Effects of Ba2+ and tetraethylammonium on cortical neurones

J. Physiol. (1971), 215, pp. 223-245 223With 15 text-ftgurePrinted in Great Britain

EFFECTS OF Ba2+ AND TETRAETHYLAMMONIUM ONCORTICAL NEURONES

BY K. KRNJEVIJ, R. PUMAIN* AND L. RENAUDtFrom the Department of Research in Anaesthesia,

McIntyre Building, McGill University, Montreal, Canada

(Received 7 January 1971)

SUMMARY

1. Ba2+, applied by micro-iontophoresis, excites most cortical neuronesthat are excitable by ACh; other neurones tend to be depressed.

2. The discharges evoked by Ba2+ resemble those evoked by ACh, butthey have an even slower time course and are characterized by firing inhigh frequency bursts.

3. The excitatory action of Ba2 , unlike that of ACh, is not abolished bymuscarine antagonists; but it can be prevented with dinitrophenol.

4. The depolarizing effect of Ba2+ is associated with a rise in membraneresistance and it has a reversal level 24 mV more negative than the restingpotential.

5. These observations suggest that, as in other tissues, Ba2+ reducedthe K+ conductance by a direct action on the cell membrane. Some diminu-tion in Na+ inactivation is indicated by the repetitive firing at high fre-quency.

6. TEA has a predominantly depressant effect on all neurones tested.Like Ba2+, it often increases greatly the duration of spikes, but thereis no regular change in resting membrane resistance and no tendency torepetitive firing. TEA probably reduces only the delayed K+ current.

7. Even in large doses neither Ba2+ nor TEA interferes with the conduc-tance increase that generates the typical prolonged JPSPs recorded incortical neurones.

INTRODUCTION

As a divalent cation, Ba2+ might be expected to have some Ca2+-likeproperties. In fact, Ba2+ can substitute at least partly for Ca2+ in main-taining or restoring the release of transmitters at some junctions (Douglas

* Present address: Laboratoire de Neurophysiologie comparee, Facult6 desSciences, Paris V, France.

t Canadian Medical Research Council Fellow.8 p H Y 215

K. KRNJEVI6, R. PUMAIN AND L. RENAUD

& Rubin, 1964; Miledi, 1966). Similarly, the effects of Ba2+ on the lobsteraxon appear to be very much like those of Ca2+ (Blaustein & Goldman,1968); and when applied to frog nerve fibres Ba2+ rapidly depressesexcitability in a Ca2+-like manner (LUttgau, 1954).

However, Ba2+ has another, slower action on nerve fibres, which leadsto a delayed rise in excitability: this effect is associated with depolariza-tion and an increase in electrical resistance (LUttgau, 1954), as well as atendency to repetitive firing in response to single stimuli (Lorente de No& Feng, 1946). These observations suggest that Ba2+ interferes with move-ments of K+, both at rest and during activity. The characteristic featuresseen in studies on a wide variety of invertebrate and vertebrate nerve ormuscle - depolarization, rise in membrane resistance, generation of spikesfrom local responses, prolonged spikes and repetitive firing - indeed con-firm the hypothesis that Ba2+ lowers the membrane conductance to K+(gK) (Fatt & Ginsborg, 1958; Werman, McCann & Grundfest, 1961;Werman & Grundfest, 1961; Narahashi, 1961; Biilbring & Kuriyama,1963; Nishi, Soeda & Koketsu, 1965; Sperelakis, Schneider & Harris, 1966).The explanation for this effect may be that Ba2+ in its hydrated state hasdimensions very similar to those of the hydrated K ion (Mullins, 1961) andthus could block sites of K+ movements (Werman & Grundfest, 1961).At a number of cholinergic junctions, Ba2+ initiates activity or strongly

potentiates transmission (Stavraky, 1932; Feng, 1937; Ambache, 1949;Douglas, Lywood & Straub, 1961; Douglas & Rubin, 1964; Takeshige &Volle, 1964; Kuriyama, Osa & Toida, 1967; Bolton, 1968). Much of thiseffect can be ascribed to prolonged or repetitive activity in presynapticnerve terminals; but a direct post-synaptic action has been demonstratedin certain places, particularly in denervated sympathetic ganglia (Ambache,1949; Takeshige & Volle, 1964). As these direct effects may be abolishedby atropine (Takeshige & Volle, 1964), it appears that Ba2+ may alsoactivate muscarinic acetylcholine (ACh) receptors, though the evidenceon this point is not conclusive.Both Ca2+ and Mg2+ have been shown to have strong depressant effects

