1. 1 Bi / CNS 150 Lecture 20 Wednesday, November 13, 2013 Olfaction: vertebrates / Worms / insects Henry Lester Reading: Kandel Chapter 32, pp 625-636 (not taste) (What is olfaction?)

2. 2 Proust, Remembrance of Things Past “as soon as I had recognized the taste of the piece of madeleine soaked in her decoction of lime-blossom which my aunt used to give me (although I did not yet know and must long postpone the discovery of why this memory made me so happy) immediately the old grey house upon the street, where her room was, rose up like a stage set to attach itself to the little pavilion opening on to the garden which had been built out behind it for my parents (the isolated segment which until that moment had been all that I could see); and with the house the town, from morning to night and in all weathers, the Square where I used to be sent before lunch, the streets along which I used to run errands, the country roads we took when the weather was fine... “ Olfactory memory The nose can detect and (in principle) classify thousands of different compounds. The ‘mapping’ of these compounds probably occurs by matching to memory templates stored in the brain; thus, a smell is categorized based on one’s previous experiences of it and on the other sensory stimuli that correlate with its appearance.

3. 3 Recognition of chemicals by the olfactory system The nose can distinguish very similar compounds as different smells. An example: the two stereoisomers of carvone smell like spearmint and caraway. This implies that there are stereoisomer-specific carvone receptors. An earlier argument for proteins! Carvone Stereo center

4. 4 Anatomy of the mammalian olfactory system In many mammals (rodent shown here), the olfactory organs within the nose are split into the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). MOE neurons project to the main olfactory bulb (MOB). VNO neurons project to the accessory olfactory bulb (AOB). MOB output neurons project to regions of cortex, while AOB output neurons project only to the (ventral) amygdala.

5. 5 Cells of the mammalian main olfactory epithelium Olfactory neurons have apical dendrites with long ciliary extensions, where the transduction components are located. Cilia are embedded in the mucus layer. Olfactory neurons turn over and are replaced every 60 days. Axon Olfactory sensory neuron Dendrite Cilia Basal cells Supporting cells Mucus To olfactory bulb Figure 32-2

6. 6 Olfactory receptor proteins in vertebrates and most other phyla Odorants bind to 7-helix (G-protein coupled) receptors. In mice, >1000 genes (2-3% of genes!) encode these receptors. Receptor sequences also are quite variable, especially in putative odorant- binding helices. Thus, the repertoire is extremely diverse. In mammals, each neuron probably expresses only a single receptor.

7. 7 GTPGDP + P i Effector: membrane-bound enzyme outside Odorant binds to receptor  activates G protein The start of the G protein pathway in vertebrate olfaction How fast? 100 ms to 10 s How far? Probably less 1  m    inside Part of Fig. 32-3

8. 8 cytosol The usual GPCR pathway kinase phosphorylated protein cAMP Ca 2+ intracellular messenger receptor tsqi G protein enzymechannel effector membrane from Lecture 12

9. 9 Intracellular messengers bind to proteins kinases phosphorylated protein A few ion channels (olfactory system, retina) Ca 2+ and but in a previous lecture, we said... intracellular messenger Ca 2+ cAMP cGMP Previous lecture

10. 10 The GPCR pathway in an olfactory cell channel receptor tolfqi G protein enzymechannel effector intracellular messenger Ca 2+ cAMP cGMP Very similar to Gs

11. 11 Olfactory neurons have cAMP-activated Na + /Ca 2+ Channels Excised “inside-out” patch allows access to the inside surface of the membrane no cAMP no channel openings +cAMP closed open receptor qi G protein channel ts enzymechannel effector intracellular messenger Ca 2+ cAMP cGMP

12. 12 More about olfactory channels and their role in olfactory transduction Olfactory cAMP-gated channels are permeable to Na + and Ca 2+ Thus, odorant binding causes depolarization of the olfactory neuron through Na + entry. Ca 2+ also enters and activates a Cl - channel, increasing depolarization (E Cl is near zero in these cells). This process stimulates the olfactory neuron to fire action potentials.

13. 13 Expression zones of 4 individual olfactory receptors (rat nose, coronal section) The olfactory turbinates display four ‘expression zones’. Each receptor is expressed in a small, randomly distributed subset of neurons within one of the 4 zones. As there are ~1000 receptors, about 1/250 of neurons within a zone express each receptor. Neurons within each expression zone send axons to a different quadrant of the olfactory bulb. Another gene class, expressed in all olfactory neurons Figure 32-5 K20 L45 A16 Olfactory receptor Olfactory epithelium Olfactory bulb

14. 14 Projections to the olfactory bulb Olfactory neurons send axons to the glomeruli (synaptic balls shielded by glia) of the olfactory bulb. Olfactory neurons excite mitral cells, which are the bulb output cells. Olfactory sensory neuron Mitral cell Periglomerular cell Tufted cell Inhibitory Figures 32-1, 32-6 To lateral olfactory tract like a bishop’s miter (hat) perforated (Latin) glomus, ball of yarn (Latin)

15. 15 Projections to specific glomeruli Neurons expressing a specific olfactory receptor project their axons to a single glomerulus in each half-bulb. Axons converge from many directions onto the target. This projection specificity is at least partly determined by the receptor itself, but the mechanisms are unknown.

16. 16 Mice: glomeruli connected to neurons expressing I7, a receptor for octanol, respond to octanol. This was also the case if the I7 glomerulus was moved to the wrong place in the bulb by transplacing the I7 gene into the genomic locus for another receptor. Glomerular odorant responses: Ca 2+ imaging in a fish Individual glomeruli are selectively activated by specific odorants. In fish, “odorants” are soluble amino acids. Imaging studies now show that specific glomeruli in mammals are also activated in response to odorants.

