The Lesioned Spinal Cord Is a "New" Spinal Cord: Evidence from Functional Changes after Spinal Injury in Lamprey

Abstract

Finding a treatment for spinal cord injury (SCI) focuses on reconnecting the spinal cord by promoting regeneration across the lesion site. However, while regeneration is necessary for recovery, on its own it may not be sufficient. This presumably reflects the requirement for regenerated inputs to interact appropriately with the spinal cord, making sub-lesion network properties an additional influence on recovery. This review summarizes work we have done in the lamprey, a model system for SCI research. We have compared locomotor behavior (swimming) and the properties of descending inputs, locomotor networks, and sensory inputs in unlesioned animals and animals that have received complete spinal cord lesions. In the majority (∼90%) of animals swimming parameters after lesioning recovered to match those in unlesioned animals. Synaptic inputs from individual regenerated axons also matched the properties in unlesioned animals, although this was associated with changes in release parameters. This suggests against any compensation at these synapses for the reduced descending drive that will occur given that regeneration is always incomplete. Compensation instead seems to occur through diverse changes in cellular and synaptic properties in locomotor networks and proprioceptive systems below, but also above, the lesion site. Recovery of locomotor performance is thus not simply the reconnection of the two sides of the spinal cord, but reflects a distributed and varied range of spinal cord changes. While locomotor network changes are insufficient on their own for recovery, they may facilitate locomotor outputs by compensating for the reduction in descending drive. Potentiated sensory feedback may in turn be a necessary adaptation that monitors and adjusts the output from the "new" locomotor network. Rather than a single aspect, changes in different components of the motor system and their interactions may be needed after SCI. If these are general features, and where comparisons with mammalian systems can be made effects seem to be conserved, improving functional recovery in higher vertebrates will require interventions that generate the optimal spinal cord conditions conducive to recovery. The analyses needed to identify these conditions are difficult in the mammalian spinal cord, but lower vertebrate systems should help to identify the principles of the optimal spinal cord response to injury.

Keywords: lamprey; neuromodulation; plasticity; regeneration; spinal cord injury.

FIGURE 1

Behavioral recovery. (Ai) Video frames from an animal that recovered good locomotor function. The bar indicates a mechanical stimulus given to the body, and shows the coordinated movement away from the stimulus. (Aii) Swimming of an animal that failed to recover. (B) Graph showing the correlation of swimming performance with the degree of regeneration, assessed from ventral root responses below the lesion site ipsilateral (A3) and contralateral (A4) to stimulation above the lesion. (Ci–Civ) Graphs comparing the swimming performance in animals that recovered well with that of unlesioned animals: the only significant difference was the duration of a swimming episode. (Di–Diii) Example of myogram activity from animals that failed to recover locomotor function. Data from Hoffman and Parker (2011). Permission granted to reproduce. ^∗Indicates statistical significance at p < 0.05.

FIGURE 2

Properties of regenerated synapses. (Ai) The initial EPSP amplitude from reticulospinal axons in unlesioned and lesioned spinal cords. The inset shows the largest response seen in an unlesioned spinal cord (Aii), and the occasional very large inputs seen in lesioned animals (Aiii). (B) Proportions of connections showing different forms of activity-dependent plasticity in unlesioned and lesioned spinal cords. (C) Graph comparing facilitation in unlesioned spinal cords and from above the lesion site. The inset shows facilitation above the lesion. (D) The effect of high and low Ca²⁺ on the properties of reticulospinal EPSPS. (E) Variance-mean plots in high and low calcium for four connections.

