RESUMEN
Electrical synapses formed by connexin36 (Cx36)-containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter-based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36-puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild-type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36-puncta co-localized with Cx45-puncta, which in turn was associated with the scaffolding protein zonula occludens-1. In rats, Cx45-puncta were also co-localized with Cx36-puncta and additionally occurred along Bergmann glial processes adjacent to Cx43-puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.
Asunto(s)
Corteza Cerebelosa/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Corteza Cerebelosa/irrigación sanguínea , Corteza Cerebelosa/citología , Conexinas/genética , Técnica del Anticuerpo Fluorescente , Uniones Comunicantes/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/citología , Neuronas/citología , Ratas Sprague-Dawley , Especificidad de la Especie , Proteína delta-6 de Union ComunicanteRESUMEN
Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Glándula Pineal/metabolismo , Animales , Conexinas/genética , Uniones Comunicantes/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Glándula Pineal/ultraestructura , Ratas , Ratas Sprague-Dawley , Proteína delta-6 de Union ComunicanteRESUMEN
Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at "large myelinated club ending (LMCE)" synapses on Mauthner cells (M-cells) of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical "pre-potentials" immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified "mixed" (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with connexin35 (Cx35) restricted to axon terminal hemiplaques and connexin34.7 (Cx34.7) restricted to apposing M-cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of connexin36 (Cx36) on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins.
Asunto(s)
Encéfalo/metabolismo , Uniones Comunicantes/metabolismo , Ácido Glutámico/metabolismo , Carpa Dorada/metabolismo , Sinapsis/metabolismo , Animales , Axones/metabolismo , Dendritas/metabolismo , Proteínas de Peces/metabolismo , Inmunohistoquímica , Microscopía Confocal , Microscopía ElectrónicaRESUMEN
Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) are a common feature in mammalian brain circuitry, but less is known about their deployment in spinal cord. It has been reported based on connexin mRNA and/or protein detection that developing and/or mature motoneurons express a variety of connexins, including Cx26, Cx32, Cx36 and Cx43 in trigeminal motoneurons, Cx36, Cx37, Cx40, Cx43 and Cx45 in spinal motoneurons, and Cx32 in sexually dimorphic motoneurons. We re-examined the localization of these connexins during postnatal development and in adult rat and mouse using immunofluorescence labeling for each connexin. We found Cx26 in association only with leptomeninges in the trigeminal motor nucleus (Mo5), Cx32 only with oligodendrocytes and myelinated fibers among motoneurons in this nucleus and in the spinal cord, and Cx37, Cx40 and Cx45 only with blood vessels in the ventral horn of spinal cord, including those among motoneurons. By freeze-fracture replica immunolabeling, > 100 astrocyte gap junctions but no neuronal gap junctions were found based on immunogold labeling for Cx43, whereas 16 neuronal gap junctions at postnatal day (P)4, P7 and P18 were detected based on Cx36 labeling. Punctate labeling for Cx36 was localized to the somatic and dendritic surfaces of peripherin-positive motoneurons in the Mo5, motoneurons throughout the spinal cord, and sexually dimorphic motoneurons at lower lumbar levels. In studies of electrical synapses and electrical transmission between developing and between adult motoneurons, our results serve to focus attention on mediation of this transmission by gap junctions composed of Cx36.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Médula Espinal/crecimiento & desarrollo , Transmisión Sináptica/fisiologíaRESUMEN
Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over 40 years ago. Because gap junctions formed by connexins are the morphological correlate of electrical synapses, and with demonstrations of widespread expression of the gap junction protein connexin36 (Cx36) in neurons, we investigated the distribution and cellular localization of electrical synapses in the adult and developing rodent vestibular nuclear complex, using immunofluorescence detection of Cx36 as a marker for these synapses. In addition, we examined Cx36 localization in relation to that of the nerve terminal marker vesicular glutamate transporter-1 (vglut-1). An abundance of immunolabeling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabeling was associated with somata and initial dendrites of medium and large neurons, and was absent in vestibular nuclei of Cx36 knockout mice. Cx36-puncta were seen either dispersed or aggregated into clusters on the surface of neurons, and were never found to occur intracellularly. Nearly all Cx36-puncta were localized to large nerve terminals immunolabeled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labeling for Cx36 was already present in the vestibular nuclei at postnatal day 5, where it was only partially co-localized with vglut-1, and did not become fully associated with vglut-1-positive terminals until postnatal day 20-25. The results show that vglut-1-positive primary afferent nerve terminals form mixed synapses throughout the vestibular nuclear complex, that the gap junction component of these synapses contains Cx36, that multiple Cx36-containing gap junctions are associated with individual vglut-1 terminals and that the development of these mixed synapses is protracted over several postnatal weeks.
Asunto(s)
Conexinas/biosíntesis , Neuronas Aferentes/metabolismo , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/biosíntesis , Núcleos Vestibulares/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neurogénesis/fisiología , Ratas , Ratas Sprague-Dawley , Núcleos Vestibulares/crecimiento & desarrollo , Proteína delta-6 de Union ComunicanteRESUMEN
Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.
