Local and target-derived brain-derived neurotrophic factor exert opposing effects on the dendritic arborization of retinal ganglion cells in vivo

Abstract

The dendritic and axonal arbors of developing retinal ganglion cells (RGCs) are exposed to two sources of BDNF: RGC dendrites are exposed to BDNF locally within the retina, and RGC axons are exposed to BDNF at the target, the optic tectum. Our previous studies demonstrated that increasing tectal BDNF levels promotes RGC axon terminal arborization, whereas increasing retinal BDNF levels inhibits RGC dendritic arborization. These results suggested that differential neurotrophic action at the axon versus dendrite might be responsible for the opposing effects of BDNF on RGC axonal versus dendritic arborization. To explore this possibility, we examined the effects of altering BDNF levels at the optic tectum on the elaboration of RGC dendritic arbors in the retina. Increasing tectal BDNF levels resulted in a significant increase in dendritic branching, whereas neutralizing endogenous tectal BDNF with function-blocking antibodies significantly decreased dendritic arbor complexity. Thus, RGC dendritic arbors react in opposing manners to retinal- versus tectal-derived BDNF. Alterations in retinal BDNF levels, however, did not affect axon terminal arborization. Thus, RGC dendritic arborization is controlled in a complementary manner by both local and target-derived sources of BDNF, whereas axon arborization is modulated solely by neurotrophic interactions at the target. Together, our results indicate that developing RGCs modulate dendritic arborization by integrating signals from discrete sources of BDNF in the eye and brain. Differential integration of spatially discrete neurotrophin signals within a single neuron may therefore finely tune afferent and efferent neuronal connectivity.

Fig. 1.

Altering endogenous retinal and tectal BDNF levelsin vivo. Diagrams representing a transverse view of aXenopus tadpole brain and eye illustrate experimental procedures (see Materials and Methods). RGCs are depicted inred, relative endogenous BDNF expression levels (Cohen-Cory et al., 1996) are depicted in blue, and exogenously applied factors are depicted in green.A, Effects of altered tectal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated green fluorescent microspheres were injected into the stage 38 tadpole tectum. At stage 42, RGCs were retrogradely labeled by injecting rhodamine–dextran in the contralateral tectum. At stage 45, dendritic morphologies of double-labeled RGCs were evaluated. A low-power view of a tadpole eye shows green fluorescent microspheres retrogradely transported to the retinal ganglion cell layer, where a rhodamine–dextran-labeled RGC soma can also be visualized (lines denote lens and eye periphery). Scale bar, 50 μm. A single-plane, high-power view of a stage 45 retina reveals a rhodamine–dextran-labeled RGC with internalized green fluorescent microspheres. Scale bar, 5 μm. B, Effects of altered retinal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 38 tadpole retina, and then RGCs were retrogradely labeled at stage 42. The low-power view shows rhodamine–dextran-labeled RGCs and green fluorescent microspheres restricted within the tadpole eye. Scale bar, 200 μm. The single-plane, high-power view of a stage 45 retina reveals the morphology of a rhodamine–dextran-labeled RGC surrounded by green fluorescent microspheres. Scale bar, 5 μm. C, Effects of altered retinal neurotrophins on RGC axonal arborization in the tectum. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 43 tadpole retina, and the morphology of DiI- or YFP-labeled RGC axon arbors was visualized 24 and 48 hr later. Confocal microscope images of a control RGC axon at 0 and 24 hr demonstrate normal RGC axon arborization dynamics. Scale bar, 20 μm.

Fig. 2.

Tectal BDNF retrogradely enhances RGC dendritic arborization. To determine whether tectal BDNF influences RGC dendritic arborization within the retina, tadpoles received tectal injections of microspheres treated with control, BDNF, or anti-BDNF function-blocking antibodies. Microsphere-containing neurons colabeled with rhodamine–dextran were analyzed morphologically (Fig.1A). A, Image reconstructions of two rhodamine-labeled RGCs with simple and complex dendritic arbors illustrate differences in dendritic arbor morphologies.B, Images of RGC dendritic arbors reveal that increasing tectal BDNF enhances RGC dendritic arborization, whereas neutralizing endogenous tectal BDNF with function-blocking antibodies reduces RGC dendritic arborization. C, Quantitative analysis reveals that primary dendrite number, branch tip number, branch tips per primary dendrite, and overall dendritic length were significantly enhanced by increasing tectal BDNF and reduced by injecting anti-BDNF into the optic tectum. Scale bar, 5 μm. Error bars indicate SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 3.

