Anatomical, behavioral and functional characterization of novel zebrafish mutant lines for nlgn2, a high confidence ASD risk-gene

Neuroligin-2 (NLGN2) is a postsynaptic structural component of GABAergic synapses, and it is predicted as high-confidence autism spectrum disorder risk-gene. To investigate the relevance of this gene in the development of the brain circuits and functions, two newly-generated CRISPR-CAS9 nlgn2(a/b) mutant lines were characterized focusing on viability, neuronal anatomy and functional aspects. Both nlgn2 mutant fish exhibit decreased survival rate, gross anatomical alterations in head morphometry and body length. From the functional point of view, mutant fish show alterations in basic photo-response and in the nlgn2a brain activity, with higher power in the infra-slow range (<0.1 Hz). At the anatomical level, in nlgn2a mutants, the population of periventricular pyramidal neurons in the tectum show differences in dendritic spine densities, retinotopic areas and neuronal branching properties. An electron microscopy analysis revealed a reduced extension of the active zones at the synapse for both mutants. To better characterize the spatial distribution of PSD95- and gephyrin-positive synapses during development, two Gal4/UAS-transgenic lines were established. In parallel, a transcriptomic analysis highlighted differential expression patterns for genes involved in synaptic and axonal structural organization. In conclusion, the developed approaches allowed to identify a number of alterations consistent with the pathological traits of a neurodevelopmental disease.