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Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats

Abstract

Cholera toxin subunit B (CTB) is a highly sensitive retrograde neuroanatomical tracer. With the new availability of fluorescent Alexa Fluor (AF) conjugates of CTB, multiple neuroanatomical connections can be reliably studied and compared in the same animal. Here we provide a protocol that describes the use of AF-CTB for studying connections in the central nervous system of rats. The viscous properties of CTB allow small and discreet injection sites yet still show robust retrograde labeling. Furthermore, the AF conjugates are extremely bright and photostable, compared with other conventional fluorescent tracers. This protocol can also be adapted for use with other neuroanatomical tracers. Including a 7-d survival period, this protocol takes 11 to 12 d to complete in its entirety.

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Figure 1: Photographs of key surgical steps (ax) from the PROCEDURE (Steps 9–25).
Figure 2: Example injection sites and labeling patterns of AF 488 (green) and 594 (red) CTB.

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Acknowledgements

We greatly thank Maggie Stoll for her continued technical help. We also thank the help of Dr. Stephen B. Lewis, Robin Barry and Bryan Thomasey for providing equipment for the video clips. We also appreciate further technical help from Elyse Morin and Rachel Tai. This study was supported in part by the NIMH Grant MH60399.

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Correspondence to William L Conte.

Supplementary information

Supplementary Video 1

Positioning of the animal in a stereotaxic device (Step 11). (MOV 13794 kb)

Supplementary Video 2a

Opening of the scalp and exposure of skull surface (Step 13). (MOV 14344 kb)

Supplementary Video 2b

Opening of the scalp and exposure of skull surface (Step 13). (MOV 14325 kb)

Supplementary Video 3a

Drilling of two separate burr holes (Steps 15-16). (MOV 13694 kb)

Supplementary Video 3b

Drilling of two separate burr holes (Steps 15-16). (MOV 14392 kb)

Supplementary Video 4a

Excision of the meninges (Step 17). (MOV 13682 kb)

Supplementary Video 4b

Excision of the meninges (Step 17). (MOV 13481 kb)

Supplementary Video 5a

Cleaning the surgical site, placement of gel foam, and closing of the wound (Step 25). (MOV 10188 kb)

Supplementary Video 5b

Cleaning the surgical site, placement of gel foam, and closing of the wound (Step 25). (MOV 10275 kb)

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Conte, W., Kamishina, H. & Reep, R. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats. Nat Protoc 4, 1157–1166 (2009). https://doi.org/10.1038/nprot.2009.93

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