The discerning fruit fly: Linking brain-cell activity and behavior in smell recognition

Behind the common expression “you can’t compare apples to oranges” lies a fundamental question of neuroscience: How does the brain recognize that apples and oranges are different? A group of neuroscientists at Cold Spring Harbor Laboratory (CSHL) has published new research that provides some answers.

In the fruit fly, the ability to distinguish smells lies in a region of the brain called the mushroom body (MB). Prior research has demonstrated that the MB is associated with learning and memory, especially in relation to the sense of smell, also known as olfaction.

CSHL Associate Professor Glenn Turner and colleagues have now mapped the activity of brain cells in the MB, in flies conditioned to have Pavlovian behavioral responses to different odors. Their results, outlined in a paper published today by the Journal of Neuroscience, suggest that the activity of a remarkably small number of neurons — as few as 25 — is required to be able to distinguish between different odors.

They also found that a similarly small number of nerve cells are involved in grouping alike odors. This means, for instance, that “if you’ve learned that oranges are good, the smell of a tangerine will also get you thinking about food,” says Robert Campbell, a postdoctoral researcher in the Turner lab and lead author on the new study.

These intriguing new findings are part of a broad effort in contemporary neuroscience to determine how the brain, easily the most complex organ in any animal, manages to make a mass of raw sensory data intelligible to the individual — whether a person or a fly — in order to serve as a basis for making vital decisions.

Looking closely at Kenyon cells

The neurons in the fly MB are known as Kenyon cells, named after their discoverer, the neuroscientist Frederick Kenyon, who was the first person to stain and visualize individual neurons in the insect brain. Kenyon cells receive sensory inputs from organs that perceive smell, taste, sight and sound. This confluence of sensory input in the MB is important for memory formation, which comes about through a linking of different types of information.

Kenyon cells make up only about 4% of the entire fly brain and are extremely sensitive to inputs triggered by odors, in which only two connections between neurons, called synapses, separate them from the receptor cells at the “front end” of the olfactory system.

But in contrast to other regions of the brain, such as the vertebrate hippocampus, the sensory responses in the MB are few in number and relatively weak. It is the sparseness of the signals in the Kenyon cell neurons that makes studying memory formation in flies so promising to Turner and his team. “We set out to learn if these signals were really informative to the animal’s learning and memory with regard to smell,” Turner says.

In particular, Turner’s group wanted to see if they could link these signals with actual behavior in flies. The team used an imaging technique that allowed them to view the responses of over 100 Kenyon cells at a time and, importantly, quantify their results. They found that even the very sparse responses in these cells that are triggered by odors provide a large amount of information about odor identity. Turner suspects the very selectiveness of the response helps in the accurate formation and recall of memories.

When the researchers used two odors blended together in a series of increasingly similar concentrations, they found that two very similar smells could be distinguished as a result of the activity of as few as 25 Kenyon cells. This correlated well with the behavior of the flies: when brain activity suggested the flies had difficulty discerning the odors, their behavior also showed they could not choose between them.

The activity of these cells also accounts for flies’ ability to discern novel odors and group them together. This was determined in a “generalization” test, in which the degree to which flies learned a generalized aversion to unfamiliar test odors could be predicted based upon the relatively similar activity patterns of Kenyon cells that the odors induced.

“Being able to do this type of ‘mind-reading’ means we really understand what signals these activity patterns are sending,” says Turner. Ultimately, he and colleagues hope to be able to relate their findings in the fly brain with the operation of the brain in mammals.

Researchers Develop Novel Drug That Reverses Loss of Brain Connections in Models of Alzheimer’s

The first experimental drug to boost brain synapses lost in Alzheimer’s disease has been developed by researchers at Sanford-Burnham Medical Research Institute. The drug, called NitroMemantine, combines two FDA-approved medicines to stop the destructive cascade of changes in the brain that destroys the connections between neurons, leading to memory loss and cognitive decline.

The decade-long study, led by Stuart A. Lipton, M.D., Ph.D., professor and director of the Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, who is also a practicing clinical neurologist, shows that NitroMemantine can restore synapses, representing the connections between nerve cells (neurons) that have been lost during the progression of Alzheimer’s in the brain. The research findings are described in a paper published June 17 by the Proceedings of the National Academy of Sciences of the United States of America (PNAS).

