( A) Complement C3 was queried on the STRING Protein-Protein Association Network and the top 30 interaction partners are displayed. ( K) Microglia depletion increases the escape probability to bright looming stimuli. Sidak’s multiple comparisons test **p<0.01. ( J) Presentation of bright looming stimuli increases distance traveled over 3 s in microglia-depleted animals (red) but not vehicle-treated animals (blue). ( I) After the bright looming stimulus is presented (0 s), microglia-depleted animals (red) increase in velocity, whereas vehicle-treated animals (blue) do not. ( H) Representative contrails to bright looming stimuli. ( G) Microglia depletion reduces the escape probability to dark looming stimuli. Sidak’s multiple comparisons test ****p<0.0001. ( F) Presentation of dark looming stimuli increases distance traveled over 3 s in vehicle-treated animals (blue) but not microglia-depleted animals (red). ( E) After the dark looming stimulus is presented (0 s), vehicle-treated animals (blue) increase in velocity, whereas microglia-depleted animals (red) do not. ( D) Representative contrails of the escape responses to dark looming stimuli in a single animal (10 trials). A contrail is drawn from 0 to 2 s post-stimulus. ( C) Representative response to dark looming stimulus (presented at 0 s) in a vehicle-treated animal. ( B) Exponentially expanding dark and bright circles were presented as looming stimuli. Stage 47 animals were presented looming stimuli and free-swimming escape responses were recorded. ( A) Schematic of a looming behavioral task to assess visuomotor responses in Xenopus laevis tadpoles. ( F) Microglial depletion with PLX5622 increased axon branch number. ( E) Microglial depletion with PLX5622 did not affect axon arbor length. ( D) Monitoring of individual RGC axons in vehicle control and PLX5622-treated animals. ( C) PLX5622 reduces the number of processes per microglia. Sidak’s multiple comparison post-hoc test ****p<0.0001. ( B) PX5622 depletes microglia in the optic tectum. The white dotted line indicates the border of the optic tectum. Brain structures and microglia were labeled using CellTracker Green BODIPY (green) and IB4-isolectin (red), respectively. ( A) Animals were treated with vehicle or 10 μM PLX5622. ( F) Microglial green fluorescence does not significantly correlate with the number of SYP-pHtdGFP-labeled axons in the optic tectum on day 4 (n = 57, Pearson’s r = 0.063, p=0.64), and weakly correlates on day 5 (n = 57, Pearson’s r = 0.34, p=0.010). ( E) SYP-pHtdGFP fusion protein localizes pHtdGFP to presynaptic puncta. ( D) Microglial green fluorescence weakly correlates with the number of pHtdGFP-labeled axons in the optic tectum on day 4 (n = 47, Pearson’s r = 0.30, p=0.038) and moderately correlates on day 5 (n = 47, Pearson’s r = 0.52, p=0.0002). ( C) Presence of pHtdGFP-labeled axons in the optic tectum increases microglial green fluorescence between day 4 and day 5. The average microglial green fluorescence was quantified from the population of microglia sampled in the z-stack. 3D microglia ROIs were automatically generated (magenta outlines). pHtdGFP-labeled axons (green) were imaged concurrently with microglia (magenta). ( B) Measurement of green fluorescence signal from microglia. 2-photon imaging was performed on day 4 and day 5 post-labeling. Axons were labeled with pH-stable GFP (pHtdGFP).
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