The corticospinal (CS) tract is essential for voluntary movement, but what we know about the organization and development of the CS tract remains limited. To determine the total cortical area innervating the seventh cervical spinal cord segment (C7), which controls forelimb movement, we injected a retrograde tracer (fluorescent microspheres) into C7 such that it would spread widely within the unilateral gray matter (to Ͼ80%), but not to the CS tract. Subsequent detection of the tracer showed that, in both juvenile and adult mice, neurons distributed over an unexpectedly broad portion of the rostral two-thirds of the cerebral cortex converge to C7. This even included cortical areas controlling the hindlimbs (the fourth lumbar segment, L4). With aging, cell densities greatly declined, mainly due to axon branch elimination. Whole-cell recordings from spinal cord cells upon selective optogenetic stimulation of CS axons, and labeling of axons (DsRed) and presynaptic structures (synaptophysin) through cotransfection using exo utero electroporation, showed that overgrowing CS axons make synaptic connections with spinal cells in juveniles. This suggests that neuronal circuits involved in the CS tract to C7 are largely reorganized during development. By contrast, the cortical areas innervating L4 are limited to the conventional hindlimb area, and the cell distribution and density do not change during development. These findings call for an update of the traditional notion of somatotopic CS projection and imply that there are substantial developmental differences in the cortical control of forelimb and hindlimb movements, at least in rodents.

The GluN2B (GluRε2/NR2B) and GluN2A (GluRε1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [ Grin2b −/− ( 2b −/− )] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [ 2b −/− , Grin2a −/− ( 2a −/− ), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)- 2b −/− (SpC) pairs but not in WT(Cx)- 2a −/− (SpC) or 2b −/− (Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in 2a −/− SpC slices, where Ca 2+ entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in 2b −/− slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca 2+ entry through GluN2B-containing channels.

. In corticospinal synapses reconstructed in vitro by slice co-culture, we previously showed that the synapses were distributed across the gray matter at 6 -7 days in vitro (DIV). Thereafter, they began to be eliminated from the ventral side, and dorsal-dominant distribution was nearly complete at 11-12 DIV. The synapse elimination is associated with retraction of the corticospinal (CS) terminals. We studied whether this specific type of synapse elimination is a physiological phenomenon rather than in vitro artifact. The rat corticospinal tract was stimulated at the medullary pyramid, and field potentials were recorded at the cervical cord along an 200-m interval lattice on the axial plane. Clearly defined negative field potential were identified as field excitatory postsynaptic potentials (fEPSPs) generated by corticospinal synapses. They were recorded from the entire spinal gray matter at postnatal day 7 (P7). These negative fEPSPs reversed to positive in the most ventrolateral part at P8. Reversal extended to the more mediodorsal area at P10, indicative of progressive synapse elimination in the ventrolateral area. To verify that regression of the axons in vivo paralleled the changes in spatial distribution of fEPSPs as observed in vitro, corticospinal axons were anterogradely labeled. Redistribution of the labeled terminals closely paralleled the fEPSP distribution, being present in the ventrolateral spinal cord at P7, decreased at P8, further deceased at P10, but unchanged at P11. Furthermore, double immunostaining for labeled terminals and synaptophysin observed under a confocal microscope suggests that corticospinal fibers at P7 possess presynaptic structures in the ventrolateral area as well as the dorsomedial area. These findings suggest that corticospinal synapses are widely formed in the spinal gray matter at P7, are rapidly eliminated from the ventrolateral side from P8 to P10, a time-course very similar to that observed in vitro, and are associated with axonal regression.

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.

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