Minireview opiates, opioid peptides and single neurones

Life Sciences, Vol . 24, pp . 1527-1546 Printed in the U .S .A .

Pergamon Press

MINIREVIEW OPIATES, OPIOID PEPTIDES AND SINGLE NEORONES ß . A . North Neurophysiology Laboratory, Department of Pharmacology, Loyola üaivereity Medical Center, Maywood, Illinois 60153 (Received in final form October 18, 1978) The use of electrophyeiological techniques to study the mechanisms of action of opiates has three distinct advantages . Ln the first place, they measure directly the important functional parameter of the nervous system the electrical activity of single neurones . Second, they have a high resolution in tine which facilitates arguing from effect to cause . Third, they have a high resolution is space because single neurones, or single loci on the eamn neurone, can be ezsmined selectively . The present review deals ezclueively with recordings at the level of the single neurone . In vivo liecordinge The effects of morphine oa single neurones recorded in vivo are snmtarised is Table 1 . There are three important advantages to the is vivo recording te chnique . First, the cells are more or less close to their milieu interisur . If anesthesia and other ezperisental factors (hypotension, carbon diozide retention, pathological sensory input, etc .) are controlled then the action of opiates may be studied on neurones which are ín a relatively normal condition and which are receiving and giving rise to relatively normal synaptic oonaections . The second major advantage is that the cells can be identified by their aaatamital coordinates . The anatomical location is important because it enables studies to be made of regions known to be high in their density of etereospetifit opiate binding sites - studies which involve the caudate nucleus, locus coeruleue or periaqueductal gray are likely to be mre relevant sad easier to interpret than those involving the observations made where opiate binding sites are sparse (eg . cortez, cerebellua, anterior horn of spinal cord) (96, 97) . Third, functional indentifitation of the neurones is poaeible sad is much preferable to simple anatomical ldentificatioa . This can be done, for ezaaple, by showing that the neurone under study is involved ín the response to pain (eg . thalamus, dorsal horn) or by demonstrating that opiate effects are clearly restricted in site to a group of cells of relatively hamogeaeous sad understood function (eg . locus coeruleus, nucleus raphe nagnus) rather than raadamly dispersed as in the cortez . The major limitations of eztracellular recording techniques lie in the method of administering the opiates . The manifold effects of narcotic analgaeics slake systemic administration fraught with difficulties . The effects of opiates on the neurones in question must be clearly distinguished froo effects secondary to the hypotension and hypercapnia which are hallmarks of opiate action . liven when such factors can be limited or ezcluded, it can seldom be established that the opiate ie acting directly on the neurone whose activity is being recorded . There ie a need to continue stable recordings from single neurones for long periods in order to study the effects of opiates applied systemically . In the case of the opioid peptides rapid 0024-3205/79/171527-1902 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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degradation and poor penetration to the nervous system virtually preclude their systemic administration . For these reasons, iontophoretic application of opiates and opioid peptides is widely used . Application of opiates and opioids by iontophoresis is full of pitfalls ( inter alia , ref . 12) . The opiate or opioid peptide is expelled from the tip of a microelectrode by passage of electrical current ; the main deter minants of the amount of drug leaving the electrode in a given period of time (usually 10 - 100 s) are its concentration within the barrel (usually 5 - 100 mM), its transport number at the given pH (which is a measure of the proportion of the current passed which is carried by the ionized form of the drug), and the size of the current passed . It is now customary to use coastaat current sources for ejecting drugs so that changes in electrode tip resistaaces which frequently occur will less seriously interfere with the ejection . In the majority of studies cited, but by no ~~a all, gross effects due to current density or pH changes in the vicinity of the electrode tip were excluded or controlled . Despite all such precautions, the concentration of agonist at the receptor site remains unknown . The steady state concentration of expelled drug falls with distance linearly from the tip of the iontophoretic electrode - in the absence of factors such as inhomogenous diffusion coefficients, dissolution in membranes and specific uptake process (see ref . 23) . It is likely that this concentration gradient will be much steeper in the inhomogeaeous environment offered by the compact neuropil of the central nervous system, and it is worth considering that the tissue actually adjacent to the tip will theoretically be subject to a concentration equal to that within the barrel multiplied by the transport number (eg . for 50 mM morphine multiplied by 0 .05 the concentration is the infinitesimally thin boundary layer at the electrode tip would be 2 .5 ml~ . The ideal circumstances, therefore, are to show that similar effects can be observed with low doses given systemically and known currents applied iontophoretically . This confirms that one is recording from a morphine-sensitive region, but also enables a crude sort of biological calibration of the iontophoretic application . For example, the cells of the locus cceruleus have been shown to be inhibited by both iontophoretic application (10) and systemic administration (71) of morphine at doses which cause analgesia ; this contrasts with, for example, the nucleus raphe magma where the effects of systemic application of opiates could not readily be mimicked by iontophoretic application, thus suggesting a site of action outwith the raphe magma itself (2), The actual currents used to eject opiates from iontophoretic electrodes vary markedly from study to study . Opiates have marked local anesthetic effects (eg . 46, 72), and it is clearlq important to observe carefully the spikes recorded for evidence of this . Such effects have been reported with iontophoretic application of morphine at currents as low as 40 nA (87) yet several studies have used currents much higher than this ( with similar concentrations in the electrodes) . Similarly there is a great discrepancy among the dotations of application of opiates from study to study . Those findings which are obtained 10 - 30 a of beginning the iontophoretic applicati~n are easier to interpret and are more likely to be relevant than those resulting from very long periods of iontophoretic application (eg . 5, 6) . Just as the concentration of agoniat is not known with iontophoretic application, nor is its precise site of action indicated . The finding of inhibition with this iontophoretic technique does not allow one to distinguish readily between direct effects an the neurone under study, presynaptic inhibition of ongoing excitatory synaptic inputs, or even excitation of closely adjacent inhibitory interneuronea . A selective

