Extensive GJD2 Expression in the Song Motor Pathway Reveals the Extent of Electrical Synapses in the Songbird Brain

SIMPLE SUMMARY: Electrical synapses are ubiquitous in nervous systems, where they coordinate the activity of neural networks in time. However, their role in the execution and learning of complex behaviors remains unknown. Electrical synapses have remained unexplored in the songbird brain, which provides a model to link the production and learning of a complex behavior to the synaptic structure of defined neural circuits. Here, we show that GJD2 mRNA, coding for the major channel-forming electrical synapse protein connexin 36 (Cx36), is extensively expressed in the two nuclei that control song production, HVC and RA and their embedding regions. Our in situ hybridizations, together with the analysis of published transcriptomics data, demonstrate that electrical synapses are a general and widespread feature of song premotor nuclei in songbirds, where they show brain region-specific, cell type-specific expression patterns, dynamic during neuronal differentiation. We propose songbirds as a suitable model to investigate the contribution of the major vertebrate electrical synapse protein, Cx36, to the production and learning of motor skills in vertebrates. ABSTRACT: Birdsong is a precisely timed animal behavior. The connectivity of song premotor neural networks has been proposed to underlie the temporal patterns of neuronal activity that control vocal muscle movements during singing. Although the connectivity of premotor nuclei via chemical synapses has been characterized, electrical synapses and their molecular identity remain unexplored. We show with in situ hybridizations that GJD2 mRNA, coding for the major channel-forming electrical synapse protein in mammals, connexin 36, is expressed in the two nuclei that control song production, HVC and RA from canaries and zebra finches. In canaries’ HVC, GJD2 mRNA is extensively expressed in GABAergic and only a fraction of glutamatergic cells. By contrast, in RA, GJD2 mRNA expression is widespread in glutamatergic and GABAergic neurons. Remarkably, GJD2 expression is similar in song nuclei and their respective embedding brain regions, revealing the widespread expression of GJD2 in the avian brain. Inspection of a single-cell sequencing database from zebra and Bengalese finches generalizes the distributions of electrical synapses across cell types and song nuclei that we found in HVC and RA from canaries, reveals a differential GJD2 mRNA expression in HVC glutamatergic subtypes and its transient increase along the neurogenic lineage. We propose that songbirds are a suitable model to investigate the contribution of electrical synapses to motor skill learning and production.