SPINAL V1 INHIBITORY INTERNEURON CLADES DIFFER IN BIRTHDATE, PROJECTIONS TO MOTONEURONS AND HETEROGENEITY

Abstract

ABSTRACTSpinal cord interneurons play a crucial role in shaping motor output, but their precise identity and circuit connectivity remain unclear. Focusing on the cardinal class of inhibitory V1 interneurons, we define the diversity of four major V1 subsets according to timing of neurogenesis, genetic lineage-tracing, synaptic output to motoneurons, and synaptic inputs from muscle afferents. Birthdating delineates two early-born (Renshaw and Pou6f2) and two late-born V1 clades (Foxp2 and Sp8) suggesting sequential neurogenesis gives rise to different V1 clades. Neurogenesis did not correlate with motoneuron targeting. Early-born Renshaw cells and late-born Foxp2-V1 interneurons both tightly coupled to motoneurons, while early-born Pou6f2-V1 and late-born Sp8-V1 interneurons did not. V1-clades also greatly differ in cell numbers and diversity. Lineage labeling of the Foxp2-V1 clade shows it contains over half of all V1 interneurons and provides the largest inhibitory input to motoneuron cell bodies. Foxp2-V1 subgroups differ in neurogenesis and proprioceptive input. Notably, one subgroup defined by Otp expression and located adjacent to the lateral motor column exhibits substantial input from proprioceptors, consistent with some Foxp2-V1 cells at this location forming part of reciprocal inhibitory pathways. This was confirmed with viral tracing methods for ankle flexors and extensors. The results validate the previous V1 clade classification as representing unique interneuron subtypes that differ in circuit placement with Foxp2-V1s forming the more complex subgroup. We discuss how V1 organizational diversity enables understanding of their roles in motor control, with implications for the ontogenetic and phylogenetic origins of their diversity.SIGNIFICANCE STATEMENTSpinal interneuron diversity and circuit organization represents a key challenge to understand the neural control of movement in normal adults and also during motor development and in disease. Inhibitory interneurons are a core element of these spinal circuits, acting on motoneurons either directly or via premotor networks. V1 interneurons comprise the largest group of inhibitory interneurons in the ventral horn and their organization remains unclear. Here we present a comprehensive examination of V1 subtypes according to neurogenesis, placement in spinal motor circuits and motoneuron synaptic targeting. V1 diversity increases during evolution from axial-swimming fishes to limb-based mammalian terrestrial locomotion and this is reflected in the size and heterogeneity of the Foxp2-V1 clade which is closely associated to limb motor pools. We show Foxp2-V1 interneurons establish the densest and more direct inhibitory synaptic input to motoneurons, especially on cell bodies. This is of further importance because deficits on motoneuron cell body inhibitory V1 synapses and on Foxp2-V1 interneurons themselves have recently been shown to be affected at early stages of pathology in motor neurodegenerative diseases like amyotrophic lateral sclerosis.