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3D Neuroscience at the Speed of Life

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This fluorescent worm makes for much more than a mesmerizing video. It showcases a significant technological leap forward in our ability to capture in real time the firing of individual neurons in a living, freely moving animal.

As this Caenorhabditis elegans worm undulates, 113 neurons throughout its brain and body (green/yellow spots) get brighter and darker as each neuron activates and deactivates. In fact, about halfway through the video, you can see streaks tracking the positions of individual neurons (blue/purple-colored lines) from one frame to the next. Until now, it would have been technologically impossible to capture this “speed of life” with such clarity.

With funding from the NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative, Elizabeth Hillman at Columbia University, New York, has pioneered the pairing of a 3D live-imaging microscope with an ultra-fast camera. This pairing, showcased above, is a technique called Swept Confocally Aligned Planar Excitation (SCAPE) microscopy.

Since first demonstrating SCAPE in February 2015 [1], Hillman and her team have worked hard to improve, refine, and expand the approach. Recently, they used SCAPE 1.0 to image how proprioceptive neurons in fruit-fly larvae sense body position while crawling. Now, as described in Nature Methods, they introduce SCAPE “2.0,” with boosted resolution and a much faster camera—enabling 3D imaging at speeds hundreds of times faster than conventional microscopes [2]. To track a very wiggly worm, the researchers image their target 25 times a second!

As with the first-generation SCAPE, version 2.0 uses a scanning mirror to sweep a slanted sheet of light across a sample. This same mirror redirects light coming from the illuminated plane to focus onto a stationary high-speed camera. The approach lets SCAPE grab 3D imaging at very high speeds, while also causing very little photobleaching compared to conventional point-scanning microscopes, reducing sample damage that often occurs during time-lapse microscopy.

Like SCAPE 1.0, since only a single, stationary objective lens is used, the upgraded 2.0 system doesn’t need to hold, move, or disturb a sample during imaging. This flexibility enables scientists to use SCAPE in a wide range of experiments where they can present stimuli or probe an animal’s behavior—all while imaging how the underlying cells drive and depict those behaviors.

The SCAPE 2.0 paper shows the system’s biological versatility by also recording the beating heart of a zebrafish embryo at record-breaking speeds. In addition, SCAPE 2.0 can rapidly image large fixed, cleared, and expanded tissues such as the retina, brain, and spinal cord—enabling tracing of the shape and connectivity of cellular circuits. Hillman and her team are dedicated to exporting their technology; they provide guidance and a parts list for SCAPE 2.0 so that researchers can build their own version using inexpensive off-the-shelf parts.

Watching worms wriggling around may remind us of middle-school science class. But to neuroscientists, these images represent progress toward understanding the nervous system in action, literally at the speed of life!

References:

[1] . Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Bouchard MB, Voleti V, Mendes CS, Lacefield C, et al Nature Photonics. 2015;9(2):113-119.

[2] Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0. Voleti V, Patel KB, Li W, Campos CP, et al. Nat Methods. 2019 Sept 27;16:1054–1062.

Links:

Using Research Organisms to Study Health and Disease (National Institute of General Medical Sciences/NIH)

The Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)

Hillman Lab (Columbia University, New York)

NIH Support: National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute


Exploring the Universality of Human Song

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Analysis of Music-Internationally

It’s often said that music is a universal language. But is it really universal? Some argue that humans are just too culturally complex and their music is far too varied to expect any foundational similarity. Yet some NIH-funded researchers recently decided to take on the challenge, using the tools of computational social science to analyze recordings of human songs and other types of data gathered from more than 300 societies around the globe.

In a study published in the journal Science [1], the researchers conclude that music is indeed universal. Their analyses showed that all of the cultures studied used song in four similar behavioral contexts: dance, love, healing, and infant care. What’s more, no matter where in the world one goes, songs used in each of those ways were found to share certain musical features, including tone, pitch, and rhythm.

