Referee 1's review:

Comments on the manuscript entitled "Ultrasonic vocalizations in M2, M4, M5 muscarinic and D2 dopamine receptor knockout mice"

Rodents produce a variety of social vocalizations, including vocalizations audible to humans, like postpartum sounds and distress calls, as well as ultrasonic vocalizations (USVs). When male mice encounter female mice or their pheromones, they emit USVs with frequencies ranging over 30-110 kHz. This was experimentally shown in the previous reports. Studying these USVs during different phases of sexual behavior is very important as mice communicate largely through USVs during this period. Previously, scientists have studied this type communication by mice by giving several stimuli (such as urine stimuli). In the present study authors studied the effects of M2, M4 and M5 muscarinic and D2 dopamine receptor gene deletion and dopamine activator in USV production. The idea is novel. Appropriate materials and methods have been used and presented in sufficient detail. However the results presented along with previous findings makes it little difficult to follow.

Major findings of the paper
-Analyzed the effect of mascarinic receptor (M2, M4 and M5) and dopaminic receptor gene deletion on USV production in mice.
-Found M2 and M5 muscarinic, but not M4 and D2 receptors, play crucial roles in the male-emitted USVs.
-Amphetamine induced male wild-type USVs dose-dependently during male-female sexual interactions.

Below are my comments on the manuscript

1) After going through the manuscript and a few related previous papers, I strongly feel that first 2 objectives and their subsequent results of the present paper are overlapping with the previous reports (Please see: Holy and Guo, 2005 Plos Biol). If authors are still able to justify that 2 objectives and their results are different from those of the previous works, then they have to discuss their results in comparison with the already reported ones.

2) In my opinion the work on muscarinic and dopaminic receptor knockout (KO) mice is interesting and worth publishing. It is better to concentrate more on knockout mice experiments.

3) As the text of the paper runs through many pages, reader may find it difficult to follow. I feel that, this manuscript needs to be shortened by removing some of the unwanted/repetitious sentences and lengthy explanation, highlighting the major findings of the study. Number of figures can also be reduced accordingly.

4) I am surprised to know why D2 receptor deletion did not have any effect on USV production, though it is shown to have a role in sexual and morphine reward. I am not convinced by the authors' explanation on reasons for the above. It would be interesting to know possible reasons why D2 has no role in USV production. Please discuss with more information regarding this, in discussion part.

5) Any change in pattern of USVs, observed in KO mice (especially in M2 and M5)? It would be interesting to know about it.


Minor comments

1) Page 7, 1st para: Authors mention "This result further strengthens the notion .... USV induction". I am bit puzzled about this statement. I wonder whether the authors are confirming the previously published results. If so please cite previous paper.

2) Page 9: Please expand CNS when it is mentioned for the first time in the text.

3) In Introduction, it is argued that 'little is known about the underlying molecular and genetic basis for USVs of different types in mice'. I agree with this, but at the same time I am afraid that the situation remains essentially the same also after the present study. Because, not much explanation is given for non-difference of USV production in M4 KO and D2 KO mice. In my opinion it is better to avoid such expressions.

4) Please structure the abstract according to PLoS ONE format.

5) While reading the manuscript I found a few wrong English expressions. As I am not a native English speaker, I leave this to academic editor to suggest authors to check for grammatical errors once again, if there is such a need.

6) Abstract: Text from 5th to 13th line can be removed, as it is less important to be mentioned in the abstract.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.