(Image caption: Calcium imaging of neurons in a rat hippocampal slice through transparent graphene electrode. Black square at the center is transparent graphene electrode and neurons are shown in green. Yellow traces shows a representative example of electrophysiological recordings with graphene electrode. Credit: Hajime Takano and Duygu Kuzum)

See-Through, One-Atom-Thick, Carbon Electrodes are a Powerful Tool for Studying Epilepsy, Other Brain Disorders

Researchers from the Perelman School of Medicine and School of Engineering at the University of Pennsylvania and The Children’s Hospital of Philadelphia have used graphene — a two-dimensional form of carbon only one atom thick — to fabricate a new type of microelectrode that solves a major problem for investigators looking to understand the intricate circuitry of the brain.

Pinning down the details of how individual neural circuits operate in epilepsy and other neurological disorders requires real-time observation of their locations, firing patterns, and other factors, using high-resolution optical imaging and electrophysiological recording. But traditional metallic microelectrodes are opaque and block the clinician’s view and create shadows that can obscure important details. In the past, researchers could obtain either high-resolution optical images or electrophysiological data, but not both at the same time.

The Center for NeuroEngineering and Therapeutics (CNT), under the leadership of senior author Brian Litt, PhD, has solved this problem with the development of a completely transparent graphene microelectrode that allows for simultaneous optical imaging and electrophysiological recordings of neural circuits. Their work was published this week in Nature Communications.

"There are technologies that can give very high spatial resolution such as calcium imaging; there are technologies that can give high temporal resolution, such as electrophysiology, but there’s no single technology that can provide both," says study co-first-author Duygu Kuzum, PhD. Along with co-author Hajime Takano, PhD, and their colleagues, Kuzum notes that the team developed a neuroelectrode technology based on graphene to achieve high spatial and temporal resolution simultaneously. 

Aside from the obvious benefits of its transparency, graphene offers other advantages: “It can act as an anti-corrosive for metal surfaces to eliminate all corrosive electrochemical reactions in tissues,” Kuzum says. “It’s also inherently a low-noise material, which is important in neural recording because we try to get a high signal-to-noise ratio.”          

While previous efforts have been made to construct transparent electrodes using indium tin oxide, they are expensive and highly brittle, making that substance ill-suited for microelectrode arrays. “Another advantage of graphene is that it’s flexible, so we can make very thin, flexible electrodes that can hug the neural tissue,” Kuzum notes.

In the study, Litt, Kuzum, and their colleagues performed calcium imaging of hippocampal slices in a rat model with both confocal and two-photon microscopy, while also conducting electrophysiological recordings. On an individual cell level, they were able to observe temporal details of seizures and seizure-like activity with very high resolution. The team also notes that the single-electrode techniques used in the Nature Communications study could be easily adapted to study other larger areas of the brain with more expansive arrays.

The graphene microelectrodes developed could have wider application. “They can be used in any application that we need to record electrical signals, such as cardiac pacemakers or peripheral nervous system stimulators,” says Kuzum. Because of graphene’s nonmagnetic and anti-corrosive properties, these probes “can also be a very promising technology to increase the longevity of neural implants.” Graphene’s nonmagnetic characteristics also allow for safe, artifact-free MRI reading, unlike metallic implants.

Kuzum emphasizes that the transparent graphene microelectrode technology was achieved through an interdisciplinary effort of CNT and the departments of Neuroscience, Pediatrics, and Materials Science at Penn and the division of Neurology at CHOP.

Ertugrul Cubukcu’s lab at Materials Science and Engineering Department helped with the graphene processing technology used in fabricating flexible transparent neural electrodes, as well as performing optical and materials characterization in collaboration with Euijae Shim and Jason Reed. The simultaneous imaging and recording experiments involving calcium imaging with confocal and two photon microscopy was performed at Douglas Coulter‘s Lab at CHOP with Hajime Takano.  In vivo recording experiments were performed in collaboration with Halvor Juul in Marc Dichter’s Lab. Somatosensory stimulation response experiments were done in collaboration with Timothy Lucas’s Lab, Julius De Vries, and Andrew Richardson.

As the technology is further developed and used, Kuzum and her colleagues expect to gain greater insight into how the physiology of the brain can go awry. “It can provide information on neural circuits, which wasn’t available before, because we didn’t have the technology to probe them,” she says. That information may include the identification of specific marker waveforms of brain electrical activity that can be mapped spatially and temporally to individual neural circuits. “We can also look at other neurological disorders and try to understand the correlation between different neural circuits using this technique,” she says.

