Regressive events in rat corticospinal axons during development in in vitro slice cocultures: Retraction, amputation, and degeneration

Abstract

Axonal regression is utilized to refine neuronal circuits during development, but the dynamic properties of such regression remain largely unknown. We used confocal time-lapse imaging to examine the regression of single enhanced yellow fluorescent protein-labeled axons in corticospinal slice cocultures. By acquiring images at long (1 day) and short (30-60 min) intervals on days 5-13 in vitro, we detected three types of regressive events: 1) retractions, 2) amputations (referred to as autoaxotomy), and 3) degeneration. Retractions proceeded at some constant rate for up to 3 hours and then paused or switched to another constant rate, apparently shifting stepwise among three retraction rates (6, 12, 17 microm/hours). Autoaxotomy was a previously unreported strategy for regression. It occurred spontaneously, either at a proximal branch neck or at a distal end. Axons also underwent a form of degeneration that had several novel characteristics. Degenerating axons showed bright bead-like spots arranged at 3-9-microm intervals. The gaps were much larger than the spot size, and there was no prior sign of damage (e.g., swelling). Each spot's fluorescence intensity often waxed and waned, with its position unchanged. Degeneration progressed without clear proximal-to-distal directionality and was complete within 3-4 hours. Quantitative analysis of daily branch regression showed that branches almost always regressed up to their branch point or stopped before it, thereby keeping the branch point stable. This branch-point stability was retained for all three regression strategies observed, suggesting that the fate of each branch is determined relatively independently during the development of axonal arborization.

Corticospinal Tract Development and Spinal Cord Innervation Differ between Cervical and Lumbar Targets

“…Several studies have shown that almost none of the ectopic axons enters the spinal gray matter (Joosten et al, 1987;Oudega et al, 1994). On the other hand, in the previous studies of the development of CS projections in rodents, we found that CS axons initially form synapses throughout the spinal gray matter (Ohno et al, 2004;Kamiyama et al, 2006;Maeda et al, 2007;Yoshioka et al, 2009), but those on the ventral side are largely eliminated in an NMDAand GluN2B-dependent manner (Ohno et al, 2010). Many of these observations were made in vitro, however.…”

“…9B). Despite a shorter expression time than usually used in optogenetic experiments (Zhang et al, 2010), YFP immunoreactivity was widely discernible in the spinal gray matter, suggesting that ChR2-EYFP was expressed at considerable levels within 1 week and was transported to the distal portion of CS axons (Fig. 9C).…”

“…ChR2 levels in CS axon terminals are dependent on the interval between vector injection and performance of the photostimulation experiments, as well as on the transport distance from the somata, where the protein is synthesized, to the axon terminals. For stimulation experiments, it is recommended that one wait 3 weeks after injection of the AAVChR2 expression system (Zhang et al, 2010). But because this was a developmental study, we waited only ϳ1 week after injection (experiments on P7-P10).…”

A Retrograde Tracing Study of Compensatory Corticospinal Projections in Rats with Neonatal Hemidecortication

“…Therefore, the bilaterally projecting axons observed in our study might be a temporary compensatory system and could be eliminated after a certain period. Previous studies of normal rats reported that corticospinal synapses were widely formed in the spinal gray matter at P7, and rapidly eliminated from the ventrolateral side, indicating that the decrease in the number of synapses on the ventral side is due primarily to axon branch elimination rather than to cortical neuron death [37,38,39]. The elimination of axon branches found in normally developing rats might also occur in neonatal rats with unilateral brain injury.…”

Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral, Neuroanatomical, and DNA Microarray Approaches

“…Electrophysiological studies have also indicated that corticospinal axon temporarily project over all gray matter in the spinal cord, but superfluous axons existing in the ventral gray matter are eliminated with growth [ 19 ]. This axonal elimination has been demonstrated by slice co-culture [ 20 ]. Although not much has been done on the molecular aspects of brain injury in context of motor function, molecular mechanisms of axon guidance in the developing CST have been reported.…”