Abstract
Astroglia are vital facilitators of brain development, homeostasis, and metabolic support. In addition, they are also essential to the formation and regulation of synaptic circuits. Due to the extraordinary complex, nanoscopic morphology of astrocytes, the underlying cellular mechanisms have been poorly understood. In particular, fine astrocytic processes that can be found in the vicinity of synapses have been difficult to study using traditional imaging techniques. Here, we describe a 3D three-colour super-resolution microscopy approach to unravel the nanostructure of tripartite synapses. The method is based on the SMLM technique direct stochastic optical reconstruction microscopy (dSTORM) which uses conventional fluorophore-labelled antibodies. This approach enables reconstructing the nanoscale localisation of individual astrocytic glutamate transporter (GLT-1) molecules surrounding presynaptic (bassoon) and postsynaptic (Homer1) protein localisations in fixed mouse brain sections. However, the technique is readily adaptable to other types of targets and tissues.
Highlights
We describe a protocol for super-resolution imaging of tripartite synapses
Super-resolution microscopy revealed clusters of GLT-1, bassoon and Homer1 that were hardly identifiable in the epifluorescence images
The protocol is adapted to any three-colour imaging and can be used in a wide range of biological applications
Summary
Protoplasmic astrocytes reside in the grey matter where they occupy non-overlapping territories [4,5,6] These cells derive their name from their star-like appearance as they extend several primary processes from the soma, which in turn give off secondary branches. The bulk of an astrocyte is made up by the nanoscopic protrusions which mediate key astrocytic functions In addition to their involvement in neuronal signal transduction, astrocytes play essential roles in brain homeostasis [9]. Astroglia are involved in chemosensing and pH regulation [15], regulate overall energy balance and food intake [15], as well as sleep homeostasis [16] These cells regulate neurogenesis, neuronal development and guidance [17] as well as synaptogenesis, and synaptic maintenance, elimination and plasticity [18,19]. Owing to their critical role in normal brain function and health, astroglia dysfunction can lead to, and is a hallmark of, most if not all neurodegenerative disorders; in the context of pathophysiology, astrocytes are a promising target for therapeutic intervention [26,27,28]
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