Figures and data
Anatomical characterization of Purkinje cell outputs to the brainstem using PC/synaptophysin-tdTomato mice.
Aa. First column. Diagram showing parasagittal slice location.
Second column. Low magnification views of tdTomato (tdT) fluorescence (red) in the cerebellar cortex and brainstem.
Third column. Medium magnification views of the regions indicated in Ab.
Fourth column. Anatomical regions corresponding to the preceding column.
Last column. i-iv. High magnification views of the regions indicated in the preceding column. Ab-d. As in Aa, but for subsequent parasagittal slices.
B. As in A, but for coronal slices.
Quantification of Purkinje cell synapses in the brainstem.
GABAergic boutons were detected using a vGAT antibody and PC presynaptic boutons were detected based on tdT-synaptophysin labelling in PC/synaptophysin-tdTomato mice.
A. (upper left) Confocal image of td-Tomato fluorescence.
(upper right) GFP fluorescence in CGRP-GFP mouse in which the locus coeruleus and facial nerve are labelled. (lower left) vGAT labelling of GABAergic boutons.
(lower right) Allen Atlas coronal section corresponding to confocal images with landmarks used for registration highlighted in cyan.
B. (upper left) vGAT labelling in a single confocal section. (upper right) tdT labelling in a single confocal section.
(lower left) z-stack of vGAT and tdT labelling in 5 sections (1.5 μm thick).
(lower right) Detected GABAergic boutons based on vGAT labelling (grey) and PC boutons (red).
C. (left) Detected GABAergic boutons corresponding to PCs (red), and neurons other than PCs (grey) from six example coronal sections.
(right) Labelled anatomic regions for each coronal section.
D. Number of PC synapses detected per section in each region. Individual section values are represented with symbol colors correspond to the sections in C, right (bottom right corner color bars). Violin plot of the region shown in grey with average and quartile values in black.
E. Density of PC boutons in each region. Individual section and region average as represented in D.
F. Percentages of GABAergic boutons that correspond to PCs for each region. Individual section and region average as represented in D.
Characterization of functional properties of PC synapses in the brainstem.
Light-evoked PC-IPSCs were measured in the brainstem of PC/ChR2-YFP mice.
A. (left) Averaged PC synapse density from previous quantification of brainstem regions (red, from Figure 2). Synapses were binned in voxels of 144×144×4 µm. The locations of all recorded neurons are shown with the symbols coded for the light-evoked IPSC amplitudes (n=310)
A. (right) Labeled anatomical regions are indicated and the position of each slice is color coded, as in Figure 2.
B. Evoked amplitudes of the responding neurons (n=113) in each region are shown. Symbol colors correspond to the sections in A.
C. Fraction of responding cells in each brainstem region and total number of neurons recorded (n=300). Bar graph colors correspond to the sections in A.
D. (left) Average current in each region for all cells. (right) Average current of responding cells in each region.
E. (left) A violin plot of the average PC synapses densities for neurons where a light-evoked PC-IPSC was detected (responding) and for cells were such a response was not observed (nonresponding).
(right) The probability of observing a light-evoked PC-IPSC in neurons is plotted as a function of the density of PC synaptic boutons in that voxel.
Minimal PC inputs to locus coeruleus and mesencephalic trigeminal neurons
A. Example confocal image of PC presynaptic boutons labeled by synaptophysin-tdT showing cases where labeling was near locus coeruleus and the mesencephalic trigeminal nuclei.
B. (left) Biocytin was included in the pipette to label MEV cells during whole-cell recordings. (middle) MEV cells visualized with a PV antibody.
(right) Colocalization of biocytin and PV confirms that patched neurons were PV+ MEV neurons.
C. Locations of MEV cells in two coronal slices and one horizontal slice. No PC-IPSC responses were detected in any of the cells (n=23) at any location.
D. As in B, but for LC cells labelled with a TH antibody
E. As in C, but for two coronal levels of the LC (n=67).
F. Summary of the amplitudes of PC IPSCs recorded in LC cells.
G. Average PC-LC neuron IPSCs for all cells (n=65 left), and for cells where a synaptic response was detected (n=2, right).
