Extracellular ionic variations during spreading depression

Neurosciencr, 0 IBRO

Vol.

3. pp. 1045-1059.

Pergamon

Press

Ltd. 1978.Printed in GreatBritain.

0306-4522,7X,

EXTRACELLULAR IONIC VARIATIONS SPREADING DEPRESSION

IIOI-1045

SOZoO/

DURING

R. P. KRAIG and C. NICHOLSCIN Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue. New York, NY 10016, U.S.A.’ Abstract-Changes in the concentrations of K+, Ca* +, Na+ and Cl- were measured during spreading depression in the exposed lissencephalic cerebellar molecular layer of the catfish, Corydoras aneus. Liquid ion exchanger ion-selective microelectrodes were used in pairs to monitor simultaneously changes in the concentrations of two ionic species in the extracellular space. Normothermic spreading depression in the catfish cerebellum consists of a slow negative potential shift that develops at a rate of 2-3 mV/s, reaches an amplitude of -25 mV, lasts 16 min and propagates at a rate of OS-l.5 mm/min. [K’], rises from a resting level of 2.3 35 mM at the peak of spreading depression. Between 20 and 40 s later [Ca”], falls from 2.2 mM to 0.8 mM and [Na’], and [Cl-], decrease from 149 to 57 mM and 137 to 47 mM respectively at approximately the same time. [Ca’+&, [Na’], and [Cl-], decreases begin when [K’], exceeds 10 mM. These results establish the magnitude and temporal sequence of the major ion concentration changes in extracellular space during spreading depression. The earliest extracellular precursor of spreading depression is a rise in [K+],. In light of the equality of [Na’], and [Cl-], changes and the rise in [K’]., electroneutrality in extracellular space involved may be maintained by the net accumulation of some unidentified anion equivalent to the rise in [K’],. Alternatively, no net accumulation is needed if some extracellular anions are impermeant and the volume of extracellular space decreases. The sum of the extracellular ion concentrations suggests that the ionic strength of extracellular space decreases by greater than one-third during spreading depression. These results demonstrate that the brain is capable of establishing and recovering from local ionic inhomogeneities.

SPREADINGdepression is a pathological phenomenon associated with susceptible brain cell ensembles which was initially characterized electrophysiologically in the rabbit cerebrum (LEXO, 1944). It is defined as a cessation of electrical activity which lasts for minutes, propagates slowly and is accompanied by a large, predominantly negative potential in extracellular space (LE~o, 1947). Spreading depression can be evoked by mechanical, electrical or chemical stimuli and can occur spontaneously (BuRES,BURES~VA& K~~IV~NEK, 1974). The mechanism of spreading depression is still unknown; two theories suggesting primary roles for different ion species have been proposed. The first, by GRAFSTEIN(1956a,b), attributes a primary role to the neuronal release of K+. The second, by VAN HARREVELD(1959; 1966), assumes that the release of the excitatory amino acid, glutamate, triggers neuronal Na+ conductance changes. Neither theory alone can account for all the known phenomena associated with spreading depression (BURESet al., 1974; VAN HARRE-

& WAIXH, 1968; SUGAYA, TAKATO& NODA, 1975) and of glia (HIGASHIDA,MIYAKE, TARO & WATANABE, 1971;

HIGASHIDA, MITARI &

SUGAYA et al.,

WATANABE, 1974;

1975; MORI, MILLER& TOMITA,1976) occurs. At the same time as these events, brain tissue impedance rises (LE;~o & MARTINS-FERREIRA,1953; FREYGANG& LANDAU, 1955; VAN HARREVELD& OCHS, 1957; RANCK, 1964; HOFFMAN& CLARK, 1974; HAVSTAD,1977). When the peak of the slow extracellular potential is taken into account, it appears that neurons and glia are depolarized to about 0 mV (COLLEWIJN& VAN HARREVELD, 1966; SOMJEN,1975). The extracellular negative shift and depolarization of brain cells has long been thought to be associated with large ion fluxes in and out of cells (for reviews, see MARSHALL,1959; OCHS, 1962; VAN HARREVELD, 1966; LE~o, 1972; BUR& et al., 1974) but for many years this remained unconfirmed. Recently, evidence for such ion changes has begun to accumulate and this will now be summarized. Since no net changes in water or electrolyte content VELD, 1977). have been detected in the whole cortex after a During the slow, negative, extracellular potential sequence of repeated spreading depression (BURES et shift, depolarization both of neurons (GOLDENSCHON al., 1974) it is assumed that ion changes consist of transient fluxes in and out of the brain or between brain compartments. Early attempts to detect ion I This work was commenced in the Department of shifts focussed on K+ and relied on the use of flame photometry of fluid samples (BURES & KRIV~~NEK. Physiology and Biophysics, University of Iowa, Iowa City, 1960) or radiolabels (BRINLEY,KANDEL& MARSHALL, IA 52242, U.S.A. 1960; BURES& KRIV~~NEK,1960). These experiments Ahhreoiation: ISM, ion selective micropipette. 1045

