Gene found to be crucial for formation of certain brain circuitry

Identified using new technique that can speed identification of genes, drug candidates Using a powerful gene-hunting technique for the first time in mammalian brain cells, researchers at Johns Hopkins report they have identified a gene involved in building the circuitry that relays signals through the brain. The gene is a likely player in the aging process in the brain, the researchers say. Additionally, in demonstrating the usefulness of the new method, the discovery paves the way for faster progress toward identifying genes involved in complex mental illnesses such as autism and schizophrenia — as well as potential drugs for such conditions. A summary of the study appears in the Dec. 12 issue of Cell Reports. "We have been looking for a way to sift through large numbers of genes at the same time to see whether they affect processes we're interested in," says Richard Huganir, Ph.D., director of the Johns Hopkins University Solomon H. Snyder Department of Neuroscience and a Howard Hughes Medical Institute investigator, who led the study. "By adapting an automated process to neurons, we were able to go through 800 genes to find one needed for forming synapses — connections — among those cells." Although automated gene-sifting techniques have been used in other areas of biology, Huganir notes, many neuroscience studies instead build on existing knowledge to form a hypothesis about an individual gene's role in the brain. Traditionally, researchers then disable or "knock out" the gene in lab-grown cells or animals to test their hypothesis, a time-consuming and laborious process. In this study, Huganir's group worked to test many genes all at once using plastic plates with dozens of small wells. A robot was used to add precise allotments of cells and nutrients to each well, along with molecules designed to knock out one of the cells' genes — a different one for each well. "The big challenge was getting the neurons, which are very sensitive, to function under these automated conditions," says Kamal Sharma, Ph.D., a research associate in Huganir's group. The team used a trial-and-error approach, adjusting how often the nutrient solution was changed and adding a washing step, and eventually coaxed the cells to thrive in the wells. In addition, Sharma says, they fine-tuned an automated microscope used to take pictures of the circuitry that had formed in the wells and calculated the numbers of synapses formed among the cells. The team screened 800 genes in this way and found big differences in the well of cells with a gene called LRP6 knocked out. LRP6 had previously been identified as a player in a biochemical chain of events known as the Wnt pathway, which controls a range of processes in the brain. Interestingly, Sharma says, the team found that LRP6 was only found on a specific kind of synapse known as an excitatory synapse, suggesting that it enables the Wnt pathway to tailor its effects to just one synapse type. "Changes in excitatory synapses are associated with aging, and changes in the Wnt pathway in later life may accelerate aging in general. However, we do not know what changes take place in the synaptic landscape of the aging brain. Our findings raise intriguing questions: Is the Wnt pathway changing that landscape, and if so, how?" says Sharma. "We're interested in learning more about what other proteins LRP6 interacts with, as well as how it acts in different types of brain cells at different developmental stages of circuit development and refinement." Another likely outcome of the study is wider use of the gene-sifting technique, he says, to explore the genetics of complex mental illnesses. The automated method could also be used to easily test the effects on brain cells of a range of molecules and see which might be drug candidates Continuing Education for Social Workers ### Other authors on the paper are Se-Young Choi, now of Seoul National University School of Dentistry; Yong Zhang, Shunyou Long and Min Li of Johns Hopkins University School of Medicine; and Thomas J.F. Nieland, now of the Broad Institute of Harvard and MIT. This work was supported by grants from the Howard Hughes Medical Institute and the National Institute of Mental Health (grant numbers P50MH084020 and 5U54MH084691). Related stories: Gene Found to Foster Synapse Formation in the Brain http://www.hopkinsmedicine.org/news/media/releases/gene_found_to_foster_synapse_formation_in_the_brain Study Refutes Accepted Model of Memory Formation http://www.hopkinsmedicine.org/news/media/releases/study_refutes_accepted_model_of_memory_formation____ Newly Discovered "Switch" Plays Dual Role in Memory Formation http://m.hopkinsmedicine.org/news/media/releases/newly_discovered_switch_plays_dual_role_in_memory_formation

