Table 1

Maternal Sickness Behavior (G15)

Friedman’s non-parametric repeated measures

Main effect of time for Saline-LPS dams: X²(5) = 11.970, p = 0.001

Figure 2A

(Ex. 1)

Saline-Saline: N.S

Met-Saline: N.S

MET-LPS: N.S

Post hocs:

Kruskal-Wallis follow-up test at each time point:

Saline-Saline vs Saline-LPS:

baseline & 30 minutes = N.S

60 min: p = 0.001

90 min: p = 0.001

120min: p = 0.001

240 min: p = 0.010

360 min: p = 0.030

Saline-LPS vs MET-LPS

baseline & 30 minutes = N.S

60 min: p = 0.001

90 min: p = 0.001

120 min: p = 0.001

240 min: p = 0.035

360 min: p = 0.001

Saline-Saline vs MET-Saline

120 min: p = 0.005

Maternal plasma IL-17A, 3-hr post LPS challenge (Ex. 1)

Kruskal-Wallis non-parametric ANOVA

Main effect of G15 LPS vs G15 Saline: X²(1) = 8.669, p = 0.003

Figure 2B

Main effect of G15 MET vs G15 Saline: N.S

Maternal plasma IL-6, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA (G15 MET by G15 LPS)

G15 MET by G15 LPS interaction: F(1, 24) = 18.437, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.434

Figure 2C

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: p = 0.024

Saline-Saline vs MET-Saline: N.S

Maternal plasma corticosterone, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA (G15 MET by G15 LPS)

G15 MET by G15 LPS interaction: F(1, 24) = 10.401, p = 0.004, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.302

Figure 2D

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.012

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: N.S

Maternal plasma corticosterone validation of MET (Ex. 2)

Repeated Measures ANOVA

Time by G15 MET by G15 LPS: F(7.38, 14) = 5.05, p < .001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.444

Figure 2G

(G15 MET by G15 LPS across time)

Post hocs:

One-way ANOVA for 60 min: N.S.

Skewed so used Kruskal Wallis for 90 min: p = 0.004

Saline-Saline vs Saline-LPS: p < 0.001

MET-LPS vs Saline-LPS: p = 0.006

Saline-Saline vs MET-Saline (p = 0.004)

One-way ANOVA for 120min: F(3,19) = 13.46, p < .001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.680

Saline-Saline vs Saline-LPS: p < 0.001

Saline-LPS vs MET-LPS: p < 0.001

One-way ANOVA for 240 min: F(3, 19) = 3.89, p = .025, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.381

Saline-Saline vs Saline-LPS: p = 0.040

Saline-LPS vs MET-LPS: p = 0.045

Skewed so used Kruskal Wallis for 360 min: X²(3) = 13.55, p = .004.

Saline-Saline vs Saline-LPS: p = 0.006

MET-LPS vs Saline-LPS: p < 0.001

Maternal 3-hour plasma corticosterone correlations and maternal factors

Pearson Correlations

Maternal plasma corticosterone concentrations on G15, 3 hrs after challenge

Figure 2F

versus

maternal 3hr plasma interleukin-6: r = 0.789, p = 0.001

maternal 3hr plasma interleukin-17A: r = 0.517, p = 0.001

maternal sickness scores at 60 minutes: r = 0.325, p = 0.015

maternal sickness scores at 90 minutes: r = 0.431, p = 0.001

maternal sickness scores at 120 minutes: r = 0.479, p = 0.001

Maternal plasma corticosterone concentrations on G15, 3 hrs after challenge

versus maternal MET-Saline maternal sickness scores at 30 minutes: r = 0.538, p = 0.047

maternal Saline-LPS G15 dam body weight: r = −0.775, p = 0.001

maternal Saline-LPS 3 hr plasma interleukin-6: r = 0.887, p = 0.001

maternal Saline-LPS maternal sickness scores at 120 minutes: r = −0.557, p = 0.038

maternal Saline-Saline maternal sickness scores at 90 minutes: r = −0.625, p = 0.017

Maternal 3-hour plasma corticosterone correlations and offspring factors

Pearson Correlations

Maternal plasma corticosterone concentrations on G15, 3 hrs after challenge

Data Not shown

versus

offspring 3hr placental corticosterone: r = 0.485, p = 0.001

offspring 3hr fetal brain corticosterone: r = 0.499, p = 0.001

offspring 3hr placental 11HSD2: r = −0.317, p = 0.017

Placental 11HSD1, 3-hrs post LPS challenge (Ex. 1)

Three-way ANOVA

G15 MET by G15 LPS interaction: F(1, 47) = 4.234, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.083

Figure 2G

(Sex by G15 MET by G15 LPS) with litter as covariate

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: N.S

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: p = 0.001

Placental 11HSD2, 3-hrs post LPS challenge (Ex. 1)

Three-way ANOVA

Sex by G15 MET by G15 LPS interaction: F(1, 47) = 4.622, p = 0.037, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.090