I< 1’ K;KAI(, ‘mtt C. Nlc~flol

1046

so2

t:XPERIMENTAL t’RC)(‘l-111 RI,\ showed a large increase in [K I,, during spreading depression, but lacked spatio-temporal resolution. Single-barrelled ISMs were siliconired with .! ‘,, 11 ‘. f These methodological inadequacies wcrc obviated Dow Corning 1107 fluid in trichloroethylenc tW41 ~I:R through the use of ion-selective micropipettes (ISMs) 1971; KHIIKI. AGLILIA~& WISI. 19-i : KKAIO.iif:Ol. SIIIto continuously measure [K ‘I,, rises, from a normal conized pipettes tilled wtth approprtatc electrl)iytes .md baseline value of 2~~4mM to levels of 20 50 rnM during broken to a tip diameter of 7 3 ,m’ were made ion \electrv<, spreading depression. which are transient and correby dipping mto appropriate ion exchanger ~uII~\. ion lated with the negative excursion of the extracellular exchanger columns were 200 500 itm m length N,t -ISMI monensin liquid ion exchanger IKKUG & potential (VYSKOi’lL. Km’% & Btt& 1972; PKINC’I contained NICHOLSON. 1976) and were backfilled with I50 mkt NaC‘l Lux & NEHEK, 1973; FL:I-AMA~HI,MUTAIVI& PKINU.. K I-ISMs contained Corning 477117 ton exchanger and 1974; MORRIS & KKNJEVI~‘. 1974; NI(.HOLSO~ & KRAIG, 1975; MORI er (II.. 1976; NIC‘HOLSON.TEN w’ere backfilled with 150 mM KC’]. (‘I -lSMs contained Corning 477315 ion exchanger and were backfilled with BRUGGENCATE, STEINBEKG, & Srijck~~,, 1977). 150mr~1 KC]. Reference electrodes were tilled with I50 IIIV Measurements of changes in [Na +J,, by flame phoNa acetate and 2 rnbi CaCI, (ALI\ & HOI_TIMA\. 19661 tometry of superfused samples showed that Na’ to minimize their sensitivity to changes m iomc strength. release was not significantly changed during spreadDouble-barrelted ISMs (Lt:x. 1974) were fabricated with ing depression (KRIVANEK & BURES. 1960). the same electrolytes as descrtbed abov~c for single barrel Chloride was shown by a histochemical technique ISMs. These electrodes were also broken to an overall ttp diameter of 7 3 )nn. Ca’* -1SMs contained ‘t neutral ion to accumulate in cerebral apical dendrites during (OUIM~. KESSL~K Kr SIXION. 1976. ,AMMAN\. spreading depression (VAT HARREVELD & SCHADI:. exchanger Gt.c-(I. PRFTSCH & SIMON. 1975) and were backtillcd with 1959) and in cerebellar Golgi epithelial ceils (Bergmann glia) during asphyxia (VAN HAKRI VL:LD.196 I ). 100m%t CaCII. Diffusion from the reference barrel did nut influence the ion sensitive barrel ISMs wcrc stored tn a process phenomenologically similar to spreading mammalian Ringer until thetr use depression (VAN HARREVIJLD. 1961; H~IRES ct ~1.. Dual ion sensing arrays were constructed Iron1 smgie 1974). Light (VAN HARR~VELD & SCHADI'.1959) and barrel lSMs by positioning a smgle reference electrode electron microscopic (VAN HAKRL:VIJLD & KHA'I‘TAN. between two single barrel ISMs with a Narashige MT5 1967)examination of tissue frozen at the peak of triple holder micromanipulator. The electrodes were glued spreading depression suggested that the compartwith rapid-setting epoxy so that the Inter-tip dtstance was IO ,irn or less. Double-barrelled ISMa were stmilarly posttments that take up chloride swell while the extracellutoned and glued lar space shrinks. These histochemical techniques. Electrodes were connected vta AgAgC‘l wires to unity though, are susceptible to errors (BRINLO. 1963) and gain tow capacity. low basis currcnt (incorporating Philprovide only limited dynamic information. brick Model No 1429 operational amplifiers). high impeThe purpose of this investigation was to monitor dance buffer amplifiers (K~M)Ts~L. & IOHNSON. f97.3) the the extracellular ionic milieu of the brain during low impedance side of which was connected to rl Ag AgC‘I, spreading depression and establish a comprehensive agar KC1 indifferent macroelectrode placed on the foredescription of the ionic events. This information is brain (Fig. I ). The outputs of the buffer amplifiers were indispensable for understanding the mechanism of electronically separated to yield electrochemtcal and referspreading depression. Measurements of [K’],,. ence potenttal signals. These signals were filtered using low pass filters with upper limitmg frequencies of II) I37 and CCa’+l,. INa ’ I,, and [Cl I,, were made during recorded on a four channel chart recorder. Field potentials spreading depression with EMS. Spreading depression was studied in the exposed lissencephalic cerebellum of the teleost. Cor_vdoms u~~c’~~s. We chose to examine spreading depression in the cerebellum because of the wsealth of anatomical and physiologica data available for this structure (Ecc~~s. Ire & SZL.NT.iwmfAi,1967;LLINAS,1969) and the great simplicity of this structure compared with the cerebral cortex. We chose to study the cerebellum of a fish because in our preparation spreading depression was easily and reliably initiated there and because the ionic events are slowed down compared to those of a mammal,

thus

as we can discover.

improving the basic

time

resolution.

characteristics

So

far

of spread-

ing depression in the catfish cerebellum are the same as those in the cerebellum of rat (NIOrorso~ ef (II.. 1977: KRAIG. NICHOLSON & P~tu.Ln~s. 1978) and cat (NICHOLSON. TEN BRUGGENCATL..STOCKLF & STFINHEKG. 197X). Brief accounts of some aspects of the present studies have already been published (NK‘HOLSO& & KRAIG, 1975: KKAIG & NI(.HOI,SOY. 1976).

were derived from the unfiltered reference signals ,md were photographed from an oscilloscope. The ISMs used here are only slightly influenced by mterfertng ions likely to he found in oxtracellular fluid. The major interfering ion for the K ‘-ISM is Na ‘. to which the K ’ -ISM displays a selecttvity of X0 100: I under ideal conditions (WALUK. 1971). Thts was corrected for by adding NaC‘I to the K’ catibrattng solutions used Tot- the K ‘-ISM (set below). Quaternary ammonium compounds cause significant interference atth the K’-ISM (NEH~:R& LLX. 1973) but their concentration should be very low in the cercbellar molecular layer. which apparently lacks cholinergic synapses (PHILLIS, 1970). The selecttvtty of the Na -ISM against K ’ is 15: I (KKAIG& NICHOLSOh. 1976). The selectivity of the Ca’ +-EM agamst K + IS better than 600: I and against Na. is better than 1OOO:t. The sclectivity of (‘I -ISMs is ahout 5: 1 agatnst bicarbonate and 200: 1 agamst glutamate (Lr.x. 1974; NICHOLSO\ 6i KRAI~;. 1975). Against y-aminobutyrtc acid the sclcctivity is 600: I (KKAIG, 1976). No attempt was made to correct any of the ISMs for changes in thetr mterfering ion\ during spreading depression since the clfects would not h,tvc hecn

Ion changes in spreading depression

Ioo < ~

1047

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Ion (a) < ~ exchanger barrelX ~

o V~ o Ib

LOC - ~ Pc.

