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Imaging axonal transport in the rat visual pathway

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.


Introduction
Axonal transport is critical to the growth, function and survival of neurons, as it enables metabolic messages (such as neurotrophic factors and neurotransmitters) to be passed between the cell soma and its axon terminals. Active axonal transport is performed primarily by molecular motor proteins (such as the family of kinesins for anterograde transport, and dynein for retrograde transport), which use cytoskeletal polymer tracks (microtubules or actin) to support the unidirectional movement of cargo molecules between proximal and distal regions of the axon [1][2][3][4]. Disruption of active axonal transport in retinal ganglion cells (RGCs) is thought to be a critical pathophysiological element of glaucomatous optic neuropathy and may even represent one of the earliest functional abnormalities affecting these cells . Active axonal transport can be assayed using histopathological methods, but this requires sacrifice of the animal and typically time-and labor-intensive protocols involving tissue blocking, cutting and imaging of serial sections (see, e.g., [5,27].) Hence, development of more rapid axonal transport assays is desirable for use in experimental models of optic neuropathy, particularly if such assays can be accomplished in vivo. Indeed, development of an in vivo assay of axonal transport within RGCs could enable assessment of the pathophysiological role and sequence of transport obstruction in models of neurodegeneration such as glaucoma, and may allow evaluation of potential treatments to reverse transport deficits.
Recent reports have demonstrated development of novel techniques to examine active axonal transport in vivo and in vitro. For example, axonal transport of mitochondria in peripheral nerves of living transgenic mice and in acute explants has been imaged with timelapse recordings [31] and live-cell imaging has revealed the transport dynamics of brainderived neurotrophic factor (BDNF) in RGC axons and dendrites in vitro [32]. In addition, the availability of imaging tools such as confocal scanning laser ophthalmoscopy (CSLO) provide an opportunity to detect and quantify axonal transport tracers within the visual system in vivo.
One requirement for such an assay is that the tracer is carried primarily by active axonal transport rather than by simple diffusion through the neuronal/axonal cytosol or membrane. Work over the past two decades has raised the profile of cholera toxin subunit B (CTB) as an excellent candidate for use in axonal transport assays because of its high sensitivity, its capability to move in either anterograde or retrograde direction from the site of injection within a given neuronal tract, its restriction from labeling fibers of passage and its ability to label the entire neuron, even fine processes and terminals in the mammalian nervous system including the visual pathway [33][34][35][36][37][38][39][40][41][42][43]. Its high sensitivity is thought to be due to the numerous binding sites (monosialoganglioside receptors, Gm1) on the surface of neurons [44,45].
Although CTB is often presumed to be transported by fast active mechanisms in vivo, there is actually considerable variability across the literature regarding the estimated rate of anterograde and retrograde transport of CTB [35,36,42,46]. Moreover, to our knowledge, there are no reports about the sensitivity of CTB to colchicine, which is a fundamental experiment for whether a tracer travels by active transport [47]. Colchicine is a compound that disrupts microtubules [48][49][50], therefore it can be used to assay the integrity of active axonal transport [32,47,[51][52][53][54]. Microtubules within RGC axons are rapidly disrupted when colchicine is administered by intravitreal injection [55][56][57][58]. Hence, determining both the sensitivity of CTB axonal transport to intravitreal colchicine injection and the bulk rate of transport along the length of the axon are important experiments to confirm the transport mechanism of CTB.
Thus the overall aim of this study was to develop techniques imaging anterograde and retrograde active axonal transport in rodent RGC axons. Primarily, we used the well-known neuronal tract tracer CTB conjugated to a fluorescent marker appropriate for use with a commercially available CSLO, but also tested latex microspheres and a carbocyanine dye for comparison. Specifically, we tested the hypothesis that CTB is transported in both anterograde and retrograde directions by fast active mechanisms within RGC axons of the rat optic nerve. We performed two fundamental experiments to test this hypothesis, which have not previously been done: 1) we tested the dependence of CTB transport on intact microtubules by evaluating its sensitivity to colchicine treatment and 2) we tested the rate of transport (to be contrasted with slow active mechanisms and/or passive diffusion) [1,59].

