Unsupervised classification of brain-wide axons reveals the presubiculum neuronal projection blueprint.

We present a quantitative strategy to identify all projection neuron types from a given region with statistically different patterns of anatomical targeting. We first validate the technique with mouse primary motor cortex layer 6 data, yielding two clusters consistent with cortico-thalamic and intra-telencephalic neurons. We next analyze the presubiculum, a less-explored region, identifying five classes of projecting neurons with unique patterns of divergence, convergence, and specificity. We report several findings: individual classes target multiple subregions along defined functions; all hypothalamic regions are exclusively targeted by the same class also invading midbrain and agranular retrosplenial cortex; Cornu Ammonis receives input from a single class of presubicular axons also projecting to granular retrosplenial cortex; path distances from the presubiculum to the same targets differ significantly between classes, as do the path distances to distinct targets within most classes; the identified classes have highly non-uniform abundances; and presubicular somata are topographically segregated among classes. This study thus demonstrates that statistically distinct projections shed light on the functional organization of their circuit.

Unsupervised classification of brain-wide axons reveals neuronal projection blueprint.

Long-range axonal projections are quintessential determinants of network connectivity, linking cellular organization and circuit architecture. Here we introduce a quantitative strategy to identify, from a given source region, all "projection neuron types" with statistically different patterns of anatomical targeting. We first validate the proposed technique with well-characterized data from layer 6 of the mouse primary motor cortex. The results yield two clusters, consistent with previously discovered cortico-thalamic and intra-telencephalic neuron classes. We next analyze neurons from the presubiculum, a less-explored region. Extending sparse knowledge from earlier retrograde tracing studies, we identify five classes of presubicular projecting neurons, revealing unique patterns of divergence, convergence, and specificity. We thus report several findings: (1) individual classes target multiple subregions along defined functions, such as spatial representation vs. sensory integration and visual vs. auditory input; (2) all hypothalamic regions are exclusively targeted by the same class also invading midbrain, a sharp subset of thalamic nuclei, and agranular retrosplenial cortex; (3) Cornu Ammonis, in contrast, receives input from the same presubicular axons projecting to granular retrosplenial cortex, also the purview of a single class; (4) path distances from the presubiculum to the same targets differ significantly between classes, as do the path distances to distinct targets within most classes, suggesting fine temporal coordination in activating distant areas; (5) the identified classes have highly non-uniform abundances, with substantially more neurons projecting to midbrain and hypothalamus than to medial and lateral entorhinal cortex; (6) lastly, presubicular soma locations are segregated among classes, indicating topographic organization of projections. This study thus demonstrates that classifying neurons based on statistically distinct axonal projection patterns sheds light on the functional organizational of their circuit.