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depression of ezcitatioas caused by glutamate (leaving the spontaneous firing rate little affected) is sasptiaes considered to constitute evidence for a direct action on the cell whose activity is being recorded . Such a distinction may not be infallible because glutamate may cause release of another excitatory transmitter and thin could not be diatinguishad with eztracellular recording; on the other hand, the fact that glutamate ghost always excites central neurones does suggest a predominantly direct action on the neurone whose activity ie recorded . A presynaptic action may be best excluded by selective physical or biochemical deaffereatation prior to the e:parimats; few brain regions lead themselves to this although one which does, the dorsal horn of the spinal cord, has not yet been investigated . Otherwise, presynaptic effects nay then be inferred if the response to one type of input (eg . C fibers in the dorsal horn) is depreaeed when the respanse of the same cell to another i~ut is unaffected (76) ; but such a distinction could not exclude selective poateynaptic interference with the action of the released transmitter when the traasni.tter used by thn two input pathways ie different, or when different iaterneuraaea era interposed in the two pathways . Bzcitation of a covertly situated inhibitory interneurone nay be a mchaniea of inhibition ; indeed, the converse phenomenon, inhibition of closely adjacent inhibitory interneurane has recently been shown to ezplaia the excitatory effects of opiatna on hippocampal neurones (see below) (122) . In the absence of any knowledge of the precise concentration of opiates at the receptor site, the experimenter using iontophoresia mist find other methods to demonstrate that the observed effects are mdia~dd by opiate receptors . üithout doubt, the bast of these is etereospecificity . Ae it ie unlikely that there would be significant differences between the transport nusbere and rates of diffusion of optical eaaatiaagrs, the testing of the relative seaeitivity to anantioaeric agoniets is a simple way of deaenstratiag that the site iavolvad is affected only by the pharmacologically active isomer . ühilet antagonain by naloxone is a prerequisite for the deaioastrstion of opiate receptor mediated events, simple reversal or blockade at ~mimown concentrations is of limited value . The recent demonstration that iontophoretic application of nalozone antagonizes the effects of Y-a=inobutyric acid (GABA) as well se those of morphine is germane (30, 34) . The currents used for the ejection of nalozone is these studies were nodeet by comparison with those used in meat' studies which purport to demonstrate specific opiate effects . The fact that the opiate receptor diacriainatee cell between optical enantiomrs can also be exploited by testing for aar~ {~ by pairs of isomrlc antagonists (53, 116) . The doee~ratio method of Gaddum (51) and Schild (106) is the appropriate way to test whether the antagonien is caupatitiva at a comooa site because by canparing equal responses it nliminatee con8lderation of the processes occurring between receptor occupation and the effect as firing . But such analyses are difficult with ioatophoretic application of both agoniet and antagonist because the dose-ratio method requires a knowledge of the equilibrium concentration of antagonist at the receptor Bite . The next most eatiafactory mthod to demonstrate a specific action of the opiates may be to teat for aatagoaism with the eystemi.c e~+~i~ tration of a dose of nalozaae known to be auffiaient to reverse the affects of opiates in the whole animal (eg. 39, 40) gad to see whether this aatagonisa ie eurmimtable by increasing the amount of agoaíst ejected from thn electrode . IInfortuaately, the ignorance of the concentration of antagonist at the receptor site makes it difficult to use the iontophoratíc tectmique to provide elactrophysiological evidence for the different receptor subtypes as the basis of different antagonist aquilibrius constaata (82) .

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A further problem of the iontophoretic technique ie the difficulty in studying deaénaitizatiaa pheno~aa because one can never be sure that the save current ejected from the iaatophoresis electrode is carrying the same number of moles of opiate with repeated ejections (24) . Factors which must be controlled carefully are the amplitude of any retaining current, sad the time since the last ejection current . One clever way to avoid such problems is to use the save electrode in different brain regions, with the other factors such as retaining current and ejection interval held constant - such studies indicate that repeated applications to etriatal neurones leads to a rapid lose in the effect of morphine or enkephalia whilst similar experiments on morphine-sensitive cells in the cortez indicate ao desensitization (50) . In spite of these limitations, the in vivo approach has produced several clear and important insights into the actions of opiates and opioid peptides (Table 1) . It is Convenient to divide further discussion of their described effects into those which deal with spontaneous (or glutamate~voked) activity, those which deal with responses to specific forms of atiuuli sad those which have utilized the greater resolution afforded by intracellular recording . Bffects on Spontaneous Activity The most consistent feature of the experiments is that the action of the opiates which is reversed by naloaone is as inhibition of neuronal firing . At all sites so far examined, there has bean good correlation between the effects of vorphine and the opioid peptides, although it often seems that the latter are lees readily antagonized by nalozone . The effects of ßeadorphin appear to be similar in time course to those of morphine, whereas the enkephalina usuallq haws a somewhat more rapid onset sad offset of action . Interestingly, several of the studies in which opiates (29, 115) or enkephalin (54, 115) caused inhibition of neuronal firing which was not antagonized by naloaone were made in the cat . In the hippocampua of the rat, excitations by uorphine predóminate over inhibitions . These excitations must be distinguished as those which are not atereospacific nor antagonized by naloaone (49, 107) and those which are antagonized bq naloaone whether applied iontophoretically (61, 87) or in rather large doses systemically (87) . Many of the nalaaone-reversible excitations of pyramidal cells appear to be due to a prisary action of the opiates to inhibit the firing of basket cells . This ezcitation by diainhibition is indicated by the facts that it is blocked by iontophoretic application of magnesium or bicuculline, sad by direct simultaneous recording of the activity of the two neurones involved (122) . The other site at which ezcitation has been described is the Renahaw cell (6, 25, 26, 29, 36, 37, 81) . Here the situation is pore sample : because iontophoretic application of naloaone has been reported to antagonize not only the excitation caused by morphine (26) and enkephalin (26), but also that produced by acetylcholine (29) and substance P (27) . Furtheruore, the only experiments which have studied the response of ßenshaw cells is a species other than the cat (25) indicated that a clear excitation by morphine is sees is onlq a small proportion of neurones, even though morphine often enhanced the responses to iontophoretic application of acetylcholine . Indeed, in the rat, aalozana antagonized only two of the four cells suited by morphine, although it did effectively antagonize the excitations caused by acetylcholine . Although neither enkephalia levels nor the density of stereospecific opiate binding Bites are high is the anterior horn of the spinal cord ( inter alia , 96, 97, 109, 110) the possibility has to be considered that acetylcholine and

