Female-specific myoinhibitory peptide neurons regulate mating receptivity in Drosophila melanogaster

Upon mating, fruit fly females become refractory to further mating for several days. An ejaculate protein called sex peptide (SP) acts on uterine neurons to trigger this behavioural change, but it is still unclear how the SP signal modifies the mating decision. Here we describe two groups of female-specific local interneurons that are important for this process—the ventral abdominal lateral (vAL) and ventral abdominal medial (vAM) interneurons. Both vAL and vAM express myoinhibitory peptide (Mip)-GAL4. vAL is positive for Mip neuropeptides and the sex-determining transcriptional factor doublesex. Silencing the Mip neurons in females induces active rejection of male courtship attempts, whereas activation of the Mip neurons makes even mated females receptive to re-mating. vAL and vAM are located in the abdominal ganglion (AG) where they relay the SP signal to other AG neurons that project to the brain. Mip neuropeptides appear to promote mating receptivity both in virgins and mated females, although it is dispensable for normal mating in virgin females.

should be provided to help understand the behavioural phenotype. 4. P4: given the importance of the ovipositor extrusion result, I suggest to include representative videos showing the respective phenotypes. 5. P4: given the phenotype of ovipositor extrusion, I find it problematic to talk about a 'mating decision' in the context of this assay. Do the females 'decide' not to remate or the males cannot mate with them, because they extrude their ovipositor. This should be rephrased to allow for different interpretations. 6. P4: 'Because there were no Mip-GAL4-positive ppk neurons in the uterus (not shown)…'. This result should be shown given its importance. 7. P5: 're-mating frequency in mated females (Fig. 2c)'… The label above the figure panel states 'mating' and not 're-mating'. This should be corrected. 8. P6: 'We used 27 °C for thermal activation to minimize the activation of the subpopulation of Mip neurons that inhibits mating (i.e., those that express Mip6.0-GAL80, see Fig. 1).' I do not understand the rationale here. Please explain. Why does 27 degrees affect these neurons differentially as compared to the non-Gal80 populations? 9. Figure 2e: the result that Mip neuron activation enhances mating in the absence of SPR is very interesting. Depending on the effect of Mip on SPR signalling (activating or inhibiting (possibly depending on Mip levels or cell state), however, this result might be expected although SPR is the receptor for Mip. I suggest to include SPR/SPR mutants in the absence of Mip-TrpA1 and test them at 31 degrees to exclude that the effect of temperature in the background of SPR mutants contributes or explains this effect.
10. Figure 3b: Why was the re-mating assay not carried out for 2 copy dsx-flp animals? Otd-flp only addresses the brain neurons, but not the additional neurons labelled in the VNC… 11. P8: The authors state: 'We noted that activation of vAL alone does not induce the remating phenotype in mated females, whereas activation of all Mip neurons does.' I assume they refer to the dsx-flp results. If so, this should be made clear. If not, they should name the evidence.

Reviewer #3 (Remarks to the Author):
This is an interesting study that uses manipulation of specific peptide-containing neurons in Drosophila to examine the neural circuitry underlying female sexual receptivity. This field has produced many novel findings and the authors have previously contributed important findings, especially regarding the nature of the peptide ligands and receptors involved. I have several questions and comments that may help improve this manuscript: Lines 23-25 The summary finishes with the sentence: "Genetic analyses with a Mip-null mutant suggest that the Mip neuropeptide produced in vAL promotes mating receptivity both in virgins and mated females. But lines 144-145 states that MIP -/-vigins mate normally -so I do not understand how this conclusion (the main one) is reached.
Line 73 "Silencing of EH, capa and kinin gal4 neurons produces a marginal or non-significant phenotype…" From the Methods, I understand "marginal" to mean p<0.05 or p<0.001that sounds significant by every conventional statistical approach I am used to….so what is meant by the term 'marginal'? non-significant? The authors should explain their thinking on this.
S Figure 1 Not sure why MIP-Gal4>UAS-shi (21 and 30 degrees) in mated females is not reportedplease include or explain.
Line 95 The 6 kB gal80 line appears to be a very precise discriminator for presumed 'inhibitory' MIP neurons. Was this fragment chosen arbitrarily? Were any others tried? It would be useful to see a schematic map of MIPGAL4+ neurons and MIP-Gal80[6.0]+ neurons. I would have more confidence in the interpretation if the authors had explored the result more with additional constructs/experiments. Were all inhibitory neurons removed? Were any excitatory neurons silenced?
Lines 113-114 Please provide a reference for statement regarding egg-laying.

