May 2011
Volume 52, Issue 6
Free
Glaucoma  |   May 2011
Dynamic Imaging of Axonal Transport in Living Retinal Ganglion Cells In Vitro
Author Affiliations & Notes
  • Yuji Takihara
    From the Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto City, Japan;
  • Masaru Inatani
    From the Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto City, Japan;
  • Hideki Hayashi
    the Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto City, Japan;
  • Naoki Adachi
    the Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan; and
  • Keiichiro Iwao
    the Department of Ophthalmology, Faculty of Medicine, Saga University, Saga City, Japan.
  • Toshihiro Inoue
    From the Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto City, Japan;
  • Minako Iwao
    From the Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto City, Japan;
  • Hidenobu Tanihara
    From the Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto City, Japan;
  • Corresponding author: Masaru Inatani, Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, 1-1-1, Honjo, Kumamoto City, 860-8556, Japan; inatani@kumamoto-u.ac.jp
Investigative Ophthalmology & Visual Science May 2011, Vol.52, 3039-3045. doi:10.1167/iovs.10-6435
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      Yuji Takihara, Masaru Inatani, Hideki Hayashi, Naoki Adachi, Keiichiro Iwao, Toshihiro Inoue, Minako Iwao, Hidenobu Tanihara; Dynamic Imaging of Axonal Transport in Living Retinal Ganglion Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(6):3039-3045. doi: 10.1167/iovs.10-6435.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Purpose. Disruption of the axonal transport of neurotrophic factors plays a critical role in the apoptosis of retinal ganglion cells (RGCs) in glaucomatous optic neuropathy. Live-cell imaging in vitro was used in this study, to visualize and evaluate the axonal transport of brain-derived neurotrophic factor (BDNF) in both living and axon-damaged RGCs.

Methods. Rat RGCs were purified by a two-step immunopanning method. Cultivated RGCs were transfected with a plasmid encoding BDNF tagged with green fluorescent protein (GFP), and the dynamics of BDNF-GFP in the axons and dendrites were analyzed by time-lapse imaging. Changes in the axonal transport of BDNF-GFP were examined after treatment with 1 mM colchicine, and RGC death after treatment was evaluated with ethidium homodimer-1.

Results. The expression of BDNF-GFP showed a vesicular pattern in the axons and dendrites of cultivated RGCs. Time-lapse imaging revealed that the mean velocity of BDNF-GFP in the axons (0.86 ± 0.37 μm/s; maximum = 2.03 μm/s) was significantly greater (P < 0.0001) than that in the dendrites (mean = 0.49 ± 0.19 μm/s). Colchicine significantly inhibited the axonal transport of BDNF-GFP at 2 and 3 hours after treatment (P = 0.003 and 0.0002, respectively) without affecting cell viability; however, RGC death was detected 24 hours after treatment.

Conclusions. Live-cell imaging revealed the dynamics of the axonal transport of BDNF in living RGCs, which clearly differed from the movements in dendrites. Furthermore, it was possible to confirm the disruption of axonal transport in colchicine-treated RGCs before cell death.

