Figure 1. Imaging neuronal responses to pure tones in ferret A1.

(A) An example cortical field in A1 of ferret 1, imaged 120 µm below the pial surface. (B) Responses of one neuron in (A) to pure tones presented at different frequencies and sound levels. Top panel: single trial (gray) and trial-averaged (black) ΔF/F₀ traces, measured for 1 s from sound onset. Bottom panel: Frequency Response Area (FRA) of the same neuron. Color scale indicates the trial-averaged response of the neuron to tones presented at each frequency/level combination, calculated from the deconvolved fluorescence trace (see Materials and methods). (C) Frequency-response profile (i.e. level-averaged tuning curves) of all neurons in (A). Neurons above the dashed black line were significantly modulated by frequency (two-way ANOVA, p<0.05) and are sorted by their Best Frequency (BF). Neurons below the line were not sensitive to sound frequency and are sorted by the p-value of the frequency predictor in the two-way ANOVA (bottom neurons have the largest p-value). (D) Map of the neurons in (A) color-coded according to their BF (see color scale below). Gray dots represent neurons that were not sensitive to tone frequency. Each gray grid is 100 µm².

Figure 1—figure supplement 1. GCaMP6 expression in ferret auditory cortex.

Tangential sections of the auditory cortex are shown to visualize the locations and spread of GCaMP6 injections. Sections were 50 µm thick and were acquired at 200 µm below the cortical surface. (A) The auditory cortex of ferret 3 was injected with a 1:1 solution of AAV1.Syn.GCaMP6f.P2A-nls-dTomato.WPRE.SV40 and AAV1.mDlx.GFP-GCaMP6f-Fishell-2.WPRE.SV40 in four cortical locations. The typical spread of these injections was ~800 µm in diameter per site. (B) Auditory cortex shown as in (A), but for ferret 7. Here, a 1:1 solution of AAV1.Syn.GCaMP6m.WPRE.SV40 and PBS was injected at seven injection sites, and the typical spread was ~400 µm in diameter per site.

Figure 1—figure supplement 2. Imaging in layer 2/3 of ferret A1.

(A) A series of z-projections acquired in vivo using two-photon imaging in ferret 7. The projections have the same fronto-caudal and dorso-ventral axis as the imaged field shown in Figure 1A. Projections were selected to show 10 µm increments in cortical depth. While imaging, the most superficial neuronal bodies were visible ~ 60 μm below the cortical surface. (B) A coronal section of the A1 of ferret 4 showing the extent of GCaMP6 labeling throughout the cortical depth. (C) Distribution of the number of fields imaged at each cortical depth.