With an overdose of isoflurane. Surgery procedure A total of ten male animals, aged amongst ten and 14 weeks, weighing 204 g were anesthetized with isoflurane and health-related oxygen (4 isoflurane for induction, 1.0.0 for upkeep, flow rate: 1 l/min). For analgesia, 0.1 mg/kg of buprenorphine hydrochloride was subcutaneously injected prior and 8 h after surgery. Anesthetized animals were head-fixed on a stereotaxic apparatus (David Kopf Instruments) having a custom-made inhalation tube. Physique temperature was kept stable at 378 with a heating pad underneath the animal’s body (ATC-2000, Planet Precision Instruments). Soon after exposure from the skull, holes of a diameter of 0.6 mm have been drilled based on stereotaxic coordinates (Paxinos Franklin, 2001). Two octrodes (eight twisted wires) had been implanted into the correct and left dorsal CA1 area (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) were implanted into the cerebellum to serve as reference and 61970-00-1 Purity & Documentation ground electrode, respectively. After surgery, animals were permitted to recover for 7 days. Electrophysiology We recorded LFPs in the dorsal hippocampal CA1 area of freely moving mice. Along with electrophysiological recordings, 3 accelerometers registered translational movements in x, y, and z directions. A single week just after surgery, recordings were performed for 5 h inside the animal’s residence cage (see Fig 4A). Extracellular signals have been filtered (one hundred Hz), amplified (RHA2116, Intan Technologies), digitized (2.five kHz), and stored for offline analyses with customwritten MATLAB routines (The MathWorks). Behavioral staging We restricted our analyses to chosen fast eye movement sleep phases (REM), exactly where both characteristic and robust theta-nested gamma oscillations (42 Hz and 3040 Hz) occur. REM sleep was identified by the absence of movement on all accelerometers accompanied by prominent normal theta oscillations. Spectral evaluation Power spectral density evaluation was calculated by signifies of the Welch periodogram technique employing the pwelch.m function from the Signal Processing Toolbox of Matlab (50 overlapping, 4-s Hamming windows). Cross-frequency coupling analysis To measure the intensity of coupling between the phase of a slow oscillation as well as the amplitude of a speedy oscillation, we computed the modulation index (MI, for particulars see Tort et al (2008)). In short, the MI is a measure to evaluate the divergence of phase mplitude coupling from a uniform distribution, normalized such that values variety in between 0 (no coupling) and 1 (maximal coupling). We next selected the MI peaks to evaluate the maximum MI values of low gamma and high gamma to theta coupling, respectively, for further statistical analyses. Immunostaining in hippocampal neurons Mass neuronal cultures had been fixed for 30 mins at space temperature in PBS containing four paraformaldehyde and four sucrose. Cultures were quenched for 10 mins with 50 mM NH4Cl in PBS, blocked for 1 h in PBS containing 3 BSA, and 0.1 Triton X-100. Major and secondary antibodies were diluted in three BSA, 0.1 Triton X-100 in PBS. Cells had been incubated with major antibodies anti-synaptophysin (mouse monoclonal, Synaptic Systems, Germany) overnight. After rinsing, they were incubated when with PBS containing four M NaCl, then washed briefly in PBS. Alexa-Fluor 543 (Invitrogen) was applied for two h. Afte.
