to na and the tail current from to. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20,1M) fully prevented these actions

Transcription

1 Journal of Physiology (1991), 435, pp With 8 figures Printed in Great Britain DEPRESSION OF A SUSTAINED CALCIUM CURRENT BY KAINATE IN RAT HIPPOCAMPAL NEURONES IN VITRO BY ANDREA NISTRI* AND ENRICO CHERUBINIt From INSERM, Unite' 29, 123 Boulevard de Port Royal, Paris, France (Received 26 April 1990) SUMMARY 1. High-threshold, slow inactivating inward Ca2+ currents were studied in CAl pyramidal neurones from rat hippocampal slices using the single-electrode voltage clamp technique. 2. Kainate ( nm) induced a dose-dependent depression of the amplitude of the slow Ca2+ current. At a dose of 200 nm the current amplitude was reduced from to -0O na. Such an effect of kainate was associated with the development of a small inward current ( na). Kynurenic acid (1 mm) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20,1M) fully prevented these actions of kainate. 3. The structurally related kainate analogue a-amino-3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA; 200 nm) depressed the slow Ca21 current by 30±7 %, an effect also blocked by CNQX. 4. In low-na+ medium slow Ca2+ currents were followed by sustained inward tail currents. Kainate reduced both the steady-state Ca21 current (from to na) and the tail current (from to -0' na). 5. The inactivation process of the slow Ca2+ current was tested by a double-pulse protocol and was found to be enhanced by kainate. 6. Equimolar replacement of Ca2+ by Ba2+ produced larger inward currents followed by prolonged tails. Kainate reduced the Ba2+ steady-state current from to na and the tail current from to na. 7. In current clamp experiments Ca2+ action potentials were recorded from cells loaded with the Ca2+ chelator BAPTA. In these conditions kainate failed to reduce the Ca2+ action potential, while in the absence of BAPTA kainate shortened the Ca2+ action potentials by 30 %. 8. It is suggested that low concentrations of kainate reduced the slow Ca2+ current by promoting its inactivation perhaps through a rise in free intracellular Ca2+. * To whom reprint requests should be sent at: Department of Pharmacology, Queen Mary and Westfield College, University of London, London El 4NS. t Present address: Department of Biophysics, S.I.S.S.A., Strada Costiera, Trieste, Italy. MS 8457

2 466 A. NISTRI AND E. CHER UBIVI INTRODUCTION Kainate, an analogue of the transmitter amino acid glutamate, is a potent excitant of hippocampal neurones. In vivo studies have shown that systemic or intracerebral injections of kainate produce convulsive activity originating from the hippocampus and limbic structures, spreading to most cortical brain areas and resulting in neuropathological lesions reminiscent of those found in patients with temporal lobe epilepsy (Ben-Ari, 1985). In vitro electrophysiological studies using micromolar concentrations of this substance have confirmed that kainate directly depolarizes hippocampal neurones (Robinson & Deadwyler, 1981; Westbrook & Lothman, 1983), probably by increasing their membrane permeability to Na' and K+ (Mayer & Westbrook, 1987). Often such an effect of kainate is cytotoxic and produces neuronal death (Monaghan, Bridges & Cotman, 1989). In contrast, binding studies have demonstrated a much higher potency (in the low nanomolar range) of kainate as a specific ligand for two membrane receptor sites on hippocampal cells (Simon, Contrera & Kuhar, 1976; London & Coyle, 1979; Berger, Tremblay, Nitecka & Ben-Ari, 1984). When electrophysiological experiments have used concentrations of kainate only one order of magnitude larger than those found to be effective for binding studies, a more subtle effect consisting of a depression of the spike after-hyperpolarization and its underlying Ca2"-dependent K+ currents was detected (Gho, King, Ben-Ari & Cherubini, 1986; Cherubini, Rovira, Ben-Ari & Nistri, 1990). This effect of kainate has been observed in association with a reduced amplitude and duration of Ca2+ action potentials (Cherubini et al. 1990). This phenomenon is not mimicked by N-methyl-D-aspartate (NMDA), a glutamate analogue acting via a different receptor type, and is not due to a fall in cell input resistance which might have decreased Ca2` spikes simply by making the neuronal membrane more 'leaky'. These findings therefore suggest the existence of a powerful and specific electrophysiological action of kainate on hippocampal neurones, and raise the possibility that clarification of these phenomena might provide some insight into the processes regulating the excitability of these cells. To this end we examined whether low concentrations of kainate might influence voltage-dependent Ca2` currents in hippocampal CAI neurones in vitro. We found that kainate consistently depressed a persistent Ca2` current probably by raising intracellular free Ca2+ which in turn reduced the transmembrane Ca2+ influx. Part of this study has been reported in a preliminary form (Cherubini, Nistri & Ruiz de Le6n, 1989). METHODS Experiments were performed on hippocampal slices obtained from adult male Wistar rats. The technique for preparing the slices has been reported previously (Cherubini et al. 1990). Briefly, transverse 500,um thick hippocampal slices were cut and immediately incubated at room temperature (20-22 C) in artificial cerebrospinal fluid (ACSF) containing (mm): NaCl, 126; KCl, 3 5; CaCl 2 2 NaH 2PO 4'1 1-2; MgCl2.6H ~ 1 3; NaHCO3, 20'3Eulbrtn 25; glucose, II. Equilibrating the ACSF with 95 % 02 and 5 % CO2 gave a ph of The slices were allowed to recover for 1 h before being transferred to a recording chamber in which they were continuously superfused at room temperature with oxygenated ACSF at a rate of ml min'. Intracellular recordings were obtained from hippocampal CAI neurones using microelectrodes filled in most cases with 3 M-CsCl