P'f"/~~~~ ~ ~ ~

~

.~

exchanger (b) l°n barrel Indifferent elec~,,~r~] =

~

Cerebellum

F-orebrain

FIG. 1. Experimental technique. Ion selective micropipettes (ISMs) were glued together with an intertip spacing of 10#m or less. Single-or double-barrelled ISMs were used. Output potentials were buffered with high impedance buffer amplifiers (1 x) and the reference signals (V~, and Vb) were subtracted from the ISM signals to yield the electrochemical signals (I, and Ib). The reference signals also provided slow potentials and field potentials. The indifferent agar bridge macroelectrode was placed on the forebrain. The tips of the microelectrodes were located about 50 #m below the surface of the cerebellum. A local bipolar electrode (LOC) stimulated parallel fibers (p.f.) the axons of granule cells (g.c.) which synaptically excite Purkinje cells (P.c.). Pressure microinjection of 1 M KC1 saturated with Fast Green FCF (for visibility) elicited spreading depression. significantly greater than inherent random errors in the system. ISMs were calibrated before and at the conclusion of experiments in a series of principal ion solutions. Na ÷and C1--ISMs were calibrated in pure NaC1 solutions (25-250mM). K+-ISMs were calibrated in KC1 solutions (2-50mM). CaZ+-ISMs were calibrated in CaClz solutions (0.5-16mM). The latter two sets of calibrating solutions also contained 150 mM NaCI to simulate the ionic strength of the extracellular microenvironment and to allow for the Na + interference in the K+-ISM (PRINCE et al., 1973). Experiments were discarded if the slope of the ISMs changed by more than 5 mV from start to finish of an experiment or if reference electrodes displayed more than a 5 mV change for a decade change in principal ion concentration. Corydoras aneus, the green (or bronze) catfish, was purchased locally at tropical aquarium stores and prepared as previously described (NICHOLSON& KRAIG, 1975). Fish were anesthetized with tricaine methane-sulfonate (Sigma) and paralyzed with tubocurarine chloride. Respiration was maintained by infusing water in through the mouth and allowing it to exit via the gill slits. Anesthesia was maintained by adding tricaine to the water. The brain was exposed from the telencephalon caudally to include the cerebellum. Spreading depression was initiated by pressure injection via a micropipette of 1 M KC1 saturated with Fast Green FCF for visibility (Fig. 1). Spreading depression initiation and recording were at a depth of about 50ffm down in the cerebellar molecular layer under normothermic conditions (25°C). After each spreading depression the ISM array was withdrawn and calibrated in small agar blocks containing 100 and 150 mM NaCI. For the K+-ISM the agar blocks were made with 2, 4, 7 and 10ram KC1 together with 150mM NaCI. For the Ca2+-ISM, the agar blocks were made with 0.5, 1, 2, 4 and 8 mM CaCI2 together with 150mM NaCI.

RESULTS

Characteristics of the extracellular potentials associated with spreading depression The p h e n o m e n o n which has been classified as cerebellar spreading depression in Corydoras aneus exhibits all the characteristics that define spreading depression in other preparations. In Fig. 2 it is seen that during spreading depression in Corydoras aneus there is a large, predominantly negative potential in extracellular space. The slow negative potential shift develops at a rate of 2-3 mV/s, reaches an amplitude of a b o u t - 2 5 mV relative to uninvolved extracellular space, and lasts 1-6 min before returning to baseline. A small (0-3 mV) positivity usually precedes the negative slow potential in spontaneous and electrically or chemically induced spreading depression (Fig. 2). Field potentials were evoked by local bipolar stimulation (Fig. 1) of parallel fibers every 5 s prior to and t h r o u g h o u t spreading depression (Fig. 2). Such field potentials at a depth of 50 # m exhibit the typical fast initial positive-negative c o m p o n e n t of the presynaptic parallel fiber volley and subsequent slower negative wave associated with the postsynaptic depolarization of Purkinje cell dendrites (ECCLES et al., 1967; LLINA.S, 1969). D u r i n g spreading depression, locally evoked field potentials are rapidly extinguished as the slow negative potential develops and progressively return minutes after the slow potential has returned to baseline (Fig. 2). D u r i n g the early positive phase of the slow potential there is often an e n h a n c e m e n t of the parallel fiber c o m p o n e n t of the field potential. The postsynaptic c o m p o n e n t is ext-

104x

R. P KI
b I I

FIG. 2. Fast and slow potential changes during spreading depression. Upper trace (VI shows slow potentials during spreading depression elicited by KC1 microinjection (KCI). Lower records show field potentials evoked every 5 s and recorded from the same electrode that recorded the slow potentials; time of stimulus corresponds to position on slow potential trace. Field potentials consist of an initial fast positive-negative presynaptic parallel fiber volley followed by a slower postsynaptic negativity associated with Purkinje cell dendritic depolarizalion. Most of the evoked response is depressed at (a) and begins to return at (b). Recording electrode located within 50 pm of the surface of the cerebellum Note difference in time calibrations for slow potential and field potentials.

