, 2005), and kept in artificial cerebrospinal fluid in the dark to avoid ChR2 activation. Electrophysiological

Recordings. Whole-cell patch-clamp recordings were visually guided by infrared videomicroscopy (DM-LFS; Leica), using 4–9 MOhm borosilicate pipettes filled with 140 mM KCl, 10 mM HEPES, 2 mM MgCl2, 0.1 mM CaCl2, 0.1 mM BAPTA, 2 mM ATP Na salt, 0.3 mM GTP Na salt (pH 7.3), 300 mOsm, and amplified with an Axopatch 200B (Axon Instruments). For cell-attached recordings, KCl was replaced with KMeSO4. ChR2-mCherry expression was identified by fluorescent PD173074 microscopy and post hoc immunohistochemistry. Blue-Light Stimulation. For in vitro experiments, optical stimulation was done via a mercury lamp (Short Arc 103W/2, Osram; ∼5 mW/mm2) in combination with a shutter (VS25S22M1R1, Uniblitz) or a TTL-pulsed LED source (LXHL-LB3C, Roithner; ∼10 mW/mm2), both yielding similar results. Following recovery from guide cannulae implantation (1 week), female virgin rats of random hormonal cycle were exposed to a contextual fear-conditioning protocol. This consisted of three sessions

on consecutive days (Figure 5B). On day 1, rats were individually introduced in Panobinostat cost the conditioning box (45 × 18 × 25 cm) and after 10 min received a first series of seven electric shocks (0.8 mA) at random intervals (15–120 s) over 7 min. After the last these shock, the rats were left in the box for 3 more min. BL was applied in 10-ms pulses at 30 Hz via a glass fiber protruding 2 mm beyond the lower end of the cannulae and delivered light intensity of ∼10 mW. OTA injection was done via two injectors (cut to fit the 5.8 mm

guide cannulae protruding 2 mm beyond the lower end of the cannulae) that were bilaterally lowered into the guide cannulae, connected via polyten tubing to two Hamilton syringes that were placed in an infusion pump, and 0.5 μl of liquid containing 21 ng OTA was injected in each hemisphere at 0.25 μl/min over a 2 min period. Application of BL during Fear-Context Exposure. On day 2, the rats were habituated to the glass fibers by inserting them into the cannulae for the complete duration of the protocol, which was similar to the one of day 1 ( Figure 5C). On day 3, the rats were tested in the context for 10 min before receiving bilateral BL stimulation for either 20 s or 120 s. The effect was video recorded for the complete 20 min of the test (see Figure 5B). Freezing time was analyzed per 10 s intervals. Application of BL prior to Fear-Context Exposure. On day 2, rats received in addition a sham blue-light application during 6 min of very light isoflurane (5% induction, 1% for maintenance), during which time the glass fiber was inserted into the guide cannulae, as on day 3, without exposure to BL ( Figure 5D).