Hypothalamic neuron cultures were prepared from rat embryos as described elsewhere (Gao et al. 1998). Use of rat tissue was approved by the university committee on animal use. Pregnant rats were given an overdose of Nembutal (100 mg kg−1), embryos were collected, and then the pregnant rats were given a second lethal dose of Nembutal (150 mg kg−1). Briefly, lateral hypothalami were dissected out of the brains of Sprague-Dawley rats at embryonic days (E) 15-18 and cut into small pieces (smaller than 1 mm3). The tissue was digested at 37 °C for 15 min in an enzyme solution (containing 20 units ml−1 papain, 0.5 mm EDTA, 1.5 mm CaCl2, and 0.2 mg ml−1l-cysteine) and mechanically triturated in culture medium to obtain dissociated cells. After washing with culture medium (containing 10 % fetal calf serum), LH cells were plated in 35 mm culture dishes at a density of 200 000-300 000 cells per dish and maintained at 37 °C and 5 % CO2. Serum-containing medium was replaced by serum-free medium 1-2 h after plating. The serum-free culture medium contained neurobasal medium (Gibco), 5 % B27 supplement (Gibco), 0.5 mml-glutamine, 100 units ml−1 penicillin-streptomycin and 6 g ml−1 glucose. Neurons were fed twice a week.
All experiments were performed at room temperature in neurons between 14 and 21 days in vitro. The recording chamber was continuously perfused at a rate of 2 ml min−1 with a bath solution containing (mm): NaCl 150, KCl 2.5, MgCl2 2, CaCl2 2, Hepes 10 and glucose 10; pH 7.3 with NaOH. Whole-cell current and voltage clamp (at -60 mV) were used to observe action potentials, spontaneous, miniature and evoked postsynaptic currents with a L/M EPC-7 amplifier. The patch pipette was made of borosilicate glass (World Precision Instruments) with a Narashige puller (PP-83). The tip resistance of the recording pipettes was 4-6 MΩ after filling with a pipette solution containing (mm): KCl (or KMeSO4) 145, MgCl2 1, Hepes 10, EGTA 1.1, Mg-ATP 2, Na2-GTP 0.5; pH adjusted to 7.3 with KOH. In experiments on calcium currents, the recorded neuron was clamped at -80 mV and then given a voltage step to +20 mV. During these experiments, the bath solution contained (mm): NaCl 100, TEA-Cl 40, KCl 2.5, BaCl2 5, Hepes 10, glucose 10, TTX 1 (μm); pH adjusted to 7.3 with KOH. The pipette solution contained (mm): CsCl 135, MgCl2 1, Hepes 10, BAPTA-Cs 5, Mg-ATP 4, Na2-GTP 0.5; pH adjusted to 7.3 with CsOH. After a gigohm seal and whole-cell access were achieved, the series resistance was between 20 and 40 MΩ and partially compensated by the amplifier. Both input resistance and series resistance were monitored throughout the experiments. Only those recordings with seal resistance higher than 0.8 GΩ and stable series resistance were accepted.
The response of postsynaptic glutamate receptors was tested during voltage clamp by brief pressure application (20 ms duration, 8 p.s.i, 5 s interval between each application) of glutamate (100 μm) in the absence or presence of MCH; MCH was applied continuously for 4 min prior to glutamate application. Under current clamp, negative currents (20 pA, 100 ms) were injected to monitor membrane conductance for control purposes. To study voltage-dependent sodium currents, 40 mm TEA-Cl (an equal amount of NaCl was removed) and 200 μm CdCl2 were added to all extracellular solutions and CsCl was used in the pipette solution. To examine voltage-dependent potassium currents, 1 μm TTX and 200 μm CdCl2 were added to all extracellular solutions.
All data were sampled at 3-10 kHz and filtered at 1-3 kHz with an Apple Macintosh computer using AxoData 1.2.2 (Axon Instruments). Data were analysed with Axograph 3.5 (Axon Instruments) and plotted with Igor Pro software (WaveMetrics, Lake Oswego, OR, USA). Both miniature and spontaneous postsynaptic currents were detected and measured with an algorithm in Axograph 3.5 (Clements & Bekkers, 1997; Gao & van den Pol, 1999). Briefly, a simulated template with the width and time course of a typical synaptic event was moved along the recorded data trace one point at a time. At each position, this template was optimally scaled and offset to fit the data so that an event could be detected. The template function used to approximate the time course of miniature and spontaneous synaptic events had a flat baseline region followed by an exponential rise and decay typical of a synaptic event:
where τrise is the time constant of the rising phase, τdecay is the time constant of the falling phase of the template and t is the time from onset of an idealised synaptic event.
The same criteria were applied during control periods, drug treatment and washout. To eliminate electronic noise, we only used signals > 5 pA. All data were reported as means ±s.e.m. The median and cumulative probability of miniature EPSC (mEPSC) and IPSC amplitudes were analysed based on more than 50 events per condition/cell detected with the above method. In each tested neuron, mEPSCs were obtained by analysing at least 50 detected events from control, MCH and washout groups. The rise time was measured from 10 to 90 % of the maximum amplitude using Axograph software (Axon Instruments). The decay time constant of mEPSC events was determined by fitting the averaged mEPSC events with a monoexponential function with a simplex optimisation procedure in Axograph software. A likelihood estimator was used to generate a goodness of fit with a precision of 1 %. Student's t test and ANOVA were used to compare two and more groups of data, respectively. The Kolmogorov-Smirnov test was used to evaluate the significance of change in the amplitude of miniature postsynaptic currents.
MCH was obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA, USA) The lyophilised peptide was reconstituted in DMSO to a concentration of 1 mm and stored at -70 °C. The stock solution was diluted 1000-fold to the working concentration of 1 μm just prior to use. DMSO (0.1 %) was used in control buffers and did not contribute to the actions of MCH. 2-Amino-5-phosphono-pentanoic acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and bicuculline methobromide (BIC) were obtained from Sigma-RBI (Research Biochemicals International, MO, USA). Tetrodotoxin (TTX) was obtained from Alomone Labs, Ltd (Jerusalem, Israel). Pertussis toxin (PTX) was obtained from Sigma (St Louis, MO, USA).