Posts tagged calcium ions
Articles and news from the latest research reports.
A faster vessel for charting the brain
Princeton University researchers have created “souped up” versions of the calcium-sensitive proteins that for the past decade or so have given scientists an unparalleled view and understanding of brain-cell communication.
Reported July 18 in the journal Nature Communications, the enhanced proteins developed at Princeton respond more quickly to changes in neuron activity, and can be customized to react to different, faster rates of neuron activity. Together, these characteristics would give scientists a more precise and comprehensive view of neuron activity.
The researchers sought to improve the function of proteins known as green fluorescent protein/calmodulin protein (GCaMP) sensors, an amalgam of various natural proteins that are a popular form of sensor proteins known as genetically encoded calcium indicators, or GECIs. Once introduced into the brain via the bloodstream, GCaMPs react to the various calcium ions involved in cell activity by glowing fluorescent green. Scientists use this fluorescence to trace the path of neural signals throughout the brain as they happen.
GCaMPs and other GECIs have been invaluable to neuroscience, said corresponding author Samuel Wang, a Princeton associate professor of molecular biology and the Princeton Neuroscience Institute. Scientists have used the sensors to observe brain signals in real time, and to delve into previously obscure neural networks such as those in the cerebellum. GECIs are necessary for the BRAIN Initiative President Barack Obama announced in April, Wang said. The estimated $3 billion project to map the activity of every neuron in the human brain cannot be done with traditional methods, such as probes that attach to the surface of the brain. “There is no possible way to complete that project with electrodes, so you have to do it with other tools — GECIs are those tools,” he said.
Despite their value, however, the proteins are still limited when it comes to keeping up with the fast-paced, high-voltage ways of brain cells, and various research groups have attempted to address these limitations over the years, Wang said.
“GCaMPs have made significant contributions to neuroscience so far, but there have been some limits and researchers are running up against those limits,” Wang said.
One shortcoming is that GCaMPs are about one-tenth of a second slower than neurons, which can fire hundreds of times per second, Wang said. The proteins activate after neural signals begin, and mark the end of a signal when brain cells have (by neuronal terms) long since moved on to something else, Wang said. A second current limitation is that GCaMPs can only bind to four calcium ions at a time. Higher rates of cell activity cannot be fully explored because GCaMPs fill up quickly on the accompanying rush of calcium.
The Princeton GCaMPs respond more quickly to changes in calcium so that changes in neural activity are seen more immediately, Wang said. By making the sensors a bit more sensitive and fragile — the proteins bond more quickly with calcium and come apart more readily to stop glowing when calcium is removed — the researchers whittled down the roughly 20 millisecond response time of existing GCaMPs to about 10 milliseconds, Wang said.
The researchers also tweaked certain GCaMPs to be sensitive to different types of calcium ion concentrations, meaning that high rates of neural activity can be better explored. “Each probe is sensitive to one range or another, but when we put them together they make a nice choir,” Wang said.
The researchers’ work also revealed the location of a “bottleneck” in GCaMPs that occurs when calcium concentration is high, which poses a third limitation of the existing sensors, Wang said. “Now that we know where that bottle neck is, we think we can design the next generation of proteins to get around it,” Wang said. “We think if we open up that bottleneck, we can get a probe that responds to neuronal signals in one millisecond.”
The faster protein that the Princeton researchers developed could pair with work in other laboratories to improve other areas of GCaMP function, Wang said. For instance, a research group out of the Howard Hughes Medical Institute reported in Nature July 17 that it developed a GCaMP with a brighter fluorescence. Such improvements on existing sensors gradually open up more of the brain to exploration and understanding, said Wang, adding that the Princeton researchers will soon introduce their sensor into fly and mammalian brains.
“At some level, what we’ve done is like taking apart an engine, lubing up the parts and putting it back together. We took what was the best version of the protein at the time and made changes to the letter code of the protein,” Wang said. “We want to watch the whole symphony of thousands of neurons do their thing, and we think this variant of GCaMPs will help us do that better than anyone else has.”
(Source: blogs.princeton.edu)
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.”
