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1 Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; 2 Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; 3 Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; 4 Research Division for Life Sciences, Kumamoto Health Science University, Kumamoto 861-5533, Japan
Submitted 26 December 2002; accepted in final form 30 May 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate (KA) receptors in the mammalian CNS. KA receptors play important physiological roles in synaptic transmission and also in its presynaptic regulation. At hippocampal mossy fiber synapses, KA receptors mediate a small and slow component of the glutamatergic synaptic response (Cossart et al. 2002
). However, much attention has been made to elucidate the functional significance of presynaptic KA receptors as presynaptic modulators of fast synaptic transmission (for review see Frerking and Nicoll 2000; Lerma et al. 2001). For example, presynaptic KA receptor activation can modulate either glutamatergic (Casassus and Mulle 2002; Kamiya and Ozawa 2000; Schmitz et al. 2000, 2001) or GABAergic transmission (Cossart et al. 2001; Jiang et al. 2001; Kerchner et al., 2001) at various CNS regions.
The substantia nigra pars compacta (SNc) as well as the ventral tegmental area possesses a dense area of dopamine-containing neurons in the CNS. SNc dopaminergic neurons are well known to be crucial in normal and pathological motor control (for review see Bunney et al. 1991). Degeneration of these neurons is primary to the etiology of Parkinson's disease (for review see Olanow and Tatton 1999), and dysfunction of dopaminergic transmission has been implicated in the symptom as schizophrenia (Creese et al. 1976). SNc dopaminergic neurons in vivo display three different firing patterns: a pacemaker-like regular firing pattern, a random pattern, and a burst firing pattern (Grace and Bunney 1985; Tepper et al. 1995). Among them, the pacemaker-like pattern is the only one that occurs spontaneously in dopaminergic neurons recorded in vitro (Grace 1987; Kita et al. 1986), suggesting afferent inputs play an important role in the control of the firing pattern of SNc dopaminergic neurons. SNc dopaminergic neurons receive both excitatory glutamatergic afferents inputs from pedunculopontine nucleus, subthalamic nucleus, and prefrontal cortex (Futami et al. 1995; Kita and Kitai 1987; Sesack and Pickel 1992), and inhibitory GABAergic afferents from the striatum, globus pallidus, and the substantia nigra pars reticulata (SNr) (Hajós and Greenfield 1994; Oertel et al. 1981; Ribak et al. 1976; Smith and Bolam 1990). In particular, GABAergic afferents are thought to play an important role in the control of the firing pattern of SNc dopaminergic neurons because of their abundance within the SNc (Oertel et al. 1981; Ribak et al. 1976; Smith and Bolam 1990).
On the other hand, neurons in the striatum, globus pallidus, and SNr, the GABAergic afferents of which innervate the SNc, express KA receptor subunit mRNAs in a high level (Bischoff et al. 1997). In addition, there is a differential expression pattern of KA receptor subunit mRNAs in the basal ganglia circuitry (Bischoff et al. 1997), suggesting that KA receptors may be involved in the functions associated with the basal ganglia, with a key role in the control of the dopaminergic output pathway. In the present study, therefore, we investigated whether GABAergic presynaptic nerve terminals projecting to SNc dopaminergic neurons express functional KA receptors and whether presynaptic KA receptor activation can directly modulate GABAergic synaptic transmission.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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All experiments conformed to the guiding principles for the care and use of animals approved by the Council of the Physiological Society of Japan and all efforts were made to minimize both the number of animals used and their suffering.
Wistar rats (12–15 days old) were decapitated under pentobarbital anesthesia [50 mg/kg, intraperitoneally (ip)]. The brain was dissected and transversely sliced at a thickness of 370 µm using a microslicer (VT1000S; Leica, Nussloch, Germany). Slices containing the SNc were kept in the control incubation medium (see following text) saturated with 95% O2–5% CO2 at room temperature (23–25°C) for 
1 h before the mechanical dissociation. For dissociation, slices were transferred into a 35-mm culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ) containing the standard external solution (see following text), and the region of the SNc was identified under a binocular microscope (SMZ-1; Nikon, Tokyo). Details of the mechanical dissociation were described previously (Rhee et al. 1999). Briefly, mechanical dissociation was accomplished using a custom-built vibration device and a fire-polished glass pipette oscillating at about 50–60 Hz (0.3–0.5 mm). The tip of the fire-polished glass pipette was lightly placed on the surface of the SNc region with a micromanipulator. The tip of the glass pipette was vibrated horizontally for about 2 min. Slices were removed and the mechanically dissociated neurons allowed to settle and adhere to the bottom of the dish for 15 min. Such neurons undergoing dissociation retained a short portion of their proximal dendrites (Fig. 1A).
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For the slice preparation, Wistar rats (12–15 days old) were decapitated under pentobarbital anesthesia (50 mg/kg, ip). The brain was dissected and transversely sliced at a thickness of 270 µm by using a microslicer (VT1000S; Leica) in a cold low-Na+ medium (see following text). The slices were kept in an external bath solution (see following text) saturated with 95% O2–5% CO2 at 34–35°C for 1 h. Thereafter the slices were transferred into a recording chamber, and the SNc and SNr were identified under an upright microscope (DM LFSA; Leica). Drug was perfused by bath application at 3–4 ml/min.
