Emergence of the sex difference in calbindin D-28K neurons and the role of calbindin D-28K in clustering neurons of the preoptic area in mice.

Neurons expressing Calb1 that encodes calbindin D-28K (Calb), a calcium-binding protein, are significant components of the sexually dimorphic nucleus (SDN) of the preoptic area in mice. Calb is therefore used as a marker to study the SDN. The number of Calb neurons in the SDN is greater in males than in females, and this sex difference emerges before puberty. However, the timing of emergence of this difference and the role of Calb1 in the SDN remain unclear. In this study, we investigated when the sex difference in Calb neurons appears and whether Calb1 is required for organizing the SDN. Profiling the temporal changes in Calb neurons revealed that these neurons dramatically increased in the SDN during postnatal days 15 to 20 in males but not in females, resulting in a significant difference between sexes. However, the Calb1 mRNA in the SDN remained unchanged during the late postnatal period and did not differ between sexes. Calb1 knockdown (KD) was performed by injecting an adeno-associated virus vector into the preoptic area of neonatal mice. The analysis of postnatal Calb1-KD mice revealed that Calb1 KD reduced not only Calb1 expression but also the number of neurons comprising the SDN. These findings suggest that compared with the female SDN, more Calb neurons migrate to and are incorporated into the male SDN, thereby yielding the sex difference in the number of Calb neurons at the late postnatal period, and that Calb1 plays a significant role in clustering neurons in the SDN.