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Bicuculline increases the intracellular calcium response of CA1 hippocampal neurons to synaptic stimulation
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,: 1993 Elsevier Scientific Publishers heland Ltd. All rights reserved f)304-394fl/93/S [)6 0(~
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Bicuculline increases the intracellular calcium response of CA1 hippocampal neurons to synaptic stimulation J.A.M. van der Linden, M. Jo6ls, H. Karst, A.J.A Juta and W.J. W a d m a n Department ~)[Experimental Zoology. U)tiversityof Amsterdam. Amsterdam (The Netherlands) (Received 11 September 1992: Revised version received 11 March 1993; Accepted 11 March 1993)
Key words. lntracellular Ca2+: Fura-2; Hippocampal slice: CAI pyramidal neuron; Bicuculline; GABA; Epilepsy lntracellular calcium levels were measured by ratio imaging of Fura-2-injected CA1 pyramidal neurons during repetitive electrical stimulation ot" the Schaffer collaterals in rat hippocampal slices. Baseline intracellular calcium levels of 102 nM increased to 190 nM during a 3-s stimulus train of 5 Hz. Bicuculline (20/,tM) significantly enhanced this stimulus-dependent rise in intracellular calcium, while the baseline calcium levels remained unchanged. Concomitantly performed extra- and intracellular electrophysiological recordings indicate that the increased calcium response in the presence of bicuculline is linked to a prolongation of the excitatory postsynaptic potential and the induction of multiple action potentials. The bicuculline-induced increased calcium response could have long-term implications for cell function and eventually lead to cell degeneration.
Glutamate is the main excitatory transmitter mediating synaptic transmission between Schaffer collaterals and CA1 pyramidal neurons (see for reviews ref. 14 and 18), Glutamate activates several subtypes of ligand-activated cation channels (NMDA, AMPA, kainate) of which at least the NMDA-type allows calcium influx into the cell [6, 15, 16, 19, 22, 23, 25]. Additionally, metabotropic receptors are activated which may cause release of calcium from intracellular stores [17]. Glutamate-induced calcium influx can also occur through activation of voltage-dependent calcium currents secondary to the transmitter-evoked depolarization of the membrane [23]. In addition to the glutamate-mediated input, CAI neurons receive a GABAergic input, either via feedforward or feedback inhibitory pathways [1, 2, 8, 13, 24]. As a result, activation of the Schaffer collaterals induces an excitatory postsynaptic potential (EPSP) in CA1 cells, followed by an early inhibitory postsynaptic potential (IPSP) (mediated by GABAA receptors) and a late IPSP via GABAB receptors [11, 13]. The GABA Aantagonist bicuculline blocks the early IPSP and, consequently, the late phase of the glutamate-induced depolarization becomes unmasked allowing the generation of multiple action potentials [7, 11, 26]. In extracellular studies, bicuculline treatment was shown to induce multiple population spikes in response to stimulation of the Corre6pondenee: W.J. Wadman, Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
afferents and, at higher concentrations, spontaneous discharges. In this study we investigated the calcium influx in CA 1 pyramidal neurons, evoked by repetitive low-frequency stimulation of the Schaffer collaterals, under control conditions and in the presence of bicuculline. To this end, CA1 pyramidal neurons in thin hippocampal slices were injected with the fluorescent calcium (Ca2+)-sensi tive dye Fura-2. Field potentials and changes in intracellular Ca 2+ induced by stimulation of the Schaffer collaterals were simultaneously measured. In separate experiments we recorded membrane potential and synaptic responses with conventional intracellular microelectrodes before and during bicuculline administration. Male wistar rats of 160-180 g were decapitated under ether anesthesia, the brain was rapidly removed and the hippocampus was dissected free. Thin hippocampal slices (150-200/lm) were made with a Sorvall TC-2 tissue chopper and maintained in artificial cerebrospinal fluid (ACSF gassed with 95% O2, 5% CO2; composition in raM: NaC1 120, KCI 3.5, NaH2PO4 1.25, NaHCO3 25, MgSO4 1.3, CaCIz 2.5, glucose 10; pH 7.4). CA1 pyramidal neurons were impaled with glass microetectrodes containing 12 mM of the Ca~+-sensitive dye Fura-2 (pentapotassium salt, Molecular Probes Inc.) in 100 mM potassium chloride (impedance 400 MD). The cells were filled with the dye by applying 0.5 nA hyperpolarizing pulses of 500 ms duration at 1 Hz superimposed on a steady hyperpolarizing current of 0.5-1 nA, for 10 min.
