Ultrastructure-function properties of recycling vesicle pools in native central synapses

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

Abstract

The basic function of the brain is to process information: receiving sensory input, generating appropriate responses and learning/remembering. The information is encoded in the form of small electrical signals, which are passed between specialized cells called neurons wired up together to form circuits. The way the information transfers from neuron to neuron is closely controlled but also adjustable, and these properties underlie the computational power of the brain. Currently, we only have a basic comprehension of transmission; determining how neurons regulate and vary information flow is central to understanding how the brain works and is a major goal in neuroscience research.

Most information transmission occurs at chemical synapses, specialized contact points where two neurons - a signalling and receiving neuron - come close together. The signalling neuron has a cluster of spherical structures called vesicles, each containing a chemical signal. Electrical activity triggers this neuron to mobilize its vesicles and release their chemical contents towards the part of the synapse belonging to the receiving neuron. This target structure has special receptors which respond to the chemical and bring about a change in the electrical activity of the neuron. In this way, information is moved from one neuron to the next.

Why does the process involve an intermediate chemical transmission step? This allows synapses to control and adjust the information transferred. Such a feature sets networks of neurons apart from digital circuits in computers, allowing them to adapt to changes in operational demand or even 'rewire' themselves to support learning.

How is this flexible nature of transmission achieved? This is a key issue in neuroscience. One possibility is that transmitter-containing vesicles themselves are involved. Most mammalian synapses have ~250 vesicles. Surprisingly, only a fraction of these are available to release their chemical signal. If the number of releasable vesicles could be varied at each synapse, this would offer a simple mechanism to allow adjustments in the information transferred. Alternatively, the physical positioning of vesicles in synapses - allowing them to be released more or less efficiently - could be an important factor. These ideas have been difficult to address, particularly in real brain circuits, because of the technical challenges in monitoring small synapses and the nanometre scale of the vesicles.

The objective of this grant is to explore these key ideas. We will use fluorescence imaging methods to directly visualize the dynamic properties of single synapses in rat brain tissue. Also, using a novel approach we will uniquely view individual releasable vesicles with ultrastructural resolution. We will characterize the numbers of functional vesicles in different synapses in large neuronal networks, and determine what molecular pathways and other properties of circuits set these parameters. By building 3d reconstructions of vesicle populations at a synapse, we will also investigate how releasable vesicles are arranged, and examine how these properties influence synaptic performance. We will also define the characteristics of vesicle organization in synapses driven by visual input in behaving animals, allowing us to explore synaptic properties in relation to real sensory signals. Additionally, we will test whether forms of electrical input, corresponding to activity experienced by neurons during learning, bring about changes in the properties of vesicle organization. Addressing these questions is a major step in understanding fundamental brain function. Moreover, synaptic transmission is a major target for neurological diseases, such as Alzheimer's. Vesicle populations represent obvious potential substrates that could underlie synaptic failure; in the future, characterizing the mechanisms that regulate their function could offer promising new strategies for disease-related drug therapies.

Technical Summary

Chemical synapses underlie most information transfer in the CNS. Elucidating the factors that determine their variable transmission strength and operational flexibility, endowing the nervous system with powerful computational capabilities, is a key issue in neuroscience.

The neurotransmitter-containing vesicles in the presynaptic terminal are a defining feature of synaptic architecture. Vesicles appear equivalent but only a limited subset participate in activity-driven signalling. As such, the properties of these recycling sub-pools - their size, nanoscale organization and recycling kinetics - are likely to be important determinants of synaptic transmission. They are also potential targets for plasticity, contributing to observed changes in transmission strength. However, in native central synapses, functional vesicle pools are almost completely undefined and these key ideas remain unexplored.

The proposed research will address this knowledge gap, exploiting novel methods to uniquely characterize the relationship between nanoscale and functional properties of recycling vesicles. Specifically, we will test the hypothesis that pool size and spatial organization are key determinants of presynaptic transmission under regulatory control. What specifies these parameters across synaptic populations and how substrates regulating pool organization influence synaptic efficacy will be examined. In vivo experiments in visual cortex will explore recycling pool organization related to activity driven by natural sensory input. We will also examine changes in functional pools using plasticity models (long-term potentiation or long-term depression) to test whether reorganization of functional vesicle pools contributes to plasticity-evoked modulation of synaptic strength. This work will shed light on a fundamental area of contemporary neuroscience bringing us towards an ultrastructure-function-based understanding of synaptic transmission.

