43 Rao, M. S., Tyrell, S., Landis, S. C. and Patterson, P. H. (1992) (1988) Neuroscience 27, 1073-1080 Dev. Biol. 150, 281-293 38 Hidaka, H. and Ishikawa, T. (1992) Cell Calcium 13,465-472 39 Wakade, A. R., Wakade, T. D., Malhotra, R. K. and Bhave, 44 Scott, B. S. (1971) Exp. NeuroL 30, 297-308 45 Lasher, R. and Zagon, I. S. (1972) Brain Res. 41,482-488 S. V. (1988) J. Neurochem. 51,975-983 40 Martin, D. P., Ito, A., Horigome, K., Lampe, P. A. and 46 Phillipson, O. T. and Sandler, M. (1975) Brain Res. 90, 273-281 Johnson, E. M., Jr J. Neurobiol. (in press) 41 Rydel, R. E. and Greene, L. A. (1988) Proc. Natl Acad. Sci. 47 Bennett, M. R. and White, W. (1979) Brain Res. 173, 549-553 USA 85, 1257-1261 42 Walicke, P. A., Carnpenot, R. B. and Patterson, P. H. (1977) 48 Eichler, M. E., Dubinsky, J. M. and Rich, K. M. (1992) J. Neurochem. 58, 263-269 Proc. Natl Acad. 5ci. USA 74, 5767-5771
Molecularmessengersofolfaction G a b r i e l e V. R o n n e t t a n d S o l o m o n H. S n y d e r
GabrieleE Ronnett Our knowledge of olfactory signal transduction has andSolomonH. been greatly clarified by several recent advances. Snyderareat Molecular cloning has revealed a large family of TheJohnsHopkins putative odorant receptors localized to olfactory epiUniversitySchoolof thelium that display a seven-transmembrane-domain Medicine,Deptsof motif suggesting an association with G proteins. Very Neuroscience, potent and rapid enhancement of both adenylyl cyclase PharmacoloEyand MolecularSciences, and phosphoinositide turnover has been demonstrated andPsychiatry,725 in response to odorants both in isolated olfactory cilia N. WolfeStreet, and primary olfactory receptor neuronal cultures. A Baltimore, Ca2 +-calmodulin-dependent phosphodiesterase has MD21205, USA. been localized to olfactory cilia. A key role for Ca 2÷ is
evident from many investigations. More recently, odorants have also been shown to affect the levels of cGMP in olfactory receptor neurons. The involvement of multiple second messengers may provide mechanisms for both fine-tuning and desensitization of olfaction. The olfactory system is ideal for the study of a number of fundamental questions in the neurosciences. Poised at the interface between the environment and the CNS, it subserves a sensory function that is chemical in nature. Thus, an understanding of transduction mechanisms involved in olfaction may provide insight into signal integration elsewhere in the CNS. The olfactory receptor neuron (ORN) is highly specialized in order to accommodate its function. These neurons are located in a pseudostratified epithelium consisting of three principal cell types 1-3. Sustentacular or supportive cells resembling glia stretch from the epithelial surface to the basal lamina4. The ORN is bipolar, extending apical dendrites to the surface of the neuroepithelium and sending unmyelinated axons through the basal lamina and the cribriform plate of the ethmoid bone to terminate in the olfactory bulb of the brain. The apical dendrites form dendritic knobs from which arise specialized, non-motile cilia where the initial events of olfactory transduction occur 3-8. Basal cells, the third type of cell, underlie the receptor neurons and serve as precursors for the generation of new ORNs throughout adult life2'3. Their ability to regenerate from a precursor population distinguishes ORNs from other neurons so that understanding the biology of these mechanisms may provide insight to neurodegenerative diseases. Interestingly, nasal biopsies of Alzheimer's disease patients reveal regenerative responses to olfactory neuronal degeneration suggesting early involvement of the olfactory neurons in Alzheimer's disease 9'1°. 508
t~) 1992, ElsevierScience Publishers Ltd. (UK)
Odorant signal transduction As with other sensory modalities, the ORN responds to appropriate odorant stimulation with the generation of a graded receptor potential u'l~ that spreads passively along the cell body to the proximal segment of the axon, at which point an action potential fires if threshold is attained. Intracellular recordings 1~-15 demonstrate that sensory neurons have a relatively high resistance and low resting potential. Two types of potential changes are recorded intracellularly in response to odorants ~3. There are monophasic spikes of approximately 10 ms duration that are superimposed on a depolarizing slow potential. This slow potential presumably represents the generator potential resulting from the initial interactions of odorants with ciliary receptors. There is a response latency of several hundred ms, suggesting that a second messenger is involved ~6. Odorant concentration determines the rate of action potential firing. Prolonged exposure to odorants results in an initially high firing rate that decays with time to a low steady state level4'1L12'17. An early step in understanding the molecular events in olfaction was the identification of the olfactory receptor neuronal cilia as the initial site of transduction 4,7,18,19. Selective removal of the cilia abolishes olfactory responses 2°. Thereafter, Cagan and associates 21'22 demonstrated specific binding of amino acids to isolated fish cilia. The first direct studies of odorant influences on second messengers ~3 reported a robust odorantsensitive adenylyl cyclase (AC) present in isolated frog olfactory sensory cilia. Odorant activation of this AC was tissue specific and occurred only in the presence of GTP, suggesting the involvement of receptors coupled to guanine nucleotide binding proteins (G proteins). More detailed characterization of this enzyme 24 showed that it was best stimulated by fruity, floral and herbaceous agents whose effects are generally regarded as pleasant. However, some features of the system did not fit with cAMP as being the physiological mediator for olfaction. Thus, little enhancement of AC was apparent at an odorant concentration of less than 100 ~tM. Moreover, while some odorants were modestly more potent than others in stimulating AC, the differences were not nearly as pronounced as the variation in detection thresholds. Considerable electrophysiologic evidence substantiates biochemical findings implicating cyclic TINS, Vol. 15, No. 12, 1992
nucleotides in olfaction 18'25-31. In excised patches of amphibian ciliary plasma membrane, cAMP directly gates an ion chalme126. This resembles the cGMPgated conductance that mediates phototransducfion in rods but differs in being activated by both cAMP and cGMP. This channel has also been demonstrated in mammalian olfactory cells (Manis, P. B. and Ronnett, G. V., unpublished observations). The reversal potential for the cAMP-gated channel is close to zero, resembling the reversal potential for odorant-stimulated conductanceslS; the absolute value of the reversal potential indicates that this conductance does not select between Na ÷ and K ÷ ions, which is also characteristic of the cGMP-gated conductance of rod and cone outer segments. More recently, Firestein and associates 16'28'29'31 demonstrated that the conductance gated by cyclic nucleotides appears to be identical to that activated by odorant stimulation. An involvement of G proteins is supported by evidence that GTP-y-S prolongs the stimulus response. Blocking phosphodiesterase activity with isobutylmethylxanthine prolongs the odor-elicited current, suggesting that the rate-limiting step in the decline of the current may be the hydrolysis of cAMP by phosphodiesterase. Molecular cloning revealed that the appropriate signal transducfion proteins are enriched in olfactory sensory cilia. The Gs-like G protein designated Golfis greatly enriched in olfactory cilia and stimulates AC in a heterologous system 32'33. More recently, Bakalyar and Reed34-36 cloned a Type III AC that is selectively concentrated in olfactory cilia and is Ca2+ dependent. A cyclic nucleotide-gated channel specific to the olfactory neuron has been cloned37,38 by homology to the cGMP-gated channel of the rod outer segment 39'4°. Two of these components, Golfand type Ill AC, have been ultrastructurally localized to the olfactory sensory cilia41. More recent studies have demonstrated much more potent effects of odorants on AC. Using rapidmixing techniques, Breer and collaborators a2'43 found extremely rapid enhancement of cAMP levels by odorants in isolated rat olfactory cilia, with increases evident as early as 25 ms. Certain odorants stimulated cAMP production at concentrations of 0.01 or 0.1 9M, while others had no effect. To evaluate signal transduction in intact cells, we developed a primary culture system of neonatal rat ORNs 44. By immunocytochemical analysis and polymerase chain reaction (PCR), these cells have been found to express olfactory marker protein (OMP), vimentin, neuronspecific enolase (NSE), synaptophysin, nerve growth factor receptors, neuron-specific tubulin, Golf, odorant receptors (Cunningham, A. and Ronnett, G. V., unpublished observations) and the olfactory cyclic nucleotide-gated channel 37, and are negative for $100 protein and glial fibrillary acidic protein (GFAP) 44. Thus, cultures of primary ORNs express those transduction proteins that occur in mature neurons in vivo. Preliminary data (Manis, P. B. and Ronnett, G. V., unpublished observations) demonstrate that these cells depolarize in response to odorants and display a conductance resembling the cyclic nucleotidegated conductance. AC can be assayed in intact cells maintained in monolayer culture by pretreatment of cells with staphylococcal ec-toxin4s, which introduces extremely small pores, allowing introduction of TINS, Vol. 15, No. 12, 1992
