Blotnote

Sequencing of oligonucleotide barcodes holds promise as a high-throughput approach for reconstructing synaptic connectivity at scale. Rabies viruses can act as a vehicle for barcode transmission, thanks to their ability to spread between synaptically connected cells. However, applying barcoded rabies viruses to map synaptic connections in vivo has proved challenging. Here, we develop Barcoded Rabies In Situ Connectomics (BRISC) for high-throughput connectivity mapping in the mouse brain. To ensure that the majority of post-synaptic "starter" neurons are uniquely labeled with distinct barcode sequences, we first generated libraries of rabies viruses with sufficient diversity to label >1000 neurons uniquely. To minimize the probability of barcode transmission between starter neurons, we developed a strategy to tightly control their density. We then applied BRISC to map inputs of single neurons in the primary visual cortex (V1). Using in situ sequencing, we read out the expression of viral barcodes in rabies-infected neurons, while preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections. The resulting connectivity matrix revealed layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs to V1. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons while preserving spatial information, enabling reconstruction of neural circuits at an unprecedented scale.

Sensory processing involves information flow between neocortical areas, assumed to rely on direct intracortical projections. However, cortical areas may also communicate indirectly via higher-order nuclei in the thalamus, such as the pulvinar or lateral posterior nucleus (LP) in the visual system of rodents. The fine-scale organization and function of these cortico-thalamo-cortical pathways remains unclear. We find that responses of mouse LP neurons projecting to higher visual areas likely derive from feedforward input from primary visual cortex (V1) combined with information from many cortical and subcortical areas, including superior colliculus. Signals from LP projections to different higher visual areas are tuned to specific features of visual stimuli and their locomotor context, distinct from the signals carried by direct intracortical projections from V1. Thus, visual transthalamic pathways are functionally specific to their cortical target, different from feedforward cortical pathways, and combine information from multiple brain regions, linking sensory signals with behavioral context.

Excitatory synaptic activity in the brain is shaped and balanced by inhibition. Because inhibition cannot propagate, it is often recruited with a synaptic delay by incoming excitation. Cerebellar Purkinje cells are driven by long-range excitatory parallel fiber inputs, which also recruit local inhibitory basket cells. The axon initial segment of each Purkinje cell is ensheathed by basket cell axons in a structure called the pinceau, which is largely devoid of chemical synapses. In mice, we found at the single-cell level that the pinceau mediates ephaptic inhibition of Purkinje cell firing at the site of spike initiation. The reduction of firing rate was synchronous with the presynaptic action potential, eliminating a synaptic delay and allowing granule cells to inhibit Purkinje cells without a preceding phase of excitation. Axon-axon ephaptic intercellular signaling can therefore mediate near-instantaneous feedforward and lateral inhibition.