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iabla 1 Tha e!laca o! Opiatp aoa Opioida m einela lauraw letivlq apaeias

opiatp

apioia paptidaa

aalosooa rawsaal

notaa

rafaranaa

owcortas eat

73

-

9

ataraoapaciíie aliact rn

-

atarawpacilic afíact, ascitstion vitti aoch bichas tartana

7r

104

105

rn

gatade nalosoos 2 ~/~ and somi e7 l0e aas mia not antaeonisad b 20

rat

47 bZ

nt

123 124

nt aaadaa/patamn nt

apentanaooa i OLD iadnad lirine

7aa

rat

_.

nt

rat

7aa

aa

~aa

nnelaaa aee~aoa

30 47 e7

ataraoapacüie afiact

u e3

rn

-

_

T,a

'

rn

+

++

7~ no

~

w e7 moat °cortai'

7w

apontaaaoaai~ Hrine ea1L a:citad ; QLD ea1L iahibitad

61

apantaaaoaa lirine ca1L not ailaetad ; raapooaaa a 4Ch, OLD, 18! and 1e bloctad

107

rn

+

+

+/-

nt rat

+/no

both laaorphanol and da:trorphao asaitad

e7

49

nt

0/-

est

+

7aa

est

+

f~

partial antyoaian b nalosona

21

~at

-

no

apontanaoaa, OLD indaaad aad nosiosa ati~ly inaoosd Brine

3e

0

21

1532

Opiates-Opioid Peptidas.~-Single Neurons

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cabla 1 continaad species

opiate I

rat

-

ra[

-

cat

0

rat

hspotbaluo~ rat rat

aubatanti.a niara rat

opioid paptida

8

-

nalosooa rawraal

I

I

8

-*

yea

-

yea

ya

-

lea

yea

-

aria affect when s+orphine ws placed 66 directly into caudate

yu

no:ioua edenlos indncad firm;

58

spontaneous and CLII induced firing

47 48

yea

-

-

-

no

0 +

araballoa

-/0/+

pooa/ssidalla -

rat rat

yea

-/0/+

ya

-

yea

-

yea

yu -

yea

apantanao~r firing

58

affecta variable but all antagonised by nalosona

87

nalozona antagonised ayatadc anrphiaa only

locus coeralaua

yea

-

68

115

dorsal rapbe nnclea~

rat

43

yo

cat

rat

64

+

yea

rat

37

(D-ala2-D-lees-antaphalia)

auitatiau in vantr~a+~~ inhibi[iona is lateral, both mtagonised

-

0

nalomw bloctad ACh aaitatiooa

63

yea

-

0

87

apontaaaour, Q.D i ICh induced and nosioua atiaulua induced firing

yea

rat

rat

apantanaoua firing

+/-

psriagnaductal isa7

rat

reference

-

aiaa anc~p~a~i c reticular forsrtian ra[

yea

nota

logo ooarnlana, starawpacific affect

59

117 10

locus coeraiees; ipontanaoua and nozious etiauh induced firing

71

uoatly IüGC i 3~C ; aacitatioss wra not antagonised by nalo~ona

14 16 17

locus coeralaua

57

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Opiates-Opioid Peptides-Single Neurons

Lbla 1 coatisned apeciee

opiaW

opioid aalorone peptidee rawreai I I S B

I cat

-

_*

no

rai

-

-{

7aa

7aa

rat rat cat

-

-e -

pas

~a

-

no

-

lea

-/0

-

cat cat

anet >AOC i pontas cand~m "

7p pas

+ -

te~ita~daal mclene cat dorsal hose noetl~ leans cat (S) cat (S)

v

3'p

(eaartiaee I, Iv and vI) vee )p

rat (S)

>"

cat (D)

0

cat (S)

-

cat (S)

-

cat (S)

o -

rat (S)

0

t

lea fee

o -

notaa

~p

rabbit (I)

-

7eae

eat (S)

-

lea

relerence

54 lu 13 13 87 62 2

soatlp 1000 i lip= etereoepeeilic died aoatlp 1iI. dorsal rdulia (4 rats aaip) ]Aí11= gatad.c leata~l ras >uch tura s!lectiw the lontophosetie sarphine liaL (saati identilied ee bnlboepinal) 3 aa1.o®ne alme inhibited Hrias bulbar wpbs 115 epantaoeoaa i msiov atiaulna induced farina

