PROBING THE DEVELOPMENTAL ROLE OF EARLY EXCITATION/INHIBITION IMBALANCE IN AUTISM

Autism and related developmental disorders encompass a wide range of heterogeneous conditions with an overall prevalence of 1 % in the human population. Although autism varies in symptomatology and severity, its two main core symptom domains (i.e. social and communicative impairment, as well as restricted and repetitive behaviors) appear to be consistently affected across the spectrum. However, how diverse etiological mechanisms converge to produce the relatively narrow set of behavioral and etiopathological manifestations that characterize autism remains unclear. A popular theory posits that an imbalance between excitatory and inhibitory (E/I) activity might contribute to (or underlie) the etiology of autism (Rubenstein and Merzenich, 2003). However, controversy exists about whether the markers of E/I imbalance measured in autistic individuals and animal models are indicative of a direct, causal contribution to the underlying pathology, or if they instead represent a compensatory, epiphenomenal phenotype of limited etiological relevance. In my dissertation I test the hypothesis that alterations in E/I ratio occurring during early critical developmental windows can alter the developmental trajectory of the brain, leading to lasting, autism-relevant, behavioral, transcriptomic and connectivity alterations. To this aim, we used chemogenetics in mice to transiently increase the neocortical E/I balance during early postnatal phase. Cell-type specific increase of neuronal excitability was obtained using intersectional genetics. Specifically, we expressed excitatory designer receptor exclusively activated by designer drugs (DREADD) receptors hM3Dq in Vglut1-cre mice (Giorgi et al., 2017) and induced E/I imbalance by chronically treating mice with clozapine N-oxide (CNO) during the first two postnatal weeks (Control N = 29, Vglut1-gq N = 27, mixed sexes). Longitudinal behavioral tests, resting state functional magnetic resonance imaging (rsfMRI) and RNA-sequencing were performed at multiple developmental stages (late infancy, adolescence and adulthood). Our results show that this simple manipulation results in permanently impaired social behavior, as well as transcriptional and connectivity alterations of relevance for autism. Specifically, chemogenetically increasing E/I balance during early development resulted in robustly impaired sociability as measured with two different tests, in two different cohorts of animals, a trait that lasted throughout adulthood. Importantly, socio-behavioral alterations were not associated with overt anxiety-like phenotypes, or impairments in olfactory abilities, tactile sensitivity, motor coordination and working or long-term memory. Corroborating the developmental specificity of these results, control studies in which the same manipulation was applied to adolescent mice did not replicate any of these behavioral alterations. Transcriptomic analyses showed that transient cortical hyperexcitability during early development results in permanent alterations in the expression level of a large set of ribosomal and synaptic related transcripts. Notably, the list of differentially expressed genes was significantly enriched for known autism-associated synaptic genes, thus implicating activity-dependent transcriptional alterations in synaptic coupling in the generation of the observed phenotypes. We next interrogated brain circuit organization of the manipulated mice using task-free fMRI in adult animals. We found that chemogenetically-manipulated animals exhibited profoundly disrupted functional connectivity in socially-relevant fronto-hippocampal regions, but preserved fMRI coupling in sensory networks. Corroborating the behavioral relevance of these findings, multivariate modelling revealed that fMRI hypo-connectivity was highly predictive of behavioral disruption in manipulated animals, an effect that specifically involved subcortical components of the reward system. Taken together, our findings document that E/I imbalance during early development is sufficient to produce multi-omic (i.e. phenomic, connectomic and transcriptomic) autism-relevant phenotypes in rodents. These results support a causal (as opposed to epiphenomenal) contribution of E/I imbalance to the pathogenesis of autism and related developmental conditions. Our study also elucidates a possible mechanism by which early hyperexcitability introduces epigenetic alterations in activity-dependent expression of synaptic genes that lead to abnormal functional network coupling and behavior.