differential effects of general anaesthetics on identified molluscan

Gen Pharmac Vol 23, No 6, pp 985-992, 1992

0306-3623/92 $5 00 + 0 00 Copyright © 1992Pergamon Press Ltd

Pnnted m Great Britain All rights reserved

D I F F E R E N T I A L EFFECTS OF GENERAL ANAESTHETICS ON IDENTIFIED MOLLUSCAN NEURONES I N SITU AND IN C U L T U R E W WINLOW,T YAR, G SPENCER,D GIRDLESTONE*and J HANCOXt Department of Physmlogy, Umverslty of Leeds, Leeds LS2 9NQ, England

(Recewed 12 June 1992) Abstract--1 The only umfymg pnnclple of general anaesthesm ~s that general anaestheucs interact with membrane components and no single cellular mechamsm appears to explain their w~despread effects m the central nervous system 2 The gastropod mollusc, Lymnaea stagnahs, prowdes an excellent model system for studies on general anaesthettcs because it has large, umquely ldenUfiablenerve cells Severalof these cells are mterneurones w~th identified neurotransmltters and monosynapt~c connectmns to other cells 3 Recent work on Lymnaea neurones suggests that calcium currents are depressed by volatile general anaesthetics apphed in the chmcal range, whist ewdence from other preparations m&cates that there ~s a rise m mtracellular calcmm concentratmn follovang apphcatmn of these substances 4 Identified Lymnaea neurones have &fferent responses to apphed anaesthetics, ~rrespectwe of the anaesthetic used Following apphcatmn of halothane, barbiturates and several other anaesthettc agents, some cells gradually become qmescent after a short period, whdst m others a series of paroxysmal depolanmng shifts occur pnor to qmescence 5 Cultured neurones of Lymnaea, Hehsoma and related species retain their characteristic action potentml types and neurotransmttter ~dentlty Their responses to anaesthetics are s~mllar to those m the intact brain They may also form synapses m culture Thus, they are a useful tool for studymg the cellular and subcellular actmns of general anaesthetics

INTRODUCTION In recent years there has been an increase of interest m the cellular mechamsms underlying general anaesthesm, but no single cellular mechanism appears to explain ~ts widespread effects m the central nervous system (CNS) It is probable that anaesthetics have a number of different actmns on neurones and that these effects vary from one cell type to another What is clear is that both mn channels (Ken&g, 1989, Franks and Lleb, 1988) and mtracellular 1on concentrations (Ialzzo et al, 1990b, Herland et al, 1990) may be altered by general anaesthetics and it is probable that the interplay of these effects, depending on their relatwe magmtudes, can produce differential changes m the actwmes of ln&wdual neurones or groups of neurones and their synaptlc lnteractmns The only umfymg pnnc~ple of anaesthetic action is that anaesthetics somehow interact vath membrane components, whether these components are found mtracellularly or at the cell-surface In vertebrates it can prove difficult to specify the &fferences of response of particular neurone types to anaestheUcs gwen the very large numbers of neurones m the CNS For this reason we have estabhshed the gastropod mollusc Lymnaea stagnahs as a model system for detailed investigations of the mode(s) and *Present address Rhone-Poulenc Rorer, CRVA, B P 14, 94403 Vltry-sur-Seme, France tPresent address Department of Physiology, School of Me&cal Sciences, Umverslty Walk, Bristol BS8 ITD, England 985

site(s) of action of general anaesthetics Lymnaea and other gastropod molluscs have proved to be excellent model systems for stu&es on general anaestheUcs since they have large, umquely ~dentlfiable, nerve cells and ~t is possible to study the cellular (Franks and Lleb, 1988, Wmpenny et al, 1991), network (McCrohan et al, 1987) and behavloural actions (Glrdlestone et al, 1989b, Barron et al, submitted) of anaesthetics dehvered at chmcal concentrations (Glrdlestone et al, 1989a) Here we wdl explore the view that neurones are &fferenUally affected by general anaesthetics and discuss the acUons of general anaesthetics on calcmm currents and mtracellular calcmm concentration We will also discuss the feaslbihty of using cultured molluscan neurones to study the cellular effects of general anaesthetics CALCIUMCHANNELSARE AFFECTED BY GENERALANAESTHETICS Many ion channels are affected by general anaesthetics [eg K+channels (Franks and Lleb, 1988, Southan and Warm, 1989)] However, commonly used mhalaUonal and barbiturate anaesthetics produce dose-dependent effects on the spontaneous discharge of ldenUfied neurones (Glrdlestone et al, 1989c, Wmlow and Glrdlestone, 1988), abohsh the calcmm dependent components of action potenUals and block chemical synapt~c transrmsslon (Glrdlestone et al, 1989c) These stu&es rmply that calcmm channels may be selectwely affected by anaesthetic agents, but other channels are clearly affected as well Many

