R ES E A RC H | R E S EA R C H A R T I C LE S
observed among different treatment groups were not due to differences in activity or arousal, as total distances moved during the sociability test
were indistinguishable (Fig. 3D). Moreover, different treatment groups displayed comparable gender ratios, litter sizes, and weights (fig. S5). Taken
together, these data indicate that the IL-17a pathway in pregnant mice is crucial in mediating the MIA-induced behavioral phenotypes in offspring.
Marble Burying Index
TBR1+ cells (Count)
8
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Fig. 4. RORgt expression in maternal T cells is required for manifestation of ASD-like phenotypes in the MIA model. (A) SATB2 and TBR1 staining in the cortex of E18.5 male fetal brains after MIA induction with poly(I:C) in mothers with the indicated genotypes. Cortical layers: II to IV, V, and VI. Images are representative of three independent experiments. Scale bar, 100 mm. (B) Quantification of TBR1- and SATB2-positive cells in a 300 × 300 mm2 ROI centered on the malformation in the cortical plate of E18.5 male fetal brains (n = 6 for all groups). (C) Number of USVs emitted by P9 pups. Total USVs emitted during test period (3 min) are plotted on the y axis [n = 16, 18, and 15 offspring from PBS-treated WT, RORgt HET, and RORgt TKO mothers, respectively; n = 15, 11, and 28 from poly(I:C)-treated WT, RORgt HET, and RORgt TKO mothers, respectively; data from four to seven independent dams]. (D) Social approach behavior is shown as a social preference index as in Fig. 3. [n = 21, 15, and 15 26 FEBRUARY 2016 • VOL 351 ISSUE 6276
**
*** ***
80 60 40 20 0
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Total Distance Moved (cm)
Marble Burying Index
O TK
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an Co ti- nt IL -1 7a an Co ti- nt IL -1 7a
C on an t tiIL -1 7a
C on an t tiIL -1 7a
an C ti- on IL t -1 7a an C ti- on IL t -1 7a
an Co ti- nt IL -1 7a C an o ti- nt IL -1 7a
% Interaction
USV index
Total distance moved (cm)
Fig. 3. The IL-17a ** Social pathway promotes 2500 100 ** ** 400 100 Inanimate ** ASD-like phenotypes 2000 80 80 ** ** in the MIA offspring. 300 ** * ** 1500 (A) USV assay. At P9, 60 60 200 pups from the indi1000 40 40 cated experimental 100 500 groups were 20 20 separated from their 0 0 0 0 mothers to elicit USV calls. The number of pup calls is plotted on the y axis {n = 25 PBS Poly(I:C) PBS Poly(I:C) PBS Poly(I:C) PBS Poly(I:C) (PBS, Cont); n = 28 (PBS, anti–IL-17a); n = 38 [poly(I:C), Cont]; n = 34 [poly(I:C), anti–IL-17a]; from six or seven Percentage of the number of buried marbles is plotted on the y axis {n = 15 independent experiments}. (B) Social approach behavior. Graphed as a (PBS, Cont), n = 15 (PBS, anti–IL-17a), n = 15 [poly(I:C), Cont]; n = 20 [poly(I:C), social preference index (% time spent investigating social or inanimate anti–IL-17a]; from six or seven independent experiments}. (D) Total distance stimulus out of total object investigation time) {n = 15 (PBS, Cont); n = 15 traveled during social approach behavior. (A), (C), and (D) One-way ANOVA (PBS, anti–IL-17a); n = 16 [poly(I:C), Cont]; n = 20 [poly(I:C), anti–IL-17a]; with Tukey’s post hoc tests. (B) Two-way ANOVA with Tukey’s post hoc tests. from six or seven independent experiments}. (C) Marble-burying behavior. **P < 0.01 and *P < 0.05. Means ± SEM.
PBS
Poly(I:C)
adult offspring from PBS-treated WT, RORgt HET, and RORgt TKO mothers, respectively; n = 36, 15, and 21 from poly(I:C)-treated WT, RORgt HET, and RORgt TKO mothers, respectively; data from four to seven independent dams]. (E) Marble-burying behavior as in Fig. 3 [n = 14, 19, and 15 adult offspring from PBS-treated WT, RORgt HET, and RORgt TKO mothers, respectively; n = 32, 15, and 25 from poly(I:C)-treated WT, RORgt HET, and RORgt TKO mothers, respectively; data from four to seven independent dams]. (F) Total distance moved by offspring tested for social behavior. RORgt HET and RORgt TKO refer to RORg Neo/+; CD4-Cre/+ and RORgtFL/RORg Neo; CD4-Cre/+, respectively. (C) One-way ANOVA with Holm-Sidak post hoc tests. (B) and (D) Two-way ANOVA with Tukey’s post hoc tests. (E) and (F) Oneway ANOVA with Tukey’s post hoc tests. ***P < 0.001, **P < 0.01, and *P < 0.05. Means ± SEM.
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