Using the whole-cell voltage-clamp configuration of the patch-clamp technique, ionic currents were recorded from isolated neurons at -80 mV holding potential after 18–32 hours as previously published
[15 (link), 58 (link)]. The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2 (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck). Borosilicate glass micropipettes (Science Products, Hofheim, Germany) pulled with a horizontal puller (Sutter Instruments Company, Novato, CA, USA) were filled with internal solution (ICS, in mM): 148 KCl, 2 MgCl2, 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2, 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck). After filling, electrode resistance was 4–6 MΩ. Currents were filtered at 2.9 kHz, sampled at 3 kHz and recorded using an EPC-9 (HEKA, Germany) and the Pulse v8.74 software (HEKA) without Rs compensation. Experiments were performed at room temperature and only one neuron was tested per Petri dish. An automated seven-barrel system with common outlet positioned at 100 μm distance from the recorded cell was used for fast drug administration
[15 (link)]. S1P (1.0 μM) was used as intermittent conditioning stimuli (60s). S1P, capsaicin, PTX and GDPβS were purchased from Sigma Aldrich. All other chemicals were purchased from Merck-Calbiochem.
[15 (link), 58 (link)]. The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2 (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck). Borosilicate glass micropipettes (Science Products, Hofheim, Germany) pulled with a horizontal puller (Sutter Instruments Company, Novato, CA, USA) were filled with internal solution (ICS, in mM): 148 KCl, 2 MgCl2, 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2, 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck). After filling, electrode resistance was 4–6 MΩ. Currents were filtered at 2.9 kHz, sampled at 3 kHz and recorded using an EPC-9 (HEKA, Germany) and the Pulse v8.74 software (HEKA) without Rs compensation. Experiments were performed at room temperature and only one neuron was tested per Petri dish. An automated seven-barrel system with common outlet positioned at 100 μm distance from the recorded cell was used for fast drug administration
[15 (link)]. S1P (1.0 μM) was used as intermittent conditioning stimuli (60s). S1P, capsaicin, PTX and GDPβS were purchased from Sigma Aldrich. All other chemicals were purchased from Merck-Calbiochem.
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