Brain scans might predict future criminal behavior

Low anterior cingulate activity linked to repeat offenses ALBUQUERQUE, NM and DURHAM, NC--A new study conducted by The Mind Research Network in Albuquerque, N.M., shows that neuroimaging data can predict the likelihood of whether a criminal will reoffend following release from prison. The paper, which is to be published in the Proceedings of the National Academy of Sciences, studied impulsive and antisocial behavior and centered on the anterior cingulate cortex (ACC), a portion of the brain that deals with regulating behavior and impulsivity. You can view the paper by clicking here: http://www.pnas.org/cgi/doi/10.1073/pnas.1219302110. The study demonstrated that inmates with relatively low anterior cingulate activity were twice as likely to reoffend than inmates with high-brain activity in this region. "These findings have incredibly significant ramifications for the future of how our society deals with criminal justice and offenders," said Dr. Kent A. Kiehl, who was senior author on the study and is director of mobile imaging at MRN and an associate professor of psychology at the University of New Mexico. "Not only does this study give us a tool to predict which criminals may reoffend and which ones will not reoffend, it also provides a path forward for steering offenders into more effective targeted therapies to reduce the risk of future criminal activity." The study looked at 96 adult male criminal offenders aged 20-52 who volunteered to participate in research studies. This study population was followed over a period of up to four years after inmates were released from prison. "These results point the way toward a promising method of neuroprediction with great practical potential in the legal system," said Dr. Walter Sinnott-Armstrong, Stillman Professor of Practical Ethics in the Philosophy Department and the Kenan Institute for Ethics at Duke University, who collaborated on the study. "Much more work needs to be done, but this line of research could help to make our criminal justice system more effective." The study used the Mind Research Network's Mobile Magnetic Resonance Imaging (MRI) System to collect neuroimaging data as the inmate volunteers completed a series of mental tests. "People who reoffended were much more likely to have lower activity in the anterior cingulate cortices than those who had higher functioning ACCs," Kiehl said. "This means we can see on an MRI a part of the brain that might not be working correctly -- giving us a look into who is more likely to demonstrate impulsive and anti-social behavior that leads to re-arrest." The anterior cingulate cortex of the brain is "associated with error processing, conflict monitoring, response selection, and avoidance learning," according to the paper. People who have this area of the brain damaged have been "shown to produce changes in disinhibition, apathy, and aggressiveness. Indeed, ACC-damaged patients have been classed in the 'acquired psychopathic personality' genre." Kiehl says he is working on developing treatments that increase activity within the ACC to attempt to treat the high-risk offenders. ### The four-year study was supported by grants from the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and pilot funds by the John D. and Catherine T. MacArthur Foundation Law and Neuroscience Project. The study was conducted in collaboration with the New Mexico Corrections Department. ABOUT THE MIND RESEARCH NETWORK The Mind Research Network (MRN), headquartered in Albuquerque, N.M., is committed to advancing the diagnosis and treatment of mental illness and other brain disorders. MRN is a 501(c)3 non-profit organization consisting of an interdisciplinary association of scientists located at universities, national laboratories and research centers around the world and is focused on imaging technology and its emergence as an integral element of neuroscience investigation. The Mind Research Network is a part of the Lovelace Respiratory Research Institute family of companies Professional Counselor Continuing Education Learn more at http://www.mrn.org