1

no~oue etirulne indoaed firins ti0 apontaoeoua i no>ione stia~lae 78 indacad liríss lirins induced b7 C-Hbre etir 79 nlatioa spcataneow i no~doua aeimlua 77 induced Hrins epontaneone, (d.II indmed i noaioae 18 strains induced firms inhibition o! reepoose to Ib and 76 C Hbra bnt not to da atiaalatim rhea applied into l.aadaa v 39 rhea applied into leadna II there 41 rue a eelecti~e inhibition of the 42 reeponee to nozioae atiaol.atian up to 80 n~ had no effect m OLII 11 induced Hrins 0.3-2 ss/ics rorphiw, bra~kiaia 114 iadueed Hrins (+~oalorphine) apaataoeoue i electrlcall~ stir 9S slated but not bra~tinin iadopd firias

Opiates-Opioid Peptides-Single Neurons

1534

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Table 1 confirmed :patio

opiates

cat (S)

-

cat (S)

opioid peptides

nalozone ravaraal

-

cat (S)

yae*

cat (S)

+

cat (S)

0 -

?no 7yes -

yes

Rombar calla + rat

cat

+

rat

+

cat

+

cat

+

yea

+

yes

yea

notas

refersnca

spoatanavus i nosiow sti~ulua induced firing blocked CLII i ASP azcitatioms ; high iontophorstic currants spontaaeow i nosious stiailna induced firing (*nalorphine) notiaeptive neurones, non'rotiteptivs nsurmu ; high currenU 6 long dmratioms

69 70

whoa applied close to call róen applied 30-100 1a from the call, nosi.ous inputs selectively depressed

28

ezcitation vas stereospecific ; nalozone antagaaisad AC6 bnt not Q.D eztitatiom

25

ezcitation vas steraospacific ; nalosone aatagonised ACh but aot Di.H euitatiam norphim rewrsad inhibition by glycim, but not that by G11~A 4 tata only tA(:h ascitation aLo blocked) SP escited calls, thL was blocked by nalosoae, DI.H azcitatiom wre not blocked

37

3S 8 6

81 26 27 28

- inhibition ; + ~ eztitatien ; 0 ~ no affect on firing I - iantophoretit application ; S ' eysteait addni.etration * ~ ß-endorphin is addition to eakaphalins (S) ' sptw" t anirt ; (D) ' dacarabrate enrol ; (I) ~ intact enrol Q.II ~ glutasiata ; SP ' aubstante P ; DLH ~ dl-he~ocyetaate ; ACh ~ atatylcholine ; ASP ~ aepartate ; 1fE ~ norepimphrim ; è1gGC ' nutLus retítalaris gigantoeallmlasis ; 3~C ~ nuclew retitnlaria parasrdianus ; lOtL ~ nucleue reticalaria lataralie ; äilS ' nutleue raphe satgnv

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substance P are causing the release of enknphalia . änwever, there ie no direct evidence that these opiate receptors are located on the Renshaw cells themselves, and the likelihood that their activation causes inhibition of local inhibitory interneuronee must be seriously considered . E:peri~eats similar to those perforoed is the hippoaampus with local blockade of synaptic traasaission may help to clarify the issue. The practice ie widespread of applying curreate of glutamate is order to suite otherwise silent neurones, or to speed up the discharge of slowly firing epoataneously active cells . There is considerable variability in 21eg1the effect of morphine and enkephalin on glutamate induced activity . g~nsberger's group (118, 124,126) have found that morphine and enkephalia inhibit spontaneous firing of vertical neurones as well as that induced by Evidence was presented that the neurone was acetylcholine and glutamate . more sensitive to inhibition by morphine when its firing rate was elevated by acetylcholine or glutamate, in that some cells were unaffected by morphine ezcept when they were thus suited . Other ezcitant subataacea such ae homocysteate have also been used to suite neurones prior to application of morphine (83), and it may be that when the neurone is firing at a higher rate it is more likely to manifest a depressant action of the opiates . Nicoll et al . (87) found no evidence for a selective antiglutamate effect of opts-tes and opioida - ezcitations by glutamate were inhibited by opiates In the cat in the same manner as the spontaneous firing of the neurones . thalamus (38), however, the response of neurones to glutamate was reduced is some cases by iontophoretic application of morphine and in many cases by systemic administration of morphine ; these effects occurred with little or no change is spontaneous firing rates or sensitivity to GABA . It is is these particular respects that the lack of insight afforded by eatracellular recording and iontophoretic application of drugs is most obvious . The interactioas of opiates with the glutamate depolarization has been pursued further at the intracellular level (see below) . Reduction in Evoked Responses Numerous studies have sham that the excitatory response of single neurones to stimulation of their peripheral receptive field are inhibited by The response of thalamic neurones evoked by opiates and opioid peptides . electrical stimulation of the paw was depressed by systemic morphine (38, 63) ; and a similar depression of the respaise of rat thalamic neurones to 'painful' tail stimuli was sham to be produced by iontophoretic application of eakephalin and one of its analogues (62, 64) . The excitation of locus coeruleus cells by noxious stimulation was also antagonized by systemic morThe response of neurones in the mesencephalic reticular formation phine (71) . of the rat to foot pinch stimulation was also blocked by iontophoretic or In all the above studies, the systemic administration of morphine (58, 59) . effect of morphine was antagonized by naloxone given systemically . In the lamina V of the dorsal horn of the spinal cord, neurones can be activated either by noxious or non-noxious stimuli applied to their peripheral receptive fields . A great deal of useful information in the response of these neurones to noxious stimulation is available snd has been reviewed recently (67) . Moat workers agree that the response of neurones in lamina V of the spinal cord to noxious stimulation is inhibited by systemic adminietratíon of morphine . The response to C and Aó fibre input ie much more These effectively antagonized than is the response to Aa fibre input (76) . Determination of the site of action of effects are antagonized by aalozone . morphine is especially important in these experiments in view of the contributing roles of local dorsal horn sites of action and actions in the brain stem For eaample, Takagi's to enhance descending inhibitory influences (44, 56) .