Electrical measurements
All electrical measurements were performed using the conventional whole cell patch recording mode at a holding potential (VH) of 0 mV (CEZ-2300; Nihon Kohden, Tokyo), except where indicated. Patch pipettes were made from borosilicate capillary glass (1.5 mm OD, 0.9 mm ID; G-1.5; Narishige, Tokyo) in two stages on a vertical pipette puller (PB-7; Narishige). The resistance of the recording pipettes filled with internal solution was 5–6 M
. Electrode capacitance and liquid junction potential were compensated, but series resistance was not. Neurons were viewed under phase contrast on an inverted microscope (Diapot; Nikon). Current and voltage were continuously monitored on an oscilloscope (VC-6023; Hitachi) and a pen recorder (RECTI-HORIT-8K; Sanei, Tokyo), and recorded on a digital-audio tape recorder (RD-120TE; TEAC). Membrane currents were filtered at 1 kHz (E-3201A Decade Filter; NF Electronic Instruments, Tokyo), digitized at 4 kHz, and stored on a computer equipped with pCLAMP 8.02 (Axon Instruments). When recording, 10-mV hyperpolarizing step pulses (30 ms in duration) were periodically delivered to monitor the access resistance. All experiments were performed at room temperature (23–25°C), except for the slice preparation (34–35°C).
Electrical stimulation used to obtain GABAergic evoked IPSCs (eIPSCs) was performed by applying short current pulses (100 µs, 30–50 µA) at 0.1 Hz through a glass pipette (ID, 7–8 µm). The pipette was placed around the dorsomedial or peripeduncular region of the SNr, 500–700 µm distally from the recorded neurons (Hajós and Greenfield 1994) and filled with the external bath solution, using a stimulator (SEN-7203; Nihon Kohden) with an isolator unit (SS-701J; Nihon Kohden). The signals were filtered at 3 kHz and digitized at 10 kHz, and stored on a computer equipped with pCLAMP 8.0.
Data analysis
Miniature IPSCs (mIPSCs) were counted and analyzed using the MiniAnalysis program (Synaptosoft, Decatur, GA) as described previously (Jang et al. 2002). Briefly, spontaneous events were screened automatically using an amplitude threshold of 10 pA and then visually accepted or rejected based on the rise and decay times. The average values of mIPSC frequency and amplitude during the control period (5–10 min) were calculated, and the frequency and amplitude of all the events during KA application (2 min) were normalized to these values. The effect of KA was quantified as a percentage increase in mIPSC frequency compared with the control values. The interevent intervals and amplitudes of a large number of mIPSCs obtained from the same neuron were examined by constructing cumulative probability distributions and compared using Kolmogorov–Smirnov test with Stat View software (SAS Institute, Cary, NC). The amplitude of evoked IPSCs (eIPSCs) was analyzed using pCLAMP 8.0. Numerical values are provided as means ± SE using values normalized to the control. Possible differences in amplitude and frequency distribution were tested by Student's paired 2-tailed t-test using their absolute rather than normalized values. Values of P < 0.05 were considered significant.
Immunocytochemistry
To determine whether the large neurons (>25 µm in soma diameter) used for electrophysiological recordings in the present experiments indeed belong to SNc dopaminergic neurons, we performed immunocytochemical examinations using anti-tyrosine hydroxylase antibody. Neurons were mechanically dissociated on the glass coverslips coated with polyethylenimine (PEI) in a 35-mm culture dish. After most neurons were settled down and adhered to the coverslips, each coverslip was moved to a parafilm sheet for immunocytochemistry. Neurons were washed with phosphate-buffered saline (PBS, pH 7.4) and fixed with 4% paraformaldehyde in PBS for 30 min. After treatment with 0.2% Triton-X 100 for 5 min, neurons were incubated with PBS containing rabbit anti-tyrosine hydroxylase (TH) antibody (1:2000; Chemicon International) and 1% normal bovine serum for 2 h. Then neurons were incubated with fluorescein isothiocyanate (FITC)–conjugated donkey anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories) for 1 h. Images of the neurons were collected with a digital camera (Carl Zeiss, Germany) attached to a fluorescent microscope (Carl Zeiss) and stored in a computer using AxoVision 2.05 (Carl Zeiss). All procedures were performed at room temperature.
