Extracellular calcium and potassium changes in hippocampal slices

Brain Research, 187 (1980) 165-182 © Elsevier/North-Holland Biomedical Press

165

E X T R A C E L L U L A R CALCIUM A N D POTASSIUM C H A N G E S IN HIPPOCAMPAL SLICES

C. BENNINGER*, J. KADIS and D. A. PRINCE** Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.)

(Accepted August 23rd, 1979) Key words: extracellular calcium and potassium - - changes -- hippocampus

SUMMARY Ca 2+ and K + ion sensitive microelectrodes were used to measure changes in ionic activities in the CA1 region of hippocampal slices during orthodromic (stratum radiatum) stimulation. Baseline levels of [K+]0 and [Ca2+]0 were those of the bathing medium which contained 5 mM K + and 2.0 mM Ca 2+. During stimulation [K+]0 rose to maximal levels of 12 mM while [Ca2+]0 decreased to as low as 1.4 mM. Systematic alterations in extracellular field potentials in stratum pyramidale accompanied the ionic shifts. Following stimulation K + undershoots occurred. An active K + uptake mechanism was demonstrated using iontophoretic K + pulses. [K+]0 and [CaZ+]0 changes occurred in parallel and in a laminar distribution with maximal changes recorded in stratum pyramidale. Maximal [K+]0 changes occurred from baselines of 5 mM and declined progressively at higher baseline levels. During epileptiform activity produced by exposure of slices to penicillin, la/ger ionic shifts with a more rapid onset occurred. The alterations in [K+]0 and [CaZ+]0 in the hippocampal slice are similar in some respects to those obtained by stimulation in vivo, making this preparation a potentially useful one for determination of mechanisms and effects of alterations in the ionic microenvironment. INTRODUCTION Since the introduction of ion sensitive microelectrodes (ISMs) for measuring extracellular potassium activity ([K+]o) in the mammalian braina,16,22,24,a4, 47 and the more recent use of Ca 2+ sensitive microelectrodes11,27,2s, it has become obvious that the ionic microenvironment in cortex changes substantially with normal and pathological cellular activities. Major questions with respect to these changes include (1) * Present address: Klinikum Der Universit~it Heidelberg, Kinderklinik, 6900 Heidelberg, Hofmeisterweg 1-9, G.F.R. ** To whom correspondence and reprint requests should be addressed.

166 their source in terms of the types and distribution of ionic conductances over the cell membrane; and (2) the potential effects of such alterations in ionic rnicroenvironment on the excitability of neurons involved in their generation and other nearby elements '~1, 35,43. Such issues are difficult to approach in vivo where baseline extracellular ionic concentrations cannot be easily manipulated. However, the hippocampal slice preparation 50 provides unique advantages for the study of mammalian cortical neurophysiology, including the opportunity to change the ionic environment. It has been demonstrated that excitability changes occur in neuronal aggregates within the slice as a result of manipulations of extracellular ionic concentrations 3°,31,33,35,3s,39,5°. It has also been reported that changes in [K+]0 may be induced in the hippocampal slice by electrical stimulation 6. We elected to use K + and Ca2+-ISMs in this preparation in order to examine several issues relevant to regulation of these extracellular ions. MATERIALS AND METHODS Transverse slices of guinea pig hippocampus with a thickness of 350-400 #m were prepared and maintained using techniques previously described 37,5°. Standard perfusion solution contained NaC1 124 mM; KC1 5 mM; NaHzPO4 1.25 mM; MgSO4 2.0 mM; CaC12 2 mM; NaHCO3 26 mM; dextrose 10 mM. Solutions containing lower (3.0 mM) or higher (10.0 mM) concentrations of K ÷ were prepared by adjusting concentrations of NaCI and KCI in the bath. In some experiments bath solutions containing 3 mM K ÷ and 1.2 mM Ca 2÷ were used (e.g. Figs. 3 and 7), to more closely mimic ionic conditions in the in vivo preparation. We found that slices could easily be maintained in either medium. As noted below, evoked changes in [K+]0 were larger from a baseline of 5 mM [K÷]0. It has also been our experience that stable neuronal recordings are obtained more easily if [CaZ+]0 in the bath is 2.0 mM. Penicillin solutions were prepared by adding 2000 IU/ml (3.4 mM) sodium penicillin G to the bathing solution, pH was 7.40. The above ionic manipulations produced insignificant changes in osmolality which was 305 mOsm. Experiments were carried out at 37 °C. All recordings were done in the CA1 region. Ion sensitive microelectrodes were made using Corrting potassium ion exchanger resin 477314 and calcium neutral carrier 29 supplied by Professor W. Simon, Laboratory for Organic Chemistry, University of Zurich. These electrodes were prepared using standard procedures and theta tube glass according to the technique of Lux and co-workers21, ~2. Electrode tips were 2-4 #m in diameter. In experiments where calcium and potassium were measured simultaneously, the tips of the ion sensitive electrodes were manipulated so that they were about 40 # m apart at the site of penetration, and each oriented at approximately the same distance from the alvear surface as verified using a calibrated eyepiece and dissecting microscope. Measurements were made at various sites in the hippocampus at a depth of about 150 # m below the cut surface of the slice. Potassium ISMs were calibrated in solutions containing 150 mM NaCI and various concentrations of K ÷. Similar calibrations were made with calcium ISMs. Potassium electrodes had slopes of greater than 40 mV for a 10 x change in [K+]0, and

