Posts tagged calcium
Articles and news from the latest research reports.
Mouse model for epilepsy, Alzheimer’s gives window into the working brain
University of Utah scientists have developed a genetically engineered line of mice that is expected to open the door to new research on epilepsy, Alzheimer’s and other diseases.
The mice carry a protein marker, which changes in degree of fluorescence in response to different calcium levels. This will allow many cell types, including cells called astrocytes and microglia, to be studied in a new way.
"This is opening up the possibility to decipher how the brain works," said Petr Tvrdik, Ph.D., a research fellow in human genetics and a senior author on the study.
The research was published Aug. 14, 2014, in Neuron, a world-leading neuroscience journal. The work is the result of a three-year study involving multiple labs connected with The Brain Institute at the University of Utah. The lead author is J. Michael Gee, who is pursuing both a medical degree and a graduate degree in bioengineering at the university.
"We’re really in the era of team science," said John White, Ph.D., professor of bioengineering, executive director of the Brain Institute and the study’s corresponding author.
With the new mouse line, scientists can use a laser-based fluorescence microscope to study the calcium indicator in the glial cells of the living mouse, either when the mouse is anesthetized or awake. Calcium is studied because it is an important signaling molecule in the body and it can reveal how well the brain is functioning.
Using this method, the scientists are essentially creating a window into the working brain to study the interactions between neurons, astrocytes and microglia.
"We believe this will give us new insights for treatments of epilepsy and for new views of how the immune system of the brain works," White said.
About one-third of the 3 million Americans estimated to have epilepsy lack adequate treatment to manage the disease.
Describing a long-standing collaboration with fellow university researcher and professor of pharmacology and toxicology Karen Wilcox, Ph.D., White said, “We believe the glial cells are malfunctioning in epilepsy. What we’re trying to do is find out in what ways astrocytes participate in the disease.”
This research is expected to lead to new classes of drugs.
The ability to track calcium changes in microglial cells will also open up the possibility of studying inflammatory diseases of the brain. Every neurological disease, including Multiple Sclerosis and Alzheimer’s, appears to include components of inflammation, the scientists said.
"Live imaging and monitoring microglial activity and responses to inflammation was not possible before," said Tvrdik, particularly in living animals. In the past, researchers studied post-mortem tissue or relied on invasive approaches using synthetic dyes.
(Source: eurekalert.org)
Neurons Can Use Local Stores for Communication Needs
Researchers reveal that neurons can utilize a supremely localized internal store of calcium to initiate the secretion of neuropeptides, one class of signaling molecules through which neurons communicate with each other and with other cells. The study appears in The Journal of General Physiology.
(Image caption: Localization of ryanodine receptors (red) in an isolated nerve terminal from the posterior pituitary gland. Image credit: McNally et al., 2014)
Neuropeptides are released from neurons through a process that—like other secretory events—is triggered primarily by the influx of calcium into the neuron through voltage-gated channels. Although neuropeptides are stored in large dense core vesicles (LDCVs) that also contain large amounts of calcium, it has been unclear whether these locally based calcium supplies can also be used to modulate secretion.
A team of researchers led by José Lemos from the University of Massachusetts Medical School examined the mechanisms at play during secretion of vasopressin from nerve terminals in the posterior pituitary gland, which releases the neuropeptide into the blood so that it can make its way to the kidney and regulate water retention. The researchers found that certain intracellular calcium channels known as ryanodine receptors are likely responsible for mobilizing calcium from LDCVs to facilitate vasopressin release. The findings indicate that neurons have a greater capacity than previously appreciated to fine-tune the release of neuropeptides and thereby their communications with other cells.
(Source: newswise.com)
Role of Calcium in Familial Alzheimer’s Disease Clarified in Penn Study, Pointing to New Therapeutic Options
In 2008, researchers at the Perelman School of Medicine at the University of Pennsylvania showed that mutations in two proteins associated with familial Alzheimer’s disease (FAD) disrupt the flow of calcium ions within neurons. The two proteins interact with a calcium release channel in an intracellular compartment. Mutant forms of these proteins that cause FAD, but not the normal proteins, result in exaggerated calcium signaling in the cell.
Now, the same team, led by J. Kevin Foskett, PhD, chair of Physiology, and a graduate student, Dustin Shilling, has found that suppressing the hyperactivity of the calcium channels alleviated FAD-like symptoms in mice models of the disease. Their findings appear this week in the Journal of Neuroscience.
