Pre-motor neuronal networks, from connectivity to function

The human central nervous system contains more than 100 billion neurons, each one of them transmitting and receiving information from thousands of other neurons through small contacts called synapses. Decoding the exact wiring diagram of such a complicated machine is one of the most fascinating tasks in modern neuroscience and it could be said that our cognitive and motor functions, who we are and what we do, are strictly related to the pattern of connectivity between our nerve cells and the strength of their connections, in the same way as the performance of a computer is determined by the wiring of its microcircuits. Until a few years ago the task of deciphering the neural code might have seemed unapproachable in the case of complex organisms and in fact it was achieved with some success only on small animals whose brain is composed of a few tens or at most hundreds of neurons. The study of the so called "connectome", or in other words the map of the brain connections over a large scale, has made giant steps in the past four years thanks to the introduction of novel modified viral compounds attached to fluorescent proteins that can spread to all of the neurons that make synaptic contacts with the cell or group of cells that were initially infected, and only to those cells. We plan to use this novel and powerful tool to infect specific populations of motoneurons in the spinal cord. Motoneurons are the only cells in the central nervous system that excite non neuronal cells (the muscles) and they are responsible for the initiation and coordination of every single movement we make. Following infection of motoneurons associated with specific muscles, we will be able to trace down all of the cells that communicate with them and determine their positions relative to their target motoneurons. We will also record the electrical signal transmitted to the motoneuron from each of the connected cells and we will be able to measure their relative contribution to the execution of motor tasks. Mapping the connectivity one cell at a time is extremely accurate, but it could be time consuming and not very effective, so we will use another recently developed technique and attach to the viral construct a protein that once expressed in the target cell, can excite it when exposed to an intense blue light and therefore imitate the normal mode of transmission between neurons. With the aid of this light activated protein we will be able to hop quickly from one cell to the other using a blue laser beam and map the entire connectivity of single motoneurons. The information that we will collect from this large scale connectivity study will tell us how the motor circuits in the spinal cord are wired together to produce the complex and multifaceted motor tasks that we execute every day.
Our proposed research will give us an unprecedented level of knowledge of the circuits underlying the control of movement. This is certainly one of the prerequisites to design interventions aimed at repairing damages that occurs due to injuries or diseases. Furthermore, we will exploit new methods for transferring specific coding genes into population of cells and for controlling their activity with light. These methods have been already used with spectacular success for controlling seizures in an animal model of epilepsy and it is possible that their application to the spinal cord could be a future avenue towards an improvement in the quality of life of subjects with impaired motor control.

The timing of motoneuron firing is regulated by the strength and density of synaptic inputs they receive from an intricate network of excitatory and inhibitory premotor interneurons, whose characteristics, spatial distribution and relative effect on motoneurons are largely unknown. The recent development of viral trans-synaptic tracers that can selectively label individual motor nuclei and retrogradely cross a single synaptic step gives us a unique opportunity to target last order interneurons for expression of fluorescent proteins to label them and of light activated opsins, to excite them selectively. We will obtain a connectivity map for motor nuclei corresponding to specific pairs of antagonist or synergist hindlimb muscles and determine whether muscles with different functions are controlled by spatially separated groups of premotor interneurons. Reconstruction of fixed and labelled tissue, together with immunocytochemistry will be used to identify the position of known classes of premotor cells, including neurons with proprioceptive inputs, Renshaw cells and commissural interneurons. Using multiple patch clamp recordings we will measure the strength of individual excitatory and inhibitory synapses and determine whether synaptic strength differs systematically among cells belonging to different classes or those contacting motor nuclei with different functions. We will also test whether premotor interneurons contacting synergist or antagonist motor pools have a stereotyped pattern of connectivity and whether this depends on their excitatory/inhibitory phenotype.
Our experiments will produce a large scale connectivity map of premotor circuits and will provide a quantitative measure of the strength and distribution of inputs to motoneurons. Knowledge of the circuitry of networks involved in motor control is a strict requirement for understanding and possibly repairing, the damage occurring due to disease or injury, and our proposal is a step in this direction.

Understanding the wiring diagrams in the central nervous system is basic research, but there are many reasons why the work we are proposing has the potential for being exploited for the general well-being of society. While some of the outcomes might take time to come to fruition, others might be achieved within the time scale of our proposed project.
Health sector
One pathway towards impact is represented by potential contributions to the health sector: in the UK alone there are more than 50,000 people suffering severe disabilities due to spinal cord injury and the social and economic cost is enormous. Any research that can improve our knowledge of motor function in a healthy organism has potential impact on their well being.
Traditionally, research strategies have focussed on fibre regeneration or on electrical stimulation in an attempt to bridge across the damaged connections. Both approaches require:
1) knowledge of the circuits
2) safe and locally efficient drug delivery methods
3) electrical control of the neural network.
These frontier themes are fully developed in our research program, since we will describe motor circuits in an intact organism and exploit a novel method of gene transfer using viral constructs. Recently, a similar method of gene delivery by means of viral constructs has been successfully used to control epilepsy in mice. Our techniques have a high potential of being translated into animal models, with the future prospect of an application to humans with spinal cord injuries. We are in an advantageous position to promote the translation, due to the overlap of interests of the PI with the neighbouring Sobell Department of Motor Neuroscience and Movement Disorders at UCL, one of the UK's leading institutions in motor research. The PI is already interacting with the Sobell Unit, through joint meetings and seminars. We will extend this interaction to include informal lab presentations and explore the applicability of our techniques to current animal models of spinal cord injury and disease. The co-PI (DJM) is currently involved in a collaborative study of the reorganisation of corticospinal tract terminations in a rat stroke model, with basic scientists and clinicians. In this study we are examining the potential for information from the corticospinal tract to be conveyed to motoneurons via novel 'detour' circuits. This project will ultimately involve the use of stem cells to promote new growth; a technique that is currently being pursued in clinical trials in Glasgow. Greater knowledge of premotor interneurons will enable us to understand their role in motor networks and ultimately identify suitable candidates for detour circuits. In particular, we need much more information about commissural interneurons which have the potential to convey information from the corticospinal tract to contralateral regions of the cord that have become denervated as a consequence of stroke.
Industry
During the past 4 years the PI has worked in close contact with suppliers of optical and electrophysiological instruments and has offered advice on the design of new tools and the optimization of existing ones. Especially fruitful has been the interaction with Scientifica, a leading UK provider of specialized tools for research and recent winner of the Queen's Award for Enterprise. The PI designed a custom accessory to avoid transmitting vibration during delicate recordings that is currently commercially available and widely used in the community. Furthermore, the PI contributed to the design, troubleshooting and testing of a novel amplifier (ELC-03), that is the only commercially available instrument designed to perform simultaneously recordings and stimulations of neurons. The PI has currently a pending application for a Case Studentship (jointly funded by UCL and Scientifica) to develop a new tool for selective optical stimulation. Its development is in progress and the first bench tests are expected within a year.