4% biocytin. The brain was continuously superfused with an extracellular

solution containing 103 mM NaCl, 3 mM KCl, 5 mM TES, 8 mM trehalose, 10 mM glucose, 7 mM sucrose, 26 mM NaHCO3, 1 mM NaH2PO4, 1.5 mM CaCl2, 4 mM MgCl2 (pH 7.3), and continuously equilibrated with 95% O2-5% CO2. Signals were recorded with a MultiClamp 700B Microelectrode Amplifier, filtered at 6–10 kHz, and digitized at 10–20 kHz with an ITC-18 data acquisition board controlled by the Nclamp and NeuroMatic packages. Data were analyzed with NeuroMatic (http://neuromatic.thinkrandom.com) and custom procedures in Igor Pro (WaveMetrics). The membrane time constant was determined by fitting a single exponential to the voltage deflection caused by a 200-ms-long hyperpolarizing check details current pulse. Input resistances were estimated from linear fits of the subthreshold voltage deflections elicited by small current pulses of increasing amplitude and a duration of 1 s. Excitability

was quantified by holding cells at resting potentials of –60 ± 2 mV and injecting sequences of depolarizing current pulses (5 pA increments, 1 s duration). Spikes were detected by finding minima in the second derivative of the membrane potential record. The spike rate was calculated by dividing this website the number of action potentials discharged by the time elapsed between the first and the last spike. Cells that fired only a single action potential per current pulse are denoted as such in Figure 7. The current amplitude at which each cell reached a given frequency threshold (5–50 Hz) was used to construct cumulative distribution functions. For statistical analyses, the distributions were fit with logistic Naka-Rushton functions (Albrecht and Hamilton, 1982, Naka and Rushton, 1966 and Sclar et al., 1990) of the form F=FmaxInIn+I50n,where FF is the percentage of cells

reaching threshold at a given current level II, FmaxFmax is the percentage of cells reaching threshold at maximal current, I50I50 indicates the half-maximal or semisaturation current, and the exponent nn determines Florfenicol the steepness of the curve. With only two free parameters (I50I50 and nn, given that FmaxFmax is measured experimentally), this simple model provided a satisfying fit to all WT distributions (R2 > 0.98), irrespective of sleep history. Statistical significance between pairs of distributions was measured with pairwise Kolmogorov-Smirnov (K-S) tests. Because multiple K-S tests were performed, Bonferroni step-down corrections were used. The 20%–80% slope of each cumulative probability distribution was calculated by comparing the 20th and 80th percentiles of the population of cells that reached a particular frequency threshold. For imaging of native GFP fluorescence, brains were dissected in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, and 175 mM NaCl) and fixed for 20 min in 4% paraformaldehyde in PBS at 4°C. Brains containing biocytin fills were incubated in 1:200 streptavidin conjugated to Alexa Fluor 568 (Invitrogen) in PBS containing 0.