Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 123-127, January 1992 Neurobiology Intracellular injection of Ca2+ chelators blocks induction of long-term depression in rat visual cortex (/slices/synaptic plasticity) SUSANNE BROCHER, ALAIN ARTOLA*, AND WOLF SINGER Max-Planck-Institut ffir Hirnforschung, Deutschordenstrasse 46, 6000 Frankfurt 71, Federal Republic of Germany Communicated by Leon N Cooper, October 3, 1991

ABSTRACT In a variety of brain structures repetitive METHODS activation of synaptic connections can to long-term po- tentiation (LTP) or long-term depression (LTD) of synaptic Slices (350 ,.m) of the visual cortex of adult rats were prepared by standard techniques (19) and were allowed to transmission, and these modifications are held responsible for recover at room temperature for at least 1 h. A single slice memory formation. Here we examine the role of postsynaptic was then transferred to the recording chamber where it was Ca2+ concentration in the induction of LTD in the neocortex. held completely submerged. The slice was maintained at In layer HI cells of the rat visual cortex, LTD can be induced 290C-300C and continuously perfused with a solution con- by tetanic stimulation of afferent fibers ascending from the taining 124 mM NaCl, 5 mM KCl, 1.25 mM NaHPO4, 2 mM white matter. We show that LTD induction is reliably blocked MgSO4, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM D-glu- by intracellular injection of either EGTA or BAPTA [bis(2- cose, saturated with 95% 02/5% CO2. aminophenoxy)-N,N,N',N'-tetraacetate], two different Intracellular recordings were obtained with 3 M potassium Ca2+ chelators. This confirms that the processes underlying the acetate-filled electrodes (80-120 MW) from regular spiking induction of LTD in neocortex are located postsynaptically and cells in layer III (20). Only cells with resting membrane indicates that they depend on intracellular Ca2+ concentration. potentials more negative than -69 mV were used. To exam- Thus, both LTP and LTD induction appear to involve calcium- ine the role of postsynaptic Ca2+ concentration in LTD, we mediated processes in the postsynaptic neuron. We propose injected neurons with high concentrations of one of the two that LTD is caused by a surge of calcium either through Ca2+ chelators EGTA or BAPTA. Intracellular electrodes voltage-gated Ca2+ conductances and/or by transmitter- containing either 500 mM EGTA or 100 mM BAPTA and 3 M induced release of calcium from intracellular stores. potassium acetate were used. For injection, hyperpolarizing current pulses (1.5 nA, 0.5 s, 0.5 Hz) were applied for 10-30 Long-term potentiation (LTP) of synaptic transmission has be- min; an additional 30 min was allowed to elapse between come a classical model for investigation of use-dependent syn- injection and LTD induction to ensure diffusion of the aptic plasticity and is considered a substrate of learning and chelators to the dendrites. The efficacy of the Ca2+ chelators memory (1, 2). More recently, evidence has been obtained in the was assessed by measuring the amplitude changes of the cerebellum (3), hippocampus (4), and neocortex (5, 6) that afterhyperpolarizing potentials (AHPs) following current- synaptic transmission can also undergo long-term depression induced depolarizing steps (500 ms). According to Schwindt (LTD) and theoretical considerations have demonstrated the et al. (21, 22), two of these are Ca2' dependent and mediated usefulness of LTD in learning processes (7, 8). by K+ conductances. The first has a very fast rise time and The biochemical cascades that lead to the associative form a short duration. The second has a slower rise time and a of LTP in cortical structures appear to be triggered by a surge longer duration (see legend to Fig. 1A). In addition, there is of free Ca2+ in the postsynaptic neuron (9-11) after a very slowly decaying AHP (or slow AHP) of small ampli- activation of N-methyl-D-aspartate (NMDA) receptor-gated tude, which is Ca2+ independent (22). The early AHP, which Ca2+ conductances (12, 13) and voltage-dependent Ca2+ is contingent with the offset of the current pulse, is thus channels (14, 15). In the visual cortex, evidence has been mainly due to Ca2+-dependent conductances. Hence, we obtained that LTD induction also depends on postsynaptic measured changes in the amplitude ofthis early AHP in order mechanisms. It requires a critical level of postsynaptic de- to assess the efficacy of Ca2+ chelators. polarization but unlike associative LTP does not depend on Synaptic responses were elicited by electrical stimulation NMDA receptor activation (5). This excludes Ca2+ ions through bipolar tungsten electrodes that were placed in the entering through NMDA receptor-gated channels as messen- underlying white matter (w.m.) and intracortically (i.c.) in gers for translation of electrical activity into chemical pro- layer II adjacent to the recording site. The test stimuli cesses. However, Ca2+ entry remains an attractive possibil- consisted of 200-pus pulses whose intensity was adjusted to ity because recent evidence suggests that in the cerebellum 50-70o of the intensity required for the elicitation of spikes. LTD induction depends on an increase of intracellular Ca2+ The tetanus for LTD induction was applied through the w.m. (16). Changes of intracellular Ca2+ could result from activa- electrode and consisted of five 2-s-long trains (50 Hz) that tion of voltage-dependent Ca2+ channels (for review, see ref. were delivered at 10-s intervals. The intensity of this tetanic 17) or of metabotropic quisqualate receptors (18). As a direct stimulus was set above threshold for spike generation in test of this hypothesis, we examined whether LTD induction order to reach the threshold for LTD induction (5). The in slices of the rat visual cortex is influenced by intracellular intensity required for LTD induction depended on the tip injection of the Ca2+ chelators EGTA or bis(2-aminophen- separation of the bipolar stimulation electrode. In the series oxy)ethane-N,N,N',N'-tetraacetate (BAPTA). Abbreviations: LTD, long-term depression; LTP, long-term potentia- tion; AHP, afterhyperpolarizing potential; PSP, postsynaptic potential; The publication costs of this article were defrayed in part by page charge w.m., white matter; i.c., intracortical; BAPTA, bis(2-aminophen- payment. This article must therefore be hereby marked "advertisement" oxy)ethane-N,N,N',N'-tetraacetate; NMDA, N-methyl-i -aspartate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

