Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100 µm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

Accelerated Growth, Differentiation, and Ploidy with Reduced Proliferation of Right Ventricular Cardiomyocytes in Children with Congenital Heart Defect Tetralogy of Fallot.

The myocardium of children with tetralogy of Fallot (TF) undergoes hemodynamic overload and hypoxemia immediately after birth. Comparative analysis of changes in the ploidy and morphology of the right ventricular cardiomyocytes in children with TF in the first years of life demonstrated their significant increase compared with the control group. In children with TF, there was a predominantly diffuse distribution of Connexin43-containing gap junctions over the cardiomyocytes sarcolemma, which redistributed into the intercalated discs as cardiomyocytes differentiation increased. The number of Ki67-positive cardiomyocytes varied greatly and amounted to 7.0-1025.5/106 cardiomyocytes and also were decreased with increased myocytes differentiation. Ultrastructural signs of immaturity and proliferative activity of cardiomyocytes in children with TF were demonstrated. The proportion of interstitial tissue did not differ significantly from the control group. The myocardium of children with TF under six months of age was most sensitive to hypoxemia, it was manifested by a delay in the intercalated discs and myofibril assembly and the appearance of ultrastructural signs of dystrophic changes in the cardiomyocytes. Thus, the acceleration of ontogenetic growth and differentiation of the cardiomyocytes, but not the reactivation of their proliferation, was an adaptation of the immature myocardium of children with TF to hemodynamic overload and hypoxemia.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

Presynaptic coupling by electrical synapses coordinates a rhythmic behavior by synchronizing the activities of a neuron pair.

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

Interphotoreceptor coupling: an evolutionary perspective.

In the vertebrate retina, signals generated by cones of different spectral preference and by highly sensitive rod photoreceptors interact at various levels to extract salient visual information. The first opportunity for such interaction is offered by electrical coupling of the photoreceptors themselves, which is mediated by gap junctions located at the contact points of specialised cellular processes: synaptic terminals, telodendria and radial fins. Here, we examine the evolutionary pressures for and against interphotoreceptor coupling, which are likely to have shaped how coupling is deployed in different species. The impact of coupling on signal to noise ratio, spatial acuity, contrast sensitivity, absolute and increment threshold, retinal signal flow and colour discrimination is discussed while emphasising available data from a variety of vertebrate models spanning from lampreys to primates. We highlight the many gaps in our knowledge, persisting discrepancies in the literature, as well as some major unanswered questions on the actual extent and physiological role of cone-cone, rod-cone and rod-rod communication. Lastly, we point toward limited but intriguing evidence suggestive of the ancestral form of coupling among ciliary photoreceptors.

Connexin43 is differentially distributed within renal vasculature and mediates profibrotic differentiation in medullary fibroblasts.

Connexins (Cxs) form gap junctions for intercellular exchange of inorganic ions and messenger molecules. In the kidney, Cxs play essential roles within its compartments, but data on the precise cellular localization and cell type-related function of their isoforms are scarce. We tested whether Cx43 distribution is restricted to vascular and interstitial cells and whether medullary fibroblasts express Cx43 to coordinate profibrotic signaling. Confocal immunofluorescence techniques, ultrastructural labeling, and functional experiments in cell culture were performed. Cx43 was chiefly expressed in the vasculature but was absent from tubular epithelia. All arterial, arteriolar, and lymphatic endothelia showed continuous Cx43 signal along their borders. In the inner medulla, only the interstitium showed Cx43 signals, which were assigned to fibroblasts and their processes. Cultured Cx43-expressing medullary fibroblasts served to study the role of gap junctions in a profibrotic context. In a dye spreading assay, Cx43-sensitive diffusion of Lucifer yellow was dependent on gap junctional passage. The addition of transforming growth factor-ß1 (5 ng/mL for 48 h) activated Cx43 biosynthesis and caused Cx43-sensitive transformation of the fibroblasts into a myofibroblast phenotype. This suggested that Cx43 gap junctional channels enable the coordination of profibrotic signaling between cells of the medullary interstitium. In summary, we demonstrate the presence of Cx43-expressing gap junctions within the two major renal compartments, the vasculature and interstitium. Endothelial Cx43 likely provides functions of an earlier-defined "electrical syncytium" within the vascular wall. Additionally, Cx43 facilitates profibrotic signaling between medullary interstitial fibroblasts.

A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad.

In Caenorhabditis elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. A strong loss-of-function mutation (T239I) affects the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. We conducted a genetic screen for suppressor mutations that restore germ cell proliferation in the T239I mutant background and isolated seven intragenic mutations, located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky channels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three of the suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. The mutations described here may be useful for elucidating the biochemical pathways that produce the active biomolecules transiting through soma-germline gap junctions.

