Genetic variation across the human olfactory receptor repertoire alters odor perception.

Humans use a family of more than 400 olfactory receptors (ORs) to detect odors, but there is currently no model that can predict olfactory perception from receptor activity patterns. Genetic variation in human ORs is abundant and alters receptor function, allowing us to examine the relationship between receptor function and perception. We sequenced the OR repertoire in 332 individuals and examined how genetic variation affected 276 olfactory phenotypes, including the perceived intensity and pleasantness of 68 odorants at two concentrations, detection thresholds of three odorants, and general olfactory acuity. Genetic variation in a single OR was frequently associated with changes in odorant perception, and we validated 10 cases in which in vitro OR function correlated with in vivo odorant perception using a functional assay. In 8 of these 10 cases, reduced receptor function was associated with reduced intensity perception. In addition, we used participant genotypes to quantify genetic ancestry and found that, in combination with single OR genotype, age, and gender, we can explain between 10% and 20% of the perceptual variation in 15 olfactory phenotypes, highlighting the importance of single OR genotype, ancestry, and demographic factors in the variation of olfactory perception.

How the brain sees smells.

Genetically encoded transneuronal tracers provide the first glimpse into the logic of olfactory processing in higher cortical areas of the brain. The results suggest a complex distributed coding scheme, which is remarkably similar in different individuals.

Block in nuclear localization of period protein by a second clock mutation, timeless.

In wild-type Drosophila, the period protein (PER) is found in nuclei of the eyes and brain, and PER immunoreactivity oscillates with a circadian rhythm. The studies described here indicate that the nuclear localization of PER is blocked by timeless (tim), a second chromosome mutation that, like per null mutations, abolishes circadian rhythms. PER fusion proteins without a conserved domain (PAS) and some flanking sequences are nuclear in tim mutants. This suggests that a segment of PER inhibits nuclear localization in tim mutants. The tim gene may have a role in establishing rhythms of PER abundance and nuclear localization in wild-type flies.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Circadian rhythms in Drosophila can be driven by period expression in a restricted group of central brain cells.

Neural tissues controlling circadian rhythmicity have been identified in a variety of organisms and are often closely associated with the visual system. In Drosophila, the clock gene period (per), which is required for circadian rhythms, is expressed in many neurons and glia throughout the eye and brain. We asked whether biological rhythms could be generated if per expression were restricted to a subset of these cells that is involved in photoreception. Here we demonstrate that expression of per under the control of the glass promoter confers both behavioral and molecular rhythmicity. glass is required for development of Drosophila photoreceptors, and this promoter is active in eyes, ocelli, and certain cells of the central brain. When we genetically removed all external photoreceptor cells, rhythms persisted in these transgenic animals. This suggests that a few central brain cells producing glass and per are capable of generating biological rhythms.

New short period mutations of the Drosophila clock gene per.

Earlier work has indicated that the period length of Drosophila circadian behavioral rhythms is dependent on the abundance of the period (per) gene product. Increased expression of this gene has been associated with period shortening for both the circadian eclosion (pupal hatching) rhythm and circadian locomotor activity rhythms of adult Drosophila. In this study it is shown that a wide variety of missense mutations, affecting a region of the per protein consisting of approximately 20 aa, predominantly generate short period phenotypes. The prevalence of such mutations suggests that short period phenotypes may result from loss or depression of function in this domain of the per protein. Possibly mutations in the region eliminate a regulatory function provided by this segment, or substantially increase stability of the mutant protein.

Olfaction in Drosophila.

The fruit fly, Drosophila melanogaster, is equipped with a sophisticated olfactory sensory system that permits it to recognize and discriminate hundreds of discrete odorants. The perception of these odorants is essential for the animal to identify relevant food sources and suitable sites for egg-laying. Advances in the last year have begun to define the molecular basis of this insect's discriminatory power. The identification of a large multi-gene family of candidate Drosophila odorant receptors suggests that, as in other animals, a multitude of distinct odorants is recognized by a diversity of ligand-binding receptors. How olfactory signals are transduced and interpreted by the brain remains an important question for future analysis. The availability of genetic tools and a complete genome sequence makes Drosophila a particularly attractive organism for studying the molecular basis of olfaction.

Humans can discriminate more than 1 trillion olfactory stimuli.

Humans can discriminate several million different colors and almost half a million different tones, but the number of discriminable olfactory stimuli remains unknown. The lay and scientific literature typically claims that humans can discriminate 10,000 odors, but this number has never been empirically validated. We determined the resolution of the human sense of smell by testing the capacity of humans to discriminate odor mixtures with varying numbers of shared components. On the basis of the results of psychophysical testing, we calculated that humans can discriminate at least 1 trillion olfactory stimuli. This is far more than previous estimates of distinguishable olfactory stimuli. It demonstrates that the human olfactory system, with its hundreds of different olfactory receptors, far outperforms the other senses in the number of physically different stimuli it can discriminate.

The molecular logic of olfaction in Drosophila.

Drosophila fruit flies display robust olfactory-driven behaviors with an olfactory system far simpler than that of vertebrates. Endowed with 1300 olfactory receptor neurons, these insects are able to recognize and discriminate between a large number of distinct odorants. Candidate odorant receptor molecules were identified by complimentary approaches of differential cloning and genome analysis. The Drosophila odorant receptor (DOR) genes encode a novel family of proteins with seven predicted membrane-spanning domains, unrelated to vertebrate or nematode chemosensory receptors. There are on the order of 60 or more members of this gene family in the Drosophila genome, far fewer than the hundreds to thousands of receptors found in vertebrates or nematodes. DOR genes are selectively expressed in small subsets of olfactory neurons, in expression domains that are spatially conserved between individuals, bilaterally symmetric and not sexually dimorphic. Double in situ RNA hybridization with a number of pairwise combinations of DOR genes fails to reveal any overlap in gene expression, suggesting that each olfactory neuron expresses one or a small number of receptor genes and is therefore functionally distinct. How is activation of such a subpopulation of olfactory receptor neurons in the periphery sensed by the brain? In the mouse, all neurons expressing a given receptor project with precision to two of 1800 olfactory bulb glomeruli, creating a spatial map of odor quality in the brain. We have employed DOR promoter transgenes that recapitulate expression of endogenous receptor to visualize the projections of individual populations of receptor neurons to subsets of the 43 glomeruli in the Drosophila antennal lobe. The results suggest functional conservation in the logic of olfactory discrimination from insects to mammals.

