3 Hz; low pass, 100 Hz), and sampled at 200 Hz. To filter out the low-frequency artefacts, EEG signals were digitally processed through a high-pass filter (1.0 Hz) with spike2 software (version 5.11; Cambridge Electronic Devices, Cambridge, UK). EEG recordings were manually scored in 4-s epochs for wakefulness, non-rapid eye movement sleep, and rapid eye movement sleep, which were distinguished

as follows: wakefulness – low-amplitude desynchronized EEG activity and high-amplitude EMG activity; non-rapid eye movement sleep – high-amplitude δ-wave (1–4 Hz) EEG activity and low-amplitude or absent EMG activity; and rapid eye movement sleep – regular θ-wave (5–9 Hz) EEG activity and decreased or absent EMG activity. Dabrafenib order We calculated EEG power spectra by using fast Fourier transformation Torin 1 solubility dmso (FFT) with the following parameters: frequency range, 1–50 Hz; FFT block size 256; Hanning window resolution, 0.5 Hz. Two or three days after the start of EEG/EMG recording, a microdialysis probe (CMA 7, 1-mm membrane; CMA/Microdialysis) was implanted in the posterior hypothalamus. The stereotaxic coordinates of the probe tip (relative to bregma) were: anterior, −2.14 to −3.07; lateral, +0.5; and vertical, −5.4 (Paxinos & Franklin, 2004). The probe was connected

to a sample collection system, and continuous perfusion (1 μL/min) with artificial cerebrospinal fluid (147 mm NaCl, 3 mm KCl, 1.2 mm CaCl2, 1 mm MgCl2) was then started. Sample collection was started 1 day after probe implantation, with 30-min intervals, for five consecutive days. After the experiment, the mice were killed

by decapitation, and the brains were removed and sectioned with a cryostat in the coronal plane according to the stereotaxic atlas (Paxinos & Franklin, 2004) to verify the probe position. The probe location was selected for several methodological and anatomical reasons. The TMN sends projections to all brain areas without Elongation factor 2 kinase anatomically distinct subgroups (Ericson et al., 1987), and this region of the posterior hypothalamus contains a very dense network of histaminergic fibres. Histamine recovery in vitro with the CMA 7-1 probe from the standard solutions was 10–12% (data not shown), which motivated the use of a terminal-rich area for study of long-term release. Therefore, to enable reliable and reproducible detection of histamine with our experimental setup, the TMN region with the adjacent supramamillary region was chosen as the preferential site for the microdialysis. Each cage was equipped with a CAMZWMBLAH2N video camera (Velleman, Gavere, Belgium) combined with an infrared light source. The video stream was captured and recorded continuously with GeoVision surveillance software (GeoVision, Taiwan) from 5 days before surgery until the end of the experiment. The recorded video data were converted and prepared for tracking with virtualdub 1.9.2 (www.virtualdub.