Ultrasensitive Calcium Sensors Shine New Light on Neuron Activity

A new protein engineered by scientists at the Janelia Farm Research Campus fluoresces brightly each time it senses calcium, giving the scientists a way to visualize neuronal activity. The new protein is the most sensitive calcium sensor ever developed and the first to allow the detection of every neural impulse.

Every time you say a word, take a step, or read a sentence, a collection of neurons sends a speedy relay of messages throughout your brain to process the information. Now, researchers have a new way of watching those messages in action, by watching each cell in the chain light up when it fires.

When a neuron receives a signal from one of its neighbors, the impulse sets off a sudden series of electrochemical events geared toward passing the message along. Among the first events: calcium ions rush into the neurons when a set of channels opens. Scientists at the Howard Hughes Medical Institute’s Janelia Farm Research Campus have engineered a new protein that brightly fluoresces each time it senses these calcium waves, giving the scientists a way to visualize the activity of every neuron throughout the brain. The new protein is the most sensitive calcium sensor ever developed and the first to allow the detection of every neural impulse, rather than just a portion. The results are reported in the July 18, 2013 issue of the journal Nature.

“You can think of the brain as an orchestra with each different neuron type playing a different part,” says Janelia lab head Karel Svoboda, a neurobiologist and member of the team that developed the new sensor. “Previous methods only let us hear a tiny fraction of the melodies. Now we can hear more of the symphony at once. Improving the molecule and imaging methods in the future could allow us to hear the entire symphony.”

Detecting which neurons in the brain are firing, and when, is a key step in learning which areas of the brain are linked to particular activities or disorders, how memories are formed, how behaviors are learned, and basic questions about how the brain organizes neurons and stores information in this organization.

Two decades ago, scientists who wanted to use calcium to pinpoint neural activity relied on synthetic calcium-indicator dyes, first developed by HHMI Investigator Roger Tsien. The dyes lit up when neurons fired, but were difficult to inject and highly toxic—an animal’s brain could only be imaged once using the dyes.

In 1997, researchers led by Tsien developed the first genetically encoded calcium indicator (GECI). GECIs were made by combining a gene for a calcium sensor with the gene for a fluorescent protein in a way that made the calcium sensor fluoresce when it bound calcium. The GECI genes could be integrated into the genomes of model organisms like mice or flies so that no dye injection was necessary. The animals’ own brain cells would produce the proteins throughout their lives, and brain activity could be studied again and again in any one animal, allowing long-term studies of processes like learning and development. But GECIs weren’t as accurate as the cumbersome dyes had been, and improving them was a slow process.

“New versions were developed in a very piecemeal way,” says Svoboda, explaining that after chemists developed the sensors, it might be years before biologists had an opportunity to test them in the brains of living animals. “It was a very slow process of getting feedback.”

Svoboda, along with Janelia lab heads Loren Looger, Vivek Jayaraman and Rex Kerr formed the Genetically Encoded Neural Indicator and Effector (GENIE) project at Janelia to speed up the innovation. The GENIE project, led by Douglas Kim, an HHMI program scientist, is one of several collaborative team projects online at Janelia. The project developed a higher-throughput and more accurate way of testing new variants of the best-working GECI, called GCaMP. Steps included simple tests that could easily be performed on many proteins at once, like measuring how much fluorescence the protein gave off when exposed to calcium in a cuvette, as well as early tests of function in different types of neurons and final experiments in genetically engineered mice, flies, and zebrafish.

“When people developed previous GECIs, they would test somewhere between ten and twenty variants very carefully. We were able to screen a thousand in a highly quantitative neuronal assay,” Looger says. “And when you can look at that many constructs, you’re going to make better and more interesting observations on what makes the ideal sensor.”

The team made successive rounds of tweaks to the structure of the GCaMP so that it accurately sensed calcium, shone brightly in response, and worked in model organisms. After that work they settled upon a version of the sensor that performed better in all aspects than previous GECIs. Their new sensor, dubbed GCaMP6, produced signals seven times stronger than past versions. Surprisingly, its sensitivity even outperformed synthetic dyes.

“People had assumed that the synthetic dyes were letting us see every event in neurons,” says Looger. “But we’ve now shown that not only are these dyes hard to load and quite toxic, but they weren’t even recording every event.”

