Long-term precision editing of neural circuits in mammals using engineered gap junction hemichannels

Abstract

The coordination of activity between brain cells is a key determinant of neural circuit function; nevertheless, approaches that selectively regulate communication between two distinct cellular components of a circuit, while leaving the activity of the presynaptic brain cell undisturbed remain sparce. To address this gap, we developed a novel class of electrical synapses by selectively engineering two connexin proteins found in Morone americana (white perch fish): connexin34.7 (Cx34.7) and connexin35 (Cx35). By iteratively exploiting protein mutagenesis, a novel in vitro assay of connexin docking, and computational modeling of connexin hemichannel interactions, we uncovered the pattern of structural motifs that broadly determine connexin hemichannel docking. We then utilized this knowledge to design Cx34.7 and Cx35 hemichannels that dock with each other, but not with themselves nor other major connexins expressed in the human central nervous system. We validated these hemichannels in vivo, demonstrating that they facilitate communication between two neurons in Caenorhabditis elegans and recode a learned behavioral preference. This system can be applied to edit circuits composed by pairs of genetically defined brain cell types across multiple species. Thus, we establish a potentially translational approach, Long-term integration of Circuits using connexins (LinCx), for context-precise circuit-editing with unprecedented spatiotemporal specificity.

Prefrontal Interneurons: Populations, Pathways, and Plasticity Supporting Typical and Disordered Cognition in Rodent Models

“…By pairing these advances with more sophisticated tools to monitor and manipulate interneuron populations, we stand to build a detailed guide to their interconnections, plasticity, and behavioral contributions. As tools emerge that enable synapse-specific manipulations (e.g., altered connectivity between defined presynaptic and postsynaptic elements) (Ransey et al, 2021;Prakash et al, 2022), we will similarly need innovation in behavioral analyses sensitive to the subtle effects these targeted manipulations may yield. Last, alongside efforts to identify and target molecules and synaptic processes unique to select cell types and circuits in the rodent brain, we must advance similar efforts in nonhuman primates with due consideration of the anatomical, functional, and behavioral homology across species.…”

On the Diverse Functions of Electrical Synapses

“…A recent innovation has created novel connexin genes through an iterative mutation approach. Mutated perch connexin34.7 and connexin35 proteins form exclusively heterotypic gap junctions ( Ransey et al, 2021a ) resulting in asymmetrical electrical synapses ( Ransey et al, 2021b ), and expression of each protein can be directed toward specific cell types. This technique could be useful for interrogating how the addition or substitution of asymmetric electrical synapses modifies a circuit and behavior.…”