[a2p]ATP for AC assays. In such preparations odorants are extremely potent in stimulating AC, with as little as 0.1 nM isobutylmethoxypyrazine (IBMP) elevating enzyme activity46. Other odorants are less potent, but all activate AC in the low nanomolar range. Responses are biphasic for odorants, with enhancement of AC disappearing at elevated concentrations, sometimes to reappear at still higher (1-10 ~M) levels. Odorant stimulation of AC is rapid in cultured cells, with peak effects observed at 10-15 s of odorant exposure and responses vanishing by 20-30 s. The diminished AC response at increasing concentrations of odorants, as well as the brevity of response, suggests desensitization. Behaviorally, desensitization to many odorants is both pronounced and rapid. Re-exposure of cultures to the same odorant at the same concentration within 15s results in the absence of an AC response 46. Strikingly, reapplication after 1 rain produces a substantially greater response than with the first application, suggesting supersensitization may occur as well. Such assays may eventually permit study of the mechanisms of homologous and heterologous desensitization in olfaction. A role for Ca 2+ in odorant signal transduction is suggested from many studies, with some conflicting results possibly involving methodologic or interspecies differences4'14'26'47-49. Although monovalent cations are generally regarded as being responsible for the olfactory potentials, electrophysiologic studies demonstrate that Ca2+ plays a role either in the initial depolarization or in adaptive responses. Using ORNs of the lamprey, Suzuki 14 showed that Ca2+ channel blockers inhibit spike responses. Nakamura and Gold26 demonstrated that rectification is abolished in the absence of divalent cations, suggesting a voltagedependent channel-blocking effect. Using the wholecell patch-clamp technique, Kurahashi and Shibuyaz° observed inactivation of the odorant-activated conductance only when the external medium contained Ca 2+. Although Zufall et al. Zl found evidence of Ca2+activated K + channels in insect olfactory receptor neurons, they could not identify a Ca 2+ current. On the other hand, divalent cations were found to be necessary for generation of the electro-olfactogram (EOG) in amphibia 4s'52. Single-channel recordings performed on dendritic membranes of isolated salamander ORNs show no voltage dependence in divalent cation-free buffer. In olfactory cilia isolated from the catfish, Restrepo et ai.49 have demonstrated an IP3gated cation channel that is dependent upon Ca2+. Biochemical studies have similarly demonstrated a role for Ca 2+ in olfaction. Calmodulin mRNA is expressed at high levels in both mature and immature olfactory neurons 53. By direct measurement, olfactory cilia contain significant amounts of calmodulin, which activates olfactory AC in isolated bullfrog olfactory cilia47. In o~-toxin-permeabilized monolayer cultures of ORNs, AC activation by odorants is dependent upon Ca 2+, with minimal odorant responses seen below 0.1 ~LMfree Ca 2+, and maximal effects at 10 ~LMfree Ca2+ in the assay buffer46. This dependency fits with the Ca 2+ requirement for the Type III AC localized to the olfactory receptor neuronal cilia34. In isolated rat olfactory cilia elevated Ca2+ levels were not found to be necessary for odorant-induced increases in cAMP levels42, although using the same system, we detect cAMP increases that are as great in the presence as in 509
the absence of Ca2+ (Ronnett, G. V., unpublished observations). Calcium levels may be augmented in chemosensory cilia through a phosphoinosifide mechanism. In the brain and peripheral tissues, receptor-mediated stimulation of phospholipase C generates inositol 1, 4, 5-trisphosphate (IP3), which releases intraceUular Ca2+ (Refs 54, 55). Huque and Bruch56 demonstrated phospholipase C activity in isolated catfish olfactory cilia. This enzyme activity was stimulated by L-alanine, which is an odorant for catfish, as well as by GTP and its non-hydrolysable analogues. Restrepo et al. 49 showed that amino acids enhance Ca2+ influx in isolated catfish olfactory receptor neurons from the catfish. Utilizing the same rapid-mixing system employed in cAMP studies, Breer and colleagues42'43 demonstrated very rapid and potent effects of odorants upon IP3 formation. In primary cultures of olfactory receptor neurons, we also observed low nanomolar effects of odorants upon phosphoinositide turnover57 (Ronnett, G. V., Cho, H., Hester, L. D. and Snyder, S. H., unpublished observations). The response of phosphoinosifide turnover to odorant stimulation is even more rapid than AC responses, with peak production of inositol phosphates occurring by 1 s after odorant stimulation, and responses markedly diminishing thereafter. In isolated rat cilia42, odorantenhancement of the levels of IPa and cAMP appears mutually exclusive. By contrast, the same odorants stimulate AC and phosphoinositide turnover in primary cultures of rat ORNs, although with different potencies, which perhaps suggests interactions with different receptors (Ronnett, G. V., Cho, H., Hester, L. D. and Snyder, S. H., unpublished observations). Reasons for these discrepancies are unclear but may be related to the different systems employed. Breer and associates 42'4a used isolated cilia prepared by 10rr~ Ca2+ shock, which may uncouple transducing elements for certain odorants. The site of IP3-induced Ca2÷ release in olfactory cilia may be unique. In most tissues, IP3 is thought to release Ca2+ from specific sites in the endoplasmic reticulum. Immunohistochemistry at the electron microscope level has localized IPa receptors to the endoplasmic reticulum of neurons58. Kalinoski et aL 59 have demonstrated an IP3 receptor in isolated catfish cilia. Utilizing immunohistochemistry at confocal and electron microscope levels, we showed that in rat olfactory cilia, IP3 receptors are localized to the plasma membrane (Cunningham, A. M. et al., unpubfished observations). Thus, whereas IP3 releases Ca2+ from intracellular stores in most tissues, in olfactory cilia IPa presumably triggers an influx of extracellular Ca2+. The ability of odorants to augment both phosphoinositide turnover and AC activity raises the possibility of crosstalk between the two second messenger systems which may modulate the olfactory response. Such crosstalk is well documented in hormone receptor systems 6~63. This notion fits with the colocalization of IPa receptors and Golf immunoreactivities across the olfactory ciliary layer (Cunningham, A. M. et al., unpublished observations). Following odorant stimulation, cAMP levels decline as rapidly as they rise. This may reflect the rapid decline in odorant-stimulated AC activity, activation of a phosphodiesterase, or both. Recently, we observed that .510