GCaMP6 will be a boon to researchers at Janelia, and around the world, who want to get a full picture of the activity of every neuron in the brain. Meanwhile, the team plans to continue to continue to improve it, developing entirely new versions for specific uses. For example, they hope to make a GECI that gives off red fluorescence rather than green, because red is easier to see in deeper tissues.

“One of the stated goals of Janelia Farm is to develop an atlas of every neuron in the Drosophila brain,” says Looger. “The most practical way I can think of to assign functions to such an atlas is with calcium sensors. With this new sensor, I think people will feel much more comfortable that they’re really getting all the information they can.”

Scientists Develop New Way to Measure Cumulative Effect of Head Hits in Football

Scientists at Wake Forest Baptist Medical Center have developed a new way to measure the cumulative effect of impacts to the head incurred by football players.

The metric, called Risk Weighted Cumulative Exposure (RWE), can capture players’ exposure to the risk of concussion over the course of a football season by measuring the frequency and magnitude of all impacts, said senior author of the study Joel Stitzel, Ph.D., chair of biomedical engineering at Wake Forest Baptist and associate head of the Virginia Tech - Wake Forest University School of Biomedical Engineering and Sciences.

The study is published in the current online edition of the Annals of Biomedical Engineering.

Based on data gathered throughout a season of high school football games and practices, the researchers used RWE to measure the cumulative risk of injury due to linear and rotational acceleration separately, as well as the combined probability of injury associated with both.

“This metric gives us a way to look at a large number of players and the hits they’ve incurred while playing football,” Stitzel said. “We know that young players are constantly experiencing low-level hits that don’t cause visible injury, but there hasn’t been a good way to measure the associated risk of concussion.”

Concussion is the most common sports-related head injury, with football players having the highest rate among high school athletes, according to the study. It is estimated that nearly 1.1 million students play high school football in the United States. However, research on the biomechanics of football-related head impacts traditionally has concentrated on the collegiate level rather than on the high school level.

With such a large number of players in the sport, it is critical to understand the risk associated with different levels of impact and accurately estimate cumulative concussion risk over the course of a practice, game, season or lifetime, Stitzel said.

In the Wake Forest Baptist study, the researchers measured the head impact exposure in 40 high school football players by using sensors placed in their helmets to record linear and rotational acceleration. A total of 16,502 impacts were collected over the course of one football season and the data were analyzed as a group and as individual players.

Impacts were weighted according to the associated risk from linear acceleration and rotational acceleration alone, as well as to the combined probability of injury associated with both. This is an improved method of capturing the cumulative effects from each impact because it accounts for nonlinear relationships between impact magnitude and the associated risk of injury, Stitzel said.

“All hits involve both linear and rotational acceleration, but rotation coveys the idea that your head is pivoting about the neck whereas linear acceleration is experienced from a direct blow in more of a straight line through the center of mass of the head,” Stitzel said.

The median impact for each player ranged from 15.2 to 27.0 g, with an average value of 21.7 g, which shows the wide variability in the force of impacts.

The study found that impact frequency was greater during games (15.5) than during practices (9.4). However, overall exposure over the course of the season was greater during practices.

This information may help teams reduce exposure to head impacts during practices by teaching proper tackling techniques that could reduce exposure to impacts that may result in a higher concussion rate, the researchers reported.

Additionally, the study found a wide variation in player exposure within the team, with a 22-fold variation in the exposure per impact for practices and a 47-fold variation in the exposure for impact for games.

Studies like this are vital to understanding the biomechanical basis of head injuries related to football, Stitzel said. The metric fully captures a player’s exposure over the course of the season and will be used in conjunction with other pre- and post-season evaluations, including MRI and neurological tests conducted as part of this study.

The research team hopes that this work may ultimately improve helmet safety and design to make football a safer sport.

(Image: Getty Images)

Brain imaging study reveals our brains ‘divide and conquer’

University of Queensland (UQ) researchers have found human brains ‘divide and conquer’ when people learn to navigate around new environments.

The research by UQ’s Queensland Brain Institute (QBI) could provide hope for people with spatial memory impairments.

The study found that the mental picture people create to help navigate to a new location is split into two sections.

The size of the environment is coded by one area of the brain and its complexity is coded in another.

QBI postdoctoral research fellow and lead researcher Dr Oliver Baumann said the work shed new light on how learning the layout of a new environment, and then accessing this information from memory, was represented in the brain.

“We’ve known for some time that a part of the brain called the hippocampus is important for building and maintaining cognitive maps,” he said.

“The results of our study have shown for the first time that different aspects of a learned environment – specifically its size and complexity – are represented by distinct areas within the hippocampus.”

