Supplementary Figure 13: Young Cx3cr1^CreErt2/+;Eed^loxP/loxP mice and aged Cx3cr1 haploinsufficient Cx3cr1CreErt2/+ mice display normal behavior.

(a) 3-month-old Cx3cr1^CreErt2/+;Eed^loxP/loxP mice display normal thigmotaxis in the open field. Bar graphs with individual data points show that mutant mice spend equal time in the center vs periphery (mean=0.1372, SEM=0.02633) compared with control mice (mean=0.2046, SEM=0.06382) p=0.3764, F=5.876, t₅=0.9704, n=6/genotype. (b) Left: 3-month-old Cx3cr1^CreErt2/+;Eed^loxP/loxP mice display normal behavior in the elevated plus maze at young age. 3-month-old mice spend equal time in the open arms (left: control: mean=20.88, SEM=13.27; mutant: mean=35.83, SEM=35.57; p=0.6592, F=2.789, t₈=0.4579) and in closed arms (middle: control: mean=171.6, SEM=35.03; mutant: mean=171.0, SEM=16.88; p=0.9897, F=6.459, t₈=0.01330); and travel equal distances (right: control: mean=1564, SEM=212.7; mutant: mean=2092, SEM=210.9; p=0.1311, F=1.527, t₈=1.682). n=4 control, 5 mutant mice. Right: 15-month-old Cx3cr1^CreErt2/+;Eed^loxP/loxP mice travel equal distances in the elevated plus maze paradigm (control: mean=1758, SEM=163.8; mutant: mean=1430, SEM= 201.8) p=0.2210, F=1.365, t₁₇=1.271; n=10/genotype. (c) Cx3cr1 haploinsufficiency in 15-month-old Cx3cr1^CreErt2/+ mice does not affect normal behavior in the elevated plus maze. Mutant and control mice spend equal time in the open arms (left: control: mean=22.17, SEM=4.888; mutant: mean=21.83, SEM=7.682; p=0.9713, F=2.778, t₁₅=0.03657) and in closed arms (middle: control: mean=178.6, SEM=10.98; mutant: mean=171.7, SEM=6.651; p=0.5910, F=2.422, t₁₅=0.5491); and travel equal distances (right: control: mean=1845, SEM=(212.7; mutant: mean=1683, SEM=210.9; p=0.1250, F=1.238, t₁₅=1.625; n=8 control, 9 mutant mice). (d) 15-month-old Cx3cr1^CreErt2/+;Eed^loxP/loxP mice show a deficit in cued memory in the fear conditioning paradigm. Bar graphs with individual data points show that control and mutant mice freeze equal amounts at baseline (control: mean=2.831, SEM=1.131; mutant: mean=8.024, SEM=3.445; p=0.1820, F=1.384, MWU=31.50; Shapiro Wilk p-value (SWp)=0.0039 & 0.0010), in training (control: mean=40.89, SEM=8.172; mutant: mean=67.78, SEM=10.04; p=0.0500, F=1.372, t₁₉=2.093), in the old context (control: mean=36.59, SEM=6.040; mutant: mean=36.48, SEM= 7.900; p=0.9918, F=1.555, t₁₉=0.01041), and in the new context (control: mean=16.00, SEM=4.083; mutant: mean=18.03, SEM=5.824; p=0.7786; F=2.035, t₁₈=0.2854). However, mutant mice freeze less (mean=23.17, SEM=9.154) than control mice (mean=46.24, SEM=7.778) after the tone cue (p=0.0448, F=1.259, MWU=26.00; SWp=0.0111 for mutant, t₁₉=1.932). n=11 control, 10 mutant mice. (e) 15-month-old Cx3cr1^CreErt2/+;Eed^loxP/loxP mice show a deficit in novel object recognition test. Bar graphs with individual data points show that mutant mice spend equal time (object 1: mean=1.742, SEM=1.181; object 2 mean=2.916, SEM=1.113; p=0.3896, F= 1.127, t₉=0.9039) sniffing two identical objects during training as 15-month-old control Eed^loxP/loxP mice (object 1: mean=1.655, SEM=0.5399; object 2 mean=2.856, SEM=0.5316; p=0.0610, F=1.032, t₉=2.141) and 15-month-old Cx3cr1 haploinsufficient Cx3cr1^CreErt2/+ mice (object 1: mean=3.329, SEM=0.7976; object 2: mean=5.314, SEM=1.218; p=0.0660, F=2.071, t₇=2.176). However, control mice spent more time sniffing a novel object (novel: mean=4.338, SEM=1.485; familiar: mean=1.421, SEM= 0.4844; p=0.0222, F=9.396, t₉=2.758), as do Cx3cr1 haploinsufficient mice (novel: mean=6.521, SEM=1.094; familiar: mean=2.812, SEM=0.8749; p=0.0137, F=2.823, t₈=3.146); whereas, mutant mice (right) do not (novel: mean=2.763, SEM=1.126; familiar: mean=2.176, SEM=0.6447; p=0.4640, F=3.049, t₉=0.7648). Two-tailed paired t-tests (n=9 for Cx3cr1^CreErt2/+, n=10 for Eed^loxP/loxP and Cx3cr1^CreErt2/+;Eed^loxP/loxP). Grubbs’ Test was used to identify and exclude an outlier. (f) Cx3cr1 haploinsufficiency is not associated with the development of seizures in Cx3cr1^CreErt2/+ mice Kaplan-Meier curve for n=9 mice/genotype; p=0.8491; χ²=0.03618 from log-rank Mantel–Cox test). (g) Cx3cr1^CreErt2/+;Eed^loxP/loxP mice show a normal survival rate compared to control mice. Kaplan-Meier curve for n=28 mice/genotype; p=0.8491; χ²=0.03618 from log-rank Mantel–Cox test). (h) 12-month-old microglia-depleted (PLX) mice show no anxiety-like phenotype in the open field. PLX mice spend equal time in the center vs periphery (mean=0.3400, SEM=0.06352) compared with control mice (mean=0.3973, SEM=0.06133) p=0.4605, F=1.515, t₁₉=0.6165; n=8 PLX-treated / 13 control mice. (i) 12-month-old PLX mice show no anxiety-like phenotype in the elevated plus maze. PLX mice spend equal time in the open arms (mean=22.65, SEM=7.520) compared with control mice (mean=19.83, SEM=6.603) p=0.7814, F=1.297, t₁₈=0.2817; and in closed arms (control: mean=179.6, SEM=12.24; PLX: mean=182.3, SEM=6.801) p=0.8532, F=3.241, t₁₈=0.1877; and travel equal distances (control: mean=2490, SEM=104.5; PLX: mean=2689, SEM=77.91; p=0.1448, F=1.798, t₁₈=1.525; n=10/group. (j) PLX mice are not prone to development of a seizure phenotype. Kaplan-Meier curve (n=7/group; p=1.00; χ²=0.00 (log-rank Mantel–Cox test). Bar graphs with individual data points show mean ± SEM. All t-tests were two-tailed unpaired.