“Before you know, you must imagine”
Curiosity, intellectual intensity and collaboration – these words define Dr. Richard Axel’s distinguished scientific career. His pioneering work on the olfactory system won him and his fellow investigator, Linda Buck, the Nobel Prize for Physiology and Medicine in 2004. This award reflected over a decade of work accomplished in his laboratory, discovering the large multigene family which encodes odorant receptors and using this knowledge to decipher the organization of the olfactory system. The nature of the receptor molecules involved in olfaction long remained elusive; as they were thought to be quite diverse, experiments in the 80s approached the problem in a more unconventional way than the usual ligand-binding techniques1. Pace et al. (1985) probed for receptors by looking for common transductory components; they discovered an odorant-sensitive adenylate cyclase, which was only activated in the presence of GTP. This suggested the involvement of an olfactory G-protein, which was characterized by Jones and Reed in 1989; mRNA for this protein, termed Golf, was restricted to sensory neurons in the olfactory epithelium. It shared 88% amino acid identity with the G-protein, Gsα2. This protein generally functions to stimulate adenylate cyclase, which elevates levels of cyclic AMP (cAMP), a second messenger that activates downstream effector enzymes and ion channels. Nakamura and Gold (1987) used the patch-clamp technique on the olfactory cilia and discovered the presence of cyclic nucleotide-gated conductance, which they suggested could initiate membrane depolarization in response to odorants. Together, these and other studies presented a picture of signal transduction mediated by G-protein coupled receptors (GPCRs) (Fig. 1) The similarity with proteins involved in visual transduction, as well as neurotransmitter and hormone systems which rely on specific G-proteins for signal transduction, helped form the basis of the assumption that odorant receptors were part of the superfamily of GPCRs4. This played a key role in identifying and characterizing the receptors, and disentangling the mechanisms of olfactory perception.
The growth of a scientist
Richard Axel was born and raised in New York, where he continues to work, holding several professor titles at Columbia University, as well as being an investigator at the Howard Hughes Medical Institute. He considers New York ‘his world’ and grew up appreciating the culture and aesthetics of Manhattan, developing a passion for art, books and music5. He was in fact studying literature, when chance struck, as often happens in the progress of science. To support himself, he found a job washing glassware in Bernard Weinstein’s lab at Columbia, who was investigating the universality of the genetic code5. This was in the early 60s, soon after the revolutionary discoveries about DNA, and the establishment of the central dogma of molecular biology. He became fascinated by the “enormous explanatory power” of this field, was hired as a research assistant and decided to pursue further education in genetics5. After graduating then obtaining a medical degree in order to avoid conscription, he ended up back at Columbia in the microbiology lab of Sol Spiegelman, who taught him “how to dissect a scientific problem”5. He then moved to the National Institute of Health, where biophysicist Gary Felsenfield encouraged him to embrace technology, and use a variety of techniques to solve a problem5. After returning to Columbia to study the structure of genes in chromatin, he helped developed new techniques that enabled DNA-mediated gene transfer to mammalian cells6. Not only did this provide an assay to study gene regulation and function; there were also significant commercial implications for recombinant DNA technology7. Patents held by the university were licensed to pharmaceutical, biotechnology and agricultural companies, earning them millions7. Meanwhile, his laboratory used these techniques to characterize T-cells and made a fortuitous discovery; by isolating clones encoding the human surface glycoprotein, CD4, and expressing the gene in different cellular environments, they uncovered its role as a high affinity receptor for HIV8,9. The most striking aspect of Axel’s development as a scientist is his naturally inquisitive, creative approach to scientific questions, with a deep appreciation of the contribution of other scientists.