Current therapies for Alzheimer’s include drugs that treat the symptoms of cognitive loss and dementia, and drugs that address the pathology of Alzheimer’s are experimental. These new observations suggest that approaches based on modulating calcium signaling could be explored, says Foskett.
The two proteins, called PS1 and PS2 (presenilin 1 and 2), interact with a calcium release channel, the inositol trisphosphate receptor (IP3R), in the endoplasmic reticulum. Mutant PS1 and PS2 increase the activity of the IP3R, in turn increasing calcium levels in the cell. “We set out to answer the question: Is increased calcium signaling, as a result of the presenilin-IP3R interaction, involved in the development of familial Alzheimer’s disease symptoms, including dementia and cognitive deficits?” says Foskett. “And looking at the findings of these experiments, the answer is a resounding ‘yes.’”
Robust Phenomenon
Exaggerated intracellular calcium signaling is a robust phenomenon seen in cells expressing FAD-causing mutant presenilins, in both human cells in culture and in mice. The team used two FAD mouse models to look for these connections. Specifically, they found that reducing the expression of IP3R1, the dominant form of this receptor in the brain, by 50 percent, normalized the exaggerated calcium signaling observed in neurons of the cortex and hippocampus in both mouse models.
(Image caption: Amyloid-beta (antibody 12F4) and hyper-phosphorylated tau (antibody AT180) immunostaining of hippocampus from 18-month-old mice. Amyloid plaques (top row) and intracellular tau tangles (bottom row) in the 3xTg mouse were strongly reduced by genetic deletion of 50% of the IP3R1 in the 3xTg/Opt mouse. Wild-type (WT) and Opt mice expressing 50% of InsP3R exhibited no pathology. Credit: J. Kevin Foskett, PhD & Dustin Shilling, Perelman School of Medicine, University of Pennsylvania)
In addition, using 3xTg mice – animals that contain presenilin 1 with an FAD mutation, as well as expressed mutant human tau protein and APP genes — the team observed that the reduced expression of IP3R1 profoundly decreased amyloid plaque accumulation in brain tissue and the hyperphosphorylation of tau protein, a biochemical hallmark of advanced Alzheimer’s disease. Reduced expression of IP3R1 also rescued defective electrical signaling in the hippocampus, as well and memory deficits in the 3xTg mice, as measured by behavioral tests.
“Our results indicate that exaggerated calcium signaling, which is associated with presenilin mutations in familial Alzheimer’s disease, is mediated by the IP3R and contributes to disease symptoms in animals,” says Foskett. “Knowing this now, the IP3 signaling pathway could be considered a potential therapeutic target for patients harboring mutations in presenilins linked to AD.”
The ‘calcium dysregulation’ hypothesis
“The ‘calcium dysregulation’ hypothesis for inherited, early-onset familial Alzheimer’s disease has been suggested by previous research findings in the Foskett lab. Alzheimer’s disease affects as many as 5 million Americans, 5 percent of whom have the familial form. The hallmark of the disease is the accumulation of tangles and plaques of amyloid beta protein in the brain.
“The ‘amyloid hypothesis’ that postulates that the primary defect is an accumulation of toxic amyloid in the brain has long been used to explain the cause of Alzheimer’s”, says Foskett. In his lab’s 2008 Neuron study, cells that carried the disease-causing mutated form of PS1 showed increased processing of amyloid beta that depended on the interaction of the PS proteins with the IP3R. This observation links dysregulation of calcium inside cells with the production of amyloid, a characteristic feature in the brains of people with Alzheimer’s disease.
Clinical trials for AD have largely been directed at reducing the amyloid burden in the brain. So far, says Foskett, these trials have failed to demonstrate therapeutic benefits. One idea is that the interventions started too late in the disease process. Accordingly, anti-amyloid clinical trials are now underway using asymptomatic FAD patients because it is known that they will eventually develop the disease, whereas predicting who will develop the common form of AD is much less certain.
“There has been an assumption that FAD is simply AD with an earlier, more aggressive onset,” says Foskett. “However, we don’t know if the etiology of FAD pathology is the same as that for common AD. So the relevance of our findings for understanding common AD is not clear. What’s important, in my opinion, is to recognize that AD could be a spectrum of diseases that result in common end-stage pathologies. FAD might therefore be considered an orphan-disease, and it’s important to find effective treatments, specifically for these patients - ones that target the IP3R and calcium signaling.”
