Synaptic Proteins and Structure Defects in Adolescent Rats Following Prenatal Exposure to Valproic Acid

Background: Autism Spectrum Disorders (ASDs) are a heterogenous group of neurodevelopmental brain diseases. The number of cases is steadily increasing, currently 1 child in 100 is diagnosed with autism, while the prevalence in the U.S. is estimated to be 1 in 50 children. Their typical symptoms include impaired communication (verbal and non-verbal) as well as social interactions along with stereotyped, repetitive behaviors and restricted interests. Recent genetic studies in affected individuals with ASDs indicate the exist of the mutations in a number of genes coding proteins involved in the development and function of synapses, suggested the synapses as a possible site of autism origin. However, the cause of the defects of synaptic proteins and their roles in the pathogenesis of idiopathic autism remain obscure.

Objectives: The aim of this study was to investigate the effect of prenatal exposure to valproic acid (VPA), - rodent model of environmentally triggered ASDs - on the synaptic structure as well as the synaptic protein level in adolescent rat offspring.

Methods: Pregnant Wistar rats received a single intraperitoneal (i.p.) injection of VPA (400 mg/kg of body weight) on gestational day (GD) 12.5. Controls received a single i.p. dose of solvent (sterile 0.9 % NaCl). On postnatal day (PND) 22 to 23, rat pups were separated. In order to study social disorders at neonatal stage, we analyzed vocalization of the offspring in response to maternal isolation in all infant rats at PND 11 and anxiety-related behavior (open field test) in adolescent male offspring (PND 40). Males offspring were decapitated at PND 52, their brains removed and cerebral cortex including hippocampus isolated. We determined the gene expression of neuroinflammatory factors, changes in levels of key pre- and postsynaptic proteins, as well as abnormalities of synaptic morphology in cerebral cortex and hippocampus.

Results: Our data showed that embryological exposure to VPA impairs early communication (lower vocalization) in neonatal and enhances anxiety-like behavior in adolescent rats. Prenatal exposure to VPA promotes pathological changes in synapses in both analyzed structures including nerve endings swelling, blurred and thickened synaptic cleft structure as well as swelling the pre- and postsynaptic parts accompanied with lysis and disruption of their synaptic membranes. Together with ultrastructural changes we observed alterations in proteins involved in the synaptic vesicles regulation and neurotransmitter release, increased expression of synaptobrevin (VAMP1/2), synaptophysin and synapsin-1 while the level of presynaptic membrane protein SNAP25 was decreased. In addition, analysis of postsynaptic proteins revealed the lowering level of PSD95 in both hippocampus and cerebral cortex whereas neuroligin-1 (NLGN1) decreased exclusively in cortex. Moreover, we observed changes in the expression of Shank family proteins (SHANK1, SHANK2, and SHANK3) compared to control rats. Prenatal exposure to VPA also upregulated the expression of pro-inflammatory cytokine interleukin 6 (IL-6) and cyclooxygenase-2 (COX-2) in adolescent rat offspring.

Conclusions: These results demonstrate that prenatal VPA exposure affected the synaptic proteins level which could be responsible for synaptic structure and plasticity changes and subsequently contributing to behavioral abnormalities in ASDs.

Supported by the NSC grant 2017/25/B/NZ4/01969