Extrasynaptic transmission: investigating synaptic vesicle fusion at non-conventional release sites in hippocampal neurons

Lead Research Organisation: University of Sussex
Department Name: Sch of Life Sciences

Abstract

Overview: The brain of a mammal is arguably the most sophisticated of all biological structures and scientists are very motivated to understand, in detail, how it works. When we talk about brain activity, the essence of this is the communication of information between individual brain cells, or 'neurons'. Although a great deal is known about this process, new experimental findings which add to our understanding of brain function are emerging all the time. This proposal will examine an important and only recently identified aspect of neuron-to-neuron communication which impacts on our knowledge of brain operation. To investigate this phenomenon, state-of-the-art methods will be employed which allow movements of molecules in living brain cells to be directly observed. Research like this adds greatly to our knowledge of how the brain works at the most fundamental level and could have important implications for understanding forms of brain disease or even inspiring the design of new and more sophisticated computer technology. Detail: Most neuron-to-neuron information transfer relies on release of a chemical 'transmitter' from a source neuron to a target neuron. Transmitter release is a complex process requiring lots of different types of specific molecules. These are concentrated at specialized sites in neurons known as presynaptic terminals, and the established view is that these terminals are therefore the only location within a brain cell where transmitter release can occur. Recently, however, researchers have found this idea to be inaccurate: in certain types of neuron, transmitter can be released at other 'non-specialized' sites within a source neuron. This is called 'extrasynaptic' release, meaning literally 'release away from the presynaptic terminal'. The mechanism allowing this process to occur, however, is not understood. The most likely explanation for how extrasynaptic release is achieved is that molecular machines needed for transmitter release can be readily moved from normal presynaptic terminals to new parts of the neuron. Confirmation of this idea, the manner in which it is controlled and what types of machinery are actually necessary, though, remains to be established. A knowledge of this would greatly expand our understanding of the necessary and sufficient components of chemical-neurotransmission, and will be investigated in this proposal. Experiments will rely on an important system for studying transmitter release: neurons grown on glass to form miniature brain circuits. With this system, presynaptic molecules can be visualized in living cells with microscopes and sensitive cameras. A second type of experimental approach will even allow neurons to be viewed at a resolution beyond the limits of a light microscope so that detailed information about the structure of extrasynaptic release sites can be obtained. Using these methods, experiments will focus on investigating how release sites are constructed, characterizing their properties, and comparing them to normal presynaptic terminals. The idea of extrasynaptic release is an emerging theme in neurobiology and has important potential roles in contributing to the communication between brain cells. This makes it a very worthwhile area of study. Moreover, incorrect control of transmitter release machinery is thought to be associated with some brain-related diseases; therefore, understanding this fundamental process may even offer clues about the treatment of forms of brain-disorders.

Technical Summary

The classical view of fast neurotransmitter release at central neurons is that exocytosis occurs exclusively at specialized ultrastructurally-defined active zones in presynaptic terminals. However, recent work has challenged this idea suggesting that at some central connections, vesicle fusion can also take place at extrasynaptic or ectopic release sites with important functional consequences for neuronal signalling. In hippocampal cultured neurons, an established system for studying neurotransmission, synaptic vesicles arising from stable synapses can readily aggregate at extrasynaptic sites and undergo Ca2+-triggered fusion with normal release kinetics. Moreover, preliminary experiments suggest that vesicles can even exocytose as they transit along the axon. How the highly-regulated and coordinated process of vesicle fusion can occur at such non-specialized sites remains unclear, however. To address this issue, fluorescence tagging and time-lapse imaging methods will be employed here to examine the composition of extrasynaptic release sites and the dynamics of their formation and operation. Experiments will also utilize immunocytochemistry, newly-developed optical glutamate sensors and Ca2+-imaging methods to explore the functional consequences of extrasynaptic transmitter release. Using acute hippocampal slices and ultrastructural investigation, the facility for extrasynaptic release in native tissue will also be examined. The capability of neurons to undergo extrasynaptic vesicle fusion undermines the conventional view of highly compartmentalized neurotransmission, suggesting that release machinery can be readily available across presynaptic structures. Understanding how this is achieved will shed new light on what constitutes the minimal complement of active zone machinery necessary for vesicle exocytosis, and further our knowledge of the fundamental mechanisms underlying information transfer.

