Resolving the morphology of individual neurons in densely packed brain regions remains challenging. Sparse labeling is essential for delineating cellular architecture, yet achieving reproducible low-density labeling e.g. < 1% has been a persistent technical hurdle/problem. We present a genetically encoded two-step strategy, GT-SPARCL (Genetic Two-Step Sparse Cre/FLPe Combination Labeling), which leverages two independent stochastic recombination events to deliver stable, tunable low-density labeling in mice. As a proof of concept, we applied this method to visualizing arginine vasopressin (AVP) neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker composed of ~10,000 neurons per side in mice. Using two-photon imaging of whole-mount SCN blocks, we reconstructed individual AVP neurons and uncovered previously under-resolved morphological heterogeneity. Based on axonal trajectories, we distinguished five structural types, including subclasses with commissural projections to the contralateral SCN and others extending projections beyond the nucleus. The majority (~70%) exhibited projections both within and outside the ipsilateral SCN, whereas the second most dominant subset (~20%) projected exclusively outside the SCN, representing an output-biased type. In contrast, neurons projecting exclusively within the ipsilateral SCN were exceptionally rare, suggesting that 'dedicated' local-circuit AVP neurons do not form a major structural subtype. Collectively, our data indicate that AVP neurons are not structurally uniform but instead comprise diverse projection-defined subtypes, implying subtype-dependent contributions to intra-SCN communication, bilateral coupling, and circadian output. Beyond the SCN, our GT-SPARCL method may be applicable for achieving low-density labelling of neurons that can be defined by other specific Cre mouse lines.
Hsiao, S.-W., Yamaguchi, Y., Moriyasu, D., Doi, S., Zhou, H., Mizuno, S., Takahashi, S., Yukinaga, H., Macpherson, T., Hasegawa, E., Doi, M.
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