Optic nerve injury leads to the death of retinal ganglion cells (RGCs) through apoptosis. 1,2 RGC apoptosis after axonal injury has been attributed to disruption of the supply of neurotrophic factors from the axonal terminals to the cell bodies. In glaucomatous optic neuropathy, the depletion of neurotrophic factors caused by axonal damage has been suggested as a mechanism of RGC apoptosis. 3,4 Several histologic studies using radioactive materials showed that elevated intraocular pressure (IOP) disrupts the axonal transport of RGCs in monkeys. 5, 7 Brain-derived neurotrophic factor (BDNF) is thought to be depleted in RGC bodies in glaucomatous optic neuropathy. 8,9 Histologic studies revealed that acute and chronic IOP elevation in glaucomatous animal models interrupted the retrograde axonal transport of radiolabeled BDNF in RGCs. 10,11 By contrast, exogenous BDNF administration provided partial neuroprotection to RGCs in rat chronic hypertensive eyes after the cauterization of episcleral vessels. 12 Furthermore, overexpression of the BDNF gene in RGCs transfected with an adeno-associated viral vector had a neuroprotective effect in an experimental model of glaucoma. 13 Understanding BDNF dynamics in the axons of living RGCs might thus help to predict the viability of axon-damaged RGCs and detect conditions that threaten their survival in glaucoma patients. However, so far, investigations of axonal transport in RGCs have been limited to observations made using fixed sections. To our knowledge, no live-imaging data on the axonal transport of BDNF in RGCs have been published. 
In the present study, we investigated the dynamics of the axonal transport of BDNF in both living and axon-damaged RGCs. To visualize the axonal transport of BDNF in living RGCs, we transfected purified rat RGCs with a plasmid encoding BDNF tagged with green fluorescent protein (GFP) in vitro. Our live-cell imaging system revealed the dynamics of the axonal transport of BDNF in living RGCs, which significantly differed from those in the dendrites, and was able to detect disruption of this activity in colchicine-treated RGCs before their death. 
Methods
Preparation of RGCs and Transfection of Plasmid cDNA
As previously described, 14,15 RGCs from 3-day-old Sprague-Dawley rats were purified by a two-step immunopanning procedure with anti-macrophage antibodies (Accurate Chemical and Scientific Corp., Westbury, NY) and anti-Thy 1.1 antibodies derived from T11D7e2 cells (ATCC, Manassas, VA). This method has been reported to yield RGCs with >99.5% purity. 15 The experimental procedures used were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The RGCs were plated at a low density (7000 cells/cm2) on dishes coated with poly-d-lysine (Sigma-Aldrich, St. Louis, MO) and laminin (Invitrogen, Carlsbad, CA). As described previously, 16 the RGCs were cultured in defined, serum-free medium (Neurobasal; Invitrogen) without phenol red, BDNF (50 ng/mL; PeproTech, Rocky Hill, NJ), ciliary neurotrophic factor (50 ng/mL; PeproTech), basic fibroblast growth factor (50 ng/mL; PeproTech), forskolin (10 μM; Sigma-Aldrich), B27 supplement (Invitrogen), glutamine, insulin, sodium pyruvate, Sato medium, triiodothyronine, and N-acetylcysteine. The medium did not include antibiotics to reduce the cellular toxicity associated with the plasmid transfection. To support the RGCs, one half of the medium was replaced with a mixture of fresh medium and glia-conditioned medium every 3 days. The glia-conditioned medium was obtained from cerebral glial cells that were prepared as published elsewhere. 17 After 12 days in vitro, the RGCs were transfected with the plasmid cDNA of mouse recombinant BDNF tagged with GFP at the C terminus (BDNF-GFP) provided by Masami Kojima. 18 The BDNF cDNA was inserted into the multiple cloning site of the EGFP-N1 vector (Clontech, Palo Alto, CA). The EGFP-N1 vector without BDNF was used as the control plasmid for the transfection. Transfection of the BDNF-GFP cDNA was performed with transfection reagent, according to the manufacturer's protocols (Lipofectamine 2000; Invitrogen). 
Immunolabeling
The RGCs were fixed by treatment with 4% paraformaldehyde for 20 minutes at room temperature, and were incubated with 0.2% Triton-X in phosphate-buffered saline (PBS) for 5 minutes. The RGCs were blocked by 10% goat serum in PBS for 1 hour at 37°C. The following antibodies were applied overnight at 4°C: monoclonal antibody to τ-1 (1:500; Millipore); polyclonal antibody to MAP2 (1:4000, Millipore); polyclonal antibody to GFP (1:200; Invitrogen); polyclonal antibody to synapsin I (1:500; Millipore); and monoclonal antibody to MAP2 (1:1000, Sigma-Aldrich). The RGCs were then incubated with anti-rabbit secondary antibody conjugated with Alexa 488 (1:2000, Invitrogen) and anti-mouse secondary antibody conjugated with Alexa 594 (1:2000, Invitrogen) for 1 hour at room temperature. 
Time-Lapse Imaging
Time-lapse imaging was conducted for RGCs in which dynamics of BDNF-GFP vesicles could be evaluated using a cooled charged-coupled device camera (C10600-10B; Hamamatsu Photonics, Hamamatsu, Japan, on the AS MDW system; Leica, Deerfield, IL) after 30 hours of transfection. Image-analysis software (Metamorph; MDS Analytical Technologies, Downingtown, PA) was used to obtain and analyze the data. To study the axonal transport of BDNF-GFP, sequential images were acquired using the 63× or 100× objectives of an inverted microscope at 5-second intervals in a chamber kept at 37°C and 5% CO2. To evaluate the effects of 50 mM KCl, the time-lapse imaging was performed at 1-minute intervals before and after the treatment. To evaluate the effects of 1 mM colchicine (Sigma-Aldrich), the time-lapse imaging was carried out at 5-second intervals for 3 minutes before the treatment, and at 1, 2, and 3 hours after treatment. Colchicine diluted in serum-free medium without phenol red (1 mM final concentration; Neurobasal; Invitrogen) was added through a custom-made cover of culture dishes with an openable hole. Controls were treated with serum-free medium without phenol red. We evaluated the effects of colchicine on axonal transport by counting the number of BDNF-GFP vesicles passing across a line plotted to mark the middle of the axons in the images displayed on screen, in a masked fashion, as previously described. 19 The cell viability was evaluated 20 minutes after the application of ethidium homodimer-1 (1 μM) of a live/dead cell assay kit (Invitrogen). Ethidium homodimer-1 is not taken up by living cells; it can enter cells only through damaged membranes and binds to nucleic acids, thereby producing a bright red fluorescence. Statistical analyses were performed with Mann-Whitney nonparametric test to compare the velocity of axonal transport and movements in dendrites, and the Student's t-test to compare the passage of BDNF-GFP vesicles between negative controls and RGCs treated with colchicine. 
Results
Low-Density Culture System for Live Imaging of Axonal Transport in RGCs
In high-density cultures, it is difficult to differentiate axons from dendrites and to confirm the origin of axons, because they often overlap one another. The live imaging of axonal transport in RGCs thus requires a low-density culture system in addition to high-yield purification. Previously, Brewer et al. 20 reported on the survival and growth of hippocampal neurons at densities as low as 5000 cells/cm2. We plated RGCs at a density of 7000 cells/cm2 and stained them using antibodies against τ-1 (an axonal marker) and MAP2 (a somatodendritic marker) to distinguish axons and dendrites. As shown in Figures 1A–C, a distinct axon was stained by the anti-τ-1 antibody and was negative for MAP2, indicating that an axon from an RGC could be differentiated from dendrites in this low-density culture. 
Figure 1.
 
