Advancing Basic Science for Humanity
Frontiers in Neuroscience
Published June, 2007
One of the strangest and most wondrous things in the universe is the wrinkled lump in every person’s head: the human brain. Weighing about three pounds for the average adult, within the brain are 100 billion neurons that give us the ability to see, smell and move, as well as think, weep, talk and read. Furthermore, all we experience and remember – in essence, every little thing that makes us who we are – is rooted in the neocortex, the seat of the "thinking" brain.
Understanding how such a miracle is possible is the vast mission of the relatively young field of neuroscience.
In the past few decades, researchers have learned much about the fundamental workings of the brain, with tremendous gains in knowledge about the molecules that make it run. Scientists identified genes for receptor proteins that detect smell and taste. They determined that the stuff of memories is, literally, a cascade of biochemical changes at the connections, or synapses, between neurons. And belying an old view that the nervous system is hardwired from birth, experts found that its cells retain some capacity to adapt and reorganize in response to experience.
Now armed with the human genome and a combination of cutting-edge genetic methods and brain imaging techniques, lab scientists are exploring the neural circuitry of living animals in ways they could likely have never dreamed of 20 years ago. Rather than scrutinizing one or two neurons at a time, they aim to study how networks or systems of the cells function to influence behavior. Such efforts promise to bridge the gap between studies of the cognitive powers of the mind – traditionally the turf of psychologists and linguists – and investigations of the physical brain by neurobiologists.
"We're at the point now... where we can put together these two disciplines and understand the mind in terms of the operations of the nerve cells in the brain,” says Nicholas Spitzer, co-director of the Kavli Institute of Brain and Mind at the University of California, San Diego.
Truly fathoming how the whole nervous system functions will ultimately require building powerful computer simulations that can predict the behavior of millions to billions of neurons working together. The budding subspecialty of computational neurobiology is thus “a hugely important domain for the future," says David Van Essen, president of the Society for Neuroscience and a researcher at Washington University in St. Louis, Missouri.
Researchers will no doubt be busy for years to come before they can pull together a unifying theory that explains the miracle of the brain. But step-by-step, they’re making headway in many areas, including these developments on several fascinating fronts.
Mapping the Neural Wilderness
Neuroscientists need a diagram of the brain’s internal wiring, but mapping neural circuits isn’t easy. The human neocortex houses dozens of types of neurons, all intimately intertwined. Each nerve cell is like a tree, with a head of fine branches – known as dendrites – that receive messages from several hundred to thousands of neighbors, and with a complex array of roots that pass the signals on to other cells across synapses. Small wonder that 1906 Nobel laureate Santiago Ramon y Cajal famously described the cerebral cortex as an "impenetrable jungle.” But modern-day researchers can finally see how to survey that wilderness, with some nifty genetic tools.
“We still have to hack through some vines here and there, but we have sharper machetes now,” says neuroscientist Edward Callaway of the Salk Institute for Biological Studies in La Jolla, California. He and colleagues have invented a method that should make it possible for the first time to pick any cell in the cortex of a lab animal and then label “every single neuron in the brain that connects to exactly that one cell,” he says.
Figure 1. Early image of stained pyramidal neurons by laureate Santiago Ramon y Cajal.
Using a clever set of genetic tricks, Callaway's team modified a rabies virus so that it can invade only a brain cell of a pre-selected type and move across its synapses to infiltrate all cells linked to it. But the microbe is prevented from then spreading further. Because the virus is also engineered to produce green fluorescent protein, the scientists can see under a microscope all the glowing-green neighbors that the original brain cell (or group of cells) directly talks to.
In addition, neuroscientists are mastering the art of turning neurons on and off, which will also help with tracing circuits. The standard means of activating nerve cells is to gently zap them with an electrode, but that stimulates all cells in the area. Research labs have devised a number of ingenious ways of genetically introducing molecular switches into neurons that can control their activity more precisely. Lately, neuroscience circles have been abuzz over one new breakthrough technique in particular: photo-sensitive proteins, called channelrhodopsin-2 and halorhodopsin, that can trigger neurons to respectively fire or shut down within milliseconds when exposed to light.
Stanford University investigators and their collaborators created transgenic mice that produce channelrhodopsin throughout their brains, without ill effect. The researchers quickly scanned with blue light over huge regions of an anesthetized rodent’s exposed brain; then, using electrodes, they monitored responses triggered in other areas. With this strategy, says Stanford bioengineer and psychiatrist Karl Deisseroth, scientists “can start to map circuitry much faster than you could before.” In this case, they examined a key neural pathway involved in processing smells.
