Posted on by Dr. Francis Collins
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 . 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 . 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.
 Classification of electrophysiological and morphological neuron types in the mouse visual cortex. N Gouwens NW, et al. Neurosci. 2019 Jul;22(7):1182-1195.
 Shared and distinct transcriptomic cell types across neocortical areas. Tasic B, et al. Nature. 2018 Nov;563(7729):72-78.
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
Posted on by Dr. Francis Collins
The NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative is revolutionizing our understanding of how the brain works through its creation of new imaging tools. One of the latest advances—used to produce this rainbow of images—makes it possible to view dozens of proteins in rapid succession in a single tissue sample containing thousands of neural connections, or synapses.
Apart from their colors, most of these images look nearly identical at first glance. But, upon closer inspection, you’ll see some subtle differences among them in both intensity and pattern. That’s because the images capture different proteins within the complex network of synapses—and those proteins may be present in that network in different amounts and locations. Such findings may shed light on key differences among synapses, as well as provide new clues into the roles that synaptic proteins may play in schizophrenia and various other neurological disorders.
Synapses contain hundreds of proteins that regulate the release of chemicals called neurotransmitters, which allow neurons to communicate. Each synaptic protein has its own specific job in the process. But there have been longstanding technical difficulties in observing synaptic proteins at work. Conventional fluorescence microscopy can visualize at most four proteins in a synapse.
As described in Nature Communications , researchers led by Mark Bathe, Massachusetts Institute of Technology (MIT), Cambridge, and Jeffrey Cottrell, Broad Institute of MIT and Harvard, Cambridge, have just upped this number considerably while delivering high quality images. They did it by adapting an existing imaging method called DNA PAINT . The researchers call their adapted method PRISM. It is short for: Probe-based Imaging for Sequential Multiplexing.
Here’s how it works: First, researchers label proteins or other molecules of interest using antibodies that recognize those proteins. Those antibodies include a unique DNA probe that helps with the next important step: making the proteins visible under a microscope.
To do it, they deliver short snippets of complementary fluorescent DNA, which bind the DNA-antibody probes. While each protein of interest is imaged separately, researchers can easily wash the probes from a sample to allow a series of images to be generated, each capturing a different protein of interest.
In the original DNA PAINT, the DNA strands bind and unbind periodically to create a blinking fluorescence that can be captured using super-resolution microscopy. But that makes the process slow, requiring about half an hour for each protein.
To speed things up with PRISM, Bathe and his colleagues altered the fluorescent DNA probes. They used synthetic DNA that’s specially designed to bind more tightly or “lock” to the DNA-antibody. This gives a much brighter signal without the blinking effect. As a result, the imaging can be done faster, though at slightly lower resolution.
Though the team now captures images of 12 proteins within a sample in about an hour, this is just a start. As more DNA-antibody probes are developed for synaptic proteins, the team can readily ramp up this number to 30 protein targets.
Thanks to the BRAIN Initiative, researchers now possess a powerful new tool to study neurons. PRISM will help them learn more mechanistically about the inner workings of synapses and how they contribute to a range of neurological conditions.
 Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes. Guo SM, Veneziano R, Gordonov S, Li L, Danielson E, Perez de Arce K, Park D, Kulesa AB, Wamhoff EC, Blainey PC, Boyden ES, Cottrell JR, Bathe M. Nat Commun. 2019 Sep 26;10(1):4377.
 Super-resolution microscopy with DNA-PAINT. Schnitzbauer J, Strauss MT, Schlichthaerle T, Schueder F, Jungmann R. Nat Protoc. 2017 Jun;12(6):1198-1228.
Schizophrenia (National Institute of Mental Health)
Mark Bathe (Massachusetts Institute of Technology, Cambridge)
Jeffrey Cottrell (Broad Institute of MIT and Harvard, Cambridge)
NIH Support: National Institute of Mental Health; National Human Genome Research Institute; National Institute of Neurological Disorders and Stroke; National Institute of Environmental Health Sciences
Posted on by Dr. Francis Collins
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 . 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 .
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.
 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.
 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.
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
Posted on by Dr. Francis Collins
A major aim of the NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative is to develop new technologies that allow us to look at the brain in many different ways on many different scales. So, I’m especially pleased to highlight this winner of the initiative’s recent “Show Us Your Brain!” contest.
Here you get a close-up look at pyramidal neurons located in the hippocampus, a region of the mammalian brain involved in memory. While this tiny sample of mouse brain is densely packed with many pyramidal neurons, researchers used new ExLLSM technology to zero in on just three. This super-resolution, 3D view reveals the intricacies of each cell’s structure and branching patterns.
The group that created this award-winning visual includes the labs of X. William Yang at the University of California, Los Angeles, and Kwanghun Chung at the Massachusetts Institute of Technology, Cambridge. Chung’s team also produced another quite different “Show Us Your Brain!” winner, a colorful video featuring hundreds of neural cells and connections in a part of the brain essential to movement.
Pyramidal neurons in the hippocampus come in many different varieties. Some important differences in their functional roles may be related to differences in their physical shapes, in ways that aren’t yet well understood. So, BRAIN-supported researchers are now applying a variety of new tools and approaches in a more detailed effort to identify and characterize these neurons and their subtypes.
