Inside Salk; Salk Insitute

Brain Gain

Kathleen Quach

Kathleen Quach, a graduate student in the Salk laboratory of Sreekanth Chalasani, studies C. elegans to learn how its simple nervous system gives rise to complex behaviors. P. pacificus roundworm (right) biting C. elegans roundworm (left)

Watching the tiny worms under Kathleen Quach’s microscope glide gracefully around their petri dish, it’s hard to imagine their lives are anything but serene. Named in part for their elegant motion–the “elegans” in C. elegans–the dozen or so roundworms undulate across a white circle of light, gobbling up E. coli bacteria without a care in the world. But life isn’t always so carefree for Quach’s worms.

“These don’t have any predators in with them,” says Quach, pointing to the round dish on the microscope’s brightly lit stage. “If a predator worm is nearby, they can back up pretty quickly to get away.”

P. pacificusroundworm (right) biting C. elegans roundworm (left)

P. pacificus roundworm (right) biting C. elegans roundworm (left)

To demonstrate this, Quach, a graduate student in the laboratory of Salk neuroscientist Sreekanth Chalasani, goes to a laptop next to the microscope and pulls up a video she recently shot. In the video, a group of C. elegans is joined by a brawnier roundworm, a toothy member of the P. pacificus species that proceeds to rush one of the C. elegans and nip the smaller worm on its side. Getting the message loud and clear, the C. elegans puts itself in reverse, avoiding a fight by wiggling away from the P. pacificus.

It’s clear from this tempest in a microscopic teapot that roundworms can be both predator and prey. But what’s more intriguing are the questions at the center of Quach and Chalasani’s research. What, they ask, is the role of aggression and fear in this relationship? How does the roundworm nervous system, one of the simplest in nature, give rise to such behaviors? And what can these tiny creatures tell us about ourselves–about human aggressions and fears, emotions and behaviors at once necessary for our survival but also sources of great suffering?

In the past, such questions were exceedingly difficult, if not impossible, to answer. But in recent years, neuroscientists have started looking in odd places–Quach and Chalasani’s worm brains, for instance–and using unexpected new tools to explain how the human brain works. From studying cells once overlooked as simply “brain glue,” to buddying up to rabies virus and pond scum, brain researchers are making rapid progress thanks to unlikely allies. As a result, now perhaps more than ever, Salk scientists exude excitement about the state of neuroscience and their ability to ask big questions–and actually find answers.

Tiny Creatures Big Ideas

The complexity of the human brain is–pardon the pun–utterly mind blowing. The brain is thought to contain around 86 billion neurons that are wired together to communicate through approximately 100 trillion–yes, trillion–connections called synapses. Each neuron can connect with thousands of other neurons and, to make matters even more complicated, connections are broken and new ones formed constantly.

Given the difficulty of hitting this moving target, one could forgive scientists for just throwing in the towel and seeking a more straightforward pursuit–quantum physics maybe. Fortunately, many have persevered. Over the past century, scientists have made terrific strides in describing the nervous system and explaining how it works, and for the past fifty years the Salk Institute has been in the vanguard of this quest. Beginning with early pioneers–including Francis Crick, who turned his focus to the brain after winning the Nobel Prize with James Watson for describing the structure of DNA, and Sydney Brenner, who won a Nobel for his work studying neural development in C. elegans–Salk scientists have helped lay the groundwork for understanding what makes nervous systems tick in creatures big and small.

Sreekanth Chalasani

Sreekanth Chalasani

Shrek Chalasani decided to go small. Chalasani’s studies provide an example of how decades of foundational research on a particular organism–C. elegans roundworms in this case–combined with powerful technologies are breathing new life into neuroscience. Seen through the eyes of a scientist, the beauty of C. elegans lies in its familiarity. This species of roundworms is one of a handful of creatures dubbed “model organisms,” meaning they have been extensively studied both as the primary representatives of a class of organisms–invertebrates, in the case of C. elegans–and as manageable surrogates of more complex organisms.

It was Sydney Brenner who first proposed that the roundworms be used as a model organism in 1963, noting that they are one of the simplest organisms with a nervous system. Since then, the worms have been studied extensively. They were the first multicellular organisms to have their entire genome sequenced, and they are the only creatures whose entire nervous system—302 neurons in total—has been mapped. Charts in hand, Chalasani and his team can now observe exactly what’s going on in these compact nervous systems, from the activity of genes to the signals traveling through the worms’ neural circuits. For Chalasani, what’s most intriguing is how those observations connect with the worms’ behavior, particularly in situations where these behaviors align with those of humans and other organisms.

