(Image caption: During development, nerve cells (shown in blue, green, violet and yellow) extend their axons to target leg muscles. If the EphA4 receptors of the growing nerve cells no longer encounter freely accessible ephrins, the axons of many nerve cells (violet) are no longer able to find their partner cells. Credit: © MPI of Neurobiology / Gatto)

Neurons in a forest of signposts

Our ability to move relies on the correct formation of connections between different nerve cells and between nerve and muscle cells. Growing axons of nerve cells are guided to their targets by signposts expressed on the surface of other cells. Very prominent are “do not enter” signs that push axons away. Cell culture studies suggest that protein-cutting enzymes (proteases) remove these signs as soon as they are recognized by the growing axons. In this way, the “bond of recognition” between the axon and the sign is quickly broken, and the axons are more easily guided in a new direction. Scientists from the Max Planck Institute of Neurobiology in Martinsried and the Institut de Recherches Cliniques de Montréal have now shown that proteases indeed control the navigation of growing axons. However, contrary to the current belief, they do so by regulating the number of existing signs. Without proteases, the signposts would be masked and the axons would grow in the wrong direction. These findings clarified how cells form connections during development and may also improve our understanding of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

The human brain consists of about 100 billion nerve cells. During embryonic development each of these cells connects with other cells by means of a long extension, known as axon. Some axons need to navigate long distances through the body to find their correct targets, for example from the spinal cord down to the foot. Only if all these connections are correctly established we can perform basic and fine-tuned movements, such as walking or playing the piano.

It is therefore essential that each nerve cell finds its correct target. But how does an axon navigate and find the appropriate partner cells among billions of other possible targets? “We have now identified a few dozen guidance molecules and their receptors that help axons orient themselves,” says Rüdiger Klein, Director at the Max Planck Institute of Neurobiology. “However, these few receptor-guidance molecule pairs need to control a very large number of navigational decisions. Therefore, there must be some mechanisms to amplify and modulate the effects of these protein pairs”.

Cutting for speed?

Over the last decade, Rüdiger Klein and his team have been studying how nerve cells find their way during development. They are focusing on “do not enter” signs, e.g. ephrin guidance molecules and their Eph receptors. Ephrins and Eph receptors, being present on almost all cell surfaces: on axons as well as on cells in the surrounding tissues, help the growing axons to explore their surroundings and locate their partner cells.

As an axon travels through the body, it docks again and again to other cells via the ephrin/Eph system. This triggers cellular processes, in one or both cells, that eventually cause the connection to be severed and the cells to repel each other, preventing the axon to grow in the wrong direction. It has been hypothesised that this cellular repulsion is accelerated by proteases. Proteases are enzymes that cut Eph receptors and/or ephrins, thus by severing the Eph/ephrin bond between two opposing cells they might expedite the repulsion process. “In this way, proteases could contribute to changes in the guidance process – but this has not yet been experimentally proven.” says Rüdiger Klein.

Not faster, but better

To address this question, the neurobiologists studied how proteases affect the rate of cellular repulsion controlled by EphA4 receptors and ephrins. “Although the experiments in cell culture initially appeared to confirm the theory, we discovered something quite different in living organisms,” states Rüdiger Klein. Contrary to expectations, cellular repulsion proceeded with undiminished accuracy in animals whose axons expressed EphA4 receptors resistant to protease severing. On the other hand, in animals whose axons and muscles expressed EphA4 receptors resistant to protease cutting many axons grew in the wrong direction. Because no cutting occurred, more and more functional EphA4 receptors accumulated on cell surfaces of the leg tissues. This accumulation caused EphA4 receptors to bind to the ephrins on the same cell surface, a phenomena termed as “masking”. Consequently, the ephrins could no longer act as “do not enter” signs for the growing axons. Thus, axons, being no longer repelled, are misguided in a “no entry zone” and are unable to find their correct targets.

These results show that the cleavage of Eph receptors by proteases does not, as expected, accelerate the repulsion reaction. Instead, it regulates the number of functioning receptors and indirectly the number of available ephrins on cells, where they serve as navigational aids. If the balance is disrupted, growing axons are misdirected.