Distinct axons and synapse formation in a low-density RGC culture. (AC) RGC axons could be differentiated from dendrites in the low-density culture. The distinct axon (arrows) of an RGC was stained by anti-τ-1 antibody (red) but was negative for MAP2 (green). (DF) RGCs formed synapses in the low-density culture. An axon that originating from the left side of the images included vesicles that stained positive for synapsin I (green). The vesicles formed synapses (arrows) where the axon came into contact with the cell body and a dendrite of another RGC that stained positive for MAP2 (red). Scale bars, 10 μm.
Figure 1.
 
Distinct axons and synapse formation in a low-density RGC culture. (AC) RGC axons could be differentiated from dendrites in the low-density culture. The distinct axon (arrows) of an RGC was stained by anti-τ-1 antibody (red) but was negative for MAP2 (green). (DF) RGCs formed synapses in the low-density culture. An axon that originating from the left side of the images included vesicles that stained positive for synapsin I (green). The vesicles formed synapses (arrows) where the axon came into contact with the cell body and a dendrite of another RGC that stained positive for MAP2 (red). Scale bars, 10 μm.
The survival of RGCs is highly dependent on their density. 16 To visualize the dynamics of axonal BDNF transport in RGCs, it is necessary for them to be in good condition and to form synapses, because this factor is thought to be transported to the nerve terminals and released in an activity-dependent manner. 18,21,22 Using a low-density culture, we stained RGCs with an anti-MAP2 antibody and an antibody against synapsin I, which is a marker of presynaptic vesicles in axons. As shown in Figures 1E and 1F, the cell body and dendrites of a RGC were stained with the anti-MAP2 antibody. Then, synapsin I-positive vesicles were found in the MAP2-positve somatodendritic region (Figs. 1D, 1F), indicating that the dendrites formed synapses with an axon originating from another RGC. The data confirmed that RGCs could interact with each other and form synapses in the low-density culture. 
Properties of BDNF-GFP Expressed in RGCs
Next, we examined whether RGCs could express BDNF-GFP in a vesicular pattern in axons, similar to endogenous BDNF, which would allow us to evaluate the dynamics of axonal transport. 23 RGCs transfected with plasmid cDNA for BDNF-GFP were stained using anti-GFP and -MAP2 antibodies. As shown in Figures 2A and 2B, an axon negative for MAP2 included BDNF-GFP which was positive for GFP. Fluorescein-positive vesicles were seen in the axon (Fig. 2C). This result suggested that BDNF-GFP was expressed in a vesicular pattern in the axons of RGCs. 
Figure 2.
 
