Figure 1. Measures of sickness behavior, litter loss, maternal IL-6, and offspring neuronal sMHCI are predictive of a disease-relevant dose of poly(I:C).

(A) Sickness behavior in pregnant female Charles River C57BL/6 mice at gestational day (GD) 12.5 was observed 4 hr post-injection with all 3 poly(I:C) doses—20, 30, and 40mg/kg (F_3,184 = 102.3, P < 0.0001). (B) However, body temperature of GD12.5 dams 4hr post-injection was decreased at 30 and 40mg/kg, but not 20mg/kg, compared to saline, with only the 30mg/kg dose reaching significance (F_3,35 = 4.289, P < 0.05). (C) Although all 3 doses caused significant elevations in maternal serum IL-6 at 2.5 and 4 hr post-injection, IL-6 levels were much higher at 2.5hr than at 4hr post-injection and only the 30 and 40mg/kg doses reached the 10,000 pg/ml IL-6 MIA threshold (F_3,35 = 25.54, P < 0.0001). (D) The 30 and 40, but not 20, mg/kg doses caused significant weight loss in dams 24hr after poly(I:C) injection compared to saline (F_7,187 = 26.93, P < 0.0001). (E) Poly(I:C) caused litter loss in a dose-dependent manner. For the pregnancies that resulted in pups, there was no change in litter size (not shown). (F) Representative images of glutamatergic synapses on dendrites cultured from the frontal cortex (FC) of newborn offspring of saline-injected (saline) or poly(I:C)-injected (MIA) mothers. Neurons were immunostained at 8 DIV for excitatory synapse density using antibodies against PSD-95 (green) and VAMP2 (red). Scale bar = 5 μm. (G) All doses of poly(I:C) were associated with a significant decrease in synapse density (SD) (F_3,43 = 11.01, P < 0.0001). Values were normalized to saline control (n ≥ 7 litters). (H) Surface MHCI (H2-Kb) was significantly increased on acutely dissociated neurons from FC of P0 offspring following MIA elicited by 30, but not 20 or 40, mg/kg poly(I:C), as assessed using flow cytometry (n ≥ 5 mice per group, 2 experiments) (F_3,19 = 5.156, P < 0.01). (I-J) The effects of poly(I:C) dose on male offspring grooming (secs/10 min) behavior were assessed between P60-80. (I) Although there was a strong trend toward increased grooming in the MIA offspring from the 30 mg/kg group, the results were not significant due to the high variance (P = 0.06) (n ≥ 19 mice per group from at least 6 litters, 3 experiments). (J) The lack of significance and high variance remained after accounting for litter effects by averaging the grooming behavior of individuals within each litter (P = 0.22) (n ≥ 6 litters, 3 experiments). Bars represent mean ± s.e.m *p < 0.05, **p < 0.01, ***p < 0.001.