This is an important finding, as EphA4 receptors perform essential functions during the development of neural networks in the brain and in the spinal cord. They are also involved in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In the absence of EphA4 receptors, ALS manifests itself later and develops more slowly in a number of animal models. “It’s possible that the number of EphA4 receptors is kept low by the regulatory activity of proteases,” Rüdiger Klein reflects. “This could provide a way to exert a positive influence on the course of ALS.”

Neurons in human muscles emphasize stimulation from the outside world

Stretch sensors in our muscles participate in reflexes that serve the subconscious control of posture and movement. According to a new study published in the Journal of Neuroscience, these sensors respond weakly to muscle stretch caused by one’s voluntary action, and most strongly to stretch that is imposed by external forces. The ability to reflect causality in this manner can facilitate appropriate reflex control and accurate self-perception.  

“The results of the study show that stretch receptors in our muscles indicate more than which limb is moving or how fast; these sensors also adjust their signals according to who caused the movement,” says Michael Dimitriou, who conducted this study and is currently a post doc at the Department of Integrative Medical Biology, Umeå University, Sweden.

Normally, we can easily distinguish between movements we make ourselves and movements that are imposed on our body by external forces. The ability to discriminate between self-generated and externally generated sensory events is crucial for accurate perception and the control of posture and movement. This ability is also believed to form the foundation on which conscious self-awareness is built.

Such discrimination between self and other has previously been thought to arise as a result of complex computations performed in the brain, that use prior knowledge or memories of the consequences of own actions. But the study by Michael Dimitriou shows that information on the cause of a sensory effect can be provided in real-time by so-called ‘muscle spindles’, a class of stretch receptors found in most of our skeletal muscles.

Muscle spindles differ from other sensory receptors, such as stretch receptors in the skin, because their sensitivity can be controlled by the nervous system via specialized motor neurons. The purpose of this control has been unclear. The neural data presented by Michael Dimitriou indicates that these specialized motor neurons increase the sensitivity of stretch receptors when the body is exposed to an externally imposed stretch stimulus, such as when a falling ball is caught in the hand. Because amplified spindle responses mean stronger stretch reflexes, the resulting muscle activity instantly counteracts movement of the hand. When making a voluntary movement, however, the nervous system ‘automatically’ reduces the sensitivity of spindles in the stretching muscles, thereby making it possible for us to move without setting off strong stretch reflexes that would otherwise counteract movement. Uncontrollably strong stretch reflexes are commonly referred to as ‘spasticity’.

“These results provide an explanation of how reflexes can be functionally adjusted to help us achieve our everyday tasks, without requiring conscious control of reflex sensitivity or complex computations in the brain for predicting the sensory consequences of our actions,” says Michael Dimitriou.

He believes that these new findings are important both for understanding the neural mechanisms that underlie movement control and self-perception, but also for understanding pathological states where these mechanisms are disturbed.

“With these findings, we also get new insights into mechanisms whose malfunction may contribute to neuromuscular problems such as spasticity or alien hand syndrome (also known as ‘Dr. Strangelove syndrome’), and help identify potential treatment targets for these conditions,” says Michael Dimitriou.

New research offers help for spinal cord patients

In a study on rats, researchers at the University of Copenhagen have discovered the cause of the involuntary muscle contractions which patients with severe spinal cord injuries frequently suffer. The findings have just been published in the Journal of Neuroscience and, in the long run, can pave the way for new treatment methods.

Three thousand Danish patients suffer from severe spinal cord injuries after being involved in traffic accidents or accidents at work. An injury to the spinal cord is a catastrophe for the individual, and often results in complete or partial paralysis of the person’s arms and legs. Despite the paralysis, several patients experience problems with involuntary muscle contractions or spasms which impair the patient’s quality of life.

The movements are due to the neurotransmitter serotonin, which normally plays a crucial role in relation to our voluntary control of movements by reinforcing the level of activity in the motor neurones when they have to activate the muscles to an extraordinary degree. Research shows that a group of cells in the spinal cord start supplying serotonin in an uncontrolled way following an injury, and this knocks the motor system out of control.

“We now have a qualified idea of why the serotonin level goes out of control, and we have documented that a special serotonin-producing enzyme plays a key role. By targeting the specific enzyme, in the long term we will be able to devise new methods of treatment when we are trying to impact functions in the nervous system,” says associate professor and neurophysiologist Jacob Wienecke.

