The entorhinal cortex (EC) plays a pivotal role in memory function and spatial navigation, connecting the hippocampus with the neocortex. The EC integrates a wide range of cortical and subcortical inputs, but its synaptic organization in the human brain is largely unknown. We used volume electron microscopy to perform a 3D analysis of the microanatomical features of synapses in all layers of the medial EC (MEC) from the human brain. Using this technology, 12,974 synapses were fully 3D reconstructed at the ultrastructural level. The MEC presented a distinct set of synaptic features, differentiating this region from other human cortical areas. Furthermore, ultrastructural synaptic characteristics within the MEC was predominantly similar, although layers I and VI exhibited several synaptic characteristics that were distinct from other layers. The present study constitutes an extensive description of the synaptic characteristics of the neuropil of all layers of the EC, a crucial step to better understand the connectivity of this cortical region, in both health and disease.

Purkinje cells (PCs) primarily project to cerebellar nuclei but also directly innervate the brainstem. Some PC-brainstem projections have been described previously, but most have not been thoroughly characterized. Here, we use a PC-specific cre line to anatomically and electrophysiologically characterize PC projections to the brainstem. PC synapses are surprisingly widespread, with the highest densities found in the vestibular and parabrachial nuclei. However, there are pronounced regional differences in synaptic densities within both the vestibular and parabrachial nuclei. Large optogenetically evoked PC-IPSCs are preferentially observed in subregions with the highest densities of putative PC boutons, suggesting that PCs selectively influence these areas and the behaviors they regulate. Unexpectedly, the pontine central gray and nearby subnuclei also contained a low density of putative PC boutons, and large PC-IPSCs are observed in a small fraction of cells. We combined electrophysiological recordings with immunohistochemistry to assess the molecular identities of two potential PC targets: PC synapses onto mesencephalic trigeminal neurons were not observed even though these cells are in close proximity to PC boutons; PC synapses onto locus coeruleus neurons are exceedingly rare or absent, even though previous studies concluded that PCs are a major input to these neurons. The availability of a highly selective cre line for PCs allowed us to study functional synapses, while avoiding complications that can accompany the use of viral approaches. We conclude that PCs directly innervate numerous brainstem nuclei, and in many nuclei they strongly inhibit a small fraction of cells. This suggests that PCs selectively target cell types with specific behavioral roles in the brainstem.