(Image caption: A series of three MRI images (top row) shows how dopamine concentrations change over time in the brain’s ventral striatum. Photocollage: Christine Daniloff/MIT, with images courtesy of the researchers)

Delving deep into the brain

MRI sensor allows neuroscientists to map neural activity with molecular precision

Launched in 2013, the national BRAIN Initiative aims to revolutionize our understanding of cognition by mapping the activity of every neuron in the human brain, revealing how brain circuits interact to create memories, learn new skills, and interpret the world around us.

Before that can happen, neuroscientists need new tools that will let them probe the brain more deeply and in greater detail, says Alan Jasanoff, an MIT associate professor of biological engineering. “There’s a general recognition that in order to understand the brain’s processes in comprehensive detail, we need ways to monitor neural function deep in the brain with spatial, temporal, and functional precision,” he says.

Jasanoff and colleagues have now taken a step toward that goal: They have established a technique that allows them to track neural communication in the brain over time, using magnetic resonance imaging (MRI) along with a specialized molecular sensor. This is the first time anyone has been able to map neural signals with high precision over large brain regions in living animals, offering a new window on brain function, says Jasanoff, who is also an associate member of MIT’s McGovern Institute for Brain Research.

His team used this molecular imaging approach, described in the May 1 online edition of Science, to study the neurotransmitter dopamine in a region called the ventral striatum, which is involved in motivation, reward, and reinforcement of behavior. In future studies, Jasanoff plans to combine dopamine imaging with functional MRI techniques that measure overall brain activity to gain a better understanding of how dopamine levels influence neural circuitry.

“We want to be able to relate dopamine signaling to other neural processes that are going on,” Jasanoff says. “We can look at different types of stimuli and try to understand what dopamine is doing in different brain regions and relate it to other measures of brain function.”

Tracking dopamine

Dopamine is one of many neurotransmitters that help neurons to communicate with each other over short distances. Much of the brain’s dopamine is produced by a structure called the ventral tegmental area (VTA). This dopamine travels through the mesolimbic pathway to the ventral striatum, where it combines with sensory information from other parts of the brain to reinforce behavior and help the brain learn new tasks and motor functions. This circuit also plays a major role in addiction.

To track dopamine’s role in neural communication, the researchers used an MRI sensor they had previously designed, consisting of an iron-containing protein that acts as a weak magnet. When the sensor binds to dopamine, its magnetic interactions with the surrounding tissue weaken, which dims the tissue’s MRI signal. This allows the researchers to see where in the brain dopamine is being released. The researchers also developed an algorithm that lets them calculate the precise amount of dopamine present in each fraction of a cubic millimeter of the ventral striatum.

After delivering the MRI sensor to the ventral striatum of rats, Jasanoff’s team electrically stimulated the mesolimbic pathway and was able to detect exactly where in the ventral striatum dopamine was released. An area known as the nucleus accumbens core, known to be one of the main targets of dopamine from the VTA, showed the highest levels. The researchers also saw that some dopamine is released in neighboring regions such as the ventral pallidum, which regulates motivation and emotions, and parts of the thalamus, which relays sensory and motor signals in the brain.

Each dopamine stimulation lasted for 16 seconds and the researchers took an MRI image every eight seconds, allowing them to track how dopamine levels changed as the neurotransmitter was released from cells and then disappeared. “We could divide up the map into different regions of interest and determine dynamics separately for each of those regions,” Jasanoff says.

He and his colleagues plan to build on this work by expanding their studies to other parts of the brain, including the areas most affected by Parkinson’s disease, which is caused by the death of dopamine-generating cells. Jasanoff’s lab is also working on sensors to track other neurotransmitters, allowing them to study interactions between neurotransmitters during different tasks.

Controlling Brain Waves to Improve Vision

Have you ever accidently missed a red light or a stop sign? Or have you  heard someone mention a visible event that you passed by but totally missed seeing?

“When we have different things competing for our attention, we can only be aware of so much of what we see,” said Kyle Mathewson, Beckman Institute Postdoctoral Fellow. “For example, when you’re driving, you might really be concentrating on obeying traffic signals.”

But say there’s an unexpected event: an emergency vehicle, a pedestrian, or an animal running into the road—will you actually see the unexpected, or will you be so focused on your initial task that you don’t notice?