231
Subsequently, the slices were left for 30 rain at room temperature in ACSF to allow diffusion of the dye. For calcium imaging and extracellular electrophysiology, slices were transferred to a superfusion chamber with a thin glass cover slip as a bottom. During all experiments, this chamber was perfused continuously with ACSF (32°C) at 1 ml/min. The GABAA blocker bicuculline ( 2 0 / J M bicuculline methiodide, Sigma) was bath applied. Bipolar stainless steel stimulation electrodes ( 100 y m in diameter) were placed in the stratum radiatum, and biphasic current pulses (10M00/JA) were applied to the Schaffer collateral afferent pathway. Field potentials were recorded with glass microelectrodes (165 mM NaC1, impedance 1 2 MD) which were placed close to the Fura-2filled neuron in the pyramidal cell layer. Calcium imaging was done with an inverted microscope equipped for epifluorescence (Nikon T M D - E F , Fluor objective 20x, n.a. 0.75), Excitation was provided by a mercury lamp (Osram HBO-100) at 340 nm and 380 nm wavelength [5]. Fluorescence images were collected at 510 nm {20 nm bandpass) by a cooled C C D camera (Photometrics series 200). The exposure time for each subsequent image was 500 ms, and the total time needed to acquire a 340 380 nm image pair was 1270 ms. Ratio images were calculated off-line after subtraction of background fluorescence. Ratios were converted to Ca =+ concentrations with the calibration formula of Grynkiewicz et al [9]: [Ca 2+] = ,fl'K~¢(R-Rmi.O/(R ..... - R ) with Rmi n = 0.4, R ..... = 12.4, fl 10, Kd 225 nM. Rmi n and R ...... were obtained from an in-vitro calibration and a 30% viscosity correction was applied [20, 23]. For each stimulus sequence 9 ratio measurements were taken with an interval of 2 s: two before, one during and the rest after the 3-s repetitive stimulation. Two to three such stimulus sequences were given before and during bicuculline treatment. Field potentials were recorded concomitantly. We did not observe a significant reduction of the Fura-2 signals during the time course of an experiment. In separate experiments, 4 M potassium acetate-filled electrodes (without Fura-2; impedance 80-150 M.Q) was used for intracellular recording from CA I ceils using conventional methods as described in detail elsewhere
[10]. In control experiments the Ca =+ resting level in the soma of CA1 pyramidal cells amounted to 102_+17 nM (mean _+ S.E.M., n 6). Cells with relatively high resting Ca>values (> 300 nM) were incidentally encountered: they did not show a reversible transient response to electrical stimulation and were not incorporated in the present study. A single electrical stimulus to the Schaffer collaterals was not sufficient to significantly enhance the intracellular Ca x+ level. Application of 15 stimuli at 5 Hz, however, markedly increased the intracellular Ca =+ level
(example in Fig. 1A). The m a x i m u m level of C a 2+ reached immediately after termination of the stimulus train was 190 + 29 nM (n 6), which was significantly larger than the resting level (P < 0.05, paired t-test), lntracellular Ca > levels quickly recovered to pre-stimulus levels with an exponential decay of 3.3 _+ 0.5 s. Concomitant with the calcium signal, we recorded field potentials in response to stimulation of the Schafl'er collaterals (example in the inset of Fig. I A). Field potentials measured 20 s after the train were identical to those measured just before. Therefore, the stimulus frequency and intensity that were used were insufficient to induce long-term potentiation. Intracellular recordings from CA1 cells in another set of slices typically showed a last EPSP (duration around 20 ms) followed by a subsequent fast and slow IPSP in response to synaptic stimulation (n 5: Fig. 1B). The cells always returned to resting membrane potential (between - 6 5 and -7(1 inV, H 5) within 700 ms. The repetitive stimulation generated a hyperpolarization of 3 5 mV (Fig. 1B). Each stimulus in the train evoked an EPSP/IPSP sequence superimposed on this hyperpolarization.