‘The interface of molecular biology and neuroscience’
The second half of the twentieth century saw growing interest in neuroscience; advances in technology helped establish the idea that the mind emerges from cellular activities. Axel saw this as the perfect opportunity to use his experience as a molecular biologist to investigate the “tenuous relationship” whereby genes influence behaviour and cognitive functions5. He was inspired by Eric Kandel, who was investigating the cellular basis of memory in the mollusc Aplysia, elucidating what occurs at the level of the synapse10. Alongside Richard Scheller, they decided to isolate genes responsible for innate behaviours, reasoning that since these behaviours are inherited, and mostly unchanged by experience and learning, they would be dictated by genes. In 1982, they published a paper characterizing the organization and expression of the gene for the egg-laying hormone (ELH) in Aplysia; they found that transcription resulted in several distinct RNA transcripts, which were differentially expressed in various tissues. They suggested that these transcripts were involved in creating different combinations of active neuropeptides from the same gene, in this way mediating the various behavioural patterns associated with egg-laying11.
With the arrival of Tom Jessel to his laboratory in 1986, attention was turned to the genes encoding neurotransmitter receptors5. They managed to produce a functional cDNA clone encoding the serotonin receptor, 5HT1c, without protein sequence information, by combining cloning in RNA vectors with an electrophysiological assay in Xenopus oocytes12. It was shown that 5HT1c shared many sequence and structural properties of GPCRs (Fig. 2).
To prove that the clone was functional, they introduced it into mammalian fibroblasts and used pharmacological approaches to demonstrate high-affinity binding to agonists, whilst antagonists inhibited binding. They also used a calcium-sensitive dye and FACS analysis to show that intracellular calcium increased in response to serotonin, further demonstrating the functionality of the receptor. Minute amounts of RNA were sufficient to produce membrane currents in the electrophysiological assay, which was likely due to the amplification generated by second messenger systems; in this case, phospholipase C and calcium12. This made their cloning strategy applicable to the isolation of genes encoding neurotransmitter and growth factor receptors which involve similar mechanisms of signal transduction, even if the mRNA constituted only a tiny portion of the total RNA population, and importantly, without any information about protein sequence12. Previous pharmacological studies and gene cloning confirmed that most neurotransmitters have multiple receptor subtypes; this provides a mechanism for the diverse neural actions of serotonin and other neurotransmitters12. Using the techniques established in this paper, additional receptor subtypes could be characterized to help elucidate the mechanisms of neurotransmitter action. By this point, Axel declared, “there was no departure from neuroscience” 5.
The perception problem
Axel’s interest in sensory perception seemed to stem from a mind inclined to embrace the ‘two cultures’ rather than view them in opposition. In his Nobel Lecture, he opens with René Magritte’s ‘La bonne aventure’, to demonstrate the tension between image and reality13. He recognizes that the problem of how the brain represents the external world is not only a source of creativity for art, but also central to philosophy, psychology and neuroscience13. Organisms have evolved mechanisms to sense the external world dependent on what is important for their own survival and reproduction. Perception, Axel argues, is not a direct recording of the world but rather an internal construct; a “biological reality” determined by the brain, therefore laid down by genes13. Relatively little was known about olfaction in the 80s; although in humans it is viewed as more of an aesthetic sense, it is relied on by many organisms to detect food and choose mates13. The diversity of odours presented a fascinating problem for molecular neuroscientists, in terms of molecular recognition and perceptual discrimination13. The fact that the sensory input consists of defined chemical structures, in contrast with the complexities of visual images, meant that understanding the representation of olfactory stimuli in the brain would be a simpler matter of how ‘chemical space’ is transformed into ‘brain space’13,14.
In the late 80s, after reading a paper by Sol Snyder discussing hypotheses about odorant receptors, Linda Buck, then a postdoctoral student in Axel’s lab, decided to begin her own investigations15,16. The first significant biological question under investigation was how the diversity and specificity of odor recognition was accomplished. This problem was addressed by isolating the genes thought to encode odorant receptors, with the experimental design based on three main assumptions; firstly, that the receptors were likely to belong to the GPCR superfamily (outlined above), secondly, due to the diversity of odorants, they were likely to be encoded by a multigene family and finally, they would be expressed only in the olfactory epithelium4. Axel said that had only one of these criteria been used, thousands more genes would have had to be sorted through17. The experiment involved using polymerase chain reaction (PCR) to amplify homologs of the GPCR superfamily from rat olfactory epithelium RNA4. Restriction digestion of a single PCR product was then performed; multiple species of DNA, representative of a multigene family, would result in DNA fragments whose molecular weight exceeded that of the original PCR product4. The results were more than expected; they confirmed the existence of a multigene family encoding GPCRs expressed only in olfactory sensory neurons, identifying at least 100 genes4. However, they suggested this was probably an underestimate, as the probes used probably did not detect all possible subfamilies and it was likely many genes were linked, so a single clone could contain multiple genes4. They were proved correct; subsequent studies identified over 1000 olfactory receptor genes in mice, and around 900 in humans, of which most appear to be pseudogenes18,19,20.
