Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
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.
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 35–46 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 35–36 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.