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Calcium-independent inhibition of GABAA current by caffeine in hippocampal slices
Brain Research 1016 (2004) 229 – 239 www.elsevier.com/locate/brainres
Research report
Calcium-independent inhibition of GABAA current by caffeine in hippocampal slices M. Taketo a,*, H. Matsuda a, T. Yoshioka b a
Department of Physiology 1, Faculty of Medicine, Kansai Medical University, 10-15 Fumizono-cho Moriguchi, Osaka 570-8506, Japan b Department of Molecular Neurobiology, Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1, Ohkubo, Shinjuku, Tokyo 169-8555, Japan Accepted 1 May 2004 Available Available online online 24 June 2004
Abstract Although inhibitory postsynaptic currents (IPSCs) mediated by GABAA receptor is thought to be affected by intracellular calcium ion concentration ([Ca2 +]i), origin or route of [Ca2 +]i increment has not been well elucidated. Reports on the effect of [Ca2 +]i elevation on GABAAergic IPSCs per se are also controversial. In this study, effects of caffeine and several other [Ca2 +]i-mobilizing drugs were examined on the IPSCs in acute slices of rat hippocampus. Using the patch clamp recording method, spontaneous and evoked currents were recorded from CA3 neurons. Caffeine strongly inhibited both extra-synaptic and synaptic GABAergic IPSCs, regardless of the presence or absence of extracellular Ca2 +. This inhibition was not relieved by the intracellular application of EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,NV,NV-tetraacetic acid (BAPTA). This inhibition by caffeine was not prevented by preequilibration with caffeine. Ca2 + store depletion caused by thapsigargin or repetitive stimulation by caffeine could not prevent the inhibition. Moreover, ruthenium red and ryanodine could not overcome the inhibition. On the contrary, GABAAergic currents were not inhibited by stimulation with several Ca2 +mobilizing agonists. Forskolin could not mimic the effect of caffeine on the IPSC, and caffeine inhibited the IPSC in the presence of adenosine. These results suggest that intracellular Ca2 + mobilization through ryanodine-sensitive store stimulation does not significantly affect GABAergic IPSCs, and most of the inhibitory effect of caffeine is independent of [Ca2 +]i elevation under the present experimental conditions. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: GABA receptors Keywords: GABAA receptor; Hippocampus; Caffeine; Intracellular Ca2+
1. Introduction GABAA receptor mediates major inhibitory postsynaptic current (IPSC) in the central nervous system. Modulations of the efficacy of GABAAergic synaptic transmission have
Abbreviations: ACSF, artificial cerebrospinal fluid; BAPTA, 1,2-bis(2aminophenoxy)-ethane-N,N,NV,NV-tetraacetic acid; CNQX, 6-cyano-7nitroquinoxaline-2,3-dione; cADPR, cyclic adenosine diphosphate ribose; D-AP5, D( )-2-amino-5-phosphonovaleric acid; IPSC, inhibitory postsynaptic current; sIPSC, spontaneous inhibitory postsynaptic current; TTX, tetrodotoxin * Corresponding author. Tel.: +81-669-93-9422; fax: +81-669-921409. E-mail address: [email protected] (M. Taketo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.008
been reported in several brain regions, and the elevation of intracellular calcium concentration ([Ca2 +]i) has been suggested to underlie the modulations [9]. Effects of [Ca2 +]i elevation on GABAergic IPSC have been studied in several systems, but the results are controversial [1,2]. Even whether [Ca2 +]i elevation regulates the IPSC positively or negatively has not been decided. One of the generally used reagent to increase [Ca2 +]i in these experiments is caffeine, which releases calcium from ryanodine-sensitive intracellular store. Inhibitory effect of caffeine on GABAergic postsynaptic currents has been reported and regarded as the consequence of increasing [Ca2 +]i [2,7,20]. In several reports however, caffeine was suggested to exert its effect on inhibitory transmission, unrelated to releasing Ca2 + from ryanodine-sensitive Ca2 + store [12,19].
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The conflicting results may partly be caused by difference in the stimulation methods employed for elevating [Ca2 +]i in each experiment. Tissue distribution and subcellular localization of the receptor with different subunit subtypes may also contribute to the inconsistency of the results, because many subunit subtypes of heteropentameric GABAA receptor have been cloned and demonstrated to have different pharmacological properties when reconstructed [4]. It has been reported that synaptic and extra synaptic GABAergic IPSCs are mediated by receptors of different subclasses with different pharmacological and kinetical properties. In hippocampal neurons, calcium-dependent [7] and -indepedent [19] inhibitions of the IPSC have been described. The discrepancy may be ascribed to the different nature of the synaptic IPSC and extra synaptic IPSC, because the former report investigated miniature IPSC and the latter dealt with agonist-evoked IPSC. Besides these factors, methods of tissue preparation have been suggested to cause the variation of experimental results [7]. Among previous reports, there are few studies determining the inhibitory effect on IPSCs which are mediated by different subclasses of receptors in same tissue preparation. In addition, [Ca2 +]i dependency of the inhibition by caffeine was mainly determined only by the application of a chelating agent [such as 1,2-bis(2-aminophenoxy)-ethane-N,N,NV,NV-tetraacetic acid (BAPTA)] in previous studies. In this study, the effect of caffeine on both the synaptic and extra synaptic GABAergic current was accordingly examined in acute slices of young rat hippocampus, using the patch clamp recording technique. Calcium dependency of the effect on the IPSC was further investigated using several methods. Spontaneous and evoked GABAAergic IPSCs were also recorded in the presence of other calcium mobilizing reagents: acetylcholine, bradykinin, or ATP. Based on the experimental analyses, mechanism of the calcium-independent GABAAergic current inhibition was discussed.
2. Materials and methods 2.1. Slice preparation Hippocampal slices were prepared from Wister rats aged 4 to 12 days, as reported previously [18]. All experimental protocols were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals revised 1996 and the guidelines for animal research of the Physiological Society of Japan. All efforts were made to minimize animal suffering and to reduce the number of animals used. Following diethyl ether anesthesia, the animals were decapitated and the brains were removed and cooled in an artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 138.6, KCl 3.35, NaHCO 3 21.0, NaH2PO4 0.6, CaCl2 2.5, MgCl2 1.0, glucose 10.0) bubbled
to equilibrium with 95% O2/5% CO2 for 2 min. Then, the brains were dissected, and the removed hippocampi were sliced to a thickness of 250 –400 Am using a vibrating tissue slicer (model DTK1000, D.S.K., Japan). Slices were placed in a storage chamber filled with ACSF, and then kept at least 1 h at room temperature. Before recording, the slices were individually transferred to a recording chamber and continuously perfused with ACSF during experiment. In some specified experiments, NaCl concentration in the extracellular solution was reduced to 118.6 mM, and CaCl2 concentration was increased to 12.5 mM. 2.2. Whole cell recordings Whole cell patch clamp recordings were performed in pyramidal neurons of the CA3 region. Each cell was identified morphologically using cooled charge coupled device (CCD) camera (model C2400-79, Hamamatsu Photonics, Japan) connected to a microscope with infrared filter (model E600-UD, Nikon, Japan). Patch electrodes (2– 4 MV after filling with intrapipette solution) were pulled from borosilicate glass using a twostage vertical puller (model PP-83, Narishige, Japan) and filled with symmetrical chloride-intracellular solution [composition in mM: CsCl 140, CaCl2 1, MgCl2 2, ethyleneglycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) 10, HEPES 10, ATP 2, adjusted to pH 7.3 using CsOH]. In some experiments, CaCl2 was omitted and EGTA in intracellular solution was increased or replaced by 10 or 30 mM BAPTA. Under the experimental conditions, the GABAergic IPSCs were reversed at f0 mV equivalent to the ECl predicted by Nernst equation. Cells were voltage-clamped at 70 mV, and the GABAergic IPSC appeared as inward current. Electrosignals were measured with an EPC7 patch clamp amplifier (List, Germany) and filtered at 3 kHz. The signals were digitized at 10 kHz and stored using DigiData 1200 with the pClamp6 data collection and analysis software (Axon Instruments, USA). Data recorded from cells with significant change in series resistance during experiment were discarded. Isoguvacine was focally applied by iontophoresis (3– 15 ms) through a microelectrode placed near cell body or proximal dendrite of recorded neuron, in the presence of 0.5 AM tetrodotoxin (TTX). Electrostimulation was applied by glass microelectrode filled with ACSF (3 –10 V and 200 As) placed in stratum pyramidale close to the soma of the recorded neuron. All experiments were carried out at room temperature (f23 jC). 2.3. Calcium imaging Whole cell current recording was performed as described above, and calcium imaging was performed simultaneously. Cells were loaded with calcium indicator through a patch pipette (filled with standard CsCl solution of the same composition as described above, except for the addition of
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10 AM fura-2). The GABAergic IPSC was evoked by isoguvacine. Fluorescence image was obtained through an objective lens (CFI Fluor, Nikon) and a cooled CCD camera (model C4742-95-12ER, Hamamatsu Photonics) under alternating excitation wavelengths of 340 and 380 nm. Images were stored and analyzed using a digital image processor (Aquacosmos, Hamamatsu Photonics). Calcium concentration was expressed as the ratio of the fura-2 fluorescence intensities excited at 340 and 380 nm. A normalized ratio value was calculated for the temporal analysis of [Ca2 +]i
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alteration (average of values during the first 3 min was used as baseline ratio). The acquisition rate was usually 0.1 Hz. 2.4. Data analysis Average of interevent interval and amplitude distributions of spontaneous IPSCs obtained in control and in test conditions were calculated during 3 or 5 min. Statistical analyses were carried out using unpaired t-test with Welch’s correction (a level of significance of P V 0.05).