167 calcium electrodes had slopes of greater than 20 mV for a 10 × change in [CaZ+]o. Since K + ISMs are known to be sensitive to quarternary amines they were also calibrated in solutions containing 5 mM K + and various concentrations of acetylcholine (ACh) ranging from 10-6 to 10 -3 M. Threshold for producing changes in electrode potential was about 10-5 M ACh. Electrodes were calibrated before and after recordings and data accepted if no significant drift or change in sensitivity had occurred. We estimated the speed of response of ISMs by manipulating an iontophoresis electrode containing KC1 of CaC12 close to the tip of the ISM in a saline filled dish in a mannel similar to that previously described for K ÷ (ref. 22) and Ca 2+ (ref. 11) ISMs. Under these circumstances half rise times for recording K ÷ and Ca z+ iontophoretic pulses were between 50-100 msec. It is likely that the speed of response is faster than this, given the presumed distance between the tips of the sensing and iontophoretic electrodes. Earlier studies using another approach showed response times for K + ISMs of about 3-5 msec 24. Iontophoresis electrodes filled with 0.1 m KCI and beveled to about 20 M,Q were used in experiments to focally alter [K÷]o in the sliceZL These electrodes were positioned using a dual electrode carrier so that there was a 20 # m separation between iontophoresis and ISM tips. Iontophoretic pulses of 200 nA were used. In some experiments intracellular activities were recorded together with the potassium and calcium signals using standard intracellular techniques and 4 M potassium acetate-filled micropipettes. Bipolar orthodromic and antidromic stimuli were delivered through pairs of tungsten electrodes which had been electrolytically sharpened and insulated to their tips. Fig. 1 shows the typical experimental set-up with Ca 2+ and K + ion sensitive electrodes located in stratum pyramidale of the CA1 region and an orthodromic stimulating electrode in the stratum radiatum. Orthodromic stimuli consisted of trains of 0.1 msec pulses with frequencies of 1-30 Hz and intensities up to 0.1 mA. In most experiments it was found that small changes in stimulus intensity and in the position of the stimulating electrode had significant effects on the recorded field potentials and the the evoked changes in K ÷ and Ca z+. Therefore, as a control, an independent microelectrode was placed in stratum radiatum of CA1 to monitor the field potentials produced by the afferent volleys. Signals from ISMs were recorded differentially, ion sensitive barrel against reference barrel, in order to cancel field potentials. Amplifiers had input resistance of 1013 fL RESULTS

Resting ionic concentrations The baseline levels of [K÷]o and [Ca2+]0 in well maintained slices were the same as those in the bathing solutions. Later in some experiments, when slices showed electrophysiological signs of deterioration, the tissue level of [K+]0 became higher than that of the perfusate. [K+]0 was iatrogenieally changed in some experiments by bathing slices with media containing 3 or 10 mM K +. Under these circumstances simultaneous K+ recordings in the bath and in the slice showed that tissue potassium reached a new steady state level within 10 min. Brief changes in local potassium concentration also

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Fig. 1. Schematic representation of the experimental setup with calcium and potassium ion sensitive electrodes situated in stratum pyramidale (SP) of the CA1 region of a hippocampal slice and a bipolar stimulating electrode in stratum radiatum (SR). SO, stratum oriens. occurred when the potassium electrode pair injured a neuron and high frequency cell discharges were recorded through the reference barrel.

