Cholinesterase activity of ganglia of gastropoda, Lymnaea stagnalis and Planorbarius corneus—I. Effect of anti- cholinesterase agents on giant neurone depolarization by acetylcholine and its analogues

Comp. Biochem. Physiol., 1973, VoL 45A, pp. 45 to 60. PergamonPress. Printed in Great Britain

CHOLINESTERASE ACTIVITY IN GANGLIA OF GASTROPODA, L YMNAEA STAGNALIS AND P L A N O R B A R I U S CORNEUS--I. EFFECT OF ANTICHOLINESTERASE A G E N T S O N G I A N T N E U R O N E DEPOLARIZATION BY ACETYLCHOLINE AND ITS ANALOGUES *OLGA P. JURCHENKO, *CATHERINE A. VULFIUS and tELLA V. ZEIMAL *Laboratory of Nerve Cell Biophysics, Institute of Biological Physics, Academy of Sciences of the U.S.S.R., Puschino on Oka, Moscow Region; and tLaboratory of Pharmacology, Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R. (Received 20 ~uly 1972)

Abstract--1. The action of eserine and two organophosphorous cholinesterase inhibitors on the sensitivity of identified neurones of Lymnaea stagnalis and Planorbarlus corneus to cholinergic agonists was studied with microeleetrodes. 2. Eserine did not affect the action of ACh on Lymnaea neurones but did enhance the depolarizing potency of BuChE substrates, BuCh and D-6, as much as 1.6-fold and 13-fold respectively. 3. On Lymnaea neurones the BuChE selective inhibitor, compound GT-165, potentiated D-6, BuCh, ACh and AChE substrate, acetyl-fl-methylcholine (rneeholyl), as much as 33-8-, 3.7-, 4- and 3.2-fold respectively. The selective inhibitor of ACHE, compound Gd-42, increased the action of ACh 6"9 times and that of mecholyl four times, but did not change the D-6 and BuCh depolarization. 4. From these findings a conclusion may be drawn about high cholinesterase activity in Lymnaea ganglia; there seems to exist at least two different enzymes. This was emphasized by the dam of experiments with a consecutive application of Gd-42 and GT-165. 5. With the inhibitors used we failed to reveal any essential cholinesterase activity in Planorbarius ganglia. INTRODUCTION

THERe are some arguments for ACh being one of the transmitters in the nervous system of Gastropoda. ACh and choline acetyltransferase were found in ganglia homogenates of Helix aspersa (Kerkut & Cottrell, 1963) and Aplysia californica (Giller & Schwartz, 1968, 1971a; McCaman & Dewhurst, 1970). ACh and its analogues decrease membrane resistance and change the resting potential of Helix, Abbreviations used are: ACh, acetylcholine; BuCh, butyrylcholkne; D-6, suberyldicholine; 'rMA, tetramethylammonium; ACHE, acetylcholinesterase (E.C. 3.1.1.7); BuChE, butyrylcholinesterase (E.C. 3.1.1.8); Gd-42, O-ethyl-S-fl-ethylmercaptoethyl)-methylthlophosphonate methylsulfomethylate; GT-165, O,O-diethyl-S-[fl-(cyclohexylmethylamin0) ethyl] thiophosphate iodide. 45

46

OLGA P. JURCHI~NKO,CATHERINEA. VULFIUSAND ELLA V. ZEIMAL

Aplysia, Lymnaea and other mollusc neurones (Tauc & Gerschenfeld, 1960, 1962; Kerkut & Walker, 1961; Gerschenfeld, 1964; Gerschenfeld & Stefani, 1966, 1967; Vulfius & Zeimal, 1967), the reversal potential of the ACh effect on H-neurones (hyperpolarizing) being coincidental with that of IPSP's (Taue & Gerschenfeld, 1962; Kerkut & Thomas, 1963, 1964). Ionic mechanism of the response of H-neurones to ACh appears to be the same as that of IPSP's just as it is in the case with D-neurones (depolarizing) and EPSP's (Kerkut & Thomas, 1964; Kerkut & Meech, 1966; Sato et al., 1968). n-Tubocurarine was shown to decrease not only ACh depolarization or hyperpolarization of Aplysia neurones, but also the PSP amplitude of the same cells (Tauc & Gerschenfeld, 1962; Kandel et al., 1967). The treatment of Helix ganglia with the inhibitor of ACh synthesis, hemicholinium, induces a decrease in the height of cholinergic EPSP's (Walker et al., 1971). The existence of cholinesterase activity in molluscan central nervous system appears to be another argument for cholinergic transmission. Cholinesterase in Helix pomatia, Aplysia caIifornica, Lyrnnaea stagnalis ganglia and Pila globosa nerves was studied with the help of biochemical methods (Augustinsson, 1946; Dettbarn & Rosenberg, 1962; Varanka, 1968; Korn, 1969; Murali & Murati, 1969; Giller & Schwartz, 1971b; McCaman & Dewhurst, I971; Grigor'eva, 1972). Cholinesterase activity was also found in Lymnaea ganglia with a histochemical method (Zs.-Nagy & Sal~nki, 1965).

