Electrical coupling and innexin expression in the stomatogastric ganglion of the crab Cancer borealis.
ABSTRACT: Gap junctions are intercellular channels that allow for the movement of small molecules and ions between the cytoplasm of adjacent cells and form electrical synapses between neurons. In invertebrates, the gap junction proteins are coded for by the innexin family of genes. The stomatogastric ganglion (STG) in the crab Cancer borealis contains a small number of identified and electrically coupled neurons. We identified Innexin 1 (Inx1), Innexin 2 (Inx2), Innexin 3 (Inx3), Innexin 4 (Inx4), Innexin 5 (Inx5), and Innexin 6 (Inx6) members of the C. borealis innexin family. We also identified six members of the innexin family from the lobster Homarus americanus transcriptome. These innexins show significant sequence similarity to other arthropod innexins. Using in situ hybridization and reverse transcriptase-quantitative PCR (RT-qPCR), we determined that all the cells in the crab STG express multiple innexin genes. Electrophysiological recordings of coupling coefficients between identified pairs of pyloric dilator (PD) cells and PD-lateral posterior gastric (LPG) neurons show that the PD-PD electrical synapse is nonrectifying while the PD-LPG synapse is apparently strongly rectifying.
Project description:Gap junctions are physical channels that connect adjacent cells, permitting the flow of small molecules/ions between the cytoplasms of the coupled units. Innexin/innexin-like proteins are responsible for the formation of invertebrate gap junctions. Within the nervous system, gap junctions often function as electrical synapses, providing a means for coordinating activity among electrically coupled neurons. While some gap junctions allow the bidirectional flow of small molecules/ions between coupled cells, others permit flow in one direction only or preferentially. The complement of innexins present in a gap junction determines its specific properties. Thus, understanding innexin diversity is key for understanding the full potential of electrical coupling in a species/system. The decapod crustacean cardiac ganglion (CG), which controls cardiac muscle contractions, is a simple pattern-generating neural network with extensive electrical coupling among its circuit elements. In the lobster, Homarus americanus, prior work suggested that the adult neuronal innexin complement consists of six innexins (Homam-Inx1-4 and Homam-Inx6-7). Here, using a H. americanus CG-specific transcriptome, we explored innexin complement in this portion of the lobster nervous system. With the exception of Homam-Inx4, all of the previously described innexins appear to be expressed in the H. americanus CG. In addition, transcripts encoding seven novel putative innexins (Homam-Inx8-14) were identified, four (Homam-Inx8-11) having multiple splice variants, e.g., six for Homam-Inx8. Collectively, these data indicate that the innexin complement of the lobster nervous system in general, and the CG specifically, is likely significantly greater than previously reported, suggesting the possibility of expanded gap junction diversity and function in H. americanus.
Project description:Mosquitoes are vectors of pathogens that cause diseases of medical and veterinary importance. Female mosquitoes transmit these pathogens while taking a blood meal, which most species require to produce eggs. The period after a blood meal is a time of extreme physiological change that requires rapid coordination of specific tissues. Gap junctions (GJ) are intercellular channels that aid in the coordination of cells within tissues via the direct transfer of certain small molecules and ions between cells. Evolutionarily distinct groups of proteins form the gap junctions of vertebrate and invertebrate animals (connexins and innexins, respectively). Aedes aegypti mosquitoes possess six genes encoding innexins: inx1, inx2, inx3, inx4, inx7, and inx8. The goal of this study was to identify potential roles of innexins in the physiology of mosquitoes after a blood meal by using qPCR to quantify their mRNA expression in adult females at 3 h and 24 h post-blood meal (PBM) relative to non-blood-fed controls. We found that at 24 h PBM, expression levels of inx2, inx3, and inx4 mRNAs increased; inx2 was the most highly upregulated innexin in key tissues associated with blood-meal digestion and egg production (i.e., the midgut and ovaries, respectively). However, knocking down inx2 mRNA levels by over 75% via RNA interference had no significant effect on fecundity. Altogether, our results suggest that a blood meal influences the molecular expression of innexins in mosquitoes, but their specific physiological roles remain to be elucidated.