The prospects of the study are interesting for both spinal cord patients and patients suffering from Parkinson’s disease.

Emergency response kicks in

The enzyme aromatic L-amino acid decarboxylase (AADC) plays an important role in the production of the neurotransmitter serotonin:

“In the first few days after an injury to the spinal cord, we can see there is a very rapid regulation of AADC which results in the uncontrolled production of serotonin. It is our guess that this is the spinal cord’s emergency response trying to boost the enzyme’s capacity,” says Jacob Wienecke.

According to the researchers, it may be the same emergency response which causes the involuntary movements – dyskinesia – that are also experienced by patients with Parkinson’s disease. However, for Parkinson’s patients, it is the dopamine system which is affected, but the enzyme which activates the emergency response is the same.

“It is an interesting perspective, which will hopefully focus efforts on targeting drugs specifically at the AADC cells. Perhaps in the future we can regulate the undesired neural activity in this way so that the unnecessary ‘disturbance on the line’ disappears for the affected patients,” says Jacob Wienecke.

Existing treatment puts a damper on learning

Existing forms of treatment for spinal cord patients currently involve, for example, using the drug baclofen, which suppresses neural activity, and thereby the motor neurones which cause the involuntary movements. The problem with baclofen though is that it impacts motor learning – and thus the patients’ rehabilitation. However, there is still a long way to go. Developing new drugs is a protracted process, and the way is paved with obstacles. Injuries to the spinal column are extremely complex, and primarily result in interruptions to the signalling between the brain and the body.

“Finding a solution to the problem is no easy task. However, a lot suggests that regulating serotonin production more precisely could mitigate undesirable spasms while also supporting the rehabilitation of controlled movements. So far, the study has been carried out on rats, but we have reason to believe that the same mechanisms apply in humans,” says Jacob Wienecke in conclusion.

Neurons at work

Film editors play a critical role by helping shape raw footage into a narrative. Part of the challenge is that their work can have a profound impact on the finished product — with just a few cuts in the wrong places, comedy can become tragedy, or vice versa.

A similar process, “alternative splicing,” is at work inside the bodies of billions of creatures — including humans. Just as a film editor can change the story with a few cuts, alternative splicing allows cells to stitch genetic information into different formations, enabling a single gene to produce up to thousands of different proteins.

Harvard scientists say they’ve now been able to observe that process within the nervous system of a living creature.

Read more

(Image caption: The presence of p45 (green staining) and p75 (red staining) indicates that motor neurons increase both p45 and p75 expression after sciatic nerve injury in an animal. Image credit: Courtesy of the Salk Institute for Biological Studies)

Scientists uncover new clues to repairing an injured spinal cord

Frogs, dogs, whales, snails can all do it, but humans and primates can’t. Regrow nerves after an injury, that is—while many animals have this ability, humans don’t. But new research from the Salk Institute suggests that a small molecule may be able to convince damaged nerves to grow and effectively rewire circuits. Such a feat could eventually lead to therapies for the thousands of Americans with severe spinal cord injuries and paralysis.

"This research implies that we might be able to mimic neuronal repair processes that occur naturally in lower animals, which would be very exciting," says the study’s senior author and Salk professor Kuo-Fen Lee. The results were published today in PLOS Biology.

For a damaged nerve to regain function, its long, signal-transmitting extensions known as axons need to grow and establish new connections to other cells.

In a study published last summer in PLOS ONE, Lee and his colleagues found that the protein p45 promotes nerve regeneration by preventing the axon sheath (known as myelin) from inhibiting regrowth. However, humans, primates and some other more advanced vertebrates don’t have p45. Instead, the researchers discovered a different protein, p75, that binds to the axon’s myelin when nerve damage occurs in these animals. Instead of promoting nerve regeneration, p75 actually halts growth in damaged nerves.

"We don’t know why this nerve regeneration doesn’t occur in humans. We can speculate that the brain has so many neural connections that this regeneration is not absolutely necessary," Lee says.

In the study published today, the scientists looked at how two p75 proteins bind together and form a pair that latches onto the inhibitors released from damaged myelin.

By studying the configurations of the proteins in solutions using nuclear magnetic resonance (NMR) technology, the researchers found that the growth-promoting p45 could disrupt the p75 pairing.