New Information about Neurons Could Lead to Advancements in Understanding Brain and Neurological Disorders

Neurons are electrically charged cells, located in the nervous system, that interpret and transmit information using electrical and chemical signals. Now, researchers at the University of Missouri have determined that individual neurons can react differently to electrical signals at the molecular level and in different ways—even among neurons of the same type. This variability may be important in discovering underlying problems associated with brain disorders and neural diseases such as epilepsy.

“Genetic mutations found in neurological disorders create imbalances in the inward and outward flow of electrical current through cells,” said David Schulz, associate professor in the Division of Biological Sciences in the College of Arts and Science and a researcher in the Interdisciplinary Neuroscience Program at MU. “Often, neurons react to electrical signals, or voltage, and compensate by altering their own electrical outputs. The variability in these imbalances, even among multiple cells of the same kind within the brain, is one of the major problems scientists face when trying to design therapeutics for disorders like epilepsy. Seizures in individuals can be caused by different imbalances—therefore getting to the root of how neurons act individually makes our studies important.”

Schulz and his team previously proved that two identical neurons can reach the same electrical activity in different ways. In his new study, Schulz hypothesized that neurons might use the cell’s genetic code, or its messenger RNA (mRNA), to “fine tune” the production of proteins, helping individual cells react accordingly.

Using clusters of neurons obtained from Jonah crabs, Schulz and his team experimentally altered electrical input and output in the neurons and measured the messenger RNA (mRNA) levels found within the cells. Invertebrates like crabs are useful in neuroscience research because their neurons are simple enough to observe and study, but advanced enough that they can be “scaled up” to apply to higher organisms, Schulz said.

They found that when normal patterns of stimulation were maintained, cells engaged the correct ratios of mRNA to produce the proteins needed to help keep electrical impulses in order; however, when normal patterns of activity were not maintained, this fundamentally changed the cells at the molecular level.

“We were the first to show that the correct ratios of mRNAs are actively maintained by the actual activity or voltage of the cell, and not chemical feedback,” Schulz said. “These results represent a novel aspect of regulation that might be useful for developing therapeutics for neuronal disorders later.”

Schulz’ study, “Activity-dependent feedback regulates correlated ion channel mRNA levels in single identified motor neurons,” was published in the August 18^th edition of Current Biology.

MagLab MRI machine provides in-depth analysis of strokes

New research conducted at the Florida State University-based National High Magnetic Field Laboratory has revealed a new, innovative way to classify the severity of a stroke, aid in diagnosis and evaluate potential treatments.

“Stroke affects millions of adults and children worldwide,” said Sam Grant, MagLab researcher and associate professor of chemical and biomedical engineering at the FAMU-FSU College of Engineering. “This research offers a new technique for the chemical analysis of metabolites during stroke and a means of evaluating dynamic changes in cell processes and size in living tissue.”

The research is detailed in two papers, “Metabolic properties in stroked rats revealed by relaxation-enhanced magnetic resonance spectroscopy at ultrahigh fields,” in Nature Communications and “Metabolic T1 dynamics and longitudinal relaxation enhancement in vivo at ultrahigh magnetic fields on ischemia” in the Journal of Cerebral Blood Flow and Metabolism.

The new technique is a way of narrowly applying energy to the metabolites of a specimen exposed to a very high magnetic field. Metabolites are the biological compounds used in the chemical process of breaking down food or other chemicals into energy and producing new materials.

By selectively “exciting” these metabolites and analyzing their distribution and confinement in brain tissue, the research team can investigate the metabolic microenvironment and tell whether cells were shrinking or expanding, a critical tool to understanding the severity of stroke, Grant said.

That information could help medical professionals better treat patients.

“Strokes cause an interruption of blood and oxygen to flow to the brain,” explained Jens Rosenberg, another MagLab researcher and one of Grant’s co-authors. “Through this research, we can see how neurons and other neural cells respond to the disruption of blood flow after stroke and use that information to better understand the full impacts of stroke.”   

The MagLab’s flagship 900 MHz Ultra Widebore NMR magnet system was a critical component to the research. Utilizing this powerful magnet, the research team, which included scientists from the Champaulimod Center in Portugal and the Weizmann Institute of Science in Israel, were able to acquire localized chemical signatures of metabolites from 125-microliter volumes within the brain with high sensitivity and fidelity in six seconds.

Typical MRIs at hospitals or doctor’s offices measure around 1.5 – 3 tesla (the unit of magnetic field strength), while the 900 MHz measures a whopping 21.1 tesla, providing at least seven times the sensitivity.