Line 135
The use of tethered SP is useful. Have the authors tried a tethered MIP? That would seem to be a useful reagent in this analysis. What evidence exists to indicate the transmitter phenotype of the SPSN neurons that project from the uterus?

Line 152
The text does not reference the correct panels of Figure 2. Not sure the significance of Figure 2C, as MIP cell activation is supposed to increase receptivity, but in virgin females, it is already near 100% -therefore this experiment appears hampered by a ceiling effect.
Line 162 Unexpectedly, the design switches protocols for thermal activation: previously it used 30 degrees (e.g. Figure 1), now it switches to 27 degrees. The text explains that use of 27 degrees was intended to "minimize the activation" of the inhibitory MIP cells -on what basis was this predicted? How do the authors know the inhibitory neurons require a higher thermal threshold for activation -is there some evidence? Without some plausible explanation, I find this set of experiments very difficult to evaluate.

Figure 2
In Figure 2b, why not add the Mip-Gal80 to test the contribution of the MIP gene to the "full" complement of activating MIP+ neurons? In Figure 2d, mipGal4>TrpA1 at 27 degrees produces 100% cumulative mating in less than 20 min. However, that genotype at 30 degrees produces ~60% copulation within 1 hr. Aren't these the same behavioral measure, and if so why are the outcomes discrepant? If different behavioral measures, please explain the difference in the assay, and also why two different assays were used.

Line 192
Silencing Mip/dsx neurons decreased virgin female receptivity ( Figure 3a); yet the text states these neurons "maintains female sexual receptivity". I disagree -at most, the evidence supports the hypothesis that they elevate virgin female receptivity. BTW, 3a uses Kir to silence neurons whereas other experiments us UAS-shi: any explanation for using different reagents to the same end? This adds design variables that lessen the strength of the conclusion regarding neuronal function.
Lines 201-210 The section using confocal microscopy to interpret synaptic interconnections between SPSN and vAL and SAG neurons is not credible. Apparent proximity at the LM level interpreted to represent "reciprocal connections" is not a conservative interpretation.
Lines 211-250 I was confused by this section in that the text no longer referred to dsx neurons -why not?
Line 215 EGFP labeled Gal4 neurons are not specified as to age or mating status.  The low power images are of little value without some description. A high power view is offered of the vAM region (I cannot really see the yellow dashed box): How were these two examples (the ones illustrated for us) scored quantitatively in panel e? I think I see 2 cells in the virgin and 2 cells in the mated -is that what the authors scored? In the lateral aspects of T3, the virgin tissue contains 1-2 dark cells and the mated tissue appears to contain these same cells, but the latter are much less intensely stained. That raises the question -is diminished TRIC staining exclusively displayed by vAM neurons? If not, does diminished staining in the vAM retain as much significance? What are the criteria employed here to score a positive cell body? Were all labeled cell groups scored? Is it fair to only compare cell body staining with this method? There appears to be considerable staining of processes. Finally, can the authors demonstrate the validity of the assay (the staining and scoring methods) with some positive control set of neurons, for which there is independent evidence of activity in a given interval? i.e., a positive control.
> We performed additional experiments to examine the functional relationships between the Mip neurons (vAL/vAM), SPSNs, and SAG (See new Fig. 6). Our functional epistasis experiments show that Mip neuron activations can override SPSN silencing, but not SAG silencing. This indicates the Mip neurons are positioned between SPSNs and SAG neurons in this neuronal circuit. This observation, together other anatomical and behavioral data suggest that Mip neurons likely relay signals from SPSNs to SAGs, which project to the brain.
In response to the second comment on characterizing the Mip-GAL4 and Mip6.0-GAL80 subsets, we prepared a new Fig. 2 that shows Mip-GAL4, Mip6.0-GAL4 and Mip6.0-GAL80 expression in the CNS stained with anti-MIP. Schematic diagrams are also included in Fig. 2. For more details, please see our response to comment 2 of Reviewer 1 below.

