NTRK2 Gene Expression Levels in Laser Captured Glutamatergic Neurons From Animal Models of Social Behavior Deficits

Autism spectrum disorder (ASD) is a neurodevelopmental disability affecting communication and social behaviors. Research is needed because the percentage of children affected by ASD is 1 in 59, and it is diagnosed in males at a rate of 1 in 42. Animal models must be used, because the neurological changes that lead to ASD occur during prenatal development. In this study, three mouse models were used to represent possible causes of ASD. The BTBR model is a genetically engineered model that displays social behavior deficits and has neuroanatomical findings similar to ASD. The other models include the Poly-IC and valproic acid injected mice which exposes the pregnant mother to a virus activating her immune system or a drug thought to affect brain development, respectively. In all three models the effects of brain-derived neurotrophic factor or BDNF, which is an important cytokine in the brain responsible for synaptic plasticity, maintenance and recognition, are being studied via expression levels of NTRK2. BDNF activates cell signaling cascades in glutamatergic neurons via the TrkB receptor which is encoded by the NTRK2 gene. It was previously found that NTRK2 expression was reduced in glutamatergic cells in people affected by ASD. The first outcome of the study is to determine gene expression differences in glutamatergic neurons captured from the cingulate cortex in all three models as well as in wild type control mice. Additionally, a second outcome of the study is to optimize a new protocol for single cell gene expression using a nested PCR method. This was done by comparing the previously used method for relative end-point PCR with the nested method to identify gene expression alterations. To prepare for the two PCR methods, samples were dehydrated and laser capture microdissection was performed on mouse brain tissue to obtain pyramidal neurons from the cingulate area. This area is highly connected to the limbic system and plays a role in personality and communication. All animal procedures were approved by the ETSU animal care committee. RNA isolation was performed on 1000 cells after which RNA was reverse transcribed into cDNA using the Superscript III cDNA synthesis system. Initial optimization experiments included using various amounts of starting cDNA and determining expression differences using relative end-point PCR and Agilent tape station. The same starting cDNA was used and initially 20 cycles of PCR were performed using Prime5 HotStart Master Mix followed by a quantitative PCR reaction using Powerup on the BioRad CFX96 RT detection system. Gene expression was performed using NTRK2 as the target gene and GAPDH as the reference gene for each method. Both methods will allow the detection of changes in the expression levels of NTRK2 and GAPDH when different sample concentrations are used. This data could help establish a link between maternal immune system activation or exposure to certain drugs during pregnancy with the occurrence of ASD