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B
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Fig. 1. A: somatic calcium concentration in a CA1 pyramidal neuron as a function of time. Repetitive stimulation {3 s. 5 Hz, 400/JA} was given between t 3 and t 6 (horizontal bar} to the Schaf[er collaterals. Open symbols represent data measured under control conditions. Closed symbols are data from the same cell during tile application of 20 ~M bicuculline. Inset shows field potentials recorded in stratum pyramidale in the same slice (upper trace under control conditions, lower trace during 20//M bicuculline). B: imracellular recording from a CAI pyramidal neuron in a different slice during low frequency stimulation. under control conditions (upper trace) and in the presence of 20 ,aM bicuculine (middle trace). Resting membrane potential was in both cases -69 inV. The action potential amplitude is truncated. In the lower trace, the response to the first stimulus of the train is plotted on an expanded time scale and superimposed for both conditions. Note the disappearance of the hyperpolarizing fast IPSP and the appearance of multiple action potentials during bicuculline treatment. Horizontal calibration bar is 10 ms t\w insets in A, 420 ms for tipper and middle trace in B and 12 ms for the lower trace in B: vertical calibration is 1 mV for insets in A. 3(I mV for tipper and middle trace in B and 27 mV for the lower trace in B.
232
400-
300" 20~M
200" 100"
.
Control -100
I
0
I
4
I
I
I
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8 12 T i m e (s)
I
I
16
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20
Fig. 2. Mean (n 6) normalized Ca 2+ response of CA1 pyramidal cells to repetitive Schaffer collateral stimulation (5 Hz, 3 s, indicated by the horizontal bar). The resting values varied little between cells, but the maximal increase reached during repetitive stimulation could vary considerably, most likely due to a difference in stimulus efficacy. The rise in control buffer was set at 100%; the increase during bicuculline was normalized to this value and then averaged. Vertical bars indicate standard error of the mean.
We next investigated the Ca2+-response and synaptic potentials during application of the GABAA-antagonist bicuculline (20 pM). In the presence of bicuculline, baseline Ca 2+ levels (106 + 17 nM, n 6) were not changed in comparison to control conditions. Synaptic stimulation with 15 pulses evoked a transient increase in Ca 2+ level, which reached a peak of 346 + 94 nM immediately after termination of the stimulus. The maximal increase in Ca 2+ (238 + 96 nM; peak level minus resting level) was significantly larger (P < 0.05, paired t-test, n 6) than the corresponding Ca 2+ increase in controls (88 + 28 nM). Averaged calcium levels, normalized to baseline level before the stimulus train, are shown in Fig. 2 before and during bicuculline treatment. The Ca 2+ signal recovered to resting level with an exponential decay of 3.4 + 0.5 s (examle in Fig. 1A), which is not significantly different from controls. Extracellular population spikes recorded together with the Ca signal revealed the well-known appearance of multiple population spikes in the presence of bicuculline (inset in Fig. 1A). Intracellular measurements in another set of slices showed that the resting membrane potential was not significantly affected by the addition of 20/zM bicuculline, but that fast IPSPs evoked after stimulation of the Schaffer collaterals were abolished (n 4).
