Gap Junction-mediated Coupling Research Articles

A crystallin mutant cataract with mineral deposits

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a non-connexin mutant mouse cataract model. By co-segregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins and of connexin46 and connexin50, but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions of fiber cell connexins were associated with a scarcity of immunoreactive gap junction punctae as detected by immunofluorescence, and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses, but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses also stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection micro-Fourier transform infrared spectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of gap junctional coupling between lens fiber cells leads to formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to formation of cataracts of different etiologies.

Age-Related Changes in Neurons and Satellite Glial Cells in Mouse Dorsal Root Ganglia.

The effects of aging on the nervous system are well documented. However, most previous studies on this topic were performed on the central nervous system. The present study was carried out on the dorsal root ganglia (DRGs) of mice, and focused on age-related changes in DRG neurons and satellite glial cells (SGCs). Intracellular electrodes were used for dye injection to examine the gap junction-mediated coupling between neurons and SGCs, and for intracellular electrical recordings from the neurons. Tactile sensitivity was assessed with von Frey hairs. We found that 3-23% of DRG neurons were dye-coupled to SGCs surrounding neighboring neurons in 8-24-month (Mo)-old mice, whereas in young adult (3 Mo) mice, the figure was 0%. The threshold current for firing an action potential in sensory neurons was significantly lower in DRGs from 12 Mo mice compared with those from 3 Mo mice. The percentage of neurons with spontaneous subthreshold membrane potential oscillation was greater by two-fold in 12 Mo mice. The withdrawal threshold was lower by 22% in 12 Mo mice compared with 3 Mo ones. These results show that in the aged mice, a proportion of DRG neurons is coupled to SGCs, and that the membrane excitability of the DRG neurons increases with age. We propose that augmented neuron-SGC communications via gap junctions are caused by low-grade inflammation associated with aging, and this may contribute to pain behavior.

The IgCAM CAR Regulates Gap Junction-Mediated Coupling on Embryonic Cardiomyocytes and Affects Their Beating Frequency.

The IgCAM coxsackie-adenovirus receptor (CAR) is essential for embryonic heart development and electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analyzed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wild type and CAR knockout (KO) embryos. Surprisingly, in the absence of the CAR, cultured cardiomyocytes showed increased frequencies of beating and calcium cycling. Increased beatings of heart organ cultures were also induced by the application of reagents that bind to the extracellular region of the CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed size increases but decreased in the total numbers of membrane-localized Cx43 clusters. This was accompanied by improved cell-cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that the CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions.

Satellite Glial Cells and Neurons in Trigeminal Ganglia Are Altered in an Itch Model in Mice.

Itch (pruritus) is a common chronic condition with a lifetime prevalence of over 20%. The mechanisms underlying itch are poorly understood, and its therapy is difficult. There is recent evidence that following nerve injury or inflammation, intercellular communications in sensory ganglia are augmented, which may lead to abnormal neuronal activity, and hence to pain, but there is no information whether such changes take place in an itch model. We studied changes in neurons and satellite glial cells (SGCs) in trigeminal ganglia in an itch model in mice using repeated applications of 2,4,6-trinitro-1-chlorobenzene (TNCB) to the external ear over a period of 11 days. Treated mice showed augmented scratching behavior as compared with controls during the application period and for several days afterwards. Immunostaining for the activation marker glial fibrillary acidic protein in SGCs was greater by about 35% after TNCB application, and gap junction-mediated coupling between neurons increased from about 2% to 13%. The injection of gap junction blockers reduced scratching behavior, suggesting that gap junctions contribute to itch. Calcium imaging studies showed increased responses of SGCs to the pain (and presumed itch) mediator ATP. We conclude that changes in both neurons and SGCs in sensory ganglia may play a role in itch.

The effects of beta-cell mass and function, intercellular coupling, and islet synchrony on {text {Ca}}^{2+} dynamics

