4 aminopyridin 4 ap

To isolate presynaptic Ca²⁺ currents, aCSF was supplemented with 1 µM tetrodotoxin (TTX, Alomone labs, Jerusalem, Israel), 100 µM 4-aminopyridin (4-AP, Tocris, Bristol, UK) and 20 mM tetraethylammonium chloride (TEA, Sigma Aldrich, Darmstadt, Germany) to block Na⁺ and K⁺ conductance. Calyxes were whole-cell voltage-clamped at −80 mV. Current-voltage relationships were recorded in the presence of 1 mM CaCl₂, pharmacological isolation of VGCC subtypes was performed in 2 mM CaCl₂. We used 200 nM ω-agatoxin IVA (Alomone labs) to selectively block Ca_V2.1 and 2 µM ω-conotoxin GVIA (Alomone labs) for Ca_V2.2 VGCCs. Remaining current was blocked by 50 µM CdCl₂. All experiments to isolate Ca_V2 subtypes were conducted in presence of cytochrome c (0.1 mg/ml). Presynaptic patch pipettes with open tip diameters 4–6 MΩ resistance were pulled from 2.0 mm thin-walled borosilicate glass (Hilgenberg, Malsfeld, Germany) and were filled with the following (in mM): 145 Cs-gluconate, 20 TEA-Cl, 10 HEPES, 2 Na₂-phosphocreatine, 4 MgATP, 0.3 NaGTP, and 0.5 EGTA, pH 7.2, 325–340 mOsm). Pipettes were coated with Sylgard. Presynaptic series resistance was between 6 and 20 MΩ (usually between 10–15 MΩ) and was compensated online to 6 MΩ. Leak and capacitive currents were subtracted online with a P/5 routine. Cells with series resistance >20 MΩ and leak currents >100 pA were excluded from the analysis.