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The Giovanni Armenise-Harvard Foundation Session 1: Neurobiology I Overview So many Armenise-sponsored investigators are engaged in exciting neurobiology research that this year's Symposium devoted two full sessions to this vital and fast-moving field. The conference's opening session introduced four issues that are fundamental for understanding neuronal and brain function, according to Dr. Jacopo Meldolesi, who moderated the program. As distinct as these areas of research are, all are in fast-moving areas where even more interesting news can be expected in the near future. The first presentation explores how the human nervous system transduces smell from the binding of odorants to special receptors in the nose to the transmission of information to the brain. The second speaker homed in on a large family of receptors, the SEX/plexins, that in mammals appear to play essential roles in the development of nerve tissue. The focus shifted to the cell membrane for the third talk, which introduced a new process of membrane regulation in neurons that molds the excitability of those cells. The final paper dealt with a fascinating transcription factor that appears to exert itself during embryogenesis by suppressing neuronal fate determination.
Smells have shapes. But instead of having one receptor dedicated to smelling bananas and another to pine trees, Dr. Buck's research demonstrates that individual odorant receptors (ORs) recognize specific parts of an odorant's structure, rather than the whole molecule. The olfactory system combines data from numerous ORs to determine what it's smelling, which explains how humans and other mammals can discriminate an enormous variety of odors as well as pheromones (chemicals that elicit sexual or other innate behaviors). This combinatorial coding scheme relies on about 1,000 ORs expressed by the millions of sensory neurons in the olfactory epithelium lining the nose. Each neuron expresses one OR gene and neurons expressing the same OR are scattered throughout one of four nasal zones, an arrangement that decentralizes information processing. When a volatile chemical enters the nose, different parts of its structure are recognized by scattered neurons equipped with various ORs. Axons from these neurons travel to the olfactory bulb, where input from neurons with the same OR-no matter where they may be located in the nasal epithelium-is channeled into specific glomeruli with fixed locations, much as scattered light is focused by a lens. The vomeronasal organ, where the subconscious detection of pheromones begins, is equipped with two distinct families of candidate pheromone receptors, one of which was first identified by Dr. Buck's laboratory. Inputs from these receptors appear to be processed separately from ORs, ensuring that signals for primal behaviors such as mating and aggression won't be confused with commonplace smells. In recent experiments, Dr. Buck and her colleagues have combined calcium imaging and single cell RT-PCR (a technique for amplifying parts of the genome that encode proteins) to identify ORs for odorants that people perceive quite differently even though they are structurally similar. The combination of receptors that codes for a floral aroma, they found, may overlap substantially with the combination that picks up a rancid smell-but the two will be channeled to different glomeruli in the olfactory bulb. These observations of single cells support three important conclusions: a single OR recognizes multiple odorants, a single odorant is recognized by different ORs, and most odorants can be detected by different combinations of ORs.
Several years ago, when Dr. Tamagnone discovered an unusual family of receptor tyrosine kinase genes, he named the prototype SEX because it was located on the X chromosome. Additional research has expanded the universe of human SEX/plexins to include nine members grouped in four families. These genes were first identified in the nervous system, and they contain highly conserved sequences that are the same in nematodes as in humans. The SEX/plexins encode cell surface proteins homologous to the extracellular domain of the receptor tyrosine kinase that binds hepatocyte growth factor (HGF), a key growth factor in embryonic development. Early on, researchers determined that SEX/plexins appear to influence the migration of axon processes toward synapse formation. Now other roles are coming to light. Dr. Tamagnone's team has observed that the extracellular domains of SEX/plexins (the parts that protrude from the cell surface) bear a strong resemblance to semaphorins, a large family of soluble and membrane-bound ligands. In Cell, they described an interaction between plexin-A and semaphorin-1 in Drosophila; since that report they have identified two semaphorins that bind to human SEX/plexin receptors. This is the first time that ligands for the SEX/plexins have been identified. More recently, Dr. Tamagnone's laboratory has explored the possibility that the plexins are important in cell-to-cell signaling in epithelial and endothelial cells, in addition to their established role in neuronal development. When they used semaphorin protein as a probe, the researchers found that it bound to plexins that interact with neuropilins in cell signaling. Future experiments will explore whether semaphorin-binding plexins may also collaborate with neuropilins in signal transduction, acting via some novel cytoplasmic (inside the cell) structure.
