Mechanisms controlling the number and location of synaptic AMPARs
Lead Research Organisation:
University of Bristol
Department Name: Anatomy
Abstract
Glutamate is a molecule in the brain that transfers messages from one neurone to the next. In the receiving neurone, glutamate binds to proteins called receptors that activate that neurone which, in turn, often stimulates it to release glutamate itself to activate the next neurone in the chain. We are particularly interested in the glutamate receptor subtype called AMPA receptors (AMPARs). Information passing through AMPARs represents the vast majority of information flow in the brain.
To function correctly, AMPARs need to be precisely located on the surface of the neurone. The focus of our work is how neurones achieve the remarkable feat of getting the AMPARs to the right place at the right time. In addition, the number and location of AMPARs can be rapidly changed. It has been discovered that there are proteins that direct and anchor AMPARs; they determine the level and location of AMPAR expression and they connect to specific signalling pathways inside the neurone.
This project aims to provide a more complete picture of how AMPARs are transported (trafficked) around the neurone and define the specific roles of interacting proteins. We shall use a combination of methods that takes full advantage of the technological advances made over the last few years to elucidate the principles that govern the turnover and surface expression of AMPARs.
To do this we will look at what happens in both resting, unstimulated neurones and also in neurones that have been stimulated with physiologically relevant stimuli. We will use new microscopes and special fluorescent protein labels that allow us to actually see AMPARs moving in the cell and how this is changed by different conditions. We will then use biochemical methods to work out what proteins are involved in orchestrating these movements and what happens when we prevent them from working properly.
The public can access neuroscience research in the University of Bristol via Bristol Neuroscience (BN). There is an extensive website that allows the public to gain access to the research going on in our labs. BN is also active in arranging events to increase public understanding of science. Via BN and the widening participation scheme the PI gives talks about ?how the brain works? to A-level students in schools.
To function correctly, AMPARs need to be precisely located on the surface of the neurone. The focus of our work is how neurones achieve the remarkable feat of getting the AMPARs to the right place at the right time. In addition, the number and location of AMPARs can be rapidly changed. It has been discovered that there are proteins that direct and anchor AMPARs; they determine the level and location of AMPAR expression and they connect to specific signalling pathways inside the neurone.
This project aims to provide a more complete picture of how AMPARs are transported (trafficked) around the neurone and define the specific roles of interacting proteins. We shall use a combination of methods that takes full advantage of the technological advances made over the last few years to elucidate the principles that govern the turnover and surface expression of AMPARs.
To do this we will look at what happens in both resting, unstimulated neurones and also in neurones that have been stimulated with physiologically relevant stimuli. We will use new microscopes and special fluorescent protein labels that allow us to actually see AMPARs moving in the cell and how this is changed by different conditions. We will then use biochemical methods to work out what proteins are involved in orchestrating these movements and what happens when we prevent them from working properly.
The public can access neuroscience research in the University of Bristol via Bristol Neuroscience (BN). There is an extensive website that allows the public to gain access to the research going on in our labs. BN is also active in arranging events to increase public understanding of science. Via BN and the widening participation scheme the PI gives talks about ?how the brain works? to A-level students in schools.
Technical Summary
Our aim is to understand how the number and specific localization of synaptic AMPARs is determined. Our hypothesis is that AMPARs with specific subunit compositions and interacting proteins undergo targeted, activity-dependent delivery to, stabilisation at and removal from pre- and postsynaptic sites. For presentation purposes we have divided the application into four objectives but these are highly interrelated in terms of conceptual and experimental design.
Our major objectives are to:
1. Define trafficking of postsynaptic AMPARs in resting and stimulated neurones to compare subunit composition, delivery pathways and properties and sites of constitutive and evoked exocytosis
2. Distinguish the roles of AMPAR interactors in intracellular transport, endocytosis, exocytosis and lateral diffusion. In particular we shall focus on GRIP and ABP isoforms and Protein kinase Mzeta (PKM?) in the regulation of synaptic AMPARs.
3. Elucidate the molecular mechanisms underlying the rapid regulation of the GluR2 content of AMPARs and define the source of GluR2-lacking AMPARs
4. Identify the mechanisms that regulate trafficking and function of presynaptic AMPARs. We shall identify their subunit compositions, interacting proteins and the parameters of their activity-dependent regulation
This is an integrated proposal that will use a combination of established and emergent molecular, cellular and imaging techniques to address questions fundamental to the basic understanding how neurones and neuronal networks modulate information transfer and storage via synaptic plasticity. Furthermore, because AMPAR function and dysfunction underlies a wide range of clinically important neurological diseases our results will influence future research into novel drug targets and therapeutic intervention strategies.
We will use rat brain tissue and cultured neurones and slices. In all cases the tissue preparation(s) most appropriate for each specific series of experiments will be used. For example, to visualise AMPAR and AMPAR-associated protein trafficking we exploit dispersed cell culture systems to optimise the tools and techniques. Then, based on advances in the equipment, reagents and experimental methods we will progress to the use of acute slice cultures and ex vivo slices that better represent the physiological situation in vivo.
Finally, an important aspect of this application is our support our MRC Centre colleagues and the wider neuroscience community. We will continue to share our existing and new tools (e.g. viral vectors, fluorophore-tagged proteins, specific targeted mutants etc) with our electrophysiology and behavioural collaborators to facilitate the focus of multiple levels of investigation on defined problems of common interest.
Our major objectives are to:
1. Define trafficking of postsynaptic AMPARs in resting and stimulated neurones to compare subunit composition, delivery pathways and properties and sites of constitutive and evoked exocytosis
2. Distinguish the roles of AMPAR interactors in intracellular transport, endocytosis, exocytosis and lateral diffusion. In particular we shall focus on GRIP and ABP isoforms and Protein kinase Mzeta (PKM?) in the regulation of synaptic AMPARs.
3. Elucidate the molecular mechanisms underlying the rapid regulation of the GluR2 content of AMPARs and define the source of GluR2-lacking AMPARs
4. Identify the mechanisms that regulate trafficking and function of presynaptic AMPARs. We shall identify their subunit compositions, interacting proteins and the parameters of their activity-dependent regulation
This is an integrated proposal that will use a combination of established and emergent molecular, cellular and imaging techniques to address questions fundamental to the basic understanding how neurones and neuronal networks modulate information transfer and storage via synaptic plasticity. Furthermore, because AMPAR function and dysfunction underlies a wide range of clinically important neurological diseases our results will influence future research into novel drug targets and therapeutic intervention strategies.
We will use rat brain tissue and cultured neurones and slices. In all cases the tissue preparation(s) most appropriate for each specific series of experiments will be used. For example, to visualise AMPAR and AMPAR-associated protein trafficking we exploit dispersed cell culture systems to optimise the tools and techniques. Then, based on advances in the equipment, reagents and experimental methods we will progress to the use of acute slice cultures and ex vivo slices that better represent the physiological situation in vivo.
Finally, an important aspect of this application is our support our MRC Centre colleagues and the wider neuroscience community. We will continue to share our existing and new tools (e.g. viral vectors, fluorophore-tagged proteins, specific targeted mutants etc) with our electrophysiology and behavioural collaborators to facilitate the focus of multiple levels of investigation on defined problems of common interest.
