Ttx citrate

To isolate L-type Ca²⁺ currents from other neuronal Ca²⁺ currents, 4–5 DIV neurons were treated for 30 min prior to recording with blockers of N- and P/Q-type channels, ω-CTx-GVIA (1 μM) and ω-CTx-MVIIC (5 μM). Neurons were used within 1 hr of blocker pre-treatment to minimize adulteration of L current as N- and P/Q-type channels became unblocked (Oliveria et al., 2007 (link)). The whole-cell pipet contained (mM): 120 CsMeSO₄, 30 tetraethylammonium-Cl, 10 ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 5 MgCl₂, 5 Na₂ATP and 10 HEPES; pH 7.2 with TEA-OH. The bath solution contained 1 μM tetrodotoxin (TTX) and (mM): 125 NaCl, 10 CaCl₂, 5.85 KCl, 22.5 TEA-Cl, 1.2 MgCl₂, 10 HEPES(Na), 11 D-glucose; pH 7.4 with HCl. Glutamate and other agents were applied using continuous perfusion (~1 ml/min) via a 3-barrel system (SF-77B, Warner Instruments) that allowed rapid solution exchange between control solution, test (e.g., glutamate + glycine), and when needed, antagonist (e.g., glutamate + glycine + MK801). (+)-MK801 maleate, DNQX disodium salt (6,7-dinitroquinoxaline-2,3-dione), MTEP hydrochloride (3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride), and TTX citrate were obtained from Tocris Bioscience. Nimodipine was obtained from Sigma-Aldrich.

MK801 (M107, Sigma Aldrich) and TTX citrate (1069, Tocris) was applied to the surface of the S1 after removing a small bone flap (∼200 μm in diameter) adjacent to a thinned skull window. MK801 (100 μM) and TTX (100 nM) were dissolved directly in ACSF to final concentrations. The bone flap for drug delivery was made during awake head mounting and covered with a silicone elastomer such that it could be easily removed at the time of imaging. Because small molecules diffuse rapidly in the cortex, we estimated that the drug concentration was reduced ∼10 times in the imaged cortical region, such that the final effective concentration would be ∼10 μM and 10 nM for MK801 and TTX, respectively. As a control, we applied ACSF after removing a bone flap. In Figure 1f, TTX (100 nM) was locally delivered to the surgical site of sciatic nerve. In Supplementary Figure 1, MK801 (15 μg/ml in saline; 0.1 or 0.25 μg/g body weight) was delivered by intraperitoneal injection.