Dalfampridine

For recordings from brain slices, ACSF was supplemented with bicuculline (10 μM) and strychnine (1 μM) to block inhibitory inputs. To isolate K⁺ currents, tetrodotoxin (TTX, 0.5 μM) and cadmium chloride (CdCl₂, 20 μM) were added to block Na⁺ and Ca²⁺ channels. Intracellular solution for recording K⁺ currents and action potentials (APs) contained the following (in mM): K-gluconate (97.5), KCl (32.5), EGTA (0.5) or BAPTA (30), HEPES (40), MgCl₂ (1), pH 7.3. To isolate presynaptic Ca²⁺ currents, TTX (0.5 μM), tetraethylammonium (TEA, 10 mM), and 4-aminopyridine (0.3 mM) were applied extracellularly to inhibit Na⁺ and K⁺ channels. Intracellular solution for recording presynaptic Ca²⁺ currents included (in mM): CsCl (110), HEPES (40), EGTA (0.5), MgCl₂ (1), ATP (2), GTP (0.5), phosphocreatine (12), TEA (20), K-glutamate (3), pH adjusted to 7.3 with CsOH. For postsynaptic recordings, intracellular solution contained (in mM): K-gluconate (97.5), CsCl (32.5), EGTA (5), HEPES (10), MgCl₂ (1), TEA (30), and lidocaine N-ethyl bromide (3), pH 7.3. Patch electrodes typically had resistances of 4–6 and 2.5–3 MΩ for presynaptic and postsynaptic recordings, respectively. For whole-cell voltage clamp recordings, presynaptic and postsynaptic series resistances were 6–15 MΩ (<10 MΩ in most cases) and 3–6 MΩ respectively, and compensated to 90%. The respective holding potential for presynaptic or postsynaptic neurons was −80 and −60 mV. Presynaptic K⁺ and Ca²⁺ currents were evoked by the voltage command protocols indicated in the text and leak subtraction was done with an on-line P/4 procedure. To validate the on-line leak P/N subtraction, we recorded raw currents evoked by a typical AP train at 400 Hz without P/N subtraction after blocking Na⁺ channels with TTX and Ca²⁺ channels with CdCl₂. We subtracted these currents with those obtained with additional blockers for K-HT (TEA) and K-LT (DTX). By comparing the subtracted TEA and DTX sensitive currents with K⁺ currents recorded with on-line P/N correction, we found that they were almost identical in the amplitude and more importantly in the profile of facilitation over the train stimuli (Supplementary Fig. 4). For experiments where real APs were used as voltage-command (Fig. 3, Supplementary Fig. 3,4), we first recorded APs from a P17 calyx at a sampling rate of 50 kHz in current-clamp configuration by stimulating afferent axon fibers using a bipolar platinum electrode delivered through a stimulator (Master-8, A.M.P. Instruments). After manually removing stimulation artifacts preceding the APs, the digitized values were fed back into the amplifier as voltage-command templates (Axon Text File) through pClamp 9 software at the same sampling frequency as their acquisition. Similarly, the current-command templates in Figure 8 were generated by reversing the polarity of previously obtained EPSCs in response to 400 Hz train stimuli with normal or attenuated K-LT facilitation (Fig. 7) and converting these currents to conductance by G = I/(V_m−E_rev) where V_m and E_rev were set at −70 mV and 0 mV respectively for postsynaptic neurons. The digitized conductance templates (Text File) were injected into these neurons to evoke APs with an amplifier (AXOPATCH 200B, Molecular Devices) and a digitizer (CED 1401, Cambridge Electronic Design).
CHO cells transfected with Kv1.1&1.2 were positively identified with red/green fluorescence illuminated briefly by a mercury burner (Olympus). Recordings from CHO cells expressing Kv3.1 or Kv1.1&1.2 (Fig. 4 & Supplementary Fig. 5) were made in the extracellular solution containing (in mM): NaCl (140), KCl (2.5), CaCl₂ (1.3), HEPES (10), glucose (33), pH 7.3. And the intracellular solution was the same as used for recording K⁺ currents from brain slices. The holding potential was set at −90 mV and series resistance was 3–6 MΩ, compensated to 90%.
Most of the electrophysiological experiments were performed at room temperature (~22°C), except for several sets of recordings of K⁺ currents from CHO cells or cerebellar neurons obtained at 35°C using an inline heater with a feedback thermistor (TC-324B, Warner Instruments). All the recordings (except for those in Fig. 8) were acquired on-line, filtered at 4 kHz, digitized at 50 or 100 kHz with a dual-channel amplifier (MultiClamp 700A, Molecular Devices) and digitizer (Digidata 1322A, Molecular Devices). Reagents were from Sigma (St. Louis, MO), Tocris Cookson (Bristol, UK) and Alomone Labs (Jerusalem, Israel).