Long-term editing of brain circuits using an engineered electrical synapse | Nature

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Subjects

- Emotion

- Molecular neuroscience

Abstract

Electrical signalling across distinct populations of brain cells underpins cognitive and emotional function. However, approaches that selectively regulate electrical signalling between two cellular components of a mammalian neural circuit remain sparse. Here we engineered an electrical synapse composed of two connexin proteins1 found in Morone americana (white perch fish)—connexin 34.7 and connexin 35—to accomplish mammalian circuit modulation. By exploiting protein mutagenesis, devising a new in vitro system for assaying connexin hemichannel docking, and performing computational modelling of hemichannel interactions, we uncovered a structural motif that contributes to electrical synapse formation. Targeting this motif, we designed connexin 34.7 and connexin 35 hemichannels that dock with each other to form an electrical synapse but not with other major connexins expressed in the mammalian central nervous system. We validated this electrical synapse in vivo using worms (Caenorhabditis elegans) and mice (Mus musculus). We demonstrate that it can strengthen communication across neural circuits composed of pairs of distinct cell types and modify behaviour accordingly. Thus, we establish ‘long-term integration of circuits using connexins’ (LinCx) for precision circuit editing in mammals.

Main

Electrical synapses enable the direct flow of ions and small molecules between two cells and play a prominent part in coupling electrical activity in multiple organs, including the brain2,3,4. Electrical synapses comprise multiple gap junction channels, each composed of two docked hemichannels embedded in the membranes of two touching cells. Each hemichannel is an oligomer that consists of six monomeric proteins called connexins, of which there are 21 isoforms in humans5,6. Most connexins can form single-isoform hemichannels that dock with themselves to create homotypic gap junctions (Fig. 1a, left).

Fig. 1: Screen to identify a mutant connexin hemichannel pair with exclusively heterotypic docking.The alternative text for this image may have been generated using AI.Full size image

a, Left, schematic outlining the limitation of introducing heterologous WT connexin hemichannels (pink rectangles) to modulate specific neural circuits composed of neurons (brown and yellow). Note that connexin hemichannels produce off-target electrical synapses between presynaptic neurons (left). Right, strategy for using exclusively heterotypic docking hemichannels (green and red rectangles) to selectively modulate specific neural circuits. b, Depiction of red (iRFP670) and green (mEmerald) fluorescence-exchange profiles (left) and representative flow cytometry plots (right) for hemichannel pairs with (Cx36–Cx36; top) and without (Cx36–Cx45; bottom) docking compatibility. The pink dashed squares in the flow cytometry plots highlight the proportion of cells that express two distinct fluorescent proteins. c, Left, proportion of dual fluorescence-labelled cells for connexin pairs with known docking compatibility profiles. Right, FETCH scores for Cx43(F199L)–Cx43(F199L) and Cx26(K168V N176H)–Cx43 (ref. 26). Blue lines on the right-hand graph are the mean ± s.e.m. score for the known-negative distribution of connexin pairs with docking incompatibility. d, Schematic of M. americana Cx34.7 and Cx35 mutations in EL1 and EL2 used to screen for heterotypic-exclusive hemichannels. Positions and mutations specific to Cx34.7 or Cx35 or common to both proteins, are shown in green, red and black, respectively. e,f, Plots showing homotypic FETCH results for Cx34.7 (e) and Cx35 (f) mutations. The locations of these mutations can be mapped back to the structure for EL2 in d by the mutation number and colour. Circular bar graphs show the Cohen’s D effect size of FETCH scores for homotypic mutant combinations compared with the heterotypic pairing of human Cx36 and Cx45, which fails to dock. The black horizontal line in the centre is the scale bar for effect sizes. Targeted residues are listed around the circle rim; substituted amino acids are listed in the interior. The intermittent black circle segregates each targeted residue, and the light purple circle corresponds to a Cohen’s D of zero. Mutations that disrupted docking are also highlighted by black arrows and letters. g, Heterotypic FETCH results for Cx34.7 and Cx35 mutant protein combinations. Bar graphs show the effect size of heterotypic mutant combinations relative to the WT Cx34.7 and Cx35 pair. The purple circle provides the reference point for an effect size of zero. The green intermittent circle corresponds to the Cx34.7 mutations identified by green labels in the outermost level around the rim of the plot. The black horizontal line in the centre is the scale bar for effect size. For n values and statistical tests, see main text. For definitions of box plots, see Methods.

Neural circuit editing using gap junctions is well esta