2.3. Signal Acquisition

Spontaneous LFP activity was acquired by a linear 32-electrode-silicon probe (ATLAS Neuro Probe: E32-100-S1-L6-NT; pointy tip feature; 100 µm spaced electrodes; mean impedance 0.28 MΩ in Krebs’ solution) connected by a 32-channel head stage (Intant, RHD2000) and an SPI cable to the acquisition system (Open Ephys, OEps Tech, Lisbon, Portugal). The probe was inserted into the PPC and lowered up to 2.3 mm, so that the first 24 channels of the probe were recording from the deepest layer o α,f dentate gyrus (DG) up to the cortical layers. The LFP signals were visualized, recorded, and digitalized at 10 kHz through the open Graphic User Interface software supplied with the Open Ephys acquisition system. Together with the LFP signals, other physiological signals were simultaneously recorded to determine the animal’s health conditions. Heartbeat was monitored through electrocardiogram (ECG) recording, 10X amplified and filtered between 1 and 100 Hz by means of a DAM 50 Amplifier (World Precision Instruments, Friedberg, Germany). ECG positive and negative derivations were subcutaneously inserted into the forelimbs. Respiration-induced movements of the chest wall were converted in voltage fluctuations by the piezoelectric properties of the temperature probe (IT-23, World Precision Instruments, Friedberg, Germany). The respiration signal was amplified 100X and band-passed between 0.1 and 100 Hz by means of a DP-301 amplifier (Warner Instruments, Crisel, Rome, Italy). Physiological signals were digitalized at 10 kHz by a PCI-6071E I/O card (−0.5 to 0.5 V input range) combined with a BNC-2090 terminal block (National Instruments, Rome, Italy) in differential mode and recorded through a custom-made LabView (National Instruments, Rome, Italy) script. Upon reaching a stable level of anesthesia (see below for quantitative evaluation of anesthesia level), we recorded the spontaneous brain activity for 30–40 min.