Jose Ernesto Canton-Josh - US grants

SUMMARY The cerebellum is characterized by stereotyped cytoarchitecture across all of its lobules. However, recent evidence has revealed new complexity in these circuits, including variation in cells types, connectivity patterns, and receptor expression. Within the vestibulocerebellum, there is an excitatory glutamatergic interneuron cell type known as the unipolar brush cell (UBC). UBCs are heavily enriched within regions of the cerebellum controlling eye movements and vestibular processing. UBCs receive input from mossy fibers entering the cerebellum and form a local recurrent feed- forward excitatory network. This connectivity suggests that UBCs may perform large scale transformations on inputs to the cerebellum. Several distinct functions for these cells within the cerebellum have been proposed, but the resulting hypotheses have been difficult to test without selective genetic tools for targeting UBCs. We have found a transgenic mouse line that selectively labels the most common subtype of UBCs. In addition, our preliminary data show that vestibulocerebellar Purkinje cells make functional inhibitory synapses onto UBCs. The goals of this project are to examine how UBCs modulate information flow to the cerebellum, using a combination of optogenetic, electrophysiology, and imaging techniques and to characterize a novel feedback circuit from Purkinje cells to UBCs. Aim 1 is to characterize the responses of Purkinje cells to optogenetic activation of UBCs. This will be accomplished by using light-gated opsin ChR2 to activate UBCs directly and measuring either the firing responses or synaptic currents in Purkinje cells. We will also use inhibitory opsins to transiently inactivate UBCs and examine how Purkinje cells respond to mossy fiber input in the presence or absence of UBC activity. Aim 2 will examine new synaptic connections between Purkinje cells and UBCs, using promoter-restricted specific optogenetic activation of Purkinje cells with recordings from genetically identified UBCs. Using stimulation of mossy fiber inputs, we will record the spatiotemporal dynamics of UBC activity. This will be done with both cell-attached recordings and rapid 3D light-sheet imaging of calcium dynamics to image activity in dozens of UBCs simultaneously. To measure how circuit level Purkinje cell inhibition of the granular layer neurons alters UBC activity, in interleaved trials, we will combine mossy fiber activation with optogenetically evoked Purkinje cell firing. Carrying out these experiments will build a conceptual framework to explain how UBCs operate within feedback circuits to regulate cerebellar output, relevant to motor control in physiological and pathological conditions.