The focus on a downstream target to treat Alzheimer’s, rather than on amyloid beta plaques and neurofibrillary tangles—approaches which have shown little success—“is very exciting because everyone is now looking for an earlier treatment of the disease,” Lipton said. “These findings actually mean that you might be able to intercede not only early but also a bit later.” And that means that an Alzheimer’s patient may be able to have synaptic connections restored even with plaques and tangles already in his or her brain.

Targeting lost synapses

In their study, conducted in animal models as well as brain cells derived from human stem cells, Lipton and his team mapped the pathway that leads to synaptic damage in Alzheimer’s. They found that amyloid beta peptides, which were once thought to injure synapses directly, actually induce the release of excessive amounts of the neurotransmitter glutamate from brain cells called astrocytes that are located adjacent to the nerve cells.

Normal levels of glutamate promote memory and learning, but excessive levels are harmful. In patients suffering from Alzheimer’s disease, excessive glutamate activates extrasynaptic receptors, designated eNMDA receptors (NMDA stands for N-methyl-D-aspartate), which get hyperactivated and in turn lead to synaptic loss.

How NitroMemantine works

Lipton’s lab had previously discovered how a drug called memantine can be targeted to eNMDA receptors to slow the hyperactivity seen in Alzheimer’s. This patented work contributed to the FDA approval of memantine in 2003 for the treatment of moderate to severe Alzheimer’s disease. However, memantine’s effectiveness has been limited. The reason, the researchers found, was that memantine—a positively charged molecule—is repelled by a similar charge inside diseased neurons; therefore, memantine gets repelled from its intended eNMDA receptor target on the neuronal surface.

In their study, the researchers found that a fragment of the molecule nitroglycerin—a second FDA-approved drug commonly used to treat episodes of chest pain or angina in people with coronary heart disease—could bind to another site that the Lipton group discovered on NMDA receptors. The new drug represents a novel synthesis connecting this fragment of nitroglycerin to memantine, thus representing two FDA-approved drugs connected together. Because memantine rather selectively binds to eNMDA receptors, it also functions to target nitroglycerin to the receptor. Therefore, by combining the two, Lipton’s lab created a new, dual-function drug. The researchers developed 37 derivatives of the combined drug before they found one that worked, Lipton said.

By shutting down hyperactive eNMDA receptors on diseased neurons, NitroMemantine restores synapses between those neurons. “We show in this paper that memantine’s ability to protect synapses is limited,” Lipton said, “but NitroMemantine brings the number of synapses all the way back to normal within a few months of treatment in mouse models of Alzheimer’s disease. In fact, the new drug really starts to work within hours.”

To date, therapies that attack amyloid plaques and neurofibrillary tangles have failed. “It’s quite disappointing because I see really sick patients with dementia. However, I’m now optimistic that NitroMemantine will be effective as we advance to human trials, bringing new hope to both early and later-stage Alzheimer’s patients,” Lipton said.

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.

Scientists Map Process by Which Brain Cells Form Long-Term Memories

Scientists at the Gladstone Institutes have deciphered how a protein called Arc regulates the activity of neurons – providing much-needed clues into the brain’s ability to form long-lasting memories.

These findings, reported Sunday in Nature Neuroscience, also offer newfound understanding as to what goes on at the molecular level when this process becomes disrupted.

Led by Gladstone senior investigator Steve Finkbeiner, MD, PhD, this research delved deep into the inner workings of synapses. Synapses are the highly specialized junctions that process and transmit information between neurons. Most of the synapses our brain will ever have are formed during early brain development, but throughout our lifetimes these synapses can be made, broken and strengthened. Synapses that are more active become stronger, a process that is essential for forming new memories.

However, this process is also dangerous, as it can overstimulate the neurons and lead to epileptic seizures. It must therefore be kept in check.

Neuroscientists recently discovered one important mechanism that the brain uses to maintain this important balance: a process called “homeostatic scaling.” Homeostatic scaling allows individual neurons to strengthen the new synaptic connections they’ve made to form memories, while at the same time protecting the neurons from becoming overly excited. Exactly how the neurons pull this off has eluded researchers, but they suspected that the Arc protein played a key role.

“Scientists knew that Arc was involved in long-term memory, because mice lacking the Arc protein could learn new tasks, but failed to remember them the next day,” said Finkbeiner, who is also a professor of neurology and physiology at UC San Francisco, with which Gladstone is affiliated. “Because initial observations showed Arc accumulating at the synapses during learning, researchers thought that Arc’s presence at these synapses was driving the formation of long-lasting memories.”