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W WINtX)wet al

of these effects could be due in part to blockage or partial mactwatlon of calcmm channels (Glrdlestone et al, 1989c, Nlshl and Oyama, 1983, Wlnlow and Girdlestone, 1988, Winlow et al, 1987) This hypothesis is supported by work with calcmm channel lnhlbltors (Dohn and Little, 1986a) and activators (Dohn and Little, 1986b), on several preparations (Terrar and Victory, 1986, Caldwell and Hams, 1985, Tas et al, 1989) in which a wide variety of lnhalat~onal and systemac anaesthetic agents was used However, one should not assume that anaesthetics always block or partmlly mactwate channels, since m some Lymnaea neurones an anaesthetic reduced potassium current (I~(A.)) causes hyperpolanzatlon (Franks and Lleb, 1988) In vertebrates the T-, N-, and L-type Ca :÷ channels are commonly described (Carbone and Lux, 1987, Fox et al, 1987, Tslen et al, 1988) and these are both electrophysiologlcally and pharmacologically &stlnct from one another The T-channels are low voltage activated (LVA) channels whdst the N and L channels are high voltage activated (HVA) More recently P-type channels from cerebellar Purkmje cells and the presynaptlc terminals of the squid grant synapse have been described and found to be very similar to one another (Lhnas et al, 1989, Regan, 1989, Sah et al, 1989) Thus it is doubtful whether the whole range of calcium channels has yet been discovered Furthermore minor species varmtions m calcmm receptors clearly exist and more are likely to be found However, both agonlst and voltage gated Ca 2+ channels occur m all highly evolved groups of animals, including vertebrates, and may be potentially regulated by secondary messenger systems (HIUe, 1989) Both LVA and HVA channels are also known to exist in molluscan neurones (Haydon and Man-Son-Hmg, 1988) which are easdy accessible, physically large, identifiable cells whose biophysical and pharmacological properties can be determined with relative ease INTRACELLULARCALCIUMCONCENTRATION,ICa2+]I, IS ELEVATEDBY INHALATIONANAESTHETICS Recent work on rat hepatocytes (Imzzo et al, 1990a, b), skmned myocar&al cells of rats (Herland et al, 1990) and CA1 hlppocampal cells of rats (Mody et al, 1991) demonstrates that volatile anaesthetics produce a rise m [Ca2+]~probably due to a reversible efflux of calcium from the endoplasmac retxculum Molluscan neurones are varmble m terms of ultrastructure (Coggeshall, 1967) and electrophysiological properties (Wlnlow et al, 1982) and preliminary data on apphcatlon of 10-5M caffeme and 10-6M ryanodme, both of which increase [Ca2+]~, reveal varied responses, which are consistent for particular neurone types (Ahmed, Hopkins and Wmlow, m press) In some cell types there are no marked differences m action potentml shape m the presence of these drugs, whilst m others the pseudoplateau and undershoot both vary, presumably due to alterations m [Ca2+], Previous stu&es on related species in&cate that rising [Ca2+]~may reactivate both K + currents and Ca 2+ dependent K + currents (Alkon, 1984) By lmphcatlon Ca 2+ dependent Ca 2+ channels should also be reactivated and specific neurones will have &fferentml alterations of

