Locomotor training maintains normal inhibitory influence on both alpha- and gamma-motoneurons after neonatal spinal cord transection

Abstract

Spinal cord injuries lead to impairments, which are accompanied by extensive reorganization of neuronal circuits caudal to the injury. Locomotor training can aid in the functional recovery after injury, but the neuronal mechanisms associated with such plasticity are only sparsely known. We investigated ultrastructurally the synaptic inputs to tibialis anterior motoneurons (MNs) retrogradely labeled in adult rats that had received a complete midthoracic spinal cord transection at postnatal day 5. A subset of the injured rats received locomotor training. Both γ- and α-MNs were studied. The total number of boutons apposing γ-MNs, but not α-MNs, was reduced after neonatal spinal cord transection. The proportion of inhibitory to excitatory boutons, however, was increased significantly in both α-MNs and γ-MNs in spinally transected rats, but with locomotor training returned to levels observed in intact rats. The specific densities and compositions of synaptic boutons were, however, different between all three groups. Surprisingly, we observed the atypical presence of both C- and M-type boutons apposing the somata of γ-MNs in the spinal rats, regardless of training status. We conclude that a neonatal spinal cord transection induces significant reorganization of synaptic inputs to spinal motoneurons caudal to the site of injury with a net increase in inhibitory influence, which is associated with poor stepping. Spinal cord injury followed by successful locomotor training, however, results in improved bipedal stepping and further synaptic changes with the proportion of inhibitory and excitatory inputs to the motoneurons being similar to that observed in intact rats.

Figures

Figure 1.

Distribution of all MNs based on mean diameter (μm), including intermediary diameter range, which was excluded from synaptology analysis. A, Note the bimodal distribution with a clear trough at the 30–35 μm range for all groups. B, There were no significant differences in the mean diameter of γ- or α-MNs between the three groups. C, D, The total soma membrane synaptic coverage and number of boutons significantly decreased for γ-MNs after transection injury with and without step training, but were unchanged between all groups for α-MNs. *p < 0.05.

Figure 2.

Bouton types in apposition with α- and γ-MNs in neonatally transected rats. A–D, Excitatory S-type (A; green), inhibitory F-type (B; red), large cholinergic C-type (C; yellow), large M-type (D; blue) with associated Taxi-bodies (arrows). E, F, F/S ratios for percentage coverage and number of boutons for γ- and α-MNs. **p < 0.005.

Figure 3.

A, C, Percentage of the membrane covered by each bouton type for γ-MNs (A) and α-MNs (C). B, D, Number of boutons per 100 μm of soma membrane for each bouton type for γ-MNs (B) and α-MNs (D). *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 4.

Electron micrographs of M-type bouton apposing a γ-MN in a neonatally transected rat. A, High magnification of the M-bouton (blue). Note multiple active zones (arrowheads) and HRP product crystals (arrow). B, Detail of box in A showing HRP product crystals (arrow) and active sites (arrowheads). C, C-type bouton (yellow) apposing a γ-MN in a neonatally transected rat. D, Detail of subsynaptic cistern (arrows) associated with the postsynaptic element. Note the presence of a single large dense core vesicle near the presynaptic active zone region. IC, Motoneuron intracellular space; P, P-type bouton.

Figure 5.

A, Kinematics analysis was performed during bipedal locomotion. Retro-reflective markers placed on bony landmarks were digitized to reconstruct movements. B, ST-Tr rats performed a greater number of steps than ST-Non-Tr rats. C–F, Analyses from a ST-Non-Tr rat (C, E,) and a ST-Tr rat (D, F). Note that the ST-Non-Tr rat performed fewer steps, which were not alternating (C) and showed poor intralimb coordination (E). In contrast, the ST-Tr rat performed a greater number of alternating steps (D), which were more consistent and better coordinated (F). *p < 0.05.