Fig. 1. Extra-synaptic GABAergic IPSC recorded from CA3 neurons. GABAergic current was evoked by iontophoretically applied 100 AM isoguvacine (indicated by arrow). (A)-a Sensitivity of the GABAAergic IPSC to caffeine. (A)-b Amplitudes of baseline, peak, and net current plotted against time. (B) The inhibitory effect of caffeine on the IPSC in nominally Ca2 +-free ACSF. Normalized net current amplitudes were plotted against the time. The average amplitude of the current during the first 5 min was used to the normalization.
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2.5. Materials Isoguvacine was purchased from Tocris Cookson (MO, USA). TTX and fura-2 were purchased from Wako Pure Chemical Industries (Osaka, Japan). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was obtained from Tocris Cookson and D- or D/L-amino-5-phosphonovaleric acid (D- or D/LAP5) was from Research Biochemicals International (MA, USA). BAPTA was purchased from Sigma (MO, USA).
3. Results 3.1. Inhibition of GABAergic inhibitory postsynaptic currents by caffeine Caffeine releases Ca2 + from ryanodine-sensitive intracellular Ca2 + store. The GABAAergic IPSC evoked by iontophoretically applied isoguvacine (mean F S.E.M.; 94.9 F 11.1 pA, n = 8) was inhibited by the perfusion of 10 mM caffeine (17.1 F 3.0% of control, n = 8; Fig. 1A) or 1 mM caffeine (55.3 F 5.1% of control, n = 6). This inhibition was transient, and the GABAAergic response recovered after washing out caffeine (Fig. 1A). The inhibitory effect of caffeine was still observed when normal extracellular solution was replaced by nominally Ca2 +-free solution (Fig. 1B); amplitude of the IPSC after the application of caffeine (10 mM) in nominally Ca2 +-free ACSF was comparable with that of the control (29.6 F 6.6% of control, n = 5). Increasing extracellular calcium concentration up to 12.5 mM per se (which might lead to facilitation of calcium influx through leak channels) did not affect the amplitude of control IPSCs (96.5 F 0.8% of control, n = 5). It has been reported that agonist-induced IPSC and synaptic IPSC have different kinetic properties, and these currents are thought to be mediated by a different subtype of receptors of different subcellular localization [3,14]. To examine the modulation of the synaptic IPSC by caffeine, miniature GABAAergic IPSCs were also recorded in the presence of CNQX, D-AP5, and TTX (Fig. 2A). Caffeine (10 mM) was applied extracellularly. Amplitude distribution of the miniature IPSCs demonstrated that the synaptic IPSCs were inhibited similarly (Fig. 2B; 60.1 F 2.6% of control, n = 3). Decrease in event frequency was also observed during caffeine-induced inhibition of the current. This decrement however, probably resulted from strong postsynaptic reduction of the current amplitude to undetectable magnitude.
Fig. 2. Inhibition of synaptic IPSC by caffeine. Miniature GABAAergic current was separated from excitatory currents by superfusing CNQX and D-AP5. Tetrodotoxin was also added to the perfusing solution. (A) Current traces before, during, and after application of caffeine. (B) Amplitude distributions of the miniature IPSCs.
near 0 mV. Application of caffeine (1 mM) does not change the reversal potential but inhibited the IPSCs at each holding potential between 80 and 40 mV (Fig. 3A). Current – voltage relationships for synaptical IPSCs were also determined. Like agonist- evoked IPSC, IPSC caused by electrostimulation was reversibly inhibited by caffeine (Fig. 3B; 28.7 F 6.0%, n = 10). Current –voltage curves of electrically evoked IPSCs are shown in Fig. 3C. No changes of the reversal potential were observed after application of caffeine, and the inhibition did not show voltage dependency. Current – voltage relationship of miniature IPSC was also determined and similar result was obtained, although average of the miniature IPSC amplitude, especially recorded at near 0 mV, was not so accurate because of failure in event detection (data not shown).
3.2. Current – voltage relationships of the GABAA current
3.3. Caffeine inhibited the inhibitory postsynaptic current independently of intracellular calcium mobilization
To elucidate the nature of inhibition by caffeine, current– voltage curves of GABAergic IPSCs were obtained with or without caffeine. With intrapipette solution of symmetrical Cl concentration, isoguvacine-evoked current reversed at
Caffeine has been thought to exert its inhibitory effect on IPSCs by releasing calcium from intracellular ryanodinesensitive store [2,7,20]. To examine the contribution of intracellular calcium mobilization to the inhibition by caf-
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Fig. 3. Current – voltage relationships for GABAergic IPSC in the presence or absence of caffeine. The GABAergic current was evoked by the application of isoguvacine (A). IPSCs were recorded before (A)-a and after (A)-b the application of 1 mM caffeine. (A)-c I – V relationship of agonist-evoked IPSCs before ( , solid line) and after (n, broken line) the application of caffeine. The IPSC amplitudes were normalized to the amplitude of the IPSC recorded at 80 mV. Data points represent the normalized mean current amplitudes (F S.E.M., n = 8). Electrically evoked IPSC was reversibly inhibited by 10 mM caffeine [(B)-a raw traces and (B)-b temporal pattern of normalized peak current amplitude]. The GABAergic current was evoked by electrical stimulation, and IPSCs were recorded before (C)-a and after (C)-b application of 1 mM caffeine. I – V relationship of electrostimulated IPSCs before ( ) and after (n) application of caffeine was shown in (C)-c. The IPSC amplitudes were normalized to the amplitude of the IPSC recorded at 80 mV. Data points represent the normalized mean current amplitudes (F S.E.M., n = 9).
