With an overdose of isoflurane. Surgery process A total of ten male animals, aged between 10 and 14 weeks, weighing 204 g have been anesthetized with isoflurane and healthcare oxygen (four isoflurane for Laminaran supplier induction, 1.0.0 for upkeep, flow price: 1 l/min). For analgesia, 0.1 mg/kg of buprenorphine hydrochloride was subcutaneously injected prior and eight h just after surgery. Anesthetized animals had been head-fixed on a stereotaxic apparatus (David Kopf Instruments) having a custom-made inhalation tube. Body temperature was kept stable at 378 using a heating pad underneath the animal’s body (ATC-2000, World Precision Instruments). Just after exposure from the skull, holes of a diameter of 0.six mm were drilled in line with stereotaxic coordinates (Paxinos Franklin, 2001). Two octrodes (eight twisted wires) have been implanted in to the appropriate and left dorsal CA1 region (AP +/1.94 mm, ML +/1.25 mm, DV .1 mm), and two brass watch stainless steel screws2017 The AuthorsThe EMBO Journal Vol 36 | No 18 |The EMBO JournalSignaling by hippocampal TRPC1/C4/C5 channelsJenny Br er-Lai et al(M1x3) have been implanted in to the cerebellum to serve as reference and ground electrode, respectively. Following surgery, animals have been allowed to recover for 7 days. Electrophysiology We recorded LFPs from the dorsal hippocampal CA1 region of freely moving mice. Along with electrophysiological recordings, 3 accelerometers registered translational movements in x, y, and z directions. A single week after surgery, recordings were performed for 5 h inside the animal’s residence cage (see Fig 4A). Extracellular signals were filtered (100 Hz), amplified (RHA2116, Intan Technologies), digitized (two.5 kHz), and stored for offline analyses with customwritten MATLAB routines (The MathWorks). Behavioral staging We restricted our analyses to selected fast eye movement sleep phases (REM), where each characteristic and robust theta-nested gamma oscillations (42 Hz and 3040 Hz) take place. REM sleep was identified by the absence of movement on all accelerometers accompanied by prominent frequent theta oscillations. Spectral evaluation Energy spectral density evaluation was calculated by implies with the Welch periodogram technique working with the pwelch.m function in the Signal Processing Toolbox of Matlab (50 overlapping, 4-s Hamming windows). Cross-frequency coupling evaluation To measure the intensity of coupling involving the phase of a slow oscillation plus the amplitude of a rapid oscillation, we computed the modulation index (MI, for particulars see Tort et al (2008)). In short, the MI is actually a measure to evaluate the divergence of phase mplitude coupling from a uniform distribution, normalized such that values range between 0 (no coupling) and 1 (maximal coupling). We subsequent selected the MI peaks to evaluate the maximum MI values of low gamma and high gamma to theta coupling, respectively, for additional statistical analyses. Immunostaining in hippocampal neurons Mass neuronal cultures were fixed for 30 mins at room temperature in PBS containing four paraformaldehyde and four sucrose. Cultures have been quenched for 10 mins with 50 mM NH4Cl in PBS, blocked for 1 h in PBS containing three BSA, and 0.1 Triton X-100. Major and secondary antibodies had been diluted in three BSA, 0.1 Triton X-100 in PBS. Cells had been incubated with key antibodies anti-synaptophysin (mouse monoclonal, Synaptic Systems, Germany) 85118-33-8 Protocol overnight. Soon after rinsing, they have been incubated once with PBS containing four M NaCl, then washed briefly in PBS. Alexa-Fluor 543 (Invitrogen) was applied for two h. Afte.