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group have indicated that the morphine inhibition of the excitation of lamina V cells induced by intro-arterial bradykinin injection does not occur in spinal rabbits except when the dose is substantially increased (114) . Indeed, they have localized the site of action of morphine in the brain stem by microinjection techniques - only when morphine was applied in the nucleon reticularis gígantocellularis (NRGC) was an inhibition of bradykinin induced firing observed (113) . Naloxone administered to the same site prevented this effect . These findings are particularly significant because it is known that the cells of the medullary reticular formation, and particularly the NRGC, respond readily and rather selectively to noxious stimulation or Ad fibre input (7, 19, 20, 55, 80) . The experimental conditions of the animal are clearly important. In decerebrate cats (77), morphine had virtually no effect on either spontaneous of bradykinin induced activities whilst in the npinalized state morphine reduced spontaneous firing rate and strongly depressed or abolished bradykinin evoked activity . The interpretation is that the dorsal horn neurones are already strongly inhibited in the decerebrate state prior to the administration of an opiate - because of the absence of lover trannection of the neuraxis - and are thereby lean likely to reveal inhibitions due to opiates . Attempts to replicate these inhibitory effects of morphine on lamina V cells by applying it in the region of the cell bodies have met with mixed success . Calvillo et _al . (18) reported that iontophoretic application of morphine affected only those cells which responded to noxious stimulation; in these cells it blocked spontaneous activity and activity evoked by stimulation in approximately half of the cells and it blocked glutamate-evoked excitation in all cells . However, the effect of iontophoretic application of morphine was blocked by iontophoretic naloaone in only 2 of 7 cells, and by intravenous naloaone in only 2 of 4 cells . The depression of firing of these cells first reported by Doatrovsky and Pomeranz (35) has also been shown to be insensitive to naloaone (37) . Zieglgâneberger's group, however, did find that naloaone antagonized the morphine inhibition of firing of dorsal horn neurones - whether this firing was spontaneous, evoked by tactile stimulation or evoked by glutamate (119, 126) . In an attempt to reconcile these discrepancies, Duggaa's group recorded from neurones in lamina V and applied morphine by iontophoretic either into lamina V (from another barrel of the recording electrode) or into lamina II (from a second separate iontophoretis electrode) (39, 40) . When applied to the region of the cell bodies, morphine and enkephalin had little effect on the responses of the neurones to noxious stimulation. But when applied into the aubstantia gelatinosa, both morphine and enkephalin caused a selective inhibition of the response of the lamina V cells to noxious stimulation while leaving unchanged the response to innocuous stimulation . Administration of naloaone into the subatantia gelatinosa reversed these effects of the opiates and enkephalia, as did intravenous injection of rather low concentrations of naloaone . The selectivity for sensory modality is in keeping with the findings of other workers and agrees with what in known about the effects of morphine given systemically . The authors argue that their findings might be due to a presynaptic inhibition of transmitter release from the texminals of nociceptive afferents, or to a blockade of the poataynaptic action of the primary afferent transmitter by an action in the dendrites of the lamina V cells, or possibly mediated by an action on interneuronea of the substantia gelantinoea itself . The selectivity for noaioua stimuli might be construed to favour the first or third of these alternatives . In any event, it ie clear that morphine is more effective when applied directly into the aubstantia gelatinosa - which is both the region of the dorsal horn known to be richest in stereospecific opiate binding sites, and close to the region of teradnation of the fine calibre lateral division primary afferent fibres (for review see ref . 67) .

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Intracellular recording-in vivo The advantages of intracellular recording lie in the increased likelihood of being able to distinguish between direct actions on the neurone mmd those mediated by trmmsaynaptic influences, and the greater understanding of the mechanism of the changes at the membrane level. On the other head, it must be constantly borne ín mind that intracellular recording gives information on events which take place oa the soma membrane - either directly or by electrotonic spread from more distant regions . It is well known that the propertiea of the soma membrane are sometimes different from those of the cellular processes, and there is considerable evidence that activation of the soma membrane need not even occur in the normal function of some neurones . It is unfortunate that the central neurones whose activity it is simplest to record with intracellular electrodes in vivo are situated in a region is which opiate binding sites are not particularly dense (75, 96, 97) . However, although the majority of intracellular recordings have been made from spinal motoaeurones, essentially similar findings have also been obtained from neuroses in lamina IV and V. Iontophoretic application of morphine at a distance of about 100 um from the cell soma (which is presumed to be the site of the intracellular recording electrode) leads to no significant chmmges is membrane potential or input resiatmmce (119, 120) . Morphine so applied reduces the rate of rise and amplitude of the excitatory poataynaptic potential (e .p .s .p .) obtained by stimulating a dorsal rootlet . Such mm observation could reflect either a depression of trmmsmitter release from the presynaptic elements or a direct posteynaptic action . The former is rendered less likely by the finding that the rate of rise and amplitude of the depolarization induced by iontophoretic . application of glutamate is also diminished by morphine . The possibility should be considered that glutamate is acting to release the excitatory trmmsmítter onto the motoneuroaes, although this would seem to be unlikely in view of the observation that the glutamate depolarization is sot affected by iontophoretic application of tetrodotozia . However, the glutamate is not acting on the soma membrane of the motonaurone and the potential change recorded in the soma is the electrotonic remnant of the glutamate effect on relatively distal parts of the deadritic tree (121, 125) . The blockade by morphine of the glutamate depolarization may be occurring by one or both of two mechanisms which are very difficult to distinguish experimentally . Tha first possibility is a direct blockade of the im~ard sodium current induced by glutamate at the aubsynaptic membrane . However, morphine reduces the rate of rise and amplitude not only of the glutamate potential and e.p .s .p . (which may not be mediated by glutamate) but also of the acetylcholine potential mmd i .p .e .p . (which ie certainly not mediated by glutamate) . Acetylcholine depolarizes spinal motoneuronea by a mechanism which is quite different from that of glutamate ; whereas glutamate increases the conductance to sodium (and perhaps other ions) (125), acetylcholine appears to reduce the resting conductance to potassium ions (see review by Rrnjevic (74)) . It is therefore puzzling that both the glutamate and acetylcholine depolarizations are depressed by opiates in a similar aaloxoae-reversible manner . The second possible mechanism of action of morphine is a hyperpolarization or increased coaductmmce of the dendritic membrane close to or more proximal (ie. nearer to the soma) than the site of action of the glutamate. Either membrmme action, but especially the latter, would be effective in attenuating the potential change occurring at the soma and a sensitive measure of such attenuation would be the rate of rise of the This mechanism ie difficult to demonstrate potential change (65, 99) . directly . Iontophoretic application of morphine results in its acting on only a small part of the deadritic tree and intracellular current iajectian into the soma would fail to detect mmy change in resistmmce (most of the currnnr rrnnwna the mamhrane of the soma and other parts of the proximal deadri-

