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First published online 1 September 2005
doi: 10.1242/dev.02017

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BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo

Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA

View larger version (156K): [in a new window]   Fig. 1. GFP-synaptobrevin specifically localizes to presynaptic sites in RGC axon terminals. (A-D) The localization of GFP-synaptobrevin was determined by examining the distribution of GFP immunoreactivity by electron microscopy. Morphologically mature synapses (black arrows), containing presynaptic terminals with numerous synaptic vesicles (v) and clearly defined pre- and postsynaptic specializations, are present in the tectal neuropil of stage 45 tadpoles. (B-D) Electron photomicrographs of tadpole brains immunostained with an antibody to GFP show the localization of gold particles (open arrows) to presynaptic terminals in the tectal neuropil. Silver enhancement of the secondary antibody coupled to 1 nm gold particles shows that the GFP immunolabel is preferentially associated to synaptic vesicles in morphologically mature retinotectal synapses (B,D), as well as in presynaptic terminals near contact sites (C). Scale bar: 0.2 µm. (E) Regions of an individual RGC axon arbor imaged at 5 minute intervals illustrate the distribution of GFP-synaptobrevin puncta. The majority of the GFP-synaptobrevin puncta remain constant throughout time. This is in contrast to motile GFP-synaptobrevin puncta present in small transport packets, prevalent in axon terminals of neurons grown in culture (Ahmari et al., 2000). Scale bar: 20 µm.

View larger version (109K): [in a new window]   Fig. 2. Neutralizing endogenous BDNF influences presynaptic sites in individual RGC arbors. Time-lapse confocal imaging of individual DsRed2-labeled RGC axons expressing GFP-synaptobrevin illustrates the effects of neutralizing endogenous tectal BDNF on synapse number and axon arbor morphology. (A) Image reconstructions of a RGC axon in a vehicle-treated (control) tadpole show the localization of GFP-synaptobrevin clusters (yellow) within specific regions of the arborizing, DsRed2-labeled axons (red). (B) The number and distribution of GFP-synaptobrevin clusters was significantly altered in RGC arbors in tadpoles that received a single injection of anti-BDNF following the first imaging session (0h). Anti-BDNF not only influences axon arbor complexity but also decreases the number and density of GFP-synaptobrevin clusters per axon arbor. (C) Magnified region of the arbor shown in B illustrates the localization of GFP-synaptobrevin clusters to branch points and branch termini, and their disappearance after anti-BDNF treatment. By separating the green component (middle panel, GFP fluorescence) from the red component (overlay DsRed2 and GFP fluorescence; top panel) one can clearly distinguish specific GFP-synaptobrevin puncta from the background fluorescence signal. The line scans (bottom panels) obtained from raw confocal data show the intensity of the DsRed2 and GFP-synaptobrevin signals at the level of the axon branch demarcated by the light-blue hairlines (top panels). The green arrowheads (middle panels) indicate sites containing GFP-synaptobrevin clusters that are crossed by the line scan. In the 0 and 4 hour images, the proximal part of the line scan (1 pixel width) travels near GFP-synaptobrevin puncta but only crosses the arbor area where background fluorescent signal is observed. Background fluorescence intensity values remain similar after repeated imaging and that fluorescence intensity values of specific GFP-synaptobrevin puncta are at least twice as great as those of background signals. Scale bar: 20 µm in A,B; 10 µm in C. Posterior is upwards, anterior is downwards.

View larger version (28K): [in a new window]   Fig. 3. Anti-BDNF significantly decreases GFP-synaptobrevin cluster number and influences axon arbor complexity. Several morphological parameters illustrate the dynamic changes in GFP-synaptobrevin labeled presynaptic sites and axon arborization in control and anti-BDNF treated tadpoles followed every 2 hours for 8 hours and again at 24 hours. All parameters are expressed as percent change from their initial value at the time of treatment. (A) Total GFP-synaptobrevin cluster number per axon terminal in control- and anti-BDNF-treated tadpoles. Anti-BDNF significantly decreases GFP-synaptobrevin cluster number versus control 4 hours after treatment. (B) The complexity of both control and anti-BDNF-treated arbors is illustrated by the net increase in total branch number per axon terminal. A significant decrease in branch number by 4 hours in anti-BDNF treated tadpoles versus control parallels the decrease in synaptic cluster number. (C,D) A measure of synapse density in both control- and anti-BDNF-treated tadpoles is provided by comparing the increase in GFP-synaptobrevin cluster number with the increase in (C) branch number or (D) total arbor length (expressed as a ratio). In controls, there is a one-to-one relationship in the increase in GFP-synaptobrevin cluster number to arbor length, while in anti-BDNF-treated tadpoles GFP-synaptobrevin cluster density is decreased to 50-60%. This difference is significant from 4 to 8 hours after treatment. Bars indicate mean±s.e.m. n=14 axon arbors in control and n=10 arbors in anti-BDNF; *P0.05; **P0.005; ***P0.0005.