Subjects
The subjects of this study were ninety-five adult male Brown-Norway rats (Rattus norvegicus; Charles River Laboratories Inc. Wilmington, MA). Seven rats were excluded from analysis due to poor injections or complications such as large vitreal hemorrhage or retinal detachment: anterograde colchicine experiment (n = 1), retrograde colchicine experiment (n = 5), anterograde rate experiment (n = 1). The remaining eighty-eight rats were used and analyzed as follows; development of the anterograde axonal transport assay technique (n = 8), development of the retrograde axonal transport assay technique (n = 6), anterograde CTB assay with colchicine experiment (n = 10), bilateral CTB controls for anterograde colchicine experiment (n = 4), retrograde CTB assay with colchicine experiment (n = 5), bilateral CTB controls for retrograde colchicine experiment (n = 10), rate of anterograde axonal transport (n = 26), rate of retrograde axonal transport (n = 10), naïve controls for rate experiments (n = 5), retrograde transport of latex microspheres (n = 2), retrograde transport of carbocyanine dye (n = 2). Rats were maintained under a 12-hour light/12-hour dark cycle with normal rat chow and water available ad libitum. The rats ranged in weight from 174 to 360 g and were 8 to 20 weeks old. All experimental methods and animal care procedures adhered to the Guide for the Care and Use of Laboratory Animals and were approved and monitored by the Institutional Animal Care and Use Committee at Legacy Health.

Imaging protocol
Rats were placed on a custom-built imaging stage and kept warm with a thermostatically controlled system (TP650; Gaymar Industries, Inc., Orchard Park, NY). Proparacaine hydrochloride (0.5%; Alcon Laboratories Inc., Fort Worth, TX) was instilled for topical anesthesia, and tropicamide (0.5%; Alcon Laboratories Inc.) and phenylephrine (2.5%; Bausch and Lomb Pharmaceuticals Inc., Tampa, FL) for topical mydriasis. Custom rigid gaspermeable contact lenses (3.5 mm posterior radius of curvature, 5.0 mm optical zone diameter, and +5.0 diopter back vertex power) were used to maintain corneal hydration and clarity. CSLO images of in vivo retina and post-mortem brain structures were obtained (Spectralis HRA + OCT; Heidelberg Engineering GmbH, Heidelberg, Germany) with an additional +25 diopter lens mounted to the camera objective. The infrared and BluePeak TM blue laser (488 nm) autofluorescence imaging modes (standard contrast settings) were used with 100 images averaged using the automatic real-time (ART) eye tracking software.

Development of the anterograde axonal transport assay
The anterograde transport assay was performed with a 2 µl intravitreal injection of 1% cholera toxin b-subunit conjugated to AlexaFluor488 (CTB; Molecular Probes C22841) dissolved in sterile PBS. In development of the assay, four animals had 5 µl intravitreal injections of 1% CTB, and one animal had 2 µl intravitreal injections of 0.5% CTB. Antibiotic ointment (neomycin, polymyxin B sulfates and dexamethasone, Falcon Pharmaceuticals Ltd, Fort Worth, Texas) was applied topically after injections. Animals were sacrificed at time points varying from 2 h to 34 days after intravitreal injection to determine the success and approximate time course of CTB transport to the optic nerve and superior colliculus. In vivo retinal imaging by CSLO was performed longitudinally at various time points (for up to 34 days after CTB injection) prior to sacrifice to confirm successful injection and uptake of CTB by RGCs. Animals were overdosed with pentobarbital sodium and phenytoin sodium (intraperitoneal 0.7-1.4 ml/kg; Euthasol Solution, Virbac Animal Health Inc., Fort Worth, Texas). Subsequently, the eyes were enucleated and the animal transcardially perfused with 125 ml of cold 4% paraformaldehyde in 0.5 M phosphate buffer (PB, pH 7.35) following an intracardiac injection of 0.1 ml heparin sodium (10,000 USP Units/ml, APP Pharmaceuticals). The retinas were dissected and mounted in 4% paraformaldehyde in 0.5 M PB for immediate fluorescence microscopy. The brain was dissected from the skull with the pre-chiasmal optic nerves attached and the cortices were splayed apart at the midline to reveal the dorsal aspect of the midbrain for imaging the superior colliculi.
The brain was immersion fixed in 4% paraformaldehyde in 0.5 M PB for at least 30 minutes prior to CSLO imaging. The patency of axonal transport was assessed from both post-mortem CSLO and microscopy images of the optic nerves and the superior colliculi to compare these two methods. CSLO has the potential for providing faster results and a field size and depth better suited to the task of imaging the nerves and colliculi than microscopy, particularly if the latter involves tissue blocking, cutting and imaging of serial sections. The CSLO images were obtained by positioning the brain on a custom-made mount strapped onto the stage. After CSLO imaging, the optic nerves and superior colliculi were dissected from the brain and mounted on a slide in phosphate buffer solution (PBS). Micrographs of the retinal flat-mounts (5x, 10x, 20x or 40x air objective), the ventral surface of the optic nerves (5x air objective) and the superior colliculi dorsal surface (5x air objective) were taken using a digital camera (QImaging Retiga 1300, Canada), mounted onto either a DMRXE or a DMLB microscope (Leica, Germany). Images in a single plane of best-focus were acquired with filter set #513808 (FITC; 450-490 nm excitation, 515 nm long pass emission; Chroma) for all specimens.