Table 1 DifferentialacUonsof general anaestheticson Lymnaea neurones PDS Quiescence AnaestheUcused(n) A gp J cells M gp RPDI RPeDIVV1/2 Halothane(20) 8 2 4 2 4 Pentobarbttone(28) 5 6 5 4 3 5 n = 48 13 8 9 6 7 5 Isoflurane(10) Allcellsquiescent,n = 10 [CaZ+],, which will profoundly effect whole cell calcmm currents DIFFERENTIALRESPONSES OF IDENTIFIEDNEURONES TO ANAESTHETICS Application of inhalation or systemic anaesthetics to the intact, isolated brain of Lymnaea has revealed important differences in the neuronal responses of individual, identified cells to the anaesthetics (Table 1), irrespective of the drug used A series of specific cell types have been studied (see Fig 1 for locations) and there is a clear correlation between each cell type and its response irrespective of the drug used, with the exception of lSOflurane (Glrdlestone and Wlnlow, unpublished observations) This correlation consists of either a gradual decline in discharge frequency and eventual quiescence or the occurrence of paroxysmal depolarizing shifts (PDS) and oscillatory behavlour dunng either spontaneous or evoked activity (see Fig 2) Slmdar effects on these neurones have also been demonstrated with enflurane, ketamme thlopentone and menthol (Haydon et al, 1982) In lSOflurane all cell types rapidly became silent The significance of PDS to anaesthetic actions IS unclear, but three types of neurone consistently demonstrated spontaneous PDS m the presence of anaesthetic agents, that is the A group cells, J cells and M cells (Table 1) PDS has been descnbed elsewhere, especially in relation to epilepsy (Speckmann et al, 1972, Pnnce, 1968, 1971, Lux, 1984), and ~t has been investigated to determine whether it is an endogenous or synapt~cally mediated phenomenon (Speckmann & Caspers, 1973) It appears that an increase in excitatory synaptlc input plays a part m producing PDS but these authors have shown that it can be evoked in isolated neurones of Hehx and they have concluded that paroxysmal depolarizations anse from changes in intnnsic membrane properties The unorthodox anaesthetic menthol, which is widely used to anaesthetize small aquaUc invertebrates, causes s~mdar relaUonsh~ps between cell type and the occurrence of PDS to emerge (Haydon et al, 1982) It seems that PDS can be triggered by strong synapUc inputs [e g by the action of input 3 on J cells (Benjamin and Winlow, 1981)], so presumably anaesthetic-induced changes at excitatory synapses might m turn produce PDS On the other hand, direct anaesthetic action on membrane channels of the somatic membrane channel could also cause PDS (see Fig 5) A number of drugs other than anaesthetics are capable of reducing PDS Among them is pentylenetrazol (PTZ), which is believed to activate adenylate cyclase which leads to an increased concentratmn of cychc AMP (Onozuka et al, 1983, McCrohan and Gdlette, 1988) This m turn mobilizes mtracellular

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Fig 1 The locatmns of some of grant neurones and major cell groups on the dorsal surface of the brain of Lymnaea stagnahs from wh]ch electrophysmloglcal recordings have been made m the presence of superfused general anaesthetics The buccal and cerebral gangha are not shown Abbrewatlons L, left, R, right, G, ganglion, Pa, pataetal, Pc, pedal, Pl, pleural, Vise, visceral The scheme of neurone nomenclature ]s outhned by Benjamin and Wmlow (1981) and by Slade el al (1981)

a

d

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Fig 2 Effects of superfused ketarmne on spontaneous neuronal actwtty m RPD1 (a,b,c) and an A group neurone (d,e,f) recorded m vwo from the isolated brain (a) Normal acuvlty of RPDI, (b) 1 mm after addmon of 0 2 mM ketanune to the preparation the frequency of finng was depressed, but subthreshold actavity, possibly synapt~c m ortbqn, appeared to be enhanced, (c) 40 nun after nnsmg normal actwRy returned (d) Normal actlvaty of an A cell, (e) after 6 nun m ketanune the A cell &splayed a dramaUc example of PDS, (f) 30 nun after rmsmg normal actwlty Is restored GP

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free calcmm which is followed by calcmm-dependent phosphorylatlon of membrane proteins and subsequent alteration of membrane permeabdlty In Lymnaea differential responses to PTZ may be mduced in particular neurones and this is due to &stmctly different Ca2+-actwated conductances in the two cells, one of which was a N a ÷ conductance and the other a calcmm-dependent K ÷ conductance (Brown and McCrohan, 1991, in press) Ttus clearly demonstrates the importance of knowing the density of specific channel types in particular cells and the lntracellular free calemm concentraUon necessary to modify particular conductances Because of their hpld solublhty anaesthetics must have numerous sites of action both at the cell membrane and at mtracellular sites The effects on mtracellular calcmm stores wdl vary according to the size of those stores and the resulting levels of mtracellular free calcaum vail themselves have effects on membrane permeability as well as on many other metabolic processes The recent paper by M o d y et al (1991) indicates that halothane raises mtracellular calcium of patch-clamped neurones from hlppocampal shces and this may be a general phenomenon Thus with respect to calcium, it seems probable that voltagedependent calcium channels m particular are directly altered by anaesthetic action, and also that effects on mtracellular calcmm release may then adjust the permeabdmes of both these and other channels