As exciting as the new findings may be for those who love music (like me), the implications may extend far beyond music itself. The work may help to shed new light on the complexities of the human brain, as well as inform efforts to enhance the role of music in improving human health. The healing power of music is a major focus of the NIH-supported Sound Health Initiative.

Samuel Mehr, a researcher at Harvard University, Cambridge, MA, led this latest study, funded in part by an NIH Director’s Early Independence Award. His multi-disciplinary team included anthropologists Manvir Singh, Harvard, and Luke Glowacki, Penn State University, State College; computational linguist Timothy O’Donnell, McGill University, Montreal, Canada; and political scientists Dean Knox, Princeton University, Princeton, NJ, and Christopher Lucas, Washington University, St. Louis.

In work published last year [2], Mehr’s team found that untrained listeners in 60 countries could on average discern the human behavior associated with culturally unfamiliar musical forms. These behaviors included dancing, soothing a baby, seeking to heal illness, or expressing love to another person.

In the latest study, the team took these initial insights and applied them more broadly to the universality of music. They started with the basic question: Do all human societies make music?

To find the answer, the team accessed Yale University’s Human Relations Area Files, an internationally recognized database for cultural anthropologists. This rich resource contains high-quality data for 319 mostly tribal societies across the planet, allowing the researchers to search archival information for mentions of music. Their search pulled up music tags for 309 societies. Digging deeper in other historical records not in the database, the team confirmed that the remaining six societies did indeed make music.

The researchers propose that these 319 societies provide a representative cross section of humanity. They thus conclude that it is statistically probable that music is in fact found in all human societies.

What exactly is so universal about music? To begin answering this complex question, the researchers tapped into more than a century of musicology to build a vast, multi-faceted database that they call the Natural History of Song (NHS).

Drawing from the NHS database, the researchers focused on nearly 5,000 vocally performed songs from 60 carefully selected human societies on all continents. By statistically analyzing those musical descriptions, they found that the behaviors associated with songs vary along three dimensions, which the researchers refer to as formality, arousal, and religiosity.

When the researchers mapped the four types of songs from their earlier study—love, dance, lullaby, and healing—onto these dimensions, they found that songs used in similar behavioral contexts around the world clustered together. For instance, across human societies, dance songs tend to appear in more formal contexts with large numbers of people. They also tend to be upbeat and energetic and don’t usually appear as part of religious ceremonies. In contrast, love songs tend to be more informal and less energetic.

Interestingly, the team also replicated its previous study in a citizen-science experiment with nearly 30,000 participants living in over 100 countries worldwide. They found again that listeners could tell what kinds of songs they were listening to, even when those songs came from faraway places. They went on to show that certain acoustic features of songs, like tempo, melody, and pitch, help to predict a song’s primary behavioral function across societies.

In many musical styles, melodies are composed of a fixed set of distinct tones organized around a tonal center (sometimes called the “tonic,” it’s the “do” in “do-re-mi”). For instance, the researchers explain, the tonal center of “Row Your Boat” is found in each “row” as well as the last “merrily,” and the final “dream.”

Their analyses show that songs with such basic tonal melodies are widespread and perhaps even universal. This suggests that tonality could be a means to delve even deeper into the natural history of world music and other associated behaviors, such as play, mourning, and fighting.

While some aspects of music may be universal, others are quite diverse. That’s particularly true within societies, where people may express different psychological states in song to capture their views of their culture. In fact, Mehr’s team found that the musical variation within a typical society is six times greater for that reason than the musical diversity across societies.

Following up on this work, Mehr’s team is now recruiting families with young infants for a study to understand how they respond to their varied collection of songs. Meanwhile, through the Sound Health Initiative, other research teams around the country are exploring many other ways in which listening to and creating music may influence and improve our health. As a scientist and amateur musician, I couldn’t be more excited to take part in this exceptional time of discovery at the intersection of health, neuroscience, and music.