Development of the retrograde axonal transport assay
The retrograde transport assay was performed with 2 µl stereotactic injections of 1% CTB into both superior colliculi. The rat was affixed into a stereotactic frame (Kopf Instruments, CA), and then the head was shaved and sterilized with povidone-iodine prior to performing a 1.5 mm midline incision. The head tilt was adjusted so that the skull landmarks lambda and bregma were level. Bilateral holes were drilled through the skull using a Dremel bit and a Hamilton syringe was used to inject the CTB centered at co-ordinates corresponding to the center of each superior colliculus. The co-ordinates were optimized during development of the retrograde assay for successful CTB injection in Brown-Norway rats of this age and weight range (Table 1) and determined to be: −5.5 mm anterior-posterior (AP; relative to bregma), ±1.25 mm medial-lateral (ML), and −4.5, −4.25, −4.0, and −3.75 mm dorsal-ventral (DV; from the skull surface). At each of these four dorsal-ventral locations 0.5 µl of CTB was injected, with each injection made over the course of 30 seconds and a 30 second delay prior to moving the cannula to the next location. The cannula was slowly removed from the brain five minutes after the final injection. The incision was sutured or stapled closed, and buprenorphine (0.3 mg/ml) analgesic administered intra-muscularly. Retinas were imaged in vivo by CSLO for up to 5 hours immediately after CTB injection and/or at longer post-injection time points of 1 to 68 days to determine the success of transport and time course of CTB fluorescence ( Table 1). As described in the anterograde protocol, rats were sacrificed by transcardial perfusion at different time points (3 h to 68 days; Table 1), the retinas and brains were dissected, and post-mortem retinal micrographs and CSLO fluorescence images of the superior colliculus were taken to confirm injection success. The bilateral retrograde assay of axonal transport was also tested using markers alternative to CTB, namely latex microspheres (size range 0.05-0.2 µm diameter, green retrobeads IX, Lumafluor) and the carbocyanine dye 3-octadecyl-2-[3-(3-octadecyl-2(3H)benzoxazolylidene)-1-propenyl] (Di-O, Vybrant DiO cell-labeling solution, #V22886, Invitrogen). After injection (2 µl injection volume), retinas were imaged with in vivo CSLO for follow up periods of 3 to 15 weeks.