B

Fio. 1. Schematic representation of L. stagnalis (A) and P. corneus (B) ganglionic rings (ventral surface). P, Pedal ganglia; PI, pleural ganglia; RP, right parietal ganglion; LP, left parietal ganglion; V, visceral ganglion. Identified neurones are designated by figures. In .this investigation cholinesterase activity in Lymnaea and Planorbarius ganglia was estimated according to an increase in the neurone response to ACh and other choline esters after treatment with eserine and organophosphorous inhibitors. Preliminary data have been reported (Vulfius et al., 1969; Vulfius & Jurchenko, 1972).

47

CHOLINESTERAS]E ACTIVITY OF GASTROPODA, L. S T # I G N A L I S AND P. C O R N E U S - - I

MATERIALS AND METHODS The experiments were performed on identified neurones of Lyranaea stagr~lis right parietal and visceral ganglia and on neurones of Planorbarius corneus left parietal, visceral and pedal ganglia (Fig. l a and b), All the neurones studied were D cells. The isolated ganglionic ring or isolated ganglion was continuously perfused with the following salt solution: NaCI, 50 raM; KCI, 1"6raM; CaCI=, 4 m M ; MgCIs, 8 raM; Tris-HCl up to pH 7-5. The rate of perfusion was about 15 ml/min and the chamber volume was I nil. ACh and its analogues were added to the perfusion fluid and their effect was estimated on the changing of cell resting potential registered with conventional microelectrodes and a d.c. amplifier on a pen recorder. Microelectrodes with a tip diameter of < 0-5/~ were filled with 2.5 M KC1 and had a resistance of 30-100 M~. (al

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FiG. 2. Influence of the organophosphorous BuChE inhibitor, GT-165, on the action of ACh and its analogues on neurones of L. stagnalis. (a). Responses of a RP4 neurone to ACh, D-6, mecholyl (Mech) and TMA before and after treatment of the ganglia with 1 x 10 -5 M GT-165. Upward arrows indicate addition of the drugs to the perfusion fluid; downward arrows indicate washing them out. A voltage calibration (10 mV) is given as a rectangular deflection at the end of the records. (b). Depolarization of another RP4 neurone (ordinate) plotted against concentrations (abscissa) of ACh (©,O) D-6 (O,1) or carbachol (A,A). After treatment with 1 x 10 -~ M GT-165 (filled symbols), there is a shift of curves in the direction of low concentrations as compared to control testing (open symbols). The 10 mV depolarization level is shown by an interrupted horizontal line. Concentration of drugs to induce 10 mV depolarization are found from the points of intersection of the 10 mV line by the effect-concentration curves.