Planned Impact

The research will reveal key mechanisms regulating synaptic transmission in central mammalian neurons in native tissue. The fundamental relevance of this research topic means that the findings will be of benefit to the broad academic research community where understanding the underlying principles of neuronal signalling is important; for example researchers studying synaptic operation, neural circuit function, learning and memory, sensory processing, neuropharmacology and neurological disease. Findings from this research will be published in high-profile peer-reviewed journals, disseminated at international meetings and communicated to the public via engagement events such as Café Scientifique, open labs and various forms of media. Together, these benefits will enhance the knowledge economy starting in 2-4 years, with clear relevance for worldwide academic advancement.

Additionally, the research plan includes the use of new and innovative technical approaches - for example an in vivo method for elucidating synaptic transmission properties down to single vesicle resolution. Such methods are beneficial for driving advances in understanding in many fields of neuroscience-related research. The impact of these developments will start over 2-4 years.

The work will also deliver and train highly-skilled researchers (PDRA, Technician, PhD students) with expertise in organization, analysis, oral communication, and formal scientific writing skills, relevant to many employment sectors. Moreover, the techniques used in this proposal are cutting-edge; potential recipients of this expertise could include other academic research institutes, pharmaceutical companies, biotechnology/imaging companies and even computer technology enterprises exploring neural-digital interfaces. The timecourse of this benefit will start after the end of the grant.

The research could also have societal impact. An expected outcome is the identification of new target substrates that help determine synatic strength. These could be highly relevant to understanding forms of disease-related synaptic dysfunction - for example in Alzheimer's and Parkinson's disease - which have such catastrophic consequences for cognition and adversely affect society. Pharmaceutical companies looking to develop new approaches to treat such disorders will benefit from a clearer understanding of the regulatory mechanisms and molecular substrates that determine transmission characteristics. Ultimately, success in the development of new treatments would substantially benefit the broader public, impacting on the economy and wealth and health of the nation. Of course, the development of new therapeutics goes well beyond the specific aims of this grant; perhaps over a timecourse of ~10 years such benefits may start to be realized.
 
Description We are still analyzing some of the data related to this grant. The key aim is to understand the link between structural and functional properties of synapses - the contact points between neurons where information transfer takes place. Each synapse contains vesicles - neurotransmitter containing organelles. There is great interest in how these are organized in the synapse, what impact this organization has on synaptic function, and how their properties might undergo changes under certain conditions (application of drugs, plasticity etc.). Key findings include:
1. Characterization of the properties of one population of vesicles - the readily releasable pool - and its relationship to synaptic function. In particular we have revealed major new understanding of the fate of vesicles as they are released, and their relevance to re-use in further rounds of transmission. This has been published in a key primary research paper (Rey et al., 2015, Nature Comms). We have also published a paper on some of the key methodological approaches that we have developed (Nature Protocols, Marra et al., 2014).
2. Related study that is exploring the impact of vesicle pool modulation in supporting mechanisms of synaptic plasticity (potentiation) - the basis of learning and memory. Specifically, we show that the number of vesicles available for use by the synapse increases under potentiation. Moreover, the specific physical positions of those vesicles changes so that they are more advantageously placed to allow efficient neuron-to-neuron signalling. We also characterized the molecular pathways that control this important mechanism of modulation - in particular function rearrangement of vesicle pools at the active zone. This manuscript has just been published (Rey et al. 2020, Cell Reports). A related dataset examined a pathway for regulating vesicle pools that relies on the key protein, synapsin. This manuscript is in preparation.
3. Identification of an important feature of synapses where properties of their fundamental vesicle recycling properties are conserved from one round of stimulation to the next at specific terminals; this suggests a novel form of synapse-specific regulation. This manuscript is currently in preparation.
4. We have linked our core findings with examples of whole animal behaviour - in particular exploring mechanisms of functional vesicle recruitment in defensive behaviours (collaboration T. Branco, SWC, UCL) - and in visual processing in visual cortex using FIBSEM technology (with M. Hausser, UCL). We have presented the latter work at a major meeting (SFN, 2019).
5. We also considered presynaptic activity in the context of regulation of ongoing signalling, collaborating with a Moscow lab and publishing a new paper that revealed a key feedback pathway linking synaptic function to action potential tuning (Roshchin et al., Science Advances, 2018).
Collectively, our work has significant consequences for understanding the fundamental processes of brain function.
Exploitation Route This work provides an important foundation for further studies that are focused on fundamental mechanisms of information transmission. Likely beneficiaries could include pharmaceutical companies who are interested in modulating vesicle properties to influence synaptic transmission associated with novel therapies or correction of disease states. In the long-term, this could impact on clinical treatment approaches and thus patient welfare. Primary research academics will benefit from a more complete understanding of synaptic transmission which will inform their future research plans. Our research informs undergraduate and postgraduate teaching-related activities. A new grant that builds on these ideas is has been funded by BBSRC looking at whole-animal feeding behaviour.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our findings have revealed fundamental mechanisms of synaptic function that underpin control mechanisms in brain function and substrates for disease. Our findings have informed public understanding of principles of nervous system operation. We have also worked in collaboration with industry - in particular Janssen Pharmaceutica - to define the molecular control mechanisms and look at the key target substrates in the context of disease states such as epilepsy and Alzheimer's. We have now also set up a new collaboration with Syndesi Therapeutics to look at some of the pathways that relate to disease-related neuronal dysfunction in more detail. These findings are in the early stages but we see potential future benefits for understanding and influencing control of important brain circuits. In particular, our future work plans will define new mechanisms of whole animal behavioural regulation underlying appetite control, associated with healthy eating and disorders. Such approaches will be of significant societal value with implications for health and well-being and we have applied for new BBSRC funding to support this key aim.
First Year Of Impact 2014
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description ARUK equipment Grant
Amount £76,825 (GBP)
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2018 
End 06/2018
 
