Friday, 27 January 2012

New Fluorescent Dyes Highlight Neuronal Activity

Researchers at the University of California, San Diego School of Medicine have created a new generation of fast-acting fluorescent dyes that optically highlight electrical activity in neuronal membranes. The work is published in this week’s online Early Edition of the Proceedings of the National Academy of Sciences.

The ability to visualize these small, fast-changing voltage differences between the interior and exterior of neurons – known as transmembrane potential – is considered a powerful method for deciphering how brain cells function and interact.

However, current monitoring methods fall short, said the study’s first author Evan W. Miller, a post-doctoral researcher in the lab of Roger Tsien, PhD, Howard Hughes Medical Institute investigator, UC San Diego professor of pharmacology, chemistry and biochemistry and 2008 Nobel Prize co-winner in chemistry for his work on green fluorescent protein.

“The most common method right now monitors the movement of calcium ions into the cell,” said Miller. “It provides some broad indication, but it’s an indirect measurement that misses activity we see when directly measuring voltage changes.”


The new method employs dyes that penetrate only the membrane of neurons, either in in vitro cells cultured with the dye or, for this study, taken up by neurons in a living leech model. When the dyed cells are exposed to light, neuronal firing causes the dye momentarily to glow more brightly, a flash that can be captured with a high-speed camera.

“One of the tradeoffs with using voltage-sensing dyes in the past is that when they were reasonably sensitive to voltage changes, they were slow compared to the actual physiological events,” said Miller. “The new dye gives big signals but is much faster and doesn’t perturb the neurons. We essentially see no lag time between the optical signal and electrodes (used to double-check neuronal activity).”

The new method provides a wider view of neuronal activity, said Miller. More importantly, it makes it possible for neuroscientists to do accurate, single trial experiments. “Right now, you have to repeat experiments with cells, and then average the results, which is physiologically less relevant and meaningful.”

For Tsien, the new dyes address a career-long challenge.
“These results are the first demonstration of a new mechanism to sense membrane voltage, which is particularly satisfying to me because this was the first problem I started working on as a graduate student in 1972, with little success back then,” said Tsien. “Later, we devised indirect solutions such as calcium imaging or dyes that gave big but slow responses to voltage. These techniques have been very useful in other areas of biology or in drug screening, but didn’t properly solve the original problem. I think we are finally on the right track, four decades later.”

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Wednesday, 25 January 2012

Alzheimer's neurons induced from pluripotent stem cells

Stem-cell-derived neurons, made from patients with Alzheimer's disease, provide a new tool for unraveling the mechanisms underlying the neurodegenerative disease. In this image, DNA is shown in blue, dendrites and cell bodies in red and endosomal markers Rab5 and EEA1 in green and orange, respectively. Credit: UC San Diego School of Medicine

Led by researchers at the University of California, San Diego School of Medicine, scientists have, for the first time, created stem cell-derived, in vitro models of sporadic and hereditary Alzheimer's disease (AD), using induced pluripotent stem cells from patients with the much-dreaded neurodegenerative disorder.
"Creating highly purified and functional human Alzheimer's neurons in a dish– this has never been done before," said senior study author Lawrence Goldstein, PhD, professor in the Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute Investigator and director of the UC San Diego Stem Cell Program. "It's a first step. These aren't perfect models. They're proof of concept. But now we know how to make them. It requires extraordinary care and diligence, really rigorous quality controls to induce consistent behavior, but we can do it."
The feat, published in the January 25 online edition of the journal Nature, represents a new and much-needed method for studying the causes of AD, a progressive dementia that afflicts approximately 750,000 in the UK. More importantly, the living cells provide an unprecedented tool for developing and testing drugs to treat the disorder.
"We're dealing with the human brain. You can't just do a biopsy on living patients," said Goldstein. "Instead, researchers have had to work around, mimicking some aspects of the disease in non-neuronal human cells or using limited animal models. Neither approach is really satisfactory."
Goldstein and colleagues extracted primary fibroblasts from skin tissues taken from two patients with familial AD (a rare, early-onset form of the disease associated with a genetic predisposition), two patients with sporadic AD (the common form whose cause is not known) and two persons with no known neurological problems. They reprogrammed the fibroblasts into induced pluripotent stem cells (iPSCs) that then differentiated into working neurons.
The iPSC-derived neurons from the Alzheimer's patients exhibited normal electrophysiological activity, formed functional synaptic contacts and, critically, displayed tell-tale indicators of AD. Specifically, they possessed higher-than-normal levels of proteins associated with the disorder.
With the in vitro Alzheimer's neurons, scientists can more deeply investigate how AD begins and chart the biochemical processes that eventually destroy brain cells associated with elemental cognitive functions like memory. Currently, AD research depends heavily upon studies of post-mortem tissues, long after the damage has been done.
"The differences between a healthy neuron and an Alzheimer's neuron are subtle," said Goldstein. "It basically comes down to low-level mischief accumulating over a very long time, with catastrophic results."
The researchers have already produced some surprising findings. "In this work, we show that one of the early changes in Alzheimer's neurons thought to be an initiating event in the course of the disease turns out not to be that significant," Goldstein said, adding that they discovered a different early event plays a bigger role.
The scientists also found that neurons derived from one of the two patients with sporadic AD exhibited biochemical changes possibly linked to the disease. The discovery suggests that there may be sub-categories of the disorder and that, in the future, potential therapies might be targeted to specific groups of AD patients.
Though just a beginning, Goldstein emphasized the iPSC-derived Alzheimer's neurons present a huge opportunity in a desperate fight. "At the end of the day, we need to use cells like these to better understand Alzheimer's and find drugs to treat it. We need to do everything we can because the cost of this disease is just too heavy and horrible to contemplate. Without solutions, it will bankrupt us – emotionally and financially."

