Intraspinal injection of adeno-associated viruses into the adult mouse spinal cord - PubMed (original) (raw)
Intraspinal injection of adeno-associated viruses into the adult mouse spinal cord
Shrivas Chaterji et al. STAR Protoc. 2021.
Abstract
Genetic dissection of neural circuits has been accelerated by recent advances in viral-based vectors. This protocol describes an effective approach to performing intraspinal injections of adeno-associated viruses, which can be used to label, manipulate, and monitor spinal and supraspinal neurons. By avoiding invasive laminectomies and restrictive spinal-clamping and by adopting injectable anaesthetics and tough quartz glass micropipettes, our protocol presents a time-saving and efficient approach for genetic manipulation of neural circuits nucleated in the spinal cord. For complete details on the use and execution of this protocol, please refer to Sathyamurthy et al. (2020).
Keywords: Microscopy; Model organisms; Neuroscience.
© 2021 The Authors.
Conflict of interest statement
The authors declare no competing interests.
Figures
Graphical abstract
Figure 1
Intraspinal delivery of viral vectors presents a powerful approach for addressing basic questions regarding the organization and function of spinal circuits Genetically-defined groups of spinal neurons and the circuits that they are embedded in can be manipulated using two different groups of AAVs – anterogradely transporting (A and B) and retrogradely transporting AAVs (C). By injecting recombinant anterogradely transporting AAVs into different segments of the spinal cord in wildtype mice or genetic reporter lines, it is possible to acutely manipulate select spatially defined populations of spinal neurons, specifically in adulthood (Dougherty et al., 2013; Fink et al., 2014; Bourane et al., 2015; Foster et al., 2015; Ruder, Takeoka and Arber, 2016; Choi et al., 2020; Sheahan et al., 2020; Barik et al., 2021; Gatto et al., 2021) (A). By combining spinal injections of anterogradely transporting AAVs with stereotactic injections of retrogradely transporting AAVs in brain regions that are targeted by ascending spinal neurons, and/or by the placement of optic fibers or drug cannulae in specific brain regions, it is possible to manipulate ascending pathways in a circuit-specific manner (Conner et al., 2021; Fink et al., 2014; Bouvier et al., 2015; François et al., 2017; Sheahan et al., 2020) (B). Spinal injections of retrogradely transporting AAV (AAVretro) (Tervo et al. 2016) combined with stereotactic injections of anterogradely transporting viral vectors in the brain enables the targeting of functionally and somatotopically distinct groups of supraspinal neurons that communicate with specific segments of the spinal cord (Esposito, Capelli and Arber, 2014; Basaldella et al., 2015; Murray et al., 2018; Sathyamurthy et al., 2020; Usseglio et al., 2020).
Figure 2
Photograph of the surgery set-up (A) Photograph showing the surgery bench. (B) Photograph showing the surgery tools.
Figure 3
Setup of the spinal injection station Picture showing assembly of the Hamilton syringe and micropipette (A), cut and uncut micropipettes (B), and the injection set-up without (C) and with (D) the tilted stereozoom microscope.
Figure 4
Photograph of the mouse preparation station
Figure 5
Representative images of a mouse at different stages of cervical injection (A) Mouse is kept taut along the rostro-caudal axis by taping the limbs and tail to the metal plate. Scale bar – 1 cm. (B) A wedge is inserted below the mouse to make access to the spinal cord easier. Scale bar – 1 cm. (C) Image showing skin incision. Scale bar – 1 cm. (D and D′) (D) Retractors are used to hold open the incision on the skin. A magnified image of the skin incision depicting the musculature and adipose tissue underlying the skin (D′). Scale bar – 3 mm. (E) Image showing T2 vertebra. Scale bar – 3 mm. (F and F′) (F) Image showing exposed cervical segments (magnified in F′). Scale bar – 3 mm.
Figure 6
Representative images of a mouse at different stages of lumbar injection (A and B) An incision is made on the dorsal hump skin to expose the underlying musculature. (C) The musculature is gently separated to expose the vertebrae corresponding to lumbar spinal segments. (D) The exposed spinal segments with the corresponding vertebrae labelled. Scale bar – 1 cm.
