For whole-cell recordings, the pipette solution contained (in mM): 140 KMeS, 10 NaCl, 10 HEPES, 1 MgCl2, and 0.1 EGTA (osmolality 295 mmol/kg; pH 7.4), supplemented with 0.3 mM Na3GTP and either 0.3 or 4 mM MgATP. A voltage ramp protocol was used to measure input resistance and slope conductance: from the −90 mV holding potential followed two 100 ms steps Panobinostat cell line first to −100 mV then to −120 mV before initiation of a 1 s ramp from −120 to −65 mV.
The prepulses were used to calculate the cell’s input resistance. Voltage ramps were applied every 10 s and fitted with a straight line from −120 to −80 mV to provide a running assessment of whole-cell slope conductance. Tolbutamide was diluted in ACSF from a 200 mM stock solution in ethanol. Ethanol as a vehicle control was added to ACSF at 0.1%. All chemicals are from Sigma-Aldrich, unless otherwise noted. Data and statistical analysis were performed with Patchmaster v2x43 (HEKA Instruments Inc.) and Origin 8 (OriginLab, Northampton, MA, USA). Single-channel PF-06463922 cell line analysis was performed using QuB (http://www.qub.buffalo.edu). Open and closed levels for channel activity were detected
using a 50% threshold criterion. Raw data sets featuring excessive baseline shift, sufficient high- or low-frequency noise to preclude effective idealization of channel openings, or too few data traces (<3 min of recording) were discarded without idealization or further analysis. The number of independent experiments (n, Figure 5) corresponds to the number of patches or whole-cell experiments each from a different hippocampal slice. In all cases, each experimental condition was tested in slices from at least three different animals. In all cases, mean ± SEM is presented. The number of independent experiments is indicated in the figure or figure legend in each case. Statistical significance was determined using two-tailed
Student’s t test. We thank Marina Godes and Juan unless Quijada for technical assistance and animal husbandry; Gregory Holmes for advice on EEG acquisition and analysis; Rosalind Segal, Bernardo Sabatini, Qiufu Ma, S. Robert Datta, and members of the Danial and Yellen laboratories for critical reading of this manuscript and valuable discussions; and E. Smith for manuscript preparation. A.G.C. was supported by a postdoctoral fellowship from the Ministerio de Educación y Ciencia (MEC, Spain). N.N.D. is a recipient of the Burroughs Wellcome Fund Career Award in Biomedical Sciences. This work was supported by the U.S. National Institutes of Health grants (K01CA106596 to N.N.D., R01 NS055031 to G.Y., and R56 NS072142 to N.N.D. and G.Y.), Harvard Catalyst Pilot Award (based on NIH UL1 RR025758 to N.N.D. and G.Y.), and a CURE Epilepsy Challenge Award (G.Y. and N.N.D.). ”
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