Volume 6, Issue 44 May 2009
On the Scent of Smell
John Ngai is the Coates Family Professor of Neuroscience and Director of the Functional Genomics Laboratory at UC Berkeley. Photo credit: Kirstie Tweed (courtesy UC Berkeley University Relations)
The next time you stop to smell the roses, take a moment to consider how you are able to enjoy their perfume. Sniff the velvet petals, and a cocktail of aromatic molecules wafts across odor receptors within your nasal cavity. Each is located on an olfactory neuron that extends into the brain. Chemical compounds that tumble past will bind to and activate a variety of receptors and their neurons. Your brain decodes this pattern as the unmistakable fragrance of a rose.
The neurons that link olfactory receptors to the brain are laid down according to a precise developmental blueprint, says Berkeley professor of neurobiology John Ngai. "When you and I smell a lemon, the same olfactory sections of the brain light up in the same pattern. There is a spatial representation of chemical sensation in the brain that is stereotyped from person to person."
The nose contains more than 5 million olfactory neurons, each of which expresses just one of more than 1,000 possible receptor types. Neurons bearing the same receptor types all extend their axons to the same areas of the brain's olfactory bulb. For the system to work, each neuron must connect to precisely the right bulb sections.
Ngai wants to know how the body controls this monumental wiring job. To that end, his lab is identifying the genetic and molecular cues involved in olfactory system development. This work is yielding new ways to control neural growth and direct stem cell development. Meanwhile, their analyses of olfactory receptors promise a tastier future for food.
The head of a zebrafish embryo showing the olfactory sensory neurons and their axonal projections (green) innervating the olfactory bulb (red) of the brain. Photo credit: Shannon DeMaria, MCB graduate student
To understand how olfactory neurons reach their targets, Ngai is searching for the molecular cues they follow. Just last year, his graduate student Jonathan Scolnick reported that growing olfactory neurons express receptors for a molecule called insulin-like growth hormone, or IGF. These receptors enable neurons to find their way to the olfactory bulbs. In mouse mutants lacking IGF signaling, it prevented an important symmetry from forming within the bulb. The discovery establishes IGF as one of a handful of axon guidance molecules known to help neurons establish brain connections.
Ngai is studying the genes regulating this process with the help of DNA microarrays. These gene chips indicate which genes are active at any given moment within a cell. Some of the proteins produced during neural growth are likely involved in steering neural axons.
The olfactory system may also provide insights into regenerative medicine. While most neurons grow only during development, the olfactory system keeps producing replacement neurons throughout life. These arise from a population of neural stem cells that multiply and then differentiate into mature olfactory neurons.
Using a computational model of a goldfish olfactory receptor to screen novel odorants, John Ngai discovered that goldfish can smell the bacterial metabolite diaminopimelic acid (DAP). In the fish's natural environment, DAP be a cue for the presence of food. Photo credit: Jake Osborne, University of Minnesota
"It's a possible model for understanding how neural stem cells in general regenerate," Ngai says. "If we understand how this process is regulated in the nose, the closer we are to being able to use neural stem cells to treat diseases" such as spinal cord injuries and Parkinson's disease.
Ngai's group is characterizing both the genes and molecules driving this process. By surveying all the genes that are expressed in olfactory stem cells, they are identifying the genes that regulate neuronal stem cell maintenance and differentiation. The information should help advance stem cell science toward future medical therapies.
Another important aspect of smell is how chemical odorants interact with olfactory receptors. Collaborating with colleagues at the University of Paris and the Accelrys company, Ngai uses detailed three-dimensional computer models to observe how well odorants fit a given receptor. "We look for common features of a chemical's structure that the receptor must like, to determine what makes that chemical a good odorant," Ngai says.
A molecular model of the binding pocket of the goldfish DAP olfactory receptor. The ligand binds in the cleft and the wings close around it. The image shows arginine, one of the receptor's natural ligands, docked in the binding pocket. Photo credit: Hugues-Olivier Bertrand, Accelrys
As a proof of concept, Ngai performed this analysis on an amino acid receptor in the fish nose. "It looks like a Venus flytrap — instead of catching and holding a fly in its binding pocket, it docks an amino acid," he says. This receptor shares many structural similarities to human receptors for the amino acid glutamate, which include receptors for neurotransmitters in the brain and for the flavor enhancer monosodium glutamate on the tongue.
After using a computational program to search over 1 million chemical structures, Ngai discovered one, di-amino pimelic acid, or DAP, that is present in rivers at concentrations fish can smell. DAP is a metabolite only found in certain types of bacteria. "A high local concentration of DAP probably signals high local concentrations of bacteria. That means the bacteria must be eating something. So DAP could be a proxy for available fish food," Ngai says
Much of our sense of taste depends on our sense of smell. So Ngai is now screening vast computer databases to find other ligands that bind to DAP-type receptors found in tongue taste buds. He hopes to identify flavorings that could, for example, increase the palatability of bland, restricted-calorie diets. These could help address the twin epidemics of obesity and diabetes-and keep us all enjoying the roses over long, healthy lives.