the most prominent phosphodiesterase in olfactory epithelium is a Ca2+-calmodulin form of the enzyme with a uniquely high affinity (KM < 1 ~tM) for cAMP (Ref. 64). Immunohistochemical analysis reveals this phosphodiesterase highly localized to olfactory cilia64. Thus, Ca2+ released by IP3 may activate the phosphodiesterase to degrade the cAMP formed by AC, which itself was activated by Ca2+. Desensitization of signal transduction may occur through a variety of processes, which include receptor internalization, receptor-effector uncoupling and receptor phosphorylation65~7. Utilizing the I52-adrenergic receptor as a model, the functional consequences of G protein-coupled receptor phosphorylation have been extensively characterized6s, and shown to result from the interaction of multiple systems, including cAMP-activated protein kinases and protein kinase C (PKC); homologous desensitization proceeds through agonist-activated receptor phosphorylation catalysed by a specific kinase called [3-adrenergic receptor kinase (~ARK)69. Complete quenching of signal transduction requires the binding of a protein called ~-arrestin (~ARR) to phosphorylate the receptor7°. The specific isoforms ~ARK-2 and [3ARR-2 have been localized to olfactory neurons, specifically the olfactory cilia and dendritic knobs (Dawson, T. M., Arriza, J. L., Lefkowitz, R. J., Jaworsky, D. and Ronnett, G. V., unpublished observations). Additionally, preincubation of isolated olfactory cilia with neutralizing antibodies to ~ARK-2 and [3ARR-2 enhances the absolute level of odorantinduced cAMP 5-10 fold and completely blocks desensitization (Dawson, T. M., Arriza, J. L., Lei'kowitz, R. J., Jaworsky, D. and Ronnett, G. V., unpublished observations). Cyclic AMP-dependent protein kinase has also been shown to play a possible role in olfactory desensitization71. Cyclic GMP may also be involved in odorant responses. In primary olfactory neuronal cultures, odorants augment cGMP levels with peak effects at 30s to 2m in, a lower timecourse than occurs for cAMP (Ret 72). In brain areas such as the cerebellum, nitric oxide mediates certain types of cGMP enhancement73. In olfactory neurons and other brain areas carbon monoxide (CO) appears to be the principal mediator. Thus, inhibitors of heme oxygenase, the CO-forming enzyme, block odorant enhancement of cGMP in olfactory neurons, while specific inhibitors of nitric oxide synthesis are ineffective. Odorant receptors Ligand-binding studies have enhanced our understanding of transmitter receptors, revealing multiple subtypes and elucidating the coupling of receptors to second messengers and ion channels. Similarly, labeling of odorant receptor recognition proteins might clarify the processes involved in olfaction. Several groups have attempted to identify directly putative receptors by the binding of tritiated odorants. Cagan and co-workers2L22'74 demonstrated specific saturable binding of tritiated amino acid odorants to purified cilia, and others have shown membrane binding sites for tritiated amino acids that can be discriminated into various classes 75'76. Utilizing oligonucleofide probes targeted to conserved regions of the genes encoding G-proteinTINS, Vol. 15, No. 12, 1992
coupled receptors with seven transmembrane domains, Buck and Axe177'78 cloned a large family of putative odorant receptors. They used these degenerative oligonucleotides in various combinations in PCR techniques to amplify homologous sequences in cDNAs prepared from rat olfactory epithelium mRNA. They postulated that odorant receptors are members of a large family of genes with closely similar properties and thus would lie in the same PCR band. They digested PCR bands and focused upon those that produced sets of fragments whose molecular weights greatly exceeded the size of the original DNA, hence representing mixtures of multiple DNA sequences. The sequences of ten of these cDNAs reveal a number of interesting properties. All contain seven hydrophobic stretches, the presumed transmembrane domains in which maximal sequence similarity is observed to other members of this superfamily. A number of features of the putative odorant receptors differ from G-protein-linked neurotransmitter receptors. For neurotransmitter receptors, the transmitter appears to bind within the plane of the membrane, with maximal sequence conservation within the transmembrane domain reflecting interactions with the same or similar transmitters. In contrast, striking divergences occur within the third, fourth and frith transmembrane domains of the odorant receptors, suggesting a mechanism for discriminating large numbers of odorants of different structures. The putative odorant receptors already sequenced appear to comprise a number of distinct families, each of which contain subfamilies. Within the subfamilies about 90% identity is apparent between various members, while the homology between the different families is substantially less. Genomic Southern blotting and screens of genomic libraries indicate that the multi-gene family comprises several hundred member genes. Since the cDNA probes used to isolate the genes may not be representative of the full complement of subfamilies, the total number of odorant receptors may be substantially larger, perhaps as large as 1000. Buck and Axe177 have established that DNA rearrangement is not responsible for the diversity of odorant receptors, all of which are encoded by distinct genes. It should be emphasized that expressed proteins for the putative odorant receptor genes have not yet been shown to respond to odorants, although their structural properties strongly imply a receptor function.
Odorant binding protein Initial efforts to identify odorant receptors by the binding of tritiated odorants resulted instead in the identification of specific odorant binding proteins (OBP) in our own79-81 and othera2 laboratories. Purified OBP is a homodimer comprised of two identical 19 kD subunits. OBP can bind virtually all odorants examined. Its affinity for odorants is in the micromolar range. In contrast to odorant receptors, OBP is abundant, constituting at least 1% of soluble nasal protein. Again, in contrast to odorant receptors, there do not appear to be multiple forms of OBP, as Southern blot analysis indicates a very limited potential family size with probably no more than two to four members at most 83. Insight as to the function of OBP comes from its amino acid sequence determined by molecular TIN& Vol. 15, No. 12, 1992
0 o
0 o
¢'=r"P,c
I
CoZ+
//.-A' k
Odorant
~-OBP
G
o
/\
e--Q_
\
1
I olfactorycilium Fig. 1. Model of olfactory signal
/
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transduction. The molecular mechanism of olfaction may be quite compficared. Odorants may initially interact with olfactory binding protein (OBP), which may facilitate odorant binding to odorant receptors. Odorant receptors are coupled to either phospholipase C (PLC) via a Gq-like G protein, or to adenylyl cyclase (AC) via Golf, a Gs-like protein. The former reaction would result in the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2), releasing inositol 1,4,5-trisphosphate (IP3), which may act at the ciliary plasma membrane via an inositol 1,4,5trisphosphate receptor (IPzR) to mediate influx of extracellular Ca2+. Calcium may then augment the activities of phosphodiesterase (PDE) or AC, or perhaps directly depolarize the cell. The AC activated by the initial interaction with odorants produces cAMP, which can gate a nonspecific cation channel leading to the generator potential.
clonings3. OBP is a member of a family of proteins all of which serve as carriers for small lipophilic molecules. Other examples include the retinal-binding proteins, which convey retinal from retinal pigmented epithelium to rods and cones where it is incorporated into rhodopsin 84. These observations suggest that OBP is a carder for odorants and may deliver them to odorant receptors or conceivably remove them from the receptor sites. The former possibility is supported by studies of OBP localization81 . Immunohistochemistry of OBP, as well as [3H]odorant autoradiography and in situ hybridization visualization of OBP mRNA, reveals a selective concentration in the lateral nasal gland, the largest of 20 discrete nasal glands in mammals. The lateral nasal gland in rodents occurs in the posterior area of the nose and extends a long duct to the tip of the nose where its water secretions are atomized in order to humidify inspired air. OBP in these secretions might trap odorants and carry them with inspiration to olfactory receptor neurons. As an alternative, OBP might protect receptors from excessively high concentrations of odorants or remove odorants from receptors. Recently, Rabbitts and colleagues 85 have used oligonucleotides based on the OBP sequence to clone cDNA for new forms of OBP. Some of the new forms of mRNA have been localized in Bowman's glands, located just beneath the olfactory neurons, and in olfactory neurons
511
themselves. OBPs have also been described and their cDNAs cloned from insects 86'87.