Colchicine protocol
To assess the dependence of anterograde transport on intact microtubules, animals had an intravitreal injection of colchicine (vitreal concentration 1.0 mM) in one eye and vehicle control in the fellow eye, followed by bilateral intravitreal injections of CTB between 2 -4.5 hours later. The vehicle control was dimethyl sulphoxide (DMSO) in three animals and was deionized water passed through a 0.2 µm filter (#PN4192, Acrodisc syringe filter, Pall Life Sciences) in the remaining seven. Bilateral control animals had intravitreal CTB but not colchicine injections. Retinas were imaged in vivo by CSLO for up to 4 hours immediately after intravitreal injection of CTB and again at 24 hours, to assess uptake of CTB by the RGCs, and to confirm successful injections. Animals were sacrificed 24 hours after CTB injection for post mortem CSLO of the optic nerves and superior colliculi, and microscopy of retinal flat-mounts, as described above. The fluorescence intensity of the right and left optic nerves and superior colliculi was compared to determine whether CTB had reached these structures by anterograde axonal transport.
To assess the dependence of retrograde transport on microtubules, an intravitreal injection of colchicine (1.0 mM) was performed in one eye and a vehicle injection (all deionized water passed through a 0.2 µm filter) was given in the fellow eye, followed by bilateral stereotactic injections of CTB into the superior colliculi 2-2.75 hours later. Bilateral control animals had the CTB injections into the superior colliculi only. Retinas were imaged in vivo by CSLO at 24 hours after CTB injection into the superior colliculi. All animals were sacrificed at 24 hours for post mortem CSLO of the superior colliculi, and microscopy of retinal flat-mounts as described above. Successful superior colliculi injections for this purpose were defined as requiring both the central 10% of the superior colliculus surface filled by CTB, and an indication from CSLO retinal imaging that CTB had reached the optic disc (positive disc fluorescence).

Rate of anterograde axonal transport
The rate of anterograde transport of CTB was calculated based on the time taken for the CTB to travel from the retina to the superior colliculus. The rat visual pathway length (eye to superior colliculus) was measured at 19.9 ± 0.5 mm (n = 24 eyes) with calipers (including optic nerve stump, pre-chiasmal optic nerve, chiasm, and optic nerve tract to the middle of the superior colliculus). The time taken to first detect fluorescence in the optic nerve and the superior colliculus after intravitreal injection of CTB was quantitatively measured (as described below) to allow calculation of the bulk rate of transport (mm per day).
Three to four animals were sacrificed at each time point of 2, 3, 4, 5, 6, 7, 24 and 48 hours after successful unilateral intravitreal CTB injection (anterograde assay). Five animals were additionally sacrificed as naïve animals without injection of CTB or vehicle, two of which had no CSLO imaging to provide absolute naïve controls for microscopy, while the remaining three had CSLO imaging prior to microscopy.
CSLO images of the optic nerves and the superior colliculi and corresponding microscopy images underwent quantitative analysis of CTB fluorescence intensity using ImageJ software (NIH). For the optic nerve CSLO data, six square bins of size 75x75 pixels (120x120µm) positioned with equal spacing along each nerve and at the same locations between right and left, were measured for fluorescence intensity in arbitrary units (0 = black, 255 = white). For the optic nerve microscopy data, eleven square bins of size 150x150 pixels (320x320µm) were similarly measured for fluorescence intensity. The normalized percent difference between the experimental and control eyes (for each bin position and in total for each time point) were determined for both the CSLO and the microscopy data. By normalizing the signal to the control, we control for autofluorescence. For the superior colliculi data, the right and left superior colliculi were outlined with the same "D" shaped polygon (matching the shape of each colliculus), the average fluorescence intensity at the surface was measured and the mean normalized difference between experimental and control superior colliculi determined for each time point. Note that in rat, approximately 95% of axons cross at the chiasm [39, [60][61][62], so the experimental superior colliculus is of opposite laterality to the experimental pre-chiasmal optic nerve and eye.

Rate of retrograde axonal transport
The retrograde transport rate was determined from the time taken for CTB to first reach the eye after injection into the superior colliculus. Ten animals were imaged with fluorescent CSLO every 30 minutes for up to 5 hours after bilateral CTB injections into the superior colliculi (retrograde assay) to note the time taken to first detect fluorescence in vivo in the optic disc and retina (see Table 1 for precise imaging times). Since the length of the rat visual pathway is known, the rate of transport (mm per day) can be calculated. To obtain postmortem microscopy results, rats were sacrificed at various time points between 3 and 5 hours after injections ( Table 1). The three naive animals that had retinal CSLO and microscopy performed for the anterograde rate calculation were also used as naïve controls for the retrograde rate experiment. For this retrograde bulk rate experiment, successful injections required at least the central 30% of the superior colliculus surface filled by CTB on postmortem CSLO imaging. This criterion was stricter than for the colchicine retrograde protocol because it involved fluorescence detection at threshold, rather than at supra-threshold.