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feine, neurons were pretreated with several reagents which prevent calcium release from internal store, prior to the application of caffeine. Depletion of the calcium store by
thapsigargin, a Ca2 +-ATPase blocker, did not affect the inhibition by caffeine (14.4 F 1.0%, n = 4; Fig. 4A-a). Ryanodine binds to its specific receptor on membrane of calcium
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Fig. 4. Inhibition of the IPSCs and its independence on Ca2 + release from ryanodine sensitive intracellular store. (A)-a GABAergic IPSC was evoked under the same experimental conditions as described in Fig. 1, except for extracellular application of thapsigargin (10 AM). (A)-b and (A)-c Effect of caffeine on isoguvacine-evoked current was examined with ryanodine (20 AM, b) or ruthenium red [Ru red, 20 AM, (A)-c] introduced intracellularly. (B)-a, effect of caffeine on isoguvacine-evoked GABAergic current was examined with intracellular application of BAPTA (10 mM). (B)-b Effect of caffeine on miniature IPSCs was examined in the presence of intracellularly applied BAPTA. Miniature IPSC was recorded as described in Fig. 2. (C) Caffeine was repetitively applied during Ca2 +-free ACSF perfusion. IPSC was induced by agonist application.
store and, in the micromolar range, causes persistent activation of the receptor leading to the blockade of calcium release. Effect of caffeine on isoguvacine-evoked IPSC was still observed in neurons treated with 20 AM of ryanodine (10.2 F 1.5%, n = 5; Fig. 4A-b). Ruthenium red (20 AM), an inhibitor of calcium-induced calcium release, did not prevent the caffeine-induced inhibition of GABAAergic IPSC when added to the intracellular solution (Fig. 4A-c; 11.1 F 1.5% of control, n = 6). The difference between the IPSC inhibition by caffeine with or without thapsigargin, ryanodine, or ruthenium red was not significant statistically.
Effect of caffeine on the IPSC was then examined by preventing [Ca2 +]i elevation. Intracellularly applied BAPTA (10 mM) could not significantly affect the inhibition by caffeine (Fig. 4B-a; 13.6 F 2.8% of control, n = 7). The caffeine-induced inhibition could not be blocked even by a higher concentration of BAPTA (30 mM). Inhibition of miniature IPSC by caffeine was also observed in the presence of intracellular BAPTA (10 mM, 59.3 F 3.0%, n = 3; Fig. 4B-b). Statistical analysis revealed that the inhibition of GABAergic IPSCs by caffeine was not significantly affected by treatment with BAPTA.
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The relationship between the caffeine-induced inhibition and Ca2 + elevation was further examined under the condition causing Ca2 + store depletion. When Ca2 + influx is prevented by perfusing Ca2 +-free ACSF, Ca2 + store will be depleted by repetitive stimulation of the store. Actually, in CA1 pyramidal neurons, it has been reported that caffeine-depleted Ca2 + stores did not refill when extracellular Ca2 + was removed [10]. During perfusion with nominally Ca2 +-free ACSF, caffeine was applied repetitively (Fig. 4C). In spite of prevention of the intracellular Ca2 + store refilling, extent of the second inhibition of the IPSC by caffeine was comparable to that of the first inhibition (the IPSC inhibition caused by the second application of caffeine was 99.7 F 0.8% of that caused by the first application; n = 3). 3.4. GABAAergic inhibitory postsynaptic current was insensitive to Ca2 +-mobilizing reagents Several reagents increasing [Ca2 +]i were tested for their ability to inhibit the GABAAergic IPSC. In the hippocampal CA3 region, expression of B2 bradykinin receptor, m1, m3, and m5 muscarinic Ach receptors and P2X ATP receptor, has been demonstrated histochemically [6,11,15]. Ligands of these receptors were therefore employed to examine the effect of ligand-evoked calcium mobilization on hippocampal IPSCs. It is known that the activation of B2 bradykinin receptor or m1, m3 or m5 types of muscarinic acetylcholine receptors causes calcium release from IP3-sensitive calcium store and, if the cell is current-clamped, induces subsequent Ca2 + influx through voltage-sensitive calcium channels. ATP stimulates P2X purinergic receptors expressed in this brain region, leading to calcium influx. Neither bradykinin (500 nM), ATP (200 AM), nor acetylcholine (100 AM), that increase [Ca2 +]i by releasing Ca2 + from IP3-sensitive store and/or causing Ca2 + influx, significantly inhibited the GABAAergic current (Fig. 5). Under the same experimental conditions, caffeine inhibited the IPSC distinctly. For the examination of the effect of Ca2 + release from ryanodine-sensitive store on GABAAergic IPSCs, miniature IPSCs were recorded in the presence or absence of cyclic adenosine diphosphate ribose (cADPR, 5 AM) in intrapipette solution. Averages of amplitudes of the IPSCs within each period recorded from both control and cADPR-introduced neuron were calculated and mutually compared. Decrease of the IPSC amplitude recorded by cADPR-containing pipette is, however, not significantly different from that of the control current (data not shown). 3.5. Study of other inhibiting mechanism than elevation of calcium concentration Except for elevating intracellular calcium, caffeine modulates neuronal activity through alteration of adenosine receptor function [8]. Under our experimental conditions, adenosine (100 AM) itself did not affect the
Fig. 5. Insensitivity of GABAergic currents to acetylcholine (Ach), bradykinin (BK), and ATP. (A) Bradykinin (500 nM), ATP (200 AM), or (B) Ach (100 AM) was applied to extracellular solution. The isoguvacineevoked GABAAergic current was recorded as described above. Caffeine (10 mM) was also applied as positive control under the same experimental conditions.
isoguvacine-evoked current (91.4 F 3.9% of control, n = 4; Fig. 6A-a), and pretreatment with adenosine could not prevent the inhibition by caffeine (14.0 F 2.7% of control, n = 3; Fig. 6A-b). The inhibitory effect of caffeine was also observed even when the intracellular solution was preequilibrated with caffeine (10 mM, in pipette solution; Fig. 6B). In the caffeine-preequilibrated neurons, amplitude of the isoguvacine-evoked (Fig. 6B-a) and miniature (Fig. 6B-b) IPSCs was also decreased by the application of caffeine (15.7 F 3.0% of control, n = 7; 68.7 F 3.64% of control, n = 3). Caffeine is known to exert its effect through inhibiting cyclic nucleotide phosphodiesterase followed by the ac-
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Fig. 6. Adenosine or PKA activation did not mimic the effect of caffeine, and preequilibration by caffeine does not prevent the inhibition by externally applied caffeine. Agonist-evoked [(A)-a, (A)-b, (B)-a and (C)] or spontaneously occurred [(B)-b] IPSCs were recorded. (B)-a Effect of extracellularly applied caffeine on the isoguvacine-evoked IPSC was examined after preequilibration of the intracellular solution with caffeine (10 mM, in pipette solution). (B)-b Effect of extracellular application of caffeine on the synaptic IPSC was also examined after preequilibration of the intracellular solution with caffeine. Miniature IPSCs were recorded as described in Fig. 2 legend. The possibility that caffeine exerts its inhibitory effect through activating PKA was tested by using 20 AM forskolin. The IPSC was evoked by postsynaptic application of isoguvacine.
cumulation of cyclic AMP [8] leading to the activation of protein kinase A (PKA). To determine whether the inhibition by caffeine was mediated by PKA activation or not, the experiment was repeated using forskolin instead of caffeine. As shown in Fig. 6C, the amplitude of isoguvacine-evoked IPSC was not affected by extracellular perfusion of 20 AM forskolin (91.1 F 5.4% of control, n = 5).
3.6. Calcium imaging and simultaneous current recording We tried to measure intracellular calcium concentration in hippocampal slice simultaneously with current recording. Fluorescence imaging revealed distinct increment of calcium after caffeine application (Fig. 7A). The caffeine-induced elevation of fluorescence ratio was prevented by the intracellular application of BAPTA (10 mM) or ryanodine
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Fig. 7. Fluorescent Ca2 + imaging revealed calcium-independent inhibition of the IPSC by caffeine. Cells were loaded with calcium indicator fura-2 (10 AM) intracellularly, and fura-2 fluorescence intensity and IPSC amplitude were simultaneously measured. (A) Caffeine (10 mM) increased 340 nm/380 nm ratio of fura-2 fluorescence intensity. Simultaneous current recording demonstrated inhibition of isoguvacine-evoked IPSC by caffeine. Increment of fluorescent ratio was prevented by treatment with 10 mM BAPTA (B) and 20 AM ryanodine (C). The IPSCs however were inhibited by application of caffeine.
