Cl-out: a novel cooperative-optogenetic strategy to control neuronal chloride
Lead Research Organisation:
Newcastle University
Department Name: Biosciences Institute
Abstract
Chloride is a very important ion in the brain, because neurons use it to give negative (inhibitory) signals. It is a negatively charged ion, and for the most important type of synaptic inhibition, it provides the negative signal to neurons. For this to work, however, chloride needs to be kept very low inside neurons. On occasions, however, neurons lose the ability to remove chloride, and when this happens, synaptic inhibition no longer works well. This can present serious problems for brain function, and may give rise to seizures, spasticity, severe pain and multiple other neurological conditions.
Despite the clear importance of maintaining a good chloride balance, we know little about how this is achieved in healthy brains, or why it might go wrong. Even worse, we have very limited tools to treat this, or even do research on this important topic. We therefore designed a new way to manipulate chloride levels, both up and down, that could transform this field and even offers the potential for clinical use.
Our new technology is what is called an optogenetic protein. Optogenetics is a revolution in progress in neuroscience. It involves introducing into neurons light sensitive proteins that, when illuminated, can directly modulate the neuron's activity. Because it is such a powerful technique, allowing precise manipulation of selected neurons, it is considered to be the future of brain-machine interfaces. It is also transforming the type of research questions that can be asked. We created a new optogenetic protein with multiple beneficial effects: when illuminated, it directly stops neurons from firing, but more importantly, it also removes chloride and thereby provides a persistent protective effect by improving the brain's own inhibitory systems. We call the new protein "Cl-out", standing for "chloride (Cl) out".
So far, we have preliminary data showing Cl-out can extrude chloride. This though is just the start; our proposal describes how we will develop this further.
We have already identified a way to modify Cl-out: our original solution couples chloride movement to the removal of a hydrogen ion, but we now have identified a way of doing this differently, coupling it instead to the movement of a potassium ion. This offers a fundamentally different "quality" of effect, which will extend the utility of this tool. We will learn how to express the protein in different types of neurons, and explore what is the optimal strategy for activating it to give the best chloride-correction effect, and whether this is further boosted by the additive action of certain drugs.
We will also create a powerful set of resources for expression of Cl-out, which we will make available to the research community. These will allow Clout to be expressed in subpopulations of neurons with fine control, and will facilitate its uptake by other researchers.
In addition to the work developing Cl-out as a research tool, we will conduct parallel studies to investigate how chloride regulation affects neuronal behaviour. In this aim, we will also use another light sensitive membrane protein, Halorhodopsin, which we have demonstrated can be used to drive chloride in the opposite direction, into neurons. We thus have light-sensitive mechanisms which can drive chloride in either direction very rapidly, opening all manner of research opportunities. For instance, we will be able to learn about how ionic regulation in neurons affects whole brain behaviour. Second, and perhaps more important is our technical developments will offer simple experimental model systems which can be used for drug development, exploration and screening.
Despite the clear importance of maintaining a good chloride balance, we know little about how this is achieved in healthy brains, or why it might go wrong. Even worse, we have very limited tools to treat this, or even do research on this important topic. We therefore designed a new way to manipulate chloride levels, both up and down, that could transform this field and even offers the potential for clinical use.
Our new technology is what is called an optogenetic protein. Optogenetics is a revolution in progress in neuroscience. It involves introducing into neurons light sensitive proteins that, when illuminated, can directly modulate the neuron's activity. Because it is such a powerful technique, allowing precise manipulation of selected neurons, it is considered to be the future of brain-machine interfaces. It is also transforming the type of research questions that can be asked. We created a new optogenetic protein with multiple beneficial effects: when illuminated, it directly stops neurons from firing, but more importantly, it also removes chloride and thereby provides a persistent protective effect by improving the brain's own inhibitory systems. We call the new protein "Cl-out", standing for "chloride (Cl) out".
