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Mapping the brain at high resolution

Posted: 17 Jan 2019 11:00 AM PST

Researchers have developed a new way to image the brain with unprecedented resolution and speed. Using this approach, they can locate individual neurons, trace connections between them, and visualize organelles inside neurons, over large volumes of brain tissue.

The new technology combines a method for expanding brain tissue, making it possible to image at higher resolution, with a rapid 3-D microscopy technique known as lattice light-sheet microscopy. In a paper appearing in Science Jan. 17, the researchers showed that they could use these techniques to image the entire fruit fly brain, as well as large sections of the mouse brain, much faster than has previously been possible. The team includes researchers from MIT, the University of California at Berkeley, the Howard Hughes Medical Institute, and Harvard Medical School/Boston Children's Hospital.

This technique allows researchers to map large-scale circuits within the brain while also offering unique insight into individual neurons' functions, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology, an associate professor of biological engineering and of brain and cognitive sciences at MIT, and a member of MIT's McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research.

"A lot of problems in biology are multiscale," Boyden says. "Using lattice light-sheet microscopy, along with the expansion microscopy process, we can now image at large scale without losing sight of the nanoscale configuration of biomolecules."

Boyden is one of the study's senior authors, along with Eric Betzig, a senior fellow at the Janelia Research Campus and a professor of physics and molecular and cell biology at UC Berkeley. The paper's lead authors are MIT postdoc Ruixuan Gao, former MIT postdoc Shoh Asano, and Harvard Medical School Assistant Professor Srigokul Upadhyayula.

Large-scale imaging

In 2015, Boyden's lab developed a way to generate very high-resolution images of brain tissue using an ordinary light microscope. Their technique relies on expanding tissue before imaging it, allowing them to image the tissue at a resolution of about 60 nanometers. Previously, this kind of imaging could be achieved only with very expensive high-resolution microscopes, known as super-resolution microscopes.

In the new study, Boyden teamed up with Betzig and his colleagues at HHMI's Janelia Research Campus to combine expansion microscopy with lattice light-sheet microscopy. This technology, which Betzig developed several years ago, has some key traits that make it ideal to pair with expansion microscopy: It can image large samples rapidly, and it induces much less photodamage than other fluorescent microscopy techniques.

"The marrying of the lattice light-sheet microscope with expansion microscopy is essential to achieve the sensitivity, resolution, and scalability of the imaging that we're doing," Gao says.

Imaging expanded tissue samples generates huge amounts of data — up to tens of terabytes per sample — so the researchers also had to devise highly parallelized computational image-processing techniques that could break down the data into smaller chunks, analyze it, and stitch it back together into a coherent whole.

In the Science paper, the researchers demonstrated the power of their new technique by imaging layers of neurons in the somatosensory cortex of mice, after expanding the tissue volume fourfold. They focused on a type of neuron known as pyramidal cells, one of the most common excitatory neurons found in the nervous system. To locate synapses, or connections, between these neurons, they labeled proteins found in the presynaptic and postsynaptic regions of the cells. This also allowed them to compare the density of synapses in different parts of the cortex.

MIT researchers have developed a method to perform large-scale, 3D imaging of brain tissue.  Here, they image the entire fruit fly brain.

Using this technique, it is possible to analyze millions of synapses in just a few days.

"We counted clusters of postsynaptic markers across the cortex, and we saw differences in synaptic density in different layers of the cortex," Gao says. "Using electron microscopy, this would have taken years to complete."

The researchers also studied patterns of axon myelination in different neurons. Myelin is a fatty substance that insulates axons and whose disruption is a hallmark of multiple sclerosis. The researchers were able to compute the thickness of the myelin coating in different segments of axons, and they measured the gaps between stretches of myelin, which are important because they help conduct electrical signals. Previously, this kind of myelin tracing would have required months to years for human annotators to perform.

This technology can also be used to image tiny organelles inside neurons. In the new paper, the researchers identified mitochondria and lysosomes, and they also measured variations in the shapes of these organelles.

Circuit analysis

The researchers demonstrated that this technique could be used to analyze brain tissue from other organisms as well; they used it to image the entire brain of the fruit fly, which is the size of a poppy seed and contains about 100,000 neurons. In one set of experiments, they traced an olfactory circuit that extends across several brain regions, imaged all dopaminergic neurons, and counted all synapses across the brain. By comparing multiple animals, they also found differences in the numbers and arrangements of synaptic boutons within each animal's olfactory circuit.

In future work, Boyden envisions that this technique could be used to trace circuits that control memory formation and recall, to study how sensory input leads to a specific behavior, or to analyze how emotions are coupled to decision-making.

"These are all questions at a scale that you can't answer with classical technologies," he says.

The system could also have applications beyond neuroscience, Boyden says. His lab is planning to work with other researchers to study how HIV evades the immune system, and the technology could also be adapted to study how cancer cells interact with surrounding cells, including immune cells.

The research was funded by John Doerr, the Open Philanthropy Project, the National Institutes of Health, the Howard Hughes Medical Institute, the HHMI-Simons Faculty Scholars Program, the U.S. Army Research Laboratory and Army Research Office, the US-Israel Binational Science Foundation, Biogen, and Ionis Pharmaceuticals.