Gap junctions: historical discoveries and new findings in the Caenorhabditiselegans nervous system.

Gap junctions are evolutionarily conserved structures at close membrane contacts between two cells. In the nervous system, they mediate rapid, often bi-directional, transmission of signals through channels called innexins in invertebrates and connexins in vertebrates. Connectomic studies from Caenorhabditis elegans have uncovered a vast number of gap junctions present in the nervous system and non-neuronal tissues. The genome also has 25 innexin genes that are expressed in spatial and temporal dynamic pattern. Recent findings have begun to reveal novel roles of innexins in the regulation of multiple processes during formation and function of neural circuits both in normal conditions and under stress. Here, we highlight the diverse roles of gap junctions and innexins in the C. elegans nervous system. These findings contribute to fundamental understanding of gap junctions in all animals.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.

Recombinant Human Soluble Thrombomodulin Suppresses Monocyte Adhesion by Reducing Lipopolysaccharide-Induced Endothelial Cellular Stiffening.

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.

The protective effect of ginsenoside Rg1 on depression may benefit from the gap junction function in hippocampal astrocytes.

Studies have shown that the ginsenoside Rg1 can improve depressive symptoms in vitro and in vivo. However, the efficacy of Rg1on the hippocampal astrocyte gap junctions in depression are unclear. We mainly aimed to explore the relationship between Rg1, hippocampal astrocyte gap junctions and depression. Using primary cultured astrocytes, corticosterone (CORT) was used to induce stress. CORT (100 µM) significantly reduced the survival rate in astrocytes, and this effect was prevented by additional Rg1 administration. Interestingly, the gap junction blocker carbenoxolone (CBX) was able to revert this Rg1 effect. In in vivo models, one group was exposed to chronic unpredictable stress (CUS) for 47 days, while another group was bilaterally injected with CBX (100 µM) into the hippocampal CA1 region. Rats treated with Rg1 (20 mg/kg) showed an improvement in the sucrose preference and the forced swimming test in both models, indicating an antidepressive activity of Rg1. The levels of astrocyte gap junction connexin 43 (Cx43) were detected by immunofluorescence (IF) and western blotting. The levels of glial fibrillary acidic protein (GFAP) were detected by IF. The gap junctions in the hippocampal CA1 area were evaluated using dye transfer and electron microscopy. The reduction in Cx43 expression, the decrease in the Cx43 to GFAP ratio, the shorter dye diffusion distance, and the abnormal ultrastructure of gap junctions in rats exposed to CUS were markedly alleviated by concomitant Rg1 treatment. Taken together, the ginsenoside Rg1 could improve depression-like behavior in rats induced by astrocyte gap junction dysfunction in the hippocampus.

Spatio-temporal patterning of different connexins in developing and postnatal human kidneys and in nephrotic syndrome of the Finnish type (CNF).

Connexins (Cxs) are membrane-spanning proteins which enable flow of information important for kidney homeostasis. Changes in their spatiotemporal patterning characterize blood vessel abnormalities and chronic kidney diseases (CKD). We analysed spatiotemporal expression of Cx37, Cx40, Cx43 and Cx45 in nephron and glomerular cells of developing, postnatal kidneys, and nephrotic syndrome of the Finnish type (CNF) by using immunohistochemistry, statistical methods and electron microscopy. During kidney development, strong Cx45 expression in proximal tubules and decreasing expression in glomeruli was observed. In developing distal nephron, Cx37 and Cx40 showed moderate-to-strong expression, while weak Cx43 expression gradually increased. Cx45/Cx40 co-localized in mesangial and granular cells. Cx43 /Cx45 co-localized in podocytes, mesangial and parietal epithelial cells, and with podocyte markers (synaptopodin, nephrin). Different Cxs co-expressed with endothelial (CD31) and VSMC (α -SMA) markers in vascular walls. Peak signalling of Cx37, Cx43 and Cx40 accompanied kidney nephrogenesis, while strongest Cx45 signalling paralleled nephron maturation. Spatiotemporal Cxs patterning indicate participation of Cx45 in differentiation of proximal tubules, and Cx43, Cx37 and Cx40 in distal tubules differentiation. CNF characterized disorganized Cx45 expression in proximal tubules, increased Cx43 expression in distal tubules and overall elevation of Cx40 and Cx37, while Cx40 co-localized with increased number of interstitial myofibroblasts.

