All recordings were taken at physiological temperature (36°C ± 1°C). Brains were frozen in “Lamb OCT” compound (Thermo Fisher Scientific) and CP-690550 ic50 cryostat sectioned at 12 μm in the transverse plane. Sections were incubated with primary Abs to Kv3.1b (1:1000; NeuroMab), Kv3.3 (1:1000; Alomone), Kv3.4 (1:100; Alomone), Kv2.1 (1:100;

Alomone), and diluted in PBS-T containing 1% BSA and 10% NGS overnight at 4°C. After three washes in PBS-T, sections were incubated with secondary Abs (1:1000, Invitrogen; Molecular Probes anti-goat Alexa Fluor 488 and 546 depending on primary Ab), and diluted in PBS-T, 1% BSA, and 10% NGS for 2 hr at room temperature. Images were acquired with a Zeiss laser-scanning confocal microscope (LSM 510; Carl Zeiss International). Tissue samples from the CA3 soma region of the hippocampus were excised Ivacaftor solubility dmso from the same batch of frozen cryostat sections used for immunostaining using laser microdissection (PALM laser system; Zeiss). PCR primers were designed using the Primer Express Software version 2.0 program (Applied Biosystems, Foster City, CA, USA). Primers were designed to cross exon-exon regions, and the gene of interest was normalized against a housekeeping

gene (β-actin) (see Supplemental Experimental Procedures). Statistical analyses utilized unpaired two-tailed Student’s t test and analysis of variance (ANOVA) with posttest to test for significance at p < 0.05. Data were tested for normality distributions. Data are denoted as mean ± SEM; “n” indicates number of neurons tested. Activation plots were fit by a Boltzmann function (I = Imax/(1+exp(V-V1/2/k)), with variables Imax, V1/2, and k (the slope factor).

Fits were performed using Clampfit 9.2 (Molecular Devices) or Excel (Microsoft) with least-squares minimization. Input resistance (Rs) was determined Lenvatinib ic50 using Ohm’s law applied to the voltage deflection in response to a 180 ms injection of 50 pA hyperpolarizing current applied at the resting potential. The membrane time constant was determined from a single exponential fit to the membrane-charging curve. This work was funded by the MRC, Deafness Research UK (M.D.H., PhD scholarship), and The Wellcome Trust’s Knockout Mouse Resource Committee for provision of the transgenic mouse lacking kcnb2 (LEX1551). Thanks to David Read for assistance with confocal imaging, and to Andy Randall, Timothy O’Leary, and David Wyllie for comments on a draft manuscript. “
“Neurons are rarely silent in the intact brain. Rather, intrinsic and network mechanisms interact to drive action potential firing, even in the absence of external stimuli. For example, multiple classes of inhibitory interneuron exhibit spontaneous spiking behavior in vivo (Gentet et al., 2010, Klausberger et al., 2003 and Ruigrok et al., 2011) and in vitro (Parra et al., 1998).