As a consequence, the late phase of the glutamate-induced depolarization became unmasked (duration up to 100 instead of 20 ms in control) and reached a higher amplitude (Fig. 1B). While single pulse stimulation in control buffer was often subthreshold tor the initiation of action potentials, the same stimulus in bicucullinecontaining medium could easily produce repetitive spiking (Fig. 1B). In this study we have shown that baseline intracellular Ca 2÷ levels in Fura-2-injected CA1 pyramidal cells are around 100 nM, which is similar to the values reported previously by others in comparable preparations [12, 23, 25]. Repetitive, low-frequency stimulation of the Schaffer collaterals induced a transient, 2-fold increase of the baseline Ca 2+ level. The transient rise in intracellular calcium is probably caused by Ca2+-influx through nonN M D A receptor-associated channels and particularly through voltage-dependent calcium channels [22, 23]; the contribution of NMDA-receptors to synaptic responses evoked by low-frequency stimulation has been reported to be relatively small [4]. The intracellularly recorded resting membrane potential was not changed by bicuculline. Its application also did not affect the baseline intracellular Ca 2+ level. But repetitive stimulation in the presence of bicuculline resuited in a more than 3-fold increase o[" the intracellular Ca 2+ level, instead of the 2-fold increase observed under control conditions. Similar observations were made with the GABAA receptor channel blocker picrotoxin (n 3). Eiectrophysiologically it was shown that bicuculline blocked the fast IPSP and that, consequently, the EPSP was increased both in amplitude and duration, allowing the induction of multiple action potentials. The bicuculline-induced increase of the Ca2+-response is likely to be caused by an increased Ca 2+ influx particularly through voltage-gated calcium channels, which are now activated to a greater extent during the enlarged EPSP and multiple spikes. Given the similar baseline level and time constant of decay for the C a 2+ response, it is suggested that Ca extrusion is similar under bicuculline and control conditions. Many models of experimental epilepsy are based on a reduction of GABA-ergic inhibition [7]. They all show an enhanced excitability of neurons. We have here shown that these conditions lead to an enhanced influx of Ca >, which could have long-term implications for cell function and may eventually lead to cell degeneration [3]. This research was supported by the Foundation for Biological Research (BION, Grant 811-425-241) of the Netherlands Organization for Scientific Research (NWO).
233 1 Alger, B.E. and Nicoll, R.A., Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro, J. Physiol., 328 (1982) 105 123. 2 Andersen, P., Eccles, J.C. and Loyning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608 619. 3 Choi, D.W., Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci.. 11 (1988) 465469. 4 Collingridge, G.L., Herron, C.E. and Lester, R.A.J., Synaptic activation of N-methyI-D-aspartate receptors in the Schaffer collateralcornmisural pathway of rat hippocampus, J. Physiol., 399 (1988) 283 3O0. 5 Connor, J.A., Digital imaging of free calcium changes and of spinal gradients in growing processes in single mammalian CNS cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 6179 6183. 6 Connor. J.A., Wadman, W.J., Hockberger, RE. and Wong, R.K.S., Sustained dendritic gradients of Ca 2+ induced by excitatory amino acids in CAI hippocampal neurons, Science, 240 (1988) 649 653. 7 Dingledine, R. and Gjerstad, L., Reduced inhibition during epileptiform activity in the in vitro hippocampal slice, J. Physiol., 305 (1980) 297 313. 8 Dingledine, R. and Langmoen, I.A., Conductance changes and inhibitory actions of hippocampal recurrent IPSP's, Brain Res., 185 (1991) 277 287. 9 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca :+ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260 (1985) 3440 3450. 10 Joels, M.. Hesen, W. and de Kloet, E.R., Mineralocorticoid hormones suppress serotonin-induced hyperpolarization of rat hippocampal CA1 neurons, J. Neurosci., 11 (1991) 2288-2294. I 1 Knowles, W.D. and Schwartzkroin, RA., Local circuit synaptic interactions in hippocampal brain slice, J. Neurosci., 1 (1981) 318 322. 12 Kudo, Y., lto, K., Miakawa, H., lzumi, Y., Ogura, A. and Kato, H., Cytoplasmic calcium elevation in hippocampal granule cell induced by perlorant path stimulation and e-glutamate application, Brain Res., 407 (1987) 168 172. 13 Lacaille, J., Mueller, A.L., Kunkel, D.D. and Schwartzkroin, RA., Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology, J. Neurosci., 7 (1987) 1979 1993.