Concluding remarks
22 Rhein, L. D. and Cagan, R. H. (1980) Proc. Natl Acad. 5cL USA 77, 4412-4416 23 Pace, U., Hanski, E., Salomon, Y. and Lancet, D. (1985) Nature 316, 255-258 24 Sklar, P. B., Anholt, R. R. H. and Snyder, S. H. (1986) J. Biol. Chem. 261, 15538-15543 25 Menevse, A., Dodd, G. and Poynder, M. T. (1977) Biochem. Biophys. Res. Commun. 77, 671-677 26 Nakamura, T. and Gold, G. H. (1987) Nature 325,442--444 27 Lowe, G., Nakamura, T. and Gold, G. H. (1989) Proc. Natl Acad. 5ci. USA 86, 5641-5645 28 Firestein, S., Zufall, F. and Shepherd, G. M. (1991) J. Neurosci. 11, 3565-3572 29 Firestein, S., Darrow, B. and Shepherd, G. M. (1991) Neuron 6, 825-835 30 Firestein, S. and Werblin, F. S. (1987) Proc. Natl Acad. 5ci. USA 84, 6292-6296 31 Zufall, F., Firestein, S. and Shepherd, G. M. (1991) J. Neurosci. 11, 3573-3580 32 Jones, D. T. and Reed, R. R. (1989) Science 244, 790-795 33 Jones, D. T. and Reed, R. R. (1987) J. BioL Chem. 262, 14241-14249 34 Bakalyar, H. A. and Reed, R. R. (1990) Science 250, 1403-1406 35 Bakalyar, H. A. and Reed, R. R. (1991) Curr. Opin. NeurobioL 1,204-208 36 Bakalyar, H. A. and Reed, R. R. (1991) Curr. Opin. Neurobiol. 1,284-285 37 Dhatlan, R. S., Yau, K., Schrader, K. and Reed, R. (1990) Nature 347, 184-187 38 Ludwig, J., Marglit, T., Eismann, E., Lancet, D. and Kaupp, 8. (1990) FEB5 Lett. 270, 24-29 39 Kaupp, U. 8. (1991) Trends Neurosci. 14, 150-15,7 40 Kaupp, U. 8. et al. (1989) Nature 342,762-766 41 Menco, B. P. M., Bruch, R. C., Dau, 8. and Danho, W. (1992) Neuron 8, 441-453 42 Boekhoff, I., Tareilus, E., Strotmann, J. and Breer, H. (1990) EMBO J. 9, 2453-2458 43 8reer, H., 8oekhoff, I. and Tareilus, E. (1990) Nature 345,
The model of olfaction that emerges from these recent studies is intriguing (Fig. 1). Odorants may first be recognized by OBPs, which carry them to the region of olfactory neuronal receptors. There, odorant molecules are delivered to receptors. How the large number of putative odorant receptor proteins handle recognition remains a mystery. Are the receptors localized according to topographic patterns that reflect psychophysical properties or are odorant receptors disposed in a random fashion with informational integration taking place in the olfactory bulb? Even if each olfactory neuron possesses only a single odorant receptor, the smell quality of odorants still presumably reflects relative affinities of a given odorant for a broad range of receptors whose 'population response' determines the psychophysical response. After odorants bind to receptors, the phosphoinositide and AC messenger systems are triggered. Odorant activation of phosphoinositide turnover and generation of IP3 elevates intracellular Ca2+ that may augment AC activity as well as Ca2+-calmodulin PDE activity localized to the olfactory receptor neuron. This model would fit with a cyclic nucleotide-gated ion channel as the final determinant of olfactory neuronal depolarization. Alternatively, the Ca2+ influx could itself depolarize the olfactory receptor neuron. Olfactory neurons then synapse with mitral cells in the olfactory bulb where a complex integration involving 65-68 multiple interneurons and synapses takes place. At 44 Ronnett, G. V., Hester, L. D. and Snyder, S. H. (1991) the central level very little is known of the molecular J. Neurosci. 11, 1243-1255 mechanisms of olfaction, as even the identities of the 45 Bernheimer, A. W. (1968) Science 159, 847-851 transmitters released at the primary synapse of the 46 Ronnett, G. V., Parfitt, D. J., Hester, L. D. and Snyder, S. H. (1991) Proc. Natl Acad. Sci. USA 88, 2366-2369 ORN onto the bulb have not been determined.
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69 Benovic, J. L., DeBlasi, A., Stone, W. C., Caron, M. G. and Lefkowitz, R. J. (1989) Science 246, 235-240 70 Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. and Lefkowitz, R. J. (1990)Science 248, 1547-1550 71 Boekhoff, I. and Breer, H. (1992) Proc. Natl Acad. Sci. USA 89, 471-474 72 Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V. and Snyder, S. H. Science (in press) 73 Bredt, D. S. and Snyder, S. H. (1989) Proc. Nail Acad. Sci. USA 86, 9030-9033 74 Cagan, R. H. and Zeiger, W. N. (1978) Proc. NatlAcad. Sci. USA 75, 4679-4683 75 Boyle, A. J., Park, Y. S., Huque, T. and Bruch, R. C. (1987) Comp. Biochem. Physiol. 88, 767 76 Wegert, S. and Caprio, J. (1991) J. Cornp. Physiol. A168, 201-211 77 Buck, L. and Axel, R. (1991) Cei165, 175-187 78 Buck, L. B. (1992) Curr. Biol. 2, 467-473
Vision and Visual Dysfunction edited by J. R. Cronly-Dillon, Macmillan Press, 1991. £1250 (17 volumes); £82.50 (individual volumes) ISBN 0 333 52713 5
Never bury your magnum opus in someone else's handbook. Knowles and Dartnall did that with their authoritative book on visual pigments: as Volume 2B of Davson's The Eye, it largely lost its own identity. Within the 17 volumes of Cronly-Dillon's new handbook, Vision and Visual Dysfunction, one volume separates itself from the r e s t - that devoted to the visual agnosias and other central disturbances of perception. It is wholly written by O-J. Griisser and T. Landis (although their names are not on the spine), it is the largest volume in the set (some 600 pages), and, being equipped with its own historical survey of visual science, it can stand alone as an important book in its own right. Yet, so far, it is not well known among neuropsychologists and we should like to draw attention to its virtues. Griisser and Landis's book is timely, for agnosia has regained the central role in perceptual theory that it enjoyed at the end of the 19th century. This fascinating disorder is one in which the patient sees, but does not recognize. He may exhibit normal acuity and may localize stimuli accurately, but he is unable to identify what is before him. A classical distinction, assessed in detail by Griisser and Landis, is that between apperceptive and associative agnosias: in apperceptire agnosia the patient appears TINS, Vol. 15, No. 12, 1992
79 Pevsner,J., Trifiletti, R. R., Strittmatter, S. S. and Snyder, S. H. (1985) Proc. Nail Acad. Sci. USA 82, 3050-3054 80 Pevsner,J., Sklar, P. B. and Snyder, S. H. (1986) Proc. Natl Acad. Sci. USA 83, 4942-4946 81 Pevsner,J., Hwang, P. M., Sklar, P. B., Venable, J. C. and Snyder, S. H. (1988) Proc. Natl Acad. Sci. USA 85, 2383-2387 82 Pelosi, P., Baldaccini, N. E. and Pisanelli, A. M. (1982) Biochern. J. 201,245-248 83 Pevsner,J., Reed, R. R., Fernstein, P. G. and Snyder, S. H. (1988) Science 241,336-339 84 Hellner, J. (1975) J, Biol. Chem. 250, 3613-3619 85 Dear, T. N., Boehm, T., Keverne, E. B. and Rabbitts, T. H. (1991) EMBOJ. 10, 2813-2819 86 Vogt, R. G., Prestwich, G. D. and Lerner, M. R. (1990) J. Neurobiol. 22, 74-84 87 Vogt, R. G., Rybczynski, R. and Lerner, M. R. (1991) J. Neurosci. 1I, 2972-2984
unable to integrate the elements or the attributes of the visual array, whereas the associative agnosic can accurately draw a complex stimulus without knowing what it is. Especially intriguing are cases whose difficulty is confined to a particular category: a patient may have disproportionate difficulty with faces, say, or may be unable to recognize everyday objects while yet being able to read readily. Such cases, for example the rare patients with selective blindness for colour or motion (also reviewed in this volume), are prized today for what they can tell us about the modular nature of visual analysis. Griisser and Landis, with intimidating scholarship but somewhat discursive style, give us a guide to both the glorious old German neurology and the burgeoning modern Anglo-Saxon neuropsychology. There are some 5000 items in their bibliography. Although all the other volumes in the set are edited collections, each deserves to be reviewed as a book in its own right. One or two volumes exhibit the traditional vices of handbooks, being collections of papers that have been written by different authors at different times for different purposes and different audiences. However, most of the individual editors have worked hard to collect a set of distinguished chapters that give coherent coverage of a specialist area of visual science. Initially, it was only possible to buy the complete set of volumes, and libraries were the only candidates as buyers; but now individual volumes can be bought separately, and all of
them can be recommended to the specialist reader. The cake of visual science has been cut by several different categorical knives - by visual subsystem (colour, form, depth and eye movements), by discipline (optics, psychophysics, anatomy, neurophysiology, ergonomics and computation), by pathology, and by phylogeny and ontogeny. Inevitably, the result is both redundancy and omission. There are, for example, two independent accounts of cerebral achromatopsia; there are two accounts of the neural basis of saccadic eye movements; and one set of cone absorbances is replotted at least four times. Yet whereas colour perception, one of the visual system's luxuries, attracts two and a half volumes to itself, the perception of motion, which is arguably more fundamental, earns only three or four chapters and a few paragraphs scattered over several volumes. The publishers have provided a useful index volume, but the student will not find 'Korte's Laws', 'common fate' or 'transparency' in the master index. If, unable to remember the name 'Labidocera', she looks up 'scanning' in the index, she will find Mike Land's paragraph on these curious copepods, but not Richard Gregory's whole chapter on scanning eyes. Most of the handbook must exist in electromagnetic format, if only at the printers: how powerful an instrument it would be if the publishers were to sell it on disc. When we come to consider Visual Dysfunction, it is clear that the handbook is delimited by a trade demarcation. The Editor-in-
Acknowledgements Supportedby USPHS grantsDA-OO266to S.H.S.,N5-01231to G.V.R.,aMcKnight ScholarsA wardand JohnsHopkins ClinicianScientist Awardto G.V.R., ResearchScientist A wardDA-00074to S.H.S.,agrantof IntemationalFlavors andFragrances,anda grantfromtheKeck Foundation.