Effects of stimulation The only regular recurrent spontaneous activity in the slice, which is sufficiently stereotyped to be useful as an index response in making measurements with ISMs, is the extracellularly recorded field potential burst which occurs after convulsant drug applicationZS,89, s0. Since such bursts did not usually persist over long periods we elected to perform most manipulations in normal solutions, using orthodromic stimuli. Single orthodromic stimuli delivered in stratum radiatum evoked extracellular field potentials and small increases in [K+]0 of up to 0.2 mM, which were dependent on the stimulus intensity. Such stimuli did not evoke measurable changes in [Ca2+]0. Trains of stimuli with frequencies greater than 2 Hz produced measurable alterations in both [K+]0 and [Ca2+]0. Optimal frequencies ranged between 5 and 30 Hz in different preparations6, ~s. Typical changes ale shown in Fig. 2 where effects of trains lasting 5 sec and 25 sec are shown. With the shorter train, [K+]o increased to a peak of 10 mM and there was a concurrent fall in [Ca2+]0 from 2.0 to 1.45 mM. The time courses of the [K+]0 and [CaZ+]0 changes were very similar. With trains of longer duration (second train of Fig. 2) both the [K+]0 and [Ca2+]0 levels tended to reach a peak and then assume a steady state plateau, while the stimulus was continuously applied (see refs. 11, 21a and 24). With such long trains, undershoots in [K+]0 of up to 0.2 mM below the baseline lasting as long as 60 see were apparent, however no Ca 2+ overshoots were obvious (but see ref. 28). The maximum [K+]0 levels reached during

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Fig. 2. Recordings from stratum pyramidale in one hippocampal slice during stratum radiatum stimulation. The effectsof 5 sec (first segment) and 25 sec (second segment) trains of 10 Hz stimulation are shown. Dotted lines in K + and Ca2+ traces: baseline activity levels. UPlcer trace: low gain DC recording from K + reference micropipette. A-C: representative evoked field potentials taken at the beginning of the train (A), near the peak of the ionic shifts (B), and during the later portion of the stimulus train (C). stratum radiatum stimulation were about 12 mM; [Ca2+]o was decreased to as low as 1.4 mM by similar stimuli. The ceiling levels for [K+]0 were influenced by baseline [K÷]0 (see below and Fig. 6); effects of varying baseline [Ca~÷]0 were not examined. Field potentials A systematic alteration in the appearance of extracellular field potentials in stratum pyramidale occurred during the course of the stimulus train. There was a tendency for the field potential to become larger in amplitude and develop multiple peaks during a period beginning shortly after stimulus onset and extending to the peak of the [K+]0 rise (cf. A and B in Fig. 2). At the peak of the ionic alteration, multispiked field potentials were the rule (Fig. 2B). However some decrease in the number of peaks occurred toward the end of the stimulus train when the [K÷]0 level had declined to a plateau below the peak (Fig. 2C). The ionic alterations and development of the multipeaked field potential appeared more rapidly with higher frequency, more intense stimuli. The development of multipeaked field potentials could also be conditioned by changes in baseline [K+]0 (see below and Fig. 6). K + uptake Decreases in K + release or increases in clearance from the extracellular space

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Fig. 3. Interactions between iontophoretically evoked [K+]0 increases and those produced by trains of orthodromic stimuli. A: iontophoretic K + pulse of 200 nA, 500 msec evokes a control [K+]0 increase of 2.0 mM. Test pulse evokes larger changes in [K+]0 when it is delivered near the peak or early falling phase of the orthodromically evoked increase (B: 2.8 mM; C" 2.3 mM). Iontophoretic pulses falling late during the orthodromically evoked rises (D : 1.4 mM), or during the recovery phase (E: 1.4 mM), or [K+]0 undershoot (F: 1.7 mM), evoked [K+]0 increases which are smaller than control. Arrowheads: onset of 500 msec iontophoretic pulses. Calibration for K-- ISM following segment (B) is for all traces. Baseline at the beginning of each segment except (F) : 3 raM.

