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1Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee, Memphis, Tennessee; and 2The Japan Society for the Promotion of Science, Tokyo, Japan
Submitted 10 March 2005; accepted in final form 25 April 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, 1994; Kita and Kitai 1991; Parent and Hazrati 1995). The GP sends GABAergic outputs to many nuclei of the basal ganglia including the Str, STN, entopeduncular nucleus, and the substantia nigra (Bevan et al. 1998; Hazrati et al. 1990; Kita 1994; Kita and Kita 2001; Kita and Kitai 1994).
Studies have shown that GABAergic inputs play crucial roles in controlling GP neural activity (Kita 1992, 1994; Kita and Kitai 1991; Kita et al. 2004; Nambu and Llinás 1994). Although previous studies focused mainly on the ionotropic GABAA receptor-mediated actions, accumulating evidence suggests that metabotropic GABAB receptors may also be active in the GP. The expression of both the GABABR1 and GABABR2 subunits in the GP has been revealed by immunohistochemical and in situ hybridization studies in rat, monkey, and humans (Charara et al. 2000, 2004; Chen et al. 2004; Smith et al. 2000; Waldvogel et al. 2004). Electron microscopic analysis showed both pre- and postsynaptic expressions of GABAB receptors in the GP (Chen et al. 2004). In other brain areas, activation of postsynaptic GABAB receptors induces a slow hyperpolarization through the opening of potassium conductances and activation of receptors located at presynaptic terminals inhibits neurotransmitter release (Misgeld et al. 1995). In spite of these findings, electrophysiological studies investigating the functional roles of GABAB receptors in the GP are scarce (Chen and Yung 2003; Chen et al. 2002; Stefani et al. 1999). Because previous studies were performed using the exogenous application of the GABAB receptor agonist baclofen, it remains to be determined whether synaptically released GABA can activate GABAB receptors in the GP. In rat GP slices, single stimulation-induced inhibitory postsynaptic potentials and currents (IPSPs/IPSCs) were completely blocked by a GABAA receptor antagonist (Matsui and Kita 2003; Ogra and Kita 2000), suggesting very little or no involvement of GABAB receptors in the IPSPs/IPSCs induction. In other brain regions, repetitive stimulation is required to observe synaptically induced GABAB responses (Isaacson et al. 1993; Johnson et al. 1992; Mitchell and Silver 2000; Saitoh et al. 2004). Thus the aim of the present study was to investigate whether synaptically released GABA by repetitive stimulation can activate pre- and postsynaptic GABAB receptors in the GP using rat brain slice preparations.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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This study was performed in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Sprague-Dawley juvenile rats (15–19 days old; 20–42 g) of both sexes were anesthetized with an intraperitoneal injection of a mixture of Ketamine (85 mg/kg) and Xylazine (15 mg/kg) and decapitated. The brains were rapidly removed and blocks containing the GP were obtained. Parasagittal slices (300–350 µm thick) were cut from the blocks on a vibrating blade microtome, Leica VT1000S (Leica Microsystems, Nussloch, Germany) in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 126 choline chloride, 3 KCl, 1.24 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 6.3 MgSO4, 0.2 thiourea, 0.2 ascorbic acid, and 20 D-glucose, pH 7.4. The slices were then incubated in ACSF containing (in mM) 126 NaCl, 3 KCl, 1.24 NaH2PO4, 26 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, and 10 D-glucose, pH 7.4 at 33°C for 
1 h before recording.