So far, we have preliminary data showing Cl-out can extrude chloride. This though is just the start; our proposal describes how we will develop this further.
We have already identified a way to modify Cl-out: our original solution couples chloride movement to the removal of a hydrogen ion, but we now have identified a way of doing this differently, coupling it instead to the movement of a potassium ion. This offers a fundamentally different "quality" of effect, which will extend the utility of this tool. We will learn how to express the protein in different types of neurons, and explore what is the optimal strategy for activating it to give the best chloride-correction effect, and whether this is further boosted by the additive action of certain drugs.
We will also create a powerful set of resources for expression of Cl-out, which we will make available to the research community. These will allow Clout to be expressed in subpopulations of neurons with fine control, and will facilitate its uptake by other researchers.
In addition to the work developing Cl-out as a research tool, we will conduct parallel studies to investigate how chloride regulation affects neuronal behaviour. In this aim, we will also use another light sensitive membrane protein, Halorhodopsin, which we have demonstrated can be used to drive chloride in the opposite direction, into neurons. We thus have light-sensitive mechanisms which can drive chloride in either direction very rapidly, opening all manner of research opportunities. For instance, we will be able to learn about how ionic regulation in neurons affects whole brain behaviour. Second, and perhaps more important is our technical developments will offer simple experimental model systems which can be used for drug development, exploration and screening.
Technical Summary
These studies build upon preliminary work to create a new optogenetic strategy to pump chloride out of cells, called Cl-out. This new technology provides the first means of reducing intracellular chloride, and complements other recent work demonstrating how a different optogenetic tool, Halorhodopsin can be used to drive chloride in the opposite direction. Together, these new tools have the potential to revolutionise research into chloride regulation, which is integral to synaptic inhibition throughout the nervous system.
Cl-out works by fusing two optogenetic proteins: the hyperpolarising proton pump Archaerhodopsin, and one of the new optogenetic chloride channels. The Archaerhodopsin serves as an optical voltage clamp, using light to clamp the cell's membrane potential below the chloride channel reversal potential. This provides the driving force to pump chloride from the cytosol by simultaneously opening Cl- channels. Because both proteins are activated by light, we can achieve Cl-clearance in millions of cells simultaneously simply by expressing the protein generally, and then bathing the tissue in light.
This proposal has two facets:
1. The validation and development of ClouT. We will first develop a variant of Cl-out, which couples Cl extrusion to the movement of a K+ ion (instead of a proton). We will further test different expression strategies, and create plasmids, viral vectors, and a floxed animal line to facilitate future work using this resource. We will investigate different illumination strategies to achieve efficient and persistent GABAergic improvement for the lowest levels of illumination (important for efficient brain-machine interface function).
2. The demonstration of the impact of chloride manipulation on neuronal function in simple in vitro model systems that can subsequently be developed for assays for drug development.
Cl-out works by fusing two optogenetic proteins: the hyperpolarising proton pump Archaerhodopsin, and one of the new optogenetic chloride channels. The Archaerhodopsin serves as an optical voltage clamp, using light to clamp the cell's membrane potential below the chloride channel reversal potential. This provides the driving force to pump chloride from the cytosol by simultaneously opening Cl- channels. Because both proteins are activated by light, we can achieve Cl-clearance in millions of cells simultaneously simply by expressing the protein generally, and then bathing the tissue in light.
This proposal has two facets:
1. The validation and development of ClouT. We will first develop a variant of Cl-out, which couples Cl extrusion to the movement of a K+ ion (instead of a proton). We will further test different expression strategies, and create plasmids, viral vectors, and a floxed animal line to facilitate future work using this resource. We will investigate different illumination strategies to achieve efficient and persistent GABAergic improvement for the lowest levels of illumination (important for efficient brain-machine interface function).
2. The demonstration of the impact of chloride manipulation on neuronal function in simple in vitro model systems that can subsequently be developed for assays for drug development.
