Na3gtp

Sagittal slices (350 μm) containing the hippocampus were obtained as mentioned above. After recovery, the slices were transferred to a recording chamber on the stage of an Olympus microscope with infrared digital interference contrast optics for visualizing whole-cell patch-clamp recordings. sEPSCs were recorded from CA1 pyramidal neurons with an Axon 200B amplifier (Axon Instruments). The recording pipettes (3–5 MΩ) were filled with a solution containing 145 mM K-gluconate, 5 mM NaCl, 1 mM MgCl₂, 0.2 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, and 0.1 mM Na₃-GTP (all from Sigma-Aldrich) (pH adjusted to pH 7.2 with KOH; 290 mOsm). Neurons were clamped at −70 mV. Access resistance (5–30 MΩ) was monitored throughout the experiment. The data were discarded if the change in access resistance was over 15% during an experiment.

Cells were voltage clamped in the whole-cell recording configuration at room temperature (22–25°C). The resistance of electrodes pulled from glass micropipette capillaries (Sutter Instrument Co., Novato, CA, USA) was 2–2.5 MΩ with >60% compensation of series resistance errors. Fast and slow capacitances were compensated before application of the test pulse. Membrane currents were recorded using a HEKA EPC-10 amplifier with pulse software (HEKA Elektronik, Lambrecht, Germany). The pipette solution used for recording hTRPA1 currents contained 140 mM KCl, 5 mM MgCl₂, 10 mM HEPES, 0.1 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 3 mM Na₂ATP, and 0.1 mM Na₃GTP (adjusted to pH 7.4 with KOH). The external Ringer’s solution used for recording hTRPA1 currents contained 160 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM EGTA, 10 mM glucose, and 10 mM HEPES (adjusted to pH 7.4with NaOH). hTRPA1 currents were recorded by holding the cell at -70 mV. The following reagents were obtained: BAPTA, Na₂ATP, Na₃GTP, and EGTA (Sigma-Alrich), HEPES (Calbiochem, San Diego, CA, USA), and other chemicals (Merck, Township, NJ, USA).

Hippocampal organotypic slices were used for electrophysiological recordings shown in Fig. 7. Slices made from PKCα WT and M489V littermates were interleaved. Simultaneous whole-cell recordings were obtained from two neurons, one infected and one neighboring control CA1 pyramidal neurons under visual guidance using differential interference contrast and fluorescence microscopy. One stimulating electrode (contact Pt/Ir cluster electrodes (Frederick Haer)) was placed between 100 and 300 μm down the apical dendrite. Whole-cell recordings were obtained with Axopatch-1D amplifiers (Molecular Devices) using 3–5 MΩ pipettes with an internal solution containing 115 mM cesium methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl₂, 4 mM Na₂ATP, 0.4 mM Na₃GTP, 10 mM sodium phosphocreatine (Sigma), and 0.6 mM EGTA (Amresco), at pH 7.25. External perfusion consisted of artificial cerebrospinal fluid containing 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl₂, 4 mM MgCl₂, 26 mM NaHCO₃, 1 mM NaH₂PO₄, 11 mM glucose, 0.004 mM 2-chloroadenosine (Sigma), and 0.1 mM picrotoxin (Sigma) (pH 7.4), and gassed with 5% CO₂/95% O₂ at 27 °C. The AMPAR-mediated excitatory post-synaptic current (EPSC) was measured as peak inward current at a holding potential of −60 mV. Evoked responses were analyzed by averaging 30–100 sweeps using Igor Pro 4.04 software, blind to experimental conditions.

Human primary neurons and host neurons from the human organotypic cultures were identified by infrared differential interference contrast microscopy, not expressing mCherry under fluorescent light. For a subset of experiments, these cells were identified by GFP + expression (Fig. 6G). The patch pipette was backfilled with a solution containing in mM: 140 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.4 Na₃GTP, and 10 NaCl, pH 7.2–7.4 (mOsm 290–300; all from Sigma-Aldrich).