Gender–specific Single Transcript Level Atlas of Vasopressin and its Receptor (AVPR1a) in the Mouse Brain

Reviewer #1 (Public review):

Summary:

Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains. The findings are important, given that: (1) a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking, and (2) the study offers valuable new insights into Avpr1a expression across the mouse brain. The findings are solid, and with improved data presentation and analysis, this work could serve as an important resource for the neuroscience community.

Strengths:

This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.

Weaknesses:

A few concerns remain to be addressed. The primary weakness of this manuscript lies in the robustness of its data presentation and analysis.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, limited use of photomicrographs, and low number of subjects all combine to limit the impact of the dataset. The finding of Avp-expressing cells outside of the hypothalamus and extended amygdala is poorly documented but would be novel. The Avpr1a data suggest expression in numerous brain regions. However, the data presented are difficult to interpret, with every value being an extremely small density value for a large swath of the brain. How many cells are impacted? Are puncta spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield? For a descriptive study, there is minimal statistical inference and relatively little description. The authors make a case for the novel nature of the work but do not seem, at times, to recognize a robust literature developed over the last 50 years. In conclusion, the experimental data are important and informative; however, the low number of subjects, lack of statistical power, limited description of individual brain regions, and poor quality and design of data figures reduce the overall impact.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring the function of vasopressin in the mammalian brain and developing hypotheses about cell - and circuit-level function.

Weaknesses:

(1) The style and type of data presentation, focusing on the density of individual mRNA transcript across a whole brain region, seemed incomplete in so far as the data presentation did not provide a clear visualization of the distribution of Avpr1a-expressing cells or transcript itself. However, knowing which brain regions do express transcript is itself informative.

(2) The manuscript strongly emphases on the possibility of sex differences in Avp and Avpr1a expression. However, the low number of animals used does not provide adequate statistical power to make strong inferences regarding sex differences in the data.

(3) The manuscript's methods are minimal but adequate to understand data acquisition. The description of how quantitative analyses were conducted is inadequate and would be impossible to replicate beyond identifying the program used.