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The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.

Three experiments were conducted to evaluate the role of drug-dosage and stimulus-specificity parameters on the tolerance of drug-induced (poly I:C) natural killer (NK) cell activity. In the first experiment a protocol which provided mice with four weekly 20 micrograms/mouse ip injections of the immunostimulatory synthetic polynucleotide (poly I:C) following exposure to either a simple odor cue or a complex cue resulted in tolerance of NK cell activity. The identical protocol with a higher drug dose (50 micrograms/mouse) did not produce tolerance. In a second experiment, the stimulus specificity of tolerance was assessed by giving two groups of mice repeated signaled drug injections. For one of these groups the final poly I:C injection of the series was signaled, while for the other group it was not. Although both groups were tolerant relative to controls not previously exposed to the drug, indirect evidence of conditioning was obtained. Specifically, it was found that tolerance among mice receiving the signal on the test was such that they were not different from undrugged controls, while uncued mice had significantly higher levels of NK cell activity. The third experiment evaluated the role of stimulus specificity within an extinction paradigm. It was found that tolerance was reversed in mice provided with repeated nonreinforced reexposure to drug-signaling cues, while mice exposed to novel cues remained tolerant. These results further support the hypothesis that associative factors contribute to the tolerance of a drug-induced immune response.