Figure 4. Functional imaging using auto-head-fixed mice.

(a) Timeline for head-fixation trial with water reward delivery for Ai94 GCaMP6 mice. The mice initially get a water reward for entering the head fixation tube (valve open for 400 ms). The rewards are then given at 10-s intervals. For the third reward, the images of fluorescence from the cortex are shown. A large calcium increase is observed in the sensory and motor cortex peaking at 500 ms after reward delivery (the colour heat-map indicates change in fluorescence %ΔF/F₀ and the white dot the bregma landmark). The plot on the right indicates the average time course for 18 trials within a single mouse (P value=0.0023 post-water versus baseline variation, t-test). The data were obtained from mouse #115 86 days after cranial window surgery. (b) Functional connectivity derived from imaging activity during both spontaneous activity and water reward delivery. Left: brain atlas view showing specific regions of interest in the sensory and motor cortex (adapted from the Allen Mouse Brain Connectivity Atlas). Green raw RGB image (fluorescence) of mouse cortex is shown. Far right: seed pixel-based correlation maps for barrel cortex (BC), visual cortex (V1) and hind-limb (HL) are shown. These maps are from nine concatenated trials from mouse #377 78 days from cranial window surgery. (c) Seed pixel correlation maps made from a single mouse spanning several months of repeated auto-head-fixation for the cortical point indicated by the plus sign (+); the heat-map indicates correlation. The maps reflect co-activated areas over nine concatenated 30 s trials obtained from mouse #115 (28, 66 and 86 days from cranial window surgery, respectively). These points for seed-pixel maps were determined on the basis of anatomical markers, such as bregma (marked by white dots) and knowledge of visually evoked map locations. Corrections for small brain movements (within trials) were implemented as described in these data sets for correlation maps only.