View larger version (32K): [in a new window]   Fig. 4. Anti-BDNF rapidly influences presynaptic site and axon branch stability. (A) Diagrammatic representation of GFP-synaptobrevin cluster dynamics and arbor growth. The number of GFP-synaptobrevin clusters stabilized and eliminated, and the number of new GFP-synaptobrevin clusters added between observation intervals was calculated and normalized for each time interval to obtain a dynamic measure of synapse addition and stabilization over time. As new GFP-synaptobrevin clusters are added, the absolute number of clusters that are stabilized increases, but as a proportion it remains relatively constant. The hypothetical axon depicted here exhibits rates of synapse stabilization that are slightly higher than those observed for RGC axon arbors in vehicle-treated tadpoles (control). (B) Detailed analysis of the number and distribution of GFP-synaptobrevin clusters per axon branch, and of the lifetimes of individual GFP-synaptobrevin clusters for every observation period reveal the effects of neutralizing endogenous BDNF on synapse stabilization. Anti-BDNF significantly reduced the stability of GFP-synaptobrevin clusters by 2 hours (0-2 hours), an effect that was maintained through every observation period. (C) Analysis of the number of axon branches that are retained or eliminated from one observation interval to the next provides a measure of the effects of anti-BDNF on axon branch stability. Axon branches are significantly destabilized and eliminated 0-2 hours after treatment and this effect is maintained for the first 6 hours following treatment. On average, 60.2±2.6% of branches are stable every 2 hours in anti-BDNF treated arbors versus 73.3±1.6% in controls. *P0.05; **P0.005.

View larger version (94K): [in a new window]   Fig. 5. Distribution and dynamics of GFP-synaptobrevin labeled presynaptic sites along RGC axon terminals. (A) Time-lapse sequence of a region of a control arbor illustrates the dynamic relationship between presynaptic site location and axon branch formation. New axonal branches originate from sites rich in GFP-synaptobrevin puncta (arrowheads), while new GFP-synaptobrevin clusters appear along an axon branch (Alsina et al., 2001). (B) Magnified region of an arbor illustrates the localization of GFP-synaptobrevin puncta to a nascent branch (arrows) in a DsRed2 labeled axon and its disappearance after anti-BDNF treatment (overlay, top panel; GFP-synaptobrevin fluorescence only, bottom panel). In some branches, GFP-synaptobrevin cluster dismantling precedes axon branch elimination (arrow), as indicated by the significant decrease in GFP fluorescence at the 2 hour time point. (C) Time-lapse sequence of a region of an anti-BDNF treated axon arbor shows the disappearance of GFP-synaptobrevin clusters and the retraction of an axon branch (arrow). The arrowhead indicates a site where a decrease in punctuate GFP-synaptobrevin fluorescence correlates with the shortening of the distal region of the axon branch. Asterisks indicate arbor sites with stable GFP-synaptobrevin clusters. Scale bars: 20 µm in A; 10 µm in B,C. Posterior is upwards, anterior is downwards.