17. 17 Maps of mitral cell projections to higher olfactory areas Piriform cortex neurons receive projections from mitral cells corresponding to many glomeruli that receive input from ORNs expressing different receptors. Mitral cells also project to olfactory tubercle and other areas. Integration of odorant responses and odorant identification may take place in cortex, although some integration is also likely to occur in the bulb.

18. 18 The vomeronasal organ The VNO is thought to respond to pheromones. It is a cup-shaped organ near the front of the rodent nose; its neurons are divided into basal and apical (near the lumen) layers. The microvilli of the VNO neurons face the lumen. Neurons in the apical layer express the G protein α subunit G αi2, while those in the basal layer express G αo. The transduction channel and the receptors are located on the microvilli at the edge of the lumen.

19. 19 VNO receptor molecules The 2 distinct families of VNO G protein-coupled receptors are all unrelated to MOE receptors. Each VNO neuron probably expresses only one receptor, as in the MOE. Figure 32-9 V2Rs (~100 genes in the rodent) are expressed in a random pattern by basal layer neurons (Go-expressing neurons). V2Rs have large N-terminal extracellular domains. V1Rs (~180 genes) are expressed by different subsets of neurons within the apical layer (Gi-expressing neurons).

20. 20 The GPCR pathway in a VNO cell channel receptor tsqi G protein enzymechannel effector intracellular messenger Ca 2+ cAMP cGMP IP 3 DAG

21. 21 VNO signal transduction Like Alberts 15-36 © Garland phosphatidyl inositol 4,5 bisphosphate = PI(4,5)P 2 TRPC2 channel (like the GPCR lecture)

22. 22 Response characteristics of VNO neurons VNO neurons respond to urine. Some neurons selectively respond to urine from mice of the same sex, others to urine of the opposite sex. Unlike ORNs, their responses are narrowly tuned; no neurons were ever observed to respond to more than one compound. A behavioral assay: mice produce ultrasonic calls (‘whistling’) in response to contact with urine from the opposite sex; production of these calls requires both the VNO and the MOE. In TRPC2 knockout mice, VNO neurons do not respond to urine; and mice do not vocalize in response to urine

23. 23 AOB projections to the brain Mitral cells in the AOB have apical dendrites that arborize in multiple glomeruli. The AOB projects to the amygdala (directly), and the hypothalamus (via the amygdala). The projections from the rostral and caudal AOB halves are superimposed in the amygdala. This implies that integration of pheromone signals may take place primarily in the AOB.

24. 24 Generalities about main olfactory system and vomeronasal system function The main olfactory system mediates cortical responses to volatile odorants, and these cortical responses are used to drive conscious behavior (food- seeking, predator avoidance, etc). The VN system is thought to mediate unconscious responses to water-soluble pheromone compounds found in urine and secretions of other individuals.

25. 25 Loss of VNO signaling eliminates aggressive responses to intruders TRPC2 knockout males mate normally with females. Remarkably, though, they also mount males, which control mice never do. The TRPC2 knockout phenotype suggests that the ‘default’ pathway in the absence of VNO input is to mate with everything. VNO input causes male mice to fight rather than attempt to mate Normal male mice attack intruders introduced into their territory, especially intruders swabbed with male pheromone. TRPC2 knockout mice lack this response. TRP2 = TRPC2

26. 26 Chemical nature of pheromones The various pheromones include prostaglandins in fish, androstenone in pigs, and protein ligands such as hamster aphrodisin. In most cases, however, individual pure compounds don’t elicit strong responses. Natural pheromones are mixtures of many substances, perhaps combinations of (protein carriers) plus (bound small organic compounds).

27. 27 A genetic model system: nematode olfaction C. elegans can chemotax toward and away from volatile attractants and repellents. It uses only two pairs of neurons, AWA and AWC, to respond to volatile attractants. It has many olfactory receptors, however, so each chemosensory neuron must express many of these. The ODR-10 receptor is expressed in AWA and localized to its dendrite. ODR-10 is a receptor for the odorant diacetyl (2,3 butanedione). Worms lacking ODR-10 are not attracted to diacetyl. Figure 32-11

28. 28 ODR-10 phenotype ODR-10 is specific for diacetyl and does not respond to 2,3-pentanedione, which differs by only one methylene group. ODR-10 mutants still chemotax to 2,3-pentanedione The AWC cell has receptors for 2,3-pentanedione Deletion of AWC destroys chemotaxis to 2,3-pentanedione

29. 29 ODR-10 recognizes components of a metabolic pathway What is the selective advantage of a worm’s response to diacetyl? Diacetyl results from respiration by certain bacteria. These bacteria often use citrate as a carbon source. ODR-10 also recognizes the metabolic intermediates citrate and pyruvate. Diacetyl is a volatile signature compound for certain bacterial species. Many other bacteria do not make diacetyl but do make acetoin or lactate as respiratory endproducts. Diacetyl attraction thus allows the worm to recognize specific food sources at a distance in the soil. Citrate and pyruvate (nonvolatile) interactions with ODR-10 may provide taste-like recognition of these bacteria after the worm arrives at their colony.

30. 30 A Recent Surprise: Insect Olfactory Receptors are Probably Ligand-gated Channels Encoded by one of ~ 60 genes An auxiliary subunit, common to most insect olfactory receptors. Also called Or83b, Or1, Or2, and Or7. Terminology is converging on “Orco” For structure of the Drosophila olfactory system, see Fig. 32-10

31. 31 “Metabotropic signalling in vertebrates provides a rich panoply of positive and negative regulation, whereas ionotropic signalling in insects enhances processing speed.” Kaupp, Nature Revs. Neuro, 2010

32. 32 End of Lecture 20