Planned Impact
The immediate beneficiaries will be amongst the academic community. Such is the widespread importance of chloride-dependent inhibitory mechanisms across the brain, that we predict our research to have relevance to laboratories working in all areas of neuroscience. It will be of particular interest to the large and growing optogenetic community, as the first demonstration of combinatorial opsin function, whereby emergent properties arise only from co-activation of multiple opsins. And by extension, also for the field of brain-machine interfaces (BMIs), which will feed heavily off the continued developments in optogenetics; our new constructs, or derivatives of these, will, we believe, be among the principle optogenetic tools used in such BMIs. Dissemination to this interest group will be helped by the major optogenetic research initiatives in Newcastle ("CANDO").
We will make the resources generated through this project - plasmids, viral vectors, transgenic mice expressing the new optogenetic constructs - available to the research community through Addgene, Viral Vector Cores (e.g. U.Penn and UNC) and mouse line repositories (Jackson Laboratories and Harwell, MRC), and promote their use by collaborative efforts.
The Biopharma Industry can also benefit from the utilising our new techniques for manipulating chloride levels in neurons, for future drug development in this field. The successful completion and publication of our research will be the basis for engaging with the Biopharma Industry through future collaborative funded projects including CASE studentships and industry placements.
Novel research findings will be incorporated into the various teaching and outreach activities of the team. There will be obvious benefits for the postgraduate and early career researchers in the Institute, where there is growing interest in implementing optogenetics into research programmes.
We will engage with the wider academic and non-academic communities through various routes including the University of Newcastle, local schools, and through our involvement in patient outreach programs. These have mainly focussed on our specialist research interests of epilepsy (AT: through the annual North East Epilepsy Research Meetings, Epilepsy Interest Group, which is part of the Northern England Stategic Clinical Network, Epilepsy Research UK and International League Against Epilepsy meetings, local and national patient groups, including through Epilepsy Action (contributions to Epilepsy Professional magazine)) and mitochondrial disease (RL, through his involvement as founding member and PI of the Mitochondrial Research Group - see numerous local and national level work, leading ultimately to the recent change in the law regarding so-called "three parent families"). These events also stimulate interest and understanding of science in children and young adults, providing academic role models and roadmaps for their own career ambitions.
Medical charities, such as Epilepsy Research UK and Epilepsy Action who have both funded topics related to this project, will benefit by communicating to the public about recent scientific discoveries and new breakthrough technologies. This boosts fundraising, by providing tangible evidence of the effective use of the benefactions, which in turn supports further research and services for people suffering with these conditions.
Through these various pathways, these research efforts will help raise the profile of the North East and the UK generally, as leaders of biomedical and health care innovation.
We will make the resources generated through this project - plasmids, viral vectors, transgenic mice expressing the new optogenetic constructs - available to the research community through Addgene, Viral Vector Cores (e.g. U.Penn and UNC) and mouse line repositories (Jackson Laboratories and Harwell, MRC), and promote their use by collaborative efforts.
The Biopharma Industry can also benefit from the utilising our new techniques for manipulating chloride levels in neurons, for future drug development in this field. The successful completion and publication of our research will be the basis for engaging with the Biopharma Industry through future collaborative funded projects including CASE studentships and industry placements.
Novel research findings will be incorporated into the various teaching and outreach activities of the team. There will be obvious benefits for the postgraduate and early career researchers in the Institute, where there is growing interest in implementing optogenetics into research programmes.
We will engage with the wider academic and non-academic communities through various routes including the University of Newcastle, local schools, and through our involvement in patient outreach programs. These have mainly focussed on our specialist research interests of epilepsy (AT: through the annual North East Epilepsy Research Meetings, Epilepsy Interest Group, which is part of the Northern England Stategic Clinical Network, Epilepsy Research UK and International League Against Epilepsy meetings, local and national patient groups, including through Epilepsy Action (contributions to Epilepsy Professional magazine)) and mitochondrial disease (RL, through his involvement as founding member and PI of the Mitochondrial Research Group - see numerous local and national level work, leading ultimately to the recent change in the law regarding so-called "three parent families"). These events also stimulate interest and understanding of science in children and young adults, providing academic role models and roadmaps for their own career ambitions.
