Improving our understanding of the anatomy of spinal locomotor networks, how they physiologically interact with proprioception to execute locomotion in vivo, and how ES combined with physical training mechanistically reorganizes spinal networks will provide crucial information in how to use ES optimally to improve clinical outcomes along with inspiring future design of epidural electrodes and training programs. This thesis aims to adapt an engineering and big data workflow to answer these pressing biological problems, namely finding a way to generate large sets of data from current immunohistochemistry (IHC) techniques known to generate very little and to find a way to analyze this large amount of data in a time efficient manner when the current gold standard has been manual analysis.

A complete spinal cord transection results in loss of all supraspinal motor and bladder control below the level of the injury. The neural circuitry in the lumbosacral spinal cord, however, can generate locomotor patterns in the hindlimbs of rats and cats with the aid of motor training, epidural stimulation and/or administration of monoaminergic agonists. Gerasimenko et al., (2003) first reported the use of electrical stimulation to facilitate locomotion in chronic decerebrated cats. Ichiyama et al (2005) then demonstrated that epidural electrical stimulation of the spinal cord can induce rhythmic, alternating hindlimb locomotor activity in chronic spinal rats. Stimulation at the L2 spinal segment at frequencies between 30 and 50 Hz consistently produced successful bilateral stepping. Similar epidural stimulation at other spinal segments were less effective, e.g., epidural stimulation at the T13 or L1 evoked rhythmic activity in only one leg and stimulation at the L3, L4, or L5 produced mainly flexion movements.

More recently, completely paralyzed (motor complete, sensory incomplete) human subjects were implanted with a commercially available spinal cord electrode array and stimulation package originally designed for pain suppression (Harkema et al., 2011). Stimulation of specific spinal segments (caudal electrodes, ~ S1 spinal level) in combination with the sensory information from the lower limbs and weeks of stand training was sufficient to generate full weight-bearing standing. These subjects also recovered some voluntary control of movements of the toe, ankle, and the entire lower limb, but only when epidural stimulation was present. Thus it appears that the epidural stimulation provided excitation of lumbosacral interneurons and motoneurons that, when combined with the weak excitatory activity of descending axons that were not otherwise detectable, achieved a level of excitation that was sufficient to activate the spinal motor circuits. These results demonstrate that some patients clinically diagnosed as having complete paralysis can use proprioceptive input combined with some synaptic input from descending motor signals, perhaps residual but functionally silent without epidural stimulation to the spinal motor circuits to generate and control a range of motor functions during epidural stimulation.

The mechanisms of pharmacological and/or epidural electrical stimulation that enable motor control (eEmc) in the spinal circuitry for locomotion are still not clearly understood. During standing, a single bipolar epidural stimulus between L2 and S1 produces three types of evoked responses, i.e., early (ER, latency 1-3 ms), middle (MR, latency 4-6 ms), and late (LRs, latency >7 ms) in the hindlimb muscles in both intact (Gerasimenko et al., 2006) and spinal (Lavrov et al., 2006) rats. Similar responses were observed during rhythmic locomotor-like EMG activity in the hindlimb muscles of spinal rats while stepping on a motorized treadmill in the presence of epidural stimulation (40 Hz) between L2 and S1 (Lavrov et al., 2008). In addition, the time course of the re-emergence of the LRs was similar to that for the recovery of stepping after a complete spinal cord injury (SCI), indicating that LRs are a potential biomarker of functional recovery (Lavrov et al., 2006).

The results demonstrate that spinal rats can stand and step when the spinal cord is stimulated (tonic 40 Hz stimulation) by electrodes located at specific sites on the spinal cord and at specific frequencies of stimulation. The quality of stepping and standing was dependent on the location of the electrodes on the spinal cord, the specific stimulation parameters, and the orientation of the cathode and anode. spinal cord stimulation triggered evoked potentials in flexor and extensors muscles form a 'foot print' of the physiological state of the spinal cord.

Chronic subthreshold stimulation enabled greater activity in completely transected rats but only with stimulation. Spinal cord stimulation at specific frequencies resulted in partial bladder control.

Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the curry spice curcumin (Cur) could affect spinal cord learning in adult spinal mice. Using Paw Withdrawal instrumental learning paradigm (PaWL) to assess spinal learning we observed that mice fed a diet containing DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. Previous studies in the cortex have demonstrated that both DHA and curcumin elevate the expression of brain derived neurotrophic factor (BDNF) and plays a major role in learning and neural plasticity. Here we examined the effect of DHA and curcumin on the Paw Withdrawal learning. The enhanced performance was accompanied by increases in mRNA of molecular markers of learning, i.e., BDNF, cyclic AMP response element-binding protein (CREB), Calcium/Calmodulin Kinase II (CaMKII), and syntaxin 3. Sequestering BDNF with TrkB IgG in the lumbar spinal cord of DHA/Cur fed mice showed significant decrease in spinal learning, and levels of mRNA of BDNF, CaMKII, CREB and syntaxin 3. These results emphasize the capacity of select dietary factors to foster spinal cord learning and suggest the BDNF pathway may be a key mediator of spinal plasticity. Given the non-invasiveness and safety of the modulation of diet, this intervention should be considered in light of their potential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after spinal cord injury.

Sensory afferents to the spinal cord is the main source for therapeutic intervention and rehabilitation that can generate and coordinate complex movements even without supraspinal control. Using the activity-dependent marker c-fos, we identified cholinergic lumbar interneurons around the central canal that are activated during load bearing stepping in intact and complete spinal cord transected adult rats. We observed activation of cholinergic central canal cluster and partition neurons in intact rats after quadrapedal stepping and in spinal rats step trained bipedally for 5 weeks with epidural and pharmacological stimulation. Furthermore, increased load imposed during treadmill stepping activated more cholinergic interneurons in adult rats. Intact adult mice whose tibialis anterior (TA) hindlimb muscle was injected with transynaptic pseudorabies virus, showed activated cholinergic interneurons in the TA spinal circuitry after quadrapedal stepping. These data demonstrate the role of sensorimotor pathways and specific interneurons in the recovery after spinal cord injury.