on central neurones (Krnjevic, 1965; Kato & Somjen, 1969; Kelly, Krn-jevic & Somjen, 1969). Other divalent cations are also mainly depressant(Rozear, de Groof & Somjen, 1970). But there appears to have been nosystematic study of the effects of Ba2+ on the vertebrate central nervoussystem, though some unusual properties are suggested by the observationthat injections of 'minute' amounts of Ba2+ into cerebrospinal fluid have astrong stimulating action, leading to convulsions (Chou & Chin, 1943).In the experiments described here, we have applied Ba2+ directly ontocortical neurones by iontophoresis from micropipettes, and compared itsaction with that of some relevant compounds such as ACh; since tetra-

224

Ba2+ AND TEA ON NEURONESethylammonium (TEA) is also known to block gE in nerve membranes(Schmidt & Stampfli, 1966; Koppenhofer, 1966; Hille, 1967) we have alsoexamined its effects. Two brief reports of the results described in this paperhave been presented orally (Krnjevic, Pumain & Renaud, 1970a, b).

METHODS

Experiments were done on twenty cats. In the first eight, only extracellularobservations were made, using five-barrelled micropipettes for simultaneous record-ing and extracellular microiontophoresis of Ba2+ (from 0-2 M solutions of BaCl2),TEA (from 1 M-TEA bromide, Eastman) and other agents of interest (0.05 M hemi-cholinium-3-dibromide, Aldridge; 0-2M atropine sulphate, Mann; 0-2 M hyoscinehydrobromide, Nutritional Biochemicals; 0-1 M-2,4-dinitrophenol Na, pH 9-0,British Drug Houses; 0-1 M pentachlorophenol Na, pH 9, Dow; 0-1 M dicumarol(bishydroxycoumarin), Nutritional Biochemicals). Five of these cats were anaesthe-tized with a mixture of Penthrane and N20; two others were anaesthetized withalpha-chloralose (Merck, 80 mg/kg i.P.), and the third with Dial compound (CibaLtd., 0-70 ml/kg i.P.).

In the second series of experiments, performed on cats anaesthetized with Penth-rane-N20 or Dial compound and paralysed with succinylcholine, intracellularelectrodes were used to record membrane potentials and changes in resistance,while Ba2+, TEA or other relevant agents were applied outside the cells. The tech-nique was thus largely similar to that described in the previous paper (Godfraind,Kawamura, Krnjevic & Pumain, 1971). The membrane resistance was measured with20-30 msec current pulses, provided by a Linc-8 computer, and delivered through therecording micro-electrode, whose resistance was balanced out by means of a resis-tance bridge. The computer displayed on-line as a voltage-current plot and recordedon magnetic tape the applied currents and the maxima of the resulting displace-ments of membrane potential (Kelly, 1970). The pulses were in groups of eight,varying regularly in intensity but mainly in the hyperpolarizing direction; andeach series of eight was repeated at intervals of 4-5 sec. Subsequently, the computerderived from the recorded data the voltage-current lines of best fit (by the methodof least squares) and the corresponding values of membrane potential and resistance,as well as the reversal levels of IPSPs obtained from the intercepts of the appro-priate voltage-current lines. The IPSPs were evoked by surface shocks. The IPSPresistance was measured 20-50 msec after the stimulus.

RESULTS

Extracellular observationsEffects of Ba2+

On cells excitable by AChTests of Ba2+ were made on a total of eighty-two neurones. Out of

forty-eight cells which fired spontaneously in the characteristic manner(Krnjevic & Phillis, 1963 a) and were readily excited by ACh, 45 (or 93 %)could also be excited with iontophoretic applications of Ba2+. This effectis illustrated in Fig. 1. The first trace A, shows a characteristic prolonged

8-2

225

K. KRNJEVIC, R. PUMAIN AND L. RENAUD

excitation by ACh. In B, the release of Ba2+ (by a larger iontophoreticcurrent) resulted in a more delayed, but strong discharge, only the firstpart ofwhich is shown in the Figure; an increased rate of firing persisted forover 1 min longer. An excitatory effect of this kind, resembling that ofACh but having an even slower time course, can be seen also in Figs. 2-6.The amount of Ba2+ required for excitation varied a good deal. The

iontophoretic current applied was usually somewhat greater than thatneeded to elicit a comparable effect with ACh. But equal currents of ACh