“In the car, we may see something so brief or so faint, while we’re paying attention to something else, that the event won’t come into our awareness,” says Mathewson. “If you present this scenario hundreds of times to someone, sometimes they will see the unexpected event, and sometimes they won’t because their brain is in a different preparation state.”

By using a novel technique to test brain waves, Mathewson and colleagues are discovering how the brain processes external stimuli that do and don’t reach our awareness. A paper about their results, “Dynamics of Alpha Control: Preparatory Suppression of Posterior Alpha Oscillations by Frontal Modulators Revealed with Combined EEG and Event-related Optical Signal,” published this month in the Journal of Cognitive Neuroscience, reveals how alpha waves, typically thought of as your brain’s electrical activity while it’s at rest, can actually influence what we see or don’t see.

The researchers used both electroencephalography (EEG) and the event-related optical signal (EROS), developed in the Cognitive Neuroimaging Laboratory of Gabriele Gratton and Monica Fabiani, professors of psychology and members of the Beckman Institute’s Cognitive Neuroscience Group, and authors of the study.

While EEG records the electrical activity along the scalp, EROS uses infrared light passed through optical fibers to measure changes in optical properties in the active areas of the cerebral cortex. Because of the hard skull between the EEG sensors and the brain, it can be difficult to find exactly WHERE signals are produced. EROS, which examines how light is scattered, can noninvasively pinpoint activity within the brain.

“EROS is based on near-infrared light,” explained Fabiani and Gratton via email. “It exploits the fact that when neurons are active, they swell a little, becoming slightly more transparent to light: this allows us to determine when a particular part of the cortex is processing information, as well as where the activity occurs.”

This allowed the researchers to not only measure activity in the brain, but also allowed them to map where the alpha oscillations were originating. Their discovery: the alpha waves are produced in the cuneus, located in the part of the brain that processes visual information.

The alpha can inhibit what is processed visually, making it hard for you to see something unexpected.

By focusing your attention and concentrating more fully on what you are experiencing, however, the executive function of the brain can come into play and provide “top-down” control—putting a brake on the alpha waves, thus allowing you to see things that you might have missed in a more relaxed state.

“We found that the same brain regions known to control our attention are involved in suppressing the alpha waves and improving our ability to detect hard-to-see targets,” said Diane Beck, a member of the Beckman’s Cognitive Neuroscience Group, and one of the study’s authors.

“Knowing where the waves originate means we can target that area specifically with electrical stimulation” said Mathewson. “Or we can also give people moment-to-moment feedback, which could be used to alert drivers that they are not paying attention and should increase their focus on the road ahead, or in other situations alert students in a classroom that they need to focus more, or athletes, or pilots and equipment operators.”

The study examined 16 subjects and mapped the electrical and optical data onto individual MRI brain images.

(Image caption: Channelrhodopsins before (upper left) and after (lower right) molecular engineering, shown superimposed over an image of a mammalian neuron. In the upper left opsin, the red color shows negative charges spanning the opsin that facilitated the flow of positive (stimulatory) ions through the channel into neurons. In the newly engineered channels (lower right), those negative charges have been changed to positive (blue), allowing the negatively charged inhibitory chloride ions to flow through. Credit: Andre Berndt, Soo Yeun Lee, Charu Ramakrishnan, and Karl Deisseroth.)

Researchers Build New “Off Switch” to Shut Down Neural Activity

Nearly a decade ago, the era of optogenetics was ushered in with the development of channelrhodopsins, light-activated ion channels that can, with the flick of a switch, instantaneously turn on neurons in which they are genetically expressed. What has lagged behind, however, is the ability to use light to inactivate neurons with an equal level of reliability and efficiency. Now, Howard Hughes Medical Institute (HHMI) scientists have used an analysis of channelrhodopsin’s molecular structure to guide a series of genetic mutations to the ion channel that grant the power to silence neurons with an unprecedented level of control.

The new structurally engineered channel at last gives neuroscientists the tools to both activate and inactivate neurons in deep brain structures using dim pulses of externally projected light. HHMI early career scientist Karl Deisseroth and his colleagues at Stanford University published their findings April 25, 2014 in the journal Science. “We’re excited about this increased light sensitivity of inhibition in part because we think it will greatly enhance work in large-brained organisms like rats and primates,” he says.