14 Lopes da Silva, F.H., Witter, M.P., Boeijinga, EH. and Lohman, A.H.M., Anatomic organization and physiology of the limbic cortex, Physiol. Rev., 70 (1990) 453 511. 15 Mayer, M.L. and Miller, R.J.. Excitatory amino acid receptors, second messengers and regulation ofintracellular Ca :+ in mammalian neurons, Trends Pharmacol. Sci., 11 (1990) 254 260. 16 Mayer, M.L. and Westbrook, G.L., The physiology of excitatory amino acids in the vertebrate central nervous system, Prog. Neurobiol., 28 (1987) 197 276. 17 Murphy. S.N. and Miller, R.J., Two distinct quisqualate receptors regulate Ca:" homeostasis in hippocampal neurons in vitro, Mol. Pharmacoh, 38 (1989) 671 680. 18 Nicoll, R.A., Malenka, R.C and Kauer, J.A.. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system, Physiol. Rev., 70 (1990) 513 565. 19 Ogura, A., Akita, K. and Kudo, Y., Non-NMDA receptor mediates cytoplasmic Ca "+ elevation in cultured hippocampal neurones, Neurosci. Res., 9 (1990) 103 113. 20 Poenie, M., Alderton. J., Steinhardt, R. and Tsien, R.. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase, Science, 233 11986) 886 889. 21 Regehr, W.G., Connor, J.A. and Tank, D.W., Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation, Nature, 341 (1989) 533 536. 22 Regehr, W.G. and Tank. D.W.. Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites. Nature, 345 (1990) 807 810. 23 Regehr. W.G. and Tank. D.W., Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells, J. Neurosci., 12 (1992) 4202 4223, 24 Turner. D.A., Feed-forward inhibitory potentials and excitatory interactions in guinea-pig hippocampal pyramidal cells, J. Physiol., 422 (1990) 333 350. 25 Wadman, W.J. and Connor, J.A., Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated CAI neurons, Neuroscience, 48 (1992) 293 305. 26 Zhang, L., Spigelman, 1. and Carlen. P.I., Whole-cell patch study of GABAergic inhibition in CA I neurons of immature rat hippocampal slices, Dev. Brain Res.. 56 (1990) 127 130.
230
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?::5
,: 1993 Elsevier Scientific Publishers heland Ltd. All rights reserved f)304-394fl/93/S [)6 0(~
NSL 09578
Bicuculline increases the intracellular calcium response of CA1 hippocampal neurons to synaptic stimulation J.A.M. van der Linden, M. Jo6ls, H. Karst, A.J.A Juta and W.J. W a d m a n Department ~)[Experimental Zoology. U)tiversityof Amsterdam. Amsterdam (The Netherlands) (Received 11 September 1992: Revised version received 11 March 1993; Accepted 11 March 1993)
Key words. lntracellular Ca2+: Fura-2; Hippocampal slice: CAI pyramidal neuron; Bicuculline; GABA; Epilepsy lntracellular calcium levels were measured by ratio imaging of Fura-2-injected CA1 pyramidal neurons during repetitive electrical stimulation ot" the Schaffer collaterals in rat hippocampal slices. Baseline intracellular calcium levels of 102 nM increased to 190 nM during a 3-s stimulus train of 5 Hz. Bicuculline (20/,tM) significantly enhanced this stimulus-dependent rise in intracellular calcium, while the baseline calcium levels remained unchanged. Concomitantly performed extra- and intracellular electrophysiological recordings indicate that the increased calcium response in the presence of bicuculline is linked to a prolongation of the excitatory postsynaptic potential and the induction of multiple action potentials. The bicuculline-induced increased calcium response could have long-term implications for cell function and eventually lead to cell degeneration.
