An analysis of synaptic plasticity at single synapses using the photolytically active AMPA receptor antagonist ANQX.

Lead Research Organisation: University of Oxford
Department Name: Pharmacology

Abstract

Perhaps the greatest challenge in contemporary biology is to understand the workings of the brain. The implications for such an understanding are almost boundless: From therapies with which to treat brain disorders through to recognizing the basis of self-awareness or consciousness, things that are the very essence of being human. The difficulties in achieving such goals are, predictably, as great as the rewards for success.

In recent years significant advances in the field of light microscopy have revolutionised our ability to measure fundamental signalling processes within the cells the brain, the neurones. These measurements are achieved by introducing fluorescent dyes into individual brain cells and stimulating these dyes using precisely guided laser light. The dyes we use are specifically designed to change intensity in response to metabolic changes within the neurone, thus we are able to gather detailed information about activity within these cells simply by measuring changes in intensity of fluorescent light.

In our current work we are using a fluorescent dye that changes intensity in response to a change in the calcium ion concentration within the neurone. An increase in calcium ions accompanies activity within neurones and so we are able to obtain a measure the level of activity by measuring increases in fluorescence. We also have a further method by which we can use light to measure biological processes. In this method we transform an inert compound into an active one. This is achieved by cutting a chemical bond with high intensity laser light. Using this approach we will investigate how neurones encode changes to their inputs, the synapses. The biological significance of changes at synapses is more profound than at first it may appear as these changes are thought to occur as an animal learns and stores new information to memory. Thus by examining these processes we hope to gain insight into the basic cellular mechanisms that underlie learning and memory.

Technical Summary

In this study we wish will utilise a unique a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) antagonist 6-azido-7-nitro-1,4-dihydoquinoxaline-2,3-dione (ANQX). ANQX becomes irreversibly bound to AMPARs on photolytic activation. ANQX will be used to investigate specific features of synaptic transmission associated with long-term potentiation (LTP), an activity-dependent form of plasticity widely regarded to be the synaptic basis of learning and memory. ANQX is not commercially available and will be synthesised ?in house?. We intend to enhance the utility of ANQX by appending a carboxy-fluorescein side-chain. This modification will permit us to identify the region where ANQX-AMPAR binding had occurred. As carboxy-flourescein is quenched at low pH, internalised receptors loose fluorescence, ensuring that only surface receptor expression contributes to the image. By combining the unique features of ANQX with high-resolution Ca2+ imaging we propose to address two specific questions. The first directly examines the widely held view that the addition of AMPARs accompanies LTP at active (AMPAR- containing) as well as silent synapses. The second examines the synaptic contribution made by a recently activated input such as occurs when silent synapses are unmasked by LTP.

Publications

10 25 50
 
Description Responsive Mode
Amount £462,247 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2013 
End 03/2016
 
Title ANQX 
Description A series of light sensitive glutamate receptor antagonists have been developed for use in this project. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This material will become available for scientific research once characterisation is complete. 
 
Description Chemistry 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We are applying novel pharmacological tools to single synapse imaging applications.
Collaborator Contribution Synthesis of novel pharmacological research tools.
Impact New reagents.
Start Year 2008