1. Lecture 21 : Taste I - Receptors
11/16/09

2. Final project background - 1 pg
What are you studying?
What is known about it?
Why is it interesting?
What do you want to find out?
What methods / approach are you using?
List 4 references you are using
Debate if this should be done for Wed or for next Monday
This is graded for your having completed it and not for it’s scientific quality. It is a chance to get some feedback and keep moving.

3. Background This is due either 11/18 OR 11/23
Choose the date based on when you can make substantial progress to move your project forward

4. S M T W Th F S November 4 5 6 7 8 9 10 11 Idea 12 13 14 15 16 Taste I17 18
Taste II 19 20 21 22
23 Project
Backgnd
Unusual senses 24 25
Help (no class) 26
Thanks- 27
giving 28 29
30 Gains and losses
Dec 1
2 Prelim results
Summary 3
Present1
9 Prjct due
Present 2

5. Final projects 2 weeks to develop some preliminary results
Detailed summary of one or two papers or
Preliminary analysis of some molecules …

6. Difference between taste and smell
Fewer receptors
Diversity?
Take it in or send it out
Smell - find food; find mate; detect friend/foe; find territory
Taste - food only
Not a difference in water- vs airborne

7. Drosophila taste Sensory cells all over body Legs taste first
Extend proboscis and taste
Intake and taste with taste papillae on oral surface

8. Proboscis extension Sensory bristles
on feet
Labellum of probuscis
Easy behavioral test for preference and for taste sensitivity

9. Gustatory hairs Single hair has multiple cell types 1 mechanoreceptor
2-4 chemoreceptors
Fig 2.8

10. Multiple sensory responses
Mechanoreception
S neuron
Sugar, amino acids
L1 neuron
Salt (NaCl)
Anion receptor
- fatty acids ??
Pollack and Balakrishnan 1997Record from pure mechanoreceptor (A) or combined mechano-chemoreceptor (B). Mechanoreception occurs more quickly than chemoreception
Pollack and Balakrishnan 1997

11. Fly sensory perception
Mechanoreception - ionotropic
Salt - ionotropic
Anion receptor - ionotropic
S neuron - ionotropic
Sugar gated ion channel
Pollack and Balakrishnan 1997

12. Sucrose detection is ionotropic
Cut sensillum and expose to low Ca2+ so process swells up and can patch onto it
Apply sucrose and see direct response either from whole cell or from pulled off patch
Get response even when inhibit G protein signaling so not metabotropic

13. Search Drosophila genome
Find 43 GPCRs
No homology to known odorant receptors
No homologs in vertebrates
Highly divergent from each other
8-20% similar
Used computer program to screen the emerging drosophila genome for sequences with 7 TM structure - did not screen by blasting so could cast a wider net. Only able to screen first 60% of genome.
At this time, only 2 mammalian taste receptors were available.

14. High divergence - only TM7 is similar
Clyne
Fig 1

15. Several clusters of splice variants All have same 7th TM but 1-6 are different
Clyne fig 3 A few of the genes were in arrays were first 6 TM’s were in one block which then differentially splices with 3 exons containing the last TM
Clyne et al 2000 fig 3

16. How could they test whether these are for taste?
See if expressed in right tissue - taste organs and not someplace else
See if when knock them out Drosophila loses taste sensation.

17. RT-PCR to show gene is expressed in labellum and not elsewhere
Labellum is the major gustatory organ for the fly
Note: They tried to do in situ’s but none of the transcripts were detected. They suggest that in situ’s are not sensitive enough.

18. All expressed in labellum suggesting are gustatory
Note: they made a mutant Drosophila called poxn^70 which lacks the 4 chemosensory neurons in the multi chemo (4)- mechano (1) receptor neuron. In the mutant, 18 of 19 GR’s were not expressed, as they must be expressed just in the missing chemosensory cells. So this suggests they are for gustation.
32D.1 is one of the genes that Scott et al 2001 also shows in their fig 1

19. Scott et al found new genes and tested where all genes were expressed
Able to do the in situ’s that Clyne et al 2000 were not! Used Gal4 expression system. Found some more genes and then tested the prevous ones as well.

20. Took advantage of Gal4-UAS system of gene expression
UAS = Upstream Activating Sequence
See also:
Duffy 2002 Gal4 System in Drosophila - A fly geneticist’s swiss army knife
Dow, J. A. T. J Exp Biol 2007;210:

21. Express Gal4 and UAS in separate lines and then cross them
Duffy 2002
Gal4 expression driven in tissue of interest
UAS sites upstream of report like GFP

22. Different receptors in different cells
Proboscis
proboscis
antennae
Did not see any in situe results for 80% of receptors. So switched to transgenic Gal4-UAS system. Used kb of promoter for any given GR. Mated to flies expressing either b-galactosidase or GFP. Here are some of the results for b-galatosidase reporter in the proboscis.
Transgenic expression of Gal4 driven by gene specific promoter
In situ hybridization of mRNA

23. Proboscis Mouth parts Legs
With GFP you can see the axons and dendrites as well as the receptor part of the cell
GFP driven by GR promoter
Scott et al 2001

24. Receptors expressed in several important body parts
Those in the antennae are considered olfactory. Those in labral, cibarial organs and legs are considered gustatory.
Several of GR genes are expressed in the antenna. These project to antennal lobe and not the subesophageal ganglion (SOG). They also occur in cells which do not contain known olfactory receptors. So this GR family is actually a GOR family as it encodes both gustatory and olfactory receptors!