Project description:Approximately 10% of Caenorhabditis elegans nervous system synapses are electrical, that is, gap junctions composed of innexins. The locomotory nervous system consists of several pairs of interneurons and three major classes of motor neurons, all with stereotypical patterns of connectivity that include gap junctions. Mutations in the two innexin genes unc-7 and unc-9 result in identical uncoordinated movement phenotypes, and their respective gene products were investigated for their contribution to electrical synapse connectivity.unc-7 encodes three innexin isoforms. Two of these, UNC-7S and UNC-7SR, are functionally equivalent and play an essential role in coordinated locomotion. UNC-7S and UNC-7SR are widely expressed and co-localize extensively with green fluorescent protein-tagged innexin UNC-9 in the ventral and dorsal nerve cords. A subset of UNC-7S/SR expression visualizes gap junctions formed between the AVB forward command interneurons and their B class motor neuron partners. Experiments indicate that expression of UNC-7S/SR in AVB and expression of UNC-9 in B motor neurons is necessary for these gap junctions to form. In Xenopus oocyte pairs, both UNC-7S and UNC-9 form homomeric gap junctions, and together they form heterotypic channels. Xenopus oocyte studies and co-localization studies in C. elegans suggest that UNC-7S and UNC-9 do not heteromerize in the same hemichannel, leading to the model that hemichannels in AVB:B motor neuron gap junctions are homomeric and heterotypic.UNC-7S and UNC-9 are widely expressed and contribute to a large number of the gap junctions identified in the locomotory nervous system. Proper AVB:B gap junction formation requires UNC-7S expression in AVB interneurons and UNC-9 expression in B motor neurons. More broadly, this illustrates that innexin identity is critical for electrical synapse specificity, but differential (compartmentalized) innexin expression cannot account for all of the specificity seen in C. elegans, and other factors must influence the determination of synaptic partners.
Project description:Intercellular gap junction channels and single-membrane channels have been reported to regulate electrical synapse and the brain function. Innexin is known as a gap junction-related protein in invertebrates and is involved in the formation of intercellular gap junction channels and single-cell membrane channels. Multiple isoforms of innexin protein in each species enable the precise regulation of channel function. In molluscan species, sequence information of innexins is still limited and the sequences of multiple innexin isoforms have not been classified. This study examined the innexin transcripts expressed in the central nervous system of the terrestrial slug Limax valentianus and identified 16 transcripts of 12 innexin isoforms, including the splicing variants. We performed phylogenetic analysis and classified the isoforms with other molluscan innexin sequences. Next, the phosphorylation, N-glycosylation, and S-nitrosylation sites were predicted to characterize the innexin isoforms. Further, we identified 16 circular RNA sequences of nine innexin isoforms in the central nervous system of Limax. The identification and classification of molluscan innexin isoforms provided novel insights for understanding the regulatory mechanism of innexin in this phylum.
Project description:Gap junction channels are formed by two unrelated protein families. Non-chordates use the primordial innexins, while chordates use connexins that superseded the gap junction function of innexins. Chordates retained innexin-homologs, but N-glycosylation prevents them from forming gap junctions. It is puzzling why chordates seem to exclusively use the new gap junction protein and why no chordates should exist that use non-glycosylated innexins to form gap junctions. Here, we identified glycosylation sites of 2388 innexins from 174 non-chordate and 276 chordate species. Among all chordates, we found not a single innexin without glycosylation sites. Surprisingly, the glycosylation motif is also widespread among non-chordate innexins indicating that glycosylated innexins are not a novelty of chordates. In addition, we discovered a loss of innexin diversity during early chordate evolution. Most importantly, lancelets, which lack connexins, exclusively possess only one highly conserved innexin with one glycosylation site. A bottleneck effect might thus explain why connexins have become the only protein used to form chordate gap junctions.