Electrophysiological recordings
The slices were transferred to a recording chamber with oxygenated ACSF continuously perfused at a flow rate of 1–2 ml/min. The temperature of recording chamber was kept at 33 ± 1°C. Whole cell patch-clamp and cell-attached recording pipettes with a tip diameter of 
1.5 µm were pulled from 1.5 mm, thin-wall, borosilicate glass capillaries on a horizontal electrode puller (P-97; Sutter Instruments, Novato, CA). For the recording of GABAA-mediated IPSCs, the pipettes were filled with high-Cl electrolyte to increase the driving force of the IPSCs. The electrolyte contained (in mM) 90 K-gluconate, 50 KCl, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, 0.2% Neurobiotin, and 3 QX-314 with the pH adjusted to 7.2 with KOH. The chloride equilibrium potential of the recorded neurons was estimated to be –26 mV by the Nernst equation when the cytoplasm of the neurons was fully equilibrated with 50 mM chloride. QX-314 was included to block action potential generation and postsynaptic GABAB responses (Andrade 1991; McLean et al. 1996; Nathan et al. 1990). For other recordings, the pipettes were filled with low-Cl electrolyte containing (in mM) 135 K-gluconate, 5 KCl, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, and 0.2% Neurobiotin with the pH adjusted to 7.2 with KOH. The membrane potential recorded was corrected by the liquid junction potential of –10 mV. The resistance of these recording pipettes ranged from 4 to 8 M
. Neurons and recording pipettes were visualized using an infrared-differential interference contrast microscope BX50WI (Olympus, Tokyo), with a x40 water-immersion objective LUM Plan PL (Olympus) and a CCD camera (4990 series; COHU, San Diego, CA). Data were collected using an Axopatch 200B amplifier and AxoGraph 4.6 (Axon Instruments, Foster City, CA). Signals were filtered at 2 kHz, digitized at 5 kHz with a computer interface ITC-18 (InstruTECH, Port Washington, NY), and stored on the hard disc drive of a Macintosh G4 computer. For later off-line analysis, signals were also digitized and stored on a data recorder CDAT4 (Cygnus Technology, Delaware Water Gap, PA).
To evoke IPSPs/IPSCs or excitatory postsynaptic currents (EPSCs), a bipolar stimulating electrode with a tip distance of 0.2–0.3 mm was placed into the GP 300 µm ventrocaudal to the recording neuron. In some experiments, the Str or the internal capsule (IC) was also stimulated. Electrical stimulation (200 µs in duration) with 2–50 pulses (usually 20 pulses) at 25–200 Hz (usually 50 Hz) was delivered through the bipolar electrode at 2–3-min intervals. To isolate GABAergic responses from glutamatergic ones, the N-methyl-D-aspartate (NMDA) receptor antagonist, 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid (CPP, 30 µM), and the AMPA/kainate receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX; 5–10 µM), were applied to the bath. To isolate EPSCs, the GABAA receptor antagonist gabazine (10 µM) was applied to the bath. To record action potential-independent miniature IPSCs (mIPSCs), tetrodotoxin (TTX; 1 µM) was applied to the bath in addition to the glutamate antagonists.
Histology
After recording, the slices were fixed overnight with a mixture of 4% paraformaldehyde and 0.2% picric acid. The fixed slices were rinsed several times with phosphate-buffered saline, incubated overnight with avidin-biotin-horseradish peroxidase complex (1% in buffered saline with 0.4% Trition X-100), rinsed, and then reacted with 3,3-diaminobenzidine. The slices were postfixed with 0.5% osmium tetroxide, dehydrated, infiltrated with a plastic resin, and mounted onto glass slides.
Data analysis and statistics
The amplitude of the slow IPSPs induced by repetitive stimulation was determined in the following manner. The peak of the slow IPSP occurred 100 ms after the end of the repetitive stimulation. To suppress noise error, a mean response amplitude for a 200-ms period starting at the end of the stimulation was obtained from digitized data. This value was then subtracted from the mean prestimulus membrane potential for 5 s. We also normalized the amplitude of the slow IPSP by comparing the amplitude of the response to the amplitude of the current pulse injections. A current pulse (–10 pA, 300 ms) was injected 6 s prior to the stimulation to monitor the input resistance and the mean amplitude of the current pulse-induced response for the last 200 ms of the pulse was obtained. To obtain the normalized amplitude, the amplitude of the slow IPSP measured with the method described in the preceding text was divided by the amplitude of the current pulse-induced response.
mIPSCs were analyzed using the Mini Analysis Program (Synaptosoft, Decatur, GA). Events were ranked by amplitude and inter-event interval for the preparation of the cumulative probability distribution within 2-min epochs for control and drug conditions. The cumulative probability distributions were compared by the Kolmogorov-Smirnov test. All group data were expressed as means ± SD and analyzed statistically using the Student's t-test or an ANOVA with a post hoc Bonferroni test.
Chemicals
(R)-Baclofen, CGP52432 CGP55845 and (S)-(+)-
-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) were obtained from Tocris Cookson (Ellisville, MO). CPP, NBQX and gabazine (SR-95531) were obtained from Sigma-Aldrich RBI (St. Louis, MO). QX-314 was obtained from Alomone Labs (Jerusalem, Israel).