Figure 2H

(Sex by G15 MET by G15 LPS) with litter as covariate

Post hocs:

Males

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-saline: N.S

Females

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: N.S

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: N.S

Placental corticosterone, 3-hrs post LPS challenge (Ex. 1)

Three-way ANOVA

G15 MET by G15 LPS interaction: F(1, 47) = 160.380, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.773

Figure 2I

(Sex by G15 MET by G15 LPS) with litter as covariate

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: N.S; p = 0.052

Saline-Saline vs MET-Saline: p = 0.001

Fetal CNS corticosterone, 3-hrs post LPS challenge (Ex. 1)

Three-way ANOVA

G15 MET by G15 LPS interaction: F(1, 47) = 14.736, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.239

Figure 2J

(Sex by G15 MET by G15 LPS) with litter as covariate

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: N.S

Fetal CNS IL-6R, 3-hrs post LPS challenge (Ex. 1)

Three-way ANOVA

Sex by G15 MET by G15 LPS interaction: F(1, 47) = 4.044, p = 0.050, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.079

Figure 2K

(sex by G15 MET by G15 LPS)

Males:

Main effect of LPS: F(1, 23) = 8.772, p = 0.007, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.276

Post hoc:

LPS vs Saline: t(25) = p = 0.004

Saline: 1.65 ± 0.13

LPS: 2.19 ± 0.14

Females:

N.S

Male fetal CNS CRFR1, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1A

(G15 MET by G15 LPS)

N.S

Female fetal CNS CRFR1, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1A

(G15 MET by G15 LPS)

N.S

Male fetal CNS GR, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1B

(G15 MET by G15 LPS)

N.S

Female fetal CNS GR, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1B

(G15 MET by G15 LPS)

N.S

Male fetal CNS IL-17A, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1C

(G15 MET by G15 LPS)

N.S

Female fetal CNS IL-17A, 3-hr post LPS challenge (Ex. 1)

Two-way ANOVA

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 1C

(G15 MET by G15 LPS)

N.S

Maternal P4 retrieval test

Two-way ANOVA

Latency to retrieve first pup: N.S

Data not shown

(G15 MET by G15 LPS) with litter used as a covariate

Latency to retrieve whole litter; main effect of ME/Saline: F(1, 77) = 4.271, p = 0.042, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.053; independent t-test as post hoc test since fewer than three levels = N.S

Male P12 Maternal Separation Potentiation of USVs on P12 – Total Number of Syllables (Ex. 3)

Repeated Measures ANOVA (G15 MET by G15 LPS across time)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3A

Time (baseline vs potentiation) x G15 MET interaction: F(1, 37) = 5.361, p = 0.026, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.127

Post hocs:

Baseline G15 Saline versus Baseline G15 MET: N.S

Potentiation G15 Saline versus Potentiation G15 MET: p = 0.020

Main effect of G15 LPS F(1, 37) = 5.762, p = 0.022, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.135

Main effect of time (baseline vs potentiation): F(1, 37) = 5.361, p = 0.026, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.0127

Female P12 Maternal Separation Potentiation of USVs on P12 – Total Number of Syllables (Ex. 3)

Repeated Measures ANOVA (G15 MET by G15 LPS across time)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3A

Main effect of G15 LPS F(1, 37) = 9.344, p = 0.004, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.188

Male P12 Maternal Separation Potentiation of USVs on P12 – Mean Syllable Duration (msec) (Ex. 3)

Repeated Measures ANOVA (G15 MET by G15 LPS across time)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 2A

N.S

Female P12 Maternal Separation Potentiation of USVs on P12 – Mean Syllable Duration (msec) (Ex. 3)

Repeated Measures ANOVA (G15 MET by G15 LPS across time)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 2A

N.S

Male P22 Social Play USVs – Total Number of Syllables (Ex.3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3B

G15 MET by G15 LPS interaction: F(1, 37) = 5.392, p = 0.030, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.204

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.011

Saline-LPS vs MET-LPS: p = 0.048

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: N.S

Female P22 Social Play USVs – Total Number of Syllables (Ex.3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3B

G15 MET by G15 LPS interaction: F(1, 37) = 8.184, p = 0.009, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.280

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.017

Saline-LPS vs MET-LPS: N.S

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: p = 0.025

Male P22 Social Play USVs – Mean Syllable Duration (msec) (Ex.3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 2B

Main effect of G15 LPS: F(1, 37) = 8.944, p = 0.007, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.299

G15 saline: 44.54 ± 1.62 versus G15 LPS: 37.42 ± 1.83

Female P22 Social Play USVs – Mean Syllable Duration (msec) (Ex.3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 2B

N.S

Male P30 Percent Time in Center of Open Field (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3A

Main effect of G15 MET F(1, 37) = 9.690, p = 0.004, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.208

G15 saline: 5.14 ± 0.57 versus G15 MET: 2.87 ± 0.47

Female P30 Percent Time in Center of Open Field (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3A

N.S

Male P90 Percent Time in Center of Open Field (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3B