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48

OLGA P. JURCHENKO, CATHERINEA. VULFIU$AND ELLA V. ZEIMAL

An increase in drug potency as a result of treatment with anticholinesterase agents was expressed quantitatively from the changes in the curves relating depolarization to log molar drug concentration before and after perfusion of the ganglia with antieholinesterases. Concentrations required to cause a 10 mV depolarization were found by interpolation and were used as a standard (Fig. 2). Tetramethylammonium (TMA) and carbaehol, not destroyed by cholinesterases were tested as the control of possible nonspecific action of anticholinesterase agents. Coefficients for enhancing cholinesterase substrate depolarizing action were corrected for nonspecific action according to an increase in T M A or carbaehol potency. Ganglia were perfused with 1 x 10-s-1 x 10 -4 M eserine for 3 0 - 4 0 m in and then cholinergic drugs were tested in the presence of the same concentration of eserine. T h e ganglia were treated with one of the organophosphorous irreversible inhibitors for a short time (1 × 10 -s M GT-165 for 20-30 min, 5 x 10-e M Gd-42 for 10-20 min) and cholinergic drugs were tested after washing the inhibitor out of the chamber. T h e following compounds were used: acetylcholine iodide, acetyl-/~-methylcholine bromide (mecholyl), propionylcholine iodide, butyrylcholine iodide, suberyldicholine diiodide, tetramethylammonium bromide, carbaminoylcholine chloride (carbachol), eserine salicylate, organophosphorous inhibitors, compounds GT-165 and Gd-42 (see Table 1). T h e experiments were performed at room temperature of 18-22°C.

RESULTS Lymnaea neurones Most of the experiments were carried out on three identified cells of the right parietal ganglion: RP1, RP2 and RP4 (Fig. l a). These cells were silent, generating two to three spikes only when impaled bya microdectrode. During the experiment single or summated (1-2 and 25-30 mV) EPSP's were observed. Resting potential was an average 60--65 mV and varied insignificantly during the experiment. ACh and its analogues induced depolarization of the neurones (Figs. 2 and 3) sometimes with spike firing.

The action of BuChE selective inhibitor, compound GT-165 Perfusion of the ganglionic ring with GT-165 did not change the resting potential of Lymnaea neurones, but led to a significant increase in the responses to drugs, destroyed by cholinesterases. In Fig. 2(a) the effects of ACh, D-6, mecholyl and TMA on a neurone RP4 before and after GT-165 treatment are compared. In order to produce the same depolarization as that of control testing (20 mV) the ACh concentration must be reduced ten times after perfusion with GT-I65. An increase in D-6 efficiency is much more pronounced: the ratio of equieffective concentrations is equal to 50, the factor for mecholyl is about 5, whereas the response to TMA (2 x 10 -6 M) does not change. The results of another experiment are presented as concentration-effect lines in Fig. 2(b). After treatment with GT-165 there is a shift of curves of ACh and D-6 to the left in the direction of !ower concentrations. In this experiment a small increase in carbachol efficiency is also observed. The ratios of drug concentrations inducing 10 mV depolarization before GT-165 to equieffective drug concentrations after treatment with GT-165 were found on the plot. The ratio for ACh = 8,

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FIo. 3. Influence of the organophosphorous AChE inhibitor, Gd-42, on the action of ACh and its analogues on Lymnaea neurones. (a). The action of ACh, mecholyl (Mech) and D-6 on a RPI neurone before and after perfmion of the ganglia with Gd-42 (1 x 10 -5 M). (b). Depolarization of a RP4 neurone induced by ACh, D-6, mecholyl and carbadaol relating to their concentrations. Treatment with Gd-42 (1 x I0 -5 M). <~,O, Mecholyl; other symbols as in Fig. 2(b).

D-6 = 119 and carbachol = 1.5. Taking into account nonspecific GT-165 action (increase in carbachol activity), the coefficients of ACh and D-6 potentiation are equal to 5 and 79 respectively. These coefficients appear to characterize an increase in the cell sensitivity to choline esters due to chotinesterase inhibition. GT-165 also enhanced the action of BuCh and propionylcholine. The mean coefficients of potentiation of different choline esters on Lymnaea neurones are summarized in Table 2a and b. GT-165 is a selective inhibitor of BuChE (Table 1). D-6 and BuCh are substrates of the same enzyme; ACh and: propionylcholine are destroyed by both BuChE and ACHE; however, mecholyl is a selective substrate of ACHE. Two different explanations may be advanced for potentiation of all these compounds: (1) in Lymnaeastagnalisganglia there are two cholinesterases, differing in substrate specificity, both being inhibited by GT-165 at the concentrations used; (2) there is one enzyme, but it is less specific to substrates than BuChE and ACHE. To solve this question a selective inhibitor of ACHE, compound Gd-42 (Table I), was used.

