(Image caption: Three-dimensional reconstruction of a synapse in the mouse brain. Readily releasable fusionable synaptic vesicles (blue, around 45 millionths of a millimetre in diameter) are docked at the cell membrane. Credit: © MPI f. Experimental Medicine/ Benjamin H. Cooper)

Synapses always on the starting blocks

While neurons rapidly propagate information in their interior via electrical signals, they communicate with each other at special contact points known as the synapses. Chemical messenger substances, the neurotransmitters, are stored in vesicles at the synapses. When a synapse becomes active, some of these vesicles fuse with the cell membrane and release their contents. To ensure that valuable time is not lost, synapses always have some readily releasable vesicles on standby. With the help of high-resolution, three-dimensional electron microscopy, scientists at the Max Planck Institute of Experimental Medicine in Göttingen succeeded in demonstrating that these fusionable vesicles have a very special characteristic: they already have close contact with the cell membrane long before the actual fusion occurs. In addition, the research team also decoded the molecular machinery that facilitates the operation of this docking mechanism.

The fusion of the neurotransmitter vesicles with the cell membrane involves close cooperation between numerous protein components, which monitor each other and ensure that every single ‘participant’ is always in the right place. This is referred to as the fusion machinery and the comparison is an apt one: if a cogwheel in a clock mechanism is broken, the hands do not move. In a similar way, faulty or missing molecules impair synaptic operations.

In research studies carried out some years ago, Nils Brose and his colleague JeongSeop Rhee from the Max Planck Institute of Experimental Medicine in Göttingen already demonstrated that the transmission of information at the synapses in genetically modified mice, in which all known genes of the Munc13 or CAPS proteins had been switched off, is severely defective. Although the neurons of the genetically modified mice do not differ from those of healthy mice when examined under an optical microscope, if Munc13 is missing, the release of neurotransmitters actually grinds to a halt completely. Brose and Rhee’s findings showed that to be able to react immediately to signals at all times, each synapse must keep a small number of ‘readily releasable’ fusionable vesicles on standby.

But how do Munc13 and CAPS convert the vesicles to this kind of fusionable state? To answer this question, the Göttingen-based scientists studied the synaptic contacts in the minutest possible detail. To do this, neurobiologists Cordelia Imig and Ben Cooper, who have been working with Brose and Rhee for many years, used a high-pressure freezing process. This involves the rapid freezing of neurons in the brain tissue under high pressure so that no disruptive ice crystals are formed and the fine structure of the cells is particularly well conserved. The samples obtained in this way were then analysed using electron tomography. Using this method, electron microscope images of a structure are recorded from many different angles, in a similar way to the process used in medical computed tomography. The individual images can then be combined on the computer to give a high-resolution three-dimensional image – of a synapse in this case (see image).

“Our results showed that readily releasable vesicles in healthy synapses touch the cell membrane,” explains Cooper. “However, if Munc13 and CAPS proteins are missing, the vesicles do not reach the active zone and accumulate a few nanometres away from it.” To their astonishment, the researchers also observed that SNARE proteins, which collaborate with Munc13 and CAPS in the nerve endings, are also involved in this docking process. SNARE proteins are found in the cell and vesicle membranes of healthy synapses and control the fusion of the two membranes during neurotransmitter release. When a vesicle approaches the cell membrane, the individual SNARE molecules line up opposite each other like the sides of a zip and pull the membranes close to each other in this way. The vesicles await the starting gun for their fusion in this state – in the starting blocks, so to speak.

The findings of the neurobiologists in Göttingen prove that Munc13, CAPS and SNARE proteins closely align the vesicle and cell membrane in the synapse, long before the signal for fusion is given. This is the only way that the fast and controlled transmission of information at the synapse can be guaranteed, thanks to which we can react specifically to information from our environment. “It had long been clear that synapses have to be extremely fast to carry out all of the many complex brain functions. Our study shows for the first time how this is managed at the molecular level and on the level of the synaptic vesicles,” says Brose. Because almost all of the protein components involved in this process also play a role in neurological and psychiatric diseases, the Göttingen-based scientists believe that their discovery will soon benefit medical research.