Table 2 The majority of neurones studied maintain their normal action potential shapes m culture

Neurone type

Type of action potential

M group II RPD1 II VVI/2 II RPeD 1 II Total number of cellsstudied

No cells studied m culture

No cells retaining spike shape in culture

16 8 4

14 7 4

8

5

36

30 (83 33%)

CULTURED, IDENTIFIED NEURONES ARE ADVANTAGEOUS FOR STUDIES ON T H E CELLULAR BASIS O F GENERAL ANAESTHESIA

Since neurones are differentially responsive to anaestheUcs, but these responses are often difficult to & s c n m m a t e against the background actwlty of the intact nervous system, it is important to study small numbers of Identified neurones, under controlled c o n d m o n s and m the absence of complex synapt~c inputs Molluscan nervous systems are advantageous for this work since it is possible to identify and isolate specific cells Simple mechanical Isolation of neurones Is inadequate for our studies, since mechanical techtuques often yield very few undamaged cell bodies and even these usually have very low membrane potentials Using culture techniques most isolated neurones survive and appear to retain their normal

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Fig 3 The giant neurone W l retains its baste actaon potential charactensttcs m cell culture (a) Recording o f W l m the mtact bram, finng spontaneously at a rate of about 2 spikes/see (b) One of the spikes from (a) expanded to show the "ealcmm plateau" on the falhng phase of the action potential and its duration (c) Recording of a W l cell after 2 days in culture and finng spontaneously at a frequency of about 1 spike/see Despite the lower finng frequency the duration of the action potential is longer (d)

Differential effects o f general anaesthettcs

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Table 3 Action potential half-width (I e width of action potentaal at half the amplitude) and action potential amplitude (i e the distance between the highest and the lowest points in the action potential) in neurones in culture and in neurones in intact brains There are no significant differences in these two parameters at 5% level (Student's t-test) Action potential half-width (msec) Neurone type

Splke-amphtud¢ (mV)

Cultured

Intact

Cultured

Intact

RPDI

1564+874 (n = 16) 1660+529

21 2 7 + 8 0 8 (n = 7) 1185+590

8023+864 (. = 16) 7682_+1193

7842+831 (n = 7) 9061_+920

(n = 8)

( . = 5)

(n = 8)

( . = 5)

RPeD1

1307_+337

74_+42

7670_+1097

9139_+715

(n = 9)

(n = 10)

( . = 9)

M group

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Fig 4 A 2 day cultured RPeD1 cell becomes qmescent m 1 0% halothane v/v as it does in the intact brain (a) Cell finng spontaneously at a rate of 2 sptkes/sec (b) A smgle spike from (a) expanded to demonstrate the actton potental shape and the plateau on its falling phase (c) In 1% halothane the cell gradually became quiescent and the calcium dependent plateau was abohshed (d) This was not due to the lower frequency of discharge of the cell, but to a genmne loss o f the plateau as demonstrated in (e) and (f), where a r a m p depolarization was apphed in the presence o f halothane to m a m t a m a steady discharge N o frequency dependent broadening o f the action potential was discernible m comparison with (d), clearly mdtcatmg that the plateau had been abohshed

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b

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Fig 5 Paroxysmal depolarizing shifts demonstrated on an isolated M group neurone of Lymnaea m 1% halothane v/v after 3 days m culture (a) Halothane was introduced into the recording chamber at the arrow and wxthm 20 sec the discharge altered from a regular spiking &scharge to PDS-hke actwlty in&catmngthat thxs type of discharge is endogenous rather than a network phenomenon (b) The fifth burst from (a) on an expanded Ume base to demonstrate the membrane osexllatlonswhich are similar to those prevxously reduced by general anaesthehcs xn recordings from the whole brain of the ammal (Modified from Wmnlowet al, 1991 ) physiological characteristics (see below) Techmques for the preparation of either spherical neurones (Haydon, 1988) or extended neurones with neurmc trees are now available and synapses may be prepared tn vttro between both cell types DIFFERENTIAL DISTRIBUTION OF CALCIUM CHANNELS IN CULTURED NEURONES