inguished

as the slow

potential

crosses

its null

point

and swings negative. All field potentials are extinguished as the slow potential reaches its negative peak. Several minutes later the presynaptic component returns to be followed later by the postsynaptic component. Often the latter component is enhanced in amplitude for some time after its return. During the period when the field potentials are abolished, the stimulus artifact is enhanced. This is probably caused by the increased tissue impedance, known to occur during spreading depression (see Introduction). Using three micropipettes spaced 150 pm apart in a line with tips at the same depth (50 pm). the conduction velocity of the slow potential was measured at 25°C. (Fig. 3). The results shown in Fig. 3 indicate a delay in the arrival of the potential wavefront at the three positions. Since there is some change in wave shape, the velocity was determined and the delay was measured at the point of 250, maximum height of the leading edge of the waveform. This resulted in a conduction velocity of between 0.5 and 1.5 mm/min for different preparations. Since the duration of the negativity at half maximum height is

about

1 min,

this

gives

a

spatial

extent

geneously. since propagation is ubiquitous throughout this region. Below the molecular layer, the slow potential is diminished and is absent in the white matter. Chanyrs

in [K ‘1,

und

[Ca’*-),,

duriny

spreuding

dcptmsion

ISMs exhibit a logarithmic sensitivity to ions. Consequently, in the presence of low ambient ion levels. EMS are very sensitive. Since K’ and Ca”’ are minority ions of the extracellular microenvironment, in comparison to Na’ and Cl-. fluctuations in K’ and Cali can be measured with comparable sensitivity. As will be shown, these ions exhibit a striking dissimilarity in behavior during spreading depression. During spreading depression in the catfish, [K’],, begins to rise from a baseline of 2.3 _t 0.2 mM (mean t_ standard error: JI = 30) to a level of

2”rn”

[+

of

0.5-I .5 mm for this phase of the spreading depression. The Corydoras cerebellum is only about 3 mm in diameter, so that a propagating spreading depression occupies a significant proportion of the surface area and its propagation must be affected by part of the wave encountering a boundary soon after initiation. This factor probably accounts for the variation in wave shape seen at the different positions in Fig. 3. Other experiments show that the cerebellar molecular layer is able to sustain spreading depression homo-

FIG. 3. Slow negative potential propagation. Three potential recording electrodes spaced in a line as indicated and 5Opm below the surface, show that slow potential propagates through the cerebellar molecular layer at a velocity of about 0.5- I .5 mm/min under normothermic (25 ‘C) conditions. Vertical dotted lines are placed at time to 25”,, peak amplitude.

Ion changes in spreading depression

1049

1

ZQmV

-*

+

7 08

-

02

rnY

‘Inn

40

D f

__________-------------_____--________

_JlL_[

2

20mV

v

L

FIG. 4. [Ca”],. [K’],, and potential changes during spreading depression. Double-barrelled ISMs were glued with an intertip spacing of IOpm. The upper trace shows the slow potential recorded from the reference barrel of the Ca’+-ISM and the lowest trace the slow potential recorded from the reference barrel of the K+-ISM during spreading depression elicited by KCI microinjection (KCI). The second and third traces respectively show [Ca”],, decrease and [K’], increase. The amplification of the [K’], signal was half that of the [Cal+],, signal so the resolution of the two records is equal.

35 k 3 mM (n = 18) at the peak negative slow poten-

tial shift (this point defines the peak of spreading depression). The elevation of [K’], apparently reaches a steady state which lasts for about a minute before the concentration begins to return to baseline (Figs 4 and 6). Field potentials recorded during spreading depression are completely extinguished when the [K’],, reaches a level of about Lomb (Fig. 6) the same point where the slow potential swings negative (Fig. 5) and changes in the other ions begin (see later). In contrast to the rises in [K’],, our measurements

K’

I _:____ I

;

/

j

2

X)mV

v An

I --_ _--_ +

FIG. 5. Delay between [K’],, rise and [Ca”], fall during spreading depression. Records shown are an enlargement of Fig. 4. The resolution of the Ca’+ and K+ records is k0.l mM. A [K’], rise occurs at the beginning of the positivity in the potential waveform. A [Ca”], decrease occurs at about IO mM [K’],. Note that the slow potential waveforms recorded from the two separate ISMs are virtually identical.

FIG. 6. [K ‘I,, [Cl -1. and potential changes during spreading depression. Single-barrelled ISMs and common reference electrode with an intertip spacing of IO pm were used. The upper trace shows slow potential during spreading depression elicited by KC1 microinjection (KCI). The second trace shows [Cl-], decrease and the third trace shows [K’],, increase. The lowest records show field potentials recorded on a fast time scale by reference electrode at times indicated.

revealed a drop in [Ca”], during spreading depression (Figs 4 and 5). We found that [Ca”], fell from a baseline value of 2.2 + 0.1 mM (n = 20) to 0.8 + 0.1 mM (n = 10) at the peak of spreading depression. Temporal

disparity

in onsets of changes in

[K’],

and

CCaz+lo Baseline [Ca”], is about equal to that of [K’]‘, and the slope of an ISM for a univalent ion is twice that for a divalent ion. Thus K+-ISMs are twice as sensitive as Ca’+-ISMs. However, in practice the resolution of these ISM recordings are equal since [Ca*‘], recordings were made at twice the amplification of those for [K+],. This enables a direct comparison of the times of onset of the respective ion changes during spreading depression to be made. Previous attempts to examine such time relationships in rat (NICHOLSON et al., 1977) and cat (NICHOLSON et al., 1978) were hampered by the rapidity of spreading depression in those species. In Corydoras, spreading depression is a slower phenomenon than that in warm-blooded animals and time differences were successfully resolved. Simultaneous measurements of [K’], and [Ca”], with paired, double-barrelled theta-ISMs revealed that [K’], begins to rise about 2@4Os (n = 10) before the [Ca”], begins to falI (Fig. 5). The resolution of the [Ca”], and [K’], recordings is +O.l IIIM.In no case was [Ca”], seen to fall prior to the increase in [K’],. In Fig. 5, it is seen that the fall in [Ca”], commences at a [K’], level of about 1Om~ and coincides with the instant at which the extracellular slow potential, V, crosses its null point and swings negative; the delay between [K’], rise and [Ca2’], fall is 26 s in this case.

