Temporal Course of Transient Direct Corticomotoneuronal Connections during Development in Rodents.

We previously observed in rodents that during the 2nd postnatal week corticospinal axons make monosynaptic connections with motoneurons. Prior to that finding, it had been believed that such contacts only occur in higher primates. Although an in vitro electrophysiological study is prerequisite for studying the developmental time course of synaptic connections, the technical difficulty of reliably recording synaptic responses from spinal motoneurons in animals over 2 weeks old hampered the study. Instead, we used retrograde transsynaptic labeling with a genetically modified rabies virus to confirm the presence of direct corticomotoneuronal connections at an early developmental stage and to show that these connections were subsequently eliminated. However, determination of an accurate elimination time course and quantitative evaluation of synaptic connectivity cannot be achieved through viral-tracing experiments. For the present study, we improved the slice preparation procedure and maintenance of slice viability, which enabled us to record postsynaptic responses using the whole cell patch-clamp technique from retrogradely labeled forearm motoneurons up until postnatal week 7. We examined the extent of corticomotoneuronal monosynaptic connections and studied the time course of their accumulation and loss. Positive ratios of monosynaptic corticomotoneuronal EPSCs increased from P6 to P8 and then plateaued (P8-P13: 65%). Thereafter, the monosynaptic connections declined until P21, at which time they were no longer detected. The time course of the falling phase and elimination was confirmed by experiments using optogenetic stimulation. The timing of the elimination fell within the same range (P18-22) estimated in our earlier study using retrograde transsynaptic labeling.

Higher primate-like direct corticomotoneuronal connections are transiently formed in a juvenile subprimate mammal.

The corticospinal (CS) tract emerged and evolved in mammals, and is essentially involved in voluntary movement. Over its phylogenesis, CS innervation gradually invaded to the ventral spinal cord, eventually making direct connections with spinal motoneurons (MNs) in higher primates. Despite its importance, our knowledge of the origin of the direct CS-MN connections is limited; in fact, there is controversy as to whether these connections occur in subprimate mammals, such as rodents. Here we studied the retrograde transsynaptic connection between cortical neurons and MNs in mice by labeling the cells with recombinant rabies virus. On postnatal day 14 (P14), we found that CS neurons make direct connections with cervical MNs innervating the forearm muscles. Direct connections were also detected electrophysiologically in whole cell recordings from identified MNs retrogradely-labeled from their target muscles and optogenetic CS stimulation. In contrast, few, if any, lumbar MNs innervating hindlimbs showed direct connections on P18. Moreover, the direct CS-MN connections observed on P14 were later eliminated. The transient CS-MN cells were distributed predominantly in the M1 and S1 areas. These findings provide insight into the ontogeny and phylogeny of the CS projection and appear to settle the controversy about direct CS-MN connections in subprimate mammals.

The decline in synaptic GluN2B and rise in inhibitory neurotransmission determine the end of a critical period.

Neuronal plasticity is especially active in the young, during short windows of time termed critical periods, and loss of a critical period leads to functional limitations in the adults. The mechanism that governs the length of critical periods remains unknown. Here we show that levels of the NMDA receptor GluN2B subunit, which functions as a Ca2+ channel, declines in spinal cord synapses toward the end of the critical period for activity-dependent corticospinal synapse elimination. This period could be prolonged by blocking the decline of GluN2B, and after its termination the critical period could be reopened through upregulation of GluN2B. It is known that inhibitory neural activity increases with development in the CNS including the spinal cord. Suppression of the increasing inhibitory activity using low-dose strychnine also prolonged this critical period. During the strychnine-widened time window, Ca2+ influx through GluN2B channels returned to a level comparable to that seen during the critical period, though the level of GluN2B was slightly reduced. These findings indicate that loss of GluN2B subunits and the associated reduction in Ca2+ influx determines the end of the critical period in our in vitro CS system.

Specific involvement of postsynaptic GluN2B-containing NMDA receptors in the developmental elimination of corticospinal synapses.

The GluN2B (GluRepsilon2/NR2B) and GluN2A (GluRepsilon1/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.

Characterization of the VP1 loop mutations widespread among JC polyomavirus isolates associated with progressive multifocal leukoencephalopathy.

Recently, we found that JC polyomavirus (JCPyV) associated with progressive multifocal leukoencephalopathy (PML) frequently undergoes amino acid substitutions (designated VP1 loop mutations) in the outer loops of the major capsid protein, VP1. To further characterize the mutations, we analyzed the VP1 region of the JCPyV genome in brain-tissue or cerebrospinal fluid samples from 20 PML patients. VP1 loop mutations occurred far more frequently than silent mutations. Polymorphic residues were essentially restricted to three positions (55, 60, and 66) within the BC loop, one (123) within the DE loop, and three (265, 267, and 269) within the HI loop. The mutations at most polymorphic residues showed a trend toward a change to specific amino acids. Finally, we presented evidence that the VP1 loop mutations were associated with the progression of PML. These findings should form the basis for elucidating the biological significance of the VP1 loop mutations.

Regionally specific distribution of corticospinal synapses because of activity-dependent synapse elimination in vitro.

We have shown previously that the corticospinal tract (CST) with functional connections can be reconstructed in vitro in slice cocultures. Using that system, we stimulated the deep cortical layer and recorded field EPSPs (fEPSPs) along a 100 microm-interval lattice in the spinal gray matter. The specific, spatial synapse distribution on the dorsal side at 14 d in vitro (DIV) basically corresponded to the in vivo area in which CST axons terminate. Anterograde labeling of corticospinal axons with biocytin showed a similar terminal distribution on that side. In vitro development of synapse spatial distribution was investigated. fEPSPs were recorded all across the gray matter at 7 DIV, but amplitudes began to decrease on the ventral side at 9 DIV, dorsal-dominant distribution being nearly complete at 14 DIV. Anterograde labeling showed that the decrease in fEPSP amplitudes was associated with a decrease in the number of axon terminals on the ventral area. Decreases in the synaptic responses and terminals were blocked by applications of D-2-amino-5-phosphonovaleric acid and tetrodotoxin, whereas 6-cyano-7-nitroquinoxaline-2,3-dione had a partial effect. These findings suggest that this regressive event, which occurs during development, is activity and NMDA dependent. Retrograde labeling with two colors of beads and an electrophysiological study that investigated the axon reflex showed that at 7 DIV most corticospinal neurons project to both the ventral and dorsal spinal cord, indicating that synapse decrease on the ventral side is attributable primarily to axon branch elimination rather than to death of cortical cells that send axons solely to that side.