8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose. Recording electrode pipettes had resis tances of 2 4 M and were filled with an internal pipette solution containing, 130 K gluconate, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES and 0. 4 Na2GTP. Action potentials were evoked by injecting depolarization current into primary little cortical neurons under normal conditions and recovery after treatment with 1. 5 mM NaCN for 30 mins. Recordings were not performed during NaCN treat ment because the Ag AgCl reference electrode can be oxidized by NaCN which causes a pseudo neuronal mem brane depolarization artifact. Data were collected via a patch clamp amplifier, stored on a PC, and analyzed by pClamp 9. 0 software. After whole cell configu Inhibitors,Modulators,Libraries ration was achieved, series resistance was compensated Inhibitors,Modulators,Libraries by 80 90% and monitored periodically.

Most cultured cortical Inhibitors,Modulators,Libraries neurons had series resistance around 7 8 M. A small percentage of cortical neurons were considered as unhealthy and discarded due to rest ing membrane potentials less than 55 mV or gradual changes in membrane potential, input resistance, or action potential amplitudes. For current clamp record ings, a depolarizing current step was injected to induce multiple action potentials. To quantitatively measure the changes in cultured neuronal network activities, the duration of plateau depolarization was monitored in batches of three minute recordings. Dendrite spine count Cortical cells were Inhibitors,Modulators,Libraries seeded on a poly D lysine coated cover slip in 25 mm diameter at a density of 3 �� 105 in 6 well plate. Cultured cortical cells were transfected with 2 Inhibitors,Modulators,Libraries ug of green fluorescent protein plasmid mixed with 0.

5 M CaCl2 and HEBS solution for 40 min, washed with DMEM, then normal neurobasal medium was added. Two weeks following GFP transfection, cultured cells were placed on a microscope stage incubation chamber with 37 C and 5% www.selleckchem.com/products/arq-197.html CO2 control then filled with ACF buffer 2 hours before image capture. Images were acquired by using an inverted Zeiss LSM 510 META confocal micro scope with a 40�� oil immersion objective and a digital zoom of 3��. Image stack was generated by reconstructing 8 sections at an interval 0. 4 um from each slide. The mea surements of spine density were determined by counting spines from the length of a 20 um secondary dendrite from each individual neuron. The rate of N N0 was used in the statistics. N corresponds to the total number of dendritic spines at each time point and N0 indicates the number before NaCN administration. Statistics Statistic analysis was performed using commercially available software. Differences among the groups were determined by one way ANOVA fol lowed by Newman Keuls test. Data are expressed as mean S. E. M. and p value 0. 05 was considered signifi cant.