On 2017 Feb 06, GARRET STUBER commented:

*This review was completed as part of a graduate level circuits and behavior course at UNC-Chapel Hill. The critique was written by students in the class and edited by the instructor, Garret Stuber.

Comments and critique

Written by Li et al., this paper investigated a class of oxytocin receptor interneurons (OxtrINs) on which the same group first characterized in 2014 [1]. OxtrINs are a subset of somatostatin positive interneurons in the medial prefrontal cortex (mPFC) that seem to be important for sociosexual behaviors in females, specifically during estrus and not diestrus. To complement their previous story, here the authors concluded that OxtrINs in males regulate anxiety-related behaviors through the release of corticotropin releasing hormone binding protein (Crhbp). While we agree that these neurons could be mediating sexually dimorphic behaviors, it is unclear how robust these differences really are.

We had some technical issues with this paper. First, it is unclear exactly how many mice were allotted to each experimental group, and it would have been useful to see individual data in each of the behavioral experiments, so that we can better understand some of the variability in the authors’ graphs. Even among different experiments, there were variable sizes of n (e.g. Fig. 5F-H, “n = 8-14 mice per group”). There was also no mention of how many cells per animal were tested for each brain slice experiment; instead, we received total numbers of cells tested per group. This paper did not include the complementary female data to Fig. 4F-G and Fig. 5A-B, the experiments pairing blue light with Crhr1 antagonist or Crhbp antagonist. We would have appreciated seeing this data adjacent to that for the males. In addition, there was no mentioned control for the optogenetic experiments. The authors only compared responses between light on and light off trials. Typically in optogenetic approaches, a set of control mice are also implanted with optic fibers and flashed with blue light in the absence of virus to test whether the light alone influences behavior. Incidentally, there is evidence that blue light influences blood flow, which may affect neuronal activity [2]. It was also unclear during the sociosexual behavioral testing whether the males were exposed to females in estrus or diestrus. In all, lack of detailed sample sizes and controls made it difficult to assess how prominent these sex differences were.

These issues aside, knocking out endogenous Oxtr in their targeted interneuron population was a key experiment, as it demonstrated that oxytocin signaling in OxtrINs is important in anxiety-related behaviors in males, but not in females regardless of the estrus stage. They did this using a floxed Oxtr mouse and deleted OxtR using a Cre-inducible virus, allowing for temporal and cell-type-specific control of this deletion, and subsequently measured the resulting phenotype using an elevated plus maze and open field task. The authors also validated that changes in exploration were not due to hyperactivity. We think these experiments are convincing.

TRAP profiling, which the same research group pioneered in 2014 [3], provided a set of genes enriched in OxtrINs. TRAP targets RNAs while they are translated into proteins, so we think their results here are particularly relevant. Moreover, the authors provided a list of genes enriched in sex-specific OxtrINs, a useful resource for those interested in gene expression differences in males and females. Once they identified Crhbp, an inhibitor of Crh, they hypothesized that OxtrINs were releasing Crhbp to modulate anxiogenic behaviors in males. The authors next measured Crh levels in the paraventricular nucleus of the hypothalamus and found that Crh levels are higher in females than males. They thus concluded Crh levels were driving sex differences associated with OxtrINs. We wonder whether Crh levels are also higher in the female mPFC, but we agree here too.

To demonstrate that Crhbp expressed by OxtrINs is important in modulating anxiety-like behaviors in males, the authors targeted Crhbp mRNA using Cre-inducible viral delivery of an shRNA construct and subsequently tested anxiety-related behaviors. They found that knocking down Crhbp was anxiogenic in males and not in females. This was a critical experiment, but the shRNA constructs targeting Crhbp were validated solely in a cell line. It would have been more appropriate to perform a western blot on mPFC punches of adult mice, showing whether this lentiviral construct knocked down Crhbp expression in the mouse brain prior to behavioral testing. In fact, it also would have been useful to see a quantification of the shRNA transfection rate, as well as its specificity in vivo. As stated above, we also do not know the distribution of behavioral responses here either. Without these pieces of information, it is difficult to assess how reliable or robust their knockdown was.