N.S

Female P90 Percent Time in Center of Open Field (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3B

G15 MET by G15 LPS interaction: F(1, 37) = 11.597, p = 0.002, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.239

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: N.S

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: p = 0.003

Male P30 Distance Traveled (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3C

N.S

Female P30 Distance Traveled (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3C

N.S

Male P90 Distance Traveled (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3D

N.S

Female P90 Distance Traveled (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Supplemental Results Figure 3D

N.S

Maternal Sickness Behavior and Offspring USV correlations

Pearson Correlations

Maternal Sickness Behavior (120-min) and male P12 Offspring USVs Total Syllables (potentiated): r = −0.417, p = 0.007

Figure 3D

(120-min maternal sickness correlations displayed)

Maternal Sickness Behavior (240-min) and male P12 Offspring USVs Total Syllables (potentiated): r =- 0.341, p = 0.029

Maternal Sickness Behavior (120-min) and P22 Offspring Social USVs Total Syllables: r = 0.449, p = 0.024

Male P30 Social Preference Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3E

Main effect of G15 LPS: F(1, 37) = 10.863, p = 0.02, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.227

G15 saline: 0.28 ± 0.02 versus G15 LPS: 0.16 ± 0.03

Female P30 Social Preference Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3E

N.S

Male P90 Social Preference Score

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3F

(Ex. 3)

N.S

Female P90 Social Preference Score

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3F

(Ex. 3)

Main effect of G15 LPS: F(1, 37) = 12.216, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.248

Main effect of G15 MET: F(1, 37) = 11.830, p = 0.001

Male P31 Social Discrimination Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3G

G15 MET by G15 LPS interaction: F(1, 37) = 4.644, p = 0.038, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.112

Post hocs:

Saline-Saline vs Saline-LPS: N.S

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: p = 0.025

Saline-Saline vs MET-Saline: N.S

Female P31 Social Discrimination Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3G

G15 MET by G15 LPS interaction: F(1, 37) = 10.000, p = 0.003, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.21

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.040

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: p = 0.001

Saline-Saline vs MET-Saline: N.S

Male P91 Social Discrimination Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3H

G15 MET by G15 LPS interaction: F(1, 37) = 7.661, p = 0.009, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.172

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: p = 0.001

MET-Saline vs MET-LPS: p = 0.002

Saline-Saline vs MET-Saline: N.S

Female P91 Social Discrimination Score (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences

Figure 3H

G15 MET by G15 LPS interaction: F(1, 37) = 18.622, p = 0.001, \({{{{{{\rm{n}}}}}}}_{{{{{{\rm{p}}}}}}}^{2}\) = 0.335;

Post hocs:

Saline-Saline vs Saline-LPS: p = 0.001

Saline-LPS vs MET-LPS: N.S

MET-Saline vs MET-LPS: N.S

Saline-Saline vs MET-Saline: p = 0.001

Maternal Sickness Behavior and Offspring Social Discrimination Index

Pearson Correlations

FEMALES

Figure 3I

P91 social discrimination index correlated to maternal sickness at 30 minutes: r = −0.379, p = 0.014

(120-min maternal sickness correlations displayed)

P91 social discrimination index correlated to maternal sickness at 90 minutes: r = −0.502, p = 0.001

P91 social discrimination index correlated to maternal sickness at 120 minutes: r = −0.420, p = 0.006

P91 social discrimination index correlated to maternal sickness at 240 minutes: r = −0.384, p = 0.013

P91 social discrimination index correlated to maternal sickness at 360 minutes: r = −0.325, p = 0.038

MALES

P31 social discrimination index correlated to maternal sickness at 30 minutes: r = −0.340, p = 0.030

P91 social discrimination index correlated to maternal sickness at 60 minutes: r = −0.623, p = 0.001

P91 social discrimination index correlated to maternal sickness at 90 minutes: r = −0.691, p = 0.001

P91 social discrimination index correlated to maternal sickness at 120 minutes: r = −0.6080, p = 0.001

P91 social discrimination index correlated to maternal sickness at 240 minutes: r = −0.524, p = 0.001

Male P94 Adult Ventral Hippocampal GR (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 8A

N.S

Female P94 Adult Ventral Hippocampal GR (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 8A

N.S

Male P94 Adult Ventral Hippocampal CRFR1 (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 8B

N.S

Female P94 Adult Ventral Hippocampal CRFR1 (Ex. 3)

Two-way ANOVA (G15 MET by G15 LPS)

*males and females analyzed separately as this measure is not powered to detect sex differences. Males and females also ran on separate blots so cannot be compared directly.

Supplemental Results Figure 8B

N.S

Offspring adult behavior and brain correlations

Pearson Correlations

Male Saline-LPS:

Data not shown

Adult ventral hippocampal CRFR1 and P90 distance traveled: r = −0.738, p = 0.037

Male MET-LPS:

Adult ventral hippocampal GR and P90 distance traveled: r = 0.816, p = 0.025