Gap Junction Subunits Research Articles

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

The response of retinal ganglion cells to optical defocused visual stimuli in mouse retinas

Myopia and astigmatism are two primary types of refractive errors characterized by inaccurate focusing images on the retina. This study aimed to investigate the response characteristics of Retinal Ganglion Cells (RGCs), represented by alpha (α) RGCs, when exposed to focused, simulated spherically defocused images and astigmatically defocused images projected onto mouse retinas.Negative pressure was applied to stretch the soma of RGC in vitro to simulate myopia using a 7–8 μm diameter glass microelectrode, resulting in a 5% increase in the cell's diameter. A custom-made device was utilized to project spherically (equal to ±10 and ± 20 D) and astigmatically (+6.00 D) defocused images onto the retinas. As a control for a deficient intact retinal circuit, αRGCs of connexin 36 knockout (Cx36 KO) mice were used.The response of αRGCs varied significantly in terms of spikes, excitatory postsynaptic currents (EPSCs) and capacitances under stretching conditions to mimic myopia. Significant differences in the amplitudes of EPSCs were observed in the majority of αRGCs when exposed to focused and spherically defocused images in normal and mechanically simulated myopic retinas. However, this difference was not observed in αRGCs of Cx36 KO mice. αRGCs demonstrated significant differences in response between focused and astigmatically defocused images. Once again, αRGCs of Cx36 KO mice did not display differences.αRGCs have the ability to detect focused, spherically, and astigmatically defocused images and exhibit differential responses ex vivo. Gap junction subunit Cx36 may play a crucial role in transmitting visual signals associated with developing and perceiving refractive errors.

Retinal ganglion cells encode differently in the myopic mouse retina?

The etiology of myopia remains unclear. This study investigated whether retinal ganglion cells (RGCs) in the myopic retina encode visual information differently from the normal retina and to determine the role of Connexin (Cx) 36 in this process. Generalized linear models (GLMs), which can capture stimulus-dependent changes in real neurons with spike timing precision and reliability, were used to predict RGCs responses to focused and defocused images in the retinas of wild-type (normal) and Lens-Induced Myopia (LIM) mice. As the predominant subunit of gap junctions in the mouse retina and a plausible modulator in myopia development, Cx36 knockout (KO) mice were used as a control for an intact retinal circuit. The kinetics of excitatory postsynaptic currents (EPSCs) of a single αRGC could reflect projection of both focused and defocused images in the retinas of normal and LIM, but not in the Cx36 knockout mice. Poisson GLMs revealed that RGC encoding of visual stimuli in the LIM retina was similar to that of the normal retina. In the LIM retinas, the linear-Gaussian GLM model with offset was a better fit for predicting the spike count under a focused image than the defocused image. Akaike information criterion (AIC) indicated that nonparametric GLM (np-GLM) model predicted focused/defocused images better in both LIM and normal retinas. However, the spike counts in 33% of αRGCs in LIM retinas were better fitted by exponential GLM (exp-GLM) under defocus, compared to only 13% αRGCs in normal retinas. The differences in encoding performance between LIM and normal retinas indicated the possible amendment and plasticity of the retinal circuit in myopic retinas. The absence of a similar response between Cx36 KO mice and normal/LIM mice might suggest that Cx36, which is associated with myopia development, plays a role in encoding focused and defocused images.

Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma.

Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma. We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas. Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas. Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool.

Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions-lacking somatic gonadal cell coverage of germ cells-are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base.

The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein α1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

Connexin 32 induces pro-tumorigenic features in MCF10A normal breast cells and MDA-MB-231 metastatic breast cancer cells

Connexins (Cx), the basic subunit of gap junctions, play important roles in cell homeostasis, and their abnormal expression and function are associated with human hereditary diseases and cancers. In tumorigenesis, connexins were observed to have both anti-tumorigenic and pro-tumorigenic roles in a context- and stage-dependent manner. Initially, Cx26 and Cx43 were thought to be the only connexins involved in normal breast homeostasis and breast cancer. Later on, association of Cx32 expression with lymph node metastasis of breast cancer and subsequent demonstration of its expression in normal breast tissue suggested that Cx32 contributes to breast tissue homeostasis. Here, we aimed to determine the effects of Cx32 on normal breast cells, MCF10A, and on breast cancer cells, MDA-MB-231. Cx32 overexpression had profound effects on MCF10A cells, decreasing cell proliferation by increasing the doubling time of MCF10A. Furthermore, MCF10A cells acquired mesenchymal-like appearance upon Cx32 expression and had increased migration capacity and expression of both E-cadherin and vimentin. In contrast, Cx32 overexpression altered the EMT markers of MDA-MB-231 by increasing the expression of mesenchymal markers, such as slug and vimentin, and decreasing E-cadherin expression without affecting their proliferation and morphology. Our results indicate, for the first time in the literature, that Cx32 has tumor-promoting roles in MCF10A and MDA-MB-231 cells.

Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange

BackgroundChanges in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (Vmem) and intracellular pH (pHi). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation.ResultsWe expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K+channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium.ConclusionsThe genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.

Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Betacellulin enhances ovarian cancer cell migration by up-regulating Connexin43 via MEK-ERK signaling

Epithelial ovarian cancer is the fifth common cause of cancer death in women and the most lethal gynecological malignancies. Our previous studies have shown that up-regulation of Connexin43, a gap-junction subunit crucial for cell-cell communication, enhances ovarian cancer cell migration. Betacellulin is a member of the epidermal growth factor (EGF) family which can bind to multiple EGF family receptors. Overexpression of betacellulin is found in a variety of cancers and is associated with reduced survival. However, the specific roles and molecular mechanisms of betacellulin in ovarian cancer progression are poorly understood. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by up-regulating Connexin43. Our results showed that treatment with betacellulin significantly increased Connexin43 expression and cell migration in both OVCAR4 and SKOV3 ovarian cancer cell lines. Moreover, betacellulin induced the activation of MEK-ERK signaling, and its effects on Connexin43 were inhibited by pre-treatment with U0126. Pre-treatment with AG1478 totally blocked the activation of MEK-ERK signaling but only partially inhibited betacellulin-induced Connexin43 expression and cell migration. Most importantly, betacellulin-induced cell migration was attenuated by knockdown of Connexin43, and co-treatment with gap junction inhibitor carbenoxolone did not alter this effect. Our results suggest a bilateral role of Connexin43 in ovarian cancer migration, and also demonstrate a gap junction-independent mechanism of betacellulin.

Connexin 43-Mediated Astroglial Metabolic Networks Contribute to the Regulation of the Sleep-Wake Cycle

Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by invivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions.

Prostaglandin E2 and Connexin 43 crosstalk in the osteogenesis induced by extracorporeal shockwave

As a type of mechanical stimulation, extracorporeal shockwave (ESW) has been widely used in the clinic to treat bone fracture delayed union and non-unions. A large number of studies have shown beneficial effects of ESW in promoting fracture healing by inducing bone regeneration; however, the underlying mechanisms remain unclear. ESW has been shown to induce the production of prostaglandin E2 (PGE2), which is essential for gap junction intercellular communication in response to mechanical stress. Among the 19 known gap junction subunits, connexin43 (Cx43) is the most prevalent for mediating the response of mechanical stress. However, to our knowledge, the effect of ESW on Cx43 expression has not been reported before. Herein, we propose that a crosstalk between PGE2 and Cx43 is involved in the enhancement of osteogenesis induced by ESW. We review the currently available data to propose an unrevealed, but important mechanism via which ESW treatment affects osteogenic differentiation of bone marrow stromal cells.

Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina.

Retinal degeneration (RD) encompasses a family of diseases that lead to photoreceptor death and visual impairment. Visual decline due to photoreceptor cell loss is further compromised by emerging spontaneous hyperactivity in inner retinal cells. This aberrant activity acts as a barrier to signals from the remaining photoreceptors, hindering therapeutic strategies to restore light sensitivity in RD. Gap junctions, particularly those expressed in AII amacrine cells, have been shown to be integral to the generation of aberrant activity. It is unclear whether gap junction expression and coupling are altered in RD. To test this, we evaluated the expression and phosphorylation state of connexin36 (Cx36), the gap junction subunit predominantly expressed in AII amacrine cells, in two mouse models of RD, rd10 (slow degeneration) and rd1 (fast degeneration). Using Ser293-P antibody, which recognizes a phosphorylated form of connexin36, we found that phosphorylation of connexin36 in both slow and fast RD models was significantly greater than in wildtype controls. This elevated phosphorylation may underlie the increased gap junction coupling of AII amacrine cells exhibited by RD retina.

Connexin 43 expression is associated with increased malignancy in prostate cancer cell lines and functions to promote migration

Impaired expression of connexins, the gap junction subunits that facilitate direct cell-cell communication, have been implicated in prostate cancer growth. To elucidate the crucial role of connexins in prostate cancer progression, we performed a systematic quantitative RT-PCR screening of connexin expression in four representative prostate cancer cell lines across the spectrum of malignancy. Transcripts of several connexin subunits were detected in all four cell lines, and connexin 43 (Cx43) showed marked elevation at both RNA and protein levels in cells with increased metastatic potential. Analysis of gap-junction-mediated intercellular communication revealed homocellular coupling in PC-3 cells, which had the highest C x 43 expression, with minimal coupling in LNCaP cells where C x 43 expression was very low. Treatment with the gap junction inhibitor carbenoxolone or connexin mimetic peptide ACT-1 did not impair cell growth, suggesting that growth is independent of functional gap junctions. PC-3 cells with C x 43 expression reduced by shRNA showed decreased migration in monolayer wound healing assay, as well as decreased transwell invasion capacities when compared to control cells expressing non-targeting shRNA. These results, together with the correlation between C x 43 expression levels and the metastatic capacity of the cell lines, suggest a role of C x 43 in prostate cancer invasion and metastasis.