Project description:Locating circuit neurons and recording from them with single-cell resolution is a prerequisite for studying neural circuits. Determining neuron location can be challenging even in small nervous systems because neurons are densely packed, found in different layers, and are often covered by ganglion and nerve sheaths that impede access for recording electrodes and neuronal markers. We revisited the voltage-sensitive dye RH795 for its ability to stain and record neurons through the ganglion sheath. Bath-application of RH795 stained neuronal membranes in cricket, earthworm and crab ganglia without removing the ganglion sheath, revealing neuron cell body locations in different ganglion layers. Using the pyloric and gastric mill central pattern generating neurons in the stomatogastric ganglion (STG) of the crab, Cancer borealis, we found that RH795 permeated the ganglion without major residue in the sheath and brightly stained somatic, axonal and dendritic membranes. Visibility improved significantly in comparison to unstained ganglia, allowing the identification of somata location and number of most STG neurons. RH795 also stained axons and varicosities in non-desheathed nerves, and it revealed the location of sensory cell bodies in peripheral nerves. Importantly, the spike activity of the sensory neuron AGR, which influences the STG motor patterns, remained unaffected by RH795, while desheathing caused significant changes in AGR activity. With respect to recording neural activity, RH795 allowed us to optically record membrane potential changes of sub-sheath neuronal membranes without impairing sensory activity. The signal-to-noise ratio was comparable with that previously observed in desheathed preparations and sufficiently high to identify neurons in single-sweep recordings and synaptic events after spike-triggered averaging. In conclusion, RH795 enabled staining and optical recording of neurons through the ganglion sheath and is therefore both a good anatomical marker for living neural tissue and a promising tool for studying neural activity of an entire network with single-cell resolution.
Project description:Electrical synapses are formed by two unrelated gap junction protein families, the primordial innexins (invertebrates) or the connexins (vertebrates). Although molecularly different, innexin- and connexin-based electrical synapses are strikingly similar in their membrane topology. However, it remains unclear if this similarity extends also to more sophisticated functions such as long-term potentiation which is only known in connexin-based synapses. Here we show that this capacity is not unique to connexin-based synapses. Using a method that allowed us to quantitatively measure gap-junction conductance we provide the first and unequivocal evidence of long-term potentiation in an innexin-based electrical synapse. Our findings suggest that long-term potentiation is a property that has likely existed already in ancestral gap junctions. They therefore could provide a highly potent system to dissect shared molecular mechanisms of electrical synapse plasticity.
Project description:Innexins in invertebrates are considered to play roles similar to those of connexins and pannexins in vertebrates. However, it remains poorly understood how innexins function in biological phenomena including their function in the nervous systems. Here, we identified inx-4, a member of the innexin family in C. elegans, by a forward screening of thermotaxis-defective mutants. The inx-4 mutants exhibited abnormal migration to a temperature slightly higher than the cultivation temperature, called mild thermophilic behavior. Rescue experiments revealed that INX-4 acts in the major thermosensory neuron AFD to regulate thermotaxis behavior. INX-4::GFP fusion protein localized exclusively along axons in AFD neurons. In addition, over-expression of INX-4 in AFD neurons induced a cryophilic behavior, which is opposite to inx-4 mutants. Our findings suggest that INX-4/Innexin in AFD may fine-tune the execution of thermotaxis behavior when moving to desired temperatures.
Project description:C. elegans body-wall muscle cells are electrically coupled through gap junctions. Previous studies suggest that UNC-9 is an important, but not the only, innexin mediating the electrical coupling. Here we analyzed junctional current (I j ) for mutants of additional innexins to identify the remaining innexin(s) important to the coupling. The results suggest that a total of six innexins contribute to the coupling, including UNC-9, INX-1, INX-10, INX-11, INX-16, and INX-18. The I j deficiency in each mutant was rescued completely by expressing the corresponding wild-type innexin specifically in muscle, suggesting that the innexins function cell-autonomously. Comparisons of I j between various single, double, and triple mutants suggest that the six innexins probably form two distinct populations of gap junctions with one population consisting of UNC-9 and INX-18 and the other consisting of the remaining four innexins. Consistent with their roles in muscle electrical coupling, five of the six innexins showed punctate localization at muscle intercellular junctions when expressed as GFP- or epitope-tagged proteins, and muscle expression was detected for four of them when assessed by expressing GFP under the control of innexin promoters. The results may serve as a solid foundation for further explorations of structural and functional properties of gap junctions in C. elegans body-wall muscle.
Project description:Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.