Type 2 diabetes (T2D) is a challenging metabolic disorder characterized by a substantial loss of beta -cell mass and alteration of beta -cell function in the islets of Langerhans, disrupting insulin secretion and glucose homeostasis. The mechanisms for deficiency in beta -cell mass and function during the hyperglycemia development and T2D pathogenesis are complex. To study the relative contribution of beta -cell mass to beta -cell function in T2D, we make use of a comprehensive electrophysiological model of human beta -cell clusters. We find that defect in beta -cell mass causes a functional decline in single beta -cell, impairment in intra-islet synchrony, and changes in the form of oscillatory patterns of membrane potential and intracellular {text {Ca}}^{2+} concentration, which can lead to changes in insulin secretion dynamics and in insulin levels. The model demonstrates a good correspondence between suppression of synchronizing electrical activity and published experimental measurements. We then compare the role of gap junction-mediated electrical coupling with both beta -cell synchronization and metabolic coupling in the behavior of {text {Ca}}^{2+} concentration dynamics within human islets. Our results indicate that inter-beta -cellular electrical coupling depicts a more important factor in shaping the physiological regulation of islet function and in human T2D. We further predict that varying the whole-cell conductance of delayed rectifier text {K}^{+} channels modifies oscillatory activity patterns of beta -cell population lacking intercellular coupling, which significantly affect {text {Ca}}^{2+} concentration and insulin secretion.

Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

Glucose Tightly Controls Morphological and Functional Properties of Astrocytes.

The main energy source powering the brain is glucose. Strong energy needs of our nervous system are fulfilled by conveying this essential metabolite through blood via an extensive vascular network. Glucose then reaches brain tissues by cell uptake, diffusion and metabolization, processes primarily undertaken by astrocytes. Deprivation of glucose can however occur in various circumstances. In particular, ageing is associated with cognitive disturbances that are partly attributable to metabolic deficiency leading to brain glycopenia. Despite the crucial role of glucose and its metabolites in sustaining neuronal activity, little is known about its moment-to-moment contribution to astroglial physiology. We thus here investigated the early structural and functional alterations induced in astrocytes by a transient metabolic challenge consisting in glucose deprivation. Electrophysiological recordings of hippocampal astroglial cells of the stratum radiatum in situ revealed that shortage of glucose specifically increases astrocyte membrane capacitance, whilst it has no impact on other passive membrane properties. Consistent with this change, morphometric analysis unraveled a prompt increase in astrocyte volume upon glucose deprivation. Furthermore, characteristic functional properties of astrocytes are also affected by transient glucose deficiency. We indeed found that glucoprivation decreases their gap junction-mediated coupling, while it progressively and reversibly increases their intracellular calcium levels during the slow depression of synaptic transmission occurring simultaneously, as assessed by dual electrophysiological and calcium imaging recordings. Together, these data indicate that astrocytes rapidly respond to metabolic dysfunctions, and are therefore central to the neuroglial dialog at play in brain adaptation to glycopenia.

Role of satellite glial cells in gastrointestinal pain.

Gastrointestinal (GI) pain is a common clinical problem, for which effective therapy is quite limited. Sensations from the GI tract, including pain, are mediated largely by neurons in the dorsal root ganglia (DRG), and to a smaller extent by vagal afferents emerging from neurons in the nodose/jugular ganglia. Neurons in rodent DRG become hyperexcitable in models of GI pain (e.g., gastric or colonic inflammation), and can serve as a source for chronic pain. Glial cells are another element in the pain signaling pathways, and there is evidence that spinal glial cells (microglia and astrocytes) undergo activation (gliosis) in various pain models and contribute to pain. Recently it was found that satellite glial cells (SGCs), the main type of glial cells in sensory ganglia, might also contribute to chronic pain in rodent models. Most of that work focused on somatic pain, but in several studies GI pain was also investigated, and these are discussed in the present review. We have shown that colonic inflammation induced by dinitrobenzene sulfonic acid (DNBS) in mice leads to the activation of SGCs in DRG and increases gap junction-mediated coupling among these cells. This coupling appears to contribute to the hyperexcitability of DRG neurons that innervate the colon. Blocking gap junctions (GJ) in vitro reduced neuronal hyperexcitability induced by inflammation, suggesting that glial GJ participate in SGC-neuron interactions. Moreover, blocking GJ by carbenoxolone and other agents reduces pain behavior. Similar changes in SGCs were also found in the mouse nodose ganglia (NG), which provide sensory innervation to most of the GI tract. Following systemic inflammation, SGCs in these ganglia were activated, and displayed augmented coupling and greater sensitivity to the pain mediator ATP. The contribution of these changes to visceral pain remains to be determined. These results indicate that although visceral pain is unique, it shares basic mechanisms with somatic pain, suggesting that therapeutic approaches to both pain types may be similar. Future research in this field should include additional types of GI injury and also other types of visceral pain.