Eavesdropping on brain cell chatter
Everything we do — all of our movements, thoughts and feelings – are the result of neurons talking with one another, and recent studies have suggested that some of the conversations might not be all that private. Brain cells known as astrocytes may be listening in on, or even participating in, some of those discussions. But a new mouse study suggests that astrocytes might only be tuning in part of the time — specifically, when the neurons get really excited about something. This research, published in Neuron, was supported by the National Institute of Neurological Disorders and Stroke (NINDS), part of the National Institutes of Health.
For a long time, researchers thought that the star-shaped astrocytes (the name comes from the Greek word for star) were simply support cells for the neurons.
It turns out that these cells have a number of important jobs, including providing nutrients and signaling molecules to neurons, regulating blood flow, and removing brain chemicals called neurotransmitters from the synapse. The synapse is the point of information transfer between two neurons. At this connection point, neurotransmitters are released from one neuron to affect the electrical properties of the other. Long arms of astrocytes are located next to synapses, where they can keep tabs on the conversations going on between neurons.
In recent years, it has been shown that astrocytes may also play a role in neuronal communication. When neurons release neurotransmitters, levels of calcium change within astrocytes. Calcium is critical for many processes, including release of molecules from the cell, and activation of a host of proteins within the cell. The role of this astrocytic calcium signaling for brain function remains a mystery.
In this study, Baljit S. Khakh, Ph.D., of the University of California, Los Angeles and his colleagues wanted to know when astrocytes responded to neuron activity with changes in their internal calcium levels. Using calcium indicator dyes, the researchers were able to image, for the first time, changes in calcium levels in the entire astrocyte. Previously, it was only possible to look at certain areas of the cell at one time, which provided an incomplete picture of what was happening.
Dr. Khakh said one of the most important outcomes of this work was in the methods that were used. “What our use of these calcium indicators shows is that we can image calcium throughout the entire astrocyte. This provides a new set of tools for the research community to use and to extend these findings,” he said.
“There has been intense interest in understanding how astrocytes facilitate communication between neurons, but it is only recently that studies with this level of precision have been possible,” said Edmund Talley, Ph.D., program director at NINDS. “Dr. Khakh’s study is an example of an exciting basic, or fundamental, research project that could have an important contribution to the shifting field of astrocyte biology,” he added.
For these experiments, researchers focused on the mossy fiber pathway, which connects two areas of the hippocampus, the structure involved in learning and memory. “This pathway has a unique architecture and although it has been very well studied, the role of astrocytes in this circuit has not been previously explored. This study provides one of the first really detailed understandings of astrocytes within this particular circuit,” said Dr. Khakh.
Dr. Khakh’s team activated neurons (getting them to release neurotransmitter by a variety of techniques) and then looked for a response in the neighboring astrocyte. As calcium levels rose, the astrocyte would light up quickly. They discovered that two neurotransmitters, glutamate and GABA, triggered the astrocytes to release calcium from their internal stores. Importantly, the researchers discovered that calcium levels increased through the entire astrocyte only if there was a large burst of neurotransmitter being released.
“We found that astrocytes in the mossy fiber pathway do not listen to the constant, millisecond by millisecond synaptic chatter that neurons engage in. Instead, they listen when neurons get excessively excited during bursts of activation,” said Dr. Khakh.
These findings suggest that astrocytes in the mossy fiber system may act as a switch that reacts to large amounts of neuronal activity by raising their levels of calcium. These calcium increases occur over multiple seconds, a relatively long time period compared to that seen in neurons. The spatial extent of the astrocyte calcium increases was also relatively large in comparison to the size of the synapse.
“Astrocytes may be sitting there quietly and when there is excessive activation in the neuronal circuit, they immediately respond with an increase in calcium which we could detect. And the next big question becomes, what they do with that calcium?” said Dr. Khakh.
Dr. Khakh’s results in the mossy fiber system differ from those others have described in other brain regions. This raises the intriguing possibility that astrocytes are not all the same and may serve various roles throughout the brain.
“It would be really interesting and important to find that astrocytes function differently in different areas of the brain, in a circuit-specific manner. This study gives a hint that this might be true,” said Dr. Talley.
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.”