could account for potassium potentials such as those of Fig. 2 which fell from a peak to a plateau and developed undershoots. This phenomena has been investigated in vivo 9A0 using applied iontophoretic pulses of K + during the orthodromically evoked rise to assess possible active clearance mechanisms. Similar experiments wele done in the slice by applying iontophoretic pulses of K + from an electrode immediately adjacent to the K + ISM. Fig. 3 shows a typical result of such an experiment, in which a bathing solution containing 3 mM K + and 1.2 mM Ca 2+ was employed, to more nearly mimic extracellular ionic concentrations found in vivo. Iontophoretic pulses of K ÷ lasting 500 msec produced transient rises in the levels measured by the K + electrode (first segment of Fig. 3A). When such iontophoretic pulses were superimposed upon [K+]0 changes produced by a train of orthodromic stimuli, it was found that pulses falling close to the peak of the [K+]0 rise would evoke larger changes in [K+]0 (Fig. 3B, C), whereas pulses falling during the declining phase of the orthodromic increase, when the [K÷]o level was moving toward a plateau, would evoke smaller rises than control (Fig. 3D). Iontophoretic pulses which fell after the end of the orthodromic train, when [K÷]0 was returning toward baseline (Fig. 3E), or pulses during the undershoot (Fig. 3F), would also evoked smaller changes in [K+]0 than control

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Fig. 4. Laminar distribution of [K+]0 and [Ca2+]0changes during trains of 10 Hz stimulation. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Baseline levels at the beginning of each segment are 5.0 mM and 2.0 mM for K + and Ca2+ respectively. Calibrations at the end of SR segment are the same for each segment. pulses*. Because iontophoretic K ' transients were delivered while the orthodromically-evoked change in [K+]0 was returning to baseline, it was not possible to accurately assess their fall time course in comparison to control. In any case these findings suggest that an active uptake mechanism is present in the slice preparation as it is in vivo 9, and that alterations in excitability may be present at the peak of the K + rise (see discussion below).

Laminar distribution of ionic changes Previous studies have shown that there is a laminar distribution for changes in [K+]04 and [Ca++]08 in the hippocampus during interictal epileptiform discharges. We studied the laminar distribution of ionic changes in the hippocampal slice by recording in stratum radiatum (SR), stratum pyramidale (SP), and stratum oriens (SO) with potassium ISMs or simultaneously with potassium and calcium ISMs, during trains of submaximal olthodromic stimuli lasting up to 25 see. The position of the electrodes was confirmed visually and with photographs made during the experiment. The architecture of the layered hippocampus could be easily seen in the transilluminated slice a2 so that these placements were reproducible in the same experiment and from experiment to experiment within 50/~m or less. A typical result is shown in Fig. 4. Rises in [K+]0 and decreases in [Ca~]0 were always largest in amplitude when the recording electrodes were located in SP. Data from 10 experiments with K + electrodes alone and 10 experiments in which [Ca2+]o and [K+]0 were simultaneously measured at * Note that the logarithmic scale makes it difficult to compare amplitudes of events evoked at different baseline [K+]oswithout attention to calibration marks. See legends.

172 various sites in the slice are summarized in the graph of Fig. 5. This shows that increases in [K~]0 paralleled decreases in [Ca2+]0 and that the maximum change was always in SP with a fall-off as electrodes were moved into SO and alveus on the one hand, and SR and stratum lacunosum on the other. Relationships between A K ~ and baseline K ÷

Because previous results have suggested a relationship between the transient increases in [K+]0 produced by stimulation or epileptiform discharges and the baseline [K+]0 from which such changes arise10,24, 26, we performed experiments in which the baseline [K+]0 was altered iatrogenically either by changing [K ÷] in the bathing medium, or applying iontophoretic pulses of K ÷. A typical experimental result is shown in Fig. 6. Here the same orthodromic stimulus train is presented when slices are equilibrated in solutions containing [K÷]s of 3, 5, and 10 mM (left column). The largest change in this experiment occurred when [K+]0 was 5 mM. When [K+]0 was increased to 10 mM, the rise during stimulation was larger than in 3 mM, but smallel than in 5 mM. Fig. 6 also shows that the initial rise time of the [K+]0 change during stimulation was faster at higher baseline [K+]0 levels (cf. change in 3 mM with that in 10 raM). We measured the field potentials evoked by trains of orthodromic stimuli by recording from the reference barrel of the K + ISM, and found that there were systematic alterations which occurred with changes in [K+]0 level, and the sequence of responses in the train. For example in Fig. 6, repesentative evoked field potentials at the beginning (A) and the end (B) of the stimulus train are shown at each baseline level (right columns). In 3 mM [K-]0 there was a gradual increment in the amplitude of the