First discovered in unicellular green algae in 2002, channelrhodopsins function as photoreceptors that guide the microorganisms’ movements in response to light. In a landmark 2005 study, Deisseroth and his colleagues described a method for expressing the light-sensitive proteins in mouse neurons. By shining a pulse of blue light on those neurons, the researchers showed they could reliably induce the ion channel at channelrhodopsin’s core to open up, allowing positively charged ions to rush into the cell and trigger action potentials. Channelrhodopsins have since been used in hundreds of research projects investigating the neurobiology of everything from cell dynamics to cognitive functions.

A few years later came the deployment of halorhodopsins, light-sensitive proteins selective for the negatively charged ion chloride. These proteins, derived from halobacteria, provided researchers with a tool for the light-controlled inactivation of neurons. A major limitation of these proteins, however, is their inefficiency. Unlike channelrhodopsin, halorhodopsin is an ion pump, meaning that only one chloride ion moves across the neuron’s membrane per photon of light. “What that translates into is you get partial inhibition,” Deisseroth says. “You can inhibit neurons, but in the living animal it’s not always complete.”

Searches for a naturally occurring light-sensitive channel with a pore permeable to negatively charged ions have come up empty handed. “We searched,” Deisseroth says. “We did big genomic searches and found many interesting channelrhodopsins and lots of pumps, but we never found an inhibitory channel in nature.”

The team’s fruitless exploration led them to try modifying the molecular structure of channelrhodopsin so that its pore would shuttle negative ions into the cell. “To do that you need to know what the channel pore looks like at the angstrom level,” Deisseroth says. “What we really needed was the high-resolution crystal structure.” In 2012, working with a group in Japan, Deisseroth and his colleagues captured the structure of a chimera of channelrhodopsin called C1C2 using X-ray crystallography.

A molecular analysis of channelrhodopsin’s pore suggested that swapping out certain negatively charged amino acid residues lining the pore with positive residues could reverse the electrostatic potential of the channel, making it more conductive to negatively charged ions such as chloride. To achieve this molecular switcheroo, the researchers performed dozens of single site-directed mutations. Several mutations conferred selectivity for chloride, but the channels failed to conduct current. So, the team screened hundreds of combinations of mutations. “In a systematic process we found first a combination of four mutations, and then a group of five mutations, that seemed to change selectivity,” says Deisseroth. “We put those together into a nine-fold mutated channel and that one, amazingly, was chloride selective.”

Not only does the new channel—dubbed iC1C2 for “inhibitory C1C2”—allow the selective passage of chloride ions, it greatly reduces the likelihood of action potentials by making the neuron more “leaky,” a function not possible in ion pumps like halorhodopsin.

Deisseroth’s team made a final mutation to a cysteine residue in iC1C2 that makes the channel both bi-stable and orders of magnitude more sensitive to light. When activated by blue light, the mutated channels remain open for up to minutes at a time, while exposing the channels to red light makes them close quickly. This level of long-term control is useful in developmental studies where events play out over minutes to hours. The long channel open times also mean that neurons can essentially integrate chloride currents over longer time scales and, therefore, weaker light can be used to inhibit the neurons. Increased light sensitivity translates to less light-induced damage to neural tissue, the ability to reach deep brain structures, and the possibility of controlling brain functions that involve large regions of the brain.

“This is something we’ve sought for many years and it’s really the culmination of many streams of work in the lab—crystal structure work, mutational work, behavioral work —all of which have come together here,” Deisseroth says.

Rapid whole-brain imaging with single cell resolution

Eavesdropping on brain cell chatter

Everything we do — all of our movements, thoughts and feelings – are the result of neurons talking with one another, and recent studies have suggested that some of the conversations might not be all that private. Brain cells known as astrocytes may be listening in on, or even participating in, some of those discussions. But a new mouse study suggests that astrocytes might only be tuning in part of the time — specifically, when the neurons get really excited about something. This research, published in Neuron, was supported by the National Institute of Neurological Disorders and Stroke (NINDS), part of the National Institutes of Health.

For a long time, researchers thought that the star-shaped astrocytes (the name comes from the Greek word for star) were simply support cells for the neurons.

It turns out that these cells have a number of important jobs, including providing nutrients and signaling molecules to neurons, regulating blood flow, and removing brain chemicals called neurotransmitters from the synapse. The synapse is the point of information transfer between two neurons. At this connection point, neurotransmitters are released from one neuron to affect the electrical properties of the other. Long arms of astrocytes are located next to synapses, where they can keep tabs on the conversations going on between neurons.