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RESULTS |
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To verify the existence of GABAB receptor-mediated inhibition, unitary responses of GP neurons to repetitive local stimulation (20 pulses at 50 Hz) were recorded using the cell-attached recording technique. The neurons examined (n = 6) exhibited regular spiking activity (6–13 Hz). Repetitive local stimulation increased the firing rate of the action potentials during the stimulation in all six neurons examined (7.0 ± 2.7 Hz at prestimulation to 15.2 ± 4.5 Hz during stimulation, n = 6, P < 0.02, paired t-test). In four of the six neurons, the excitation was followed by a pause in the firing for 200–500 ms (Fig. 1A). The two remaining neurons responded with an excitation only. To isolate the GABAB receptor-mediated response, a mixture of the AMPA/kainate receptor antagonist NBQX (10 µM), the NMDA receptor antagonist CPP (30 µM), the GABAA receptor antagonist gabazine (10 µM), and the metabotropic glutamate receptor subtype 1 (mGluR1) antagonist LY367385 (10 µM) were applied. The antagonist mixture blocked the excitation and prolonged the duration of the pause but did not alter the basal firing activity of any neuron (8.9 ± 3.8 Hz in control and 8.4 ± 2.8 Hz in the antagonist mixture, n = 6, P > 0.7, paired t-test; Fig. 1B). An additional application of the GABAB receptor antagonist CGP55845(3 µM) completely blocked the prolonged pause (Fig. 1C). These results suggested that the pause was due to activation of postsynaptic GABAB receptors. To further characterize the GABAB receptor-mediated response, we performed the following experiments.
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To characterize the GABAB receptor-mediated responses, whole cell patch-clamp recordings were performed. The neurons triggered repetitive firing without prominent spike accommodation on depolarizing current injection and had either prominent or moderate Ih-like sags on hyperpolarizing current injection, corresponding to both type A and B neurons reported by Cooper and Stanford (2000) and to the most numerous type in adult rodent GP (Kita and Kitai 1991; Nambu and Llinás 1994). Intracellular staining with Neurobiotin revealed that these neurons were of medium size and had fusiform- or multipolar-shaped somata with long, slowly tapering, smooth dendrites (data not shown).
Current-clamped neurons were continuously hyperpolarized (–65 to –72 mV) to prevent spontaneous spiking. The repetitive local stimulation (20 pulses at 50 Hz) induced fast excitatory postsynaptic potentials (EPSPs) with or without accompanying action potentials followed by fast inhibitory postsynaptic potentials (IPSPs) or slow IPSPs in the hyperpolarized neurons (Fig. 2A). In some neurons, these responses were followed by a small, slow depolarization. The slow depolarization was mediated in part by mGluR1 (unpublished observations). Application of gabazine (10 µM) eliminated the fast IPSPs, significantly increased the amplitudes of the fast EPSPs (Fig. 2D) and increased the number of the action potentials triggered by the EPSPs (Fig. 2B). Gabazine did not change the amplitude of the slow IPSPs (Fig. 2E). Additional application of NBQX (10 µM) and CPP (30 µM) abolished the fast EPSPs (Fig. 2, C and D) and significantly enhanced the amplitude of the slow IPSPs (Fig. 2, C and E). In 3 of 12 neurons tested, the slow IPSPs were undetected in control conditions but were observed after application of the antagonist mixture. The blockade of ionotropic glutamate receptors also caused a faster onset of the slow IPSPs. The latency of the slow IPSPs was 334.9 ± 185.7 ms in control and 152.2 ± 65.6 ms in the presence of gabazine, NBQX, and CPP (P < 0.05, paired t-test; n = 9).
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The induction of CGP55845sensitive slow IPSPs was also tested by repetitive stimulation of the Str and the IC. Although both Str and IC stimulation induced CGP55845sensitive slow IPSPs in GP neurons, the number of slow IPSP inducing neurons with the standard stimulation (100 µA, 20 pulses at 50 Hz) was lower for the Str (40%, n = 10) and slightly lower for the IC (61.4%, n = 102) as compared with GP stimulation. The slow IPSPs to IC stimulation were usually preceded by large fast EPSPs (data not shown).