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On the other hand, tic tree which are not reached by iontophoretic morphine) . systemic administration of morphine would affect all the deadritic tree which bears opiate receptors . It is of interest that most spinal motoneurones were hyperpolarized by systemic administration of opiates (119) . In vitro recordings These experiments have the advantage of permitting the application of opiates in precisely known concentrations ; they allow intracellular recordings to be made from single neurones, transsynaptic influences to be excluded, anesthesia to be avoided and ionic mechanisms to be elucidated . In the case of autonomic neurones removed from mammals, there is good evidence that the neuronal properties are not greatly altered by their translocation to as in vitro environment (112) ; similar evidence exists for explants of No reports fetal tissue removed from the central nervous system (22, 86) . have appeared on the action of opiates on single neurones of the adult central nervous system maintained in _in vitro , although such experiments are now in progress . Unfortunately, most neurones of the peripheral nervous system of mammals appear to be insensitive to any specific action of opiates . Although it is clear that opiate-sensitive nerve terminals occur in some selected sites, and have been of enormous value in advancing opiate and opioid peptide research, cell bodies which respond to morphine have been found almost only in the myenteric plexus of the guinea-pig ileum. Even in this site, there is evidence that the primary site of action is not the neuronal soma (see below) . A survey of several other autonomic ganglia has revealed only a few neurones in the superior cervical ganglion of the cat which are hyperpolarized by morphine . Superior cervical ganglion cells of the rabbit, guinea-pig, rat and mouse were not affected by normorphiae (1 uM) and nor were superior mesenteric ganglion cells of the guinea-pig or dorsal root ganglion (DRG) cells of the frog (Hsu 6 North, unpublished) or cat (Gal lagher, unpublished) . The lack of sensitivity of cells of the DRG correlates with the observations that atereospecific opiate binding sites are found predominantly on the neuritic out-growth from cultured DRGs (111) and with the observation that the density of such binding sites in the dorsal horn of the cord falls following rhizotomy (75) . The possibility might also be considered that intracellular recordings were predominantly from large somata, whilst the morphine sensitivity may be restricted to the smaller somata of the Ad and C fibres . The action of opiates has been investigated on isolated nerve fibre preparations such as squid axon but the concentrations used were very high and the effects reported non-specific (45, 46) . Myenteric Neurones The first report of the effect of opiates oa single myenteric neurones was made by Sato _et _al . (102) . They used glass suction electrodes to record unit activity from mgeateric neurones is the guinea-pig ileum and found that the frequency of firing was reduced by morphine . In a subsequent paper (103) they showed that morphine reduced the firing of these neurones whether this was spontaneous (probably induced by the recording electrode), or elevated by 5-hydrozytryptamine (5-HT), caerulein, nicotine or sodium piccate . The inference from these studies was that morphine probably acted directly on the myenteric neurones to reduce their excitability . A similar conclusion was made by Dinglediae and Goldstein (31, 32, 33) oa much firmer grounds . They showed quite clearly that the effects of opiates on the neurones were stereospecific and antagonized by naloxone, and that the effects persisted in conditions in which all synaptic transmission in the plexus was blocked .