Their experiments also identified subfamilies within the protein family, based on significant sequence divergence in the transmembrane domains (Fig. 3)4.
Figure 3 shows that high variability occurs in transmembrane regions 3, 4 and 5, which could represent sites of direct contact with odorous ligands; this is supported by previous studies revealing that ligand-binding in other GPCRs, for example the β-adrenergic receptor, involves residues in the hydrophobic core of the protein4,21. Axel and Buck proposed a model whereby individual subfamilies encoded receptors that bind distinct structural classes of odorants4. Within subfamilies, sequence differences were restricted to a small number of residues, suggesting a mechanism for recognising subtle variations within odorant groups4. The contrast with other sensory systems, particularly vision which only involves three different receptors, reflects the fact that olfactory stimuli are not continuously variable in a single parameter, such as wavelength of light, or the frequency of sound13. Rather than processing quantitative differences, the cumulative effort of a multitude of receptors, each recognising a small number of ligands, underlies olfactory perception.
The topographic map
The second significant question under investigation in Axel’s lab was conceptually more difficult than the first; how does the olfactory system discriminate between odorant stimuli, once they had been recognized by the nose? The identification of odorant receptor genes allowed for a molecular approach, further simplified by cloning experiments which showed that individual sensory neurons express only one odorant receptor22. In situ hybridization experiments demonstrated that neurons expressing a given receptor are randomly dispersed in the olfactory epithelium but project to specific glomeruli in the olfactory bulb of the brain, hence creating a topographic map23,24. Genetic manipulations were used to directly visualize the pattern of projections in the brain; the topographic map was conserved amongst individuals of a species, presenting a model whereby the quality of an olfactory stimulus was encoded by spatial patterns of glomerular activity25.
Further experiments in Axel’s lab characterized the development of the olfactory sensory map. Antibody staining revealed that odorant receptors are expressed on both the dendrites and axons of sensory neurons, whilst genetic modifications of receptor coding sequences altered patterns of projection, demonstrating that receptors play an instructive role in establishing the topographic map26,27. Transgene experiments were used to show that the expression of a single gene in each neuron was determined stochastically13. Using lineage tracing to map the fate of neurons expressing mutant receptors, they found that neurons could switch receptors at a low frequency, and proposed a mechanism whereby the expression of a functional receptor triggers a feedback system that suppresses switching, ensuring stable receptor choice for the life of the cell28. By cloning mice from a single olfactory neuron expressing the P2 receptor, they demonstrated that the mechanism for stochastic receptor choice does not involve irreversible DNA changes; the pattern of receptor gene expression was normal – if DNA recombination had occurred, the clones would preferentially express the P2 receptor gene29. Axel suggested the existence of transcriptional machinery only capable of accommodating one receptor gene13.
To explore the functionality of the anatomic map, Drosophila was employed as a model organism; they exhibit complex olfactory driven behaviours mediated by a nervous system several orders of magnitude simpler than mammals13. The principles of organization were found to be remarkably similar to that of mammals, however, attesting to the evolutionary advantages of an efficient system to recognize and discriminate an array of odors13. Two-photon calcium imaging was used to examine odor-elicited changes in the antennal lobe (anatomic equivalent of olfactory bulb), by expressing the calcium sensitive fluorescent protein, G-CaMP, in olfactory neurons and projection neurons30. Different odors were found to create distinct, sparse patterns of glomerular activation, which were conserved between individuals and faithfully transmitted to higher brain centres30. Axel concluded that different odors had unique signatures represented by spatial patterns of neural activity in the brain30. They then attempted to relate these patterns to specific behaviours and found a single population of olfactory neurons innervating one glomerulus to be involved in the detection of CO2, which elicits avoidance behaviour in Drosophila31. Inhibiting neurotransmission in this glomerulus blocked the avoidance response to CO2, providing solid evidence of the neural circuitry underlying an innate behaviour31.