Optimal stimulus condition to evoke slow IPSP
The optimal number and frequency of repetitive stimulations to evoke slow IPSPs were examined in five neurons that had been identified as being able to evoke slow IPSPs. The neurons were current-clamped at approximately –70 mV during recording in the ACSF containing the aforementioned glutamate and GABAA blockers. The mGluR1 receptor antagonist LY367385 (50 µM) was also included to suppress the slow depolarizations evoked in some neurons. In the first test, the number of stimulus pulses was increased stepwise from 2 to 50 or 100 with the standard stimulus frequency of 50 Hz and intensity of 100 µA. Stimulation with double pulses failed to evoke identifiable slow IPSPs in all neurons. The minimum, but also sufficient, stimulus pulse number needed to evoke distinct slow IPSPs was five. As the pulse number increased, the amplitude and duration of the responses increased (n = 5; Fig. 3A). Fifty pulse stimulations at 50 Hz evoked a nearly saturated response (Fig. 3, A and B). In the second test, the stimulus frequency was changed to a constant 10 pulses with an intensity of 100 µA. Three of the five neurons evoked the largest slow IPSPs by 50-Hz stimulation and remaining two neurons by 100-Hz stimulation (Fig. 3, C and D). The results indicated that the amplitude and the duration of the slow IPSPs were dependent on both the number and the frequency of stimulus pulses.
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Examination of GABAB receptor antagonist effects on the pharmacologically isolated slow IPSPs was initiated with 3 µM CGP55845 the concentration expected to block GABAB receptors sufficiently according to a number of previous in vitro studies on other brain areas (Mott et al. 1999; Pham et al. 1998). CGP55845(3 µM) significantly suppressed the slow IPSPs (Fig. 4B). Application of 0.3 µM CGP55845also significantly inhibited the slow IPSPs (Fig. 4, A and B). The effects were not significantly different between the two concentrations (P > 0.05; Fig. 4B). In contrast, 0.03 µM CGP55845had no effect (Fig. 4B). CGP55845
3 µM did not alter the membrane potential and the conductance. Application of another GABAB receptor-selective antagonist CGP52432(3 µM) also abolished the slow IPSPs (Fig. 4B). Application of TTX (1 µM) totally abolished the slow IPSPs (data not shown). These data suggested that the slow IPSPs induced by the repetitive local stimulation were due to the activation of postsynaptic GABAB receptors.
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Recording from hyperpolarized neurons revealed that, in some neurons, repetitive stimulation evoked slow depolarizing responses that were resistant to the antagonists of ionotropic glutamate, GABAA receptors, and of mGluR1 (Figs. 4B and 5A). Because of the existence of pharmacologically unblockable slow depolarizing responses, the estimate of slow IPSP reversal potentials was performed in the following manner. First, we measured the amplitude of the slow IPSPs evoked at three different membrane potentials (–70, –90, and –110 mV, Fig. 5A). After application of CGP55845(3 µM), the same experiment was repeated. The CGP55845sensitive components of the slow responses were estimated by subtracting the corresponding responses (Fig. 5B). The reversal potential, estimated by plotting the holding potentials (Vh) versus the slow IPSP amplitudes, was –94.9 ± 6.6 mV (n = 5; Fig. 5, C and D), which was close to the potassium equilibrium potential (–101.3 mV) obtained by the Nernst equation.
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GABAB receptors are localized on the GABAergic and glutamatergic presynaptic terminals as well as on postsynaptic elements in the rat GP (Chen et al. 2004). We examined whether synaptically released GABA can activate presynaptic GABAB receptors and control GABA release. The ACSF contained the ionotropic glutamate receptor blockers NBQX (10 µM) and CPP (30 µM). The GP neurons were voltage-clamped at –80 mV with electrodes filled with an electrolyte containing high-Cl and QX-314 (3 mM). QX-314 is known to inhibit the postsynaptic GABAB response as well as the sodium current (Andrade 1991; McLean et al. 1996; Nathan et al. 1990). To confirm the effect of QX-314 on the postsynaptic GABAB response, we examined the effects of baclofen (1–30 µM), a selective agonist of GABAB receptors, on the GP neurons. The neurons were voltage-clamped at –65 mV in the normal potassium concentration (3 mM). Bath application of baclofen dose-dependently elicited an outward current in all GP neurons recorded with the normal pipette solution (Fig. 6Aa). The effect of baclofen was saturated at 10 µM because 30 µM baclofen showed no further increase in the current amplitude (Fig. 6B). When the recordings were performed with pipettes containing 3 mM QX-314, 10 µM baclofen evoked significantly smaller outward currents (Fig. 6, Ab and B).
