Effect of elevated potassium ion concentrations on electrically evoked release of [3H]acetylcholine in slices of rat hippocampus

Vol. 3,

Neuroscience

0 IBRO

pp. 427434.

Press Ltd.1978.Printed inGreatBritain.

Pergamon

0306-4522,78/0501-0427SO2.00/0

EFFECT OF ELEVATED POTASSIUM ION CONCENTRATIONS ON ELECTRICALLY EVOKED RELEASE OF C3H]ACETYLCHOLINE IN SLICES OF RAT HIPPOCAMPUS J. C. SZERB, P. HAD&Y

Department

and J. D. DUDAR

of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Abstract-To establish the effect of raising the concentration of extracellular potassium ions on axonal conduction and transmitter release in a mammalian central pathway, the septohippocampal cholinergic tract, the rate of [3H]acetylcholine release evoked by electrical stimulation was measured in rat hippocampal slices superfused with Krebs’ solution containing 3-15 mM K+. The evoked release of C3H]acetylcholine was abolished by the presence of tetrodotoxin or by the omission of Car+ in the superfusion medium, indicating that it originated from terminals depolarized by conducted action potentials. Potassium concentrations between 3 and 8 mM had no effect but l&15 mM Kf reduced the rate of evoked release and decreased the size of the releasable pool of C3H]acetylchohne. Decreasing the sodium content of the Krebs’ solution to 97 mM or less had effects similar to those of elevated [K’ 1. Elevated K+ (lO-15mht) reversibly reduced the size of compound action potentials in the fimbria and the alveus. The results suggest that extracellular potassium concentrations occurring under physiological conditions do not affect axonal conduction and transmitter release but that both are reduced in pathological states when extracellular [K+] above 8 mM occur. THERE

is now general

cortex

and spinal cord the extracellular

concentration similar 3 mu.

agreement

([K’],)

during

In the hippocampus was found

1975).

During

somewhat

is

ture end-plate

near

to a decrease

state

fluid, namely higher

neuronal

activity

[K’],

[K’],

(LILEY, 1956)

junctions

10 mM approximately

ion

WILLIS, in-

doubles

potentials.

1968;

This increase

TEL, 1973). The enhanced (ACh)

1972; KRNJEVIk & MORRIS, 1972;

been

directly

measured

release by

1973;

TENNENBAUM & QUASTEL, 1939;

MORRIS, 1974;

1975; VYKLICKL, SYKOVA.& KiW, trical

stimulation

higher

[K’],

&

LIEBL, 1974;

LQTHMAN & activity

The second

elec-

result

in

(LOTHMAN, LA MANNA,

of 8-11 mu

function, action

CORDINGLEY,ROSENTHAL& SOMIEN, 1975; LOTHMAN

known The

spreading large

depression

or anoxia

KRNJEVI~:& MORRIS, 1974;

The above observations, selective similar activity using

electrodes findings

BURES, 1972;

following

in [K’],

nervous potential the

action

system of glia

by

with neural of the

leech,

cells

or the

potential

as

release

of transmitters

[K’],

indi-

[K’].

also in

(MANN,

BERTELS-MEEUWS &

of elevated reduction

in the peripheral time

on neuronal

in the axon

size

of the

has been also

(HUXLEY & STXMPFLI,

is presumably

steady-state

[K’],

potential

which

inactivation

size

of

presynaptic

at

the

in turn

of regenerative

action

leads

sodium

potentials

leads to a reduced neuromuscular

by

release

junction

neuro-

of a transmitter

evoked

electrical

has been tested

2 to 7.5m~ giant

427

synapse

squid

(ERULKAR junction

and &

increasing

at the

WEIGHT, transmis-

was raised from

(TAKEUCHI & TAKEUCHI, 1961). of the squid

[K’],

on the frog neuromuscular

1977). In the frog neuromuscular

extracellular

&

by an action

(TAKEUCHI & TAKEUCHI, 1961) of the

chanin the

of the squid

potential

synapse

in

to a

(HUBBARD

junction giant

by

of transmit-

1968) and at the giant synapse

on the release

1951).

potential

also due to a decrease

sion failed in some cases when [K’], Abbreviations: ACh, acetylcholine; potassium ion concentration.

slices

(MILEDI & SLATER, 1966). The effect of elevated

at amphibian

(TAKEUCHI& TAKEUCHI, 1961) or mammalian

brain

such

has workers

in the size of the action

the membrane

ter

on the spontaneous

is welJ known:

high [K’],

WILLIS,

1966; KUFFLER,1967; BAYLOR & NICHOLLS, 1969).

