Global Connectivity and Function of Descending Spinal Input Revealed by 3D Microscopy and Retrograde Transduction - PubMed (original) (raw)

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

Zimei Wang et al. J Neurosci. 2018.

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 >90% 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.SIGNIFICANCE STATEMENT 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.

Keywords: DREADD; corticospinal; light-sheet microscopy; motor control; retrograde gene therapy; supraspinal centers.

Copyright © 2018 the authors 0270-6474/18/3810566-16$15.00/0.

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Figures

Figure 1.

Figure 1.

AAV2-Retro shows limited retrograde transduction of cervical propriospinal neurons. A, Mixed AAV2-Retro-tdTomato and CTB-488 were injected bilaterally to C4/5 gray matter, and transverse spinal sections at the injury site and 1 or 2 mm rostral were examined 3, 7, or 14 d later. B–D, Transverse sections at the level of spinal injection show readily detectable CTB-488 and tdTomato. E–G, In cervical spinal cord 2 mm rostral to the injection, CTB-488 retrogradely labels cell bodies that project axons to the injection site (arrows). H–M, tdTomato signal is rarely detectable in CTB-488+ cell bodies, indicating low transduction. N, Quantification of tdTomato detection in CTB-488+ cell bodies showed that <20% of propriospinal neurons projecting to C4/5 were transduced 1 mm rostral to the injury, and <10% at 2 mm. **_O_**, By 14 d postinjection, tdTomato signal is apparent in locations corresponding to ascending and descending axon tracts. _N_ > 100 individual cells from each of at least four animals at each time point. Error bars show SEM. Scale bars, 1 mm. Statistical comparisons of transduction efficiencies between supraspinal populations are provided in Figure 1-1.

Figure 2.

Figure 2.

AAV2-Retro effectively transduces brainstem-spinal projection neurons. A, Mixed AAV2-Retro-tdTomato and CTB-488 were injected bilaterally to C4/5 gray matter, and transverse sections of brainstem examined 3, 7, or 14 d later. In vestibulospinal (B–D), reticulospinal (E–G), and cerebellar-spinal (E–G) populations, spinally projecting neurons identified by CTB-488 also express tdTomato as early as 3 d postinjection. H, Quantification of the percentage of CTB-488+ neurons that are virally transduced shows a majority of vestibulospinal, reticulospinal, and cerebellar-spinal neurons are retrogradely labeled. N > 100 individual cells from each of at least four animals at each time point. No significant differences between groups existed (p > 0.05, two-way ANOVA with post hoc Tukey's). I–L, Two weeks after spinal injection of mixed CTB-488 and AAV2-Retro-tdTomato, transverse brainstem sections were immunostained for 5HT. Raphe-spinal neurons (arrows) were identified by CTB-488 (I) to confirm an axon projection to the C4/5 injection site, and expression of 5HT (K). These neurons were invariably negative for tdTomato, although nearby non-serotonergic neurons were effectively transduced. Scale bars: B–G, 1 mm; I–L, 500 μm. Error bars show SEM.

Figure 3.

Figure 3.

Effective retrograde transduction of rubrospinal neurons. A, Mixed CTB-488 and AAV2-Retro-tdTomato were injected to cervical spinal cord and transverse sections of midbrain prepared 3, 7, or 14 d later. B–J, The magnocellular population and (K–S) the parvocellular. T, A majority of CTB-488+ neurons in both parvo- and magnocellular nuclei were colabeled with tdTomato, indicating rapid and effective transduction. Groups did not differ significantly (p > 0.05, two-way ANOVA with post hoc Tukey's). N > 100 individual cells from each of at least four animals at each time point. Scale bars, 500 μm. Error bars show SEM.

Figure 4.

Figure 4.

Effective retrograde transduction of corticospinal tract neurons. A, B, Adult mice received cervical injection of mixed AAV2-Retro-tdTomato and CTB-488, and the percentage of double-labeled cells quantified in transverse sections of cortex 3, 7, or 14 d later. C–E, CTB-488 identifies CST neurons. F–H, tdTomato is readily detectable as early as 3 d postinjection). I–N, Overlays show the great majority of CTB-488+ cells also express tdTomato, indicating efficient retrograde transduction. Groups did not differ significantly (p > 0.05, ANOVA with post hoc Sidak's). N ≥ 4 animals per time point, >100 cells/section, 3 sections per animal. Scale bar, 2 mm. Error bars show SEM.

Figure 5.

Figure 5.

Effective retrograde transduction of corticospinal tract neurons after spinal injury. A, Adult female mice received unilateral cervical dorsal hemisection or sham injury, followed by bilateral injection of mixed Retro-AAV-tdTomato and CTB-488. B–E, In sagittal sections of spinal cord 28 d postinjection, GFAP immunohistochemistry (B) and the absence of CTB-488 (C) and tdTomato (D) signal caudal to the injury confirmed complete dorsal transections. F, G, Coexpression of CTB-488 and tdTomato was high in CST neurons in injured animals (F) and in both cortices of animals that received unilateral spinal injury (G). H, Quantification of coexpression rates showed no difference in the left cortex of injured animals, the location of axotomized CST cell bodies (p = 0.82, two-way ANOVA with post hoc Tukey's). N = 4 animals per group, >200 individual cells quantified per animal. Error bars show SEM. Scale bars, 1 mm.

