Mice P13–23 were urethane-anesthetized (1.2 g/kg), and a small (1–2 mm) cranial window was created over the barrel cortex. All in vivo experimental procedures were in accordance with national regulation and institutional guidelines and follow previously described methods (Crochet and Petersen, 2006 and Glazewski et al., 2007). Juxtacellular, loose patch recordings

were performed with glass microelectrodes. Internal solution contained 50 μm Alexa 594 for shadow patching (Kitamura et al., 2008). For imaging, the laser was Nutlin-3a mouse tuned to 930 nm for GFP visualization or 820 nm for Alexa 594 emission during electrode positioning. To verify that electrodes could detect APs from a target, cells were only included in the analysis if they fired at least once during the recording period. Firing rates were calculated over ∼300+ s. Coronal or thalamocortical brain slices (350 μm thick) from mice P12–P15 (wild-type C57Bl6, fosGFP heterozygotes, or arcGFP heterozygotes) were prepared. Slices were recovered in regular ACSF at 35°C for 30 min

and maintained at room temperature in low-divalent ACSF (0.5 mM MgSO4, 1 mM CaCl2, 3.5 mM KCl). Spiny, pyramidal layer 2/3 neurons in primary somatosensory cortex were targeted for recording based upon fosGFP+/− expression. Internal pipette solution contained a K-gluconate internal solution (Supplemental Experimental Procedures). Because the whole-cell recording configuration unambiguously indicated that a single neuron was targeted for Ibrutinib molecular weight recording, pairs in which one cell did not exhibit any firing (0 Hz) were included in analysis of whole-cell recordings. APs were elicited by injection of minimal current (using 20, 60, 80 pA steps), and trials that yielded a single AP were used for analysis. AP threshold, peak, and half-width (threshold to peak) were determined using custom-written Igor Pro macros. Spontaneous firing activity was collected where Vrest was maintained at −50 mV to normalize differences in resting potential between cells, a technique that did not appreciably alter firing

rates (data not shown). Onset and offset of synchronized network activity were determined using membrane potential mean and standard deviation over 500 ms, with a 10 ms sliding Mannose-binding protein-associated serine protease window (Gerkin et al., 2010). Cells were maintained at their normal resting potential, which was approximately −60 mV, the experimentally determined reversal potential for Cl−. One cell was assigned as the trigger cell and a series of 10 pulses (500 pA, 5 ms duration) at 20 Hz were delivered across 20 separate trials. Bidirectional connectivity was assessed sequentially for each pair. Spontaneous EPSP frequency was calculated in the 500 ms time window preceding the stimulus, and evoked EPSP frequency was calculated across the 10-pulse series including 50 ms after the last pulse (500 ms total).