Watching Molecules Morph into Memories

In two studies in the January 24 issue of Science (1, 2), researchers at Albert Einstein College of Medicine of Yeshiva University used advanced imaging techniques to provide a window into how the brain makes memories. These insights into the molecular basis of memory were made possible by a technological tour de force never before achieved in animals: a mouse model developed at Einstein in which molecules crucial to making memories were given fluorescent “tags” so they could be observed traveling in real time in living brain cells.

Efforts to discover how neurons make memories have long confronted a major roadblock: Neurons are extremely sensitive to any kind of disruption, yet only by probing their innermost workings can scientists view the molecular processes that culminate in memories. To peer deep into neurons without harming them, Einstein researchers developed a mouse model in which they fluorescently tagged all molecules of messenger RNA (mRNA) that code for beta-actin protein – an essential structural protein found in large amounts in brain neurons and considered a key player in making memories. mRNA is a family of RNA molecules that copy DNA’s genetic information and translate it into the proteins that make life possible.

"It’s noteworthy that we were able to develop this mouse without having to use an artificial gene or other interventions that might have disrupted neurons and called our findings into question," said Robert Singer, Ph.D., the senior author of both papers and professor and co-chair of Einstein’s department of anatomy & structural biology and co-director of the Gruss Lipper Biophotonics Center at Einstein. He also holds the Harold and Muriel Block Chair in Anatomy & Structural Biology at Einstein.

In the research described in the two Science papers, the Einstein researchers stimulated neurons from the mouse’s hippocampus, where memories are made and stored, and then watched fluorescently glowing beta-actin mRNA molecules form in the nuclei of neurons and travel within dendrites, the neuron’s branched projections. They discovered that mRNA in neurons is regulated through a novel process described as “masking” and “unmasking,” which allows beta-actin protein to be synthesized at specific times and places and in specific amounts.

"We know the beta-actin mRNA we observed in these two papers was ‘normal’ RNA, transcribed from the mouse’s naturally occurring beta-actin gene," said Dr. Singer. "And attaching green fluorescent protein to mRNA molecules did not affect the mice, which were healthy and able to reproduce."

Neurons come together at synapses, where slender dendritic “spines” of neurons grasp each other, much as the fingers of one hand bind those of the other. Evidence indicates that repeated neural stimulation increases the strength of synaptic connections by changing the shape of these interlocking dendrite “fingers.” Beta-actin protein appears to strengthen these synaptic connections by altering the shape of dendritic spines. Memories are thought to be encoded when stable, long-lasting synaptic connections form between neurons in contact with each other.

The first paper describes the work of Hye Yoon Park, Ph.D., a postdoctoral student in Dr. Singer’s lab at the time and now an instructor at Einstein. Her research was instrumental in developing the mice containing fluorescent beta-actin mRNA—a process that took about three years.

Dr. Park stimulated individual hippocampal neurons of the mouse and observed newly formed beta-actin mRNA molecules within 10 to 15 minutes, indicating that nerve stimulation had caused rapid transcription of the beta-actin gene. Further observations suggested that these beta-actin mRNA molecules continuously assemble and disassemble into large and small particles, respectively. These mRNA particles were seen traveling to their destinations in dendrites where beta-actin protein would be synthesized.

In the second paper, lead author and graduate student Adina Buxbaum of Dr. Singer’s lab showed that neurons may be unique among cells in how they control the synthesis of beta-actin protein.

"Having a long, attenuated structure means that neurons face a logistical problem," said Dr. Singer. "Their beta-actin mRNA molecules must travel throughout the cell, but neurons need to control their mRNA so that it makes beta-actin protein only in certain regions at the base of dendritic spines."

Ms. Buxbaum’s research revealed the novel mechanism by which brain neurons handle this challenge. She found that as soon as beta-actin mRNA molecules form in the nucleus of hippocampal neurons and travel out to the cytoplasm, the mRNAs are packaged into granules and so become inaccessible for making protein. She then saw that stimulating the neuron caused these granules to fall apart, so that mRNA molecules became unmasked and available for synthesizing beta-actin protein.