The effect of elevated

including

the

for some

reduction

depolarization

(KUFFLER& NICHOLLS,

in [K’],

[K’]

nels (HODGKIN & HUXLEY, 1952). A reduction

with potassium-

(WALKER, 1971), were preceded

in the central

of changes

as

L~THMAN et al., 1975).

obtained

on an increase

the membrane

undershoot cators

while

lead to increases

70 mM (VYSKOCIL, KRiZ &

as

1977)

effect

namely

potential

& SOMJEN, 1976; HEINEMANN&

Lux,

of transmitters

SOMJEN, POLAK, 1968).

1975). Strong

or epileptiform

to a direct

numerous

various

due

(COOKE & QUAS-

by elevated

NEHER, 1973; Lux & NEHER, 1973; SINGER & Lux, TEN BRUGGENGATE, Lvx

preparations,

release

to

of minia-

(HUBBARD &

KATZ, 1969) and partially

as acetylcholine

KRNJEVIC &

[K’],

is partially

potential

on transmitter

creases to 5-8 mM (VYKLICK~, SYKOVA, KRiZ & UJEC, PRINCE, LUX &

raising

the frequency

in membrane

rest (LEWIS & SCHLJEITE, effect of K+

during

intense

potassium

the quiescent

to that of the cerebrospinal

(4.6m~)

muscular

that in the cerebral

At the

[K’ 1, from 9

428

J. C. SZERB,P. HADHAZY and J. D.

to 10.5 or to 12m~ decreased the size of the pre-

synaptic action potential and of the postsynaptic potential (ERULKAR& WEIGHT, 1977). These results would suggest that small increases of [K’],, such as those reported to occur in the mam~~an central nervous system, may affect transmitter release either by increasing the spontaneous release or by reducing the release induced by conducted action potentials. Since no data are available on the effect of elevated [K”], on the release of transmitter evoked by conducted action potentials in the mammalian CNS, this study was undertaken to find out the influence of [K”],, in a range reported to occur in the CNS, on the overall effectiveness of axonal conduction and transmitter release in a central mammalian pathway, namely the septohippocampal cholinergic tract. There is now extensive evidence based on histochemica1 (LEWB & SHUTE, 1967), neurochemical (LEWIS, SHUTE& SILVER,1967; SETHY,ROTH, KUHAR & VAN (SMITH, 1974; WOERT, 1973) and physiological DUDAR. 1975) methods that the cholinergic projection to the hippocampus originates in the medial septum. Recently it has been shown that this septohipp~ampal tract is the origin of the evoked release of acetylcholine (ACh) in slices of rat hippocampus since this release disappeared after a lesion had been made in the septum (SZERB, HADHAZY& DUDAR, 1977). The release of labelled ACh evoked by field stimulation of slices of rat cortex is abolished in the absence of Ca2+ or by tetrodotoxin (SOMOGYI& SZERB, 1972). This suggests that the evoked release of ACh occurs from nerve terminals activated by conducted action potentials. However, there are reports on the presence of intrinsic cholinergic neurons in the cortex (KRNJEvtc’ & SILVER, 1965; MCGEER, MCGEER, SINGH & CHASE, 1974). Therefore, the release of ACh evoked by field stimulation of cortical slices could have occurred, at least in part, through the synaptic activation of these intrinsic cholinergic neurons. The demonstration of a similar sensitivity of electrically evoked release of ACh to tetrodotoxin and to the lack of Ca’+ in hippocampal slices where the cholinergic innervation is purely extrinsic, could provide a preparation in which the release of a transmitter from ter_mjnals is activated only by conducted action potentials. ‘The...,~elease of ACh from hippocampal slices stimulated &ectrically can therefore be a useful tool for establishing the influence of ionic changes on axonal conduction and transmitter release. The direct measurement of transmitter release is independent of the postsynaptic effect of solutions with an altered ionic content and in contrast to in uivo, where it is difficult to maintain a constant known [K’], because of active removal of K+ (FISHER,PEDIEY & PRINCE, 1976; HEINEMANN & Lux, 1977), in vitro the [K’], can be maintained at a constant level. However, in slices of the hippocampus it is impossible to identify presynaptic cholinergic

neurons and measure their membrane potential. Therefore, the influence of elevated [K’], on the

