Sections were treated with glycine

(100 mM) in PBS for 15 min. Antigen retrieval was carried out by microwaving sections at 50°C for 5 min, resting them for 5 min, and repeating, in 10 mM Na-citrate buffer (pH 6.0). Blocking buffer (1.5% normal goat serum, 1% bovine serum albumin in PBS) was applied for 30 min. To immunostain PV-tdTomato neurons and thalamic afferents, the following reagents (Vector Labs except as noted) were applied in sequence with PBS washes between steps: Rabbit anti-RFP (1:1000, 14 hr, 4°C; Abcam); biotinylated goat anti-rabbit buy Ku-0059436 (0.5%, 2 hr); avidin-biotin reaction (2 hr; Vector ABC kit); the chromogen VIP (10 min) (Zhou and Grofova, 1995); 0.5% Na azide; guinea pig anti-VGluT2 (1:2000, 14 hr, 4°C; Chemicon); biotinylated goat anti-guinea pig secondary (0.5%, 2 hr); DAB (10 min). Slices were fixed in 1% OsO4 on ice for 1 hr. Slices were washed in cold ddH2O and passed through a series of cold ethanol solutions of (70%, 90%, 100% (2×); 10 min each). The slices were then treated with cold dry acetone for 10 min and room temperature acetone for 10 min,

followed by 50:50 acetone:Durcupan ACM resin overnight and then 100% Durcupan overnight. The slices were flat-embedded between glass slides c-Met inhibitor treated with liquid release agent (Ted Pella) and left in oven at 60°C for 2 days. A block of layer 4 in barrel cortex was trimmed and ribbons of serial thin sections (80 nm) were cut on a Leica Ultracut UCT using a diamond knife (Diatome). Ribbons were collected on formvar-coated slot grids and poststained with 1% aqueous uranyl acetate and Sato lead. TEM images were collected on a 1200EX JEOL microscope at 20,000× magnification. Negatives were digitized with a

Flextight 3-mercaptopyruvate sulfurtransferase scanner at 1500 dpi, contrast and brightness were adjusted in Adobe Photoshop, and volume reconstruction was performed using Reconstruct (Fiala, 2005). Three neurons were selected based on physiology and completeness of fill for further reconstruction, including process thickness, of the dendritic arbor and the initial ∼1/3 of the axon. Tracings were imported into NEURON 7 (Hines and Carnevale, 1997) using the Import3D tool and endowed with specific membrane capacitance of 1 μF/cm2, intracellular resistivity of 170 Ω-cm, and passive leak conductance of 130 μS/cm2 (soma and dendrites) or 6 μS/cm2 (axon) (Nörenberg et al., 2010). The d-λ method was used to set segment partitioning such that the length of each segment was <1% of the alternating current length constant at 1 kHz (Carnevale and Hines, 2006). Synaptic conductances were simulated using a custom dynamic clamp (A. Gartland).