30933
Maternal and Offspring Mthfr Deficiency As a Risk Factor for Social Deficits and Alternation in Cortical Interneurons Populations in Rodents.
A significant environmental risk factor for autism spectrum disorder (ASD) is prenatal environment. Methylenetetrahydrofolate reductase (MTHFR) shows high prevalence of polymorphism in ASD patients and their mothers. Excitation – inhibition imbalance has been suggested in the pathogenesis of ASD, supported by significant alternations in the GABA pathway. In a previous study, we showed both maternal and offspring Mthfr+/- genotype predisposes mice toward ASD-like behavior. Therefore, it is possible that these mice present alterations in cortical interneurons, as shown in human and mouse models of ASD.
Objectives:
To investigate the interaction between maternal and offspring Mthfr+/- genotype, ASD-like behavior and cortical interneuron distribution were examined
Methods:
Mthfr+/+ and Mthfr-/+ female mice on a Balb/cAnNCrlBR background were mated with male tg-GAD65-tdTomato on a C57/Bl6 background to generate Mthfr+/+(WT) and Mthfr+/-(HT), all carrying GAD65-tdTomato. Three groups representing maternal:offspring genotypes were created: 1.WT:WT 2.HT:WT 3.HT:HT. Six male mice per group were evaluated for ASD-like behavior using the following tests: Nest building, Marble burying and Social proximity (pairs with similar maternal and offspring genotype). Tests were recorded and analyzed offline by an observer blind to mouse identity. After the last test, mice were anesthetized and perfused with 4% PFA. Immunofluorescent staining was performed on sagittal brain sections for the detection of GAD, NeuN and Parvalbumin positive neurons.
Results:
A social deficit was found in the social proximity test where maternal HT genotype was associated with shorter sniffing time by mice of the HT:WT and HT:HT groups (51% and 41%, respectively), compare to WT:WT (F1,13=10.35, p<0.01). In addition, offspring HT genotype suppressed dominant behavior, represented by whisker trimming (F1,14=9.6, p<0.01). Mice were categorized as social or asocial, on the basis of their behavior (sniffing and non-aggressive behavior), regardless of their genotype, in order to evaluate the relation between phenotype and interneuron laminar distribution. Social phenotype correlated with GAD/NeuN ratio, so that social mice had higher GAD/NeuN ratio in deep layers of the cingulate cortex (layers 4-6, for example, 0.38 vs. 0.12 for layer 6), as represented by the interaction between layer x social phenotype (F1,13=10.43 p=0.007). Mice with repetitive behavior phenotype differed in the laminar distribution of parvalbumin neurons in the cingulate cortex (layer x repetitive behavior phenotype, F1,13=4.4 p=0.05). Mice presenting the repetitive behavior phenotype had a high density of parvalbumin neurons in layers 2-3 compared to mice that did not have this phenotype.
To further evaluate the excitation–inhibition balance associated with maternal and offspring genotype, seizure susceptibility was tested. Maternal genotype increased susceptibility to seizures induced by PTZ (50mg/kg, i.p). WT:HT pups exhibiting a greater amount of convulsion episodes in response to PTZ injection (F1,33=13.25 p=0.001). Moreover, an interaction between genotype x maternal genotype was found (F1,33=19.76 p<0.001).
Conclusions:
The interaction found between laminar distribution of interneurons and behavioral phenotype emphasizes a possible causal relation between cortical circuit organization and the pathophysiology of the social deficit seen in Mthfr+/- mice.