Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging
© Adachi et al; licensee BioMed Central Ltd. 2005
Received: 01 February 2005
Accepted: 21 June 2005
Published: 21 June 2005
Brain-derived neurotrophic factor (BDNF), which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP)-tagged BDNF in axons and dendrites of living cortical neurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP) was compared with that of nerve growth factor (NGF) tagged with yellow fluorescent protein (YFP), to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin.
We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD) μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s).
The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.
Neurotrophins have been considered to play roles in the differentiation, neurite outgrowth and survival of a certain group of neurons [1–4]. In addition to these well-known functions, most neurotrophins are involved in rapid changes in the function of neural circuits [5, 6]. In particular, brain-derived neurotrophic factor (BDNF) plays a role in activity-dependent changes in synaptic function [7–9].
To serve such a broad-ranging function, BDNF produced in the nucleus of neurons is sorted into a regulated secretory pathway through the trans-Golgi network, and transported to release sites in neurites [10, 11]. In axons it is suggested that BDNF is transported through the fast axonal flow and then released and transferred to postsynaptic neurons in an activity-dependent manner [10–15]. In dendrites or dendrite-like neurites also, the targeting of BDNF to distal parts and its release were suggested to occur in an activity-dependent manner [16–22]. Since these previous studies were carried out using immunohistochemical and/or in situ hybridization technique after the fixation of neurons, or by observing the decrease in fluorescence intensity of neurons expressing BDNF tagged with green fluorescent protein (GFP), the actual dynamics of BDNF trafficking was not analyzed in dendrites. Thus, a question of whether the trafficking of BDNF is different between axons and dendrites of neurons is not answered yet.
An answer to this question will give a clue that may resolve the controversial issue of whether BDNF acts retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. To address this question, it is desirable to perform a real-time analysis of movements of BDNF tagged with GFP in both axons and dendrites of living neurons. However, such an analysis of BDNF trafficking has not been successfully carried out except for two recent studies on its retrograde transport in dorsal root ganglion neurons  and bidirectional transport in cortical cell neurites . However, the observation was restricted to axons in the former study, and axons and dendrites were not distinguished in the latter study.
In the present study we carried out a real-time analysis of movements of BDNF tagged with GFP in both axons and dendrites of living cortical neurons using the method of direct injection of their plasmid cDNAs into the nucleus . We found that most of BDNF-GFP moves smoothly in the anterograde direction in axons while it does not move in dendrites in most cases. Even if it moves in dendrites, its velocity is slower than that in axons.
Parts of the present results were published in an abstract form .
Plasmids encoding BDNF tagged with GFP or CFP at the COOH-terminus were injected into the nucleus of cultured cortical neurons through a micropipette under visual control, as reported previously . GFP- or CFP-tagged BDNF resulting from these plasmids were confirmed to be biologically active and mimic the releasing properties of untagged BDNF . In the present experiments, 20–30% of the neurons into which plasmids had been injected expressed fluorescent signals, as reported previously . The expression of signals was already detected 16 h after the injection, but recordings were usually carried out about 24 h after the injection.
In the present study we attempted to inject plasmids into pyramidal cell-like neurons which are excitatory. To confirm that neurons expressing fluorescent signals were excitatory, they were stained immunocytochemically with anti-glutamic acid decarboxylase 65 (GAD65) antibody. As shown in Figure 1C and 1D, a neuron that expressed BDNF-GFP was not stained with the antibody. This was confirmed by the superposition of the two images (Fig. 1E). This result suggests that the BDNF-GFP-injected cell was an excitatory neuron. All of the 12 BDNF-GFP-injected neurons that were immunocytochemically tested were negative to anti-GAD65 antibody, indicating that the neurons analyzed in the present study were most likely excitatory. We also stained neurons by immunocytochemistry using anti-microtubule-associated protein 2 (MAP2) antibody to differentiate axons from dendrites. As shown in Figure 1F–I, we could identify axons on the basis of their negativity to anti-MAP2 antibody (arrowheads in Fig. 1F, H and 1I). Fluorescent signals were detected also in MAP2-negative neurites (Fig. 1H and 1I), indicating that BDNF-GFP was expressed in axons as well as somatodendritic regions.