Reviewers' comments:
Reviewer #1 (Remarks to the Author): 1. Authors performed well-controlled genetic experiments to identify the interneuronal microcircuits that are difficult to dissect when they are associated with complex behavioural traits. As authors already pointed out, the SPSN is the primary sensory neurons located in the female uterus. Authors need to show that SPSN postsynaptic terminals are connected to vAL or vAM. This can be done simply by using ppk-CD4-tdGFP lines (Bloomington 35843) crossed to Mip-GAL4>UAS-tdTomato flies. If these flies can be combined to fru-GAL80, they can confirm that SPSN synaptically connected to vAL or vAM. > Indeed, we performed a GFP Reconstitution Across Synaptic Partners (GRASP) experiment to visualize potential synaptic contacts between Mip-GAL4 neurons (including vAL and vAM) and ppk neurons (see Figure P1 below). The GRASP results, however, were difficult to interpret because the ppk-LexA and Mip-GAL4 expression were not limited to the processes of the SPSNs and vAL (or vAM), respectively. Mip-GAL4 expression occurs not only in vAL and vAM, but also several additional AG neurons (Fig. P1A). Likewise, ppk-LexA is expressed in SPSNs and many additional ppk-positive sensory neurons, many of the processes of which enter the AG (Fig.  P1B). Although we detected robust GRASP signals between ppk-LexA and Mip-GAL4 processes (Fig. P1C), we were unable to distinguish the GRASP signal coming from the contact points between vAL (or vAM) and SPSNs and that coming from other functionally irrelevant ppk-LexA and Mip-GAL4 contacts. We, therefore, are unable to offer new data in response to this point because of a technical reason. The signal from these neurons is restricted to the distal tip of the AG. TG, thoracic ganglion; AG, abdominal ganglion. Scale bars, 50 μm. Fig. 1d-j. All the works authors performed are impressive and promising results that will help the Drosophila research community to increase the knowledge of how the neuropeptide signalling can slowly and reversibly modulate interneuronal network and results in significantly altered behaviour. I strongly suggest that Editor should publish this manuscript in Nature Communications.