Figure 6.

Figure 6.

AAV2-Retro and 3D imaging reveal CST cell body distribution, axon trajectories, and collateralization. A–D, Adult mice received bilateral cervical injection of AAV2-Retro-tdTomato. Two weeks later brains were optically cleared by 3DISCO and imaged with light-sheet microscopy. Images showed rotated views of whole brain, with rostral to the left and initially viewed from the dorsal surface in A. Three distinct groups of CST cell bodies are apparent, and to clear points of collateral branching from the main CST are visible (arrows). E, Individual tracings of CST neurons from the Mouselight Neuron Browser. Consistent with the 3D reconstruction, collateral branches to tectal areas (orange arrow) and to Basilar Pontine Nuclei (green arrow) are visible. Videos of cleared brains are available in Movies 1, 2, 3, and 4. Scale bars, 1 mm.

Figure 7.

Figure 7.

AAV2-Retro and 3D imaging reveal supraspinal populations and axon trajectories. A–G, Adult mice received bilateral cervical injection of AAV2-Retro-tdTomato, and 2 weeks later brains were optically cleared by 3DISCO and imaged with light-sheet microscopy. A, B, Imaging in the diencephalon shows scattered cell bodies likely corresponding to previously described hypothalamic-spinal populations. C–E, 3D imaging in the midbrain shows magnocellular (RMC) and parvocellular (PRC) populations of the red nucleus, as well as the centrally projecting component of the Edinger Westphal nucleus (Ewcp) and the interstitial nucleus of Cajal (INC). F–H, 3D imaging shows multiple brainstem populations and axon tracts, including the dorsal cerebellar nuclei (DCN), vestibular nuclei, reticulospinal populations, the CST, rubrospinal tract (RST) and cerebellospinal tract. I–K, Confocal imaging of sagittal sections of brainstem and cerebellum reveals axons that enter the cerebellar cortex and form large terminals, likely corresponding to mossy fiber terminals of spinocerebellar origin. Videos of cleared tissue are available in Movies 5, 6, and 7.

Figure 8.

Figure 8.

Dual AAV labeling from cervical and lumbar spinal cord shows distinct patterns of innervation. Adult mice received bilateral injections of AAV2-Retro-EGFP to cervical spinal cord, and AAV2-Retro-tdTomato to lumbar spinal cord. Brains were cleared and imaged 4 weeks later. A–C, Both CST and rubrospinal cell bodies segregated into distinct lumbar versus cervically-projecting populations. D, E, Longer-exposure images of the brainstem reveal distinct populations of lumbar-projecting neurons, including a prominent cluster located in the dorsal pons. a, CST, lumbar projecting; (b) CST, cervical projecting; (c) CST, rostral forelimb area; (d) CST, secondary sensory; (e) red nucleus, lumbar projecting; (e′) red nucleus, cervical projecting; (f) mixed dorsal pontine nuclei; (g) hypothalamic-spinal nuclei; (h) vestibular nuclei; (i) reticular nuclei; (j) cerebellospinal nuclei; and (k) pyramids of corticospinal tract. Error bars: A–C, 2 mm; D, E, 1 mm. A video of cleared tissue is available in Movie 8.

Figure 9.

Figure 9.

Gi-DREADD-mediated silencing of supraspinal input to cervical spinal cord. A, Mixed AAV2-Retro-Flex-Gi-DREADD-mCherry and AAV2-Retro-Cre was injected to the cervical spinal cord of wild-type mice, and Retro-Flex-Gi-DREADD-mCherry alone was injected to the cervical spinal cord of CaMKII-Cre animals. Four weeks later, mCherry signal was readily detectable in the brainstem, red nucleus, and cortex of wild-type animals, but was cortically enriched in CamkII-Cre animals. B, Clozapine produce reproducible forelimb deficits in DREADD-injected wild-type animals. C, Dose–response and timing curves for clozapine- and CNO-triggered motor deficits. Both ligands triggered reversible forelimb paralysis in Gi-DREADD injected wild-type animals, but not CamkII-Cre animals or non-injected controls. D, Mice were tested on a horizontal ladder task atop a wheel with irregularly spaced rungs and errors in forelimb placement were scored. E, In CaMKIIa-Cre animals, clozapine produced significant reduction in correctly targeted steps, consistent with selective silencing of corticospinal tract input to the spinal cord. N = 5 wild-type, 4 CamkII-Cre, and 3 non-injected controls. **p < 0.01, two-tailed paired t test. Error bars show SEM.

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