J. D. Mollon anti B. C Regan Universityof Cambridge, ThePsychological Laboratory, DowningSt, Cambridge,UK CB23EB.
513
Molecularmessengersofolfaction G a b r i e l e V. R o n n e t t a n d S o l o m o n H. S n y d e r
GabrieleE Ronnett Our knowledge of olfactory signal transduction has andSolomonH. been greatly clarified by several recent advances. Snyderareat Molecular cloning has revealed a large family of TheJohnsHopkins putative odorant receptors localized to olfactory epiUniversitySchoolof thelium that display a seven-transmembrane-domain Medicine,Deptsof motif suggesting an association with G proteins. Very Neuroscience, potent and rapid enhancement of both adenylyl cyclase PharmacoloEyand MolecularSciences, and phosphoinositide turnover has been demonstrated andPsychiatry,725 in response to odorants both in isolated olfactory cilia N. WolfeStreet, and primary olfactory receptor neuronal cultures. A Baltimore, Ca2 +-calmodulin-dependent phosphodiesterase has MD21205, USA. been localized to olfactory cilia. A key role for Ca 2÷ is
evident from many investigations. More recently, odorants have also been shown to affect the levels of cGMP in olfactory receptor neurons. The involvement of multiple second messengers may provide mechanisms for both fine-tuning and desensitization of olfaction. The olfactory system is ideal for the study of a number of fundamental questions in the neurosciences. Poised at the interface between the environment and the CNS, it subserves a sensory function that is chemical in nature. Thus, an understanding of transduction mechanisms involved in olfaction may provide insight into signal integration elsewhere in the CNS. The olfactory receptor neuron (ORN) is highly specialized in order to accommodate its function. These neurons are located in a pseudostratified epithelium consisting of three principal cell types 1-3. Sustentacular or supportive cells resembling glia stretch from the epithelial surface to the basal lamina4. The ORN is bipolar, extending apical dendrites to the surface of the neuroepithelium and sending unmyelinated axons through the basal lamina and the cribriform plate of the ethmoid bone to terminate in the olfactory bulb of the brain. The apical dendrites form dendritic knobs from which arise specialized, non-motile cilia where the initial events of olfactory transduction occur 3-8. Basal cells, the third type of cell, underlie the receptor neurons and serve as precursors for the generation of new ORNs throughout adult life2'3. Their ability to regenerate from a precursor population distinguishes ORNs from other neurons so that understanding the biology of these mechanisms may provide insight to neurodegenerative diseases. Interestingly, nasal biopsies of Alzheimer's disease patients reveal regenerative responses to olfactory neuronal degeneration suggesting early involvement of the olfactory neurons in Alzheimer's disease 9'1°. 508
t~) 1992, ElsevierScience Publishers Ltd. (UK)
Odorant signal transduction As with other sensory modalities, the ORN responds to appropriate odorant stimulation with the generation of a graded receptor potential u'l~ that spreads passively along the cell body to the proximal segment of the axon, at which point an action potential fires if threshold is attained. Intracellular recordings 1~-15 demonstrate that sensory neurons have a relatively high resistance and low resting potential. Two types of potential changes are recorded intracellularly in response to odorants ~3. There are monophasic spikes of approximately 10 ms duration that are superimposed on a depolarizing slow potential. This slow potential presumably represents the generator potential resulting from the initial interactions of odorants with ciliary receptors. There is a response latency of several hundred ms, suggesting that a second messenger is involved ~6. Odorant concentration determines the rate of action potential firing. Prolonged exposure to odorants results in an initially high firing rate that decays with time to a low steady state level4'1L12'17. An early step in understanding the molecular events in olfaction was the identification of the olfactory receptor neuronal cilia as the initial site of transduction 4,7,18,19. Selective removal of the cilia abolishes olfactory responses 2°. Thereafter, Cagan and associates 21'22 demonstrated specific binding of amino acids to isolated fish cilia. The first direct studies of odorant influences on second messengers ~3 reported a robust odorantsensitive adenylyl cyclase (AC) present in isolated frog olfactory sensory cilia. Odorant activation of this AC was tissue specific and occurred only in the presence of GTP, suggesting the involvement of receptors coupled to guanine nucleotide binding proteins (G proteins). More detailed characterization of this enzyme 24 showed that it was best stimulated by fruity, floral and herbaceous agents whose effects are generally regarded as pleasant. However, some features of the system did not fit with cAMP as being the physiological mediator for olfaction. Thus, little enhancement of AC was apparent at an odorant concentration of less than 100 ~tM. Moreover, while some odorants were modestly more potent than others in stimulating AC, the differences were not nearly as pronounced as the variation in detection thresholds. Considerable electrophysiologic evidence substantiates biochemical findings implicating cyclic TINS, Vol. 15, No. 12, 1992
nucleotides in olfaction 18'25-31. In excised patches of amphibian ciliary plasma membrane, cAMP directly gates an ion chalme126. This resembles the cGMPgated conductance that mediates phototransducfion in rods but differs in being activated by both cAMP and cGMP. This channel has also been demonstrated in mammalian olfactory cells (Manis, P. B. and Ronnett, G. V., unpublished observations). The reversal potential for the cAMP-gated channel is close to zero, resembling the reversal potential for odorant-stimulated conductanceslS; the absolute value of the reversal potential indicates that this conductance does not select between Na ÷ and K ÷ ions, which is also characteristic of the cGMP-gated conductance of rod and cone outer segments. More recently, Firestein and associates 16'28'29'31 demonstrated that the conductance gated by cyclic nucleotides appears to be identical to that activated by odorant stimulation. An involvement of G proteins is supported by evidence that GTP-y-S prolongs the stimulus response. Blocking phosphodiesterase activity with isobutylmethylxanthine prolongs the odor-elicited current, suggesting that the rate-limiting step in the decline of the current may be the hydrolysis of cAMP by phosphodiesterase. Molecular cloning revealed that the appropriate signal transducfion proteins are enriched in olfactory sensory cilia. The Gs-like G protein designated Golfis greatly enriched in olfactory cilia and stimulates AC in a heterologous system 32'33. More recently, Bakalyar and Reed34-36 cloned a Type III AC that is selectively concentrated in olfactory cilia and is Ca2+ dependent. A cyclic nucleotide-gated channel specific to the olfactory neuron has been cloned37,38 by homology to the cGMP-gated channel of the rod outer segment 39'4°. Two of these components, Golfand type Ill AC, have been ultrastructurally localized to the olfactory sensory cilia41. More recent studies have demonstrated much more potent effects of odorants on AC. Using rapidmixing techniques, Breer and collaborators a2'43 found extremely rapid enhancement of cAMP levels by odorants in isolated rat olfactory cilia, with increases evident as early as 25 ms. Certain odorants stimulated cAMP production at concentrations of 0.01 or 0.1 9M, while others had no effect. To evaluate signal transduction in intact cells, we developed a primary culture system of neonatal rat ORNs 44. By immunocytochemical analysis and polymerase chain reaction (PCR), these cells have been found to express olfactory marker protein (OMP), vimentin, neuronspecific enolase (NSE), synaptophysin, nerve growth factor receptors, neuron-specific tubulin, Golf, odorant receptors (Cunningham, A. and Ronnett, G. V., unpublished observations) and the olfactory cyclic nucleotide-gated channel 37, and are negative for $100 protein and glial fibrillary acidic protein (GFAP) 44. Thus, cultures of primary ORNs express those transduction proteins that occur in mature neurons in vivo. Preliminary data (Manis, P. B. and Ronnett, G. V., unpublished observations) demonstrate that these cells depolarize in response to odorants and display a conductance resembling the cyclic nucleotidegated conductance. AC can be assayed in intact cells maintained in monolayer culture by pretreatment of cells with staphylococcal ec-toxin4s, which introduces extremely small pores, allowing introduction of TINS, Vol. 15, No. 12, 1992