A turbocharger for nerve cells
Locating a car that’s blowing its horn in heavy traffic, channel-hopping between football and a thriller on TV without losing the plot, and not forgetting the start of a sentence by the time we have read to the end – we consider all of these to be normal everyday functions. They enable us to react to fast-changing circumstances and to carry out even complex activities correctly. For this to work, the neuron circuits in our brain have to be very flexible. Scientists working under the leadership of neurobiologists Nils Brose and Erwin Neher at the Max Planck Institutes of Experimental Medicine and Biophysical Chemistry in Göttingen have now discovered an important molecular mechanism that turns neurons into true masters of adaptation.
Neurons communicate with each other by means of specialised cell-to-cell contacts called synapses. First, an emitting neuron is excited and discharges chemical messengers known as neurotransmitters. These signal molecules then reach the receiving cell and influence its activation state. The transmitter discharge process is highly complex and strongly regulated. Its protagonists are synaptic vesicles, small blisters surrounded by a membrane, which are loaded with neurotransmitters and release them by fusing with the cell membrane. In order to be able to respond to stimulation at any time by releasing transmitters, a neuron must have a certain amount of vesicles ready to go at each of its synapses. Brose has been studying the molecular foundations of this stockpiling for years.
The problem is not merely academic. “The number of immediately releasable vesicles at a synapse determines its reliability,” explains Brose. “If there are too few and they are replenished too slowly, the corresponding synapse becomes tired very quickly in conditions of repeated activation. The opposite applies when a synapse can quickly top up its immediately available vesicles under pressure. In fact, such a synapse may even improve with constant activation.”
This synaptic adaptability can be observed in practically all neurons. It is known as short-term plasticity and is indispensable for a large number of extremely important brain processes. Without it, we would not be able to localise sounds, mental maths would be impossible, and the speed and flexibility with which we can alter our behaviour and turn our attention to new goals would be lost.
Some years ago, Brose and his team discovered a protein with the cryptic name of Munc13. Not only is this protein indispensable for the replenishment of vesicles for immediate release at synapses; neuron activity regulates it in such a way that the fresh supply of vesicles can be adjusted in line with demand. This regulation occurs by means of a complex consisting of the signal protein calmodulin and calcium ions that build up in the synapses during intense neuron activity.
“Our earlier work on individual neurons in culture dishes showed that the calcium-calmodulin complex activates Munc13 and consequently ensures that immediately releasable vesicles are replenished faster,” says Noa Lipstein, an Israeli guest scientist in Brose’s lab. “But many colleagues were not convinced that this process also played a role in neurons in the intact brain.”
So Lipstein and her Japanese colleague Takeshi Sakaba created a mutant mouse with genetically altered Munc13 proteins that could not be activated by calcium-calmodulin complexes. The two neurophysiologists first studied the effects of this genetic manipulation on synapses involved in the localisation of sound, which are typically activated several hundred times every second. “Our study shows that the sustained efficiency of synapses in intact neuron networks is critically dependent on the activation of Munc13 by calcium-calmodulin complexes,” explains Lipstein.
The Göttingen-based scientists are convinced of the significance of their study. After all, leading neuroscientists of the past described the calcium sensor responsible for synaptic short-term plasticity and its target protein as the Holy Grail. “I am confident that we have discovered a key molecular mechanism of short-term plasticity that plays a role in all synapses in the brain, and not only in cultivated neurons, as many colleagues believed,” affirms Lipstein. And if she is, in fact, proved right about the interpretation of her findings, Munc13 could even be an ideal pharmacological target for drugs that influence brain function.
Neurochemical Traffic Signals May Open New Avenues for the Treatment of Schizophrenia
Researchers at Boston University School of Medicine (BUSM) have uncovered important clues about a biochemical pathway in the brain that may one day expand treatment options for schizophrenia. The study, published online in the journal Molecular Pharmacology, was led by faculty within the department of pharmacology and experimental therapeutics at BUSM.
Patients with schizophrenia suffer from a life-long condition that can produce delusions, disordered thinking, and breaks with reality. A number of treatments are available for schizophrenia, but many patients do not respond to these therapies or experience side effects that limit their use.