QBI Cognitive Neuroscience Laboratory Head Professor Jason Mattingley said the findings could have important implications for people suffering from spatial memory impairments.

“This research is important for understanding how our brain normally stores and manages spatial information,” Professor Mattingley said.

“It also gives us clues as to why people with memory loss due to Alzheimer’s disease often become lost in new or previously familiar surroundings.”

Dr Baumann said 18 people navigated their way through three virtual mazes that differed either in the number of corridors through which they could travel or the length of the corridors.

After learning the task, the participants were asked to recall mental maps from each of the mazes while their brain activity was measured using functional magnetic resonance imaging.

“We found that one region in the hippocampus was more active when participants recalled a complex maze in which there were many corridors to choose from, irrespective of the overall size of the maze,” Dr Baumann said.

“Conversely, we found that a separate area of the hippocampus was more active when the overall size of the maze increased, regardless of the number of corridors.”

The study, “Dissociable representations of environmental size and complexity in the human hippocampus”, is published in The Journal of Neuroscience.

(Image: iStockphoto)

Uncovering a Healthier Remedy for Chronic Pain

Physicians and patients who are wary of addiction to pain medication and opioids may soon have a healthier and more natural alternative.

A Duke University study revealed that a derivative of DHA (docosahexaenoic acid), a main ingredient of over-the-counter fish oil supplements, can sooth and prevent neuropathic pain caused by injuries to the sensory system. The results appear online in the Annals of Neurology.

The research focused on a compound called neuroprotectin D1=protectin D1 (NPD1=PD1),  a bioactive lipid produced by cells in response to external stimuli. NPD1=PD1 is present in human white blood cells, and was first identified based on its ability to resolve abdominal and brain inflammation. 

"These compounds are derived from omega-3 fatty acids found in fish oil, but are 1,000 times more potent than their precursors in reducing inflammation," said Ru-Rong Ji, professor of anesthesiology and neurobiology at Duke University Medical Center and principal investigator of the study.  

The team used laboratory mouse models of nerve injuries to simulate pain symptoms commonly associated with post-surgical nerve trauma. They treated these animals with chemically synthesized NPD1=PD1, either through local administration or injection, to investigate whether the lipid compound could relieve these symptoms. 

Their findings revealed that NPD1=PD1 not only alleviated the pain, but also reduced nerve swelling following the injuries. Its analgesic effect stems from the compound’s ability to inhibit the production of cytokines and chemokines, which are small signaling molecules that attract inflammatory macrophages to the nerve cells. By preventing cytokine and chemokine production, the compound protected nerve cells from further damage. NPD1=PD1 also reduced neuron firing so the injured animals felt less pain.

Ji believes that the new discovery has clinical potential. “Chronic pain resulting from major medical procedures such as amputation, chest and breast surgery is a serious problem,” he said. Current treatment options for neuropathic pain include gabapentin and various opioids, which may lead to addiction and destruction of the sensory nerves. 

On the other hand, NPD1=PD1 can relieve neuropathic pain at very low doses and, more importantly, mice receiving the treatment did not show signs of physical dependence or enhanced tolerance toward the lipid compound. 

"We hope to test this compound in clinical trials," Ji said. The initial stages of the trial could involve  DHA administration through diet and injection. "DHA is very inexpensive, and can be converted to NPD1 by an aspirin-triggered pathway," he said. The ultimate goal is to develop a safer approach to managing chronic pain.

Low doses of psychedelic drug erases conditioned fear in mice

Low doses of a psychedelic drug erased the conditioned fear response in mice, suggesting that the agent may be a treatment for post-traumatic stress disorder and related conditions, a new study by University of South Florida researchers found.

The unexpected finding was made by a USF team studying the effects of the compound psilocybin on the birth of new neurons in the brain and on learning and short-term memory formation. Their study appeared online June 2 in the journal Experimental Brain Research, in advance of print publication.

Psilocybin belongs to a class of compounds that stimulate select serotonin receptors in the brain.  It occurs naturally in certain mushrooms that have been used for thousands of years by non-Western cultures in their religious ceremonies.

While past studies indicate psilocybin may alter perception and thinking and elevate mood, the psychoactive substance rarely causes hallucinations in the sense of seeing or hearing things that are not there, particularly in lower to moderate doses.

There has been recent renewed interest in medicine to explore the potential clinical benefit of psilocybin, MDMA and some other psychedelic drugs through carefully monitored, evidence-based research.

