Na phosphocreatine

For action potential recordings, the membrane potential was held at −70 mV and ACSF was supplemented with 20 µM DNQX, 100 µM APV, and 50 µM picrotoxin. The internal solution contained (in mM): 100 K-gluconate (Sigma, G4500), 20 KCl (Sigma, P3911), 0.2 EGTA (Fluka, 03778), 10 HEPES, Na-phosphocreatine (Sigma, P7936), 4 Mg-ATP, 0.3 Na-GTP, and 0.025 Alexa Fluor 594. Internal solutions also contained 0.05% Neurobiotin Tracer, pH was adjusted to 7.22 with 1 M KOH, and osmolarity adjusted ∼292 mOsM by addition of sucrose. Current was injected with 10 pA steps and average action potential (AP) frequency was calculated for each current injection. The spike adaptation ratio was calculated by dividing the average of the last two inter-event intervals by the first inter-event interval in the spike train. Spike trains from 10–15 Hz were averaged to calculate the spike adaptation. The voltage of spike threshold for AP generation was determined when dVm/dt reached close to 10 V/s. Resting membrane potential (V_m) was determined before holding current was injected.

Brain slices were transferred to a recording chamber constantly perfused with 32 to 34°C warm ACSF containing (in mM): 119 NaCl (Sigma-Aldrich), 2.5 KCl, 1 NaH₂PO₄, 1.3 MgCl₂, 26 NaHCO₃, 10 D (+)-glucose and 2.5 CaCl₂ (all from Merck, Darmstadt, Germany) bubbled with a gas mixture of 95% O₂ and 5% CO₂. Cortical pyramidal neurons were visualized in layer 5 with an Axioskop 2 FS + microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) equipped with infrared differential interference contrast. Patch pipettes were pulled (P-97 micropipette puller, Sutter Instruments, Novato, CA, USA) to a resistance of 3 to 5 MΩ. For recordings with 4β-PMA and Bryostatin1 intracellular solution comprised (in mM) 120 K-gluconate, 10 Na-phosphocreatine, 11 EGTA, 2 Mg²⁺ATP, 0.3 Tris-GTP (Sigma-Aldrich), 10 KCl, 1 MgCl₂, 1 CaCl₂ and 10 HEPES. For experiments with IFN-β, pipette solution contained (in mM): 120 K-methylsulphate (KMeSO₄) (ICN Biomedical Inc, California, USA), 20 KCl, 14 Na-phosphocreatine, 4 NaCl, 0.5 EGTA, 10 HEPES, 4 Mg²⁺-ATP, 0.3 Tris-GTP and 0.1 cAMP (Sigma-Aldrich). The pH of intracellular solutions was adjusted with KOH (Carl Roth, Karlsruhe, Germany) to 7.2.

Optical imaging and electrophysiological recording of HEK293 cells were performed at 48 h post-transfection in a perfusion chamber with the bath temperature kept at 22 ± 2 °C and perfused at a constant rate of 3 mL/min. A coverslip with HEK293 cells was placed in the recording chamber of an upright fluorescence microscope and superfused with bath solution (in mM: 125 NaCl, 25 NaHCO₃, 27.5 glucose, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂ and 1.5 MgCl₂, pH 7.4) pre-aerated with 95% O₂, 5% CO₂. Electrophysiological recordings were taken in voltage-clamp mode. Borosilicate glass electrodes (resistance of 5 MΩ) were filled with a solution containing (in mM) 132 K-Gluconate, 20 KCl, 4 Mg-ATP, 0.3 Na₂GTP, 10 Na-Phosphocreatine, 10 HEPES, pH 7.25 (all from Sigma, St. Louis, MO, USA). Voltage steps were applied in whole-cell voltage clamp mode with npi ELC-03XS amplifier driven with a DigiData 1440A ADC board (Molecular Devices, San Jose, CA, USA). For precise positioning of the patch pipette, the rig was equipped with a motorized micromanipulator (Luigs and Neumann, Ratingen, Germany) mounted on an air table.