FIGURE 3

(Ai) Experimental approach to examine changes in spinal cord excitability. A₁–C₄ are ventral root locations along the body relative to a lesion site (or where the lesion would be in an unlesioned spinal cord). A₁–A₂ are ipsilateral or contralateral ventral root responses recorded one segment above the lesion in response to stimulation three segments above the lesion (A_Stim); A₃ and A₄ are ipsilateral and contralateral responses, respectively, evoked by A_Stim two segments below the lesion site. B₁–B₈ are ventral root locations 2–20 segments below the lesion site (B_1/2, 2 segments below; B_3/4, 5 segments below; B_5/6, 10 segments below B_7/8, 20 segments below) ipsilateral to stimulation one segment below the lesion (B_Stim). C_1-4 are ventral root locations 10–20 segments below the lesion site (C_1/2, 11 segments below; C_3/4, 20 segments below) in response to stimulation 10 segments below the lesion (C_Stim). (Aii) Changes in excitability in larvae that showed good recovery. The colored squares represent increased excitability for stimulation in the regions indicated by the boxes. (Aiii) Excitability changes in larvae that showed poor recovery and regeneration, and (Aiv) in larvae that showed poor recovery and no regeneration. (Av) Excitability changes in juvenile adults that showed poor recovery. (Avi) Excitability changes in juvenile adults that showed good recovery. (Bi) The relationship between the extent of regeneration and the excitability below the lesion site. With good recovery sub-lesion excitability increased as regeneration increased, but in poor recovery excitability decreased as regeneration increased (Bii).

FIGURE 4

Lesion-induced changes in cellular properties. (Ai) Graph showing changes in excitability in unlesioned larvae, and in lesioned larvae showing good and poor recovery. Traces show excitability changes in a lesioned (Aii) and unlesioned animal (Aiii). (B) Spontaneous synaptic inputs in an unlesioned spinal cord, and below the lesion site in animals that showed good or poor recovery. (C) Graph and traces showing “double” spontaneous miniature EPSPs. (Di) Graph showing the number of vesicles in synaptic terminals in an unlesioned and a lesioned spinal cord. (Dii) Electron micrographs showing examples of putative glutamatergic synapses in an unlesioned (top) and a lesioned spinal cord (below). (E) Example of an excitatory interneuron to motor neuron connection in an unlesioned animal and the slow depolarization that occurs at the same type of connection in a lesioned animal. (F) Changes in spontaneous synaptic inputs above and below the lesion site in juvenile adult animals. (G) Changes in excitatory and inhibitory synaptic inputs above and below the lesion site in juvenile animals that recovered well or poorly. Data is presented as a boxplot to indicate the variability. Data from Cooke and Parker (2009) and Becker and Parker (2015). No permission is required to reproduce this material. ^∗Indicates statistical significance at p < 0.05.

FIGURE 5

Changes in reticulospinal-evoked synaptic inputs to motor neurons in unlesioned juvenile animals, and lesioned animals above and below the lesion site. (A) Note the facilitation above, but depression below the lesion site and in unlesioned animals. (B) Spontaneous synaptic inputs above and below the lesion site in juvenile adult animals. (C,D) Evidence for polysynaptic excitatory synaptic interactions (indicated by ^∗) above the lesion site. (E,F) Polysynaptic inhibition below the lesion site in juvenile adult animals (in this case ^∗ refers to inputs that failed to evoke an input, IPSPs only developing during the spike train as a result of activation of the feedforward inhibitory pathway). Data from Becker and Parker (2015). No permission is required to reproduce this material.

FIGURE 6

Changes in sensory feedback after lesioning. (A) Diagram showing the experimental procedure for evoking and monitoring proprioceptive feedback to the spinal cord. The spinal cord is fixed except at a free end that is attached to a computer-driven motor that imposes sinusoidal movements of the cord. Movement-dependent activity is recorded from the lateral tract where edge cell axons run. Stretch-evoked responses to a 1 Hz bending command in an unlesioned (Bi) and lesioned spinal cord (Bii). (Ci) Graph showing the significant reduction of the post-spike slow afterhyperpolarization (sAHP) in an edge cell in a lesioned spinal cord. The inset shows an edge cell action potential in an unlesioned and lesioned spinal cord (thick line). (Cii) Traces showing the increase in edge cell excitability in response to depolarizing current injection in the unlesioned and lesioned spinal cord. (Di) Traces showing spontaneous synaptic inputs in an edge cell in an unlesioned and lesioned spinal cord. (Dii) Graph showing the significant increase in the integrated spontaneous synaptic input in edge cells after lesioning. Data from Hoffman and Parker (2011). Permission granted to reproduce. ^∗Indicates statistical significance at p < 0.05.

