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SummaryThe complex morphology of axons presents a challenge in understanding axonal responses to injury and disease. By in vivo two-photon imaging of spinal dorsal column sensory axons, we systematically examined the effect of injury location relative to the main bifurcation point on axon degeneration and regeneration following highly localized laser injuries. Retrograde but not anterograde degeneration was strongly blocked at the bifurcation point at both the acute and subacute phases. Eliminating either the ascending or descending branch led to a poor regenerative response, while eliminating both led to a strong regenerative response. Thus, a surviving intact branch suppresses both retrograde degeneration and regeneration of the injured branch, thereby preserving the remaining axon architecture. Regenerating axons exhibited a dynamic pattern with alternating phases of regeneration and pruning over a chronic period. In vivo imaging continues to reveal new insights on axonal responses to injury in the mammalian spinal cord.

In this issue of Neuron, Tedeschi et al. (2016) describe the voltage-gated calcium channel subunit alpha2delta2 as a developmental switch from axon elongation to synapse formation and transmission that doubles as a suppressor of axon regeneration, providing a molecular clue for the synaptic stabilization hypothesis of CNS regeneration failure.

SummaryIt is a long-standing question how developing motor and sensory neuron projections cooperatively form a common principal grid of peripheral nerve pathways relaying behavioral outputs and somatosensory inputs. Here, we explored this issue through targeted cell lineage and gene manipulation in mouse, combined with in vitro live axon imaging. In the absence of motor projections, dorsal (epaxial) and ventral (hypaxial) sensory projections form in a randomized manner, while removal of EphA3/4 receptor tyrosine kinases expressed by epaxial motor axons triggers selective failure to form epaxial sensory projections. EphA3/4 act non-cell-autonomously by inducing sensory axons to track along preformed epaxial motor projections. This involves cognate ephrin-A proteins on sensory axons but is independent from EphA3/4 signaling in motor axons proper. Assembly of peripheral nerve pathways thus involves motor axon subtype-specific signals that couple sensory projections to discrete motor pathways.

SummaryA central hypothesis for the limited capacity for adult central nervous system (CNS) axons to regenerate is the presence of myelin-derived axon growth inhibitors, the role of which, however, remains poorly understood. We have conducted a comprehensive genetic analysis of the three major myelin inhibitors, Nogo, MAG, and OMgp, in injury-induced axonal growth, including compensatory sprouting of uninjured axons and regeneration of injured axons. While deleting any one inhibitor in mice enhanced sprouting of corticospinal or raphespinal serotonergic axons, there was neither associated behavioral improvement nor a synergistic effect of deleting all three inhibitors. Furthermore, triple-mutant mice failed to exhibit enhanced regeneration of either axonal tract after spinal cord injury. Our data indicate that while Nogo, MAG, and OMgp may modulate axon sprouting, they do not play a central role in CNS axon regeneration failure.

Department of Neurosciences, University of California at San Diego, La Jolla, CA 92093, USA*Correspondence: osteward@uci.eduDOI 10.1016/j.neuron.2007.04.004IntroductionNogohasbeenproposedtobeamajormyelin-derived inhibitor of axon re-generation in the mammalian centralnervous system (CNS). Three studiestested this hypothesis by assessingregeneration in various Nogo-deficientmice following spinal cord injury (Kimet al., 2003; Simonen et al., 2003;Zhengetal.,2003),yetdifferentregen-erative responses were reported. Instudies of a Nogo gene trap mutant,Kim et al. (2003) reported dramaticallyenhanced sprouting above the lesion,reflected by the presence of CSTaxons in ectopic locations (the lateralfuniculus ipsilateral to the injection) inall (12/12) Nogo knockout mice. Evi-dence of striking long-distance regen-eration was the presence of ectopicBDA-labeled axons more than 5 mmcaudal to the injury in 7/11 Nogoknockout mice that received spinalcord injuries at 7.5–9 weeks of age.Three mice that underwent surgery at11–14 weeks of age did not exhibit ev-idence of long-distance regeneration.The labeled axons in caudal segmentsseemed to be especially compellingevidence of robust but disorderly re-generationoftheCSTbecauselabeledaxons were present bilaterally in thelateral funiculus (an ectopic location).In contrast, Simonen et al. (2003)reported increased sprouting nearthe lesion site in a Nogo-A-targetedknockout(inwhichNogo-Bexpressionwas upregulated) and evidence foren-hanced regeneration below the lesionin a subset (4/16) of mutants, althoughthere was no statistically significantdifferencebetweenknockoutandcon-trols. Our group found no statisticallysignificant difference between Nogo-A,B(orNogo-A,B,C)mutantsandwild-type controls in the extent of CSTsprouting above the lesion or regener-ation past the lesion (Zheng et al.,2003). One enigmatic finding was thatectopic axons similar to those re-ported by Kim et al. were observed inone control mouse (see Figure S2 inthe Supplemental Data of Zhenget al., 2003).Possible reasons for the discrepantresults have been discussed (Woolf,2003), but there has been no satisfac-tory explanation of the apparently ro-bust regeneration in one study andmodest or no enhanced regenerationin the others. Accordingly, we set outto perform a full replication of the ex-periments in the knockout line usedin our study (Zheng et al., 2003) andthe gene trap line used by Kim et al.(2003), which were reported to exhibitstriking regeneration. In the course ofour studies, we discovered that axonsin the white matter stain artifactuallywhenBDAleaksintothecerebrospinalfluid (CSF) in the cerebral ventricle,producing a pattern of ectopic labeledaxons that is remarkably similar towhat was interpreted as massivesprouting/regeneration by Kim et al.Inthisreport,wedocumenttheartifac-tual labeling, show that it occurs asa result of BDA entering the CSF, anddemonstrate that the artifact may beavoided by delaying the tract tracinginjections for 4 weeks after spinalcord lesions.ResultsIn all three studies of Nogo knockoutmice, regeneration was assessed fol-lowing transection of the dorsal halfof the spinal cord (a dorsal hemi-section) atT8, which in mice interruptsdescending CST axons while sparingwhite matter and gray matter in ventralportions of the spinal cord. Possibleregeneration of cortico-spinal tract(CST) axons was then assessed byinjecting biotinylated dextran amine(BDA) into the sensorimotor cortex.Importantly, all three studies used asimilar, short-term protocol: the corti-cal BDA injection was made in thesame operative procedure as the spi-nal cord lesion, and regeneration wasassessed 2–3 weeks later.In our replication experiment, weusedthesameprotocoltoassesspos-sibleCSTregenerationinhomozygousmutant and control (wild-type and het-erozygotes). BDA was injected usingthe coordinates described in Zhenget al. (2003) (Kim et al. [2003] did notreport their coordinates). One smallprocedural difference that turned outto be important was that, in our previ-ous study (Zheng et al., 2003), BDAwas injected at a depth of 0.5 mm inthe cortex, whereas in the repeatexperiment we lowered the syringebelow 0.5 mm and then withdrew tothe final location. This was done toachieve greater accuracy becausethe cortex is frequently displacedslightlyasthemicrosyringe islowered.As discussed below, this had theunin-tended consequence that the syringesometimes penetrated the ventricle.In intact mice (that is, without spinalcord injuries), injections of BDA intothe sensorimotor cortex producerobust labeling of dorsal main CST inthe ventral part of the dorsal columnNeuron 54, April 19, 2007 a2007 Elsevier Inc. 191

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.