Electrophysiological recordings and FM1–43 experiments

Cultured neurons were used for electrophysiological experiments 10–21 days after plating. All electrophysiological recordings were obtained using WC patch clamp (current-clamp or voltage-clamp configuration) and neurons were continuously superfused (1–2 ml min⁻¹) at room temperature (24 °C) with a Tyrode solution containing (in mM): 119 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 25 HEPES, and 30 D-glucose (osmolarity 305 mOsm, pH 7.4). For miniature currents recordings, this solution was supplemented with the voltage-gated sodium channel blocker TTX (1 µM; Latoxan, Valence, France). For SB uptake experiments in current-clamp, bath was supplied with the NMDA receptor blocker D-2-amino-phosphonovalerate (APV; 25 µM; Tocris Cookson, Bristol, UK) and the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM; Tocris Cookson, Bristol, UK) to inhibit network activity. Patch pipette electrodes (resistance 5–10 MΩ) were filled with an intracellular solution containing (in mM): 110 D-gluconic acid, 5 MgCl₂, 10 NaCl, 0.6 Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 2 ATP, 0.2 GTP, and 49 HEPES (pH adjusted to 7.2 with CsOH; osmolarity 290 mOsm). When testing evoked transmission between neuron pairs CNQX was not applied to the bath and the pipette patching the presynaptic neuron was filled with a K-gluconate solution containing (in mM): 120 K-D-Gluconate, 20 KCl, 2 MgCl₂, 0.2 EGTA, 10 HEPES, 0.2 GTP-Na₂, 2 ATP-Na₂ (pH adjusted to 7.2 with KOH; osmolarity 290 mOsm), while QX-314 was added to the pipette patching the postsynaptic neuron (1.5 mg ml⁻¹, Tocris Cookson, Bristol, UK). Current-clamp recordings were obtained with an EPC7 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany; membrane potential maintained at ~ −75 mV to reduce spontaneous action potential firing). Evoked and miniature currents were acquired in voltage-clamp (holding potential −70 mV; Axopatch 200B or 200A amplifier; Axon Instruments, Foster City, CA). Membrane and series resistances were constantly monitored by applying 2–5 mV depolarizing pulses (recordings with series resistance higher than 20 MΩ were discarded). All traces were filtered at 2–5 kHz and digitally acquired at 20 kHz using a 16-bit analog-to-digital interface (ITC-18; HEKA Elektronik, Lambrecht/Pfalz, Germany) controlled by custom acquisition software (Labview and C/C++). Analysis of recordings was performed with custom code developed in Matlab (Mathworks; miniature detection algorithm as previously described⁵¹). In vivo recordings of visual evoked potentials (PEVs) were performed on Sprague Dawley rats (300–350 g) under general anesthesia (sevoflurane 3.5%) by measuring electro-cortical activity through screws implanted in the skull²⁶. For FM1–43 experiments, synapses were labeled for 1–2 min with 10 μM FM1–43 (Molecular Probes—Thermo Fisher) dissolved in modified Tyrode solution (isotonic 90 mM KCl; 10 μM CNQX; APV 25 μM). After 15 min of perfusion with a washing Tyrode solution (10 μM CNQX; APV 25 μM; TTX 1 μM), unloading was obtained in a standard Tyrode solution (10 μM CNQX; APV 25 μM) by field stimulation using two parallel Platinum electrodes (constant current stimulation, 40 mA/cm; 10 APs trains at 10 Hz, 4 s inter-train interval).