Large-scale imaging of cortical dynamics during sensory perception and behavior

Abstract

Sensory-driven behaviors engage a cascade of cortical regions to process sensory input and generate motor output. To investigate the temporal dynamics of neural activity at this global scale, we have improved and integrated tools to perform functional imaging across large areas of cortex using a transgenic mouse expressing the genetically encoded calcium sensor GCaMP6s, together with a head-fixed visual discrimination behavior. This technique allows imaging of activity across the dorsal surface of cortex, with spatial resolution adequate to detect differential activity in local regions at least as small as 100 μm. Imaging during an orientation discrimination task reveals a progression of activity in different cortical regions associated with different phases of the task. After cortex-wide patterns of activity are determined, we demonstrate the ability to select a region that displayed conspicuous responses for two-photon microscopy and find that activity in populations of individual neurons in that region correlates with locomotion in trained mice. We expect that this paradigm will be a useful probe of information flow and network processing in brain-wide circuits involved in many sensory and cognitive processes.

Keywords: functional imaging; orientation selectivity; visual cortex.

Fig. 1.

A transgenic GCaMP6s reporter mouse. A: tetO construct used to generate transgenic mouse line. Ptight, Ptight Tet-responsive promoter; SV40, Simian virus 40. B: isolated brain from a GCaMP6s mouse crossed to a CAMK2 driver, viewed under a fluorescence dissecting scope, demonstrating strong expression restricted to cortex. Scale bar, 5 mm. C: low-magnification coronal view, showing broad expression throughout cortex. Scale bar, 2 mm. D: high-magnification coronal view, showing expression throughout the layers of cortex. Scale bar, 500 μm. E: confocal section showing that the majority of neurons expresses GCaMP6s, with negligible filling of cell nuclei. Blue, 4′,6-diamidino-2-phenylindole (DAPI) counterstain. Scale bar, 50 μm.

Fig. 2.

Widefield imaging of neural activity. A: diagram of imaging and head-fixation setup. B: widefield fluorescence image of GCaMP6s fluorescence in a cranial window. Inset shows positioning of cranial window. C: fluorescence trace from a single, 26-μm pixel after blue − green subtraction, showing large step increases in fluorescence locked to timing of a 5-s on, 5-s off noise stimulus. D: cycle-averaged traces from the data in C, before (green) and after (black) temporal deconvolution, showing agreement with multiunit electrophysiological recording (blue). Stimulus duration shown in gray. sp/sec, spikes per second.

Fig. 3.

Correction for hemodynamic signals. A–C: Fourier phase maps in response to a 5-s on, 5-s off binary noise stimulus (as in Fig. 2), showing significant vascular artifact present in green (A) and blue (B) illumination but greatly reduced in the subtraction (C). Scale bar, 1 mm. D–F: time course of signals for point shown by circles in A–C, demonstrating that blue − green subtraction eliminates a delayed, slow dip, evident in the green reflectance signal. Stimulus duration is shown in gray.

Fig. 4.

Cortex-wide mapping of sensorimotor modalities. A: individual frames from 2 cycles (top and bottom rows) of a block-presentation visual noise stimulus, showing visually evoked activity superimposed on spontaneous activity. White arrowhead denotes location of visual cortex. Inset shows positioning of cranial window. B: average response to presentation of visual noise stimulus. C: average response to tactile whisker stimulation. D: average activity during periods of locomotion. E: overlay of responses during visual stimulus, whisker stimulus, and locomotion, demonstrating partitioning of cortex by functional properties. Average maps are based on 5 min imaging sessions. Scale bars, 2 mm.

Fig. 5.