As mentioned before, the single injection of plasmid cDNAs encoding GFP-, CFP- or YFP-tagged neurotrophins also showed the difference in the expression pattern between BDNF and NGF (Fig. 2E). In this analysis the proportion of cells with vesicular expression was calculated for each session of injection trials and then the mean values were calculated from 21 and 20 sessions of trials for fluorescent protein-tagged BDNF and NGF, respectively. The means ± SEMs were 71.2 ± 1.5 and 29.2 ± 5.0%, respectively. The difference between these two values was statistically significant (P <0.001, unpaired t-test). These results indicate that the granular expression of BDNF-GFP was not common to other neurotrophins, although we did not observe the expression of fluorescent protein-tagged neurotrophin-3 and neurotrophin-4/5 in the present study.
In the live-cell imaging of GFP- or CFP-tagged BDNF, we observed that most of fluorescent protein-tagged BDNF which were expressed as vesicular puncta in axons moved rapidly in the anterograde direction, as reported previously . It is possible to argue that because of the direct injection of the plasmids into the nucleus of neurons, the anterograde movements of BDNF-GFP from the soma were dominant. This possibility seems unlikely, however, because the movements of BDNF-GFP vesicles in axons were already observed 16 h after the injection and the vesicles were calculated to move over a distance of 20 mm by the observation time (24 h after the injection) assuming that the mean velocity of 0.73 μm/s was maintained. Thus, BDNF-GFP is expected to have reached terminals of neurites long before the observation time. Indeed we observed that a substantial proportion of vesicles were moving in the retrograde direction in axons and dendrites, as shown in Figure 4A.
BDNF-GFP vesicles were expressed in dendrites as well as in axons. The number of expressed vesicles in dendrites was much larger than that in axons. This difference seems to reflect the difference in areas between dendrites and axons. Thus, we did not find a strongly polarized expression of BDNF-GFP in either structural domain of neurons. In dendrites, however, the trafficking pattern of BDNF-GFP was markedly different from that in axons. Most of the BDNF-GFP vesicles in dendrites did not move at all or just fluttered within a small distance. Only a small number of puncta in dendrites moved continuously, while most of the puncta in axons moved smoothly over a long distance. There is a possibility that the directed movement of the fluorescent vesicles was not seen in dendrites, because BDNF-GFP had already arrived at dendritic sites where it was packed for release machinery. This possibility seems unlikely, since we did not observe a higher mobility of the puncta in dendrites at 16 h after the injection than at the standard observation time (24 h after the injection).
We further found that the velocity of transport of a minor group of BDNF-GFP puncta in dendrites was slower than that of BDNF-GFP puncta in axons. The mean velocity in axons (0.73 ± 0.26 μm/s) is comparable to the reported values for the anterograde transport of a synaptic vesicle protein, synaptophysin tagged with GFP, in the axons of mouse dorsal root ganglion cells (0.69 ± 0.33 μm/s, ) and of 125I-labelled BDNF in the optic nerve of neonatal rats (0.47–0.92 μm/s, ). The present observations that the velocity and the pattern of movements of BDNF-GFP in dendrites were different from those in axons seem consistent with the previous results that dendrites have microtubules of mixed polarity orientation, while axonal microtubules have a uniform polarity orientation, with their plus ends toward the axonal terminal  and that motor proteins, microtubule-based kinesin superfamily proteins (KIFs), are differentiated in axons and dendrites . The question of what kinds of KIFs or other motor proteins are responsible for the difference in the pattern of trafficking of BDNF-GFP between axons and dendrites will be clarified in future studies.
In the present study using the plasmid cDNA injection method, there is a possibility that BDNF-GFP was expressed at much higher level than that of endogenous BDNF, and thus the sorting and trafficking characteristics would be different from those of endogenous BDNF. Although we cannot completely exclude this possibility, we believe that it is unlikely, based on the two observations. 1) The intracellular localization of expressed BDNF-GFP is similar to that of endogenous BDNF when BDNF-GFP is expressed in a punctated manner. 2) As discussed above, the mean velocity of trafficking of BDNF-GFP in axons is similar to the reported value for the anterograde transport of 125I-labelled BDNF in the optic nerve of neonatal rats. This suggests that the trafficking properties of BDNF-GFP are not significantly different from those of endogenous BDNF. Furthermore we observed that the expression pattern of BDNF-GFP or -CFP was different from that of NGF-GFP or -YFP in most cases. These results altogether suggest that the expression and trafficking of BDNF-GFP may not be substantially different from those of endogenous BDNF. This further suggests that the GFP tag may not notably affect sorting and trafficking of secretory proteins, as reported previously .
