Fig. 3: Distant brain regions remain structurally intact.

a Schematic showing the experimental protocol for reverse brain clearing and immunolabeling. b Two rabies-labeled input neurons (red) identified in a sagittal optical section of NDB in an intact control brain. Representative animal from n = 3 uninjured controls. 50 μm maximum intensity projection obtained by whole-brain light-sheet imaging. c Sagittal section containing the same input neurons (magenta) labeled for CHAT (green) after processing for traditional immunostaining and confocal imaging. Representative animal from n = 3 uninjured controls. Arrow indicates co-labeled cell. d NDB of uninjured control (left) and brain-injured animal (right) labeled for CHAT (green). Representative animals from n = 3 uninjured and 4 TBI mice. e Proportion of mCherry+ rabies-labeled input neurons that expressed CHAT. f Quantification of CHAT+ neuron density in ipsilateral and contralateral NDB in uninjured controls and brain-injured animals. n = 3 uninjured and 5 TBI animals. g Sagittal section of dentate gyrus in uninjured control (left) and brain injured animal (right) labeled for CHAT (green) and DAPI (blue). Representative animals from n = 3 uninjured and 4 TBI mice. h, hilus; gcl, granule cell layer; ml, molecular layer. h. Quantification of CHAT+ axon density in ipsilateral and contralateral dentate gyrus in uninjured controls and brain-injured animals. n = 3 uninjured and 4 TBI animals. i ENTm of uninjured control (left) and brain-injured animal (right) labeled for reelin (green) and mCherry (magenta). Representative animals from n = 3 uninjured and 5 TBI animals. j Proportion of mCherry+ rabies-labeled input neurons that expressed reelin. k Quantification of reelin+ cell density in ipsilateral and contralateral ENTm. n = 3 uninjured and 5 TBI animals. Error bars, s.e.m.; scale bars, 100 μm. See also Supplementary Fig. 8 and Supplementary Data 1. Source data are provided as a Source Data file.