Single and dual voltage-clamp recordings were performed using the whole-cell configuration of the patch-clamp technique at a pipette voltage of −60 mV using the Axopatch 200B and 1D amplifiers (Molecular Device Co., Sunnyvale CA, USA). Access resistance was monitored during the recordings, and experiments with >20% change were discarded. The baseline membrane potential for current-clamp recordings was set at −70 mV before each series of current step injection protocols. Rheobase current was defined as the first current step, within a series of increasing 20 pA steps, that elicited an action potential.
Stock solutions of bicuculline methobromide (BMR), tetrodotoxin (TTX), 4,5,6,7-tetrahydroisoxazolo{5,4-c}pyridine-3-ol, (THIP), SKF-81297, quinpirole, sulpiride, SCH 23390, and GABA (all from Sigma) were prepared in water. Etomidate (Sigma) was dissolved in dimethylsulfoxide (<0.0001% final concentration). All stock solutions were diluted to the desired concentration in aCSF and applied locally through a Y tube (Murase et al., 1989 (link)) modified for optimal solution exchange in brain slices (Hevers and Luddens, 2002 ).
Currents were filtered at 2 kHz with a low-pass Bessel filter and digitized at 5–10 kHz using a personal computer equipped with Digidata 1322A data acquisition board and pCLAMP9 software (both from Molecular Devices). Off-line data analysis, curve fitting, and figure preparation were performed with Clampfit 9 software (Molecular Devices). Spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) were identified using a semi-automated threshold based mini detection software (Mini Analysis, Synaptosoft Inc., Fort Lee, NJ) and were visually confirmed as in Ade et al. (2008) (link). Briefly, IPSC averages were based on >60 events and the decay kinetics were determined using double exponential curve fittings and reported as weighted time constants (tau). All detected events were used for event frequency analysis, but superimposing events were eliminated for the amplitude, rise time, and decay kinetic analysis. Tonic current measurements were made as in Ade et al., 2008 (link). Briefly, an all-points histogram was plotted for a 10 s period immediately before and during BMR application. Tonic currents are represented as the change in baseline amplitude. When PKA or PKI was included in the internal solution, events were analyzed at least four minutes after break-in to allow the peptide to function and equilibrate with the internal components of the cell.
Statistical significance was determined using the two-tailed Student’s t test (unpaired when comparing two populations of cells and paired when comparing results within the same cell). All values are expressed as mean ± SEM. In all figures, *p < 0.05, **p < 0.005, and ***p < 0.0005.
Stock solutions of bicuculline methobromide (BMR), tetrodotoxin (TTX), 4,5,6,7-tetrahydroisoxazolo{5,4-c}pyridine-3-ol, (THIP), SKF-81297, quinpirole, sulpiride, SCH 23390, and GABA (all from Sigma) were prepared in water. Etomidate (Sigma) was dissolved in dimethylsulfoxide (<0.0001% final concentration). All stock solutions were diluted to the desired concentration in aCSF and applied locally through a Y tube (Murase et al., 1989 (link)) modified for optimal solution exchange in brain slices (Hevers and Luddens, 2002 ).
Currents were filtered at 2 kHz with a low-pass Bessel filter and digitized at 5–10 kHz using a personal computer equipped with Digidata 1322A data acquisition board and pCLAMP9 software (both from Molecular Devices). Off-line data analysis, curve fitting, and figure preparation were performed with Clampfit 9 software (Molecular Devices). Spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) were identified using a semi-automated threshold based mini detection software (Mini Analysis, Synaptosoft Inc., Fort Lee, NJ) and were visually confirmed as in Ade et al. (2008) (link). Briefly, IPSC averages were based on >60 events and the decay kinetics were determined using double exponential curve fittings and reported as weighted time constants (tau). All detected events were used for event frequency analysis, but superimposing events were eliminated for the amplitude, rise time, and decay kinetic analysis. Tonic current measurements were made as in Ade et al., 2008 (link). Briefly, an all-points histogram was plotted for a 10 s period immediately before and during BMR application. Tonic currents are represented as the change in baseline amplitude. When PKA or PKI was included in the internal solution, events were analyzed at least four minutes after break-in to allow the peptide to function and equilibrate with the internal components of the cell.
Statistical significance was determined using the two-tailed Student’s t test (unpaired when comparing two populations of cells and paired when comparing results within the same cell). All values are expressed as mean ± SEM. In all figures, *p < 0.05, **p < 0.005, and ***p < 0.0005.
