Imaging axonal transport in the rat visual pathway

Abstract

A technique was developed for assaying axonal transport in retinal ganglion cells using 2 µl injections of 1% cholera toxin b-subunit conjugated to AlexaFluor488 (CTB). In vivo retinal and post-mortem brain imaging by confocal scanning laser ophthalmoscopy and post-mortem microscopy were performed. The transport of CTB was sensitive to colchicine, which disrupts axonal microtubules. The bulk rates of transport were determined to be approximately 80-90 mm/day (anterograde) and 160 mm/day (retrograde). Results demonstrate that axonal transport of CTB can be monitored in vivo in the rodent anterior visual pathway, is dependent on intact microtubules, and occurs by active transport mechanisms.

Keywords: (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging.

Fig. 1

Anterograde assay showing the time-course of CTB uptake into RGCs and their axons within the eye in vivo by CSLO. (a) CSLO infrared reflectance image (“CSLO-IR”) provides orientation to the ocular fundus. (b)–(c) CSLO fluorescence (“CSLO-FL”) images taken at 0.5 h (b), and 24 h (c) after intravitreal CTB injection demonstrate increasing uptake and transport of CTB in RGC axons over time. RGC uptake of CTB is first seen superiorly near the injection site; axon bundle filling typically progresses toward the optic disc and throughout the rest of the fundus. (d) High magnification CSLO-FL image obtained in a different eye 24 h after CTB injection and (e) flat-mount retinal microscopy (x20) corresponding to inset box in (d) demonstrate fluorescent RGC soma (arrow) and axons. Scale bar in (a) applies (a)–(c).

Fig. 2

Results of anterograde axonal transport 24 h after unilateral CTB injection. The right eye (a), (b) received an intravitreal injection of CTB, while the left eye (c), (d) was a non-injected control. CSLO images in vivo (a), (c) and post-mortem micrographs (x5) of flat-mount retina (b), (d) show strong CTB fluorescence in RGCs and axons of the right eye only. Post-mortem imaging of the optic nerves and chiasm (ventral view, (e), (f) and superior colliculi (dorsal view, (g), (h)) obtained either by CSLO (e), (g) or by epi-fluorescence microscopy (x5) montages (f), (h) show unilateral fluorescence of the ipsilateral optic nerve and contralateral superior colliculus. This indicates that CSLO and microscopy are both able to clearly detect successfully transported CTB to the optic nerves and superior colliculi. Scale bars: (a) applies (a)–(d); (g) applies (g), (h). Abbreviations: fluorescence (FL), right eye (OD), left eye (OS), right (R), left (L), optic nerves (ON), superior colliculi (SC).

Fig. 3

Results of retrograde axonal transport 24 h after CTB injection bilaterally into the superior colliculi. (a)–(d) CSLO fundus images in vivo and post-mortem micrographs (x5) of flat-mount retinas from the right and left eyes demonstrate strong CTB fluorescence of RGC somas and optic discs bilaterally, indicating successful axonal transport of CTB. Higher magnification CSLO fundus image in vivo (e) and post-mortem microscopy (x20) of the retina (f) shows the fluorescent RGC somas and dendrites. Box in (e) indicates region shown in (f). Post-mortem imaging of the dorsal midbrain by CSLO (g) reveals bilateral fluorescence of both superior colliculi indicating that CTB diffuses throughout the superior colliculi from the central injection sites. Scale bars: (a) applies (a)–(d). Abbreviations: fluorescence (FL), right eye (OD), left eye (OS), right (R), left (L), optic nerves (ON), superior colliculi (SC).

Fig. 4

Effect of colchicine on anterograde axonal transport. (a)–(d) Fluorescence mode CSLO fundus images obtained in vivo 24 h after CTB injections from the right and left eyes of a bilateral CTB positive control rat (a), (b) and another bilateral CTB rat that had unilateral (OD) pre-treatment with intravitreal colchicine (c), (d). (e)-(h) High magnification post-mortem fluorescence micrographs of flat-mount retinas from the bilateral positive control (x20; (e), (f)) and the unilateral colchicine animal (x10; (g), (h)). The CSLO images and the micrographs demonstrate strong CTB fluorescence of RGC somas, RNFL and optic discs bilaterally, indicating successful uptake of CTB by RGCs across all retinas. Box in (a)–(d) indicates region shown in (e)–(h). Post-mortem imaging of the ventral and dorsal midbrain by CSLO (i)–(l) reveals bilateral fluorescence of both optic nerves and tracts (i) and both superior colliculi (j) in the bilateral positive control rat, indicating patent axonal transport of CTB in both pathways. However for the unilateral colchicine rat, the ipsilateral optic nerve and contralateral optic tract (k) and contralateral superior colliculus (l) to the colchicine-injected eye exhibit minimal fluorescence, indicating disruption of axonal transport in the colchicine-treated pathway. The fellow control eye (OS) in the unilateral colchicine rat shows patent axonal transport (CTB fluorescence) at its corresponding brain structures (k), (l). Scale bars: (a) applies (a)–(d), (e) applies (e)–(h), (i) applies (i)–(l). Abbreviations: fluorescence (FL), right eye (OD), left eye (OS), right (R), left (L), optic nerves (ON), superior colliculi (SC).

Fig. 5

Effect of colchicine on anterograde axonal transport. Average fluorescence intensity (±SEM) is shown for the group of rats (n = 7, bars with small checks) in which one eye was pre-treated with an intravitreal injection of either vehicle or colchicine prior to intravitreal injection of CTB; a unilateral control group of rats (n = 3, bars with larger checks) in which the intravitreal injection of CTB was unilateral with the fellow eye serving as a non-injected control (CTB–); a bilateral positive control group of rats (n = 4, open bars) in which CTB was injected into the vitreous bilaterally (CTB+)); and a negative control group of naïve rats (n = 3, solid bars) which were sacrificed without any CTB injection in either eye (CTB–). Colchicine reduced the fluorescence intensity of the contralateral superior colliculus 24 h after CTB injection to the level of non-injected controls (CTB–); contrast between hemispheres was nearly as great as that in the group of unilateral controls. Abbreviations: right superior colliculus (R) and left superior colliculus (L).