The video featured here took advantage of Chung’s new method for preserving brain tissue samples . Another secret to its powerful imagery was a novel suite of mouse models developed in the Yang lab. With some sophisticated genetics, these models make it possible to label, at random, just 1 to 5 percent of a given neuronal cell type, illuminating their full morphology in the brain . The result was this unprecedented view of three pyramidal neurons in exquisite 3D detail.
Ultimately, the goal of these and other BRAIN Initiative researchers is to produce a dynamic picture of the brain that, for the first time, shows how individual cells and complex neural circuits interact in both time and space. I look forward to their continued progress, which promises to revolutionize our understanding of how the human brain functions in both health and disease.
 Protection of tissue physicochemical properties using polyfunctional crosslinkers. Park YG, Sohn CH, Chen R, McCue M, Yun DH, Drummond GT, Ku T, Evans NB, Oak HC, Trieu W, Choi H, Jin X, Lilascharoen V, Wang J, Truttmann MC, Qi HW, Ploegh HL, Golub TR, Chen SC, Frosch MP, Kulik HJ, Lim BK, Chung K. Nat Biotechnol. 2018 Dec 17.
 Genetically-directed Sparse Neuronal Labeling in BAC Transgenic Mice through Mononucleotide Repeat Frameshift. Lu XH, Yang XW. Sci Rep. 2017 Mar 8;7:43915.
Chung Lab (Massachusetts Institute of Technology, Cambridge)
Yang Lab (University of California, Los Angeles)
Show Us Your Brain! (BRAIN Initiative/NIH)
NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute of Biomedical Imaging and Bioengineering
Posted on by Dr. Francis Collins
Wow! Click on the video. If you’ve ever wondered where those pesky flies in your fruit bowl come from, you’re looking at it right now. It’s a fruit fly larva. And this 3D movie offers never-before-seen details into proprioception—the brain’s sixth sense of knowing the body’s location relative to nearby objects or, in this case, fruit.
This live-action video highlights the movement of the young fly’s proprioceptive nerve cells. They send signals to the fly brain that are essential for tracking the body’s position in space and coordinating movement. The colors indicate the depth of the nerve cells inside the body, showing those at the surface (orange) and those further within (blue).
Such movies make it possible, for the first time, to record precisely how every one of these sensory cells is arranged within the body. They also provide a unique window into how body positions are dynamically encoded in these cells, as a segmented larva inches along in search of food.
The video was created using a form of confocal microscopy called Swept Confocally Aligned Planar Excitation, or SCAPE. It captures 3D images by sweeping a sheet of laser light back and forth across a living sample. Even better, it does this while the microscope remains completely stationary—no need for a researcher to move any lenses up or down, or hold a live sample still.
Most impressively, with this new high-speed technology, developed with support from the NIH’s BRAIN Initiative, researchers are now able to capture videos like the one seen above in record time, with each whole volume recorded in under 1/10th of a second! That’s hundreds of times faster than with a conventional microscope, which scans objects point by point.
As reported in Current Biology, the team, led by Elizabeth Hillman and Wesley Grueber, Columbia University, New York, didn’t stop at characterizing the structural details and physical movements of nerve cells involved in proprioception in a crawling larva. In another set of imaging experiments, they went a step further, capturing faint flashes of green in individual labeled nerve cells each time they fired. (You have to look very closely to see them.) With each wave of motion, proprioceptive nerve cells light up in sequence, demonstrating precisely when they are sending signals to the animal’s brain.
From such videos, the researchers have generated a huge amount of data on the position and activity of each proprioceptive nerve cell. The data show that the specific position of each cell makes it uniquely sensitive to changes in position of particular segments of a larva’s body. While most of the proprioceptive nerve cells fired when their respective body segment contracted, others were attuned to fire when a larval segment stretched.
Taken together, the data show that proprioceptive nerve cells provide the brain with a detailed sequence of signals, reflecting each part of a young fly’s undulating body. It’s clear that every proprioceptive neuron has a unique role to play in the process. The researchers now will create similar movies capturing neurons in the fly’s central nervous system.
A holy grail of the BRAIN Initiative is to capture the brain in action. With these advances in imaging larval flies, researchers are getting ever closer to understanding the coordinated activities of an organism’s complete nervous system—though this one is a lot simpler than ours! And perhaps this movie—and the anticipation of the sequels to come—may even inspire a newfound appreciation for those pesky flies that sometimes hover nearby.
 Characterization of Proprioceptive System Dynamics in Behaving Drosophila Larvae Using High-Speed Volumetric Microscopy. Vaadia RD, Li W, Voleti V, Singhania A, Hillman EMC, Grueber WB. Curr Biol. 2019 Mar 18;29(6):935-944.e4.
Using Research Organisms to Study Health and Disease (National Institute of General Medical Sciences/NIH)
Hillman Lab (Columbia University, New York)
Grueber Lab (Columbia University, New York)
NIH Support: National Institute of Neurological Disorders and Stroke; Eunice Kennedy Shriver National Institute of Child Health and Human Development