Fig. 1. A platform for labeling MLI morphologies in the cerebellum.

a Single MLI labeling with Cre-dependent Brainbow AAVs encoding multiple fluorescent proteins injected into Gad2-Cre mice between P0 to P10. b Cross-sections of mature cerebellar cortex show enriched labeling of Gad2-Cre positive subpopulations. AAV delivery at P0 predominantly labels Purkinje cells (PC); P5, lower MLIs; P10, upper MLIs. Dashed lines denote molecular layer (ML) boundaries. c Schematic of canonical innervation patterns of basket (BC, light blue) and stellate (SC, dark blue) cells residing in the lower and upper ML, respectively (adapted with permission from ref. ¹¹⁰). d Mouse cerebellum (pink), and its cross-sectional morphology. ML outlined in cyan. Immunostaining of MLIs (anti-Parvalbumin (PV), cyan) shows distribution throughout the ML. PCs are co-labeled with PV (cyan) and calbindin (red). ML was divided into four strata to record the laminar position of labeled MLI soma. e Percent laminar distribution of AAV-labeled single MLIs. Representative images (b) and quantifications (e) are from at least three animals per injection time point. f Percent laminar distribution of the 79 mature single MLI reconstructions. g–i Canonical MLI morphologies. g Inverted fluorescent image of four labeled MLIs. The lower MLI (pink arrowhead) displays canonical BC morphology, including axonal basket terminals targeting the PC somas and axon initial segments (AIS; asterisks). The upper MLIs (teal arrowhead) have canonical SC morphologies. The faintly labeled MLI (blue arrowhead) has SC morphological characteristics but resides within the lower ML. h Reconstruction of canonical BC and, i SC from panel g with dendritic (orange) and axonal arbor traces (blue). j–l Representative images (top) and reconstructions (bottom) of MLIs showing mixtures of BC and SC characteristics. j MLI located in the middle ML with SC dendritic features and a long axon (arrow) with collaterals that reach the PC soma base or AIS (asterisks). k MLI in the lower ML with SC-like arbors. l MLI in the upper ML with a long axon (arrow) and two collaterals enveloping PC somas (asterisks). Images (g–l) and quantifications (f) are from 79 cells, N = 9 animals. Scale bars are 50 µm.