Medical charities, such as Epilepsy Research UK and Epilepsy Action who have both funded topics related to this project, will benefit by communicating to the public about recent scientific discoveries and new breakthrough technologies. This boosts fundraising, by providing tangible evidence of the effective use of the benefactions, which in turn supports further research and services for people suffering with these conditions.
Through these various pathways, these research efforts will help raise the profile of the North East and the UK generally, as leaders of biomedical and health care innovation.
Publications
Burman RJ
(2019)
Excitatory GABAergic signalling is associated with benzodiazepine resistance in status epilepticus.
in Brain : a journal of neurology
Currin CB
(2020)
Chloride dynamics alter the input-output properties of neurons.
in PLoS computational biology
Lodovichi C
(2022)
Genetically encoded sensors for Chloride concentration.
in Journal of neuroscience methods
Mackenzie-Gray Scott C
(2022)
PV-specific loss of the transcriptional coactivator PGC-1a slows down the evolution of epileptic activity in an acute ictogenic model.
in Journal of neurophysiology
Papasavvas CA
(2020)
Divisive gain modulation enables flexible and rapid entrainment in a neocortical microcircuit model.
in Journal of neurophysiology
Parrish R
(2018)
Simultaneous profiling of activity patterns in multiple neuronal subclasses
in Journal of Neuroscience Methods
Parrish R
(2023)
Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization
in The Journal of Neuroscience
Parrish RR
(2018)
Stress-testing the brain to understand its breaking points.
in The Journal of physiology
Parrish RR
(2023)
Optogenetic ion pumps differ with respect to the secondary pattern of K+ redistribution.
in Physiological reports
Pracucci E
(2023)
Daily rhythm in cortical chloride homeostasis underpins functional changes in visual cortex excitability.
in Nature communications
Ridler T
(2018)
Initiation and slow propagation of epileptiform activity from ventral to dorsal medial entorhinal cortex is constrained by an inhibitory gradient
in The Journal of Physiology
Trevelyan AJ
(2023)
Synergistic Positive Feedback Mechanisms Underlying Seizure Initiation.
in Epilepsy currents
Zhang J
(2020)
Modulation of brain cation-Cl- cotransport via the SPAK kinase inhibitor ZT-1a.
in Nature communications
Title | Deep Mind |
Description | A theatre piece, that was due to be performed live, in a theatre in Newcastle, but this was cancelled due to Covid. It was inspired by research work that I'm involved with, aimed at treating epilepsy using brain-machine interfaces and optogenetics. |
Type Of Art | Film/Video/Animation |
Year Produced | 2020 |
Impact | Viewed many times on youtube |
URL | https://www.youtube.com/watch?v=dnR05r1QkLg |
Title | Susan Aldworth and Andrew Carnie |
Description | Installation of art inspired by people's lived experience of epilepsy, created in the Hatton Gallery in Newcastle, by two artists, Susan Aldworth and Andrew Carnie |
Type Of Art | Artwork |
Year Produced | 2020 |
Impact | The launch event was attended by over 200 people, and many more visitors came to see the installation at the Hatton Gallery, before it was closed due to Covid restrictions. |
URL | http://www.cando.ac.uk/illuminatingtheself/hattongalleryexhibition/ |
Description | March 2023: creation of two new co-opsins March 2023: creation of new mouse line, with flexed ClopHensor, allowing conditional expression of this new biosensor December 2022: publication of paper, in Journal of Neuroscience, demonstrating that strong persistent activation of the chloride pump, Halorhodopsin, causes a secondary inward movement also of K+ ions. This then can lead to initiation of cortical spreading depression events. This is a completely novel way of inducing such events, and strongly suggests that they actually arise from osmotic stress, induced by the net movement of K-Cl ions into neurons. March 2022: This last year, a new K+ channelrhodopsin (KChR) has been created, by John Spudich's group (who has developed / described multiple other opsin in recent years). This thus offers a substitute to BLINK, which was the previous one we attempted to use, and was in the original proposal. We tried a number of BLINK variants, and even though the postdoc on the team, Laura Alberio, was one of the people working o the original