3 KAINATE ANVD CALCIUTM CURRENTS (resistance MQ). In some experiments microelectrodes contained the Ca2" chelator 1,2-bis(2- aminophenoxy)ethane N,N,N',V'-tetracetic acid (BAPTA, 200 mm dissolved in 3 M-CsCl; ph was adjusted to 7 2 by adding KOH; resistance MDI) and were protected from light. In voltage clamp experiments membrane currents originating from the neuronal soma were recorded via a single-electrode voltage clamp amplifier (Axoclamp 2A), switching between voltage recording and current injection at khz (30 % duty cycle). The voltage signal at the head-stage amplifier was continuously monitored on a separate oscilloscope to ensure correct operation of the voltage clamp system. Responses were digitized and displayed on a Nicolet digital oscilloscope and a computerdriven chart recorder. Ca2+ currents were routinely studied in conditions in which fast Na' currents were abolished by tetrodotoxin (1 /tm) and K+ currents were minimized by intracellular Cs' and extracellular Cs' (2 mm) plus tetraethylammonium (TEA, 10 mm). Ba2+ currents were studied in the same conditions except that the ACSF contained equimolar concentrations of Ba2+ instead of Ca2". Co2" (3 mm) and Cd2+ (0 5 mm) were applied via ACSF from which NaH2PO4 was omitted to prevent precipitation of divalent cations. In some experiments Ca2" currents were recorded in a low-na' ACSF (NaCl was substituted by equimolar concentrations of TEA-Cl, corresponding to 82 % Na' substitution), in the presence of extracellular TTX and Cs': for these tests microelectrodes containing 2 M-potassium methylsulphate or 4 M-potassium acetate (resistance MQ) were also used. Ca21 currents, generated by applying depolarizing test potentials, were elicited every 30 s to minimize frequency-dependent changes in their amplitude. Current-voltage (I-V) plots were constructed by stepping the voltage to various potentials and measuring the current when it had reached a steady value (usually at 1 s). The I-V relation was found to be linear between -40 and -70 mv (this linearity was maintained when potassium methylsulphate or potassium acetate microelectrodes were used). A least-squares routine was fitted to the linear part of the I-V curve, the slope of which was taken to calculate passive 'leak' conductance. Assuming that the leak conductance was time- and voltage-independent, the I-V relation of Ca2+ currents was plotted in most cases after subtracting the observed currents from the extrapolated leak currents at the same level of test potential (chart records of responses are, however, shown without leak subtraction). Drugs were added to the superfusate to give the final concentration stated in the text. Kainate was purchased from Sigma; quisqualate, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were from Tocris; kynurenic acid was a gift of Dr P. L. Herrling (Sandoz, Berne). Data are expressed as means +S.E.M. Statistical analysis was performed with Student's one-tailed or two-tailed t test. 467 RESULTS Stable intracellular recordings were obtained from seventy-four CAI pyramidal neurones which shortly after impalement had a resting membrane potential more negative than -62 mv, action potentials greater than 75 mv and resting input resistance ranging from 28 to 64 MQ. Kainate depressed a high-threshold Ca21 current The classical three types of voltage-dependent calcium channel (Fox, Nowycky & Tsien, 1987) have been found to generate three distinctive currents in rat hippocampal neurones (Blaxter, Carlen & Niesen, 1989; Ozawa, Tsuzuki, lino, Ogura & Kudo, 1989). Since the low- and high-threshold transient currents are mainly of dendritic origin and therefore more difficult to clamp (see Brown & Griffiths, 1983), the present study examined quantitatively the effects of kainate on the highthreshold, slowly inactivating calcium current which can be more effectively clamped in view of its somatic origin (Brown & Griffiths, 1983; Blaxter et al. 1989). Following pharmacological block of Na+ and K+ currents (see Methods) and voltage inactivation of the low-threshold Ca21 current by holding cells at -40 mv (Ozawa et al. 1989), depolarizing steps from this level of membrane potential activated net inward

