Maternal immune activation alters social affective behavior and sensitivity to corticotropin releasing factor in male but not female rats

Abstract

Prenatal infection increases risk for neurodevelopmental disorders such as autism in offspring. In rodents, prenatal administration of the viral mimic Polyinosinic: polycytidylic acid (Poly I: C) allows for investigation of developmental consequences of gestational sickness on offspring social behavior and neural circuit function. Because maternal immune activation (MIA) disrupts cortical development and sociability, we examined approach and avoidance in a rat social affective preference (SAP) task. Following maternal Poly I:C (0.5 mg/kg) injection on gestational day 12.5, male adult offspring (PN 60-64) exhibited atypical social interactions with stressed conspecifics whereas female SAP behavior was unaffected by maternal Poly I:C. Social responses to stressed conspecifics depend upon the insular cortex where corticotropin releasing factor (CRF) modulates synaptic transmission and SAP behavior. We characterized insular field excitatory postsynaptic potentials (fEPSP) in adult offspring of Poly I:C or control treated dams. Male MIA offspring showed decreased sensitivity to CRF (300 nM) while female MIA offspring showed greater sensitivity to CRF compared to sham offspring. These sex specific effects appear to be behaviorally relevant as CRF injected into the insula of male and female rats prior to social exploration testing had no effect in MIA male offspring but increased social interaction in female MIA offspring. We examined the cellular distribution of CRF receptor mRNA but found no effect of maternal Poly I:C in the insula. Together, these experiments reveal sex specific effects of prenatal infection on offspring responses to social affective stimuli and identify insular CRF signaling as a novel neurobiological substrate for autism risk.

Keywords: Corticotropin releasing factor; Insular cortex; Rat; Social affect.

Fig. 1.

Maternal immune activation and fever verification. A. Timeline of MIA experiments. Dams were mated with a male partner overnight and checked for the presence of a sperm plug in the morning. Positive identification of the sperm plug was treated as day 0.5. On embryonic day 12.5 dams were injected with either 0.5 mg/kg Poly I:C (n = 7 dams) or saline (n = 5 dams) and allowed to return to their cages. The presence of a fever response was verified by taking temperatures at baseline (prior to injection) and 4 and 24 h post injection. Mothers carried out their pregnancy until parturition and were allowed to raise their pups normally. Offspring of Poly I:C and saline treated mothers were randomly assigned to treatment groups and cannula implantation occurred on PN50. Behavioral testing began on PN64. B. Mean (+/−) SEM) rectal temperatures. Mothers treated with Poly I:C developed significant fever responses with temperatures significantly increased in dams treated with Poly I:C compared to saline injected dams at 4 h post injection. ***p < 0.001.

Fig. 2.

Maternal immune activation altered social affective behavior. A. Mean (+/− SEM) time spent interacting with naive conspecifics on day 2 of the SAP test (A, n = 20 per sex). Males spent more time investigating naive juveniles (PN30) compared to naive adults (PN50), **ps < 0.01 Sex by Age interaction. B–C. Mean (+/− SEM) time spent interacting with naive and stressed juvenile conspecifics on Day 3 of the SAP test with juvenile (B, n = 20 per sex) or adult (C, n = 19 males, 20 females) conspecifics. Male and Female control rats (saline) and Female MIA rats (Poly I:C) spent more time investigating stressed juveniles but Male MIA rats did not differentiate between naive and stressed conspecifics. D. Mean (+/− SEM) preference for interaction with the stressed conspecifics (n = 19–20 per group). Values >50 % indicate more time spent with stressed conspecific; horizontal line at 50 % indicates no preference. Poly I:C male offspring did not show preference for PN30 or avoidance of PN50 stressed conspecifics. Dots indicate individual replicates. *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001 Sidak post hoc comparisons; see text for additional statistics.

Fig. 3.

Effect of CRF on insular synaptic efficacy after maternal immune activation in males and females. A. Photograph of coronal section with insular cortex placed over the perforated multiple electrode array. B. Representative fEPSPs were recorded in aCSF then after CRF (300 nM) by biphasic stimuli from 0 to 5 V. Data were analyzed as peak fEPSP amplitude normalized to the 5 V response at each channel observed in aCSF. C–D. Mean (+/− SEM) fEPSP amplitude before and after CRF application in acute slices from adult males (C, ns = 10–11 slices per condition) and females (D, n = 10 slices per condition). In slices from male saline offspring and from female Poly I:C offspring CRF increased fEPSP amplitude at higher voltages (* indicate significant differences between aCSF and CRF fEPSP, see text for additional statistics). CRF had no effect on slices from Poly I:C male or saline female offspring. E. Mean (+SEM) normalized fEPSP in response to CRF at the highest stimulation voltages (2.5 to 5) from males and females. A significant Sex by CRF by MIA interaction is present (p = 0.042). *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 4.

Effect of CRF on social interaction after maternal immune activation in males and females. A. Diagram of experimental procedure. B. Mean (+/− SEM with individual replicates, ns = 10–12/treatment) time spent interacting with a naive juvenile conspecific in a 5 min social interaction test. Male offspring of saline treated dams (n = 11) and female offspring of Poly I:C treated dams spent more time investigating the juvenile after CRF injection to the insular cortex (***p < 0.001, ****p < 0.0001). C. Representative locations of insula cannula implants.

Fig. 5.

Distribution of CRF₁ mRNA in insula, central amygdala and sensory cortex. (A). CRF₁, vGlut1, and gad1 mRNAs were visualized with fluorescent multiplex in situ hybridization in male and female adult offspring of control or MIA treated mothers, n = 8 treatment. Composite image from posterior insular cortex. Scale bar = 50 μm. (B) Percentage of cells in the regions of interest expressing CRF₁. MIA increased the number of CRF₁ cells in the sensory cortex (p = 0.013). (C). Percentage of cells in the region of interest expressing vGlut1. MIA increased the number of vGlut1 cells in the sensory cortex (p = 0.017). (D) Percentage of cells in the region of interest expressing gad1. MIA increased the number in the sensory cortex (p = 0.017). (E) Percentage of vGlut1 neurons coexpressing CRF₁. (F) Percentage of gad1 neurons coexpressing CRF₁. MIA increased the number of CRF₁ + gad1 neurons in the CEA of males. MIA increased the number of CRF₁ + gad1 neurons in the sensory cortex of females. Bars depict the mean +/− SEM with individual replicates. *p < 0.05.