2–7.3 and osmolarity of ∼270–280 mOsm. Series resistances ranging from 7 to 40 MΩ were not compensated for during recordings but were monitored throughout the experiments. Recordings were discarded when a significant

(>20%) change of series PD0325901 molecular weight resistance was detected. Data were digitized with Digidata 1322A, collected with CLAMPEX, and analyzed with Clampfit. EPSCs were recorded under voltage clamp at −60 mV. Evoked EPSCs were recorded from hippocampal CA1 pyramidal neurons by electrically stimulating the SC pathway. Cholinergic terminals were activated by electrically stimulating the SO. The stimulation intensity was adjusted to evoke a postsynaptic current of about 50–100 pA in amplitude, and the intensity was usually around 20–100 μA for 0.1 ms for SC and 50–200 μA for SO pathway. For LTP the amplitude of EPSCs at the 40 min time point after the pairing protocol was compared with that before the pairing protocol. For STD the amplitude at the 5 min time

point after pairing was compared with Vemurafenib ic50 before. Bath-applied cholinergic receptor antagonists or other chemicals were applied 5 min before and during the pairing protocol, and were washed away immediately after the pairing procedure. Calcium imaging was done with the calcium indicator fluo-4 (200 μM included in recording pipette). Alexa 594 (100 μM) was also included in the recording pipette to visualize the dendrites of neurons under recording. Images were acquired with a Zeiss LSM 510 NLO META system coupled to an Axioskop 2FS microscope (Carl Zeiss, Inc., Thornwood, NY, USA) using a Ti:sapphire Chameleon two-photon laser system (Coherent, Inc., Auburn, CA, USA). A wavelength of 810 nm was used to excite both fluo-4 and Alexa 594. An IR Achroplan

63× objective lens (N.A. 1.1) was used. Emissions were collected using band-pass filters BP 500–550 IR and BP 640–720 IR, respectively (Chroma Technology Corp., Rockingham, VT, USA). Image acquisition and online analysis were performed using Zeiss LSM 510 software. ChR2 was expressed in the medial septum in ChAT-Cre mice Isotretinoin (expressing Cre under ChAT promoter). AAV-FLEX-rev-ChR2(H134R)-mCherry carrying double-floxed ChR2 with reversed sequence (Addgene plasmid 20297 from Karl Deisseroth) was packaged with AAV serotype 9 by the virus core facility at NIEHS. Virus (0.5 μl) was injected into 21-day-old mice (anesthetized with 75 mg/kg ketamine and 7.5 mg/kg xylazine) with the following coordinates: bregma, 0.5 mm; lateral, 0.3 mm tilted at 8° toward the midline; and dorsal-ventral, 4.0 mm. Animals were allowed to recover for at least 12 days, and then hippocampal slices for recordings were prepared as described above. The Zeiss LSM 510 NLO META system was also used to generate the light to activate ChR2-expressing cholinergic terminals in the hippocampal SO region, which was coexpressed with mCherry and visualized with 543 nm light that does not activate ChR2. ChR2 was activated with 488 nm laser.