Publications

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Kemenes I (2011) Dynamic clamp with StdpC software in Nature Protocols

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Orenbuch A (2012) Synapsin selectively controls the mobility of resting pool vesicles at hippocampal terminals. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Ratnayaka A (2011) Extrasynaptic vesicle recycling in mature hippocampal neurons in Nature Communications

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Samu D (2012) Single electrode dynamic clamp with StdpC. in Journal of neuroscience methods

 
Description The rapid transfer of information between neurons is a key element of brain function. Most neurons communicate through the release of chemical neurotransmitter at specialized contact points called synapses. The neurotransmitter is contained in synaptic vesicles - small spherical structures - which fuse with the membrane of the signalling neuron when active. Released neurotransmitter diffuses to synaptic structures on receiving ('postsynaptic') neurons, propagating information around neural networks. The established convention is that synapses are the only sites for vesicle fusion and transmission. Nonetheless, the contrary idea - that vesicle fusion could occur at sites outside synaptic terminals - would represent a novel form of information transmission which could signal unique and dynamic aspects of neuronal function. In this project we set out to test this idea.

Key findings include the following:

1. The 'superpool'. To examine the potential for non-synaptic vesicle fusion we used fluorescence imaging methods to track synaptic vesicles. Importantly, we found that vesicles were not stably located at individual presynaptic terminals but instead moved along adjoining tracks, or axons, between synaptic neighbours. We coined the term 'superpool' to describe the novel concept of a synapse-spanning vesicle population.

2. Extrasynaptic fusion. The presence of a superpool revealed how vesicles were continuously moved between presynaptic terminals. Next we used specialized fluorescent markers that change their signal as vesicles fuse, to show that mobile neurotransmitter-containing vesicles were also fusion-competent along axons in both cultures and native brain tissue. Key features of this novel finding were: a) mobile fusion required calcium and occurred at speeds comparable with fusion in synaptic terminals, b) important elements of synaptic machinery were recruited to or available at extrasynaptic sites to support fusion, c) mobile vesicles were 'retrieved' after fusion and re-used, d) mobile vesicle fusion occurred in cultured neurons and native tissue from brain slices. We also characterized the detailed organization of extrasynaptic sites using approaches to capture high-resolution images at the moment of fusion: vesicles were organized in small clusters near the membrane and opposite postsynaptic target structures containing receptors. Thus mobile vesicle fusion provides a dynamic and relevant mechanism to support novel forms of neuron-neuron signalling outside conventional terminals.

3. Regulation of the extrasynaptic pool. We showed that synapsin, a key synaptic protein was a critical regulator of the extrasynaptic vesicle pool size. Synapsin acts to tether vesicles at synapses but in cultures made from mice without synapsin, this tethering was disrupted and vesicle numbers outside terminals were substantially elevated. Another signalling molecule, brain-derived neurotrophic factor (BDNF), was similarly important in promoting increases in the extrasynaptic pool size.

4. Extrasynaptic vesicles and plasticity. We demonstrated that synaptic terminals can undergo a novel form of adaptive change in function - or plasticity - which could underlie forms of learning and memory. This involved intrasynaptic changes in vesicle pools but did not depend on the trafficking of vesicles from the extrasynaptic superpool.

Our findings reveal a new non-conventional and dynamic feature of information transmission. Our findings are highly relevant to a complete understanding of neuron-neuron signalling, cellular/molecular mechanisms of learning and memory and as disease targets. Indeed, our pilot experiments are revealing that robust changes in vesicle traffic and extrasynaptic fusion occur in a disease model of Alzheimer's, offering possible substrates for new therapeutic development. We are now using our newly-developed approaches to examine relationships between synaptic pool architecture and function in native tissue.
Exploitation Route These findings offer fundamental insight into elementary brain function. They will stimulate further research by other researchers aimed at exploring basic mechanisms of nervous system operation. In the longer-term they may aid our understanding of disease mechanisms. Evidence for this is starting to appear in the scientific literature where the 'superpool' and extrasynaptic release mechanisms are being mooted as possible disease substrates. In the future, understanding the cellular and molecular underpinnings of these processes could inform the development of targeted therapies aimed at alleviating or halting disease processes.
Sectors Digital/Communication/Information Technologies (including Software),Education,Pharmaceuticals and Medical Biotechnology

