Temporary Depletion of Microglia during the Early Postnatal Period Induces Lasting Sex-Dependent and Sex-Independent Effects on Behavior in Rats

Microglia numbers are reduced after clodronate treatment and rapidly repopulate. Coronal sections of brains from PN5 VEH-treated (a, d, g, j) and LC-treated (b, e, h, k) rats. Representative images from sections labeled for Iba1 and counterstained with hematoxylin from the hippocampus (a, b), cortex (d, e), amygdala (g, h), and hypothalamus (j, k). Quantification of Iba1⁺ cell density in the hippocampus (c), cortex (f), amygdala (i), and hypothalamus (l) at PN5, 10, and 15. Scale bars = 500 μm (a, b, g, h, j, k) or 250 μm (d, e). Bars indicate group means ± SEM for VEH-treated (black) and LC-treated (green) rats of both sexes. Open circles indicate data from individual female rats, and triangles, from male rats. *p < 0.05, **p < 0.01. n = 2–3 per treatment.

Microglia depletion alters the development of early reflexes, behaviors, and body weight throughout life. Body weight (in grams) in female (a) and male (b) rats measured daily from PN0 to PN90. c, Time (in seconds) for rat pups to retreat from an edge (cliff aversion). d, Time (in seconds) for rat pups to successfully right themselves (surface righting). e, Duration (in seconds) rat pups hung from their forepaws before falling (wire hang). f, Latency (in seconds) for rat pups to reverse orientation and face upward on an incline (negative geotaxis). g, Latency (in seconds) for rat pups to leave a designated area (locomotion). Solid lines indicate group means for VEH-treated (black; a–g) and LC-treated (red; a) females, LC-treated males (blue; b), and LC-treated rats of both sexes (green; c–g). Shaded regions indicate ± SEM. **p < 0.01, ***p < 0.001. n = 18–20 per treatment.

Postnatal microglia depletion impairs nest-seeking behavior and decreases USV emission. Quantification of data from the two-choice nest-seeking task represented as average score per day (a), average total score summed across all testing days (b), and average latency (in seconds) to reach either goal arm of the testing apparatus (c). d, Quantification of the number of USVs produced by pups when separated from the mother on PN8. Solid lines and bars indicate group means for VEH-treated (black) and LC-treated (green) rats of both sexes. Shaded regions and error bars indicate ± SEM. Open circles represent data from individual rats. Horizontal dashed line indicates score equal to chance (a, b). ***p < 0.001. n = 18–20 (a–c) and 13–14 (d) per treatment.

Postnatal microglia depletion induces selective memory impairment in juvenile rats. a, Percentage successful spontaneous alternations in a T-maze from PN25 to PN29. b, Social recognition index calculated on PN30 after a 30-min retention interval. c, Novel object recognition index calculated after a 1-h (PN26) and 24-h (PN27) retention interval. Bars indicate group means ± SEM for VEH-treated (black) and LC-treated (green) rats of both sexes. Open circles represent data from individual rats. Horizontal dashed line indicates score equal to chance (a–c). *p < 0.05. #p < 0.01 compared with chance. n = 18–20 (a) and 13–16 (b, c) per treatment.

Postnatal microglia depletion increases locomotion, center time in the open field, and open arm time in the elevated plus maze in juvenile rats. Center time (a; in seconds) and gridline crosses (b) in an open field arena on PN25. c, Percentage of time in the open arms of an elevated plus maze on PN32. Bars indicate group means ± SEM for VEH-treated (black) and LC-treated (green) rats of both sexes. Open circles represent data from individual rats. **p < 0.01, ***p < 0.001. n = 18–20 per treatment.

Microglia-depleted animals exhibit decreased fear and avoidance but display risk-assessing behaviors. Quantification of data from the predator odor exposure test (PN33) for time (in seconds) spent in various behaviors including time behind the barrier (a), freezing behavior (b), stretch-attend behavior (c), stretch-locomotion behavior (d), number of stimulus cloth approaches (e), and stimulus cloth interaction duration (f). Bars indicate group means ± SEM for VEH-treated (black) and LC-treated (green) rats of both sexes. Open circles represent data from individual rats. *p < 0.05, **p < 0.01, ***p < 0.001. n = 18–20 per treatment.

Postnatal microglia depletion induces deficits in adult male sex behaviors. Quantification of various components of male sex behavior including number of mounts (a), latency to first mount (b), number of intromissions (c), latency to first intromission (d), number of ejaculations (e), and latency to first ejaculation (f). Solid bars indicate group means ± SEM for VEH-treated (black) and LC-treated (blue) male rats. Open circles represent data from individual rats. *p < 0.05, **p < 0.01. n = 7–10 per treatment.

Adult female sex behavior is unchanged after postnatal microglia depletion. Quantification of female sexual proceptivity: number of hops and darts (a) and solicitations (b) and receptivity lordosis quotient (c). Solid bars indicate group means ± SEM for VEH-treated (black) and LC-treated (red) female rats. Open circles represent data from individual rats. n = 9–10 per treatment.