4 468 A. NISTRI AND E. CHER UBINI currents, which peaked within ms and slowly declined (over 1 s) to a more sustained level (see Fig. 1A). These responses were followed at the end of the pulse by a brief inward tail current. The current-voltage plot of Fig. lb shows that the amplitude of the steady-state current (calculated at the end of a 1 s pulse after A Control Kynurenic acid kainate Kainate ~1 I 1 s -I--- 1 s I 1 na B Potential (mv) L -0.6 Fig. 1. Kainate-induced depression of high-threshold Ca2+ currents, and its antagonism by kynurenic acid. A, upper traces from left to right: inward currents elicited by 30 mv depolarizing voltage steps from a holding potential of -40 mv in control conditions, during application of kainate (200 nm) in the presence of kynurenic acid (1 mm) and during application of kainate 30 min after wash-out of kynurenic acid. Lower traces: inward currents evoked by hyperpolarizing voltage steps of 20 mv from the same holding potential to measure leak conductance. B, I-V relation for the amplitude of the steadystate inward current shown in A (measured at the end of 1 s depolarizing pulses after subtraction of the leak conductance) in control conditions (0), during bath application of kynurenic acid and kainate (A) and during kainate superfusion following wash-out of kynurenic acid (@). subtraction of the passive 'leak' current) peaked at approximately -15 mv and was still inward at + 10 mv. In eighteen cells the peak amplitude of the slow current was na and had an extrapolated reversal potential between + 20 and + 30 mv. Both the fast and slow components of the inward current were apparently carried by Ca2, since they were reversibly abolished in a Ca2+-free solution, or by the c a1) U3

5 KAINATE AND CALCIUM CURRENTS 469 inorganic Ca2+ antagonists Co2+ (3 mm) or Cd21 (0 5 mm). Superfusion with 200 nmkainate depressed (within 2-5 min) both the fast and slow components of the voltage-activated inward current (compare left- and right-hand responses in Fig. 1A; see also Fig. 4A) in 16/18 cells. On average the amplitude of the slow component A - B Kainate (400 nm) C 0.~~~ ~(20) Kainate 4H-402mV 1 > S ~~~1 na 0. 0 ( (4) Kainate a) (400 nhm) Kainate (100 nm) - (4) 75 wcontrot 25 o (2) L~~1 ~130 mv C4 0 VH -40hmV is Kainate (nm) Fig. 2. Dose-related depression of high-thresholdcai2a currents by kainate. A, superimposed inward currents produced by hyperpolarizing and depolarizing voltage steps from a holding potential (VH) of -40 mv in control conditions (curved arrow) and in the presence of 100 nm- (open arrow) or 400 nm- (filled arrow) kainate. B, log dose-effect relation for the depression by kainate of the amplitude of the slow Ca2e current. Numbers in parentheses indicate number of cells. was reduced from - 0' to na (49% reduction: P < ) without apparent shifts in the current-voltage relation (Fig. I B). Figure 2 shows that within the concentration range of nm, kainate elicited a dose-related depression of the maximum amplitude of the slow, inward current. This phenomenon was associated with small and inconsistent changes in cell leak conductance. In fact, out of eighteen cells exposed to kainate in control solution, nine displayed no measurable change in their leak conductance, one a 8 % decrease and the remaining eight an increase of 12-25%. Furthermore, the effects of 200 nm-kainate were accompanied by only small changes in the steady inward current (-,01 ± 0-03 na). In accordance with previous work on hippocampal neurones (Cherubini et al. 1990), these effects of kainate were reversible only slowly as recovery required at least 30 mm of washing. The depression of Ca2+ currents was not due to their gradual rundown as such an effect was not observed in the absence of kainate. In four cells application of the broad-spectrum excitatory amino acid antagonist kynurenic acid (imm; Cherubini et al. 1990) for at least 15 mm prior to and during kainate administration, completely prevented the action of kainate on the Ca2+ currents although the effect of kainate was fully manifested after wash-out of kynurenic acid (Fig. 1A). Kynurenic acid per se did not induce any change in the steady current, leak conductance or amplitude of the inward currents (Fig. IB). In three neurones bath-application of CNQX (20,sm), a preferential antagonist of non-nmda receptors (Watkins, Krogsgaard-Larsen & Honore', 1990), fully prevented the effects of

6 470 A. NISTRI AND E. CHERUBINI 200 nm-kainate on the steady inward current and on the slow Ca2+ current (the latter was % of its control). These data indicate that activation of an excitatory amino acid receptor was necessary to observe the effect of kainate on the Ca 2+ current and that these currents were highly reproducible if the action of kainate was pharmacologically prevented. CNQX CNQX + AMPA CNQX B -50 Potential (mv) C Control AMPA Wash D 50 0otential (mv) 1 na 'c Fig. 3. Reduction of high-threshold Ca2+ current by AMPA, and its antagonism by CNQX. A, inward currents induced by depolarizing steps (+ 30 mv; top traces) or hyperpolarizing steps (-20 mv; bottom traces) from a holding potential of -43 mv in the presence of CNQX (20 lam) or of CNQX plus AMPA (200 nm). B, I-V relation for the same cell in the presence of CNQX alone (A) or following addition of AMPA (A). C, responses recorded from the same neurone after 30 min wash-out of CNQX. Hyperpolarizing steps are now -25 mv. Note depression of the amplitude of the Ca2+ current after 3 min application of AMPA (200 nm). D, I-V relation for the responses recorded during tests as shown in C. 0, control conditions; *, in the presence of AMPA. For further details see legend to Fig. 1. AMPA or quisqualate depressed Ca2+ currents Since some responses to kainate may be mediated by a particular population of non-nmda receptors activated by AMPA (Watkins et al. 1990), the latter agent was tested in the present experiments. AMPA ( nm) was applied to six neurones. At a 200 nm concentration AMPA reduced the amplitude of the slow inward current by % in four cells (see Fig. 3 C and D). This effect was associated with steady inward current of pa; no change in leak conductance was observed in two cells while a 25 % increase was found in the remaining two. Higher concentrations of AMPA induced is

