Abstract

Mice lacking the homeobox-containing transcription factor En2 (En2-/- mice) are considered as a reliable animal model for investigating the neurodevelopmental basis of autism spectrum disorders, as they display some ASD-related anatomical deficits, including cerebellar hypoplasia (Joyner A L, et al., 1991), reduced Purkinje neurons numbers (Kuemerle B, et al., 1997), altered anatomy of the amygdala (Kuemerle B, et al., 2007), and a significant loss of forebrain GABAergic interneurons (Sgadò P et al., 2013). En2-/- mice also display autistic-like behavioral defects, including decreased sociability (Brielmaier J, et al., 2012), enhanced seizure susceptibility (Tripathi P P, et al., 2009) and impaired spatial learning and memory (Provenzano G, et al., 2014) (Cheh M A, et al., 2006). A recently published study from our laboratory revealed that En2-/- mice displayed an altered disposition of GABAergic circuits in the visual cortex, that might contribute to alter binocularity and plasticity of the visual system, while leaving other visual functional properties (acuity, response latency, receptive field size) unaffected (Allegra M, et al., 2014). Moreover, preliminary experiments performed in our laboratory suggested that, at cellular level, the retina of En2-/- mice showed alterations in the number of specific cell subtypes compared with the retina of WT littermates. More importantly, an increasing number of studies published in the past years indicated that difficulties in sensory processing might somehow contribute to the pathogenesis of autism (Robertson C E, Simon B C, 2017). Therefore, we decided to investigate the possible retinal defects in the En2-/- mice. We showed for the first time that the En2 gene is expressed in all nuclear layers of the adult mouse retina. We also found that En2-/- adult mice showed a significantly decreased number of Calbindin (Calbindin+) positive horizontal cells, and a significantly increased number of Calbindin+ amacrine/ganglion cells. The number of Brn-3a (Brn-3a+) positive ganglion cells displayed no difference with respect to wild-type littermates. In addition, En2-/- adult mice showed a significantly reduced expression of the rod photoreceptor marker rhodopsin at both mRNA and protein levels, and a significant reduction of the S-cone photoreceptor S opsin, the bipolar cells marker pcp2, and the GABAergic interneurons marker parvalbumin. Electroretinogram (ERG) analysis revealed that the amplitude of b-wave in En2-/- mice scotoptic ERG was significantly reduced as compared with controls. Together, all these data indicate that En2-/- mice exhibit retinal defects at molecular, cellular and functional level, whose significance needs to be further investigated.