Pharmacological characteristics of the two largest neurons symmetrically situated in the suboesophageal ganglia of an African giant snail (Achatina fulica férussac)

Comp. Biochem. Physiol. Vol. 73C, pp. 339 346, 1982

0306-4492/82/060339-08503.00/0 © 1982 Pergamon Press Ltd

Printed in Great Britain

PHARMACOLOGICAL CHARACTERISTICS OF THE TWO LARGEST NEURONS SYMMETRICALLY SITUATED IN THE SUBOESOPHAGEAL GANGLIA OF AN AFRICAN GIANT SNAIL (ACHATINA FULICA FI~RUSSAC) HIROSHI TAKEUCHI a n d NORIKO YAMAMOTO Department of Physiology, Gifu University School of Medicine, Gifu 500, Japan

(Received 14 January 1982) A b s t r a c t - - l . Pharmacological characteristics of the two largest neurons, r-PLN (right-parietal large

neuron) and d-VLN (dorsal-visceral large neuron), situated symmetrically on the anterior-dorsal surface in the suboesophageal ganglia of an African giant snail (Achatinafulica F6russac), were determined. 2. Of the catecholamines examined, dopamine (DA) showed marked but complex effects on the two neurons. DA produced a transient excitation followed by either hyperpolarization or depolarization of the neuromembranes. L-Norepinephrine produced a slight depolarization of the neurons. 3. 5-Hydroxytryptamine (5-HT) produced marked excitation of both neurons. Bufotenine showed the same effects, but they were weaker than those of 5-HT. 4. Histamine showed inhibitory effects on the two neurons examined, whereas L-histidine had rather slight or no excitatory effects. 5. Of the amino acids examined, L-homocysteic acid (L-HCA) and erythro-fl-hydroxy-L-glutamic acid had marked excitatory effects on r-PLN and d-VLN. L-Homocysteine sulfinic acid also showed the excitatory effects, but they were weaker than those of L-HCA. Glycine and L-methionine had slight excitatory effects on both neurons, whereas L-aspartic acid, L-glutamic acid and GABA had none. 6. Acetylcholine (ACh) exhibited marked and complex effects on r-PLN and d-VLN. In trials, ACh produced either excitation or inhibition of both neurons. Propionylcholine and butyrylcholine had slightly less effect, producing a slight inhibition of r-PLN and either excitation or inhibition of d-VLN in trials.

INTRODUCTION N e u r o p h a r m a c o l o g i c a l studies, especially on the putative n e u r o t r a n s m i t t e r s a n d their derivatives, have been carried out using two spontaneously firing giant neurons, P O N (periodically oscillating neuron) a n d T A N (tonically autoactive neuron), identified in the suboesophageal ganglia of the African giant snail, Achatinafulica F6russac (Miyamoto et al., 1979; Takeuchi et al., 1974a, b, c, 1975, 1976a, b, 1977; Takeuchi & Tamura, 1980; W a t a n a b e & Takeuchi, 1977). Two other extremely large neurons, the diameters of which measure a b o u t 500 pm, are symmetrically situated on the anterior-dorsal surface in the same ganglia, as shown in Fig. 1. The one is called r - P L N (right-parietal large neuron) a n d the other d - V L N (dorsal-visceral large neuron). Since these n e u r o n s are large a n d easily identifiable, they are good materials for electrophysiological or biochemical experiments at the n e u r o n a l level. In this study, the effects of putative neurotransmitters of molluscan neurons a n d their related substances on the excitability of the two largest n e u r o n s in the suboesophageal ganglia, r - P L N a n d d-VLN, were investigated a n d c o m p a r e d with those on P O N a n d TAN. MATERIAL AND METHODS

African giant snails were flown in from Okinawa, and the suboesophageal ganglia were dissected from them. When the connective tissue covering the ganglia was re339

moved, the two giant neurons under study, r-PLN (rightparietal large neuron) and d-VLN (dorsal-visceral large neuron), were found to be situated symmetrically on the anterior-dorsal surface in the dissected ganglia (Fig. l). R-PLN is located in the right-parietal ganglion near the border of the right-pleural ganglion, and d-VLN is found in the visceral ganglion near the border of the left-parietal ganglion. The diameter of these two neurons is about 500/~m. The neurons do not have much colouring, but they are easily distinguished, because of their size and location. Usually, they did not display spontaneous spike discharges. The electrophysiological methods used in this study have been described in detail in the previous reports cited above. The substances examined are listed in Table 1. They were dissolved in the snail's physiological solution (pH 7.5) (Takeuchi et al., 1973) at 10-4kg/1, except for DL-OCtOpamine, which was dissolved at 2 x 10-4kg/I. The solutions were applied directly to the dissected ganglia (bath application). The temperature of the experimental room was kept at 22 + I°C.

RESULTS The effects of the substances examined at 10 - 4 kg/1 (DL-OCtopamine only at 2 × 10 - 4 kg/l) on r - P L N a n d d-VLN, were summarized in Table 1. The effects on the two n e u r o n s were quite similar.