In recent years, it has been shown that astrocytes may also play a role in neuronal communication. When neurons release neurotransmitters, levels of calcium change within astrocytes. Calcium is critical for many processes, including release of molecules from the cell, and activation of a host of proteins within the cell. The role of this astrocytic calcium signaling for brain function remains a mystery.

In this study, Baljit S. Khakh, Ph.D., of the University of California, Los Angeles and his colleagues wanted to know when astrocytes responded to neuron activity with changes in their internal calcium levels. Using calcium indicator dyes, the researchers were able to image, for the first time, changes in calcium levels in the entire astrocyte. Previously, it was only possible to look at certain areas of the cell at one time, which provided an incomplete picture of what was happening.

Dr. Khakh said one of the most important outcomes of this work was in the methods that were used. “What our use of these calcium indicators shows is that we can image calcium throughout the entire astrocyte. This provides a new set of tools for the research community to use and to extend these findings,” he said.

“There has been intense interest in understanding how astrocytes facilitate communication between neurons, but it is only recently that studies with this level of precision have been possible,” said Edmund Talley, Ph.D., program director at NINDS. “Dr. Khakh’s study is an example of an exciting basic, or fundamental, research project that could have an important contribution to the shifting field of astrocyte biology,” he added.

For these experiments, researchers focused on the mossy fiber pathway, which connects two areas of the hippocampus, the structure involved in learning and memory. “This pathway has a unique architecture and although it has been very well studied, the role of astrocytes in this circuit has not been previously explored. This study provides one of the first really detailed understandings of astrocytes within this particular circuit,” said Dr. Khakh.

Dr. Khakh’s team activated neurons (getting them to release neurotransmitter by a variety of techniques) and then looked for a response in the neighboring astrocyte. As calcium levels rose, the astrocyte would light up quickly. They discovered that two neurotransmitters, glutamate and GABA, triggered the astrocytes to release calcium from their internal stores. Importantly, the researchers discovered that calcium levels increased through the entire astrocyte only if there was a large burst of neurotransmitter being released.

“We found that astrocytes in the mossy fiber pathway do not listen to the constant, millisecond by millisecond synaptic chatter that neurons engage in. Instead, they listen when neurons get excessively excited during bursts of activation,” said Dr. Khakh.

These findings suggest that astrocytes in the mossy fiber system may act as a switch that reacts to large amounts of neuronal activity by raising their levels of calcium. These calcium increases occur over multiple seconds, a relatively long time period compared to that seen in neurons. The spatial extent of the astrocyte calcium increases was also relatively large in comparison to the size of the synapse.

“Astrocytes may be sitting there quietly and when there is excessive activation in the neuronal circuit, they immediately respond with an increase in calcium which we could detect. And the next big question becomes, what they do with that calcium?” said Dr. Khakh.

Dr. Khakh’s results in the mossy fiber system differ from those others have described in other brain regions. This raises the intriguing possibility that astrocytes are not all the same and may serve various roles throughout the brain.

“It would be really interesting and important to find that astrocytes function differently in different areas of the brain, in a circuit-specific manner. This study gives a hint that this might be true,” said Dr. Talley.

Researchers Close In On The Most Important Question In Neuroscience With Fly Study

By scrutinizing the twists, turns, wiggles and squirms of 37,780 fruit fly larvae, neuroscientists have created an unprecedented view of how brain cells create behavior. The results, published March 27 in Science, draw direct connections between neurons and specific movements.

"Understanding how neural activity gives rise to behavior is the most important question in neuroscience," says neuroscientist Kay Tye of MIT, who was not involved in the research. The new study provides a way for scientists to start answering that question, she says. "I think this is a really important approach that ‘s going to be very influential."

Scientists led by Marta Zlatic of the Howard Hughes Medical Institute ‘s Janelia Farm Research Campus in Ashburn, Va., took advantage of an existing set of specially mutated flies. In each animal, small groups of neurons, usually between 2 and 15 cells, were engineered to respond to blue light. By activating handfuls of neurons with light and analyzing videos of the resulting behaviors, the researchers systematically explored most of the 10,000 neurons in Drosophila melanogaster larvae’s brain.

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Lack of coronin 1 protein causes learning deficits and aggressive behavior

Learning and memory relies on the proper processing of signals that stimulate neuronal cells within the brain. Researchers at the Biozentrum of the University of Basel, together with an international team of scientists, has uncovered an important role for the protein coronin 1 in cognition and behavior. They found that a lack of coronin 1 in mouse and in man is associated with poor memory, defective learning and aggressive behavior. The results, recently published in PLOS Biology, identify a novel risk factor for neurobehavioral dysfunction and reveal a molecular pathway involved in transferring information within neurons.