Statistical analysis
For the effect of colchicine on anterograde transport, fluorescence intensity of the superior colliculus was assessed by analysis of variance (ANOVA) with Bonferroni-corrected post hoc comparisons across experimental groups. For the effect of colchicine on retrograde transport, the density of CTB-positive fluorescent RGCs was assessed by ANOVA with Bonferronicorrected post hoc comparisons across experimental groups. To determine the time point that showed the first departure from background fluorescence, two separate approaches were undertaken: 1) one-sample t-tests with a theoretical mean of zero were applied to each postinjection time point and 2) the aggregate data for relative fluorescence intensity versus time after intravitreal CTB injection was fit using nonlinear regression to determine the time of departure from baseline (no relative fluorescence difference; see details and equation below). To compare CSLO and microscopy methods of measuring relative fluorescence intensity of the superior colliculus, nonparametric Spearman's correlations were performed. Statistics were calculated using Prism software (Version 5, GraphPad Software, Inc., La Jolla, CA).

Anterograde axonal transport assay
Intravitreal CTB injection resulted in rapid uptake and filling of retinal nerve fiber layer (RNFL) axon bundles in normal eyes, typically beginning nearest the injection 15-20 min after injection and filling the entire fundus within 1-2 hrs. Figure 1 provides an example of longitudinal CSLO imaging in vivo after intravitreal CTB injection as well as comparison to ex vivo microscopy of the flat-mount retina. Fluorescent axon bundles were first visible by CSLO superiorly (nearest the injection site) within 10 minutes after the injection and filled most of the superior retina by 0.5 hours (Fig. 1(b)). By 24 hours (Figs. 1(c), 1(d)) the neuroretinal rim of the optic disc fluoresced strongly; suggesting much of the CTB had entered the nerve by this time. Retinal microscopy ( Fig. 1(e)) confirmed uptake into the RGCs by revealing brightly fluorescent somas, as well as dendrites and proximal axons in most instances. With the epi-fluorescent microscope and CSLO, the somas are only visible in the regions between axon bundles since the axons also fluoresce strongly. Figure 2 provides an example of post-mortem CSLO imaging and microscopy of the ventral and dorsal aspects of the midbrain 24 h after unilateral intravitreal CTB injection, which revealed a brightly fluorescent ipsilateral optic nerve through the chiasm, contralateral optic tract and contralateral superior colliculus after transport of CTB to these structures. The fellow control eye showed minimal auto-fluorescence of the retina, optic nerve, optic tract and superior colliculus. These results show that both CSLO and microscopy techniques are able to clearly detect successful anterograde transport of CTB to the superior colliculus. To evaluate the longer term time-course and persistence of CTB fluorescence in RGCs, one retina from one rat was imaged in vivo by CSLO immediately after intravitreal CTB injection, then subsequently at 1 day, 28 days, 32 days and 34 days follow up. At 24 hours the in vivo fluorescent CSLO image showed the usual pattern of CTB uptake with strong filling of ganglion cells and retinal nerve fiber bundles, in similar fashion to Figs. 1(c), 1(d) and Fig.  2(a). However, by 28 days the in vivo image had become more punctate, portraying individual RGCs only and no longer the nerve fiber bundles. By 34 days there was considerable depletion in the RGC numbers seen in vivo, indicating that CTB fades from RGCs at 1 month after intravitreal injection.