Solutions
The ionic composition of the incubation medium consisted of (in mM) 124 NaCl, 3 KCl, 1.5 KH2PO4, 24 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose saturated with 95% O2–5% CO2. The pH was about 7.45. The low-Na+ medium consisted of (in mM) 230 sucrose, 3 KCl, 1.5 KH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 10 glucose. The standard external solution was (in mM) 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 Hepes. The Na+-free external solution consisted of (in mM) 150 N-methyl-D-glucamine-Cl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 Hepes. The Ca2+-free external solution consisted of (in mM) 150 NaCl, 5 KCl, 5 MgCl2, 2 EGTA, 10 glucose, and 10 Hepes. These external solutions were adjusted to a pH of 7.4 with Tris-base. For recording mIPSCs, external solutions routinely contained 500 nM tetrodotoxin (TTX) and 50 µM D-2-amino-5-phosphonovaleric acid (AP5) to block voltage-dependent Na+ channels and NMDA receptors, respectively, except where indicated. In the slice experiments, the external bath solution consisted of (in mM) 124 NaCl, 3 KCl, 1.5 KH2PO4, 26 NaHCO3, 2 CaCl2, 1 MgCl2, and 10 glucose saturated with 95% O2–5% CO2. The pH was about 7.5. The ionic composition of the internal (patchpipette) solution for the whole cell patch recording consisted of (in mM) 145 Cs-methanesulfonate, 5 TEA-Cl, 5 CsCl, 2 EGTA, 10 Hepes, and 4 ATP-Mg with a pH adjusted to 7.2 with Tris-base. In the current-clamp experiment, the internal solution for the nystatin-perforated patch recording consisted of (in mM) 80 K-methanesulfonate, 70 KCl, 5 MgCl2, and 10 Hepes with pH adjusted to 7.2 with Tris-base. Nystatin was dissolved in acidified methanol at 10 mg/ml. This stock solution was diluted with the internal solution just before use to a final concentration of 100–200 µg/ml.
Drugs
The drugs used in the present study were TTX, bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), AP5, NMDA, AMPA, nystatin, GYKI52466, (RS-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), KA, dopamine, EGTA, (±)-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM2206), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (+)--methyl-4-carboxyphenylglycine (MCPG), ATP-Mg (from Sigma, St. Louis, MO), (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (from Tocris, UK). All solutions containing drugs were applied using the "Y-tube system" for rapid solution exchange (Akaike and Harata 1994), except for the slice preparation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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After brief mechanical dissociation of the SNc region, we found large (>25 µm in somatic diameter) and small neurons (15–20 µm) (Fig. 1A). Large neurons displayed a variety in the shape of the somata including fusiform, triangular, and multipolar forms, and were comparable to those previously described types (Grace and Onn 1989; Katayama et al., 2003; Lacey et al. 1989; Uchida et al. 2000). To confirm whether these large neurons dissociated mechanically belong to SNc dopaminergic neurons, we examined immunoreactivity against tyrosine hydroxylase (TH), a marker of dopaminergic neurons, and electrophysiological membrane properties. As shown in Fig. 1A, large neurons were TH-positive, whereas small ones were TH-negative. In the current-clamp condition, large neurons fired spontaneously in a very regular pacemaker fashion without bursting activity (Fig. 1, B and C). Membrane responses to injection of hyperpolarizing currents were distinctive inward rectification, which is one of the distinctive physiological membrane properties of SNc neurons (Fig. 1B). Because SNc dopaminergic neurons are known to express dopamine D2-like receptors (Uchida et al. 2000), we also tested the effects of dopamine on SNc neurons. As shown in Fig. 1C, dopamine (1 µM) hyperpolarized SNc neurons and significantly reduced action potential firing rate. All of such immunocytochemical and electrophysiological properties were indistinguishable from those described previously in vitro as identified dopaminergic neurons (Grace and Onn 1989; Katayama et al. 2003; Lacey et al. 1989; Uchida et al. 2000). Therefore we used large neurons in all subsequent experiments.
In the presence of 500 nM TTX and 50 µM AP5, the spontaneous outward miniature postsynaptic currents were recorded from mechanically dissociated SNc neurons at a VH of 0 mV. These currents were completely and reversibly blocked by 3 µM bicuculline (Fig. 2A). Figure 2B shows typical events at various VH values and their I–V relationship. The reversal potential of the miniature currents, as estimated from the I–V relationship, was –68.1 mV (n = 4), which was almost identical to the theoretical Cl– equilibrium potential (ECl) of –69.9 mV calculated by the Nernst equation using extra- and intracellular Cl– concentrations (161 and 10 mM, respectively). These results indicate that the spontaneous postsynaptic currents are GABAergic mIPSCs.
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Effect of KA on GABAergic mIPSCs
To elucidate whether GABAergic presynaptic nerve terminals projecting to SNc dopaminergic neurons express functional KA receptors, and whether presynaptic KA receptor activation can directly modulate GABAergic synaptic transmission, first we observed the effect of exogenously applied KA on GABAergic mIPSCs. In the majority of SNc neurons tested (62 out of 76 cells; 82%), 3 µM KA increased GABAergic mIPSC frequency. In 14 neurons in which this effect was fully analyzed, KA increased the mean mIPSC frequency to 222 ± 14% of the control (P < 0.01), without affecting the mean mIPSC current amplitude (108 ± 4% of the control, P = 0.09) (Fig. 3, A and B insets). The KA-induced increase in mIPSC frequency sustained throughout the period of application and was rapidly reversed on washout (Fig. 3Ab). In addition, as shown in Fig. 3B, KA (3 µM) significantly shifted the distribution of interevent interval to the left, but did not affect the amplitude of mIPSCs. Taken together, these results suggest that KA acts presynaptically to increase spontaneous GABA release onto SNc neurons.
