25, 285 mOsm). With cortical neuron recording, the extracellular solution contained 1 μM TTX. Light intensity was measured with a calibrated photometer with an integrating sphere detector (International Light Technologies) placed on the objective. A glass slide with a semispherical lens was used to direct the light into the integrating sphere. The area of illumination was measured with a stage micrometer for illumination intensity. Organotypic hippocampal slices were prepared from 2 days old rat pups as described previously (Shi et al., 1999). The various constructs were expressed using rAAV virus in CA3 neurons in 2 DIV slice cultures.

Cells were allowed to express for 14–17 days before being used for recordings. CA3 region was surgically removed to prevent stimulus induced bursting. Recordings were done in ACSF containing 119 mM NaCl, Pazopanib nmr 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose, 4 mM MgCl2, and 4 mM CaCl2, 100 μM picrotoxin and 4 μM 2-chloroadenosine (pH 7.4) at 24°C–28°C. Organotypic hippocampal slices were placed in a recording chamber on an Olympus BX50WI microscope with 60× water immersion objective (Olympus). Extracellular field potential was recorded in stratum radiatum with glass electrodes (1–2 MΩ) filled

with the perfusion solution. Synaptic responses were evoked by stimulating two independent pathways using bipolar stimulating electrodes (Frederick Haer) placed 150–250 μm down the apical dendrites, 100 μm apart, and 150–200 μm laterally in opposite directions. Field EPSP was measured by averaging the response 3-Methyladenine over a 5 ms fixed window covering

the peak amplitude. Results from two pathways were averaged and analyzed as n = 1. Whole-cell patch-clamp recordings were done with intracellular solution containing 115 mM Cs-Methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM Na-phosphocreatine, 0.6 mM EGTA (pH 7.25). Electrically evoked EPSCs and miniature EPSCs were recorded under voltage clamping (Vhold = −60 mV; junctional potential not corrected). Recording and analysis were done with IGOR Pro (WaveMetrics). For the analysis of mEPSC frequency, EPSCs that had the characteristic excitatory EPSC profile (Bekkers et al., 1990) was manually identified over 1 min period of recording. Light illumination crotamiton was provided from a 100 W mercury arc lamp filtered through a eGFP filter set with 480/40 nm excitation filter (Olympus). The illumination area was 360 μm in diameter. Hippocampal organotypic slices infected with rAAV and Sindbis virus were imaged under low magnification with a MVX10 Macroview microscope (Olympus). Citrine fluorescence was imaged with the eGFP filter set and tdTomato was imaged with a Texas Red filter set. For high magnification, the slices were fixed with 4% paraformaldehyde and imaged with a Zeiss LSM 780 confocal microscope (Zeiss).