(Image caption: The structure determines the function: AMPA receptors in the nerve cells of the brain are composed of a range of more than 30 different proteins. Source: Bernd Fakler)

Understanding the Components of Memory

Dr. Uwe Schulte, Dr. Jochen Schwenk, Prof. Dr. Bernd Fakler, and their team have elucidated the enormous spatial and temporal dynamics in protein composition of the AMPA-type glutamate receptors, the most important excitatory neurotransmitter receptors in the brain. These receptors are located in the synapses, the contact points between two nerve cells, where they are responsible for the rapid signal transduction and information processing. The results illustrate that the receptors are far more diverse than previously anticipated and pave the way for research into their functions in the various regions of the brain. The biologists published their findings in the journal Neuron.

The researchers have thus opened up the possibility to investigate the properties and functions of the AMPA receptors in the various regions of the brain at the level of their protein components. This is of particular significance as the AMPA receptors and their dynamics are regarded as central elements for memory formation. The researchers succeeded in elucidating the subunit structure of the AMPA receptors in various regions of the brain and even in different groups of distinct nerve cells. It became clear that the receptors exhibit an enormous range of variation in structure and molecular architecture and can evidently be precisely adapted to the function of the nerve cells and brain region in which they are located. In addition, the researchers demonstrated that this diversity in protein composition of the receptors is also exploited during the development of the brain.

In 2012, Fakler’s research team already used novel proteomic technologies to show that AMPA receptors in the brain are assembled from a pool of more than 30 different proteins - whose primary function(s) is are most parts as yet unknown. In fact in another recent study, also published in Neuron, the researchers demonstrated just how significant these unknown components are or can be: They showed that the cornichon protein dictates the time course of the AMPA receptor-mediated synaptic transmission and thus accounts for the difference between various types of nerve cells in the brain.

Scientists Link Alcohol-Dependence Gene to Neurotransmitter

Scientists at The Scripps Research Institute (TSRI) have solved the mystery of why a specific signaling pathway can be associated with alcohol dependence.

This signaling pathway is regulated by a gene, called neurofibromatosis type 1 (Nf1), which TSRI scientists found is linked with excessive drinking in mice. The new research shows Nf1 regulates gamma-aminobutyric acid (GABA), a neurotransmitter that lowers anxiety and increases feelings of relaxation.

“This novel and seminal study provides insights into the cellular mechanisms of alcohol dependence,” said TSRI Associate Professor Marisa Roberto, a co-author of the paper. “Importantly, the study also offers a correlation between rodent and human data.”

In addition to showing that Nf1 is key to the regulation of the GABA, the research, which was published recently in the journal Biological Psychiatry, shows that variations in the human version of the Nf1 gene are linked to alcohol-dependence risk and severity in patients.

Pietro Paolo Sanna, associate professor at TSRI and the study’s corresponding author, was optimistic about the long-term clinical implications of the work. “A better understanding of the molecular processes involved in the transition to alcohol dependence will foster novel strategies for prevention and therapy,” he said.

A Genetic Culprit

Researchers have long sought a gene or genes that might be responsible for risk and severity of alcohol dependence. “Despite a significant genetic contribution to alcohol dependence, few risk genes have been identified to date, and their mechanisms of action are generally poorly understood,” said TSRI Staff Scientist Vez Repunte-Canonigo, co-first author of the paper with TSRI Research Associate Melissa Herman.

This research showed that Nf1 is one of those rare risk genes, but the TSRI researchers weren’t sure exactly how Nf1 affected the brain. The TSRI research team suspected that Nf1 might be relevant to alcohol-related GABA activity in an area of the brain called the central amygdala, which is important in decision-making and stress- and addiction-related processes.

“As GABA release in the central amygdala has been shown to be critical in the transition from recreational drinking to alcohol dependence, we thought that Nf1 regulation of GABA release might be relevant to alcohol consumption,” said Herman.

The team tested several behavioral models, including a model in which mice escalate alcohol drinking after repeated withdrawal periods, to study the effects of partially deleting Nf1. In this experiment, which simulated the transition to excessive drinking that is associated with alcohol dependence in humans, they found that mice with functional Nf1 genes steadily increased their ethanol intake starting after just one episode of withdrawal. Conversely, mice with a partially deleted Nf1 gene showed no increase in alcohol consumption.