References:

[1] Universality and diversity in human song. Mehr SA, Singh M, Knox D, Ketter DM, Pickens-Jones D, Atwood S, Lucas C, Jacoby N, Egner AA, Hopkins EJ, Howard RM, Hartshorne JK, Jennings MV, Simson J, Bainbridge CM, Pinker S, O’Donnell TJ, Krasnow MM, Glowacki L. Science. 2019 Nov 22;366(6468).

[2] Form and function in human song. Mehr SA, Singh M, York H, Glowacki L, Krasnow MM. Curr Biol. 2018 Feb 5;28(3):356-368.e5.

Links:

Sound Health Initiative (NIH)

Video: Music and the Mind—A Q & A with Renée Fleming & Francis Collins (YouTube)

The Music Lab (Harvard University, Cambridge, MA)

Samuel Mehr (Harvard)

NIH Director’s Early Independence Award (Common Fund)

NIH Support: Common Fund


What a Memory Looks Like

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Engram Image
Credit: Stephanie Grella, Ramirez Group, Boston University

Your brain has the capacity to store a lifetime of memories, covering everything from the name of your first pet to your latest computer password. But what does a memory actually look like? Thanks to some very cool neuroscience, you are looking at one.

The physical manifestation of a memory, or engram, consists of clusters of brain cells active when a specific memory was formed. Your brain’s hippocampus plays an important role in storing and retrieving these memories. In this cross-section of a mouse hippocampus, imaged by the lab of NIH-supported neuroscientist Steve Ramirez, at Boston University, cells belonging to an engram are green, while blue indicates those not involved in forming the memory.

When a memory is recalled, the cells within an engram reactivate and turn on, to varying degrees, other neural circuits (e.g., sight, sound, smell, emotions) that were active when that memory was recorded. It’s not clear how these brain-wide connections are made. But it appears that engrams are the gatekeepers that mediate memory.

The story of this research dates back several years, when Ramirez helped develop a system that made it possible to image engrams by tagging cells in the mouse brain with fluorescent dyes. Using an innovative technology developed by other researchers, called optogenetics, Ramirez’s team then discovered it could shine light onto a collection of hippocampal neurons storing a specific memory and reactivate the sensation associated with the memory [1].

Ramirez has since gone on to show that, at least in mice, optogenetics can be used to trick the brain into creating a false memory [2]. From this work, he has also come to the interesting and somewhat troubling conclusion that the most accurate memories appear to be the ones that are never recalled. The reason: the mammalian brain edits—and slightly changes—memories whenever they are accessed.

All of the above suggested to Ramirez that, given its tremendous plasticity, the brain may possess the power to downplay a traumatic memory or to boost a pleasant recollection. Toward that end, Ramirez’s team is now using its mouse system to explore ways of suppressing one engram while enhancing another [3].

For Ramirez, though, the ultimate goal is to develop brain-wide maps that chart all of the neural networks involved in recording, storing, and retrieving memories. He recently was awarded an NIH Director’s Transformative Research Award to begin the process. Such maps will be invaluable in determining how stress affects memory, as well as what goes wrong in dementia and other devastating memory disorders.

References:

[1] Optogenetic stimulation of a hippocampal engram activates fear memory recall. Liu X, Ramirez S, Pang PT, Puryear CB, Govindarajan A, Deisseroth K, Tonegawa S. Nature. 2012 Mar 22;484(7394):381-385.

[2] Creating a false memory in the hippocampus. Ramirez S, Liu X, Lin PA, Suh J, Pignatelli M, Redondo RL, Ryan TJ, Tonegawa S. Science. 2013 Jul 26;341(6144):387-391.

[3] Artificially Enhancing and Suppressing Hippocampus-Mediated Memories. Chen BK, Murawski NJ, Cincotta C, McKissick O, Finkelstein A, Hamidi AB, Merfeld E, Doucette E, Grella SL, Shpokayte M, Zaki Y, Fortin A, Ramirez S. Curr Biol. 2019 Jun 3;29(11):1885-1894.

