Epc 300

A microdialysis probe (1mm CMA-7, 6 kDa, CMA) connected to an infusion pump was inserted through the cannula placed in the striatum. Artificial cerebrospinal fluid (ACSF) was perfused at 1.2 μL/min. After the probe insertion we waited for 40 minutes to avoid artifacts evoked by mechanical manipulation. Each sample was collected for 5 minutes in awake animals moving freely on a custom designed jetball system. Immediately after collection, each sample was quantified by an HPLC system (Eicom). Chromatograms were analyzed with the software EPC-300 (Eicom). Dopamine concentration was determined using a dopamine solution of 0.5 pg/μL (Sigma-Aldrich). The temporal course of uncaged dopamine was normalized to the maximum peak evoked by LED irradiation. We used such normalization because the measurements of uncaged dopamine in the samples varied as a function of the distance between the fiber optic and the microdialysis probe.

We measured the levels of 5-HT, 5-HIAA, NE, and MHPG in the hippocampus and striatum by HPLC-ECD (HTEC 500; Eicom, Kyoto, Japan) following a previous protocol^{6 (link),7 (link)}. In the experiment 6, mice were maintained at each feeding schedule for 4 weeks before sacrifice. At ZT 1 and 13, mice were sacrificed with isoflurane without or after the FST, and the hippocampus and striatum were removed for HPLC-ECD. Samples for 5-HT, 5-HIAA, NE, and MHPG measurement received 0.2 M perchloric acid (including 100 μM EDTA·2Na) containing 20 ng of isoproterenol. Samples were homogenized using a micro-homogenizer before being centrifuged at 15,000 × g at 4 °C for 15 min. The supernatant for each sample was collected and filtered using a 0.45 μm filter. The quantity of monoamine in each 20 μL sample was measured by HPLC-ECD with the following conditions: 85% of the transfer phase was composed of 0.1 M acetate citric acid buffer (pH 3.5), including 5 mg/L EDTA·2 Na, 190 mg/L 1-octanesulfonic acid sodium salt, and 15% methanol. The velocity of the flow was 500 μL/min. The column temperature was 25 °C and the applied voltage was + 750 mV versus Ag/AgCl. The data were evaluated by EPC-300 software (Eicom). Dopamine could not be detected in the hippocampus.

Tissue monoamine contents (NE and MHPG) were measured by HPLC-ECD (HTEC 500; Eicom, Kyoto, Japan) as described previously.^{32 (link)} Data were analyzed using EPC-300 software (Eicom).