But that observation raised a question: How do neurons prevent these newly liberated mRNAs from making more beta-actin protein than is desirable? “Ms. Buxbaum made the remarkable observation that mRNA’s availability in neurons is a transient phenomenon,” said Dr. Singer. “She saw that after the mRNA molecules make beta-actin protein for just a few minutes, they suddenly repackage and once again become masked. In other words, the default condition for mRNA in neurons is to be packaged and inaccessible.”

These findings suggest that neurons have developed an ingenious strategy for controlling how memory-making proteins do their job. “This observation that neurons selectively activate protein synthesis and then shut it off fits perfectly with how we think memories are made,” said Dr. Singer. “Frequent stimulation of the neuron would make mRNA available in frequent, controlled bursts, causing beta-actin protein to accumulate precisely where it’s needed to strengthen the synapse.”

To gain further insight into memory’s molecular basis, the Singer lab is developing technologies for imaging neurons in the intact brains of living mice in collaboration with another Einstein faculty member in the same department, Vladislav Verkhusha, Ph.D. Since the hippocampus resides deep in the brain, they hope to develop infrared fluorescent proteins that emit light that can pass through tissue. Another possibility is a fiberoptic device that can be inserted into the brain to observe memory-making hippocampal neurons.

Researchers reveal more about how our brains control our arms

Ready, set, go.

Sometimes that’s how our brains work. When we anticipate a physical act, such as reaching for the keys we noticed on the table, the neurons that control the task adopt a state of readiness, like sprinters bent into a crouch.

Other times, however, our neurons must simply react, such as if someone were to toss us the keys without gesturing first, to prepare us to catch.

How do the neurons in the brain control planned versus unplanned arm movements?

Krishna Shenoy, a Stanford professor of electrical engineering, neurobiology (by courtesy) and bioengineering (affiliate), wanted to answer that question as part of his group’s ongoing efforts to develop and improve brain-controlled prosthetic devices.

In a paper published today in the journal Neuron, Shenoy and first author Katherine Cora Ames, a doctoral student in the Neurosciences Graduate Program, present a mathematical analysis of the brain activity of monkeys as they make anticipated and unanticipated reaching motions.

Monitoring the neurons

The experimental data came from recording the electrical activity of neurons in the brain that control motor and premotor functions. The idea was to observe and understand the activity levels of these neurons during experiments in which the monkeys made planned or reactive arm movements. What the researchers found is that when the monkeys knew what arm movement they were supposed to make and were simply waiting for the cue to act, electrical readings showed that the neurons went into what scientists call the prepare-and-hold state – the brain’s equivalent of ready, set, waiting for the cue to go.

But when the monkeys made unplanned or unexpected movements, the neurons did not go through the expected prepare-and-hold state. “This was a surprise,” Ames said.

Before the experiment, the researchers had believed that a prepare-and-hold state had to precede movement. In short, they thought the neurons had to go into a “ready, set” crouch before acting on the “go” command. But they discovered otherwise in three variations of an experiment involving similar arm movements.

Experimental design

In all three cases, the monkeys were trained to touch a target that appeared on a display screen.

During each motion, the researchers measured the electrical activity of the neurons in control of arm movements.

In one set of experiments, the monkeys were shown the target but were trained not to touch it until they got the “go” signal. This is called a delayed reach experiment. It served as the planned action.

In a second set of experiments the monkeys were trained to touch the target as soon as it appeared. This served as the unplanned action.

In a third variant, the position of the target was changed. It briefly appeared in one location on the screen. The target then reappeared in a different location. This required the monkeys to revise their movement plan.

Monkey see, then monkey do

Ames said that, in all three instances, the first information to reach the neurons was awareness of the target.

“Perception always occurred first,” Ames said.

Then, about 50 milliseconds later, some differences appeared in the data. When the monkeys had to wait for the go command, the brain recordings showed that the neurons went into a discernable prepare-and-hold state. But in the other two cases, the neurons did not enter the prepare-and-hold state.

Instead, roughly 50 milliseconds after the electrical readings showed evidence of perception, a change in neuronal activity signaled the command to touch the target; it came with no apparent further preparation between perception and action. “Ready, set” was unnecessary. In these instances, the neurons just said, “Go!”

