Anatomical Characterisation of Prolactin Receptor Expression in the Developing Mouse Brain

Abstract

Prolactin signalling through the prolactin receptor plays a significant role in many physiological processes. The diverse biological functions attributed to prolactin are largely dependent on the distribution of its receptor. In particular, the distribution of the prolactin receptor has been well characterised in the adult brain of rodents, but has yet to be fully documented in the developing brain of neonates.

To determine the role of the prolactin receptor in the developing brain of neonates, we have developed a transgenic mouse in which cre-recombinase is expressed under the control of an internal ribosomal entry site (IRES) in the long form of the prolactin receptor gene (PrlrL-IRES-Cre). The PrlrL-IRES-Cre mouse has been crossed with a cre-dependent tau-GFP reporter line. In this study tau-GFP is expressed wherever the prolactin receptor gene has been transcribed. The tau-GFP associates with microtubules in neuronal processes, which is important, because in addition to identifying neuronal cell bodies expressing the prolactin receptor, we were able to study the projections of prolactin-responsive neurons for the first time.

Post natal day (PND) 1 (n = 8), PND 14 (n = 8) and PND 28 (n = 6) male and female mouse brains were perfused using 4% paraformaldahyde, cut in 20 micron serial sections and thaw mounted onto slides. To visualise PrlrL expressing cells, immunohistochemistry was performed. Sections were incubated with rabbit polyclonal anti-GFP (green fluorescent protein) (A-6455, Life Technologies), for 48 hours at 4℃, followed by an incubation with goat anti-rabbit biotinylated secondary antibody (BA-1000, Vector) for 3 hours. Labelled cells were visualised using nickle enhanced 3,3'-diaminobenzidine (DAB), and identified using an Olympus AX70 light microscope.

Staining showed minimal expression in any part of the brain of PND 1 mice, whereas staining could be identified in the choroid plexus, anterior ventral periventricular nucleus and arcuate nucleus of PND 14 and 28 mice. Interestingly, minimal staining was observed in the amygdala and no staining was observed in the paraventricular nucleus in any of the ages examined. These are areas that exhibit high levels of PrlrL expression in adult mice. The increase in PrlrL expression may indicate an age dependent upregulation of the prolactin receptor through the availability of the PrlrL gene, likely driven by hormonal changes associated with puberty. The age-associated increase in PrlrL expression in the anteroventral periventricular nucleus and arcuate nucleus in prepubertal animals suggests a developmental role for prolactin in these regions.