“This very high field coupled with the RF pulse sequence design by our collaborators and homebuilt RF probes offer a unique non-invasive way of evaluating stroke evolution and potential treatments,” Rosenberg said.

The team also sees exciting possibilities to use this technique to further investigate debilitating diseases.

“By evaluating spectral regions previously undetectable, we hope to fingerprint certain diseases, like ischemic stroke, so that we can identify new characteristics that are specific to pathological conditions at the metabolic level in vivo,” Grant said. “There is a lot of work to be done to identify these dynamic changes and decide when and how our treatments can be most effective.”

Further research on metabolites using this technique could also be used for analysis of neurological disorders such as dementia, schizophrenia, Lou Gehrig’s, Parkinson’s, Alzheimer’s and Huntington’s diseases.

(Image credit)

On the frontiers of cyborg science

No longer just fantastical fodder for sci-fi buffs, cyborg technology is bringing us tangible progress toward real-life electronic skin, prosthetics and ultraflexible circuits. Now taking this human-machine concept to an unprecedented level, pioneering scientists are working on the seamless marriage between electronics and brain signaling with the potential to transform our understanding of how the brain works — and how to treat its most devastating diseases.

Their presentation is taking place at the 248^th National Meeting & Exposition of the American Chemical Society (ACS), the world’s largest scientific society. The meeting features nearly 12,000 presentations on a wide range of science topics and is being held here through Thursday.

“By focusing on the nanoelectronic connections between cells, we can do things no one has done before,” says Charles M. Lieber, Ph.D. “We’re really going into a new size regime for not only the device that records or stimulates cellular activity, but also for the whole circuit. We can make it really look and behave like smart, soft biological material, and integrate it with cells and cellular networks at the whole-tissue level. This could get around a lot of serious health problems in neurodegenerative diseases in the future.”

These disorders, such as Parkinson’s, that involve malfunctioning nerve cells can lead to difficulty with the most mundane and essential movements that most of us take for granted: walking, talking, eating and swallowing.

Scientists are working furiously to get to the bottom of neurological disorders. But they involve the body’s most complex organ — the brain — which is largely inaccessible to detailed, real-time scrutiny. This inability to see what’s happening in the body’s command center hinders the development of effective treatments for diseases that stem from it.

By using nanoelectronics, it could become possible for scientists to peer for the first time inside cells, see what’s going wrong in real time and ideally set them on a functional path again.

For the past several years, Lieber has been working to dramatically shrink cyborg science to a level that’s thousands of times smaller and more flexible than other bioelectronic research efforts. His team has made ultrathin nanowires that can monitor and influence what goes on inside cells. Using these wires, they have built ultraflexible, 3-D mesh scaffolding with hundreds of addressable electronic units, and they have grown living tissue on it. They have also developed the tiniest electronic probe ever that can record even the fastest signaling between cells.

Rapid-fire cell signaling controls all of the body’s movements, including breathing and swallowing, which are affected in some neurodegenerative diseases. And it’s at this level where the promise of Lieber’s most recent work enters the picture.

In one of the lab’s latest directions, Lieber’s team is figuring out how to inject their tiny, ultraflexible electronics into the brain and allow them to become fully integrated with the existing biological web of neurons. They’re currently in the early stages of the project and are working with rat models.

“It’s hard to say where this work will take us,” he says. “But in the end, I believe our unique approach will take us on a path to do something really revolutionary.”

(Image caption: Tracer dye (red) leaked through capillaries (green) in the brains of mice that lacked the gene Mfsd2a, helping to reveal the gene’s role in regulating blood-brain barrier permeability. Credit: Gu Lab)

Breaking Through the Barrier

Like a bouncer at an exclusive nightclub, the blood-brain barrier allows only select molecules to pass from the bloodstream into the fluid that bathes the brain. Vital nutrients get in; toxins and pathogens are blocked. The barrier also ensures that waste products are filtered out of the brain and whisked away.

The blood-brain barrier helps maintain the delicate environment that allows the human brain to thrive. There’s just one problem: The barrier is so discerning, it won’t let medicines pass through. Researchers haven’t been able to coax it to open up because they don’t know enough about how the barrier forms or functions.

Now, a team from Harvard Medical School has identified a gene in mice, Mfsd2a, that may be responsible for limiting the barrier’s permeability—and the molecule it produces, Mfsd2a, works in a way few researchers expected.