The ma.jor ions of the brain cell microenvironment. Na’ and I‘1 . can also be monitored with tSM techionic hasrlinc niques. ~~IthoLigh the high ambient reduces the sensitivity to small changes tn concentration. WC have previously briefly described [Na ’ I,, changes (KKAIG & NI(~HOI.SO~. 1976) and [<‘I I,, changes (NIUXXSOK & KKAIC;. 1975) during sprewding depression. We present here a more complete dcscription and analysis. During spreading depression in ~~~~~~~~~~~~.s [Na ’ 1, rapidlq falls from ;I baseline of 149 2 3 rnM (II = 26) to 57 h 3 rn>t (II = 14) at the peak of spreading depression (Fig. 7). The [Na-‘I,, change never plateaus. but immediately begins to return to baseline. The [Cl I,, change. on the other hand. does reach an apparent steady state at its peak depression (Figs h and 7) which lasts about a minute before it returns to baseline. The [Cl-J,, typically decreases from a baseline of 1.37 k 5 rnM (II = 26) to 47 F 4 rnM (II = 13) during spreading depression. The resolutlon of [Na 1,. was &h mv and that of [Cl- 1, + 7 m&i in those recordings, which is too low to define prccisely the beginning of these concentration changes. However. as noted above for the [Cal +I,, changch. both begin approximately as the slow potential crosses its null point. Simultaneous measurements of [Na ‘1, and [Cl J,, changes reveal that. within ex~rimental error. the absolute shifts in [Nn ’ .],, and [Cl J,, mx equal in magnitude and time course throughoLlt spreading depression, Figure 7 shows such a simultaneou< single-barrelled Na A_ and measurement with Cl ~-ISMs. The equalit) of the changes is not readily apparent due to the logarithmic nature of ISM sig-

I%. 7. [Na’],. [Cl -1. and potential changes during spreading depression. The recording paradigm is similar to that in Fig. 6. The upper trac? shows slow potential recorded from common reference electrode. The second and third traces respectively show [Na’],, and [Cl-],. decreases recorded from single-barrelled ISMs. The lowest records demonstrate the behavior of evoked presynaptic and postsynaptic activity during spreading depression.

Sources of error in fhc rccordmg paratt~ym used to establish the equality of the changes III [Na ‘J,, and [Cl -I,, during spreading dcprcssion were considcrcd to be less than the rcsolut~on ( I I III\,‘) of ISM recordings. [K ‘I,, interfercncc in jNa * 1,. riicordlngs would ne%er have been more than II mu ouing to the 15: I Na’ :K 1 sclectivith 01’ Na *-ISMs (KRAIC; Ji Nlf‘HOLSOiX. 1976). EutraccAlulur bicarbonate interfcrence in Cl,, rccordinga WI\ more ditticult to prcdiet. since accurate extraccllular pH measurements during spreading depression ha\:c not bcc‘n ma&. However. LIVCI~if the cntirc l)rcdicted ;m~n Jcticit (see later) consisted of ii<‘O, [_C’l ]:, recordings would not be in error by more than + 9 m&f owing to the 5: I. (.“I : HCOg selectivq of C’I -iSMs (Lr x. 1974: NICHOLSON bt KKAIC;, 1975). Furthermore a large increase in [HCO,],, is unlikely since it would necessitate an extracellular alkaline shift. Surface pH n~easurements have shown that in fact the shift during spreading depression is cithcr acidic (Tst I-IIKW, IXANAGA, TAYLOK. WA1.U K c1: ~W+it.NS(-Ht.iS.

i%s)

Of’

Insignificant (RAIOWK I & ~ARSt1Al.I. iYh4). ,1 second possible source of error III single-barrelled ISM electrode array recordings could come from inaccurate reference signal subtraction due to $htz small. but finite. spatial sep~~raii~~Iioi the common reference electrode from the ion sensing electrodes. This source of error was shown to be inqnificant h> the close similarity of referencesignals in double-barrcllcd EM arrays (Figs 4 and 5). In addition [Na ’ I,, end [Cl I,, changes wcrc found to be c~iual using double-harrelied LSM arrays.

Although simultaneous measurements of changes in [Na’],,, [K ‘I,, and [Cl -I,, during spreading depression were neter made. changes in the extracellular ion content can hc predicted beoausc of the equality of [Na-1, and [Cl J,, changes. The results of such a prediction are shown in Fig, 8 which. for the first time. allows changes in the cxtracellular anion deficit and ion content to be estimated during spreading depression. Changes in [Na’],, were calculated by the subtraction of the absolute change in measured [Cl --I,, from the average baseline [Na ‘3, of I49 mM. The extracellular anion deficit, [A-J,,, was calculated from the sum of equivalents of extracellular cations (Na’ and K ) minus the equivalents of [Cl-‘],, at various times. For simplicity. changes in [Ca”“],

Ion changes in spreading depression

1051

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ccc**PIL-____________------------~_

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0

0

0

.

.

- 120

0

.

0

-60

..

O. o l. . . ..m.* noo 0 I 0.*le.e:.o” . on . 0. . . . l. . -0.. 00.. l *..... .* Ct)~~~_-___--_------------_______--_-________--. . AA. . .. ~~~~~~~~~~~~~~~~___________~___~~~~~~~~ .

-40

.

.

.

- omM

c 1.0

. .

*

t

a9

FIG. 8. Estimation of total extracellular ion content changes during spreading depression for one experiment. In contrast to other ion data, these graphs are plotted on a linear concentration scale. The extracellular anion deficit concentration ([A-],) was calculated from the sum of the equivalents of K+ and Naf minus equivalents of Cl; at various times. [Na’], was calculated by the subtraction of the change in [Cl-], from the average baseline [Na’], since [Na’], and [Cl-], changes were equal during spreading depression. [Cl-], and [K’], were plotted on a mM scale from experimental values. [Ca’+], changes not shown. Lower-most graph represents an estimation of the total extracellular ion content (I) changes during spreading depression. Here the normalized sum of cations and anions, shown above, were plotted as a function of time. were not included in Fig. 8 but were used in Table 1. Within experimental error, the changes in [A-], mirror the changes in [K’], since [Na’], and [Cl-], changes are equal. Normalized total ion content changes were estimated by adding the measured changes in [K’], and [Cl-], to the calculated shifts in [Na’], and [A-], for a typical spreading depression. Such information suggests that the extracellular ion content is decreased by 40 f 3% (n = 13) in extracellular space involved in spreading depression (Fig. 8; Table 1).

