Data were filtered at 9–15 kHz and sampled at 50 kHz with a Digidata 1440 interface controlled by pClamp Software (Molecular Devices, Union City, CA). Electrode resistance in the bath ranged from 2 to 5 MΩ and series resistance ranged from 8 to 20 MΩ. Only cells with an input resistance <300 MΩ were selected to exclude recordings from newly generated granule cells (Liu et al., 1996 and Schmidt-Hieber et al., 2004). The internal solution contained [in mM] 130 K-gluconate,
20 KCl, 10 HEPES-acid, 0.16 EGTA, 2 Mg-ATP, 2 Na2-ATP, and 200 μM Alexa 488 or 594 (Invitrogen) (pH 7.2), osmolality 295 mOsm. Voltages were corrected for the calculated liquid-junction potential of +14.5 mV. Dendritic recording electrodes were see more made from thick walled borosilicate glass capillaries (GB200-8F, Science Products) on a horizontal puller (P-97, Sutter Instruments) and used without further modifications. Dual-whole cell recordings were performed from the soma (2–5 MΩ electrode resistance) and dendrites (20–30 MΩ electrode resistance) using a BVC-700 amplifier (Dagan Corporation). The series resistance
of the dendritic recordings was 100.7 ± 5.2 MΩ (60–135 MΩ, not correlated with distance, Pearson’s r = 0.31, p = 0.15). Dendritic input resistance was 374 ± 45 MΩ (correlated with distance, Pearson’s r = 0. 64, p = 0.003, control recordings see Supplemental Experimental Procedures). Drug application was carried out either via a local application via www.selleckchem.com/products/pf-06463922.html a glass microelectrode or via bath application. In addition, in some experiments, dual dendritic and somatic patch-clamp recordings were obtained and EPSPs were evoked both by current injection and sucrose puff application. Electrical two-pathway stimulation was performed with two theta-glass pipettes filled with ACSF, positioned near the same granule cell in the middle and outer molecular layers, and connected to two stimulus isolators (AM-Systems) operating in bipolar constant current mode. Two-photon excitation fluorescence Resminostat microscopy
was combined with IR-SCG using an ultrafast Ti:Sa laser (950 nm, Chameleon Ultra, Coherent) coupled to a microscope (BX-51, Olympus) equipped with a galvanometer-based scanning system (Ultima, Prairie Technologies). IR-SCG images were generated by spatially filtering the forward scattered infrared laser light with an oblique illumination field stop in the condenser and subsequent detection with a substage photomultiplier tube. An enhanced frequency of unitary EPSPs was evoked by local application of high-osmolar external solution, consisting of normal ACSF with 300 mOsm sucrose and 1 μM TTX added. Two-photon glutamate uncaging at dendrites of dentate granule cells was performed using a microscope equipped with a galvanometer-based scanning system (Prairie Technologies) to photorelease MNI-caged-L-glutamate (Biozol; 12 mM applied via a patch pipette above slice) at multiple dendritic spines.