Article Figures & Data
Figures
- Figure 1.
Sex differences and hormonal dependence of microglia in the POA. Iba1 staining in the POA on postnatal day 2 of a male pup (A, B) and a female pup (C, D) at 4× magnification and 20× magnification. Scale bar: A, B, 500 μm; C, D, 50 μm. Black arrows indicate examples of ameboid microglia; gray arrows indicate examples of transitioning microglia; white arrows indicate examples of ramified microglia. Males had significantly more total microglia (E) and ameboid microglia specifically (F) in the POA on PN2 than females. Estradiol masculinized microglial counts in females, and cotreatment of females with estradiol and minocycline prevented this masculinization. Among transitioning microglia, females cotreated with minocycline and estradiol had significantly fewer cells than males or estradiol-treated females (G). *Significantly different from males (ANOVA: p < 0.05). +Significantly different from females + E2 (ANOVA: p < 0.05). There was no sex difference or hormonal dependence on the number of ramified microglia (H).
- Figure 2.
Three dimensional morphometric analysis of microglia (A–C). Males and estradiol-treated females had significantly larger microglial cell bodies (A), shorter processes (B), and fewer process branch points (C) than vehicle-treated females in the POA on PN2. Cotreatment with estradiol and minocycline prevented estradiol's effects on process length and number of branch points. *Significantly different from males (ANOVA: p < 0.05). +Significantly different from females + E2 (ANOVA: p < 0.05). D, Microglia in the neonatal POA do not stain for estrogen receptor α. Confocal imaging at 40× magnification of POA sections costained for tomato lectin (a pan-macrophage stain) and estrogen receptor α in males (pictured) and females show no colocalization. Scale bar, 150 μm. Arrows indicate examples of labeled microglia that are negative for estrogen receptor α staining.
- Figure 3.
Microglia in the neonatal POA are positive for markers of classical (M1) and alternative (M2) microglial activation. A total of 100% of Iba1-stained microglia in males and females were positive for the M1 marker interleukin 1β (A), the M1 marker tumor necrosis factor α (B), the M2 marker arginase-1 (C), and the M2 marker CD206 (D). Confocal imaging performed at 40× magnification. Scale bar, 150 μm.
- Figure 4.
PGE2 induces ameboid microglial morphology in the POA. A, Effects of PGE2 and the COX inhibitor, indomethacin, on total microglial counts in the POA. B, Effects of PGE2 and indomethacin on ameboid microglial counts in the POA. Males had significantly more ameboid microglia in the POA on PN2 than females. Indomethacin prevented masculinization of ameboid counts in males and PGE2 masculinized microglial counts in females. *Significantly different from vehicle males (ANOVA; p < 0.05). +Significantly different from PGE2-treated females (ANOVA; p < 0.05). There was no sex difference or prostaglandin dependence on ramified microglia (C).
- Figure 5.
Effects of microglial inhibition on dendritic spine-related proteins in vivo. A, The dose of minocycline used for all in vivo experiments (0.2 μg i.c.v.) significantly decreased levels of the microglial-associated protein Iba1 in the POA at PN2 relative to vehicle-treated controls. B, Estradiol treatment on PN0 and PN1 in females increased levels of the spine-related protein spinophilin in the POA by PN2 relative to vehicle-treated control females; coadministration of minocycline with estradiol prevented that increase in spinophilin. C, PGE2 produced similar increases in spinophilin to estradiol, and coadministration of PGE2 and minocycline similarly prevented this increase. Minocycline administration alone had no effect on spinophilin levels. *Significantly different from vehicle (ANOVA; p < 0.05).
- Figure 6.
Effects of microglial inhibition on dendritic spine-related proteins in vitro. A, Representative images of MAP2 labeled neurons from primary POA cultures used to quantify dendritic spine-like protrusions at 100× magnification. Scale bar, 20 μm. B, Estradiol increased the density of spine-like protrusions relative to vehicle-treated controls. Estradiol-treated cultures depleted of microglia did not show an increase in protrusions, nor did cultures cotreated with estradiol and minocycline. There was no change in spine density in cultures treated only with minocycline. *Significantly different from vehicle (ANOVA; p < 0.05). C, Iba1 staining in control cultures and cultures depleted of microglia. Depleted cultures had ∼80% fewer microglia.
- Figure 7.
Effects of microglial inhibition on PGE2 levels in the POA. Female pups treated with estradiol on PN0 and PN1 showed a significant increase in PGE2 levels in the POA at PN2 relative to vehicle-treated females, and cotreatment with estradiol and minocycline prevented this increase. *Significantly different from vehicle female (ANOVA; p < 0.05).
- Figure 8.
Effects of neonatal microglial inhibition on adult masculine sex behavior. A, After adult gonadectomy and testosterone replacement, females had significantly fewer mounts relative to males, and neonatal estradiol treatment masculinized mount number. Cotreatment neonatally with estradiol and minocyline prevented this masculinization in females. B, All female groups showed significantly fewer intromission-like behaviors than males. When female groups were compared without males (inset), estradiol-treated females had an increased number of intromission-like behaviors than vehicle females, and cotreatment with minocycline prevented the estradiol-induced increase. C, Females had significantly longer latency to first mount than males; neonatal estradiol treatment masculinized latencies, and cotreatment neonatally with estradiol and minocyline prevented estradiol-induced masculinization in females. D, Control females had significantly longer latencies to first intromission-like behavior than males; estradiol-treated females had decreased latencies relative to vehicle-treated females, and minocycline prevented estradiol-induced decreases in latency to intromit. *Significantly different from male (p < 0.05). +Significantly different from vehicle female (p < 0.05). ∧Significantly different from estradiol-treated female (p < 0.05).