Non-propagating events It is important to consider whether the various ion changes described above are by necessity concomitant, or whether cases arise where dissociation occurs, so that one or more of the ion changes are absent during spreading depression. We can give a partial answer to this question by describing the ionic changes seen during a relatively rare occurrence; the non-propagating event. Occasionally 1 M KC1 microinjections induced events having some characteristics of spreading depression but which did not

1052

* Values listed are mean + standard t Calculated as the aniondeficit.

error:

1~) is the sample

propagate (Fig. 9A). Field potentials during such events were never fully extinguished but consisted of a widened parallel fiber component at the peak of the event (KRAIG,1976). These field potentials rapidly returned to their pre-event form. consisting of the typical pre- and postsynaptic components, in a few seconds as opposed to minutes for normal spreading depressions. Such events were sometimes the initial ones induced in animals which later went on to give full propagating spreading depressions. The ionic changes during non-propagating events are of particular note. During the event, [K+], rises by only 8 mM to a level of around 10 mM while [Na’], falls by only 8 mM (Fig. 9B) and no change at all occurs in [Ca”],, (Fig. 9C). In addition no large negative slow potential is seen (Fig. 9B and C). The rise in [K ‘1, to 10 mM thus seems lo be associated with a reciprocal fall in [Na’],, while the very large and precipitous falls in [Na’],,. [Ca’ ‘I,, and [Cl-], and the large negative slow potential shift only occur when a normal spreading depression takes place. This transition to a regular propagating spreading depression seems to occur only when [K ‘I,, exceeds 10 mM. DISCUSSION

Spreading

depression

irl Corydoras

crrc+rllunl

cots-

pared to other preparations

Our experiments confirm that spreading depression in the cerebellum of Corydoras has all the characteristics of spreading depression in the mammalian cerebral cortex. In the Corydoras cerebellum. however, spreading exhibits a slower time course and propagation velocity, compared to the mammalian cerebrum. The propagation velocity of 0.5. 1.5 mmjmin in Corydoras is similar to the value of I 2 mm/‘min reported for spreading depression in the cerebellum of the skate (Ruju erinacea and Rocellata) (YOUNG. 1975) but less than the velocity of 3.3 mm/min found in the rat cerebellum (FIFKOVA, BLJRES,KOSHTOYANTS, KRIVA~~EK& WEISS, 1961) and 9 mm/min in the cat cerebellum (NICHOLSON et al., 1978). In the mammalian cerebral cortex, the mean conduction velocity is 3 mm/min (BURES et al., 1974). These data indicate that the low conduction velocity in the Corydoras cerebellum is not an inherent cerebellar characteristic. but probably due to body temperature (BuRES, BURE-

size

SOVA & ZACHAROVA, 1957) and

species differences. The slow time course of spreading depression in Carydoras has been an asset in the present experiments since it has offered us better resolution of events. Increases in [K’], during spreading depression have been measured with ISMs in the mammalian cerebral cortex (VYSKO~L (or ul., 1972; PRIN~L ct ,A.. 1973; MORRIS & KRNJEVI~‘. 1974; F~ITAMACHI rt uI., 1974; SUGAYA et ul.. 1975: MURI et al.. 1976) and mammalian cerebellum (NICHOLSON rr ul., 19?7: I978). Reported values ranged between 30 mM and 80m~. Thus, the [K’], rise in Corydorus of 35 mM is somewhat small. but if the lower baseline of [K’],, in our preparation is taken into account. then the rise is by a factor of 15 in Cor>,doras, which is quite similar to that in many mammalian examples. .4 drop in [Ca’ ‘I,, by as much as 1 mM has been reported in the rat (NICHOLSON et 01..1977) and cat cerebellum (NIcHOLSoh. et d., 1978). In Corydoras the tnM tiecrease is even larger. Large decreases in [Na’],, and [Cl I,, during spreading depression, comparable 10 those seen in Corydorm, have now been seen in the rat cerebellum (KRAIG CI ul.. 197X). Validity o/’ meusurewnts

and irtterpretutiorr

Before discussing in more detail the nature of the ionic fluctuations during spreading depression, we would like to comment on two assumptions upon which our interpretation of the data is based. The first is that the tips of our ISMs reside in a small artificial space, created by the electrode, which is at all times in chemical equilibrium with the ionic milieu that interpenetrates the narrow space (of the order of 200 A) between immediately surrounding cellular elements. This assumption would be seriously violated if physical forces acting in the narrow clefts between cells were such as to create a microscopic environment which differed from the one that our ISMs measure. Such a situation might arise to some extent due to charge separation (FINKELsTE~N & MAURO, 1977) and the possible effects of charged macromolecules in the intercellular regions. Since we are unable to characterize such effects realistically we must assume them to be small enough to be neglected at the level of resolution of our present techniques. Our second qualification is that we assume that ions which appear in. or disappear from, the extracellular microenvironment move across the membranes

Ion changes in spreading depression

1053

XJmV

V

I: +

(A)

-

I

4

lrnin

KCI

2OmV

t +

_ 2OIN

V

[

_e-

+

148

(B)

I loo

NO+ KCI

’

mh4

lmin ’

IO 4

K+

1

-_-____________^____-_---_

2OmV

V

Ca2+

[ + [ :8 -

flit4

lmin

KCI K+

2

-_-___--________________L____

IO I

4 2

FIG. 9. Non-propagating event. Occasionally an event was observed after 1 M KC1 microinjection (KCI) which did not propagate. (A) the event recorded by three potential recording electrodes spaced 150 gm apart, 5Ogm down in the cerebellar molecular layer. Decremental spread was seen in depth as well as horizontally (not shown). Field potentials were unique in that only the postsynaptic Purkinje cell dendrite component was briefly extinguished at the peak of the d.c. signal (V). (B) and (C) represent ion changes during events with similar field potential changes. In (B), [Na’],, fell by about 8rn~ while [IL+], rose by about 8m~. In (C), no change occurred in [Ca”], while [K+], again rose by about 8 mM.

of cellular elements. The alternative hypotheses are that ions leave or enter the blood stream or that they are released or bound by extraccllular macromolecules. Although ionic movement with respect to the blood stream during spreading depression has been postulated (GRAFSTEIN, 1%3) several pieces of evidence argue against this playing a major role. First, spreading depression occurs in the isolated retina without any blood supply (MARTINS-FERRERIRA & DE

OLIVE~RACASTRO,1966; MORIer al., 1976) and seems normal as regards potentials, propagation and CR*], changes. Thus the blood supply is not necessary for a normal spreading depression. Second, the measured permeability between blood and brain for K* and Naf seems very low (HANSEN,LUND-ANDERSON & CRONE,1977) and is probably similar to that of neuronal membranes (RAPOWRT, 1976). With regard to the binding of ions in the extracellular space, there

is no cvidencc

for it except

possibly in the case 01 that <‘a’* is bound to neuronal surface molecules. but the data and interpretation are inconclusiv*e. Moreover. there is clcal evidence that Ca’ ’ enters cells during synaptic transmission and action potentials (Ralir:a. 1972: ROUTER, 1973; LIJNAS. 1978). Thus the simplest assumption at present is that ions translocate across cell membranes. either by movement down an electrochemical gradient or by a molecular pump moving them against such a gradient.