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The e:peri~ata with extracellular recording have subsequently bemm repeated (94) and eztended to enkaphalin (93) and ß-endorphin (116) . These peptides also cause a dose-related nalozoae reversible inhibition of neuronal firing which persists in solutions completely devoid of calcium ions (116) . Szperiments with intracellular recording first failed to show mmy specific effects of morphine oa the neuronal properties (88), but it svbaeqummtly became clear that a proportion of cells within the plazas were hyperpolarized by morphine and enkephalin . However, the primary site of opiates mmd opioid peptides appeared not to be the neuronal soma, which presumably accounts for the relatively small proportion of morphine sensitive cells and the variability in the effects recorded with electrodes in their soma (91, 92) . Two pieces of evidence now point to a non-somatic site of action (90) . First, the hyperpolarization was sometimes but not always associated with fall in neuronal input resistance ; if one presumes that the mechanism underlying the opiate action is the same in all neurones, then other factors must affect the detection of any resistance change . One such factor would be the distance from the eons at which the opiate or opioid peptide is acting, others are the geometrical and electrical properties of the neurone . Second, when enkephalin was applied to the soma membrane by íontophoresis, hyperpolarizations were never observed - even though application of enkephalin by perfusion in the same neurone causes a hyperpolarization . Such a non-somatic . site of action could form a basis for the well-established effects of morphine mmd enkephalin in reducing the evoked release of acatylchoine frog the ayenteric plexus - although it aeema that the mechmmisn of such inhibition would be presynaptic inhibition by hyperpolarization (leading to propagation block) or conductmmce increase (leading to a shunting of action potential amplitude) (see ref . 90) . The pheno~noa whereby opiates inhibit the firing of myenteric neurones in a stereospecific and naloaone-reversible Banner has bemm investigated further . in a study of th~ chmmges which accompmmy the development of tolerance and dependence . When guinea-pigs are pretreated with morphine for several days (by pellet ímplmmtation or repeated iajectiose) and the myenteric neurones removed into a Krebs solution which contains morphine, the neuronal firing is no longer inhibited by adding additional morphine to the perfusiog solution - that is to esy, tolerance has developed (95) . If morphine is removed from the perfusiog solution, or nalozone added to it, the firing rate of thn neurones increases markedly ; in such circumstmmces the firing rate reaches levels never observed in normal neurones removed from naive mmimals, and even a short e:poaure of such a neurone to nalozone sometimes triggers mm increase in its firing rate which far outlasts the period of application of the nalozone . Moreover, ezactly similar chmmgee can be induced in myenteric neurones by ezpoaing pieces of ileum to morphine _ia vitro for periods of 24 hours (89) . In these conditions the chmmges are not quite as florid as when the animal is made dependent, but the fact that essentially identical changes can occur in vitro argues strongly that the changes are taking place directly withinthe~myenteric pleaua and not as a aecoadary consequence of humoral or other systemic chmmges. Neurones in Culture The other nervous tissue which ie suitable for in vitro investigation of the action of opiates are eaplmmts grown in tissue culture. Thia technique has the further advmmtage of allowing correlative electrophyeiological and biochemical eaperimente oa a relatively homogenous population of cells although full advantage of this has yet to be made . It hoe the potential disadvmmtage that the neurones are in a condition of relatively abnormal synaptic connections and this may greatly influence their sensitivity to transmitter substances . However, remarkable progress is now being made with

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dissociated cultures which have been allowed to produce functional and anatomiThere have been oalq a few studies cal syaapsee between different components . of opiate action at the level of the single cultured neurones . There has been extensive investigation of the actions of opiates and opioid peptides on the adenylate cyclase of neuroblaetoma a glioma hybrid cells, but these cells have been the subject of only one electrophysiological study (85) . Morphine had little effect on the resting membrane potential or input resistance of these cells, but it did affect the depolarizing response Low concentrations of the cells to the iontophoretic application of dopamine .

of morphine (200 - 600 nM) increased the amplitude of the dopamine potential up to 75x but this effect was transient and difficult to repeat in the same cell . Higher concentrations of morphine (8 - 12 uM) reversibly depressed the dopamine depolarization and this action was antagonized by~equal concentrations of naloaone . The concentration of morphine needed to block the dopamine response was 100 - 1000 times that needed to inhibit the adenylate cyclase in the same cells, and this makes it difficult to assess the relation between the two findings .

Dissociated dorsal root ganglion (DRG) and spinal cord (SC) cell cultures have also been used to investigate the action of opiates . MacDonald and Nelson (84) recorded simultaneously from the DRG cell and the SC cell which it had grown to innervate. Electrical stimulation of the single DRG cell resulted in as e.p .s .p . in the SC cell . Low iontophoretic currents (5 nA) o£ etorphine applied to the regions of contact between the DRG and SC cells reversibly depressed the e .p .s .p . and this effect was reversed by simultaneous iontophoretic application of aaloxose (20 - 40 nA) . On the assumption that the release of transmitter at this site was quaatal and that the release of quanta could be described by the Poisson distribution, the authors concluded that etorphine acted preeynaptically to reduce the number of quanta which comprise each e .p .s .p . Unfortunately, transmitter release has not been demonstrated to be Poisson distributed at this site, ant it may be invalid to assume, ae the authors do, that the variance of the amplitudes of the e .p .s .p .s is equal to their mean quaatal content (100) . On the other hand, one can argue that as the e .p .s .p . amplitud e was depressed without change in postsynaptic membrane potential or resistance, a likely mechanism of action is a depression of transmitter release. The best way to show this would be to demonstrate that etorphine does not interfere with the posteynaptic actions of the transmitter when this is applied by focal iontophoretis - however, the transmitter between the DRG and SC cells is not knows with certainty. There is evidence that the transmitter between the DRG and SC cells is not glutamate (101) . This makes it difficult to place in perspective the observations that enkephalin does depress the responses of the SC cells to iontophoretic application of glutamate, independent of any other effects which it has on neuronal membrane properties (4) . Voltage-clamp studies indicated that in the presence of enkephalin (itself applied by iontophoreie) less ionic current flowed as a result of the iontophoretic application of the same amount of glutamate . The inference is that enkephalin can impede the activation by glutamate of the voltage-independent but chemically aeasitive ionic conductance - an interpretation identical to that proposed by Zieglg~asberger for his findiage in the cat spinal cord . It would be helpful to know what concentrations of agonists and antagonists are effective when the agents are added directly to the culture medium, and in this way information can be obtained on the nature of the receptors on the spinal cord cells .