(20 AM). Simultaneously recorded isoguvacine-evoked IPSCs however were inhibited by caffeine irrespective of blockers (Fig. 7B and C).
4. Discussion In this study, GABAAergic IPSC inhibition by caffeine was still observed after the elimination of extracellular calcium (Fig. 1B) or pretreatment with inhibitors of calcium release (Fig. 4A). Possible involvement of calcium release from internal store in the inhibition is also contradictory to the experimental result shown in Fig. 4C. Ca2 + independency of the inhibitory effect of caffeine was also supported by the experiment performed with a fast calcium chelator BAPTA to preclude [Ca2 +]i elevation (Fig. 4B). Furthermore, Ca2 + fluorescence imaging with simultaneous current recording indicated that IPSC inhibition by caffeine occurred independent of intracellular calcium mobilization (Fig. 7). Occurrence of the [Ca2 +]i-indepedent modulation of GABAAergic current by caffeine has not so far been established. From BAPTA sensitivity of the inhibition, several investigators concluded that the effect of caffeine on the IPSC was Ca2 +-dependent (GABA-induced current in retinal amacrine cells [20], GABA-induced and in isolated turtle retina ganglion cells [2], miniature GABAAergic current in hippocampal slice preparation [7]) or Ca2 +-
indepedent (agonist-evoked glycinergic current in dissociated hippocampal neurons [19]). Ca2 + dependency has been also suggested by the experiment using other chelating reagent, EGTA (isoguvacine-evoked IPSC in melanotrophs [16]). There is no direct evidence settling why the inhibition of IPSC is susceptible to [Ca2 +]i increment in some cases but not in other cases. A minor fraction of the inhibition by caffeine may possibly be sensitive to [Ca2 +]i elevation. At a higher temperature, Ca2 + (a diffusible messenger)-mediated fraction of the inhibition might be facilitated when compared with the fraction in the present experiment performed at room temperature. Nevertheless, the [Ca2 +]i elevationindependent fraction of inhibition certainly exists and is outstanding, whereas [Ca2 +]i elevation-mediated fraction is feeble. Alternatively, [Ca2 +]i dependency may differ between the IPSCs through synaptic receptors and those via extrasynaptic ones. Kinetically different classes of GABAAergic IPSCs were reported in the hippocampus and other tissues [3,14]; the IPSCs are mediated by synaptic or extrasynaptic receptors. The receptors are also classified into somatic (which mediate GABAA,fast IPSC) or dendritic groups (which mediate GABAA,slow IPSC), and GABAA,slow IPSC is suggested to be mediated by extrasynaptic type of receptors which are located at the soma. In our experiment, both agonist-evoked and synaptic IPSCs were inhibited by caffeine in the presence of
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BAPTA, suggesting that the receptor class does not always decide the calcium dependency of the inhibition by caffeine. Difference in tissue preparation may also contribute to the discrepancy among these experimental results, because some treatments of neurons may lower the Ca2 +buffering capacity of neurons. If the inhibitory effect on GABAergic IPSC taken from dissociated neurons is compared to the effect on synaptic IPSC measured from neurons in slice preparation, the different capacities for calcium buffering between the neurons of the two preparations have to be taken into consideration. In this experiment however, both synaptic and extrasynaptic IPSC were recorded from slice preparation, and difference in methods of cell preparation did not exist at all. Variance of the BAPTA sensitivities between dissociated neuron and slice preparation may be derived from other factors. Other possibility is that a network-driven and calciumdependent modulation of the IPSC might occur in some brain region after caffeine application. The mechanism by which caffeine inhibits the IPSC independently of [Ca2 +]i-elevation has been unknown. Besides releasing calcium from the intracellular store, caffeine variously modulates intracellular signal transduction. There is a possibility that IPSC inhibition by caffeine might be caused via these modulations, such as the activation of PKA, through increasing cyclic AMP level. Activation of adenylate cyclase by forskolin however could not mimic the inhibition by caffeine (Fig. 6C), suggesting that caffeine did not exert its inhibitory effect through increasing cyclic AMP level. Caffeine has also been reported to bind to adenosine receptors and antagonize the action of agonists at these receptors in the central nervous system [8]. Pretreatment by adenosine however could not obstruct the effect of caffeine on the GABAergic IPSC (Fig. 6A), indicating that caffeine does not exert its effect through the blockade of adenosine receptors on the IPSC, in this case. In several previous reports, second messenger-independent mechanisms of the IPSC inhibition by caffeine have been proposed. In the presence of caffeine, the concentration – response curves for glycinergic currents were shifted in the hippocampal dissociated neurons, indicating that caffeine acted as a competitive antagonist [19]. Lopez et al. [12] demonstrated that caffeine did not interact with the benzodiazepine site but decreased GABA-induced Cl uptake in murine cortical synaptosomes, and they suggested the possibility that caffeine may interact with GABAA receptor by the alteration of coupling between GABA and the Cl channel. Sugimoto et al. [17] reported that theophilline inhibited GABAAergic IPSC in a competitive but voltage-dependent manner. Same as in our experimental result (shown in Fig. 3), voltage-independent inhibition by caffeine of glycinergic IPSC [19] and GABAAergic IPSC [20] was also reported, although in
the latter case, the inhibition was not competitive. Furthermore, experimental result on the hippocampal neurons preequilibrated by caffeine (Fig. 6B) indicates that this xanthine derivative exerts its effect extracellularly. Although direct evidence is still deficient, these results suggest that the inhibition is competitive at least in our system. Calcium-independent inhibition by caffeine however might not necessarily exclude a possible contribution of [Ca2 +]i in modulation of GABAAergic IPSCs. According to several reports, elevation of [Ca2 +]i by other methods decreased GABAergic IPSCs in a Ca2 +-dependent manner. Aguayo et al. [1] demonstrated that dialysing the neurons with very low concentration of intracellular Ca2 + (10 mM BAPTA, 0 Ca2 +) decreased amplitude of the GABAAergic currents. It has also been reported that the IPSCs were modulated by calcium mobilization from intracellular calcium stores ([11] in visual cortex) or calcium influx across plasma membrane (through NMDA channel, [1] in cortical neurons, [5], [16] in CA1 pyramidal cells). Ineffectiveness of several calcium-mobilizing ligands on the IPSCs (Fig. 5) however, suggested that caffeine- or other reagentinduced [Ca2 +]i mobilization hardly affects the GABAAergic currents in the present experimental conditions. Effect of secondary-occurring Ca2 + influx was negligible under the voltage-clamped condition, but it has been reported that calcium influx through voltage-dependent calcium channels did not inhibit the GABAAergic IPSC in retina [2] and in hippocampal neurons [16]. In some systems, extensive cytosolic [Ca2 +]i elevation caused by general incentive, or local [Ca2 +]i elevation by specific Ca2 + influx through proximal channels, might inhibit GABAAergic current. Both extra-synaptic and synaptic GABAAergic IPSCs were reversibly inhibited by application of caffeine. The inhibition of the IPSCs by caffeine was still observed even when [Ca2 +]i increase was prevented. Other reagents which increase [Ca2 +]i could not inhibit the GABAergic IPSCs. These results suggest that the elevation of [Ca2 +]i by store release does not exert major effect on GABAergic transduction in this brain region, and the most part of the inhibition by caffeine is caused independently of intracellular calcium. Inhibition of GABAAergic currents however might be forced by artificially higher [Ca2 +]i elevation or calcium influx. Studies are in progress for the direct demonstration of the inhibitory effect of caffeine on hippocampal GABAA receptor.
Acknowledgements This work was supported in part by Grant-in-Aid (07279105) for Scientific Research on Priority Areas on ‘‘Functional Development of Neural Circuits’’, the Ministry of Education, Science, Sports and Culture of Japan, and also by Research for the Future Project 96L00310
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program from the Japan Society for the Promotion of Science.
