Sparse labelling with AAV-PHP.eB, a noninvasive gene delivery method: Optimization of a protocol for morphological and anatomical connectivity analyses.

L. TERESITA TRIBBIA; DIEGO E. PAFUNDO; CARLOS A. PRETELL ANNAN; IRENE RE. TARAVINI; GERMÁN LA IACONA; JUAN E. BELFORTE

Congreso; XXXV Reunión Anual SAN; 2020

Sociedad Argentina de Investigación en neurociencias

Since its inception, neuroscience has been captivated by the morphological understandingof neural elements and their intertwining into complex circuits. Studies concerning structure-function relationships at the single cell level and those aimed at delineating anatomicalconnectivity at neural network level are fundamental for the elucidation of both physiologicaland pathophysiological processes of the central nervous system (CNS). Although variousmethods exist for studying neuronal morphology, drawbacks can be encountered in certainsettings: e.g., heavy metal staining can hamper simultaneous neurochemical analyses, andgenetically-directed labelling can result in insufficient sparseness for adequate reconstructionof complex arborizations. If a detailed study of neuroanatomical projections with conventionalor viral tracers is to be combined with a morphological analysis of the cellular elementsreceiving these inputs, a sparse labelling allowing visualization of axonal processes andperformance of immunofluorescence (IF) would be favored. Hence, we optimized a protocolfor sparse neuronal labelling taking advantage of a commercially available adeno-associatedvirus (AAV) variant developed for efficient noninvasive CNS gene delivery: AAV-PHP.eB. Wepresent data supporting its use for 3D neuronal reconstruction, spine density analysis, and thecomplementation with AAV tracers and IF to analyze local and distal afferents to individualneurons in prefrontal cortex of mice.