Gabaergic Interneuron Cell Loss in Mice Lacking Autism-Associated Gene Slit3

Background: Altered neuronal connectivity reported in Autism Spectrum Disorder (ASD) leads to alterations in brain function and multisensory integration. Human studies reported that SLIT3, an axon guidance molecule, may be associated with neurological conditions, such as major depressive disorder, schizophrenia and ASD. SLIT proteins bind to the receptor Roundabout (ROBO) and have a role in axon guidance, cell migration, cell proliferation and differentiation of various cell types during embryogenesis. Taken together, these findings suggest that Slit3 may be an attractive candidate to analyze altered neuronal connectivity in ASD.

Objectives: The purpose of the current study is to investigate how Slit3 mutation affects neuronal connectivity in ASD. Since SLIT3 is involved in cell migration, we hypothesized that during brain development the migration of GABAergic interneurons is affected by the loss of SLIT3 gene, leading to alterations in the Excitation/Inhibition (E/I) balance, as seen in ASD.

Methods: Slit3 mutant mice were crossed with either GAD (Glutamate decarboxylase) 65 or GAD67-GFP (Green Fibrillary Protein) mouse lines to study GABAergic interneurons in different key areas of the brain affected in ASD. Inserting these GAD knock-in mouse lines within the Slit3 knock-out mouse line allows for reliable tracking of specific cell populations to investigate the contribution of Slit3 toward GABAergic interneuron migration.

Results: Analysis of the Slit3 mutant mice crossed with either GAD65-GFP or GAD67-GFP mouse lines revealed a reduced number of GABAergic interneurons in the cortex, hippocampus, reticular thalamic nucleus and cerebellum in Slit3 knockout (KO) adult mice compared to SLIT3 wild type (WT) mice. This cell reduction in GABAergic interneurons was seen early during brain development suggesting that the loss of SLIT3 gene affects either the production, proliferation or migration of GABAergic interneurons. When we analyzed the different populations of GABAergic interneurons our results showed that Parvalbumin interneuron number was reduced in Slit3 KO mice cortex, hippocampus, reticular thalamic nucleus and cerebellum (KO, n= 14, WT, n=15), whereas Calretinin and Calbindin interneurons were not affected, suggesting that SLIT3 gene loss affects only the fast spiking-PV population.

Conclusions: Interneurons are known to synchronize neuronal activity and this synchronization is essential for cortical network function. Our results show a loss of GABAergic interneurons, especially the fast spiking-PV population, in mice lacking autism-associated gene Slit3. These findings reveal the importance of Slit3, an axon guidance molecule, in the formation of GABAergic neuronal networks and provide insight into the molecular pathways that may lead to altered neuronal connectivity in ASD.