Researchers Identify “Switch” for Long-term Memory
Calcium signal in neuronal cell nuclei initiates the formation of lasting memories
Neurobiologists at Heidelberg University have identified calcium in the cell nucleus to be a cellular “switch” responsible for the formation of long-term memory. Using the fruit fly “Drosophila melanogaster” as a model, the team led by Prof. Dr. Christoph Schuster and Prof. Dr. Hilmar Bading investigates how the brain learns. The researchers wanted to know which signals in the brain were responsible for building long-term memory and for forming the special proteins involved. The results of the research were published in the journal “Science Signaling”.
The team from the Interdisciplinary Center for Neurosciences (IZN) measured nuclear calcium levels with a fluorescent protein in the association and learning centres of the insect’s brain to investigate any changes that might occur during the learning process. Their work on the fruit fly revealed brief surges in calcium levels in the cell nuclei of certain neurons during learning. It was this calcium signal that researchers identified as the trigger of a genetic programme that controls the production of “memory proteins”. If this nuclear calcium switch is blocked, the flies are unable to form long-term memory.
Prof. Schuster explains that insects and mammals separated evolutionary paths approximately 600 million years ago. In spite of this sizable gap, certain vitally important processes such as memory formation use similar cellular mechanisms in humans, mice and flies, as the researchers’ experiments were able to prove. “These commonalities indicate that the formation of long-term memory is an ancient phenomenon already present in the shared ancestors of insects and vertebrates. Both species probably use similar cellular mechanisms for forming long-term memory, including the nuclear calcium switch”, Schuster continues.
The IZN researchers assume that similar switches based on nuclear calcium signals may have applications in other areas – presumably whenever organisms need to adapt to new conditions over the long term. “Pain memory, for example, or certain protective and survival functions of neurons use this nuclear calcium switch, too”, says Prof. Bading. This cellular switch may no longer work as well in the elderly, which Bading believes may explain the decline in memory typically observed in old age. Thus, the discoveries by the Heidelberg neurobiologists open up new perspectives for the treatment of age- and illness-related changes in brain functions.
Ion selectivity in neuronal signaling channels evolved twice in animals
July 26, 2012
Excitation of neurons depends on the selected influx of certain ions, namely sodium, calcium and potassium through specific channels. Obviously, these channels were crucial for the evolution of nervous systems in animals. How such channels could have evolved their selectivity has been a puzzle until now. Yehu Moran and Ulrich Technau from the University of Vienna together with Scientists from Tel Aviv University and the Woods Hole Oceanographic Institution (USA) have now revealed that voltage-gated sodium channels, which are responsible for neuronal signaling in the nerves of animals, evolved twice in higher and lower animals. These results were published in Cell Reports.
Close-up of nervous system of a transgenic polyp of the sea anemone Nematostella vectensis, in which a red fluorescent reporter gene (mCherry) is driven by the regulatory sequence of the neuronal ELAV gene. The picture shows the diffuse structure of the nervous system, but also reveals the accumulation of longitudinal axonal tracts along the eight gastric tissue folds (mesenteries). Credit: Copyright: U. Technau
The opening and closing of ion channels enable flow of ions that constitute the electrical signaling in all nervous systems. Every thought we have or every move we make is the result of the highly accurate opening and closing of numerous ion channels. Whereas the channels of most lower animals and their unicellular relatives cannot discern between sodium and calcium ions, those of higher animals are highly specific for sodium, a characteristic that is important for fast and accurate signaling in complex nervous system.
Surprising results in sea anemones and jellyfish
However, the researchers found that a group of basal animals with simple nerve nets including sea anemones and jellyfish also possess voltage-gated sodium channels, which differ from those found in higher animals, yet show the same selectivity for sodium. Since cnidarians separated from the rest of the animals more than 600 million years ago, these findings suggest that the channels of both cnidarians and higher animals originated independently twice, from ancient non-selective channels which also transmit calcium.
Since many other processes of internal cell signaling are highly dependent on calcium ions, the use of non-selective ion channels in neurons would accidently trigger various signaling systems inside the cells and will cause damage. The evolution of selectivity for sodium ions is therefore considered as an important step in the evolution of nervous systems with fast transmission. This study shows that different parts of the channel changed in a convergent manner during the evolution of cnidarians and higher animals in order to perform the same task, namely to select for sodium ions.
This demonstrates that important components for the functional nervous systems evolved twice in basal and higher animals, which suggests that more complex nervous systems that rely on such ion-selective channels could have also evolved twice independently.
Source: PHYS.ORG