Neurons were held in current-clamp using a patch-clamp amplifier (HEKA, EPC10) in the whole-cell configuration. Intracellular solution composition was (in mM): 130 K-methylSO4, 5 KCl, 5 NaCl, 10 HEPES, 2.5 Mg-ATP, 0.3 GTP, and 0.5% neurobiotin. No correction for liquid junction potential was applied. The osmolarity was 265–275 mOsm, pH 7.3. Microelectrodes resistance PD-0332991 mouse was 4–8 MOhms. Uncompensated access resistance was monitored throughout the recordings. Values below 20 MOhms were considered acceptable and the results were discarded if it changed by more than 20%. Whole cell measurements were filtered at 3 kHz

using a patch-clamp amplifier. Recordings were digitized online (10 kHz) with an interface card to a personal check details computer and acquired using Axoscope 7.0 software. Spontaneous EPSPs were detected and analyzed using the MiniAnalysis software. For most stimulation experiments, the movie acquisition time was separated evenly between (1) an ∼3 min resting period during which the cell was held close to Vrest (i.e., zero current injection); (2) an ∼3 min stimulation period during which phasic stimulation protocols were applied; and (3) an ∼3 min recovery period where the cell was brought back to resting membrane potential. Stimulation protocol: suprathreshold current pulses (amplitude: 100–200 pA, duration: 200 ms) repeated at 0.1–0.2 Hz. Vrest was measured

as the membrane potential baseline value obtained in current-clamp mode in the absence of current injection. The action potential threshold (Vthreshold) and amplitude, membrane capacitance (Cm), and resistance (Rm) were tuclazepam measured offline using Clampfit. Vrest and Vthreshold were not corrected for errors due to high input resistance values in developing neurons. Slices were fixed overnight at 4°C in Antigenfix, rinsed

in PBS containing 0.3% Triton X-100 (PBST) and incubated overnight at room temperature in cy3-streptavidin (1/1000 in PBST). Post hoc analysis was performed using a confocal microscope. Stacks of optical sections were collected for computer-assisted neuron reconstructions. Animals were deeply anesthetized with a ketamine (50 mg /ml) and xylazine (7.5 mg /ml) solution at a dose of 2 ml/kg (i.p.) and perfused transcardially with 4% paraformaldehyde in PB (1 ml/g) at a constant flow (2 ml/min). Brains were postfixed overnight in fixative and washed. Horizontal brain sections (40 μm) were routinely processed for multiple immunofluorescence. Briefly, after preincubation in 5% normal donkey serum, sections were incubated in a mixture of primary antibodies from different species, then incubated in appropriate secondary antibodies conjugated with either Al488 (1:500, Molecular Probes; Invitrogen), Cy3, Cy5, DL488, DL549, DL649 (Jackson ImmunoResearch Laboratories) for 2 hr. The dilutions, characteristics, specificity, and sources of primary antibodies are presented in Table S1.