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To quantify KA-induced presynaptic responses, we examined the effects of the repeated applications of KA on GABAergic mIPSC frequency. The results might be important particularly for determining concentration–response relationships or analyzing the pharmacological properties of KA receptor subtypes. KA-induced presynaptic responses were reproducible during the repeated applications with a time interval of 10 min (first: 232 ± 37%; second: 216 ± 37%; third: 212 ± 49%, n = 4). Figure 3C shows the concentration–response relationship for both the KA-induced increase in mIPSC frequency. KA even at lower concentrations significantly increased mIPSC frequency (1 µM: 181 ± 23%; 3 µM: 215 ± 38%; 10 µM: 173 ± 22%). We also tested the effect of ATPA, a potent agonist of KA receptor containing GluR5 subunit, on GABAergic mIPSCs, and found that ATPA had little effect on mIPSC frequency (Fig. 3C).
Pharmacological properties of presynaptic KA receptors
KA can activate both KA and AMPA receptors (for review see Lerma et al. 2001). To investigate the receptor subtypes involved in the facilitatory action of KA on GABAergic mIPSCs, we tested the effects of GYKI52466 or SYM2206, selective AMPA receptor antagonists, and CNQX, a nonselective AMPA/KA receptor blocker. In the presence of 20 µM GYKI52466, 3 µM KA increased mIPSC frequency to 221 ± 18% of the control (P < 0.05, n = 5; Fig. 4, A and B). This facilitation ratio was almost identical to that induced by KA in the absence of GYKI52466. In addition, KA also increased mIPSC frequency in the presence of 30 µM SYM2206 (198 ± 39% of the control, n = 4; data not shown), thereby indicating that AMPA receptors are not involved in the facilitatory actions of mIPSCs. On the other hand, KA-induced facilitation of mIPSC frequency was completely suppressed in the presence of 20 µM CNQX to 111 ± 23% of the control (P = 0.89, n = 5; Fig. 4, A and B). In a subset of experiments, we also tested the effects of AMPA and NMDA on GABAergic mIPSCs. Neither AMPA nor NMDA (0.1, 1 and 10 µM, n = 3, respectively) affected GABAergic mIPSCs (data not shown).
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Ca2+-impermeable KA receptors modulate spontaneous GABA release
Next, we studied the mechanisms involved in the KA-induced increase in mIPSC frequency. The application of 200 µM Cd2+, a general voltage-dependent Ca2+ channel (VDCC) blocker, itself reduced the frequency (32 ± 9%, n = 7, P < 0.01) and amplitude (59 ± 6%, n = 7, P < 0.01) of mIPSCs. In the presence of Cd2+, KA action on mIPSC frequency (Fig. 5Aa) was completely abolished to 113 ± 12% of the control (n = 7, P > 0.05; Fig. 5Ba). We also examined the effect of Ca2+-free external solution on KA-induced facilitation of mIPSC frequency. In a Ca2+-free external solution, both frequency and amplitude of mIPSCs were greatly reduced (to 25 ± 6%, n = 5, P < 0.01, or to 63 ± 2%, n = 5, P < 0.01, respectively) (Fig. 5Ab). The facilitatory effect of KA on mIPSC frequency was also completely inhibited to 106 ± 12% of the Ca2+-free condition (n = 5, P = 0.79; Fig. 5, Ab and B). These results suggest that KA-induced facilitation of mIPSC frequency is mediated by the Ca2+ influx from the extracellular space by VDCC activation or directly through KA receptors. To distinguish these two possibilities, we observed the effect of Na+-free external solution on KA-induced facilitation of mIPSC frequency. In a Na+-free external solution, GABAergic mIPSC frequency slightly increased, but the effect was not statistically significant (147 ± 35%, n = 6, P = 0.29). In a Na+-free external solution, the facilitatory effect of KA on mIPSC frequency was not observed (to 97 ± 5% of the control, P = 0.85, n = 6) (Fig. 5, Ac and B).
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Effect of KA on action potential-dependent GABA release
To elucidate a physiological significance of the present finding, we observed the effect of KA on the mIPSCs and evoked GABAergic IPSCs (eIPSCs), by use of slice preparation. Because SNc dopaminergic neurons receive GABAergic afferents from the striatum, globus pallidus, and the axon collaterals of the SNr (Hajós and Greenfield 1994; Oertel et al. 1981; Ribak et al. 1976; Smith and Bolam 1990), the stimulus pipette was placed around the SNr. We found that bath application of KA increases spontaneous IPSC frequency dose-dependently (0.3 µM: 123 ± 18%; 1 µM: 251 ± 22%; 3 µM: 283 ± 37% of the control, n = 6, respectively). However, KA (0.3 to 3 µM) significantly reduced eIPSC amplitude in a dose-dependent manner (0.3 µM: 75 ± 5%; 1 µM: 60 ± 7%; 3 µM: 44 ± 9% of the control, n = 6, respectively; Fig. 6A). The inhibitory action of KA on eIPSC amplitude is probably not attributable to either vesicle depletion or the postsynaptic mechanism, given that during the KA application, a sustained increase in spontaneous IPSC frequency was observed and the amplitude distribution of spontaneous IPSC was not affected (data not shown). We also examined the effect of KA on GABAergic mIPSCs in the presence of TTX, and found that KA (3 µM) also increases mIPSC frequency to 173 ± 25% of the control (n = 4, P < 0.05; Fig. 6B), thereby indicating that KA acts on presynaptic nerve terminals of GABAergic neurons that innervate SNc.