“How do fear, aggression and other behaviors manifest at the very basic level in the nervous system?” asks Chalasani. “Somehow, with just 302 neurons, these worms appear capable of these behaviors–which means they offer an opportunity to map the circuitry of fear and anxiety. Nature likes to repurpose its innovations, so chances are that we’re not as different from worms in this respect as we might like to think. What we find in worms will probably tell us a lot about the same behaviors in people.”

At the core of Chalasani’s research and that of many of Salk’s neuroscience labs, are new technologies that connect the dots between genes, neurons, neural circuits and behaviors. For instance, using genetic engineering, his team was able to breed worms with neurons that glow when calcium levels rise inside the cell, an indication of increased neural activity. This allows the team to observe precisely which neurons light up during a certain behavior. Another technique lets Chalasani turn off genes that code for certain neurotransmitters, molecules that allow neurons to communicate. This helps him and colleagues identify, through a process of elimination, which neurotransmitters stimulate certain behaviors.

“Serotonin is a good example of a neurotransmitter in humans that kicks off a large behavioral change,” Chalasani says. “We know that drugs that increase serotonin levels in our bodies can alleviate anxiety. It’s a neurotransmitter found in both worms and humans, so understanding how this system works in the worm might tell us how to better treat anxiety disorders or prevent violent outbursts in soldiers with post-traumatic stress disorder, for example.”

We know exactly how many neurons C. elegans possesses and how they connect with one another, but charting the nervous systems of other creatures has proven more challenging. Here too, however, Salk scientists are making rapid progress, thanks to new technologies.

Rabies Virus - The Neuroscientist's Little Helper

At the opposite end of the complexity continuum from C. elegans’ simple nervous system is the cerebral cortex, the outer layer of the brain of humans and other mammals. It’s here that we mammals really shine in terms of neural circuitry and sheer computing power. Each of the two halves of the cortex is divided into four deeply grooved lobes that are subdivided into areas responsible for processing the deluge of information streaming in from our sensory organs. The cortex is also responsible for the higher-level functions that make humans different from roundworms and fruit flies, such as the capacity for language, memory and planning.

A Spaniard named Santiago Ramón y Cajal provided the first glimpses of the astoundingly complex neural architecture of the human cortex more than 100 years ago. Using a technique developed by his contemporary, the Italian scientist Camillo Golgi, he used a silver-based compound to stain individual neurons in slices of brain tissue. This allowed Ramón y Cajal, who’d originally wanted to become an artist before his father pushed him into medicine, to pen beautiful renderings of neurons organized into columns just under the outer surface of the cortex. The drawings illustrated a forest of living circuitry that Ramón y Cajal would call an “impenetrable jungle.”

Since he made his observations around the turn of the twentieth century, scientists have been exploring this neural “jungle,” hunting for the big game of neuroscience. One of the most sought-after prizes is the “canonical cortical circuit,” a pseudo-mythical beast that may only exist in the minds of neuroscientists. The thinking goes that the functional unit of the cortex–its basic hardware, in computer speak–is a circuit composed of about 100,000 neurons, known as the “cortical column.” Each of these blocks of cells takes up a millimeter square under the surface of the cortex and is repeated thousands of times.

John Reynolds

John Reynolds

“In concept, it’s similar to the transistors found in a computer chip,” says John Reynolds, a professor of neuroscience at Salk who studies how the visual cortex processes information. “The cortical circuit is the basic unit of computation, working in tandem with all the other units to process information, make decisions and produce action and thought.”

A “canonical cortical circuit” is an archetype, a theoretical model of real-life cortical circuits. But it’s only that, because life is messier than theory. The architecture of the visual cortex, for instance, which is responsible for processing information coming from the eyes, is somewhat different than that of the auditory cortex, responsible for processing data streaming in from our ears. Yet, while different, they are probably riffs on a theme.

“There is a degree of variation in the organization of this circuit across the cortex,” Reynolds says, “but based on what we’ve seen to date, it is a neural circuit motif that is repeated throughout the cortex.”

Mapping various real-world examples of these circuits has occupied scientists since Ramón y Cajal first glimpsed them, but the sheer numbers and types of neurons, as well as the difficulty in untangling their webs of connections, has made progress agonizingly slow. Recently, however, neuroscientists have recruited a powerful, if unlikely, ally: the rabies virus. When a rabid animal bites another animal or a human, rabies virus found in the animal’s saliva invades nearby nerves in the victim’s body, then travels along the fibers of the peripheral nervous system to infiltrate the spinal cord and brain, where it spreads rapidly nerve to nerve, leaving a swath of destruction. The infection leads to terrible symptoms, including anxiety, confusion, paralysis, hallucinations, agitation and a fear of water. Left untreated, it typically leads to a very unpleasant death within a few weeks.