7 KAINATE AND) CALCI.TM ClURRENTS steady inward currents whose negativity exceeded -200 pa and rises in leak conductance of more than 40 % which made it difficult to ascertain any direct effect of AMPA on Ca21 currents. At the concentration of 100 nm, AMPA produced only a 19 % decrease in the slow Ca21 current without changing either the steady inward 471 A Control Kainate B Potential (mv) IlnA / 30 mv s Fig. 4. Kainate depresses the high-threshold Ca2+ currents recorded in low-na' medium. A, inward currents elicited by depolarizing voltage steps of 30 mv from a holding potential of -30 mv. Note the lack of effect of kainate on the leak conductance (measured from hyperpolarizing voltage steps of -20 mv from the same holding p)otential). Recording with potassium acetate-containing microelectrode. B, I-V plot for the amplitude of the steady-state current (after subtraction of the leak conductance) before (0) and durinig (0) superfusion with kainate (200 nm). current or the leak conductance. The action of AMPA was fully developed in 3-5 min and (unlike that of kainate) was readily reversible after 10 min washing. Figure 3 (A and B) shows that application of 20 /am-cnqx fully prevented the effect of 200 nm- AMPA on the slow Ca21 current and on the leak conductance. Similar results were obtained in two other neurones. Quisqualate, a non-nmda receptor agonist structurally related to AMPA, was tested on three cells. At the concentration of 1-2 am, quisqualate reversibly reduced the amplitude of the slow Ca2" current by 26+9 %, increased the steady inward current by pa and changed by less than 10% the leak conductance. Lower concentrations ( nm) of quisqualate were ineffective. Kainate depressed Ca2+ currents recorded in low-na+ solution The kainate-induced depression of Ca2+ currents might have been secondary to: (i) increased permeability to Na+ and K+ via the monovalent cation channels activated by this excitatory amino acid analogue (Mayer & Westbrook, 1987), or (ii) a Na+-Ca2+ exchange mechanism similar to the one found in other excitable cells (Baker & Reuter, 1975). A simple way to explore these issues was to perform

8 472 A. NISTRI AND E. CHER UBINI experiments with reduced extracellular levels of Na+ to minimize any enhancement by kainate of Na+ or K+ permeability. In eight experiments 126 mm of Na+ in the ACSF was replaced by TEA, a procedure which corresponded to 82 % Na+ substitution. In low-na+ solution (containing TTX and Cs+) depolarizing voltage steps from a holding potential of -40 mv activated a high-threshold slow Ca2+ current whose peak amplitude occurred between -10 and 0 mv (Fig. 4B) and was on average na (n = 8), a value significantly different (P < 0'01) from similar responses in control ACSF. The estimated reversal potential of the slow current was similar to the control one (about + 20 mv: Fig. 4B). The Ca2+ currents recorded in low-na+ media clearly differed from control ones in displaying a more conspicuous inward tail current (Fig. 4A). In order to examine whether the tail currents were due to activation of a Ca2+_ dependent Cl- current (Owen, Segal & Barker, 1984), the reversal of the tail current was investigated in three cells recorded with microelectrodes filled with either potassium methylsulphate or potassium acetate. Figure 5 shows that, when varying voltage command pulses were applied immediately after the end of a 30 mv test pulse used to elicit the inward current, the value of the tail reversal potential appeared to be similar to the one for the steady-state slow current measured at the end of the pulse (compare open circle plots in Figs 4B and 5B). In Na+-deficient media kainate reduced the voltage-sensitive Ca2+ currents (Fig. 4A) and their inward tails (see also Fig. 5A). This effect was not accompanied by changes in the reversal potential for the slow current (Fig. 4B) or its tail (Fig. 5B). On average the maximal amplitude of the slow current in the presence of kainate was na (38 % less than control in low-na+ solution; P < 0-005). The tail current amplitude (measured 1 s after the end of a + 30 mv voltage command pulse from holding potential of -40 mv) was na, a value significantly smaller (P < 0-01) than its control ( na) before kainate application. These reductions in the inward current responses were associated with only slight changes in the steady current: in fact, in four cells there was an actual decrease in the inward steady current (range pa) while in three neurones the inward steady current increased by -50 pa. Changes in leak conductance following kainate administration mainly consisted of decreases (8-50 % reductions) although in two neurones an increase (15-25 % rise) was observed. The persistence of kainate-induced reductions in Ca2+ currents in spite of very low extracellular Na+ levels, a rather high concentration of the K+ channel blocker TEA and the use of microelectrodes which should not have altered the intracellular Clconcentration, indicated that such an effect of kainate was a direct one and not mediated via changes in Na+, K+ or Cl- permeabilities. In the absence of obvious alterations in the Ca2+ current reversal potential (Figs 1, 4 and 5) consistent with large increases in intracellular Ca2, it seemed possible that the action of kainate on the Ca2+ current was more subtle and perhaps involved the current inactivation mechanism which itself is known to be dependent on intracellular Ca2+ (Eckert & Chad, 1984; Nistri & Cherubini, 1990). In order to test this hypothesis, the Ca2+ current inactivation was next investigated.