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We also examined whether the synaptically released GABA could modulate glutamate release by activating the GABAB receptors located on the glutamatergic axon terminals. The GP neurons were voltage-clamped at –80 mV with the electrodes filled with the low-Cl intracellular electrolyte including QX-314 (3 mM). To block the GABAA receptor-mediated IPSCs, gabazine (10 µM) was applied to the bath. Under these conditions, the repetitive stimulation (20 pulses at 50 Hz) delivered to the GP evoked EPSCs in GP neurons (n = 5; data not shown). In contrast to the augmenting effect of CGP55845on the IPSC amplitudes, CGP55845failed to affect the amplitudes of the EPSCs (Fig. 7F).
Effect of baclofen on miniature IPSCs
Finally, we examined the effects of the GABAB receptor agonist baclofen on TTX-insensitive miniature IPSCs (mIPSCs). mIPSCs were recorded in the presence of NBQX (10 µM), CPP (30 µM), and TTX (1 µM) using pipettes containing the high-Cl and QX-314 (3 mM). Gabazine (10 µM) application blocked the mIPSCs, confirming that they were mediated via GABAA receptors (data not shown). Neurons exhibiting relatively high-frequency mIPSCs were chosen for this experiment. Bath application of both 10 and 30 µM baclofen significantly reduced the mIPSCs frequency (Fig. 8, A–D). The cumulative distributions of the inter-mIPSC intervals showed that baclofen significantly increased the width of the distribution curve (P < 0.01, Kolmogorov-Smirnov test, Fig. 8E). In either concentration, baclofen had no effect on the mean amplitude (Fig. 8G) or the amplitude distribution (Fig. 8F) of the mIPSCs. The effect of baclofen was blocked when CGP55845(3 µM) was co-applied with baclofen (Fig. 8D). Thus these results suggested that activation of the presynaptic GABAB receptors reduces the release of GABA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Responses to intra-pallidal repetitive stimulation
Repetitive local stimulation evoked a series of fast EPSPs followed by a slow IPSP and, in some neurons, a slow depolarization. We are currently investigating the nature of the slow depolarization. Preliminary results suggest that the response is partially mediated by mGluR1 (unpublished observations). The fast EPSPs were glutamatergic and thus were considered to be due to the activation of glutamatergic afferents including those from the STN (Kita 1992, 1994; Kita and Kitai 1991; Parent and Hazrati 1995). Gabazine application significantly increased the amplitudes of the fast EPSPs, suggesting that GABAA-mediated IPSPs overlapped with the fast EPSPs. GABAA receptors are unlikely to be involved in the induction of the slow IPSP because gabazine did not affect the amplitudes of the slow IPSPs. In the presence of gabazine, the NBQX/CPP mixture augmented the amplitude and shortened the latency of the slow IPSPs. This suggests that the fast EPSPs partially overlapped with the slow IPSPs and decreased the amplitude of the slow IPSPs or that activation of ionotropic glutamate receptors at the synaptic terminals was suppressing the GABA release (Rodriguez-Moreno et al. 1997). Thus the slow IPSP induction was attributable to a direct activation of GABAergic axons that did not require glutamatergic activations in other intermediate neurons.
The finding that a repetitive stimulation of five pulses, delivered at 50 Hz, was required to evoke the slow IPSPs suggests that a relatively large amount or a prolonged release of GABA is necessary to effectively activate postsynaptic GABAB receptors. This is consistent with previous observations that a single stimulation of the Str evoked fast GABAA-mediated IPSPs/IPSCs but not GABAB-mediated responses in GP neurons (Matsui and Kita 2003; Ogra and Kita 2000). Similar requirements of repetitive stimulation for the induction of GABAB-mediated responses have been reported in other neurons (Johnson et al. 1992; Saitoh et al. 2004). The abundantly expressed GABA transporters in the GP might regulate the extent of GABAB receptor activation (Chen and Yung 2003; Ikegaki et al. 1994; Yasumi et al. 1997).