Links:

The Ramirez Group (Boston University, MA)

Ramirez Project Information (Common Fund/NIH)

NIH Director’s Early Independence Award (Common Fund)

NIH Director’s Transformative Research Award (Common Fund)

NIH Support: Common Fund


Defining Neurons in Technicolor

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Brain Architecture
Credit: Allen Institute for Brain Science, Seattle

Can you identify a familiar pattern in this image’s square grid? Yes, it’s the outline of the periodic table! But instead of organizing chemical elements, this periodic table sorts 46 different types of neurons present in the visual cortex of a mouse brain.

Scientists, led by Hongkui Zeng at the Allen Institute for Brain Science, Seattle, constructed this periodic table by assigning colors to their neuronal discoveries based upon their main cell functions [1]. Cells in pinks, violets, reds, and oranges have inhibitory electrical activity, while those in greens and blues have excitatory electrical activity.

For any given cell, the darker colors indicate dendrites, which receive signals from other neurons. The lighter colors indicate axons, which transmit signals. Examples of electrical properties—the number and intensity of their “spikes”—appear along the edges of the table near the bottom.

To create this visually arresting image, Zeng’s NIH-supported team injected dye-containing probes into neurons. The probes are engineered to carry genes that make certain types of neurons glow bright colors under the microscope.

This allowed the researchers to examine a tiny slice of brain tissue and view each colored neuron’s shape, as well as measure its electrical response. They followed up with computational tools to combine these two characteristics and classify cell types based on their shape and electrical activity. Zeng’s team could then sort the cells into clusters using a computer algorithm to avoid potential human bias from visually interpreting the data.

Why compile such a detailed atlas of neuronal subtypes? Although scientists have been surveying cells since the invention of the microscope centuries ago, there is still no consensus on what a “cell type” is. Large, rich datasets like this atlas contain massive amounts of information to characterize individual cells well beyond their appearance under a microscope, helping to explain factors that make cells similar or dissimilar. Those differences may not be apparent to the naked eye.

Just last year, Allen Institute researchers conducted similar work by categorizing nearly 24,000 cells from the brain’s visual and motor cortex into different types based upon their gene activity [2]. The latest research lines up well with the cell subclasses and types categorized in the previous gene-activity work. As a result, the scientists have more evidence that each of the 46 cell types is actually distinct from the others and likely drives a particular function within the visual cortex.

Publicly available resources, like this database of cell types, fuel much more discovery. Scientists all over the world can look at this table (and soon, more atlases from other parts of the brain) to see where a cell type fits into a region of interest and how it might behave in a range of brain conditions.

References:

[1] Classification of electrophysiological and morphological neuron types in the mouse visual cortex. N Gouwens NW, et al. Neurosci. 2019 Jul;22(7):1182-1195.

[2] Shared and distinct transcriptomic cell types across neocortical areas. Tasic B, et al. Nature. 2018 Nov;563(7729):72-78.

Links:

Brain Basics: The Life and Death of a Neuron (National Institute of Neurological Disorders and Stroke/NIH)

Cell Types: Overview of the Data (Allen Brain Atlas/Allen Institute for Brain Science, Seattle)

Hongkui Zeng (Allen Institute)

NIH Support: National Institute of Mental Health; Eunice Kennedy Shriver National Institute of Child Health & Human Development


Gene Therapy Shows Promise Repairing Brain Tissue Damaged by Stroke

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Glial Gene Therapy
Caption: Neurons (red) converted from glial cells using a new NeuroD1-based gene therapy in mice. Credit: Chen Laboratory, Penn State, University Park

It’s a race against time when someone suffers a stroke caused by a blockage of a blood vessel supplying the brain. Unless clot-busting treatment is given within a few hours after symptoms appear, vast numbers of the brain’s neurons die, often leading to paralysis or other disabilities. It would be great to have a way to replace those lost neurons. Thanks to gene therapy, some encouraging strides are now being made.

