Locomotion is an innate behaviour that is controlled by different areas of the central nervous system, which allow for effectiveness of movement. The spinal cord is an important centre involved in the generation and maintenance of rhythmic patterns of locomotor activity such as walking and running. Interneurons throughout the ventral horn of the spinal cord form the locomotor central pattern generator (CPG) circuit, which produces rhythmic activity responsible for hindlimb movement. Motoneurons within the lumbar region of the spinal cord innervate the leg muscles to convey rhythmic CPG output to drive appropriate muscle contractions. Intrinsic modulators, such as acetylcholine acting via M2 and M3 muscarinic receptors, regulate CPG circuitry to allow for flexibility of motor output. Using electrophysiology and genetic techniques, this work characterized the receptors involved in cholinergic modulation of locomotor networks and the role and mechanism of action of a subpopulation of genetically identified cholinergic interneurons in the lumbar region of the neonatal mouse spinal cord. Firstly, the effects of M2 and M3 muscarinic receptors on the output of the lumbar locomotor network were characterised. Experiments in which fictive locomotor output was recorded from the ventral roots of isolated spinal cord preparations revealed that M3 muscarinic receptors are important in stabilizing the locomotor rhythm while M2 muscarinic receptor activation seems to increase the irregularity of the locomotor frequency whilst increasing the strength of the motor output. This work then explored the cellular mechanisms through which M2 and M3 muscarinic receptors modulate motoneuron output. M2 and M3 receptor activation exhibited contrasting effects on motoneuron function suggesting that there is a fine balance between the activation of these two receptor subtypes. M2 receptor activation induces an outward current and decreases synaptic drive to motoneurons while M3 receptors are responsible for an inward current and increase in synaptic inputs to motoneurons. Despite the different effects of M2 and M3 receptor activation on synaptic drive and subthreshold properties of MNs, both M2 and M3 receptors are required for muscarine-induced increase in motoneuron output. CPG networks therefore appear to be subject to balanced cholinergic modulation mediated by M2 and M3 receptors, with the M2 subtype also being important for regulating the intensity of motor output. Next, using Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology, the impact of the activation or inhibition of a genetically identified group of cholinergic spinal interneurons that express the Paired-like homeodomain 2 (Pitx2) transcription factor was explored. Stimulation of these interneurons increased motoneuron output through the activation of M2 muscarinic receptors and subsequent modulation of Kv2.1 channels. Inhibition of Pitx2+ interneurons during fictive locomotion decreased the amplitude of locomotor bursting. Genetic ablation of these cells confirmed that Pitx2+ interneurons increase the strength of locomotor output by activating M2 muscarinic receptors. Overall, this work provides new insights into the receptors and mechanisms involved in intraspinal cholinergic modulation. Furthermore, this study provides direct evidence of the mechanism through which Pitx2+ interneurons regulate motor output. This work is not only important for advancing understanding of locomotor networks that control hindlimb locomotion, but also for dysfunction and diseases where the cholinergic system is impaired such as Spinal Cord Injury and Amyotrophic Lateral Sclerosis.

Locomotion persists despite changes in external and internal circumstances. Motor responses to gait impairment exhibit commonalities across various taxa and types of injury, yet we lack a systematic understanding of compensation strategies. The objective of this dissertation is to uncover principles governing implicit goals within the control of locomotion. I propose that coordination of injured locomotion will demonstrate that these goals follow a hierarchical organization of the neuromuscular system. Accurate quantification of gait deficits in rodents demands sophisticated measurement techniques. I utilize X-ray technology to examine intralimb and interlimb coordination after unilateral injury in rats. My findings indicate that compensation to injury involves the coordination of lower-order motor elements to preserve the pre-injury behaviors of higher-order elements. Specifically I present evidence that preservation of limb angle and limb length are critical task goals that transcend injury states and afferent sensory feedback conditions. Broadening my investigation to include interlimb coordination revealed that task goals may change to satisfy the goals of a higher hierarchical level. This work is a necessary precursor to study locomotor coordination and injury compensation in more complex rodent injury models such as self-reinnervation, sciatic nerve, and spinal cord injury. These results could also translate to clinical gait rehabilitation through future protocols that address motor patterns of the entire limb over the behavior of individual joints.

The present study examined the role of group I metabotropic glutamate receptors (mGluRs) in mammalian spinal motor networks and investigated the potential role of mGluRs in the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Group I mGluR activation was found to modulate locomotor-related activity recorded from ventral roots of in vitro mouse spinal cord preparations. Activation of group I mGluRs led to an increase in the frequency of locomotor-related bursts and a decrease in their amplitude. The cellular mechanisms underlying group I mGluR-mediated modulation were investigated using whole-cell patch-clamp recordings from spinal neurons. Recordings from motoneurons revealed a wide range of effects, some of which were expected to increase motoneuron excitability, such as membrane depolarisation and hyperpolarisation of action potential thresholds. However, the net modulatory effect of group I mGluR activation was a reduction in motoneuron excitability, likely reflecting a reduction in the density of fast inactivating Na+ currents. The activation of group I mGluRs also reduced excitatory synaptic input to motoneurons, suggesting that modulation of motoneuron properties and synaptic transmission both contribute to group I mGluR-mediated reductions in locomotor motoneuron output. Recordings from spinal interneurons revealed a smaller range of modulatory effects for group I mGluRs. The clearest effect on interneurons, membrane depolarisation, may underlie group I mGluR-mediated increases in the frequency of locomotor activity. Finally, the potential role of group I mGluRs in the pathogenesis of ALS was investigated using a mouse model of the disease. Although no major perturbations in group I mGluR-mediated modulation were demonstrated in ALS affected spinal cords, there appeared to be a difference in the intrinsic excitability of spinal interneurons between wild type and ALS affected animals. Together these data highlight group I mGluRs as important sources of neuromodulation within the spinal cord and potential targets for the treatment of ALS.