Postsynaptic GABAB responses
Intra-pallidal repetitive local stimulation was more effective in inducing slow IPSPs compared with Str and IC stimulation probably because it activated the local collateral axons and the striatal afferent axons more effectively. Unfortunately, the results obtained from Str and IC stimulation do not identify the origins of the GABAB responses. Stimulation of the Str activates both Str-GP and GP-Str axons along with its intra-pallidal collaterals (Kita and Kitai 1994; Ogura and Kita 2000). Similarly, stimulation of the IC activates the local collaterals and intra-pallidal collaterals of Str-entopeduncular/substantia nigra axons (Kita and Kitai 1994; Wu et al. 2000). Receptor localization studies suggest both striatal and local collateral synapses can evoke GABAB responses (Chen et al. 2004).
All repetitive stimulation at frequencies of 25–200 Hz could induce the slow IPSPs in GP neurons, although the most effective stimulus frequency was 50 or 100 Hz. The firing pattern of the major type of rat GP neurons in vivo is high frequency with pause and bursts (Gardiner and Kitai 1992; Kita and Kitai 1991; Magill et al. 2000; Ni et al. 2000). Thus it can be speculated that GABAB receptors in the GP would be sufficiently activated even with the spontaneous firing of GP neurons in vivo and be maximally activated by bursting activities. In contrast, projection neurons in rat Str are usually quiescent but show movement or task related activities of various intensities (Carelli et al. 1997; Gardiner and Kitai 1992; Wilson and Groves 1981). Thus the GABAB receptors associating with striatal synapses may be activated only when Str neurons were activated by strong cortical inputs.
The induction of postsynaptic GABAB responses requires intracellular GTP (Couve et al. 2000; Misgeld et al. 1995). When the cell-attached recordings were performed to preserve the intracellular milieu, repetitive stimulation induced a GABAB-mediated pause in spontaneous firings that had a similar duration to the slow IPSP. This finding suggests that the GTP contained in the pipette did not significantly alter the nature of postsynaptic GABAB responses.
The postsynaptic GABAB responses are mainly mediated by potassium conductances through the activation of G-protein-gated inwardly rectifying potassium (GIRK) channels (Jones et al. 1998; Kaupmann et al. 1998; White et al. 1998). Immunohistochemical and in situ hybridization studies suggested the expression of four subtypes of GIRK proteins and mRNAs in rodent GP neurons (Murer et al. 1997; Ponce et al. 1996; Wickman et al. 2000). In support of the functional expression of these channels, the reversal potential of the slow IPSP estimated in the present study was close to the potassium equilibrium potential as calculated by the Nernst equation. It is possible that the large variation in the amplitude of the GABAB response observed among GP neurons could be due to differences in the expression patterns of the GIRK channels in individual GP neurons.
Presynaptic GABAB responses
To study GABAB-mediated presynaptic modulations, the postsynaptic GABAB response was blocked by the intracellular injection of QX-314 (Andrade 1991; McLean et al. 1996; Nathan et al. 1990) and by the elevation of the extracellular potassium concentration that shifted the potassium equilibrium potential close to the holding membrane potential. These treatments were very effective as repetitive stimulation or baclofen application evoked no postsynaptic GABAB-mediated responses in these neurons. Under these conditions, the GABAB antagonist CGP55845increased the amplitudes of repetitive local stimulation-induced fast IPSCs, suggesting that synaptically released GABA activated presynaptic GABAB autoreceptors and decreased GABA release. Consistent with these findings, baclofen significantly reduced mIPSCs frequency without affecting their amplitudes.
The CGP55845induced increase of the fast IPSCs reached maximum at about five stimulus pulses, suggesting that a repetitive stimulation with five pulses at 50 Hz could fully activate presynaptic GABAB receptors. This was in contrast with evoking saturated-slow IPSPs that require a repetitive stimulation of 50 pulses at 50 Hz. The possible reasons for the difference include that GABAB autoreceptors are largely localized at the main body of the synaptic specializations that are closest to GABA release site, while postsynaptic GABAB receptors are distributed more distantly (Chen et al. 2004). Different mechanisms between pre- and postsynaptic actions may also be involved. Possible presynaptic mechanisms for the reduction of GABA release include an activation of potassium conductance (Thompson and Gähwiler 1992) and a direct inhibition of Ca2+ entry and/or of the secretion process itself (Dittman and Regehr 1996; Takahashi et al. 1998; Thompson et al. 1993; Wu and Saggau 1997).