“Right now, 98 percent of small-molecule drugs and 100 percent of large-molecule drugs and antibodies can’t get through the blood-brain barrier,” said Chenghua Gu, associate professor of neurobiology at HMS and senior author of the study. “Less than 1 percent of pharmaceuticals even try to target the barrier, because we don’t know what the targets are. Mfsd2a could be one.”

Most attempts to understand and manipulate blood-brain barrier function have focused on tight junctions, seals that prevent all but a few substances from squeezing between barrier cells. Gu and her team discovered that Mfsd2a appears to instead affect a second barrier-crossing mechanism that has received much less attention, transcytosis, a process in which substances are transported through the barrier cells in bubbles called vesicles. Transcytosis occurs frequently at other sites in the body but is normally suppressed at the blood-brain barrier. Mfsd2a may be one of the suppressors.

“It’s exciting because this is the first molecule identified that inhibits transcytosis,” said Gu. “It opens up a new way of thinking about how to design strategies to deliver drugs to the central nervous system.”

Because Mfsd2a has a human equivalent, blocking its activity in people could allow doctors to open the blood-brain barrier briefly and selectively to let in drugs to treat life-threatening conditions such as brain tumors and infections.

Conversely, because researchers have begun to link blood-brain barrier degradation to several brain diseases, boosting Mfsd2a or Mfsd2a could allow doctors to strengthen the barrier and perhaps alleviate diseases such as Alzheimer’s, amyotrophic lateral sclerosis (ALS) and multiple sclerosis. The findings may also have implications for other areas of the body that rely on transcytosis, such as the retina and kidney.

The study was published May 14 in Nature.

Back to the beginning

As developmental biologists, Gu and her colleagues believed watching the barrier develop in young organisms would reveal molecules important for its formation and function.

The team introduced a small amount of dye into the blood of embryonic mice at different stages of development and watched whether it leaked through the walls of the tiny capillaries of the mice’s brains, suggesting that the blood-brain barrier hadn’t formed yet, or stayed contained within the capillaries, indicating that the barrier was doing its job. This allowed them to define a time window during which the barrier was being built.

The team was able to do this by devising a new dye injection technique. Researchers studying blood-brain barrier leakage in adult organisms can inject dye directly into blood vessels, but the capillaries of embryos are too small and delicate. Instead, researchers typically inject dye into the heart. However, according to Gu, this can raise blood pressure and burst brain capillaries, making it difficult to tell whether leakage is due to blood-brain barrier immaturity or the dye procedure itself. She and her team used their vascular biology expertise to identify an alternate injection site that would avoid such artifacts: the liver.

“This allowed us to provide definitive evidence that the blood-brain barrier comes into play during embryonic development,” said Ayal Ben-Zvi, a postdoctoral researcher in the Gu lab and first author of the study. “That changes our understanding of the development of the brain itself.”

Telltale pattern

Now that they knew when the barrier formed in the mice, the team compared endothelial cells—the cells that line blood vessel walls and help form the blood-brain barrier—from peripheral blood vessels and cortical (brain) vessels and looked for differences in gene expression. They made a list of genes that were expressed only in the cortical endothelial cells. From that list, they validated about a dozen in vivo.

The team could have studied any of the genes first, but they were most intrigued by Mfsd2a because of its expression pattern. In addition to being switched on in brain vessels, it was active in the placenta and testis, two other organs that have barrier-type functions. Also, the gene is shared across vertebrate organisms that have blood-brain barriers, including humans.

Gu and the team then conducted experiments in mice that lacked the Mfsd2a gene. They found that without Mfsd2a, the blood-brain barrier leaked (although it didn’t prevent the blood vessels themselves from forming in the first place). The next question was why.

“We focused on two basic characteristics: tight junctions between cells, which prohibit passage of water-soluble molecules, and transcytosis, which happens all the time in peripheral vessels but very little in the cortical vessels,” said Gu. “We found the surprising result that Mfsd2a regulates transcytosis without affecting tight junctions. This is exciting because conceptually it says this previously unappreciated feature may be even more important than tight junctions.”

“At first we were looking at tight junctions, because we were also biased by the field,” said Ben-Zvi, who will be starting his own lab later this year at The Hebrew University of Jerusalem. “We weren’t finding anything on the electron micrographs even though we knew the vessels leaked. Then we noticed there were tons of vesicles.

“It really shows that if you do systematic science and see something strange, you shouldn’t dismiss it, because maybe that’s what you’re looking for.”

