All procedures were done at 4°C. ECS was induced through a constant-current generator (ECT unit; Ugo Basile, Comerio, Italy) (Cole et al., 1990), in accordance with the guidelines see more of the Johns Hopkins Animal Care and Use Committee. The brain was dissected 2 hr after ECS and placed immediately into cold (2.5°C) modified CSF composed of the following (in mM): 110 choline chloride, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 2.4 Na-pyruvate, 1.3 Na-ascorbic acid, 1.2 NaH2PO4, 25 NaHCO3, and 20 glucose. Coronal brain slices of the prefrontal cortex (250 μm)

were prepared from P20-22 WT and Homer1a KO mice using a Vibratome 3000 (Leica Biosystems, St. Louis LLC, St. Louis, MO). After cutting, slices were incubated for 15 min at 32°C and then for up to 3 hr at 25°C in ACSF. Whole-cell patch-clamp recordings from cortical cultures and slices were carried out at 30°C–32°C. Pyramidal neurons in cortical cultures and the layer II-III region of the prefrontal cortex were visually identified using Dodt Gradient Contrast.

Transfected neurons were also visually identified under epifluorescence. learn more The recording chamber was continuously perfused with artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 10 glucose, equilibrated with 95% O2 and 5% CO2 (pH 7.4, 305 ± 5 mmol/kg). The bath solution also contained both 1 μM TTX and 10 μM GABAzine to block action potential dependent EPSCs and GABAA receptors, respectively. The pipette solution contained (in mM): 90 Cs-methansulfonate, 48.5 CsCl, 5 ethylene glycol tetraacetic acid,

2 MgCl2, 2 Na-ATP, 0.4 Na-GTP, and 5 HEPES (pH 7.2, 290 ± 2 mmol/kg). Patch pipettes were pulled from borosilicate glass (4–5 MΩ) using a horizontal puller (Sutter Instruments, Novato, CA). Signals were recorded with a Multiclamp 700B (Molecular Devices, Union City, CA) amplifier, filtered at 2 kHz and sampled at 10 kHz. To detect a sufficient number of events (200 events per neuron), recordings were performed on gap 17-DMAG (Alvespimycin) HCl free mode (sweeps of 30 s without any latency). Data were acquired 3 min after achieving the whole-cell configuration. Series resistances (Rs) of recordings ranged between 10 and 15 MΩ. Cells were rejected from analysis if Rs changed by more than 15%. mEPSCs were analyzed by Mini Analysis Software (Synaptosoft, NJ). All group data are shown as mean ± standard error of the mean (SEM). Statistical comparison was performed by the independent t test, ANOVA for multiple comparison (see Figures 1F and 5G), or Fisher’s exact test (see Figure 7F). All drugs were purchased from Tocris (Ellisville, MO) except for TTX (Ascent Scientific LLC, Princeton, NJ). All the data were analyzed by two-tailed Student’s t test except the analysis of the multiple comparisons (Figures 1F and 5G). Error bars indicate the SEM. We thank Dr. Alison Barth of Carnegie Mellon University for Fos-GFP mice.