Fig. 1. Extracellular recording of discharges evoked with ACh (A) andBa2+ (B) released by iontophoretic currents indicated in nA. Excitatoryeffect of Ba2+ had a slower onset and a much longer duration, only initialpart of discharge being shown here. Glutamate (Glut) gave an unusuallyprolonged after-discharge for several minutes after application of Ba2+(cf. 2 records in C obtained respectively before, and 3 min after release ofBa2+).

and Ba2+ were not infrequently equipotent (cf. Fig. 6) and in some casesmuch lower currents of Ba2+ were effective, though longer applicationswere commonly necessary. In the two cats under chloralose, continuousspontaneous discharges were much less well developed and both AChand Ba2+ evoked only rather poorly maintained and unusually unpredict-able responses.When both ACh and Ba2+ were applied simultaneously, there was

marked occlusion. For example, in Fig. 2, the combined effect of Ba2+and ACh was rather less than that of ACh alone. This was clearly notbecause the neurone was responding maximally, since glutamate couldevoke a much stronger response.

226

Ba2+ AND TEA ON NEURONES

The action of Ba2+ differed from that of ACh in one respect: under itsinfluence, there was a strong tendency for discharges to occur in sharpbursts of 5-20 impulses, at a frequency of > 100 Hz. Such bursts arevisible as sharp deflexions in the paper traces illustrating the excitatoryaction of Ba2+, in Fig. 3A, and even more clearly in the intracellularrecords of Fig. 12H-I. They are remarkably similar to the bursts seen inthe presence of gallamine (Galindo, Krnjevic & Schwartz, 1968). Most cellsshowed some tendency to fire in bursts when excited by Ba2+; but this wasuncommon when cells were excited with ACh (cf. Galindo et al. 1968),

Ba 28 Ba 28ACh28 Ba28 ACh28 ACh56 ACh56GIut42

80

4oL I I X30 sec

Fig. 2. Occlusion between excitatory actions of ACh and Ba2 , appliedseparately or together as indicated. Paper records of neuronal dischargefrequency.

except in a few cases where much less intense bursts were encountered(Fig. 12C-D). A residual facilitatory action of Ba2+ was sometimes mani-fested, well after the end of any overt excitation, by a prolongation ofglutamate-evoked discharges (Fig. 1C); but this was not seen as readily asafter applications of ACh (Krnjevic et al. 1971).

On cells not clearly excitable by AChOnly three cells out of thirty-four of this type showed an increase in

mean firing rate, mostly owing to the appearance of occasional highfrequency bursts; only one cell gave a continuous discharge comparableto the effect seen with ACh-sensitive cells.Not infrequently Ba2+ had a clear depressant action on cells not excited

by ACh. As usual, these cells were found relatively near the surface(Krnjevic & Phillis, 1963 a; Crawford, 1970); and, as they typically did notdischarge spontaneously, the depressant effect of Ba2+ was most easilydemonstrated against a background of discharges evoked with glutamate,as in the example of Fig. 3C. The intensity of this depression variedgreatly with different cells: it was usually more pronounced than thedepressant effect of ACh (as in Fig. 3B-C), but less so than that of Ca2+or Mg2+ (Kato & Somjen, 1969).

227


In presence of some blockers of cholinergic transmissionWe used several agents to try to obtain further evidence about the

mechanism of excitation by Ba2+.Hemicholinium-3. This compound inactivates cholinergic transmission

by interfering with the metabolism of ACh (Birks & MacIntosh, 1961);and it has been found to reduce the output of ACh from the cerebral

300j A

200

ACh56 100 BaI00 1 min

B C

-II111111 - -- IGlut 42 ACh 100 Ba 100

Fig. 3. A: excitation of ACh-sensitive cell by Ba2+; note slower time courseand early appearance of high-frequency bursts. B: neurone insensitive toACh; responses evoked by regular applications of glutamate (42 nA, indi-cated initially by vertical bars) were very much depressed by Ba2+.

cortex (Szerb, 1965). The aim was to eliminate the possibility that themain action of Ba2+ might be presynaptic, causing a great enhancement ofspontaneous or active release of ACh.