Investigating further, the researchers found that in mice with partially deleted Nf1 genes, alcohol consumption did not further increase GABA release in the central amygdala. In contrast, in mice with functional Nf1 genes, alcohol consumption resulted in an increase in central amygdala GABA.

In the second part of the study, a collaboration with a distinguished group of geneticists at various U.S. institutions, the team analyzed data on human variations of the Nf1 gene from about 9,000 people. The results showed an association between the gene and alcohol-dependence risk and severity.

The team sees the new findings as “pieces to the puzzle.” Sanna believes future research should focus on exactly how Nf1 regulates the GABA system and how gene expression may be altered during early development.

(Image caption: Positron-Emission-Tomography (PET) of a depressive patient without medication (left) with elevated monoamine-oxidase-A-levels (green, yellow, red) and after a six-week-treatment with the monoamine-oxidase-A-inhibitor moclobemid (right). Credit: © Sacher et al., 2011, J Psy Neurosci.)

Monoamine oxidase A: biomarker for postpartum depression

Many women suffer from baby blues after giving birth. Some even develop full-blown postpartum depression in the weeks that follow. Monoamine oxidase A, an enzyme responsible for the breakdown of neurotransmitters like dopamine and serotonin, plays an important role in this condition. In comparison to healthy women, women who experience postpartum depression present strongly elevated levels of the enzyme in their brains. This was discovered by a Canadian-German research team including Julia Sacher from the Max Planck Institute for Human Cognitive and Brain Sciences in Leipzig. Their findings could help in the prevention of postpartum depression and in the development of new drugs for its treatment.

For most women, the birth of their baby is one of the most strenuous but also happiest days in their lives. However, joy and happiness are often followed by fatigue and exhaustion. The vast majority of women experience a temporary drop in mood for a few days after birth. These symptoms of “baby blues” are not an illness; however, in some cases they can represent early signs of an imminent episode of depression: in 13 percent of mothers, the emotional turmoil experienced after childbirth leads to the development of a full-blown postpartum depression. Postpartum depression is harmful not only to the mother, but also to the baby. It is difficult to treat this condition effectively, as its precise neurobiological causes have remained unidentified to date.

The new study shows that postpartum depression is accompanied by strongly elevated monoamine oxidase A in the brain, particularly in the prefrontal cortex and in the anterior cingulate cortex. In women with postpartum depression, the values recorded were 21 percent higher than those of women who were not plagued by negative feelings after giving birth. Women who did not develop full-blown depression but found themselves crying more often than usual due to depressed mood also presented moderately elevated values.

“Therefore, we should promote strategies that help to reduce monoamine oxidase A levels in the brain, and avoid everything that makes these values rise,” explains Sacher. Such factors include heavy smoking, alcohol consumption and chronic stress, for example when the mother feels neglected and abandoned by her partner and family. “My ultimate goal is to provide women and their families with very concrete lifestyle recommendations that will enable them to prevent postpartum depression,” explains the psychiatrist.

A new generation of long-established drugs could also play an important role in the treatment of postpartum depression in future. Up to now, depressed mothers are mainly given drugs that increase the concentration of serotonin in the brain. However, because monoamine oxidase A breaks down not only serotonin but also other monoamines like dopamine and noradrenaline, a treatment that directly targets monoamine oxidase A could have a higher success rate, particularly in very serious cases: this alternative is provided by selective and reversible monoamine-oxidase- A inhibitors. “The first monoamine oxidase inhibitors often had severe side effects, for example hypertensive crises, which necessitated adherence to a strict diet,” explains Sacher. “However, the new selective and reversible drugs are better tolerated,” she adds. In the next stage of this research involving clinical trials, the scientists intend to test the effectiveness of these reversible monoamine oxidase A inhibitors in the treatment of postpartum depression.

Because the measurement of this enzyme in the brain requires complex technology, it is not suitable for routine testing. Thus, the researchers are also looking for a peripheral marker of this enzyme that can be detected in saliva or blood.