Vesicular pattern of BDNF-GFP expression in axons of RGCs and response to depolarization. (AC) An axon negative for MAP2 (red) included BDNF-GFP, which was stained by anti-GFP antibody (A, green; arrows and box). Magnification of the area shown in the box revealed that BDNF-GFP was expressed in a vesicular pattern. (D, E) Time-lapse imaging (at 1-minute intervals) of the release of BDNF-GFP after treatment with 50 mM KCl. Magnification of the area shown in the box revealed that the BDNF-GFP vesicles were stable before treatment with KCl, but decreased gradually after the treatment. Many BDNF-GFP vesicles disappeared, although some persisted. This confirmed that the z coordinate remained the same during the recording. Scale bars: (A, B, D, E) 5 μm; (C) 1 μm.
Figure 2.
 
Vesicular pattern of BDNF-GFP expression in axons of RGCs and response to depolarization. (AC) An axon negative for MAP2 (red) included BDNF-GFP, which was stained by anti-GFP antibody (A, green; arrows and box). Magnification of the area shown in the box revealed that BDNF-GFP was expressed in a vesicular pattern. (D, E) Time-lapse imaging (at 1-minute intervals) of the release of BDNF-GFP after treatment with 50 mM KCl. Magnification of the area shown in the box revealed that the BDNF-GFP vesicles were stable before treatment with KCl, but decreased gradually after the treatment. Many BDNF-GFP vesicles disappeared, although some persisted. This confirmed that the z coordinate remained the same during the recording. Scale bars: (A, B, D, E) 5 μm; (C) 1 μm.
To check whether the BDNF-GFP expressed in RGCs was released in an activity-dependent manner, we studied the response to depolarization with 50 mM KCl. We obtained sequential images of the BDNF-GFP vesicles before and after the application of KCl (Figs. 2D, 2E). The intensity of the BDNF-GFP vesicles was stable before the treatment, but decreased gradually after depolarization with KCl. 
To exclude the possibility that BDNF translated from the plasmid modified the RGC shape or the condition, we examined the comparison of cell morphology between RGCs transfected with empty vector control plasmids (Figs. 3A–D) and BDNF-GFP-transfected RGCs (Figs. 3E–H). Consistent with the previous report, 18 GFP without BDNF was diffusely expressed in cell structure including axons (Figs. 3A, 3C, arrows) in a smearlike pattern. The BDNF-GFP-transfected RGCs exhibited fluorescein signals in their cell structure, whereas the signals in the axon were weaker than those in the cell body. Immunocytochemistry confirmed that no apparent morphologic difference was observed between the control GFP plasmid-transfected RGCs and BDNF-GFP-plasmid transfected RGCs. 
Figure 3.
 
Comparison of the control GFP plasmid-transfected RGCs with BDNF-GFP plasmid-transfected RGCs. (AD) GFP without BDNF was diffusely expressed in cell structure including axons (arrows) in a smearlike pattern. (EH) The BDNF-GFP-transfected RGCs exhibited fluorescein signals in their cell structure, whereas the signals in the axon (arrows) were weaker than those in the cell body. No apparent morphologic difference was observed between the control GFP plasmid-transfected RGCs and BDNF-GFP-plasmid transfected RGCs. Scale bar, 20 μm.
Figure 3.
 
Comparison of the control GFP plasmid-transfected RGCs with BDNF-GFP plasmid-transfected RGCs. (AD) GFP without BDNF was diffusely expressed in cell structure including axons (arrows) in a smearlike pattern. (EH) The BDNF-GFP-transfected RGCs exhibited fluorescein signals in their cell structure, whereas the signals in the axon (arrows) were weaker than those in the cell body. No apparent morphologic difference was observed between the control GFP plasmid-transfected RGCs and BDNF-GFP-plasmid transfected RGCs. Scale bar, 20 μm.
Live Imaging of Axonal Transport in Purified RGCs
To analyze the dynamics of the BDNF-GFP vesicles in the axons, we conducted time-lapse imaging of transfected RGCs in a low-density culture. A BDNF-GFP vesicle was seen to move in a retrograde direction (Figs. 4A, 4B). By contrast, a BDNF-GFP vesicle was seen to move in an anterograde direction (Figs. 4C, 4D). Time-lapse images of the axonal transport in both retrograde and anterograde directions are shown in Supplementary Movies S1 and S2, respectively. The axonal transport was compared with the movements of BDNF-GFP vesicles in dendrites (Figs. 4E, 4F). 
Figure 4.
 
Time-lapse imaging (at 5-second intervals) of the axonal transport of BDNF-GFP and its movements in dendrites. (A, B) An example of retrograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the distal side to the proximal side. (C, D) An example of anterograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the proximal side to the distal side. (E, F) An example of movement of BDNF-GFP in a dendrite. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported in a dendrite with a diameter that clearly differed from that of axons. Scale bars, 5 μm.
Figure 4.
 