The ghost in the machine
Axel’s lab is now investigating further stages of olfactory processing. Recent experiments have shown that odors activate a sparse subpopulation of neurons in the piriform cortex, without the spatial segregation or chemotopy previously described in active glomeruli32. Different odors therefore activate unique ensembles of cortical neurons, suggesting the piriform may only be an associative cortex33. His lab continues to use mouse and Drosophila to elucidate the sensory coding of odors in the brain and the pathways by which sensory responses are converted to behaviour; his most recent paper examined the glomerular organization of the Drosophila mushroom body, suggesting a mechanism for contextualizing novel sensory experience34,35 . The ultimate problem that remains is how electrical information in the brain is finally decoded to allow the perception of a particular odor; who reads the map13? In his Nobel lecture, he describes this as the old problem of the “ghost in the machine”13. The astounding pace at which neuroscience is moving may soon reveal this ghost, bearing in mind a saying of Axel’s which highlights the importance of creativity in science – “before you know, you must imagine” 7.
- Pace, U., Hanski, E., Salomon, Y., & Lancet, D. (1985). Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature, 316(6025), 255–258.
- Jones, D. T., & Reed, R. R. (1989). Golf: an olfactory neuron specific-G protein involved in odorant signal transduction. Science, 244(4906), 790–795.
- Nakamura, T., & Gold, G. H. (1987). A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature, 325(6103), 442–444.
- Buck, L., & Axel, R. (1991). A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell, 65(1), 175–187.
- Axel, R. (2004). Richard Axel – Biographical. In: Frängsmyr, T. ed. 2005. Les Prix Nobel. The Nobel Prizes 2004, The Nobel Foundation, Stockholm.
- Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G., & Chasin, L. (1979). DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proceedings of the National Academy of Sciences, 76(3), 1373–1376.
- Eisner, R. 2005, Richard Axel: One of the Nobility in Science, P&S, 25(1), [online] Available at: <http://www.cumc.columbia.edu/psjournal/archive/winter-2005/nobility.html> [Accessed 5 January 2014]
- Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A., & Axel, R. (1986). The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell, 47(3), 333–348.
- Gay, D., Maddon, P., Sekaly, R., Talle, M. A., Godfrey, M., Long, E. Goldstein, G., Chess, L., Axel, R. & Kappler, J. (1987). Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature, 328(6131), 626–629.
- Castellucci, V. F., & Kandel, E. R. (1974). A Quantal Analysis of the Synaptic Depression Underlying Habituation of the Gill-Withdrawal Reflex in Aplysia. Proceedings of the National Academy of Sciences, 71(12), 5004–5008.
- Scheller, R. H., Jackson, J. F., McAllister, L. B., Schwartz, J. H., Kandel, E. R., & Axel, R. (1982). A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior in Aplysia. Cell, 28(4), 707–719.
- Julius, D., MacDermott, A. B., Axel, R., & Jessell, T. M. (1988). Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science, 241(4865), 558–564.
- Nobel Media AB, 2004. ‘Richard Axel – Nobel Lecture: Scents and Sensibility: A Molecular Logic of Olfactory Perception’ [online] Available at: < http://www.nobelprize.org/nobel_prizes/medicine/laureates/2004/axel-lecture.html> [Accessed 6 January 2014]
- Axel, R & Buck, L. P. 2004, ‘Interview with Richard Axel and Linda B. Buck’, Interviewed by Peter Sylwan, [webcasting] Nobel Media AB. Available at: <http://www.nobelprize.org/mediaplayer/index.php?id=582> [Accessed 5 January 2014]
- Nobel Media AB, 2004. ‘Nobel Laureates 2004 – Physiology or Medicine’ (documentary) [online] Available at: <http://www.nobelprize.org/mediaplayer/index.php?id=638> [Accessed 5 January 2014]
- Snyder, S. H., Sklar, P. B., Hwang, P. M., & Pevsner, J. (1989). Molecular mechanisms of olfaction. Trends in neurosciences, 12(1), 35–38.