In contrast to the results of IPSCs amplitude modulation, the GABAB receptor blockade failed to affect the fast EPSCs; this is in agreement with the report by Hanson and Jaeger (2002). Presynaptic GABAB receptors are localized at asymmetric synapses as well as at symmetric synapses in the rat GP, and the bath application of baclofen reduced the mEPSCs frequency without changing their amplitudes in GP neurons (Chen et al. 2002, 2004). Thus functional GABAB receptors might exist at the glutamatergic synapses. The failure of the GABAB antagonist to affect the amplitudes of evoked EPSCs under our recording conditions might be attributable to the difference in the distance between the GABA release sites and the location of presynaptic GABAB receptors. GABAB autoreceptors are largely localized at the main body of the synaptic specializations, whereas GABAB receptors at asymmetric synapses are mainly localized at perisynaptic sites (Chen et al. 2004). It is tempting to speculate that the autoreceptors and the GABAB receptors at asymmetric synapses might play different roles such as that the former operates in normal conditions and the latter in pathologic conditions such as Parkinson's disease in which a large number of pallidal neurons fire synchronously (Nini et al. 1995).
Functional implications
The results presented here imply that GABAB receptor actions contribute significantly to the control of the level and pattern of GP neuronal activity. Through pre- and postsynaptic GABAB receptors, GABAergic inputs with various strengths might dynamically modulate GP neuron activity. For instance, when prolonged high-frequency repetitive activation of GABAergic terminals takes place, released GABA would activate both post- and presynaptic GABAB receptors, the former inducing slow IPSPs and the latter leading to the suppression of GABAergic transmission. These actions would dampen GABAA-mediated actions following the burst inputs. On the other hand, a short bursting activity of GABAergic terminals would mainly dampen the succeeding GABA release.
The present findings also have implications for our understanding of the rhythmic activities generated by the GP-STN network in normal and pathophysiological conditions such as Parkinson's disease (Bevan et al. 2002b). Rhythmic activities could be induced by both cortical excitatory and pallidal inhibitory synaptic inputs and could be supported or enhanced by membrane properties of these neurons and also by the reciprocal connections between the GPe and STN (Bevan et al. 2002b; Magill et al. 2000, 2004). High-frequency firing of some GP neurons would induce GABAB-mediated slow IPSPs in both GP (present finding) and STN neurons (N. E. Hallworth and M. D. Bevan, personal communication). The slow IPSP in the GP neurons may disinhibit postsynaptic STN neurons. In addition, the slow IPSP evoked in the STN neurons may be followed by rebound burst firings (Bevan et al. 2000, 2002a ). The bursting activities in the STN neurons might, in turn, cause burst firings in recipient GP neurons. Taken together, these GABAB-contributed feedforward and feedback mechanisms might be involved, at least in part, in the rhythm generation in the GP-STN network.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Kita, Dept. of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, 855 Monroe Ave., Memphis, TN 38163 (E-mail: hkita{at}utmem.edu)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) in standard ACSF. B: bath application of gabazine (10 µM) increased the amplitude of the EPSPs and the number of action potentials triggered by the EPSPs. Gabazine did not change the amplitude of the slow IPSP in this neuron. C: an additional application of an NBQX (10 µM) and CPP (30 µM) mixture completely suppressed the EPSPs and, at the same time, shortened the onset and increased the duration of the slow IPSP. The action potentials and stimulus artifacts were truncated. D: a summary of the effects of GABAA and ionotropic glutamate receptor antagonists on normalized amplitudes of the fast EPSPs. Normalized amplitudes were obtained by dividing the amplitude of the fast EPSPs by the amplitude of the prestimulus test pulse (see METHODS for the detailed normalization procedure). Gabazine significantly increased the amplitude of the fast EPSPs, whereas an additional application of an NBQX and CPP mixture abolished the fast EPSPs. E: a summary of the effects of GABAA and ionotropic glutamate antagonists on the normalized amplitude of the slow IPSPs. Normalized amplitudes were obtained by the same methods as in D. Gabazine alone did not change the amplitude of the slow IPSPs, whereas an additional application of an NBQX and CPP mixture significantly increased the amplitude of the slow IPSPs.