Time-lapse imaging (at 5-second intervals) of the axonal transport of BDNF-GFP and its movements in dendrites. (A, B) An example of retrograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the distal side to the proximal side. (C, D) An example of anterograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the proximal side to the distal side. (E, F) An example of movement of BDNF-GFP in a dendrite. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported in a dendrite with a diameter that clearly differed from that of axons. Scale bars, 5 μm.
Dynamics of Axonal Transport in Purified RGCs
To determine the patterns of movement of BDNF-GFP vesicles in axons and dendrites, we classified them into four groups, as previously described 24 : Vesicles that moved in an anterograde direction for >10 μm; vesicles that moved in a retrograde direction for >10 μm; vesicles that did not move; and vesicles that fluttered ≤10 μm. The proportions of BDNF-GFP vesicles in each category, in axons and dendrites, are shown in Figures 5A and 5B, respectively. In the axons, 62.5% of the vesicles moved in either direction (Fig. 5A), whereas only 29.2% of the vesicles moved in either direction in the dendrites (Fig. 5B). The velocities of BDNF-GFP vesicles in axons and in dendrites were compared (Fig. 5C). The mean velocity of BDNF-GFP vesicles in the axons (0.86 ± 0.37 μm/s) was significantly greater (P < 0.0001) than that in the dendrites (0.49 ± 0.19 μm/s). There were no significant differences between the velocities of anterograde and retrograde transport in the axons (P = 0.96). Figure 5D shows the distribution of the BDNF-GFP transport velocity in the axons of RGCs. The RGCs included BDNF-GFP vesicles with a velocity >1.2 μm/s, which was the maximum reported for axonal transport in neurons from the visual cortex. 24 Figure 5E and Supplementary Movie S3, show sequential images of the fastest axonal transport recorded in this study, the velocity of which was 2.03 μm/s. 
Figure 5.
 
The dynamics of BDNF-GFP vesicles in axons and dendrites. (A, B) The proportion of BDNF-GFP vesicles in the four categories described in the text, in axons (A, n = 112) and dendrites (B, n = 72). (C) Comparison of the velocities of BDNF-GFP vesicles in axons and in dendrites. *P < 0.0001. (D) Histogram of the velocity of BDNF-GFP axonal transport. RGCs contained vesicles with a velocity >1.2 μm/s, which was the maximum reported in neurons from the visual cortex. (E) Sequential images of the fastest axonal transport (arrows) in the study (velocity, 2.03 μm/s). Scale bar, 20 μm.
Figure 5.
 
The dynamics of BDNF-GFP vesicles in axons and dendrites. (A, B) The proportion of BDNF-GFP vesicles in the four categories described in the text, in axons (A, n = 112) and dendrites (B, n = 72). (C) Comparison of the velocities of BDNF-GFP vesicles in axons and in dendrites. *P < 0.0001. (D) Histogram of the velocity of BDNF-GFP axonal transport. RGCs contained vesicles with a velocity >1.2 μm/s, which was the maximum reported in neurons from the visual cortex. (E) Sequential images of the fastest axonal transport (arrows) in the study (velocity, 2.03 μm/s). Scale bar, 20 μm.
Effects of Colchicine on Axonal Transport in Purified RGCs
To detect changes in the axonal transport of BDNF-GFP vesicles caused by axonal damage before RGC death, we analyzed the effects of colchicine, which disrupts the assembly and function of microtubules. 19,25, , , 29 The relative number of BDNF-GFP vesicles passing through the middle of the imaged axons (standardized to the number of passing vesicles before the application of colchicine) significantly decreased at 2 and 3 hours after treatment with 1 mM colchicine, compared with that in the negative controls (P = 0.003 and 0.0002, respectively; Fig. 6A). The BDNF-GFP vesicles remained mobile 3 hours after the application of 1 mM colchicine, but most could not move smoothly (Fig. 6B and Supplementary Movie S4). The viability of these RGCs was then evaluated using ethidium homodimer-1. All four of the RGCs treated with colchicine and all four of the negative controls were negative for ethidium homodimer-1 3 hours after the treatment. By contrast, at 24 hours after treatment, all four of the RGCs treated with colchicine were positive for ethidium homodimer-1, whereas all four of negative controls remained unstained. These results suggest that axonal transport started to decrease before the death of the RGCs. 
Figure 6.
 