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TABLE 2b--INcREASE IN DRUG POTENCLES ON L. stagnalis NEURONES AFTER TREATMENT WITH CHOLINESTERA8~. INHIBITORS (WITH CORRECTIONS FOR NONSPECIFIC ACTION OF ANTICHOLINESTERASE AGENTS ACCORDING TO POTENTIATION OF T M A OR CARBACHOL)

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The action of selective inhibitor of ACHE, compound Gd-42 In some experiments Gd-42 caused the depolarization of the neurones (approximately 4 mV), an increase in EPSP frequency and in their amplitude. The resting potential recovered completely after washing Gd-42 out of the chamber. The effect of Gd-42 on the action of ACh, mecholyl and D-6 is shown in Fig. 3(a). From this figure it can be seen that potentiation of ACh and mecholyl is equal to approximately 10, and the sensitivity of the cell to D-6 does not change. In Fig. 3(b) there is another experiment presented as a plot: ACh, meeholyl and carbachol efficiencies increase 10, 5.5 and 1.2 times respectively; the points of the cell responses to D-6 being coincidental with a control curve. The mean coefficients of different drug potentiation after Gd-42 treatment are summarized in Table 2. The action of BuChE substrates, BuCh and D-6, do not change practically. This indicates that the first suggestion about the existence of the two enzymes is more plausible. To cheek this possibility, once again some experiments with a consecutive treatment with two inhibitors were performed. To make sure that the action of the first inhibitor had completely developed, it was added twice. After 10-12 rain perfusion with 5 x 10-0-1 x 10 -5 M Gd-42, a repeated treatment did not enhance drug action. The development time of the complete GT-165 (1 x 10 -5 M) effect was 20-30 rain. Gd-42 after GT-165 treatment appeared to give a further rise in ACh, mecholyl and propionylcholine depolarizing potency, the sensitivity of neurones to D-6 remaining unchanged. GT-165 after Gd-42 treatment increased additionally D-6 and BuCh action, but not that of ACh and mecholyl (Table 2a and b). The results of two experiments with a consecutive application of GT-165 and Gd-42 are shown in Figs. 4 and 5.

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The action of eserine In Fig. 6 the curves relating to ACh, D-6, BuCh and T M A depolarization to molar concentration of drugs before and after perfusion of the ganglia with 1 x 10 4 M eserine are presented (the concentration of eserine being unchanged during drug testing). In this experiment D-6 after eserine treatment appeared 13.6 times more potent than at control testing, the potency of BuCh increased

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CHOLINI~TERASE ACTIVITY OF OASTROPODA, L. S T A G N A L I S AND P. C O R N E U S - - I

55

Application of GT-165 after eserine did not induce any further potentiation of cholinesterase substrates. Planorbarius neurones The resting potential of Planorbarius neurones was usually 55-60 mV. Most of the cells were spontaneously active, action potential averaged 75-85 inV. ACh and its analogues induced depolarization and increased spike frequency. None of the organophosphorous inhibitors used altered considerably the sensitivity of the cells to choline esters (Table 3). TABLE 3--INCREASE IN DRUG POTENCIES ON P . c o r n e ~ NEURO17£8 ~ TREATMENT WITH CHOLINESTERASE INHIBITORS (WITH CORRECTIONS FOR NONSPECIFIC ACTION OF ANTICHOLINESTERASE AGENTS ACCORDING TO POTENTIATION OF T1VIA OR CARBACHOL)

Increase in drug potency (mean __S.E.M.) Inhibitor GT-165 Gd-42 Eserine

ACh 1-3 + 0"17t (7)* 1.0 _+0.00t (3) 1 . 4 _+0.31t (5)

D-6 1.3 _+0"17t (7) -2"3 +_0.44 (5)

Mecholyl m 1.0 + 0.00t (3) --

* Number of experiments is given in parentheses. t Not significant at the 5 per cent level. Eserine at the concentration of 2 x 10 -5 M increased the efficiency of D-6 as much as 2.3 times, but did not affect ACh action. Raising the eserine concentration up to 1 × 10 -~ M did not lead to drug potentiation. DISCUSSION An increase in excitatory action of ACh and its analogues after treatment with anticholinesterase agents appears to be evidence of high cholinesterase activity in Lymnaea ganglia. This conclusion coincides with our histochemical findings (Jurchenko et al., 1972) and with Grigor'eva's data who reported that saline extracts of Lyranaea ganglia hydrolyzed 4-14 mM ACh/g wet wt. per hr (Grigor'eva, 1972). Using some specific organophosphorous inhibitors Grigor'eva determined the KII values according to the rate of hydrolysis of different substrates. She found these values to be equal for the same inhibitor. From this equality she came to the conclusion that there exists only one enzyme in Lyrnnaea ganglia. This enzyme possesses properties of both AChE (hydrolysis of mecholyl, similar values of KII for different inhibitors to those of ACHE) and BuChE (hydrolysis of BuCh, sensitivity to BuChE inhibitors).