25. Sugar GPCRs have been found
This is 2007!

26. Drosophila Gr64 family - 6 genes
Made a mutant which is missing all 6 Gr64 family genes. Each gene is composed of exons and introns. The numbered arrows below are the position of primers used in RT-PCR. The rescue line has Gr64e replaced with GFP: so it includes a, b, c, d, GFP, f

27. Knock out all Gr64 genes - Gr64 Lose sensitivity to nearly all sugars
Proboscis extension reflex

28. A new taste in Drosophila?

29. Unique Drosophila receptors
Taste receptors identified from screen
Gr5a - sweet
Gr66a - bitter
E409 - New receptor
E409 receptor is in proboscis and it projects to the SOG in the brain, just like other taste receptors

30. Use calcium imaging to determine sensitivity : CO2
Detect the cell response using Calcium imaging and then expose cell to many stimulants

31. Use calcium imaging to determine sensitivity : CO2
Detect the cell response using Calcium imaging and then expose cell to many stimulants
Respond to carbonated water. Respond to sodium bicarbonate at pH’s favoring CO2 formation but not at other pH’s
b) Shows fluorescence increase in E409 in SOG when exposed to CO2
e) Shows that other taste neurons (Gr5a and Gr66a) do not respond to carbonation

32. Response of olfaction and taste to CO2 are opposite and independent
Olfaction is in antennae. WT avoid gas phase CO2. If knock out E409, fly still avoids gas phase CO2. W/o antennae no longer avoid gas phase stimulae. So response to gas phase is in antennae and not driven by E409
Taste is in labellum. WT prefer CO2 containing. If remove antennae, still prefer. If knockout another receptor, OK. If make temp sensitive Gal4-UAS, at low temp still prefers aqueous CO2. But if heat up, this knocks out E409 receptor cell, then preference decreases.
The control they have in drosophila is AMAZING

33. Questions What is the receptor? Is it a new kind of GPCR?
Is it unique to Drosophila?

34. Questions What are mammalian taste receptors?
What mechanisms do they work by?

35. Five kinds of taste Sweet Sour Salty Bitter Umami
MSG / AA present proteins (meat broth, cheese) and Chinese food
Sweet, sour, bitter, salty, umami (delicious)

36. Three kinds of taste papillae Fungiform contain only 1-2 buds Circumvallate may contain 1000’s Multiple taste receptor cells (50-150) come together to make a taste bud

37. Taste buds connect to cranial nerves
Taste neurons do not project to brain
Synapse on cranial nerves which carry the gustatory signal to brain
Might make easier to regenerate?

38. Record from taste buds Determine sensitivity Single taste bud can be sensitive to multiple taste modalities Modalities likely controlled by different cells and transduction mechanisms, but reside in same taste bud!

39. Chandrashekar et al 2006

40. Papillae made up of many taste buds
1000
100s
few
Different locations
Circumvallate at back
Foliate at back sides
Fungiform in front

41. Diversity of taste mechanisms
Sense
To detect
Metabotropic
Sweet
Nutrients
Bitter
Harmful
Umami
Amino acids
Ionotropic
Salty
Salt balance
Sour

42. No tongue map - all taste buds detect multiple signals
Contrary to popular belief and even a neuroscience text or two

43. Neural wiring alternatives
Labeled line says each taste receptor cell is tuned to specific modality and that info is carried back to brain by specific neuron.
Across fiber model says either each receptor is multiply sensitive with multimodal info going to brain or TRCs are specific but same neurons carry info for all receptors
Chandreshakar 2006

44. Attractant taste Sweet and umami are desired substances
Sweet receptor responds to all sugars, artificial sweeteners
Umami responds to L-amino acids
Monosodium glutamate, aspartate

45. Use of mouse mutants to determine taste genes
Black peak means response to stimulus
Red means no response
Chandrashekar et al 2006

46. Use of mouse mutants to determine taste genes
Use of mouse mutants to determine taste genes Umami=T1R1+T1R3 Sweet=T1R2+T1R3 Both need T1R3 to form heterodimer receptor
Chandrashekar et al 2006

47. Sweet and umami GPCRs
Venus fly trap binding site
Xu et al 2004

48. Bitter detection Detect a diverse array of chemicals
Avoidance
Don’t need to distinguish
May require a larger family of receptors

49. Use of mouse mutants to determine taste genes
Use of mouse mutants to determine taste genes Sensitivity to bitter is a related group of genes Bitter=T2R
Chandrashekar et al 2006

50. Use of mouse mutants to determine taste genes
Use of mouse mutants to determine taste genes PLC-2 is effector TrpM5 is channel Shared by all 3
PLC - phospholipase C
TrpM
Chandrashekar et al 2006

51. Bitter, sweet and umami receptors activate G protein, gustducin which stimulates phospholipase C
Release calcium from internal stores using IP3 receptor
T1R1+T1R3
PLC

52. Salty and sour detection are different pathway
Pkd2l1 is candidate sour receptor
Member of TRP channel family
Pkd2l is polycystic kidney disease 2

53. Mammalian receptor for CO2

54. Pkd2L1 containing cells contribute to CO2 sense
Attenuated diptheria toxin (DTA) knocks out all of the PKD2L1 cell. So something in that cell is responsible. For sensing CO2.

55. Carbonic anhydrase 4 converts CO2 to HCO3 which Pkd2l cell detects
Chandrashekar et al 2009
Car4-/- does not affect sour detection
BZA and DZA inhibit Car4 so decrease CO2 response in WT

56. Each taste modality relies on different receptors
CO2 and pH

57. Salt detection likely a channel
Membrane responds to Na+ gradient

58. Taste modalities are wired so brain responds in desired way
Brain does not avoid bitter substance de novo. It responds in certain way to certain kind neurons turning on. So if put bitter receptors in sweet cells, the brain is wired to like whatever that cell responds to.
Chandrashaker 2006 fig 5
Sweet receptors wired for attraction; bitter receptors for avoidance