An olfactory sensory map in the fly brain.

We have isolated the "complete" repertoire of genes encoding the odorant receptors in Drosophila and employ these genes to provide a molecular description of the organization of the peripheral olfactory system. The repertoire of Drosophila odorant receptors is encoded by 57 genes. Individual sensory neurons are likely to express only a single receptor gene. Neurons expressing a given gene project axons to one or two spatially invariant glomeruli in the antennal lobe. The insect brain therefore retains a two-dimensional map of receptor activation such that the quality of an odor may be encoded by different spatial patterns of activity in the antennal lobe.

A spatial map of olfactory receptor expression in the Drosophila antenna.

Insects provide an attractive system for the study of olfactory sensory perception. We have identified a novel family of seven transmembrane domain proteins, encoded by 100 to 200 genes, that is likely to represent the family of Drosophila odorant receptors. Members of this gene family are expressed in topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. Sensory neurons express different complements of receptor genes, such that individual neurons are functionally distinct. The isolation of candidate odorant receptor genes along with a genetic analysis of olfactory-driven behavior in insects may ultimately afford a system to understand the mechanistic link between odor recognition and behavior.

From Beaumont to poison ivy: marine sponge cell aggregation and the secretory basis of inflammation.

We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.

Topographic organization of sensory projections to the olfactory bulb.

The detection of odorant receptor mRNAs within the axon terminals of sensory neurons has permitted us to ask whether neurons expressing a given receptor project their axons to common glomeruli within the olfactory bulb. In situ hybridization with five different receptor probes demonstrates that axons from neurons expressing a given receptor converge on one, or at most, a few glomeruli within the olfactory bulb. Moreover, the position of specific glomeruli is bilaterally symmetric and is constant in different individuals within a species. These data support a model in which exposure to a given odorant may result in the stimulation of a spatially restricted set of glomeruli, such that the individual odorants would be associated with specific topographic patterns of activity within the olfactory bulb.

Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling.

The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.

Activation of the human neutrophil by calcium-mobilizing ligands. II. Correlation of calcium, diacyl glycerol, and phosphatidic acid generation with superoxide anion generation.

Calcium and protein kinase C (Ca2+/phospholipid-dependent enzyme) have been proposed to act as signals in triggering superoxide anion (O2-) generation by neutrophils. We have probed the adequacy and necessity of calcium and diacylglycerol (DG), activators of protein kinase C, in eliciting O2- generation and degranulation. Activation of neutrophils by the ligand 10(-7) M fMet-Leu-Phe triggered elevation of cytosolic calcium (fura-2) and a rapid, biphasic increase in labeled DG in [14C]glycerol and [3H]arachidonate prelabeled cells. Buffering of the fMet-Leu-Phe-induced elevation of cytosolic calcium with MAPTAM (a cell permeant EGTA analogue) inhibited O2- generation by 90% and degranulation by 50%, concordant with a role of calcium in signaling. However, buffering the increase in calcium also decreased DG. Since phosphatidylinositol 4,5-bisphosphate breakdown in response to fMet-Leu-Phe was not inhibited and phosphatidic acid levels were enhanced in MAPTAM pretreated cells, the removal of calcium may enhance further DG metabolism. Thus, a requirement for calcium could not be differentiated from a requirement for DG, and the profound inhibition of O2- generation in the presence of MAPTAM may reflect removal of DG. Four stimuli, fMet-Leu-Phe, 10(-7) M leukotriene B4, 100 micrograms/ml concanavalin A, and 200 nM ionomycin elevated cytosolic calcium and triggered release of specific granules, but only fMet-Leu-Phe and concanavalin A triggered substantial O2- generation. Nevertheless, all four stimuli significantly increased labeled DG. Therefore, elevated DG and elevated calcium may be necessary but do not appear adequate to elicit O2- generation. Only fMet-Leu-Phe and concanavalin A triggered generation of phosphatidic acid (PA) together with DG. Correlation of O2- generation with PA may reflect a requirement for PA per se or for a specific pool of DG that can be further metabolized to PA.

Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish "triggering" from "activation" of human neutrophils.

Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses; N-formylmethionylleucylphenylalanine (fMLP) (0.1 microM) provokes brisk generation of superoxide anion; leukotriene B4 (LTB4, 0.1 microM) is a poor stimulus. In contrast, phorbol myristate acetate (PMA, 1.6 microM) acting directly via protein kinase C is a potent stimulus for O2-. We compared the kinetics of appearance of various "second messengers" with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an "early" (less than or equal to 15 s) and a "late" (greater than 15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Cytochalasin B increased the late phase of DG labeling elicited by all agonists. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Early increments of DG were also accompanied by addition of plasma membrane (ultrastructural morphometry); the ratio of surface perimeter to area increased rapidly (10 s) and persisted (60 s) in response to fMLP. Increments were more gradual in response to PMA. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 Mr protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP (early and late) and PMA (late). In contrast, LTB4 provoked only early phosphorylation of this protein. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only "triggering" (the rapid generation of a signal) but also "activation" (the maintenance of a signal) to sustain responses.