Evidence fromIn VivoImaging That Synaptogenesis Guides the Growth and Branching of Axonal Arbors by Two Distinct Mechanisms

Abstract

To explore the relationship between axon arbor growth and synaptogenesis, developing retinal ganglion cell (RGC) axon arbors in zebrafish optic tectum were imaged in vivo at high temporal and spatial resolution using two-photon microscopy. Individual RGC axons were dually labeled by expression of a cytosolic red fluorescent protein (DsRed Express) to mark arbor structure and a fusion of the synaptic vesicle protein synaptophysin with green fluorescent protein (Syp:GFP) to mark presynaptic vesicles. Analysis of time-lapse sequences acquired at 10 min intervals revealed unexpectedly rapid kinetics of both axon branch and vesicle cluster turnover. Nascent axonal branches exhibited short average lifetimes of 19 min, and only 17% of newly extended axonal processes persisted for periods exceeding 3 h. The majority (70%) of Syp:GFP puncta formed on newly extended axonal processes. Syp:GFP puncta also exhibited short average lifetimes of 30 min, and only 34% of puncta were stabilized for periods exceeding 3 h. Moreover, strongly correlated dynamics of Syp:GFP puncta and branch structure suggest that synaptogenesis exerts strong influences on both the extension and the selective stabilization of nascent branches. First, new branches form almost exclusively at newly formed Syp:GFP puncta. Second, stabilized nascent branches invariably bear Syp:GFP puncta, and the detailed dynamics of branch retraction suggest strongly that nascent synapses can act at branch tips to arrest retraction. These observations thus provide evidence that synaptogenesis guides axon arbor growth by first promoting initial branch extension and second by selective branch stabilization.

KBP is essential for axonal structure, outgrowth and maintenance in zebrafish, providing insight into the cellular basis of Goldberg-Shprintzen syndrome

“…6A-D), but the small size of axonal mitochondria precluded quantitative analysis of mitochondrial distribution and dynamics at the light microscopy level. We did not see any overt defects in the localization of the synaptic vesicle protein marker, synaptophysin:GFP (Meyer and Smith, 2006) in kbp st23 mutant axons (Fig. 6E,F).…”

“…To co-label axons and synaptophysin, embryos were injected at the one cell stage with 25 pg of syn:GFP-DSR plasmid DNA (Meyer and Smith, 2006). Embryos were screened at 96 hpf and those with suitably labeled neurons were imaged.…”

Retinocollicular Synapse Maturation and Plasticity Are Regulated by Correlated Retinal Waves

“…Numerous studies have revealed developmentally regulated synaptic maturation during periods when the expression of LTP is also robust (Crair and Malenka, 1995;Rumpel et al, 1998;Chen and Regehr, 2000), and this synaptic maturation is correlated with axon remodeling during map refinement (Meyer and Smith, 2006;Ruthazer et al, 2006). Whether the experimentally induced LTP is physiologically relevant to circuit formation is unclear, however, because of the wide variety of induction protocols used and the lack of information about natural activity patterns during development.…”

“…Interestingly, chronic NMDAR blockade serves to increase RGC arbor branch dynamics, suggesting that NMDAR activity and synaptic maturation normally stabilizes retinotectal projections during circuit formation (Rajan et al, 1999). Recent in vivo imaging of zebrafish and tadpole retinotectal axons reveals that synapse formation and maturation are tightly correlated with branch dynamics and may play an instructive role in arbor remodeling during map formation (Meyer and Smith, 2006;Ruthazer et al, 2006). Indeed, correlated activity is known to regulate RGC axon branch dynamics during development (Ruthazer et al, 2003;Hua et al, 2005), offering additional evidence that the refinement of retinotopic projections proceeds through activitydependent instruction and synaptic competition.…”

Two-photon imaging of synaptic plasticity and pathology in the living mouse brain

“…Thus, TPM allows imaging of thick biological specimens such as brain slices, intact embryos, and brains up to ϳ800 m deep into the tissues. 25,26 Rather than reviewing the extensive field of structural synaptic plasticity on which there are many excellent imaging studies and reviews, 3,18,23,[27][28][29] in this paper we will mainly focus on recent findings obtained by TPM in living mice that relate to structural changes of synapses in the normal and degenerating brain. Because live imaging of synapses is a relatively new field, there is still no consensus regarding the degree of synaptic dynamism at baseline and in response to experience.…”