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

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 vo-cal muscle movements during singing. Although the connectivity of premotor nuclei via chemical synapses has been characterized, electrical synapses and their molecular identity remain unex-plored. We show with in situ hybridizations that GJD2 mRNA, coding for the major channel-form-ing 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 data-base 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.

Regional Variation of Gap Junctional Connections in the Mammalian Inner Retina

The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8–10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Convergent NMDA receptor—Pannexin1 signaling pathways regulate the interaction of CaMKII with Connexin-36

Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation. Ectopic Panx1 expression in a Panx1 knock-down cell line is required to restore CaMKII mediated opening of Cx36. Furthermore, blocking of Src-family kinase activation of Panx1 is sufficient to prevent the opening of Cx36 channels. Our research demonstrates that the efficacy of Cx36 channels requires convergent calcium-dependent signaling processes in which activation of ionotropic N-methyl-D-aspartate receptor, Src-family kinase, and Pannexin1 open Cx36. Our results add to the best of our knowledge a new twist to mounting evidence for molecular communication between these core components of electrical and chemical synapses.

The Roles of Calmodulin and CaMKII in Cx36 Plasticity

Anatomical and electrophysiological evidence that gap junctions and electrical coupling occur between neurons was initially confined to invertebrates and nonmammals and was thought to be a primitive form of synaptic transmission. More recent studies revealed that electrical communication is common in the mammalian central nervous system (CNS), often coexisting with chemical synaptic transmission. The subsequent progress indicated that electrical synapses formed by the gap junction protein connexin-36 (Cx36) and its paralogs in nonmammals constitute vital elements in mammalian and fish synaptic circuitry. They govern the collective activity of ensembles of coupled neurons, and Cx36 gap junctions endow them with enormous adaptive plasticity, like that seen at chemical synapses. Moreover, they orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie the fundamental integrative processes, such as memory and learning. Here, we review the available mechanistic evidence and models that argue for the essential roles of calcium, calmodulin, and the Ca2+/calmodulin-dependent protein kinase II in integrating calcium signals to modulate the strength of electrical synapses through interactions with the gap junction protein Cx36.

Effects of Constitutive and Acute Connexin 36 Deficiency on Brain-Wide Susceptibility to PTZ-Induced Neuronal Hyperactivity

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: (a) does Cx36 deficiency affect seizure susceptibility, (b) does seizure-like activity affect Cx36 expression patterns, and (c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol [PTZ; a GABA(A) receptor antagonist] induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within 30 min. This Cx36 reduction persists after 1-h of recovery but recovered after 3–6 h. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.

Connexin-36-expressing Gap Junctions in VTA GABA Neurons Sustain Opiate Dependence

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance becomes negatively reinforcing. Ventral tegmental area (VTA) GABA neurons form a point of divergence between two double dissociable pathways responsible for these respective motivational states. Here we show that this switch from drug-naïve to opiate-dependent and withdrawn (ODW) motivation is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve motivational state and a loss of opiate withdrawal aversions. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36fl(CFP)/fl(CFP)) were perpetually drug-naïve and never experienced opiate withdrawal aversions. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to ODW motivation. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.SignificanceThe motivation to seek drugs can vary depending on prior exposure. For instance, recreational and habitual drug use can stem from a desire to experience the pleasurable or relieving properties of the substance, respectively. Here we identify a subpopulation of midbrain neurons that dictate opiate-seeking motivation via expression of the gap junction protein, connexin-36. We show that connexin-36 expression increases upon opiate dependence and withdrawal. We then demonstrate that this is not merely a correlation, as pharmacological or genetic manipulations that interfere with connexin-36 function prevent the development of opiate dependence in rats and mice. Our results identify gap junctions as a critical node in the pathogenesis of opiate addiction, and a potential new target for substance use disorder pharmacotherapies.

Enhanced differentiation of human pluripotent stem cells into pancreatic endocrine cells in 3D culture by inhibition of focal adhesion kinase

Background Generation of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood. Methods We established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell–cell and cell–extracellular matrix interactions which are fundamentally different between 2D and 3D cultures. Results The expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression. Conclusion We developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.