DUDAR

synaptic transfer function, independently from axonal conduction, cannot be analyzed as it was in the giant synapse of the squid (KUSANO,1970). However, to see whether the effect of elevated [K’& on action potentials was at least qualitatively the same in hippocampal slices as in other preparations, such as the squid giant synapse (ERULKAR& WEIGHT, 1977), conducted compound action potentials resulting from focal stimulation were measured with extracellular electrodes. EXPERIME~AL PROCEDURES Measurement

of acetylchotine

release

The release of ACh was estimated by measuring the efflux of tritiated substances from hippocampal slices superfused at a constant rate following incubation with [3HJcholine. Freshly prepared brain slices incubated with labelled choline form labelled ACh (COLLIER,POON & SA~~M~HADDAM, 1972; MOLENAAR,POLAK & NICKOLSON, 1973). After such incubation brain slices superfused in the presence of a cholinesterase inhibitor spontaneously release both labelled choline and labelled ACh but electrical stimulation causes the release of only labelled ACh and the increased efflux of the label measured in the absence of a cholinesterase inhibitor reflects accurately the evoked release of C3H]ACh (RICHARDSON & SZERB,1974). Therefore in this study, the difference between the observed and spontaneous efflux during stimulation was used as a measure of the evoked release of C3H]ACh as has been done in previous studies (BOLJRDOI$MITCHELL,SOMCGYI & SZERB, 1974; HADHIZY & SZERB, 1977; SZERB et al., 1977). The hippocampus of the rat was sliced in a sagittal direction into strips 0.4mm wide by means of a McIlwain tissue chopper and were incubated for 40 min in 25 ml Krebs’s solution at 37°C. bubbled with 95% 0, and 5% CO2 and containing 4 &i/ml [methyi-3H]choline (AmershamSearie, 10.1 Ci/mmol) and unlabelled choline giving a final con~ntration of 1 PM choline. After removing the incubation fluid by suction, the strips were placed into a superfusion bath (volume 0.5ml) and the slices were superfused at a rate of 1ml/min with a Krebs’s solution containing 10 pM hemicholinium-3 to prevent the re-uptake of released choline. After 75 min superfusion, the collection of 27 4-min samples was begun. During the collection of samples 6-17, electrical pulses (1 Hz, 5 ms, 40 mA, alternating polarity) were applied by means of electrodes at the top and botton of the bath. Superfusion with solutions of different ionic composition was begun 15 min prior to the start of collection. Therefore the tissue was exposed to a solution of changed composition for 35 min prior to the start of stimulation. Su~rfusion with the changed solution was continued until the end of the experiment. The effects of solutions of changed ionic composition on spontaneous release was investigated by applying them only during samples 617, when normal electrical stimulation would have been delivered. In each experiment the expected spontaneous outfiow was calculated from the first 5 samples (prior to stimulation) and from the last 5 samples, when the effect of stimulation had subsided, by plotting the logarithm of radioactivity in these samples and interpolating the antilog of the calculated values by the method of least squares. In the absence of stimulation, the observed efflux and the spontaneous efflux so calculated, based on a single