[redacted]
Reviewer #2 (Remarks to the Author): Jang and colleagues investigate the neural circuit underpinning Drosophila melanogaster female receptivity to re-mating. They focus on neurons that express the neuropeptide Mip based on results of a screen for neuromodulators involved in this behavioral switch. The question is interesting, the approach is very good, the results are of high quality. Using a combination of genetic tools including a Mip-Gal4 and a Mip-Gal80 line they genetically dissect out two sets of neurons in the fly's VNC. Based on another set of results (with dsx-flp and otd-flp) the authors propose that in particular the so-called vAL neurons promote female mating receptivity. While this manuscript clearly has the potential to extend our knowledge of the circuit that controls female receptivity, it, unfortunately, also leaves several open questions and knots untightened. The model (Figure 4f) therefore appears quite vague, because the functional connectivity of the neurons is still somewhat elusive. This carries over into the discussion, which is brief and to the point, but again shows that many issues remain unresolved, including the role of SPR, the relative role of vAL and mAL, the role of Mip itself in these different neuron populations and its relationship to SP. It is clear that the authors will not be able to solve the puzzle completely, but at this point, this reviewer feels left with more questions than answers. I suggest to either solidify the role of SPR or remove this part altogether, because the results are not fully convincing (to their credit the authors point this out themselves in the discussion).
> In this revision, we show the functional relationships among the Mip neurons (vAL/vAM), SPSNs, and SAG neurons (See new Fig. 6). Using functional epistasis experiments, we found that Mip neurons function downstream of SPSNs, and upstream of SAGs. This supports the model that Mip neurons relay SPSN-born SP signal to SAGs. In addition, we removed our speculation on the possibility that Mip competes against SP from the haemolymph for SPR (lines 283-289 in the first submission), because Mip activation is capable of promoting mating even when the SPSNs have been silenced. This means Mip's action on the SPSNs appears to be negligible. Technically, the paper is of high quality with careful behavioral analysis including the statistics, beautiful images and state-of-the-art methods. > We revised the text to remove the term 'mating decision.' > In our first submission, we did not exclude the possibility that Mip neurons are simply involved in motor activities generating ovipositor extrusion behaviour. In this revision, we now show virgin females with silenced Mip neurons show rejection behavior only when they are courted (Fig. 1l). We showed this in two ways. First, we showed that Mip neuron silencing itself does not induce ovipositor extrusion in isolated virgin females. Next, we repeated the same result in females kept with noncourting males, such as fru F males. Males homozygous for fru F show very little courtship toward females 6 .

P4: 'Because there were no Mip-GAL4-positive ppk neurons in the uterus (not shown)…'. This result should be shown given its importance.
It is now included as Supplementary Fig. 4a. 7. P5: 're-mating frequency in mated females (Fig. 2c)  Mip-GAL4 neurons seem to comprise two functionally opposing subsets. This is because thermal activation of Mip-Gal4 neurons elevates re-mating in mated females, but it also suppresses virgin mating. We generated Mip 6.0 -GAL4, a new Mip-GAL4 that carries a smaller genomic fragment of the Mip promoter than the original Mip-GAL4. We then found that activation of Mip 6.0 -GAL4 neurons (Mip 6.0 >dTrpA1, 30 o C) almost completely suppresses virgin mating (See Fig. 2d). Remarkably, however, we observed that Mip 6.0 >dTrpA1 virgin females incubated at 27 o C remain fully receptive (see Fig. 2d). We compared Mip 6  10. Figure 3b: Why was the re-mating assay not carried out for 2 copy dsx-flp animals? Otd-flp only addresses the brain neurons, but not the additional neurons labelled in the VNC… > We replaced the old Fig. 3b with a new one. The new one show the results for animals with 2 copies of dsx FLP (See Fig. 4b).

P8: The authors state: 'We noted that activation of vAL alone does not induce the re-mating phenotype in mated females, whereas activation of all Mip neurons does.' I assume they refer to the dsx-flp results. If so, this should be made clear. If not, they should name the evidence.
This is an interesting study that uses manipulation of specific peptide-containing neurons in Drosophila to examine the neural circuitry underlying female sexual receptivity. This field has produced many novel findings and the authors have previously contributed important findings, especially regarding the nature of the peptide ligands and receptors involved. I have several questions and comments that may help improve this manuscript: Lines 23-25

