Inra- and inter-neuronal trafficking of BDNF and its role in synaptogenesis

Tadaharu Tsumoto

Abstract

Neurotrophins such as brain-derived neurotrophic factor (BDNF) are suggested to play a role in synaptogenesis and synaptic plasticity. Important steps of processes for BDNF to exert such a role are assumed to be its trafficking in neurites to release sites, activity-dependent release and internalization with its receptors, TrkB in postsynaptic neurons or presynaptic terminals. Elucidation of mechanisms underlying such dynamic movements of BDNF is crucial for understanding of the function of BDNF. However, an actual movement of BDNF in neurites of living neurons has not fully been analyzed.

To address this issue, we developed molecular imaging techniques to demonstrate intra- and inter-cellular trafficking of BDNF tagged with green fluorescent protein (GFP) or other fluorescent proteins in living cortical neurons with the method of direct intranuclear injection of plasmid cDNAs. With this method we found that BDNF moves in presynaptic axons in the anterograde direction and transfers to postsynaptic neurons in an activity-dependent manner (Kohara et al., Science 291, 2419-2423, 2001). Then, we tested whether endogenous BDNF also transfers to postsynaptic neurons in the similar way to BDNF-GFP, using Òchimera cultureÓ preparations of visual cortical neurons obtained from two types of transgenic mice, GFP mice and BDNF knockout mice. Neurons derived from the former mice have endogenous BDNF as well as GFP. We found that endogenous BDNF transfers to postsynaptic neurons and plays a role in development of dendrites of postsynaptic neurons (Kohara et al., J. Neurosci. 23, 6123-6131, 2003).

Recent studies suggest that GABAergic neurons are supposed to be a target of such an action of BDNF. To elucidate actions of BDNF on GABAergic neurons, we examined effects of a long-term application of BDNF on solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. The chronic treatment with BDNF enhanced the amplitude of evoked inhibitory postsynaptic currents (IPSCs) and the frequency of miniature IPSCs (Parizvan et al., Neuroscience, in press). In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. The quantitative morphological analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. The increase in the number of functional active synaptic sites was confirmed by staining with FM1-43. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.

Finally we quantitatively analyzed trafficking of BDNF in axons and dendrites of living cortical neurons with the molecular imaging technique. We found that BDNF-containing vesicles moved smoothly in axons with the mean velocity of 0.73 ± 0.26 _m/s. On the other hand, movements of BDNF-containing granules in dendrites were not smooth, i.e., often stopped or moved back and forth in most cases. In the minority of granules, which moved relatively smoothly in dendrites, the mean velocity of movements (0.46 ± 0.23 _m/s) was significantly (p < 0.001, t-test) slower than that in axons. When glutamate was applied to neurons, the movement of vesicles in neurites was stopped and the intensity of their fluorescence was reduced in most cases. An application of tetrodotoxin blocked such an action of glutamate. These results suggest that the trafficking of BDNF in neurites as well as release of BDNF is regulated by neuronal activity.

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