Probing presynaptic receptor function with two-photon uncaging, Ca2+ imaging and photobleaching

Lead Research Organisation: University College London
Department Name: UNLISTED

Abstract

Synapses that transmit information among neurons are modulated in a highly diverse manner by an interplay of intrinsic and extrinsic factors. This contributes both to the richness of information processing by brain circuits and to their stability, which can however break down in disease states such as epilepsy. Several methods are available to study rather indirectly how chemical receptors interact with calcium signals within individual neurotransmitter-releasing nerve processes. However, they cannot be readily integrated with one another: some can only be used with living neurons, while others require the tissue to be frozen or chemically treated. We propose to achieve a much more detailed insight into the function of individual synapses by incorporating a novel method to manipulate the local concentration of chemically active substances with laser-evoked excitation of molecules. This method relies on hitting individual electrons with two photons in very rapid succession, thus evoking a highly localised and brief change in the state of the atoms. Such localised manipulations are necessary to study synapses, because they normally function on a space- and time-scale of microns and milliseconds. We will apply this method to several topical questions that are relevant to normal brain function and to epilepsy. Specifically, we will investigate how the spatial distribution of neurotransmitter receptors and different proteins mediating calcium movements into nerve processes interact with one another. We will also ask whether some changes that occur in epilepsy are caused by alterations in the local expression of these receptors. The work will shed light on how neuronal signalling underlies cognitive processes and how abnormal synaptic function contributes to seizures.

Technical Summary

Presynaptic receptors exert a powerful influence on evoked release of neurotransmitters. This project aims to determine the molecular mechanisms and organising principles that govern presynaptic receptor function at individual central synapses. We will achieve this by combining state-of-the-art methods developed by the applicants and collaborators. We will complement an existing multi-photon imaging system (coupled with single-cell electrophysiology) with a two-photon uncaging facility. This will allow us to probe presynaptic receptor function at sub-micron resolution in acute brain slices. We will focus on several distinct synapses in the hippocampus (mossy fibre synapses in CA3 and in the hilus, s. lacunosum-moleculare interneuron -interneuron and s.oriens - s. lacunosum-moleculare inetrneuron connected pairs) and address the identity and location of different types of presynaptic glutamate receptors and Ca2+ channels. We will also use two-photon photobleaching to study how activity and neurotransmitter receptors can affect chemical compartmentalisation and structural changes in axonal varicosities. The anticipated results will provide important insights into the extent, consequences and heterogeneity of presynaptic receptor actions in the brain.

Publications

10 25 50
 
Description Chair, Physiology and Medicine Section, Academia Europaea
Geographic Reach Asia 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
Impact Influenced academic science policies and award criteria in Europe.
 
Description Brain Research Trust Research Grant
Amount £53,000 (GBP)
Organisation Brain Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2007 
End 02/2008
 
Description Wellcome Trust Enhancement Grant
Amount £102,612 (GBP)
Funding ID 5772 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2012 
End 09/2013
 
Description Wellcome Trust Senior Fellowhip (Renewal)
Amount £1,350,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2008 
End 09/2013
 
Title Single-bouton recordings and imaging 
Description A technique to carry out patch-clamp recording and and simultaneously image Ca2+ in small axonal boutons 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Publications and new techniques 
 
Title The neurophysiological role of cannabinoids in cannabis users 
Description The neurophysiological role of cannbinoid in cannabis users 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact The finding could help to understand mechsnism of cannabis abuse 
 
Title point-source line-scan diffusion measurement 
Description A new point-source line-scan imaging technique has been developed to assess extracellular molecular mobility (diffusivity) in brain tisssue. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This technique has allowed measurements of extracellular molecular mobility in the hippocampus providing an important alternative to the only existing technique. 
 
Title A large scale biophysical computer model of synaptic microenvironment 
Description A large scale biophysical computer model of synaptic microenvironment is designed for Monte-Carlo simulations of rapid molecular evemts inside and outside the synaptic cleft, ion 3D brain tissue. 
Type Of Material Computer model/algorithm 
Year Produced 2010 
Provided To Others? Yes  
Impact High-impact publications. 
 