There's more to a resting neuron than meets the eye. Although the conventional view is that nothing much goes on when the membrane potential is below -60mV, Dr. Fesce and Oscar Sacchi, a biologist at the University of Ferrara, have detected a series of active conductances in the -60 to -80mV range. They now say that interactions between fluctuating currents of potassium and chloride mold the excitability of the cell and help determine how much synaptic input is required to trigger an action potential. Working with the isolated, intact superior cervical ganglion of rats, Dr. Sacchi used a two-electrode, voltage-clamp system to characterize the conductances and electrical properties of these cells. Dr. Fesce used these experimental measurements to build a computational model of a complete sympathetic neuron, which revealed surprising interactions between a potassium current that acts just above the -60mV threshold and a subthreshhold chloride current. A potassium current called IA is activated at about -60mV and becomes fully activated at about -30/-40mV, causing transient depolarizations that rapidly relax to the initial value. During the upstroke of excitatory potential, Dr. Fesce said that this potassium current apparently gives rise to a counter current of chloride ions, which push toward repolarization. All this happens in a neuron that appears to be "resting," with the result that a higher level of synaptic input will be required to trigger an action potential. When a neuron is at rest, there is supposedly no net flow of ions across the plasma membrane. Nevertheless, Dr. Fesce found remarkable chloride currents that operate below -60mV. These currents respond to transient changes in membrane potential and return to a steady state after hundreds of seconds. Chloride conductance increases with depolarization, and at -60/-80mV the chloride equilibrium potential sustains a small inward current. Whatever happens to membrane resting potential sparks changes in chloride redistribution and conductances, Dr. Fesce said, which challenges the notion that membrane potential reflects a simple balance between sodium and potassium.
Scientists have known for years that regulation of tissue-specific genes during embryonic development is controlled primarily at the level of transcription. Dr. Shi's experiments demonstrate that in Caenorhabditis elegans, certain enzymes are involved both in normal transcription and in abnormal cell growth. Nature has many strategies for releasing DNA from the compact chromatin packaging that enables it to fit into the cell nucleus. One such method is the attachment of an acetyl group to histones, which bind the chromatin package. Acetylation is determined by give-and-take between histone acetyltransferases (HATs), such as mammalian CBP and p300, and histone deacetylases (HDAs). Earlier work revealed that the adenovirus oncoprotein E1A cannot induce immature cells to divide endlessly so long as CBP/p300 are functioning, suggesting that these proteins are needed for normal cell growth and differentiation. More recently, Dr. Shi expanded on this idea by manipulating cbp-1, the gene encoding CBP-1, the C. elegans protein that corresponds to CBP/p300. C. elegans may be a simple organism with less than 1,000 cells, "but it does all the things that we do, pretty much," Dr. Shi said of his favorite model. Many of its 20,000 protein-coding genes are homogolous with human genes. As in humans, cell fate is determined in part by transcription factors. When the researchers used a new technique called RNA-mediated Interference (RNAI) to inhibit expression of CBP-1, they saw undifferentiated embryos with no sign of normal morphology. At the stage when gut and muscle tissue are apparent, no such differentiation had occurred. As expected, C. elegans genes that correspond to mammalian histone deacetylase appear to repress somatic differentiation. In a kind of tug of war, CPB-1 appears to promote endoderm differentiation by antagonizing the repressive effects of HDA. These experiments are the first to show how a homolog of the human proteins CPB and p300 functions in a live animal. These results also provide critical in vivo evidence that the histone acetylase activity of CBP-1 may be important for its biological activity. In addition to confirming Dr. Shi's hypothesis, these experiments yielded an unexpected result as well. When monoclonal antibodies were used to study the seemingly undifferentiated cells in worms without CPB-1 activity, those cells appeared to be neurons Ð suggesting that neuronal differentiation may be a kind of default setting for C. elegans cells in the absence of CBP-1. Similar observations have been made recently in Xenopus laevis, suggesting that HATs may be a highly conserved, essential player in the differentiation of non-neural tissue.
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Deconstructing smell
