LR‐1 Antibody Staining in Kisspeptin Receptor Knockout Mice Brains Reveals Axonal Diameter Differences in GnRH Neurons Due to Reduced GnRH Protein Levels

Kisspeptin, through its receptor, GPR54, regulates gonadotropin releasing hormone (GnRH) mRNA. However, the relationship between mRNA and protein is controversial and confounded by the lack of a method for analyzing protein levels from tissue sections. GnRH mRNA is markedly suppressed in GPR54 GnRH knockout (KO) mice. We determined whether reduced mRNA dictated less protein expression in the neurons. We examined two features: axon diameter and somal size. We hypothesized that the absence of GPR54 would produce decreased GnRH levels reflected by the thickness of the immunostained axons and the size of the cell bodies. Sections from global and conditional GPR54 KO mice were stained using the ABC immunoperoxidase method and compared to sections from wild type controls. We first learned that the optimum staining conditions for the KO mice grossly overstained the WT. When conditions were adjusted to be in the linear part of the titration curve, obvious differences in protein levels emerged. Axons containing GnRH were imaged at a magnification of 200x, then skeletonized to 1 µm width, enabling calculation of total axon length in the field. The axon area divided by the skeletonized length would then be equivalent to the axon diameter. Axon volume was calculated. For cells, somal width was determined. Our results demonstrate that the GnRH + axons in GPR54 KO mice are approximately three times thinner than WT axons and somal diameter was proportionately reduced. The results stress the importance of primary antibody titrations. Funded by NIH grants R01HD370246, U01HD66435 and U01HD66432 to SR and GEH