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Tuesday, 17 January 2012

Molecular Visualizations of DNA

This video features stunning animation representing the formation of DNA and how it is coiled into chromosomes. The video also looks at how DNA is split and copied in DNA replication. 

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Sunday, 15 January 2012

MS damage washed away by stream of young blood

A fountain of youthful cells reverses the damage found in diseases like multiple sclerosis, a study in mice reveals.

Nerve cells lose their electrically insulating myelin sheath as MS develops. New myelin-generating cells can be produced from stem cells, but the process loses efficiency with age.

Julia Ruckh at the University of Cambridge, and colleagues, have found a way to reverse the age-related efficiency loss. They linked the bloodstreams of young mice to old mice with myelin damage. Exposure to youthful blood reactivated stem cells in the old mice, boosting myelin generation.

White blood cells called macrophages from the young mice gathered at the sites of myelin damage. Macrophages engulf and destroy pathogens and debris, including destroyed myelin (Cell Stem Cell, DOI: 10.1016/j.stem.2011.11.019)

"We know this debris inhibits regeneration, so clearing it up is important," says team member Amy Wagers of Harvard University.

Neil Scolding at the University of Bristol, UK, who was not involved in the new work, says reactivating ageing stem cells may be a more realistic approach for treating MS than transplanting stem cells from a donor.

(from NewScientist, 14 January 2012, Issue 2847)

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Monday, 9 January 2012

Images of a real human brain

This website features real images of a human brain and some of its sections (including the cerebrum, cerebellum and hippocampus), detailed and labelled photos from different angles, as well as MRI scans.

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Friday, 6 January 2012

Neuroscientists Identify a Master Controller of Memory

When you experience a new event, your brain encodes a memory of it by altering the connections between neurons. This requires turning on many genes in those neurons. Now, MIT neuroscientists have identified what may be a master gene that controls this complex process.
The findings, described in the Dec. 23 issue of Science, not only reveal some of the molecular underpinnings of memory formation — they may also help neuroscientists pinpoint the exact locations of memories in the brain.
The research team, led by Yingxi Lin, a member of the McGovern Institute for Brain Research at MIT, focused on the Npas4 gene, which previous studies have shown is turned on immediately following new experiences. The gene is particularly active in the hippocampus, a brain structure known to be critical in forming long-term memories.
Lin and her colleagues found that Npas4 turns on a series of other genes that modify the brain’s internal wiring by adjusting the strength of synapses, or connections between neurons. “This is a gene that can connect from experience to the eventual changing of the circuit,” says Lin, the Frederick and Carole Middleton Career Development Assistant Professor of Brain and Cognitive Sciences.
To investigate the genetic mechanisms of memory formation, the researchers studied a type of learning known as contextual fear conditioning: Mice receive a mild electric shock when they enter a specific chamber. Within minutes, the mice learn to fear the chamber, and the next time they enter it, they freeze.
The researchers showed that Npas4 is turned on very early during this conditioning. “This sets Npas4 apart from many other activity-regulated genes,” Lin says. “A lot of them are ubiquitously induced by all these different kinds of stimulations; they are not really learning-specific.”
Furthermore, Npas4 activation occurs primarily in the CA3 region of the hippocampus, which is already known to be required for fast learning.
“We think of Npas4 as the initial trigger that comes on, and then in turn, in the right spot in the brain, it activates all these other downstream targets. Eventually they’re going to modify synapses in a way that’s likely changing synaptic inhibition or some other process that we’re trying to figure out,” says Kartik Ramamoorthi, a graduate student in Lin’s lab and lead author of the paper.
So far, the researchers have identified only a few of the genes regulated by Npas4, but they suspect there could be hundreds more. Npas4 is a transcription factor, meaning it controls the copying of other genes into messenger RNA — the genetic material that carries protein-building instructions from the nucleus to the rest of the cell. The MIT experiments showed that Npas4 binds to the activation sites of specific genes and directs an enzyme called RNA polymerase II to start copying them.
“Npas4 is providing this instructive signal,” Ramamoorthi says. “It’s telling the polymerase to land at certain genes, and without it, the polymerase doesn’t know where to go. It’s just floating around in the nucleus.”
When the researchers knocked out the gene for Npas4, they found that mice could not remember their fearful conditioning. They also found that this effect could be produced by knocking out the gene just in the CA3 region of the hippocampus. Knocking it out in other parts of the hippocampus, however, had no effect. Though they focused on contextual fear conditioning, the researchers believe that Npas4 will also prove critical for other types of learning.
Gleb Shumyatsky, an assistant professor of genetics at Rutgers University, says that an important next step is to identify more of the genes controlled by Npas4, which should reveal more of its role in memory formation. “It’s definitely one of the major players,” says Shumyatsky, who was not involved in this research. “Future experiments will show how major a player it is.”
The MIT team also plans to investigate whether the same neurons that turn on Npas4 when memories are formed also turn it on when memories are retrieved. This could help them pinpoint the exact neurons that are storing particular memories.
“We’re hunting for the memory, and we think we can use Npas4 to mark where it is,” Ramamoorthi says. “That’s because it’s turned on specifically and now we can label the cells and maybe fish out where in the brain the memory is sitting.”

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