Excitatory or inhibitory currents (EPSCs or IPSCs) were evoked by stimulating the dorsal root attached to each slice using a suction electrode. Stimulus duration was 0.1 ms, stimulus intensities were determined by performing extracellular recordings of compound action potentials from the dorsal root (see Results and Fig. 5A). Monosynaptic vs polysynaptic EPSCs were identified following the procedure described by Torsney and MacDermott44 and Daniele and MacDermott38. In particular, EPSCs were considered to be monosynaptic if there were no failures during high frequency stimulation (20 pulses at 20 Hz for Aβ, at 10 or 2 Hz for Aδ and 1 Hz for C fibers). Onset latency varied <1 ms for monosynaptic A fiber mediated EPSCs.
Active and passive membrane properties were determined by applying current steps (10 or 20 pA of amplitude) in current clamp, starting from a membrane potential of −60/−65 mV. Membrane resistance was calculated from responses to hyperpolarizing pulses, rheobase was defined as the current step amplitude able to elicit the lowest number of action potentials64. Action potential threshold was determined for the first action potential evoked at rheobase, at the point where the increase of membrane potential exceeds 20 mV/ms.
Drugs were bath-applied for 1 min. All drugs were obtained from Sigma-Aldrich (Saint Louis, USA), except for GRP, that was purchased from Genscript (Piscataway, USA), and tetrodotoxin (TTX) from Tocris (Bristol, UK). Data were analyzed off-line using pClamp10 or MiniAnalysis (Synaptosoft, Decatur, USA). Graphs were obtained using Sigmaplot 11 (Systat software, San Jose, USA).
Experimental design and statistical analysis
Electrophysiology experiments were performed on spinal cord slices from GRPR eGFP mice of either sex. A total of 92 mice have been used. Typically, 2–3 viable slices, with well-preserved synaptic connections, were obtained from the lumbar segment of the spinal cord (L3-L5). One to three neurons were recorded from each slice (only one cell if drugs were applied). Only neurons showing stable recording conditions (constant series resistance and membrane potential) were included in this study. Sample sizes were established according to similar studies of firing pattern and synaptic input characterization33,38.