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Research report
Calcium-independent inhibition of GABAA current by caffeine in hippocampal slices M. Taketo a,*, H. Matsuda a, T. Yoshioka b a
Department of Physiology 1, Faculty of Medicine, Kansai Medical University, 10-15 Fumizono-cho Moriguchi, Osaka 570-8506, Japan b Department of Molecular Neurobiology, Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1, Ohkubo, Shinjuku, Tokyo 169-8555, Japan Accepted 1 May 2004 Available Available online online 24 June 2004
Abstract Although inhibitory postsynaptic currents (IPSCs) mediated by GABAA receptor is thought to be affected by intracellular calcium ion concentration ([Ca2 +]i), origin or route of [Ca2 +]i increment has not been well elucidated. Reports on the effect of [Ca2 +]i elevation on GABAAergic IPSCs per se are also controversial. In this study, effects of caffeine and several other [Ca2 +]i-mobilizing drugs were examined on the IPSCs in acute slices of rat hippocampus. Using the patch clamp recording method, spontaneous and evoked currents were recorded from CA3 neurons. Caffeine strongly inhibited both extra-synaptic and synaptic GABAergic IPSCs, regardless of the presence or absence of extracellular Ca2 +. This inhibition was not relieved by the intracellular application of EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,NV,NV-tetraacetic acid (BAPTA). This inhibition by caffeine was not prevented by preequilibration with caffeine. Ca2 + store depletion caused by thapsigargin or repetitive stimulation by caffeine could not prevent the inhibition. Moreover, ruthenium red and ryanodine could not overcome the inhibition. On the contrary, GABAAergic currents were not inhibited by stimulation with several Ca2 +mobilizing agonists. Forskolin could not mimic the effect of caffeine on the IPSC, and caffeine inhibited the IPSC in the presence of adenosine. These results suggest that intracellular Ca2 + mobilization through ryanodine-sensitive store stimulation does not significantly affect GABAergic IPSCs, and most of the inhibitory effect of caffeine is independent of [Ca2 +]i elevation under the present experimental conditions. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: GABA receptors Keywords: GABAA receptor; Hippocampus; Caffeine; Intracellular Ca2+
1. Introduction GABAA receptor mediates major inhibitory postsynaptic current (IPSC) in the central nervous system. Modulations of the efficacy of GABAAergic synaptic transmission have
Abbreviations: ACSF, artificial cerebrospinal fluid; BAPTA, 1,2-bis(2aminophenoxy)-ethane-N,N,NV,NV-tetraacetic acid; CNQX, 6-cyano-7nitroquinoxaline-2,3-dione; cADPR, cyclic adenosine diphosphate ribose; D-AP5, D( )-2-amino-5-phosphonovaleric acid; IPSC, inhibitory postsynaptic current; sIPSC, spontaneous inhibitory postsynaptic current; TTX, tetrodotoxin * Corresponding author. Tel.: +81-669-93-9422; fax: +81-669-921409. E-mail address: [email protected] (M. Taketo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.008
been reported in several brain regions, and the elevation of intracellular calcium concentration ([Ca2 +]i) has been suggested to underlie the modulations [9]. Effects of [Ca2 +]i elevation on GABAergic IPSC have been studied in several systems, but the results are controversial [1,2]. Even whether [Ca2 +]i elevation regulates the IPSC positively or negatively has not been decided. One of the generally used reagent to increase [Ca2 +]i in these experiments is caffeine, which releases calcium from ryanodine-sensitive intracellular store. Inhibitory effect of caffeine on GABAergic postsynaptic currents has been reported and regarded as the consequence of increasing [Ca2 +]i [2,7,20]. In several reports however, caffeine was suggested to exert its effect on inhibitory transmission, unrelated to releasing Ca2 + from ryanodine-sensitive Ca2 + store [12,19].
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The conflicting results may partly be caused by difference in the stimulation methods employed for elevating [Ca2 +]i in each experiment. Tissue distribution and subcellular localization of the receptor with different subunit subtypes may also contribute to the inconsistency of the results, because many subunit subtypes of heteropentameric GABAA receptor have been cloned and demonstrated to have different pharmacological properties when reconstructed [4]. It has been reported that synaptic and extra synaptic GABAergic IPSCs are mediated by receptors of different subclasses with different pharmacological and kinetical properties. In hippocampal neurons, calcium-dependent [7] and -indepedent [19] inhibitions of the IPSC have been described. The discrepancy may be ascribed to the different nature of the synaptic IPSC and extra synaptic IPSC, because the former report investigated miniature IPSC and the latter dealt with agonist-evoked IPSC. Besides these factors, methods of tissue preparation have been suggested to cause the variation of experimental results [7]. Among previous reports, there are few studies determining the inhibitory effect on IPSCs which are mediated by different subclasses of receptors in same tissue preparation. In addition, [Ca2 +]i dependency of the inhibition by caffeine was mainly determined only by the application of a chelating agent [such as 1,2-bis(2-aminophenoxy)-ethane-N,N,NV,NV-tetraacetic acid (BAPTA)] in previous studies. In this study, the effect of caffeine on both the synaptic and extra synaptic GABAergic current was accordingly examined in acute slices of young rat hippocampus, using the patch clamp recording technique. Calcium dependency of the effect on the IPSC was further investigated using several methods. Spontaneous and evoked GABAAergic IPSCs were also recorded in the presence of other calcium mobilizing reagents: acetylcholine, bradykinin, or ATP. Based on the experimental analyses, mechanism of the calcium-independent GABAAergic current inhibition was discussed.
2. Materials and methods 2.1. Slice preparation Hippocampal slices were prepared from Wister rats aged 4 to 12 days, as reported previously [18]. All experimental protocols were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals revised 1996 and the guidelines for animal research of the Physiological Society of Japan. All efforts were made to minimize animal suffering and to reduce the number of animals used. Following diethyl ether anesthesia, the animals were decapitated and the brains were removed and cooled in an artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 138.6, KCl 3.35, NaHCO 3 21.0, NaH2PO4 0.6, CaCl2 2.5, MgCl2 1.0, glucose 10.0) bubbled
to equilibrium with 95% O2/5% CO2 for 2 min. Then, the brains were dissected, and the removed hippocampi were sliced to a thickness of 250 –400 Am using a vibrating tissue slicer (model DTK1000, D.S.K., Japan). Slices were placed in a storage chamber filled with ACSF, and then kept at least 1 h at room temperature. Before recording, the slices were individually transferred to a recording chamber and continuously perfused with ACSF during experiment. In some specified experiments, NaCl concentration in the extracellular solution was reduced to 118.6 mM, and CaCl2 concentration was increased to 12.5 mM. 2.2. Whole cell recordings Whole cell patch clamp recordings were performed in pyramidal neurons of the CA3 region. Each cell was identified morphologically using cooled charge coupled device (CCD) camera (model C2400-79, Hamamatsu Photonics, Japan) connected to a microscope with infrared filter (model E600-UD, Nikon, Japan). Patch electrodes (2– 4 MV after filling with intrapipette solution) were pulled from borosilicate glass using a twostage vertical puller (model PP-83, Narishige, Japan) and filled with symmetrical chloride-intracellular solution [composition in mM: CsCl 140, CaCl2 1, MgCl2 2, ethyleneglycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) 10, HEPES 10, ATP 2, adjusted to pH 7.3 using CsOH]. In some experiments, CaCl2 was omitted and EGTA in intracellular solution was increased or replaced by 10 or 30 mM BAPTA. Under the experimental conditions, the GABAergic IPSCs were reversed at f0 mV equivalent to the ECl predicted by Nernst equation. Cells were voltage-clamped at 70 mV, and the GABAergic IPSC appeared as inward current. Electrosignals were measured with an EPC7 patch clamp amplifier (List, Germany) and filtered at 3 kHz. The signals were digitized at 10 kHz and stored using DigiData 1200 with the pClamp6 data collection and analysis software (Axon Instruments, USA). Data recorded from cells with significant change in series resistance during experiment were discarded. Isoguvacine was focally applied by iontophoresis (3– 15 ms) through a microelectrode placed near cell body or proximal dendrite of recorded neuron, in the presence of 0.5 AM tetrodotoxin (TTX). Electrostimulation was applied by glass microelectrode filled with ACSF (3 –10 V and 200 As) placed in stratum pyramidale close to the soma of the recorded neuron. All experiments were carried out at room temperature (f23 jC). 2.3. Calcium imaging Whole cell current recording was performed as described above, and calcium imaging was performed simultaneously. Cells were loaded with calcium indicator through a patch pipette (filled with standard CsCl solution of the same composition as described above, except for the addition of
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10 AM fura-2). The GABAergic IPSC was evoked by isoguvacine. Fluorescence image was obtained through an objective lens (CFI Fluor, Nikon) and a cooled CCD camera (model C4742-95-12ER, Hamamatsu Photonics) under alternating excitation wavelengths of 340 and 380 nm. Images were stored and analyzed using a digital image processor (Aquacosmos, Hamamatsu Photonics). Calcium concentration was expressed as the ratio of the fura-2 fluorescence intensities excited at 340 and 380 nm. A normalized ratio value was calculated for the temporal analysis of [Ca2 +]i
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alteration (average of values during the first 3 min was used as baseline ratio). The acquisition rate was usually 0.1 Hz. 2.4. Data analysis Average of interevent interval and amplitude distributions of spontaneous IPSCs obtained in control and in test conditions were calculated during 3 or 5 min. Statistical analyses were carried out using unpaired t-test with Welch’s correction (a level of significance of P V 0.05).
