All experiments were done in accordance with the regulations of the Italian Animal Welfare act (DL 27/1/92 n. 116) following the European Community directives no. 86/609 and 93/88 (Italian Ministry of Health authorization for the local animal care facility in Trieste: D. 69/98-B), and approved by the local authority veterinary service.
Under deep urethane anaesthesia (2 g (kg body wt)−1), i.p. injection) neonatal (1- to 6-day-old; P1–6) Wistar rats were decapitated, their brainstems dissected out and fixed to an agar block (Donato & Nistri, 2000). Transverse slices 200 μm thick were cut in ice-cold Krebs solution. After 1 h recovery at 32°C, slices were kept for at least 1 h at room temperature before recording.
Whole-cell patch recording
HMs were visualized within nucleus hypoglossus with an infrared video-camera, patched and recorded under voltage and current clamp mode. A few cells were also injected with neurobiotin through patch pipette (0.2% in intracellular solution) and processed for histology (Lape & Nistri, 2001). All electrophysiological recordings (in voltage or current clamp mode) were carried out as described before in detail (Sharifullina et al. 2004). For voltage clamp experiments HMs were clamped within the range of −60 to −70 mV holding potential to minimize the leak current at rest. For current clamping, cells were initially kept at their resting level of membrane potential without injecting intracellular current which was applied for certain tests only.
Analysis of a sample of cells voltage clamped with a Cs+-filled pipette gave an average holding potential of −62 ± 1 mV (input resistance = 148 ± 8 MΩ; n= 62), while for a pool of cells recorded with intracellular K+ solution the corresponding holding potential was −67 ± 2 mV (input resistance = 163 ± 13 MΩ; n= 26; P= 0.35 between cell groups). For double-patch recordings two neighbour cells were simultaneously patch clamped (average distance ≤ 30 μm). To elicit synaptic glutamatergic responses we electrically stimulated premotoneurones in dorsomedullary reticular column (DMRC; Cunningham & Sawchenko, 2000) as detailed earlier (Sharifullina et al. 2004). Single stimuli were applied at 10 s interval (0.1 ms, 10–100 V intensity). All electrophysiological responses were filtered at 3 kHz, sampled at 5–10 kHz, acquired and analysed with pCLAMP 9.0 software (Axon Instruments).
Solutions and drugs
The external solution for cutting and maintaining slices contained (mm): NaCl, 130; KCl, 3; NaHPO4, 1.5; CaCl, 1; MgCl2, 5; glucose 15 (315–320 mosm), and was continuously oxygenated with O2 95%–CO2 5%. In the recording chamber slices were superfused with gassed solution containing (mm): NaCl, 130; KCl, 3; NaHPO4, 1.5; CaCl2, 1.5; MgCl2, 1; glucose 15 (315–320 mosmol l−1), pH 7.4. Unless otherwise stated, all experiments were done in the continuous presence of bicuculline (10 μm) and strychnine (0.4 μm) to block GABA and glycine-mediated transmission (Donato & Nistri, 2000; Marchetti et al. 2002) so that glutamatergic effects could be observed in isolation. Patch pipettes contained (mm): KCl, 130; NaCl, 5; MgCl2, 2; CaCl2 0.1; Hepes, 10; EGTA, 5; ATP-Mg, 2; GTP-Na, 1 (pH 7.2 with KOH, 280–300 mosmol l−1). In about 50% of recordings K+ was replaced with equimolar Cs+. The pipette solution used for voltage clamp experiments often also contained QX-314 (300 μm) to block voltage-activated Na+ currents and slow inward rectifier (Ih) (Marchetti et al. 2003) as indicated in the Results. In a group of experiments 20 mm BAPTA was added to patch pipette to buffer intracellular Ca2+.
Drugs were applied via the recording saline (2–3 ml min−1 superfusion rate) with the exception of AMPA, which was applied by pressure pulses (10–50 ms; 6 p.s.i. pressure; 100 μm solution; once every 45 s to minimize desensitization) via a closely positioned puffer pipette as described before (Sharifullina et al. 2004). General reagents were of analytical grade. The following drugs were purchased from Tocris (Bristol, UK): 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; selective antagonist for mGlu1 receptors), (RS)-3,5-dihydroxyphenylglycine (DHPG; selective agonist for group I receptors and equipotent on mGlu1 and mGlu5 subtypes), 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; selective antagonist for mGlu5 receptors); (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX),d-amino-phosphonovalerate (APV), N-(2,6-dimethylphenylcar-bamoylmethyl) triethylammonium bromide (QX-314), 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimi-dinium chloride (ZD 7288), glibencl-amide, apamin, (aS)-a-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-5-iodo-2,4-dioxo-1(2H)-pyrimid-inepropanoic acid (UBP 301; selective antagonist for kainate receptors), (+/−)-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcar-bamoyl-6,7-methylenedio-xyphthalazine (SYM 2206; selective antagonist for AMPA receptors), (2S)-3- [[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxy-propyl](phenyl-methyl)phosphinic acid (CGP 55845; selective antagonist for GABAB receptors).
Bicuculline methiodide, 4-hydroxyquinoline-2-carboxylic acid (kynurenic acid), dihydro-β-erythroidine hydrobromide, atropine sulphate, 1,2-bis(2-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrapotassium salt (BAPTA), tolbutamide, strychnine hydrochloride, and carbenoxolone (disodium salt) were purchased from Sigma; tetrodotoxin (TTX) was purchased from Latoxan, and caesium chloride from Calbiochem. Full details about receptor specificity and concentrations of DHPG and antagonists on mGluR1s are given by Schoepp et al. (1999).
Cell input resistance (Rin) was measured as mentioned previously (Sharifullina et al. 2004) while cell capacitance was monitored on-line using pCLAMP 9.0 software. To estimate the strength of electrical coupling between cell pairs, rectangular pulses (±0.3 nA, 1000 ms) were applied to one cell under current clamp and responses recorded simultaneously from both cells: the coupling coefficient was then estimated from the ratio between the response of the stimulated cell and the one of its coupled neighbour (Long et al. 2004).
Each oscillatory cycle comprised a cluster of fast inward currents followed by a slow outward component. To quantify the oscillatory period we measured the time between the first fast discharge in each cycle and the first discharge in the following cycle. For each cell at least 50 cycles were measured and averaged. Analysis of all current responses (synaptic as well as oscillatory ones) was carried out using a template search protocol (pCLAMP 9.0) applied to at least 5-min-long consecutive records. For each cell, templates of various events (synaptic, oscillatory components, etc.) were obtained from electrophysiological records and checked for adequacy when compared with actual responses. Coefficient of variation (standard deviation/mean; CV) was expressed as a percentage value. Data for response rise and decay times were obtained from the 10–90% time interval of the response peak. To fit slow oscillatory outward currents we used the sum of two exponents with pCLAMP 9.0 software. To quantify oscillation periods from current clamp traces when oscillations were associated with intermittent firing, we used their fast Fourier transform obtained with pCLAMP 9.0 software. Whenever current clamp oscillations were accompanied by spikes or devoid of any spike activity, we instead used the standard template constructing protocol reported above to obtain period values.
To check cross correlation between two simultaneously recorded cells, standard cross-correlation analysis was performed using pCLAMP 9.0 for 1 s epochs. Linear regression analysis was carried out with Origin 6.1 (OriginLab Corporation, Northampton, MA, USA). Data are given as mean ±s.e.m. and n is the number of cells, unless otherwise indicated. Statistical significance was assessed with Student's paired or unpaired t test, applied only to raw data with parametric distribution; P < 0.05 was considered as significant.