Glutamate is the main excitatory transmitter mediating synaptic transmission between Schaffer collaterals and CA1 pyramidal neurons (see for reviews ref. 14 and 18), Glutamate activates several subtypes of ligand-activated cation channels (NMDA, AMPA, kainate) of which at least the NMDA-type allows calcium influx into the cell [6, 15, 16, 19, 22, 23, 25]. Additionally, metabotropic receptors are activated which may cause release of calcium from intracellular stores [17]. Glutamate-induced calcium influx can also occur through activation of voltage-dependent calcium currents secondary to the transmitter-evoked depolarization of the membrane [23]. In addition to the glutamate-mediated input, CAI neurons receive a GABAergic input, either via feedforward or feedback inhibitory pathways [1, 2, 8, 13, 24]. As a result, activation of the Schaffer collaterals induces an excitatory postsynaptic potential (EPSP) in CA1 cells, followed by an early inhibitory postsynaptic potential (IPSP) (mediated by GABAA receptors) and a late IPSP via GABAB receptors [11, 13]. The GABA Aantagonist bicuculline blocks the early IPSP and, consequently, the late phase of the glutamate-induced depolarization becomes unmasked allowing the generation of multiple action potentials [7, 11, 26]. In extracellular studies, bicuculline treatment was shown to induce multiple population spikes in response to stimulation of the Corre6pondenee: W.J. Wadman, Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
afferents and, at higher concentrations, spontaneous discharges. In this study we investigated the calcium influx in CA 1 pyramidal neurons, evoked by repetitive low-frequency stimulation of the Schaffer collaterals, under control conditions and in the presence of bicuculline. To this end, CA1 pyramidal neurons in thin hippocampal slices were injected with the fluorescent calcium (Ca2+)-sensi tive dye Fura-2. Field potentials and changes in intracellular Ca 2+ induced by stimulation of the Schaffer collaterals were simultaneously measured. In separate experiments we recorded membrane potential and synaptic responses with conventional intracellular microelectrodes before and during bicuculline administration. Male wistar rats of 160-180 g were decapitated under ether anesthesia, the brain was rapidly removed and the hippocampus was dissected free. Thin hippocampal slices (150-200/lm) were made with a Sorvall TC-2 tissue chopper and maintained in artificial cerebrospinal fluid (ACSF gassed with 95% O2, 5% CO2; composition in raM: NaC1 120, KCI 3.5, NaH2PO4 1.25, NaHCO3 25, MgSO4 1.3, CaCIz 2.5, glucose 10; pH 7.4). CA1 pyramidal neurons were impaled with glass microetectrodes containing 12 mM of the Ca~+-sensitive dye Fura-2 (pentapotassium salt, Molecular Probes Inc.) in 100 mM potassium chloride (impedance 400 MD). The cells were filled with the dye by applying 0.5 nA hyperpolarizing pulses of 500 ms duration at 1 Hz superimposed on a steady hyperpolarizing current of 0.5-1 nA, for 10 min.
231
Subsequently, the slices were left for 30 rain at room temperature in ACSF to allow diffusion of the dye. For calcium imaging and extracellular electrophysiology, slices were transferred to a superfusion chamber with a thin glass cover slip as a bottom. During all experiments, this chamber was perfused continuously with ACSF (32°C) at 1 ml/min. The GABAA blocker bicuculline ( 2 0 / J M bicuculline methiodide, Sigma) was bath applied. Bipolar stainless steel stimulation electrodes ( 100 y m in diameter) were placed in the stratum radiatum, and biphasic current pulses (10M00/JA) were applied to the Schaffer collateral afferent pathway. Field potentials were recorded with glass microelectrodes (165 mM NaC1, impedance 1 2 MD) which were placed close to the Fura-2filled neuron in the pyramidal cell layer. Calcium imaging was done with an inverted microscope equipped for epifluorescence (Nikon T M D - E F , Fluor objective 20x, n.a. 0.75), Excitation was provided by a mercury lamp (Osram HBO-100) at 340 nm and 380 nm wavelength [5]. Fluorescence images were collected at 510 nm {20 nm bandpass) by a cooled C C D camera (Photometrics series 200). The exposure time for each subsequent image was 500 ms, and the total time needed to acquire a 340 380 nm image pair was 1270 