Description BBSRC CASE studentship award
Amount £94,126 (GBP)
Funding ID BB/L016982/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 09/2018
 
Description Doctoral Training Centre award
Amount £349,982 (GBP)
Funding ID AS-DTC-2014-003 
Organisation Alzheimer’s Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description Higher Education Innovation Fund - University of Sussex
Amount £10,000 (GBP)
Organisation University of Sussex 
Sector Academic/University
Country United Kingdom
Start 05/2018 
End 07/2018
 
Description MRC Discovery Award
Amount £117,040 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2016 
End 06/2018
 
Description MRC Programme Grant
Amount £2,056,454 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2022
 
Description Multi-User Equipment Award
Amount £337,311 (GBP)
Funding ID WT208348/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2017 
End 11/2022
 
Description Presynaptic substrates in hypothalamus as pivotal regulators of feeding behaviour
Amount £525,587 (GBP)
Funding ID BB/S00310X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2021
 
Title Collaboration in developing new microscopy platform 
Description In neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing 'divergent beam optics' (DBO), we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation increases both the space-bandwidth product and effective excitation power, and allows for straight-forward tailoring of the point-spread-function. Moreover, rapid laser-focus control via an electrically tunable lens now allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and fully compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in mice, zebrafish and fruit flies. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Major interest by the scientific community as indicate by the reading metrics on BioRxiv 
 
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 Eisuke Koya, Sussex University 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established a collaboration with Eisuke Koya, who has significant expertise in viral injection methods and feeding control circuits that we would like to use in our new BBSRC grant: Presynaptic substrates in hypothalamus as pivotal regulators of feeding behaviour
Collaborator Contribution At this stage, advising, but the Koya lab will help support and oversee experiments aimed at targeting reporters in feeding circuits.
Impact Advisory role at present.
Start Year 2018
 
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 Janssen Pharmaceuticals 
Organisation Janssen Research & Development
Country Global 
Sector Private 
PI Contribution BBSRC CASE studentship looking at pharmacology of synaptic vesicle recycling with Janssen as Industrial Partner. PhD student is currently on industrial placement in Belgium.
Collaborator Contribution Writing of successful award of CASE studentship. Dialogue during reciprocal visits to Belgium and Sussex between teams.
Impact Preliminary evidence on effects of Levetiracetam on vesicle recycling.
Start Year 2014
 