The summary finishes with the sentence: "Genetic analyses with a Mip-null mutant suggest that the Mip neuropeptide produced in vAL promotes mating receptivity both in virgins and mated females. But lines 144-145 states that MIP -/-vigins mate normally -so I do not understand how this conclusion (the main one) is reached.
> Mip neuron activation promotes mating in virgins and re-mating in mated females. In the Mip mutant background, however, the mating-promoting effects of Mip neuron activation are largely abolished. Nevertheless, as pointed by the reviewer, the Mip gene is dispensable for normal mating receptivity. To clarify these points in the summary, we added "although it is not required for normal virgin mating receptivity" (see line 25) Line 73 "Silencing of EH, capa and kinin gal4 neurons produces a marginal or nonsignificant phenotype…" From the Methods, I understand "marginal" to mean p<0.05 or p<0.001 -that sounds significant by every conventional statistical approach I am used to….so what is meant by the term 'marginal'? non-significant? The authors should explain their thinking on this.
> As seen in Figure S1, the p values for the comparisons between test groups (GAL4>UAS-Shi ts , 30 o C) and parental groups (GAL4>+ or UAS-Shi ts >+, 30 o C) are higher than 0.05. Thus, we removed "marginal."  Corrected.

Line 135
The use of tethered SP is useful. Have the authors tried a tethered MIP? That would seem to be a useful reagent in this analysis. What evidence exists to indicate the transmitter phenotype of the SPSN neurons that project from the uterus? > Indeed, we attempted to generate a membrane-tethered Mip (mMIP) in the same way mSP was constructed. We prepared two mMIP constructs: one with and one without an amidation signal (GRK) at its C-terminus (see slide below). Most neuropeptide precursors have a GRK motif where C-terminal amidation occurs. Unlike with mSP, ppk neuron-specific expression of either mMIP or mMIP-GRK has little effect on virgin female mating receptivity (Fig. P3A). We suspect that mMIP is not active because it lacks C-terminal amidation, which is essential for MIP's agonism of SPR 7 . We also noted that neither ppk>mMIP nor ppk>mMIP-GRK show anti-Mip staining in ppk processes (Fig. P3B). This is consistent with our suspicion that our UAS-mMIPs produce mMip lacking C-terminal amidation because the anti-Mip antibody was raised against the C-terminal domain of amidated Mip.

Figure P3 | Generation and testing of membrane tethered Mip (mMIP) constructs.
A. The mating rate for virgin females of the indicated genotypes. mSP-expression in ppk-Gal4 cells completely suppresses mating, whereas mMip expression has no effect. Here, ppk-GAL4 drives two copies of UAS-mMIP or UAS-mMIP-GRK. B. Anti-Mip or anti-EGFP staining of females of the indicated genotypes. Note that CNSs expressing mMIP or mMIP-GRK in ppk neurons do not show additional ppklike anti-Mip staining when compared with anti-Mip staining of control w 1118 .

Line 152
The text does not reference the correct panels of Figure 2.

> Corrected
Not sure the significance of Figure 2C, as MIP cell activation is supposed to increase receptivity, but in virgin females, it is already near 100% -therefore this experiment appears hampered by a ceiling effect.
> To address this concern, we compared the time course of the cumulative mating rate for several genotypes (See Fig. 3c). This approach allowed us to successfully demonstrate the pro-mating effect of Mip neuron activation in virgin females (Fig. 3e). Nevertheless, Mip overexpression has no discernable effect on the mating rate of virgin females.

Line 162
Unexpectedly, the design switches protocols for thermal activation: previously it used 30 degrees (e.g. Figure 1), now it switches to 27 degrees. The text explains that use of 27 degrees was intended to "minimize the activation" of the inhibitory MIP cells -on what basis was this predicted? How do the authors know the inhibitory neurons require a higher thermal threshold for activation -is there some evidence? Without some plausible explanation, I find this set of experiments very difficult to evaluate.
> See the response to comment 8 of reviewer 2.

Figure 2
In Figure 2b, why not add the Mip-Gal80 to test the contribution of the MIP gene to the "full" complement of activating MIP+ neurons? > In the limited time given for this revision, it was impossible to combine multiple transgenes with the Mip mutant to perform the suggested experiment. We hope the reviewer can understand our situation.
In Figure 2d, mipGal4>TrpA1 at 27 degrees produces 100% cumulative mating in less than 20 min. However, that genotype at 30 degrees produces ~60% copulation within 1 hr. Aren't these the same behavioral measure, and if so why are the outcomes discrepant? If different behavioral measures, please explain the difference in the assay, and also why two different assays were used.
> We used the same behavioral assay for two temperatures. For the reasons the results show discrepancies, please see the response to comment 8 of reviewer 2.