In a recent study in Molecular Therapy, researchers reported that, in their mouse and rat models of ischemic stroke, gene therapy could actually convert the brain’s support cells into new, fully functional neurons [1]. Even better, after gaining the new neurons, the animals had improved motor and memory skills.

For the team led by Gong Chen, Penn State, University Park, the quest to replace lost neurons in the brain began about a decade ago. While searching for the right approach, Chen noticed other groups had learned to reprogram fibroblasts into stem cells and make replacement neural cells.

As innovative as this work was at the time, it was performed mostly in lab Petri dishes. Chen and his colleagues thought, why not reprogram cells already in the brain?

They turned their attention to the brain’s billions of supportive glial cells. Unlike neurons, glial cells divide and replicate. They also are known to survive and activate following a brain injury, remaining at the wound and ultimately forming a scar. This same process had also been observed in the brain following many types of injury, including stroke and neurodegenerative conditions such as Alzheimer’s disease.

To Chen’s NIH-supported team, it looked like glial cells might be a perfect target for gene therapies to replace lost neurons. As reported about five years ago, the researchers were on the right track [2].

The Chen team showed it was possible to reprogram glial cells in the brain into functional neurons. They succeeded using a genetically engineered retrovirus that delivered a single protein called NeuroD1. It’s a neural transcription factor that switches genes on and off in neural cells and helps to determine their cell fate. The newly generated neurons were also capable of integrating into brain circuits to repair damaged tissue.

There was one major hitch: the NeuroD1 retroviral vector only reprogrammed actively dividing glial cells. That suggested their strategy likely couldn’t generate the large numbers of new cells needed to repair damaged brain tissue following a stroke.

Fast-forward a couple of years, and improved adeno-associated viral vectors (AAV) have emerged as a major alternative to retroviruses for gene therapy applications. This was exactly the breakthrough that the Chen team needed. The AAVs can reprogram glial cells whether they are dividing or not.

In the new study, Chen’s team, led by post-doc Yu-Chen Chen, put this new gene therapy system to work, and the results are quite remarkable. In a mouse model of ischemic stroke, the researchers showed the treatment could regenerate about a third of the total lost neurons by preferentially targeting reactive, scar-forming glial cells. The conversion of those reactive glial cells into neurons also protected another third of the neurons from injury.

Studies in brain slices showed that the replacement neurons were fully functional and appeared to have made the needed neural connections in the brain. Importantly, their studies also showed that the NeuroD1 gene therapy led to marked improvements in the functional recovery of the mice after a stroke.

In fact, several tests of their ability to make fine movements with their forelimbs showed about a 60 percent improvement within 20 to 60 days of receiving the NeuroD1 therapy. Together with study collaborator and NIH grantee Gregory Quirk, University of Puerto Rico, San Juan, they went on to show similar improvements in the ability of rats to recover from stroke-related deficits in memory.

While further study is needed, the findings in rodents offer encouraging evidence that treatments to repair the brain after a stroke or other injury may be on the horizon. In the meantime, the best strategy for limiting the number of neurons lost due to stroke is to recognize the signs and get to a well-equipped hospital or call 911 right away if you or a loved one experience them. Those signs include: sudden numbness or weakness of one side of the body; confusion; difficulty speaking, seeing, or walking; and a sudden, severe headache with unknown causes. Getting treatment for this kind of “brain attack” within four hours of the onset of symptoms can make all the difference in recovery.

References:

[1] A NeuroD1 AAV-Based gene therapy for functional brain repair after ischemic injury through in vivo astrocyte-to-neuron conversion. Chen Y-C et al. Molecular Therapy. Published online September 6, 2019.

[2] In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer’s disease model. Guo Z, Zhang L, Wu Z, Chen Y, Wang F, Chen G. Cell Stem Cell. 2014 Feb 6;14(2):188-202.

Links:

Stroke (National Heart, Lung, and Blood Institute/NIH)

Gene Therapy (National Human Genome Research Institute/NIH)

Chen Lab (Penn State, University Park)

NIH Support: National Institute on Aging; National Institute of Mental Health


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