In another study, Deisseroth’s team applied the method less intrusively by sending blue light through a very thin optical fiber inserted into the cortex of a lightly sedated mouse. The light pulses turned on certain motor neurons (which were engineered to carry channelrhodopsin), causing the animal’s whiskers to twitch. In the future, Deisseroth would like to study brain cell types that might be overactive or underactive in depression and autism.
Similarly, neuroscientist Karel Svoboda and colleagues at Cold Spring Harbor Laboratory in New York, and the Howard Hughes Medical Institute’s Janelia Farm campus in Ashburn, Virginia, have used channelrhodopsin to trace the long neurons that link the two sides of the mammalian brain through the structure known as the corpus callosum. By turning on or off parts of a neural loop and watching what happens, researchers hope to learn how specific complex circuits influence an animal’s behavior. The light-activated methods, Svoboda says, “will make a new kind of neurobiology possible.”
Tracing the Deep History of the Brain
Approximately two million years ago, the brain capacity of our ancient forebears began greatly increasing, eventually culminating in a brain that today is roughly three times larger than that of a chimpanzee, our closest evolutionary cousin. How that transformation happened, and how we acquired our impressive cognitive abilities, is a mystery that touches on the core of what made us human. Lately, scientists have been gleaning fresh clues from studying genetic data troves and the anatomy of primate brains at the cellular, molecular and genetic levels.
Using computer algorithms, researchers have compared the whole genomes of humans, chimps, rodents and other animals, and identified several hundred regions containing DNA differences that may have played a role in human evolution. Because periodically arising genetic changes are what drive evolution, our DNA is a historical record of deep ancestral secrets.
For instance, biologists at the University of California, Santa Cruz have identified 202 human DNA segments that underwent rapid changes in the 6 to 7 million years since the human and chimp lineages diverged from a common ancestor. Most of those regions aren't genes that code for proteins; instead, they are sequences that appear to regulate when or where certain genes turn on in the body – and some of those genes may be involved in neuro-development.
Figure 2. Large, spindle-shaped Von Economo neurons have a simple arrangement of two dendrites extanding from the top and bottom of the cell body (soma) - unlike the more complex pyramidal neurons of the cortex. Scientists theorize the spindle cells are parts of the human brain circuitry involved in social awareness. (Courtesy of John Allman)
In lab experiments, the research team found that one DNA region, named HAR1, is active in Cajal-Retzius neurons, which organize the initial formation of the neocortex. Although that discovery is exciting, the scientists have much to learn about HAR1's function in the brain before reaching any conclusion about its potential role in human evolution, says computational biologist Katherine Pollard, who now works at the University of California, Davis.
Indeed, understanding the early development of the cerebral cortex is another important source of information for deciphering possible mechanisms of how evolution built a bigger and more intricate brain. “We want to figure out how it was done, and the secret is in individual cells, in how they behave during embryonic development,” says Pasko Rakic, director of the Kavli Institute for Neuroscience at Yale University. Working at the molecular and genetic level, his lab has been studying how neurons born deep inside the brain, including Cajal-Retzius and pyramidal cells, know exactly where to go as they migrate upward to form the six layers of the neocortex.
Everyone thought the Cajal-Retzius cells were the earliest neurons of the cortex, but Rakic and his colleagues reported last summer that a previously unrecognized type of brain cell emerges even sooner, in 31-day-old human embryos. Rakic hypothesizes that these “predecessor neurons” also guide the organization of the cortex and instruct other precursor cells how much to multiply. The scientists didn’t observe the predecessor cells in rodents and are now planning to search for and study them in monkeys.
So far, very little is known about how neuroanatomy differs between people and other primates. But neuroscientists have found that the brains of great apes – which includes humans, chimps and gorillas – evolved a completely new kind of neuron that is absent in monkeys and baboons. More plentiful in people than chimps, these large spindle-shaped cells (called Von Economo neurons) lie in only two locations of the cortex that appear to play a role in social cognitive functioning. Patients with a dementia that destroys these neurons often behave in socially inappropriate ways, says neurobiologist John Allman of the California Institute of Technology in Pasadena.
Meanwhile, while it is relatively straightforward to identify potential genes underlying human brain evolution, the difficult next step is comparing what those genes actually do in people versus other primates, says neuroscientist Todd Preuss of the Yerkes National Primate Research Center at Emory University in Atlanta. Preuss and his associates are investigating a gene that codes for a protein named thrombospondin 4, which seems to spur neurons into building new synapses. Analyses of gene expression patterns in brain tissue samples revealed that the human cortex makes six times as much of the protein than the chimp brain does. Other experiments have detected the extra thrombospondin in human brains clustering in the spaces where a neuron’s synapses lie. Preuss speculates that our neurons are sculpting and resculpting connections at a faster rate than brain cells in our closest evolutionary relatives do. All of this supports neuroscientists’ view that the extraordinary talents of the human brain don’t arise merely from its bigness; there’s something special about the way it’s wired.