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Conclusione All 18 not known about the actions of opiates and opioid peptides on single neurones, and even the correct interpretation of the available observations is no easy matter . In terms of defining the acute actions of opiates which relate to their pharmacological effects, there remains considerable difficulty in interpreting the results of ioatophoretíc application of opiates to neurones in vivo . A clearer interpretation would be afforded if both iontophoretic and systemic application of opiates was used . ßeversal or protection by a single concentration (current) of naloaone applied by iontophoresis is not sufficient to establish an opiate receptor mediated event. Systemic administration of nalozone has the advantage that a dose can be used which is known to be appropriate for the reversal of the pharmacological actions of morphine . The vee of stereoisomere of both agonista and antagonists will provide the clearest picture . In the case of the opioid peptides, the situation is morn complez. It is known that more nalozone is required to antagonise enkephalin than morphine (82), so that the relevant concentrations of naloaone to gives systemically are not ao obvious . It must be borne in mind that the actions of enkephalin and ß-endorphin which are not reversed by aaloxoae may not be entirely without physiological significance . There is no compélling reason why opiates and opioid peptides should have the same mechanism of action oa neurones ín different regions . After . all, the activation of muscarinic receptors by acetylcholine may lead to an increase in Na, R sad Ca conductances (smooth muscle), an increase in R conductance (certain cardiac muscle) or a decrease in R conductance (some central neurones) . Electrophysiological evidence is compatible with three actions of opiates and opioid peptides at the present time . First, there is a presyaaptic action to depress the release of certain neurotransmitters . The mechanism underlying this might be relatively novel ; namely, a hyperpolarisation sad/or conductance increase in fine nerve terminals . Second, evidence is substantial that the opiates and opioid peptides can interfere directly with the posteynaptic actions of certain transmitters so ae to reduce their ability to alter ionic conductances . The third action which seems likely is a direct 'posteynaptic' action on some cells ; where this action occurs it may be predominantly inhibitory and non-somatic. The evidence does not yet allow one to conclude which one of these may be the most important action underlying the acute effects of the opiates . The incentive to resolve the bewilderment is strong, because the electrophysiological manifestations of the longer term effect of opiates which underlie tolerance and dependence have hardly begun to be studied. Acknowled$aments Work carried out in the author's laboratory was supported by NS06672, DiA01730 and the Schwappe Foundation . I am grateful to Dra . G. Henderaon and W . 2leglgänsberger for their comments an the manuscript . Referencee R.R . ANDERSEN, J.P . LUND, and E. PUTL, Canal . J . Physiol . Pharmacol . 56 216-222 (1978) . 2 . E .G. ANDERSON, M. LOSATZ and H.R . PRDUDFIT, Iontophoresis and Traasmitter McCltaais~es in the Mamalisn CentrB]. Nervous atea, (Ed. B.W . Ryall and J.S . Rally pp . 29 301, Elsevier, Austerdan (1978) . 3 . S .D . ANDERSON, A.I . HASBAUM and H .L . FIELDS, Brain Rea . 123 363-368 (1977) . 4 . J .L . BARRER, J.H . NEALE, T.G . SMITH and R.L . MACDOtiALD, Science 199 1451-1453 (1978) . l.

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Vol . 24, No . 17, 1979

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11Y . 113 . 114 . 115 . 116 . 117 . 118 . 119 . 120 . 121 . 122 . 123 . 124 . 125 . 126 .

Opiates-Opioid Peptides-Single Neurons

Vol . 24, No . 17, 1979

V. SROR, Physiology of Autonomic Ganglia , Tgaku, Shoin, Tokoyo (1973) . H . TARAGT, M . SATOH, A . ARAIRS, T . SHIBATA and Y . RIIRATSHI, Europ . J . Pharuacol . 45 91-92 (1977) . H . TARACI, M . SATOH, T . DOT, R . RAWASARI and A. ARAIRE, Arch . Intern . Phaxm . Terap . 22 1 96-104 (1976) .. J .H .-WOLSTSNCROFT, D .C . WEST, and J .P . GENT, Iontophoresis and Traae mitter Mechanisms in the Mammalian Central Nervous S stem, (Sd . R .W . Ryall and J .S . Belly pp . 341-343, Slaevier, Amsterdam (1978) . J .T.WILLIAMS aad R .A . NORTH, Brain Ree . In prese (1978) . W .S . YODNG, S .J . BIRD and M .J . ROHAR, Brain Rea . 129 366-370 (1977) . W . ZIEGLGANSBERGER and H . BAYSRL, Pflughera Archiv .355 Suppl . R85 (1975) . W . ZTEGLGANSBERGER aad H . BAYSRL, Brain Rea . _I15 111-128 (1976) . W . ZIEGLGANSBERGER and H . BAYERL, Dru and Central S a tic Tranamiseion , (Ed . P .B . Bradley and B .N . ~awan pp . 131-138, Macmillan London (1976) . W . ZIEGLGANSBSRGER and J . CEAI~GNAT, Iontophoresis aad Transmitter Mechanisms in the Mammaliaa Central NeYVOUS S atea (Ed, R.W . Ryall aad J .S . Belly pp . 403-405, Elsevier, Amsterdam (1978) . W . ZIEGLGANSBERGSR, E . FRENCH, G .R. SIGGINS and F . BLOOM, Characteristics and Functions 6f Opioida , (Ed . J .M . Van Ree and L . Terenius) in preas,'Elaevier, Amsterdam (1978) . W . ZIEGLGANSBERGER and J .P . FRY, Opiate and Endogenous Opioid Peptides , (Ed . H .W . Roaterlitz) pp . 231-238, Elsevier, Amsterdam (1976) . W . ZIEGLGANSBERGER, J .P . FRY, A . HERZ, L . MORODER and E . WONSCH, Brain Res . 115 160-164 (1976) . W . ZIEGLGÂNSBERGSR and E .A . PDTL, Eap . Brain Rea . 17 35-49 (1973) . W . ZIEGLGANSBERGER, M . SATOH and J . BAYBRL, Natmyn-Schmiedeberg's Arch . Pharmacol . 287 Suppl . R16 (1976) .