Fig 1.
Maternal probiotic supplementation reduces anxiety-like offspring behavior in light-dark box test.
(A) There was a significant interaction between the effects of sex and treatment on activity levels in the light zone (p = 0.04). (B) The difference in (A) was driven by female mice (p = 0.005). (C) Probiotic-exposed mice (male n = 9 and female n = 12) spent 10% more time than the controls ((male n = 9 and female n = 10) in the light zone, however this didn’t reach significance.
Fig 2.
Maternal probiotic supplementation differentially affects female mice in fear conditioning test.
(A) In utero exposure to probiotics did not affect conditioning of the mice. Control female mice (n = 10) showed higher levels of freezing than males (n = 8), however, the main effect of sex did not reach significance (p = 0.06). (B) Probiotic exposure did not have an effect on the contextual memory of mice. Though female mice (n = 10) showed higher levels of freezing than male mice (n = 10), separate sex analysis showed no treatment effect. (C) Probiotic exposure did not affect the first minute response to tone, but there is a significant treatment effect in female mice, shown in detail in panel (D).
Fig 3.
Increase in plasma oxytocin levels in probiotics-treated dams.
(A) Oxytocin levels are increased in the plasma of dams treated with L. lactis between day 0.5 of pregnancy and 1 day after birth (n = 5) compared to Control dams (n = 8). P = 0.0008 by Student’s t-test. (B) Plasma oxytocin levels remain unchanged in 3-month old male and female pups exposed to L. lactis between E0.5 and P1 (n = 5–6 per sex/group).
Fig 4.
Maternal exposure to probiotics modulates formation of cortical vasculature in the pups.
(A) Blood vessel outlines based on CD31 expression demonstrate increase in the density of cortical blood vessels in probiotic-exposed P1 pups compared to control pups. Counting masks are shown in red. (B) Immunofluorescent images of CD31 staining show increased staining intensity in P1 L. lactis-exposed compared to control cortices. White squares designate areas of blood vessel density analyses. (C-D) Increase in blood vessel density within the cortical wall is detected in both male (C; p = 0.0007 by Students t-test; control n = 18; L. lactis n = 9) and female (D; p = 0.0037 by Two-tailed Student’s t-test; control n = 15; L. lactis n = 20) L. lactis-exposed versus control P1 pups. (E) Average expression levels of CD31 per blood vessel is increased in L. lactis-exposed P1 pup cortices (p = 0.0045 by Two-tailed Student’s t-test; control n = 33; L. lactis n = 18). (F) Average blood vessel area is increased in P1 probiotic-exposed compared to control pups (p = 0.0045 by Two-tailed Student’s t-test; control n = 33; L. lactis n = 18). Scale bar in (B): 80 μm. CP—cortical plate; VZ—ventricular zone.
Fig 5.
Changes in cortical neuronal marker expression in P1 pups induced by maternal intake of probiotics during pregnancy.
(A) Expression of Tbr1 increased in the cortices of P1 pups exposed to L. lactis, compared to control pups. White brackets indicate Tbr1 layer thickness. (B) Density of Satb2-expressing cells in the upper layer of the cortical wall is increased in P1 L. lactis-exposed compared to control pups. Insets show higher magnification images of areas designated by white boxes. (C-D) Density of Tbr1-expressing cells is increased in male (C; p < 0.0001 by Two-tailed Student’s t-test; control n = 26; L. lactis n = 13) and female (D; p = 0.05 by Two-tailed Student’s t-test; control n = 10; L. lactis n = 18) probiotic-exposed P1 pups. (E-F) Tbr1 layer thickness was not significantly increased in probiotic-exposed compared to control P1 pups. (G-H) Density of Satb2-expressing cells is increased in the upper cortical layers (II-IV) of probiotic-exposed P1 male pups (G; p = 0.0006 by Two-tailed Student’s t-test; control n = 7; L. lactis n = 5), but not female pups (E; p = 0.16 by Two-tailed Student’s t-test; control n = 5; L. lactis n = 5) compared to control groups. Scale bar in (A): 80 μm. CP—cortical plate.
Fig 6.
Expression of mitotic marker PH3 is increased in probiotics-exposed female P1 pups.
(A) Larger numbers of PH3-expressing cells (white arrows) are observed in the cortical ventricular zone (VZ) of L. lactis-exposed versus control P1 pups. (B-C) Increase in PH3-expressing cells is significant in female pups (C; p = 0.0009 by Two-tailed Student’s t-test; control n = 8; L. lactis n = 15), but not in male pups (B) (p > 0.9 by Two-tailed Student’s t-test; control n = 18; L. lactis n = 15), compared to control groups. Scale bar in A: 60 μm.