Brain coronal sections of 50 μm were cut on a cryostat, stained with luxol fast blue—cresyl violet and the recording site was verified by light microscopy. A cleaved end of an optical fiber (200 μm diameter, Thorlabs) was inserted through the guide cannula into the VTA of anesthetized mice. After baseline

recording of the spontaneous activity of VTA cells, the laser (light power was controlled to reach no more than 5 mW at the tip of the optical fiber) was switched PFT�� on for 1 or 2 s continuously and off for 9 or 8 s. This optical stimulation was repeated 50 times. Footshock was delivered by two 30 gauge needles implanted in the lateral side of the foot controlateral to the neuronal recordings. Electrical stimulations were generated using an isolated pulse stimulator (1–5 mA, 0.1 ms single pulse duration; AM systems) and delivered at a frequency of 0.5 Hz using custom-made program within IGOR (Wavemetrics). Morphine (2 mg/kg), naloxone (1 mg/kg), apomorphine (0.05 mg/k), haloperidol (0.2 mg/kg) were prepared in 0.9% saline and administered GSK-3 assay intravenously through a 30 gauge cannula inserted

in a lateral tail vein. Injection volumes ranged between 30 and 60 μl. Injection of saline had no effect on the firing rate and discharge pattern. Bicuculline methiodide (20 mM) was either applied locally to DA-like neurons via diffusion from the recording electrode as shown previously Cell press in vivo (Ji and Shepard, 2007 and Tepper et al., 1995) or via a guide cannula (500 nM; Gavello-Baudy et al., 2008). To compare the basal firing rate and discharge pattern with and without drugs, cells were recorded after 10 min of drug diffusion and before delivering the footshocks. These parameters were stable over time indicating that the effect of the drug was rapid in onset and persistent. VTA neurons were recorded for 5 min to establish their basal firing rate and discharge

properties before drug administration, footshock delivery, or light stimulation. The processed data were displayed as event raster plots, binned color-coded raster plots, peristimulus time histograms (PSTHs), or firing rate plots. Event raster plots show the time markers of detected activity of 40–50 consecutive footshock or light responses. PSTHs (5 ms bin width) were analyzed to determine excitatory and inhibitory periods as described previously (Jodo et al., 1998). Briefly, baseline values were obtained by calculating the mean and standard deviation (SD) of counts per bin of the 500 ms preceding the footshock or 1 or 2 s preceding the light stimulation. The onset of the response was defined as the first of 5 consecutive bins for which the mean firing rate was more or less that the baseline by 2 SDs. Inhibition was defined as a period of at least 15 bins in which the mean count per bin dropped at least 35% below mean baseline.