A characterisation of last order interneurons of the rodent spinal cord with specific focus on their roles in the control of locomotor activity

It has been known since the early 1900's that networks of neurons within the mammalian spinal cord control locomotion. However, our knowledge of these neuronal networks is very limited. In order to design treatments for paralysing injuries or diseases of the spinal cord, we need to gain a better understanding of the networks of spinal neurons that control movement. This initially requires the identification of neurons which make up these networks. Following their identification we need to study the individual properties of these neurons and how it is that they connect to each other to produce the activity which controls movement. The proposed study aims to provide some of this critical information. Molecular techniques will be used to identify specific populations of neurons in the spinal cord. Using these techniques we will be able to concentrate on 'last order interneurons' which communicate directly with the neurons that send the final message to muscles to contract (motoneurons). Following their identification, last order interneurons will be studied during locomotor activity in isolated preparations of the spinal cord obtained from mice. This will enable us to investigate the roles that specific groups of last order interneurons play in the production of locomotor activity. As outlined above this information is critical toward our understanding of the neural control of movement and ultimately towards the design of treatment strategies for injury or disease which affects the spinal cord.

Despite nearly a century of research since the discovery that the isolated spinal cord can generate locomotion, the neuronal networks underlying such motor behaviour remain poorly understood. However, with the advent of new molecular labelling techniques and physiological preparations allowing the study of locomotor activity in vitro, it is now possible to identify and study components of these networks. Using such techniques the proposed study aims to identify last order interneurons of the spinal cord and elucidate their specific roles in locomotion. Last order interneurons will be identified using a pseudo rabies virus which contains the eGFP construct. The virus will be injected into the hind limb muscles of neonatal mice to retrogradely infect motoneurons and last order interneurons and label them with eGFP. We will concentrate on cholinergic last order interneurons which supply abundant C bouton inputs to motoneurons. Recent evidence suggests that these inputs play an important modulatory role, increasing motoneuron excitability so that appropriate motor output is produced during locomotion. Initially, anatomical analyses will identify cholinergic last order interneurons immunohistochemically. Utilising juxtacellular filling techniques in isolated in vitro spinal cord preparations the axonal projections of cholinergic last order interneurons will then be determined. Next, whole-cell patch-clamp recordings will be performed in spinal cord slice preparations to investigate the intrinsic properties of cholinergic last order interneurons. Finally, simultaneous electroneurographic recordings from ventral roots and paired whole-cell patch-clamp recordings of interneurons and motoneurons will be performed in isolated spinal cord preparations which can elicit fictive locomotion in vitro. These experiments will determine the activity pattern of cholinergic last order interneurons during fictive locomotion, and their effects on motoneurons during fictive locomotion.