123 Downloaded by guest on September 29, 2021 124 Neurobiology: Brocher et al. Proc. Natl. Acad. Sci. USA 89 (1992) of experiments with EGTA, tip separation was 350 ,um and RESULTS stimulation intensity required for reliable LTD induction was In neocortical neurons, brief depolarizing current pulses that 15 V, corresponding to =3 times the threshold intensity. In lead to sustained discharges are followed by long-lasting the experiments with BAPTA, tip separation was 270 Am and AHPs (Fig. 1A). The early AHPs are due to Ca2'-activated intensity for reliable LTD induction was 12 V, corresponding K+ conductances (21-24) and can therefore be used to assess to -7.5 times the threshold intensity. This reflects the the efficacy of the Ca2` chelators. Since AHPs increase with different abilities of the stimulation electrodes to recruit the membrane depolarization and firing rate (Fig. 1A and ref. 22), number of excitatory afferents required to reach the coop- they were measured at a membrane potential 20-30 mV erativity threshold for the induction of LTD. above the resting membrane potential and after current In all experiments, the stability of the synaptic responses pulses evoking high-frequency discharges. Both Ca2" chela- to test stimuli applied at 0.03-0.06 Hz was checked for 10-30 tors EGTA and BAPTA effectively blocked these early AHPs min prior to application of the tetanus. All measurements (Fig. 1B; see also Fig. 3A). This blockade lasted throughout were performed on averages of five consecutive responses the recording period (>60 min). The slow Ca2+-independent that had been digitized at a rate of 5 kHz. To assess AHP remained visible in the majority ofthe cells treated with posttetanic response modifications, we measured the ampli- BAPTA (see Fig. 3A) but was suppressed in cells treated with tude of the postsynaptic potential (PSP) at the peak (8-10 ms EGTA. We attribute this to nonspecific side effects ofEGTA. poststimulus) and expressed these amplitudes as percentages In none of the cases did injection of Ca2" chelators have any of control (value of the averaged amplitude during the whole detectable effects on the resting membrane potential or on control period) obtained before tetanic stimulation. The input resistance, nor did the injected cells differ in these time-dependent amplitude changes shown in the figures and variables from the cells that served as controls [resting the values given in the text have been obtained by averaging membrane potential: control group, -73.2 ± 1.8 mV (n = 17); the results of all the cells in each group. They are expressed injected group, -74.3 ± 1.0 mV (n = 13); input membrane as mean ± SEM. Values given in the text are those measured resistance: control group, 47.7 ± 4.0 Mfl (n = 17); injected 20 min after tetanus. Student's t test or Wilcoxon's U test was group, 42.3 ± 6.3 Mfl (n = 13)]. The Ca2+ chelators also had performed to compare mean values. no significant effect on the peak amplitude ofPSPs elicited by