ms. Ratio images were calculated off-line after subtraction of background fluorescence. Ratios were converted to Ca =+ concentrations with the calibration formula of Grynkiewicz et al [9]: [Ca 2+] = ,fl'K~¢(R-Rmi.O/(R ..... - R ) with Rmi n = 0.4, R ..... = 12.4, fl 10, Kd 225 nM. Rmi n and R ...... were obtained from an in-vitro calibration and a 30% viscosity correction was applied [20, 23]. For each stimulus sequence 9 ratio measurements were taken with an interval of 2 s: two before, one during and the rest after the 3-s repetitive stimulation. Two to three such stimulus sequences were given before and during bicuculline treatment. Field potentials were recorded concomitantly. We did not observe a significant reduction of the Fura-2 signals during the time course of an experiment. In separate experiments, 4 M potassium acetate-filled electrodes (without Fura-2; impedance 80-150 M.Q) was used for intracellular recording from CA I ceils using conventional methods as described in detail elsewhere
[10]. In control experiments the Ca =+ resting level in the soma of CA1 pyramidal cells amounted to 102_+17 nM (mean _+ S.E.M., n 6). Cells with relatively high resting Ca>values (> 300 nM) were incidentally encountered: they did not show a reversible transient response to electrical stimulation and were not incorporated in the present study. A single electrical stimulus to the Schaffer collaterals was not sufficient to significantly enhance the intracellular Ca x+ level. Application of 15 stimuli at 5 Hz, however, markedly increased the intracellular Ca =+ level
(example in Fig. 1A). The m a x i m u m level of C a 2+ reached immediately after termination of the stimulus train was 190 + 29 nM (n 6), which was significantly larger than the resting level (P < 0.05, paired t-test), lntracellular Ca > levels quickly recovered to pre-stimulus levels with an exponential decay of 3.3 _+ 0.5 s. Concomitant with the calcium signal, we recorded field potentials in response to stimulation of the Schafl'er collaterals (example in the inset of Fig. I A). Field potentials measured 20 s after the train were identical to those measured just before. Therefore, the stimulus frequency and intensity that were used were insufficient to induce long-term potentiation. Intracellular recordings from CA1 cells in another set of slices typically showed a last EPSP (duration around 20 ms) followed by a subsequent fast and slow IPSP in response to synaptic stimulation (n 5: Fig. 1B). The cells always returned to resting membrane potential (between - 6 5 and -7(1 inV, H 5) within 700 ms. The repetitive stimulation generated a hyperpolarization of 3 5 mV (Fig. 1B). Each stimulus in the train evoked an EPSP/IPSP sequence superimposed on this hyperpolarization.
A
B
E
C
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I
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l
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D,
21]
Fig. 1. A: somatic calcium concentration in a CA1 pyramidal neuron as a function of time. Repetitive stimulation {3 s. 5 Hz, 400/JA} was given between t 3 and t 6 (horizontal bar} to the Schaf[er collaterals. Open symbols represent data measured under control conditions. Closed symbols are data from the same cell during tile application of 20 ~M bicuculline. Inset shows field potentials recorded in stratum pyramidale in the same slice (upper trace under control conditions, lower trace during 20//M bicuculline). B: imracellular recording from a CAI pyramidal neuron in a different slice during low frequency stimulation. under control conditions (upper trace) and in the presence of 20 ,aM bicuculine (middle trace). Resting membrane potential was in both cases -69 inV. The action potential amplitude is truncated. In the lower trace, the response to the first stimulus of the train is plotted on an expanded time scale and superimposed for both conditions. Note the disappearance of the hyperpolarizing fast IPSP and the appearance of multiple action potentials during bicuculline treatment. Horizontal calibration bar is 10 ms t\w insets in A, 420 ms for tipper and middle trace in B and 12 ms for the lower trace in B: vertical calibration is 1 mV for insets in A. 3(I mV for tipper and middle trace in B and 27 mV for the lower trace in B.