Elevated K+ and electrically exponential

model, were in good agreement (Fig. 4C). The difference between the observed efflux and the calculated spontaneous efflux during stimulation gave the evoked release of [‘H]ACh. These calculations were based on the disintegration/min values of the samples, which were obtained by means of the external channel ration method. To obtain the rate constant of the evoked release, i.e. the rate of emptying of preformed C3H]ACh stores by stimulation and the size of the store from which this evoked release occurred, the evoked release was plotted cumulatively against time and the points were fitted by computer with the equation described by RICHARDSON & SZERB (1974). This equation represents the outflow from a sequential two-compartment system, the tissue and the bath, each having first-order kinetics. Released radioactivity was expressed as pmol.g-’ based on the specific activity of choline in the incubation medium. The slices were weighed at the end of the experiment. Their average weight was 87 (SD. 12) mg. Recording

of extracellular

compound

action

potentials

Action potentials were recorded from either the alveus or fimbria of the rat hippocampus in vitro. For this purpose, a 30(r_400prn slice was cut from the dorsal surface of the hippocampus using a guide, and the fimbria was removed and divided along its long axis. The slices were placed on a mesh of an incubation chamber similar to that described by SKREDE & WESTGAARD (1971) and SPENCER. GRIBKOFF, COTMAN & LYNCH (1976). The chamber was perfused at 1 ml/min with Krebs’s solution at 35”, gassed with 95% Oz and 5% CO* and with Krebs’ solution at 35°C at 1 ml/min. With this perfusion rate the contents of the bath could be changed completely every 6 min. Square wave pulses (0.5 Hz, 0.05 ms) were delivered through two Teflon-coated steel wires (diameter llOpm, cut Rush) 0.5 mm apart. Potentials were recorded 5-20 mm from the stimulating electrode by means of glass-coated tungsten microelectrodes with a tip diameter of 10-15 pm. The composition of the Krebs’ solution was (in tnM); NaCl, 120; KCI, 3.0; CaCI,, 2.6; NaH,PO,, 1.2; MgSO,, 1.2; NaHC03, 24; glucose, 10. To raise the potassium content of the solution (maximum 15m~) KC1 was added without reducing the NaCl content. Reduced sodium Krebs’ solution was prepared by replacing part of the NaCl with iso-osmolar sucrose.

evoked transmitter release

429

181

2 1

STIMULATION

O,2 TIME ( min 1 FIG. 1. The effect of tetrodotoxin and of Ca*+-free solution

on the evoked release of C3H]ACh. M Control; X- x in the presence of 1.5~~ tetrodotoxin; 0 .‘. 0 Ca’+-free Krebs. Each point is average f S.E.M.of four experiments. STIM indicates the period when 1 Hz stimulation was applied. Tetrodotoxin containing Krebs and Ca2+-free Krebs were applied 15 min before the start of the collection of the samples.

campal fibres to the cholinergic terminals without any synaptic interruptions. Increasing the potassium concentration of the superfusion fluid from 3 to 8 mM had no significant effect on the evoked release of C3H]ACh (Fig. 2, Table l), although the evoked release in the presence of 8 mM K+ became more variable, as shown by the large standard errors in Fig. 1B. Higher potassium concentrations (10-15 mM), however, depressed the evoked release significantly and this depression was the result of both the decrease in the rate of evoked release and a decrease in the size of the pool of C3H]ACh which was available for release by stimuRESULTS lation (Table 1). The cumulative plots of the evoked It has been shown previously (SOMOGYI& SZERB, release against time shown in Fig. 3 illustrate the 1972) that in slices from rat cortex the release of method of calculation of these two parameters of C3H]ACh evoked by electrical stimulation is blocked evoked release. While the rate of evoked release deby tetrodotoxin and by the omission of Ca2+ from creased in a concentration-dependent fashion when the superfusion medium. To see whether this was the the potassium concentration was increased from 10 case with the evoked release of C3H]ACh from the to 15mq the size of the releasable pool dropped by hippocampus, these experiments were repeated in 40% in the presence of 1OmM K+ but did not dehippocampal slices. Figure 1 shows that tetrodotoxin crease further with higher potassium concentrations and lack of Ca” suppressed the electrically evoked (Fig. 3, Table 1). release of C3H]ACh in hippocampal slices. This sugIt has been known for some time (HUXLEY & gests that the evoked release is the result of conducted STKMPFLI,1951) that increasing [K’], causes a reducaction potentials depolarizing cholinergic terminals tion in the size of action potentials recorded from which release ACh in a Ca’+-dependent manner. peripheral nerves through the inactivation of the regenerative sodium channels. Reducing [Na’],, has Since this release is also abolished by chronic lesion in the septum (SZERBet al., 1977) these action potena similar effect on the size of action potentials. As shown in Fig. 4B, reducing the sodium concentration tials are conducted along the axons of the septohippo-