This research focused on key components of the brain known as NMDA receptors. These receptors are located on nerve cells in the brain and serve as biochemical gates that allow calcium ions (electrical charges) to enter the cell when a neurotransmitter, such as glutamate, binds to the receptor. Proper activation of these receptors is critical for sensory perception, memory and learning, including the transfer of short-term memory into long-term storage. Patients with schizophrenia have poorly functioning or “hypoactive” NMDA receptors, suggesting the possibility of treatment with drugs that positively affect these receptors. Currently the only way to enhance NMDA receptor function is through the use of agents called agonists that directly bind to the receptor on the outer surface of the cell, opening the gates to calcium ions outside the cell.
In this study, the researchers discovered a novel “non-canonical” pathway in which NMDA receptors residing inside the cell are stimulated by a neuroactive steroid to migrate to the cell surface (a process known as trafficking), thus increasing the number of receptors available for glutamate activation. The researchers treated neural cells from the cerebral cortex with the novel steroid pregnenolone sulfate (PregS) and found that the number of working NMDA receptors on the cell surface increased by 60 to 100 percent within 10 minutes. The exact mechanism by which this occurs is not completely clear, but it appears that PregS increases calcium ions within the cell, which in turn produces a green light signal for more frequent trafficking of NMDA receptors to the cell surface.
Although still in the early stages, further research in this area may be instrumental in the development of treatments not only for schizophrenia, but also for other conditions associated with malfunctioning NMDA receptors, such as age-related decreases in memory and learning ability.
In our interaction with our environment we constantly refer to past experiences stored as memories to guide behavioral decisions. But how memories are formed, stored and then retrieved to assist decision-making remains a mystery. By observing whole-brain activity in live zebrafish, researchers from the RIKEN Brain Science Institute have visualized for the first time how information stored as long-term memory in the cerebral cortex is processed to guide behavioral choices.
The study, published today in the journal Neuron, was carried out by Dr. Tazu Aoki and Dr. Hitoshi Okamoto from the Laboratory for Developmental Gene Regulation, a pioneer in the study of how the brain controls behavior in zebrafish.
The mammalian brain is too large to observe the whole neural circuit in action. But using a technique called calcium imaging, Aoki et al. were able to visualize for the first time the activity of the whole zebrafish brain during memory retrieval.
Calcium imaging takes advantage of the fact that calcium ions enter neurons upon neural activation. By introducing a calcium sensitive fluorescent substance in the neural tissue, it becomes possible to trace the calcium influx in neurons and thus visualize neural activity.
The researchers trained transgenic zebrafish expressing a calcium sensitive protein to avoid a mild electric shock using a red LED as cue. By observing the zebrafish brain activity upon presentation of the red LED they were able to visualize the process of remembering the learned avoidance behavior.
They observe spot-like neural activity in the dorsal part of the fish telencephalon, which corresponds to the human cortex, upon presentation of the red LED 24 hours after the training session. No activity is observed when the cue is presented 30 minutes after training.
In another experiment, Aoki et al. show that if this region of the brain is removed, the fish are able to learn the avoidance behavior, remember it short-term, but cannot form any long-term memory of it.
“This indicates that short-term and long-term memories are formed and stored in different parts of the brain. We think that short-term memories must be transferred to the cortical region to be consolidated into long-term memories,” explains Dr. Aoki.
The team then tested whether memories for the best behavioral choices can be modified by new learning. The fish were trained to learn two opposite avoidance behaviors, each associated with a different LED color, blue or red, as a cue. They find that presentation of the different cues leads to the activation of different groups of neurons in the telencephalon, which indicates that different behavioral programs are stored and retrieved by different populations of neurons.
“Using calcium imaging on zebrafish, we were able to visualize an on-going process of memory consolidation for the first time. This approach opens new avenues for research into memory using zebrafish as model organism,” concludes Dr. Okamoto.
Researchers identify how cells control calcium influx
When brain cells are overwhelmed by an influx of too many calcium molecules, they shut down the channels through which these molecules enter the cells. Until now, the “stop” signal mechanism that cells use to control the molecular traffic was unknown.
