Chemosensation, the detection of chemicals in the environment, is among the oldest of the sensory systems. Most unicellular prokaryotic and eukaryotic organisms are capable of detecting the presence of natural chemical substances and generating attractive or repulsive responses. The chemosensory system of higher organisms, particularly vertebrates, is highly evolved and mediates the detection and identification of complex mixtures of chemical stimuli. These external stimuli are converted into nerve impulses and subsequent cognitive and behavioral responses. In mammals, the olfactory system is exquisitely sensitive, capable of detecting some odorants present at a concentration of only a few parts per trillion. Rapid progress has been made in understanding anatomical, cellular, biochemical, and genetic contributions to this overall sensitivity and selectivity. These studies are also providing considerable insight into the development, wiring, and organization of the mammalian central nervous system.
The ability of the olfactory system to discriminate thousands of different odorants results from a complex coding mechanism. The elucidation of the molecular pathways responsible for odorant signaling in mammals has provided a framework to understand the biochemical, cellular, and systems-level contributions to odor discrimination. How many receptors are responsible for the detection of the broad spectrum of odors we can detect? Unlike the visual system, where three visual pigments are sufficient to define the color along a linear frequency spectrum, olfactory information is almost certainly multidimensional and may require dozens or hundreds of receptors to define a position in "odor space." Remarkably, the 1,000 or more types of odorant receptor proteins likely funnel into a single transduction mechanism that underlies the detection of all odorant molecules.
Odorants appear to activate a second messenger cascade in olfactory neurons that leads to the propagation of an electrical signal to the brain. This signaling is achieved through a second messenger cascade consisting of the membrane-bound, G protein–coupled olfactory receptor; a specific GTP-binding protein, Golf; an adenylyl cyclase; and a cyclic nucleotide–activated ion channel expressed exclusively in olfactory sensory neurons. Previous work from our laboratory identified the genes encoding these components and has recently allowed the functional reconstitution of odorant-activated second messenger generation in a model tissue culture system. These studies are part of a concerted effort to elucidate the principles of ligand recognition and represent a powerful approach to understanding the basis of ligand recognition in other G protein–coupled receptors.
How are the genes that encode odorant receptors organized, and how is their expression regulated? We are using a genetic system in mice to explore the mechanisms that direct the expression of particular receptors in individual neurons. We have demonstrated that relatively small regions of a few thousand base pairs adjacent to individual receptor genes are sufficient to direct expression to individual neurons in a zonally restricted pattern within the epithelium. These studies have revealed a remarkable feedback mechanism in which the expression of olfactory receptor protein prevents the expression of additional receptor genes in the same cell. Additionally, we have succeeded in expressing a single olfactory receptor of known odorant specificity in all olfactory neurons. This induces dramatically altered patterns of activity in the region of the brain responsible for the initial processing of olfactory information. We are currently exploring the hypothesis that the expression of high levels of odorant receptors may perturb the expression of endogenous receptors and the highly stereotyped pattern of connections from the nose to the brain.
We are constantly exposed to a complex environment rich in odorant stimuli. Our ability to appreciate the dynamic changes in our surroundings requires efficient mechanisms for desensitizing to the existing environment and recognizing new stimuli. We have discovered that one of the cyclic nucleotide channel subunits appears to play a central role in this process. The loss of this channel component by genetic disruption in the mouse leads to an olfactory epithelium that fails to desensitize to odorant stimuli. These animals are altered in their ability to perceive changes in temporally modulated odor concentration when assessed in behavioral paradigms.
The odorant transduction apparatus is a highly specialized cellular structure consisting of cilia and a mechanism to transport protein components from their site of synthesis to the membrane surfaces exposed to the environment. We have identified olfactory-specific proteins that contribute to this localization. Additionally, we have demonstrated that humans with a complex genetic disease, Bardet-Biedl syndrome, display a here-to-for unappreciated defect in olfaction that can be studied in a mouse model system. These experiments have revealed important pathways for the development of olfactory cilia and the translocalization of proteins to these structures.
The olfactory system is also an attractive model for neuron differentiation and development. The olfactory neuroepithelium is the only neuronal tissue in the adult mammal that undergoes continual regeneration. The lifetime of sensory neurons is approximately 60 days, after which they die and are replaced from a population of neuroblast-like precursor cells. Moreover, if the nerve leading from the sensory neurons to the olfactory bulb is severed or the epithelium damaged, all 5 million receptor cells are rapidly lost and subsequently replaced in a relatively synchronous fashion. The early phases of this differentiation process are controlled by proneural genes, including mash1, and loss of this transcription factor leads to altered cell fates within the olfactory epithelium. The analysis of these transcription factors in a model where the cell fate of early stem cell populations can be examined has revealed unexpected pathways for the generation of new olfactory neurons. At later stages of neuronal differentiation, activity-dependent processes contribute to the development of this sensory tissue. We have also recently developed a genetic system where we can investigate activity-dependent competition for neuronal connections and survival. These studies suggest that cyclic nucleotide–activated channels contribute to the maturation of the neuronal connections and survival of the olfactory neurons.
Finally, our laboratory is addressing the mechanisms that regulate olfactory neuron–specific gene expression. All the olfactory neuronal genes characterized to date contain a conserved sequence in the promoter region that directs their expression. We have identified and cloned a family of proteins capable of binding to this conserved DNA sequence and activating transcription. The O/E transcription regulators may direct the expression of the entire repertoire of genes involved in olfactory signal transduction and neuronal maturation, including odorant receptors. Additional transcriptional factors identified by the laboratory appear to interact with the O/E family members to modulate the transition from proliferation to differentiation. These factors also appear to play critical roles in the development of other important brain structures, including the cerebellum.
The O/E transcription factors are also expressed transiently in the developing mouse embryo by neuronal precursors in the spinal cord. This suggests a role for O/E proteins in the differentiation of neuronal cells during development and continued expression at that site, where replacement of neurons occurs throughout adult life. Recent analysis of mice with genetic deletions of each of the O/E family members suggests that they play an important role in controlling the patterning of sensory nerve projections to the olfactory bulb. We are continuing to explore the role of the O/E protein family in neuronal differentiation and additional modulatory transcriptional regulators that may control cell fates in the developing neuronal systems.
The mammalian olfactory system provides an opportunity to study the complex mechanisms of signal transduction, signal processing, and the formation of neural connections. The molecular tools available in this system are providing valuable insights into G protein–coupled receptor structure and function as well as neuronal differentiation throughout the mammalian nervous system.
This work was supported in part by grants from the National Institute on Deafness and Other Communication Disorders.