1 2 3 4 5 6

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FIG. 1. Effects of Ca2+ chelators on AHPs (A and B) and on PSPs (C). (A) Responses of a cell to brief (500 ms) depolarizing current (current clamp mode) pulses (1-6: 0.2, 0.4, 0.6, 0.6, 0.4, and 0.2 nA) at resting membrane potential (pulses 1-3, -76 mV) and at -50 mV (pulses 4-6, depolarization maintained by continuous current injection). At resting membrane potential, there is only a slow, low-amplitude AHP and only after the strongest current pulse (pulse 3). At depolarized membrane potential, an additional fast-rising AHP appears in response to a current pulse of the same intensity (compare pulses 3 and 4), the amplitude of which decreases with decreasing intensity of the current pulse and with decreasing spike frequency (compare pulses 4, 5, and 6). At lower pulse intensities (pulses 5 and 6), the early AHP can be seen to dissociate in two components with different rise times, a fast AHP that is contingent with the offset of the current pulse (arrow in pulse 6) and a more slowly rising AHP. (B) Responses to depolarizing current pulses (+0.3 nA; 500 ms) measured before and 5 and 10 min after EGTA injection in another cell. For these measurements, the cell has been depolarized by +20 mV above resting membrane potential (Vr = -85 mV) to enhance the AHPs. After EGTA injection, the AHPs are partially blocked after 5 min and totally abolished after 10 min. In this cell, there was no detectable slow Ca2'-independent AHP. (C) Averaged (n = 5) synaptic responses to w.m. stimulation obtained before (i) and 20 min after (ii) injec- tion of EGTA are superimposed (iii). Note the slight decrease of the slope of the repolarizing phase of the PSP after EGTA injection (arrow). Downloaded by guest on September 29, 2021 Neurobiology: Brocher et al. Proc. Natl. Acad. Sci. USA 89 (1992) 125