Hemicholinium-3 (HC-3) was applied to twenty-four cortical neuronesby iontophoresis. Much to our surprise, HC-3 proved to have a strongexcitatory action on the sixteen cells which were excited by ACh. Thisaction was not unlike that ofACh itself, except that there was often a morepronounced initial depression of firing, and the after-discharge tended tobe shorter. Equipotent iontophoretic doses ofACh and HC-3 were approxi-mately similar, and the effects of both compounds were blocked by atro-pine. It is not likely that HC-3 was merely accelerating the release of AChfrom nerve terminals, because its excitatory action was well maintained

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228

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when applications were repeated several times in succession, or during avery prolonged application, for as long as 35 min. Tests of Ba2+ against abackground of such repeated or prolonged applications of HC-3 showed noevidence of any diminishing effectiveness.The eight neurones which were not excited by ACh were rather strongly

depressed by HC-3, to a much greater degree than by ACh.

60

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ACh84 Ba100

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Fig. 4. Atropine applied by iontophoresis blocks excitatory action of AChbut not that of Ba2+. Records of rate of firing, (A) before and (B) duringrelease of atropine (between arrows).

Atropine and hyoscine. If Ba2+ acts mainly by increasing the release ofACh from nerve endings, or if it activates ACh receptors on the post-synaptic neurones, the effect should be abolished by atropine or hyoscine,which block the excitatory action of ACh (Krnjevic6 & Phillis, 1963b).Tests were made on eight cells with atropine and on seven cells withhyoscine: both compounds were released locally from the micropipettes.In moderate doses, they produced the usual selective depression ofspontaneous and ACh-evoked activity, but the responses evoked by

229

I

K. KRNJEVI(, R. PUMAIN AND L. RENAUD

Ba2+ were only noticeably depressed in two instances out of fifteen.Typical experiments are illustrated in Figs. 4 and 5. The spontaneousfiring of the unit of Fig. 4 was progressively reduced after the start of therelease of atropine (B), and when ACh was tested 45 sec later there was noresponse; but Ba2+ evoked an even more powerful discharge than before.

Fig. 5. Hyoscine also fails to block excitatory action of Ba2+. A-C: controlapplications of ACh and Ba2+ (B and C are continuous). Release of hyo.scine begins at onset of D; ACh effect is abolished but not that of Ba2+ (Eand F are continuous). ACh and Ba2+ were released during periods betweenarrows.

Fig. 5 illustrates the first part of a very long application of hyoscine.Within a minute of the start, the rapid spontaneous firing ceased, andACh became quite ineffective (Fig. 5D). One minute later, the spikes weresomewhat smaller, owing to the general depressant ('local anaesthetic')action of hyosbine, but Ba2+ was strikingly effective (Fig. 5E-F). Althoughthe application of hyoscine was continued for > 10 min, and the spikeswere reduced to about 1/3 of their control size, the effect of Ba2+ was notblocked.

Uncouplers of oxidative phosphorylation. As shown in the previous paper

230


(Godfraind et al. 1971), 2, 4-dinitrophenol (DNP) and some other uncouplersstrongly antagonize neuronal excitation by ACh, apparently by causing anincrease in membrane conductance for K+. In the present experiments,we tested three compounds of this type (DNP, pentachlorophenol anddicumarol) on ten cells which were strongly excited by ACh and Ba2+.Both DNP and pentachlorophenol regularly depressed spontaneous

ACh 70 Ba 70200

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Ba 70 ACh 70Fig. 6. Pentachlorophenol blocks action of both ACh and Ba2+. A: before;B: 2 min after start of release of pentachlorophenol, which ends at sametime as release of Ba2+; C: 10 min later.

activity and the responses evoked by ACh and Ba2+ (Fig. 6). Dicumarolgave less predictable results (Godfraind et al. 1971), but a clear block ofBa2+ effects was obtained with the only cell which also showed a block ofACh responses.

Effects of tetraethylammoniumSince TEA may also block K+ movements, it seemed of interest to

examine its effects on cortical cells. In two cats under Penthrane, twenty-one cells were tested with TEA; of these, fourteen could be excited withACh.