In the new issue of the journal Neuron, UC Davis Health System scientists report that they have identified the mechanism. Their findings are relevant to understanding the molecular causes of the disruption of brain functioning that occurs in stroke and other neurological disorders.
"Too much calcium influx clearly is part of the neuronal dysfunction in Alzheimer’s disease and causes the neuronal damage during and after a stroke. It also contributes to chronic pain," said Johannes W. Hell, professor of pharmacology at UC Davis. Hell headed the research team that identified the mechanism that stops the flow of calcium molecules, which are also called ions, into the specialized brain cells known as neurons.
Hell explained that each day millions of molecules of calcium enter and exit each of the 100 billion neurons of the human brain. These calcium ions move in and out of neurons through pore-like structures, known as channels, that are located in the outer surface, or “skin,” of each cell.
The flow of calcium ions into brain cells generates the electrical impulses needed to stimulate such actions as the movement of muscles in our legs and the creation of new memories in the brain. The movement of calcium ions also plays a role in gene expression and affects the flexibility of the structures, called synapses, that are located between neurons and transmit electrical or chemical signals of various strengths from one cell to a second cell.
Neurons employ an unexpected and highly complex mechanism to down regulate, or reduce, the activity of channels that are permitting too many calcium ions to enter neurons, Hell and his colleagues discovered. The mechanism, which leads to the elimination of the overly permissive ion channel employs two proteins, α-actinin and the calcium-binding messenger protein calmodulin.
Located on the neuron’s outer surface, referred to as the plasma membrane, α-actinin stabilizes the type of ion channels that constitute a major source of calcium ion influx into brain cells, Hell explained. This protein is a component of the cytoskeleton, the scaffolding of cells. The ion channels that are a major source of calcium ions are referred to as Cav1.2 (L type voltage-dependent calcium channels).
The researchers also found that the calcium-binding messenger protein calmodulin, which is the cell’s main sensor for calcium ions, induces internalization, or endocytosis, of Cav1.2 to remove this channel from the cell surface, thus providing an important negative feedback mechanism for excessive calcium ion influx into a neuron, Hell explained.
The discovery that α-actinin and calmodulin play a role in controlling calcium ion influx expands upon Hell’s previous research on the molecular mechanisms that regulate the activity of various ion channels at the synapse.
One previous study proved relevant to understanding the biological mechanisms that underlie the body’s fight-or-flight response during stress.
In work published in the journal Science in 2001, Hell and colleagues reported that the regulation of Cav1.2 by adrenergic signaling during stress is performed by one of the adrenergic receptors (beta 2 adrenergic receptor) directly linked to Cav1.2.
"This protein-protein interaction ensures that the adrenergic regulation is fast, efficient and precisely targets this channel," Hell said.
"We showed that Cav1.2 is regulated by adrenergic signaling on a time scale of a few seconds, and this is mainly increasing its activity when needed, for example during danger, to make our brain work faster and better. The same channel is in the heart, where adrenergic stimulation increases channel/Ca influx activity, increasing the pacing and strength of our heart beat to meet the increased physical demands during danger."
(Source: universityofcalifornia.edu)
Calcium reveals connections between neurons
A team led by MIT neuroscientists has developed a way to monitor how brain cells coordinate with each other to control specific behaviors, such as initiating movement or detecting an odor.
The researchers’ new imaging technique, based on the detection of calcium ions in neurons, could help them map the brain circuits that perform such functions. It could also provide new insights into the origins of autism, obsessive-compulsive disorder and other psychiatric diseases, says Guoping Feng, senior author of a paper appearing in the Oct. 18 issue of the journal Neuron.
“To understand psychiatric disorders we need to study animal models, and to find out what’s happening in the brain when the animal is behaving abnormally,” says Feng, the James W. and Patricia Poitras Professor of Neuroscience and a member of the McGovern Institute for Brain Research at MIT. “This is a very powerful tool that will really help us understand animal models of these diseases and study how the brain functions normally and in a diseased state.”