56

OLGA P. JURCHENKO, CATHERINEA. VULFIUS AND ELLA V. ZEIMAL

Our data suggest the existence of two different cholinesterases in the Lymnaea central nervous system. Treatment of the ganglia with Gd-42 potentiates only ACh and the substrate of ACHE, mecholyl, but does not alter the sensitivity of neurones to BuChE substrate, D-6. After pretreatment of ganglia with GT-165, Gd-42 causes an additional increase in ACh and mecholyl action. Compound GT-165 potentiates not only ACh and mecholyl, but also the substrates of BuChE, D-6 and BuCh, the potentiation of D-6 being especially pronounced* (Table 2). After pretreatment with Gd-42, compound GT-165 continues to increase the action of D-6 and BuCh though less considerably. Eserine potentiates D-6 and to a lesser degree BuCh, but not ACh. Thus, one of the enzymes is able to hydrolyze ACh, propionylcholine and mecholyI, which is an AChE substrate; it is inhibited completely by the AChE inhibitor Gd-42 and partly by the BuChE inhibitor GT-165. The second enzyme hydrolyzes BuChE substrates, D-6 and BuCh, only; it is inhibited by GT-165 and eserine and is not at all sensitive to Gd-42. The difference between Grigor'eva's and our conclusions may be explained by the fact that she did not use D-6 as a substrate. As can be seen from Table 2, BuCh was slightly potentiated by Gd-42; the enhancement of BuCh efficiency by GT-165 was much less pronounced than that of D-6. That is why we suppose that using only BuCh as BuChE substrate may be insufficient to reveal the second cholinesterase. In the data obtained there are some facts which are difficult to explain. (1) GT-165 increased the action of ACh only if it was used first. After pretreatment with Gd-42, GT-165 did not change the sensitivity to ACh. Eserine did not potentiate ACh at all. Thus, the second cholinesterase seems to hydrolyze D-6 and BuCh, but not ACh. There are some data in the literature about a cholinesterase which is able to hydrolyze benzoylcholine and BuCh but is ineffective in relation to ACh. An enzyme with such properties (named benzoylcholinesterase) was found in rabbit brain when the homogenate was separated electrophoretically in agar gel (Dolgo-Saburov et al., 1970). It is possible that the second enzyme in Lymnaea ganglia is similar to benzoylcholinesterase. The fact that GT-165 after Gd-42 treatment was unable to potentiate ACh may have an alternative explanation. According to Grigor'eva's data (Grigor'eva, 1972), K,~ for Lymnaea ganglia cholinesterase using ACh as a substrate is equal to 2.86x 10-* M. It is not far from the Km for BuChE with the same substrate (Grigor'eva, 1964). The ACh concentrations used in physiological experiments are about two to three orders lower (10-¢-10 -e M). Hydrolysis of ACh at such concentrations is likely to be very slow. This may be the reason why we failed to reveal an increase in ACh action after inhibition of the second enzyme. On the contrary, K m values for BuChE hydrolysis of diacetylcholines are of the order * The rate of hydrolysis of diacetylcholines of the D-6 type (having 6 and more CH~ between esteri¢ groups) by BuChE is known to be higher than that of ACh (Pevzner, 1955; Briieke, 1956).