"For reasons that are not understood, when p45 comes in, it breaks the pair apart," says Lee, holder of the Helen McLoraine Chair in Molecular Neurobiology.

What’s more, the p45 protein was able to bind to the specific region in the p75 protein that is critical for the formation of the p75 pair, thus decreasing the amount of p75 pairs that bond to inhibitors release from myelin. With less p75 pairs available to bond to inhibitor signals, axons were able to regrow.

The findings suggest that an agent—either p45 or another disrupting molecule—that can effectively break the p75 pair could offer a possible therapy for spinal cord damage.

One method of therapy could be to introduce more p45 protein to injured neurons, but a smarter tactic might be to introduce a small molecule that jams the link between the two p75 proteins, Lee says. “Such an agent could possibly get through the blood-brain barrier and to the site of spinal cord injuries,” he says.

The next step will be to see if introducing p45 helps regenerate damaged human nerves. “That is what we hope to do in the future,” Lee says.

Complex Neural Circuitry Keeps You From Biting Your Tongue

Eating, like breathing and sleeping, seems to be a rather basic biological task. Yet chewing requires a complex interplay between the tongue and jaw, with the tongue positioning food between the teeth and then moving out of the way every time the jaw clamps down to grind it up.

If the act weren’t coordinated precisely, the unlucky chewer would end up biting more tongue than burrito.

Duke University researchers have used a sophisticated tracing technique in mice to map the underlying brain circuitry that keeps mealtime relatively painless. The study, which appears June 3 in eLife, could lend insight into a variety of human behaviors, from nighttime teeth grinding to smiling or complex vocalizations.

"Chewing is an activity that you can consciously control, but if you stop paying attention these interconnected neurons in the brain actually do it all for you," said Edward Stanek IV, lead study author and graduate student at Duke University School of Medicine. "We were interested in understanding how this all works, and the first step was figuring out where these neurons reside."

Previous mapping attempts have produced a relatively blurry picture of this chewing control center. Researchers know that the movement of the muscles in the jaw and tongue are governed by special neurons called motoneurons and that these are in turn controlled by another set of neurons called premotor neurons. But the exact nature of these connections — which premotor neurons connect to which motoneurons — has not been defined.

Senior study author Fan Wang, Ph.D., associate professor of neurobiology and a member of the Duke Institute for Brain Sciences, has been mapping neural circuits in mice for many years. Under her guidance, Stanek used a special form of the rabies virus to trace the origins of chewing movements.

The rabies virus works naturally by jumping backwards across neurons until it has infected the entire brain of its victim. For this study, Stanek used a genetically disabled version of rabies that could only jump from the muscles to the motoneurons, and then back to the premotor neurons. The virus also contained a green or red fluorescent tag, which enabled the researchers to see where it landed after it was done jumping.

Stanek injected these fluorescently labeled viruses into two muscles, the tongue-protruding genioglossus muscle and the jaw-closing masseter muscle. He found that a group of premotor neurons simultaneously connect to the motoneurons that regulate jaw opening and those that trigger tongue protrusion. Similarly, he found another group that connects to both motoneurons that regulate jaw closing and those responsible for tongue retraction. The results suggest a simple method for coordinating the movement of the tongue and jaw that usually keeps the tongue safe from injury.

"Using shared premotor neurons to control multiple muscles may be a general feature of the motor system," said Stanek. "For other studies on the rest of the brain, it is important to keep in mind that individual neurons can have effects in multiple downstream areas."

The researchers are interested in using their technique to jump even further back in the mouse brain, eventually mapping the circuitry all the way up to the cortex. But first they plan to delve deeper into the connections between the premotor and motoneurons.

"This is just a small step in understanding the control of these orofacial movements," Stanek said. "We only looked at two muscles and there are at least 10 other muscles active during chewing, drinking, and speech. There is still a lot of work to look at these other muscles, and only then can we get a complete picture of how these all work as a unit to coordinate this behavior," said Stanek.

Motor cortex shown to play active role in learning movement patterns

Skilled motor movements of the sort tennis players employ while serving a tennis ball or pianists use in playing a concerto, require precise interactions between the motor cortex and the rest of the brain. Neuroscientists had long assumed that the motor cortex functioned something like a piano keyboard.