View larger version (99K): [in a new window]   Fig. 6. BDNF prevents the effects that NMDA receptor blockade exerts on RGCs. Reconstructions of three-dimensional arbors illustrate the effects of APV and BDNF treatments on axon arbor complexity and GFP-synaptobrevin cluster number. Individual RGC axons double-labeled with GFP-synaptobrevin and DsRed2 were visualized by confocal microscopy in the live developing tadpole after tectal injection of (A) control vehicle solution, (B) APV or (C) APV plus BDNF. (B) A significant decrease in the number and density of GFP-synaptobrevin clusters in RGC axon arbors is observed 2 hours after APV treatment. GFP-synaptobrevin cluster density and arbor complexity begin to recover by 24 hours and further develop 9 days after treatment. (C) BDNF maintained GFP-synaptobrevin cluster density for most of the observation period in RGC axon arbors treated with APV. Posterior is upwards, anterior is downwards. Scale bar: 20 µm.

View larger version (32K): [in a new window]   Fig. 7. NMDA receptor blockade specifically influences presynaptic sites without altering RGC axon arbor complexity. The effects of altering NMDAR transmission in the optic tectum on GFP-synaptobrevin cluster number and axon branching in tadpoles that received single tectal injections of APV or MK801 is shown as the percent change from their initial value at the time of treatment. (A) Both APV and MK801 significantly decreased GFP-synaptobrevin cluster number versus control 2 hours after treatment. The peak cumulative effects of APV on GFP-synaptobrevin cluster number occur 4 hours after treatment (0-4 hours), while the MK801 cumulative effects peak 6 hours after treatment (0-6 hours). (B) RGC axon arbor complexity, expressed as the increase in total branch number per axon terminal, is affected by the APV and MK801 treatments by 24 hours only. (C) A measure of synapse density is provided by comparing the change in GFP-synaptobrevin cluster number with the change in total arbor length from the initial observation. In controls, there is a one-to-one relationship in the increase in GFP-synaptobrevin cluster number to arbor length, while in APV and MK801-treated tadpoles GFP-synaptobrevin cluster density is significantly decreased to 50% or below. This difference is significant for all observation time points. n=14 axon arbors in control, n=10 in APV and n=12 in MK801. *P0.05; **P0.005; ***P0.0005.

View larger version (28K): [in a new window]   Fig. 8. BDNF maintains the number and density of presynaptic specializations in RGC axon terminals affected by NMDA receptor blockade. (A) APV significantly decreased GFP-synaptobrevin cluster number versus control 2 hours after treatment. Co-injection of APV and BDNF rescued GFP-synaptobrevin labeled presynaptic sites, significantly reducing the effects of APV on GFP-synaptobrevin cluster number for a period of 8 hours. (B) Total branch number was not affected by APV+ BDNF. (C) GFP-synaptobrevin cluster density was significantly decreased in the APV-treated tadpoles. BDNF maintained GFP-synaptobrevin cluster density in RGC axons co-treated with APV. The asterisks indicate significance between APV alone and APV+BDNF. (A,C) APV+BDNF is significantly different from control at the 0-4, 0-6, 0-8, 0-24 time intervals; *P0.05; **P0.005; ***P0.0005. n=14 axon arbors in control, n=10 in APV, n=12 in MK801 and n=11 in APV+BDNF.

View larger version (20K): [in a new window]   Fig. 9. BDNF prevents the acute effects of NMDAR blockade by maintaining GFP-synaptobrevin cluster addition and stabilization rates. (A,B) Detailed analyses of the number and distribution of GFP-synaptobrevin clusters per axon branch and of the lifetimes of individual presynaptic clusters for every observation period reveal the effects of BDNF on synapse dynamics following NMDAR blockade. APV significantly decreased the stability (A) of GFP-synaptobrevin clusters during the first 4 hours after treatment (0-2 and 2-4 hour observation periods). The proportion of GFP-synaptobrevin clusters that are stabilized in the presence of APV was significantly influenced by BDNF (APV+BDNF) during the first 2 hours of treatment. (B) A significant decrease in GFP-synaptobrevin cluster addition (0-2 and 2-4 hours) preceded an increase in cluster addition following APV treatment. BDNF rescued GFP-synaptobrevin cluster number by maintaining the rate of GFP-synaptobrevin cluster addition. GFP-synaptobrevin cluster dynamics is presented for APV-treated tadpoles, and was similar for axons in MK-801-treated tadpoles. The circled asterisks indicate significant differences between APV alone and APV+BDNF (P0.05). Double asterisks indicate significant differences between control and APV (P0.005). (A) APV+BDNF was significantly different from control at 0-2 and 2-4 hours only.

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