Picrotoxin

All other drugs were bath applied: NBQX and D-AP5 were from Abcam. Picrotoxin was from Alomone. All other drugs were from Sigma-Aldrich. CNO was dissolved in DMSO for stock solutions (30 mm).

Acute responses to 5-HT were probed by bath application of 5-HT (serotonin creatinine sulfate, Sigma-Aldrich, St. Louis, MO; 10 µm; 30 s) in ACSF. To examine the effect of 5-HT on the excitability of L6 pyramidal neurons, 5-HT (10 µm) was bath applied until a steady-state response was reached, and remained in bath throughout the duration of the input–output test protocols (∼2 min total application). Selective antagonists and agonists were from Tocris (Bristol, UK), except where mentioned. Antagonists for 5-HT_1A receptors (30 nm WAY100635, 10 µm NAN-190) and 5-HT_2A receptors (30 nm MDL100907; 2 µm ketanserin; 300 nm to 3 µm ritanserin) were applied in bath for 10 min before further experiments with 5-HT. There were no significant differences between effects of 300 nm and 3 µm ritanserin, and results were grouped for analysis. TCB-2 was used as a specific agonist of 5-HT_2A receptors (300 nm to 1 µm). Other agonists and antagonists used for characterization of the 5-HT response in L6 neurons were as follows: 2 µm tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel), 20 µm 6-cyano-7-nitroquinoxaline-2,3-dione, 50 µm d-2-amino-5-phosphonovaleric acid, 100 µm picrotoxin, 1 µm CGP52432, and 10 µm 8-OH-DPAT.

A spinal cord section from T2 to L6 was dissected out as described for transverse slices. Dorsal roots L2–L4 and T11–T13 roots were preserved for afferent stimulation. The dura mater was cut on the midline with scissors, and the arachnoid and the pia mater were cut using a 21-gauge needle. The left and right spinal cord sides were then separated to obtain sagittal hemisections. After incubation in the modified aCSF solution for 15–30 min at 34°C, hemisections were placed in a submersion-recording chamber with the midline facing up. Recordings were then performed in oxygenated aCSF superfused at a rate of 2–3 ml/min, and, in most of the experiments, 100 µM picrotoxin (Alomone Labs) and 0.5–1 µM strychnine (Sigma-Aldrich) were included. The internal solution was the same as above with 2–4 mM QX314 to block Na⁺ channels from driving AP firing. In this recording configuration, only CC neurons in the Gdnf^TOM mouse model or medial neurons in the Atoh1^TOM mouse model were at depths suitable for recordings. Lateral cells are too deep to be recorded using this approach.