Effects of catecholamines O f the catecholamines examined, d o p a m i n e (DA) showed the most m a r k e d effects on r-PLN, b u t they

340

HIROSHI TAKEUCH1 and NOR[KO YAMAMOTO

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Fig. 1. (A) Photograph of suboesophageal ganglia, anterior-dorsal surface. Connective tissue has been removed. (B) Schematic drawing of (A). In (A) ant., anterior direction; post., posterior direction. Scale, 500/tm. In (B) 1-par. g., left-parietal ganglion; vis. g., visceral ganglion; r-par, g., right-parietal ganglion; r-pl. g., right-pleural ganglion, d-VLN, dorsal-visceral large neuron, r-PLN, right-parietal large neuron.

were complex (Fig. 2A & B). Immediately after DA application, the transient depolarization, which is considered to be a transsynaptic event, occurred. It was followed by either hyperpolarization of the neuromembrane or slight depolarization. Both L-norepinephrine (L-NE) and L-epinephrine (L-E) produced slight excitation of the neuron. The four substances related to the three catecholamines mentioned, L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), L-DOPA and DL-OCtopamine, had only slight excitatory effects on r-PLN that were in general weaker than those of L-NE and L-E (Fig. 3). The effects of these catecholamines on d-VLN resembled those on r-PLN. D-VLN was in general slightly less susceptible to catecholamines than

r-PLN. DA effects on d-VLN also Were complex. Immediately after the DA application, a transient depolarization of d-VLN occurred, followed by a slight hyperpolarization. However, in some cases the two effects were not so marked (Fig. 4A & B). L-NE produced a slight depolarization of the neuron, whereas L-E showed almost none. The four other catecholamines, L-Phe, e-Tyr, L-DOPA and DL-OCtopamine, had almost no effect on d-VLN.

Effects of indoleamines Of the indoleamines examined, 5-hydroxytryptamine (5-HT) had marked excitatory effects on both r-PLN and d-VLN (Fig. 5). Their silent neuromembranes showed spike discharges after the 5-HT appli-

Pharmacological characteristics of two snail neurons

341

Table 1. Effects of putative neurotransmitters of molluscan neurons and their related substances on the excitability of the two largest neurons in the suboesophageal ganglia, r-PLN (right-parietal large neuron) and d-VLN (dorsal-visceral large neuron), of an African giant snail (Achatinafulica F6russac) Substance

Effects on r-PLN

Effects on d-VLN

SE SE SEt Vat (E-I, E) SE SE SEt

(- ) (- ) (-) Var (E-SI, E) SE (- ) (- )

(B) Indoleamines L-Tryptophan L-5-Hydroxytryptophan L-5-Hydroxytryptamine Bufotenine

SE SEt E SE

(- ) (-) E SE

(C) Imidazoleamines L-Histidine Histamine

SE I

(- ) SI

(D) Amino acids Glycine fl-Alanine GABA I-GABOB d-GABOB L-Aspartic acid L-Glutamic acid Erythro-fl-hydroxy-L-glutamic acid L-Homocysteic acid L-Homocysteine sulfinic acid L-Methionine Taurine

SE (-) (-) (-) (-) (-) (-) E E SE SE (- )

SE (-) (-) (-) (-) (- ) (-) E E E SEt (- )

Var (E, I) SI SI

Var (E, 1) Var (E, I) Var (E, 1)

(A) Catecholamines L-Phenylalanine L-Tyrosine L-DOPA Dopamine L-Norepinephrine L-Epinephrine DL-Octopamine*

(E) Cholines Acetylcholine Propionylcholine Butyrylcholine

Effects of the above substances at 10 kg/l (*, at 2 × 10 kg/1) were examined in bath application. Var, variable. E, excitatory. SE, slightly excitatory. SEt, slightly excitatory or sometimes not effective. I, inhibitory. SI, slightly inhibitory. (-), not effective. E-SI, excitatory and slightly inhibitory.

cation. The other three indoleamines, L-tryptophan (L-Trp), L-5-hydroxytryptophan (5-HTP) and bufotenine (5-hydroxy-N-dimethyltryptamine), produced a slight depolarization of r-PLN. The other neuron examined, d-VLN, was less affected by the latter three indoleamines. Bufotenine produced a slight d-VLN depolarization, but the other two had almost no effect on it.

Effects of imidazoleamines Histamine at 10-4kg/1 produced marked hyperpolarization of r-PLN, whereas L-histidine (L-His) produced only rather slight excitatory effects (Fig. 6). D-VLN was in general less sensitive to the two imidazoleamines than r-PLN, evidencing a weaker hyperpolarization from histamine and no effects of L-His.