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The fact that kainate at lower concentrations suppressed the amplitude of eIPSCs tempted us to examine the possible indirect actions by metabotropic receptors, including GABAB receptors, as suggested by a recent study (Kerchner et al. 2001). Interestingly, in the presence of 10 µM CGP55845, a selective GABAB receptor antagonist, KA at lower concentrations slightly increased eIPSC amplitude (0.3 µM: 120 ± 5%, P < 0.05; 1 µM: 113 ± 7% of the control, P = 0.18, n = 6, respectively; Fig. 7, A and B). However, 3 µM KA suppressed eIPSC amplitude (to 82 ± 8% of the control, n = 6, P < 0.05), although the extent of inhibition was relatively small (Fig. 7, A and B). In another set of experiments, we also tested the possible involvement of other metabotropic receptors, such as adenosine A1 receptors, metabotropic glutamate receptors, and cannabinoid CB1 receptors, in KA receptor-mediated modulation of GABAergic eIPSCs. In two neurons tested, further application of a cocktail of CGP55845 (10 µM); DPCPX (100 nM), a selective A1 receptor antagonist; MCPG (1 mM), a nonselective metabotropic glutamate receptor antagonist; and AM-251 (10 µM), a selective CB1 receptor antagonist, hardly changed the biphasic effects of KA on eIPSCs (Fig. 7C). These results suggest that GABA released by the excitation of GABAergic nerve terminals during KA receptor activation might also act on presynaptic GABAB autoreceptors to inhibit the release of GABA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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In the present study, we isolated single SNc neurons without using any enzymes. These mechanically isolated neurons showed similar morphological, immunocytochemical, and electrophysiological properties of SNc dopaminergic neurons described previously (Grace and Onn 1989; Katayama et al. 2003; Lacey et al. 1989; Uchida et al. 2000). Although GABAergic mIPSCs were recorded from many mechanically dissociated SNc neurons, the precise sources of the GABA are unclear at this time. However, it seems that the GABAergic IPSCs shown in this study originated from GABAergic terminals arising from the striatum, globus pallidus, and the axon collaterals of the SNr (Hajós and Greenfield 1994; Oertel et al. 1981; Ribak et al. 1976; Smith and Bolam 1990).
In general, it is known that miniature currents could be recorded in the presence of TTX, which blocks Na+-dependent action potentials. Because frequency and amplitude of miniature currents were unaffected by further removal of extracellular Ca2+ or by adding Cd2+ or Co2+ (Capogna et al. 1993; Scanziani et al. 1992), these events should be independent of extracellular Ca2+ influx and/or VDCCs. In the present study, however, frequency and amplitude of SNc GABAergic mIPSCs recorded in the presence of TTX were greatly reduced by adding Cd2+ or by removal of extracellular Ca2+. It was not feasible to evaluate the effect of Cd2+ on GABAergic mIPSCs because Cd2+ itself is known to block GABAA receptors (Fisher and Macdonald 1998; Kumamoto and Murata 1995). However, the present results that frequency and amplitude of GABAergic mIPSCs were reduced in the Ca2+-free external solution suggest the possible involvement of Ca2+ influx from the external solution after the blockade of Na+ channels. One possible explanation for this phenomenon would be that GABAergic nerve terminals in mechanically dissociated SNc neurons may be depolarized membrane potentials to some extent. Because of the membrane depolarization of the nerve terminals, it is reasonable to assume that the activation of VDCCs may occur spontaneously, which may in turn result in the spontaneous and synchronous GABA release. Thus spontaneous synaptic events that were observed in Ca2+-free external solution in the present experiments may correspond to the classical miniature currents in the previous studies (Capogna et al. 1993; Scanziani et al. 1992). Alternatively, the synaptic events observed in the present preparation might be Ca2+-dependent mIPSCs. In fact, Ca2+-dependent mIPSCs have been also reported in central neurons (Doze et al. 1995; Soltesz and Mody 1995). However, further studies are warranted to understand the reasons for the dependency of mIPSCs on extracellular Ca2+ and/or VDCCs.
GABAergic presynaptic nerve terminals express functional KA receptors
There is much convincing evidence that KA by presynaptic ionotropic KA receptors potentiates spontaneous inhibitory synaptic transmission (Cossart et al. 2001; Kerchner et al. 2001; Mulle et al. 2000). For example, in hippocampal CA1 interneurons, KA increases mIPSC frequency without affecting their amplitude (Cossart et al. 2001) and causes an increase in mIPSC frequency by activating presynaptic ionotropic KA receptors in the spinal cord dorsal horn (Kerchner et al. 2001). The present results also provide an example that presynaptic ionotropic KA receptors are present on GABAergic nerve terminals projecting to SNc dopaminergic neurons, and that their activation increases the probability of spontaneous GABA release. Several lines of evidence support this conclusion that presynaptic ionotropic KA receptors are responsible for the KA-induced increase of mIPSC frequency. First KA at lower concentrations increased GABAergic mIPSC frequency without affecting the current amplitude, suggesting that KA acts presynaptically to increase spontaneous GABA release. Second, KA action on mIPSC frequency was not affected by either GYKI52466 or SYM2206, selective AMPA receptor blockers, but completely blocked by CNQX, a nonselective AMPA/KA receptor blocker. In addition, exogenously applied AMPA or NMDA did not affect GABAergic mIPSC frequency. Third, the KA-induced increase in mIPSC frequency was completely suppressed in either Na+-free or Ca2+-free external solution, supporting the involvement of an ionic mechanism in KA-mediated action. Finally, dissociated neurons used in this study have cell-free presynaptic nerve terminals, although it is still unclear whether a short portion of the axon was attached to presynaptic nerve terminals (see also Akaike et al. 2002).
