On day 1 mice were habituated to the training chamber for 12 min. Training occurred on day 2 as follows: mice were allowed to acclimate to the chamber for
4 min prior to the onset of six consecuative training blocks, each consisting of a 20 s baseline, followed by a 20 s, 2 KHz, 80 dB tone (conditioned stimulus, CS), followed by an 18 s trace interval of silence, followed by a 2 s scrambled 1 mA foot shock (unconditioned stimulus, US), followed by a 40 s intertrial interval (ITI). On day 3 mice were tested. Mice were first placed in the training chamber for 3 min to assess contextual fear conditioning, after which they were returned to their home cage for 3 min. Testing for trace fear conditioning took place in a novel chamber, which Ion Channel Ligand Library molecular weight was see more distinct from the training chamber. Mice were allowed to acclimate to the novel chamber for 3 min
prior to tone presentation to assess % freezing in the novel chamber. Next, mice were presented with four testing blocks consisting of a 20 s baseline followed by a 20 s 2 KHz, 80 dB tone followed by a 60 s ITI. Percentage of time freezing was quantified using automated motion detection software (CleverSys). Hippocampal neurons from E18 rat pups were plated onto poly-L-lysine coated dishes or coverslips in Neurobasal growth medium supplemented with 2% B27, 2 mM Glutamax, 50 U/mL penicillin, 50 μg/mL streptomycin, and 5% FBS. Neurons were switched to serum-free Neurobasal medium 24 hr postseeding and fed twice a week. Neurons were transfected at DIV 14–15 using lipofectamine 2000 (Invitrogen) and pH-GluA2 recycling live-imaging assays were performed 48 hr posttransfection as described previously (Lin and Huganir, 2007). Briefly, coverslips containing neurons were assembled onto a closed perfusion chamber and continuously perfused with recording
buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM D-glucose, 1 μM Digestive enzyme TTX, pH 7.4). After 10 min of baseline recording (F0), neurons were perfused with NMDA solution (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.3 mM MgCl2, 30 mM D-glucose, 1 μM TTX, 20 μM NMDA, 10 μM glycine, pH 7.4) for 5 min before the perfusion was switched back to recording buffer for the remainder of the session. All imaging experiments were performed at room temperature using a Zeiss LSM 510 Meta/NLO system (Carl Zeiss, Thornwood, NY). The pHluorin fluorescence was imaged at 488 nm excitation and collected through a 505–550 nm filter, while the mCherry signal was imaged at 561 nm excitation and 575–615 nm emission. Neurons were imaged through a 63× oil objective (N.A. = 1.40) at a 3 μm single optical section and collected at a rate of 1 image per min.