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Biology 404 > Discussion Paper 7 > Flashcards

Discussion Paper 7 Flashcards

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1
Q

In the Kindt et al (C. elegans) paper, what 3 behaviours are being examined?

A

Nose touch
Foraging
Head withdrawal

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2
Q

In the Kindt et al (C. elegans) paper, what 5 neurons are being studied?

A

ASH
OLQ
IL1
FLP
RMD

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3
Q

In the Kindt et al (C. elegans) paper, what 2 channels are being studied?

A

TRPA-1
TRPV

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4
Q

Which channel mediates nose touch behaviour?

A

TRPV

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5
Q

Which neurons mediate nose touch behaviour?

A

ASH
OLQ
FLP

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6
Q

What neurons mediate foraging and head withdrawal behaviour?

A

OLQ and IL1 (and RMD, but this is a motor neuron downstream)

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7
Q

Describe nose touch behaviour

A

Mechanical stimulation of c. elegans nose results in reversal motion of a forward-moving worm

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8
Q

Describe foraging behaviour

A

Moving head back and forth in search of food

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9
Q

Describe head withdrawal

A

Movement of the head (as a reflex) in response to touch on the dorsal or ventral nose

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10
Q

Why did Kindt et al decide to study TRPA-1?

A

The role of this channel in C. elegans had not yet been discovered, and it is a member of this family which is present in mammals

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11
Q

What is the significance of TRPA-1 having ankyrin repeats?

A

It means it is likely connected to the cytoskeleton

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12
Q

What are tm1402 and ok999?

A

They are both loss of function mutations to the trpa-1 channel

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13
Q

In the Kindt et al study, why were 2 mutant C. elegans strains used?

A

The more mutant strains, the easier it is to prove that a loss of function in 2 mutant strains gives the same overall result, adds validity to the experiment

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14
Q

Which neurons (sensory, not motor) in the Kindt et al study express trpa-1?

A

ASH, OLQ and IL1 (NOT FLP)

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15
Q

The C. elegans RMD neuron is downstream of (2)

A

OLQ and IL1

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16
Q

How do TRPV mutants respond to head touch while foraging?

A

Will back away/reverse, this is WILD TYPE behaviour

TRPV not involved in nose touch while foraging

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17
Q

How does a gluR RMD mutant affect head touch responses while foraging?

A

Worm will not respond to head touch

18
Q

What channel is primarily involved in reversal or head withdrawal upon head touch during foraging?

A

TRPA1

19
Q

Does restoration of TRPA1 in ASH neurons restore wild type mechanosensation in C. elegans?

A

No

20
Q

What promotor is used to restore TRPA1 in OLQ, ASH, and IL1 neurons?

A

PDEL

21
Q

What promotor is used to restore TRPA1 in ASH neurons only?

A

PSra6

22
Q

Did rescue of ASH TRPA1 channels ONLY (OLQ and IL1 are still mutant) restore wild type foraging behaviours in C. elegans?

A

No

23
Q

With repeated stimuli (more eyelash touches), how did worms number of reversals or head withdrawals change?

A

With few stimuli: more reversals

With fewer stimuli: fewer reversals and more head withdrawals – getting lazy

24
Q

This kind of neuron in C. elegans is involved in both head withdrawal and reversal after nose touch

A

OLQ

25
Q

Which channel (TRPV or TRPA1) mediates reversal behaviour?

A

Both

26
Q

Which channel (TRPV or TRPA1) mediates head withdrawal behaviour?

A

TRPA1

27
Q

What is the difference between using Mouse TRPA1 to attempt to rescue foraging/head withdrawal versus reversal behaviour. Why?

A

Mouse TRPA1 was able to rescue foraging/head withdrawal, cannot rescue reversal behaviour to nose touch

The researchers propose that this is because TRPA1 must have a different role in these neurons

28
Q

What is the role of TRPV in osmosensation and chemosensation in C. elegans?

A

Mutant TRPV C. elegans have abnormal avoidance behaviours (don’t avoid things they normally would)

29
Q

ASH neurons are involved in osmosensation and chemosensation. They express both TRPV and TRPA1. How do mutant TRPA1 ASH neurons compare to wild type ASH neurons?

A

No difference - TRPA1 does not mediate osmosensory and chemosensory avoidance behaviours

30
Q

What is Cameleon?

A

A genetically-encoded calcium sensor

31
Q

Describe the components of cameleon

A

CFP and YFP linked by calmodulin and calmodulin-binding peptide (M13) linker

32
Q

How does Cameleon work?

A

Uses FRET: when calcium is present the CFP and YFP interact closely and creates a specific emission (emit in the YFP range)

33
Q

In this study, which cells expressed Cameleon?

A

ASH and OLQ (tried to do IL1 but wasn’t bright enough)

34
Q

During the Cameleon experiments, upon the first touch, how did the mutant TRPA1 neurons react compared to the wild type?

A

No difference in the reactions

35
Q

During the Cameleon experiments, upon the second touch, how did the mutant TRPA1 neurons react compared to the wild type?

A

There was less reactivity (less calcium influx) in OLQ neurons which had mutant TRPA1

36
Q

How did the researchers control the expression of TRPA1 in CHO cells?

A

Had a strong promoter linked to rtTA, added another construct which was tetO connected to TRPA1

When the cells were given tetracycline, TRPA1 was expressed

37
Q

When suction was applied to TRPA1-expressing CHO cells, what happened?

A

The cells responded, confirming they are mechanically sensitive

38
Q

What was the purpose of using Gadolinium in the CHO experiments?

A

Gadolinium blocks mechanically sensitive channels, applied suction in the presence of Gadolinium and found no response: this further confirmed their results that TRPA1 is mechanically sensitive

39
Q

Where is TRPA1 expressed in mice? (2)

A

Dorsal root ganglion (somatosensory) cells, keratinocytes/epithelial cells

40
Q

Wasabi will activate this channel… why?

A

TRPA1
Because TRPA1 has a role in nociception

Biology 404 (14 decks)

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