But Finkbeiner and his team thought there was something else in play.

The Role of Arc in Homeostatic Scaling

In laboratory experiments, first in animal models and then in greater detail in the petri dish, the researchers tracked Arc’s movements. And what they found was surprising.

“When individual neurons are stimulated during learning, Arc begins to accumulate at the synapses – but what we discovered was that soon after, the majority of Arc gets shuttled into the nucleus,” said Erica Korb, PhD, the paper’s lead author who completed her graduate work at Gladstone and UCSF.

“A closer look revealed three regions within the Arc protein itself that direct its movements: one exports Arc from the nucleus, a second transports it into the nucleus, and a third keeps it there,” she said. “The presence of this complex and tightly regulated system is strong evidence that this process is biologically important.”

In fact, the team’s experiments revealed that Arc acted as a master regulator of the entire homeostatic scaling process. During memory formation, certain genes must be switched on and off at very specific times in order to generate proteins that help neurons lay down new memories.  From inside the nucleus, the authors found that it was Arc that directed this process required for homeostatic scaling to occur. This strengthened the synaptic connections without overstimulating them – thus translating learning into long-term memories. 

Implications for a Variety of Neurological Diseases

“This discovery is important not only because it solves a long-standing mystery on the role of Arc in long-term memory formation, but also gives new insight into the homeostatic scaling process itself – disruptions in which have already been implicated in a whole host of neurological diseases,” said Finkbeiner. “For example, scientists recently discovered that Arc is depleted in the hippocampus, the brain’s memory center, in Alzheimer’s disease patients. It’s possible that disruptions to the homeostatic scaling process may contribute to the learning and memory deficits seen in Alzheimer’s.”

Dysfunctions in Arc production and transport may also be a vital player in autism. For example, the genetic disorder Fragile X syndrome – a common cause of both mental retardation and autism, directly affects the production of Arc in neurons.

“In the future,” added Dr. Korb, “we hope further research into Arc’s role in human health and disease can provide even deeper insight into these and other disorders, and also lay the groundwork for new therapeutic strategies to fight them.”

(Image: Wikimedia)

Alzheimer’s, Schizophrenia, and Autism Now Can Be Studied With Mature Brain Cells Reprogrammed from Skin Cells

Difficult-to-study diseases such as Alzheimer’s, schizophrenia, and autism now can be probed more safely and effectively thanks to an innovative new method for obtaining mature brain cells called neurons from reprogrammed skin cells. According to Gong Chen, the Verne M. Willaman Chair in Life Sciences and professor of biology at Penn State University and the leader of the research team, “the most exciting part of this research is that it offers the promise of direct disease modeling, allowing for the creation, in a Petri dish, of mature human neurons that behave a lot like neurons that grow naturally in the human brain.” Chen added that the method could lead to customized treatments for individual patients based on their own genetic and cellular information. The research will be published in the journal Stem Cell Research.

"Obviously, we don’t want to remove someone’s brain cells to experiment on, so recreating the patient’s brain cells in a Petri dish is the next best thing for research purposes and drug screening," Chen said. Chen explained that, in earlier work, scientists had found a way to reprogram skin cells from patients to become unspecialized or undifferentiated pluripotent stem cells (iPSCs). "A pluripotent stem cell is a kind of blank slate," Chen explained. "During development, such stem cells differentiate into many diverse, specialized cell types, such as a muscle cell, a brain cell, or a blood cell. So, after generating iPSCs from skin cells, researchers then can culture them to become brain cells, or neurons, which can be studied safely in a Petri dish."

Now, in their new research, Chen and his team have found a way to differentiate iPSCs into mature human neurons much more effectively, generating cells that behave similarly to neurons in the brain. Chen explained that, in their natural environment, neurons are always found in close proximity to star-shaped cells called astrocytes, which are abundant in the brain and help neurons to function properly. “Because neurons are adjacent to astrocytes in the brain, we predicted that this direct physical contact might be an integral part of neuronal growth and health,” Chen explained.

To test this hypothesis, Chen and his colleagues began by culturing iPSC-derived neural stem cells, which are stem cells that have the potential to become neurons. These cells were cultured on top of a one-cell-thick layer of astrocytes so that the two cell types were physically touching each other.