A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

Pluripotent stem cells (PSCs) have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass) differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this review, we examine novel methods of cell engineering and the role of reprogramming transcription factors in PSC development. In addition, we explore the effect of external environmental signals on PSC cultivation and differentiation by elucidating key components of the primordial stem cell microenvironment, the blastocyst. Furthermore, we assess the effects of hypoxic conditions on DNA editing, gene expression, and protein function in PSC self-renewal and growth. Finally, we speculate on the principal use of gap junction subunit expression as relevant biomarkers of PSC fate. Improving bioreactor design and pertinent cell biomarker classification could vastly enhance manufactured stem cell yield and quality, thereby increasing the potency and safety of therapeutic cells to be used in regenerative medicine.

Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.

Interactions between all three pigment cell types are required to form the stripe pattern of adult zebrafish (Danio rerio), but their molecular nature is poorly understood. Mutations in leopard (leo), encoding Connexin 41.8 (Cx41.8), a gap junction subunit, cause a phenotypic series of spotted patterns. A new dominant allele, leo(tK3), leads to a complete loss of the pattern, suggesting a dominant negative impact on another component of gap junctions. In a genetic screen, we identified this component as Cx39.4 (luchs). Loss-of-function alleles demonstrate that luchs is required for stripe formation in zebrafish; however, the fins are almost not affected. Double mutants and chimeras, which show that leo and luchs are only required in xanthophores and melanophores, but not in iridophores, suggest that both connexins form heteromeric gap junctions. The phenotypes indicate that these promote homotypic interactions between melanophores and xanthophores, respectively, and those cells instruct the patterning of the iridophores.

Astroglial connexin 43 sustains glutamatergic synaptic efficacy.

Astrocytes dynamic interactions with neurons play an active role in neurotransmission. The gap junction (GJ) subunits connexins 43 and 30 are strongly expressed in astrocytes and have recently been shown to regulate synaptic activity and plasticity. However, the specific role of connexin 43 in the morphological and electrophysiological properties of astrocytes in situ as well as in synaptic transmission remains unknown. Here, we show that connexin 43, a major determinant of astroglial GJ coupling, regulates astrocyte cell volume, but has no impact on astroglial passive membrane properties. Furthermore, we demonstrate that connexin 43 modulates glutamatergic synaptic activity of hippocampal CA1 pyramidal cells. This regulation involves changes in synaptically released glutamate, with no alteration in neuronal excitability or postsynaptic function. These results reveal connexin 43 as a critical player in neuroglial interactions by supporting synaptic efficacy.

Calcium signaling and neuropeptide secretion in the expulsion step of the Caenorhabditis elegans defecation motor program (1129.6)

A periodic posterior to anterior intestinal calcium wave initiates the defecation motor program of Caenorhabditis elegans (C. elegans). innexin‐16 (inx‐16) encodes an intestinal pannexin gap junction subunit. inx‐16 mutants have faulty calcium waves and mostly lack the expulsion step of the motor program. Our work investigates how these defective calcium waves result in loss of expulsion. The anterior‐ventral neuron L (AVL) and dorsal‐ventral neuron B (DVB) are responsible for expulsion through a two‐step signaling process. An intestinal neuropeptide‐like protein, NLP‐40, activates AVL and DVB which in turn release neurotransmitters to stimulate expulsion. NLP‐40 release likely depends on intestinal calcium flux, as vesicular localization of NLP‐40 requires synaptotagmin‐2.We propose that the expulsion defects in the inx‐16 mutant are due to improper NLP‐40 release. To establish if inx‐16’s AVL and DVB neurons are functional, we used optogenetic methods. Activation of a light‐gated ion channel channelrhodopsin‐2 (ChR2) in AVL and DVB of inx‐16 animals resulted in normal expulsion 90% of the time. This demonstrates that an inx‐16 animal’s AVL and DVB neurons are able to signal expulsion and supports our hypothesis that defects in inx‐16’s intestinal NLP‐40 release inhibit expulsion. Current work addresses whether NLP‐40 is properly synthesized and released.

Connexin 30 sets synaptic strength by controlling astroglial synapse invasion

Astrocytes play active roles in brain physiology by dynamic interactions with neurons. Connexin 30, one of the two main astroglial gap-junction subunits, is thought to be involved in behavioral and basic cognitive processes. However, the underlying cellular and molecular mechanisms are unknown. We show here in mice that connexin 30 controls hippocampal excitatory synaptic transmission through modulation of astroglial glutamate transport, which directly alters synaptic glutamate levels. Unexpectedly, we found that connexin 30 regulated cell adhesion and migration and that connexin 30 modulation of glutamate transport, occurring independently of its channel function, was mediated by morphological changes controlling insertion of astroglial processes into synaptic clefts. By setting excitatory synaptic strength, connexin 30 plays an important role in long-term synaptic plasticity and in hippocampus-based contextual memory. Taken together, these results establish connexin 30 as a critical regulator of synaptic strength by controlling the synaptic location of astroglial processes.