In no instance did TEA excite the neurones like ACh: practically allthe cells were rather clearly depressed, so that all activity - spontaneous,glutamate- or ACh-evoked - was markedly reduced by TEA. The action

231

K. KRNJEVI6', R. PUMAIN AND L. RENAUD

was quite rapid: a clear depression was noticeable within 5 see of startingan application; but recovery was slow, full responses being obtained only1-3 min after the end of an application. This effect is clearly visible inFig. 7A-B. TEA was not obviously more potent in blocking the action ofACh than that of glutamate. If anything responses to glutamate weredepressed to a somewhat greater degree (cf. Fig. 8), probably because of

A ^

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Fig 7. Depression of glutamate and ACh-evoked discharges by release ofTEA (falling progressively fromBa2 nA to 50 nA). Lines A and B are con-tinuous. Inca there is a control run during which Na+ was released insteadof TEA.

the positive-current effect. The oscilloscope traces in Fig. 8 show that thedepressant action of TEA was associated with some reduction in spikesize, but there was no evidence of any severe depolarization or inactivation.

Intracellular observationsEffects of Ba2+

On membrane potential and resistance of neuronesIn six experiments, useful intracellular records were obtained from

twenty-three neurones. Twenty-one of these cells were clearly depolarizedby extracellular applications of Ba2+ (100-200 nA), and there was a simul-taneous increase in membrane resistance: both effects were reversible;they are illustrated in Fig. 9.

232


Fig. 8. Film records of discharges seen A: before; B: during and C: afterapplication of TEA. There is some reduction in spike height in B, but nosign of excessive depolarization.

5:1 A~~~~~h 200 nA~~~~~~Ba 200 nA

40

L1 20 3 4

Time (min)Fig. 9. Changes in membrane potential and resistance of cortical neuroneinduced by extracellular applications of ACh and Ba2+. Each point isestimate obtained from voltagecurrent line based on series of eightvoltagecurrent points, as described in Methods. First dashed line indicatesperiods during which tests were suspended, and second one interruptioncaused by electrical noise generated initially by release of Ba2+.

233

234 K. KRNJEVW6, R. PUMAIN AND L. RENAUD

Reversal levels for the action of Ba2+ (EB) were found at the inter-sections of voltage-current lines obtained during applications of Ba2+and in the preceding and succeeding control periods. In Fig. 10, thirty eightvalues of EBa are plotted against the corresponding resting potential;practically all are more negative than the resting potential, and they have

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Fig. 10. Reversal level for change in membrane potential induced by Ba2+,plotted against corresponding resting potential. Open and filled circles areintersections of voltage-current lines obtained during release of Ba2+ withpreceding and following control lines respectively.

a ceiling at about - 10 mV; on the average, they differed from theresting potential by -24-2 mV (S.E. 5-51).

In nine of these cells, we were able to get estimates of EACh as well asEBa. These values and the corresponding changes in potential and resis-tance are given in Table 1. Although A V/AR was somewhat smaller forBa2+ and EBa relatively positive, probably because resistances tended to

Ba2+ AND TEA ON NEURONESbe higher and the potentials more positive during the Ba2+ control runs,none of these differed significantly from the corresponding data for ACh.

A curious feature of these experiments was a not unusual tendency for the mem-brane potential, if initially very poor, to shift rapidly towards a 'normal' level ofabout -70 mV during the release of Ba2+: this change was usually associated with acorresponding increase in resistance; it is visible in Fig. 11, after an initial depolari-zation. A similar phenomenon was never seen during applications ofACh (cf. Fig. 11).It appears to be caused by a temporary sealing of the damaged membrane, with aconsequent sharp reduction in the shunting of the membrane potential. Previousexperiments on the cortex (Kelly et al. 1969) had indicated that extracellular Ca2+and Mg2+ tend to improve the stability of intracellular recording (cf. Prothero,Bulger, Chambers & Mack, 1970). The present observations are consistent with asimilar action of Ba2+.

