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Massachusetts Institute of Technology — Synthetic Neurobiology Group

Professor Ed Boyden (center) discussing Expansion Microscopy enablements with co-inventors/first authors Fei Chen (right) and Dr. Paul Tillberg (left). (Photo courtesy of MIT)
Organization Name 
Award Date 
3/2016
Grant Amount 
$2,970,000
Purpose 
For expanding research at the MIT Media Lab’s Center for Extreme Bionics
Topic (focus area) 

Published: May 2016

Representatives of the MIT Synthetic Neurobiology Group reviewed this page prior to publication.

The Massachusetts Institute of Technology’s Synthetic Neurobiology Group is led by Associate Professor Edward Boyden, and housed by both the MIT MediaLab’s new Center for Extreme Bionics and MIT’s McGovern Institute for Brain Research. Among other things, the group is working on developing new methods and techniques for mapping the brain.

Although we are still in the early stages of exploring science philanthropy, two areas we are interested in investigating further are fundamental basic science and improving tools and techniques within biomedical sciences. We see this gift as an opportunity to explore both of these spaces. We decided to investigate Professor Boyden’s lab specifically on the basis of a strong referral from our trusted scientific advisor, Dario Amodei.

We decided to make a gift to Professor Boyden’s lab to attempt to continue progress on his brain-mapping techniques, based mainly on our impressions that: if the project succeeds, it will have a major impact on neuroscience; success is a realistic, if highly uncertain, possibility; Professor Boyden is an outstanding scientist who has outstanding students in his group; Professor Boyden’s lab has combined economic and scientific considerations to analyze the comparative strengths and weaknesses of different approaches to mapping the brain in a way that we find both compelling and rare; and the proposal is an ambitious and unconventional project which appears to be unlikely to be funded through other sources. These impressions were formed with the assistance of Open Philanthropy Science Advisors Professor Chris Somerville and Dr. Dario Amodei (“Chris” and “Dario” throughout this page). We did not deeply investigate the possible upsides from substantial advances in neuroscience, but believe they could include reduced burden from neurodegenerative and mental disorders and other positive developments.

Based on these considerations, the Open Philanthropy Project recommended an unrestricted gift of $2,970,000 to support materials, equipment use, and salaries for as many as nine post-doctoral researchers, staff scientists, and new graduate students over two years for the Synthetic Neurobiology Group at the MIT Media Lab’s new Center for Extreme Bionics.

Our largest concern about this gift is that our initial decision to evaluate the Synthetic Neurobiology Group’s proposal relied strongly on Dario’s recommendation, rather than a systematic process (such as a request for proposals or a systematic evaluation of possible areas of basic science to fund). Despite a thorough evaluation of this particular project and conversations with several scientists in the field, we still have a somewhat limited sense of how this project compares with other projects in neuroscience, or how neuroscience compares with other fields of basic science we could support.

Gift overview

Background on the MIT Synthetic Neurobiology Group

Professor Boyden leads MIT’s Synthetic Neurobiology Group. He is credited with playing a major role in the invention of optogenetics in 2005, a technique which uses light to control activity of neurons.1 We understand optogenetics to be widely acknowledged as among the most important new tools in neuroscience.2 Professor Boyden also co-invented expansion microscopy (ExM), a technique to artificially expand cells and tissue while preserving their structure, published in Science in 2015.3 ExM represents a potential way to overcome the resolution limits of light microscopes, as well as to allow cells and tissue to be more easily examined with affordable microscopes.4 Dario advised us that many people in the neuroscience community think that this discovery could be as important for neuroscience as optogenetics.

Our understanding is that the work of the Synthetic Neurobiology Group can be broken down into two main areas:

  • “Static” mapping of brains: collecting information about the structure, chemical state, and connectivity of a brain by examining it after the animal is deceased. This includes application and development of ExM techniques.
  • “Dynamic” mapping of brains: studying the brain activity of live animals, in some cases controlling sets of individual neurons in order to study how they affect behavior or the activity of other neurons. This work incorporates work on optogenetics, including to improve the technique’s precision.

Background on brain mapping

During our investigation (see “Process” below), we developed the impression that there is a prevailing belief among neuroscientists that producing a detailed model of the brain is essential to understanding how it works. The BRAIN Initiative and the Human Brain Project are two major projects (based in the U.S. and Europe respectively) aiming, among other things, to produce a full human connectome (that is, a comprehensive map of the neural connections in the human brain).5 Some of the things that might become possible if such a model is developed include comparing diseased brains with healthy brains, looking at the effect of drugs on brain structure, and seeing how brain structure changes with age.

Our understanding is that one barrier to large-scale brain mapping is the limited resolving power of light microscopes. Conventional light microscopes are limited to 200 nanometers,6 and electron microscopes to ~0.1 nanometers.7 Neural circuitry is dense and compact (for example, a synapse can be as small as 200nm in diameter),8 meaning its component factors are sometimes beyond a conventional light microscope’s resolving power. Neuroscientists are therefore limited to electron microscopes to obtain detailed structural and molecular maps of neural circuitry beyond the resolving limit of optical microscopes.9 However, using electron microscopes in this way is both slow and expensive, such that using them in brain-wide studies would present major difficulties.10 Further, although advances have been made that increase the resolution of light microscopes, such techniques are expensive and time consuming.11 To give some context about the technology’s implications for the cost of brain-mapping, the more optimistic estimates we’ve heard are that an electron microscopy image of mouse connectome may be possible for around $200m with advances in imaging. Another estimate we’ve seen is $1 billion.12

The Synthetic Neurobiology Group’s approach and goals

The group’s view is that even if electron microscopy is able to scale to large volumes of entire brain circuits, it is less suitable for detailed studies of neural tissue than optical microscopy. According to the group, the typical treatment of specimens for the process of electron microscopy destroys important details about the neurons, their molecules, and interactions within and between cells that would be useful for understanding the fundamental nature of neural circuitry. Optical microscopy, on the other hand, 1) allows for multiple colors of light wavelengths that help to differentiate between individual neurons in clusters, as well as molecular interactions within cells, and 2) involves a more flexible treatment of the specimen, which could allow for molecular tagging and readout. They hope that successful development of a combination of techniques, such as expansion microscopy and genetic labeling, could help neuroscience meaningfully progress brain mapping in more affordable and efficient ways.13

The group believes that ExM is an important step towards making conventional light microscopy a more useful technique for brain-mapping. By expanding the specimen under study, ExM can show parts of cells and tissue that are beyond light microscopy’s resolving limit of ~200nm.14

Professor Boyden told us that his group is trying to raise at least $20 million over the next several years to pursue an ambitious research agenda within fundamental neuroscience – the connectomic mapping of small mammalian brain circuits at the highest resolution possible. Our understanding is that the group’s long-term objective is to develop techniques and technologies that create an inexpensive way to map a connectome with molecular annotations. Molecular annotation means including detailed epigenomic (the state of the cell that can impact which genes are expressed) and transcriptomic information (an inventory of the amounts of different types of mRNA in the cell) about individual neurons, clearly marking the synaptic connections between neurons, and potentially also including information about changes in proteins. More immediately, we understand that the group plans to attempt to produce a full map of a mammalian connectome (possibly starting with the Etruscan shrew, or at least a portion of mouse) and potentially also improve our ability to map neural activity and take molecular measurements of neural states (3D-mapping and measuring of live brains in real time).15

As noted above, mapping a mouse connectome (without molecular annotation) using existing techniques would cost something like $1 billion. If the Synthetic Neurobiology Group’s research is successful, scientists may be able to map a mouse connectome for tens of millions of dollars, potentially less as techniques improve. Chris and Dario think that such a decrease in cost would probably result in more connectomes being mapped sooner, both within and across species.

Proposed activities

The Open Philanthropy Project is providing support for some of the initial stages of the Synthetic Neurobiology Group’s goal of mapping a mammalian connectome. These stages focus on proving that the technology the group proposes to develop and use is feasible. It is possible that funds from the gift will also be applied to support collaborating scientists working on these issues at other universities or non-profit institutions, something that we understand can be more procedurally challenging to do flexibly using funds from grant-making government agencies.

The initial research supported by our funds has four goals:

  1. Increase expansion microscopy’s expansion range from 4x to >20x. Currently, the Synthetic Neurobiology Group infiltrates brain tissue with a polymer that expands the tissue by a factor of four while preserving structure. Further increasing the magnitude of expansion would functionally increase the resolving power of conventional light microscopes.
  2. Automate the “tracing” of neurons using images obtained through light microscopy. The Synthetic Neurobiology Group will work with a series of cross-section images of neurons. Thus, they need to have a method of determining which parts of different images belong to which neurons. It would be prohibitively expensive to “trace” neurons by hand, so the group plans to develop software to automate the task. Some software already exists for electron microscopy, and we expect they will adapt that.
  3. Develop new techniques for sectioning brain tissue that has been expanded. Chris explained to us that, during expansion, brain tissues become much less dense and resemble a soft jelly. The Synthetic Neurobiology Group needs a way to section the brain into thin slices at a thickness that gives a clean cut and also allows them to infiltrate sectioned tissue with antibodies to help distinguish synaptic connections between neurons. For example, the group believes that infiltration of resins of the type used in electron microscopy, followed by polymerization of the resins into a type of plastic, may rigidify the expanded tissues and allow thin sections to be cut.
  4. Develop improved methods for “brainbow” staining and “barcoding.” Brainbow staining is a method in which neurons are infiltrated with genetically engineered viruses which express fluorescent proteins that give individual neurons unique combinations of colors so that they can be distinguished by fluorescence microscopy.16 Barcoding attempts to infiltrate neurons with unique DNA markers. Both help scientists to determine which neuron pieces are parts of the same neuron. Chris’s view of the literature is that there is decent precedent for the feasibility of brainbow staining, but that barcoding is more novel. The proposed experiments seem to be amongst the very first attempts to use barcoding in neural tissues so there is significant risk that the method may not work well-enough to be useful in practice.

We expect to have a reasonably good sense of whether the Synthetic Neurobiology Group has achieved or is closer to achieving these outcomes two years from now, based on its published and submitted papers and our conversations with the group.

Rationale for the gift

The cause

We are in the relatively early stages of thinking about science philanthropy, and we have not set focus areas within this category (see our September 2015 update about our plans going forward). However, this gift falls within two areas we are interested in investigating further: high-risk/high-reward basic science research and research into improving existing scientific tools and techniques.

Case for this gift

We see the case for this gift as being a test of whether there is a realistic possibility of making a major advance in neuroscience that will radically reduce the cost of producing maps of connectomes. Based on the potential impacts discussed below, we think such an advance could be extremely important from a humanitarian perspective, and that the small probability of making a very large impact in this field justifies our investment.

A secondary motivation of investigating and making this gift has been as part of our learning about science philanthropy and directly funding research. We’ve written previously about how we think about learning about a new cause area via giving.

Impact

Our conversations with Professor Boyden and other neuroscientists led us to think that it would be a major advance in neuroscience if this gift were successful. We believe that advancing neuroscience is a worthwhile goal from a humanitarian perspective both because of potential near-term, foreseeable applications and because of the less foreseeable developments it may provoke. Two immediate potential applications of a major advance in mapping a connectome are:

  1. It might assist research on neurodegenerative diseases and mental disorders, such as schizophrenia, autism, and Alzheimer’s. Neurodegenerative diseases and mental disorders exact a significant humanitarian cost: for example, schizophrenia affects about 2.4 million Americans,17 and Alzheimer’s disease affects between 2.6 and 5.1 million Americans.18 Our conversations with neuroscientists while investigating this gift have led us to believe that connectomic maps that show comprehensive molecular information about diseases and disorders affecting the brain could significantly increase our understanding of them, including by potentially helping scientists to identify new clinical targets.
  2. It would constrain the set of plausible hypotheses that neuroscience needs to test. Our understanding is that the tools neuroscientists use to test hypotheses don’t usually constrain the number of plausible hypotheses about the way the brain works.19 Seeing how neural circuitry is connected could suggest a smaller number of plausible hypotheses, potentially sending neuroscience research in more productive directions.

We also considered one possible downside of funding this work. It was argued to us that faster progress in mapping the connectome could influence the speed and nature of research in artificial intelligence in a way that could increase potential risks from advanced artificial intelligence in the future. Specifically, it was argued that this research might reduce time available for technical research that could prevent potential risks from misaligned superintelligent AI, or it could lead AI development in a more neuroscience-inspired direction whose long-term risks could be harder to manage. We have chosen not to explain these arguments in detail here because they are very complicated and they did not end up influencing our decision about whether to make this gift. Some reasons that these considerations did not influence us are that (i) we see these arguments as highly speculative, (ii) Dario’s intuition is that advances in neuroscience will have limited consequences for the development of AI, and (iii) plausible arguments could also be made that this research could reduce potential risks from advanced AI.

Likelihood of success

To us, it seems highly uncertain, but plausible, that this project will achieve its aims. We formed this view on three main grounds, after Chris investigated Professor Boyden’s past work, citation record and reputation in depth.

Firstly, it is our general impression that Professor Boyden is an outstanding scientist who is well-qualified to execute the funded research and supported by a well-credentialed lab. Our view of Professor Boyden is based on his co-discovering ExM and optogenetics, Chris’s view that Professor Boyden is plausibly one of the very best researchers at developing tools for neuroscience, and Dario’s opinion that Professor Boyden is the most impressive Principal Investigator he has interacted with. Because Dario has a background in neuroscience, has a close knowledge of the Open Philanthropy Project’s mission, and is strongly aligned with our values, we accord this opinion significant weight. Further, we find it likely that Professor Boyden has a very strong research group. He has seven Hertz fellows working in or affiliated with his lab, which we believe represents a significant portion of fellows of the relevant ages in this field. Given the prestige of this fellowship and our interactions with Hertz fellows, we consider it a reasonable proxy for research talent. Further, Dario thinks highly of the researchers in Professor Boyden’s group.

Secondly, our impression is that within the neuroscience field, Professor Boyden is particularly well-suited to execute research of this kind. He and his collaborators have written several public analyses of different possible techniques for mapping the brain, arguing – in a manner convincing to both Dario and Chris – that its approaches are the most feasible for overcoming the technical barriers to producing a full map of a connectome.20 We find this encouraging because we think it shows that Professor Boyden is thinking strategically about different ways the field might scale up to a mapping a whole brain, and offering that thinking for public critique. In our investigation of this gift we did not find others publishing analyses of this kind. In addition, conversations with others in the field of neuroscience gave us the impression that Professor Boyden is unusual in his ability to work with industry to commercialize developments from his lab swiftly, increasing the likelihood of impact for discoveries from his lab.

Thirdly, Chris’s view is that the technical risks of the group’s research agenda are acceptable. We relied heavily on Chris’s judgment in assessing these risks, and so our views rely heavily on our trust in him and his process.

Risks to the success of this gift

The type of work that Professor Boyden is undertaking is new and untested, and we start from the view that making major scientific advances is extremely difficult. For the goals of this gift in particular, there are a number of possible sources of technical failure that we can anticipate, and presumably others that we cannot anticipate.

However, the Synthetic Neurobiology Group does not necessarily need to succeed on all four of the goals described above in order to make substantial progress towards a molecularly annotated map of a connectome; our impression is that progress on one goal is to some extent interchangeable with progress on another. By our understanding, the two goals which are most important to the gift’s success are:

  • Developing software to automate neuron tracing using images obtained through light microscopy; and
  • Developing a technique to infiltrate brain tissue with antibodies that will help scientists distinguish synaptic connections between neurons.

Our understanding is that the other goals that this gift is intended to support are less critical. If the group fails to develop improved “barcoding” techniques to identify individual neurons, they could fall back on relatively established “brainbow” staining techniques, and they may be able to make significant progress mapping a connectome even if they only achieve 5x expansion using ExM.

We note that Professor Boyden already has a large lab with a significant operating budget, and may therefore be unable to devote a lot of personal attention to this project. Our positive impression of the quality of the researchers in Professor Boyden’s group (discussed above) causes us to be less concerned that limited personal attention from him is a significant source of technical risk. In addition, Professor Boyden notes that he spends most days in 1:1 or sub-group meetings with lab members, with most hours spent brainstorming ideas, analyzing data, and working on publications and other products.

Room for more funding

As an academic group, the Synthetic Neurobiology Group’s operating budget varies by year and grant cycle, but is recently estimated at around $5 million per year, much of which is restricted. Though this is a large budget for an academic lab in this field, our impression is that this project would be relatively unlikely to receive funding without our gift. Some of our impressions about the dynamics in science research funding and how they relate to Professor Boyden’s work include:

  • Professor Boyden’s research agenda in this area is very speculative, possibly too speculative for many funders, who may prefer to fund incremental research extending existing discoveries rather than “breakthrough science”.
  • Many funders appear to be particularly wary of funding research into new basic science tools and techniques.
  • It seems to be difficult to get large research grants to do work of this scale, and working to combine many small grants for a project like brain mapping is difficult because each grant is restricted differently.

These observations, and our belief that they apply to Professor Boyden’s work in this case, are based both on our general observations thus far about the science funding ecosystem (which we have written about previously) and on extensive discussion with Professor Boyden about the group’s current funding situation and why he had not received/did not expect to receive funding for this work from other plausible sources. Professor Boyden asked us to keep the details of this information confidential.

If Professor Boyden’s group makes substantial progress in the next two years, our hope is that this will make this area of research appear less risky and so encourage other, more established science donors and/or the NIH to fund work in the area. In general, a working hypothesis of ours is that providing funding in a relatively thin field may create something of a self-reinforcing dynamic, because research both builds out a new field and often raises new and interesting questions, making it easier and more desirable for other funders and researchers to work in the field.


Process

Our decision process

Dario began investigating this gift in early 2015, based on the ExM discovery and his knowledge of Professor Boyden. On his recommendation, we approached Professor Boyden, who shared information with us about the Synthetic Neurobiology Group’s goals to build on ExM and its funding needs in this regard. To evaluate this funding opportunity, Dario and other Open Philanthropy Project staff spoke with both experts and funders in neuroscience. The former conversations focused on evaluating Professor Boyden’s abilities and the potential impacts of his research; the latter focused on evaluating his claim that he was unlikely to attract funding for this research from another source. We became strongly interested in funding Professor Boyden’s research mainly because of our impressions that he was an outstanding scientist, that his research could have a significant impact in neuroscience, and that high-risk/high-reward research like this seems to us to be relatively neglected in the present funding environment.

Before making the gift, we asked Chris, who had recently started working with us, to more closely evaluate Professor Boyden’s reputation and his proposed research agenda. We have heavily deferred to Chris’s technical expertise and judgment. As this is the first major project Chris has worked on with us, we do not yet have a developed sense of how much faith to put in his opinion, other than what can generally be gleaned from his professional reputation, our personal impressions, and our attempts to spot-check his reasoning related to this gift.

Chris reviewed Professor Boyden’s publication record and the associated literature, as well as the scientific literature related to Professor Boyden’s proposed work. He recommended that the Open Philanthropy Project fund Professor Boyden’s core ideas, but he identified a number of technical risks in achieving Professor Boyden’s goal of mapping the full brain of an Etruscan shrew. Chris’s impression was that the technical risks were acceptable, but that we should ask Professor Boyden to first demonstrate his ability to overcome them, by working towards the four goals outlined above, before providing a gift that would fully fund his research in this area.

In collaboration with Professor Boyden, we settled on a gift of $2,970,000 to enable his group to undertake a more limited research agenda than originally discussed, focused on overcoming key technical risks to his research into tools and techniques for brain mapping.

Our principal concern about this gift to the Synthetic Neurobiology Group is that we did not use a systematic process for arriving at the neuroscience field or this project within the field as priority areas for giving. We don’t have a strong sense of how this project would compare with other promising projects in neuroscience, because we did not consider other potentially fundable projects in neuroscience as part of this investigation (though we did attempt to get a rough sense for this in the conversations with experts described above). We are also unsure how projects in neuroscience compare with projects in other areas of basic science that we could fund.

We do not intend to use a process similar to this for our scientific grant-making in the long term. We’re currently developing our process for finding and evaluating grants, with increasing advisory capacity as our top priority (see our September 2015 update). However, we do not think that the limitations of the process we used in identifying this gift undermines the justification for making this gift within our larger funding agenda and framework.

In our view, the strongest criticism that could be made of this gift is that we chose Professor Boyden’s project over other areas for research with roughly equivalent prospects and potential for impact because of a referral from someone we trust, Dario, about a project in his (rather than our) focus area. On the referral element, we note that we contracted an independent science advisor, Chris, to conduct the final review, ultimately limiting the gift to initial research based on Chris’s advice. On the choice of neuroscience, we note that we made the gift primarily based on its capacity to have a major impact in an area of basic science, rather than neuroscience in particular, and because it focuses on tools and techniques development for biomedical science, which we suspect may be underfunded relative to other research. Given that limited capacity, particularly scientific advisor capacity, is a major limitation on our work at present, we did not think it would be best to issue a request for proposals at the time.

Plans for learning and follow-up

Key questions for follow-up

Questions we hope to answer using this gift include:

  • Did the group make progress towards any of the goals described above?
  • How was the gift spent? Did we recommend an appropriate amount for the intended purposes of the gift?
  • If the Synthetic Neurobiology Group made progress on some or all of the funded research questions, did this initial progress assist it in accessing more conventional sources of funding?
  • What do we learn from the Synthetic Neurobiology Group and its research outcomes over two years? Direct funding of scientific research is a new kind of philanthropy for us, and we expect that we will learn something about how to assess funding opportunities in the future, how to work with academic institutions, and our hypotheses that basic science research and research into new tools and techniques for biomedical sciences are underfunded.

Follow-up expectations

We expect to have a conversation with Professor Boyden every six months over the course of the two year-long gift, with public notes if the conversation warrants it.

In 18-24 months, we plan to evaluate Professor Boyden’s progress toward developing these new tools and techniques for brain mapping. If the project is successful and we find that we work well with him, we will consider supporting his further efforts.

Sources

Document Source
Boyden et al. 2005 Source (archive)
Brain Initiative, What is the Brain Initiative Source (archive)
Cai et al. 2013 Source (archive)
Callaway 2015 Source (archive)
Chen, Tillberg and Boyden 2015 Source (archive)
Chen et al. 2005 Source (archive)
Chi 2013 Source (archive)
Dodt 2015 Source (archive)
Human Brain Project, Overview Source (archive)
Marblestone and Boyden 2014 Source (archive)
Marblestone et al. 2014 Source (archive)
Marblestone et al. 2014a Source (archive)
National Institutes of Health, Alzheimer’s Disease Fact Sheet Source (archive)
National Institutes of Health, Schizophrenia Fact Sheet Source (archive)
Prevedel et al. 2014 Source (archive)
Silvestri, Sacconi and Pavone 2013 Source (archive)
Stern 2015 Source (archive)
Strack 2015 Source (archive)
Synthetic Neurobiology Group, Value Proposition Source
The Brain Prize Press Release 2013 Source (archive)
University of Utah, Electron Microscopy Tutorial Source (archive)
  • 1.
    • “The Brain Prize - Denmark’s 1 million euro brain research prize - is awarded to six leading scientists for the development of ‘optogenetics’, a revolutionary technique that advances our understanding of the brain and its disorders. The names of the prize winners, Austrian Gero Miesenböck, Germans Ernst Bamberg, Peter Hegemann, and Georg Nagel, and Americans Ed Boyden and Karl Deisseroth, were announced on Monday 11 March 2013 in Copenhagen”, The Brain Prize Press Release 2013.
    • See also: “Millisecond-timescale, genetically targeted optical control of neural activity”, Boyden et al. 2005, Pg. 1263-8.
  • 2.

    “Optogenetics, which has been called the breakthrough of the decade, involves the use of light to control neurons”, The Brain Prize Press Release 2013.

  • 3.

    “Expansion Microscopy”, Chen, Tillberg and Boyden 2015, Pg. 543-8.

  • 4.

    “Conventional optical microscopes cannot distinguish objects that are closer together than about 200 nanometres. Although super-resolution microscopy can discern objects as close together as about 20 nm, they require expensive, specialized equipment, and struggle with thick structures such as sections of brains … After stretching [using ExM], the fluorescent-tagged molecules move farther away from each other; proteins that were previously too close to distinguish with a visible-light microscope come into crisp focus. In his presentation, Boyden suggested that the technique can resolve molecules that are as close as 60 nm before expansion”, Callaway 2015, Pg. 254.

  • 5.
    • “By accelerating the development and application of innovative technologies, researchers will be able to produce a revolutionary new dynamic picture of the brain that, for the first time, shows how individual cells and complex neural circuits interact in both time and space”, Brain Initiative, What is the Brain Initiative.
    • “The HBP is organized in thirteen subprojects, spanning strategic neuroscience data, cognitive architectures, theory, ethics and society, management and the development of six new informatics-based platforms including: … Brain Simulation (building and simulating multi-level models of brain circuits and functions)”, Human Brain Project, Overview.
  • 6.

    “Conventional optical microscopes cannot distinguish objects that are closer together than about 200 nanometres “, Callaway 2015, Pg. 254.

  • 7.

    “[the] resolution of an electron microscope is theoretically unlimited for imaging cellular structure or proteins. Practically, the resolution is limited to ~0.1 nm due to the objective lens system in electron microscopes”, University of Utah, Electron Microscopy Tutorial.

  • 8.

    “The mean mass of 2-min PSDs [postsynaptic densities] was 1,100 ± 600 MDa (n = 90; mean ±SD), with diameters ranging from 200 to 526 nm.” Chen et al. 2005, Pg. 11554.

  • 9.

    “The basic strategy employed by the current EM approaches is to obtain many morphological images of thin tissue sections, segmenting those images into regions corresponding to distinct neuronal processes, and tracing individual axons from one image to another. Because axons are thin, long, and densely interspersed with other neuronal processes, tracing their entire lengths is a challenge”, Marblestone et al. 2014, 3.

  • 10.

    “One of the most popular techniques allowing nanometric resolution is electron microscopy (EM) which, however, is characterized by very slow data acquisition rates. EM is thus inappropriate for brain-wide studies; conversely, it has been used successfully to reconstruct local circuitry in small regions (Briggman et al., 2011; Helmstaedter et al., 2011), and the whole nervous system in very small organisms such as the nematode Caenorhabditis elegans, which has only 302 neurons (White et al., 1986)”, Silvestri, Sacconi and Pavone 2013, Pg. 168.

  • 11.
    • “In the past decade, important advances have been made to increase the resolution of the light microscope, as acknowledged by last year’s Nobel Prize for superresolved fluorescence microscopy (1, 2). This progress is fascinating but comes at the price of high illumination intensities or long recording times, and expensive instruments”, Dodt 2015, Pg. 474.
    • “The resolution [of ExM] does not compare badly to that of superresolution microscopy techniques (1, 2), which enable a resolution of 20 to 70 nm. The additional advantage of expansion microscopy is that it depends on conventional microscopes; hence, data can be acquired quickly”, Dodt 2015, Pg. 474.
  • 12.

    “The estimated cost for a single whole mouse brain acquisition in 3 years is roughly $1B without parallelization”, Marblestone et al. 2014, Pg. 5.

  • 13.

    “ ‘With EM you have to section into thin sections, and with optical microscopy many of the technologies either require very expensive equipment or they run very slowly,’ says Boyden. He adds that, with EM, ‘you cannot see the molecular information.’ For these reasons, Boyden and his team sought a new approach to enhance image resolution”, Strack 2015, Pg. 169.

  • 14.

    “Conventional optical microscopes cannot distinguish objects that are closer together than about 200 nanometres. Although super-resolution microscopy can discern objects as close together as about 20 nm, they require expensive, specialized equipment, and struggle with thick structures such as sections of brains … After stretching, the fluorescent-tagged molecules move farther away from each other; proteins that were previously too close to distinguish with a visible-light microscope come into crisp focus. In his presentation, Boyden suggested that the technique can resolve molecules that are as close as 60 nm before expansion.” Callaway 2015, Pg. 254.

  • 15.

    See, e.g., “Simultaneous whole-animal 3d imaging of neuronal activity using light-field microscopy”, Prevedel et al. 2014.

  • 16.

    See illustrations in Chi 2013, and generally Cai et al. 2013.

  • 17.

    “Schizophrenia is a chronic and disabling brain disorder that affects 2.4 million Americans, according to the 2005 National Comorbidity Survey-Replication”, National Institutes of Health, Schizophrenia Fact Sheet

  • 18.

    “Estimates of how many people in the United States currently have Alzheimer’s disease vary, but experts suggest that between 2.6 million and 5.1 million Americans aged 65 years and older may suffer from the disease, with annual costs estimated to exceed $100 billion”, National Institutes of Health, Alzheimer’s Disease Fact Sheet

  • 19. “Neuroscience currently has powerful tools for perturbing small numbers of specific ‘nodes’ in a neural circuit, for observing small numbers of other ‘nodes,’ and for providing stimuli to an organism and observing its behavior. These tools are powerful for testing hypotheses, but often don’t constrain the hypotheses themselves as much as researchers would wish. Perhaps this is because the relevant hypotheses, in this context, are statements about the architecture of information flow in a network of circuitry that is far larger and more complex than the set of observable/perturbable nodes. Therefore, seeing the connectivity of the circuitry itself can suggest a more targeted hypothesis space, which can then be tested using more traditional approaches.” Synthetic Neurobiology Group, Value Proposition
  • 20.

    See, eg, Marblestone et al. 2014; Marblestone et al. 2014a; Marblestone and Boyden 2014.