 
Description 1. Findings disseminated in talks, invited seminars, research publications, open lab, public talks. Work has been included in undergraduate teaching. 2. Work offers new insight into fundamental mechanisms of transmission. It has provided the grounding for further successful grant awards from BBSRC (BB/K019015/1) and MRC (MR/K022105/1). In turn this will allow us to develop these findings towards the identification of regulatory substrates that might underlie for example, dysfunction of synaptic transmission seen in disease conditions such as Alzheimer's. Clearly this would have significant benefit for society. 3. Stimulated interest from a pharmaceutical company leading to a successful BBSRC-CASE award application. This work will focus on elucidating the molecular basis of vesicle recycling, exploiting our novel methodologies and the drug development expertise of the industrial partner (Janssen). Understanding this process at the molecular level will reveal critical substrates for synaptic modulation that might account for examples of disease-related dysfunction, and provide possible pharmaceutical regulators, that could be relevant to disease states. In the future this could have significant benefit for society. 4. Winning new grant funding that attracts high-quality international postdocs and forging links with European pharmaceutical companies serves to strengthen UK economic competitiveness and growth.
First Year Of Impact 2012
Sector Digital/Communication/Information Technologies (including Software),Education,Pharmaceuticals and Medical Biotechnology
 
Description Additional funding awarded in support of the grant for tissue slicing equipment
Amount £15,000 (GBP)
Funding ID RA61 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Additional funding awarded in support of this project for short-term collaboration
Amount £8,300 (GBP)
Funding ID RA60 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Newly awarded studentship that will explore vesicle dynamics in native brain slices using correlative fluorescence-EM methods.
Amount £70,000 (GBP)
Organisation South East Biosciences Network (SEBnet) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 03/2016
 
Description Newly awarded studentship that will explore vesicle dynamics in native brain slices using correlative fluorescence-EM methods.
Amount £70,000 (GBP)
Organisation South East Biosciences Network (SEBnet) 
Sector Academic/University
Country United Kingdom
Start 09/2012 
End 09/2016
 
Description PhD studentship supporting the development of key questions stemming from grant so far. In particular, the project will examine the organization of extrasynaptic vesicle fusion sites using native tissue/correlative methods.
Amount £70,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2010 
End 09/2014
 
Title Electrophysiological recording methods 
Description SOFTWARE: Development of new electrophysiological software (Dynamic Clamp, StdpC) for improved control of neuronal recordings (Nature Protocols 6: 405-417, 2011). The software is fully open access and allows the user to impose complex stimulation profiles onto target neurons. It is relevant to general methods in the grant used to carry out whole-cell recordings of neurons. 2) SOFTWARE: Development of extension to StdpC (above) incorporating Active Electrode Compensation (AEC) allowing routine single electrode dynamic clamp (J Neurosci Meths 211:11-21). The software is fully open access and allows the user to impose complex stimulation profiles onto target neurons. It is relevant to general methods in the grant used to carry out whole-cell recordings of neurons. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Some research labs use this software for their own research. 
URL http://sourceforge.net/projects/stdpc/
 
Title New genetically-encoded fusion construct: SynaptophysinI-Dendra 
Description METHODOLOGY: A novel imaging approach for dynamic studies of protein traffic based on the use of photoswitchable fluorophores. We developed new genetically-encoded fusion constructs - SynaptophysinI-Dendra and SynaptophysinI-Kaede - for this work. The methodology was used in our recent research paper (Neuron 66: 37-44). The approach is detailed in a book chapter (Cellular Imaging Techniques for Neuroscience and Beyond. Ed. Floris Wouterlood. Elsevier). 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Researchers have used this construct in their own research. 
 
Title Optimized approach for labelling functional vesicle pools in acute brain slice 
Description We have developed and optimized an approach for fluorescence and ultrastructural imaging of recycling vesicle pools in acute brain slice. We have recently published the method: Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion. Marra V, Burden JJ, Crawford F, Staras K. Nat Protoc. 2014 Jun;9(6):1337-47. doi: 10.1038/nprot.2014.088. Epub 2014 May 15. 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact This is an important approach that is already providing significant new insight into functional vesicle pools in brain slice. We are applying this method to large tissue volumes using focussed ion beam scanning electron microscopy methods in collaboration with Michael Hausser's lab at UCL. 
 