"We found that these neural stem cells cultured on astrocytes differentiated into mature neurons much more effectively," Chen said, contrasting them with other neural stem cells that were cultured alone in a Petri dish. "It was almost as if the astrocytes were cheering the stem cells on, telling them what to do, and helping them fulfill their destiny to become neurons."

To demonstrate the superiority of the neurons grown next to astrocytes, Chen and his co-authors used an electrophysiology recording technique to show that the cells grown on astrocytes had many more synaptic events — signals sent out from one nerve cell to the others. In another experiment, after growing the neural stem cells next to astrocytes for just one week, the researchers showed that the newly differentiated neurons start to fire action potentials — the rapid electrical excitation signal that occurs in all neurons in the brain. In a final test, the team members added human neural stem cells to a mixture with mouse neurons. “We found that, after just one week, there was a lot of ‘cross-talk’ between the mouse neurons and the human neurons,” Chen said. He explained that “cross-talk” occurs when one neuron contacts its neighbors and releases a chemical called a neurotransmitter to modulate its neighbor’s activity.

"Previous researchers could only obtain brain cells from deceased patients who had suffered from diseases such as Alzheimer’s, schizophrenia, and autism," Chen said. "Now, researchers can take skin cells from living patients — a safe and minimally invasive procedure — and convert them into brain cells that mimic the activity of the patient’s own brain cells." Chen added that, by using this method, researchers also can figure out how a particular drug will affect a particular patient’s own brain cells, without needing the patient to try the drug — eliminating the risk of serious side effects. "The patient can be his or her own guinea pig for the design of his or her own treatment, without having to be experimented on directly," Chen said.

Weird: Nuclear Bomb Tests Reveal Adults Grow New Brain Cells

Aboveground nuclear bomb testing in the 1950s and 1960s inadvertently gave modern scientists a way to prove the adult brain regularly creates new neurons, research reveals.

Researchers used to believe that the brain changed little once it finished maturing. That view is now considered out of date, as studies have revealed how changeable — or plastic — the adult brain can be.

Much of this plasticity is related to the brain’s organization; brain cells can alter their connections and communications with other brain cells. What has been less clear is whether, and to what extent, the human brain grows brand-new neurons in adulthood.

"There was a lot in the literature showing there was neurogenesis in rodents and every animal studied," said study researcher Kirsty Spalding, a biologist at the Karolinska Institute in Sweden, "But there was very little evidence of whether this happens in humans."

Tantalizing clues

Scientists had reason to believe it does. In adult mice, the hippocampus, a structure deep in the brain involved in memory and navigation, turns over cells all the time. Some of the biological markers linked to this turnover are seen in the human hippocampus. But the only direct evidence of new brain cells forming in the region came from a 1998 study in which researchers looked at the brains of five people who had been injected with a compounded called BrdU that cells take up into their DNA. (The compound was once used in experimental cancer studies, but is not used anymore for safety reasons.)

The BrdU study revealed that neurons in the hippocampuses of the participants contained the compound in their DNA, indicating these brain cells had formed after the injections. The oldest person in the study was 72, suggesting new neuron creation, known as neurogenesis, continues well into old age.

The 1998 study was the only direct evidence of such neurogenesis in the human hippocampus, however. Spalding and her colleagues wanted to change that. Ten years ago, they began a project to track the age of neurons in the human brain using an unusual tool: spare molecules left over from Cold War-era nuclear bomb tests.

Learning to love the bomb

Between 1945 and 1962, the United States conducted hundreds of aboveground nuclear bomb tests. These tests largely stopped with the Limited Test Ban Treaty of 1963, but their effects remained in the atmosphere. The neutrons sent flying by the bombs reacted with nitrogen in the atmosphere, creating a spike in carbon 14, an isotope (or variation) of carbon.

This carbon 14, in turn, did what carbon in the atmosphere does. It combined with oxygen to form carbon dioxide, and was then taken in by plants, which use carbon dioxide in photosynthesis. Humans ate some of these plants, along with some of the animals that also ate these plants, and the carbon 14 inside ended up in their bodies.

When a cell divides, it uses this carbon 14, integrating it into the DNA of the new cells that are forming. Carbon 14 decays over time at a known rate, so scientists can pinpoint from that decay exactly when the new cells were born.