Description Collaboration with Keith Caldecott (Genome Damage and Stability Centre) 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution We established a collaboration following a local seminar that I gave. We are interested in using our synaptic and neuronal function readouts to explore their models of DNA damage repair, which appear to modulate synaptic function in ways that are relevant on our mechanisms of fundamental synaptic regulation.
Collaborator Contribution Caldecott lab have supplied a mouse model (XRCC1 KO) which influences neuronal/synaptic function.
Impact 1. MRC Programme Grant (to Caldecott and Staras) - starting April 2017. 2. Publication: Hoch et al. Nature (2017) 541:87-91.
Start Year 2016
 
Description Collaboration with Michael Hausser, University College London 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration exploiting focussed ion beam scanning electron microscopy (FIBSEM) technology at UCL to view ultrastructural organization of functional vesicle pools. We are providing samples using a method we developed for readout of functional vesicles in acute brain slice.
Collaborator Contribution UCL group provides access to, and expertise on, FIBSEM approaches.
Impact The initial validation of the approach we will use was published: PMID: 23141069
Start Year 2011
 
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 New collaboration with Syndesi Therapeutics funding a postdoc 
Organisation Syndesi Therapeutics
Country Belgium 
Sector Private 
PI Contribution Syndesi Therapeutics wished to extend our research collaboration providing a fully-funded 4-month part-time postdoc position in my lab. My postdoc took on this role and ran new experiments examining their SV2A-related pro-cognitive compounds in synaptic assays.
Collaborator Contribution Syndesi financed the project and supplied the compounds for testing.
Impact We have generated results which are still being analyzed as part of a consultancy agreement.
Start Year 2019
 
Description Applicant visitors day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Specialized visitor day for sixth form students and guests with firm interest in neuroscience study. Covered degree structure but also wider implications of neuroscience and some discussion of research.
Year(s) Of Engagement Activity 2017
 
Description Interaction with local school - Balfour Infants - for possible future collaboration. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Invitation to attend science lab opening event at school with plans to improve links between university research and infant school teaching.
Year(s) Of Engagement Activity 2015
 
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 Invited Research Talk at German Neuroscience Meeting, Gottingen 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to speak at major research conference; there was significant interest in my seminar. I made key new contacts in the research field topic. Through this meeting, I have also set up a collaboration with the cryo-EM group at the Max Planck Institute in Gottingen.
Year(s) Of Engagement Activity 2017
 
Description Invited platform talk at Federation for European Neurosciences meeting, Milan, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk sparked questions and discussion afterwards

Interest in lab visits, collaborations.
Year(s) Of Engagement Activity 2014
 
Description Micromachines to Cellular Networks, Invited Symposium Speaker, Sussex, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A locally organized two-day meeting to publicize our growing expertise in cryo-EM methods. My talk sparked key questions/discussion. A key outcome was that I spoke with likely reviewers for a cryo-EM Wellcome Trust equipment bid which I applied for, and was subsequently awarded.
Year(s) Of Engagement Activity 2017
 
Description Neuroscience talk (Sussex) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact A seminar on research to neuroscience community at Sussex
Year(s) Of Engagement Activity 2015
 
Description Open day visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Large-scale talk to 100+ people on broad ideas about neuroscience as a career direction. Included potential students and members of public.
Year(s) Of Engagement Activity 2015
 
Description Open lab for non-scientists 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact An open lab session, showing non-scientists from the university some examples of research approaches/principles being used in our work. It included a short talk and opportunity to engage with ongoing experiments, talk to researchers etc. Very well-received.
Year(s) Of Engagement Activity 2015
 
Description Open lab session for visiting sixth form students and general public 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Set up lab for sixth form and general public to view neuronal cultures and electron microscopy data. Lots of interest and questions.
Year(s) Of Engagement Activity 2017
 
Description Participation in an open day or visit at my research institution - Open lab session for visiting sixth form students and general public 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Open lab demonstration. Showing neurons and multimedia which explains fundamental research.
Year(s) Of Engagement Activity 2018,2019,2020
 
Description Potential undergraduate student and parent visit to university 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Talk covering background to neuroscience, studying neuroscience, research etc. to broad audience interested in a possible future in this discipline.
Year(s) Of Engagement Activity 2015
 