Fat-free See-through Brain Bares All

Method Enables 3-D Analysis of Fine Structure and Connections – NIH-funded Study Slicing optional. Scientists can now study the brain’s finer workings, while preserving its 3-D structure and integrity of its circuitry and other biological machinery. A breakthrough method, called CLARITY, developed by National Institutes of Health-funded researchers, opens the intact postmortem brain to chemical, genetic and optical analyses that previously could only be performed using thin slices of tissue. By replacing fat that normally holds the brain’s working components in place with a clear gel, they made its normally opaque and impenetrable tissue see-through and permeable. This made it possible to image an intact mouse brain in high resolution down to the level of cells and molecules. The technique was even used successfully to study a human brain. “CLARITY has the potential to unmask fine details of brains from people with brain disorders without losing larger-scale circuit perspective,” said NIH Director Francis S. Collins, M.D., Ph.D., whose NIH Director’s Transformative Research Award Program helped to fund the research, along with a grant from the National Institute of Mental Health NIMH. “CLARITY will help support integrative understanding of large-scale, intact biological systems, explained Karl Deisseroth, M.D., Ph.D., of Stanford University in California. “It provides access to subcellular proteins and molecules, while preserving the continuity of intact neuronal structures such as long-range circuit projections, local circuit wiring and cellular spatial relationships.” Deisseroth, Kwanghun Chung, Ph.D., and other Stanford colleagues report on their findings April 10, 2013 in the journal Nature. “This feat of chemical engineering promises to transform the way we study the brain’s anatomy and how disease changes it,” said NIMH Director Thomas R. Insel, M.D. “No longer will the in-depth study of our most important three-dimensional organ be constrained by two-dimensional methods.” Until now, researchers seeking to understand the brain’s fine structure and connections have been faced with tradeoffs. To gain access to deeply buried structures and achieve high enough resolution to study cells, molecules and genes, they had to cut brain tissue into extremely thin sections (each a fraction of a millimeter thick), deforming it. Loss of an intact brain also makes it difficult to relate such micro-level findings to more macro-level information about wiring and circuitry, which cuts across slices. In tackling this challenge, the researchers saw opportunity in the fact that the fats, or lipids, that physically support the brain’s working components, such as neurons and their connections, also block chemical probes and the passage of light. So replacing the lipids with something clear and permeable – that would also hold everything else in place – might make it possible to perform the same tests in an intact brain that previously could only be done with brain tissue slices. Deisseroth’s team infused into brain a high-tech cocktail, including a plastic-like material and formaldehyde. When heated, it formed a transparent, porous gel that biochemically integrated with, and physically supported, the brain’s working tissue – while excluding the lipids, which were safely removed via an electrochemical process. The result was a brain transformed for optimal accessibility. They called the new method Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue Hydrogel – CLARITY, for short. Using CLARITY, the researchers imaged the entire brain of a mouse that had been genetically engineered to express a fluorescent protein. A conventional microscope revealed glowing details, such as proteins embedded in cell membranes and individual nerve fibers, while an electron microscope resolved even ultra-fine structures, such as synapses, the connections between neurons. In a series of experiments using CLARITY in mouse brain, the researchers demonstrated that, for the first time, standard immune- and genetics-based tests can be performed repeatedly in the same intact brain. Tracer molecules, such as antibodies, can be readily delivered for staining tissue – or removed – leaving brain tissue undisturbed. The researchers found that CLARITY outperformed conventional methods across a range of previously problematic technical challenges. When they used CLARITY to analyze a post-mortem human brain of a person who had autism, even though it had been hardening in formaldehyde for six years, they were able to trace individual nerve fibers, neuronal cell bodies and their extensions. Free continuing education course material at Aspira Continuing Education Online Courses

Genetic manipulation boosts growth of brain cells linked to learning, enhances antidepressants

DALLAS -- UT Southwestern Medical Center investigators have identified a genetic manipulation that increases the development of neurons in the brain during aging and enhances the effect of antidepressant drugs.

The research finds that deleting the Nf1 gene in mice results in long-lasting improvements in neurogenesis, which in turn makes those in the test group more sensitive to the effects of antidepressants.

"The significant implication of this work is that enhancing neurogenesis sensitizes mice to antidepressants – meaning they needed lower doses of the drugs to affect 'mood' – and also appears to have anti-depressive and anti-anxiety effects of its own that continue over time," said Dr. Luis Parada, director of the Kent Waldrep Center for Basic Research on Nerve Growth and Regeneration and senior author of the study published in the Journal of Neuroscience.

Just as in people, mice produce new neurons throughout adulthood, although the rate declines with age and stress, said Dr. Parada, chairman of developmental biology at UT Southwestern. Studies have shown that learning, exercise, electroconvulsive therapy and some antidepressants can increase neurogenesis. The steps in the process are well known but the cellular mechanisms behind those steps are not.

"In neurogenesis, stem cells in the brain's hippocampus give rise to neuronal precursor cells that eventually become young neurons, which continue on to become full-fledged neurons that integrate into the brain's synapses," said Dr. Parada, an elected member of the prestigious National Academy of Sciences, its Institute of Medicine, and the American Academy of Arts and Sciences.

The researchers used a sophisticated process to delete the gene that codes for the Nf1 protein only in the brains of mice, while production in other tissues continued normally. After showing that mice lacking Nf1 protein in the brain had greater neurogenesis than controls, the researchers administered behavioral tests designed to mimic situations that would spark a subdued mood or anxiety, such as observing grooming behavior in response to a small splash of sugar water.

The researchers found that the test group mice formed more neurons over time compared to controls, and that young mice lacking the Nf1 protein required much lower amounts of anti-depressants to counteract the effects of stress. Behavioral differences between the groups persisted at three months, six months and nine months. "Older mice lacking the protein responded as if they had been taking antidepressants all their lives," said Dr. Parada.