Edward Callaway

Edward Callaway

So where’s the silver lining? Edward Callaway, a Salk neuroscientist, and John Young, a former Salk virologist, wondered whether the virus’ remarkable ability to jump between neurons might not be a blessing in disguise. First, the scientists developed a technique for inserting a genetically modified rabies virus into a neuron and allowing the virus to spread only to neurons directly connected to the first infected cell. The researchers combined this with a method of making cells fluoresce under a microscope, so that the original infected cell glows red and the cells it connected to directly glow green. The result was remarkable: like plugging in a Christmas tree for the first time, the branching neurons blazed red and green. The practical application is that scientists can now map brain circuits neuron by neuron, identifying a cell’s close connections, then in turn tracing those connections’ closest contacts.

Using the technique in mice, Callaway and colleagues have assembled brain-wide maps of neurons that link with the basal ganglia, a region of the brain that is involved in movement and decision-making. Developing a detailed anatomical understanding of this region is important, as it could explain disorders traced to basal ganglia dysfunction, including Parkinson’s and Huntington’s diseases.

“This was something we’ve dreamt of having for a long time, and now it’s a reality,” says Callaway. “Before, we could guess that a cell in a certain position in the cortical column probably connected with certain other cells, but it was really a guess. This viral tracing tool gives us an unprecedented view of the brain’s architecture, which then lets us piece together how these circuits function, and what can go wrong with them.”

The Perks of Pond Scum

If rabies virus seems like a strange bedfellow, another hot technology, dubbed “optogenetics,” owes much to an equally odd contributor–pond scum.

To understand this, recall that electrical signals of a neuron are generated by gates in the neuron’ s outer membrane that control whether and how fast the cell fires off electrical impulses. Conveniently enough, similar membrane gates, known as “channelrhodopsins,” are found on the outer surface of a type of pond algae named Chlamydomonas. The crucial difference is that the membrane gates of little green pond denizens respond to light, helping the photosynthetic algae swim to a place in the sun.

By inserting the genes from the algae into the neurons of roundworms, fruit flies and mice, scientists were able to generate neurons that incorporated Chlamydomonas’ light-sensitive gates into their outer cell membranes. Different colors of lights tell the gates to open or close, allowing researchers to control the activity of the neurons with beams of light.

Whereas Callaway’s viral tracer method helps scientists map neural circuits, optogenetics can tell them what role different neurons play in those circuits. Using beams of light, they turn neurons on or off and see what effect this has on organisms’ nervous systems and behaviors.

Samuel Pfaff

Samuel Pfaff

A number of Salk labs are now using optogenetics in their reseach. Callaway and Reynolds, for instance, are using the technology to explore how the brain identifies objects among the clutter of shapes, colors and lines in our visual field. In contrast, their colleagues Martyn Goulding and Samuel Pfaff use the technology to study motor circuits that connect the brain to the muscles.

“The beauty of optogenetics lies in its speed and specificity,” says Pfaff. “We can modify the activity of neurons almost instantly, which is important because neurons operate on the millisecond timescale. You can perturb the system and immediately see the effect.”

In a recent study, Pfaff used optogenetics to discover neurons, dubbed “synergy encoders,” in the spinal cord that act as middle managers between the brain and muscles. These cells coordinate the many muscles required for walking, running and other movements, and pinpointing their identity and function is crucial to developing better therapies for diseases and injuries that impair movement.

Martyn Goulding

Martyn Goulding

Xin Jin, an assistant professor at Salk, also uses optogenetic techniques to study movement. Jin focuses on a higher center of the nervous system than Pfaff: a region of the brain known as the striatum that serves as a middleman in the flow of information from the brain to the body, linking thought to movement. Imagine, for instance, that you decide to tie your shoe. The nervous system works somewhat like a battleship’s chain of command. The cortex, acting as captain, makes the big picture decision (“tie shoes”) and that order is conveyed to the striatum, the engineering department that manages the sequence of manuevers: bending down, grabbing the laces, tying the knot. The striatum then tells “synergy encoders” in the spinal cord to orchestrate each of these tasks in the correct order.

“We want to know how movement is organized, how we drive a car, use a pen or play ping pong,” Jin says. “We’re trying to figure out how these programs are learned and localized in the striatum.”

To study this, Jin’s team created mice with channelrhodopsins incorporated into neurons in the striatum of their brains, then taught the mice to perform complex motor tasks, such as pushing a button a certain number of times. The scientists can then turn different neurons in the striatum on and off to see how this alters the animal’s behavior. For example, turning off certain neurons will cause the mice to forget what they were doing and stop halfway through the task. Turn the neurons back on and the mice go back to pushing buttons.