Oxaliplatin enhances gap junction-mediated coupling in cell cultures of mouse trigeminal ganglia

Communications between satellite glial cells and neighboring neurons within sensory ganglia may contribute to neuropathic and inflammatory pain. To elucidate the role of satellite glial cells in chemotherapy-induced pain, we examined the effects of oxaliplatin on the gap junction-mediated coupling between these cells. We also examined whether the gap junction blocker, carbenoxolone, can reverse the coupling.Primary cultures of mice trigeminal ganglia, 24–48h after cell isolation, were used. Satellite glial cells were injected with Lucifer yellow in the presence or absence of oxaliplatin (60μM). In addition, the effect of carbenoxolone (100μM) on coupling, and the expression of connexin 43 proteins were evaluated.Dye coupling between adjacent satellite glial cells was significantly increased (2.3-fold, P<0.05) following a 2h incubation with oxaliplatin. Adding carbenoxolone to the oxaliplatin-treated cultures reversed oxaliplatin-evoked coupling to baseline (P<0.05). Immunostaining showed no difference between expression of connexin 43 in control and oxaliplatin-treated cultures.Our findings indicated that oxaliplatin-increased gap junction-mediated coupling between satellite glial cells in primary cultures of mouse trigeminal ganglia, and carbenoxolone reversed this effect. Hence, it is proposed that increased gap junction-mediated coupling was seen between satellite glial cells in TG. This observation together with our previous data obtained from a behavioral study suggests that this phenomenon might contribute to chemotherapy-induced nociception following oxaliplatin treatment.

The contribution of satellite glial cells to chemotherapy‐induced neuropathic pain

Chemotherapy-induced peripheral neuropathy is a serious side effect in cancer treatment, a major manifestation being neuropathic pain that can be debilitating and can reduce the quality of life of the patient. Oxaliplatin and taxol are common anti-cancer drugs that induce neuropathic pain by an unknown mechanism. We tested the hypothesis that satellite glial cells in dorsal root ganglia (DRGs) are altered in chemotherapy-induced peripheral neuropathy models and contribute to neuropathic pain. Mice were injected with either oxaliplatin or taxol and examined at 7-30 days. Glial fibrillary acidic protein (glial activation marker) expression was determined by immunohistochemistry. Satellite glial cells in isolated DRG were injected with the fluorescent dye Lucifer yellow and the incidence of dye coupling among these cells that surround different neurons was quantified. Taxol or oxaliplatin increased glial fibrillary acidic protein expression in satellite glial cells. Gap junction-mediated coupling between satellite glial cells was increased by up to fivefold after oxaliplatin and by up to twofold after taxol. This is consistent with work on other pain models showing that augmented satellite glial cell coupling contributes to chronic pain. Administration of the gap junction blocker carbenoxolone to chemotherapy-treated mice produced an analgesic-like effect. We propose that increased coupling by gap junctions is part of satellite glial cell activation, and that augmented coupling contributes to the lowering of pain threshold in oxaliplatin- and taxol-treated mice. We further propose that gap junction blockers may have potential in treating chemotherapy-induced neuropathic pain.

Intercellular communication in sensory ganglia by purinergic receptors and gap junctions: Implications for chronic pain

Peripheral injury can cause abnormal activity in sensory neurons, which is a major factor in chronic pain. Recent work has shown that injury induces major changes not only in sensory neurons but also in the main type of glial cells in sensory ganglia-satellite glial cells (SGCs), and that interactions between sensory neurons and SGCs contribute to neuronal activity in pain models. The main functional changes observed in SGCs after injury are an increased gap junction-mediated coupling among these cells, and augmented sensitivity to ATP. There is evidence that the augmented gap junctions contribute to neuronal hyperexcitability in pain models, but the mechanism underlying this effect is not known. The changes in SGCs described above have been found following a wide range of injuries (both axotomy and inflammation) in somatic, orofacial and visceral regions, and therefore appear to be a general feature in chronic pain. We have found that in cultures of sensory ganglia calcium signals can spread from an SGC to neighboring cells by calcium waves, which are mediated by gap junctions and ATP acting on purinergic P2 receptors. A model is proposed to explain how augmented gap junctions and greater sensitivity to ATP can combine to produce enhanced calcium waves, which can lead to neuronal excitation. Thus this simple scheme can account for several major changes in sensory ganglia that are common to a great variety of pain models.

Mixed Electrical–Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36

Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for “mixed” (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328–1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.

