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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

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Graphical abstract

Figure 1

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

Figure 2

Photograph of the surgery set-up (A) Photograph showing the surgery bench. (B) Photograph showing the surgery tools.

Figure 3

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

Figure 4

Photograph of the mouse preparation station

Figure 5

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

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

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

Figure 8

Picture showing a custom-built retractor

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