Global Connectivity and Function of Descending Spinal Input Revealed by 3D Microscopy and Retrograde Transduction

Abstract

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. This complexity presents a major challenge to interpreting functional outcomes from therapies that target supraspinal connectivity after injury or disease, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate that injection of AAV2-Retro to the cervical spinal cord of adult female mice results in highly efficient transduction of supraspinal populations throughout the brainstem, midbrain, and cortex. Some supraspinal populations, including corticospinal and rubrospinal neurons, were transduced with Ͼ90% efficiency, with robust transgene expression within 3 d of injection. In contrast, propriospinal and raphe spinal neurons showed much lower rates of retrograde transduction. Using tissue clearing and light-sheet microscopy we present detailed visualizations of descending axons tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors resulted in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde vectors were also highly efficient when injected after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs.The complexity of descending inputs to the spinal cord presents a major challenge in efforts deliver therapeutics to widespread supraspinal systems, and to interpret their functional effects. Here we demonstrate highly effective gene delivery to diverse supraspinal nuclei using a retrograde viral approach and combine it with tissue clearing and 3D microscopy to map the descending projectome from brain to spinal cord. These data highlight newly developed retrograde viruses as therapeutic and research tools, while offering new insights into supraspinal connectivity.

Rostro-caudal specificity of corticospinal tract projections in mice

“…caudal levels, but the absence of double-labeling with injections of retro-AAV/GFP at C5 and retro-AAV/Cre at lumbar levels indicates that there are few if any neurons that project to both levels. This supports conclusions from a previous study that used retro-AAVs with different fluorescent reporters (Wang et al 2018).…”

“…Our results reinforce previous studies (Wang et al 2018) documenting the power of retro-AAV, tissue clearing, and light sheet imaging for obtaining quantitative and complete identification of the components of the CST. Also, our data on the normal projection patterns of CST axons from different parts of the cortex to different levels and laminae of the spinal cord provide novel insights into topographic specificity of normal CST projections, which will aid in identifying differences in projection patterns in mice carrying mutations, in detecting changes in patterns of projection due to regenerative growth following injury (Hollis et al 2016) and form part of the basis for comparisons of CST projection patterns between strains and species.…”

“…Second, whereas FG might be taken up by axons that Note the overlap with the rostral and caudal forelimb areas (RFA and CFA, respectively). J was created using a base drawing provided by Dr. T. Jones, which is a modified version of Figure 2B in Tennant et al, 2010. pass through an injection site (Dado et al 1990), it has been reported that there is minimal uptake of retro-AAV by CST axons passing through the area of a spinal cord injection (Wang et al 2018). Third, labeling produced by retrograde expression of fluorescent proteins enables imaging by light sheet microscopy, allowing global visualization of the entire population of cortical neurons that project to the spinal cord (Wang et al 2018).…”

Abnormal Pyramidal Decussation and Bilateral Projection of the Corticospinal Tract Axons in Mice Lacking the Heparan Sulfate Endosulfatases, Sulf1 and Sulf2

“…It was apparent that the CST fibers entered the contralateral dorsal funiculus ( Figure 4B). In addition to the main tract, collateral branches and their fiber terminals were observed in the striatum, thalamus, superior colliculus, pons, and dorsal column nuclei (Figure 4A; Catsman-Berrevoets and Kuypers, 1981;O'Leary et al, 1990;Lévesque et al, 1996;Wang et al, 2018). In the DKO mice, the most prominent abnormality was the U-shaped trajectory in the midbrain (n = 21/21; Figures 4C,E; arrows).…”

“…In rodents, the CST fibers pass through the internal capsule and cerebral peduncle, extend onto the ventral medulla, cross the midline at the pyramidal decussation dorsally, and further descend in the dorsal funiculus of the spinal cord contralaterally. Before reaching the spinal cord, they send collateral branches to the striatum, superior colliculus, red nucleus, pontine gray nucleus, and dorsal column nuclei (Wang et al, 2018). In the spinal cord, the CST fibers project to the dorsal and intermediate portions of the gray matter and innervate interneurons, which in turn control motor neurons (Lemon, 2008;Welniarz et al, 2017a).…”