Review for "The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila"

Completed on 15 Dec 2015 by Leslie Vosshall . Sourced from http://biorxiv.org/content/early/2015/11/22/032540.

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Author response is in blue.

Nice work!-very cool to expand the role of IRs in biology.

Here are my comments on the paper, ordered by appearance of line number
in the manuscript not by urgency/priority

L139 what is the evidence that IR21a is endogenously expressed in
DOCCs? Anything beyond the Gal4 line? Antibody/in situ? My former PhD student Kenta
Asahina was the king of larval in situs, but that was done before the IRs were
cloned!

L166 did you do a rescue of Ir25a just in the Ir21a DOCCs? Ir25a is
*everywhere* which is a problem for claiming specificity

L192 I don’t understand how brv1 is showing such a clear phenotype, and
not necessary for DOCC responses. Must be some other cells elsewhere that
require this?

L195 I thought the conclusion here was overly strong given the strength
of the evidence offered

L226 This is a great experiment—very elegant

L216 wondered why you did pan-neuronal Ir21a expression rather than
just go with the much more selective HC>Ir21a. You could consider showing
the HC result in the main figure, and putting the pan-neuronal as data not
shown. Always makes me nervous to put a protein like that into *all neurons*

L246-253 could equally be a cell biological problem with trafficking
unrelated to any functionally relevant co-factor. I would not be so forthright
here (unless you have the answer in the form of the co-factor in hand already)

L257-259 I agree with this conclusion. I think Ir25a is receptor for
heat just as much as orco is a sensor for ethyl acetate. It’s the wrong way to
look at this. Of course a ‘co-receptor’ will have a selective phenotype, but
it’s wrong to conclude that it is the subunit responsible for the specific
sensory cue.

Figure 1 I don’t understand why you are doing huge temperature swings
of 14oc vs 20oc. You say these neurons are extremely sensitive to small changes
in temperature, why not image under those conditions. Also you have the chance
to analyze the kinetics of the response to extract party of the answer. Since
your temperature ramps are slow, you could calculate the onset of calcium
signal and the rise time, etc.

Figure 2b I found the cartoon very busy and confusing. All I cared
about was what the temperatures at the extremities were and that was not
labeled

Figure 2c what is navigational bias, not defined?

Figure 2c are the Ir21 mutants actually PREFERRING the cold?

Figure 2 general: it looks like you are not doing single animal
tracking here. Can you revise the data presentation to extract additional
information on speed, turning, path tortuosity, etc etc rather than just a
single index number.

Figure 3 very pretty! But could integrate into another figure because
it’s making a single elegant point, while the other figures are pretty crowded.

Figures 4-5 my lab had the hardest time with this experiment. We
understand what you are trying to do here, but it’s not easy to explain or
understand. Isn’t IR21a already expressed in these cells? What happens if you
overexpress Ir25a? or Ir8a? Or some other random protein? I worry that the
small bumps in Figure 5d are some nonspecific problem with the neuron. Can’t
exclude that with the current data? It looks like there is still some low
amplitude cycling in Ir21a mutants? (Figure 4d, f). Do you think that’s real?



Thanks Leslie!

I appreciate your taking the time to do this… Your comments are preceded by ">>>>" and my responses follow

">>> L139 what is the evidence that IR21a is endogenously expressed in DOCCs? Anything beyond the Gal4 line? Antibody/in situ? "

Two additional types of data involving cell-specific rescue support Ir21a acting in the DOCCs. First, cell-specific expression of a wild-type Ir21a transcript under Ir21a-Gal4 rescues the Ir21a mutant's behavior (fig. 2c). In addition, the cool-activation of the DOCCs is robustly rescued by re-expression of a wild-type Ir21a transcript in these cells (fig. 4e). We are still working on the antibody!

">>>>>L166 did you do a rescue of Ir25a just in the Ir21a DOCCs? Ir25a is
*everywhere* which is a problem for claiming specificity"

Not enough n to include. The argument is not so much that Ir25a confers specificity but that Ir21a does.

">>>>L192 I don’t understand how brv1 is showing such a clear phenotype, and
not necessary for DOCC responses. Must be some other cells elsewhere that
require this?"

Perhaps.

">>>>.L195 I thought the conclusion here was overly strong given the strength
of the evidence offered"

There is no change in DOCC response to cooling in the putative brv1 and brv2 null mutants, so if brv1 has any role in the response, it is not in the ability of DOCCs to respond to cooling.