Effects of amino acids The effects of amino acids on r-PLN and d-VLN were similar. In particular, L-homocysteic acid (L-HCA) and erythro-fl-hydroxy-L-glutamic acid

(erythro-L-BHGA) had marked excitatory effects on both (Figs 7 & 8). L-Homocysteine sulfinic acid (L-HCSA), analogous to L-HCA, showed similar but weaker effects than L-HCA. Of other sulfur containing amino acids, L-methionine (E-Met) caused a slight excitation of the two neurons, but taurine (Tau) had no effect. In spite of the marked excitatory effects of erythroL-BHGA, the two related substances, L-aspartic acid (E-Asp) and L-glutamic acid (L-Glu), had no effect on the two neurons. Glycine (Gly) produced a slight depolarization, but fl-alanine (fl-Ala), GABA, 1-GABOB and d-GABOB had no effect (Fig. 9).

Effects of cholines Acetylcholine (ACh) had marked and complex effects, on both r-PLN and d-VLN, either excitatory or inhibitory depending on the case (Figs 10 & 11). As shown in Fig. 10A & B, ACh sometimes produced both excitation and inhibition of one r-PLN in trials. Propionylcholine and butyrylcholine produced either

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that these two n e u r o n s have the same physiological roles in the suboesophageal ganglia. The present results suggest that 5-HT, L-HCA and erythro-LB H G A are putative excitatory neurotransmitters of the two neurons, a n d that histamine is a putative inhibitory one. DA a n d A C h also may be neurotransmitters of the neurons, b u t it is not clear, since their effects were complex.

excitation or inhibition of the d - V L N n e u r o m e m brane, b u t for the most part only a slight r - P L N hyperpolarization. DISCUSSION Since r - P L N a n d d - V L N evidenced only slightly different pharmacological properties, it is considered

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Pharmacological characteristics of two snail neurons

345

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While DA clearly excited P O N and inhibited TAN, as reported previously (Takeuchi et al., 1974b, 1975), it showed marked and complex effects, either excitatory or inhibitory, on r-PLN and d-VLN in the present study, suggesting that it activates more than two kinds of ionic channels in the neuromembranes of the two neurons. L-NE mimicked only the excitatory effects of DA on the same two neurons. 5-HT showed the same marked excitatory effects on both r - P L N and d-VLN which it had on P O N and T A N (Takeuchi et al., 1974c, 1975). Bufotenine, an agonist of 5-HT, produced basically the same but weaker effects than 5-HT on r - P L N and d-VLN as well as P O N and TAN. Histamine has been considered to be a putative excitatory neurotransmitter of TAN, but the receptors are different from those of H 1 and H2 (Takeuchi et al., 1976). This substance showed marked inhibitory effects on r - P L N and d-VLN, whereas L-His had only slightly excitatory effects on r-PLN. While L-HCA and erythro-L-BHGA had marked inhibitory effects on P O N (Takeuchi et al., 1974a, 1976a), they caused a marked excitation of r - P L N and d-VLN. These results suggest that one of the two substances is a strong agonist of the other which is an excitatory neurotransmitter of these neurons, or that both two substances are agonists of an unknown excitatory neurotransmitter. L-HCSA showed the same but weaker effects than L-HCA on r - P L N and d-VLN, just as on PON. GABA and its derivatives, I - G A B O B and d-GABOB, had the inhibitory effects, which is due to the activation of the chloride channels in the neuromembrane, on T A N (Takeuchi et al., 1977 ; Watanabe & Takeuchi, 1977). However, they had no effect on r-PLN and d-VLN. On the other hand, Gly and L-Met produced slightly excitatory effects on r - P L N and d-VLN. L-GIu and L-Asp had no effect on P O N

and T A N nor on the two neurons examined in this study, despite the fact that these two amino acids are reported to produce high excitation of many mammalian central neurons (Krnjevid, 1974). ACh showed marked and complex effects on r - P L N and d-VLN. This finding suggests the activation of more than two kinds of ionic channels in the neuromembranes examined, just as in the case of DA application. Pch and Bch may well act as the agonists of ACh on the neurons examined. Kerkut et al. (1970, 1975) made precise diagrams of the identifiable neurons in the suboesophageal ganglia of the European garden snail, Helix aspersa. Although their diagrams showed the identifiable neurones in detail, they did not include the extremely large neurons, like r-PLN and d-VLN. F76 and F77, in their classification, are the largest neurons in the right-parietal ganglion of Helix aspersa, but these two neurones, unlike r-PLN, are not affected by 5-HT. The locations of E l 4 and El9 in the visceral ganglion are most similar to that of d-VLN. However, differently from d-VLN, E l 4 is simply inhibited by DA and ACh, and E l 9 is inhibited by ACh and unaffected by DA. The diagrams of giant neurons in the same ganglia of two other snail species, Otala lactea (Gainer, 1972) and Helix pomatia (Hancock & Voile, 1973), did not include the large neurons corresponding to the two neurons examined in Achatina Julica F6russac.

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HIROSH! TAKEUCHI and NORIKO YAMAMOTO

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