Relative contribution of changes in sodium current versus intercellular coupling on reentry initiation in 2-dimensional preparations of plakophilin-2–deficient cardiac cells

Loss of expression of the desmosomal protein plakophilin-2 (PKP2) leads to decreased gap junction-mediated (GJ) coupling, and alters the amplitude and kinetics of sodium current in cardiac myocytes. Whether these modifications, alone or in combination, are sufficient to act as arrhythmogenic substrates remains undefined. This study sought to characterize arrhythmia susceptibility and reentry dynamics consequent to loss of PKP2 expression, and to assess the relative contribution of cell uncoupling versus alterations in sodium current in generation of reentry. Monolayers of neonatal rat ventricular myocytes were treated with oligonucleotides that either prevented or failed to prevent PKP2 expression. Numerical simulations modeled experimentally observed modifications in I(Na), GJ coupling, or both (models PKP2-Na, PKP2-GJ, and PKP2-KD, respectively). Relative roles of sodium current density versus kinetics were further explored. Loss of PKP2 expression increased incidence of rotors and decreased frequency of rotation. Mathematical simulations revealed that single premature stimuli initiated rotors in models PKP2-Na and PKP2-KD, but not PKP2-GJ. Changes in sodium current kinetics, rather than current density, were key to reentry initiation. Anatomical barriers led to vortex shedding, wavebreaks, and rotors when I(Na) kinetics, but not GJ coupling or I(Na) density, were altered. PKP2-dependent changes in sodium current kinetics lead to slow conduction, increased propensity to functional block, and vortex shedding. Changes in GJ or I(Na) density played only a minor role on reentry susceptibility. Changes in electrical properties of the myocyte caused by loss of expression of PKP2 can set the stage for rotors even if anatomical homogeneity is maintained.

Peripheral inflammation augments gap junction-mediated coupling among satellite glial cells in mouse sympathetic ganglia

Intercellular coupling by gap junctions is one of the main features of glial cells, but very little is known about this aspect of satellite glial cells (SGCs) in sympathetic ganglia. We used the dye coupling method to address this question in both a prevertebral ganglion (superior mesenteric) and a paravertebral ganglion (superior cervical) of mice. We found that in control ganglia, the incidence of dye coupling among SGCs that form the envelope around a given neuron was 10-20%, and coupling between SGCs around different envelopes was rare (1.5-3%). The dye injections also provided novel information on the structure of SGCs. Following peripheral inflammation, both types of coupling were increased, but most striking was the augmentation of coupling between SGCs forming envelopes around different neurons, which rose by 8-14.6-fold. This effect appeared to be non-systemic, and was blocked by the gap junction blocker carbenoxolone. These changes in SGCs may affect signal transmission and processing in sympathetic ganglia.

Retinoic acid

What is retinoic acid? Retinoic acid is a lipophilic molecule of low molecular weight (300 Da). Retinoic acid is synthesized from vitamin A (retinol) and found in embryos and adult vertebrates. Because animals are unable to produce vitamin A, they obtain this vitamin in the form of carotenoids from plants or retinyl esters derived from animal products. How is retinoic acid produced? In vertebrates, vitamin A, in the form of retinyl esters, is stored mainly in the liver as well as in the lungs, kidneys and bone marrow. Retinol, bound to the retinol-binding protein (RBP), enters the circulatory system in order to reach cells that require retinoic acid. The retinol–RBP complex enters the cells through the RBP receptor (STRA6). Within the cells, retinol is metabolized in a reversible step to retinal by the retinol or alcohol dehydrogenases (RODHs or ADHs) and then in an irreversible manner to retinoic acid by the retinal dehydrogenases (RALDHs). In the cytoplasm, retinoic acid is associated with the cellular retinoic acid-binding protein (CRABP). And what does it do? Simply speaking, retinoic acid functions to regulate gene expression (Figure 1). In cells, retinoic acid enters the nucleus where it binds to heterodimers formed by two different classes of nuclear receptors: Retinoic acid receptors (RARs) and retinoid X receptor (RXRs). The receptor heterodimers are bound to a specific DNA sequence known as the retinoic acid-response element (RARE) and upon retinoic acid binding, recruits co-activators (including histone acetyltransferase (HAT)), leading to transcription activation of specific retinoic acid-regulated genes. How many RARs and RXRs are there? In most vertebrates, there are three RARs (α, β and γ) and three RXRs (α, β and γ), all of which can have multiple isoforms, leading to large numbers of possible receptor combinations. How is the retinoic acid ‘signal’ turned off? In the cytoplasm, retinoic acid can be catabolized by the cytochrome P450 enzyme (CYP26) into inactive compounds that are then excreted. In the absence of retinoic acid, the RAR/RXR heterodimers bound to their target sequences recruit the nuclear receptor co-repressor complex NCOR/Sin3A/HDAC (histone deacetylase), inhibiting transcription activation. What does retinoic acid do? In adults, vitamin A and its retinoid derivatives play significant roles in a number of processes, such as vision, learning and memory, immune function, reproduction as well as the maintenance of epithelial function and differentiation. In the hippocampus, retinoic acid plays important physiological roles in synaptic plasticity, learning and memory, and adult neurogenesis. In the retina, retinoic acid acts as a light-signaling neuromodulator and regulates gap junction-mediated coupling of retinal neurons. It has also been implicated in the control of sleep and the circadian clock. Why are developmental biologists so obsessed with retinoic acid? This simple molecule has been implicated in the control of many aspects of embryo development that include cranio-facial morphogenesis, neuronal differentiation, eye, limb, lung, kidney and heart development. The patterning of the antero-posterior embryo body axis is also directly regulated by retinoic acid, which controls the expression of several HOX genes through RAREs in their regulatory/enhancer regions. Are there any human diseases related to retinoic acid deficiency? Reduction of vitamin A levels in the diet can lead to a debilitating immune system, anemia or blindness. During pregnancy, insufficient levels of vitamin A lead to fetal vitamin A deficiency-induced syndrome. This results in developmental defects exhibiting many common features with those observed in the RAR/RXR mutations in mouse, including cranio-facial and eye abnormalities. Retinoic acid as an anticancer drug…? Acute promyelocytic leukaemia is caused by a chromosomal translocation that fuses the promyelocytic leukaemia gene (PML) on chromosome 15 with the RARα gene on chromosome 17. The PML-RARα fusion protein leads to a recruitment of co-repressor complexes that epigenetically silence gene expression. Differentiation therapy with retinoic acid is being used in combination with chemotherapy for the treatment of patients with acute promyelocytic leukaemia, resulting in a 70-80% cure rate. Retinoic acidVilhais-Neto et al.Current BiologyApril 08, 2008In Brief(Current Biology 18, R191–R192, March 11, 2008) Full-Text PDF Open Archive