The family of KA receptors is composed of 5 different genes that code for the subunit GluR5/6/7 and KA1/2 (Bettler and Mulle 1995; Chittajallu et al. 1999). The subunit composition of KA receptors could be very diverse, given the large combinational possibilities suggested by KA receptor subunit mRNA distribution in the CNS (Bischoff et al., 1997; Wisden and Seeburg 1993), as well as functional expression studies of recombinant receptors (Chittajallu et al. 1999; Lerma et al. 2001). In the present experiments, ATPA failed to increase spontaneous GABA release, thereby indicating that presynaptic KA receptors on GABAergic terminals projecting to SNc dopaminergic neurons might not contain GluR5 subunit. This view is supported by the previous observations that most GABAergic neurons, which innervate SNc dopaminergic neurons, express both GluR6 and KA2, but not GluR5, subunit mRNAs (Bischoff et al. 1997).
Mechanisms involved in the KA-induced increase in spontaneous GABA release
It seems that there are several possible explanations for the KA-induced increase in GABAergic mIPSC frequency. First, activation of Na+-permeable, but Ca2+-impermeable, KA receptors might cause Na+ influx into presynaptic terminals and depolarize presynaptic membrane, and thus lead to VDCC activation (Kerchner et al. 2001). Second, activation of Ca2+-permeable KA receptors might directly increase the [Ca2+]i within the terminals (Cossart et al. 2001; Kohler et al. 1993). In the present study, however, the KA-induced increase in mIPSC frequency was completely suppressed in Na+-free or Ca2+-free external solutions, and in the presence of Cd2+, suggesting that Ca2+ influx through VDCCs from extracellular space is closely related to KA action. Thus it seems the the most plausible mechanism involved is that activation of the presynaptic KA receptors leads to direct presynaptic depolarization by permitting Na+ influx, and the depolarization causes VDCC activation to increase the Ca2+ influx.
Biphasic modulation of action potential-dependent GABA release by presynaptic KA receptors
It is rather surprising that KA at lower concentrations inhibits GABAergic eIPSC amplitude, as previous studies have reported that KA at nanomolar concentration facilitates glutamate release from mossy fiber or parallel fiber terminals (Delaney and Jahr 2002; Kamiya and Ozawa 2000; Schmitz et al. 2000, 2001). On the other hand, it was reported that synaptically released GABA in the presence of KA acts GABAB autoreceptors to inhibit action potential–dependent GABA release (Kerchner et al. 2001). In the present experiments, we found that KA-induced inhibition of GABAergic eIPSCs is suppressed in the presence of CGP55845, a GABAB receptor antagonist, thereby indicating that synaptically released GABA during KA receptor activation acts presynaptic GABAB autoreceptor to inhibit GABA release. These actions of GABA on GABAB autoreceptors may mask the facilitatory action of KA on spontaneous GABA release at lower concentrations. It should be mentioned, however, that after the blockade of GABAB receptors, KA shows biphasic modulation of action potential–dependent GABA release, that is, facilitation and inhibition at lower and higher concentrations, respectively. These biphasic effects might be attributed to the extent of presynaptic depolarization induced by KA. The recent reports have demonstrated that KA at nanomolar concentration elicits mild presynaptic depolarization and facilitates neurotransmitter release by increasing the Ca2+ influx into presynaptic terminals as well as the excitability of presynaptic volleys. On the contrary, strong presynaptic depolarization attributed to axonal and/or terminal KA receptor activation might inactivate voltage-dependent Na+ and/or Ca2+ channels or membrane shunting, thus resulting in the inhibition of action potential–dependent transmitter release (Kamiya and Ozawa 2000; Kamiya et al. 2002; Schmitz et al. 2000, 2001).
Physiological significance
SNc dopaminergic neurons receive both excitatory (Futami et al. 1995; Kita and Kitai 1987; Sesack and Pickel 1992) and inhibitory afferent inputs, but the majority (
90%) of the afferents neurons to SNc dopaminergic neurons appear to be inhibitory GABAergic inputs because most of afferent terminals are immunostained for glutamate decarboxylase, a GABA synthesizing enzyme (Oertel et al. 1981; Ribak et al. 1976; Smith and Bolam 1990). These GABAergic afferents have a crucial role in controlling the firing activity of dopaminergic neurons, as local administration of GABAA receptor antagonists in vivo increases the number of neurons that fire in the burst mode (Celada et al. 1999; Tepper et al. 1995) or shifts single neurons from a regular or random firing pattern to burst firing mode (Paladini and Tepper 1999). These findings suggest that the GABAergic system might be involved in the regulation of motor functions as well as the firing patterns of SNc dopaminergic neurons. On the other hand, the hyperactivity of the subthalamic nucleus, which leads to glutamate excitotoxicity in the SNc, might play a pivotal role in the pathophysiology of Parkinson's disease (Blandini et al. 2000). In addition, large increases in extracellular glutamate concentration could occur in the hypoxic/ischemic damage (Rothman and Olney 1986). It would be interesting to determine whether presynaptic KA receptors can be activated by synaptically released glutamate in such pathophysiological conditions.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Akaike, Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan (E-mail: akaike{at}physiol2.med.kyushu-u.ac.jp).