Figure 7
Expected outcome (A) Schematic showing unilateral injection of anterogradely transporting rAAV2/9-hSyn-GFP into the cervical cord. (B) Transverse section showing unilateral distribution of GFP in the cervical spinal cord. A six-week-old mouse was injected as shown in A. Transverse sections were collected 3 weeks following injection and examined for the presence of GFP. Scale bar – 200 μm.
Figure 8
Picture showing a custom-built retractor
Similar articles
- Neural labeling and manipulation by neonatal intraventricular viral injection in mice.
Wang M, Misgeld T, Brill MS. Wang M, et al. STAR Protoc. 2022 Jan 10;3(1):101081. doi: 10.1016/j.xpro.2021.101081. eCollection 2022 Mar 18. STAR Protoc. 2022. PMID: 35059654 Free PMC article. - Lentiviral and adeno-associated vector-based therapy for motor neuron disease through RNAi.
Towne C, Aebischer P. Towne C, et al. Methods Mol Biol. 2009;555:87-108. doi: 10.1007/978-1-60327-295-7_7. Methods Mol Biol. 2009. PMID: 19495690 - Recombinant adeno-associated virus mediated gene delivery in the extracranial nervous system of adult mice by direct nerve immersion.
Richner M, Gonçalves NP, Jensen PH, Nyengaard JR, Vægter CB, Jan A. Richner M, et al. STAR Protoc. 2022 Feb 17;3(1):101181. doi: 10.1016/j.xpro.2022.101181. eCollection 2022 Mar 18. STAR Protoc. 2022. PMID: 35243373 Free PMC article. - Comparison of high-dose intracisterna magna and lumbar puncture intrathecal delivery of AAV9 in mice to treat neuropathies.
Bailey RM, Rozenberg A, Gray SJ. Bailey RM, et al. Brain Res. 2020 Jul 15;1739:146832. doi: 10.1016/j.brainres.2020.146832. Epub 2020 Apr 11. Brain Res. 2020. PMID: 32289279 Free PMC article. Review. - Peripherally delivered Adeno-associated viral vectors for spinal cord injury repair.
Sydney-Smith JD, Spejo AB, Warren PM, Moon LDF. Sydney-Smith JD, et al. Exp Neurol. 2022 Feb;348:113945. doi: 10.1016/j.expneurol.2021.113945. Epub 2021 Dec 8. Exp Neurol. 2022. PMID: 34896114 Review.
Cited by
- Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.
Chen L, Chehade HD, Chu HY. Chen L, et al. bioRxiv [Preprint]. 2024 Aug 14:2024.07.24.604995. doi: 10.1101/2024.07.24.604995. bioRxiv. 2024. PMID: 39091827 Free PMC article. Preprint. - Morphological analysis of descending tracts in mouse spinal cord using tissue clearing, tissue expansion and tiling light sheet microscopy techniques.
Xie J, Feng R, Chen Y, Gao L. Xie J, et al. Sci Rep. 2023 Sep 30;13(1):16445. doi: 10.1038/s41598-023-43610-z. Sci Rep. 2023. PMID: 37777565 Free PMC article. - Single cell atlas of spinal cord injury in mice reveals a pro-regenerative signature in spinocerebellar neurons.
Matson KJE, Russ DE, Kathe C, Hua I, Maric D, Ding Y, Krynitsky J, Pursley R, Sathyamurthy A, Squair JW, Levi BP, Courtine G, Levine AJ. Matson KJE, et al. Nat Commun. 2022 Sep 26;13(1):5628. doi: 10.1038/s41467-022-33184-1. Nat Commun. 2022. PMID: 36163250 Free PMC article. - Viral strategies for targeting spinal neuronal subtypes in adult wild-type rodents.
Kaur J, Berg RW. Kaur J, et al. Sci Rep. 2022 May 23;12(1):8627. doi: 10.1038/s41598-022-12535-4. Sci Rep. 2022. PMID: 35606530 Free PMC article.
References
- Basaldella E., Takeoka A., Sigrist M., Arber S. Multisensory signaling shapes vestibulo-motor circuit specificity. Cell. 2015;163:301–312. - PubMed
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources
Research Materials