Ca”. ADEY (1971) has postulated

Our data indicate that spreading depression is only triggered when [K’],, exceeds approximately 10 mM. During the ascent of [K’], from its baseline value of 2.3 mM to the threshold of 10 mM, no other changes can be detected, including [Ca’ ‘I,, changes for which our recording sensitivity is comparable to that for [K’],,. This threshold effect is particularly evident in the occasional cases where the rise in [K ‘I,, fails to reach 10mM and only a non-propagating event is seen, which does not involve large ion changes. but only a drop in [Nat],, to compensate for the rise in [K’],,. The threshold value of IOmM is strikingly similar to the ceiling value of [K+],, rise during stimulation and epilepsy in a variety of tissues. Experiments in the cerebral cortex (VYSKOCIL er d., 1972: Lux & NEHER. 1973; PRINCE et d., 1973; FUTAMACHI rt ul., 1974;

HEINEMANN 8~ Lux,

1975:

1977:

LOTHMAY.

LAMANNA, CORDINGLY. ROSENTHAL & SOMJEN, 1975;

MORRIS & KRNJEVI(‘.. 1974). hippocampus SCHUETTE, 1975). lateral geniculate

1973). and spinal cord

(SINGER

(LEWIS & & Lr;x.

(SOMJEN & LOTHMAN. 1974:

BRUGGENCATI:, Lux & LEIBEL. 1974; KRi&

SYKOVA.

VYKLICK~. 1974; KW, SYKOVA& VYKLICK\;. 1975) show that [K ‘I,, never rises to more than l&l2 mM except in those instances where spreading depression or anoxia occurs. In the latter cases [K ‘1, always exceeds 20m1~1 for the duration of the event. Thus the level of IO- 12 rnbt [K’],, seems to be a critical transition point for ionic stability in all preparations so far studied. UJE~. &

Changes in ion content

during

sprending

depressior~

und

their implications

The most surprising finding from these studies is the large drop in [Na’], and [Cl-],, during spreading depression and the fact that both decrease by the same amount. Since the rise in [K’],, is considerably less than the fall in [Na’],, and it is difficult to believe that other cations are involved to any appreciable extent, it seems an unavoidable conclusion that the ion content of the extracellular space falls during spreading depression. Previously, VAN HARREVELI) (1966) had speculated that NaCl should leave the extracellular space during spreading depression, but he supposed that such a change would take place

isotonically with a concomt)ant shrink,+< ,)I 111~ extracellular space. The implications in the fall o1 Ion content art’ r’ar reaching. If we assume that ions move betucen the compartments, one intracellular and the other extracellular. and that elcctroncutralit) and f,jsmot)c balance are maintained. then IMC)extreme pos>)billti<‘\ arlse. If there is no osmotic pressure difference hetwzcn the two compartments at any time, then the two conpartments must undergo a net swelling ralanvc to uninvolved tissue. This would imply that water M.OLIICI have to move into the region and this uould he consistent with observations of local swelling during spreading depression (MARSIWI I.. 1959; DI. 01 I~III
Ion changes in spreading depression

1055

ing depression in many respects (VAN HARREVELD, 1966; BURESet al., 1974). Recent ISM measurements in cat cerebral cortex (DORA & ZEUTHEN, 1976; SILVER1977) and rat cerebellar cortex (KRAIG et al., 1978) during anoxia induced by breathing nitrogen agree well with the ion concentration and ion content changes reported here during spreading depression in Corydorns. Furthermore, surface measurements with Na+ selective macroelectrodes during no-flow anoxia in brain (HOSSMAN, SAKAKI& ZIMMERMAN, 1977) and liver (HELPER,KESSLER& SIMON,1976) also revealed similar [Na’], decreases. In addition, [Cl-], and [Na’],, decrease substantially during spreading depression induced in rat cerebellum (KRAIG et al., 1978). Finally we confirmed our original observations on Cl- in Corydoras using Ag/AgCl micro wire electrodes (NICHOLSON & KRAIG, 1975) which indicated comparable [Cl-], changes to those seen with liquid ion exchanger ISMs. Such large ion concentration and ion content changes seem to be a general phenomenon of brain regions undergoing spreading depression or tissues, in general, undergoing anoxia.

to Na+ and Cl-, during the shrinkage of the extracellular space. A second possibility is that extracellular voluIpe stays constant. In this case the rise in [K’], and fall in [Ca”], would have to be balanced by a net flux of some A- into the extracellular space. We do not know what the nature of this anion would be; possible candidates include lactate, glutamate or bicarbonate. At a constant volume some 28 mM of the anion would be required, so that if it were pure bicarbonate, the pH of the extracellular space would become alkaline by about 0.4 pH units, while if it was pure lactate or glutamate, the pH of the extracellular space might become acidic. Measurements of pH during spreading depression are probably not accurate due to methodological difficulties but indicate acidic changes of 0.4 (TSCHIRGIet al., 1957) or nonsignificant changes (RAPOFQRT& MARSHALL,1964). It thus seems that during spreading depression the pH might reflect a mixture of acid and base (KAASIK, NISSEN& SIESIG,1970). This raises the problem of determining a mechanism by which the ion fluxes could occur.