[a2p]ATP for AC assays. In such preparations odorants are extremely potent in stimulating AC, with as little as 0.1 nM isobutylmethoxypyrazine (IBMP) elevating enzyme activity46. Other odorants are less potent, but all activate AC in the low nanomolar range. Responses are biphasic for odorants, with enhancement of AC disappearing at elevated concentrations, sometimes to reappear at still higher (1-10 ~M) levels. Odorant stimulation of AC is rapid in cultured cells, with peak effects observed at 10-15 s of odorant exposure and responses vanishing by 20-30 s. The diminished AC response at increasing concentrations of odorants, as well as the brevity of response, suggests desensitization. Behaviorally, desensitization to many odorants is both pronounced and rapid. Re-exposure of cultures to the same odorant at the same concentration within 15s results in the absence of an AC response 46. Strikingly, reapplication after 1 rain produces a substantially greater response than with the first application, suggesting supersensitization may occur as well. Such assays may eventually permit study of the mechanisms of homologous and heterologous desensitization in olfaction. A role for Ca 2+ in odorant signal transduction is suggested from many studies, with some conflicting results possibly involving methodologic or interspecies differences4'14'26'47-49. Although monovalent cations are generally regarded as being responsible for the olfactory potentials, electrophysiologic studies demonstrate that Ca2+ plays a role either in the initial depolarization or in adaptive responses. Using ORNs of the lamprey, Suzuki 14 showed that Ca2+ channel blockers inhibit spike responses. Nakamura and Gold26 demonstrated that rectification is abolished in the absence of divalent cations, suggesting a voltagedependent channel-blocking effect. Using the wholecell patch-clamp technique, Kurahashi and Shibuyaz° observed inactivation of the odorant-activated conductance only when the external medium contained Ca 2+. Although Zufall et al. Zl found evidence of Ca2+activated K + channels in insect olfactory receptor neurons, they could not identify a Ca 2+ current. On the other hand, divalent cations were found to be necessary for generation of the electro-olfactogram (EOG) in amphibia 4s'52. Single-channel recordings performed on dendritic membranes of isolated salamander ORNs show no voltage dependence in divalent cation-free buffer. In olfactory cilia isolated from the catfish, Restrepo et ai.49 have demonstrated an IP3gated cation channel that is dependent upon Ca2+. Biochemical studies have similarly demonstrated a role for Ca 2+ in olfaction. Calmodulin mRNA is expressed at high levels in both mature and immature olfactory neurons 53. By direct measurement, olfactory cilia contain significant amounts of calmodulin, which activates olfactory AC in isolated bullfrog olfactory cilia47. In o~-toxin-permeabilized monolayer cultures of ORNs, AC activation by odorants is dependent upon Ca 2+, with minimal odorant responses seen below 0.1 ~LMfree Ca 2+, and maximal effects at 10 ~LMfree Ca2+ in the assay buffer46. This dependency fits with the Ca 2+ requirement for the Type III AC localized to the olfactory receptor neuronal cilia34. In isolated rat olfactory cilia elevated Ca2+ levels were not found to be necessary for odorant-induced increases in cAMP levels42, although using the same system, we detect cAMP increases that are as great in the presence as in 509
the absence of Ca2+ (Ronnett, G. V., unpublished observations). Calcium levels may be augmented in chemosensory cilia through a phosphoinosifide mechanism. In the brain and peripheral tissues, receptor-mediated stimulation of phospholipase C generates inositol 1, 4, 5-trisphosphate (IP3), which releases intraceUular Ca2+ (Refs 54, 55). Huque and Bruch56 demonstrated phospholipase C activity in isolated catfish olfactory cilia. This enzyme activity was stimulated by L-alanine, which is an odorant for catfish, as well as by GTP and its non-hydrolysable analogues. Restrepo et al. 49 showed that amino acids enhance Ca2+ influx in isolated catfish olfactory receptor neurons from the catfish. Utilizing the same rapid-mixing system employed in cAMP studies, Breer and colleagues42'43 demonstrated very rapid and potent effects of odorants upon IP3 formation. In primary cultures of olfactory receptor neurons, we also observed low nanomolar effects of odorants upon phosphoinositide turnover57 (Ronnett, G. V., Cho, H., Hester, L. D. and Snyder, S. H., unpublished observations). The response of phosphoinosifide turnover to odorant stimulation is even more rapid than AC responses, with peak production of inositol phosphates occurring by 1 s after odorant stimulation, and responses markedly diminishing thereafter. In isolated rat cilia42, odorantenhancement of the levels of IPa and cAMP appears mutually exclusive. By contrast, the same odorants stimulate AC and phosphoinositide turnover in primary cultures of rat ORNs, although with different potencies, which perhaps suggests interactions with different receptors (Ronnett, G. V., Cho, H., Hester, L. D. and Snyder, S. H., unpublished observations). Reasons for these discrepancies are unclear but may be related to the different systems employed. Breer and associates 42'4a used isolated cilia prepared by 10rr~ Ca2+ shock, which may uncouple transducing elements for certain odorants. The site of IP3-induced Ca2÷ release in olfactory cilia may be unique. In most tissues, IP3 is thought to release Ca2+ from specific sites in the endoplasmic reticulum. Immunohistochemistry at the electron microscope level has localized IPa receptors to the endoplasmic reticulum of neurons58. Kalinoski et aL 59 have demonstrated an IP3 receptor in isolated catfish cilia. Utilizing immunohistochemistry at confocal and electron microscope levels, we showed that in rat olfactory cilia, IP3 receptors are localized to the plasma membrane (Cunningham, A. M. et al., unpubfished observations). Thus, whereas IP3 releases Ca2+ from intracellular stores in most tissues, in olfactory cilia IPa presumably triggers an influx of extracellular Ca2+. The ability of odorants to augment both phosphoinositide turnover and AC activity raises the possibility of crosstalk between the two second messenger systems which may modulate the olfactory response. Such crosstalk is well documented in hormone receptor systems 6~63. This notion fits with the colocalization of IPa receptors and Golf immunoreactivities across the olfactory ciliary layer (Cunningham, A. M. et al., unpublished observations). Following odorant stimulation, cAMP levels decline as rapidly as they rise. This may reflect the rapid decline in odorant-stimulated AC activity, activation of a phosphodiesterase, or both. Recently, we observed that .510