9 KAINATE AND CALCIUM CURRENTS 473 Kainate enhanced Ca2+ current inactivation A standard two-pulse protocol was used to study inactivation of the Ca2+ current (Eckert & Chad, 1984). This consisted of applying a pre-pulse of varying amplitude (P1 in Fig. 6) to 'condition' the Ca2+ current evoked (after a brief interval of 30 ms) A Control Kainate P z <1I- 11 na 1 s B Potential (mv) n//0-o C Fig. 5. Kainate depressed steady-state inward current and its tail. A, inward tail currents recorded during 3 s voltage command steps (P2) to -10 mv (lower traces) and to +10 mv (upper traces) following a fixed conditioning pre-pulse (PI) to 0 mv from a holding potential of -30 mv, in control conditions and during superfusion with kainate (400 nm). B, I-V plot of the amplitude of the inward tail currents measured 2 s after the end of the conditioning pre-pulse shown in A, in control conditions (0), and during bath application of kainate (200 nm, *; and 400 nm, [1). Recording with potassium acetatecontaining microelectrode. by a test pulse (P2 in Fig. 6) of constant amplitude (30 mv) to produce a maximal inward current. The duration of PI was fixed at 300 ms which was long enough to activate the Ca2+ current but not unduly so to produce ionic shifts. In analogy with recent studies (Nistri & Cherubini, 1990), the slow Ca2+ current was found to undergo inactivation (45 % of control current value) which was maximal following a pre-pulse to + 10 mv (Fig. 6B); beyond this value the inactivation started declining so that the curve looked sigmoidal. Kainate (200 nm) reduced the amplitude of the Ca2+ current and increased its inactivation (Fig. 6A). Figure 6B shows that the inactivation curve was shifted downwards by kainate, so that a pre-pulse to + 10 mv now elicited a much stronger reduction in the slow current (21 % of the control amplitude without pre-pulse).

10 474 A. NISTRI AND K CHER UBINI The issue of Ca2" current inactivation in the presence of kainate was further investigated by using Ba21 (instead of Ca2+) as the charge carrier, since the Ba21 currents are known to display minimal inactivation (Hagiwara & Byerly, 1981; Eckert & Chad, 1984). A Control Kainate <f-1 4-1~~~~~~~~~1 na B P2 1 (5) (7) (2) 0 0 (7) 6~~~~~ (3 (6)) (6) (6) t E (6) 0 (6) t(4) P1 is 130 mv Pre-pulse potential (mv) Fig. 6. Kainate enhanced Ca2+ current inactivation. A, inward currents recorded during 1 s voltage command step (P2) to -10 mv from a holding potential of -40 mv before (left) and after conditioning pre-pulses (P1) to -20 mv (middle) and to 0 mv (right). Bottom pair of tracing shows responses to similar voltage protocol in the presence of kainate (200 nm). B, mean values of the steady-state current (measured just before the end of P2 pulse and normalized to the maximum inward current recorded without PI pulse) are plotted against pre-pulse potentials (PI) before (0) and during (@) superfusion with kainate. Values in parentheses refer to number of cells. Bars are S.E.M. Holding potential -40 mv. PI was to potentials indicated on abscissa. P2 was to -10 mv. Kainate depressed Ba2+ currents It has recently been observed that after equimolar replacement of Ca2+ by Ba2+, voltage-activated Ba2+ inward currents (comprising an initial fast component decaying to a lower amplitude slow phase) can be elicited in hippocampal cells (Nistri