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Fig. 6. First column: effects of various baseline levels of [K+]0 (achieved by varying bath concentrations) on K ÷ rise evoked by stimulus train. Recordings from stratum pyramidale. Ref, reference trace recorded DC. A and B: representative field potential recordings from beginning (A) and end (B) of each stimulus train for each of the three baseline concentrations of [K+]0. field potential and the development of one or more additional peaks as the stimulus train progressed. In 5 mM a much larger change in the field potential developed with multiple peaks toward the end of the train and an increment in peak amplitude of 100K or moze (of. first responses in A and B in 5 mM line). The changes in field potential in 10 mM were somewhat different. The initial responses were similar to those seen toward the end of the train in 5 mM. The amplitude and number of peaks tended to increase toward the middle of the train, and then decrease toward the end of the train (of. first and third responses of 10 mM (A) with third response of 10 mM (B)). We attempted to relate these changes in field potential to the level of [K+]0 achieved (baseline plus AK +) at various points in the stimulus train. In general there was a relationship between field potential amplitude, number of peaks and the baseline [K+]0 at the beginning and end of the stimulus so that higher [K+]0s were associated with larger multipeaked potentials. This relationship was not clear in 10 mM K + where the [K+]0 rise reached a plateau or incremented slowly toward the end of the train, at a time when the field potentials were decreasing in amplitude and complexity. A similar relationship between A [K+]0 and baseline [K+]0 was obtained when K + increases from orthodromic stimulus trains were superimposed on those produced by iontophoretic pulses of K +. This result is illustrated in Fig. 7. Control stimulus trains in the 3 mM K +, 1.2 mM Ca 2+ medium produced increments of up to 2 mM [K+]0 (A; G). As the iontophoretic pulse was added to increase baseline [K+]0, the orthodromitally evoked K + rise grew and reached a maximal change when baseline was about 10 mM (Fig. 7B-E). With still higher iontophoretically induced elevations in [K+]0 the change produced by the orthodromic stimulus decreased, becoming insignificant at baseline levels of about 15 mM (Fig. 7F).

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Fig. 7. Interactions between increases in [K+]0 produced by brief train of orthodromic stimuli (A; G = control) and [K+]0 increases produced by iontophoretic pulses lasting about 6 sec and of increasing amplitude in B--F. Relationships between iontophoretic and orthodromic stimuli are shown under segment F. [K+]o baseline at the beginning of each trace: 3 mM. Calibration following segment (B) for all segments. Note that [K+]0 measurements are on a log scale so that responses to orthodromic stimulation during increasing iontophoretic currents (B-E) are larger than control, even though their absolute amplitudes are about the same as the response in A, whereas response in (F) is much smaller than control.

These results are shown graphically in Fig. 8. The same general shape of the curves for bath ( O ) and iontophoretically ( • ) increased baseline levels is apparent. There appears to be a difference in the relationship between peak AK + and [K+]o in these two types of experiments, but the small number of data points for iontophoretic pulses make a detailed comparison difficult. Differences in the effects of bath versus iontophoretically induced increases in [K+]0 might be expected since the former technique could produce generalized effects including a redistribution of K ÷ and other ions between intracellular and extracellular compartments. In any case, it appears that there is a ceiling level for K + beyond which orthodromic stimulation produces decreasing K ÷ rises.

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in which baseline alterations were produced by changes in perfusion medium as in Fig. 6. Dark squares and dark bars: increases in baseline lK+]owere produced by iontophoretic pulses as in Fig. 7. Each point (© or HI) represents 3-9 measurements,except that squares without error bars are single values.