Molecular Diversity of Connexin and Pannexin Genes in the Retina of the ZebrafishDanio rerio

Gap junctions are among the most widely distributed cell structures involved in cell-to-cell communication. Recently completed genome sequencing projects including species from all major phyla have demonstrated the existence of three distinct gene families, the connexins, pannexins, and innexins, as molecular building blocks of gap junctional communication. In the present study, the authors have addressed the molecular complexity of gap junction gene expression in the zebrafish retina, a remarkably complex sensory organ built by diverse neuronal subtypes. Using a combination of cDNA library and genomic DNA library screening and/or RACE technology, the authors have cloned, in addition to the four previously reported connexins, seven novel connexins and four pannexin transcripts resembling two pannexin genes. This result demonstrates the presence of two distinct gap junction type gene families and indicates a remarkable molecular and functional diversity of gap junction-mediated coupling in the fish retina.

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy

In an earlier study exploring the potential of gene transfer to repair myocardial conduction defects, we observed that myotubes, generated by forced expression of MyoD, exhibit reduced excitability when also modified to express connexin43 (Cx43). We hypothesized that this effect was caused by gap junction-mediated coupling between myotubes and the underlying fibroblast feeder layer. This intriguing possibility has important implications for ongoing efforts to develop strategies for repairing myocardial conduction defects by gene transfer, and also provides novel insights into the electrophysiological function of naturally occurring heterologous cell coupling within the heart. Although a conductive function for fibroblasts through heterologous coupling has previously been reported, the current study provides novel evidence that fibroblasts can modulate cardiomyocyte excitability in a Cx43-dependent manner. In a co-culture study system, neonatal rat cardiomyocytes were grown on monolayers of mouse fibroblasts with genetically altered Cx43 expression and the effect on intrinsic beat frequency examined. Cardiomyocytes grown on wild-type (WT) fibroblasts expressing native levels of Cx43 beat significantly slower than cells grown on fibroblasts devoid of this molecule (germline knockout) or with dominant-negative functional suppression. Expression of Cx43 in fibroblasts from Cx43 knockout mice restored cardiomyocyte beat frequency, to rates comparable with those observed in co-culture with WT fibroblasts.

