In vitro electrophysiology

All slice physiology experiments were performed at least one month after virus injection (kindled: 42 ± 2.0 days after injection, range 33–51: cntl: 37 ± 2.4 days, range 30–44 days). Mice were anesthetized with isoflurane and decapitated, and the cortex was quickly removed in ice-cold artificial CSF (aCSF). Parasagittal brain slices (300 μm) were cut using a vibrating microtome (Leica) in a solution containing (in mM): 10 NaCl, 2.5 KCl, 0.5 CaCl2, 7 MgSO₄, 1.25 NaH₂PO₄, 25 NaHCO₃, 10 glucose, and 195 sucrose, equilibrated with 95% O2 and 5% CO2. Slices were incubated at 34°C for 30 min in aCSF containing (in mM): 125 NaCl, 2.5 KCl, NaH₂PO₄, NaHCO₃, glucose, CaCl₂, MgCl₂, 2 NaPyruvate. Slices were then maintained at room temperature until they were transferred to a recording chamber on an upright microscope (Olympus) equipped with a 40x objective.

For most whole-cell recordings, electrodes contained (in mM): 130 D-Gluconic acid, 130 CsOH, 5 NaCl, 10 HEPES, 12 phosphocreatine, 3 MgATP, 0.2 NaGTP, 10 EGTA, 0.05 AlexaFluor 594 cadaverine. For experiments measuring miniature IPSCs and TPMPA sensitivity, electrodes contained: 115–125 CsCl, 5 NaCl, 10 HEPES, 10 EGTA, 4 MgATP, 0.3 Na3GTP and 12 phosphocreatine, 0.05 AlexaFluor 594 cadaverine; and NBQX (10 μM) and D-APV (50 μM) were added to the perfusate. Additionally, TTX (1 μM) was added to the perfusate for mIPSC experiments. Voltage-clamp responses were recorded with a Multiclamp 700B amplifier and digitized at 10 kHz (Digidata 1440); evoked responses were low-pass filtered at 4 kHz, and miniature and spontaneous responses were low-pass filtered at 1 kHz. Series resistance was typically ~10 MΩ, always < 20 MΩ, and was compensated at 80%–95%. Data were collected and analyzed offline using AxographX and IGOR Pro (Wavemetrics). Junction potentials were not corrected. Recordings were performed at 35°C. NBQX, D-APV, TTX and gabazine were acquired from Tocris.

Recordings targeted pyramidal cells, which were visualized using a fluorescent indicator (Alexa 594 Cadaverine) to ensure that cells had pyramidal cell morphologies. We recorded in voltage-clamp from uninfected cells adjacent to the infection site. To ensure cells were uninfected we first examined responses to weak, 1 s long light pulses (470 nm, CoolLED) delivered through the 40x objective. Cells that exhibited large and sustained photocurrents were considered ChR2+ and discarded. Uninfected cells were held at either −70 mV or +5 mV to isolate excitatory or inhibitory synaptic currents, respectively. Brief (2 ms, ~10 mW) pulses were delivered every 10 s to activate ChR2+ axon terminals. A concentric bipolar electrode in the lateral olfactory tract was used to activate synaptic inputs from the olfactory bulb.

qEPSCs, uEPSCs and sIPSCs were detected using the automatic Event Detection function in Axograph. Detected events were automatically filtered (amplitude > 8 pA, 10%–90% rise-time < 2 ms) and then manually checked to ensure that all detected events had single rising phases and appropriate decay kinetics. To evoke qEPSCs, the 2 mM CaCl₂ in the perfusate was replaced with 2 mM SrCl₂. qEPSCs were detected in a window 200–700 ms after the 2 ms light pulse. To evoke uEPSCs, a weak, ramping 5 s long light pulse (0 to ~1 mW) was delivered through a 10x air objective, and events were detected in a window 100–1000 ms after light onset. mIPSC frequency was calculated by counting the average number of events in a 1–3-minute continuous recording. For each cell, qEPSC, uEPSC and mIPSC amplitudes were determined by averaging > 300 validated events per cell.