Penicillin effects Addition of penicillin to the bathing solution (3.4 mM, 2000 U/ml) produced a significant change in the evoked field potential recorded in the CA1 area as. Whereas single stimuli produced single field potential spikes prior to penicillin (Fig. 9B1), after exposure to the drug similar stimuli produced multipeaked field potenials (Fig. 9B~). Associated with this change in the effectiveness of stimulation, there were also changes in the extracellular ionic alterations. Before the addition of penicillin, as described above, trains of 1 Hz stimulation produced small rises in [K+]o (Fig. 9C1) and no measurable changes in [Ca2+]0 (Fig. 9A1). Higher frequency stimulation (10 Hz) produced more significant changes in [K+]o as well as in [Ca2+]o (Fig. 9A1). After addition of penicillin and the development of epileptiform field potential responses to single stimuli, significant increases in the A[K+]o and A[Ca2+]0 to the same stimuli occurred (cf. Fig. 9A1 and 9A2; and 9C1 and 9C2). [K+]o peaks were up to 25 ~ higher in penicillin versus control solutions. The rise rate for changes in [K+]0 and [Ca2+]0 was also faster in the penicillin solutions. During long trains, however, the higher peak of [K+]o and [Ca2+]0 was not maintained and fell back toward a plateau level which was very similar to that achieved in the control solution (cf. [K+]0 and [Ca2+]0 levels at end of I0 Hz train before and after penicillin in Fig. 9A1 and 9A2). [K+]0 undershoots were seen after penicillin application as they were in the control situation (cf. response following last stimulus train in Fig. 9A2 with that following second segment of Fig. 2 above). Since we were concerned as to whether the neuronal, aggregate continued to generate epileptiform activity during the entire train of stimulation in the penicillin

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solution, we performed several experiments in which intracellular activities were recorded together with the changes in [K+]0 and [Ca~+]0 during stimulus trains. The results of such an experiment are shown in Fig. 10. A 10 Hz train of orthodromic stimuli in a penicillin-treated slice produced typical [K+]0 and [Ca2+]0 changes as described above (Fig. 10A). Stimulation at 1 Hz produced much smaller [K+]o changes and a threshold change in [Ca~+]0 (Fig. 10B). Intracellular recordings during such trains showed that 1 and 10 Hz stimuli evoked typical depolarization shifts and associated burst discharges 38 during all phases of the train (sweeps in upper lines of Fig. 10). Changes in the configuration of the intracellular potentials occurred regularly during such trains. At the peak of the [K+]0 rise and [Ca2+]0 fail, the repolarization phase of the intracellular depolarization was prolonged and spikes were broadened (compare Fig. 10A, sweep 1 with sweep 2). Toward the end of the train the intracellular event tended to recover incompletely toward control (cf. Fig. 10A, sweeps 1 and 3). When stimuli were delivered at 1 Hz (Fig. 10B) each stimulus evoked a depolarization shift and associated spike activity, but the intracellular events changed little during the train. It is not possible for us to know whether the sequential changes in burst generation occurring in intracellular recordings during trains of stimuli relate to [K+]0 or [Ca~+]0 changes, or to other variables.

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Fig. 10. A: intracellular recordings (sv,eeps 1-3) at different times during train of 10 Hz stimulation producing K ÷ and Ca 2+ changes. Sweep 4 shows recovery several minutes after train. Dots below sweeps indicate stimulus. B: representative sweep showing intracellular changes (4) during 1 Hz simulation of stratum radiatum which produces a small increase in [K+]0 and decrease in [Ca2+]0 Recordings from stratum pyramidale. Distance from intracellular micropipette to ion sensitive electrodes; 50 ~m. Time calibration: 5 sec for K + and Ca 2+, 100 msec for intracellular traces.

DISCUSSION These experiments show that virtually all of the phenomenology described with respect to ionic changes measured with microelectrodes in vivo can be reproduced in the slice preparation, without the complicating factors of anesthesia or circulatory changes. It is also interesting to note that, although the slice is essentially injured cortical tissue, no spreading depressions are ever seen, even with the most intense direct cortical stimuli. The unique advantages of the slice preparation have allowed us to delineate some aspects of [K+]0 and [Ca2+]0 changes which have not been previously appreciated. It had been concluded from earlier in vivo measurements in neocortex that stimulated changes in [K+]0 were inversely related to the baseline [K+]0 levelT, 10, zz,24,26, although such a relationship was not apparent in the in vivo hippocampus 4. It is important to note that changes in baseline [K+]o levels in such experiments were produced by variations in epileptiform activity, or orthodromic stimulation rather than by iatrogenic manipulation. Thus the cortical population being tested for its capacity to release K + was the very population whose activity had resulted in an

