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Comments regarding LUSH(D118A) transgenic flies vis-a-vis previous report on spontaneous and cVA-evoked activity

Posted by Dronder1 on 05 May 2013 at 01:09 GMT

Gomez-Diaz et al. here report that LUSH(D118A) transgenic flies have T1 neuronal spontaneous firing rates of approximately 4.5 spikes/s for LUSH(D118A-RB) (Figure 4B, p. 5) and approximately 3 spikes/s for LUSH(D118A-DS) (Figure S1B and p.10). This is consistent with our previous work (Ronderos & Smith, 2010). But the text states:

"Spontaneous activity in OR67d neurons in [LUSH(D118A-DS)] flies was not elevated (indeed, it was slightly lower) compared to a control wild-type rescue transgenic strain (Figure S1A and S1B)" (p. 5).

However, the authors fail to mention at this point that the spontaneous activity of the "control wild-type rescue transgenic strain" is approximately 5 spikes/s for LUSH(WT-DS) according to Figure S1B, and that therefore all three of these genotypes have higher spontaneous activity compared to wild-type w1118 flies. This is demonstrated both according to their own data (approximately 1-2 spikes/s for w1118, which is included in Figure S1B, but absent from Figure 4B) and our previously published work (ibid.). Indeed, it is later acknowledged in the Figure S1B Legend:

"Although we confirmed the 2-fold higher spontaneous activity in LUSH(D118A-DS) flies compared to a wild-type control, as reported [34], the firing frequency is lower than that observed in a control transgenic LUSH(WT-DS) strain, and falls within the range of spontaneous firing frequencies observed across our new transgenic LUSH lines (Figure 4B)." (p. 10).

While I appreciate this later acknowledgement in the supplemental figure legend on p.10, nonetheless, the way the text is written within the main article's Results section, as already quoted above, seems to suggest otherwise. Therefore, I wish to clarify that this report in no way contradicts our previously published observations regarding spontaneous activity of LUSH(D118A) transgenic flies, but rather confirms them. Nor does it address whether the observed changes in spontaneous firing rates, albeit relatively small, can nonetheless elicit changes in behavior in any of these transgenic lines.

Finally, Gomez-Diaz et al. also seem to be unaware that LUSH(D118A) transgenic flies were already shown to have normal cVA responses in that same paper (Ronderos & Smith, 2010). While Gomez-Diaz et al. provide a full dose-response curve, whereas we provided only responses to 1% cVA, they nonetheless present it without proper acknowledgement of our previously published work.

The comments I make here address only those points concerning my own work (Ronderos & Smith, Activation of the T1 neuronal circuit is necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila melanogaster., J Neurosci. 2010 Feb 17;30(7):2595-9. doi: 10.1523/JNEUROSCI.4819-09.2010.)

David S. Ronderos, Ph.D.
Postdoctoral Fellow
Department of Biological Chemistry
Johns Hopkins University School of Medicine
Baltimore, MD

No competing interests declared.

RE: Comments regarding LUSH(D118A) transgenic flies vis-a-vis previous report on spontaneous and cVA-evoked activity

rbenton replied to Dronder1 on 06 May 2013 at 15:51 GMT

Dear David,

Thank you very much for your comments. We would like to make a couple of responses:

1. re: spontaneous activity - you are right that we were able to confirm the relative difference in spontaneous activity in LUSHD118A-DS and w1118 as reported in your paper (Ronderos et al J Neuro 2010). However, we respectfully disagree with your interpretation that this supports the idea that LUSHD118A is dominant-active because - as described in our paper - we found the spontaneous activity of LUSHD118A-DS is actually less than that of the LUSHwt-DS transgenic control, which we consider to be a more relevant comparison strain that the genetically-unrelated (non-transgenic) w1118 strain. We have found that spontaneous activity of OR67d neurons is somewhat variable across different genetic backgrounds (our unpublished data), making it imperative to compare this very sensitive phenotype in as genetically-close strains as possible, as we have done in our study. For clarity, we preferred to focus the main body of our paper principally on the analysis of our new, site-directed transgenic strains, but present our full (re)-analysis of your transgenes in supplemental figure 1.

2. re: cVA sensitivity restored by LUSHD118A - we do appreciate that in the Discussion section of your paper (Ronderos et al J Neuro 2010) you state “it appears that LUSHD118A expressed in the fly is cVA-sensitive” and refer to a supplemental figure showing a single electrophysiological trace for wt and LUSHD118A genotypes stimulated with 1% cVA. Given your cautious wording and the lack of any dose response analysis or quantification, we felt a more thorough investigation was warranted to establish the ability of LUSHD118A to support cVA sensitivity or not, leading us to our results as described: “we observed that LUSHD118A transgenic flies still display robust responses to a 10,000-fold range of cVA concentrations (Figure 5A and 5B). These responses are statistically indistinguishable from LUSHwt, except at the highest dose presented (Figure 5B). Analysis of the temporal dynamics of cVA-evoked neuronal activity revealed that both the onset and the decay of cVA responses are very similar for LUSHD118A and LUSHwt (Figure 5C).”

Carolina Gomez-Diaz and Richard Benton

No competing interests declared.

RE: RE: Comments regarding LUSH(D118A) transgenic flies vis-a-vis previous report on spontaneous and cVA-evoked activity

Dronder1 replied to rbenton on 08 May 2013 at 16:34 GMT

Dear Richard and Carolina,

Thank you for your responses to my comments, to which I would like to reply.

Regarding the interpretation of the effects on T1 neuronal activity in transgenic flies expressing LUSH(D118A): I would like to make a distinction between 1) the activation of the receptor and 2) the activation of the neuronal circuit.

1. Based on your analysis of basal firing rates of the several LUSH transgenics, it clearly appears that 1) all of them have increased spontaneous activity and 2) therefore, this is not an effect specifically caused by the D118A mutation. (Nota bene: This comment applies only to the transgenic flies, and not to the recombinant protein infusion. We noted this discrepancy with surprise and, like you, speculated that this might be due to differences in the secretory pathways. I no longer think desensitization is a plausible explanation, for the same reasons you note in your paper, namely cVA sensitivity mentioned previously. However, there are still other possible explanations for these differences). Obviously, we did not test any other LUSH transgenics, and so were not aware of the relative basal firing rates at that time (otherwise we would have reached similar conclusions about the cause of the increased firing being attributed to D118A in this context). After all, with such a modest effect by D118A in transgenics, one could hardly expect more from the other mutations when expressed this way (a prediction validated in your paper).

2. Nonetheless, this question is distinct from that of T1 neuronal circuit activation. When we saw the very modest difference in T1 firing in the D118A transgenics, we wondered whether this was sufficient to activate the entire T1 neuronal circuit. Indeed, Schlief and Wilson showed that similar small increases in T1 neural activity robustly activate downstream PNs (Nature Neuroscience, “Olfactory processing and behavior downstream from highly selective receptor neurons”, 2006). We used mating behavior and genetics (+/- cVA) to answer this, and concluded that indeed the T1 circuit was activated in these flies, based both on the behavior results and the electrophysiology (which I will not recap here). If our conclusion is correct, then one would predict that all of the LUSH transgenics (which have similar firing rates) would share this behavioral phenotype. This is obviously testable (although it was understandably outside the scope and objective of this current paper), and I hope someone does the experiment.

I wish you both continued success in your research endeavors.

David S. Ronderos

No competing interests declared.