Line 192
Silencing Mip/dsx neurons decreased virgin female receptivity (Figure 3a); yet the text states these neurons "maintains female sexual receptivity". I disagree -at most, the evidence supports the hypothesis that they elevate virgin female receptivity. BTW, 3a uses Kir to silence neurons whereas other experiments us UAS-shi: any explanation for using different reagents to the same end? This adds design variables that lessen the strength of the conclusion regarding neuronal function.
> We noted that temporary silencing of Mip/dsx neurons with Shi ts also markedly suppresses mating rate. The UAS-Kir2.1 data in Fig. 4a has now been replaced by UAS-Shi ts data.

Lines 201-210 The section using confocal microscopy to interpret synaptic interconnections between SPSN and vAL and SAG neurons is not credible. Apparent proximity at the LM level interpreted to represent "reciprocal connections" is not a conservative interpretation.
> The sentence has been removed in the revised manuscript.
Lines 211-250 I was confused by this section in that the text no longer referred to dsx neurons -why not?
To avoid confusion, we revised line 269 by inserting 'anti-Mip and dsx-positive' in front of 'vAL.' Line 215 EGFP labeled Gal4 neurons are not specified as to age or mating status.   > In our original manuscript, we scored TRIC-positive and -negative cells according to their relative signal intensity. If the TRIC signal in a given cell body was less than 50% of the average TRIC intensity of its group, we counted it as TRIC-negative. We used this method to compare different groups of neurons with varying basal TRIC signals. Mip neurons in the AG showed clear cell-type-specific variations in TRIClabeling intensity and frequency. For example, l-vPM and vAM show relatively robust TRIC labeling in all preparations, but s-vPM and s-mPM produce little to no signal. Although this method allows us to evaluate the differences among cell types, it does not provide separate information about labeling intensity and frequency. To address the concerns raised by the reviewer, we decided to compare the TRIC labeling intensity of cell bodies. This method, however, works well only for cell types that show high and reproducible labeling. It is less suitable for cell types that show low labeling frequency (< 50%) (i.e., s-vPM, s-mPM and vAL). Because we do not count unlabeled cells and the values are presented as an average percentage of a small number of weakly labeled cells, the values are likely overestimates.
In our revision, we compared the TRIC intensities of two groups of Mip neurons that show relatively robust signal, such as vAM and l-vPM. We also examined SAG as a positive control, because like vAM neurons, SAG neurons are also active in virgin females and silent after mating. As expected, both vAM and SAG show robust changes in TRIC signal intensity before and after mating. vAM and SAG neurons have higher TRIC signal before mating than 48 hr after mating. Ideally, we would be able to include the TRIC signals from the neural processes in the analysis. The TRIC staining panel in Fig. 5c shows SAG somas and processes also show a clear change in TRIC signal after mating. The Mip neurons in the AG, however, are packed in a relatively small space, making it difficult to discern the identity of their processes.
Authors replied all the points that I asked and showed reasonable results. I think this manuscript is ready to be published in Nature communication.

Reviewer #2 (Remarks to the Author):
I am satisfied with the responses and additional experiments carried out by the authors. They have successfully addressed my concerns.

