70µm Mouse Brain Sections for Immunohistochemistry cut with 5100mz Vibrotome

Abstract

(from PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems, Xie et al, 2021)

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Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear—suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

Method

Brain sections and immunohistochemistry

For fixation, pregnant/adult mice were anesthetized, pups removed and transcardially perfused with cold phosphate buffered saline (PBS, pH 7.4) and 4% paraformaldehyde (PFA) in PBS, and brains were post-fixed with 4% PFA at 4°C overnight. After post-fixation, mouse brains were embedded using 2% low melting agarose (BBI life science) in PBS and were sectioned to 70 μm thickness with a vibratome (5100mz, Campden Instruments).