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. In efforts to restore supraspinal connectivity after injury or disease, this complexity presents a major challenge to interpreting functional outcomes, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate highly efficient retrograde transduction by AAV2-Retro in adult female mice, and employ tissue clearing to visualize descending tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors results in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde efficacy persists even 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. SIGNIFICANCE STATEMENTThe 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.

Motor cortical output for skilled forelimb movement is selectively distributed across projection neuron classes

“…This result is in contrast to the suggestion that for cortically-dependent movements motor commands are conveyed primarily via PT neurons 5,16 . However, our observations are consistent with key roles for subcortical structures such as basal ganglia in the regulation of movement vigor 39,59 , critical functions for extrapyramidal motor pathways in regulating movement execution 60 , and the notion that corticospinal projections are primarily recruited for fine control of dexterity 2,61 rather than gross movement 59 . Our data together with previous work provide evidence that the relative recruitment of IT and PT neuron types is task specific and reflects a flexible routing of motor cortical output through downstream effectors 17 .…”

In-depth characterization of layer 5 output neurons of the primary somatosensory cortex innervating the mouse dorsal spinal cord

“…When analysing the output of S1-CST neurons, we found evidence that, although they mainly project through the CST onto the spinal dorsal horn, they also send collateral branches to the dorsal striatum and the midbrain. This confirms and further specifies previous experiments in which CST collaterals were observed when all spinally projecting neurons were labelled Wang Z et al 2018). Our data therefore indicate that while their main target is the spinal dorsal horn, S1-CST neurons also contribute to cortico-striatal projections and may thereby influence goal-directed behaviours .…”

“…To this end, it would be crucial to restrict transgene expression to the layer 5 pyramidal neurons in an area of the cortex (S1 in this case) that projects to the spinal region of interest such as the dorsal horn of the spinal cord. Recently, a new recombinant adeno-associated virus (rAAV) serotype (rAAV2-retro) (Tervo DG et al 2016) has been developed with greatly improved retrograde labelling efficiency that allows high fidelity tracing of descending inputs to the spinal cord (Haenraets K et al 2017;Wang Z et al 2018).…”

Restoration of Direct Corticospinal Communication Across Sites of Spinal Injury

“…Finally, a key finding was that delivery of KLF6 by retrograde vectors, but not direct cranial injection, improved forelimb function after spinal injury. We recently characterized retrograde transduction after spinal injections of AAV2-Retro, and showed widespread transduction of CST neurons throughout the cortex, in addition to subcortical nuclei that participate in skilled movements, including the red nucleus and reticular formation (29). The likely explanation for the difference in behavioral outcomes is that compared to focal delivery to subregions of motor cortex, retrograde vectors deliver therapeutic transgenes to a larger number and greater diversity of supraspinal cell types.…”

“…To deliver KLF6 more broadly we employed Retro-AAV, a newly developed vector with enhanced retrograde properties. We recently showed that delivery of Retro-AAV to the cervical spinal cord produces highly efficient transduction of CST neurons throughout the cortex, in addition to subcortical populations including the rubrospinal tract (29). To confirm the ability of Retro-AAV to drive KLF6 expression we co-injected Retro-AAV2-KLF6 and Retro-AAV2-tdTomato to cervical spinal cord.…”