430

J. C. SZERB,P. HADHAZYand J. D. DUDAR 8 mMK+ n-b

‘

0 0

,

,

,

lb

,

32

,

,

,

48

,

,

64

,

80

1

0-r

I

0

96

,

,

lb

,

I

32

TIME(min) 10 mMK+ 1-S

I

lb

I 32

’

’

,

‘

64

,

I

80

,

‘

%

D

n-4

I 64 TIM Inin) I

4%

1

TIMEimin)

Is 15mMK+

C

’

, 48

’

I

80

8

I

96

’

0

I

16

’

I

32

’

I

45

*

I

64

.

I

80

’

I

96

8

TIME(mini

FIG. 2. Efflux of radioactivity from slices of hippocampus evoked by 1 Hz stimulation. (A) 3 mM K’, N = 10; (B) 8mM Kf, N = 6; (C) 1OmM K+, N = 5; (D) 15mM K+, N = 4. Average & S.E.M.of observed values shown by points connected by solid lines. Dotted line connects crosses indicating expected spontaneous efflux (see Experimental Procedures).

in the su~rfusion medium by 33% decreased the rate of evoked release of c3H]ACh just as elevated potassium did. The size of the releasable pool was also decreased by lower sodium concentrations (Table 1). To see whether the decrease in the size of the TABLE 1. SODIUM

EFFECT

EVOKED

OR OF REDUCED OF C3H]ACh

OF RAISED ATRIUM

CONCENTRATIONS BY

Na+ (mW

K+ (m@

N

145

3

10

145

6

4

145

8

6

145

10

5

145

12

4

145

15

4

109

3

4

97

3

4

73

3

3

ON

ELECTRICAL

THE (1

releasable pool of E3H]ACh was due to its having been released by the various superfusion fluids prior to electrical stimulation, normal Krebs’ solution was changed to high potassium or low sodium Krebs in the absence of stimulation during the collection of samples 617. The highest con~ntration of potassium

RELEASE

Hz)

SMMULATION

Rate constant of release (min-‘) + S.E.M.

Releasable pool size (pmoI.g- ‘)

0.0555 + 0.0023 0.0496 ) 0.0030 0.046 1 + 0.0043 0.0412* + 0.0026 0.0303t * 0.0022 0.0219t f 0.0030 0.0456 *0.0048 0.0327t _t 0.0034 0.0259t +0.0016

Significance of difference from normal Krebs. * < 0.01; t < 0.001.

f S.E.M.

323 +12 280 +37 242 +65 19st +9 19s* +38 186t +17 290 *22 151t &-18 176t k24

TIME

I minutes

l

FIG. 3. Cumulative plot of the evoked release of E3H]ACh against time with different concentrations of potassium in the superfusion fluid. Points at infinity are the calculated releasable pool sizes. The same data as shown in Fig. 1. Only S.E.M. of pool sizes shown, other points have an equal or smaller ~.E.w.

431

Elevated K+ and electrically evoked transmitter release P 97mM Na+

1

cl

16

32

48

,,$

“I

TIMEtmin)

80

96

TIME(min)

. . C

l~~~~ 1

0

l6

32

48

64

80

96

0

16

I

32

48

64

80

%

TIME (mini

TIME tminl FIG. 4. EfBux of radioactivity

from slices of hippocampus. (A) Evoked release (1 Hz stim) 145mM Na+, N = IO (same data as in Fig. 2A); (B) Evoked release (1 Hz stim) 97mM Na+, N = 4; (C) Spontaneous release, 3 mM Kf present in samples 617, N = 4; (D) Spontaneous release I5 mM K+ present in samples 6-17, N = 4; (E) Spontaneous release 72.5mM Na+ present in samples 6-17, N = 4. Symbols as in Fig. 1.

