Paternal high molecular weight poly I:C administration alters the sperm small non-coding RNA profile and offspring brain development and behavioural phenotype

• High molecular weight (HMW) poly I:C viral mimic modulates sperm non-coding RNAs and offspring phenotypes. • The viral mimic alters 29 sperm small non-coding RNAs, including multiple microRNAs with gene targets enriched in early embryonic developmental pathways. • This model provides the first evidence of specific long-term brain structural changes in a paternal immune activation (PIA) model. • HMW poly I:C PIA produces sexually dimorphic behavioural and cellular offspring phenotypes, including disrupted fear conditioning and reduced striatal microglia in males, increased microglial activation in females, and delayed social interaction, somatosensory cortex and corpus callosum thinning in both sexes. Paternal immune activation (PIA), including via the viral mimic polyinosinic:polycytidylic acid (poly I:C), impacts offspring physiology and behaviour. A previous model employing low molecular weight (LMW) poly I:C showed alterations in the sperm small non-coding RNA (sncRNA) profile and offspring phenotypes. High molecular weight (HMW) poly I:C increases translatability by eliciting a robust sickness response accompanied by a significant increase of key cytokines. Here, male C57BL/6J mice (F 0 ) were intraperitoneally injected with 12 mg/kg HMW poly I:C and mated with naïve females four weeks later. Adult offspring (F 1 ) displayed reduced somatosensory cortex and corpus callosum thickness, and altered social-interaction behaviour. Male offspring displayed a reduction in gross striatum weight, reduced striatal microglia numbers and disruption to Pavlovian associative learning, while female offspring displayed increased striatal and corpus callosum microglia activation. The F 0 sperm sncRNA profile showed upregulation of miR-141-3p and miR- 200a-3p among 29 dysregulated sncRNAs. These results are distinct from previous LMW poly I:C administration studies, demonstrating sperm epigenome changes and subsequent offspring phenotypes may be highly specific to particular immune pathways and activation states. Our present data provide new insights into how PIA may be able to modulate brain development and function. These new findings contribute to our understanding of how paternal viral-like immune activation affects sperm and offspring phenotypes, with implications for the future prevention of the detrimental effects of such epigenetic inheritance.