BLINK, we simply could not get these to work in tandem with the chloride channel opsin. The new opsin however, is much smaller, has a different mode of action to BLINK, and is more similar to other opsin that we have have had success combining previously. We acquired plasmids of KChR as soon as they were available, and have now combined these with the chloride opsins and are now preparing to test this brand new co-opsin in cultured neurons. March 2021: We have established ClopHensor imaging, in vivo, now, and have discovered a remarkable, and unsuspected degree of variability in chloride levels in neurons, through the day. We are preparing this work for publication. Additionally, we have made some partial success at combining ClopHensor with Cl-out, although the delays in getting ClopHensor to work has slowed this greatly. March 2020: We have completed the calibration of our newly created variant of ClopHensor, and are preparing manuscripts on the technology and also a paper on using it to record Cl levels in select populations of neurons. March 2019: We have installed our new microscope, and are currently going through the process of calibrating the signal, using dissociated neurons. In doing so, we realised that there was a slight problem with the chloride biosensor, clopHensor, which meant that it was being broken by an enzyme commonly found in brain tissue. We have redesigned the protein, to remove the weakness, and have manufactured a new version, that has been incorporated into a system for delivering into cells (a "viral vector"). |
Exploitation Route | We have shared our new clopHensor design with the people who made the original biosensor and we will continue to collaborate to look for other relevant improvements. We aim to publish this new improved version, and at that time, we will make the protein available to others working in the field, by placing it into a repository, such as Addgene, for sharing such things. |
Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
URL | https://doi.org/10.1101/2021.05.12.443725 |
Description | We showed that ontogenetic actuators can be used to create osmotic tension within cells, which may open up new avenues of research into how this may influence pathophysiology. For instance, our new experimental model suggests that cortical spreading depression events may be best understood as a response to a rise in intracellular osmolarity with secondary water influx. |
First Year Of Impact | 2022 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | Research grant, responsive mode |
Amount | £594,000 (GBP) |
Funding ID | BB/P019854/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2020 |
Description | Collaboration with Prof Gian-Michele Ratto |
Organisation | Scuola Normale Superiore di Pisa |
Country | Italy |
Sector | Academic/University |
PI Contribution | We are working with Prof Ratto to develop strategies for imaging and manipulating chloride in neurons |
Collaborator Contribution | We are developing novel optogenetic tools for manipulating chloride, while Prof Ratto is developing tools for measuring chloride. |
Impact | We now have multiple manuscripts being considered for publication, and which we have posted on to preprint servers. Additionally, we were awarded a Royal Society Collaboration grant (in association with Consiglio Nazionale delle Ricerche, Italy) in 2021. |
Start Year | 2017 |
Description | FLAIR collaborative award: Joseph Raimondo |
Organisation | University of Cape Town |
Country | South Africa |
Sector | Academic/University |
PI Contribution | £50000 award from Royal Society to facilitate collaborative work between the UK and existing FLAIR fellows. Joe Raimondo has one of these fellowships and he and I will collaborate on a project trying to understand the source of epileptic seizures in patients with neurocystercicosis. |
Collaborator Contribution | This collaborative award is only open to FLAIR fellows, so was very much initiated by Joe. Having said that, he and I have been collaborating on a variety of projects for a few years. |
Impact | We have already published two collaborative papers: Excitatory GABAergic signalling is associated with benzodiazepine resistance in status epilepticus. Burman RJ, Selfe JS, Lee JH, van den Berg M, Calin A, Codadu NK, Wright R, Newey SE, Parrish RR, Katz AA, Wilmshurst JM, Akerman CJ, Trevelyan AJ, Raimondo JV. Brain. 2019 Nov 1;142(11):3482-3501. doi: 10.1093/brain/awz283. PMID: 31553050 Divergent paths to seizure-like events. Codadu NK, Graham RT, Burman RJ, Jackson-Taylor RT, Raimondo JV, Trevelyan AJ, Parrish RR. Physiol Rep. 2019 Oct;7(19):e14226. doi: 10.14814/phy2.14226. PMID: 31587522 |
