Cogen J and Cohen-Cory S (2000), Nitric oxide modulates retinal ganglion cell ax...

XB-ART-10219

J Neurobiol 2000 Nov 05;452:120-33. doi: 10.1002/1097-4695(20001105)45:2<120::aid-neu6>3.0.co;2-6.

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Nitric oxide modulates retinal ganglion cell axon arbor remodeling in vivo.

Cogen J , Cohen-Cory S .

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Nitric oxide (NO) has been postulated to act as an activity-dependent retrograde signal that can mediate multiple aspects of synaptic plasticity during development. In the visual system, a role for NO in activity-dependent structural modification of presynaptic arbors has been proposed based on NO's ability to prune inappropriate projections and segregate axon terminals. However, evidence demonstrating that altered NO signaling does not perturb ocular dominance map formation leaves unsettled the role of NO during the in vivo refinement of visual connections. To determine whether NO modulates the structural remodeling of individual presynaptic terminal arbors in vivo we have: 1. Used NADPH-diaphorase histochemistry to determine the onset of NO synthase (NOS) expression in the Xenopus visual system. 2. Used in vivo time-lapse imaging to examine the role of NO during retinal ganglion cell (RGC) axon arborization. We show that NOS expression in the target optic tectum is developmentally regulated and localized to neurons that reside in close proximity to arborizing RGC axons. Moreover, we demonstrate that perturbations in tectal NO levels rapidly and significantly alter the dynamic branching of RGC arbors in vivo. Tectal injection of NO donors increased the addition of new branches, but not their stabilization in the long term. Tectal injection of NOS inhibitors increased the dynamic remodeling of axonal arbors by increasing branch addition and elimination and by lengthening pre-existing branches. Thus, these results indicate that altering NO signaling significantly modifies axon branch dynamics in a manner similar to altering neuronal activity levels (Cohen-Cory, 1999). Consequently, our results support a role for NO during the dynamic remodeling of axon arbors in vivo, and suggest that NO functions as an activity-dependent retrograde signal during the refinement of visual connections.

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Species referenced: Xenopus laevis
Genes referenced: nos1 nos3 prune1

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	Figure 1 Developmental regulation of NOS expression in the optic tectum of Xenopus laevis tadpoles visualized by NADPH-diaphorase histochemistry. Longitudinal cryostat sections of tadpoles from stages 35â46 of development were reacted for NADPH-diaphorase histochemistry to determine the distribution of NOS expressing neurons in the developing optic tectum. Only a few diaphorase- positive cells were detected in 35â36 tadpoles, before RGC axons have reached the tectum (A). Diaphorase-positive tectal neurons significantly increase in frequency by stage 42 (B) when RGC axons have begun to branch in the tectum, and are more abundant at stage 45 (C), when diaphorase-positive tectal dendrites (arrowheads) can be visualized extending into the neuropil (D). The dashed line delineates the tectal neuropil. L, lateral is up; P, posterior is to the right; e, pigmented cells of the eye; n, tectal neuropil; v, ventricle. Scale bars, 20 um.
	Figure 2 NOS-expressing tectal neurons extend dendrites in close proximity to branching RGC axons. Transverse sections of stage 41 (A,B) and 43 (C,D) Xenopus tadpoles in which RGC axons were anterogradely labeled with HRP shows that NADPH-diaphorase positive tectal neurons extend their dendrites (A and C, black arrowheads) into the tectal neuropil where RGC axons arborize (B and D, white arrowheads). In these two sections, NADPH-diaphorase positive neurons were visualized by NADPH-diaphorase histochemistry (A,C), and RGC axon terminals were visualized by fluorescent HRP immunohistochemistry (B,D). Note that cell bodies are restricted to the central gray matter region, while dendrites of tectal neurons and RGC axons project to, and branch in, the tectal neuropil. D, dorsal is up; L, lateral is to the right; n, tectal neuropil; v, ventricle. Scale bar, 20 um.
	Figure 3 Perturbations in tectal NO signaling increases RGC axon arbor remodeling in vivo. The effects of tectal administration of either NO donors (SNAP or DETA-NONOate) or NOS inhibitor (3B-7-NI) on the rapid dynamics of axon arborization are illustrated by reconstructions of representative arbors. Tracings of sample arbors followed every 2 h for 6 h by confocal microscopy illustrate that both NO donors and the NOS inhibitor increase the dynamic remodeling of RGC axonal arbors. Portions of the arbor that were added are represented in green, those that were eliminated are represented in red, and those that were added at one time point and then eliminated in the next are represented in yellow. Note that axons treated with NO donors extend more branches, while those treated with the NOS inhibitor are more dynamic. Posterior is up, medial is to the left.
	Figure 4 NO donors and the NOS inhibitor alter the rapid dynamics of RGC axon branching. To quantify the effects of NO manipulations on axon morphology over the 6 h time course, four morphological parameters were analyzed for every 2 h observation period: (A) the number of branches that were added and the number of branches that were lengthened, and (B) the number of branches that were eliminated and the number of branches that were shortened. Note that while both NO donors and the NOS inhibitor significantly increased the number of branches added, only the inhibitor significantly increased the number of branches eliminated. Furthermore, only treatment with the NOS inhibitor increased the number of branches that lengthened over time. The mean 6 SEM is shown for each parameter and condition. Statistical analysis was by repeated measures ANOVA. * Significantly different from control, p , .05.
	Figure 5 NO donors and the NOS inhibitor differentially alter parameters of RGC arbor complexity in the short term. To quantify the effects of NO manipulations on total branch number and arbor length over the 6 h time course, we measured (A) the net change in branch and spike number, and (B) the net change in total arbor length, as measured by the cumulative length of all branches beyond the first branch point, for every 2 h observation period. (A) Treatment with NO donors, but not the NOS inhibitor, resulted in a significant increase in both branch and spike number every 2 h. (B) Treatment with NO donors or the NOS inhibitor increased arbor length compared to control. Note that the increase in overall arbor length elicited by NO donors correlates with the net increase in branch and spike number, whereas the increase in arbor length elicited by the NOS inhibitor correlates with the increase in individual branch length. The mean 6 SEM is shown for each parameter and condition. Statistical analysis was by repeated measures ANOVA. * Significantly different from control, p # .05.
	Figure 6 NO donors and the NOS inhibitor differentially alter the complexity of Xenopus RGC axonal arbors 24 h after treatment. Tracings of representative individual RCG axon arbors before and 24 h after tectal injection of control solution, NO donor, or NOS inhibitor illustrate variabilities in arbor morphologies for each condition. Under all conditions tested, axon arbor complexity, as measured by branch number and arbor length, is increased over time. Note, however, that arbors treated with the NOS inhibitor are longer than those treated with either control solutions or NO donors. Posterior is up, medial is to the left.
	Figure 7 NO signaling differentially influences the morphology of RGC axon arbors at 24 h. The cumulative effects of tectal administration of NO donor or NOS inhibitor on axon arbor complexity over 24 h were evaluated quantitatively by measuring branch number and arbor length. Values are presented as the change in initial value 24 h after tectal administration with either NO donor or NOS inhibitor. Treatment with the NOS inhibitor increased arbor length, without eliciting a significant increase in branch number. Treatment with NO donors did not change arbor length or branch number as compared to controls. The mean 6 SEM is shown for each parameter and condition. Statistical analysis was by one-way ANOVA using multiple comparison posthoc Tukey tests. * Significantly different from control, p # .05.