Mapping and alignment of retinotopically defined areas. A: individual frames from the cycle-averaged response to the topographic noise stimulus, showing that the moving stimulus evokes corresponding moving bands of neural activity in multiple cortical areas. See Supplemental Movie S3 for full movie. Inset shows positioning of cranial window. B: reliability of maps from short subsets of data, measured by correlation with map obtained from full 5 min of data. C and D: phase maps from 1 session in 1 mouse, demonstrating retinotopy in azimuth (C) and elevation (D). Color represents spatial position in degrees, based on phase of the Fourier response and the position and size of the monitor. Brightness represents Fourier amplitude. E: time course for an individual pixel in C before (green) and after (blue) deconvolution. F and G: average retinotopic maps (azimuth and elevation, respectively), resulting from alignment across subjects and sessions, with correspondence to known extrastriate areas based on demarcation in I. H: overlay of visual field-sign boundaries (black) with watershed transform of retinotopic location (gray) delineates known retinotopic regions. I: color-coded map shows patches of consistent gradient and sign, representing discrete retinotopic regions. Assignment of cortical area identity in H and I and wireframe boundaries in I is based on previous anatomical and imaging studies. Putative assignment to processing streams, based on Wang et al. (2012), is shown in blue (ventral) and magenta (dorsal). F–I: averaged across 15 sessions in 5 mice. Scale bars (for all panels), 500 μm. RL, rostrolateral area; AL, anterolateral area; LM, lateromedial area; P, posterior area; AM, anteromedial area; PM, posteromedial area; V1, primary visual cortex.

Fig. 6.

Head-fixed visual discrimination task. A: schematic of head-fixed behavior paradigm, where the mouse must run on the ball, left or right, depending on orientation of the stimulus. B: distribution of response times from 15 sessions in 5 mice. C: performance as a function of response time for sessions in B, showing high performance during an optimal response window. D: median response time and percent correct through session duration, showing drop in performance toward the end of session.

Fig. 7.

Imaging spatiotemporal dynamics of cortical activity during a visual discrimination task. A: tracks of locomotor response during behavior for trials where the animal responded left vs. right. Correct trials, green; incorrect trials, magenta. B: average neural activity in individual subjects averaged across multiple imaging sessions for trials when the animal responded between 400 and 600 ms after stimulus onset, showing distinct patterns of activation across cortical areas at 100 ms intervals. Each row represents an individual subject. Inset shows positioning of cranial window. C: average neural activity aligned across 15 sessions in 5 subjects, with overlay of putative area boundaries from corresponding retinotopic mapping, showing temporally specific activation of restricted cortical areas that is consistent across subjects. Scale bars, 1 mm. Arrow shows small region between lateral extrastriate, barrel, and auditory cortex active throughout the trial. D: average time course at 3 cortical locations marked by colored squares in C, corresponding to V1 (blue), RL (green), and S1 (red). Response is normalized to maximum for each location to demonstrate differences in temporal activation. Gray box shows response window. E: average time course as in D, without temporal deconvolution.

Fig. 8.

Two-photon recording of passive visual responses in V1. A: baseline fluorescence image of a typical field of view in upper layer 2/3 of V1. B: pixel-wise map of orientation selectivity as measured with drifting gratings of 2 spatial frequencies and 8 orientations, showing a large number of responsive cells, with nearly all showing orientation selectivity. Color wheel shows preferred orientation (hue) and selectivity (saturation). Scale bars, 50 μm. C: histogram of visually evoked dF/F for all cells recorded (n = 479 cells in 3 mice). D: histogram of orientation selectivity for all visually responsive cells (n = 353 cells in 3 mice). Error bars represent bootstrapped confidence intervals.

Fig. 9.

Targeted 2-photon recording in area 39 during spontaneous locomotion. A: overlaid widefield mapping of response to visual white-noise stimulus. Scale bar, 1 mm. B: widefield mapping of activity during spontaneous locomotion, superimposed on an image of the vasculature, used to identify region for 2-photon recording. Scale bar, 1 mm. C: pixel-wise correlation map between fluorescence traces and locomotion, showing neurons positively correlated with locomotion (red) and negatively correlated (blue). Scale bar, 250 μm. D and E: example fluorescence and locomotion traces from 2 neurons in C, showing activation correlated with locomotion (D) and activation correlated with stationary periods (E). F: histogram of correlation between fluorescence and locomotion for individual cells, with green representing cells that are significantly positively correlated and red representing cells that are significantly negatively correlated (n = 284 cells in 3 animals).