178 elevation in baseline level in the first place. In the slice preparation we have examined this point using both bath perfusions and iontophoretic pulses to increase baseline [K+]0 and found that, rather than an inverse relationship between the variables, there is a maximal [K+]0 rise during stimulation at baseline levels of 5-6 raM, while a smaller increase in [K+]0 occurs at higher levels. A similar relationship between [K+]o and excitability has previously been reported in hippocampal slices. Field potential duration was found to be most prolonged by a conditioning tetanization when [K +] in the bathing medium was 6-7 mM 31. The mechanisms which underlie this relationship are undoubtedly complex and depend in part upon the relative contributions of the various K ÷ currents to A[K-~]0 and upon the sensitivity of these events to [K+]o. Changes in K ÷ uptake as well as K ÷ release mechanisms may be involved. The increases in neuronal excitability produced by modest depolarizations in resting potential at higher [K+]o levels 5 and positive shifts in the equilibrium potential for calcium activated potassium currents 2,15 might be offset by factors such as fiber conduction block25,a2; decreased transmitter release44; and decreased presynaptic spike height 3,45 as [K÷]o increases. The similar distributions of [K÷]0 increases and [Ca2÷]o decreases in the hippocampus were unexpected. Previous studies suggest that calcium spikes in hippocampal neurons might be generated in dendrites 41,4s. Intracellular recordings from hippocampal pyramidal cell dendrites have been used to directly confirm this conclusion4L Also, in the cerebellum the largest decreases in [CaZ+]o during orthodromic (parallel fiber) stimulation are in the molecular layer containing presynaptic terminals and dendrites z8. We had therefore expected to see the largest [Ca~+]o decrease in stratum radiatum which contains the dendrites of pyramidal neurons and terminals of Schaffer collateral afferents, since both terminals 17 and dendrites 2°,49 are known to have inward calcium currents during activity*. Our results, however, show that there is a larger decrease of [CaZ+]o at the cell body layer than in dendritic areas during orthodromic stratum radiatum stimulation. Some of this decrease in [Ca2÷]o in stratum pyramidale might be due to calcium movement into neurons during synaptic activation 19 or into terminals of basket cells known to end on CA1 somataL Also, studies in hippocampal pyramidal neurons 14,4~,4s,49 do not rule out the possibility that somata may have significant Ca 2-- currents. The reasons for development of these particular laminar distributions of [CaZ+]o and [K+]o during stimulation are thus not clear. Such profiles could partially reflect the distribution of conductances for these ions over the soma-dendritic membrane. However, it is also possible that they are influenced by the packing density of neuronal elements (somata, dendrites, presynaptic terminals) in various layers of the hippocampus and differences in the ratio of volume to surface area of various elements. This would make the observed distribution of ionic changes an unreliable index of the actual distribution of membrane ionic fluxes. The observation that the laminar distribution of A[K+]o and A[Ca2+]0 were the * The occurrence of multipeaked field potentials, such as those which developed during trains of stimuli used to construct laminar profiles, is a reliable sign of intracetlular burst generation in hippocampal pyramidal neurons. Bursts in these cells are associated with calcium spike electrogenesis in dendrites4%

179 same even though Ca 2+ ISMs are known to be insensitive to quaternary NHa compounds and the lack of correspondence of [K +] profiles with the known distribution of cholinergic terminals in the hippocampus, suggest that the apparent K + distribution was not secondary to ACh release. Also it seems unlikely that concentrations of ACh in the presumed dead space surrounding the tip of the K + ISM reached the levels of 10-5 M required for ACh responses in control experiments. The results of these experiments show that there is a very close coupling between A [Ca2+]0 and A [K+]0 in all lamina during stimulation (e.g. Figs. 2 and 5). The envelopes of these changes were almost superimposable. The exception is that we found no overshoot for [Ca2+]0 corresponding to the [K+]0 undershoot. In these respects, our results differ from those of Nicholson et al. 2s who found [Ca2+]0 overshoots and somewhat different laminar distributions of Ca 2+ and K + in the cerebellar cortex of barbiturate anesthetized cats. On the other hand, measurements of [Ca2+]0 and [K+]0 in cat neocortex show no [Ca2+]0 overshoots, but do suggest differences in correspondence between A[K+]0 and A[Ca2+]o at different cortical depths 11. On the basis of other electrophysiological data, a close relationship between changes in [K+]0and [CaZ+]0in hippocampus might be expected. In hippocampal pyramidal neurons, sodium spike activities usually evoke depolarizing afterpotentials which are blocked by Mn 2+ and are at least in part dependent upon Ca 2+ entry (Wong and Prince, unpublished). Thus, accumulation of [K+]0 as a result of frequent spike activity would be associated with decreases in [CaZ÷]0. CA1 pyramidal cells possess calcium-activated slow potassium conductances15 similar to those desclibed in other neurons 2,12,1s,2a,46, and also generate calcium spikes (see refs. above). Also, depolarization of hippocampal neurons in the subthreshold range gives rise to both delayed rectification (which presumably is mediated by a voltage dependent K ÷ currenO z) and an inward (anomalous) rectification which is in part due to Ca 2+ entry 14. Each of these membrane events would tend to cause a coupled decrease in [CaZ+]o and increase in [K+]0. One of the critical questions raised by the results of in vivo experiments has been whether the changes in [K+]0 and [Ca~+]0 produced by stimulation can actually give rise to alterations in excitability of neuronal aggregates. Data from these and other in vitro experiments are relevant to this issue. First, the levels of [K+]0 and [Ca2+]0 achieved during electrical stimulation or seizure activity are sufficient, when introduced into the bath of non-stimulated slices, to cause generation of repetitive high voltage field potentials and neuronal burst generation resembling those seen during epileptogenesis 35,z6. When calcium concentrations of 1.0-1.5 mM are used in the slice media containing 5 mM [K+], bursting tends to occur in hippocampal pyramidal cells, presumably due to loss of the stabilizing divalent cation effect of Ca 2+ (ref. 48). It may however not be legitimate to assume that ionic changes have similar effects on neuronal aggregates in stimulated and unstimulated slices. For example, if increases in [K+]0 are derived significantly from Ca2+-activated K + currents, neuronal hyperpolarization and increased membrane conductance15 may coincide with the increase in [K+]0, thus limiting its potential excitatory effects. The results of experiments where [K+]0 increases are produced by either bath