CHOLINI~TERASE ACTIVITY OF GASTROPODA, L. S T A G N J L I S AND P. C O R N E U S ~ I

57

10-6 M (Volkova, 1972). If the K~ of the second Lymnaea cholinesterase was close to that of BuChE, its inhibition should influence very much on D-6 concentrations and efficiency in physiological experiments. (2) The GT-165 used after Gd-42 is almost an order as weak in increasing D-6 action as the GT-165 used first (Table 2). This low efficiency of GT-165 is not possible to explain by inhibition of cholinesterase by Gd-42 as it does not change the sensitivity of the neurones to D-6. Let us assume that two enzymes in Lymnaeaganglia are associated in a common structure as two subunits. While one enzyme is being inhibited by Gd-42, the activity of the second one in respect to D-6 does not change; however, its affinity to GT-165 decreases. It must lead to a reduction of GT-165 efficiency, i.e. to a lesser increase in D-6 and BuCh action in the case of treatment with GT-165 after Gd-42. (3) Eserine is believed to be a nonspecific inhibitor with respect to AChE or BuChE of vertebrates. In Grigor'eva's investigation cholinesterase of Lymnaea ganglia extract was sensitive to eserine (however, KII was about two orders lower than KII for Gd-42). In our experiments eserine did not change ACh action; thus, Lymnaea cholinesterase destroying ACh and mecholyl does not seem to be inhibited by eserine. In other aspects this enzyme is similar to the cholinesterase which was described by Grigor'eva. (4) The compound Gd-42 potentiates TMA and carbachol. TMA does not contain an esteric group. Carbachol is not destroyed by any cholinesterase due to the amino group at carboxyl Carbone. Therefore, it is impossible to explain the potentiation of these agents by inhibition of cholinesterase. The anionic site of cholinesterase can adsorb all the drugs with a positively charged atom in the molecule, including TMA and carbachol. In fact, TMA and carbachol are weak reversible inhibitors of cholinesterase (see, for example, Wilson & Alexander, 1962). Cholinesterase can be "a site of loss" for all undestroyed cholinergic agents, thus reducing their concentration in surrounding fluid. It is possible that the anionic site of phosphorylated cholinesterase has a lower affinity towards quaternary compounds than that of the native enzyme. There may certainly be other sites of loss for quaternary compounds in Lymnaea ganglia, which are blocked by Gd-42. It is possible to find another reason for the increase in TMA and carbachol action after treatment with Gd-42. We added all the drugs used to the perfusion fluid, so they could act not only on the cell under study but on other neurones in the ganglion as well. Therefore, the registered response might be a result of both a direct action on the cell and an indirect influence from other cholinoceptive neurones. If there were a cholinergic synapse in exciting a synaptic way to the cell under study, cholinesterase inhibition near this synapse should lead to an increase in indirect action of TMA and carbachol. If this is the case, it is necessary to assume that cholinesterase localized at this synapse is not blocked by GT-165, and there are cholinoreceptors on Lymnaeaneurones excited by TMA and carbachol, but not by D-6 (otherwise we should observe potentiation of D-6 under the influence of Gd-42).

58

OLGA P. JURCHENKO, CATHERINEA. VULFIU8 AND ELLA V. ZEIMAL

Regarding P. corneus ganglia, there seems to be no cholinesterase but if cholinesterase exists in Planorbarius central nervous system then it differs greatly from Lymnaea enzymes in its sensitivity,to inhibitors. In fact, organophosphorous compounds were inetfective in potentiation of choline ester action on Planorbarius neurones; eserine increased the sensitivity of cells to D-6 only twice (compared with 13.6 times in the case of Lymnaea neurones) and did not change the action of other drugs (Table 3). T h e same conclusion was drawn from a histochemical study of cholinesterase activity in Lymnaea and Planorbarius ganglia (Jurchenko et al., 1972). Cholinesterase in Planorbarius haemolymph also differs greatly from that in Lymnaea haemolymph in its weak specific activity and low affinity towards organophosphorous inhibitors (Grigor'eva & Tkachenko, 1971).