- Howard Hughes Medical Institute, 2004. ‘Richard Axel and Linda Buck Awarded 2004 Nobel Prize in Physiology or Medicine’ [online] Available at: <http://www.hhmi.org/news/richard-axel-and-linda-buck-awarded-2004-nobel-prize-physiology-or-medicine> [Accessed 5 January 2014]
- Godfrey, P. A., Malnic, B., & Buck, L. B. (2004). The mouse olfactory receptor gene family. Proceedings of the National Academy of Sciences of the United States of America, 101(7), 2156–2161.
- Young, J. M., & Trask, B. J. (2002). The sense of smell: genomics of vertebrate odorant receptors. Human Molecular Genetics, 11(10), 1153–1160.
- Zozulya, S., Echeverri, F., & Nguyen, T. (2001). The human olfactory receptor repertoire. Genome Biology, 2(6), research0018.
- Strader, C. D., Sigal, I. S., & Dixon, R. A. (1989). Structural basis of beta-adrenergic receptor function. FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 3(7), 1825–1832.
- Chess, A., Simon, I., Cedar, H., & Axel, R. (1994). Allelic inactivation regulates olfactory receptor gene expression. Cell, 78(5), 823–834.
- Vassar, R., Ngai, J., & Axel, R. (1993). Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell, 74(2), 309–318.
- Vassar, R., Chao, S. K., Sitcheran, R., Nuñez, J. M., Vosshall, L. B., & Axel, R. (1994). Topographic organization of sensory projections to the olfactory bulb. Cell, 79(6), 981–991.
- Mombaerts, P., Wang, F., Dulac, C., Chao, S. K., Nemes, A., Mendelsohn, M., … Axel, R. (1996). Visualizing an olfactory sensory map. Cell, 87(4), 675–686.
- Barnea, G., O’Donnell, S., Mancia, F., Sun, X., Nemes, A., Mendelsohn, M., & Axel, R. (2004). Odorant Receptors on Axon Termini in the Brain. Science, 304(5676), 1468–1468.
- Wang, F., Nemes, A., Mendelsohn, M., & Axel, R. (1998). Odorant receptors govern the formation of a precise topographic map. Cell, 93(1), 47–60.
- Shykind, B. M., Rohani, S. C., O’Donnell, S., Nemes, A., Mendelsohn, M., Sun, Y., … Axel, R., Barnea, G. (2004). Gene Switching and the Stability of Odorant Receptor Gene Choice. Cell, 117(6), 801–815.
- Eggan, K., Baldwin, K., Tackett, M., Osborne, J., Gogos, J., Chess, A., … Axel, R., Jaenisch, R. (2004). Mice cloned from olfactory sensory neurons. Nature, 428(6978), 44–49.
- Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., & Axel, R. (2003). Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell, 112(2), 271–282.
- Suh, G. S. B., Wong, A. M., Hergarden, A. C., Wang, J. W., Simon, A. F., Benzer, S., Axel, R., Anderson, D. J. (2004). A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature, 431(7010), 854–859.
- Howard Hughes Medical Institute, 2012. ‘Representations of Olfactory Information in the Brain’ [online] Available at: < http://www.hhmi.org/research/representations-olfactory-information-brain > [Accessed 7 January 2014]
- Stettler, D. D., & Axel, R. (2009). Representations of odor in the piriform cortex. Neuron, 63(6), 854–864.
- Axel Lab, 2013. ‘The Axel Laboratory’, [online] Available at: < http://www.axellab.columbia.edu/home.php.html> [Accessed 9 January 2014]
- Caron, S. J. C., Ruta, V., Abbott, L. F., & Axel, R. (2013). Random convergence of olfactory inputs in the Drosophila mushroom body. Nature, 497(7447), 113–117
(Originally written January 2014)