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Akaike N, Murakami N, Katsurabayashi S, Jin YH, and Imazawa T. Focal stimulation single GABAergic presynaptic boutons on the rat hippocampal neurons. Neurosci Res 42: 187–195, 2002.[ISI][Medline]
Bettler B and Mulle C. Review: neurotransmitter receptors. II. AMPA and kainate receptors. Neuropharmacology 34: 123–139, 1995.[ISI][Medline]
Bischoff S, Barhanin J, Bettler B, Mulle C, and Heinemann S. Spatial distribution of kainate receptor subunit mRNA in the mouse basal ganglia and ventral mesencephalon. J Comp Neurol 379: 541–562, 1997.[ISI][Medline]
Blandini F, Nappi G, Tassorelli C, and Martignoni E. Functional changes of the basal ganglia circuitry in Parkinson's disease. Prog Neurobiol 62: 63–88, 2000.[ISI][Medline]
Bunney BS, Chiodo LA, and Grace AA. Midbrain dopamine system electrophysiological functioning: a review and new hypothesis. Synapse 9: 79–94, 1991.[ISI][Medline]
Capogna M, Gahwiler BH, and Thompson SM. Mechanism of µ-opioid receptor-mediated presynaptic inhibition in the rat hippocampus in vitro. J Physiol 470: 539–558, 1993.
Casassus G and Mulle C. Functional characterization of kainate receptors in the mouse nucleus accumbens. Neuropharmacology 42: 603–611, 2002.[ISI][Medline]
Celada P, Paladini CA, and Tepper JM. GABAergic control of rat substantia nigra dopaminergic neurons: role of globus pallidus and substantia nigra pars reticulata. Neuroscience 89: 813–825, 1999.[ISI][Medline]
Chittajallu R, Braithwaite SP, Clarke VR, and Henley JM. Kainate receptors: subunits, synaptic localization and function. Trends Pharmacol Sci 20: 26–35, 1999.[Medline]
Cossart R, Epsztein J, Tyzio R, Becq H, Hirsch J, Ben-Ari Y, and Crepel V. Quantal release of glutamate generated pure kainate and mixed AMPA/KA EPSCs in hippocampal neurons. Neuron 35: 147–159, 2002.[ISI][Medline]
Cossart R, Tyzio R, Dinocourt C, Esclapez M, Hirsch JC, Ben-Ari Y, and Bernard C. Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons. Neuron 29: 497–508, 2001.[ISI][Medline]
Creese I, Burt DR, and Synder SH. Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs. Science 192: 481–483, 1976.
Delaney AJ and Jahr CE. Kainate receptors differentially regulate release at two parallel fiber synapses. Neuron 36: 475–482, 2002.[ISI][Medline]
Doze VA, Cohen GA, and Madison DV. Calcium channel involvement in GABAB receptor-mediated inhibition of GABA release in area CA1 of the rat hippocampus. J Neurophysiol 74: 43–53, 1995.
Fisher JL and Macdonald RL. The role of an subtype M2–M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations. J Neurosci 18: 2944–2953, 1998.
Frerking M and Nicoll RA. Synaptic kainate receptors. Curr Opin Neurobiol 10: 342–351, 2000.[ISI][Medline]
Futami T, Takakusaki K, and Kitai ST. Glutamatergic and cholinergic inputs from the pedunculopontine tegmental nucleus to dopamine neurons in the substantia nigra pars compacta. Neurosci Res 21: 331–342, 1995.[ISI][Medline]
Grace AA. (1987). The regulation of dopamine neuron activity as determined by in vivo and in vitro intracellular recordings. In: Neurophysiology of Dopaminergic System: Current Status and Clinical Perspectives, edited by Chiodo LA and Freeman AS. Detroit, MI: Lakeshore, p. 1–66.
Grace AA and Bunney BS. The control of firing pattern in nigral dopamine neurons: burst firing. J Neurosci 4: 2877–2890, 1985.
Grace AA and Onn SP. Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci 9: 3463–3481, 1989.[Abstract]
Hajós M and Greenfield SA. Synaptic connections between pars compacta and pars reticulata neurones: electrophysiological evidence for functional modules within the substantia nigra. Brain Res 660: 216–224, 1994.[ISI][Medline]
Jang IS, Jeong HJ, Katsurabayashi S, and Akaike N. Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord. J Physiol 541: 423–434, 2002.