11 KAILVATE AND CALCIUM CUIRRENTS & Cherubini, 1990; see also Fig. 7). Although the peak amplitude of these currents is greater than the control Ca2+ ones (cf. Fig. 7 C), they have a similar threshold and reversal potential to the standard Ca2" currents. One distinctive feature of the Ba2+ current is a long-lasting inward tail which peaks and reverses at a similar level like 475 A Kainate A~ ~ ~ ~ ~ ~ ~ ~ m fil--xl-k H < 7 X < ~~~~~~~~~~1 na +1.5 min +5 min _!-_X10,<l30mv 1 r B Potential (mv) -20 mv +20 mv -30 mv +30 mv C / jm~~~~~~~1na _ ' C A -3 ^^t~~~~~~~~~~~- Fig. 7. Kainate depressed Baa2+ inward currents. A, tracing of inward currents (top) elicited every 30 s by 2 s depolarizing and hyperpolarizing voltage steps (bottom) of 30 mv from a holding potential of -50 mv, before, during (filled bar) and after superfusion with kainate (200 nm). Note that kainate reduced both the voltage-dependent inward current and the tail current, but had no effect on leak conductance. B, superimposed inward currents evoked in another cell by 1 s hyperpolarizing and depolarizing voltage steps from a holding potential of -50 mv in the absence and in the presence (arrows) of kainate (200 nm). C, I-V plot of the amplitude of the steady-state current for the cell shown in B before (0) and during (@) kainate superfusion. No leak subtraction was performed. the slow Ba2+ current, an observation which suggests that both the sustained and the tail current are generated by a similar mechanism (Nistri & Cherubini, 1990). Figure 7A shows that kainate (200 nm) reduced the Ba2+ current with little change in the inward leak current produced by hyperpolarizing test pulses. Figure 7B shows that superimposition of Ba2+ currents and leak responses before and after application of kainate revealed not only a depression of the Ba2+ current but also a strong decrease in its tail response. The current-voltage plot of Fig. 7 C indicates that the threshold for the slow Ba2+ current was not altered by kainate, whereas the most obvious change was a reduction in the amplitude of the slow current at various test potentials. Mean data from five cells demonstrated that the arnplitude of the slow Ba21 current ( na) was significantly depressed (20%) by kainate ( ; P < 001). The tail current amplitude (measured 1 s after the end of

12 476 A. NISTRI AND E. CHER UBIMI the pulse) was also changed from to na (P < 005). These effects were usually accompanied by a small increase in the steady inward current (-70 to pa) although in one cell the steady inward current actually decreased by 70 pa. In the same group of cells, three did not display changes in their leak conductance following kainate application (see Fig. 7) while two showed a 25 and 30 % decrease respectively. Kainate did not depress Ca2+ responses of BAPTA-loaded cells Hippocampal pyramidal cells injected with the Ca2+ buffer BAPTA display a much reduced inactivation of their slow Ca2+ current (Nistri & Cherubini, 1990). It was therefore deemed of interest to explore whether kainate might still affect Ca2` currents of BAPTA-injected neurones. Since BAPTA- (0 2 M in 3 M-CsCl) containing electrodes often had poor current-passing properties which made it difficult to hold the cell membrane for a sufficiently long period to test the action of kainate, the effects of kainate on Ca2+ action potentials were studied under discontinuous current clamp conditions. Within min after impalement with BAPTA- and Cs+containing electrodes and in the presence of extracellularly applied TTX, TEA and Cs+, a 50 ms depolarizing pulse (0-05 Hz; 0 8 na) evoked a fast, all-or-none depolarization followed by a plateau potential (Fig. 8A). The latter lasted for several hundreds of milliseconds and did not abruptly return to the baseline but displayed a shallow delayed depolarization. In the example of Fig. 8A kainate (200 nm) depolarized the cell membrane by 8 mv (on average by 12+1 mv from -50 to -65 mv resting potential; n = 4); the cell was therefore repolarized back to -65 mv by steady current injection. Figure 8A shows that at the same level of membrane potential as in the control condition, the Ca2+ spike was not reduced during the application of kainate and was followed by a very long depolarizing tail (the baseline potential was reattained 13 s later). No obvious change in input conductance (estimated from hyperpolarizing electrotonic potentials preceding the spikes) was found. For a quantitative measurement of the effect of kainate on the Ca2+ action potential, the spike width was measured at 50 % of its amplitude. In four neurones the width of the Ca2" spike was ms in control conditions, a value not significantly different from the one ( ms) found in the presence of kainate (only 2-5 % change in spike peak amplitude from the same level of control resting potential was observed). In the presence of extracellularly applied TTX, TEA and Cs+ seven neurones were impaled with standard CsCl-filled microelectrodes. In these cells kainate reduced the width of the Ca2+ spike from to ms (30 % reduction; P < 0 005; see also Fig. 8B). In accordance with a previous study on hippocampal cells (Cherubini et al. 1990) this action of kainate was associated with an average depolarization of 7 +2 mv (from -50 to -60 mv resting potential), corrected by injecting hyperpolarizing current to repolarize the cells to their initial level. In 5/7 cells there was virtually no change in input conductance while in the remaining two there was a 20 % rise.

13 KAINATE AND CALCIUM CURRENTS 477 A Control Kainate Wash (15 min) -65 Km5 mv 1Imv a X m m_ 10.5 na 1 s B Control Kainate Wash (9 min) -56 mv K 140 mv 10.2 na 1 s Fig. 8. Kainate did not reduce Ca2+ action potentials in cells loaded with BAPTA. Electrotonic potentials and Ca2+ action potentials evoked by injection of a constant outward or inward current through the recording electrode in control conditions during and after superfusion with kainate (200 nm). A, note that the delayed depolarization, which followed the action potential, was reversibly prolonged by kainate in the absence of any effect on spike width or cell input resistance. During the application of kainate the cell membrane potential was repolarized to its initial control level. B, in the absence of intracellularly applied BAPTA kainate reversibly reduced the width of the Ca2+ action potential. Note diferent time calibration in A and B. DISCUSSION Reduction of a slow Ca21 current by kainate The principal finding of the present investigation on hippocampal neurones is the unexpected depression of a voltage-dependent inward current by low concentrations of kainate. Since this current was blocked by Co2+, Cd2+ or a Ca2+-free solution and could be recorded when Ba2+ replaced Ca2, there is little doubt that it was generated by activation of voltage-sensitive Ca2+ channels. On the basis of its threshold, peak value and time course this inward current might be attributed to activation of highthreshold L-type channels (Fox et al. 1987) which are known to be present on hippocampal cells (Kay & Wong, 1987; Blaxter et al. 1989; Ozawa et al. 1989). Since the slow Ca2+ current has a perisomatic origin (Brown & Griffiths, 1983; Kay & Wong, 1987; Blaxter et al. 1989), it was possible to voltage clamp it adequately with a single-electrode clamp, whereas the preceding inward 'spike-like' current was probably less effectively clamped (Brown & Griffiths, 1983) and was not used for data analysis.