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JURCHENKOO. P., KULTASK.'N. & VuI2IUS C. A. (1972) Cholinesterase activity in ganglia of Gastropoda, Lynrnaea"stagnalis and Planorbarius cornegt--II. Histochemical investigation. Comp. Biochera. Physiol. 4LSA, 61-68. KANDEL E. R., FsAznm W. T., WAZlRI R. & COOOmHALL R. E. (1967) Direct and common connections among identified neurones in Aplysia. jT. Neurophysiol. 30, 1352-1376. KJmKUT G. A. & COTTRZLL G. A. (1963) Acetylcholine and 5-hydro~rtryptamine in the snail brain. Comp. Biochem. Physiol. 8, 53-63. KZRKUT G. A. & NIazCH R. W. (1966) The internal chloride concentration of H and D cells in the snail brain. Comp. Biochem. Physiol. 19, 819-833. KERKUT G. A. & THOMAS R. C. (1964) The effect of anion injection and changes in the external potassium and chloride concentration on the reversal potentials of the IPSP and acetylcholine. Comp. Biochem. Physiol. 11, 199-213. KERKUT G. A. & WALKER R. J. (1961) The effects of drugs on the neurons of the snail Helix aspersa. Comp. Biochem. Physiol. 3, 14-3-160. KORN E. (1969) Choline esterase activity in the tissues of the snail Helix aspersa. Comp. Biochem. Physiol. 28, 923-929. MCCAMAN R. E. & DI~'HURST S. A. (1970) Choline acetyltransferase in individual neurons of .4plysia californica. 7. Neurochem. 17, 1421-1426. MCCAMANR. E. & DEwmmsT S. A. (1971) Metabolism of putative transmitters in individual neurones of .4plysia californica. Acetylcholinesterase and catechol-o-methyl transferase. J. Neurochem. 18, 1329-1335. MURALI M. P. & MURAL! D. P. (1969) Levels of spontaneous electrical and acetylcholinesterase activities during aestivation of the Indian apple snail Pila globosa. Veliger 12, 37-39. PEVZNERF. V. (1955) The hydrolysis of dicholinic esters of dicarboxylic acids by pseudoand true cholinesterases. Farmakol. Tolm'kol. 182, 27-31. (In Russian.) SATO M., AUSTIN G., YAI H. & MAaUHASm J. (1968) The ionic permeability changes during acetylcholine-induced responses of Aplysia ganglion cells, jT. gen. Physiol. 51, 321-345. TAuc L. & GF.gSCmm~n~LVH. M. (1960) Effet inhibiteur ou excitateur du chlorure d'acttylcholine sur le neurone d'Escargot, y. Physiol. (Paris) 52, 236. TAuc L. & G E a s c ~ v H. M. (1962) A cholinergic mechanism of inhibitory synaptic transmission in a molluscan nervous system..7. Neurophysiol. 25, 236-262. VAnp,'qI~ I. (1968) Biochemical investigation of cholinesterase in the central nervous system of Lymnaea stagnalis L. Magyar tud. akad. Tihanyi. biol. lmtatointez, evk. 35, 93-107. VOLKOVAR. I. (1972) Kinetics of hydrolysis of amino-alkyl esters of aromatic dicarbonic acids by serum cholinesterase. Biochimia (In press.) (In Russian.) VULFIUS C. A. & J v a c a m ~ o O. P. (1972) The effect of anticholinesterase agents on the action of acetylcholine and its analogues on gastropod mollusc neurones. Dokl. Akad. Nauk. Sci. SSSR 205, 1254-1257. (In Russian.) VULFIUS C. A., JIYRCHENKOO. P. & ZEIMAL E. V. (1969) On the mutual disposition of choline receptors on the neuronal membrane of gastropod molluscs and on the cholinesterase activity of the neurones. Dokl..4kad. Nauk. S t . SSSR 186, 1445-1448. (In Russian.) VULFIVS C. A. & ZEI~L E. V. (1967) The effects of acetylcholine and cholinomimetics on the giant neurons of the mollusc, Lymnaea stagnalis. In Evolutionary Neurophysiology and Neurochemistry (Edited by KR~ps E. M.), pp. 17-25. Nauka, Leningrad. (In Russian.) WALKER R. J., RALPH K. L., WOODRUFF G. N. & KERKUT G. A. (1971) Effect of drugs on excitatory and inhibitory potentials in Helix asperas. Experientia 27, 281-282.

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WILSON I. B. & 2~I.~KANDERJ, (1962) Acetylcholinesterase: reversible inhibitors, substrate inhibition..7, biol. Chem. 237, 1323-1326. Zs.-NAoY I. & SALLWKIJ. (1965) Histochemical investigation of cholinesterase in:different molluscs with reference to functional conditions. Nature, Lond. 206, 842-843. Key Word Index--Fresh-water gastropod molluscs; L ymnaea stagnalis ; Planorbarius corneus; identified giant neurones; ACh-depolarization; choline esters; cholinesterase inhibition; eserine; organophosphorous compounds.