Four years ago, Julia Sacher and her colleagues at the Centre for Addiction and Mental Health CAMH in Toronto already succeeded in showing that, in the first week postpartum, the concentration of the enzyme monoamine oxidase A in the brain is on average 40 percent higher than in women who had not recently given birth. “The monoamine oxidase A values behave in the opposite way to oestrogen levels. When oestrogen levels drop acutely after childbirth, the concentration of monoamine oxidase A rises. This drastic change also influences serotonin levels, known as the happiness hormone,” explains Dr. Sacher. In most women, the values quickly return to normal. In others, they remain raised – and thereby promote the development of depression.

Anxiety in invertebrates opens research avenues

For the first time, CNRS researchers and the Université de Bordeaux have produced and observed anxiety-like behavior in crayfish, which disappears when a dose of anxiolytic is injected. This work, published in Science on June 13, 2014, shows that the neuronal mechanisms related to anxiety have been preserved throughout evolution. This analysis of ancestral behavior in a simple animal model opens up new avenues for studying the neuronal bases for this emotion.

Anxiety can be defined as a behavioral response to stress, consisting in lasting apprehension of future events. It prepares individuals to detect threats and anticipate them appropriately so as to increase their chances of survival. However, when stress is chronic, anxiety becomes pathological and may lead to depression.

Until now, non-pathological anxiety had only been described in humans and a few vertebrates. For the first time, it has been observed in an invertebrate. To achieve this, researchers at the Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (CNRS/Université de Bordeaux) and the Institut des Maladies Neurodégénératives (CNRS/Université de Bordeaux) repeatedly exposed crayfish to an electric field for thirty minutes. They then placed the crayfish in an aquatic cross-shaped maze. Two arms of the maze were lit up (which repels the crustaceans) and two were dark—which they find reassuring.

The researchers analyzed the exploratory behavior of the crayfish. Those made anxious tended to remain in the dark areas of the maze, by contrast to control crayfish, which explored the entire maze. This behavior is an adaptive response to a felt stress: the animal aims to minimize the risk of meeting an attacker. This emotional state wore itself out after about one hour.

Anxiety in crayfish is correlated to increased serotonin concentration in their brains. Neurotransmitter serotonin is involved in regulating many physiological processes in both invertebrates and humans. It is released when stress is experienced and regulates several responses related to anxiety, such as increasing blood glucose levels. The researchers have also highlighted that injecting an anxiolytic commonly used in humans (benzodiazepine) stops the prevention behavior in crayfish. This shows how early neural mechanisms that trigger or inhibit anxiety-like behavior appeared in the evolutionary process and that they have been well preserved over time.

This work provides researchers specializing in stress and anxiety with a unique animal model. Crayfish have a simple nervous system whose neurons are easy to record, so they may shed light on the neuronal mechanisms at work when stress is experienced, as well as on the role of neurotransmitters such as serotonin or GABA. The team now plans to study anxiety in crayfish subject to social stress and the neuronal changes that occur when the anxiety is prolonged for several days.

Can Chemicals Produced by Gut Microbiota Affect Children with Autism?

Children with autism spectrum disorders (ASD) have significantly different concentrations of certain bacterial-produced chemicals, called metabolites, in their feces compared to children without ASD. This research, presented at the annual meeting of the American Society for Microbiology, provides further evidence that bacteria in the gut may be linked to autism.

“Most gut bacteria are beneficial, aiding food digestion, producing vitamins, and protecting against harmful bacteria. If left unchecked, however, harmful bacteria can excrete dangerous metabolites or disturb a balance in metabolites that can affect the gut and the rest of the body, including the brain,” says Dae-Wook Kang of the Biodesign Institute of Arizona State University, an author on the study.

Increasing evidence suggests that children with ASD have altered gut bacteria. In order to identify possible microbial metabolites associated with ASD Kang and his colleagues looked for and compared the compounds in fecal samples from children with and without ASD. They found that children with ASD had significantly different concentrations of seven of the 50 compounds they identified.

“Most of the seven metabolites could play a role in the brain, working as neurotransmitters or controlling neurotransmitter biosynthesis,” says Kang. “We suspect that gut microbes may alter levels of neurotransmitter-related metabolites affecting gut-to-brain communication and/or altering brain function.”