"In summary, this work suggests that activating neural precursor cells could directly improve depression- and anxiety-like behaviors, and it provides a proof-of-principle regarding the feasibility of regulating behavior via direct manipulation of adult neurogenesis," Dr. Parada said.

Dr. Parada's laboratory has published a series of studies that link the Nf1 gene – best known for mutations that cause tumors to grow around nerves – to wide-ranging effects in several major tissues. For instance, in one study researchers identified ways that the body's immune system promotes the growth of tumors, and in another study, they described how loss of the Nf1 protein in the circulatory system leads to hypertension and congenital heart disease social worker ceus

The current study's lead author is former graduate student Dr. Yun Li, now a postdoctoral researcher at the Massachusetts Institute of Technology. Other co-authors include Yanjiao Li, a research associate of developmental biology, Dr. Renée McKay, assistant professor of developmental biology, both of UT Southwestern, and Dr. Dieter Riethmacher of the University of Southampton in the United Kingdom.

The study was supported by the National Institutes of Health's National Institute of Neurological Disorders and Stroke, and National Institute of Mental Health. Dr. Parada is an American Cancer Society Research Professor.

This news release is available on our World Wide Web home page at www.utsouthwestern.edu/home/news/index.html

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New brain connections form in clusters during learning

Researchers track structural changes during formation of new memories

SANTA CRUZ, CA--New connections between brain cells emerge in clusters in the brain as animals learn to perform a new task, according to a study published in Nature on February 19 (advance online publication). Led by researchers at the University of California, Santa Cruz, the study reveals details of how brain circuits are rewired during the formation of new motor memories ceus for social workers

The researchers studied mice as they learned new behaviors, such as reaching through a slot to get a seed. They observed changes in the motor cortex, the brain layer that controls muscle movements, during the learning process. Specifically, they followed the growth of new "dendritic spines," structures that form the connections (synapses) between nerve cells.

"For the first time we are able to observe the spatial distribution of new synapses related to the encoding of memory," said Yi Zuo, assistant professor of molecular, cell and developmental biology at UC Santa Cruz and corresponding author of the paper.

In a previous study, Zuo and others documented the rapid growth of new dendritic spines on pyramidal neurons in the motor cortex during the learning process. These spines form synapses where the pyramidal neurons receive input from other brain regions involved in motor memories and muscle movements. In the new study, first author Min Fu, a postdoctoral researcher in Zuo's lab, analyzed the spatial distribution of the newly formed synapses.

Initial results of the spatial analysis showed that one third of the newly formed synapses were located next to another new synapse. These clustered synapses tended to form over the course of a few days during the learning period, when the mouse was repeatedly performing the new behavior. Compared to non-clustered counterparts, the clustered synapses were more likely to persist through the learning sessions and after training stopped.

In addition, the researchers found that after formation of the second spine in a cluster, the first spine grew larger. The size of the spine head correlates with the strength of the synapse. "We found that formation of a second connection is correlated with a strengthening of the first connection, which suggests that they are likely to be involved in the same circuitry," Zuo said. "The clustering of synapses may serve to magnify the strength of the connections."

Another part of the study also supported the idea that the clustered synapses are involved in neural circuits specific to the task being learned. The researchers studied mice trained first in one task and then in a different task. Instead of grabbing a seed, the mice had to learn how to handle a piece of capellini pasta. Both tasks induced the formation of clustered spines, but spines formed during the learning of different tasks did not cluster together.

The researchers also looked at mice that were challenged with new motor tasks every day, but did not repeat the same task over and over like the ones trained in seed-grabbing or capellini-handling. These mice also grew lots of new dendritic spines, but few of the new spines were clustered.

"Repetitive activation of the same cortical circuit is really important in learning a new task," Zuo said. "But what is the optimal frequency of repetition? Ultimately, by studying the relationship between synapse formation and learning, we want to find out the best way to induce new memories."

The study used mice that had been genetically altered to make a fluorescent protein within certain neurons in the motor cortex. The researchers used a special microscopy technique (two-photon microscopy) to obtain images of those neurons near the surface of the brain. The noninvasive imaging technique enabled them to view changes in individual brain cells of the mice before, during, and after learning a new behavior.


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In addition to Zuo and first author Min Fu, the coauthors of the paper include UCSC graduate student Xinzhu Yu and Stanford University biologist Ju Lu. This research was supported by grants from the Dana Foundation and the National Institute of Mental Health.