used (15mM) caused a slight increase in the spontaneous release of the label (Fig. 4D) amounting to 19pmol.g-’ during 48 min, the time period along which st~ulation would no~al~y be applied. This was very much less than the 137pmol.g-’ reduction in the releasable pool size resulting from 35 min superfusion with this solution prior to stimulation. Furthermore, 10mM K” did not enhance spontaneous release while a reduction of sodium significantly decreased the spontaneous release of the label (Fig. 4E). Thus the loss of releasable ACh into the superfusion media used cannot account for the decrease in the size of the pool which can be released by stimulation. The depressant effect of elevated potassium ion concentration on the electrically evoked C3HJACh release could be due to reduced axonal conduction or to interference with depolarization-secretion coupling at the terminals, or both. The effect of elevated [K’] on axonal conduction alone was tested by measuring the size of the extracellular compound action potentials in the alveus or fimbria of the hippocampus in vitro. An example of results from this preparation illustrates the reversible depressant effect of 15m~ potassium on axonal conduction (Fig. 5). Because of the long time-lag in changing solutions, it was not possible to show reliably graded responses to smaller increases in potassium concentration, but

it is clear from the results illustrated in Fig. 4 and from two other experiments on fimbria that 15mM potassium had a marked depressant effect on axonal conduction. DISCUSSION Evoked

release

of acetylcholine

The results indicate that 3-8.mM K+, a range in concentration which has been observed under physiological conditions in the mammalian central nervous system, has little effect on axonal conduction and transmitter release in the septohipp~ampa~ tract. This is in marked contrast with the recent findings of ERULKAR& WEIGHT (1977) on the squid giant synapse where increasing [K’], by 1.5m~ from 9 to 10.5 mM reduced the size of the presynaptic action potential and the amount of tansmitter released from the terminal, as indicated by the postsynaptic potential. The lack of sensitivity of the in vitro preparation used in this study to moderately elevated [K’], is not likely to be due to a deteriorating membrane potential. The tissue continued to respond to prolonged electrical stimulation by releasing ACh and had there been a run-down in the membrane potential, the preparation presumably would have been more and not less sensitive to the depolarizing effect of elevated [K’lO. It is known that in the leech the neuronal membrane potential, in contrast to that of glia, responds little to changes in [K’& below

J. C. SZERB, P. HADHAZY and J. D. DUDAR

432

COMPOUND ACTION POTENTIAL FROM ALVEUS IN VITRO 3 mMK+

15mMK+

5 min

8 min

3mMK+ I

6 min

mVi

1msec

FIG. 5. Compound

action potentials in a slice of alveus. Each graph shows five sweeps evoked at 0.5 Hz. Time after switching of perfusion fluid indicated at top of tracings. The initial potential is a stimulus artefact.

Pooi of rele~~le aeetyl~~l~ne 8-IOmM {KLIFFLER & NICHOLLS, 1966; BAYLOR & NICHOLLS, 1969). The same appears to be the case A decrease in the size of the pool of releasable in the mammalian hippocampus (DICHTER,HERMAN [jH]ACh caused by increasing the Kt or decreasing

& SELZER, 1972) and cord (LOTHMAN& SOMJEN, 1975). Furthermore, SINGER& Lux (1973) found that stimulation of the mesencephalic reticular formation or of the visual cortex caused an increase of up to 3 IIIM in [K+ ], in the lateral geniculate nucleus. Yet according to SINGER (1973) no change in synaptic transmission occurred in spite of the fact that an increased excitability of optic tract terminals indicated some depolarization of these terminals. In contrast, ERULKAR& WEIGHT (1977) report that slight elevation of [I(‘], reduced synaptic transmission in the giant synapse of the squid with only a small reduction in the membrane potential. This would indicate that in the squid axon slightly elevated [K”], can inactivate regenerative sodium channels independently from its effect on membrane potential, as has been found previously by ADELMAN& PALTI (1969). In contrast to elevated [K’],, reduction of [Na+& had the expected effect on the rate of transmitter release, indicating that the size of the action potential which depends on [Na’ Jo (HUXLEY& STXMPFLI,1951) is one of the determinants of transmitter release.

