Biocytin

Whole-cell patch-clamp recordings were performed with thin-wall borosilicate glass pipettes (resistances of 5–8 MΩ for neurons and 7–12 MΩ for astrocytes), back-filled with intracellular solution (140 mM K⁺ gluconate, 6 mM NaCl, 1 mM EGTA, 10 mM HEPES, 4 mM MgATP, and 0.4 mM Na₃GTP). For biocytin filling, 5 μg/mL biocytin (Sigma) was dissolved in the internal solution and left to disperse for at least 15 min. During recordings, the cultures were constantly perfused with external solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose) at a rate of 2 mL/min, heated to 33–35°C. Voltage-gated currents were elicited from cells clamped at −70 mV in voltage-clamp mode, using 10 mV voltage step protocols from −90 to +20 mV (neurons) or −140 to +30 mV (astrocytes). Action potentials were elicited in current-clamp mode by the injection of 200 ms square current pulses (5 or 10 pA steps), from a baseline Vm of −70 mV and were counted if their peak was greater than −10 mV. sEPSCs were recorded in voltage-clamp mode at the reversal potential of GABA_A receptors (E_GABA; −70 mV after +14mV junction potential correction). For further details of electrophysiological data analysis, see Supplemental Experimental Procedures.

After recordings, the brain slices containing the neurons filled with neurobiotin 350 (Vector Laboratories, Inc., Newark, CA, USA) or biocytin (Sigma-Aldrich, St. Louis, MO, USA) were fixed in paraformaldehyde (4%) and stored at 4 °C. Following extensive rinse in phosphate buffered saline (0.01 M PBS), slices with biocytin-filled neurons were incubated for 4 h in a solution containing PBS, AMCA Avidin D (1:200), and TritonX-100 (1%) at room temperature. Rinsed slices were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Inc., Newark, CA, USA). Images were taken with a confocal microscope (Nikon Ti2).

Reagents for ACSF and internal solutions, biocytin, NBQX, BaCl₂, and picrotoxin were obtained from Sigma-Aldrich. CNQX, AP-5, DHK, DL-TBOA, TTX, ouabain, and D-serine were obtained from Tocris. L-glutamic acid from BioTrend and SR-101 from Invitrogen. NBQX, CNQX, and DL-TBOA were dissolved in DMSO. picrotoxin was dissolved in EtOH. AP-5, D-serine, TTX, ouabain, and DHK were dissolved in ddH₂O.
In both patch-clamp and two-photon imaging experiments, following baseline recordings, drugs were applied in the external solution for at least 15 min prior to recordings. For the double pharmacology imaging experiments (Fig. 9) following baseline recordings, DHK (300 μM) was first applied and recordings were acquired 20 min later. Subsequently, DHK (300 μM) and DL-TBOA (68 μM) were applied for 20 min before recording. In a number of recordings in which we applied much higher doses of DL-TBOA (300 µM), we observed a change in baseline iGluSnFr fluorescence (similar to ref. ^{18 (link)}), a reduced amplitude of the evoked responses and cellular swelling often accompanied by a lateral or Z drift. These experiments had to be excluded, which explains the low n value for this set of experiments (Supplementary Fig. 9).

Fresh mice cerebella were divided into right and left cerebellum. Sagittal brain slices were further sectioned at 300 μm. 1% biocytin (sigma B4261) in 2 M KCl was ionotophoretically injected into Purkinje cells by using 0.1–0.3 nA of depolarizing current for about 30 min at alternating intervals of 20 ms on and 20 ms off [29 (link)]. Brain slices were then fixed with 4% paraformaldehyde in 0.1 M PB at 4 °C for 16–18 h. Using a sliding microtome, serial frozen sections of 50 μm thickness were prepared and incubated in 0.5% H₂O₂ for 20 min to remove endogenous peroxidase. They were further blocked with blocking solution (3% Bovine serum albumin, 1% Triton X-100, 5% FBS) for 90 min at room temperature. Finally, slices were reacted with Avidin-biotinylated complex (ABC Kit, Vector) for 1 day, and developed with 3,3-diaminobenzidine. Focus was fine-tuned and all details of the cells were then depicted. Subsequently, the soma of the Purkinje cell was used as the center to quantify the maximal sagittal extension of their dendrites. Sholl analysis was applied to investigate the dendritic branches. Centered at the soma of the Purkinje cell, concentric circles with gradually increasing radii of 10 μm formed the grid. The numbers of ring-crossings of each branch (number of intersections) were counted and represented the complexity of dendritic branches [30 (link)].

All experiments are performed at 32.0 ± 0.5°C. Single L5 pyramidal neurons are identified using infrared oblique illumination and a CCD camera (CoolSnap EZ; Roper Scientific). Slices are perfused with the same extracellular solution mentioned above. Recording pipettes are filled with intracellular solution containing the following (in mM): 130 K-gluconate, 5 KCl, 30 HEPES, 10 phosphocreatine, 4 MgATP, 0.3 GTP, pH 7.3. In addition, the somatic pipette contains the following: 10–50 μM Alexa 594 (Invitrogen) to visualize the dendritic arbor for dendritic patching, and 0.2% Biocytin (Sigma). Dual whole-cell voltage recordings are performed from the soma and dendrites (6–10 and 20–40 MΩ pipette resistances, respectively) using Axoclamp 2A (Molecular Devices) and Dagan BVC-700A amplifiers (Dagan Corporation). Access resistances for the dendritic recordings are 15–90 MΩ on break-through. Data is acquired with an ITC-18 board (Instrutech) and custom software written for the Igor environment (Wavemetrics).