Speaker
Description
Neural organoids provide a great tool to decipher human brain diseases at the molecular and physiological levels. The organoids are of great value in understanding neurodevelopmental diseases, as they can reflect changes occurring even in the prenatal period. Those diseases are often accompanied by aberrant changes in the formation of dendritic spines harboring excitatory synapses. Yet, none of the studies focused on detailed characterization of dendritic spines in organoids. Herein, we present the novel protocol to visualize and characterize single dendritic spines in matured organoids.
Human induced pluripotent stem cells were differentiated to cortical spheroids. On subsequent stages of development organoids were evaluated by whole organoids’ imaging and western blot analysis. Till day 200 the organoids were evaluated for proper differentiation: rosettes formation, cortex layering and glial/neuronal differentiation. After day 200 organoids were evaluated for their maturation properties. Live calcium imaging was performed to analyze the spontaneous activity of cells. Next, organoids older than one year were evaluated for dendritic spines formation. Spines were characterized using biolistic delivery of lipophilic dye combined with subsequent immunolabeling of pre- and postsynaptic markers.
We show that organoids’ maturation can be manifested by spontaneous activity of neurons with visible synchronization. This maturation is accompanied by changes in expression of repertoire of synaptic-related proteins (glutamate receptors, postsynaptic scaffolding proteins). Importantly, we were able to optimize protocol to successfully visualize dendritic spines in neurons within organoids. Furthermore, we were able to immunolabel dendritic spines with antibodies directed to proteins forming either pre- or postsynaptic compartments. This method enables a more detailed characterization of complex dendritic spine structure and function in human neurons in both health and disease.
| Author(s) | Bogna Badyra1*, Matylda Roszkowska1, Karolina Protokowicz1, Jan Suchomski1, Dominika Kurpiewska1, Marcin Barański1, Ewa Liszewska2, Jacek Jaworski2, Leszek Kaczmarek1 |
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| Affiliation(s) | "1 Laboratory of Neurobiology, BRAINCITY, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland. 2 Laboratory of Molecular and Cellular Neurobiology, International Institute of Molecular and Cell Biology, Warsaw, Poland." |
