Changes in motoneuron properties and synaptic inputs related to step training after spinal cord transection in rats

Abstract

Although recovery from spinal cord injury is generally meager, evidence suggests that step training can improve stepping performance, particularly after neonatal spinal injury. The location and nature of the changes in neural substrates underlying the behavioral improvements are not well understood. We examined the kinematics of stepping performance and cellular and synaptic electrophysiological parameters in ankle extensor motoneurons in nontrained and treadmill-trained rats, all receiving a complete spinal transection as neonates. For comparison, electrophysiological experiments included animals injured as young adults, which are far less responsive to training. Recovery of treadmill stepping was associated with significant changes in the cellular properties of motoneurons and their synaptic input from spinal white matter [ipsilateral ventrolateral funiculus (VLF)] and muscle spindle afferents. A strong correlation was found between the effectiveness of step training and the amplitude of both the action potential afterhyperpolarization and synaptic inputs to motoneurons (from peripheral nerve and VLF). These changes were absent if step training was unsuccessful, but other spinal projections, apparently inhibitory to step training, became evident. Greater plasticity of axonal projections after neonatal than after adult injury was suggested by anatomical demonstration of denser VLF projections to hindlimb motoneurons after neonatal injury. This finding confirmed electrophysiological measurements and provides a possible mechanism underlying the greater training susceptibility of animals injured as neonates. Thus, we have demonstrated an "age-at-injury"-related difference that may influence training effectiveness, that successful treadmill step training can alter electrophysiological parameters in the transected spinal cord, and that activation of different pathways may prevent functional improvement.

Figures

Figure 1.

Schematic of the experimental preparation demonstrates the placement of the ipsilateral VLF electrode in the T11 cord and the heteronymous and homonymous monosynaptic connections of the stretch-reflex afferents from the MG and LGS nerves onto the corresponding motoneurons at L4/5. The inset shows a Nissl-stained tissue section where an electrolytic lesion was created to reveal the area of the spinal cord stimulated by the VLF electrode (dorsal at top; midline at right). The gray lines indicate the electrode track, and the dotted lines indicate the largest possible stimulation zone.

Figure 2.

Relative joint-angle plots (left) and step reconstruction (right) reveal the effects of successful training on treadmill stepping at 21 cm/s. Each axis represents the internal angle of the designated joint, which provides measures that are independent for each joint. Flexion for each joint is in the direction of decreasing angle, whereas extension is in the direction of increasing angle. The relationship of joint angles across steps in the trained good-stepper rats is highly consistent and resembles that of normal rats (bottom plot). The points of paw placement (gray arrowhead) and toe off (black arrowhead) and the stance (gray arrow) and swing (black arrow) phases are indicated on the plots from the normal and trained good-stepper rats. These behavioral elements were too inconsistent for such indicators in the other groups. Axes of all plots have a range of 90° (hip, knee) or 160° (ankle), although the minimum–maximum (hip, knee) are slightly different for the normal animal.

Figure 3.

The effect of spinal transection on cellular and synaptic parameters depends on age at transection (neonate vs adult). Motoneuron and synaptic properties from intact, neonatal-transected nontrained (Neonate), adult-transected (Adult), and adult-transected short-term recovery (Adult-ST) groups are displayed as cumulative sum histograms [pooled data from all animals in a group are plotted to demonstrate the percentage of values (y-axis) falling below a particular value (x-axis)]. These histograms represent data from all individual cells pooled over all experiments of a given type, whereas statistical comparisons in the Results were derived from means calculated from each animal. AHPd is increased by injury with neonate > adult > adult-ST. sEPSP is decreased slightly by long-term injury (either neonate or adult) but increased slightly by short-term injury in the adult. The monosynaptic component of the VLF EPSP (cEPSP-mono) is decreased substantially after both short- and long-term transection in the adult and decreased to a lesser extent after neonatal injury.

Figure 4.

Neonatally transected trained poor-stepper rats display a unique electrophysiological signature. The top panel shows the change in peak amplitude, in all recorded motoneurons, of the fifth EPSP (expressed as a percentage of the amplitude of the first EPSP) elicited by 100 Hz burst stimulation of the VLF. Note that most EPSPs in poor-steppers are potentiated to a greater degree than the maximum level in all the other preparations. The bottom panel displays records from the cell displaying the greatest facilitation in each treatment group. Groups: D, intact; C, neonatal-TX nontrained; B, trained good-stepper; A, trained poor-stepper. Traces are normalized with respect to the first EPSP.

Figure 5.

Motoneuron and synaptic properties in neonatally transected rats are affected by training and vary with success of the training. Graphs display cumulative sum histograms of motoneuron and synaptic properties from intact, neonatal-transected nontrained (Non-Tr), trained good-stepper (Tr-good), and trained poor-stepper (Tr-poor) groups. Histograms are from all individual cells in each group (as described in Fig. 3), and symbols used for plots are shared with Figure 3. AHPd is increased by injury and is returned toward normal with successful training. sEPSP is reduced in groups that did not step well, whereas successful training increased the sEPSP above values in intact preparations. The monosynaptic component of the VLF EPSP (cEPSP-mono) was reduced in all groups (although to differing degrees), but successful training increased the polysynaptic component (cEPSP-poly) compared with nontrained animals.

Figure 6.

The relationship of behavioral and electrophysiological measures is demonstrated for rats in the neonatal-transected nontrained, trained good-stepper, and trained poor-stepper groups. Rats with the best stepping capacity tended to have both large sEPSP and small AHPd. Rats with poor-stepping capacity displayed values of sEPSP and AHPd similar to nontrained rats.

Figure 7.

The drive index correlates strongly with the stepping capacity across the different groups. The number of steps taken by each rat during a 10 s test period is plotted against the drive index for neonatally injured rats. Groups are nontrained (gray triangles), trained good-stepper (filled circles), and trained poor-stepper (open circles).

Figure 8.

Anatomical tracing demonstrates projections to the MG motoneuron pool. A, B, Assay 1 demonstrates axons in the L5 ventral horn labeled from the ipsilateral VLF at T11 (red; injection site similar to VLF electrode site). Many appear to have projections onto MG motoneurons (green). A lower-power example is shown in supplemental Figure 3 (available at www.jneurosci.org as supplemental material). Animals injured as neonates (B) appear to have more labeled axons projecting into the region of the MG motoneurons than those injured as adults (A) (see Results). Some of these projections are in close apposition to motoneuron cell bodies and dendrites and have swellings suggestive of synaptic contacts (arrows in D and E). C, Assay 2 demonstrates that neurons retrogradely labeled (red; yellow arrows) from their projection to the L5 ventral horn (area of MG motoneuron pool) were concentrated in laminas VI-IX of the lower thoracic cord (area of VLF stimulation). The ipsilateral gray matter is outlined with white dots and neurons labeled with NeuN (green). The yellow dotted line delineates the general separation between laminas IV and V. The lateral edge of the cord is left, dorsal is top, and midline is right. Scale bars, 20 μm.