From Molecules to Memory
Without the brain's knack for remembering, you would have no learning and no autobiography, crafting who you become. Our memories are what make us each unique, says neurobiologist Roger Nicoll of the University of California, San Francisco. “What identifies you is nothing other than storage of events and places and people."
Fifty years ago, psychologists reported that an epilepsy patient, H.M., could no longer make new memories after surgeons removed part of his brain, including the hippocampus. Since then, neuroscientists have detailed the basic, initial biochemical steps that convert perceptions of the world into permanent recollections of facts and occurrences. “It’s absolutely incredible how far we’ve come,” says Nicoll.
The main action happens at the connections between neurons. In the first hour of memory formation, neurotransmitters are released, receptors congregate and the signals that cross the synapse are boosted. Most scientists believe that ultimately, it is an overall persistent strengthening of synaptic activity that lays down a long-term memory.
Support for that idea comes from a decades-old observation that, when hippocampal cells are rapidly bombarded with electrical zaps, neurons on the receiving end of the stimulated cells’ synapses respond with a long-lasting jump in firing activity. But the theory that this so-called long-term potentiation (LTP) underlies real-life memory encoding has been tough to prove.
Last year, however, two separate research labs finally reported detecting LTP in learning rodents. Other intriguing news came from SUNY Downstate Medical Center in Brooklyn, New York. Researchers there suspect that an enzyme called protein kinase Mzeta helps to maintain LTP after the first hour, so that long-term remembrance can be forged. And in fact, in a startling experiment, when they blocked that enzyme in the hippocampi of rats, the animals’ LTP activity in response to electrical stimulation disappeared – and the rats forgot an electric shock-avoiding behavior they had just learned a day or even a month before. “The memory was erased and, as far as we could tell, never came back,” says Downstate neurologist Todd Sacktor.
Like Sacktor, many scientists are now focusing on later stages of memory formation. In people, as years pass, the hippocampus is apparently no longer needed to sustain a recollection, which instead becomes embedded in neurons distributed across the neocortex. Scientists know little about this consolidation process. Studies of patients with brain damage have conflicted over whether lesions in the hippocampus wipe out recent memories but spare old ones – or completely empty the memory banks.
Figure 3. A model illustrating how HCN channels on PFC spines can gate information coming into the neuron based on the level of cAMP in the spine compartment. Under conditions of high cAMP production, the open probability of HCN channels is increased, and incoming information would be shunted due to reduced membrane resistance. When cAMP production is inhibited by 2A adrenocept or stimulation near the HCN channels, the channels close and allow information to pass into the cell.(Courtesy of Amy Arnsten, Yale University)
A recent careful analysis of eight amnesia patients by Larry Squire, a neuroscientist at the University of California, San Diego and the Veterans Affairs San Diego Medical Center, helps clarify the issue. If damage is only within the hippocampus, Squire and colleagues determined, patients had trouble recalling events from the past five years, but older memories survived. With bigger lesions stretching into nearby brain areas, the amnesia extended back 30 to 50 years – yet early childhood memories remained. That supports the notion that the hippocampus and adjacent structures are not lasting repositories of memory, Squire says.
However, if the damage reaches into the lateral temporal lobe, then even the oldest autobiographical memories vanish, suggesting this brain area is essential to the permanent storing of memories. Squire is further exploring the consolidation process by temporarily inactivating the hippocampus in rats – with the anesthetic lidocaine – at different times points after learning.
On another front, neurobiologists are unraveling the molecular underpinnings of working memory, the mental scratchpad that makes it possible to retain a phone number long enough to dial it. Working memory depends on a network of cells, housed in the brain’s prefrontal cortex, that all trigger each other to fire persistently to hold onto that number. Recent research has shown that certain molecules, called HCN channels, control whether this neural network is functioning. The channels are like tiny gates in a neuron’s cell membrane that let charged molecules flow through.
When the channels are open, they weaken the ability of a neuron to receive information from other cells, and thus disconnect the circuit, says Amy Arnsten, a neurobiologist at the Kavli Institute of Neuroscience at Yale. But in monkey experiments, various drugs that shut down the HCN channels enhanced the network’s activity. One such drug, a blood-pressure medication called guanfacine, improved the performance of rats as they used working memory to navigate a maze. Arnsten is now working with a pharmaceutical firm to develop the drug for treating attention deficit hyperactivity disorder.
