Pre-motor neuronal networks, from connectivity to function

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description A new key finding has unexpectedly emerged more than two years after the end of the award. The initial application was based on published results showing that the groups of premotor interneurons associated with antagonist muscles had a distinct spatial segregation. We have recently discovered that the reported spatial segregation is probably due to an artefact introduced by the original tracing methods. A new and revised method has been devised, in collaboration with 4 different labs (see 'Collaborations and Partnership' section) and we are currently pooling data in preparation for a major publication challenging the existing literature
Exploitation Route Our findings will influence the theory and understanding of motor coordination and the general knowledge we are accumulating on the spinal circuitry responsible for locomotion. Knowledge of the connectivity among different cells in the spinal cord is an essential steps towards a better understanding of the effects of diseases afecting motor coordination at the central nervous system level.
Sectors Digital/Communication/Information Technologies (including Software),Pharmaceuticals and Medical Biotechnology

URL http://www.jneurosci.org/content/34/38/12919
 
Description Leverhulme Trust Research Grant
Amount £196,355 (GBP)
Funding ID RPG-2013-176 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 08/2016
 
Description Responsive mode grant
Amount £710,268 (GBP)
Funding ID MR/R011494/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Spinal motoneurons as active players in motor control
Amount £511,965 (GBP)
Funding ID BB/S005943/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2022
 
Title Improved G-deleted rabies production 
Description We have established a technique for improving the production and quality of a strain of modified rabies virus, used fro trans-synaptic tracing of neuronal connections. This is a modification of previously published methods (developed in the lab of Prof. Callaway, at the salk Institute). We have set up the reagents and tools needed for the production and we are currently one of only three labs who can produce large quantities of highly concnetrated rabies virus. 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact The improvement in the quality of our rabies production has resulted in two main outcomes: 1) We are sharing our virus with two other labs at UCL (Dr. MacAskill and Prof. Brownstone) and the shared use of the same reagents has given rise to new collaborations among the groups 2) Improved quality of the viral tracer has resulted in more than halving the number of animals needed 
 
Title Rabies glycoprotein-GlyT2 cross mice 
Description We have generated a new transgenic mouse line starting from two existing lines. We have crossed mice expressing the rabies glycoprotein under the control of Cre with mices expressing a green fluorescent protein (EGFP) in all glycinergic neurons. The cross mice are carriers of both transgene and are currently used for studies involving rabies mediated trans-synaptic tracing. The advantage of having generated this line is that with muscle injection of a modified rabies virus, we can label the population of premotor interneurons and immediately distinguish the inhibitory ones from the excitatory ones on the basis of their expression (or lack) of EGFP. This allows us to reduce the number of necessary immunoreaction steps and we estimate that thanks to this development we have reduced the number of necessary procedures by 50% 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact Tha main impact is on the 3Rs, namely, we managed to reduce the number of animals used by ~50% 
 
Title NeuroMorpho.org 
Description Reconstructions of Renshaw Cells and Motoneurons. http://Will be available in June 2016 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact None 
 
Title motoneuron-Renshaw cell 
Description Full anatomical reconstructions of pairs of synaptically coupled motoneurons and inhibitory interneurons 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Not yet applicable 
 
Description Autaptic inhibition in the somatosensory cortex 
Organisation ICM (Brain & Spine Institute)
Country France 
Sector Hospitals 
PI Contribution We investigated the role of autaptic inhibitory connections in the somatosensory cortex. We provided a newly developed analysis tool to quantify the relative extent of self-synaptic inhibition relative to inhibition received within local circuits in fast spiking interneurones in the neocortex. While experiments were performed in our collaborators' lab, we have established the experimental design and provided a detailed mathematical analysis of the results, based on a new methods developed during the current BBSRC award
Collaborator Contribution Our partners gathered experimental data. Together we are submitting a paper describing the results obtained
Impact A full paper is at the submission stage
Start Year 2016
 
Description Divergence of DI3 interneurons 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing training and performing experiments in collaboration with Prof Rob Brownstone at the Institte of neurology. The optimization of the technique for trans-synaptic tracing, that is the main focus of our award, has generated large interest in the nearby scientific environment. As a consequence, we have been contacted by Prof. Brownstone and asked to collaborate on a project whose aim is to determine the degree of divergence of a special class of spinal interneurones onto specified subsets of motneurons. The neurons we are studying, named DI3 interneurons, have a special role in the modulation of the grasp refles and, even more interestingly, have been shown to be a necessary player in recovery of locomor abilities following a complete spinal cord transection. We have provided to Prof. Brownstone infected tissue and he and his team are currently analyzing it to determine whether DI3 neurons exhibit a lrger than average degree of divergence onto differnt motor nuclei
Collaborator Contribution While we are producing the viral tracer and performing most of the surgeries, Prof' Brownstone inititated the project and his group is performing the analysis of the smaple we provide. Regular group meetings are held, to discuss the current state of the project and its future directions.
Impact The collaboration has only started in the late months of 2016, therfore no impact has been achieved yet.
Start Year 2016
 
Description Invited professorship at Paris Descartes University 
Organisation University of Paris - Descartes
Country France 
Sector Academic/University 
PI Contribution I have been an invited professor at Paris Descartes University. My remunerated role is to give a set of specialized lectures on synaptic transmission in the spinal cord to undergraduate students and to collaborate with local research groups
Collaborator Contribution The partners are providing an advanced optical setup, not available in my institution, that we are using for performing experiments aimed at revealing the mechanisms of synaptic transmission between motoneurons and Renshaw cells in the spinal cord
Impact Collaboration still in progress
Start Year 2017
 