180 perfusions (Fig. 6), or iontophoretic pulses (Fig. 7), indicate that such changes influence the excitability of the neuronal aggregate to orthodromic input30,~l, 39. This relationship is further supported by analysis of field potential changes. Multipeaked field potentials are easily elicited in 10 mM K + or when stimulus trains elevate lower K + levels towards this point (Fig. 6; refs. 31 and 40). However, field potentials are not an absolute index of [K+]0 levels, since the later field potentials evoked by stimulation in 10 mM K + decrease in complexity and amplitude even though a plateau level of [K+]0 has been achieved. Undershoots in [K+]0, which occur in vivo after epileptiform discharge or prolonged stimulations 9 (see ref. 43 for review), are present in the slice and thus occur independently of circulatory factors which might produce a net local deficit in [K+]0 in the in vivo situation. During perfusion, tissue potassium is fixed at the concentration of the perfusate and any local decrease below this baseline level must reflect active movement of K + into neurons and/or glia. In this regard it is interesting that glial cell recordings show undershoots which parallel those recorded with the potassium electrode 4°. Further evidence that an active transport process is turned on during stimulation is provided by results from iontophoretic K ~applications. Here again a biphasic effect occurs. At the peak of the orthodromically evoked increase in [K+]0, a K ~ pulse produces a larger increase in [K+]0 than when delivered at baseline level. This probably reflects a relative increase in excitability of the surrounding population which must depolarize further, fire, and add to the [K+]o change produced by the iontophoretic electrode. However, shortly after the peak of the orthodromically evoked [K+]0 rise the same iontophoresis produces smaller increments in [K+]0 than control and this change persists during the undershoot providing evidence for active uptake. Similar findings have been reported in cat neocortex in vivo 10. It also appears from our data that this active process is dependent on the duration of the [K+]0 rise since it seems greater with longer trains of stimulation (Fig. 2). The ceiling levels measured in the slice are similar to those recorded in vivo 1°,24 and appear then to reflect a balance between increased K + uptake and a decreased release occurring in higher [K +] solutions. The increased effectiveness of stimulation following penicillin application seems to be directly related to the capacity of stimuli in the train to evoke multiphasic epileptiform field potentials. Penicillin does not affect resting levels of K + of Ca 2+ or the generation of undershoots. The larger ionic changes which occur during epileptogenesis are presumably related to the generation of bursts in large populations of pyramidal cells. Such bursts have now been shown to depend significantly on calcium entry 4s and would be expected to activate slow potassium currents la. ACKNOWLEDGMENTS This work was supported by N I H Grants NS 06477 and NS 12151 to D.A.P. and a fellowship from the Deutsche Forschungsgemeinschaft (C.B.). We are grateful to Professor W. Simon, Swiss Federal Institute of Technology, Zurich, Switzerland for supplying the calcium neutral carrier and Ms. Cheryl Joo for secretarial assistance.

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