Description Collaboration with Daniel Gitler laboratory, Ben-Gurion University, Israel 
Organisation Ben-Gurion University of the Negev
Country Israel 
Sector Academic/University 
PI Contribution Formed a collaborative project with the Ben-Gurion University, to examine the role of synapsin in vesicle clustering. This lab has established lines of synapsin knockout mice offering us a readily amenable tool for addressing questions about the regulation of vesicle pools. The collaboration has yielded a paper in Journal of Neuroscience (2012)(see journal outcomes) and an article in Journal of Neurochemistry (2013). We are now extending this collaboration to look at vesicle pool regulation in acute brain slice. Specifically, we are providing expertise in our approach to readout functional vesicle pools at EM level.
Collaborator Contribution The Gitler lab are supplying important knockout mice to allow us to test the regulation of vesicle pool organization by synapsin.
Impact PMID: 22442064 PMID: 23496032
Start Year 2010
 
Description Collaboration with Head of electron microsopy unit, MRC LMCB, UCL 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration was set up to support the ultrastructural work in this project. So far, this collaboration has made an important contribution to five publications (Neuron, 2010; J Neurosci, 2012; J Physiol, 2012; Neuron, 2012; Nat Prot, 2014). Together, we have developed methods to examine functional vesicle pools in cultured neurons, and recently extend these readouts to native tissue. We provide all tissue for this work.
Collaborator Contribution Expert help in processing and serial sectioning for EM.
Impact PMID: 22442064 PMID: 22271866 PMID: 20399727 PMID: 23141069 PMID: 24833172
Start Year 2009
 
Description Collaboration with Tiago Branco, University College London 
Organisation University College London
Department Wolfson Institute for Biomedical Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Continuation of an ongoing collaboration with Wolfson Institute for Biomedical Research. In particular, the PI has provided support in setting up slice electrophysiology/imaging in my laboratory. This approach has been instrumentral in testing the relevance of intersynaptic vesicle trafficking and fusion in native tissue (objective 3 of the grant). He has also provided academic input to many of the research papers/project running in the lab. He was listed as a formal collaborator in the original grant application.
Start Year 2007
 
Description Collaboration with University College London 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Formed a collaborative project with UCL (formerly University of Sussex) to develop image analysis software. Daniel spent ~3 months in my laboratory supported by a small university-based grant awarded to me ('VIP award') and has worked collaboratively on writing MATLAB routines/algorithims to allow identification of synaptic staining (puncta) in fluorescence image pairs or timelapse sequences. This software is an integral element of a recent paper (J Physiol, 2012, In press) arising from the grant. We are currently writing a methods-based paper detailing this software and making it freely available for use and development by others. This collaboration has yielded an important software tool for identification of synaptic puncta. This offers a high-throughput approach to analyzing data in a non-subjective way. The code is written in MATLAB and a methods-based manuscript is being prepared for publication which will provide full free access to the source code for use and development by others.
Start Year 2009
 
Title Software for dynamic clamp electrophysiology 
Description SOFTWARE: Development of new electrophysiological software (Dynamic Clamp, StdpC) for improved control of neuronal recordings (Nature Protocols 6: 405-417, 2011). The software is fully open access and allows the user to impose complex stimulation profiles onto target neurons. It is relevant to general methods in the grant used to carry out whole-cell recordings of neurons. 
Type Of Technology Software 
Year Produced 2011 
Open Source License? Yes  
Impact Researchers using it for their own research. 
URL http://sourceforge.net/projects/stdpc/
 
Description Creation of internet video (YouTube) to publicize research area 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I worked with a professional media company to produce a YouTube video to highlight general approaches and key ideas related to my research. The video shows me and a postdoc discussing research issues and featuring footage of fluorescence imaging approaches, core equipment in the lab and animations. The video has been used to support a promotional campaign related to the Mind and Brain research theme at Sussex. It has so far attracted ~850 views on YouTube with substantial international interest. The video is used frequently at open days / admissions days / promotional days at the University of Sussex and beyond. It was featured at the Royal Society to mark the 50th anniversary of the University of Sussex. It can be found at:

It was part of a promotional campaign to raise interest in neuroscience research activities at Sussex. Undergraduate students have approached me to discuss issues related to my research on the basis of having seen the video.
Year(s) Of Engagement Activity 2011
URL http://www.sussex.ac.uk/lifesci/staraslab/research
 
Description Interviewed for article on my research 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interviewed by a journalist/science writer for the production of an article associated with my research marking the 50th anniversary of the University of Sussex (Research Review) for dissemination to a general audience. The article outlines key findings related to the grant - focussing on the discovery of the vesicle superpool (Neuron, 2010; 66:37-44). It was featured in a colour brochure and showcased at the Research Meeting at the Royal Society, London.

The article is also available online at:

Some reference to my work from students and other researchers.
Year(s) Of Engagement Activity 2011
URL http://www.sussex.ac.uk/aboutus/annualreview/2011/mindandbrain
 
Description Invited Research Seminar (King's College London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Invited seminar at King's College London to ~100 or more research scientists, PG and UG students.
Year(s) Of Engagement Activity 2016
 
Description Neuroscience talk/open afternoon for non-scientists 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Participation in an event to promote my work to a non-scientific audience. Employees joining the university (typically from non-scientific and non-academic backgrounds) can choose to participate in this event; an opportunity to enter a lab and hear a talk on research, as well as sample equipment and chat to myself and postdocs. The event was a great success and supported by the Pro-Vice Chancellor for Research who attended the session. Around 25 participants attended and spend >1.5 hours in the lab. We set up a variety of samples to visualize under microscopes including cultured neurons, fluorescent markers, brain slice tissue embedded in resin (for sectioning etc.). Open lab session for non-scientists Open lab session for non-scientists

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Research Poster at Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Research Poster presented at Society for Neuroscience Meeting, Washington, USA

Dynamic properties of recycling vesicle pools in native hippocampal tissue examined using fluorescence and ultrastructural approaches.



Discussed with research colleagues for 4 hours. Research poster

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Research Poster at Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach international
Primary Audience Participants in your research or patient groups
Results and Impact Research Poster presented at Society for Neuroscience Meeting, San Diego, USA.

Properties of fusion-competent mobile synaptic vesicles in hippocampal neurons.



Discussed with research colleagues for 4 hours. Scientific Poster.

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Research Poster at Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Research Poster presented at Society for Neuroscience Meeting, Washington, USA

Recruitment of resting vesicles into functionally recycling pools as a mechanism of synaptic potentiation at hippocampal synapses.



Discussed with research colleagues for 4 hours. Research poster

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Research talk to sixth form students / public 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Research talks for a general audience on admissions days. The audience is typically sixth-form students and parents - typically ~60-100 people. My talk includes images, animations and movies and is designed to appeal to a lay audience with a non-specialized interest in neuroscience ideas. I am available afterwards to follow up questions related to the research. Short non-specialist research talks outlining key themes in my neuroscience research. Audience ask good general questions about neuroscience as a subject.

We have seen substantial rises in student recruitment on neuroscience-specific programs in recent years.
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
Description Sixth-form work experience student 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Hosted a sixth-form student in my lab, allowing him to participate in the full range of experiments in the lab over a 7 day period. The student carried out basic imaging experiments, cell culture, electrophysiology, image processing and data analysis. The student is now undertaking a full biosciences degree. Work experience for sixth-form student

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Specialist Research Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Research seminar ('Vesicle pool dynamics at central synapses') to specialist audience at the University of Sussex (Neuroscience Seminar series) concerned with findings related to this grant. Research seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Specialist Research Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Research seminar ('Vesicle pool dynamics in hippocampal synapses') to specialist audience at the University of Edinburgh (Centre for Integrative Physiology) concerned with findings related to this grant. Research seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Specialist Research Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Research seminar ('Vesicle pool dynamics at hippocampal synapses') to specialist audience at the Queen Mary University of London (School of Biological & Chemical Sciences) concerned with findings related to this grant. Research seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description University student summer placements 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Postgraduate students
Results and Impact I took on two university students to give them experience of working in a neuroscience lab.

The students participated (at depth) in various aspects of the ongoing work in the lab, ranging from fluorescence imaging to electron microscopy and data analysis. Experience for bioscience students of working in a neuroscience lab over Summer 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011