Next steps

The team also began to study the relationship between the cortical endothelial cells and another contributor to the blood-brain barrier, cells called pericytes. So far, they have found that pericytes regulate Mfsd2a. Next, they want to learn what exactly the pericytes are telling the endothelial cells to do.

Other future work in the Gu lab includes testing the dozen other potential molecular players and trying to piece together the entire network that regulates transcytosis in the blood-brain barrier.

“In addition to Mfsd2a, there may be several other molecules on the list that will be good drug targets,” said Gu. “The key here is we are gaining tools to manipulate transcytosis either way: opening or tightening.”

As important as the molecules themselves, she added, is the concept.

“I personally hope people in the blood-brain barrier field will consider the mind-shifting paradigm that transcytosis could be targeted or modulated,” said Ben-Zvi.

Better understanding—and potentially being able to manipulate—the molecular underpinnings of transcytosis could aid in the study and treatment of diseases in tissues beyond the brain, from the intestines absorbing nutrients to the kidneys filtering waste.

Being able to open and close the blood-brain barrier also promises to benefit basic research, enabling scientists to investigate how abnormal barrier formation affects brain development and what the relationship may be between barrier deterioration and disease.

Life Stressors Trigger Neurological Disorders

When mothers are exposed to trauma, illness, alcohol or other drug abuse, these stressors may activate a single molecular trigger in brain cells that can go awry and activate conditions such as schizophrenia, post-traumatic stress disorder and some forms of autism.

Until now, it has been unclear how much these stressors have impacted the cells of a developing brain. Past studies have shown that when an expectant mother exposes herself to alcohol or drug abuse or she experiences some trauma or illness, her baby may later develop a psychiatric disorder, including some forms of autism or post-traumatic stress disorder, later in life. But the new findings, published online in Neuron, identifies a molecular mechanism in the prenatal brain that may help explain how cells go awry when exposed to certain environmental conditions.

Kazue Hasimoto-Torii, PhD, Principal Investigator of the Center for Neuroscience, Children’s National Health System, and a Scott-Gentle Foundation investigator, is lead author of the paper. Torii was previously at Yale, whose researchers were co-authors in the report. The research was funded primarily through National Institutes of Health grants.

Researchers found that mouse embryos exposed to alcohol, methyl-mercury, or maternal seizures activate a single gene, HSF1, also known as heat shock factor, in cerebral cortex. The HSF1 “plays a crucial role in the response of brain cells to prenatal environmental insults,” the researchers reported. “The gene protects and enables brain cells to survive prenatal assaults. Mice lacking the HSF1 gene showed structural brain abnormalities and were prone to seizures after birth following exposures to very low levels of toxins.”

Even in mice where the HSF1 gene was properly activated to combat environmental insults, the molecular mechanism alone may permanently change how brain cells respond, and may be a reason why someone may be more susceptible to neuropsychiatric disorders later in life.

Innovative work with stem cells also provided findings that supported the theory that stress induces vulnerable cells to malfunction, the researchers reported. For the study, researchers created stem cells from biopsies of people diagnosed with schizophrenia. Stem cells are capable of becoming many different tissue types, including neurons. In the study, genes from the stem cells of those with schizophrenia responded more dramatically when exposed to environmental insults than stem cells from non-schizophrenic individuals.

While it has been generally accepted that exposure to harmful environmental factors increase the susceptibility of the brain to neurological and psychiatric disorders, new types of environmental agents are continuingly added to the mix, requiring evolving studies, Hasimoto-Torii says.

Hashimoto-Torii notes that autism rates have increased substantially and “more people are having these exposures to environmental stressors,” she says. While there have been many studies that have identified singular stressors, such as alcohol, there have not been enough studies to focus on many different environmental factors and their impacts, such as heavy metals as well as alcohol and other toxic exposure, she adds.

Identifying many risk factors helped Hashimoto-Torii and other researchers identify the gene that may be linked to neurological problems. “Different stressors may have different stress responses,” she says. She examined risk factors specifically involving epilepsy, ADHD, autism and schizophrenia. Eventually, it may open the door “to provide therapy in the future to reduce the risk” and protect vulnerable cells.

Transplanting interneurons: Getting the right mix

Despite early optimistic studies, the promise of curing neurological conditions using transplants remains unfulfilled. While researchers have exhaustively cataloged different types of cells in the brain, and also the largely biochemical issues underlying common diseases, neural repair shops are still a ways off. Fortunately, significant progress is being made towards identifying the broader operant principles that might bear on any one disease work-around. A review just published in Science focuses on recent work on transplanting interneurons—a diverse family of cells united by their mutual love of inhibition and their local loyalty. The UCLA-based authors, reach the conclusion that the fate of transplanted neurons ultimately depends less on the influences of the new host environment, and more on the early upbringing of the cells within the donor embryo.

