Generation and characterization of Neuroligin-3 knock down mice for Autism Research

Background: The cause of autism may be due to synaptic abnormalities. Neuroligins are present in the postsynaptic membranes of neurons and play a crucial role in the synaptic transmission. Neuroligin-3 is known to be mutated in rare cases with autism spectrum disorders (ASD). 

Objectives: To investigate the possible involvement of Neuroligins in autism, neuroligin-3 (NL-3) knock down (KD) mice have been generated and characterized. 

Methods: Microinjection of a NL-3 RNAi gene construct into embryos of CD-1 mice was performed.  PCR, Western blot and behavioral assessments were analyzed.

Results: NL-3 KD mice survived without premature mortality. Genotyping of the NL-3 KD mice using PCR confirmed the construct. Phenotyping by Western blot using two different antibodies to NL-3 revealed a 42-72% knock-down reduction in NL-3 in hippocampal regions of these NL-3 KD mice. Behavioral assessments of eight NL-3 KD mice compared to 10 control mice both at 33 days using an elevated plus maze showed a significant difference (p=0.005) in anxiety and emotional expression. However, open field test at 32 days showed no difference in social interaction. Morris water maze testing at 35 days showed no difference in spatial memory. A PCR positive line of NL-3 KD CD-1 mice was bred with C57BL mice as a backcross transferring the NL-3 RNAi DNA gene into this widely used inbred strain. Ultrasonic vocalization between 11 days old pups and their mother (1 PCR positive mouse, 2 PCR negative mice) of NL-3 KD C57BL mice showed delayed and reduced communication.

Conclusions: Neuroligin-3 knock down mice manifest behavioral changes in anxiety and emotionality expression consistent with those of ASD. This knock down approach may be a useful model of autism.