Jiang L, Xu J, Nedergaard M, and Kang J. A kainate receptor increases the efficacy of GABAergic synapses. Neuron 30: 503–513, 2001.[ISI][Medline]
Kamiya H and Ozawa S. Kainate receptor-mediated presynaptic inhibition at the mouse hippocampal mossy fibre synapse. J Physiol 523: 653–665, 2000.
Kamiya H, Ozawa S, and Manabe T. Kainate receptor-dependent short-term plasticity of presynaptic Ca2+ influx at the hippocampal mossy fiber synapses. J Neurosci 22: 9237–9243, 2002.
Katayama J, Akaike N, and Nabekura J. Characterization of pre- and post-synaptic metabotropic glutamate receptor-mediated inhibitory responses in substantia nigra dopamine neurons. Neurosci Res 45: 101–115, 2003.[ISI][Medline]
Kerchner GA, Wang GD, Qiu CS, Huettner JE, and Zhuo M. Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism. Neuron 32: 477–488, 2001.[ISI][Medline]
Kita T, Kita H, and Kitai ST. Electrical membrane properties of rat substantia nigra neurons in an in vitro slice preparation. Brain Res 372: 21–30, 1986.[ISI][Medline]
Kita H and Kitai ST. Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method. J Comp Neurol 260: 435–452, 1987.[ISI][Medline]
Kohler M, Burnashev N, Sakmann B, and Seeburg PH. Determinants of Ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: diversity by RNA editing. Neuron 10: 491–500, 1993.[ISI][Medline]
Kumamoto E and Murata Y. Characterization of GABA current in rat septal cholinergic neurons in culture and its modulation by metal cations. J Neurophysiol 74: 2012–2027, 1995.
Lacey MG, Mercuri NB, and North RA. Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the action of dopamine and opioids. J Neurosci 9: 1233–1241, 1989.[Abstract]
Lerma J, Paternain AV, Rodriguez-Moreno A, and Lopez-Garcia JC. Molecular physiology of kainate receptors. Physiol Rev 81: 971–998, 2001.
MacDermott AB, Role LW, and Siegelbaum SA. Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci 22: 443–485, 1999.[ISI][Medline]
Mulle C, Sailer A, Swanson GT, Brana C, O'Gorman S, Bettler B, and Heinemann SF. Subunit composition of kainate receptors in hippocampal interneurons. Neuron 28: 475–484, 2000.[ISI][Medline]
Oertel WH, Schmechel DE, Brownstein MJ, Tappaz ML, Ransom DH, and Kopin IJ. Decrease in glutamate decarboxylase (GAD)-immunoreactive nerve terminals in the substantia nigra after kainic acid lesion of the striatum. J Histochem Cytochem 29: 977–980, 1981.[Abstract]
Olanow CW and Tatton WG. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci 22: 123–144, 1999.[ISI][Medline]
Paladini CA, Celada P, and Tepper JM. Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABAA receptors in vivo. Neurosci 89: 799–812, 1999.[ISI][Medline]
Paladini CA and Tepper JM. GABAA and GABAB antagonists differentially affect the firing pattern of substantia nigra dopaminergic neurons in vivo. Synapse 32: 165–176, 1999.[ISI][Medline]
Rhee JS, Ishibashi H, and Akaike N. Calcium channels in the GABAergic presynaptic nerve terminals projecting to Meynert neurons of the rat. J Neurochem 72: 800–807, 1999.[ISI][Medline]
Ribak CE, Vaughn JE, Saito K, Barber R, and Roberts E. Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra. Brain Res 116: 287–298, 1976.[ISI][Medline]
Rothman SM and Olney JW. Glutamate and the pathophysiology of hypoxicischemic brain damage. Ann Neurol 19: 105–111, 1986.[ISI][Medline]
Scanziani M, Capogna M, Gahwiler BH, and Thompson SM. Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus. Neuron 9: 919–927, 1992.[ISI][Medline]
Schmitz D, Frerking M, and Nicoll RA. Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses. Neuron 27: 327–338, 2000.[ISI][Medline]
Schmitz D, Mellor J, and Nicoll RA. Presynaptic kainate receptor mediation of frequency facilitation at hippocampal mossy fiber synapses. Science 291: 1972–1976, 2001.
Sesack SR and Pickel VM. Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320: 145–160, 1992.[ISI][Medline]
Smith AD and Bolam JP. The output neurons and the dopaminergic neurons of the substantia nigra receive a GABA-containing input from the globus pallidus in the rat. J Comp Neurol 296: 47–64, 1990.[ISI][Medline]
Soltesz I and Mody I. Ca2+-dependent plasticity of miniature inhibitory postsynaptic currents after amputation of dendrites in central neurons. J Neurophysiol 73: 1763–1773, 1995.
Tepper JM, Martin LP, and Anderson DR. GABAA receptor-mediated inhibition of rat substantia nigra dopaminergic neurons by pars reticulata projection neurons. J Neurosci 15: 3092–3103, 1995.[Abstract]
Uchida S, Akaike N, and Nabekura J. Dopamine activates inward rectifier K+ channel in acutely dissociated rat substantia nigra neurones. Neuropharmacology 39: 191–201, 2000.[ISI][Medline]
Wisden W and Seeburg PH. A complex mosaic of high-affinity kainate receptors in rat brain. J Neurosci 13: 3582–3598, 1993.[Abstract]
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