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Researchers survey protein family that helps the brain form synapses

Neuroscientists and bioengineers at Stanford are working together to solve a mystery: How does nature construct the different types of synapses that connect neurons – the brain cells that monitor nerve impulses, control muscles and form thoughts.

In a paper published in the Proceedings of the National Academy of Sciences, Thomas C. Südhof, M.D., a professor of molecular and cellular physiology, and Stephen R. Quake, a professor of bioengineering, describe the diversity of the neurexin family of proteins.

Neurexins help to create the synapses that connect neurons. Think of synapses as switchboards or control panels that connect specific neurons when these brain cells must work together to perform a given task.

Neurexins play a key role in the formation and functioning of synaptic connections. Past human genetics studies have linked neurexins to a variety of cognitive disorders, such as autism and schizophrenia.

Südhof, the Avram Goldstein Professor in the School of Medicine and a winner of the 2013 Nobel Prize in Medicine, has spent years studying the many different forms, or isoforms, of neurexin proteins. He has postulated that different isoforms of neurexins may help to create different types of synaptic connections with distinct properties and functions, and thus enable neurons to do so many complex tasks.

But Südhof had no way to know exactly how many isoforms of neurexins existed until he sat down last year with Quake, the Lee Otterson Professor in the School of Engineering. Quake has pioneered new ways to sequence DNA – the master blueprint that nature follows when making proteins.

The study being published in PNAS represents the results of a year-long collaboration between neuroscientists and bioengineers to better understand how different neurexin proteins affect the behavior of synapses and, ultimately, normal brain functions and neurological conditions such as autism.

Though this will not be the last word on the subject, the findings help illuminate how the brain works and improve our understanding of neurological disorders.

Inside cells, a molecular machine unzips a double-stranded DNA molecule to create an RNA molecule. The RNA molecule is a copy of all the genetic instructions encoded into the DNA. But only specific regions of this RNA molecule contain instructions for making a specific protein. The cell has ways to remove the unnecessary regions and splice the protein-coding regions into a shorter RNA molecule called messenger RNA or mRNA. Thus, each mRNA contains the full instructions for making a specific protein.

To begin this experiment, Ozgun Gokce, a postdoctoral scholar in molecular and cellular physiology in Südhof’s lab, and Barbara Treutlein, a postdoctoral scholar in Quake’s lab, extracted brain cells from the prefrontal cortex of a mouse, then isolated the RNA contained in this tissue.

From this large pool of RNAs they then identified the mRNAs for neurexins. They ran those messenger molecules through equipment designed to read the entire long sequence of chemical instructions for making a specific isoform in the neurexin family of protein.

Quake’s lab is adept at using new instruments that allow researchers to read the long sequence of chemicals in an mRNA strand, allowing them to ascertain exactly what directions this messenger is carrying to the cell’s protein-making machinery.

“This experiment couldn’t have been done even a few years ago,” Treutlein explained.

The mRNAs for neurexins are very long chains of nucleotides – the chemicals that encode genetic information. Only recently have instruments been capable of reading the exact sequence of such long nucleotide chains.

The ability to read the entire sequence of each mRNA was essential because neurexins have 25 constituent parts. But not all of these parts are used each time neurons produce a copy of the protein. Isoforms of neurexin have different combinations of these 25 possible parts. This experiment was designed to discover how many isoforms of neurexin existed and how prevalent each of these isoforms was.

The researchers analyzed more than 25,000 full-length neurexin mRNAs. They found 450 variants. Each variant omitted one or more of the 25 possible components. Most of these isoforms occurred infrequently. A handful accounted for the predominant isoforms.

Although the Stanford scientists sequenced 25,000 mRNAs to discover 450 variants, they believe that if they were to sequence even more mRNAs they would discover more isoforms – their estimate is that at least 2,500 isoforms of the neurexin family exist.

“The fact that we see so many isoforms supports the theory that these protein variants contribute to the huge diversity of synaptic connections that neuroscientists have observed,” Treutlein said.

The experiment raises many questions for future study. For instance, what functions are performed by the predominant isoforms versus the rare variants; how does the inclusion or exclusion of components affect that isoform and the synapse in which it works?

“This experiment was like a flight over the terrain,” Gokce said. “Now we have to go down and look at the details.”