Journal: bioRxiv

Article Title: Molecular and functional mechanisms of long-term visual habituation in larval zebrafish

doi: 10.1101/2022.06.17.496451

Figure Lengend Snippet: Volumetric 2-photon Ca 2+ imaging of dark flash habituation. A) Tg(elavl3:H2B-GCaMP7f) larvae were imaged across 12 z-planes at 10 μ m steps. ROIs segmented using suite2p Pachitariu et al . ( 2017 ) are overlaid in random colors. B) Density of detected ROIs registered and plotted in the Z-Brain coordinate space. n=1,050,273 ROIs across 34 larvae. C) Cropped field of view used for plotting and analyzing Ca 2+ imaging data and approximate anatomical localizations of major brain areas: dien=diencephalon, mid-b = midbrain, cb = cerebellum, hind-b = hindbrain, io = inferior olive, ret = retina, tec = tectum D) Functional responses of the population of neurons in the same fish as A), plotted as a clustered heatmap (“rastermap” Pachitariu et al. ( 2017 ) , github.com/MouseLand/rastermap ), where rows represent individual neurons ordered by the similarities in their activity. Darker shades reflect increased activity. This clustering reveals neurons that are tuned to the DF stimuli (pink box) or motor events (green box). Dashed trace above the heatmap depicts the DF stimulus convolved with a kernel approximating H2B-GCaMP7f kinetics. E) ROIs in an individual fish plotted based on their correlation and tuning to regressors defining either Motor or DF stimulus events, highlighting the spatial distributions of these tunings across the imaged population. Plotted as a maximum intensity projection. F) Same analysis as E, but across the entire population of 34 larvae. G) ROIs in an individual fish plotted based on their correlation and tuning to regressors defining either the first or last three DF stimuli. H) Same analysis as G, but across the entire population of 34 larvae. tl = torus longitudinalis . Validation of motion analysis based on image artifacts during 2-photon imaging

Article Snippet: ROIs were identified using suite2p Pachitariu et al. ( 2017 ) using the parameters outlined in RunSuite2p_BrukerData_ScreenPaper.py and RunSuite2p_MartinPhotonData_ScreenPaper.py ; scripts for the data from the Bruker Ultima microscope ( – ), and custom built 2-photon microscope ( Figure 7 D,E ), respectively.

Techniques: Imaging, Functional Assay, Activity Assay, Biomarker Discovery

Journal: bioRxiv

Article Title: Molecular and functional mechanisms of long-term visual habituation in larval zebrafish

doi: 10.1101/2022.06.17.496451

Figure Lengend Snippet: An atlas-based search for molecular markers identifies GABAergic neuronal classes. A) Model 1 , proposed to explain how potentiating GABAergic inhibition could mediate the range of depressive adaptations observed during habituation. Model posits that the sensitizing neurons are GABAergic ( , blue), and they differentially connect to functional subtypes: Strong connectivity to strongly habituating classes, weak connectivity to weakly habituating classes, etc. B) Hierarchically clustered heatmap depicting the correlation of markers aligned to the Z-Brain atlas with the spatial arrangement of the 12 functional clusters (distance = complete, correlation). Correlation values are z-scored by rows to highlight the cluster(s) most strongly correlated or anti-correlated with a given marker. The subset of the hierarchy containing the gad1b-reporters is coloured in purple. C) Normalized summed intensity projections comparing the spatial arrangements of i) , and ii) with atlas markers found within the the purple cluster iii) satb1b HCR , mapZebrain Atlas iv) nns25 , aka TgBAC(gad1b:GFP) , mapZebrain Atlas v) nns26 , aka TgBAC(gad1b:LOXP-RFP-LOXP-GFP) , mapZebrain Atlas vi) TgBAC(gad1b:GFP) , Z-Brain Atlas D) 2-photon imaging of Tg(Gad1b:DsRed);Tg(elavl3:H2B-GCaMP6s) larvae depicting the raw data for each channel (top), and the ratio of Gad1b/GCaMP6s fluorescence in each ROI functionally identified using suite2p. E) ROIs imaged in double transgenic larvae are assigned a cluster identity based on their correlation to the cluster mean trace, and classified as Gad1b-positive based on a DsRed/GCaMP6s ratio of greater than 0.25. Dotted line = expected proportion based on total number of cells classified as Gad1b-positive. *=p<0.05, Chi Square test with Bonferroni correction. Distributions underlying violin plots calculated by bootstrapping 5000 replicates. n = 1835 ROIs in 6 larvae.

Article Snippet: ROIs were identified using suite2p Pachitariu et al. ( 2017 ) using the parameters outlined in RunSuite2p_BrukerData_ScreenPaper.py and RunSuite2p_MartinPhotonData_ScreenPaper.py ; scripts for the data from the Bruker Ultima microscope ( – ), and custom built 2-photon microscope ( Figure 7 D,E ), respectively.

Techniques: Inhibition, Functional Assay, Marker, Imaging, Fluorescence, Transgenic Assay