Children with ASD had significantly lower levels of the metabolites homovanillate and N,N-dimethylglycine. Homovanillate is the breakdown product of dopamine (a major neurotransmitter), indicating an imbalance in dopamine catabolism (the breaking down in living organisms of more complex substances into simpler ones with the release of energy). N,N-dimethylglycine is a building block for proteins and neurotransmitters, and has been used to reduce symptoms of ASD and epileptic seizures.

The glutamine/glutamate ratio was significantly higher in children with ASD. Glutamine and glutamate are further metabolized to gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter. An imbalance between glutamate and GABA transmission has been associated with ASD-like behaviors such as hyper-excitation.

Using next-generation sequencing technology, the researchers also were able to detect hundreds of unique bacterial species and confirmed that children with ASD harbored distinct and less diverse gut bacterial composition. 

“Correlations between gut bacteria and neurotransmitter-related metabolites are stepping stones for a better understanding of the crosstalk between gut bacteria and autism, which may provide potential targets for diagnosis or treatment of neurological symptoms in children with ASD,” says Kang.

(Image: Thinkstock)

Brain Noise Found to Nurture Synapses

A study has shown that a long-overlooked form of neuron-to-neuron communication called miniature neurotransmission plays an essential role in the development of synapses, the regions where nerve impulses are transmitted and received. The findings, made in fruit flies, raise the possibility that abnormalities in miniature neurotransmission may contribute to neurodevelopmental diseases. The findings, by researchers at Columbia University Medical Center (CUMC), were published today in the online edition of the journal Neuron.

The primary way in which neurons communicate with each another is through “evoked neurotransmission.” This process begins when an electrical signal, or action potential, is transmitted along a long, cable-like extension of the neuron called an axon. Upon reaching the axon’s terminus, the signal triggers the release of chemicals called neurotransmitters across the synapse. Finally, the neurotransmitters bind to and activate receptors of the neuron on the other side of the synapse. Neurotransmitters are packaged together into vesicles, which are released by the hundreds, if not thousands, with each action potential. Evoked neurotransmission was first characterized in the 1950s by Sir Bernard Katz and two other researchers, who were awarded the 1970 Nobel Prize in Physiology or Medicine for their efforts.

“Dr. Katz also found that even without action potentials, lone vesicles are released now and then at the synapse,” said study leader Brian D. McCabe, PhD, assistant professor of pathology and cell biology and of neuroscience in the Motor Neuron Center. “These miniature events — or minis — have been found at every type of synapse that has been studied. However, since minis don’t induce neurons to fire, people assumed they were inconsequential, just background noise.”

Recent cell-culture studies, however, have suggested that minis do have some function and even their own regulatory mechanisms. “This led us to wonder why there would be such complicated mechanisms for regulating something that was just noise,” said Dr. McCabe.

To learn more about minis, the CUMC team devised new genetic tools to selectively up- or down-regulate evoked and miniature neurotransmission in fruit flies (a commonly used model organism for neuronal function and development). This was the first study to identify a unique role for minis in an animal model.

The researchers found that when both types of neurotransmission were blocked, synapse development was abnormal. However, inhibiting or stimulating evoked neurotransmission alone had no effect on synaptic development. “But when we blocked minis, synapses failed to develop,” said Dr. McCabe, “and when we stimulated the release of more minis, synapses got bigger.”

The study also showed that minis regulate synapse development by activating a signaling pathway in neurons involving Trio and Rac1 proteins in presynaptic neurons. These proteins are also found in humans.

It remains to be seen exactly how minis are exerting their effects. “Parallel communication occurs in computer networks,” Dr. McCabe said. “Computers communicate primarily by sending bursts of data bundled into packets. But individual computers also send out pings, or tiny electronic queries, to determine if there is a connection to other computers. Similarly, neurons may be using minis to ping connected neurons, saying in effect, ‘We are connected and I am ready to communicate.’”

The researchers are currently looking into whether minis have a functional role in the mature nervous system. If so, it’s possible that defects in minis could contribute to neurodegenerative disease.