TABLE 1. Comparing changes in potential (A V) and resistance (AR) producedby Ba2+ and ACh and corresponding reversal levels (EBa and EACh), all recorded inthe same nine cells. Means, with numbers of observations and standard errors inbrackets

Resting Restingpoten- resis- A V ARV Reversaltial (V) tance (R) AV AR AR AR potential(mV) (MW) (mV) (MQ) (nA) (mV) (mV)

Ba2+ _41F8 23-8 + 9-58 + 8-72 2-00 25-4 -66-5(26, 3.73) (26, 6-02) (26, 1.51) (26, 1.56) (26, 0.503) (26, 5.55) (24, 5-89)

ACh -49-3 14-7 +11V9 +5-38 3-20 28-3 -76*3(20, 4.42) (20, 5.03) (20, 1.63) (20, 0.800) (20, 0.526) (20, 5.38) (20, 690)

On spikesAs already mentioned, a characteristic feature of excitation by Ba2+ is

the predominance of firing in high-frequency bursts. The appearance ofsuch bursts is evident in the traces of Fig. 12. In the first column (A-E),one can see that the initial background firing, in single spikes (A-B), wasaccelerated by ACh (C-E), which also caused some brief bursts to appear;most of these consisted of only two-three spikes (cf. D). After recoveryfrom the effects of ACh, there were only single spikes in the backgrounddischarge (F). The release of Ba2+ (H-J) caused a sharp increase in firing,associated with much longer bursts, consisting of six or more spikes. Atthe peak of its action, Ba2+ can cause a cell to fire in prolonged but irregularbursts, separated by variable pauses.A change in pattern of discharge is sometimes observed even when the

mean firing rate is not obviously changed. There is an example of this inFig. 13. Neither Ba2+ nor ACh increased significantly the mean frequencyof discharge (open circles), but Ba2+ soon altered the pattern of firing:initially this consisted of single spikes; when the effect of Ba2+ was at itspeak, spikes occurred only in groups of two or more (filled circles).

235

236 K. KRNJEVIO, R. PUMAIN AND L. RENAUDAnother effect of Ba2+ is also shown by the triangles in Fig. 13. The

duration of individual spikes, measured to the end of the first after-potential (the after-depolarization, Eccles & Krnjevic, 1959, or delayeddepolarization, Granit, Kernell & Smith, 1963), was initially about 6 msec(cf. Creutzfeldt, Lux & Nacimiento, 1964). But it increased greatly during

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Time (min)Fig. 11. From above down, graphs show changes in membrane potentialat rest and during IPSP, and corresponding changes ofmembrane resistanceduring IPSP and at rest, produced by extracellular release ofBa2+ and ACh.Large negative shifts are probably due to sealing effect of Ba2+; presumed'true' potential changes are indicated by dashed lines.

the release of Ba2+; and it returned towards its control level only veryslowly, more-or-less in parallel with the recovery of the normal patternof firing.Not infrequently, the after-depolarization became a large hump, with

a plateau at a level near zero potential. Such spikes are shown in the uppertraces of Fig. 14: in A, there is a humped spike lasting 25 msec; somewhatlater, an even more extreme change occurred, so that the neurone behavedlike a non-linear oscillator alternating between two relatively stable levels:a spike was followed by a plateau of depolarization (B), which could be


maintained for > 100 msec, but was sharply terminated if an IPSP super-vened (C). A very long plateau of depolarization was more common in cellswith a comparatively poor resting potential; the duration of the plateauscould be shortened by hyperpolarizing the membrane, electrically or byinhibitory stimulation (cf. spike in C).

Fig. 12. Effects ofACh and Ba2+ on pattern of firing. A: initial spontaneousdischarge. B: IPSP evoked by surface shock (at arrow). C-E: duringrelease ofACh (140 nA). F-G: recovery from effects of ACh. H-J: duringrelease of Ba2+ (140 nA). Note faster sweeps in D and I.

On IPSPsBa2+ did not obviously interfere with IPSPs. They were well maintained,

as negative deflexions, even during prolonged applications of Ba2+ (cf.Fig. 12). In these experiments, in which K citrate electrodes were mainlyused, the reversal levels of IPSPs (mean Elpsp - 74*8 mV, s.E. 5*46, n 28)

237


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Fig. 13. Ba2+ or ACh-induced changes in spike duration, spontaneousfiring, and percentage of spikes occurring at very brief intervals ( < 10 msec).

Fig. 14. A-C: humped spikes recorded intracellularly (i.C.) during appli-cation of Ba2+ (140 nA). D-I: effects of intracellular injection of TEABr on cortical IPSPs; D, F, H were taken at start of intracellular recording;E, G, I, several minutes later, as indicated. Current pulses testing mem-brane resistance are monitored on separate trace. Arrows point to artifactsgenerated by stimuli evoking IPSPs.

