Figure 4.

Germ-free mice display aberrant synaptic density and synaptic function that is normalized by bifidobacteria colonization (a) Simplified diagram of the cerebellar circuit over postnatal development and corresponding phases of synaptic refinement. (b) Inset illustrates interaction between two cerebellar cell types: Purkinje cells (PC; main functional output of cerebellum) and climbing fibers (CF). CF synapses on PCs are initially supernumerary, but eventually reach a 1 CF: 1 PC ratio with 1 CF terminals synapsing on multiple locations of PC dendrites. The circuit diagram also shows simplified signaling input and output between the cell types in this model system. (c). Micrographs (40X magnification) of molecular layer of cerebellum in lobule II of brain slices from P20 male mice in each group stained with anti-VGLUT2 antibody. Puncta of the pre-synaptic marker VGLUT2 were counted in a 400 micron field. Inset demonstrates puncta analysis in ImageJ. The density of the pre-synaptic marker VGLUT2 per field is quantified in the right panel as VGLUT⁺ puncta per mm² *p<0.05, **p<0.01, One-way ANOVA (3 fields per section with 3 sections per animal, n = 3 animals per group) (d) Schematic of in vivo electrophysiological recording of Purkinje cells in the cerebellum, and representative extracellular single-unit recording traces from male germ-free and bifidobacteria-colonized mice. Asterisk indicates low-frequency complex spikes that are triggered by climbing fiber input, which identifies the cell as a Purkinje cell. The instantaneous firing rate is indicated by the pink line above the raw traces. (Figure 1). Firing characteristics of Purkinje cells quantified from in vivo electrophysiological recordings of GF and BIF anesthetized P20 male mice (n = 3-5 animals/group with 3 separate recordings per animal).