RatDISCO, an optical clearing and immunolabelling pipeline for large rat (and mice) tissue

Understanding brain function in health and disease requires unbiased three-dimensional mapping of its neurochemical architecture. Traditional histological methods require mechanical tissue sectioning, leading to cutting artefacts, lengthy processing times and loss of crucial three-dimensional information. To overcome that, optical clearing techniques have soared since 3DISCO allowed us to visualise neuronal networks in the whole mouse brain1. Immunolabelling in whole organs is possible; however, antibody penetration is a challenge and most clearing protocols are tailored for mice or human tissue2. Currently, no methods are customised to immunolabel rat brains. Hence, we developed RatDISCO, a whole-brain immunolabelling and optical-clearing pipeline that detects proteins of interest throughout large rat brains without compromising transparency or tissue integrity. RatDISCO achieves deep brain immunolabelling by an antigen retrieval step3 before antibody labelling, followed by stager delipidations to achieve optically-transparent samples ideal for light-sheet microscopy. In addition to allowing for successful immunolabelling of rat tissue, RatDISCO is compatible with mouse and in situ hybridisation. To test RatDISCO, we developed a pipeline to identify behaviourally-activated neurons, using the immediate-early gene cFos as a proxy for neuronal activation. We created a 3D atlas of the amygdala to map and quantify the cell density of cFos-positive neurons across wild-type and Fragile-X knockout (Fmr1-/y) rats, a model of autism spectrum disorders, during the classic fear conditioning paradigm. We identified hypo-activation in the basal and basolateral amygdala in Fmr1-/y rats in response to fear during a classical fear paradigm. These results correlate with observed amygdala-dependent processing alterations in individuals with Fragile-X syndrome.