the most prominent phosphodiesterase in olfactory epithelium is a Ca2+-calmodulin form of the enzyme with a uniquely high affinity (KM < 1 ~tM) for cAMP (Ref. 64). Immunohistochemical analysis reveals this phosphodiesterase highly localized to olfactory cilia64. Thus, Ca2+ released by IP3 may activate the phosphodiesterase to degrade the cAMP formed by AC, which itself was activated by Ca2+. Desensitization of signal transduction may occur through a variety of processes, which include receptor internalization, receptor-effector uncoupling and receptor phosphorylation65~7. Utilizing the I52-adrenergic receptor as a model, the functional consequences of G protein-coupled receptor phosphorylation have been extensively characterized6s, and shown to result from the interaction of multiple systems, including cAMP-activated protein kinases and protein kinase C (PKC); homologous desensitization proceeds through agonist-activated receptor phosphorylation catalysed by a specific kinase called [3-adrenergic receptor kinase (~ARK)69. Complete quenching of signal transduction requires the binding of a protein called ~-arrestin (~ARR) to phosphorylate the receptor7°. The specific isoforms ~ARK-2 and [3ARR-2 have been localized to olfactory neurons, specifically the olfactory cilia and dendritic knobs (Dawson, T. M., Arriza, J. L., Lefkowitz, R. J., Jaworsky, D. and Ronnett, G. V., unpublished observations). Additionally, preincubation of isolated olfactory cilia with neutralizing antibodies to ~ARK-2 and [3ARR-2 enhances the absolute level of odorantinduced cAMP 5-10 fold and completely blocks desensitization (Dawson, T. M., Arriza, J. L., Lei'kowitz, R. J., Jaworsky, D. and Ronnett, G. V., unpublished observations). Cyclic AMP-dependent protein kinase has also been shown to play a possible role in olfactory desensitization71. Cyclic GMP may also be involved in odorant responses. In primary olfactory neuronal cultures, odorants augment cGMP levels with peak effects at 30s to 2m in, a lower timecourse than occurs for cAMP (Ret 72). In brain areas such as the cerebellum, nitric oxide mediates certain types of cGMP enhancement73. In olfactory neurons and other brain areas carbon monoxide (CO) appears to be the principal mediator. Thus, inhibitors of heme oxygenase, the CO-forming enzyme, block odorant enhancement of cGMP in olfactory neurons, while specific inhibitors of nitric oxide synthesis are ineffective. Odorant receptors Ligand-binding studies have enhanced our understanding of transmitter receptors, revealing multiple subtypes and elucidating the coupling of receptors to second messengers and ion channels. Similarly, labeling of odorant receptor recognition proteins might clarify the processes involved in olfaction. Several groups have attempted to identify directly putative receptors by the binding of tritiated odorants. Cagan and co-workers2L22'74 demonstrated specific saturable binding of tritiated amino acid odorants to purified cilia, and others have shown membrane binding sites for tritiated amino acids that can be discriminated into various classes 75'76. Utilizing oligonucleofide probes targeted to conserved regions of the genes encoding G-proteinTINS, Vol. 15, No. 12, 1992
coupled receptors with seven transmembrane domains, Buck and Axe177'78 cloned a large family of putative odorant receptors. They used these degenerative oligonucleotides in various combinations in PCR techniques to amplify homologous sequences in cDNAs prepared from rat olfactory epithelium mRNA. They postulated that odorant receptors are members of a large family of genes with closely similar properties and thus would lie in the same PCR band. They digested PCR bands and focused upon those that produced sets of fragments whose molecular weights greatly exceeded the size of the original DNA, hence representing mixtures of multiple DNA sequences. The sequences of ten of these cDNAs reveal a number of interesting properties. All contain seven hydrophobic stretches, the presumed transmembrane domains in which maximal sequence similarity is observed to other members of this superfamily. A number of features of the putative odorant receptors differ from G-protein-linked neurotransmitter receptors. For neurotransmitter receptors, the transmitter appears to bind within the plane of the membrane, with maximal sequence conservation within the transmembrane domain reflecting interactions with the same or similar transmitters. In contrast, striking divergences occur within the third, fourth and frith transmembrane domains of the odorant receptors, suggesting a mechanism for discriminating large numbers of odorants of different structures. The putative odorant receptors already sequenced appear to comprise a number of distinct families, each of which contain subfamilies. Within the subfamilies about 90% identity is apparent between various members, while the homology between the different families is substantially less. Genomic Southern blotting and screens of genomic libraries indicate that the multi-gene family comprises several hundred member genes. Since the cDNA probes used to isolate the genes may not be representative of the full complement of subfamilies, the total number of odorant receptors may be substantially larger, perhaps as large as 1000. Buck and Axe177 have established that DNA rearrangement is not responsible for the diversity of odorant receptors, all of which are encoded by distinct genes. It should be emphasized that expressed proteins for the putative odorant receptor genes have not yet been shown to respond to odorants, although their structural properties strongly imply a receptor function.
Odorant binding protein Initial efforts to identify odorant receptors by the binding of tritiated odorants resulted instead in the identification of specific odorant binding proteins (OBP) in our own79-81 and othera2 laboratories. Purified OBP is a homodimer comprised of two identical 19 kD subunits. OBP can bind virtually all odorants examined. Its affinity for odorants is in the micromolar range. In contrast to odorant receptors, OBP is abundant, constituting at least 1% of soluble nasal protein. Again, in contrast to odorant receptors, there do not appear to be multiple forms of OBP, as Southern blot analysis indicates a very limited potential family size with probably no more than two to four members at most 83. Insight as to the function of OBP comes from its amino acid sequence determined by molecular TIN& Vol. 15, No. 12, 1992
0 o
0 o
¢'=r"P,c
I
CoZ+
//.-A' k
Odorant
~-OBP
G
o
/\
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transduction. The molecular mechanism of olfaction may be quite compficared. Odorants may initially interact with olfactory binding protein (OBP), which may facilitate odorant binding to odorant receptors. Odorant receptors are coupled to either phospholipase C (PLC) via a Gq-like G protein, or to adenylyl cyclase (AC) via Golf, a Gs-like protein. The former reaction would result in the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2), releasing inositol 1,4,5-trisphosphate (IP3), which may act at the ciliary plasma membrane via an inositol 1,4,5trisphosphate receptor (IPzR) to mediate influx of extracellular Ca2+. Calcium may then augment the activities of phosphodiesterase (PDE) or AC, or perhaps directly depolarize the cell. The AC activated by the initial interaction with odorants produces cAMP, which can gate a nonspecific cation channel leading to the generator potential.
clonings3. OBP is a member of a family of proteins all of which serve as carriers for small lipophilic molecules. Other examples include the retinal-binding proteins, which convey retinal from retinal pigmented epithelium to rods and cones where it is incorporated into rhodopsin 84. These observations suggest that OBP is a carder for odorants and may deliver them to odorant receptors or conceivably remove them from the receptor sites. The former possibility is supported by studies of OBP localization81 . Immunohistochemistry of OBP, as well as [3H]odorant autoradiography and in situ hybridization visualization of OBP mRNA, reveals a selective concentration in the lateral nasal gland, the largest of 20 discrete nasal glands in mammals. The lateral nasal gland in rodents occurs in the posterior area of the nose and extends a long duct to the tip of the nose where its water secretions are atomized in order to humidify inspired air. OBP in these secretions might trap odorants and carry them with inspiration to olfactory receptor neurons. As an alternative, OBP might protect receptors from excessively high concentrations of odorants or remove odorants from receptors. Recently, Rabbitts and colleagues 85 have used oligonucleotides based on the OBP sequence to clone cDNA for new forms of OBP. Some of the new forms of mRNA have been localized in Bowman's glands, located just beneath the olfactory neurons, and in olfactory neurons
511
themselves. OBPs have also been described and their cDNAs cloned from insects 86'87.
