Activity of estrogen receptor beta expressing neurons in the medial amygdala regulates preference towards receptive females in male mice

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference towards sexually receptive (RF) over non-receptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERβ) expressed in the medial amygdala (MeA) in male preference towards RF. To further delineate the role played by ERβ in the MeA in the neuronal network regulating male preference, we developed a new ERβ-iCre mouse line using the CRISPR-Cas9 system. Fiber-photometry Ca2+ imaging revealed that ERβ expressing neurons in the postero-dorsal part of the MeA (MeApd-ERβ+ neurons) were more active during social investigation towards RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERβ+ neuronal activity abolished a preference to RF in “RF vs. XF”, but not “RF vs. IM”, tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERβ+ neurons. Fiber-photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERβ+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERβ+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs XF) but not sex-based preference (i.e., RF vs IM) in male mice. Significance Statement In this study, by introducing a new Cre mice line for ERβ+ cells, we described the function of MeApd-ERβ+ neurons and characteristics of their neuronal activity during preference tests. Using fiber photometry and DREADD techniques we have found MeApd-ERβ+ neurons have a specific role in receptivity-based (receptive female vs. non-receptive female) preference but not in sexbased (receptive female vs. intact male) preference in male mice. We have also described this specific role of MeApd-ERβ+ neurons is achieved by regulating the neuronal activity of downstream BNSTp neurons during receptivity-based, but not sex-based, preference tests. Our findings contribute to a better understating of the function of estrogen receptor expressing neurons in the neuronal network for the male-typical reproductive behaviors. ### Competing Interest Statement The authors have declared no competing interest.