Organisms must be able to sense signals from the outside and translate these into biochemical cues in order to adequately respond to their environment. This capability is also required to process information that reaches the brain. Within the brain, stimulation of neurons activates genes that are required, for example for learning and memory. In collaboration with an international and interdisciplinary team the research group led by Prof. Jean Pieters from the Biozentrum, University of Basel, has now uncovered the role of an evolutionarily conserved protein, called coronin 1, in providing a link between the extracellular stimulus and neuronal activation that ultimately results in efficient learning and memory in both mice and men.

From the immune system to the brain
In earlier work, Pieters’ team discovered the protein coronin 1 as being essential for the proper transduction of signals in immune cells. In mice lacking coronin 1 the researchers further investigated the molecular mechanism. Surprisingly, these mice showed aberrant behavior. In particular, mice lacking coronin 1 appeared to be far more aggressive and display extreme grooming activity, an indication of reduced sociability. An in-depth analysis in collaboration with scientists from the Friedrich Miescher Institute in Basel and the University of Bordeaux unveiled profound learning and behavioral problems and severe defects in the ability to activate neurons in the absence of coronin 1.

Activation of a signaling cascade
But how does coronin 1 ensure proper neurobehavioral functioning? Normally, stimulation of the cell surface results in an activation of an intracellular cascade of reactions and ultimately stimulates the production of the signaling molecule cAMP which then activates a number of processes, including the transcription of gene involved in neurobehavior. “We found that in the absence of coronin 1, cell surface stimulation leads to a defective cAMP production”, explains Pieters. “This in turn affects the signal transduction which is finally responsible for the deficits in learning and memory formation.”

Of mice and men
Furthermore, the researchers analyzed the clinical history of a patient lacking coronin 1 due to a mutation: it turned out that this patient showed learning defects and aggressive behavior. With this study, Pieters and his project collaborators not only define a crucial role for coronin 1 in cognition and behavior, but also unravel a coronin 1-dependent signaling pathway that may be explored both for potential risk factors as well as future interventions to modulate neurobehavioral dysfunction.

Whole-Brain Activity Maps Reveal Stereotyped, Distributed Networks for Visuomotor Behavior

Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate but ordered pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments systematically reveal the functional architecture of neural circuits underlying a sensorimotor behavior in a vertebrate brain.

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Sensing subtle differences in the environment

The hippocampus is an important region of the brain that encodes spatial memory. It consists of a number of subfields that have specialized functions in memory storage and retrieval, but the precise role of some of the subfields remains unclear. Thomas McHugh and colleagues from the Laboratory for Circuit and Behavioral Physiology at the RIKEN Brain Science Institute have now discovered that in mice, the CA2 subfield senses small changes in the environment that are at odds with their spatial memory.

McHugh and his colleagues sought to determine the role of each subfield of the hippocampus in sensing familiar and new environments through a series of mouse experiments, focusing on the often overlooked CA2 subfield. They first exposed mice to a familiar environment, and then moved them back to their home cage. The researchers then either put the mice back in the first location or moved them to a new location that the mice had never experienced. 

The research team examined similarities and differences in the way hippocampal subfields responded to the two environments by a procedure known as catFISH—cellular compartment analysis of temporal activity by fluorescence in situ hybridization. This technique allows the timing of neuronal activity to be determined and permits the assessment of contextual memory by observing changes in response to environmental manipulations.

The researchers found that in most cases, there was more overlap in the response of hippocampal neurons in all subfields when the mice were replaced in the first location after their time in the home cage compared with placement in the new location. However, in mice with a mutation in the CA3 subfield that causes CA3 neuronal activity to be uncoupled from the animal’s sensory environment, the change in CA2 response to a novel environment did not appear. The finding suggests that the CA3 inputs to CA2 modulate the ability of CA2 to sense novel environments.

In a final experiment, the researchers introduced more subtle changes to the environments during the second placement by taking objects from one location to the other. A distinct change in CA2 neuronal activity was found during these exposure intervals as a response to more subtle changes to the animals’ environment. The CA2 subfield may therefore be the most sensitive to subtle differences between existing memories and new experiences. “In future studies, we plan to use genetic approaches to control CA2 activity in order to understand its direct effect on behavior,” says McHugh.