Iba1+ cell density and morphology classification at postnatal day 10 in four hippocampal subregions of female and male C57BL/6J mice

Abstract

ABSTRACTMicroglia maintain normal brain function and support the brain’s response to disease and injury. The hippocampus is an area of focus for microglial study due to its central role in numerous behavioral and cognitive functions. Interestingly, microglia and related cells in the hippocampus and throughout the brain are distinct in male vs. female rodents, even in early life. Indeed, postnatal day (P)-dependent sex differences in number, density, and morphology of microglia-like cells have been reported in certain hippocampal subregions. For example, P3 female mice have more phagocytic microglia in dentate gyrus (DG) molecular layer (Mol) and CA1-3 stratum oriens (SO) regions vs. male mice, while P8 — but not P15 — male rats have more volume immunoreactive for markers of microglia-like cells (Iba1 and CD68) in the CA1 stratum radiatum (SR) vs. female rats. In the mouse, P10 is roughly equivalent to human term gestation, making it a common timepoint to study for many translationally-relevant neurobiological processes. However, sex differences in hippocampal microglia have not been examined in the P10 mouse hippocampus. In addition, key subregions of the hippocampus — CA3 SR, DG hilus — have not yet been assessed for sex differences in microglia. To address these knowledge gaps, we quantified Iba1+ cell densities and classified Iba1+ cell morphology in P10 male and female C57BL/6J mice. Four subregions in the bilateral anterior hippocampus were analyzed in 40-micrometer coronal sections: DG Mol (Mol), DG Hilus, CA1 SR and stratum lacunosum moleculare (CA1), and CA3 SR and stratum lucidum (CA3). Light microscope images (40x) were analyzed offline for Iba1+ cell density and morphology by an observer blind to sex. The morphology of each Iba1+ cell was used to place cells into one of four previously-published categories: Round or ameboid (round-ish soma, no processes), Stout (round-ish soma, short process), Thick (irregular soma with few, thick processes), or Thin (irregular soma with multiple thin processes). Analysis of Iba1+ cell density shows no difference between male and female mice in Mol, Hilus, CA3, or CA1 (male n=6, female n=7). However, morphology classification shows a sex-dependent difference in the Mol and Hilus, with female mice having a greater percentage of Thick Iba1+ cells vs. male mice (Mol, Hilus), and a lower percentage of Thin Iba1+ cells vs. male mice (Mol). With our analysis, it is unclear whether this greater percentage of thick and lower percentage of thin Iba1+ cells in the female vs. male hippocampus means Iba1+ microglia in female mice are ‘younger’ or ‘more active’ than those in male mice. However, these data are important as they reveal sex differences in Iba1+ microglia in the P10 mouse hippocampus. We discuss these results in the context of the large literature on sex differences in rodent microglia in the early postnatal period.