5-E9.5) in order to label early expressing Dlx1/2 precursors with GFP in the embryos. For simplification purposes GFP expressing mice pups originating from these gavaged females are named tamoxifen-treated Dlx1/2CreERTM;RCE:LoxP. Similarly, pregnant females crossed with Mash1BACCreER/CreER/ RCE:LoxP+/+ males were gavaged at E18.5, in order to label late expressing Mash1 precursors in the embryos named Mash1CreERTM;RCE:LoxP mice. To assess the temporal precision of EGins labeling, we performed six injections FRAX597 supplier of 5-bromo-2′-deoxyuridine

(BrdU, 10 mg/ml in PBS) every 4 hr, starting 6 hr after pregnant mice were force-fed with tamoxifen at E9.5 (50 μg/g intraperitoneally [i.p.]). In another set of experiments, BrdU was injected 31 hr after tamoxifen force-feeding to check that tamoxifen action does not extend over 24 hr. Sections from E12.5 embryos were immunoreacted for both

GFP and BrdU as detailed in Figure S1. Similar results were obtained when tamoxifen was force-fed at E7.5 or E9.5 (Figure S1). Horizontal hippocampal slices (380 μm thick) were prepared from 5- to 7-day-old (P5–P7) tamoxifen-treated Dlx1/2CreERTM;RCE:LoxP (n = 56 slices) or Mash1CreERTM;RCE:LoxP (n = 39 slices) mouse pups with a Leica VT1200 S vibratome using the Vibrocheck module in ice-cold Trichostatin A in vivo oxygenated modified artificial cerebrospinal fluid (0.5 mM CaCl2 and 7 mM MgSO4; NaCl replaced by an equimolar concentration of choline). Slices were then transferred for rest (1 hr) in oxygenated normal ACSF containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 1.3 MgCl2, 2.0 CaCl2, and 10 D-glucose, pH 7.4. For AM-loading, slices were incubated in a small vial containing 2.5 ml of oxygenated ACSF with 25 μl of a 1 mM Fura2-AM solution (in 100% DMSO) for 20–30 min. Slices were incubated in the dark, and the incubation solution was maintained at 35°–37°C. Slices were perfused with continuously aerated (3 ml/min; O2/CO2-95/5%) normal ACSF at 35°C–37°C.

Imaging was performed with a multibeam multiphoton pulsed laser scanning system (LaVision Biotech) coupled to a microscope as previously described ( Crépel et al., 2007). Images were acquired either through a CCD camera, which typically resulted in a time resolution of 50–150 ms per frame. Slices were imaged using a 20×, NA 0.95 objective (Olympus). Imaging depth was on average 80 μm below the surface (range: 50–100 μm). A total of 121 neurons were recorded: 65 were recorded only for morphophysiological characterization in adult slices, whereas 56 were recorded while imaging. Out of the latter, only 32 were considered. The other experiments (n = 24) were discarded because they did not comply either one of the following criteria: (1) stable electrophysiological recordings at resting membrane potential (i.e., the holding current did not change by more than 15 pA); (2) stable network dynamics measured with calcium imaging (i.e.