Applications

“This study changes our view of how movement is controlled,” Ames said. “First you get the information about where to move. Then comes the decision to move. There is no specific prepare-and-hold stage unless you are waiting for the signal to move.”

These nuanced understandings are important to Shenoy. His lab develops and improves electronic systems that can convert neural activity into electronic signals in order to control a prosthetic arm or move the cursor on a computer screen.

One example of such efforts is the BrainGate clinical trial here at Stanford, now being conducted under U.S. Food & Drug Administration supervision, to test the safety of brain-controlled, computer cursor systems – “think-and-click” communication for people who can’t move.

“In addition to advancing basic brain science, these new findings will lead to better brain-controlled prosthetic arms and communication systems for people with paralysis,” Shenoy said.

Long-term spinal cord stimulation stalls symptoms of Parkinson’s-like disease

Researchers at Duke Medicine have shown that continuing spinal cord stimulation appears to produce improvements in symptoms of Parkinson’s disease, and may protect critical neurons from injury or deterioration.

The study, performed in rats, is published online Jan. 23, 2014, in the journal Scientific Reports. It builds on earlier findings from the Duke team that stimulating the spinal cord with electrical signals temporarily eased symptoms of the neurological disorder in rodents.

"Finding novel treatments that address both the symptoms and progressive nature of Parkinson’s disease is a major priority," said the study’s senior author Miguel Nicolelis, M.D., Ph.D., professor of neurobiology at Duke University School of Medicine. "We need options that are safe, affordable, effective and can last a long time. Spinal cord stimulation has the potential to do this for people with Parkinson’s disease."

Parkinson’s disease is caused by the progressive loss of neurons that produce dopamine, an essential molecule in the brain, and affects movement, muscle control and balance.

L-dopa, the standard drug treatment for Parkinson’s disease, works by replacing dopamine. While L-dopa helps many people, it can cause side effects and lose its effectiveness over time. Deep brain stimulation, which emits electrical signals from an implant in the brain, has emerged as another valuable therapy, but less than 5 percent of those with Parkinson’s disease qualify for this treatment.

"Even though deep brain stimulation can be very successful, the number of patients who can take advantage of this therapy is small, in part because of the invasiveness of the procedure," Nicolelis said.

In 2009, Nicolelis and his colleagues reported in the journal Science that they developed a device for rodents that sends electrical stimulation to the dorsal column, a main sensory pathway in the spinal cord carrying information from the body to the brain. The device was attached to the surface of the spinal cord in rodents with depleted levels of dopamine, mimicking the biologic characteristics of someone with Parkinson’s disease. When the stimulation was turned on, the animals’ slow, stiff movements were replaced with the active behaviors of healthy mice and rats.

Because research on spinal cord stimulation in animals has been limited to the stimulation’s acute effects, in the current study, Nicolelis and his colleagues investigated the long-term effects of the treatment in rats with the Parkinson’s-like disease.

For six weeks, the researchers applied electrical stimulation to a particular location in the dorsal column of the rats’ spinal cords twice a week for 30-minute sessions. They observed a significant improvement in the rats’ symptoms, including improved motor skills and a reversal of severe weight loss.

In addition to the recovery in clinical symptoms, the stimulation was associated with better survival of neurons and a higher density of dopaminergic innervation in two brain regions controlling movement – the loss of which cause Parkinson’s disease in humans. The findings suggest that the treatment protects against the loss or damage of neurons.

Clinicians are currently using a similar application of dorsal column stimulation to manage certain chronic pain syndromes in humans. Electrodes implanted over the spinal cord are connected to a portable generator, which produces electrical signals that create a tingling sensation to relieve pain. Studies in a small number of humans worldwide have shown that dorsal column stimulation may also be effective in restoring motor function in people with Parkinson’s disease.

"This is still a limited number of cases, so studies like ours are important in examining the basic science behind the treatment and the potential mechanisms of why it is effective," Nicolelis said.

The researchers are continuing to investigate how spinal cord stimulation works, and are beginning to explore using the technology in other neurological motor disorders.

