Plotted are all of the ROIs (grey), the mean in 7-second x-bins (blue), and the linear best-fit line (red). D) The total number of seconds an ROI was active during Ca 2+ imaging plotted against the pERK level after fixation, revealing a significant correlation (p=1.1 × 10 −66, R = 0.40, Pearson’s correlation, n=1771 ROIs). In the boxplots, red line = median, blue box = 25 th and 75 th quartiles, whiskers extend to the most extreme non-outliers, red crosses mark points considered outliers. C) ChR2 activation significantly increased pERK levels (p = 8.03×10–35, ranksum test, n =1056 neurons from 20 larvae). Shown are neurons of the tangential and median vestibular nucleus (tVN and mVN), stimulated with either blue or green light. B) ChR2-YFP was driven in multiple neuron types in Tg(−6.7FRhcrtR:gal4VP16) Tg(14xUAS-E1b:hChR2(H134R)-EYFP) atoh7 th241/th241 Tg(atoh7:GAP-RFP) larvae. Boxes depict the approximate x/y positions of neurons shown in panels B and E, but in a different z-plane. Te = Telencephalon, Me = Mesencephalon, Rh = Rhombencephalon. R = Right, L = Left, A = Anterior, P = Posterior, D = Dorsal, V = Ventral.Ī) A confocal slice of a fish stained for phosphorylated-ERK (pERK, yellow) and total-ERK (tERK, magenta), from which we calculate the normalized ‘pERK level’ (pERK/tERK). Shown is the Z and X maximum intensity projections depicting the mean signal in the Z-Brain regions. I, J) The MAP-Map is then analyzed using the Z-Brain (see Online Methods). Shown are mean Z and X projections for heat exposure (Fig. H) Voxels found exhibiting significantly higher (green) and lower (magenta) pERK levels in the stimulus group are localized to create a MAP-Map (Online Methods). G) Stacks are registered to Z-Brain, and the pERK level statistics are calculated at each voxel. Shown are maximum intensity Z and X projections of 16 different labels (left), and a color Z and X random color projection image for 21 labels (right) E) Z and X mean projections of the outlines of the segmented Z-Brain regions, drawn in colors biased towards green = Telencephalon, cyan = Diencephalon, yellow = Mesencephalon, red = Rhombencephalon, magenta = Spinal Cord, F) To create a MAP-Map, pERK/tERK confocal stacks are acquired for ~10–30 fish per condition. Shown are Z and X maximum intensity projections D) Mean-stacks from a total of 29 transgenic, antigenic, or dye labels were generated (Supplementary Table 1). C) The mean across all registered fish is calculated. B) Registrations are applied to an anatomical label (reticulospinal backfills). Shown are three fish (cyan, magenta, yellow) as maximum intensity projections. The resultant maps outline hundreds of areas associated with behaviors.Ī) tERK Confocal stacks registered to a reference brain. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system.
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