Description Research Talk (Barcelona) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact European meeting to consortium of scientists interested in plasticity mechanisms. Included grant policymakers in audience.
Year(s) Of Engagement Activity 2016
 
Description Research Talk at Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Neuroscience talk/open afternoon for non-scientists with an interest in future undergraduate studies. Talk included neuroscience research and information on specific of neuroscience degrees at Sussex. Good interests and questions on careers in neuroscience.
Year(s) Of Engagement Activity 2017,2018,2019,2020
 
Description Research posters at conference (Society for Neuroscience, Chicago) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Successful research posters at Society for Neuroscience, Chicago.
Generated substantial interest with many (~>100) interactions with international neuroscience community.
Year(s) Of Engagement Activity 2015
 
Description Research seminar at University of York 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talk sparked questions and discussion afterwards

Interest in visiting lab, future collaborations.
Year(s) Of Engagement Activity 2014
 
Description STEM ambassador activity at St. Catherine's Catholic School for Girls 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact During her time in the lab, my postdoc, Stephanie Rey has carried out training to be a STEM (science, teaching, engineering and maths) ambassador. This activity was a talk to pupils (~13 y.o.) at St. Catherine's Catholic School for Girls, Bexley, London, UK on neuroscience, studying and a scientific career.
Year(s) Of Engagement Activity 2017
 
Description Society for Neuroscience communication 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact SFN abstract:
429.06. A quantitative ultrastructural readout of in vivo presynaptic activity for connectomics
*A. SIMON1, A. ROTH1, M. FISEK1, V. MARRA2, C. RACCA3, K. STARAS4, M. HAUSSER1;
1Univ. Col. London, London, United Kingdom; 2Univ. of Leicester, Leicester, United Kingdom; 3Newcastle Univ., Newcastle-Upon-Tyne, United Kingdom; 4Univ. of Sussex, Brighton, United Kingdom
Disclosures
A. Simon: None. A. Roth: None. M. Fisek: None. V. Marra: None. C. Racca: None. K. Staras: None. M. Hausser: None.
Abstract
The patterns of synaptic input received by cortical neurons during behavior are not known. Ideally, they should be measured in the context of the wiring diagram of the circuit. How to quantitatively assess synaptic weights and synaptic activity is also an open problem for current connectomics approaches. Here we describe a strategy for measuring presynaptic activity and release probability during circuit function along with circuit connectivity at nanoscale resolution. The method relies on FM 1-43FX labelling and dye photoconversion as a marker of functionally recycled vesicles in vivo, permitting active terminals to be visualized using electron microscopy-based connectomics approaches.
FM 1-43FX was injected via a patch pipette into L2/3 of V1 in awake head-fixed mice running on treadmill. The FM bolus was monitored by 2-photon imaging while mice were shown a visual stimulus for 10 min. After transcardial perfusion fixation, the brain was postfixed overnight. Vibratome sections containing the dye-loaded presynaptic boutons were photoconverted and processed for EM. FIBSEM was chosen to acquire high-resolution 3D data (6.2 x 6.2 x 9.3 nm3 voxels), allowing each synaptic vesicle to appear on 5-6 consecutive images. Two distinct vesicle populations were found in presynaptic terminals (Fig. 1): i) dye-loaded, photoconverted (PC+) active vesicles with a dark lumen, ii) unloaded (PC-) vesicles with a clear lumen. Voxel classification using ilastik readily distinguished the two vesicle types.
A large majority of excitatory and inhibitory boutons contained PC+ vesicles, suggesting that most were active during visual stimulation. In active boutons, the fraction of vesicles classified as PC+ varied widely, indicating broad distributions of activity levels and presynaptic release probabilities. In summary, our method provides a powerful readout of presynaptic activity with ultrastructural context, enabling the organization of active synapses in vivo to be revealed for the first time. We anticipate our approach will provide a valuable new strategy for functional connectomics.
Year(s) Of Engagement Activity 2019
URL https://www.abstractsonline.com/pp8/#!/7883/presentation/44928
 
Description Speaker at teachers conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact A talk on my research topic - under 'Hot Topics' session - primarily my current BBSRC and MRC work - communicated to teacher's in science disciplines. The aim is to motivate and excite teachers and their teaching by giving them insight into the latest-breaking approaches in research. Feedback from session was outstanding.
Year(s) Of Engagement Activity 2015