Retrograde axonal transport assay
Injection of CTB into the superior colliculi resulted in subsequent fluorescence of the contralateral optic discs observed in vivo by CSLO, followed by increasing accumulation of fluorescence within individual RGCs. Figure 3 shows results 24 hours after bilateral superior colliculus injections, when accumulation of CTB fluorescence is nearing its peak (time course results are shown in subsequent figures for experiments on transport rate). Figure 3 shows that RGC somas are more visible in the retrograde assay than in the anterograde assay, presumably due to a lower CTB concentration within the overlying intraretinal axons. Baseline CSLO of retinas in vivo prior to superior colliculi CTB injection shows weak auto-fluorescence of the retina depending on the CSLO sensitivity used during image acquisition (i.e., Figure 2(c)). Post-mortem CSLO of the superior colliculi ( Fig. 3(g)) shows CTB usually diffuses well throughout the colliculi, although often not completely, and allows confirmation of a successful injection. The extent of CTB diffusion through the superior colliculi seems to correlate reasonably with the extent of retinal area with positive ganglion cell fluorescence, as would be expected since visual space is represented topographically in the superior colliculus [63].
To evaluate the extended time-course of CTB fluorescence in RGCs, the retinas of one rat were imaged by CSLO in vivo immediately after bilateral superior colliculi CTB injections, then subsequently at 1, 2, 5, 11, 49 and 68 days follow up. By 24 hours the label showed good coverage across the RGCs in the central retina of both eyes, and the fluorescence intensity and coverage was maintained over the first 11 days. By 49 days the number of fluorescent RGCs seen in vivo had declined dramatically and by 68 days there was only a small group of cells still visible near the optic disc. This indicates that CTB fluorescence after superior colliculus injection fades from RGCs by 2 months. Since both the anterograde and retrograde assays produced an unequivocally strong CTB fluorescence at their targets by 24 hours, we chose this as the assay time-point for the following retrograde assay experiments with colchicine.

Effect of colchicine on anterograde and retrograde transport of CTB
Unilateral pre-treatment with colchicine (by intravitreal injection) resulted in profound inhibition of anterograde axonal transport of CTB from the colchicine-injected eye. Figure 4 demonstrates the effect in one representative example. The results for a representative bilateral positive control are shown in the left pair of columns demonstrating brightly fluorescent axons within the RNFL of both eyes (Figs. 4(a), 4(b), 4(e), 4(f))) and symmetrically bright fluorescence of the optic nerves, chiasm and tracts (Fig. 4(i)) and superior colliculi (Fig. 4(j)) 24 hours after bilateral intravitreal CTB injections. In contrast, the right pair of columns shows the results for an animal pre-treated with a unilateral colchicine injection (1.0 mM) 2 hours prior to bilateral intravitreal CTB injection, demonstrating weakto-no fluorescence of the ipsilateral optic nerve, chiasm and contralateral optic tract (Fig.  4(k)) and colliculus (Fig. 4(l)). Interestingly, the axons within the colchicine-treated eye (OD, Fig. 4(c)) exhibit essentially normal CTB fluorescence, evident both in vivo by CSLO (Figs. 4(a)-(4d)) and post mortem by microscopy of flat-mount retinae (Figs. 4(e)-4(h)), suggesting that CTB uptake occurs all along the axons, not strictly at the RGC soma. The effect of colchicine to severely disrupt anterograde axonal transport was unequivocal for all rats in this experiment (n = 10). Figure 5 shows the results of the effect of colchicine on anterograde axonal transport of CTB: colchicine reduced the fluorescence intensity of the contralateral superior colliculus to the level of non-injected controls (CTB-). Contrast between hemispheres was nearly as great 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).
as that in the group of unilateral controls: fluorescence of the superior colliculi contralateral to the eyes pre-treated with colchicine was significantly reduced compared with the group of fellow controls pre-treated with vehicle (p < 0.05) or to the groups of unilateral or bilateral positive controls (p < 0.05). Unilateral colchicine did not appear to significantly alter CTB transport in the fellow control (vehicle-injected) eyes: there were no significant differences between the superior colliculi contralateral to the eyes pre-treated with vehicle versus those of either the bilateral control group or the unilateral control groups.
Retrograde transport of CTB was also severely disrupted (and/or delayed) by pretreatment with intravitreal colchicine in all animals studied. Figure 6 demonstrates the results for one representative example. In the eye pre-treated with intravitreal colchicine injection (Figs. 6(a), 6(b)), there was only patchy or weak fluorescence of RGCs 24 hours after bilateral CTB injections to the superior colliculi, as compared to the vehicle-injected control fellow eye (Figs. 6(c), 6(d)) or to bilateral positive controls such as that shown in Fig. 3. This result was unequivocal in all five animals. In all colchicine eyes, retrograde CTB reached the optic disc, perhaps reflecting a more limited effect of colchicine along the distal optic nerve and tract over this relatively short post-injection duration. Importantly, the success of the superior colliculus injections can be confirmed rapidly and effectively by post mortem CSLO imaging of the dorsal midbrain (Figs. 6(e), 6(f)), ruling-out the possibility of injection failure as a potential cause of apparent transport deficit. Figure 7 shows the results for all five eyes included to evaluate the effect of colchicine on retrograde axonal transport of CTB. Colchicine markedly reduced the density of CTB positive RGCs as compared to either the group of fellow (vehicle-treated) control eyes or to either group of bilateral positive control eyes (p < 0.05). Though there was no significant difference between colchicine-treated eyes and negative controls (p > 0.05), RGC density was not reduced to zero in colchicine-treated eyes indicating that retrograde transport was not completely suppressed by this pre-treatment with intravitreal colchicine. The colchicine effect was clear whether RGC density was measured in vivo by CSLO (Fig. 7(a)) or post mortem by microscopy of retinal flat-mounts ( Fig. 7(b)). There was a strong association between mean RGC density measured in vivo by CSLO and post-mortem by microscopy of retinal flatmounts (R 2 = 0.98, p < 0.0001), though the former underestimated the latter by ~30% (slope = 0.68), which is apparent in the comparison between Figs. 7(a) and 7(b).