The Inexplicable Lightness of Being
Some of life’s secrets seem so amorphous and incomprehensible as to defy any attempt at inquiry. Such is the great riddle of consciousness. Where does it come from? How can electrical buzzings of physical brain cells produce nonphysical sensations of pain or the emotion of savoring the redness of a rose? What accounts for the conscious and the inherently private state of being you?
Although it is obvious to researchers that consciousness arises from the brain, for many years the “C word” was not to be mentioned in respectable scientific company because it seemed too subjective to study. In the 1990s, however, Nobel laureates Gerald Edelman and the late Francis Crick began pushing for serious biological investigations. Crick and Caltech neuroscientist Christof Koch argued the problem could be tackled by breaking it down into smaller research questions.
Figure 4. In the Eye of the Beholder. Optical illusions are a useful tool for investigating the neural mechanisms tha govern aspects of perception and consciousness as a viewer sees and interprets an image in different ways. For instance, the Necker cube, Schroeder staircase, and folded card (panel A) can each be perceived as having two separate 3-D orientations. In a phenomenon known as binocular rivalry, if a different picture (panel B) is presented to each of your eyes, you will alternately perceive either the face or the checkerboard, not a fusion of both.(Adapted with permission from Elsevier, Leopold D.A, and Logothetis N.K.  Trends in Cognitive Sciences, 3, pp. 254-264)
One intriguing approach has been to ask how the mind becomes conscious of certain information while apparently ignoring other stimuli that bombard the senses. For instance, it’s well known that if one of your eyes is presented with a photo of, say, a house while the other eye sees a photo of a face, the two images do not blend. You alternately perceive only either picture for a few seconds each – even as each retina “sees” the same image all the while. A similar effect happens while gazing with both eyes at an outline of a 3-D cube, which flips between facing leftward and rightward. Such optical illusions are called bistable visual patterns.
In numerous experiments with monkeys (or epilepsy patients undergoing neurosurgery), scientists have inserted electrodes to monitor the brain’s responses to a bistable image. Neural areas that initially process visual data fire constantly, showing no differences when conscious perception shifts from one image to the other. But something interesting happens in the higher visual processing regions. When perception switches, roughly half the neurons in the middle temporal area – and nearly all neurons in the inferotemporal cortex – display changes in activity, says David Leopold, a neurophysiologist at the National Institute of Mental Health in Bethesda, Maryland.
Such findings supported the idea that a subset of brain cells – which Crick and Koch called “neuronal correlates of consciousness” – are specialized to relay selective visual signals to the mind’s awareness. But a follow-up report in 2007 by Leopold and associates at the Max Planck Institute in Germany challenges that notion. They monitored middle-temporal neurons in two monkeys that viewed four different bistable patterns. Disappointingly, says Leopold, there was no single fixed subset of brain cells that responded as the monkeys' perceptions oscillated for each pattern. Rather, across all four visual stimuli, 70 to 90 percent of the neurons modulated during at least one perceptual shift. In each situation, he says, vetoing one image in favor of allowing another image to reach conscious awareness requires that a network of visual areas react in a different way.
Figure 5. When the Brain Takes a Break.Using transcranial magnetic stimulation, researchers can apply magnetic pulses to the brain (left panel) and then use EEG recordings to monitor electrical activity across the cerebral cortex). University of Wisconsin investigators observe neural signals jumping across the cortex during wakefulness (right panel, top). But that connectivity appears to break down with deepening sleep (right panel, middle and bottom), as consciousness fades. (Courtesy of Giulio Tononi)
Opinions still differ over whether the results detract from the theory of neuronal correlates of consciousness. Either way, scientists need a stronger understanding of how different visual areas work together in forming perceptions, Leopold says.
A different tack for exploring consciousness is through sleep studies. Neurons are active during slumber, yet if you are roused from an early, dreamless stage of sleep, not much is on your mind, says neuroscientist and psychiatrist Giulio Tononi of the University of Wisconsin in Madison. Somehow, consciousness has faded.
To investigate why, Tononi and colleagues used magnetic pulses to stimulate the brains of awake or dozing human volunteers, and then tracked the resulting electrical activity. During dreamless snoozing, neural signals didn’t jump from one region to the next like they did during wakefulness. “The various brain areas can’t really talk to each other effectively,” says Tononi. But consciousness returns in the form of dreams, especially during REM sleep, he says, and preliminary data indicates that then the connectivity patterns are more similar to those of an awake mind.
The studies are helping Tononi fine-tune a theory that views consciousness as an integrated system of information, with parts of the cortex and underlying thalamus ideally suited for managing the integration. Although overall progress in the field is slow, he says, a growing number of scientists are now using the best neuroscientific tools to ask questions about consciousness. And that in itself, he says, is a big leap forward.
© 2007 The Kavli Foundation