Circuit Specializations of Cerebellar Molecular Layer Interneurons - Project Summary The cerebellum controls behaviors that depend on prediction, including motor learning, eye movements, balance, and cognitive-affective functions. The cerebellar cortex transforms mossy fiber (MF) inputs into Purkinje cell (PC) outputs. It is vital to delineate the elements of this circuit and their connectivity to relate circuit properties to function and disease. To improve our understanding of transformations in the cerebellar cortex, this proposal seeks to delineate the circuit properties of the most abundant type of interneuron in the cerebellum, the molecular layer interneuron (MLI). MLIs inhibit PCs and other MLIs to control the output of the cerebellar cortex. However, single nucleus RNA sequencing recently identified two molecularly distinct types of MLIs, MLI type 1 (MLI1) and MLI type 2 (MLI2). Intriguingly, MLI1s express connexin 36, whereas MLI2s do not. This suggests that MLI1s might be gap junction coupled with each other, which could promote synchronous firing and make MLI1s well-suited to controlling the timing of PC outputs. The circuit properties of MLI1 and MLI2 are not known. Here I will clarify MLI1 and MLI2 connectivity using slice electrophysiology and serial electron microscopy (EM) reconstructions in mice. The first aim of this proposal is to determine the outputs of MLI1 and MLI2 using paired whole-cell recordings in brain slice. First, I will characterize MLI1 and MLI2 synaptic connections onto PCs. I will also characterize the contributions of MLI1 and MLI2 to ephaptic inhibition, which arises from large extracellular signals near MLI specializations known as pinceaux that surround PC axon initial segments. Lastly, I will characterize synaptic connections and electrical coupling between MLI1 and MLI2. The second aim of this proposal is to characterize the ultrastructural circuit specializations of MLI1 and MLI2 and generate a comprehensive map of MLI1 and MLI2 synaptic and ephaptic connectivity using large-scale EM reconstructions. The complementary approaches of electrophysiological characterization and EM reconstructions will provide a complete picture of the functional properties and the anatomical connectivity. Preliminary findings suggest that MLI1 and MLI2 target different cells, with MLI1s mainly inhibiting PCs, whereas MLI2s primarily inhibit other MLIs. Based on these preliminary studies, my working model is that MLI1s are well suited to controlling the timing of PC outputs, whereas MLI2s promote PC excitability by disinhibiting PCs. Completion of the proposed work will define the properties of these circuit elements, and if preliminary results are confirmed, it will be necessary to revise the circuit diagram of the cerebellar cortex. These experiments will also lead to future studies that will determine the in vivo firing properties of MLI1 and MLI2 and determine how MLI1 and MLI2 contribute to processing, learning and behavior. These studies promise to shed light on mechanisms of cerebellar computations that are relevant to neurological disorders and neuropsychiatric diseases.
