biochem. Pl~ysial. 64c, 53-59. Joly R. (1966) Contribution a l’ttude du cycle de mue et son diterminisme chez les Myriapodes Chilopodes. Bull. biol. Fr. Belg. C, 3, 379480. Joly R. and Descamps M. (1969) Evolution du testicule, des vCsicules skminales et cycle spermatog&&ique chez Lithobius forjcutus L. (Myriapode Chilopode), Archs Zoat. exp. gin, 110, 341-348. Lafon-Cazal M. (1978) Les neurotransmetteurs des Insectes. Ann. Biol. 17, 489-528. Leake L. D. and Walker R. J. (1980) Inzlertebrute Neuraph~lrmacology, p. 358. Blackie, Glasgow. Walker R. J. and James V. A. (1978) The action of putative transmitters and related compounds on neurons in the abdominal ganglion of the horse-shoe crab, Limtdus polyphemus. Neuropharmarology 17, 765-769. Walker R. J. and James V. A. (1980) The central nervous system of Limulus polyphemus: physiological and pharmacological studies. Comp. Biochem. Physiol. 66C, 121-124. I