Effect of colchicine on axonal transport of BDNF-GFP vesicles. (A) The relative number of BDNF-GFP vesicles passing through the middle of axons in the images (standardized to the number of passing vesicles before the application of colchicine) was significantly reduced at 2 and 3 hours after treatment with colchicine (n = 4, □), compared with negative controls (n = 4, ■; *P = 0.003 and †P = 0.0002, respectively). (B) Representative sequential images of BDNF-GFP vesicles in an axon 3 hours after treatment with 1 mM colchicine. The BDNF-GFP vesicle (arrows) 3 hours after treatment remained mobile but could not move smoothly. Scale bar, 5 μm.
Figure 6.
 
Effect of colchicine on axonal transport of BDNF-GFP vesicles. (A) The relative number of BDNF-GFP vesicles passing through the middle of axons in the images (standardized to the number of passing vesicles before the application of colchicine) was significantly reduced at 2 and 3 hours after treatment with colchicine (n = 4, □), compared with negative controls (n = 4, ■; *P = 0.003 and †P = 0.0002, respectively). (B) Representative sequential images of BDNF-GFP vesicles in an axon 3 hours after treatment with 1 mM colchicine. The BDNF-GFP vesicle (arrows) 3 hours after treatment remained mobile but could not move smoothly. Scale bar, 5 μm.
Discussion
In the present study, we used live imaging to quantify the dynamics of the axonal transport of BDNF-GFP in living RGCs and demonstrated a reduction in this activity before the death of RGCs with disrupted microtubules. The data revealed clear differences compared with the movements of BDNF-GFP in dendrites. Previous studies have shown dysfunction of the retrograde axonal transport of BDNF using fixed sections in animal glaucoma models. 10,11 However, this is the first report to quantify the dynamics of axonal transport in living and RGCs with disrupted microtubules. 
The live-cell imaging in the present study demonstrated a vesicular pattern of GFP-tagged BDNF in the axons as well as the dendrites, which was consistent with the results reported previously in hippocampal neurons. 18 Endogenous BDNF is shuttled from the trans-Golgi network into a regulated secretory pathway. 30,31 In addition, we showed that BDNF-GFP expressed in RGCs was released in a depolarization-dependent manner, as previously reported in hippocampal neurons. 18,21 Taken together, our results indicate that expressed BDNF-GFP in RGCs mimics the properties of endogenous BDNF. 
We have shown that the mean velocity of BDNF-GFP vesicles was faster in axons (0.86 ± 0.37 μm/s) than in dendrites (0.49 ± 0.19 μm/s). The mean velocity of the BDNF-GFP vesicles in the axons in the present study was similar to that reported previously for cortical neurons in vitro (0.73 ± 0.26 μm/s). 24 The differences in the movement of BDNF-GFP vesicles between axons and dendrites might result from differences in the polarity of microtubules: those in axons are unipolar with a fast-growing plus end toward the axonal terminal, whereas those in dendrites have mixed polarity. 24,32,33 The RGC axons showed a BDNF transport velocity >1.2 μm/s, which was the maximum reported in neurons from the visual cortex. 24 The v max (2.03 μm/s) of BDNF-GFP in our study, which corresponded to 175 mm/d, was compatible with a previous report on the velocity of fast axonal transport. 34 The motor proteins responsible for axonal transport of BDNF in RGCs have not yet been identified, and the v max data in our study might facilitate their identification in future studies. 
Previous papers have suggested that axonal damage contributes to the pathomechanism of glaucoma. 35, , , , , , , 43 Furthermore, axonal damage induced by elevated IOP disrupts the axonal transport of BDNF, resulting in the apoptosis of RGCs. 10,11 We therefore examined whether live-cell imaging could detect the disruption of the axonal transport of BDNF-GFP in colchicine-treated RGCs before death. Colchicine is a well-established drug that inhibits microtubule polymerization and causes severe pathologic changes in axons. 19,25, , , 29 Our finding that 1 mM colchicine significantly inhibited the axonal transport of BDNF-GFP at 2 and 3 hours after treatment was comparable to the results of a previous report, which showed that colchicine caused a significant loss of axonal microtubules 1 hour after application and completely inhibited axonal transport by 3 to 5 hours after treatment. 19 A previous study that used electron microscopy to examine axons treated with 1 mM colchicine indicated a progressive decrease of axonal microtubules, such that ∼70% had disappeared by 3 hours after treatment. 26 Our present study also showed that the disrupted axonal transport of BDNF-GFP was not associated with ethidium homodimer-1-positive cell death at 3 hours after the application of 1 mM colchicine, but that all the RGCs treated with 1 mM colchicine had died by 24 hours after treatment, whereas those without colchicine remained alive. Our results might suggest that the live-cell imaging of the axonal transport of BDNF-GFP in RGCs should predict cell death after axonal damage. 
We did not explore the dynamic imaging of the axonal transport of BDNF-GFP in experimental glaucoma animal models in vivo. However, this approach may be useful for predicting the future loss of RGCs in glaucomatous optic neuropathy. The disruption of axonal transport could reflect the existence of conditions that threaten the survival of RGCs. The dynamic imaging of axonal transport could thus help to determine whether further IOP reduction is desirable in glaucoma patients. Future in vivo studies are necessary before the clinical application of the dynamic imaging of axonal transport for glaucomatous optic neuropathy. 
In conclusion, live-cell imaging using BDNF-GFP allowed us to demonstrate and evaluate the dynamics of the axonal transport of BDNF in living RGCs, and to detect reduced axonal transport in colchicine-treated RGCs before death. This tool may be useful for predicting the viability of RGCs after axonal damage. 
Supplementary Materials
Movie sm01, MOV - Movie sm01, MOV 
Movie sm02, MOV - Movie sm02, MOV 
Movie sm03, MOV - Movie sm03, MOV 
Movie sm04, MOV - Movie sm04, MOV 
 