Fighting Flies

When one encounters a group of fruit flies invading their kitchen, it probably appears as if the whole group is vying for a sweet treat. But a closer look would likely reveal the male flies in the group are putting up more of a fight, particularly if ripe fruit or female flies are present. According to the latest studies from the fly laboratory of California Institute of Technology (Caltech) biologist David Anderson, male Drosophilae, commonly known as fruit flies, fight more than their female counterparts because they have special cells in their brains that promote fighting. These cells appear to be absent in the brains of female fruit flies.  

"The sex-specific cells that we identified exert their effects on fighting by releasing a particular type of neuropeptide, or hormone, that has also been implicated in aggression in mammals including mouse and rat," says Anderson, the Seymour Benzer Professor of Biology at Caltech, and corresponding author of the study. "In addition, there are some recent papers implicating increased levels of this hormone in people with personality disorders that lead to higher levels of aggression."

The team’s findings are outlined in the January 16 version of the journal Cell.

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Scientists discover two proteins that control chandelier cell architecture

Chandelier cells are neurons that use their unique shape to act like master circuit breakers in the brain’s cerebral cortex. These cells have dozens, often hundreds, of branching axonal projections – output channels from the cell body of the neuron – that lend the full structure of a chandelier-like appearance. Each of those projections extends to a nearby excitatory neuron. The unique structure allows just one inhibitory chandelier cell to block or modify the output of literally hundreds of other cells at one time.

Without such large-scale inhibition, some circuits in the brain would seize up, as occurs in epilepsy. Abnormal chandelier cell function also has been implicated in schizophrenia. Yet after nearly 40 years of research, little is known about how these important inhibitory neurons develop and function.

In work published today in Cell Reports, a team led by CSHL Professor Linda Van Aelst identifies two proteins that control the structure of chandelier cells, and offers insight into how they are regulated.

To study the architecture of chandelier cells, Van Aelst and colleagues first had to find a way to visualize them. Generally, scientists try to find a unique marker, a sort of molecular signature, to distinguish one type of neuron from the many others in the brain. But no markers are known for chandelier cells. So Van Aelst and Yilin Tai, Ph.D., lead author on the study, developed a way to label chandelier cells within the mouse brain.

Using this new method, the team found two proteins, DOCK7 and ErbB4, whose activity is essential in processes that give chandelier cells their striking shape. When the function of these proteins is disrupted, chandelier cells have fewer, more disorganized, axonal projections. Van Aelst and colleagues used a series of biochemical experiments to explore the relationship between the two proteins. They found that DOCK7 activates ErbB4 through a previously unknown mechanism; this activation must occur if chandelier cells are to develop their characteristic architecture.

Moving forward, Van Aelst says she is interested in exploring the relationship between structure and function of chandelier cells. “We envisage that morphological changes are likely to impact the function of chandelier cells, and consequently, alter the activity of cortical networks. We believe irregularities in these networks contribute to the cognitive abnormalities characteristic of schizophrenia and epilepsy. As we move forward, therefore, we hope that our findings will improve our understanding of these devastating neurological disorders.”

How Vision Captures Sound Now Somewhat Uncertain

When listening to someone speak, we also rely on lip-reading and gestures to help us understand what the person is saying.

To link these sights and sounds, the brain has to know where each stimulus is located so it can coordinate processing of related visual and auditory aspects of the scene. That’s how we can single out a conversation when it’s one of many going on in a room.

While past research has shown that the brain creates a similar code for vision and hearing to integrate this information, Duke University researchers have found the opposite: neurons in a particular brain region respond differently, not similarly, based on whether the stimuli is visual or auditory.

The finding, which posted Jan. 15 in the journal PLOS ONE, provides insight into how vision captures the location of perceived sound.

The idea among brain researchers has been that the neurons in a brain area known as the superior colliculus employ a “zone defense” when signaling where stimuli are located. That is, each neuron monitors a particular region of an external scene and responds whenever a stimulus — either visual or auditory — appears in that location. Through teamwork, the ensemble of neurons provides coverage of the entire scene.

