1. Lecture 19 : Olfaction
11/9/09

2. Final project requirements
1. Must consider the senses
Compare one sense across organisms
Compare multiple senses within one organism
Consider just one sense in just one organism

3. Final project 2. It must have a molecular component
Search databases and compare genes underlying the senses
Use literature to learn about the molecular basis of a sense

4. Final project A. Hand in a formal presentation (80%) 4-5 pg paper
News and Views article à la Science or Nature
Create web pages
Make a movie or video (which could be shown for B)
B. Give a brief (3-4 min) oral presentation (20%)
Share what you’ve learned

5. Timeline Next Wednesday (11/11) 11/18 1 paragraph describing your idea
Idea will be approved
I will help with finding background info
11/18
Outline of background material
Outline of methods - what are you going to do

6. S M T W Th F S November 4 5 6 7 8 9 Today 10 11 Idea 12 13 14 15 16 1718
Outline 19 20 21 22 23 24 25
Help (no class) 26
Thanks- 27
giving 28 29 30
Dec 1
2 Prelim results 3
Present1
Present 2
All due

7. Timeline Wed 11/25 - Help session (no class)
Wed 12/2 - Some preliminary results
12/7 and 12/9
Presentations
All projects due 12/9

8. Examples from past years
The sequence and mode of visual pigment evolution in mammals: What could our mammalian ancestors see?
Drosophila taste reception
Pain in phantom limbs
TRPC2 and VNO-Dependent Pheromone Sensing
Comparison of cephalopod and human vision
How do salmon migrate?
The Dog Nose
Movie about Daphne Soares’ research on crocodile pressure detectors
Butterfly vision

9. Olfaction What are the olfactory receptors? How many OR’s are there?
How are smells encoded?

10. 1. Odor receptors Much our sense of taste comes through odors
Odors can
Trigger memories
Warn of danger
How do we detect 10,000 different odors?

11. Olfaction Sensory cells in olfactory epithelium
Neurally connected to olfactory bulb
Olfactory epithelium

12. Vertebrate olfactory epithelium
Ciliated receptors
12 million receptors
20 cm2 surface area
Primary neurons
Sustentacular cells
Secrete mucus
Basal cells
Stem cells which replace both cell types
Fain 7.7

13. Nobel Prize 2004

15. What did they know Odor stimulates adenylyl cyclase Increases cAMP
Cell depolarizes
cyclic nucleotide gated channel
Dependent on GTP
Likely involves G protein signaling
Buck and Axel fig 1

16. What can they assume? How many receptors?
Are they all alike or different?
Where are they expressed?

17. Assumptions Receptors are membrane bound GPCRs
Encoded by multigene family
Need diversity to detect so many compounds
Expression should be limited to olfactory epithelium

18. Methods
Try to amplify GPCRs from olfactory cDNA using degenerate primers
TM I II III IV V VI VII

19. Methods
Try to amplify GPCRs from olfactory cDNA using degenerate primers
Cut PCR product with restriction enzyme
If get simple product - not OR
If get complex product - could be OR
Sequence

20. PCR products (A) and their digestion (B)
cut
HinfI cuts at G^ANTC

21. PCR products (A) and their digestion (B)
cut
Cut fragment sizes had to add up to more than initial PCR size for there to be multiple products
HinfI cuts at G^ANTC

22. Sequence genes
Use PCR products to probe and screen cDNA library (olfactory epithelium)
Sequence positive clones
Found 18 unique ones

23. Sequence reveals that receptors have 7 TM
Black balls show more variable sites. These are in TM III, IV and V. This is likely where ligand binds and where specificity of different receptors lies.
This GPCR is “upside down” from rhodopsins
Identify key sites know from GPCRs
7 hydrophobic regions Lots of diversity within membrane region
Cysteines for disulfide bonds

24. Genes are only expressed in olfactory epithelium

25. No introns
OR genes do not have any introns! TM

26. Unique receptors Many different receptors
Same receptor occurs in only few cells (0.1%) and randomly across olfactory epithelium
They determine this by seeing how many cells express a given OR - not many 0.1%

27. One neuron - one receptor
Using single cell qPCR they find just one OR is present

28. All receptors for same odorant project to the same location

29. Olfaction
There are about 2000 glomeruli in nose which receive input from 10^6 neurons

30. 2. How many OR’s are there??

31. Comparison of vertebrate ORs Search genomes of zebrafish, pufferfish, frog, chicken, human and mouse
Fish have fewer genes than mammals. What is the evolutionary history?

32. Methods

33. 9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78
Human - 388
Nimura and Nei 2005
Fall in 9 classes. Class gamma is greatly expanded in certain groups. These gene duplications are within an organisms so specialized OR’s evolve within certain animals.

34. 9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78
Human - 388
Nimura and Nei 2005

35. Comparison of functional and total gene #

36. Many pseudogenes Zfish - 98 / 35 Puffer - 40 / 54 Frog - 410 / 478
Chicken - 78 / 476
Human / 414
Nimura and Nei 2005
Functional
genes / Pseudogenes

37. Conclusions Fish most diverse OR’s
Unique expansions in birds, amphibs and mammals
MRCA=most recent common ancestor

38. OR’s distributed across the genome
Difference between mammals and fish
Distributed everywhere
How does one gene - one receptor work??????

39. Species differences Different surface areas
Dog = 40x human
Different repertoire of receptors
Mouse 1000 genes active
Humans 640 genes active

40. Mammalian OR’s

41. Mammal OR’s

43. Compare gene gains / losses

44. Gene gains / losses

45. How do 387 OR’s code for 10,000 smells?

46. Olfactory stimulants
Fall in 4 groups with increasing length of the carbon chain

47. Measure response of isolated mouse olfactory neurons Decrease in fluorescence = increase in calcium = cell responds to odorant
Isolate neurons - put on slide and load with fura2 - Calcium sensitive dye

49. Single receptor recognizes multiple chemicals

50. Single odorant is recognized by multiple receptors Unique set?

51. Receptors work in combination to code odorants

52. Receptors each detect different features of odorants