Fig. 1. Extra-synaptic GABAergic IPSC recorded from CA3 neurons. GABAergic current was evoked by iontophoretically applied 100 AM isoguvacine (indicated by arrow). (A)-a Sensitivity of the GABAAergic IPSC to caffeine. (A)-b Amplitudes of baseline, peak, and net current plotted against time. (B) The inhibitory effect of caffeine on the IPSC in nominally Ca2 +-free ACSF. Normalized net current amplitudes were plotted against the time. The average amplitude of the current during the first 5 min was used to the normalization.
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2.5. Materials Isoguvacine was purchased from Tocris Cookson (MO, USA). TTX and fura-2 were purchased from Wako Pure Chemical Industries (Osaka, Japan). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was obtained from Tocris Cookson and D- or D/L-amino-5-phosphonovaleric acid (D- or D/LAP5) was from Research Biochemicals International (MA, USA). BAPTA was purchased from Sigma (MO, USA).
3. Results 3.1. Inhibition of GABAergic inhibitory postsynaptic currents by caffeine Caffeine releases Ca2 + from ryanodine-sensitive intracellular Ca2 + store. The GABAAergic IPSC evoked by iontophoretically applied isoguvacine (mean F S.E.M.; 94.9 F 11.1 pA, n = 8) was inhibited by the perfusion of 10 mM caffeine (17.1 F 3.0% of control, n = 8; Fig. 1A) or 1 mM caffeine (55.3 F 5.1% of control, n = 6). This inhibition was transient, and the GABAAergic response recovered after washing out caffeine (Fig. 1A). The inhibitory effect of caffeine was still observed when normal extracellular solution was replaced by nominally Ca2 +-free solution (Fig. 1B); amplitude of the IPSC after the application of caffeine (10 mM) in nominally Ca2 +-free ACSF was comparable with that of the control (29.6 F 6.6% of control, n = 5). Increasing extracellular calcium concentration up to 12.5 mM per se (which might lead to facilitation of calcium influx through leak channels) did not affect the amplitude of control IPSCs (96.5 F 0.8% of control, n = 5). It has been reported that agonist-induced IPSC and synaptic IPSC have different kinetic properties, and these currents are thought to be mediated by a different subtype of receptors of different subcellular localization [3,14]. To examine the modulation of the synaptic IPSC by caffeine, miniature GABAAergic IPSCs were also recorded in the presence of CNQX, D-AP5, and TTX (Fig. 2A). Caffeine (10 mM) was applied extracellularly. Amplitude distribution of the miniature IPSCs demonstrated that the synaptic IPSCs were inhibited similarly (Fig. 2B; 60.1 F 2.6% of control, n = 3). Decrease in event frequency was also observed during caffeine-induced inhibition of the current. This decrement however, probably resulted from strong postsynaptic reduction of the current amplitude to undetectable magnitude.
Fig. 2. Inhibition of synaptic IPSC by caffeine. Miniature GABAAergic current was separated from excitatory currents by superfusing CNQX and D-AP5. Tetrodotoxin was also added to the perfusing solution. (A) Current traces before, during, and after application of caffeine. (B) Amplitude distributions of the miniature IPSCs.
near 0 mV. Application of caffeine (1 mM) does not change the reversal potential but inhibited the IPSCs at each holding potential between 80 and 40 mV (Fig. 3A). Current – voltage relationships for synaptical IPSCs were also determined. Like agonist- evoked IPSC, IPSC caused by electrostimulation was reversibly inhibited by caffeine (Fig. 3B; 28.7 F 6.0%, n = 10). Current –voltage curves of electrically evoked IPSCs are shown in Fig. 3C. No changes of the reversal potential were observed after application of caffeine, and the inhibition did not show voltage dependency. Current – voltage relationship of miniature IPSC was also determined and similar result was obtained, although average of the miniature IPSC amplitude, especially recorded at near 0 mV, was not so accurate because of failure in event detection (data not shown).
3.2. Current – voltage relationships of the GABAA current
3.3. Caffeine inhibited the inhibitory postsynaptic current independently of intracellular calcium mobilization
To elucidate the nature of inhibition by caffeine, current– voltage curves of GABAergic IPSCs were obtained with or without caffeine. With intrapipette solution of symmetrical Cl concentration, isoguvacine-evoked current reversed at
Caffeine has been thought to exert its inhibitory effect on IPSCs by releasing calcium from intracellular ryanodinesensitive store [2,7,20]. To examine the contribution of intracellular calcium mobilization to the inhibition by caf-
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Fig. 3. Current – voltage relationships for GABAergic IPSC in the presence or absence of caffeine. The GABAergic current was evoked by the application of isoguvacine (A). IPSCs were recorded before (A)-a and after (A)-b the application of 1 mM caffeine. (A)-c I – V relationship of agonist-evoked IPSCs before ( , solid line) and after (n, broken line) the application of caffeine. The IPSC amplitudes were normalized to the amplitude of the IPSC recorded at 80 mV. Data points represent the normalized mean current amplitudes (F S.E.M., n = 8). Electrically evoked IPSC was reversibly inhibited by 10 mM caffeine [(B)-a raw traces and (B)-b temporal pattern of normalized peak current amplitude]. The GABAergic current was evoked by electrical stimulation, and IPSCs were recorded before (C)-a and after (C)-b application of 1 mM caffeine. I – V relationship of electrostimulated IPSCs before ( ) and after (n) application of caffeine was shown in (C)-c. The IPSC amplitudes were normalized to the amplitude of the IPSC recorded at 80 mV. Data points represent the normalized mean current amplitudes (F S.E.M., n = 9).
.
