Electrophysiological Imaging of Auditory Cortex

Three to 4 weeks after the AAV injection, mice were transcardially perfused, and mouse brains were rapidly removed. Coronal slices (300 μm) containing the right AC were prepared in a cutting solution at 1°C using a vibratome (Leica, #VT1200 S) as described previously (11 (link), 51 (link)). The slices were immediately transferred and incubated at 34°C in a holding chamber for 40 min before imaging. The holding chamber contained ACSF containing the following: 125 mM NaCl, 2.5 mM KCl, 26.25 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 1.3 mM ascorbic acid, and 3 mM sodium pyruvate (pH 7.4; ∼300 mOsm, bubbled with 95% O₂/5% CO₂). Next, ex vivo imaging was performed on brain slices bathed in carbonated ACSF identical to the incubating solution. The anatomical landmarks, such as the rhinal fissure and the underlying hippocampal formation, were used to locate the AC in brain slices. FRISZ-TM or mMaroon1-TM emission was imaged with epifluorescence optics as described above for in vivo wide-field imaging. Next, to image the stimulus-evoked change, L4 of AC was electrically stimulated with 0.3-ms-long 50-V pulses (1 to 100 pulses) at 100 Hz, starting at 3 s during each imaging session, by following a previous procedure (5 (link)). FRISZ-TM signals were imaged from L2/3 of AC. Each stimulus was presented six to eight times, and the average of each stimulus was taken for further analysis. To correct for gradual linear “run-down” in fluorescence signals with LED onset, a linear fit of the fluorescence signals from 2 s before the onset of the stimulus was subtracted from the stimulus and nonstimulus fluorescence signals. Next, fluorescence values were converted into ΔF/F₀, with F₀ taken as the average baseline fluorescence from 1 s before the onset of the stimulus. Peak fluorescence signals during the 1-s period after the electrical stimulation were quantified as the stimulus-evoked response amplitude and were defined as significant responses if the electrical-evoked changes (ΔF/F₀) were 3 SD higher than the baseline mean. After the initial recording, the ZX1 solution (100 μM) was bath applied to the brain slice over 20 min, and the stimulus-evoked change in FRISZ-TM fluorescence was remeasured.