14 478 A. NISTRI AND E. CHERUBINI Several characteristics of the action of kainate on the Ca21 current suggest that it was a powerful phenomenon mediated by activation of an excitatory amino acid receptor blocked by the antagonists kynurenic acid or CNQX. Nevertheless, the rather small amplitude of the steady inward current together with the inconsistent changes in input resistance observed during application of kainate suggest that activation of kainate receptors led to only minimal increases in membrane permeability. This finding is clearly different from the dramatic fall in cell resistance through a large rise in Na+ and K+ permeability induced by micromolar concentrations of kainate (Constanti, Connor, Galvan & Nistri, 1980; Mayer & Westbrook, 1984). As the action of kainate on hippocampal neurones was fully preserved in Na+-deficient media containing a large amount of TEA, the decrease in Ca2+ current produced by kainate could not be a mere epiphenomenon against the background of a conventional rise in cell permeability to Na+ and K+ and no correlation with leak conductance changes was found. The question then arises as to the identity of the receptor systems operating under these conditions. Comparison of the action of kainate with AMPA and quisqualate Since the depressant action of kainate on the slow Ca2" current was blocked by CNQX and mimicked by AMPA or quisqualate, it appears that non-nmda receptors (Watkins et al. 1990) were mediating this phenomenon. While non-nmda receptors are thought to be heterogenous, it is difficult with pharmacological experiments to distinguish kainate receptors from other subtypes (Watkins et al. 1990). Nevertheless, a population of kainate receptors binding AMPA is localized on hippocampal CAI neurones (Monaghan et al. 1989) and might be responsible for the effects observed in the present study. AMPA was almost equipotent with kainate in reducing Ca2+ currents, although its effects were more readily reversed on wash-out. Quisqualate was clearly less active than kainate or AMPA since micromolar concentrations were needed to elicit comparable effects. Since quisqualate possesses only moderate affinity for the kainate receptor (Watkins et al. 1990), the present study further supports the concept of a discrete class of AMPA/kainate receptors involved in the control of Ca2+ currents. Distinct 'metabotrophic' receptors sensitive to quisqualate control the activity of intracellular second messengers and, consequently, cellular excitability (Sugiyama, Ito & Hirono, 1987). In hippocampal neurones quisqualate-induced activation of such receptors can depress Ca2+ currents (Lester & Jahr, 1990). This phenomenon is characterized by its insensitivity to CNQX, kainate or AMPA, by its disappearance in a solution in which Ba2+ replaces Ca2+ and by its persistence following intracellular application of BAPTA. Such properties are clearly different from those observed in the present study and make it very unlikely that a 'metabotrophic' receptor system highly sensitive to quisqualate was responsible for the effects reported in the present investigation. Possible mechanisms responsible for Ca2+ current depression Kainate could have reduced the Ca2+ current amplitude simply by blocking Ca21 channels but this interpretation is not supported by the experiments in the presence