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feine, neurons were pretreated with several reagents which prevent calcium release from internal store, prior to the application of caffeine. Depletion of the calcium store by
thapsigargin, a Ca2 +-ATPase blocker, did not affect the inhibition by caffeine (14.4 F 1.0%, n = 4; Fig. 4A-a). Ryanodine binds to its specific receptor on membrane of calcium
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Fig. 4. Inhibition of the IPSCs and its independence on Ca2 + release from ryanodine sensitive intracellular store. (A)-a GABAergic IPSC was evoked under the same experimental conditions as described in Fig. 1, except for extracellular application of thapsigargin (10 AM). (A)-b and (A)-c Effect of caffeine on isoguvacine-evoked current was examined with ryanodine (20 AM, b) or ruthenium red [Ru red, 20 AM, (A)-c] introduced intracellularly. (B)-a, effect of caffeine on isoguvacine-evoked GABAergic current was examined with intracellular application of BAPTA (10 mM). (B)-b Effect of caffeine on miniature IPSCs was examined in the presence of intracellularly applied BAPTA. Miniature IPSC was recorded as described in Fig. 2. (C) Caffeine was repetitively applied during Ca2 +-free ACSF perfusion. IPSC was induced by agonist application.
store and, in the micromolar range, causes persistent activation of the receptor leading to the blockade of calcium release. Effect of caffeine on isoguvacine-evoked IPSC was still observed in neurons treated with 20 AM of ryanodine (10.2 F 1.5%, n = 5; Fig. 4A-b). Ruthenium red (20 AM), an inhibitor of calcium-induced calcium release, did not prevent the caffeine-induced inhibition of GABAAergic IPSC when added to the intracellular solution (Fig. 4A-c; 11.1 F 1.5% of control, n = 6). The difference between the IPSC inhibition by caffeine with or without thapsigargin, ryanodine, or ruthenium red was not significant statistically.
Effect of caffeine on the IPSC was then examined by preventing [Ca2 +]i elevation. Intracellularly applied BAPTA (10 mM) could not significantly affect the inhibition by caffeine (Fig. 4B-a; 13.6 F 2.8% of control, n = 7). The caffeine-induced inhibition could not be blocked even by a higher concentration of BAPTA (30 mM). Inhibition of miniature IPSC by caffeine was also observed in the presence of intracellular BAPTA (10 mM, 59.3 F 3.0%, n = 3; Fig. 4B-b). Statistical analysis revealed that the inhibition of GABAergic IPSCs by caffeine was not significantly affected by treatment with BAPTA.
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The relationship between the caffeine-induced inhibition and Ca2 + elevation was further examined under the condition causing Ca2 + store depletion. When Ca2 + influx is prevented by perfusing Ca2 +-free ACSF, Ca2 + store will be depleted by repetitive stimulation of the store. Actually, in CA1 pyramidal neurons, it has been reported that caffeine-depleted Ca2 + stores did not refill when extracellular Ca2 + was removed [10]. During perfusion with nominally Ca2 +-free ACSF, caffeine was applied repetitively (Fig. 4C). In spite of prevention of the intracellular Ca2 + store refilling, extent of the second inhibition of the IPSC by caffeine was comparable to that of the first inhibition (the IPSC inhibition caused by the second application of caffeine was 99.7 F 0.8% of that caused by the first application; n = 3). 3.4. GABAAergic inhibitory postsynaptic current was insensitive to Ca2 +-mobilizing reagents Several reagents increasing [Ca2 +]i were tested for their ability to inhibit the GABAAergic IPSC. In the hippocampal CA3 region, expression of B2 bradykinin receptor, m1, m3, and m5 muscarinic Ach receptors and P2X ATP receptor, has been demonstrated histochemically [6,11,15]. Ligands of these receptors were therefore employed to examine the effect of ligand-evoked calcium mobilization on hippocampal IPSCs. It is known that the activation of B2 bradykinin receptor or m1, m3 or m5 types of muscarinic acetylcholine receptors causes calcium release from IP3-sensitive calcium store and, if the cell is current-clamped, induces subsequent Ca2 + influx through voltage-sensitive calcium channels. ATP stimulates P2X purinergic receptors expressed in this brain region, leading to calcium influx. Neither bradykinin (500 nM), ATP (200 AM), nor acetylcholine (100 AM), that increase [Ca2 +]i by releasing Ca2 + from IP3-sensitive store and/or causing Ca2 + influx, significantly inhibited the GABAAergic current (Fig. 5). Under the same experimental conditions, caffeine inhibited the IPSC distinctly. For the examination of the effect of Ca2 + release from ryanodine-sensitive store on GABAAergic IPSCs, miniature IPSCs were recorded in the presence or absence of cyclic adenosine diphosphate ribose (cADPR, 5 AM) in intrapipette solution. Averages of amplitudes of the IPSCs within each period recorded from both control and cADPR-introduced neuron were calculated and mutually compared. Decrease of the IPSC amplitude recorded by cADPR-containing pipette is, however, not significantly different from that of the control current (data not shown). 3.5. Study of other inhibiting mechanism than elevation of calcium concentration Except for elevating intracellular calcium, caffeine modulates neuronal activity through alteration of adenosine receptor function [8]. Under our experimental conditions, adenosine (100 AM) itself did not affect the
Fig. 5. Insensitivity of GABAergic currents to acetylcholine (Ach), bradykinin (BK), and ATP. (A) Bradykinin (500 nM), ATP (200 AM), or (B) Ach (100 AM) was applied to extracellular solution. The isoguvacineevoked GABAAergic current was recorded as described above. Caffeine (10 mM) was also applied as positive control under the same experimental conditions.
isoguvacine-evoked current (91.4 F 3.9% of control, n = 4; Fig. 6A-a), and pretreatment with adenosine could not prevent the inhibition by caffeine (14.0 F 2.7% of control, n = 3; Fig. 6A-b). The inhibitory effect of caffeine was also observed even when the intracellular solution was preequilibrated with caffeine (10 mM, in pipette solution; Fig. 6B). In the caffeine-preequilibrated neurons, amplitude of the isoguvacine-evoked (Fig. 6B-a) and miniature (Fig. 6B-b) IPSCs was also decreased by the application of caffeine (15.7 F 3.0% of control, n = 7; 68.7 F 3.64% of control, n = 3). Caffeine is known to exert its effect through inhibiting cyclic nucleotide phosphodiesterase followed by the ac-
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Fig. 6. Adenosine or PKA activation did not mimic the effect of caffeine, and preequilibration by caffeine does not prevent the inhibition by externally applied caffeine. Agonist-evoked [(A)-a, (A)-b, (B)-a and (C)] or spontaneously occurred [(B)-b] IPSCs were recorded. (B)-a Effect of extracellularly applied caffeine on the isoguvacine-evoked IPSC was examined after preequilibration of the intracellular solution with caffeine (10 mM, in pipette solution). (B)-b Effect of extracellular application of caffeine on the synaptic IPSC was also examined after preequilibration of the intracellular solution with caffeine. Miniature IPSCs were recorded as described in Fig. 2 legend. The possibility that caffeine exerts its inhibitory effect through activating PKA was tested by using 20 AM forskolin. The IPSC was evoked by postsynaptic application of isoguvacine.
cumulation of cyclic AMP [8] leading to the activation of protein kinase A (PKA). To determine whether the inhibition by caffeine was mediated by PKA activation or not, the experiment was repeated using forskolin instead of caffeine. As shown in Fig. 6C, the amplitude of isoguvacine-evoked IPSC was not affected by extracellular perfusion of 20 AM forskolin (91.1 F 5.4% of control, n = 5).
3.6. Calcium imaging and simultaneous current recording We tried to measure intracellular calcium concentration in hippocampal slice simultaneously with current recording. Fluorescence imaging revealed distinct increment of calcium after caffeine application (Fig. 7A). The caffeine-induced elevation of fluorescence ratio was prevented by the intracellular application of BAPTA (10 mM) or ryanodine
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Fig. 7. Fluorescent Ca2 + imaging revealed calcium-independent inhibition of the IPSC by caffeine. Cells were loaded with calcium indicator fura-2 (10 AM) intracellularly, and fura-2 fluorescence intensity and IPSC amplitude were simultaneously measured. (A) Caffeine (10 mM) increased 340 nm/380 nm ratio of fura-2 fluorescence intensity. Simultaneous current recording demonstrated inhibition of isoguvacine-evoked IPSC by caffeine. Increment of fluorescent ratio was prevented by treatment with 10 mM BAPTA (B) and 20 AM ryanodine (C). The IPSCs however were inhibited by application of caffeine.