"Every time you wanted to hear a specific note, there was a specific key to press," says Andrew Peters, a neurobiologist at UC San Diego’s Center for Neural Circuits and Behavior. "In other words, every specific movement of a muscle required the activation of specific cells in the motor cortex because the main job of the motor cortex was thought to be to listen to the rest of the cortex and press the keys it’s directed to press."

But in a study published in this week’s advance online publication of the journal Nature, Peters, the first author of the paper, and his colleagues found that the motor cortex itself plays an active role in learning new motor movements. In a series of experiments using mice, the researchers showed in detail how those movements are learned over time.

"Our finding that the relationship between body movements and the activity of the part of the cortex closest to the muscles is profoundly plastic and shaped by learning provides a better picture of this process," says Takaki Komiyama, an assistant professor of biology at UC San Diego who headed the research team. "That’s important, because elucidating brain plasticity during learning could lead to new avenues for treating learning and movement disorders, including Parkinson’s disease."

With Simon Chen, another UC San Diego neurobiologist, the researchers monitored the activity of neurons in the motor cortex over a period of two weeks while mice learned to press a lever in a specific way with their front limbs to receive a reward.

"What we saw was that during learning, different patterns of activity—which cells are active, when they’re active—were evident in the motor cortex," says Peters. "This ends up translating to different patterns of activity even for similar movements. Once the animal has learned the movement, similar movements are then accompanied by consistent activity. This consistent activity moreover is totally new to the animal: it wasn’t used early in learning even with movements that were similar to the later movement."

"Early on," Peters says, "the animals will occasionally make movements that look like the expert movements they make after learning. The patterns of brain activity that accompany those similar early and late movements are actually completely different though. Over the course of learning, the animal generates a whole new set of activity in the motor cortex to make that movement. In the piano keyboard analogy, that’s like using one key to make a note early on, but a different key to make the same note later."

Studies Identify Spinal Cord Neurons that Control Skilled Limb Movement

Researchers have identified two types of neurons that enable the spinal cord to control skilled forelimb movement. The first is a group of excitatory interneurons that are needed to make accurate and precise movements; the second is a group of inhibitory interneurons necessary for achieving smooth movement of the limbs. The findings are important steps toward understanding normal human motor function and potentially treating movement disorders that arise from injury or disease.

“We take for granted many motor behaviors, such as catching a ball or flipping a coin, that in fact require considerable planning and precision,” said Columbia University Medical Center’s (CUMC’s) Thomas M. Jessell, PhD, a senior author of both studies, which were published separately in recent issues of Nature (1, 2). “While such motor acts seem effortless, they depend on intricate and carefully orchestrated communication between neural networks that connect the brain to the spinal cord and muscles.”

To move one’s hand to a desired target, the brain sends the spinal cord signals, which activate the motor neurons that control limb muscles. During subsequent movements, information from the limb is conveyed back to the brain and spinal cord, providing a feedback system that can support the control and adjustment of motor output.

“But feedback from muscles is not quick enough to permit the most rapid real-time adjustments of fine motor control,” said Dr. Jessell, “suggesting that there may be other, faster, systems at play.” Dr. Jessell is the Claire Tow Professor of Motor Neuron Disorders in the Departments of Neuroscience and of Biochemistry and Molecular Biophysics, co-director of the Mortimer B. Zuckerman Mind Brain Behavior Institute, co-director of the Kavli Institute for Brain Science, and a Howard Hughes Medical Institute investigator, all at Columbia.

Researchers had suspected that one rapid form of feedback might derive from a group of interneurons in the cervical spinal cord called propriospinal neurons (PNs). Like many other neurons, PNs send signals to motor neurons that innervate arm muscles and trigger movement. But this subset of neurons also has a distinct output branch that projects away from motor neurons towards the cerebellum. Through this dual-branched anatomy, these neurons have the potential to carry internal copies of motor output signals up to the brain.

However, the nature of this internal feedback pathway and whether it has any impact on movement have not been clear. “If PNs were indeed sending copies of outgoing motor commands to the brain, they could provide a conveniently rapid means of adjusting ongoing movements when things go awry,” said Eiman Azim, PhD, a postdoctoral fellow in Dr. Jessell’s lab and lead author of the first paper. “But without a way to selectively target the copy function of PNs, there was no way to test this theory.”