238

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Ba2+ AND TEA ON NEURONESdid not differ significantly from EBa. When cells were depolarized by Ba2+,there was no evidence of any systematic change in EIpsp but the rise inmembrane conductance observed during IPSPs (expressed as a percentageof the resting conductance) became consistently greater, by a mean valueof + 209% (S.D. 286-9, n 19).

On unresponsive cells (neuroglia)Five applications of Ba2+ on four different cells caused the membrane

resistance to increase by 42-100% (mean 62*8 %), without any significantchanges in membrane potential.

Effects of TEAExtracellular TEA. Thirteen useful tests of TEA were made on ten

neurones in three cats. There were only rather doubtful effects: some minordepolarization (by 2-5 mV, only poorly reversible) in four cases, and someincrease in membrane resistance in three cases (by 28-100 %).

Intracellular TEA. In another two experiments (performed with DrsJ. J. Dreifuss & J. S. Kelly), intracellular recordings were made fromtwelve neurones with single micro-electrodes containing 0 5 M-TEA Br,alone or with 1 M-K citrate or 1 M-KC1. The neuronal properties (restingpotential and resistance) and the strength of inhibitory action evoked bycortical stimulation were examined for periods of 2-25 min. In most casesthere was a progressive depolarization; the membrane resistance showedno systematic tendency to fall or rise; and the inhibition was very wellpreserved; cf. the pairs of traces D-E, F-G and H-I in Fig. 14, where theinhibitory potency can be gauged from the fall in resistance during theIPSP (note smaller voltage pulses). Thirteen measurements of the relativerise in conductance near the peak of the IPSP showed a just significantmean increase of +76.7% (S.D. 112.6).TEA injected either outside or inside the neurones also tended to give

prolonged spikes, with more or less pronounced humps or even maintainedplateaus (Fig. 15). The duration of these humps also appeared to be afunction of the resting potential, the plateaus being greatly shortened byartificial hyperpolarization (Fig. 15 E).

Unresponsive cells (neuroglia). Repeated extracellular applications oflarge doses of TEA ( > 200 nA) on five cells produced no detectablechange in membrane potential or resistance. But prolonged intracellularinjections of TEA by diffusion from the micro-electrode were associatedwith a gradual rise in resistance (two cells).

239

240 K. KRNJEVIO, R. PUMAIN AND L. RENAUD

Fig. 15. Humped spikes produced by intracellular administration ofTEA Br. A: initial trace, with IPSP. B: 2 min later, humped spikes begin-ning to appear. C: another min later. D: spontaneous humped spikes.E: an intracellular hyperpolarizing current (1-0 nA) reduces frequency andduration of spikes. F: at 5 min, IPSP still clearly present.

DISCUSSION

Effects of Ba2+The most interesting observation is that of a strong ACh-like excitatory

action of Ba2+ on ACh-sensitive neurones.

Site of actionSince Ba2+ may have a marked effect on the release of transmitter from

nerve endings (Douglas & Rubin, 1964), can the present results be ex-plained entirely by a presynaptic action of Ba2+? Probably not. Severalfeatures point to a post-synaptic action.For example one might expect many more cells to be excited if Ba2+

simply released transmitters from all nerve endings. To account for its


selective excitation of ACh-sensitive cells would require the assumption thatonly cholinergic endings are affected. While this is not inconceivable, itcannot be reconciled with the fact that atropine and hyoscine do notblock the effect of Ba2+ although they readily block that of ACh. Theconclusion that Ba2+ must have a significant direct post-synaptic action isalso supported by the failure to observe any reduction of its effectivenessduring prolonged applications of HC-3.

Mechanism of actionA reduction in membrane conductance for K+ (gE) must be an important

factor, as observed in many previous studies (cf. Introduction). This isindicated by the combination of a depolarizing effect with a rise in mem-brane resistance, and by the relatively negative reversal potential. Asimilar effect would result from a reduction in gcl but this would be incontradiction with the absence of any significant depression of IPSPs(cf. below). A reduction in movements of K+ is also suggested by the pro-longation of spikes and the tendency to repetitive discharges, as well asthe blocking effect of uncouplers of oxidative phosphorylation which tendto enhance GE (Godfraind, Krnjevi6 & Pumain, 1970; Godfraind et al. 1971).The action of Ba2+ on cortical neurones is in keeping with that of ACh,