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The Origins of Ouch
Diana Bautista is a professor of cell and developmental biology. Photo credit: courtesy Diana Bautista
Skinning a knee, swallowing habanero salsa, and installing snow chains bare-handed might seem pretty different at first. But all have one thing in common — they're guaranteed to hurt.
The ability to detect such noxious stimuli is known as nociception. While critical for avoiding injury, nociception is also the bane of thousands of cancer, HIV, and spinal cord injury patients. Many develop hypersensitivity to pain, making a soft caress or light breeze excruciating.
Though pain is a widespread problem, says Berkeley professor of cell and developmental biology Diana Bautista, "we know very little about the basic molecular mechanisms behind nociception."
What scientists do know is that our neurons detect noxious stimuli with the help of molecular receptors. Bautista works to identify these receptors, and describes how they relay messages of heat, cold, chemical irritants and touch to the brain.
Bautista helped prove that the receptor TRPA1 mediates pain sensations from compounds in garlic (above) and wasabi (below). Image credit: Diana Bautista
In her laboratory, Bautista exposes naked pain neurons to strongly-flavored natural compounds. Garlic, chilies, mints and other plants produce these substances to stimulate pain in animals and repel would-be browsers. As a result, Bautista's stash of reagents bears far more resemblance to a pantry than a lab bench. Her lab is stocked with tins of peppermint and cinnamon lozenges, bottles of peppers, cloves of garlic and tubes of wasabi.
"We isolate the active compounds in these foods, and look to see which subtypes of somatosensory neurons are activated. Are they targeting heat sensitive, touch sensitive or cold sensitive neurons? This gives us hints on how they function," Bautista says.
For example, the sinus burning that accompanies wasabi and mustard is caused by a compound called allyl isothiocyanate. In high doses, it causes swelling, redness and hypersensitivity to heat and touch.
Bautista discovered that mustard oil interacts with a pain receptor called TRPA1. To prove TRPA1 was indeed associated with mustard oil irritation, she engineered mice that lack the receptor gene. As expected, the mice failed to raise an inflammatory response when exposed to mustard, garlic or wasabi.
The same receptor triggers coughing and asthma attacks, and is at least partly responsible for hypersensitivity that develops after nerve injury or inflammation. For this reason, every major pharmaceutical company in the world is now investigating TRPA1 to find drugs fighting asthma, airway inflammation and pain.
The berries of the Zanthoxylem or Sichuan pepper plant are widely used in Northern Chinese cooking. The pepper's numbing and tingling effects are caused by the compound hydroxy-alpha-sanshool. Photo credit: courtesy Diana Bautista
Still other natural products, like morphine from opium poppies and aspirin from willow bark, alleviate pain. Less well known is a remedy for toothache used in traditional Chinese medicine. Chew the dried berries of the Sichuan pepper, and a numbing, tingling sensation will accompany their vaguely citrus flavor. Native American healers use a similar strategy -brewing the bark of a related plant they call the "toothache tree," or American prickly ash, into tea.
"Figuring out how the active component interacts with the somatosensory system might lead us to an important molecule we can target to alleviate pain," Bautista says.
The molecule responsible for Sichuan pepper's effects is hydroxy-alpha-sanshool. Bautista first traced sanshool's effects to a type of potassium channel. Additional experiments narrowed the field to three related receptors known to interact with anesthetics. Of those, the KCNK18 receptor is expressed at high levels by somatosensory neurons. Bautista suspects that when KCNK18 is activated, it serves as a sensory gatekeeper, regulating the forwarding of pain signals to the brain. She is now pursuing this hypothesis by studying mutant mice lacking this channel.
Sensing mechanical pressure, however, is a whole other ballgame. "Compared to other sensory systems, our sense of touch is the most mysterious," Bautista says. The main problem: it's difficult to study. Sensory neurons are usually sparsely distributed and therefore tricky to isolate.
Star-nosed moles may have the finest sense of touch in the animal kingdom. Its snout is covered with more than 100,000 tiny sensory structures called Eimer's organs containing densely packed touch receptors. Photo credit: Ken Catania, Vanderbilt University
Bautista looked to Mother Nature for an answer. She found what she was looking for in the star-nosed mole. Named for the fleshy, tentacled organ that graces its snout, this mole makes its living tunneling through swampy East Coast soils, snarfing up worms, bugs and other soil organisms along the way. Though the star is less than half an inch across, it's packed with ten times more sensory neurons than the human hand.
"Instead of trying to figure out how to isolate large quantities of touch receptors from the skin, nature's done it for us," Bautista says. There is one downside to mole dependence, however. It won't breed in captivity, forcing Bautista to acquire a few unexpected job skills. "Most of my scientific life has been spent in a dark room doing fluorescence imaging on a microscope. But now, once or twice a summer, I go to rural Pennsylvania, put on waders, and trap moles" alongside her collaborator and star-nosed mole behavior expert Ken Catania of Vanderbilt University.