15 KAINATE AND CALCIUM CURRENTS of intracellular BAPTA, a Ca2+ chelator. In fact, when intracellular Ca21 was buffered, kainate lost its ability to block Ca2+ responses. Furthermore, studies with the Ca2+ indicator Fura-2 have failed to show any block by kainate of voltagedependent Ca2+ channels (Murphy & Miller, 1989). Another possibility is that kainate might have reduced the operation of a Na+-Ca2+ membrane exchange (Baker & Reuter, 1975) and thus changed Ca2+ compartmentation and fluxes. Should this be the case, reversing the direction of the exchange mechanism by lowering extracellular Na+ ought to abolish the kainateinduced depression of the Ca21 current: this phenomenon was, however, not detected in the present study. The data from experiments in low extracellular Na+ also help to discount other explanations for the effect of kainate. In particular, the larger amplitude and the longer tail of Ca2+ currents recorded in Na+-deficient media probably reflected the nearly complete pharmacological block of K+ currents by the very high concentration of TEA replacing Na+. Hence, it is unlikely that kainate appeared to block Ca2+ currents as a consequence of its co-activation of outward K+ currents. The lack of outward tail currents together with the similar reversal potential for the slow inward current and its tail further suggest that the measured responses were not obviously 'contaminated' by persistent K+ currents and actually represented sustained activation of Ca2+ conductances. As recordings with microelectrodes containing different anions made no difference to the observed phenomena, it also appears that Cl- had a negligible role in these effects. Finally, the absence of gross alterations in the Ca2+ current reversal level during application of kainate (Figs 4 and 5) indicates that this excitatory amino acid agonist did not simply act by changing the driving force for Ca2+. Enhancement of Ca2+ current inactivation by kainate In hippocampal neurones in culture (Meyers & Barker, 1989) or in brain slices (Nistri & Cherubini, 1990) one important factor regulating inactivation of the Ca2+ current is the level of intracellular Ca2+ itself. The theory of Ca2+-dependent inactivation (for an extensive review see Eckert & Chad, 1984) predicts saturable (sigmoidal) inactivation curves (as found in Fig. 6), and minimal inactivation when either Ba2+ replaces Ca2+ or intracellular Ca2+ is buffered. In the present study kainate significantly increased current inactivation as well as prolonged the Ca`+ spike of BAPTA-injected cells: both observations are compatible with the view that kainate promoted Ca2+ channel inactivation perhaps by increasing Ca2+ influx and thus reducing the slow Ca2+ current. Nevertheless, as kainate also depressed Ba2+ currents in the absence of extracellular Ca2` and as Ba2+ itself does not turn on the inactivation mechanism (Hagiwara & Byerly, 1981; Eckert & Chad, 1984), we are led to speculate that kainate was able to release intracellular Ca2+ from subcellular stores rather than promote its trasmembrane influx. This effect might manifest itself as 'inactivation' of a Ba2+ current but might be caused by a rise in intracellular free Ca2. Mechanistically one might envisage that intracellular Ca2+ binds to a site responsible for triggering channel inactivation and that such a site might have a much lower affinity for Ba2+ (Tillotson & Gorman, 1983; Eckert & Chad, 1984). The observation that kainate reduced Ba2+ currents less than Ca2+ currents supports this 479

16 480 A. NISTRI AND E. CHERUBINI view, although more direct tests on the ability of low concentrations of kainate to release intracellular Ca2+ are required to substantiate this notion. Possible significance of kainate-evoked depression of Ca2+ current The very large depolarization of hippocampal neurones by micromolar amounts of kainate is probably relevant to the understanding of the neurotoxic action of this substance (Ben-Ari, 1985; Monaghan et al. 1989). Much lower concentrations of kainate have a more subtle excitatory effect characterized by facilitation of neuronal firing with only slight changes in membrane potential or conductance (Cherubini et al. 1990). Against this background of enhanced excitability the present investigation describes what at first might seem a paradox, i.e. a kainate-induced reduction in Ca2+ inward currents, a phenomenon expected to decrease neuronal excitability. It should, however, be considered that this effect of kainate is also associated with an attenuation of the Ca2+-dependent K+ currents and of the spike after-hyperpolarization (Gho et al. 1986; Cherubini et al. 1990). Perhaps through a combination of Ca21 channel inactivation and intracellular Ca 2+ increases elicited by kainate, variations in Ca2+ availability at discrete sites for K+ channel activation may impair the operation of this important inhibitory conductance system which normally limits cell excitability. This work is supported by an Alliance grant from the British Council and INSERM. REFERENCES BAKER, P. F. & REUTER, H. (1975). Calcium Movement in Excitable Cells, pp Pergamon Press, Oxford. BEN-ARi, Y. (1985). Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, BERGER, M. L., TREMBLAY, E., NITECKA, L. & BEN-ARI, Y. (1984). Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system. Neuroscience 13, BLAXTER, T. J., CARLEN, P. L. & NIESEN, C. (1989). Pharmacological and anatomical separation of calcium currents in rat dentate granule neurones in vitro. Journal of Physiology 412, BROWN, D. A. & GRIFFITHS, W. H. (1983). Persistent slow inward calcium current in voltageclamped hippocampal neurones of the guinea-pig. Journal of Physiology 337, CHERUBINI, E., NISTRI, A. & RuIZ DE LEON, 0. (1989). Kainate depresses a persistent Ca2+ current of rat hippocampal neurones in vitro. Journal of Physiology 415, 38P. CHERUBINI, E., ROVIRA, C., BEN-ARI, Y. & NISTRI, A. (1990). Effects of kainate on the excitability of rat hippocampal neurones. Epilepsy Research 5, CONSTANTI, A., CONNOR, J. D., GALVAN, M. & NISTRI, A. (1980). Intracellularly-recorded effects of glutamate and aspartate on neurones in the guinea-pig olfactory cortex slice. Brain Research 195, ECKERT, R. & CHAD, J. E. (1984). Inactivation of Ca channels. Progress in Biophysics and Molecular Biology 44, Fox, A. P., NOWYCKY, M. C. & TSIEN, R. W. (1987). Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones. Journal of Physiology 394, GHO, M., KING, A. E., BEN-ARI, Y. & CHERUBINI, E. (1986). Kainate reduces two voltagedependent potassium conductances in rat hippocampal neurons in vitro. Brain Research 385, HAGIWARA, S. & BYERLY, L. (1981). Calcium channel. Annual Review of Neuroscience 4,

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