The authors concluded that sexually dimorphic hormones act through the otherwise sexually monomorphic OxtrINs to regulate anxiety-related behaviors in males and sociosexual behaviors in females. We agree that OxtrINs interact with oxytocin and Crh to bring about sex-specific phenotypes, but we also think that using additional paradigms testing anxiety and social behaviors, such as a predator odor, novelty-suppressed feeding or social grooming, could shed more light on the nuances of mPFC circuitry. In addition, the authors suggested that OxtrINs are sexually monomorphic because they are equally abundant in males and females. The authors’ TRAP data however suggested that OxtrINs of males and females have different gene expression profiles (Table S2), thus indicating that these interneurons may form different connections in each sex that mediate the electrophysiological and behavioral differences we see in this study.

It would be interesting to overexpress Crhbp in female mice, preferably in a cell-type-specific manner, to see whether female mice would demonstrate the anxiety-like behavior seen in males. If the Crh:Crhbp balance is in fact mediating this sexually dimorphic behavior through OxtrINs, we would expect that doing these manipulations may “masculinize” the females’ behavior. Regardless, we believe that this study opens opportunities for future work into how oxytocin and Crh release from the hypothalamus may act together to coordinate behavior. It will also be interesting to see if single-cell RNA sequencing could provide insight into whether OxtrINs can be further divided into sexually dimorphic subtypes. As the authors pointed out, understanding the dynamics of Crh and oxytocin in the mPFC will be important for gender-specific therapy and treatment.

[1] Nakajima, M. et al. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons. Cell. 159, 295-305 (2014).

[2] Rungta, R. L. et al. Light controls cerebral blood flow in naïve animals. Nature Communications. 8, 14191 (2017).

[3] Heiman, M. et al. Cell-type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols. 9, 1282-1291 (2014).

On 2017 Feb 06, GARRET STUBER commented:

*This review was completed as part of a graduate level circuits and behavior course at UNC-Chapel Hill. The critique was written by students in the class and edited by the instructor, Garret Stuber.

Comments and critique

Written by Li et al., this paper investigated a class of oxytocin receptor interneurons (OxtrINs) on which the same group first characterized in 2014 [1]. OxtrINs are a subset of somatostatin positive interneurons in the medial prefrontal cortex (mPFC) that seem to be important for sociosexual behaviors in females, specifically during estrus and not diestrus. To complement their previous story, here the authors concluded that OxtrINs in males regulate anxiety-related behaviors through the release of corticotropin releasing hormone binding protein (Crhbp). While we agree that these neurons could be mediating sexually dimorphic behaviors, it is unclear how robust these differences really are.

We had some technical issues with this paper. First, it is unclear exactly how many mice were allotted to each experimental group, and it would have been useful to see individual data in each of the behavioral experiments, so that we can better understand some of the variability in the authors’ graphs. Even among different experiments, there were variable sizes of n (e.g. Fig. 5F-H, “n = 8-14 mice per group”). There was also no mention of how many cells per animal were tested for each brain slice experiment; instead, we received total numbers of cells tested per group. This paper did not include the complementary female data to Fig. 4F-G and Fig. 5A-B, the experiments pairing blue light with Crhr1 antagonist or Crhbp antagonist. We would have appreciated seeing this data adjacent to that for the males. In addition, there was no mentioned control for the optogenetic experiments. The authors only compared responses between light on and light off trials. Typically in optogenetic approaches, a set of control mice are also implanted with optic fibers and flashed with blue light in the absence of virus to test whether the light alone influences behavior. Incidentally, there is evidence that blue light influences blood flow, which may affect neuronal activity [2]. It was also unclear during the sociosexual behavioral testing whether the males were exposed to females in estrus or diestrus. In all, lack of detailed sample sizes and controls made it difficult to assess how prominent these sex differences were.