which is probably also mediated by a reduction in gE (Krnjevic et al.1970 a, b, 1971). But it is unlikely that Ba2+ simply activates ACh-receptors,since its action is insensitive to the muscarine antagonists. ThereforeBa2+ probably blocks sites of K+ movements directly; the observedocclusion between the effects of ACh and Ba2+ suggests that Ba2+ canblock at least some of the K+ sites controlled by ACh-receptors.The action of Ba2+ though very similar to that of ACh, differed in some

respects. Ba2+ had a stronger depressant effect on cells not excited by ACh.High frequency bursts were far more prevalent after applications of Ba2+:this suggests that Ba2+ is even more effective in reducing Na+ inactiva-tion (cf. Werman & Grundfest, 1961). The reversal potential was somewhatmore positive than for the action of ACh (cf. also EACh and EDNP in theother experiments, Krnjevic et al. 1971, Godfraind et al. 1971); but themean difference between EBa and EACh measured on the same cells(15.2 mV, S.E. 7.51, n = 9) was not significant statistically.In some degree, these membrane effects of Ba2+ can also be obtained with

Ca2+ or Mg2+ (Frankenhaeuser & Hodgkin, 1957; Frankenhaeuser, 1957);but the over-all changes are equivalent to those produced by hyperpolariza-tion. A higher threshold is thus the predominant feature of the action ofCa2+ and Mg2+ on most types of excitable tissue (Shanes, 1958; Cerf, 1963),including cortical neurones (Krnjevic 1965; Kato & Somjen, 1969; Kellyet at. 1969). The special properties of Ba2+ can be ascribed to a particularly

241

K. KRNJEVIO, R. PUMAIN AND L. RENAUD

strong depression of gY, caused by the similar dimensions of hydratedBa2+ and K+ (Werman & Grundfest, 1961; Mullins, 1961).

This leads to another question: why does Ba2+ excite only the cells ex-citable by ACh? There are several possible answers. Other cells may have avery low resting conductance for Na+, or a relatively high resting conduct-ance for Cl-; or Na+ inactivation may be too effective; it is evident that aslow depolarization can lead to firing only if Na+ inactivation (or 'accom-modation') develops very slowly (cf. Hill, 1936; Hodgkin, 1964). Since thedepolarization is caused by the influx of Na+, a very rapid inactivationcould even prevent any substantial depolarization. Hence Ba2+ may tendto reduce gE and Na+ inactivation in all cells, but perhaps only certaintypes of cells are significantly depolarized; and only those with the requiredcombination of membrane properties can be excited by this mechanism.Small differences in membrane characteristics may well explain the varietyof effects produced by Ba2+ and other blockers of K+ movements indifferent cells (cf. Werman & Grundfest, 1961). A rather general depres-sion of gK is indicated by the increase in membrane resistance shown bysome neuroglia; but it will be more difficult to obtain decisive informationon this point from the relatively superficial, non ACh-excitable neurones,whose smaller dimensions are not conducive to stable intracellularrecording.The association between the sensitivity to ACh and excitation by Ba2+

may be more than fortuitous. ACh may have tonic action on corticalneurones innervated by cholinergic fibres, possibly by permanently keep-ing qK and/or Na+ inactivation at a low level; this is indicated by the poorresponsiveness of cortical neurones to ACh when the cholinergic innerva-tion is deficient, such as in the new-born kitten (Krnjevic, Randi6 &Straughan, 1964) or in the long-isolated cortical slab (Krnjevic, Reiffen-stein & Silver, 1970).

Effects of TEAThe lack of any excitatory action of TEA comparable to that of ACh or

Ba2+ is readily explained if TEA blocks only the delayed K+ currentsresponsible for the repolarization phase ofthe action potential (cf. Koppen-h6fer, 1966; Hille, 1967), but causes no significant change in the restinggK of cortical neurones. The depressant action of TEA can be ascribed tossome interference with Na+ movements, comparable to the effects de-cribed by Koppenh6fer (1966).

Ba2+, TEA and IPSPsThe failure to detect any diminution of the inhibitory conductance

changes even during large and prolonged applications of Ba2+ or TEA

242


lends further support to an earlier conclusion that a rise in gK plays onlya minor part (if any) in the generation of cortical IPSPs (Kelly et al.1969).

This work was supported by grants from the Medical Research Council of Canadaand Hoffmann-La Roche Ltd.

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Effects of Ba2+ and tetraethylammonium on cortical neurones