Volume CLEM and conventional EM: a dual approach to investigate the mechanism of neuronal dysfunction in Angelman Syndrome

Angelman Syndrome (AS) is a rare neurodevelopmental disorder arising from the loss of the maternal copy of UBE3A, a paternally imprinted gene at neuronal level. Similarly to AS, other neurological defects due to an unproper UBE3A dosage are directly associated with alterations in brain development and synapse ultrastructure. The nanometric resolution of electron microscopy (EM) has proven essential for studying their outcome at the network level and establishing meaningful correlations between synapses geometrical features and their function. To explore whether UBE3A loss effect on the developing connectome was cell-autonomous, we in-utero electroporated CRISPR/Cas9 constructs to silence the endogenous UBE3A expression in a subset of layer 2/3 pyramidal neurons of the mouse somatosensory cortex. By creating z-confined NIRB marks we were able to relocate dendrites of interest and analyze spines complexity three-dimensionally from volumetric SBF-SEM datasets. Furthermore, we exploited conventional EM to examine excitatory synapses structure in the cortical circuit of a classic AS model pan-neuronally depleted from UBE3A expression. We identified pre-synaptic compartment defects that seem to diminish over time to make room for later changes in the post-synapse. Overall, the combined use of these two models should help clarify whether the defects originate within the neurons or their wider network.