 
The authors thank Wen-Jie Song for valuable discussion, and Yuko Nakaishi-Fukuchi and Chika Naito for excellent technical assistance. 
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Figure 1.
 
Distinct axons and synapse formation in a low-density RGC culture. (AC) RGC axons could be differentiated from dendrites in the low-density culture. The distinct axon (arrows) of an RGC was stained by anti-τ-1 antibody (red) but was negative for MAP2 (green). (DF) RGCs formed synapses in the low-density culture. An axon that originating from the left side of the images included vesicles that stained positive for synapsin I (green). The vesicles formed synapses (arrows) where the axon came into contact with the cell body and a dendrite of another RGC that stained positive for MAP2 (red). Scale bars, 10 μm.
Figure 1.
 
Distinct axons and synapse formation in a low-density RGC culture. (AC) RGC axons could be differentiated from dendrites in the low-density culture. The distinct axon (arrows) of an RGC was stained by anti-τ-1 antibody (red) but was negative for MAP2 (green). (DF) RGCs formed synapses in the low-density culture. An axon that originating from the left side of the images included vesicles that stained positive for synapsin I (green). The vesicles formed synapses (arrows) where the axon came into contact with the cell body and a dendrite of another RGC that stained positive for MAP2 (red). Scale bars, 10 μm.
Figure 2.
 
Vesicular pattern of BDNF-GFP expression in axons of RGCs and response to depolarization. (AC) An axon negative for MAP2 (red) included BDNF-GFP, which was stained by anti-GFP antibody (A, green; arrows and box). Magnification of the area shown in the box revealed that BDNF-GFP was expressed in a vesicular pattern. (D, E) Time-lapse imaging (at 1-minute intervals) of the release of BDNF-GFP after treatment with 50 mM KCl. Magnification of the area shown in the box revealed that the BDNF-GFP vesicles were stable before treatment with KCl, but decreased gradually after the treatment. Many BDNF-GFP vesicles disappeared, although some persisted. This confirmed that the z coordinate remained the same during the recording. Scale bars: (A, B, D, E) 5 μm; (C) 1 μm.
Figure 2.
 
Vesicular pattern of BDNF-GFP expression in axons of RGCs and response to depolarization. (AC) An axon negative for MAP2 (red) included BDNF-GFP, which was stained by anti-GFP antibody (A, green; arrows and box). Magnification of the area shown in the box revealed that BDNF-GFP was expressed in a vesicular pattern. (D, E) Time-lapse imaging (at 1-minute intervals) of the release of BDNF-GFP after treatment with 50 mM KCl. Magnification of the area shown in the box revealed that the BDNF-GFP vesicles were stable before treatment with KCl, but decreased gradually after the treatment. Many BDNF-GFP vesicles disappeared, although some persisted. This confirmed that the z coordinate remained the same during the recording. Scale bars: (A, B, D, E) 5 μm; (C) 1 μm.
Figure 3.
 
