2.3. Whole‐cell patch‐clamp recording in spinal cord blocks and slices

Cells used for morphological analysis in this study were all labeled during whole‐cell recordings performed in intact (i.e., nonsliced) lumbar spinal cord blocks. Details of the procedures are described in our previous works (Fernandes et al., 2018; Szucs et al., 2013). Briefly, ACSF contained (in mM) NaCl 115, KCl 3, CaCl₂ 2, MgCl₂ 1, NaH₂PO₄ 1, NaHCO₃ 25, and glucose 11 (bubbled with 95% O₂/5% CO₂). The pipettes were pulled from thick‐walled glass (BioMedical Instruments, Germany) and fire polished (resistance, 4–5 MΩ). The pipette solution contained (in mM) KCl 3, K‐gluconate 150, MgCl₂ 1, BAPTA 1, HEPES 10 (pH 7.3 adjusted with KOH, final [K⁺] was 160 mM), and 1% biocytin (Sigma). In some cases (n = 12), concentration of biocytin was reduced to 0.5% and it was complemented by 0.5% rhodamine red to help faster identification of the neuronal soma after serial sectioning.

Electrophysiological recordings aiming to test the hypothesis of the dorsoventral spread of excitation were carried out on spinal cord slices. In these experiments, the artificial cerebrospinal fluid contained (in mM) NaCl 126, KCl 3, CaCl₂ 2, MgCl₂ 2, NaH₂PO₄ 1.3, NaHCO₃ 26, and glucose 10 (bubbled with 95% O₂/5% CO₂). The pipettes were pulled from thick‐walled glass (resistance, 3–5 MΩ). The whole cell recordings in spinal cord slices aimed to record often small, subthreshold postsynaptic potentials; thus, we used an intracellular solution that tried to be as close as possible to intracellular physiological ionic concentrations. The pipette solution contained (in mM) K‐gluconate 124, NaCl₂ 14, ATP‐Mg 1, GTP‐Na 0.3, HEPES 10 (pH 7.3 adjusted with KOH), and 1% biocytin (Sigma).

Recordings were performed at room temperature and all drugs were applied in the bath with the use of a peristaltic pump (1.5–2 ml/min). Drugs used to test neuronal responses to substance P (SP) were SP (Sigma); SR140333 (Sanofi), a selective neurokinin 1 (NK1) receptor antagonist (Oury‐Donat et al., 1994); [Sar9,Met(O2)11]‐SP (Sigma), a selective NK1 receptor agonist; and TTX (Sigma). Repeated application of substance P was preceded by a 7‐ to 10‐minute wash with normal ACSF. Recorded data were digitized by a Digidata 1320 A/D board, filtered at 5 kHz, and analyzed using Clampfit 8.0, Origin (Microcal Software, Northampton, MA, USA), Whole Cell Program, and Electrophysiology Data Recorder (Dr. J. Dempster, University of Strathclyde, Glasgow, UK).

Excitatory postsynaptic potentials (EPSPs) were detected offline. Control conditions were compared with the effect of the investigated drugs by determining an average EPSP number per second for a 120‐s period at the beginning of the recording (control) and at the end of the drug application (effect).