Extended Data Fig. 6: Controls for off-target effects of 2P photoactivation.
From: Causal evidence of a line attractor encoding an affective state
a. GRIN lens changes the spatial resolution based on the axial depth. Top: imaging a calibration slide with 40 μm fluorescent squares at different axial distances below the GRIN lens. Bottom: imaging in-vivo jGCaMP7s expressing Esr1+ neurons in the VMHvl at different axial distances below the GRIN lens. b. Magnification ratio at different imaging depths calculated from the fluorescent calibration slide. c. Quantification of the relationship between imaging depth and magnification error. Linear regression is used to estimate the degree of aberration caused by the GRIN lens. d. Example field of view illustrating the experimental procedure for mapping the spatial resolution of 2P targeted photo-stimulation through the GRIN lens. Reference neurons were targeted first centred on their somata, and then again stepwise at different distances from the soma centre along each of the four cardinal directions, using 10 µm diameter stimulation spirals. N = 17 cells. e. Average response of all tested neurons to stimulation at each location from the soma. Shaded area represents standard error of the mean. The red-boxed trace indicates the response observed when the stimulation is centred on the reference cell (0 µm). f. Estimated cumulative distribution of the reference cell responses at different distances from soma. Lighter shades of red represent responses at distances progressively further from the soma. n = 17 neurons. g. Raster of neural activity of all 17 reference neurons tested using the procedure in Ex. Data Fig. e. Note that at 15 µm the average response in the reference cells is close to zero. h. Normalized average activity of all neurons at different distances from soma. Each row is a different experiment on a different reference cell. i. Representative examples of field of views from two mice. Green - all x1 neurons, Red – all x2 neurons, black - non x1 or x2 neurons. Fov - field of view. j. Example illustrating how distances are calculated for estimating the spatial clustering of x1 and x2 neurons. k. Quantification of average distance within x1 and x2 neurons and between x1 and x2 neurons, across mice (n = 8 mice, p > 0.05: Kruskal-Wallis test with Dunn’s correction for multiple comparison, error bars - sem).