Study shows how specific gene variants may raise bipolar disorder risk

Posted: 17 Jan 2019 09:30 AM PST

A new study by researchers at the Picower Institute for Learning and Memory at MIT finds that the protein CPG2 is significantly less abundant in the brains of people with bipolar disorder (BD) and shows how specific mutations in the SYNE1 gene that encodes the protein undermine its expression and its function in neurons.

Led by Elly Nedivi, professor in MIT's departments of Biology and Brain and Cognitive Sciences, and former postdoc Mette Rathje, the study goes beyond merely reporting associations between genetic variations and psychiatric disease. Instead, the team's analysis and experiments show how a set of genetic differences in patients with bipolar disorder can lead to specific physiological dysfunction for neural circuit connections, or synapses, in the brain.

The mechanistic detail and specificity of the findings provide new and potentially important information for developing novel treatment strategies and for improving diagnostics, Nedivi says.

"It's a rare situation where people have been able to link mutations genetically associated with increased risk of a mental health disorder to the underlying cellular dysfunction," says Nedivi, senior author of the study online in Molecular Psychiatry. "For bipolar disorder this might be the one and only."

The researchers are not suggesting that the CPG2-related variations in SYNE1 are "the cause" of bipolar disorder, but rather that they likely contribute significantly to susceptibility to the disease. Notably, they found that sometimes combinations of the variants, rather than single genetic differences, were required for significant dysfunction to become apparent in laboratory models.

"Our data fit a genetic architecture of BD, likely involving clusters of both regulatory and protein-coding variants, whose combined contribution to phenotype is an important piece of a puzzle containing other risk and protective factors influencing BD susceptibility," the authors wrote.

CPG2 in the bipolar brain

During years of fundamental studies of synapses, Nedivi discovered CPG2, a protein expressed in response to neural activity, that helps regulate the number of receptors for the neurotransmitter glutamate at excitatory synapses. Regulation of glutamate receptor numbers is a key mechanism for modulating the strength of connections in brain circuits. When genetic studies identified SYNE1 as a risk gene specific to bipolar disorder, Nedivi's team recognized the opportunity to shed light into the cellular mechanisms of this devastating neuropsychiatric disorder typified by recurring episodes of mania and depression.

For the new study, Rathje led the charge to investigate how CPG2 may be different in people with the disease. To do that, she collected samples of postmortem brain tissue from six brain banks. The samples included tissue from people who had been diagnosed with bipolar disorder, people who had neuropsychiatric disorders with comorbid symptoms such as depression or schizophrenia, and people who did not have any of those illnesses. Only in samples from people with bipolar disorder was CPG2 significantly lower. Other key synaptic proteins were not uniquely lower in bipolar patients.

"Our findings show a specific correlation between low CPG2 levels and incidence of BD that is not shared with schizophrenia or major depression patients," the authors wrote.

From there they used deep-sequencing techniques on the same brain samples to look for genetic variations in the SYNE1 regions of BD patients with reduced CPG2 levels. They specifically looked at ones located in regions of the gene that could regulate expression of CPG2 and therefore its abundance.

Meanwhile, they also combed through genomic databases to identify genetic variants in regions of the gene that code CPG2. Those mutations could adversely affect how the protein is built and functions.

Examining effects

The researchers then conducted a series of experiments to test the physiological consequences of both the regulatory and protein coding variants found in BD patients.

To test effects of non-coding variants on CPG2 expression, they cloned the CPG2 promoter regions from the human SYNE1 gene and attached them to a "reporter" that would measure how effective they were in directing protein expression in cultured neurons. They then compared these to the same regions cloned from BD patients that contained specific variants individually or in combination. Some did not affect the neurons' ability to express CPG2 but some did profoundly. In two cases, pairs of variants (but neither of them individually), also reduced CPG2 expression.

Previously Nedivi's lab showed that human CPG2 can be used to replace rat CPG2 in culture neurons, and that it works the same way to regulate glutamate receptor levels. Using this assay they tested which of the coding variants might cause problems with CPG2's cellular function. They found specific culprits that either reduced the ability of CPG2 to locate in the "spines" that house excitatory synapses or that decreased the proper cycling of glutamate receptors within synapses.

The findings show how genetic variations associated with BD disrupt the levels and function of a protein crucial to synaptic activity and therefore the health of neural connections. It remains to be shown how these cellular deficits manifest as biopolar disorder.

Nedivi's lab plans further studies including assessing behavioral implications of difference-making variants in lab animals. Another is to take a deeper look at how variants affect glutamate receptor cycling and whether there are ways to fix it. Finally, she said, she wants to continue investigating human samples to gain a more comprehensive view of how specific combinations of CPG2-affecting variants relate to disease risk and manifestation.

In addition to Rathje and Nedivi, the paper's other authors are Hannah Waxman, Marc Benoit, Prasad Tammineni, Costin Leu, and Sven Loebrich.

The JPB Foundation, the Gail Steel Fund, the Carlsberg Foundation, the Lundbeck Foundation and the Danish Council for Independent Research funded the study.