Journal: eLife

Article Title: Pan-cortical 2-photon mesoscopic imaging and neurobehavioral alignment in awake, behaving mice

doi: 10.7554/eLife.94167

Figure Lengend Snippet: ( a ) Mesoscope behavioral apparatus for the dorsal mount preparation. Mouse is mounted upright on a running wheel with headpost fixed to dual adjustable support arms mounted on vertical posts (1” diameter). Behavior cameras are fixed at front-left, front-right, and center-posterior, with ultraviolet and infrared light-emitting diodes aligned with goose-neck supports parallel to right and left cameras. ( b ) Mesoscope behavioral apparatus for side mount preparation. Same as in ( a ), except that the mouse is rotated 22.5 degrees to its left so that the objective lens (at angle 0) is orthogonal to the center-of-mass of the preparation across the right cortical hemisphere. The objective can rotate +/-20 degrees medio-laterally, if needed, to optimize imaging of any portion of the cortex under the cranial window. The right behavior camera is positioned more posterior and lower than in ( a ), to allow for imaging of the eye under the acute angle formed with the horizontal light shield, shown in ( c ). ( c ) Mouse running on wheel with side mount preparation receiving visual stimulation from an LED screen positioned on the left side, with linear motor-positioned dual lick-spouts in place and 3D printed vertical light shield (wok 2) attached to rim of flat shield (wok 1) to block extraneous light from entering the objective. ( d ) Overhead view of dorsal mount preparation with 3D printed titanium headpost, custom cranial window, and Allen CCF aligned to paraformaldehyde-fixed mouse brain. Motor region = light green, somatosensory = dark green, visual = dark blue, retrosplenial = light blue. Olfactory bulbs (anterior) at top, cerebellum (posterior) at bottom of image. Note ridge along perimeter of headpost for fitted horizontal light shield (wok 1) attachment. ( e ) Rotated dorsal view (22.5 degrees right) of side mount preparation with 3D printed titanium headpost, custom cranial window, and Allen CCF aligned to paraformaldehyde-fixed mouse brain. Auditory region shown in light blue, ventral and anterior to visual areas, and ventral and posterior to somatosensory areas (right side of image is lateral/ventral, and left side of headpost perimeter is medial/dorsal). Other regions shown with the same color scheme as in ( d ). UV = ultraviolet, IR = infrared, M2 = secondary motor cortex, S1b = primary somatosensory barrel cortex, V1 = primary visual cortex, A1 = primary auditory cortex.

Article Snippet: The interoperability of our preparations across widefield 1p and Thorlabs mesoscope 2p imaging rigs (see Protocol IV) allows for the straight-forward development of an experimental pipeline that first surveys widespread activity at fast timescales (widefield,~10–50 Hz) and then investigates the activity of large subregions in a serial manner at intermediate timescales (Thorlabs mesoscope,~1–10 Hz).

Techniques: Imaging, Blocking Assay

Journal: eLife

Article Title: Pan-cortical 2-photon mesoscopic imaging and neurobehavioral alignment in awake, behaving mice

doi: 10.7554/eLife.94167

Figure Lengend Snippet: Comparison of neurobehavioral alignments in back-to-back mesoscope imaging sessions either without ( a , ‘OFF’) and with ( b , ‘ON’) GPU-enabled online z-motion correction. ( a ) Neural and behavioral data are shown temporally aligned for the first session. Rastermap sorted neural activity is shown at top, with a blue/green/yellow (min/mid/max) heatmap applied to activity from each individual neuron z-scored separately. Walk speed (red), whisker motion energy (blue), and pupil diameter (gray) are shown below. Red dashed vertical lines indicate times of two major walking bouts for the purpose of alignment visualization. Note that roughly the top ~30% of neurons show strong activity time-locked to the identified walking bouts (red vertical line at right, labeled ‘walk’). ( b ) Same as in ( a ), except for a 2nd consecutive session in the same mouse. The only change was that, for this session, online GPU-enabled z-motion correction was used (“ON”) following acquisition of a 60 x 1 µm step anatomical z-stack for frame-by-frame comparison and adjustment (ScanImage/Vidrio). Note the similar percentage of cells aligned with each walking bout in the top ~30% of superneurons in Rastermap sorts in both ( a ) and ( b ) (indicated by red vertical line at right labeled ‘walk’), and the overall qualitative similarity of Rastermap clustering and activity motifs compared to that of the immediately prior session without z-motion correction shown in ( a ), despite the fact that these back-to-back sessions contained different detailed patterns of spontaneous activity. ME = motion energy, au = arbitrary units, GPU = graphics processing unit.

Article Snippet: The interoperability of our preparations across widefield 1p and Thorlabs mesoscope 2p imaging rigs (see Protocol IV) allows for the straight-forward development of an experimental pipeline that first surveys widespread activity at fast timescales (widefield,~10–50 Hz) and then investigates the activity of large subregions in a serial manner at intermediate timescales (Thorlabs mesoscope,~1–10 Hz).

Techniques: Comparison, Imaging, Activity Assay, Whisker Assay, Labeling