Network Architecture of Gap Junctional Coupling among Parallel Processing Channels in the Mammalian Retina.

Gap junctions are ubiquitous throughout the nervous system, mediating critical signal transmission and integration, as well as emergent network properties. In mammalian retina, gap junctions within the Aii amacrine cell-ON cone bipolar cell (CBC) network are essential for night vision, modulation of day vision, and contribute to visual impairment in retinal degenerations, yet neither the extended network topology nor its conservation is well established. Here, we map the network contribution of gap junctions using a high-resolution connectomics dataset of an adult female rabbit retina. Gap junctions are prominent synaptic components of ON CBC classes, constituting 5%-25% of all axonal synaptic contacts. Many of these mediate canonical transfer of rod signals from Aii cells to ON CBCs for night vision, and we find that the uneven distribution of Aii signals to ON CBCs is conserved in rabbit, including one class entirely lacking direct Aii coupling. However, the majority of gap junctions formed by ON CBCs unexpectedly occur between ON CBCs, rather than with Aii cells. Such coupling is extensive, creating an interconnected network with numerous lateral paths both within, and particularly across, these parallel processing streams. Coupling patterns are precise with ON CBCs accepting and rejecting unique combinations of partnerships according to robust rulesets. Coupling specificity extends to both size and spatial topologies, thereby rivaling the synaptic specificity of chemical synapses. These ON CBC coupling motifs dramatically extend the coupled Aii-ON CBC network, with implications for signal flow in both scotopic and photopic retinal networks during visual processing and disease.SIGNIFICANCE STATEMENT Electrical synapses mediated by gap junctions are fundamental components of neural networks. In retina, coupling within the Aii-ON CBC network shapes visual processing in both the scotopic and photopic networks. In retinal degenerations, these same gap junctions mediate oscillatory activity that contributes to visual impairment. Here, we use high-resolution connectomics strategies to identify gap junctions and cellular partnerships. We describe novel, pervasive motifs both within and across classes of ON CBCs that dramatically extend the Aii-ON CBC network. These motifs are highly specific with implications for both signal processing within the retina and therapeutic interventions for blinding conditions. These findings highlight the underappreciated contribution of coupling motifs in retinal circuitry and the necessity of their detection in connectomics studies.

Connexin/Innexin Channels in Cytoplasmic Organelles. Are There Intracellular Gap Junctions? A Hypothesis!

This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, similar in size to junctional innexons that often appear to directly interact with junctional innexons; similar particles were seen also in the outer membrane of crayfish mitochondria. Indeed, vertebrate connexins assembled into hexameric connexons are present not only in the membranes of the Golgi apparatus but also in those of the mitochondria and endoplasmic reticulum. It seems possible, therefore, that cytoplasmic organelles may be able to exchange small molecules with each other as well as with organelles of coupled cells via gap junctions.

Ultrastructural evidence for presenсe of gap junctions in rare case of pleomorphic xanthoastrocytoma.

The phenomenon of unstable expression of gap junction's proteins connexins remains a "visiting card" of astrocytic tumors with various degrees of malignancy. At the same time, it stays unclear what is detected by the positive expression of connexins in astrocytic tumors: gap junctions, hemi-channels, or connexin proteins in cytosol. In the present work, for the first time, we demonstrate an ultrastructural evidence of gap junctions in pleomorphic xanthoastrocytoma, a rare primary brain tumor, the intercellular characteristics of which are poorly studied and remain very discursive and controversial. The primary tumor mass was resected during craniotomy from a 57-old patient diagnosed with pleomorphic xanthoastrocytoma Grade II based on the histopathological analysis. The immunohistochemical study was conducted with primary antibodies: Neurofilament, Myelin basic protein, Glial fibrillary acidic protein, and Synaptophysin. For electron microscopic examination fragments of tumor tissue were fixed in a glutaraldehyde, postfixed in a 1% OsO4, dehydrated and embedded into resin. After the detailed clinical, histological, and immunohistochemical study we revealed some ultrastructural characteristics of the tumor, as well as the first evidence of direct intercellular connection between the tumor cells via gap junctions. Regularly arranged gap junctions connected the somas of xanthastrocytes with dark cytoplasm containing lipid drops. Besides the localization between the cell bodies, from one to several gap junctions were found between the branches of xanthoastrocytoma in tumor intercellular space in close proximity to tumor cell. Our results may indicate gap junctions as a possible structure for intercellular communication between pleomorphic xanthoastrocytoma cells.

Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function.

PURPOSE: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. METHODS: Twenty-five Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 µm meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. RESULTS: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with ß3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001). CONCLUSIONS: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.