Electroneutrality and changes in the volume of extracellular space

Mechanisms for ionic permeability change

The necessity to balance electroneutrality in the extracellular space implies that an unknown anion exists in the extracellular space, either as an impermeant moiety or as an anion that moves between intracellular and extracellular space. The amount of this anion is directly related to the nature of volume changes in the extracellular space. One possibility is that the apparent concentration of [A-],, reflects a shrinkage of the extracellular space by a factor of 2.47 (i.e. 47 mM [A-],/19 mM [A-],). This would of course require that the A- be impermeant, perhaps residing as negative charges on membrane-bound glycoproteins (SCHMITT & SAMSON, 1969). This would be analogous to the changes that can occur with the arrest of cellular metabolism (LEAF, 1956; MACKNIGHT& LEAF, 1977). Here the extracellular decrease in [Na’], and [Ca2+10 would be balanced by a rise in [K’], and fall in [Cl-],. We do not have any evidence for changes in the size of extracellular space ourselves, but such changes have been postulated on the basis of electron microscopy and impedance increases (VAN HARREVELD, 1972). In addition the drop in ion content during spreading depression reported here is a new factor which should, itself, raise impedance. One interesting speculation that arises concerns the possible influence of the volume change on the measured [K’], increase. If [K’], were to rise to 1&12mM prior to the reduction in volume of the extracellular space and the reduction were then to occur without further transmembrane K+ flux, [K’], would be concentrated to a value of about 30m~, which approaches the actual value of 35 mM. Such a mechanism would necessitate a low K+ membrane permeability, relative

We have not tried to investigate the nature of the permeability changes underlying the measured ion shifts. Here we shall merely review briefly the types of mechanisms which could contribute to the observed effects. Several well-established mechanisms could lead to an increase in [K’], and decrease in [Na+],. Action potentials obviously do this and indeed the original hypothesis of GRAFSTEIN(1956a, b) concerning the origin of spreading depression supposed that action potentials were the major cause. A serious objection to this is that spreading depression continues to occur in the presence of tetrodotoxin, which blocks the Na+ influx component of the action potentials (Kow & VAN HARREVELD, 1972; SUGAYA et al., 1975). Recently, however, it has been shown that action potentials occur in dendrites and are probably mediated in part by tetrodotoxin-insensitive Ca2 + currents (LLIN.& & HESS, 1976; SCHWARTZKROIN& SLAWSKY, 1977); such electrogenic mechanisms may contribute to spreading depression (NICHOL.WN, 1978; NICHOUON et al., 1978). Another contributory mechanism may be the electrotonically mediated depolarization of cellular processes ahead of the spreading depression wave leading to enhanced outward K+ flux and inward Na+ and Ca2+ as permeabilities to these ions increased, due to depolarization. Excitatory synaptic transmission also brings about ionic movements of an appropriate type and massive transmitter release by potassium, or some other mechanism, has been suggested as a mechanism of spreading depression (S~MJEN, 1973; YOUNG, 1975; MORI et al., 1976). This effect could be enhanced by diminished Na+-dependent transmitter uptake

1056

R. P.

KKAlG

and c‘. Ntcuo~sou

(LOGAN & SNYDER, 1972). Such a mechanism has similarities with the glutamate release hypothesis of V&N HARREVELD,except that glutamate need not be a specific transmitter. No conclusive evidence against these concepts exists at the present time. Another way that enhanced K + efBux and Na + influx could occur is through the cessation of activity of the Na-K adenosine triphosphatase pump (GLYNN & KARLISCH, 1975). This would allow Naf and Clto leak into cells and K* to leak out (LEAF 1956; MACKNIGHT & LEAF, 1977). A similar mechanism, which would he related to active or carrier mediated ion translocation, is the astrocytic Uptake of Naf and Cl- in the presence of HCO; and elevated [K’],, (GILL, YOUNG & TOWER. 1974: BOURKE.KIMELBERG & NELSON,1976). If our data is seen from the simplest viewpoint, it is evident that all extracelluiar ions move in the direction of their concentration gradients This is not surprising; since the intracellular volume is perhaps some five times greater than the extracellular. it would simply require an overall increase in membrane permeability to all ionic species, i.e. the transient formation of suitable ‘holes’ in the membr~e leading to a mixing of intra- and extracellular mobile ions (NICHOLSON et ai., 1977). Such ‘holes’ (TRUMP. CR~IIKER& MERGNER, 1971) and some idea of their size (PARKS, SHAY& AMIES,1976) have been suggested for anoxic tissues. With a loss of membrane perm-

selectivity, the system would presumably move toward a Donnan equilibrium based on the translocation of Na+, K+ and Cl- with some intracellular anions (and possibly a few extracellular ones) remaining fixed.

The nature CI~’spreading

drpression

Our results cannot be used to directly validate OI refute either the concept of spreading depression due to Grafstein or that due to Van Harreveld. As many others have shown, and we have ~on~rmed. [K ‘I,, is clearly involved in spreading depression. We can now say that Na+. Cl- and <‘a” changes are also an integral part of the phenomenon and that the earliest extracellular precursor of spreading depression is a rise in [K+],. We have no evidence regarding glutamate. Spreading depression may, in fact, be a sequence of interlocked phenomena which can be dissociated to some extent, as our observation on nonpropagating events and the experiments of VAN HAKREVELD (I 977) Suggest. Spreading depression presents a challenge to om understanding of the possibilities inherent in the

extra~llular space as a medium for brain cell interaction (NICHOLSON. 19781, SCHMITT & SAMSON(1969) were amongst the first to describe the extracellular space in terms of a dynamic continuum of membranerelated events whose physicochemical characteristics are more complex than would be expected for a simple saline solution. The prolonged local inhomogeneities in extracellular concentrations and ion content seen during spreading depression support this contention and indicate the complexity and subtlety inherent in the brain cell microenvironment. Acknowledget~ent.~--We are grateful to Prof SIMON.Swiss Federal Institute of Technology. Zurich, for providing the Ca’ * ion exchanger. The work was supported by the U.S. Public Health Service Research Grant NS-13742 from the National Institute of Neurological and Communicative Disorders and Stroke.

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