Discussion Paper 7 Flashcards
In the Kindt et al (C. elegans) paper, what 3 behaviours are being examined?
Nose touch
Foraging
Head withdrawal
In the Kindt et al (C. elegans) paper, what 5 neurons are being studied?
ASH
OLQ
IL1
FLP
RMD
In the Kindt et al (C. elegans) paper, what 2 channels are being studied?
TRPA-1
TRPV
Which channel mediates nose touch behaviour?
TRPV
Which neurons mediate nose touch behaviour?
ASH
OLQ
FLP
What neurons mediate foraging and head withdrawal behaviour?
OLQ and IL1 (and RMD, but this is a motor neuron downstream)
Describe nose touch behaviour
Mechanical stimulation of c. elegans nose results in reversal motion of a forward-moving worm
Describe foraging behaviour
Moving head back and forth in search of food
Describe head withdrawal
Movement of the head (as a reflex) in response to touch on the dorsal or ventral nose
Why did Kindt et al decide to study TRPA-1?
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
What is the significance of TRPA-1 having ankyrin repeats?
It means it is likely connected to the cytoskeleton
What are tm1402 and ok999?
They are both loss of function mutations to the trpa-1 channel
In the Kindt et al study, why were 2 mutant C. elegans strains used?
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
Which neurons (sensory, not motor) in the Kindt et al study express trpa-1?
ASH, OLQ and IL1 (NOT FLP)
The C. elegans RMD neuron is downstream of (2)
OLQ and IL1
How do TRPV mutants respond to head touch while foraging?
Will back away/reverse, this is WILD TYPE behaviour
TRPV not involved in nose touch while foraging
How does a gluR RMD mutant affect head touch responses while foraging?
Worm will not respond to head touch
What channel is primarily involved in reversal or head withdrawal upon head touch during foraging?
TRPA1
Does restoration of TRPA1 in ASH neurons restore wild type mechanosensation in C. elegans?
No
What promotor is used to restore TRPA1 in OLQ, ASH, and IL1 neurons?
PDEL
What promotor is used to restore TRPA1 in ASH neurons only?
PSra6
Did rescue of ASH TRPA1 channels ONLY (OLQ and IL1 are still mutant) restore wild type foraging behaviours in C. elegans?
No
With repeated stimuli (more eyelash touches), how did worms number of reversals or head withdrawals change?
With few stimuli: more reversals
With fewer stimuli: fewer reversals and more head withdrawals – getting lazy
This kind of neuron in C. elegans is involved in both head withdrawal and reversal after nose touch
OLQ
Which channel (TRPV or TRPA1) mediates reversal behaviour?
Both
Which channel (TRPV or TRPA1) mediates head withdrawal behaviour?
TRPA1
What is the difference between using Mouse TRPA1 to attempt to rescue foraging/head withdrawal versus reversal behaviour. Why?
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
What is the role of TRPV in osmosensation and chemosensation in C. elegans?
Mutant TRPV C. elegans have abnormal avoidance behaviours (don’t avoid things they normally would)
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?
No difference - TRPA1 does not mediate osmosensory and chemosensory avoidance behaviours
What is Cameleon?
A genetically-encoded calcium sensor
Describe the components of cameleon
CFP and YFP linked by calmodulin and calmodulin-binding peptide (M13) linker
How does Cameleon work?
Uses FRET: when calcium is present the CFP and YFP interact closely and creates a specific emission (emit in the YFP range)
In this study, which cells expressed Cameleon?
ASH and OLQ (tried to do IL1 but wasn’t bright enough)
During the Cameleon experiments, upon the first touch, how did the mutant TRPA1 neurons react compared to the wild type?
No difference in the reactions
During the Cameleon experiments, upon the second touch, how did the mutant TRPA1 neurons react compared to the wild type?
There was less reactivity (less calcium influx) in OLQ neurons which had mutant TRPA1
How did the researchers control the expression of TRPA1 in CHO cells?
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
When suction was applied to TRPA1-expressing CHO cells, what happened?
The cells responded, confirming they are mechanically sensitive
What was the purpose of using Gadolinium in the CHO experiments?
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
Where is TRPA1 expressed in mice? (2)
Dorsal root ganglion (somatosensory) cells, keratinocytes/epithelial cells
Wasabi will activate this channel… why?
TRPA1
Because TRPA1 has a role in nociception