Fig. 6

Effect of colchicine on retrograde axonal transport. (a)–(d) CSLO fluorescence fundus images in vivo of the right (a) and left (c) eyes and post-mortem micrographs (x10) of flat-mount right (b) and left (d) retinas, 24 h after bilateral superior colliculi injections of CTB and unilateral (OD) pre-treatment with intravitreal colchicine. There was substantially less CTB fluorescence in the RGCs of the eye pre-treated with colchicine (a), (b) than the fellow control eye (c), (d), indicating disruption of retrograde axonal transport of CTB in the colchicine eye only. The RGC fluorescence in the fellow control eye (c), (d) is similar to the that in the bilateral positive control shown in Fig. 3, indicating patent axonal transport of CTB. (e) CSLO infrared reflectance image provides orientation to the dorsal midbrain including the superior colliculi. (f) Accompanying CSLO in fluorescence mode shows that both superior colliculi fluoresce equally with near full coverage, indicating that the difference in RGC fluorescence is not due to a failed CTB injection. Scale bars: (a) applies (a), (c), (b) applies (b), (d), (e) applies (e), (f). Abbreviations: fluorescence (FL), right eye (OD), left eye (OS), right (R), left (L), infra-red (IR), superior colliculi (SC).

Fig. 7

Effect of colchicine on retrograde axonal transport. Average RGC density (±SEM) measured in vivo by CSLO (a) and post-mortem by microscopy of retinal flat-mounts (b) is shown for the group of rats (n = 5, bars with checks) in which one eye was pre-treated with an intravitreal injection of either vehicle or colchicine prior to bilateral injection of CTB into the superior colliculus; a bilateral positive control group of rats (n = 9, open bars) in which CTB was injected into the superior colliculus bilaterally (CTB+)); and a negative control group of naïve rats (n = 3, solid bars) which were sacrificed without any CTB injection (CTB–). Colchicine reduced the density of CTB–positive RGCs nearly completely (i.e., nearly to the level of non-injected controls). Abbreviations: right eye (OD) and left eye (OS).

Fig. 8

Results of anterograde transport rate experiment. Representative examples from a cross-sectional series demonstrate the time of earliest detected CTB fluorescence at the optic nerves and superior colliculi after unilateral intravitreal CTB injections into the right eye. (a)–(c) Post-mortem imaging (montages) of the ventral midbrain by CSLO at (a) 5h, (b) 6h, and (c) 7h after CTB injection reveals greater fluorescence in the ipsilateral (right) optic nerve than the left, first noticeable at 5h (a) and more obviously noticeable at 6h (b) and 7h (c). (d)–(f) Post-mortem imaging of the corresponding dorsal midbrains by CSLO at (d) 5h, (e) 6h, and (f) 7h shows greater relative fluorescence intensity in the contralateral (left) superior colliculus, first noticeable at 6h (e) and more clearly noticeable by 7h (f). These results show that CTB reaches the optic nerve by 5h after intravitreal injection and the superior colliculus by 6h, indicating that CTB travels by fast active axonal transport when compared to known rates [1]. Scale bars: (a) applies (a)–(c), (d) applies (d)–(f). Abbreviations: fluorescence (FL), right eye (OD), right (R), left (L), optic nerves (ON), superior colliculi (SC).

Fig. 9

Results of experiment to estimate bulk-rate of anterograde axonal CTB transport. Relative fluorescence intensity (CTB injected side (Exp) relative to non-injected (Ctrl) side) is plotted versus time after unilateral intravitreal CTB injection for the ipsilateral optic nerve (a) and contralateral superior colliculus (b). Solid line through data represents results of fit to the data of the equation: Y = IF(X < X₀, Y₀,Y₀ + (Plateau – Y₀)*(1 – exp(–K*(X – X₀)))), which was used as a secondary method to determine the first time after injection that fluorescence intensity began to rise above that of the opposite-side structure (i.e., the X₀ parameter corresponding to the point that exponential growth began from baseline). For optic nerve, X₀ = 1.64 (95% CI −0.07 to 3.35); for superior colliculus, X₀ = 5.48 (95% CI 2.78 to 8.19). Error bars = SEM. N ≥ 3 rats per time point. Abbreviations: Exp = Experimental, Ctrl = Control.

Fig. 10

Examples from a longitudinal series demonstrate the time of earliest detected CTB fluorescence at the optic disc and RGCs after bilateral superior colliculi CTB injections in order to determine the rate of retrograde transport of CTB. CSLO fluorescence fundus images (a)–(f) were taken in vivo at pre-injection baseline (a), (d) then every 30 min from 2 to 5 h after CTB injection. At 3h the first sign of optic disc fluorescence (arrow) was noted in the right (b) and left (e) eyes, clearly brighter than at baseline. At 4h the first sign of RGC fluorescence (asterisk) was noted predominantly superior-nasally in the right (c) and left (f) eyes. Post-mortem micrographs (x10) of flat-mount right (g) and left (h) retinas at 5h after injection confirm CTB fluorescence at the disc and RGCs in all retinal quadrants. Brightness and contrast of images were adjusted to maximize visibility in panels of this figure. Scale bars: (a) applies (a)–(f), (g) applies (g), (h).. Abbreviations: fluorescence (FL), right eye (OD), left eye (OS), superior (S), inferior (I), nasal (N), temporal (T).