Rate of anterograde axonal transport
Figures 2 and 4 demonstrated strong fluorescence of the optic nerve, chiasm, tract and superior colliculus 24 hours after intravitreal injection of CTB. That the superior colliculus, approximately 20 mm from the globe, was already exhibiting strong fluorescence 24 hours after injection indicates that anterograde transport of CTB occurs at a minimum rate of 20 mm/day, which is ~3-4x faster than membranous diffusion such as in the case of carbocyanine dyes [59,64,65]. In order to further evaluate the rate of anterograde CTB transport we examined post-mortem fluorescence of these structures at earlier (2-7 h) postinjection time points. Figure 8 shows representative examples of midbrain CSLO imaging at time points 5, 6, and 7 hours after unilateral CTB injection, around the time that fluorescence was first unequivocally brighter along the ipsilateral optic nerve but only just beginning to exceed background noise and autofluorescence in the contralateral superior colliculus. Image analysis of post-mortem CSLO (Fig. 8) and microscopy (data not shown) reveal that relative  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: differences in fluorescence intensity between experimental right and fellow control left optic nerves are first evident at 4-5 hours after CTB injection (e.g., Fig. 8(a)), while differences between experimental left and fellow control right superior colliculi are first evident 6 hours after injection (e.g., Fig. 8(e)). Figure 9 shows the data for all animals and time points evaluated in this experiment by plotting relative fluorescence intensity (injected side relative to non-injected side) versus time after unilateral intravitreal injection. Figure 9 demonstrates that after a brief lag, relative fluorescence intensity begins to increase within the optic nerve ( Fig. 9(a)) and superior colliculus ( Fig. 9(b)) approaching a plateau at 48 hours. As expected, the lag is longer for the colliculus than the optic nerve. We followed two approaches for quantitative analysis of the early time point data series. First, we applied statistical analysis of each time point to determine when fluorescence was first detectable above the 'background' level in the ipsilateral optic nerve and contralateral superior colliculus (using a one sample t-test with a theoretical mean for comparison to zero). Though relative fluorescence of the ipsilateral optic nerve was already increasing at the earliest time point (2 hours, Fig. 9(a)) relative to naïve controls, it was first significantly greater than the contralateral optic nerve (non-injected eye) 4 hours after CTB injection (p = 0.021). Relative fluorescence of the contralateral superior colliculus was first significantly greater than the opposing side (ipsilateral superior colliculus) 6 hours after CTB injection (p = 0.022, Fig. 9(b)).
We also used the results of a nonlinear regression to determine the time that relative fluorescence intensity first began to increase above background (X 0 ). The potential advantage of this latter approach is to be unconstrained by the specific experimental time points chosen a priori for animal sacrifice. Using the equation Y = IF(X < X 0 , Y 0 ,Y 0 + (Plateau-Y 0 )*(1exp(-K*(X -X 0 )))), the X 0 parameter was determined to be 1.6 hours after injection for the optic nerve and 5.5 hours for the superior colliculus. The results of the nonlinear regression are also shown in Fig. 9.
Thus, the two quantitative approaches provide a similar result of 5.5 to 6 hours to reach the superior colliculus (20 mm from the eye), so the estimate for the bulk rate of anterograde axonal transport of CTB is 80-90 mm/day.
The same analysis methods were applied to post-mortem preparations of optic nerve and superior colliculi and analyzed with microscopy (e.g., Figs. 2(f) and 2(h), respectively). 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 Although the relative fluorescence intensity data obtained by CSLO and microscopy showed a good correlation for both the optic nerves (Spearman r = 0.983, p < 0.001) and superior colliculi (Spearman r = 0.817, p = 0.011), the sensitivity of the CSLO method was superior due to higher contrast and lower variance (data not shown).