The membrane of buccal neurone B5 of Hehsoma contains both LVA and HVA calcmm channels Cultured neurone somata normally exhibit both currents until, with the development of soma-soma synapses, the cells develop their secretory capacity at which t~me only the HVA current is evident In extended cells the calcium currents are differentially distributed the non-secretory soma has both LVA and HVA channels, whde the &stal secretory processes contain only the HVA channel (Haydon and Man-Son-Hmg, 1988) and are directly accessible to experimental manipulation A detailed examination of the effects of anaesthetics on the calcium currents cf synaptlcally connected spherical cells or extended cells is thus feasible using voltage- and patch-clamp teehmques In relation to synapt~c transmission we may ask whether some synapt~c terminals sequester su~clent calcium in organdies to be released by applied anaesthetics and what the effects on transmitter release are hkely to be9

CULTURED NEURONES RETAIN THEIR CHARACTERISTIC ACTION POTENTIAL TYPES AND RESPONSES TO ANAESTHETICS

Molluscan neurones may be characterized by the shape of their soma acUon potentials (Wmlow et a l , 1982) As shown m Table 2 and Fig 3 cultured neurones (either spherical or extended) have action potentials similar to those recorded from cells m the intact brain (Yar and Wmlow, 1991, Wmlow et a l , 1991) This implies that cells cultured using our techtuques express ~on channels similar m type to those m the adult from which they were derived (Table 3) The responses of cultured neurones to superfused inhalation anaesthetics are largely similar to those of cells m the intact bram For example, the giant dopamme cell becomes qmescent m 1% halothane and loses the calcium-dependent plateau on its action potential (Fig 4) Likewise, many cultured neurones spontaneously develop PDS in the presence of halothane (Fig 5), further demonstrating that this ~s an intrinsic property of the neurones themselves and not due to synapt~c mteraet~ons between a population of neural elements CULTURED MOLLUSCAN NEURONES AS A TOOL FOR ANAESTHETICS RESEARCH

A number of neurones and mterneurones containmg ~dent~fied neurotransm~tters and making mono-

D~fferentml effects of general anaesthetics synaptlc, chemical connecUons with their follower cells m the whole b r a i n have been identified in g a s t r o p o d molluscs a n d m leeches (Benjamin, 1984, Nlcholls et al, 1990) In Lymnaea each o f these cell types has multiple postynapUc acUons (Wmlow, 1990) A n e u r o n e containing acetylchohne has been identified m the buccal ganglia o f Hehsoma a n d m a k e s novel synaptic contacts with other identified cells m culture ( H a y d o n , 1989) In Lymnaea a grant s e r o t o n m - c o n t a l n m g cell synapses w~th the buccal m o t o r neurones ( M c C r o h a n a n d Benjamin, 1980) In b o t h Lymnaea a n d Planorbts a grant d o p a m l n e c o n t a i n i n g cell, presynapt~c to m a n y neurones, has been shown to exast (Berry a n d Cottrell, 1975, Wmlow et al, 1981) a n d a large F M R F a m l d e - c o n t a m m g cell (Benjamin et al, 1988) shares m a n y of these same follower cells (Syed, 1988) All these cell types surwve well m culture a n d m a k e chemical synaptlc contacts w~th their n o r m a l follower cells (Syed et al, 1990) T h e system of cultured neurones a n d cultured synapses described a b o v e offers the o p p o r t u m t y to study anaesthetic acUons b o t h at the level of the cell m e m b r a n e a n d also their effects on mtracellular processes m uniquely ~dent~fied cells w~th k n o w n neurotransmltters SUMMARY

Anaesthetic agents interact wxth m e m b r a n e comp o n e n t s b o t h at the cell surface a n d lntracellularly It is clear t h a t they modify m a n y different conductances b o t h &rectly a n d m&rectly by alteration of m e t a b o h c processes, which themselves raise free [Ca:+], m a n u m b e r o f &fferent p r e p a r a t i o n s T h e responses of Lymnaea neurones to superfused anaesthetics vary from one cell type to another, but are consistent for each cell type with respect to the i n h a l a t i o n anaesthetics h a l o t h a n e a n d enflurane, for b a r b i t u r a t e s a n d for k e t a m m e Some cells gradually become quiescent m the presence of anaestheucs w h i s t others become qmescent only after a series of P D S P D S m a y be triggered by a n increase in free [Ca2+]1, which is capable of modifying m a n y m e m b r a n e conductances Cultured molluscan neurones retain their action potential types, transnutter 1dentines and charactensuc responses to anaesthetics B o t h isolated a n d synapt~cally connected cells m a y be cultured, thus providing p a r a & g m a t l c p r e p a r a t i o n s for future stu&es on the cellular bases o f general anaesthetic actions REFERENCES

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991

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