Fig. 5: 3D reconstruction of molecular layer interneurons and granule cells distinguished by PV-specific scFv probes.

a Representative 2D CLEM image (n = 22) showing fluorescence signals (magenta) of the parvalbumin-specific scFv probe overlapping a molecular layer interneuron (MLI) cell body. b EM image showing 2D segmentation (magenta) of the MLI in (a) (n = 1). c 3D reconstruction of MLIs (MLI a, MLI b, MLI c) (n = 3) relative to the pia and Purkinje cell layer, with axons in yellow. MLI a, farthest from the Purkinje cell layer, has an axon branching extensively and innervating a Purkinje cell’s dendritic shaft at arrowhead (dendritic shaft not shown; the synapse is shown in d and e) and forming part of the pinceau structure around another Purkinje cell’s axon initial segment at asterisk (axon initial segment not shown; the pinceau is shown in Supplementary Fig. 27e). This suggests MLI a is a deep axon stellate cell. d EM image showing 2D segmentation of the synapse (arrowhead) between MLI a’s axon (yellow) and a Purkinje cell dendrite (green) (n = 1). e 3D reconstruction of the synapse in (d) (n = 1). f Representative 2D CLEM image showing fluorescence signal (magenta) of the PV-specific scFv probe overlapping Purkinje cell dendrites (magenta asterisk) but not labeling a molecular layer granule cell (MGC) (blue plus sign) (n = 7). g EM image showing 2D segmentation (dark blue) of the MGC cell body in (f) (n = 1). h 3D reconstructions of MGCs (MGC a, MGC b, MGC c) (n = 3) relative to three Purkinje cells. Unlike granule cells in the granule cell layer, these MGCs received synapses from parallel fibers. Three such synapses between parallel fibers (red) and the MGCs are labeled. i EM image showing 2D segmentation of synapse 1 in (h) (n = 1). j 3D reconstruction of synapse 1 (n = 1) labeled by the arrowhead in (i). n indicates an example.