Start Year | 2018 |
Description | Macromolecular-Ionic-Compensation team |
Organisation | Imperial College London |
Department | Department of Infectious Disease |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have shared the DNA constructs for our novel optogenetic chloride pumps with Dr John O'Neill (LMB), and Rachel Edgar (ICL) in order to investigate the effects of chloride manipulation on protein biochemistry |
Collaborator Contribution | as above |
Impact | none to date |
Start Year | 2021 |
Description | Macromolecular-Ionic-Compensation team |
Organisation | Medical Research Council (MRC) |
Department | MRC Laboratory of Molecular Biology (LMB) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have shared the DNA constructs for our novel optogenetic chloride pumps with Dr John O'Neill (LMB), and Rachel Edgar (ICL) in order to investigate the effects of chloride manipulation on protein biochemistry |
Collaborator Contribution | as above |
Impact | none to date |
Start Year | 2021 |
Description | Translating epilepsy knowledge from C.Elegans to humans |
Organisation | University of Liverpool |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have been able to test drug candidates, identified by our collaborators, on human neurons, assessed using patch clamp recordings. |
Collaborator Contribution | My collaborators at Liverpool investigate neuronal excitability using a high throughput screening programme using C.elegans. |
Impact | A publication in Epilepsia: Jones et al. (PMID: 32797628) |
Start Year | 2019 |
Description | Columbia University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Neurology grand rounds at Columbia University Medical Center |
Year(s) Of Engagement Activity | 2017 |
Description | Deep Mind & the Great Puzzle |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | An original piece of performance theatre inspired by, and based upon the research we are doing into how we might control brain activity, and specifically epilepsy, using optogenetics. This will take place in March 2020, and I will be an expert panel member for the after-performance discussion. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.ticketsignite.com/event/2084/deep-mind---the-great-puzzle |
Description | ERUK public engagement |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Third sector organisations |
Results and Impact | Public engagement of epilepsy stake holders, coordinated by Epilepsy Research UK - fundraising initiative. |
Year(s) Of Engagement Activity | 2018 |
Description | IBRO School |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I taught at the 7th IBRO-UM5 School on Functional Neuroanatomy, in Rabat, Morocco |
Year(s) Of Engagement Activity | 2019 |
Description | ILAE talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Talk in the closing session (What is a seizure) at the International League Against Epilepsy meeting. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.ilae.org/congresses/2019-ilae-british-branch-annual-scientific-meeting |
Description | Illuminating the self |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Illuminating the Self is an exhibition of art work by Susan Aldworth and Andrew Carnie, inspired by the CANDO project (Controlling abnormal network dynamics with optogenetics), and more generally by our research into the nature of epileptic activity. |
Year(s) Of Engagement Activity | 2020 |
URL | https://hattongallery.org.uk/whats-on/illuminating-the-self |
Description | Online teaching to Masters in Neuroscience Course (Erasmus) based at Athens University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | 4 hours of teaching about epileptic pathophysiology |
Year(s) Of Engagement Activity | 2021 |
Description | Talk on Experimental Epilepsy Seminar Series |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | A talk about my latest research |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.world-wide.org/Neuro/Experimental-Epilepsy/ |
Description | Yale University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Seminar at Yale University Medical School |
Year(s) Of Engagement Activity | 2017 |