Bautista is now examining the genes expressed in the star's sensory neurons to determine what receptors make this organ so sensitive. "The idea is to come up with new candidate molecules important in touch sensation. Then we can look at those candidates in mouse and human tissue to see what role they play in normal somatosensory function," Bautista says.
Ultimately, Bautista hopes to identify good drug targets for pain therapies. Current drugs for acute pain all affect brain processing, making patients woozy and often leading to addiction. Those acting on nociceptors might sidestep these problems, and make chronic, maladaptive pain a thing of the past.
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Switching On the Lights
Richard Kramer is also a faculty member of Berkeley's Helen Wills Neuroscience Research Institute. photo credit: courtesy Richard Kramer
For those born with sight, blindness is a terrifying prospect. Unfortunately, hundreds of thousands of Americans are diagnosed with diseases such as retinitis pigmentosa and age-related macular degeneration every year. These diseases damage the photoreceptors on the outermost layer of the retina, destroying the light-collecting ability of the eye and leaving patients functionally blind.
Richard Kramer, a Berkeley professor of neurobiology, is working toward an organic way to restore sight to damaged retinas. Kramer has developed a molecular switch that responds to light. Once added to a cell's existing proteins, this device can repurpose ordinary neurons into light-sensitive tissues.
"If we can get the surviving layers of the retina to respond to light directly, we might be able to restore some sight," Kramer says.
Kramer's research into photoswitches, and how the retina transmits image data to the brain, have major implications for fields ranging from neuroscience to anesthesia, drug discovery to physiology.
Kramer has engineered a photoswitch that can turn cell signals on and off in response to light. Here, the switch arm straightens to plug a potassium channel under UV light, and bends to open the channel under green light. In this way, the switch can make ordinary neurons sensitive to light. image credit: Richard Kramer and Dirk Trauner
His photoswitch is a marvel of microscale engineering. It's made of azobenzene, a chemical that is normally stiff and straight. But when exposed to the right wavelength of light, azobenzene bends in the middle, and pulls its two ends closer together.
One end of the switch can be anchored to either an open ion channel or a protein receptor. The other end is affixed to a ligand specific to the channel or receptor. When the switch arm is fully extended, the ligand fits into the channel or receptor like a key in a lock. Exposing a cell containing this switch to light causes it to send an electrical signal or trigger a biochemical reaction.
Because the bent form of azobenzene is less stable, the molecule will relax on its own over time. It will also straighten on command under green light. Kramer is developing ways to tweak the switch to better suit different applications. For example, an arm that recovers its linear form rapidly would be most useful for vision.
The switch could be added to body cells in several ways. It binds to ion channels and receptors on its own, so all that's needed is a means to bring the chemical into contact with cells. Kramer is working with colleagues at UCSF on a polymer that could release small amounts of azobenzene over time. Placing it in the eye could supply the retina with steady doses of the switch.
The eye detects light with the aid of two types of photoreceptors: rods that are very sensitive to light and cones that can sense colors. This cross-section of the retina (red) shows synaptic vesicles (green) containing neurotransmitter in the terminals of several cones. image credit: courtesy Richard Kramer
"If you put this switch into neurons, you've installed a noninvasive way to control that part of the nervous system," Kramer says. That includes pain neurons. Kramer is now working on pairing the switch with an analgesic. One wavelength of light could turn those neurons off, while another would reawaken them. "Imagine leaving the dentist's office and not being numb for the next hour," Kramer says.
Even broader applications include neuroscience and drug research. At present, studying connections between neurons involves inserting electrodes into each individual cell. This painstaking technique is impossible to perform on large numbers of cells simultaneously. Kramer's photoswitch should eliminate this bottleneck, providing a means to stimulate many cells at once and understand neural circuits. Photoswitches can also be used to trigger electrical signals in cells used for drug assays. This is important for biotech efforts to assay vast molecular libraries for their effects on ion channels and other cell functions.
"Light is noninvasive. And you can target these switches to the right cells in several ways: genetically or chemically, but also by focusing the light to make sure only particular cells are being affected," Kramer says.
Synaptic vesicles (yellow circles) bound to the synaptic ribbon (black bar) in darkness and in light. Superimposed images from hundreds of ribbons show that vesicles are depleted from the base of the ribbon in darkness. image credit: courtesy Richard Kramer
Kramer's other research asks how photoreceptors pass visual information along to the brain. Photoreceptor neurons hand off information to the next layer of retinal cells via a mysterious structure known as a ribbon synapse.
Until recently, scientists thought the ribbon synapse was responsible for speeding the delivery of neurotransmitter to the cell membrane. Kramer has demonstrated that it does just the opposite. "It stores up the vesicles like a squirrel stashing nuts for the winter, then delivers them to the synapse when they are needed," he says. In light, the synapse releases a steady dribble of vesicles. But every time the lights go off, it dumps its vesicle stash all at once.
"What's being accentuated right at the very first synapse in the visual system is the change of light intensity that happens between light and dark. It's enhancing the representation of changing stimuli," Kramer says, and it's keeping our vision keen.
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