Agrin Converts Electrical Coupling to Chemical Coupling

Agrin is a proteoglycan secreted by nerves that is well known for its role in organizing the postsynaptic regions of the neuromuscular junction (NMJ). However, agrin is not limited to nerves that innervate muscle and is a widely expressed protein, suggesting additional roles in synaptic organization in the central and peripheral nervous systems. Martin et al . provide evidence that agrin stimulates the switch from electrical coupling of cells to synaptic transmission in the adrenal gland. The abundance of the active form of agrin (Z + -agrin) increased during postnatal development of the adrenal gland and in adults was preferentially localized at cholinergic synaptic contacts. The amplitude, but not the frequency, of spontaneous excitatory postsynaptic currents (sEPSCs) in the neonatal adrenal gland, but not the adult gland, was increased by application of Z + -agrin. The effect appeared to be postsynaptic, based on an increase in the amplitude of the current evoked by application of nicotine (a ligand of acetylcholine receptors, nAChR). In neonatal adrenal gland, pharmacological inhibition of α3-type nAChR completely blocked sEPSCs; however, application of Z + -agrin resulted in sEPSCs that could only be blocked by inhibition of both α3 and α7 nAChR, as it does in the adult adrenal gland. In the neonatal adrenal gland, decay of the sEPSC was fitted by two exponentials and appeared to result in part from synaptic coupling and in part from gap junction-mediated coupling. Application of agrin or pharmacological inhibition of gap junctions converted this to a decay with a single time constant like that observed in adult gland. Within minutes, application of agrin decreased the percentage of electrically coupled cells in the neonatal adrenal gland (based on measurements of intercellular Lucifer yellow diffusion and intercellular macroscopic junctional currents), and within 1 hour, synaptic transmission was increased (based on increased sEPSC amplitude). The conversion of electrically coupled cells to synaptic transmission was blocked by pharmacological inhibition of Src, which is also implicated downstream of muscle-specific kinase (MuSK) in NMJ organization. MuSK was also detected in the neonatal and adult chromaffin gland. Thus, agrin appears to have a role in regulating the developmental switch from electrical coupling to chemical coupling in the adrenal gland. A. O. Martin, G. Alonso, N. C. Guérineau, Agrin mediates a rapid switch from electrical coupling to chemical neurotransmission during synaptogenesis. J. Cell. Biol. 169 , 503-514 (2005). [Abstract] [Full Text]

236 NUCLEAR AND CYTOPLASMIC MODIFICATIONS OF GERMINAL VESICLE BOVINE OOCYTES IN RELATION TO CHROMATIN REMODELING

We previously reported that germinal vesicle (GV) bovine oocytes can be classified on the basis of their chromatin organization and that increased chromatin condensation is accompanied by a major incidence of gap junction-mediated coupling interruption between germ and cumulus cells and by an increase in oocyte developmental competence (Lodde et al. 2005 Reprod. Fertil. Dev. 17(2), 294-295). The aim of this study was to characterize, at the ultrastructural level, both nuclear and cytoplasmic compartments of bovine oocytes classified according to their chromatin configuration because key structural modifications, such as nucleolar inactivation and remodeling of specific ooplasmic structures, take place during the later phases of oocyte growth. Cumulus-oocyte complexes collected from 0.5-2-mm early antral (EA) and 2-6-mm mid-antral (MA) follicles were freed of cumulus cells. Denuded oocytes were stained with Hoechst 33342, classified according to the degree of chromatin condensation, and processed for light microscopy of semi-thin sections (LM; n = 10 in each class) and transmission electron microscopy (TEM; n = 5 in each class). Four classes of oocytes were identified by the Hoechst staining: GV0 with filamentous chromatin diffused in the nuclear area, GV1 with few foci of condensed chromatin, GV2 with chromatin further condensed into distinct clumps, and GV3 with chromatin condensed into a single clump. Almost all oocytes collected from EA follicles were classified as GV0. Oocytes of this class were absent in MA follicles, whereas class GV1, GV2, and GV3 oocytes occurred at similar frequency. LM confirmed the chromatin condensation found by the Hoechst staining and revealed that in class GV2 and GV3 oocytes the chromatin was mainly located close to the nucleolus. Ultrastructurally, the nucleolus was fibrillo-granular in GV0 oocytes; the oocytes in the other classes displayed an electron dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most organelles were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex, were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Our findings indicate that the process of chromatin remodeling is strictly related to structural modifications that characterize the later stages of the oocyte growth phase. Because the highest degree of chromatin condensation was combined with degenerative features of cortical granules, we hypothesize that this class of oocytes (GV3) originated from early atretic follicles, as also suggested in other species. The evaluation of oocytes on the basis of chromatin configuration may be useful for the development of new strategies for manipulating fertility in mammals. This work was supported by a COFIN Grant.

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures.

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.