Reviewer #3 (Remarks to the Author):
The revisions are generally effective and greatly improve the manuscript. I commend the authors for a great piece of detective work in deciphering the positive and negative factors in the MIP signals, and in placing the sexually dimorphic vAM and vAL neurons in a defined position in this increasingly detailed circuit.
I have a few concerns about old and new issues as follows: there is no need for additional experiments. Rather I hope textual revisions could help readers more easily follow the complex arguments inherent in this treatment.
My biggest concern is the difficulty I have as a reader (both with the original and revised versions) following the narrative through the difficulty of both MIP activation and inhibition, both virgin receptivity and the re-mating physiology. The Figures move back and forth using either both assays (Figure 1) or just one (Figure 4) or just the other ( Figure 5). Combined with considerations of both artificial activation (TrpA1) versus silencing of "normal" activity (shi[ts] or kir). I have to admit to finding it difficult to sustain the basic facts needed to follow the arguments. One suggestion I offer is to revise the Introduction with a better description of the current model for control of female receptivity (if I understand it correctly, SAG neurons promote virgin receptivity and they are inhibited by Mating (via SPSN and SPK signals). Thus virgin receptivity and post-mating physiology are linked. It would have helped me the reader to have this information clearly offered at the outset. It is now clear that EH, capa and Kinin were not different from proper secondary controls. Still I think the text is confusing as written. Suggest the following edit: "Of the 39 GAL4 lines we screened, five exhibited substantial differences when tested at restrictive versus permissive temperatures (Suppl, Figure 1A). However of these five, only two lines -Myoinhibitory peptide (Mip)-GAL4 line and the Diuretic hormone 44 (Dh44)-GAL4 line -continued to exhibit significant differences when compared to proper genetic controls ( Supplementary Fig. 1b-f). Here, we focused our analysis on Mip-GAL4, which produces the strongest mating and re-mating phenotypes." Figure 1 typo -Line 153: genotypes marked 1 and 2 appear switched in Figure 1m.
Lines 160 -180 Description of MIP and MIP-6.0 Gal4 lines. There is confusion in these descriptions. Mip Gal4 is expressed in ~230 CNS neurons", while MIP 6.0 Gal4 is expressed "in 114 brain and 80 VNC neurons" -that equals 224, essentially the same as the MIP-Gal4 number. Further virtually all MIP-Gal4 neurons are MIP-IR, and MIP-6.0 Gal4 "is expressed in most anti-MIP neurons". If all these equalities hold, why do the schematic maps of Gal4+/antibody-cells appear so different between Fig2 a and b?
It is unfortunate that neither the positive nor inhibitory activities in the MIP-GAL4 could be further defined by the tiling series.
Minor questions regarding Figure 2: In A, why does CA group # grow from 2 to 4 from left to right schematic?
In general, what is the point of the left schematic, when right schematic is showing the same data plus anti-MIP?
Line 242. "Neither silencing nor activating the Mip/otd neurons affects 9 mating in virgin or mated females ( Supplementary Fig. 5e, f). This suggests it is unlikely that the brain Mip neurons are involved. Thus, we concluded that Mip/dsx neurons in the AG constitute a neural circuit that, when active, maintains female sexual receptivity." This is a negative inference -the data suggests the vAL neurons are necessary but perhaps not sufficient -if so, they would constitute a part of circuit, not all of it.
Mip/dsx female neurons -back and forth between normal activity (inhibiting activation) versus gain of function (dTRPA1). Also back and forth between virgin female and re-mated female effects: these do not always proceed together, yet the text switches back and forth. This is very confusing. Please see my first comment above 1. Line 233 silencing double-positive neurons (hereafter Mip/dsx) markedly suppresses virgin receptivity (Fig. 4a), although their thermal activation does not increase re-mating in mated females (Fig. 4b).
2. Line 244 the brain Mip neurons are involved. Thus, we concluded that Mip/dsx neurons in the AG constitute a neural circuit that, when active, maintains female sexual receptivity. 3. Line 274 Because Mip neuron activation increases re-mating, we reasoned that the relevant Mip neurons would show higher activity levels in virgin females than mated females.
Line 316 When we silenced either the SPSNs or SAGs with Kir2.1 (SPSN-and SAG-, respectively), we found that virgin females are unreceptive. I am confused here -Why does this happen? -I thought SPSNs signal mating via SP activity to turn off SAGs (Feng et al 2014).