All experiments were carried out in accordance with the Guiding Principles for the Care and Use of Laboratory Animals as set out by the Japanese Pharmacological Society and approved by the Ethical Committee on Animal Experiments of the Pharmaceutical Research Institute, Kyowa Hakko Kogyo.
The experiments were performed on male Sprague-Dawley rats (12-16 days postnatal). Animals were anaesthetized with ether and decapitated. The brain was removed rapidly. Slices containing the globus pallidus were cut on a DTK-1000 microslicer (Dosaka, Japan) at a thickness of 250 μm in an oblique plane, about 30 deg rostral-up to the horizontal (Kawaguchi et al. 1989). Slices were then incubated in oxygenated Ringer solution at a temperature of 29-30 °C for 1 h. The standard Ringer solution had the following composition (mm): NaCl, 124; KCl, 3.0; CaCl2, 2.4; MgCl2, 1.2; NaHCO3, 26.0; NaH2PO4, 1.0; and glucose, 10.0; continuously bubbled with a mixture of 95 % O2 and 5 % CO2. After incubation, a single slice was transferred to a recording chamber placed on the stage of an upright microscope, and was continuously perfused (3-4 ml min−1) with oxygenated Ringer solution at 30 °C. The remaining slices were kept in a holding chamber containing oxygenated Ringer solution at room temperature.
Whole-cell recording and data analysis
Neurones in the globus pallidus were visualized using infrared differential interference videomicroscopy (IR-DIC) with a × 40 water immersion objective (Nikon, Tokyo, Japan). Micropipettes for whole-cell recordings were pulled with a P-97 Flaming-Brown electrode puller (Sutter Instrument Company, Novato, CA, USA) from borosilicate glass (1.5 mm outer diameter, Clark Electromedical Instruments, Reading, UK). These had a final resistance of 2-5 MΩ when filled with intracellular solution. For whole-cell current-clamp recordings, micropipettes were filled with a solution containing (mm): potassium methanesulfonate, 115; KCl, 5.0; EGTA, 0.5; MgCl2, 1.7; ATP, 4.0; GTP, 0.3; and Hepes, 8.5; pH 7.2-7.4; osmolarity, 285 mosmol l−1. Current-clamp recordings were made in the bridge mode with an Axoclamp-2B amplifier (Axon Instruments, Foster City, CA, USA). Resting potentials were measured just after the patched membranes were ruptured by suction. Input resistance of cells was determined by passing hyperpolarizing current pulses (duration, 600-900 ms), which induced voltage shifts of 6-15 mV negative to rest.
For whole-cell voltage-clamp recordings, the pipette solution contained (mm): caesium methanesulfonate, 120; KCl, 5.0; EGTA, 10.0; CaCl2, 1.0; MgCl2, 2.0; ATP, 4.0; GTP, 0.3; Hepes 8.0; and QX314, 5.0; pH 7.2-7.4; osmolarity, 295 mosmol l−1. The quaternary lidocaine derivative QX314 was included to suppress fast sodium currents. Voltage-clamp recordings were made with an Axopatch-1D amplifier (Axon Instruments). Throughout voltage recordings, series resistance was monitored via a voltage step (-5 mV, 10 ms), and typical values for the series resistance were 10-20 MΩ. Experiments were discarded if changes over 20 % of the series resistance were seen. The experiments showing no recovery of IPSC amplitude when washout of the drug was omitted. Liquid junction potentials were not corrected.
Evoked synaptic currents were elicited by focal stimulation (0.1 Hz, 200 μs, 10-100 V) via a glass micropipette filled with the extracellular solution and placed within 500 μm of the recorded neurone.
Signals were filtered at 2 kHz and digitized at 5 kHz using an ITC-16 interface (Instrutech, Great Neck, NY, USA) connected to a Power Macintosh computer running Pulse (HEKA Elektronik, Lambrecht, Germany) software. Analysis for evoked IPSCs was performed using Pulse (HEKA) and IGOR Pro (WaveMetrics, Lake Oswego, OR, USA) software. For drug effects on evoked IPSCs, a normalized value for the evoked IPSC peak amplitude under drug application was obtained by averaging the peak amplitude of eight consecutive evoked IPSCs and by subsequently dividing this mean value by the average obtained in the control period.
Spontaneous synaptic currents were automatically analysed using the Mini Analysis Program (Synaptosoft Inc., Leonia, NJ, USA). Events were ranked by amplitude and inter-event interval for preparation of cumulative probability distributions within 200-300 s epochs for control and drug conditions. The cumulative probability distributions were compared by the Kolmogorov-Smirnov test; P < 0.05 was taken as indicating statistical significance.
All data are given as means ±s.e.m., unless stated otherwise. Responses to agonists are expressed as a percentage of the control obtained before the addition of the agonists. The effects of agonists, expressed as percentages, were compared to the effects of agonist-free solution using Steel's test. The effects of antagonists were compared using Wilcoxon's rank sum test. Student's paired t test was performed to compare the raw values of the control with the responses in the presence of agonists applied on the same cell for analysis of paired-pulse facilitation and spontaneous synaptic currents.
In an attempt to identify the recorded cells morphologically, 20 mm biocytin was included in the pipette solution so that they were filled by diffusion (Horikawa & Armstrong, 1988). Slices containing biocytin-loaded cells were fixed by immersion in 4 % paraformaldehyde and 0.2 % picric acid in 0.1 m phosphate buffer (PB) overnight at 4 °C, rinsed in PB for 30 min, and incubated in PB containing 0.5 % H2O2 for 30 min to suppress endogenous peroxidase activity. They were then incubated in 10 and 20 % sucrose for 30 min and 1 h, respectively. The slices, without resectioning, were then washed with Tris-buffered saline (TBS) containing 0.5 % Triton X-100 and avidin-biotin-peroxidase complex (1:100; Vector Laboratories, Burlingame, CA, USA) for 4 h at room temperature. After rinsing, the slices were reacted with 3,3′-diaminobenzidine tetrahydrochloride (DAB; 0.05 %) and H2O2 (0.003 %) in TBS, and mounted on slides.
Drugs were applied by replacing the solution superfusing the slice with one containing a set concentration. d-2-Amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and ZM241385 were obtained from Tocris Cookson (Bristol, UK); tetrodotoxin (TTX) was from Wako (Tokyo, Japan); CGS21680 was from Research Biochemicals International (Natick, MA, USA); and (-)-bicuculline methiodide was from Sigma (St Louis, MO, USA). (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) was synthesized at the Medicinal Chemistry Department of the Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd (Shizuoka, Japan).