Concluding remarks
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The model of olfaction that emerges from these recent studies is intriguing (Fig. 1). Odorants may first be recognized by OBPs, which carry them to the region of olfactory neuronal receptors. There, odorant molecules are delivered to receptors. How the large number of putative odorant receptor proteins handle recognition remains a mystery. Are the receptors localized according to topographic patterns that reflect psychophysical properties or are odorant receptors disposed in a random fashion with informational integration taking place in the olfactory bulb? Even if each olfactory neuron possesses only a single odorant receptor, the smell quality of odorants still presumably reflects relative affinities of a given odorant for a broad range of receptors whose 'population response' determines the psychophysical response. After odorants bind to receptors, the phosphoinositide and AC messenger systems are triggered. Odorant activation of phosphoinositide turnover and generation of IP3 elevates intracellular Ca2+ that may augment AC activity as well as Ca2+-calmodulin PDE activity localized to the olfactory receptor neuron. This model would fit with a cyclic nucleotide-gated ion channel as the final determinant of olfactory neuronal depolarization. Alternatively, the Ca2+ influx could itself depolarize the olfactory receptor neuron. Olfactory neurons then synapse with mitral cells in the olfactory bulb where a complex integration involving 65-68 multiple interneurons and synapses takes place. At 44 Ronnett, G. V., Hester, L. D. and Snyder, S. H. (1991) the central level very little is known of the molecular J. Neurosci. 11, 1243-1255 mechanisms of olfaction, as even the identities of the 45 Bernheimer, A. W. (1968) Science 159, 847-851 transmitters released at the primary synapse of the 46 Ronnett, G. V., Parfitt, D. J., Hester, L. D. and Snyder, S. H. (1991) Proc. Natl Acad. Sci. USA 88, 2366-2369 ORN onto the bulb have not been determined.
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Vision and Visual Dysfunction edited by J. R. Cronly-Dillon, Macmillan Press, 1991. £1250 (17 volumes); £82.50 (individual volumes) ISBN 0 333 52713 5
Never bury your magnum opus in someone else's handbook. Knowles and Dartnall did that with their authoritative book on visual pigments: as Volume 2B of Davson's The Eye, it largely lost its own identity. Within the 17 volumes of Cronly-Dillon's new handbook, Vision and Visual Dysfunction, one volume separates itself from the r e s t - that devoted to the visual agnosias and other central disturbances of perception. It is wholly written by O-J. Griisser and T. Landis (although their names are not on the spine), it is the largest volume in the set (some 600 pages), and, being equipped with its own historical survey of visual science, it can stand alone as an important book in its own right. Yet, so far, it is not well known among neuropsychologists and we should like to draw attention to its virtues. Griisser and Landis's book is timely, for agnosia has regained the central role in perceptual theory that it enjoyed at the end of the 19th century. This fascinating disorder is one in which the patient sees, but does not recognize. He may exhibit normal acuity and may localize stimuli accurately, but he is unable to identify what is before him. A classical distinction, assessed in detail by Griisser and Landis, is that between apperceptive and associative agnosias: in apperceptire agnosia the patient appears TINS, Vol. 15, No. 12, 1992
79 Pevsner,J., Trifiletti, R. R., Strittmatter, S. S. and Snyder, S. H. (1985) Proc. Nail Acad. Sci. USA 82, 3050-3054 80 Pevsner,J., Sklar, P. B. and Snyder, S. H. (1986) Proc. Natl Acad. Sci. USA 83, 4942-4946 81 Pevsner,J., Hwang, P. M., Sklar, P. B., Venable, J. C. and Snyder, S. H. (1988) Proc. Natl Acad. Sci. USA 85, 2383-2387 82 Pelosi, P., Baldaccini, N. E. and Pisanelli, A. M. (1982) Biochern. J. 201,245-248 83 Pevsner,J., Reed, R. R., Fernstein, P. G. and Snyder, S. H. (1988) Science 241,336-339 84 Hellner, J. (1975) J, Biol. Chem. 250, 3613-3619 85 Dear, T. N., Boehm, T., Keverne, E. B. and Rabbitts, T. H. (1991) EMBOJ. 10, 2813-2819 86 Vogt, R. G., Prestwich, G. D. and Lerner, M. R. (1990) J. Neurobiol. 22, 74-84 87 Vogt, R. G., Rybczynski, R. and Lerner, M. R. (1991) J. Neurosci. 1I, 2972-2984
unable to integrate the elements or the attributes of the visual array, whereas the associative agnosic can accurately draw a complex stimulus without knowing what it is. Especially intriguing are cases whose difficulty is confined to a particular category: a patient may have disproportionate difficulty with faces, say, or may be unable to recognize everyday objects while yet being able to read readily. Such cases, for example the rare patients with selective blindness for colour or motion (also reviewed in this volume), are prized today for what they can tell us about the modular nature of visual analysis. Griisser and Landis, with intimidating scholarship but somewhat discursive style, give us a guide to both the glorious old German neurology and the burgeoning modern Anglo-Saxon neuropsychology. There are some 5000 items in their bibliography. Although all the other volumes in the set are edited collections, each deserves to be reviewed as a book in its own right. One or two volumes exhibit the traditional vices of handbooks, being collections of papers that have been written by different authors at different times for different purposes and different audiences. However, most of the individual editors have worked hard to collect a set of distinguished chapters that give coherent coverage of a specialist area of visual science. Initially, it was only possible to buy the complete set of volumes, and libraries were the only candidates as buyers; but now individual volumes can be bought separately, and all of
them can be recommended to the specialist reader. The cake of visual science has been cut by several different categorical knives - by visual subsystem (colour, form, depth and eye movements), by discipline (optics, psychophysics, anatomy, neurophysiology, ergonomics and computation), by pathology, and by phylogeny and ontogeny. Inevitably, the result is both redundancy and omission. There are, for example, two independent accounts of cerebral achromatopsia; there are two accounts of the neural basis of saccadic eye movements; and one set of cone absorbances is replotted at least four times. Yet whereas colour perception, one of the visual system's luxuries, attracts two and a half volumes to itself, the perception of motion, which is arguably more fundamental, earns only three or four chapters and a few paragraphs scattered over several volumes. The publishers have provided a useful index volume, but the student will not find 'Korte's Laws', 'common fate' or 'transparency' in the master index. If, unable to remember the name 'Labidocera', she looks up 'scanning' in the index, she will find Mike Land's paragraph on these curious copepods, but not Richard Gregory's whole chapter on scanning eyes. Most of the handbook must exist in electromagnetic format, if only at the printers: how powerful an instrument it would be if the publishers were to sell it on disc. When we come to consider Visual Dysfunction, it is clear that the handbook is delimited by a trade demarcation. The Editor-in-
Acknowledgements Supportedby USPHS grantsDA-OO266to S.H.S.,N5-01231to G.V.R.,aMcKnight ScholarsA wardand JohnsHopkins ClinicianScientist Awardto G.V.R., ResearchScientist A wardDA-00074to S.H.S.,agrantof IntemationalFlavors andFragrances,anda grantfromtheKeck Foundation.
J. D. Mollon anti B. C Regan Universityof Cambridge, ThePsychological Laboratory, DowningSt, Cambridge,UK CB23EB.
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