Tectal BDNF promotes RGC dendritic arborization by direct interaction with axon terminals. To determine whether RGC dendritic arborization requires direct axonal interaction with tectal BDNF, the dendritic arbors of RGCs from tadpoles treated with control or BDNF-treated microspheres (μspheres) were analyzed. The presence of green fluorescent microspheres in the soma of rhodamine-labeled RGCs (A, with μspheres) indicated that the axon termini of these neurons interacted directly with exogenous BDNF in the tectum, whereas the absence (A, w/o μspheres) indicated that RGC did not retrogradely transport microspheres and therefore did not interact directly with tectal BDNF. A, Quantitative analysis of dendritic morphology revealed that primary dendrites and branch tip numbers were increased by tectal BDNF only when RGC axons internalized and retrogradely transported BDNF-treated microspheres. Error bars indicate SEM. ∗p < 0.05; ∗∗∗p < 0.001. B, Analysis of neighboring RGC pairs (separated by 1–2 soma diameters) with and without retrogradely transported BDNF-treated microspheres revealed that double-labeled RGCs directly exposed to BDNF (BDNF+) had more than twice as many dendrite branches than their neighboring RGCs without microspheres (BDNF−) (in x-axis, >> equals >200%, > equals >150%, and = equals same number of total branch tips; n = 13 pairs).

Fig. 4.

Retinal BDNF inhibits RGC dendritic arborization in a dose-dependent manner. To determine whether RGCs are sensitive to the concentration of BDNF in the retina, Xenopus retinas were microinjected with 1–100 ng/μl BDNF or control microspheres at the onset of dendritic arborization. Quantitative measures of dendritic arbor morphology revealed a dose-dependent response to BDNF. The highest concentration of BDNF most dramatically decreased primary dendrite number, branch tip number, tips per dendrite, and dendrite length versus control. Error bars indicate SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 5.

RGC dendritic arborization is temporally sensitive to increased retinal BDNF levels. To determine whether RGCs were sensitive to enhanced retinal BDNF in a stage-dependent manner, control or BDNF-treated microspheres were injected into Xenopus retinas at stage 38 or 42.A, The morphology of RGC dendritic arbors revealed a stage-dependent response to increased retinal BDNF levels.B–C, Quantitative analysis of dendritic differentiation indicates that earlier exposure to exogenous BDNF (stages 38–45) inhibited dendritic arborization more dramatically than later exposure to BDNF (stages 42–45). Primary dendrite number as well as dendritic branching was significantly decreased by altering retinal BDNF starting at stage 38 (B), whereas altering retinal BDNF levels from stage 42 onward (C) selectively reduced dendritic branching without affecting primary dendrite number. Error bars indicate SEM. ∗p < 0.05; ∗∗∗p < 0.001. Scale bar, 10 μm.

Fig. 6.

RGC axon arbor complexity is unaffected by retinal BDNF levels. To determine whether retinal BDNF influences RGC axon arborization at a distance, tadpoles were intraocularly injected with control, BDNF-, or anti-BDNF-treated microspheres, and the resulting changes in RGC axon arbor dynamics were compared with tectally applied BDNF (Cohen-Cory and Fraser, 1995; Lom and Cohen-Cory, 1999). A, Individual RGC axon arbor morphologies of control, retinal BDNF, and tectal BDNF at 0 and 24 hr after treatment demonstrate that only tectally applied BDNF significantly alters RGC axon arborization. B, C, Altering retinal BDNF levels had no significant effects on RGC axon arbor complexity as measured by the increase in total branch number (B) and total arbor length (C) 24 and 48 hr after treatment (p > 0.05). Error bars indicate SEM. Scale bar, 20 μm.