Sensory filtering disruption caused by poly I:C - Timing of exposure and other experimental considerations

Abstract

Maternal immune activation (MIA) in response to infection during pregnancy has been linked through various epidemiological and preclinical studies to an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia in exposed offspring. Sensory filtering disruptions occur in both of these disorders and are typically measured using the acoustic startle response in both humans and rodents. Our study focuses on characterizing the baseline reactivity, habituation and prepulse inhibition (PPI) of the acoustic startle response following exposure to MIA. We induced MIA using polyinosinic: polycytidylic acid (poly I:C) at gestational day (GD) 9.5 or 14.5, and we tested sensory filtering phenotypes in adolescent and adult offspring. Our results show that startle reactivity was robustly increased in adult GD9.5 but not GD14.5 poly I:C offspring. In contrast to some previous studies, we found no consistent changes in short-term habituation, long-term habituation or prepulse inhibition of startle. Our study highlights the importance of MIA exposure timing and discusses sensory filtering phenotypes as they relate to ASD, schizophrenia and the poly I:C MIA model. Moreover, we analyze and discuss the potential impact of between- and within-litter variability on behavioural findings in poly I:C studies.

Keywords: Autism Spectrum Disorder; Habituation; Litter variability; Neurodevelopmental disorder; Poly I:C; Prepulse inhibition; Schizophrenia; Sensorimotor gating; Startle.

Fig. 1

Maternal poly I:C injection induces a robust cytokine response in fetal tissue. Maternal temperature was measured at 3 (A, left) and 24 (A, right) hours following poly I:C or saline injection, whereas maternal body weight was measured ar 24 hours following injection (B). poly I:C did not influence maternal temperature at 3 hours (left; p=0.923), maternal temperature at 24 hours (right, p=0.723) or maternal weight at 24 hours (p=0.455) following injection. Fetal tissue was collected 6 ​h after GD9.5 poly I:C or saline injection, with a total of 6 fetuses per group and 2 fetuses per dam. Poly I:C fetuses exhibited an increase in gene expression of Interleukin-6 (p ​< ​0.001), Interleukin-10 (p ​= ​0.007), Tumor necrosis factor α (p ​< ​0.001) and Interferon-γ (p ​= ​0.007). N ​= ​6 per group (C).

Fig. 2

Neither GD9.5 nor GD14.5 poly I:C treatment change startle reactivity in adolescent offspring. Data is shown for both males and females from saline and poly I:C offspring for each time point. A 3-way repeated measures ANOVA conducted separately for GD9.5 and GD14.5 adolescent offspring showed a significant effect of stimulus intensity (p ​< ​0.001) with no interactions or main effects associated with prenatal treatment.

Fig. 3

GD9.5 but not GD14.5 poly I:C treatment increases startle reactivity in adult offspring regardless of sex. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. For GD9.5 adult offspring, a 3-way repeated measures ANOVA showed a main effect of prenatal treatment (p ​= ​0.007), as well as an interaction between stimulus intensity and treatment (p ​= ​0.035). Post-hoc analysis with Bonferroni correction showed significant differences for stimulus intensities of 105 and 110 ​dB. There was no significant interaction between prenatal treatment and sex (p ​= ​0.808). In contrast, a 3-way repeated measures ANOVA conducted for GD14.5 adult offspring showed no main effect (p ​= ​0.709) of prenatal treatment or significant interactions associated with it.

Fig. 4

Neither GD9.5 nor GD14.5 poly I:C treatment change short-term habituation of startle in adolescent offspring. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. A 3-way repeated measures ANOVA conducted separately for GD9.5 and GD14.5 offspring showed a main effect of trial (p ​< ​0.001 for both analyses), but no significant main effect of prenatal treatment or interactions between prenatal treatment and sex or prenatal treatment and trial, indicating short-term habituation across all groups and no effects of either prenatal poly I:C exposures. Insets show quantification of short term habituation using a ratio of startle amplitude on the first 5 trials divided by startle amplitude on the last 5 trials, with values ​> ​1 indicating short term habituation of startle.