In sum, the present finding that the anterograde trafficking of BDNF-GFP in axons was dominant in cortical neurons seems consistent with the view that BDNF acts on postsynaptic neurons in the anterograde direction [13–15, 29–35].
The present results show that the pattern and velocity of the trafficking of BDNF are different between the two structural domains of neurons, axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.
Culture preparation of neurons
Neonatal mice (C57/BL6, postnatal day 0–1) were anesthetized with ketamine (> 30 mg/kg, i.p.), and then killed by cervical dislocation. The experimental procedures met the regulation of the Animal Care Committee of Osaka University Graduate School of Medicine. Neurons were cultured at a low density using conventional methods, as reported previously . A piece of the visual cortex was removed from neonatal mice, enzymatically dissociated with papain (20 U/ml), and triturated with a fire-polished glass pipette. Neurons were plated on a previously prepared glial feeder layer, and were grown in a solution based on the Neurobasal A medium (GIBCO, Rockville, MD, USA) supplemented with 5% B27 (GIBCO). All experiments were carried out 14–24 days after plating.
Injection of plasmid cDNA of BDNF or NGF tagged with fluorescent proteins
The cDNAs of mouse BDNF or NGF tagged with GFP, CFP or YFP at the C-terminus were provided by Dr. Masami Kojima. Glass micropipettes were filled with Tris-ethylene-diamine-tetraacetic acid (EDTA) buffer (pH 8.0) which contained cDNAs of BDNF-GFP, or -CFP (0.5–1 μg/μl), or NGF-GFP, or -YFP (0.5 μg/μl). Then cultured neurons were placed on the stage of an inverted epifluorescence microscope (TE300, Nikon, Tokyo, Japan), and cDNAs were injected into the nucleus of a neuron through a micropipette under visual control, using a micromanipulator (MMO-202ND, Narishige, Tokyo, Japan), as described previously .
Neurons which expressed fluorescent protein-tagged BDNF or NGF signals were observed using a 40 X /1.3 NA oil immersion objective (Plan Flour, Nikon) attached to the inverted epifluorescence microscope. The fluorescence of GFP. CFP and YFP excited by light at the wavelength of 488, 434 and 514 nm was measured using a cooled CCD camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan). Time-lapse recordings were carried out about 24 h after the transfection, and sequential images were acquired using a cooled CCD camera at about 30°C. The exposure time was 1 or 2 s, the lapse of time was about 5 s, and the total time of recordings was 15–20 min. The trafficking of BDNF was analyzed by tracking the positions of GFP fluorescence-positive vesicles in neurites as a function of time. The analyses and animations of the data were made using the AQUA COSMOS software (Hamamatsu Photonics).
For immunocytochemical staining, neurons were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA) and 4% sucrose in Dulbecco's phosphate-buffered saline (PBS) for 20 min at room temperature. The cells were incubated with PBS containing 0.2% Triton-X (Sigma) for 5 min and blocked by 10% goat serum in PBS for 1 h at 37°. Then, anti-MAP2 monoclonal antibody (isotype: IgG1, 1:250, Sigma), anti-BDNF polyclonal antibody (2 μg/μl, provided by Dr. Ritsuko Katoh-Semba), and anti-GAD65 monoclonal antibody (isotype IgG2a, 1:100, Chemicon, Temecula, CA, USA) were applied overnight at 4°. Endogenous BDNF was visualized by anti-rabbit secondary antibody conjugated with Alexa 546 (1:2000, Molecular Probe). MAP2 and GAD65 were visualized by anti-mouse secondary antibody conjugated with Alexa 647 or 350 (1:200, Chemicon), and by isotype-specific secondary antibody conjugated with Alexa 546 (1: 2000, Chemicon).
brain-derived neurotrophic factor
cyan fluorescent protein
glutamic acid decaroxylase 65
green fluorescent protein
kinesin superfamily protein
microtubule-associated protein 2
nerve growth factor
yellow fluorescent protein
We would like to thank Dr. Masami Kojima for kindly providing the cDNA of BDNF-GFP, BDNF-CFP, NGF-GFP and NGF-YFP, and Mr. Atsushi Maruyama for assisting in the preparations of cultured neurons. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (12031230) to TT from the Ministry of Education, Science, Sports and Culture, Japan.
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