Xin Jin

Xin Jin

“This is a part of the brain damaged by neurodegenerative diseases such as Parkinson’s and Huntington’s,” says Jin, “so our hope is that knowing precisely the roles of different kinds of neurons might tell us what’s going on in these diseases.”

The research might also point the way for new therapies for these disorders. “If a disease has damaged one portion of a motor pathway,” Jin says, “you might be able to stimulate neurons further down the circuit, closer to the spinal cord, to initiate sequences of action.”

Hitting a Moving Target

It wasn’t long ago that the adult brain was viewed as a physiologically static organ–as the saying goes, you can’t teach an old dog new tricks. In fact, it was only in 1998 that Fred Gage, a professor in Salk’s Laboratory of Genetics, rocked the neuroscience world by demonstrating that, contrary to existing dogma, stem cells in the adult human brain continue to generate new neurons throughout our lifetimes. Gage’s discovery and reams of other research show that throughout our lifetimes our brains actually retain some degree of “plasticity,” the general term scientists use to describe the brain’s ability to adapt and change.

Fred Gage

In another nod to the brain’s dynamism, Gage has recently turned to whole-genome sequencing technologies to chip away at another edifice of scientific dogma: that each neuron in a person’s brain possesses the same DNA code. In one recent study, Gage and colleagues obtained 100 neurons from three people posthumously. When his team sequenced the genomes of each of the cells, they discovered that as many as 41 percent of neurons had at least one unique, massive deletion or duplication that arose spontaneously, meaning it wasn’t passed down from a parent. While it’s still too early to say precisely how or why this patchwork of genomic variation arises in the brain, Gage theorizes that this genomic mosaicism might help people adapt to new and unpredicted stimuli encountered over a lifetime or help them cope with unexpected disease by providing flexibility and diversity in defense mechanisms.

Other Salk scientists are attacking another fast-crumbling bit of dogma–that neurons are the only cells important in the brain’s vast networks. Roughly half of the cells in the brain are “glia,” from the Greek word for “glue,” so named because scientists originally saw them as present only to hold other cells in place. It turns out, however, that they play a crucial part in keeping the brain healthy and in managing the trillions of ever-changing synaptic connections between neurons. Instead of playing second fiddle to neurons, they may act more like conductors, coordinating and guiding the brain’s symphony of information processing.

Glia serve a number of important roles in the brain. For example, during development astroglia, named for their star-like shape, secrete factors that guide neurons to their proper destinations and allow neurons to make the right connections at the right place. In the adult brain, they shuttle nutrients from the blood vessels to neurons. Another type, oligodendroglia, wrap the axons of neurons in myelin, an insulating material that allows electrical impulses to travel much faster. And microglia act as the watchdogs of the brain, providing the first line of defense against injury or infection and cleaning up dead cells and other waste.

Axel Nimmerjahn

Axel Nimmerjahn

“We know that glia are critical to the brain’s health. When something goes wrong with the them, it impacts the neurons and vice versa. What we know much less about is what glia do in the healthy brain,” says Axel Nimmerjahn, a glia researcher in the Waitt Advanced Biophotonics Center at Salk. A physicist by training, Nimmerjahn develops optical and genetic approaches to study microglia in the brain and miniaturized microscopes to observe how astroglia and neurons interact in the brain during behavior.

Nicola Allen, who joined the Salk faculty just a couple of years ago, focuses on astroglia, which don’t transmit electrical signals of their own, but use chemical signaling to affect nearby neurons. Their “arms,” which project outward from the cells, surround the majority of neuronal synapses.

“I want to know what controls when and where these synapses are formed, and how synaptic connections are modified to allow memories to be stored,” says Allen. “We think astroglia play a crucial role in dictating synapse formation and function via the release of specific proteins that determine the type of synapse that will form and the strength of that connection.”

Nicola Allen

In previous studies, Allen began to identify the molecular signals between neurons and astroglia and showed that proteins secreted from developing astroglia affect synapse formation. She’s now trying to figure out how these proteins promote connections between neurons, trying to identify other ways astroglia influence synapse formation and maturation. Ultimately, her goal is to understand the role of astroglia in neurodevelopmental disorders–such as autism–that are caused by defects in synapse formation and function. In turn, this will shed light on addressing diseases, such as stroke, by promoting the repair of neural connections following injury.

“This is a terrific time to study the brain,” Allen says. “Technology is really changing how we do neuroscience and the scope of the questions we can ask, and that’s going to help us really make a difference in deciphering how the nervous system operates and how we treat its diseases.”