Fig. 5

GD9.5 and GD14.5 adult offspring exhibit similar short-term habituation to controls, but GD9.5 offspring show a strong trend to increased startle reactivity in the first few trials of the habituation block. Data is shown for both malesand females from saline and poly I:C offspring for each timepoint. For GD9.5 offspring, a 3-way repeated measures ANOVA showed strong trends for a main effect of prenatal treatment (p ​= ​0.074) and an interaction between trial and prenatal treatment (p ​= ​0.064 with Greenhouse-Geisser correction). In contrast, there were no significant main effects or interactions associated with prenatal treatment for GD14.5 offspring. Insets show quantification of short term habituation using the ratio of startle amplitude of the 5 first trials divided by the last 5 trials.

Fig. 6

Neither GD9.5 nor GD14.5 poly I:C treatment change adolescent startle reactivity or long-term habituation of startle as measured by the first 5 trials of the habituation block. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. A 3-way repeated measures ANOVA conducted separately for GD9.5 and GD14.5 adolescent offspring showed no significant main effect of prenatal treatment or any interactions with day or sex. Interestingly, all adolescent offspring failed to show strong long-term habituation (p ​= ​0.797 and p ​= ​0.399 respectively for main effect of day).

Fig. 7

GD9.5 and GD14.5 adult offspring exhibit similar long-term habituation to controls, but GD9.5 offspring show higher startle reactivity across days as measured by the first 5 trials of the habituation block. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. For GD9.5 offspring, a 3-way repeated measures ANOVA revelead significant main effects of day, prenatal treatment and sex, but no interactions between them (p ​< ​0.001, p ​= ​0.036 and p ​= ​0.01 respectively), providing evidence for changes in startle reactivity but not long-term habituation. For GD14.5 offspring, a similar analysis was conducted and a 3-way interaction followed by post-hoc testing revealed a significant decrease in startle reactivity only on day 3 for male poly I:C offspring, implying that these animals exhibit stronger long-term habituation across the first 3 days of testing but do not habituate further into days 4 and 5.

Fig. 8

Neither GD9.5 nor GD14.5 poly I:C treatment change PPI in adolescent offspring across a variety of prepulse conditions. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. A 4-way repeated measures ANOVA conducted separately for GD9.5 and GD14.5 adolescent offspring showed main effects of prepulse (PP) intensity and ISI for both groups but no main effect or interactions associated with prenatal treatment (see text for detailed statistics).

Fig. 9

Neither GD9.5 nor GD14.5 poly I:C treatment change PPI in adult offspring across a variety of prepulse conditions. Data is shown for both males and females from saline and poly I:C offspring for each timepoint. A 4-way repeated measures ANOVA conducted separately for GD9.5 and GD14.5 adolescent offspring showed main effects of prepulse (PP) intensity and ISI for both groups, but no main effect or interactions associated with prenatal treatment, besides a significant three-way interaction between ISI, prenatal treatment and sex in GD9.5 offspring (see text for detailed statistics).

Fig. 10

A representation of between and within litter variability in PPImeasures in our control and poly I:C offspring. Within each group, litters are depicted by different colours and each dot represents a single animal. The black group simply contains the entire group’s data, which was used to calculate effects in the results section. Error bars represent each group’s 25th and 75th quartiles are drawn in reference to the group’s median. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

The probability of a random sample of 2–7 animals picked out from litter 1 of each group to produce a biased estimate of the litter’s PPI phenotype Data was averaged across all 4 PPI conditions (prepulse-ISI combinations) used in our experiment. A biased estimate was considered to be a sample mean falling outside of the full litter’s IQ range. A thousand random samples of size 2–7 were generated from each group’s first litter, where animals were not culled, for each PPI condition. Each sample’s average was compared to the full litter’s 25th and 75th quartiles for that PPI condition. The number of samples out of 1000 that fell above the 75th or below the 25th quartiles averaged across all 4 PP-ISI conditions is represented on the Y-axis.