*2.3. Subjects and Experiments*

Experiments were carried out on ten C57BL/6 male mice (20–25 g) in accordance with the Guide for the Care and Use of Laboratory Animals (8th ed., The National Academies Press, Washington, 2011). The protocols were approved by the Local Bioethics Commission of the Saratov State University. The mice were kept in a light/dark environment with the lights on from 8:00 to 20:00 and fed ad libitum with standard rodent food and water. The ambient temperature and humidity were maintained at 24.5 ± 0.5 ◦C and 40–60%, respectively.

A two-channel cortical EEG (Pinnacle Technology, Taiwan) was recorded (Figure 2) using two silver electrodes (tip diameter 2–3 μm) located at a depth of 150 μm in coordinates (L: 2.5 mm and D: 2 mm) from Bregma on either sides of the midline under inhalation anesthesia with 2% isoflurane at 1 L/min *N*2*O*/*O*2—70:30. The head plate was mounted and small burr holes were drilled. Thereafter, wire EEG leads were inserted into burr holes on one side of the midline between the skull and the underlying dura mater. EEG leads were fixed with dental acrylic. Ibuprofen (15 mg/kg) for relief of postoperative pain was provided in water supply for two to three days before surgery and for three or more

days after surgery. Before starting the experiment, the mice were given 10 days to recover from surgery.

**Figure 2.** Experiment design: (**a**) implantation of a two–channel cortical EEG, (**b**) SD by presenting new objects to the mouse, and (**c**) EEG recording in an awake mouse. Insert shows experimental EEG signals—voltages (μV) vs. time (seconds).

As standard sleep staging rules for mice are not currently available, we referred to the visual assessment criteria from the studies [1,38]. Sleep deprivation was carried out according to the method described in [39], with adaptation to the vivarium regime. The mice were deprived of sleep from 8:00 pm to 8:00 am and were immediately used for the experiment. Sleep deprivation was maintained by bringing new objects and sounds into the experiment room [40]. The mice were constantly monitored to make sure they were actually studying objects.

Signals were measured in awake and sleeping mice (day 1, 10-h recording), and after SD (day 2, 4-h recording). This study compares two states: (1) awake mice, background EEG activity, and (2) awake mice after SD. All recordings were done with a sampling rate of 2 kHz. At the stage of preprocessing, twelve 5-min segments with a quite homogeneous structure and less distorted by artifacts were selected for each EEG channel. Every 5-min segment was analyzed with EDFA to evaluate *σ*(*Floc*(*n*)) and the *β* exponent. The results for each mouse state were averaged over all selected segments and both channels.
