In a subset of cells, we measured the SR95531-dependent increase of spontaneous APs (from 7.4 ± 0.6 to 12.66 ± 1.2 Hz, n = 7, see Häusser and Clark, 1997) that we adjusted with DC current (7.4 ± 0.5 pA) to match the observed rate in control conditions. Experiments were performed using selleck screening library internal solutions with Alexa Fluor 488 or 568 hydrazide (100 μM; Life Technologies) or 0.2% biocytin. Slices were fixed in 4% paraformaldehyde for 1 hr and mounted with anti-fade reagent (ProLong Gold, Life Technologies), or

incubated with streptavidin-conjugated Alexa Fluor 647 prior to mounting. Digital images were acquired using a 20× (NA 0.85) oil-immersion objective on an Olympus FluoView 300 confocal microscope. Images were reconstructed in Neurolucida (MicroBrightField). Data was analyzed using AxoGraphX software. Changes

to basal spontaneous action potential rate were quantified as in Mittmann et al. (2005). Briefly, peristimulus Ibrutinib cost histograms (PSHs) were computed and integrated. A linear fit to the baseline of the integral was extrapolated over the entire sweep and subtracted from the integral to yield the cumulative spike probability plot. We averaged between 300–400 ms period after stimulation to measure the number of spikes evoked by the input. Data are displayed as means ± SEM, and significance was analyzed with two-tailed Student’s t tests (Microsoft Excel and GraphPad Prism). n values indicate number of cells. Spearman or Pearson correlations were used depending on the normality of the data. ANOVAs were followed by Bonferroni’s multiple comparison test unless noted. SR95531 (GABAAR antagonist, 5 μM), MRIP NBQX (AMPAR antagonist, 10 μM), AP5 (NMDAR antagonist,

100 μM), and QX314 (Na+-channel blocker, 5 mM) were obtained from Abcam. DL-TBOA (50 μM) was purchased from Tocris Bioscience. All other chemicals and compounds were obtained from Sigma or Fisher Scientific. This work was supported by NIH NS064025 (L.O.-W.) and NS065920 (J.I.W.). We thank Kamran Khodakhah, Ming-Chi Tsai, Anastassios Tzingounis, and members of the Wadiche laboratories for discussions and reading the manuscript. “
“Allosteric modulation can profoundly regulate the function of ion channels and G protein-coupled receptors in either a positive or negative direction (Conigrave and Franks, 2003; Schwartz and Holst, 2007) and is of increasing interest for both physiology and pharmacology. Benzodiazepines (BZs) act as allosteric modulators on type-A receptors for the inhibitory neurotransmitter γ-aminobutyric acid (GABA). BZs act as either positive allosteric modulators (PAMs) and prolong currents through GABAARs to increase the duration and strength of inhibitory signals, or as negative allosteric modulators (NAMs, or inverse agonists) (Sieghart, 1995).