J Neurosci. 2026 Jan 19. pii: e1384252026. [Epub ahead of print]
In addition to providing outputs from the cerebellar cortex, Purkinje cell (PC) axon collaterals target other PCs, molecular layer interneurons (MLIs), and Purkinje layer interneurons (PLIs). It was assumed that PC collateral to MLI synapses provide positive feedback to PCs via the PC-MLI-PC pathway, because it was thought that MLIs primarily inhibit PCs. However, it was recently shown that MLIs consist of two subtypes: MLI1s primarily inhibit PCs, whereas MLI2s mainly inhibit MLI1s and disinhibit PCs. Clarifying PC connectivity onto these MLI subtypes is vital to understanding the influence of feedback from PC collaterals. Here we use a combination of serial EM and optogenetic studies to characterize PC synapses onto MLI subtypes in mice of either sex. EM reconstructions show that PCs make 53% of their synapses onto other PCs, 32% onto PLIs, 6% onto MLI1s and 7% onto MLI2s. Since there are far more MLI1s than MLI2s, each MLI2 is expected to receive many more synapses than each MLI1. In slice experiments, optogenetic activation of PCs evokes inhibitory currents in most MLI2s, but primarily disinhibits MLI1s. We also find that candelabrum cells, a type of PLI, form many more synapses onto MLI1s than MLI2s. These findings suggest that PC-MLI synapses do not primarily disinhibit PCs, and that the PC-MLI2-MLI1-PC and PC-PLI-MLI1-PC pathways might provide negative feedback to PCs that acts in concert with PC-PC synapses to counter elevations in PC firing.Significance Statement Purkinje cells (PCs) influence processing by inhibiting neurons in the cerebellar cortex, including other PCs, molecular layer interneurons (MLIs) and Purkinje layer interneurons (PLIs). The influence of PC-MLI synapses is not known because there are recently identified MLI subtypes with opposing effects: MLI1s inhibit PCs whereas MLI2s inhibit MLI1s and disinhibit PCs. We used serial EM and optogenetic studies to characterize PC synapses onto MLI subtypes and found that PCs preferentially inhibit MLI2s and disinhibit MLI1s. We also found that candelabrum cells (a type of PLI) preferentially inhibit MLI1s. These findings suggest that PC-PC synapses, the PC-MLI2-MLI1-PC pathway and the PC-candelabrum cell-MLI1-PC pathway act together to allow alterations in PC firing to provide negative feedback to other PCs.