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. 2020 Jul 21;9:e55263. doi: 10.7554/eLife.55263

Figure 6. LB altered mPFC to BLA anatomical connectivity in females but not males.

(A) Diagram of CTB 594 retrograde injection into the BLA. (B) Tracings of CTB injections into the BLA. (C) Example picture of injection in the BLA (left), and of cells labelled in mPFC (right). (D) Graphs showing the differences in PL to BLA corrected estimated mean densities with 95% confidence intervals from infancy (PND16) into adolescence (PND 35). In males, the density of PL to BLA projecting cells in LB mice did not differ at PND 16 (F(1,6) = 2.46, p=0.16), 21 (F(1,7) = 1.69, p=0.23), 28 (F(1,6) = 0.035, p=0.85) or 35 (F(1,6) = 3.08, p=0.13) when compared to control mice. In females, a significant increase in PL to BLA projecting cells density was observed in LB mice at PND 21 (F(1,4) = 21.74, p=0.010) and 28 (F(1,10) = 7.44, p=0.021) but not at PND 16 (F(1,6) = 1.47, p=0.27) or 35 (F(1,6) = 0.001, p=0.97). (E) Graphs showing the differences in IL to BLA corrected estimated mean densities with 95% confidence intervals from infancy into adolescence. LB did not affect the density of IL to BLA projections at any age for males (16: F(1,6) = 3.71, p=0.10; 21: F(1,7) = 0.22, p=0.65; 28: F(1,6) = 0.003, p=0.958; 35: F(1,6) = 2.17, p=0.19) or females (16: F(1,6) = 0.091, p=0.77; 21: F(1,4) = 5.35, p=0.082; 28: F(1,10) = 2.212, p=0.168; 35: F(1,6) = 5.10, p=0.065). For panels D and E (Ctrl male n = 5, 5, 5, 6; LB male n = 5, 6, 5, 4; Ctrl female n = 5, 4, 7, 5; LB female n = 5, 4, 7, 5). (F) Graphs showing the differences in projection index from infancy into adolescence. LB altered the balance of PL and IL projections to BLA at PND 16 in females (t7 = 3.09, p=0.0175). No other significant differences were observed in the projection index for males (16: t8 = 1.05, p=0.32; 21: t9 = 1.47, p=0.17; 28: t8 = 0.83, p=0.42; 35: t8 = 0.60, p=0.56) or females (21: t6 = 2.22, p=0.068; 28: t11 = 0.75, p=0.46; 35: t8 = 1.61, p=0.14). (Ctrl male n = 5, 5, 5, 6; LB male n = 5, 6, 5, 4; Ctrl female n = 4, 4, 6, 5; LB female n = 5, 4, 7, 5). For D and E dots represent estimated group means with 95% confidence intervals. Estimated means were derived from ANCOVA analysis using two covariates: 1) the area of the CTB injection and 2) the area of BLA where the injection occurred. An ANCOVA was run per age per sex. Statistical significance was determined through post ANCOVA univariate comparison. For panel F, dots represent individual values, bars represent group mean + / - SEM. Unpaired two-way student t-tests were used to assess statistical significance between control and LB mice per age per sex in panel F. *=p < 0.05, **=p < 0.01, ***=p < 0.001.

Figure 6.

Figure 6—figure supplement 1. Sex differences in mPFC to BLA projections in control reared mice.

Figure 6—figure supplement 1.

(A) Control females and males had significantly different corrected estimated mean densities of cells projecting from PL to BLA at PND 16 (F(1,6) = 6.18, p=0.047). Sex differences were no longer significant at PND 21(F(1,5) = 2.23, p=0.19), 28 (F(1,8) = 3.19, p=0.11), or 35 (F(1,7) = 0.90, p=0.37). No sex differences were observed between control reared females and males at any of the ages tested for IL to BLA projections (16: F(1,6) = 4.86, p=0.069; 21: F(1,5) = 0.31, p=0.59; 28: F(1,8) = 0.25, p=0.62; 35: F(1,7) = 0.97, p=0.35). (Ctrl males n = 5, 5, 5, 6; Ctrl females n = 5, 4, 7, 5). (B) Comparison of projection index between control reared male and female mice revealed a significant difference only at PND 21 (t7 = 2.40, p=0.047) but not at any of the other ages tested (16: t7 = 0.056, p=0.95; 28: t9 = 0.36, p=0.72; 35: t9 = 0.12, p=0.90). (Ctrl males n = 5, 5, 5, 6; Ctrl females n = 4, 4, 6, 5). For panel A, corrected estimated means and the 95% confidence intervals are shown. The corrected mean 95% confidence intervals were obtained through ANCOVA analysis run per brain region per age. Statistical significance was determined through post ANCOVA univariate comparison. For panel B, dots represent individual values, bars represent group mean + / - SEM. Unpaired two-way student t-tests were used to assess statistical significance between control and LB mice per age per sex in panel F. *=p < 0.05, **=p < 0.01, ***=p < 0.001.