Over the past decade, Spalding and her colleagues have used the technique in a variety of cells, including fat cells, refining it along the way until it became sensitive enough to measure tiny amounts of carbon 14 in small hippocampus samples. The researchers collected samples, with family permission, from autopsies in Sweden.

They found the tantalizing 1998 evidence was correct: Human hippocampuses do grow new neurons. In fact, about a third of the brain region is subject to cell turnover, with about 700 new neurons being formed each day in each hippocampus (humans have two, a mirror-image set on either side of the brain). Hippocampus neurons die each day, too, keeping the overall number more or less in balance, with some slow loss of cells with aging, Spalding said.

This turnover occurs at a ridge in the hippocampus known as the dentate gyrus, a spot known to contribute to the formation of new memories. Researchers aren’t sure what the function of this constant renewal is, but it could relate to allowing the brain to cope with novel situations, Spalding told LiveScience.

"Neurogenesis gives a particular kind of plasticity to the brain, a cognitive flexibility," she said.

Spalding and her colleagues had used the same techniques in other regions of the brain, including the cortex, the cerebellum and the olfactory bulb, and found no evidence of newborn neurons being integrated into those areas. The researchers now plan to study whether there are any links between neurogenesis and psychiatric conditions such as depression.

The new findings are detailed in the journal Cell.

Study Expands Concerns About Anesthesia’s Impact on the Brain

As pediatric specialists become increasingly aware that surgical anesthesia may have lasting effects on the developing brains of young children, new research suggests the threat may also apply to adult brains.

Researchers from Cincinnati Children’s Hospital Medical Center report June 5 the Annals of Neurology that testing in laboratory mice shows anesthesia’s neurotoxic effects depend on the age of brain neurons – not the age of the animal undergoing anesthesia, as once thought.

Although more research is needed to confirm the study’s relevance to humans, the study suggests possible health implications for millions of children and adults who undergo surgical anesthesia annually, according to Andreas Loepke, MD, PhD, a physician and researcher in the Department of Anesthesiology.

“We demonstrate that anesthesia-induced cell death in neurons is not limited to the immature brain, as previously believed,” said Loepke. “Instead, vulnerability seems to target neurons of a certain age and maturational stage. This finding brings us a step closer to understanding the phenomenon’s underlying mechanism”.

New neurons are generated abundantly in most regions of the very young brain, explaining why previous research has focused on that developmental stage. In a mature brain, neuron formation slows considerably, but extends into later life in dentate gyrus and olfactory bulb.

The dentate gyrus, which helps control learning and memory, is the region Loepke and his research colleagues paid particular attention to in their study. Also collaborating were researchers from the University of Cincinnati College of Medicine and the Children’s Hospital of Fudan University, Shanghai, China.

Researchers exposed newborn, juvenile and young adult mice to a widely used anesthetic called isoflurane in doses approximating those used in surgical practice. Newborn mice exhibited widespread neuronal loss in forebrain structures – confirming previous research – with no significant impact on the dentate gyrus. However, the effect in juvenile mice was reversed, with minimal neuronal impact in the forebrain regions and significant cell death in the dentate gyrus.

The team then performed extensive studies to discover that age and maturational stage of the affected neurons were the defining characteristics for vulnerability to anesthesia-induced neuronal cell death. The researchers observed similar results in young adult mice as well.

Research over the past 10 years has made it increasingly clear that commonly used anesthetics increase brain cell death in developing animals, raising concerns from the Food and Drug Administration, clinicians, neuroscientists and the public. As well, several follow-up studies in children and adults who have undergone surgical anesthesia show a link to learning and memory impairment.

Cautioning against immediate application of the current study’s findings to children and adults undergoing anesthesia, Loepke said his research team is trying to learn enough about anesthesia’s impact on brain chemistry to develop protective therapeutic strategies, in case they are needed. To this end, their next step is to identify specific molecular processes triggered by anesthesia that lead to brain cell death.

“Surgery is often vital to save lives or maintain quality of life and usually cannot be performed without general anesthesia,” Loepke said. “Physicians should carefully discuss with patients, parents and caretakers the risks and benefits of procedures requiring anesthetics, as well as the known risks of not treating certain conditions.”

Loepke is also collaborating with researchers from the Pediatric Neuroimaging Research Consortium at Cincinnati Children’s Hospital Medical Center to examine anesthesia’s impact on children’s brain using non-invasive magnetic resonance imaging (MRI) technology.