Description Fluctuations in release probability 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution Electrophysiological data
Collaborator Contribution Simulation data
Impact artilce in Nature Neuroscience
 
Description Fluorescence anisotropy imaging 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We design and run experiments in acute brain slices
Collaborator Contribution Development of a novel method of diffusion measurement on a nano-scale
Impact To date, the outcome of the aforementioned collaboration could be summarised as follows: 1. A brand new methodology of fluorescence anisotropy imaging, which enables nano-scale diffusion and miscro-viscosity measurements in live tissue has been set up at the PI's laboratory. It is currently being tested for optics optimisation and single-cell imaging in acute brain slices. 2. The paper dealing with nano-diffusion measurements in the brain extracellular space is in its final preparation stages.
Start Year 2006
 
Description GABA signalling in neurogliaform cells 
Organisation University of Oxford
Department MRC Anatomical Neuropharmacology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Specifci research ideas, experiments and designs
Collaborator Contribution Specific experiments
Impact Two papers in the Journal of Neuroscience
Start Year 2006
 
Description LTP and D-serine release from astrocytes 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Dr Stephane Oliet, INSERM Bordeaux, France.
Collaborator Contribution Provided electropohysiological data
Impact Christian Henneberger, Thomas Papouin,3, Stéphane H. R. Oliet, & Dmitri A. Rusakov. Nature (in press).
Start Year 2006
 
Description NMDA receptor function 
Organisation University College London
Department Centre for Clinical Pharmacology & Therapeutics
Country United Kingdom 
Sector Academic/University 
PI Contribution Caried out imaging experiments
Collaborator Contribution Supplied mouse model
Impact Article in EMBO Journal
Start Year 2006
 
Description Presynaptic AMPA receptors 
Organisation Mitsibishi Kasei Institute of Life Sciences
Country Japan 
Sector Academic/University 
PI Contribution To examine presynaptic AMPARs in cerebellar basket cell axons using two-photon microscopy and electorphysiology
Collaborator Contribution Provied a new methodological approach to examine basket cell axons
Impact Publications and new experimental methodologies
 
Description Presynaptic GABAA receptors 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided Ca2+ imaging of individual mossy fibre boutons
Collaborator Contribution Provided recordigns from individual presynaptic bourons of mossy fibres
Impact Publications and newly developed methodologies
 
Description Presynaptic kainate receptors 
Organisation University of Oxford
Department MRC Anatomical Neuropharmacology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided Ca2+ imaging of individual mossy fibre synapses and related experimental designs
Collaborator Contribution Provided recordings and post-hoc reconstruction of mossy fiber cell targets
Impact Publications and newly developed methodologies of signle-synapse recording
Start Year 2006
 
Title A large-scale Monte-Carlo model of synaptic environment 
Description A library of programs for Monte Carlo simulations of rapid molecular events inside and outside the synaptic cleft. Written by Drs L Savtchenko and K. Zheng. 
Type Of Technology Software 
Year Produced 2007 
Impact Several high-profile publications 
 
Title A large-scale computer model environment for exploring realistic neuronal networks 
Description A large-scale computer model environment for exploring realistic neuronal networks. Allows inclusion of >100,000 individual NEURON-type cells. Enabled for parallel computing on the cloud. 
Type Of Technology Software 
Year Produced 2013 
Impact High-impact publications reflecting some fundamental scientific observations. 
 
Title Dedicate 64-core computer cluster 
Description A dedicated in-house 64(+) computer cluster (parallel computing enabled) for biophysical and neural network modelling including computational environment. Designed and built by Dr K. Zheng. 
Type Of Technology e-Business Platform 
Year Produced 2008 
Impact High-profile publications reflecting several scientific discoveries. 
 
Title Fluorecence anisotropy imaging 
Description An image analysis tool allowing to translate two FLIM signals (parallel and perpendicular) into local rotation correlation parameter across the imaged region of interest. Applied to map local instantaneous molecular diffusion. 
Type Of Technology Software 
Year Produced 2010 
Impact Book chapter. Being implemented to gauge molecular diffusivity at nanoscale. 
 
Description International lectures at major research conferences and university deprtments 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talks were noted internationally.

This question and example are not serious.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014
 
Description Membership of international advisory bodies and reviewing panels: 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Decisions made

Influencing science policy and funding
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014
 
Description Peer-reviewing for major funding bodies 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact N/A

Decision made
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014
 
Description Peer-reviewing for top-tier science journals 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Publications

N/A
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014