">>>>L226 This is a great experiment—very elegant

>>> L216 wondered why you did pan-neuronal Ir21a expression rather than
just go with the much more selective HC>Ir21a. You could consider showing
the HC result in the main figure, and putting the pan-neuronal as data not
shown. Always makes me nervous to put a protein like that into *all neurons* "

Both "all neuron expression" N-syb (Fig. 5) and "Hot Cell-specific expression " HC-Gal4 (Supp. Fig. 5) give the same result, so IR21a expression in all neurons is not required to get this effect. For purposes of illustration ( Fig. 5a), it is useful to show how the normal responses of Hot and Cold Cells in wild type animals are out of phase with one another, and how the Hot Cells that misexpress IR21a become "re-activated" when they get cold. Since the same Gal4 drives both UAS-IR21a misexpression and UAS-GCaMP, showing the driver expressed in both cells (N-Syb) allows this.

">>>L246-253 could equally be a cell biological problem with trafficking
unrelated to any functionally relevant co-factor. I would not be so forthright
here (unless you have the answer in the form of the co-factor in hand already)"

We don't rule out trafficking as an additional factor, but co-factors do seem key.

">>> L257-259 I agree with this conclusion. I think Ir25a is receptor for
heat just as much as orco is a sensor for ethyl acetate. It’s the wrong way to
look at this. Of course a ‘co-receptor’ will have a selective phenotype, but
it’s wrong to conclude that it is the subunit responsible for the specific
sensory cue."

">>Figure 1 I don’t understand why you are doing huge temperature swings
of 14oc vs 20oc. You say these neurons are extremely sensitive to small changes
in temperature, why not image under those conditions. Also you have the chance
to analyze the kinetics of the response to extract party of the answer. Since
your temperature ramps are slow, you could calculate the onset of calcium
signal and the rise time, etc."

A detailed analysis of the thermosensitivity of these neurons was performed in the previous paper (Klein et al. 2015, PNAS). We initiated a detailed analysis of sensitivity in the mutants, and there's basically no response when the changes are small. It didn’t seem to add much to the story, so we left it out.

As for using big temperature swings. Well, tiny signals always worry us. We used a stimulus that would give a massive fluorescence change in wild type animals (often >700% increases in signal), that wiping it out in a mutant surely meant something important happened. We also looked at smaller steps and saw those wiped out too in the mutants.

">>> Figure 2b I found the cartoon very busy and confusing. All I cared
about was what the temperatures at the extremities were and that was not
labeled"

Thanks for the feedback! My attempt at graphic minimalism.... This figure was intended to make it clear that we were doing single animal tracking, but it obviously failed.
As for the temperatures: The extremes are given in the methods section. In the figure we show midpoint and gradient temperatures because most larvae never approach the extremes, so the midpoint temperatures (where larvae are released) and gradient steepness are more important parameters.

">>>Figure 2c what is navigational bias, not defined?"

We were trying to communicate this in Fig. 2b. It is the net drift of a larva toward the warm side divided by the total path length of the larva being tracked. It is a indicator of how directed the navigation is toward warmth.

">>> Figure 2c are the Ir21 mutants actually PREFERRING the cold?"

Yes.

">> Figure 2 general: it looks like you are not doing single animal
tracking here. Can you revise the data presentation to extract additional
information on speed, turning, path tortuosity, etc etc rather than just a
single index number."

The behavior is all single animal tracking. We did break down behaviors into their constituent components. As no particular aspect of the behavior was preferentially affected (all seemed to go away in the mutants), it didn't seem critical to show.

">>Figure 3 very pretty! But could integrate into another figure because
it’s making a single elegant point, while the other figures are pretty crowded."

">>Figures 4-5 my lab had the hardest time with this experiment. We
understand what you are trying to do here, but it’s not easy to explain or
understand. Isn’t IR21a already expressed in these cells? What happens if you
overexpress Ir25a? or Ir8a? Or some other random protein? I worry that the
small bumps in Figure 5d are some nonspecific problem with the neuron. Can’t
exclude that with the current data? It looks like there is still some low
amplitude cycling in Ir21a mutants? (Figure 4d, f). Do you think that’s real?"

Figure 4 shows that the larval cold cell responses go away in the mutants and that the mutant defects can be rescued — the tiny GCAMP response remaining could be non-specific, but don’t know for sure…

As for figure 5: the ectopic expression effect is actually large: often a 150% to 300% increase in GCaMP signal in response to a drop from 20 to 14 degrees. It looks like a small bump in the trace because it is being compared to the even larger heat response of the Hot Cells (often >500%).

Thanks again for commenting on the preprint.

Sincerely,
Paul