TOPOGRAPHY AND TOPOLOGY IN A CENTRAL SENSORY NEURONAL PROJECTION

Precise topographic long‑range projection patterns are a hallmark of vertebrate sensory circuits. In-depth investigation of key circuit properties underlying this topography requires dense synapse-scale mapping of these projections. We used the medial nucleus of the trapezoid body (MNTB), an auditory brainstem relay receiving monosynaptic inputs from the cochlear nucleus (CN), as a model to uncover how topographic and topological constraints shape its wiring. Using multicolor Brainbow AAV vectors we generated stochastic, spectrally distinct tags in individual CN axons. Large‑volume two‑photon wavelength‑mixing microscopy allowed continuous imaging of the entire MNTB at near‑micrometric resolution. Manual annotation yielded >1500 retrogradely traced axons from their synaptic contacts, enabling quantitative geometrical analyses of axonal trajectories. This identified the stereotyped inflection point where axons abruptly transition from a coordinated course in the trapezoid tract to independent paths within the nucleus. This position covaries with the target neuron mediolateral (ML) tonotopic axis and anteroposterior (AP) axis, evidencing in addition to the well-studied one-dimensional tonotopy, a bidimensional topographic organization of the MNTB pre‑figured by axon pre‑ordering along its AP axis. A subpopulation of principal cells receives convergent inputs persisting into adulthood, indicating tolerated violation of the strict 1:1 pairing rule. Computational modeling of CN‑MNTB pairing predicts those instances of convergence by a wiring scheme balancing opposing topographic and topological constraints. Our pipeline provides a scalable framework for connectomic interrogation of long‑range sensory pathways toward studying conserved developmental mechanisms underlying bidimensional topography.