Gap junctions and expression of Cx36, Cx43 and Cx45 in the posterodorsal medial amygdala of adult rats.

The posterodorsal medial amygdala (MePD) has an adapted synaptic organization that dynamically modulates reproduction and other social behaviors in rats. Discrete gap junctions between glial cells were previously reported in the MePD neuropil. Connexins (Cx) are components of gap junctions and indicative of cellular electrical coupling. Here, we report the ultrastructural occurrence of gap junctions between neurons in the MePD and demonstrate the expression and immunofluorescent labeling of Cx36, Cx43 and Cx45 in this subcortical area of adult male rats. Few neuronal gap junctions were found in the MePD and, when identified, occurred between dendrites. On the other hand, there is a diffuse presence and distribution of punctate labelling for the tested Cxs. Puncta were visualized isolated or forming clusters in the same focal plane of cell bodies or along the MePD neuropil. The Cx36 puncta were found in neurons, Cx43 in astrocytes and Cx45 in both neurons and astrocytes. Our data indicate the presence of few gap junctions and different Cxs composition in the MePD. Because Cxs can assemble, form hemichannel units and/or serve as transcriptional regulator, it is likely that additional modulation of intercellular communication can occur besides the chemical transmission in the MePD of adult rats.

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

OBJECTIVE: 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. CONCLUSIONS: 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.

Unravelling the ultrastructural details of αT-catenin-deficient cell-cell contacts between heart muscle cells by the use of FIB-SEM.

The intercalated disc is an important structure in cardiomyocytes, as it is essential to maintain correct contraction and proper functioning of the heart. Adhesion and communication between cardiomyocytes are mediated by three main types of intercellular junctions, all residing in the intercalated disc: gap junctions, desmosomes and the areae compositae. Mutations in genes that encode junctional proteins, including αT-catenin (encoded by CTNNA3), have been linked to arrhythmogenic cardiomyopathy and sudden cardiac death. In mice, the loss of αT-catenin in cardiomyocytes leads to impaired heart function, fibrosis, changed expression of desmosomal proteins and increased risk for arrhythmias following ischemia-reperfusion. Currently, it is unclear how the intercalated disc and the intercellular junctions are organised in 3D in the hearts of this αT-catenin knockout (KO) mouse model. In order to scrutinise this, ventricular cardiac tissue of αT-catenin KO mice was used for volume electron microscopy (VEM), making use of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), allowing a careful 3D reconstruction of the intercalated disc, including gap junctions and desmosomes. Although αT-catenin KO and control mice display a comparable organisation of the sarcomere and the different intercalated disc regions, the folds of the plicae region of the intercalated disc are longer and more narrow in the KO heart, and the pale region between the sarcomere and the intercalated disc is larger. In addition, αT-catenin KO intercalated discs appear to have smaller gap junctions and desmosomes in the plicae region, while gap junctions are larger in the interplicae region of the intercalated disc. Although the reason for this remodelling of the ultrastructure after αT-catenin deletion remains unclear, the excellent resolution of the FIB-SEM technology allows us to reconstruct details that were not reported before. LAY DESCRIPTION: Cardiomyocytes are cells that make up the heart muscle. As the chief cell type of the heart, cardiomyocytes are primarily involved in the contractile function of the heart that enables the pumping of blood around the body. Cardiac muscle cells are connected to each other at their short end by numerous intercellular junctions forming together a structure called the intercalated disc. These intercellular junctions comprise specific protein complexes, which are crucial for both intercellular adhesion and correct contraction of the heart. Imaging by conventional electron microscopy (EM) revealed a heavily folded intercalated disc with apparently random organization of the intercellular junctions. However, this conclusion was based on analysis in two dimensions (2D). 3D information of these structures is needed to unravel their true organization and function. In the present study, we used a more contemporary technique, called volume EM, to image and reconstruct the intercalated discs in 3D. By this approach, EM images are made from a whole block of tissue what differs significantly from classical EM methods that uses only one very thin slice for imaging. Further, we analyzed in comparison to normal mice also a mouse model for cardiomyopathy in which a specific protein of the cardiac intercellular junctions, αT-catenin, is absent. Volume EM revealed that in the hearts of these mice with cardiomyopathy, the finger-like folds of the intercalated disc are longer and thinner compared to control hearts. Also the intercellular junctions on the folded parts of the intercalated disc are smaller and their connection to the striated cytoskeleton seems further away. In conclusion, our volume EM study has expanded our understanding of 3D structures at the intercalated discs and will pave the way for more detailed models of disturbed cell-cell contacts associated with heart failure.