Comparison of the control GFP plasmid-transfected RGCs with BDNF-GFP plasmid-transfected RGCs. (AD) GFP without BDNF was diffusely expressed in cell structure including axons (arrows) in a smearlike pattern. (EH) The BDNF-GFP-transfected RGCs exhibited fluorescein signals in their cell structure, whereas the signals in the axon (arrows) were weaker than those in the cell body. No apparent morphologic difference was observed between the control GFP plasmid-transfected RGCs and BDNF-GFP-plasmid transfected RGCs. Scale bar, 20 μm.
Figure 3.
 
Comparison of the control GFP plasmid-transfected RGCs with BDNF-GFP plasmid-transfected RGCs. (AD) GFP without BDNF was diffusely expressed in cell structure including axons (arrows) in a smearlike pattern. (EH) The BDNF-GFP-transfected RGCs exhibited fluorescein signals in their cell structure, whereas the signals in the axon (arrows) were weaker than those in the cell body. No apparent morphologic difference was observed between the control GFP plasmid-transfected RGCs and BDNF-GFP-plasmid transfected RGCs. Scale bar, 20 μm.
Figure 4.
 
Time-lapse imaging (at 5-second intervals) of the axonal transport of BDNF-GFP and its movements in dendrites. (A, B) An example of retrograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the distal side to the proximal side. (C, D) An example of anterograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the proximal side to the distal side. (E, F) An example of movement of BDNF-GFP in a dendrite. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported in a dendrite with a diameter that clearly differed from that of axons. Scale bars, 5 μm.
Figure 4.
 
Time-lapse imaging (at 5-second intervals) of the axonal transport of BDNF-GFP and its movements in dendrites. (A, B) An example of retrograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the distal side to the proximal side. (C, D) An example of anterograde axonal transport of BDNF-GFP. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported from the proximal side to the distal side. (E, F) An example of movement of BDNF-GFP in a dendrite. Magnification of the area shown in the box revealed that the BDNF-GFP vesicle (arrows) was transported in a dendrite with a diameter that clearly differed from that of axons. Scale bars, 5 μm.
Figure 5.
 
The dynamics of BDNF-GFP vesicles in axons and dendrites. (A, B) The proportion of BDNF-GFP vesicles in the four categories described in the text, in axons (A, n = 112) and dendrites (B, n = 72). (C) Comparison of the velocities of BDNF-GFP vesicles in axons and in dendrites. *P < 0.0001. (D) Histogram of the velocity of BDNF-GFP axonal transport. RGCs contained vesicles with a velocity >1.2 μm/s, which was the maximum reported in neurons from the visual cortex. (E) Sequential images of the fastest axonal transport (arrows) in the study (velocity, 2.03 μm/s). Scale bar, 20 μm.
Figure 5.
 
The dynamics of BDNF-GFP vesicles in axons and dendrites. (A, B) The proportion of BDNF-GFP vesicles in the four categories described in the text, in axons (A, n = 112) and dendrites (B, n = 72). (C) Comparison of the velocities of BDNF-GFP vesicles in axons and in dendrites. *P < 0.0001. (D) Histogram of the velocity of BDNF-GFP axonal transport. RGCs contained vesicles with a velocity >1.2 μm/s, which was the maximum reported in neurons from the visual cortex. (E) Sequential images of the fastest axonal transport (arrows) in the study (velocity, 2.03 μm/s). Scale bar, 20 μm.
Figure 6.
 
Effect of colchicine on axonal transport of BDNF-GFP vesicles. (A) The relative number of BDNF-GFP vesicles passing through the middle of axons in the images (standardized to the number of passing vesicles before the application of colchicine) was significantly reduced at 2 and 3 hours after treatment with colchicine (n = 4, □), compared with negative controls (n = 4, ■; *P = 0.003 and †P = 0.0002, respectively). (B) Representative sequential images of BDNF-GFP vesicles in an axon 3 hours after treatment with 1 mM colchicine. The BDNF-GFP vesicle (arrows) 3 hours after treatment remained mobile but could not move smoothly. Scale bar, 5 μm.
Figure 6.
 
Effect of colchicine on axonal transport of BDNF-GFP vesicles. (A) The relative number of BDNF-GFP vesicles passing through the middle of axons in the images (standardized to the number of passing vesicles before the application of colchicine) was significantly reduced at 2 and 3 hours after treatment with colchicine (n = 4, □), compared with negative controls (n = 4, ■; *P = 0.003 and †P = 0.0002, respectively). (B) Representative sequential images of BDNF-GFP vesicles in an axon 3 hours after treatment with 1 mM colchicine. The BDNF-GFP vesicle (arrows) 3 hours after treatment remained mobile but could not move smoothly. Scale bar, 5 μm.
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