w.m. stimulation. However, in some cells (4 of 13) the slope pretetanic controls (Fig. 2B and C). This value does not differ ofthe repolarizing phase ofthe PSP was slightly reduced after significantly from the pretetanic controls but it is significantly Ca2+ (Fig. 1C). This effect is probably due to a different (P < 0.001) from that measured in cells not treated suppression of Ca2+-dependent processes contributing to the with EGTA. As in the untreated cells, there were no modi- repolarization of the PSP, such as Ca2+-activated K+ cur- fications of responses to i.c. stimulation. rents (25) or Ca2+-dependent inactivation of Ca2l currents In a second series of experiments, we compared the effect (26). of tetanic stimulation in control cells and in cells filled with In a first series of experiments, the effect of tetanic BAPTA. In these experiments a different w.m. electrode was stimulation was compared in control cells and in cells filled used. As in the first series, homosynaptic LTD was consis- with EGTA. As described (5), high-intensity tetanic stimu- tently induced in the control cells by high-intensity tetanic lation of w.m. consistently induced homosynaptic LTD in stimulation of w.m. (70.2% +± 11.8%; n = 4). By contrast, no cells not treated with EGTA. Twenty minutes after tetanic depression was observed in five cells in which BAPTA had stimulation, the peak amplitude ofthe excitatory PSP evoked led to a complete blockade of the early AHPs (Fig. 3 A and from w.m. had dropped to 67.2% ± 2.6% of the pretetanic C). As in the previous series of experiments, the posttetanic controls (n = 13) and this depression was associated with a PSP amplitude (98.7% ± 5.1%) did not differ significantly decrease in the initial slope of the PSP (Fig. 2A). As shown from the pretetanic controls but it was significantly different in Fig. 2C, the depression was confined to the tetanized (P < 0.01) from that measured in cells not treated with afferents. Control responses evoked from the i.c. electrode BAPTA. remained unchanged. By contrast, in cells injected with These results indicate that effective buffering of intracel- EGTA (n = 8), tetanic stimulation of w.m. failed to induce lular free Ca2+ prevents LTD induction. As two recent lasting changes in PSP amplitude. After a transient post- studies (27, 28) report that LTD can be successfully induced tetanic depression, which lasted between 5 and 10 min, in cortical cells injected with EGTA or BAPTA, we per- response amplitudes recovered to 98.8% ± 2.5% of the formed two additional experiments attempting to resolve this discrepancy. In the studies that showed LTD in the presence A i ii iii of Ca2W chelators, the effectiveness of the injection proce- dure was assessed by measuring the level of spike adaptation during a depolarizing current pulse and the tetanus was applied before complete suppression of spike adaptation (see figure 2 ofref. 27). This suggested the possibility that, in these experiments, buffering of intracellular Ca2+ may have been less complete than in our study. To test this hypothesis, I tetanic stimulation was applied in two BAPTA-treated cells before early AHPs were completely blocked. Despite the fact that BAPTA had already caused a near complete blockade of B: the early AHPs (Fig. 3B), the tetanus induced a marked LTD (76.6% ± 3.0%; n = 2) that was indistinguishable from that obtained in nontreated cells (Fig. 3 B and C). To exclude the possibility that prolonged current injection I--- affected the susceptibility to undergo LTD, the injection E I protocol was repeated in five cells with electrodes containing 1! N only potassium acetate. In all five cells, the AHPs were and LTD was readily induced (data not shown). 20 ms unchanged C DISCUSSION 120 Tetanus The present results show that intracellular injection of either EGTA or BAPTA blocks the induction of LTD in the o 100 neocortex. It is unlikely that Ca2' chelators prevented LTD the to the tetanus. They had no 80 by reducing response 0 significant effect on membrane potential and membrane (A T rs~~~~~~~~~~~~~~ 0 60 resistance and never attenuated the synaptic response. On - 60 the contrary, in some cells they prolonged the decay of the PSP and in all cells they enhanced the depolarization caused by the tetanus, as can be expected from the blockade of K+ 20 conductances. The fact that the blockade of LTD was ob- tained with two different Ca2+ chelators and depended crit- -10 -5 0 5 10 15 20 25 30 Time, min ically on the complete suppression of the early Ca2+- dependent AHPs suggests that the suppression of LTD was FIG. 2. Effect of EGTA injection on induction of LTD. (A and B) due to the specific Ca2+-chelating action of EGTA and Averaged (n = 5) responses to w.m. stimulation recorded before (i) BAPTA. Thus, our data indicate that the occurrence of LTD and 20 min after (ii) tetanus from a control cell (A) and from a cell requires a minimal concentration of intracellular Ca2+. Since injected with EGTA (B). Responses i and ii are superimposed on an previous experiments have shown that LTD induction de- expanded time scale (iii) to show the change of the initial slope of the pends on a critical level of postsynaptic depolarization, it depressed PSP. Vm = -71 mV in A and -70 mV in B. (C) Time appears as the most likely assumption that the occurrence of course of posttetanic changes of the amplitude of PSPs elicited from LTD is associated with a surge of intracellular free Ca2 . w.m. in control cells (A; n = 13) and in cells injected with EGTA (m; not allow us to remove the n = 8). The corresponding empty symbols represent the amplitudes Since our recording technique did ofresponses to i.c. stimulation. Stimulus intensity during tetanus was Ca2+ chelator once it was injected, we cannot decide whether 4 times threshold intensity: 19.2 ± 0.4 V in control cells and 19.3 + the surge of Ca2+ is required only for the induction of LTD 0.5 V in cells injected with EGTA. or also for its maintenance. Downloaded by guest on September 29, 2021 126 Neurobiology: Brocher et al. Proc. Natl. Acad Sci. USA 89 (1992) i ii iii

A

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C 110 100 + %+4sK~~~s s E 8 i 7 a40

