RATDISCO: A TISSUE CLEARING PIPELINE FOR DETECTING STIMULUS-ACTIVATED NEURONS IN RAT MODELS OF DEVELOPMENTAL DISORDERS

Neurodevelopmental disorders, including autism spectrum disorders (ASD), are characterized by social impairments and hypersensitivity. Hyperactivation or hypoactivation in brain regions responsible for social and emotional processing, such as the amygdala, has been evaluated in both ASD patients and rodent models using neuroimaging and conventional histological techniques. But these approaches are limited by resolution and artefacts introduced by tissue slicing. Advances in tissue clearing and light-sheet microscopy enable whole-organ imaging but require optical tissue clearing. The large size of rat brains presents challenges for antibody penetration and quantitative analysis, compounded by the lack of suitable 3D atlases.
To address these limitations, we developed RatDISCO, an immunolabelling and clearing method for intact rat brain, which is compatible with spinal cord, heart, and skin, as well as mouse tissues and organoids. It enables immunolabelling of deep brain structures through an antigen retrieval prior to antibody labelling, followed by sequential delipidation steps to produce optically transparent samples.
RatDISCO was employed to identify differentially activated neurons during behavioural tasks, using the immediate-early gene cFos as a marker for neuronal activation. We generated a three-dimensional atlas of the rat amygdala in commercially available software (Arivis4D) to map and quantify the density of cFos-positive neurons in both wild-type and Fragile-X knockout (Fmr1-/y) rats, a model of ASD. Hypoactivation was observed in the basal and basolateral amygdala of Fmr1-/y rats in response to fear during a classical fear conditioning paradigm. These findings are consistent with amygdala-dependent emotional processing alterations reported in individuals with Fragile-X syndrome.