The CUMC team, in collaboration with Bror Alstermark, PhD, a professor in integrative medical biology at Umeå University in Sweden, overcame this technical barrier by developing a genetic method for accessing and eliminating PNs in mice, abolishing both motor-directed and copy signals sent by the neurons. When the researchers quantified the limb movements of the PN-deprived mice in three dimensions as they reached for food pellets, they found that the mice’s ability to reach for the target accurately was badly compromised. “Basically, their movements were uncoordinated,” said Dr. Azim. “The PN-deprived mice consistently over- or under-reached.”

But with both PN output signals gone, the precise role of the PN copy signal remained unclear. The researchers then turned to optogenetics—the use of light to control neuronal activity. They selectively activated the copy axonal branch alone, decalibrating this copy signal from the version sent to motor neurons. With the copy signal altered, the animals’ ability to reach was severely compromised, indicating that the PN copy pathway is capable of influencing the outcome of goal-directed reaching movements.

The PN copy signal also works blazingly fast. It takes just 4 to 5 milliseconds for motor neuron activity to be altered after transmission of a PN copy signal. “These reaching movements typically take 200 to 300 milliseconds, so the PN copy signal pathway appears well equipped to correct arm movements,” said Dr. Azim. The researchers think that this copy signal represents just one of many similar internal feedback pathways that the spinal cord and brain use to validate and correct movements throughout the body.

Are these findings relevant to human motor performance? Many of the pathways and circuits that influence reach and grasp in monkeys and humans are conserved in mice. “We need to learn more about these pathways before we can evaluate how their dysfunction contributes to deficits seen after spinal cord injury and neurodegenerative disease,” said Dr. Azim.

In the second Nature study, CUMC researchers examined how spinal circuits regulate sensory feedback from muscles to control movement. The simplest form of this feedback system involves a reflex pathway (such as the knee-jerk reflex), in which sensory endings in muscles convey signals to the motor system through direct monosynaptic connections with motor neurons. Signals from motor neurons, in turn, cause muscles to contract, completing the reflex cycle.

Researchers have long wondered how the strength of this sensory signal might be regulated. Studies had shown that spinal interneurons—in particular those that release the neurotransmitter GABA, inhibiting neuronal activity—play a key role in this process. But most GABA-releasing interneurons exert their effects postsynaptically, by blocking the excitation of neurons on the receiving end of a synapse (the gap across which two neurons communicate).

“We knew that such neurons are unlikely to be responsible for fine-tuning the sensory signal,” said lead author Andrew J. P. Fink, PhD, a former graduate student in Dr. Jessell’s lab. “Postsynaptic inhibition affects the entire neuron, and motor neurons receive many different inputs. So a mechanism that shut down the motor neuron to all of its inputs would lack refinement.”

Researchers have long speculated that one subset of GABAergic interneurons might regulate movement by controlling the strength of sensory feedback signals from muscles. “These particular neurons are known to work presynaptically, by forming direct connections with the terminals of sensory neurons and suppressing the release of sensory neurotransmitter,” said Dr. Fink. For technical reasons, the function of these interneurons, if any, in motor behavior has remained elusive.

Dr. Fink and his colleagues identified a way to access this subset of interneurons genetically in mice and then devised approaches to manipulate their function in a selective manner. In one experiment, they activated presynaptic inhibitory interneurons optogenetically, decreasing the strength of sensory-motor transmission. They also ablated these interneurons by making them selectively sensitive to a lethal toxin, abolishing their control over sensory feedback strength. Without sensory feedback regulation, forelimb movements were dominated by severe oscillatory tremors, drastically diminishing motor accuracy.

This finding, along with parallel modeling studies, indicates that presynaptic inhibitory neurons normally adjust the “gain” of sensory feedback at synapses with motor neurons and are therefore crucial for the smooth execution of movement. Understanding how these basic microcircuits regulate sensory input and motor output may, in the long run, provide insight into ways to combat the movement instability and tremor seen in many neurological disorders.

“These two studies shed new light on how discrete classes of spinal interneurons empower the nervous system to direct motor behaviors in ways that match the particular task at hand,” said Dr. Jessell.