“Researchers want to find out if, at lower doses, these drugs could be safe and effective additions to psychotherapy for treatment-resistant psychiatric disorders or adjunct treatments for certain neurological conditions,” said Juan Sanchez-Ramos, MD, PhD, professor of neurology and Helen Ellis Endowed Chair for Parkinson’s Disease Research at the USF Health Morsani College of Medicine.

Dr. Sanchez-Ramos and his colleagues wondered about psilocybin’s role in the formation of short-term memories, since the agent binds to a serotonin receptor in the hippocampus, a region of the brain that gives rise to new neurons. Lead author for this study was neuroscientist Briony Catlow, a former PhD student in Dr. Sanchez-Ramos’ USF laboratory who has since joined the Lieber Institute for Brain Development, a translational neuroscience research center located in the Johns Hopkins Bioscience Park.

The USF researchers investigated how psilocybin affected the formation of memories in mice using a classical conditioning experiment. They expected that psilocybin might help the mice learn more quickly to associate a neutral stimulus with an unpleasant environmental cue.

To test the hypothesis, they played an auditory tone, followed by a silent pause before delivering a brief shock similar to static electricity. The mice eventually learned to link the tone with the shock and would freeze, a fear response, whenever they heard the sound.

Later in the study, the researchers played the sound without shocking the mice after each silent pause. They assessed how many times it took for the mice to resume their normal movements, without freezing in anticipation of the shock.

Regardless of the doses administered, neither psilocybin nor ketanserin, a serotonin inhibitor, made a difference in how quickly the mice learned the conditioned fear response.  However, mice receiving low doses of psilocybin lost their fearful response to the sound associated with the unpleasant shock significantly more quickly than mice getting either ketanserin or saline (control group). In addition, only low doses of psilocybin tended to increase the growth of neurons in the hippocampus.

“Psilocybin enhanced forgetting of the unpleasant memory associated with the tone,” Dr. Sanchez-Ramos said. “The mice more quickly dissociated the shock from the stimulus that triggered the fear response and resumed their normal behavior.”

The result suggests that psilocybin or similar compounds may be useful in treating post-traumatic stress disorder or related conditions in which environmental cues trigger debilitating behavior like anxiety or addiction, Dr. Sanchez-Ramos said.

Birds and humans have similar brain wiring

You may have more in common with a pigeon than you realise, according to new research.

It shows that humans and birds have brains that are wired in a similar way.

A researcher from Imperial College London and his colleagues have developed for the first time a map of a typical bird brain, showing how different regions are connected together to process information. By comparing it to brain diagrams for different mammals such as humans, the team discovered that areas important for high-level cognition such as long-term memory and problem solving are wired up to other regions of the brain in a similar way. This is despite the fact that both mammal and bird brains have been evolving down separate paths over hundreds of millions of years.

The team suggest that evolution has discovered a common blueprint for high-level cognition in brain development.

Birds have been shown in previous studies to possess a range of skills such as a capacity for complex social reasoning, an ability to problem solve and some have even demonstrated the capability to craft and use tools.

Professor Murray Shanahan, author of the study from the Department of Computing at Imperial College London, says:

“Birds have been evolving separately from mammals for around 300 million years, so it is hardly surprising that under a microscope the brain of a bird looks quite different from a mammal. Yet, birds have been shown to be remarkably intelligent in a similar way to mammals such as humans and monkeys. Our study demonstrates that by looking at brains that are least like our own, yet still capable of generating intelligent behaviour, we can determine the basic principles governing the way brains work.”

The team developed their map by analysing 34 studies of the anatomy of the pigeon brain, which is typical for a bird. They focussed on areas called ‘hub nodes’, which are regions of the brain that are major centres for processing information and are important for high level cognition.

In particular, they looked at the hippocampus, which is important for navigation and long-term memory in both birds and mammals. They found that these hub nodes had very dense connections to other parts of the brain in both kinds of animal, suggesting they function in a similar way.

They also compared the prefrontal cortex in mammals, which is important for complex thought such as decision making, with the nidopallium caudolaterale, which has a similar role in birds. They discovered that despite both hub nodes having evolved differently, the way they are wired up within the brain looks similar.

The long-term goal of the team is to use the information generated from the wiring diagram to build computer models that mimic the way that animal brains function, which would be used to control a robot.

The study was published this month in the Frontiers in Computational Neuroscience journal.