These issues aside, knocking out endogenous Oxtr in their targeted interneuron population was a key experiment, as it demonstrated that oxytocin signaling in OxtrINs is important in anxiety-related behaviors in males, but not in females regardless of the estrus stage. They did this using a floxed Oxtr mouse and deleted OxtR using a Cre-inducible virus, allowing for temporal and cell-type-specific control of this deletion, and subsequently measured the resulting phenotype using an elevated plus maze and open field task. The authors also validated that changes in exploration were not due to hyperactivity. We think these experiments are convincing.

TRAP profiling, which the same research group pioneered in 2014 [3], provided a set of genes enriched in OxtrINs. TRAP targets RNAs while they are translated into proteins, so we think their results here are particularly relevant. Moreover, the authors provided a list of genes enriched in sex-specific OxtrINs, a useful resource for those interested in gene expression differences in males and females. Once they identified Crhbp, an inhibitor of Crh, they hypothesized that OxtrINs were releasing Crhbp to modulate anxiogenic behaviors in males. The authors next measured Crh levels in the paraventricular nucleus of the hypothalamus and found that Crh levels are higher in females than males. They thus concluded Crh levels were driving sex differences associated with OxtrINs. We wonder whether Crh levels are also higher in the female mPFC, but we agree here too.

To demonstrate that Crhbp expressed by OxtrINs is important in modulating anxiety-like behaviors in males, the authors targeted Crhbp mRNA using Cre-inducible viral delivery of an shRNA construct and subsequently tested anxiety-related behaviors. They found that knocking down Crhbp was anxiogenic in males and not in females. This was a critical experiment, but the shRNA constructs targeting Crhbp were validated solely in a cell line. It would have been more appropriate to perform a western blot on mPFC punches of adult mice, showing whether this lentiviral construct knocked down Crhbp expression in the mouse brain prior to behavioral testing. In fact, it also would have been useful to see a quantification of the shRNA transfection rate, as well as its specificity in vivo. As stated above, we also do not know the distribution of behavioral responses here either. Without these pieces of information, it is difficult to assess how reliable or robust their knockdown was.

The authors concluded that sexually dimorphic hormones act through the otherwise sexually monomorphic OxtrINs to regulate anxiety-related behaviors in males and sociosexual behaviors in females. We agree that OxtrINs interact with oxytocin and Crh to bring about sex-specific phenotypes, but we also think that using additional paradigms testing anxiety and social behaviors, such as a predator odor, novelty-suppressed feeding or social grooming, could shed more light on the nuances of mPFC circuitry. In addition, the authors suggested that OxtrINs are sexually monomorphic because they are equally abundant in males and females. The authors’ TRAP data however suggested that OxtrINs of males and females have different gene expression profiles (Table S2), thus indicating that these interneurons may form different connections in each sex that mediate the electrophysiological and behavioral differences we see in this study.

It would be interesting to overexpress Crhbp in female mice, preferably in a cell-type-specific manner, to see whether female mice would demonstrate the anxiety-like behavior seen in males. If the Crh:Crhbp balance is in fact mediating this sexually dimorphic behavior through OxtrINs, we would expect that doing these manipulations may “masculinize” the females’ behavior. Regardless, we believe that this study opens opportunities for future work into how oxytocin and Crh release from the hypothalamus may act together to coordinate behavior. It will also be interesting to see if single-cell RNA sequencing could provide insight into whether OxtrINs can be further divided into sexually dimorphic subtypes. As the authors pointed out, understanding the dynamics of Crh and oxytocin in the mPFC will be important for gender-specific therapy and treatment.

[1] Nakajima, M. et al. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons. Cell. 159, 295-305 (2014).

[2] Rungta, R. L. et al. Light controls cerebral blood flow in naïve animals. Nature Communications. 8, 14191 (2017).

[3] Heiman, M. et al. Cell-type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols. 9, 1282-1291 (2014).