FIGURE 7

Modulation of proprioceptive activity. (Ai,Aii) The effect of GABA on bending-evoked activity recorded from the lateral margin of the spinal cord. Notice that GABA effects are weaker in lesioned animals. Traces showing proprioceptive activity in an unlesioned (Bi) and a lesioned animal (Bii). (Ci,Cii) The effects of bicuculline on bending-evoked activity depended on the degree of recovery: note that bicuculline effects are only significant in lesioned animals that showed good recovery. Traces showing the effects of bicuculline on bending-evoked activity in animals that showed poor (Di) and good recovery (Dii). Data from Svensson et al. (2013). Permission granted to reproduce. ^∗Indicates statistical significance at p < 0.05.

FIGURE 8

5-HT-mediated modulation after lesioning. (A) Graph showing the effects of 5-HT on the resting membrane potential in unlesioned animals, and lesioned animals that showed good and poor recovery. (B) Graph showing the effects of 5-HT on the sAHP in good and poor recovery: note the amplitude was only significantly reduced in animals that showed good recovery. (C) Graph showing the effects of 5-HT on glutamatergic synaptic inputs in unlesioned animals and lesioned animals above and below the lesion site. The inset shows the effect of 5-HT in an unlesioned spinal cord. Data from Becker and Parker (2015). No permission is required to reproduce this material. ^∗Indicates statistical significance at p < 0.05.

FIGURE 9

Effects of 5-HT on swimming. (Ai,Aii) EMG traces from swimming animals showing activity above and below the lesion site in an animal that failed to recover locomotor function, and the effects of 5-HT in improving activity in the same animal. (Bi,Bii) EMG traces showing activity in an unlesioned animal, and in the same animal after incubation in PCPA for 72 h to deplete 5-HT. (Ci) EMG traces showing the poor activity in an animal that was incubated in PCPA after lesioning. (Cii) EMG traces showing activity in a lesioned animal after incubation in PCPA after it had recovered. Note that after recovery incubation in PCPA was without effect. Data from Becker and Parker (2015). No permission is required to reproduce this material.

FIGURE 10

Summary of changes after lesioning. (A) The unlesioned network. Descending inputs project to the locomotor network. The network scheme is simplified to focus on known aspects, essentially limited to one half-center or hemisegmental network (open circles are glutamatergic synapses, filled circles are glycinergic synapses). Two hemisegmental networks in each spinal segment control activity on the left and right sides of the body, and are coupled by reciprocal inhibitory connections, the nature of which remains uncertain (Parker, 2006). The hemisegmental network contains EINs that provide glutamatergic inputs to other EINs, motor neurons, and the small ipsilateral inhibitory interneurons (SiIN): the latter provide feedback inhibition to the EINs and feedforward inhibition to motor neurons (this circuitry has been characterized in adults (Jia and Parker, 2016)). Movement is detected by proprioceptive edge cells that provide feedback to the locomotor network. (B) General summary of the changes after injury: see text for details of above/below, good/poor recovery, and larval/adult changes. Thicker lines represent increased activity, thinner reduced. Descending inputs to the spinal cord are reduced in number but individual connections are unaltered after lesioning. In the locomotor network there are changes in the cellular properties of EINs and motor neuron (larvae) and the connectivity and synaptic properties of the EINs (larvae and juveniles). Connections to the SiINs seem a key difference, with increased activity associated with poor recovery in larvae but with good recovery in adults. Sensory inputs are also increased after lesioning.