232
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300" 20~M
200" 100"
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Control -100
I
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I
I
I
I
8 12 T i m e (s)
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I
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I
20
Fig. 2. Mean (n 6) normalized Ca 2+ response of CA1 pyramidal cells to repetitive Schaffer collateral stimulation (5 Hz, 3 s, indicated by the horizontal bar). The resting values varied little between cells, but the maximal increase reached during repetitive stimulation could vary considerably, most likely due to a difference in stimulus efficacy. The rise in control buffer was set at 100%; the increase during bicuculline was normalized to this value and then averaged. Vertical bars indicate standard error of the mean.
We next investigated the Ca2+-response and synaptic potentials during application of the GABAA-antagonist bicuculline (20 pM). In the presence of bicuculline, baseline Ca 2+ levels (106 + 17 nM, n 6) were not changed in comparison to control conditions. Synaptic stimulation with 15 pulses evoked a transient increase in Ca 2+ level, which reached a peak of 346 + 94 nM immediately after termination of the stimulus. The maximal increase in Ca 2+ (238 + 96 nM; peak level minus resting level) was significantly larger (P < 0.05, paired t-test, n 6) than the corresponding Ca 2+ increase in controls (88 + 28 nM). Averaged calcium levels, normalized to baseline level before the stimulus train, are shown in Fig. 2 before and during bicuculline treatment. The Ca 2+ signal recovered to resting level with an exponential decay of 3.4 + 0.5 s (examle in Fig. 1A), which is not significantly different from controls. Extracellular population spikes recorded together with the Ca signal revealed the well-known appearance of multiple population spikes in the presence of bicuculline (inset in Fig. 1A). Intracellular measurements in another set of slices showed that the resting membrane potential was not significantly affected by the addition of 20/zM bicuculline, but that fast IPSPs evoked after stimulation of the Schaffer collaterals were abolished (n 4).
As a consequence, the late phase of the glutamate-induced depolarization became unmasked (duration up to 100 instead of 20 ms in control) and reached a higher amplitude (Fig. 1B). While single pulse stimulation in control buffer was often subthreshold tor the initiation of action potentials, the same stimulus in bicucullinecontaining medium could easily produce repetitive spiking (Fig. 1B). In this study we have shown that baseline intracellular Ca 2÷ levels in Fura-2-injected CA1 pyramidal cells are around 100 nM, which is similar to the values reported previously by others in comparable preparations [12, 23, 25]. Repetitive, low-frequency stimulation of the Schaffer collaterals induced a transient, 2-fold increase of the baseline Ca 2+ level. The transient rise in intracellular calcium is probably caused by Ca2+-influx through nonN M D A receptor-associated channels and particularly through voltage-dependent calcium channels [22, 23]; the contribution of NMDA-receptors to synaptic responses evoked by low-frequency stimulation has been reported to be relatively small [4]. The intracellularly recorded resting membrane potential was not changed by bicuculline. Its application also did not affect the baseline intracellular Ca 2+ level. But repetitive stimulation in the presence of bicuculline resuited in a more than 3-fold increase o[" the intracellular Ca 2+ level, instead of the 2-fold increase observed under control conditions. Similar observations were made with the GABAA receptor channel blocker picrotoxin (n 3). Eiectrophysiologically it was shown that bicuculline blocked the fast IPSP and that, consequently, the EPSP was increased both in amplitude and duration, allowing the induction of multiple action potentials. The bicuculline-induced increase of the Ca2+-response is likely to be caused by an increased Ca 2+ influx particularly through voltage-gated calcium channels, which are now activated to a greater extent during the enlarged EPSP and multiple spikes. Given the similar baseline level and time constant of decay for the C a 2+ response, it is suggested that Ca extrusion is similar under bicuculline and control conditions. Many models of experimental epilepsy are based on a reduction of GABA-ergic inhibition [7]. They all show an enhanced excitability of neurons. We have here shown that these conditions lead to an enhanced influx of Ca >, which could have long-term implications for cell function and may eventually lead to cell degeneration [3]. This research was supported by the Foundation for Biological Research (BION, Grant 811-425-241) of the Netherlands Organization for Scientific Research (NWO).
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