Morphological pseudotime ordering and fate mapping reveals diversification of cerebellar inhibitory interneurons

Abstract

Understanding how diverse neurons are assembled into circuits requires a framework for describing cell types and their developmental trajectories. Here, we combined genetic fate mapping and pseudo-temporal profiling to resolve the diversification of cerebellar inhibitory interneurons based on morphology. The molecular layer interneurons (MLIs) derive from a common progenitor but comprise a diverse population of dendritic-, somatic-, and axon initial segment-targeting interneurons. MLIs are classically divided into two types. However, their morphological heterogeneity suggests an alternate model of one continuously varying population. Through clustering and trajectory inference of 811 MLI reconstructions at maturity and during development, we show that MLIs divide into two discrete classes but also present significant within-class heterogeneity. Pseudotime trajectory mapping uncovered the emergence of distinct phenotypes during migration and axonogenesis, well before neurons reach their final positions. Our study illustrates the utility of quantitative single-cell methods to morphology for defining the diversification of neuronal subtypes. Figure 2. Morphological heterogeneity among MLIs. (A) Left: Immunostaining of MLIs (Parvalbumin, cyan) and PCs (co-labeled by Parvalbumin and Calbindin, cyan and red, respectively) show the ML space in which MLIs reside. PC somata are enveloped by BC axons (white arrowhead). Right: Inverted image of four fluorescently-labelled MLIs. The lower MLI (pink arrowhead) has canonical BC morphologies, including axonal basket terminals around the PC somata (asterisks). The two upper MLIs (teal arrowhead) have canonical SC morphologies and reside within the upper ML. The faintly labelled MLI (blue arrowhead) has SC morphologies but reside within the lower ML. (B) Reconstruction of canonical BC from panel A, with complete dendritic arbor traced in orange, and complete axonal arbor traced in blue. (C) Reconstruction of canonical SC from panel A (cell on far right). (D-F) Representative images of MLIs with mixtures of BC and SC characteristics. Top: inverted fluorescence image; Bottom: reconstruction with dendritic (orange) and axonal (blue) traces. (D) MLI located in the middle ML with SC dendritic features, and a long horizontal axon (arrow) with descending basket collaterals (asterisks). (E) MLI located in the lower ML with SC-like dendritic and axonal arbors. (F) MLI located in the upper ML with a long horizontal axon (arrow) and two descending axon collaterals with basket formations around PC somas (asterisks). All scale bars are 50 μm.

A rapid and efficient learning rule for biological neural circuits

“…S5A). Given the axonal spread of MLIs [84,85], the analysis was confined to single interneurons that were located within 150 um (rostrocaudally) and 50 um (mediolaterally) from a given Purkinje cell dendrite. The extent of suppression varied between PC dendrites and also within individual PC dendrites recorded at different depths (Fig.…”

“…After identifying these states, we compared the magnitudes of isolated dendritic events in these two conditions for Purkinje cells within a 300 × 100 μm ellipse centered on each MLI whose major axis is parallel to that of Purkinje cell dendrites in the field of view (approximately rostrocaudal). Ellipse dimensions were chosen to approximate the known rostro-caudal and mediolateral spread of MLI axons [84, 85]. Only isolated events in Purkinje cell dendrites (defined as those that occurred more than 500 ms before and after any other events) were analyzed, and fluorescence event magnitudes were calculated over the 5 frames (~500 ms) after each event for initial identification of MLI-modulated dendrites.…”