But the study by Duke researchers found that auditory neurons don’t behave that way. When the target was a sound, the neurons responded as if playing a game of tug-of-war, said lead author Jennifer Groh, a professor of psychology and neuroscience at Duke.   

"The neurons responded to nearly all sound locations. But how vigorously they responded depended on where the sound was," Groh said. "It’s still teamwork, but a different kind. It’s pretty cool that the neurons can use two different strategies, play two different games, at the same time."

Groh said the finding opens up a mystery: if neurons respond differently to visual and auditory stimuli at similar locations in space, then the underlying mechanism of how vision captures sound is now somewhat uncertain.

"Which neurons are ‘on’ tells you where a visual stimulus is located, but how strongly they’re ‘on’ tells you where an auditory stimulus is located," said Groh, who conducted the study with co-author Jung Ah Lee, a postdoctoral fellow at Duke.

"Both of these kinds of signals can be used to control behavior, like eye movements, but it is trickier to envision how one type of signal might directly influence the other." 

The study involved assessing the responses of neurons, located in the rostral superior colliculus of the midbrain, as two rhesus monkeys moved their eyes to visual and auditory targets.

The sensory targets — light-emitting diodes attached to the front of nine speakers — were placed 58 inches in front of the animals. The speakers were located from 24 degrees left to 24 degrees right of the monkey in 6-degree increments.  

The researchers then measured the monkey’s responses to bursts of white noise and the illuminating of the lights.

Groh said how the brain takes raw input of one form and converts it into something else “may be broadly useful for more cognitive processes.”

"As we develop a better understanding of how those computations unfold it may help us understand a little bit more about how we think," she said.

[Figure 1: Synaptic signaling occurs when neurotransmitter molecules (glutamate) released by the presynaptic neuron travel through the synaptic cleft to activate glutamate receptors, including NMDA receptors, on the postsynaptic neuron. Image courtesy of the National Institute on Aging]

Amplifying communication between neurons

Neurons send signals to each other across small junctions called synapses. Some of these signals involve the flow of potassium, calcium and sodium ions through channel proteins that are embedded within the membranes of neurons. However, it was unclear whether the flow of potassium ions into the synaptic cleft had a physiological purpose. An international team of researchers including Alexey Semyanov from the RIKEN Brain Science Institute has now revealed that potassium ions that leak out of channel proteins and spill into the synapse augment synaptic signaling between neurons, potentially fulfilling a reinforcement mechanism in learning and memory.

Synaptic communication between neurons begins when calcium ions enter the axon terminal of one neuron—the presynaptic neuron—causing the release of neurotransmitter molecules, such as glutamate, which travel across the synaptic cleft and bind to receptor proteins on the surface of the receiving or postsynaptic neuron (Fig. 1). When the glutamate binds to a receptor known as the NMDA receptor, a channel in the receptor protein opens and calcium flows in, which initiates activation of the postsynaptic neuron.

Semyanov and his colleagues found that the opening of the NMDA receptor channel on the postsynaptic neuron also allows potassium ions to flow out of that neuron and into the synaptic cleft. Blocking the NMDA receptor prevented the rise in potassium ions within the synaptic cleft.

The NMDA receptor is generally blocked by magnesium ions, but these ions can be released from the receptor channel upon repetitive stimulation of the postsynaptic neuron. Through mathematical modeling and subsequent experiments, Semyanov and his colleagues found that potassium levels in the synaptic cleft could increase dramatically on removal of magnesium or during repeated activation of the postsynaptic neuron.

The rise in potassium in the synaptic cleft was shown to increase calcium entry into the presynaptic neuron axon terminal when the postsynaptic neuron was stimulated, and enhanced the probability that the glutamate neurotransmitter would be released from the presynaptic neuron. In this way, repeated activation of a given neuronal network, such as during learning, could augment the strength of communication between neurons, making it more likely that a given stimulus would trigger the activation of postsynaptic neurons.

"New memories are associated with long-term changes in synaptic strength following repetitive activation of the synapse, commonly known as synaptic plasticity," explains Semyanov. "Potassium accumulation and the consequent increase in probability of glutamate release can potentially aid the induction of synaptic plasticity, thus facilitating learning and memory," he says.