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FIG. 3. Effect of BAPTA injection on induction of LTD in two different cells (A and B). (A i and ii and B i and ii) Responses to brief (500 ms) depolarizing current pulses (A, +0.7 nA; B, +0.8 nA) before (i) and after (ii) BAPTA injection. In the cell in A, blockade of early AHPs is complete except for the slow Ca2+-independent AHP. Comparison of PSPs recorded before and 20 min after the tetanus shows no evidence of LTD (Aiii). In the cell in B, BAPTA injection has been interrupted before complete disappearance ofthe fast AHP (arrow in Bii). Comparison of PSPs recorded before and 20 min after (arrow) tetanus reveals that LTD was still elicitible in this cell (Bidi). For the AHP measurements in A and B, cells have been depolarized to -49 (A) and -50 (B) mV by current injection (resting membrane potential, -79 mV in A and -81 mV in B). (C) Time course of posttetanic changes of the amplitude of PSPs elicited from w.m. in control cells (0; n = 4) and in cells injected with BAPTA (c; n = 5). Open circles refer to the cell shown in B. Stimulus intensity during tetanus was 10 times the threshold intensity (18.0 ± 2.0 V in control cells and 16.3 ± 1.4 V in injected cells).

Because LTD can be induced while NMDA receptors are Indications for an involvement of this receptor system in blocked (4, 5), we exclude the possibility that the Ca2+ LTD induction are available from cerebellum (33) and hip- changes we assume to be responsible for LTD result from pocampus (34). activation of NMDA receptor-gated channels. Another pos- Our finding that LTD induction depends on intracellular sibility is Ca2+ entry through voltage-dependent Ca2+ chan- Ca2l is somewhat surprising since this is also true for LTP nels. This hypothesis is attractive fortwo reasons. First, LTD (9-11). One possibility is that Ca2l has a general and non- induction in the neocortex requires substantial depolarization specific role in synaptic modifications and that specific of the postsynaptic neuron (5) and some of the voltage- messages leading to LTD and LTP, respectively, are coded dependent Ca2+ conductances have a high activation thresh- by other signals. However, previous studies have shown that, old (29, 30). Second, there is evidence in the cerebellum that in neocortex, the same afferent activity can lead either to LTD of the synaptic connection between parallel fibers and LTD or to LTP, solely depending on the level ofpostsynaptic Purkinje cells depends on the activation ofvoltage-dependent depolarization attained during the tetanus. If depolarization Ca2l conductances in the postsynaptic Purkinje cells. LTD is strong enough to reach the activation threshold of NMDA induction requires that Purkirje cells are depolarized above receptor-gated channels the tetanus causes LTP, if depolar- the threshold ofvoltage-dependent Ca2+ conductances either ization remains below this level, LTD is induced (5). This by concomitant climbing fiber activation (3) or by intracel- suggests as one variable a postsynaptic voltage-dependent lular current injection (31). As in neocortex, LTD induction signal and together with the present results raises the possi- is blocked if the resulting surge of intracellular Ca2+ is bility that Ca2l may actually serve as a trigger for both LTP buffered with EGTA (16). Finally, it needs to be considered and LTD. In that case, it would have to matter where the that part ofthe intracellular Ca2' required for LTD may result Ca2" ions are released from and in what concentrations. from activation of metabotropic quisqualate receptors (18). Lisman (35) has proposed that intracellular free Ca2+ can Activation ofthis receptor by excitatory amino acids to have opposite effects depending on concentration because an increase in intracellular inositol 1,4,5-trisphosphate and Ca2+-regulated kinases and phosphatases have rather differ- this in turn causes a release of Ca2+ from internal stores (32). ent affinities for Ca2l ions. Thus, there is the possibility that Downloaded by guest on September 29, 2021 Neurobiology: Br6cher et al. Proc. Natl. Acad. Sci. USA 89 (1992) 127 in neocortex very high concentrations of intracellular Ca2+ 14. Grover, L. M. & Teyler, T. J. (1990) Nature (London) 347, lead to LTP, while lower concentrations cause LTD. This is 477-479. consistent with our result that LTD was still inducible when 15. Aniksztejn, L. & Ben-Ari, Y. (1991) Nature (London) 349, 67-69. buffering of intracellular Ca2W was substantial but incom- 16. Sakurai, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3383-3385. plete. It is also compatible with the recent finding (27, 28) that 17. Carbone, E. & Lux, H. D. (1986) in Calcium Electrogenesis intracellular injection ofEGTA or BAPTA blocked induction and Neural Functioning, eds. Heinemann, V., Klee, M., Ne- of LTP but not of LTD, if-as we have argued above-Ca2' her, E. & Singer, W. (Springer, Heidelberg), pp. 1-8. buffering was not complete in these experiments. 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