Description Rabies consortium 
Organisation Emory University
Country United States 
Sector Academic/University 
PI Contribution Establishing the distribution of flexor and extensor related premotor interneurons in the spinal cord. The collaboration started when comparing different sets of data obtained through similar experiments performed in different labs. My group initially observed a strong discrepancy between our data and some previously published evidence. We therefore teamed up with other labs to share and compare our findings. Through distributed team work, coordinated by my lab, we are in the process of gathering sufficient data in order to challenge and correct the existing literature.
Collaborator Contribution The collaboration consists in repeating similar experiments across different labs and pooling the resulting data. Each participant is repeating experiments in identical conditions following a written established protocol.
Impact No relevant outcomes yet
Start Year 2018
 
Description Rabies consortium 
Organisation Helmholtz Association of German Research Centres
Department The Max Delbrück Center for Molecular Medicine (MDC)
Country Germany 
Sector Academic/University 
PI Contribution Establishing the distribution of flexor and extensor related premotor interneurons in the spinal cord. The collaboration started when comparing different sets of data obtained through similar experiments performed in different labs. My group initially observed a strong discrepancy between our data and some previously published evidence. We therefore teamed up with other labs to share and compare our findings. Through distributed team work, coordinated by my lab, we are in the process of gathering sufficient data in order to challenge and correct the existing literature.
Collaborator Contribution The collaboration consists in repeating similar experiments across different labs and pooling the resulting data. Each participant is repeating experiments in identical conditions following a written established protocol.
Impact No relevant outcomes yet
Start Year 2018
 
Description Rabies consortium 
Organisation University College London
Department The Sainsbury Wellcome Centre for Neural Circuits and Behaviour
Country United Kingdom 
Sector Academic/University 
PI Contribution Establishing the distribution of flexor and extensor related premotor interneurons in the spinal cord. The collaboration started when comparing different sets of data obtained through similar experiments performed in different labs. My group initially observed a strong discrepancy between our data and some previously published evidence. We therefore teamed up with other labs to share and compare our findings. Through distributed team work, coordinated by my lab, we are in the process of gathering sufficient data in order to challenge and correct the existing literature.
Collaborator Contribution The collaboration consists in repeating similar experiments across different labs and pooling the resulting data. Each participant is repeating experiments in identical conditions following a written established protocol.
Impact No relevant outcomes yet
Start Year 2018
 
Description Rabies consortium 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Establishing the distribution of flexor and extensor related premotor interneurons in the spinal cord. The collaboration started when comparing different sets of data obtained through similar experiments performed in different labs. My group initially observed a strong discrepancy between our data and some previously published evidence. We therefore teamed up with other labs to share and compare our findings. Through distributed team work, coordinated by my lab, we are in the process of gathering sufficient data in order to challenge and correct the existing literature.
Collaborator Contribution The collaboration consists in repeating similar experiments across different labs and pooling the resulting data. Each participant is repeating experiments in identical conditions following a written established protocol.
Impact No relevant outcomes yet
Start Year 2018
 
Description Rabies consortium 
Organisation University of Glasgow
Department School of Life Sciences Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Establishing the distribution of flexor and extensor related premotor interneurons in the spinal cord. The collaboration started when comparing different sets of data obtained through similar experiments performed in different labs. My group initially observed a strong discrepancy between our data and some previously published evidence. We therefore teamed up with other labs to share and compare our findings. Through distributed team work, coordinated by my lab, we are in the process of gathering sufficient data in order to challenge and correct the existing literature.
Collaborator Contribution The collaboration consists in repeating similar experiments across different labs and pooling the resulting data. Each participant is repeating experiments in identical conditions following a written established protocol.
Impact No relevant outcomes yet
Start Year 2018
 
Description UCL-Glasgow 
Organisation University of Glasgow
Department Institute of Neuroscience and Psychology
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of computational and electrophysiological methods to measure the strength and features of synaptic connection in the spinal cord
Collaborator Contribution anatomical experiments to validate independently the findings derived form electrophysiological experiments in living tissue
Impact Publication of scientific papers, applications for grant funding. We apply different techniques (electrophysiology and anatomy) to address the same research questions using a multidisciplinary approach
Start Year 2008
 
Title BQA 
Description Software suite using a novel computational method to determine the parameters governing the operation of identified synaptic connections 
Type Of Technology Software 
Year Produced 2013 
Open Source License? Yes  
Impact Not applicable yet 
URL http://sourceforge.net/projects/pyclamp
 
Description Berlin talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited talk delivered at the Max Delbruck Center in Berlin
Year(s) Of Engagement Activity 2018
 
Description Poster presentation at the American Society for Neuroscience meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of new scientific results to the wide audience of the largest Neuroscience meeting. The audience comprises scientists as well as policymakers (from grant funding agencies), Journal editors and media representatives.
Year(s) Of Engagement Activity 2015
 
Description Presentation of research to undergraduate students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of our research driven teaching, Every year I give talks to 3 groups of 5-6 undergraduate students explaining the research performed in my lab and describing the techniques in use and the outcomes and aims of the experiments. This practice gives the student a direct exposure to advanced research and help them grow their interest in specific research themes, guiding them towards the choice of their final year lab placement
Year(s) Of Engagement Activity 2015,2016,2017,2018