Multiscale optical Ca2+ imaging of tonal organization in mouse auditory cortex.

Animal Surgery and General Procedures

All animal procedures approved by Johns Hopkins Institutional Animal Care and Use Committee. Floxed GCaMP3 mice (R26-lsl-GCaMP3, Ai38 from Jackson Labs, JAX no. 014538) ( Zariwala et al., 2012 ) were crossed with Syn1-Cre mice (JAX no. 003966) ( Zhu et al., 2001 ) or Emx1-Cre mice (JAX no. 005628) ( Gorski et al., 2002 ), resulting in GCaMP3-Syn1 or GCaMP3-Emx1 mice used for experiments. Anesthesia, surgery, and imaging methods detailed in online extended experimental procedures . All imaging performed on unanesthetized mice. Ca 2+ Dye Injection and Cranial Window Preparation In a minority of mice (e.g., Figures 4G–I ), bulk-loaded Fluo-2 (TEFLabs) was used for Ca 2+ imaging. For Ca 2+ imaging of neurons (GCaMP3 or Fluo-2), a glass coverslip was often affixed atop craniotomy to dampen pulsations. Wide field Transcranial Imaging of GCaMP3 We typically performed wide field imaging through a thinned skull, using 460-nm excitation focused 0–200 microns beneath dura through a 10× 0.25 NA objective (Olympus). 540-nm emission collected onto a CCD camera at 20 Hz. Two-Photon Ca 2+ Imaging Imaging was performed with an Ultima system (Prairie Technologies). Excitation at 950 nm from a mode-locked laser was raster scanned at 5–12 Hz with emission collected in red (607/45 nm) and green (525/70 nm) channels. 40× 0.8 NA objective (Olympus) used. As noted, 25× objective (Olympus XLPlan N) was used in some instances to afford a larger field of view.

Auditory Stimulation

Microscope located in sound-attenuated room (Acoustical Solutions, Audio Seal ABSC-25) with noisy equipment placed outside. Sounds delivered by free-field speaker (Tucker-Davis Technologies, ES1) 12 cm from right ear. Ambient background noise significantly weaker than the softest stimuli presented ( Figure S4G–H ).

Immunohistochemistry

Mice were anesthetized with pentobarbital and perfused with freshly prepared 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA overnight at 4°C and stored in phosphate buffered saline (PBS) at 4°C until processed. 35-μm thick coronal brain sections cut with vibratome were incubated overnight with primary antibodies against GFAP and NeuN. After rinsing, sections further incubated with secondary antibodies, rinsed, and mounted on super frost glass slides. Confocal imaging was then performed. Data Analysis Transcranial Ca 2+ images were processed by a structured sparse encoding algorithm ( Haeffele et al., 2014 ). For two-photon Ca 2+ imaging of neurons, fluorescence signals were directly used to calculate output-versus-frequency response profiles (e.g., Figure 3C ). A deconvolution method was subsequently applied to estimate spike probabilities ( Vogelstein et al., 2010 ), used for analyzing FRAs as well as BF and Q metrics described below. For registration, transcranial maps and vascular fiduciaries were used to localize individual neurons. To compare across mice, elastic registration of each animal’s coordinates to a canonical coordinate system was performed. Best frequency ( BF ) was the frequency evoking strongest responses at threshold. Bandwidth was calculated as the average of the half-maximal width of Gaussian fits to output-versus-frequency plots, and the frequency range eliciting greater than half maximum responses. ‘BF spread’ (Δ BF ) was defined as the absolute difference in best frequency for each pair of tone-responsive neurons in a field, measured in octaves. Q factor was the best frequency at threshold ÷ maximum bandwidth at any sound level.

Extended Methods

More complete methods appear in the online extended experimental procedures .

Animal Surgery and General Procedures

All animal procedures approved by Johns Hopkins Institutional Animal Care and Use Committee. Floxed GCaMP3 mice (R26-lsl-GCaMP3, Ai38 from Jackson Labs, JAX no. 014538) ( Zariwala et al., 2012 ) were crossed with Syn1-Cre mice (JAX no. 003966) ( Zhu et al., 2001 ) or Emx1-Cre mice (JAX no. 005628) ( Gorski et al., 2002 ), resulting in GCaMP3-Syn1 or GCaMP3-Emx1 mice used for experiments. Anesthesia, surgery, and imaging methods detailed in online extended experimental procedures . All imaging performed on unanesthetized mice.

Extended Methods

More complete methods appear in the online extended experimental procedures .