Lactobacillus reuteri normalizes altered fear memory in male Cntnap4 knockout mice

EBioMedicine. 2022 Dec:86:104323. doi: 10.1016/j.ebiom.2022.104323. Epub 2022 Nov 15.


Background: Autism spectrum disorder (ASD) is a common neurodevelopmental disease, characterized by deficits in social communication, restricted and repetitive behaviours, and impaired fear memory processing. Severe gastrointestinal dysfunction and altered gut microbiome have been reported in ASD patients and animal models. Contactin associated protein-like 4 (CNTNAP4) has been suggested to be a novel risk gene, though its role in ASD remains unelucidated.

Methods: Cntnap4-/- mice were generated to explore its role in ASD-related behavioural abnormalities. Electrophysiological recording was employed to examine GABAergic transmission in the basolateral amygdala (BLA) and prefrontal cortex. RNA-sequencing was performed to assess underlying mechanisms. 16S rDNA analysis was performed to explore changes in faecal microbial composition. Male Cntnap4-/- mice were fed with Lactobacillus reuteri (L. reuteri) or faecal microbiota to evaluate the effects of microbiota supplementation on the impaired fear conditioning mediated by Cntnap4 deficiency.

Findings: Male Cntnap4-/- mice manifested deficiency in social behaviours and tone-cue fear conditioning. Notably, reduced GABAergic transmission and GABA receptor expression were found in the BLA but not the prefrontal cortex. In addition, gut Lactobacillus were less abundant in male Cntnap4-/- mice, and L. reuteri treatment or faecal microbiota transplantation rescued abnormal tone-cued fear memory and improved local GABAergic transmission in the BLA of male Cntnap4-/- mice.

Interpretation: Cntnap4 shapes GABAergic transmission of amygdala and fear conditioning, and microbial intervention represents a promising therapy in ASD intervention.

Funding: National Natural Science Foundation of China, Science and Technology Planning Project of Guangzhou, Guangzhou Medical University, and China Postdoctoral Science Foundation.

Keywords: Autism spectrum disorder; Cntnap4; Fear memory; GABAergic transmission; Gut microbiome.

MeSH terms

  • Amygdala / metabolism
  • Animals
  • Autism Spectrum Disorder* / genetics
  • Autism Spectrum Disorder* / microbiology
  • Disease Models, Animal
  • Fear / physiology
  • Fecal Microbiota Transplantation
  • Limosilactobacillus reuteri*
  • Male
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Memory / physiology
  • Mice
  • Mice, Knockout
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism


  • Cntnap4 protein, mouse
  • Membrane Proteins
  • Nerve Tissue Proteins