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Tiffany E. Choe, Lin Wang, Theresa A. Lusardi, Cory J. Szybala, Claude F. Burgoyne, Brad Fortune; Axonal Transport of Cholera Toxin B Subunit (CTB) in the Rodent Visual Pathway Is Dependent on Intact Axonal Microtubules. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2448.
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We previously showed that both anterograde and retrograde axonal transport of CTB could be monitored in vivo in the rodent visual system and presented evidence that their rates were consistent with active (fast axonal transport) mechanisms. This study tests the hypothesis that axonal transport of CTB is dependent on intact microtubules.
Fifteen adult male Brown-Norway rats were studied. All procedures were performed under anesthesia (ketamine, xylazine, acepromazine 55:5:1 mg/kg IM). To assess the dependence of anterograde transport on microtubules, N=4 animals had an intravitreal injection of colchicine (vitreal concentrations from 0.1 to 1.0 mM) in one eye each and vehicle control injection in the fellow eye, followed by bilateral intravitreal injections of AlexaFluor488-conjugated CTB either 1, 2 or 4 hours later; N=2 were bilateral controls. To assess retrograde transport, N=4 animals had an intravitreal injection of colchicine (1 mM) in one eye and vehicle in the fellow eye, followed by bilateral stereotactic injections of CTB-488 into the superior colliculus (SC) within 1 hour; N=5 were bilateral controls. Retinas were imaged in vivo by confocal scanning laser ophthalmoscopy (CSLO, Spectralis HRA, Heidelberg Engineering, GmbH) for up to 4 hrs and again 24 hrs after intravitreal CTB and 24 hrs after SC CTB. Animals were sacrificed at 24 hours for post mortem microscopy of retinal, optic nerve and brain tissues.
Intravitreal injection of CTB resulted in rapid uptake and filling of retinal nerve fiber layer (RNFL) axon bundles in control eyes as visualized in vivo by CSLO and ex vivo by microscopy. In colchicine-injected eyes, RNFL bundles were only weakly fluorescent but brighter background represented intact CTB uptake into retinal ganglion cell (RGC) soma and dendrites, confirmed ex vivo. CSLO imaging of the ventral and dorsal aspects of the midbrain revealed brightly fluorescent control optic nerves, contralateral chiasm and SC, but only weak or no fluorescence of those structures for colchicine-injected eyes. Retrograde transport of CTB was either delayed or disrupted by intravitreal colchicine as evidenced by patchy/weaker fluorescence of RGCs imaged by CSLO in vivo and by microscopy ex vivo, compared to controls.
These results demonstrate that axonal transport of CTB in the rodent anterior visual pathway is dependent on intact microtubules, a further indication that it occurs by active transport mechanisms.
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