(20 AM). Simultaneously recorded isoguvacine-evoked IPSCs however were inhibited by caffeine irrespective of blockers (Fig. 7B and C).
4. Discussion In this study, GABAAergic IPSC inhibition by caffeine was still observed after the elimination of extracellular calcium (Fig. 1B) or pretreatment with inhibitors of calcium release (Fig. 4A). Possible involvement of calcium release from internal store in the inhibition is also contradictory to the experimental result shown in Fig. 4C. Ca2 + independency of the inhibitory effect of caffeine was also supported by the experiment performed with a fast calcium chelator BAPTA to preclude [Ca2 +]i elevation (Fig. 4B). Furthermore, Ca2 + fluorescence imaging with simultaneous current recording indicated that IPSC inhibition by caffeine occurred independent of intracellular calcium mobilization (Fig. 7). Occurrence of the [Ca2 +]i-indepedent modulation of GABAAergic current by caffeine has not so far been established. From BAPTA sensitivity of the inhibition, several investigators concluded that the effect of caffeine on the IPSC was Ca2 +-dependent (GABA-induced current in retinal amacrine cells [20], GABA-induced and in isolated turtle retina ganglion cells [2], miniature GABAAergic current in hippocampal slice preparation [7]) or Ca2 +-
indepedent (agonist-evoked glycinergic current in dissociated hippocampal neurons [19]). Ca2 + dependency has been also suggested by the experiment using other chelating reagent, EGTA (isoguvacine-evoked IPSC in melanotrophs [16]). There is no direct evidence settling why the inhibition of IPSC is susceptible to [Ca2 +]i increment in some cases but not in other cases. A minor fraction of the inhibition by caffeine may possibly be sensitive to [Ca2 +]i elevation. At a higher temperature, Ca2 + (a diffusible messenger)-mediated fraction of the inhibition might be facilitated when compared with the fraction in the present experiment performed at room temperature. Nevertheless, the [Ca2 +]i elevationindependent fraction of inhibition certainly exists and is outstanding, whereas [Ca2 +]i elevation-mediated fraction is feeble. Alternatively, [Ca2 +]i dependency may differ between the IPSCs through synaptic receptors and those via extrasynaptic ones. Kinetically different classes of GABAAergic IPSCs were reported in the hippocampus and other tissues [3,14]; the IPSCs are mediated by synaptic or extrasynaptic receptors. The receptors are also classified into somatic (which mediate GABAA,fast IPSC) or dendritic groups (which mediate GABAA,slow IPSC), and GABAA,slow IPSC is suggested to be mediated by extrasynaptic type of receptors which are located at the soma. In our experiment, both agonist-evoked and synaptic IPSCs were inhibited by caffeine in the presence of
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BAPTA, suggesting that the receptor class does not always decide the calcium dependency of the inhibition by caffeine. Difference in tissue preparation may also contribute to the discrepancy among these experimental results, because some treatments of neurons may lower the Ca2 +buffering capacity of neurons. If the inhibitory effect on GABAergic IPSC taken from dissociated neurons is compared to the effect on synaptic IPSC measured from neurons in slice preparation, the different capacities for calcium buffering between the neurons of the two preparations have to be taken into consideration. In this experiment however, both synaptic and extrasynaptic IPSC were recorded from slice preparation, and difference in methods of cell preparation did not exist at all. Variance of the BAPTA sensitivities between dissociated neuron and slice preparation may be derived from other factors. Other possibility is that a network-driven and calciumdependent modulation of the IPSC might occur in some brain region after caffeine application. The mechanism by which caffeine inhibits the IPSC independently of [Ca2 +]i-elevation has been unknown. Besides releasing calcium from the intracellular store, caffeine variously modulates intracellular signal transduction. There is a possibility that IPSC inhibition by caffeine might be caused via these modulations, such as the activation of PKA, through increasing cyclic AMP level. Activation of adenylate cyclase by forskolin however could not mimic the inhibition by caffeine (Fig. 6C), suggesting that caffeine did not exert its inhibitory effect through increasing cyclic AMP level. Caffeine has also been reported to bind to adenosine receptors and antagonize the action of agonists at these receptors in the central nervous system [8]. Pretreatment by adenosine however could not obstruct the effect of caffeine on the GABAergic IPSC (Fig. 6A), indicating that caffeine does not exert its effect through the blockade of adenosine receptors on the IPSC, in this case. In several previous reports, second messenger-independent mechanisms of the IPSC inhibition by caffeine have been proposed. In the presence of caffeine, the concentration – response curves for glycinergic currents were shifted in the hippocampal dissociated neurons, indicating that caffeine acted as a competitive antagonist [19]. Lopez et al. [12] demonstrated that caffeine did not interact with the benzodiazepine site but decreased GABA-induced Cl uptake in murine cortical synaptosomes, and they suggested the possibility that caffeine may interact with GABAA receptor by the alteration of coupling between GABA and the Cl channel. Sugimoto et al. [17] reported that theophilline inhibited GABAAergic IPSC in a competitive but voltage-dependent manner. Same as in our experimental result (shown in Fig. 3), voltage-independent inhibition by caffeine of glycinergic IPSC [19] and GABAAergic IPSC [20] was also reported, although in
the latter case, the inhibition was not competitive. Furthermore, experimental result on the hippocampal neurons preequilibrated by caffeine (Fig. 6B) indicates that this xanthine derivative exerts its effect extracellularly. Although direct evidence is still deficient, these results suggest that the inhibition is competitive at least in our system. Calcium-independent inhibition by caffeine however might not necessarily exclude a possible contribution of [Ca2 +]i in modulation of GABAAergic IPSCs. According to several reports, elevation of [Ca2 +]i by other methods decreased GABAergic IPSCs in a Ca2 +-dependent manner. Aguayo et al. [1] demonstrated that dialysing the neurons with very low concentration of intracellular Ca2 + (10 mM BAPTA, 0 Ca2 +) decreased amplitude of the GABAAergic currents. It has also been reported that the IPSCs were modulated by calcium mobilization from intracellular calcium stores ([11] in visual cortex) or calcium influx across plasma membrane (through NMDA channel, [1] in cortical neurons, [5], [16] in CA1 pyramidal cells). Ineffectiveness of several calcium-mobilizing ligands on the IPSCs (Fig. 5) however, suggested that caffeine- or other reagentinduced [Ca2 +]i mobilization hardly affects the GABAAergic currents in the present experimental conditions. Effect of secondary-occurring Ca2 + influx was negligible under the voltage-clamped condition, but it has been reported that calcium influx through voltage-dependent calcium channels did not inhibit the GABAAergic IPSC in retina [2] and in hippocampal neurons [16]. In some systems, extensive cytosolic [Ca2 +]i elevation caused by general incentive, or local [Ca2 +]i elevation by specific Ca2 + influx through proximal channels, might inhibit GABAAergic current. Both extra-synaptic and synaptic GABAAergic IPSCs were reversibly inhibited by application of caffeine. The inhibition of the IPSCs by caffeine was still observed even when [Ca2 +]i increase was prevented. Other reagents which increase [Ca2 +]i could not inhibit the GABAergic IPSCs. These results suggest that the elevation of [Ca2 +]i by store release does not exert major effect on GABAergic transduction in this brain region, and the most part of the inhibition by caffeine is caused independently of intracellular calcium. Inhibition of GABAAergic currents however might be forced by artificially higher [Ca2 +]i elevation or calcium influx. Studies are in progress for the direct demonstration of the inhibitory effect of caffeine on hippocampal GABAA receptor.
Acknowledgements This work was supported in part by Grant-in-Aid (07279105) for Scientific Research on Priority Areas on ‘‘Functional Development of Neural Circuits’’, the Ministry of Education, Science, Sports and Culture of Japan, and also by Research for the Future Project 96L00310
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program from the Japan Society for the Promotion of Science.
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