Nociceptin: Nature’s Balm for the Stressed Brain

Collaborating scientists at The Scripps Research Institute (TSRI), the National Institutes of Health (NIH) and the University of Camerino in Italy have published new findings on a system in the brain that naturally moderates the effects of stress. The findings confirm the importance of this stress-damping system, known as the nociceptin system, as a potential target for therapies against anxiety disorders and other stress-related conditions.

“We were able to demonstrate the ability of this nociceptin anti-stress system to prevent and even reverse some of the cellular effects of acute stress in an animal model,” said biologist Marisa Roberto, associate professor in TSRI’s addiction research department, known as the Committee on the Neurobiology of Addictive Disorders.

Roberto was a principal investigator for the study, which appears in the January 8, 2014 issue of the Journal of Neuroscience.

A Variety of Effects

Nociceptin, which is produced in the brain, belongs to the family of opioid neurotransmitters. But the resemblance essentially ends there. Nociceptin binds to its own specific receptors called NOP receptors and doesn’t bind well to other opioid receptors. The scientists who discovered it in the mid-1990s also noted that when nociceptin is injected into the brains of mice, it doesn’t kill pain—it makes pain worse.

The molecule was eventually named for this “nociceptive” (pain-producing) effect. However, subsequent studies demonstrated that, by activating its corresponding receptor NOP, nociceptin acted as an antiopioid and not only affected pain perception, but also blocked the rewarding properties of opioids such as morphine and heroin.

Perhaps of greatest interest, several studies in rodents have found evidence that nociceptin can act in the amygdala, a part of the brain that controls basic emotional responses, to counter the usual anxiety-producing effects of acute stress. There have been hints, too, that this activity occurs automatically as part of a natural stress-damping feedback response.

Scientists have wanted to know more about the anti-stress activity of the nociceptin/NOP system, in part because it might offer a better way to treat stress-related conditions. The latter are common in modern societies, including post-traumatic stress disorder as well as the drug-withdrawal stress that often defeats addicts’ efforts to kick the habit.

Reducing the Stress Reaction

For the new study, Roberto and her collaborators looked in more detail at the nociceptin/NOP system in the central amygdala.

First, Markus Heilig’s laboratory at the National Institute on Alcohol Abuse and Alcoholism (NIAAA), part of the NIH, measured the expression of NOP-coding genes in the central amygdala in rats. Heilig’s team found strong evidence that stress changes the activity of nociceptin/NOP in this region, indicating that the system does indeed work as a feedback mechanism to damp the effects of stress. In animals subjected to a standard laboratory stress condition, NOP gene activity rose sharply, as if to compensate for the elevated stress.

Roberto and her laboratory at TSRI then used a separate technique to measure the electrical activity of stress-sensitive neurons in the central amygdala. As expected, this activity rose when levels of the stress hormone CRF rose and started out at even higher levels in the stressed rats. But this stress-sensitive neuronal activity could be dialed down by adding nociceptin. The stress-blocking effect was especially pronounced in the restraint-stressed rats—probably due to their stress-induced increase in NOP receptors.

Finally, biologist Roberto Ciccocioppo and his laboratory at the University of Camerino conducted a set of behavioral experiments showing that injections of nociceptin specifically into the rat central amygdala powerfully reduced anxiety-like behaviors in the stressed rats, but showed no behavioral effect in non-stressed rats.

The three sets of experiments together demonstrate, said Roberto, that “stress exposure leads to an over-activation of the nociceptin/NOP system in the central amygdala, which appears to be an adaptive feedback response designed to bring the brain back towards normalcy.”

In future studies, she and her colleagues hope to determine whether this nociceptin/NOP feedback system somehow becomes dysfunctional in chronic stress conditions. “I suspect that chronic stress induces changes in amygdala neurons that can contribute to the development of some anxiety disorders,” said Roberto.

Compounds that mimic nociceptin by activating NOP receptors—but, unlike nociceptin, could be taken in pill form—are under development by pharmaceutical companies. Some of these appear to be safe and well tolerated in lab animals and may soon be ready for initial tests in human patients, Ciccocioppo said.