Rate of retrograde axonal transport
Because CTB fluorescence within the eye can be evaluated in vivo by CSLO after retrograde transport from the superior colliculus, the experiment to estimate the bulk rate of retrograde axonal transport could be accomplished using longitudinal imaging (rather than by sacrificing groups of animals at specific time points such as in the previous experiment on anterograde transport rate). We imaged the ocular fundus by CSLO every 15-30 min after CTB injection into the superior colliculus. Figure 10 provides an overview of the results for one individual representative example. We found that the first sign of increased fluorescence (above background 'noise' and autofluorescence) typically occurred at the optic disc within 2.5 to 3.5 hours (in 17 out of 20 eyes, see, e.g., Figs. 10(b), 10(e)), representing a fast axonal transport rate of 136 to 191 mm per day (159 mm per day on average). Optic disc fluorescence might have been brighter than adjacent retinal nerve fiber bundle fluorescence because the axons (and their CTB concentration) are being viewed axially rather than transversely within the disc and because the background (autofluorescence) of the retinal pigment epithelium is stronger than that of the optic nerve head. Following optic disc fluorescence, individual RGCs generally begin to exhibit fluorescence above background 3 to 4 hours (Figs. 10(c), 10(f)) after CTB injection (in 15 of 20 eyes) and reach full coverage and brightness at 24 to 48 hours. Occasionally CTB fluorescence was detected in the RGC somas before the optic disc (in 3 out of 20 eyes), but given the relatively short amount of time between appearance at the optic disc and appearance in the retina (generally 0.5h), this variability may not be surprising; it depends on the degree of (radial) axon bundle filling for the fastest CTB transported as well as the relative image quality of the optic disc margin versus the surrounding retina.
Post-mortem microscopy of the retinal flat-mounts (Figs. 10(g), 10(h)) confirmed the results obtained in vivo by CSLO, with CTB fluorescence detected in all 20 eyes when compared to the six naïve control retinas. The retrograde injection was successful as judged by post-mortem CSLO imaging of the superior colliculi in all replicates (e.g., Fig. 10(i)), but with varying coverage and intensity, which probably contributed a degree of variability in the rate estimates.

Retrograde transport of latex microspheres and Di-O
To determine if microbeads or carbocyanine dye could serve as suitable alternatives for RGC labeling (and/or to be used in the axonal transport assay), bilateral injections of green retrobeads IX and Di-O dye into the superior colliculi were performed and the fundi were subsequently monitored in vivo by CSLO for retrograde transport. For both tracers, RGC fluorescence was much slower to accumulate (at least 3-4 times slower) as compared with CTB, though it also persisted longer than CTB, for up to 15 weeks. However, for both tracers only a limited region of the retina showed positive RGC fluorescence, and post-mortem CSLO of the superior colliculi revealed limited diffusion at the injection sites. This lack of diffusion away from the injection site has been previously reported in latex microbeads [66,67] and carbocyanine dyes [65]. The results indicate that microbeads and Di-O are not appropriate markers for the axon transport assay as transport is only able to be assessed for a small minority of RGC axons corresponding to the precise injection site rather than the majority of axons in the optic nerve, plus the transport rates are slower than for CTB. Given these results, we did not subsequently test colchicine dependence of either potential tracer.