Rhythmic movementssuch as swimming, flying, and walkingare ubiquitous in nature. Intrinsically active neural networks called central pattern generators (CPGs) provide the feedforward signals to actuate these movements, but the preferred movement frequency is often equivalent to the resonant frequency of the musculoskeletal system. Sensory feedback is essential to synchronize the neural and musculoskeletal systems to the mechanical resonant frequency, a phenomenon called resonance tuning. In this dissertation, we use a simple computational model of rhythmic movement to understand how the configuration of sensory feedback affects both the sensitivity of resonance tuning to parameter variation and the resiliency of resonance tuning to perturbation. Although previous studies have shown that resonance tuning is limited to frequencies that are above the intrinsic CPG frequency, we demonstrate that this limitation is only valid with negative feedback and with endogenously bursting CPG neurons. Specifically, we show that with positive feedback, resonance tuning occurs at frequencies that are below the intrinsic CPG frequency. Moreover, when the synaptic connections within the CPG are required for bursting activity, resonance tuning occurs both above and below the intrinsic CPG frequency with negative feedback and does not occur with positive feedback. Using Floquet analysis, we then demonstrate that perturbations decay more quickly when resonance tuning is realized with positive than with negative proportional feedback. Finally, we evaluate how the intrinsic CPG frequency, feedback gain, and mechanical damping affect the stability and range of resonance tuning with negative and positive feedback. Overall, these results indicate that the configuration of sensory feedback dramatically affects both the parameter space in which resonance tuning occurs and the stability of the resultant periodic motion.

Every movement has a goal. For reaching, the goal is to move the hand to a specific location. For locomotion, however, goals for each step cycle are unclear and veiled by the automatic nature of lower limb control. What mechanical variables does the nervous system "care" about during locomotion? Abundant evidence from the biomechanics literature suggests that the force generated on the ground, or endpoint force, is an important task variable during hopping and running. Hopping and running are called bouncing gaits for the reason that the endpoint force trajectory is like that of bouncing on a pogo stick. In this work, I captured kinematics and kinetics of human bouncing gaits, and tested whether structure in the inherent step-to-step variability is consistent with control of endpoint force. I found that joint torques covary from step to step to stabilize only peak force. When two limbs are used to generate force on the ground at the same time, individual forces of the limbs are not stabilized, but the total peak force is stabilized. Moreover, passive dynamics may be exploited during forward progression. These results suggest that the number of kinetic goals is minimal, and this simple control scheme involves goals for discrete times during the gait cycle. Uncovering biomechanical goals of locomotion provides a functional context for understanding how complex joints, muscles, and neural circuits are coordinated.

Based on the belief that it is a locomotor control structure, the pedunculopontine tegmental nucleus (PPTg) has been considered a potential target for deep brain stimulation (DBS) for Parkinson's disease (PD) patients with symptoms refractory to medication and/or stimulation of established target sites. To date, a number of patients have been implanted with PPTg electrodes with mostly disappointing results. Exact target site in PPTg, possible mechanisms of PPTg-DBS and likely potential benefits need to be systematically explored before consideration of further clinical application. The research described here approaches these questions by (i) investigating the role of the PPTg in gait per se; (ii) developing a refined model of PD that mimics the underlying pathophysiology by including partial loss of the PPTg itself; (iii) adapting a wireless device to let rats move freely while receiving DBS; and (iv) investigating the effect of DBS at different sites in the PPTg on gait and posture in the traditional and refined model of PD. Underlining the concern that understanding the PPTg as a locomotor control structure is inadequate, the experiments showed that neither partial nor complete lesions of PPTg caused gait deficits. The refined model showed hardly any differences compared to the standard one, but the effect of DBS in each was very different, highlighting the need to take degeneration in the PPTg into consideration when investigating it as a DBS target. The differential results of anterior and posterior PPTg-DBS show the critical importance of intra-PPTg DBS location: Anterior PPTg electrodes caused severe freezing and worsened gait while some gait parameters improved with stimulation of posterior PPTg. The results suggest mechanisms of PPTg-DBS beyond the proposed activation of over-inhibited PPTg neurons, including aggravation of already dysfunctional inhibitory input by anterior PPTg-DBS and activation of ascending projections from posterior PPTg to the forebrain.

Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq-linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq-linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks.