Method offers inexpensive imaging at the scale of virus particles

Using an ordinary light microscope, MIT engineers have devised a technique for imaging biological samples with accuracy at the scale of 10 nanometers — which should enable them to image viruses and potentially even single biomolecules, the researchers say.

The new technique builds on expansion microscopy, an approach that involves embedding biological samples in a hydrogel and then expanding them before imaging them with a microscope. For the latest version of the technique, the researchers developed a new type of hydrogel that maintains a more uniform configuration, allowing for greater accuracy in imaging tiny structures.

This degree of accuracy could open the door to studying the basic molecular interactions that make life possible, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology, a professor of biological engineering and brain and cognitive sciences at MIT, and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.

“If you could see individual molecules and identify what kind they are, with single-digit-nanometer accuracy, then you might be able to actually look at the structure of life.”

“And structure, as a century of modern biology has told us, governs function,” says Boyden, who is the senior author of the new study.

The lead authors of the paper, which appears today in Nature Nanotechnology, are MIT Research Scientist Ruixuan Gao and Chih-Chieh “Jay” Yu PhD ’20. Other authors include Linyi Gao PhD ’20; former MIT postdoc Kiryl Piatkevich; Rachael Neve, director of the Gene Technology Core at Massachusetts General Hospital; James Munro, an associate professor of microbiology and physiological systems at University of Massachusetts Medical School; and Srigokul Upadhyayula, a former assistant professor of pediatrics at Harvard Medical School and an assistant professor in residence of cell and developmental biology at the University of California at Berkeley.

Low cost, high resolution

Many labs around the world have begun using expansion microscopy since Boyden’s lab first introduced it in 2015. With this technique, researchers physically enlarge their samples about fourfold in linear dimension before imaging them, allowing them to generate high-resolution images without expensive equipment. Boyden’s lab has also developed methods for labeling proteins, RNA, and other molecules in a sample so that they can be imaged after expansion.

“Hundreds of groups are doing expansion microscopy. There’s clearly pent-up demand for an easy, inexpensive method of nanoimaging,” Boyden says. “Now the question is, how good can we get? Can we get down to single-molecule accuracy? Because in the end, you want to reach a resolution that gets down to the fundamental building blocks of life.”

Other techniques such as electron microscopy and super-resolution imaging offer high resolution, but the equipment required is expensive and not widely accessible. Expansion microscopy, however, enables high-resolution imaging with an ordinary light microscope.

In a 2017 paper, Boyden’s lab demonstrated resolution of around 20 nanometers, using a process in which samples were expanded twice before imaging. This approach, as well as the earlier versions of expansion microscopy, relies on an absorbent polymer made from sodium polyacrylate, assembled using a method called free radical synthesis. These gels swell when exposed to water; however, one limitation of these gels is that they are not completely uniform in structure or density. This irregularity leads to small distortions in the shape of the sample when it’s expanded, limiting the accuracy that can be achieved.

To overcome this, the researchers developed a new gel called tetra-gel, which forms a more predictable structure. By combining tetrahedral PEG molecules with tetrahedral sodium polyacrylates, the researchers were able to create a lattice-like structure that is much more uniform than the free-radical synthesized sodium polyacrylate hydrogels they previously used.

Three-dimensional (3D) rendered movie of envelope proteins of an herpes simplex virus type 1 (HSV-1) virion expanded by tetra-gel (TG)-based three-round iterative expansion. The deconvolved puncta (white), the overlay of the deconvolved puncta (white) and the fitted centroids (red), and the extracted centroids (red) are shown from left to right. Expansion factor, 38.3×. Scale bars, 100 nm.
Credit: Ruixuan Gao and Boyden Lab

The researchers demonstrated the accuracy of this approach by using it to expand particles of herpes simplex virus type 1 (HSV-1), which have a distinctive spherical shape. After expanding the virus particles, the researchers compared the shapes to the shapes obtained by electron microscopy and found that the distortion was lower than that seen with previous versions of expansion microscopy, allowing them to achieve an accuracy of about 10 nanometers.

“We can look at how the arrangements of these proteins change as they are expanded and evaluate how close they are to the spherical shape. That’s how we validated it and determined how faithfully we can preserve the nanostructure of the shapes and the relative spatial arrangements of these molecules,” Ruixuan Gao says.

Single molecules

The researchers also used their new hydrogel to expand cells, including human kidney cells and mouse brain cells. They are now working on ways to improve the accuracy to the point where they can image individual molecules within such cells. One limitation on this degree of accuracy is the size of the antibodies used to label molecules in the cell, which are about 10 to 20 nanometers long. To image individual molecules, the researchers would likely need to create smaller labels or to add the labels after expansion was complete.

Left, HeLa cell with two-color labeling of clathrin-coated pits/vesicles and microtubules, expanded by TG-based two-round iterative expansion. Expansion factor, 15.6×. Scale bar, 10 μm (156 μm). Right, magnified view of the boxed region for each color channel. Scale bars, 1 μm (15.6 μm). Image: Boyden Lab

They are also exploring whether other types of polymers, or modified versions of the tetra-gel polymer, could help them realize greater accuracy.

If they can achieve accuracy down to single molecules, many new frontiers could be explored, Boyden says. For example, scientists could glimpse how different molecules interact with each other, which could shed light on cell signaling pathways, immune response activation, synaptic communication, drug-target interactions, and many other biological phenomena.

“We’d love to look at regions of a cell, like the synapse between two neurons, or other molecules involved in cell-cell signaling, and to figure out how all the parts talk to each other,” he says. “How do they work together and how do they go wrong in diseases?”

The research was funded by Lisa Yang, John Doerr, Open Philanthropy, the National Institutes of Health, the Howard Hughes Medical Institute Simons Faculty Scholars Program, the Intelligence Advanced Research Projects Activity, the U.S. Army Research Laboratory, the US-Israel Binational Science Foundation, the National Science Foundation, the Friends of the McGovern Fellowship, and the Fellows program of the Image and Data Analysis Core at Harvard Medical School.

A high-resolution glimpse of gene expression in cells

Using a novel technique for expanding tissue, MIT and Harvard Medical School researchers have devised a way to label individual molecules of messenger RNA within a tissue sample and then sequence the RNA.

This approach offers a unique snapshot of which genes are being expressed in different parts of a cell, and could allow scientists to learn much more about how gene expression is influenced by a cell’s location or its interactions with nearby cells. The technique could also be useful for mapping cells in the brain or other tissues and classifying them according to their function.

“Gene expression is one of the most fundamental processes in all of biology, and it plays roles in all biological processes, both healthy and disease-related. However, you need to know more than just whether a gene is on or off,” says Ed Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering, media arts and sciences, and brain and cognitive sciences at MIT.

“You want to know where the gene products are located. You care what cell types they’re in, which individual cells they play roles in, and even which parts of cells they work in,” says Boyden.

In a study appearing today in Science, the researchers showed that they could use this technique to locate and then sequence thousands of different messenger RNA molecules within the mouse brain and in human tumor samples.

The senior authors of the study are Boyden, an investigator at the MIT McGovern Institute and the Howard Hughes Medical Institute; George Church, a professor of genetics at Harvard Medical School; and Adam Marblestone, a former MIT research scientist. The paper’s lead authors are Shahar Alon, a former MIT postdoc who is now a senior lecturer at Bar-Ilan University; Daniel Goodwin, an MIT graduate student; Anubhav Sinha ’14 MNG ’15, an MIT graduate student; Asmamaw Wassie ’12, PhD ’19; and Fei Chen PhD ’17, who is an assistant professor of stem cell and regenerative biology at Harvard University and a member of the Broad Institute of MIT and Harvard.

Tissue expansion

The new sequencing technique builds on a method that Boyden’s group devised in 2015 for expanding tissue samples and then imaging them. By embedding water-absorbent polymers into a tissue sample, researchers can swell the tissue sample while keeping its overall organization intact. Using this approach, tissues can be expanded by a factor of 100 or more, allowing scientists to obtain very high-resolution images of the brain or other tissues using a regular light microscope.

In 2014, Church’s lab developed an RNA sequencing technique known as FISSEQ (fluorescent in situ sequencing), which allows thousands of mRNA molecules to be located and sequenced within cells grown in a lab dish. The Boyden and Church labs decided to join forces to combine tissue expansion and in situ RNA sequencing, creating a new technique they call expansion sequencing (ExSeq).

Expanding the tissue before performing RNA sequencing has two main benefits: It offers a higher-resolution look at the RNA in cells, and it makes it easier to sequence those RNA molecules. “When you separate these molecules in the expanding sample, and move them away from each other, that gives you more room to actually perform the chemical reactions of in situ sequencing,” Marblestone says.

Once the tissue is expanded, the researchers can label and sequence thousands of RNA molecules in a sample, at a resolution that allows them to pinpoint the molecules’ locations not only within cells but within specific compartments such as dendrites — the tiny extensions of neurons that receive communications from other neurons.

“We know that the location of RNA in these small regions is important for learning and memory, but until now, we didn’t have any way to measure these locations because they are very small, on the order of nanometers,” Alon says.

Using an “untargeted” version of this technique, meaning that they are not looking for specific RNA sequences, the researchers can turn up thousands of different sequences. They estimate that in a given sample, they can sequence between 20 and 50 percent of all of the genes present.

In the mouse hippocampus, this technique yielded some surprising results. For one, the researchers found mRNA containing introns, which are sections of RNA that are normally edited out of mRNA in the nucleus, in dendrites. They also discovered mRNA molecules encoding transcription factors in the dendrites, which may help with novel forms of dendrite-to-nucleus communication.

“These are just examples of things that we never would have gone looking for intentionally, but now that we can sequence RNA exactly where it is in the neuron, we’re able to explore a lot more biology,” Goodwin says.

Cellular interactions

The researchers also showed that they could explore gene expression in a more targeted way, looking for a specific set of RNA sequences that correspond to genes of interest. In the visual cortex of the mouse, the researchers used this approach to classify neurons into different types based on an analysis of 42 different genes that they express.

In situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution throughout intact tissues. Top: schematics of physical expansion and in situ sequencing (left), and image analysis (right). Bottom: characterization of nanoscale transcriptomic compartmentalization in mouse hippocampal neuron dendrites and spines (left, middle), and maps of cell types and states in a metastatic human breast cancer biopsy (right). Image courtesy of the researchers.

This technology could also be useful to analyze many other kinds of tissues, such as tumor biopsies. In this paper, the researchers studied breast cancer metastases, which contain many different cell types, including cancer cells and immune cells. The study revealed that these cell types can behave differently depending on their location within a tumor. For example, the researchers found that B cells that were near tumor cells expressed certain inflammatory genes at a higher level than B cells that were farther from tumor cells.

“The tumor microenvironment has been studied in many different contexts for a long time, but it’s been difficult to study it with any depth,” Sinha says. “A cancer biologist can give you a list of 20 or 30 marker genes that will identify most of the cell types in the tissue. Here, since we interrogated 297 different RNA transcripts in the sample, we can ask and answer more detailed questions about gene expression.”

The researchers now plan to further study the interactions between cancer cells and immune cells, as well as gene expression in the brain in healthy and disease states. They also plan to extend their techniques to allow them to map additional types of biomolecules, such as proteins, alongside RNA.

The research was funded, in part, by the National Institutes of Health and the National Science Foundation, as well as by Lisa Yang, John Doerr, the Open Philanthropy Project, Cancer Research UK, the Chan Zuckerberg Initiative Human Cell Atlas pilot program, and HHMI.

Powered by viruses

View the interactive version of this story in our Winter 2021 issue of Brain Scan.

Viruses are notoriously adept invaders. The efficiency with which these unseen threats infiltrate tissues, evade immune systems, and occupy the cells of their hosts can be alarming — but it’s exactly why most McGovern neuroscientists keep a stash of viruses in the freezer.

In the hands of neuroscientists, viruses become vital tools for delivering cargo to cells.

With a bit of genetic manipulation, they can instruct neurons to produce proteins that illuminate complex circuitry, report on activity, or place certain cells under scientists’ control. They can even deliver therapies designed to correct genetic defects in patients.

“We rely on the virus to deliver whatever we want,” says McGovern Investigator Guoping Feng. “This is one of the most important technologies in neuroscience.”

Tracing connections

In Ian Wickersham’s lab, researchers are adapting a virus that, in its natural form, is devastating to the mammalian nervous system. Once it gains access to a neuron, the rabies virus spreads to connected cells, killing them within weeks. “That makes it a very dangerous pathogen, but also a very powerful tool for neuroscience,” says Wickersham, a Principal Research Scientist at the Institute.

Taking advantage of its pernicious spread, neuroscientists use a modified version of the rabies virus to introduce a fluorescent protein to infected cells and visualize their connections (above). As a graduate student in Edward Callaway’s lab at the Salk Institute for Biological Studies, Wickersham figured out how to limit the virus’s passage through the nervous system, allowing it to access cells that are directly connected to the neuron it initially infects, but go no further. Rabies virus travels across synapses in the opposite direction of neuronal signals, so researchers can deliver it to a single cell or set of cells, then see exactly where those cells’ inputs are coming from.

Labs around the world use Wickersham’s modified rabies virus to trace neuronal anatomy in the brains of mice. While his team tinkers to make the virus even more powerful, his collaborators have deployed it to map a variety of essential connections, offering clues into how the brain controls movement, detects odors, and retrieves memories.

With the newest tracing tool from the Wickersham lab, moving from anatomical studies to experiments that reveal circuit function is seamless, because the lab has further modified their virus so that it cannot kill cells. Researchers can label connected cells, then proceed to monitor their signals or manipulate their activity in the same animals.

Researchers usually conduct these experiments in genetically modified mice to control the subset of cells that activate the tracing system. It’s the same approach used to restrict most virally-delivered tools to specific neurons, which is crucial, Feng says. When introducing a fluorescent protein for imaging, for example, “we don’t want the gene we deliver to be activated everywhere, otherwise the whole brain will be lighting up,” he says.

Selective targets

In Feng’s lab, research scientist Martin Wienisch is working to make it easier to control this aspect of delivery. Rather than relying on the genetic makeup of an entire animal to determine where a virally-transported gene is switched on, instructions can be programmed directly into the virus, borrowing regulatory sequences that cells already know how to interpret.

Wienisch is scouring the genomes of individual neurons to identify short segments of regulatory DNA called enhancers. He’s focused on those that selectively activate gene expression in just one of hundreds of different neuron types, particularly in animal models that are not very amenable to genetic engineering. “In the real brain, many elements interact to drive cell specific expression. But amazingly sometimes a single enhancer is all we need to get the same effect,” he says.

Researchers are already using enhancers to confine viral tools to select groups of cells, but Wienisch, who is collaborating with Fenna Krienen in Steve McCarroll’s lab at Harvard University, aims to create a comprehensive library. The enhancers they identify will be paired with a variety of genetically-encoded tools and packaged into adeno-associated viruses (AAV), the most widely used vectors in neuroscience. The Feng lab plans to use these tools to better understand the striatum, a part of the primate brain involved in motivation and behavioral choices. “Ideally, we would have a set of AAVs with enhancers that would give us selective access to all the different cell types in the striatum,” Wienisch says.

Enhancers will also be useful for delivering potential gene therapies to patients, Wienisch says. For many years, the Feng lab has been studying how a missing copy of a gene called Shank3 impairs neurons’ ability to communicate, leading to autism and intellectual disability. Now, they are investigating whether they can overcome these deficits by delivering a functional copy of Shank3 to the brain cells that need it. Widespread activation of the therapeutic gene might do more harm than good, but incorporating the right enhancer could ensure it is delivered to the appropriate cells at the right dose, Wienisch says.

Like most gene therapies in development, the therapeutic Shank3, which is currently being tested in animal models, is packaged into an AAV. AAVs safely and efficiently infect human cells, and by selecting the right type, therapies can be directed to specific cells. But AAVs are small viruses, capable of carrying only small genes. Xian Gao, a postdoctoral researcher in the Feng lab, has pared Shank3 down to its most essential components, creating a “minigene” that can be packaged inside the virus, but some things are difficult to fit inside an AAV. Therapies that aim to correct mutations using the CRISPR gene editing system, for example, often exceed the carrying capacity of an AAV.

Expanding options

“There’s been a lot of really phenomenal advances in our gene editing toolkit,” says Victoria Madigan, a postdoctoral researcher in McGovern Investigator Feng Zhang’s lab, where researchers are developing enzymes to more precisely modify DNA. “One of the main limitations of employing these enzymes clinically has been their delivery.”

To open up new options for gene therapy, Zhang and Madigan are working with a group of viruses called densoviruses. Densoviruses and AAVs belong to the same family, but about 50 percent more DNA can be packed inside the outer shell of some densoviruses.

A molecular model of Galleria mellonella densovirus. Image: Victoria Madigan / Zhang Lab

They are an esoteric group of viruses, Madigan says, infecting only insects and crustaceans and perhaps best known for certain members’ ability to devastate shrimp farms. While densoviruses haven’t received a lot of attention from scientists, their similarities to AAV have given the team clues about how to alter their outer capsids to enable them to enter human cells, and even direct them to particular cell types. The fact that they don’t naturally infect people also makes densoviruses promising candidates for clinical use, Madigan says, because patients’ immune systems are unlikely to be primed to reject them. AAV infections, in contrast, are so common that patients are often excluded from clinical trials for AAV-based therapies due to the presence of neutralizing antibodies against the vector.

Ultimately, densoviruses could enable major advances in gene therapy, making it possible to safely deliver sophisticated gene editing systems to patients’ cells, Madigan says — and that’s good reason for scientists to continue exploring the vast diversity in the viral world. “There’s something to be said for looking into viruses that are understudied as new tools,” she says. “There’s a lot of interesting stuff out there — a lot of diversity and thousands of years of evolution.”

Imaging method reveals a “symphony of cellular activities”

Within a single cell, thousands of molecules, such as proteins, ions, and other signaling molecules, work together to perform all kinds of functions — absorbing nutrients, storing memories, and differentiating into specific tissues, among many others.

Deciphering these molecules, and all of their interactions, is a monumental task. Over the past 20 years, scientists have developed fluorescent reporters they can use to read out the dynamics of individual molecules within cells. However, typically only one or two such signals can be observed at a time, because a microscope cannot distinguish between many fluorescent colors.

MIT researchers have now developed a way to image up to five different molecule types at a time, by measuring each signal from random, distinct locations throughout a cell.

This approach could allow scientists to learn much more about the complex signaling networks that control most cell functions, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering, media arts and sciences, and brain and cognitive sciences at MIT.

“There are thousands of molecules encoded by the genome, and they’re interacting in ways that we don’t understand. Only by watching them at the same time can we understand their relationships,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.

In a new study, Boyden and his colleagues used this technique to identify two populations of neurons that respond to calcium signals in different ways, which may influence how they encode long-term memories, the researchers say.

Boyden is the senior author of the study, which appears today in Cell. The paper’s lead authors are MIT postdoc Changyang Linghu and graduate student Shannon Johnson.

Fluorescent clusters

Shannon Johnson is a graduate fellow in the fellow in the Yang-Tan Center for Molecular Therapeutics.

To make molecular activity visible within a cell, scientists typically create reporters by fusing a protein that senses a target molecule to a protein that glows. “This is similar to how a smoke detector will sense smoke and then flash a light,” says Johnson, who is also a fellow in the Yang-Tan Center for Molecular Therapeutics. The most commonly used glowing protein is green fluorescent protein (GFP), which is based on a molecule originally found in a fluorescent jellyfish.

“Typically a biologist can see one or two colors at the same time on a microscope, and many of the reporters out there are green, because they’re based on the green fluorescent protein,” Boyden says. “What has been lacking until now is the ability to see more than a couple of these signals at once.”

“Just like listening to the sound of a single instrument from an orchestra is far from enough to fully appreciate a symphony,” Linghu says, “by enabling observations of multiple cellular signals at the same time, our technology will help us understand the ‘symphony’ of cellular activities.”

To boost the number of signals they could see, the researchers set out to identify signals by location instead of by color. They modified existing reporters to cause them to accumulate in clusters at different locations within a cell. They did this by adding two small peptides to each reporter, which helped the reporters form distinct clusters within cells.

“It’s like having reporter X be tethered to a LEGO brick, and reporter Z tethered to a K’NEX piece — only LEGO bricks will snap to other LEGO bricks, causing only reporter X to be clustered with more of reporter X,” Johnson says.

Changyang Linghu is the J. Douglas Tan Postdoctoral Fellow in the Hock E. Tan and K. Lisa Yang Center for Autism Research.

With this technique, each cell ends up with hundreds of clusters of fluorescent reporters. After measuring the activity of each cluster under a microscope, based on the changing fluorescence, the researchers can identify which molecule was being measured in each cluster by preserving the cell and staining for peptide tags that are unique to each reporter.  The peptide tags are invisible in the live cell, but they can be stained and seen after the live imaging is done. This allows the researchers to distinguish signals for different molecules even though they may all be fluorescing the same color in the live cell.

Using this approach, the researchers showed that they could see five different molecular signals in a single cell. To demonstrate the potential usefulness of this strategy, they measured the activities of three molecules in parallel — calcium, cyclic AMP, and protein kinase A (PKA). These molecules form a signaling network that is involved with many different cellular functions throughout the body. In neurons, it plays an important role in translating a short-term input (from upstream neurons) into long-term changes such as strengthening the connections between neurons — a process that is necessary for learning and forming new memories.

Applying this imaging technique to pyramidal neurons in the hippocampus, the researchers identified two novel subpopulations with different calcium signaling dynamics. One population showed slow calcium responses. In the other population, neurons had faster calcium responses. The latter population had larger PKA responses. The researchers believe this heightened response may help sustain long-lasting changes in the neurons.

Imaging signaling networks

The researchers now plan to try this approach in living animals so they can study how signaling network activities relate to behavior, and also to expand it to other types of cells, such as immune cells. This technique could also be useful for comparing signaling network patterns between cells from healthy and diseased tissue.

In this paper, the researchers showed they could record five different molecular signals at once, and by modifying their existing strategy, they believe they could get up to 16. With additional work, that number could reach into the hundreds, they say.

“That really might help crack open some of these tough questions about how the parts of a cell work together,” Boyden says. “One might imagine an era when we can watch everything going on in a living cell, or at least the part involved with learning, or with disease, or with the treatment of a disease.”

The research was funded by the Friends of the McGovern Institute Fellowship; the J. Douglas Tan Fellowship; Lisa Yang; the Yang-Tan Center for Molecular Therapeutics; John Doerr; the Open Philanthropy Project; the HHMI-Simons Faculty Scholars Program; the Human Frontier Science Program; the U.S. Army Research Laboratory; the MIT Media Lab; the Picower Institute Innovation Fund; the National Institutes of Health, including an NIH Director’s Pioneer Award; and the National Science Foundation.

Controlling drug activity with light

Hormones and nutrients bind to receptors on cell surfaces by a lock-and-key mechanism that triggers intracellular events linked to that specific receptor. Drugs that mimic natural molecules are widely used to control these intracellular signaling mechanisms for therapy and in research.

In a new publication, a team led by McGovern Institute Associate Investigator Polina Anikeeva and Oregon Health & Science University Research Assistant Professor James Frank introduce a microfiber technology to deliver and activate a drug that can be induced to bind its receptor by exposure to light.

“A significant barrier in applying light-controllable drugs to modulate neural circuits in living animals is the lack of hardware which enables simultaneous delivery of both light and drugs to the target brain area,” says Frank, who was previously a postdoctoral associate in Anikeeva’s Bioelectronics group at MIT. “Our work offers an integrated approach for on-demand delivery of light and drugs through a single fiber.”

These devices were used to deliver a “photoswitchable” drug deep into the brain. So-called “photoswitches” are light-sensitive molecules that can be attached to drugs to switch their activity on or off with a flash of light ­– the use of these drugs is called photopharmacology. In the new study, photopharmacology is used to control neuronal activity and behavior in mice.

Creating miniaturized devices from macroscale templates

The lightweight device features two microfluidic channel and an optical waveguide, and can easily be carried by the animal during behavior

To use light to control drug activity, light and drugs must be delivered simultaneously to the targeted cells. This is a major challenge when the target is deep in the body, but Anikeeva’s Bioelectronics group is uniquely equipped to deal with this challenge.  Marc-Joseph (MJ) Antonini, a PhD student in Anikeeva’s Bioelectronics lab and co-first author of the study, specializes in the fabrication of biocompatible multifunctional fibers that house microfluidic channels and waveguides to deliver liquids and transmit light.

The multifunctional fibers used in this study contain a fluidic channel and an optical waveguide and are comprised of many layers of different materials that are fused together to provide flexibility and strength. The original form of the fiber is constructed at a macroscale and then heated and pulled (a process called thermal drawing) to become longer, but nearly 70X smaller in diameter. By this method, 100’s of meters of miniaturized fiber can be created from the original template at a cross-sectional scale of micrometers that minimizes tissue damage.

The device used in this study had an implantable fiber bundle of 480µm × 380µm and weighed only 0.8 g, small enough that a mouse can easily carry it on its head for many weeks.

Synthesis of a new photoswitchable drug

To demonstrate effectiveness of their device for simultaneous delivery of liquids and light, the Anikeeva lab teamed up with Dirk Trauner (Frank’s former PhD advisor) and David Konrad,  pharmacologists who synthesized photoswitchable drugs.

They had previously modified a photoswitchable analog of capsaicin, a molecule found in hot peppers that binds to the TRPV1 receptor on sensory neurons and controls the sensation of heat. This modification allowed the capsaicin analog to be activated by 560 nm wave-length of light (visible green) that is not damaging to tissue compared to the original version of the drug that required ultraviolet light. By adding both the TRPV1 receptor and the new photoswitchable capsaicin analog to neurons, they could be artificially activated with green light.

This new photopharmacology system had been shown by Frank, Konrad and their colleagues to work in cells cultured in a dish, but had never been shown to work in freely-moving animals.

Controlling behavior by photopharmacology

To test whether their system could activate neurons in the brain, Frank and Antonini tested it in mice. They asked whether adding the photoswitchable drug and its receptor to reward-mediating neurons in the mouse brain causes mice to prefer a chamber in which they receive light stimulation.

The multifunctional fiber-inspired neural implant was implanted into a phantom brain (left), and successfully delivered light and a blue dye (right).

The miniaturized multifunctional fiber developed by the team was implanted in the mouse brain’s ventral tegmental area, a deep region rich in dopamine neurons that controls reward-seeking behavior. Through the fluidic channel in the device, the researchers delivered a virus that drives expression of the TRPV1 receptor in the neurons under study.  Several weeks later, the device was then used to deliver both light and the photoswitchable capsaicin analog directly to the same neurons. To control for the specificity of their system, they also tested the effects of delivering a virus that does not express the TRPV1 receptor, and the effects of delivering a wavelength of light that does not switch on the drug.

They found that mice showed a preference only for the chamber where they had previously received all three components required for the photopharmacology to function: the receptor-expressing virus, the photoswitchable receptor ligand and the green light that activates the drug. These results demonstrate the efficacy of this system to control the time and place within the body that a drug is active.

“Using these fibers to enable photopharmacology in vivo is a great example of how our multifunctional platform can be leveraged to improve and expand how we can interact with the brain,” says Antonini. “This combination of technologies allows us to achieve the temporal and spatial resolution of light stimulation with the chemical specificity of drug injection in freely moving animals.”

Therapeutic drugs that are taken orally or by injection often cause unwanted side-effects because they act continuously and throughout the whole body. Many unwanted side effects could be eliminated by targeting a drug to a specific body tissue and activating it only as needed. The new technology described by Anikeeva and colleagues is one step toward this ultimate goal.

“Our next goal is to use these neural implants to deliver other photoswitchable drugs to target receptors which are naturally expressed within these circuits,” says Frank, whose new lab in the Vollum Institute at OHSU is synthesizing new light-controllable molecules. “The hardware presented in this study will be widely applicable for controlling circuits throughout the brain, enabling neuroscientists to manipulate them with enhanced precision.”

Tool developed in Graybiel lab reveals new clues about Parkinson’s disease

As the brain processes information, electrical charges zip through its circuits and neurotransmitters pass molecular messages from cell to cell. Both forms of communication are vital, but because they are usually studied separately, little is known about how they work together to control our actions, regulate mood, and perform the other functions of a healthy brain.

Neuroscientists in Ann Graybiel’s laboratory at MIT’s McGovern Institute are taking a closer look at the relationship between these electrical and chemical signals. “Considering electrical signals side by side with chemical signals is really important to understand how the brain works,” says Helen Schwerdt, a postdoctoral researcher in Graybiel’s lab. Understanding that relationship is also crucial for developing better ways to diagnose and treat nervous system disorders and mental illness, she says, noting that the drugs used to treat these conditions typically aim to modulate the brain’s chemical signaling, yet studies of brain activity are more likely to focus on electrical signals, which are easier to measure.

Schwerdt and colleagues in Graybiel’s lab have developed new tools so that chemical and electrical signals can, for the first time, be measured simultaneously in the brains of primates. In a study published September 25, 2020, in Science Advances, they used those tools to reveal an unexpectedly complex relationship between two types of signals that are disrupted in patients with Parkinson’s disease—dopamine signaling and coordinated waves of electrical activity known as beta-band oscillations.

Complicated relationship

Graybiel’s team focused its attention on beta-band activity and dopamine signaling because studies of patients with Parkinson’s disease had suggested a straightforward inverse relationship between the two. The tremors, slowness of movement, and other symptoms associated with the disease develop and progress as the brain’s production of the neurotransmitter dopamine declines, and at the same time, beta-band oscillations surge to abnormal levels. Beta-band oscillations are normally observed in parts of the brain that control movement when a person is paying attention or planning to move. It’s not clear what they do or why they are disrupted in patients with Parkinson’s disease. But because patients’ symptoms tend to be worst when beta activity is high—and because beta activity can be measured in real time with sensors placed on the scalp or with a deep-brain stimulation device that has been implanted for treatment, researchers have been hopeful that it might be useful for monitoring the disease’s progression and patients’ response to treatment. In fact, clinical trials are already underway to explore the effectiveness of modulating deep-brain stimulation treatment based on beta activity.

When Schwerdt and colleagues examined these two types of signals in the brains of rhesus macaques, they discovered that the relationship between beta activity and dopamine is more complicated than previously thought.

Their new tools allowed them to simultaneously monitor both signals with extraordinary precision, targeting specific parts of the striatum—a region deep within the brain involved in controlling movement, where dopamine is particularly abundant—and taking measurements on the millisecond time scale to capture neurons’ rapid-fire communications.

They took these measurements as the monkeys performed a simple task, directing their gaze in a particular direction in anticipation of a reward. This allowed the researchers to track chemical and electrical signaling during the active, motivated movement of the animals’ eyes. They found that beta activity did increase as dopamine signaling declined—but only in certain parts of the striatum and during certain tasks. The reward value of a task, an animal’s past experiences, and the particular movement the animal performed all impacted the relationship between the two types of signals.

Multi-modal systems allow subsecond recording of chemical and electrical neural signals in the form of dopamine molecular concentrations and beta-band local field potentials (beta LFPs), respectively. Online measurements of dopamine and beta LFP (time-dependent traces displayed in box on right) were made in the primate striatum (caudate nucleus and putamen colored in green and purple, respectively, in the left brain image) as the animal was performing a task in which eye movements were made to cues displayed on the left (purple event marker line) and right (green event) of a screen in order to receive large or small amounts of food reward (red and blue events). Dopamine and beta LFP neural signals are centrally implicated in Parkinson’s disease and other brain disorders. Image: Helen Schwerdt

“What we expected is there in the overall view, but if we just look at a different level of resolution, all of a sudden the rules don’t hold,” says Graybiel, who is also an MIT Institute Professor. “It doesn’t destroy the likelihood that one would want to have a treatment related to this presumed opposite relationship, but it does say there’s something more here that we haven’t known about.”

The researchers say it’s important to investigate this more nuanced relationship between dopamine signaling and beta activity, and that understanding it more deeply might lead to better treatments for patients with Parkinson’s disease and related disorders. While they plan to continue to examine how the two types of signals relate to one another across different parts of the brain and under different behavioral conditions, they hope that other teams will also take advantage of the tools they have developed. “As these methods in neuroscience become more and more precise and dazzling in their power, we’re bound to discover new things,” says Graybiel.

This study was supported by the National Institute of Biomedical Imaging and Bioengineering, the National Institute of Neurological Disorders and Stroke, the Army Research Office, the Saks Kavanaugh Foundation, the National Science Foundation, Kristin R. Pressman and Jessica J. Pourian ’13 Fund, and Robert Buxton.

New molecular therapeutics center established at MIT’s McGovern Institute

More than one million Americans are diagnosed with a chronic brain disorder each year, yet effective treatments for most complex brain disorders are inadequate or even nonexistent.

A major new research effort at MIT’s McGovern Institute aims to change how we treat brain disorders by developing innovative molecular tools that precisely target dysfunctional genetic, molecular, and circuit pathways.

The K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience was established at MIT through a $28 million gift from philanthropist Lisa Yang and MIT alumnus Hock Tan ’75. Yang is a former investment banker who has devoted much of her time to advocacy for individuals with disabilities and autism spectrum disorders. Tan is President and CEO of Broadcom, a global technology infrastructure company. This latest gift brings Yang and Tan’s total philanthropy to MIT to more than $72 million.

Lisa Yang (center) and MIT alumnus Hock Tan ’75 with their daughter Eva (far left) pictured at the opening of the Hock E. Tan and K. Lisa Yang Center for Autism Research in 2017. Photo: Justin Knight

“In the best MIT spirit, Lisa and Hock have always focused their generosity on insights that lead to real impact,” says MIT President L. Rafael Reif. “Scientifically, we stand at a moment when the tools and insights to make progress against major brain disorders are finally within reach. By accelerating the development of promising treatments, the new center opens the door to a hopeful new future for all those who suffer from these disorders and those who love them. I am deeply grateful to Lisa and Hock for making MIT the home of this pivotal research.”

Engineering with precision

Research at the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience will initially focus on three major lines of investigation: genetic engineering using CRISPR tools, delivery of genetic and molecular cargo across the blood-brain barrier, and the translation of basic research into the clinical setting. The center will serve as a hub for researchers with backgrounds ranging from biological engineering and genetics to computer science and medicine.

“Developing the next generation of molecular therapeutics demands collaboration among researchers with diverse backgrounds,” says Robert Desimone, McGovern Institute Director and Doris and Don Berkey Professor of Neuroscience at MIT. “I am confident that the multidisciplinary expertise convened by this center will revolutionize how we improve our health and fight disease in the coming decade. Although our initial focus will be on the brain and its relationship to the body, many of the new therapies could have other health applications.”

There are an estimated 19,000 to 22,000 genes in the human genome and a third of those genes are active in the brain–the highest proportion of genes expressed in any part of the body.

Variations in genetic code have been linked to many complex brain disorders, including depression and Parkinson’s. Emerging genetic technologies, such as the CRISPR gene editing platform pioneered by McGovern Investigator Feng Zhang, hold great potential in both targeting and fixing these errant genes. But the safe and effective delivery of this genetic cargo to the brain remains a challenge.

Researchers within the new Yang-Tan Center will improve and fine-tune CRISPR gene therapies and develop innovative ways of delivering gene therapy cargo into the brain and other organs. In addition, the center will leverage newly developed single cell analysis technologies that are revealing cellular targets for modulating brain functions with unprecedented precision, opening the door for noninvasive neuromodulation as well as the development of medicines. The center will also focus on developing novel engineering approaches to delivering small molecules and proteins from the bloodstream into the brain. Desimone will direct the center and some of the initial research initiatives will be led by Associate Professor of Materials Science and Engineering Polina Anikeeva; Ed Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT; Guoping Feng, the James W. (1963) and Patricia T. Poitras Professor of Brain and Cognitive Sciences at MIT; and Feng Zhang, James and Patricia Poitras Professor of Neuroscience at MIT.

Building a research hub

“My goal in creating this center is to cement the Cambridge and Boston region as the global epicenter of next-generation therapeutics research. The novel ideas I have seen undertaken at MIT’s McGovern Institute and Broad Institute of MIT and Harvard leave no doubt in my mind that major therapeutic breakthroughs for mental illness, neurodegenerative disease, autism and epilepsy are just around the corner,” says Yang.

Center funding will also be earmarked to create the Y. Eva Tan Fellows program, named for Tan and Yang’s daughter Eva, which will support fellowships for young neuroscientists and engineers eager to design revolutionary treatments for human diseases.

“We want to build a strong pipeline for tomorrow’s scientists and neuroengineers,” explains Hock Tan. “We depend on the next generation of bright young minds to help improve the lives of people suffering from chronic illnesses, and I can think of no better place to provide the very best education and training than MIT.”

The molecular therapeutics center is the second research center established by Yang and Tan at MIT. In 2017, they launched the Hock E. Tan and K. Lisa Yang Center for Autism Research, and, two years later, they created a sister center at Harvard Medical School, with the unique strengths of each institution converging toward a shared goal: understanding the basic biology of autism and how genetic and environmental influences converge to give rise to the condition, then translating those insights into novel treatment approaches.

All tools developed at the molecular therapeutics center will be shared globally with academic and clinical researchers with the goal of bringing one or more novel molecular tools to human clinical trials by 2025.

“We are hopeful that our centers, located in the heart of the Cambridge-Boston biotech ecosystem, will spur further innovation and fuel critical new insights to our understanding of health and disease,” says Yang.


How general anesthesia reduces pain

General anesthesia is medication that suppresses pain and renders patients unconscious during surgery, but whether pain suppression is simply a side effect of loss of consciousness has been unclear. Fan Wang and colleagues have now identified the circuits linked to pain suppression under anesthesia in mouse models, showing that this effect is separable from the unconscious state itself.

“Existing literature suggests that the brain may contain a switch that can turn off pain perception,” explains Fan Wang, a professor at Duke University and lead author of the study. “I had always wanted to find this switch, and it occurred to me that general anesthetics may activate this switch to produce analgesia.”

Wang, who will join the McGovern Institute in January 2021, set out to test this idea with her student, Thuy Hua, and postdoc, Bin Chen.

Pain suppressor

Loss of pain, or analgesia, is an important property of anesthetics that helps to make surgical and invasive medical procedures humane and bearable. In spite of their long use in the medical world, there is still very little understanding of how anesthetics work. It has generally been assumed that a side effect of loss of consciousness is analgesia, but several recent observations have brought this idea into question, and suggest that changes in consciousness might be separable from pain suppression.

A key clue that analgesia is separable from general anesthesia comes from the accounts of patients that regain consciousness during surgery. After surgery, these patients can recount conversations between staff or events that occurred in the operating room, despite not feeling any pain. In addition, some general anesthetics, such as ketamine, can be deployed at low concentrations for pain suppression without loss of consciousness.

Following up on these leads, Wang and colleagues set out to uncover which neural circuits might be involved in suppressing pain during exposure to general anesthetics. Using CANE, a procedure developed by Wang that can detect which neurons activate in response to an event, Wang discovered a new population of GABAergic neurons activated by general anesthetic in the mouse central amygdala.

These neurons become activated in response to different anesthetics, including ketamine, dexmedetomidine, and isoflurane. Using optogenetics to manipulate the activity state of these neurons, Wang and her lab found that they led to marked changes in behavioral responses to painful stimuli.

“The first time we used optogenetics to turn on these cells, a mouse that was in the middle of taking care of an injury simply stopped and started walked around with no sign of pain,” Wang explains.

Specifically, activating these cells blocks pain in multiple models and tests, whereas inhibiting these neurons rendered mice aversive to gentle touch — suggesting that they are involved in a newly uncovered central pain circuit.

The study has implications for both anesthesia and pain. It shows that general anesthetics have complex, multi-faceted effects and that the brain may contain a central pain suppression system.

“We want to figure out how diverse general anesthetics activate these neurons,” explains Wang. “That way we can find compounds that can specifically activate these pain-suppressing neurons without sedation. We’re now also testing whether placebo analgesia works by activating these same central neurons.”

The study also has implications for addiction as it may point to an alternative system for central pain suppression that could be a target of drugs that do not have the devastating side effects of opioids.

Fan Wang joins the McGovern Institute

The McGovern Institute is pleased to announce that Fan Wang, currently a Professor at Duke University, will be joining its team of investigators in 2021. Wang is well-known for her work on sensory perception, pain, and behavior. She takes a broad, and very practical approach to these questions, knowing that sensory perception has broad implications for biomedicine when it comes to pain management, addiction, anesthesia, and hypersensitivity.

“McGovern is a dream place for doing innovative and transformative neuroscience.” – Fan Wang

“I am so thrilled that Fan is coming to the McGovern Institute,” says Robert Desimone, director of the institute and the Doris and Don Berkey Professor of Neuroscience at MIT. “I’ve followed her work for a number of years, and she is making inroads into questions that are relevant to a number of societal problems, such as how we can turn off the perception of chronic pain.”

Wang brings with her a range of techniques developed in her lab, including CANE, which precisely highlights neurons that become activated in response to a stimulus. CANE is highlighting new neuronal subtypes in long-studied brain regions such as the amygdala, and recently elucidated previously undefined neurons in the lateral parabrachial nucleus involved in pain processing.

“I am so excited to join the McGovern Institute,” says Wang. “It is a dream place for doing innovative and transformative neuroscience. McGovern researchers are known for using the most cutting-edge, multi-disciplinary technologies to understand how the brain works. I can’t wait to join the team.”

Wang earned her PhD in 1998 with Richard Axel at Columbia University, subsequently conducting postdoctoral research at Stanford University with Mark Tessier-Lavigne. Wang joined Duke University as a Professor in the Department of Neurobiology in 2003, and was later appointed the Morris N. Broad Distinguished Professor of Neurobiology at Duke University School of Medicine. Wang will join the McGovern Institute as an investigator in January 2021.

A mechanical way to stimulate neurons

In addition to responding to electrical and chemical stimuli, many of the body’s neural cells can also respond to mechanical effects, such as pressure or vibration. But these responses have been more difficult for researchers to study, because there has been no easily controllable method for inducing such mechanical stimulation of the cells. Now, researchers at MIT and elsewhere have found a new method for doing just that.

The finding might offer a step toward new kinds of therapeutic treatments, similar to electrically based neurostimulation that has been used to treat Parkinson’s disease and other conditions. Unlike those systems, which require an external wire connection, the new system would be completely contact-free after an initial injection of particles, and could be reactivated at will through an externally applied magnetic field.

The finding is reported in the journal ACS Nano, in a paper by former MIT postdoc Danijela Gregurec, Alexander Senko PhD ’19, Associate Professor Polina Anikeeva, and nine others at MIT, at Boston’s Brigham and Women’s Hospital, and in Spain.

The new method opens a new pathway for the stimulation of nerve cells within the body, which has so far almost entirely relied on either chemical pathways, through the use of pharmaceuticals, or on electrical pathways, which require invasive wires to deliver voltage into the body. This mechanical stimulation, which activates entirely different signaling pathways within the neurons themselves, could provide a significant area of study, the researchers say.

“An interesting thing about the nervous system is that neurons can actually detect forces,” Senko says. “That’s how your sense of touch works, and also your sense of hearing and balance.” The team targeted a particular group of neurons within a structure known as the dorsal root ganglion, which forms an interface between the central and peripheral nervous systems, because these cells are particularly sensitive to mechanical forces.

The applications of the technique could be similar to those being developed in the field of bioelectronic medicines, Senko says, but those require electrodes that are typically much bigger and stiffer than the neurons being stimulated, limiting their precision and sometimes damaging cells.

The key to the new process was developing minuscule discs with an unusual magnetic property, which can cause them to start fluttering when subjected to a certain kind of varying magnetic field. Though the particles themselves are only 100 or so nanometers across, roughly a hundredth of the size of the neurons they are trying to stimulate, they can be made and injected in great quantities, so that collectively their effect is strong enough to activate the cell’s pressure receptors. “We made nanoparticles that actually produce forces that cells can detect and respond to,” Senko says.

Anikeeva says that conventional magnetic nanoparticles would have required impractically large magnetic fields to be activated, so finding materials that could provide sufficient force with just moderate magnetic activation was “a very hard problem.” The solution proved to be a new kind of magnetic nanodiscs.

These discs, which are hundreds of nanometers in diameter, contain a vortex configuration of atomic spins when there are no external magnetic fields applied. This makes the particles behave as if they were not magnetic at all, making them exceptionally stable in solutions. When these discs are subjected to a very weak varying magnetic field of a few millitesla, with a low frequency of just several hertz, they switch to a state where the internal spins are all aligned in the disc plane. This allows these nanodiscs to act as levers — wiggling up and down with the direction of the field.

Anikeeva, who is an associate professor in the departments of Materials Science and Engineering and Brain and Cognitive Sciences, says this work combines several disciplines, including new chemistry that led to development of these nanodiscs, along with electromagnetic effects and work on the biology of neurostimulation.

The team first considered using particles of a magnetic metal alloy that could provide the necessary forces, but these were not biocompatible materials, and they were prohibitively expensive. The researchers found a way to use particles made from hematite, a benign iron oxide, which can form the required disc shapes. The hematite was then converted into magnetite, which has the magnetic properties they needed and is known to be benign in the body. This chemical transformation from hematite to magnetite dramatically turns a blood-red tube of particles to jet black.

“We had to confirm that these particles indeed supported this really unusual spin state, this vortex,” Gregurec says. They first tried out the newly developed nanoparticles and proved, using holographic imaging systems provided by colleagues in Spain, that the particles really did react as expected, providing the necessary forces to elicit responses from neurons. The results came in late December and “everyone thought that was a Christmas present,” Anikeeva recalls, “when we got our first holograms, and we could really see that what we have theoretically predicted and chemically suspected actually was physically true.”

The work is still in its infancy, she says. “This is a very first demonstration that it is possible to use these particles to transduce large forces to membranes of neurons in order to stimulate them.”

She adds “that opens an entire field of possibilities. … This means that anywhere in the nervous system where cells are sensitive to mechanical forces, and that’s essentially any organ, we can now modulate the function of that organ.” That brings science a step closer, she says, to the goal of bioelectronic medicine that can provide stimulation at the level of individual organs or parts of the body, without the need for drugs or electrodes.

The work was supported by the U.S. Defense Advanced Research Projects Agency, the National Institute of Mental Health, the Department of Defense, the Air Force Office of Scientific Research, and the National Defense Science and Engineering Graduate Fellowship.

Full paper at ACS Nano