the Na+ content in the superfusion fluid indicates that when the size of the action potential is reduced, part of the preformed C3HJACh present in the terminals cannot be released by st~u~tion, probably because conducted action potentials fail to reach some of the terminals under these conditions. This reduction in the size of the releasable pool occurred in a step-wise fashion. This would suggest that conduction failure occurred at the site of a fairly regular branching of the septohipp~pal cholinergic fibres. Extensive branching of this tract has been reported by MOSKO,LYNCN& COTMAN(1973). Partial block of axonal conduction due to high-frequency stimulation has been described in several invertebrate preparations and this block has been ascribed to the a~umulation of extracellular potassium (GRCB~MAN, SPIRA& PARNAS,1973; Sr.rrrrt & HAIT, 1976;SPIRA, YAROM& PARNAS,1976). Spontaneous release of acetylcholine

It appears that the septohippocampal tract is at least as sensitive to the axonal conduction blocking

Elevated K’ and electrically evoked transmitter release effect as to the enhancing effect of elevated [K’], on the spontaneous release of ACh. At 15 mM, when axonal conduction was severely affected, as indicated both by the reduction in the rate of evoked release of C3H]ACh and by the depression of action potentials, the spontaneous release of C3H]ACh was increased only a little. It is possible, however, that the small enhancement of spontaneous release by 1Om~ K+ reported by LILEY (1956) at the neuromuscular junction might not have been detected. Higher (25 mM) [K’],, however, causes a marked increase in spontaneous C3H]ACh release from hippocampal slices (HADHAZY& SZERB,1977). While these observations suggest that presynaptic processes (axonal conduction and spontaneous transmitter release) are rather insensitive to [K’], in the physiological range, they do not exclude the stimulatory consequences of a small depolarizing effect of

433

such [K’]. on neurons. These postsynaptic influences can account for the enhanced firing of neurons in hippocampal slices caused by increasing [K’], from 3 to 6rn~ reported by FRITZ & GARDNER-MEDWIN (1976). Similarly the appearance of epileptiform discharges in hippocampal neurons due to the perfusion of the lateral ventricle with solutions with high [K’] may be the result of depolarization of basal dendrites of pyramidal cells as was suggested by ZUCKERMANN & GLASER (1968). This stimulatory effect of high [K’& would not manifest itself in the preparation employed in this study which was concerned only with axonal conduction and transmitter release.

Acknowledgements-The careful technical assistance of Mr GARY GILL is gratefully acknowledged. This work was supported by the Medical Research Council of Canada.

REFERENCES ADELMAN W. J., JR. & PALTI Y. (1969) The influence of external potassium on the inactivation of sodium currents in the giant axon of the squid, Loligo pealei. J. gen. Physiol. 53, 683-703. BAYLCIR D. A. & NICHOLLSJ. G. (1969) Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech. .I. Physiol., Lond. 203, 555569. BERTELS-MEEUWS M. M. & POLAKR. L. (1968) Influence of antimuscarinic substances on in vitro synthesis of acetylcholine by rat cerebral cortex. Br. J. Pharmac. 33, 368-380. BOURDOISP. S., MITCHELLJ. F., SOMOGYIG. T. L SZERBJ. C. (1974) The output per stimulus of acetylcholine from cerebral cortical slices in the presence or absence of cholinesterase inhibition. Br. J. Pharmac. 52, 509-517. COLLIERB., POON P. & SALEHMOGHADDAM S. (1972) The formation of choline and of acetylcholine by brain in vitro. J. Neurochem. 19, 51-60. COOKEJ. D. & QUASTEL D. M. J. (1973) The specific effect of potassium on transmitter release by motor nerve terminals and its inhibition by calcium. J. Physiol., Lond. 228, 435458.

DICHTERM. A., HERMANC. J. & SELZERM. (1972) Silent cells during interictal discharges and seizures in hippocampal penicillin foci. Evidence for the role of extracellular K + in the transition from interictal state to seizures. Brain Res. 48, 173-183. DUDAR J. D. (1975) The effect of septal nuclei stimulation on the release of acetylcholine from rabbit hippocampus. Brain Res. 83, 123-133.

ERULKARS. D. & WEIGHTF. F. (1977) Extracellular potassium and transmitter

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