Asunto(s)
Encéfalo/anatomía & histología , Conexinas/metabolismo , Ratones Transgénicos , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Encéfalo/metabolismo , Línea Celular Tumoral , Conexina 26 , Conexina 43/metabolismo , Conexinas/genética , Femenino , Uniones Comunicantes/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , RatasRESUMEN
The panglial syncytium maintains ionic conditions required for normal neuronal electrical activity in the central nervous system (CNS). Vital among these homeostatic functions is "potassium siphoning," a process originally proposed to explain astrocytic sequestration and long-distance disposal of K(+) released from unmyelinated axons during each action potential. Fundamentally different, more efficient processes are required in myelinated axons, where axonal K(+) efflux occurs exclusively beneath and enclosed within the myelin sheath, precluding direct sequestration of K(+) by nearby astrocytes. Molecular mechanisms for entry of excess K(+) and obligatorily-associated osmotic water from axons into innermost myelin are not well characterized, whereas at the output end, axonally-derived K(+) and associated osmotic water are known to be expelled by Kir4.1 and aquaporin-4 channels concentrated in astrocyte endfeet that surround capillaries and that form the glia limitans. Between myelin (input end) and astrocyte endfeet (output end) is a vast network of astrocyte "intermediaries" that are strongly inter-linked, including with myelin, by abundant gap junctions that disperse excess K(+) and water throughout the panglial syncytium, thereby greatly reducing K(+)-induced osmotic swelling of myelin. Here, I review original reports that established the concept of potassium siphoning in unmyelinated CNS axons, summarize recent revolutions in our understanding of K(+) efflux during axonal saltatory conduction, then describe additional components required by myelinated axons for a newly-described process of voltage-augmented "dynamic" potassium siphoning. If any of several molecular components of the panglial syncytium are compromised, K(+) siphoning is blocked, myelin is destroyed, and axonal saltatory conduction ceases. Thus, a common thread linking several CNS demyelinating diseases is the disruption of potassium siphoning/water transport within the panglial syncytium. Continued progress in molecular identification and subcellular mapping of glial ion and water channels will lead to a better understanding of demyelinating diseases of the CNS and to development of improved treatment regimens.
Asunto(s)
Axones/metabolismo , Neuroglía/metabolismo , Potasio/metabolismo , Animales , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Modelos Neurológicos , Neuromielitis Óptica/metabolismoRESUMEN
Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.
Asunto(s)
Conexinas/fisiología , Uniones Comunicantes/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Conexinas/genética , Detergentes/farmacología , Electrofisiología , Técnica de Fractura por Congelación , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Neuroglía/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Dodecil Sulfato de Sodio/farmacología , Proteína beta1 de Unión Comunicante , Proteína delta-6 de Union ComunicanteRESUMEN
Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Locus Coeruleus/citología , Neuronas/citología , Roedores/metabolismo , Animales , Animales Recién Nacidos , Conexinas/clasificación , Conexinas/deficiencia , Técnica de Fractura por Congelación/métodos , Uniones Comunicantes/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo/métodos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Roedores/crecimiento & desarrollo , Proteína delta-6 de Union ComunicanteRESUMEN
Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.
Asunto(s)
Uniones Comunicantes/ultraestructura , Vías Nerviosas/ultraestructura , Neuronas/ultraestructura , Retina/ultraestructura , Animales , Comunicación Celular/fisiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Femenino , Técnica de Fractura por Congelación , Uniones Comunicantes/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Inmunoelectrónica , Modelos Neurológicos , Vías Nerviosas/fisiología , Neuronas/fisiología , Estimulación Luminosa , Ratas , Ratas Sprague-Dawley , Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Visión Ocular/fisiologíaRESUMEN
Most gap junctions between neurons in mammalian retina contain abundant connexin36, often in association with the scaffolding protein zonula occludens-1. We now investigate co-association of connexin36, zonula occludens-1, zonula occludens-2 and Y-box transcription factor 3 (zonula occludens-1-associated nucleic acid-binding protein) in mouse and rat retina. By immunoblotting, zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were both detected in retina, and zonula occludens-2 in retina was found to co-immunoprecipitate with connexin36. By immunofluorescence, the four proteins appeared as puncta distributed in the plexiform layers. In the inner plexiform layer, most connexin36-puncta were co-localized with zonula occludens-1, and many were co-localized with zonula occludens-1-associated nucleic acid-binding protein. Moreover, zonula occludens-1-associated nucleic acid-binding protein was often co-localized with zonula occludens-1. Nearly all zonula occludens-2-puncta were positive for connexin36, zonula occludens-1 and zonula occludens-1-associated nucleic acid-binding protein. In the outer plexiform layer, connexin36 was also often co-localized with zonula occludens-1-associated nucleic acid-binding protein. In connexin36 knockout mice, labeling of zonula occludens-1 was slightly reduced in the inner plexiform layer, zonula occludens-1-associated nucleic acid-binding protein was decreased in the outer plexiform layer, and both zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were markedly decreased in the inner sublamina of the inner plexiform layer, whereas zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein puncta persisted and remained co-localized in the outer sublamina of the inner plexiform layer. By freeze-fracture replica immunogold labeling, connexin36 was found to be co-localized with zonula occludens-2 within individual neuronal gap junctions. In addition, zonula occludens-1-associated nucleic acid-binding protein was abundant in a portion of ultrastructurally-defined gap junctions throughout the inner plexiform layer, and some of these junctions contained both connexin36 and zonula occludens-1-associated nucleic acid-binding protein. These distinct patterns of connexin36 association with zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein in different sublaminae of retina, and differential responses of these proteins to connexin36 gene deletion suggest differential regulatory and scaffolding roles of these gap junction accessory proteins. Further, the persistence of a subpopulation of zonula occludens-1/zonula occludens-2/zonula occludens-1-associated nucleic acid-binding protein co-localized puncta in the outer part of the inner plexiform layer of connexin36 knockout mice suggests close association of these proteins with other structures in retina, possibly including gap junctions composed of an as-yet-unidentified connexin.
Asunto(s)
Conexinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Uniones Comunicantes/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Retina/metabolismo , Animales , Calbindina 2 , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Conexinas/genética , Proteínas de Unión al ADN/genética , Femenino , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Uniones Comunicantes/ultraestructura , Inmunohistoquímica , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Neuronas/metabolismo , Neuronas/ultraestructura , Fosfoproteínas/genética , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Proteínas de Unión al ARN/genética , Ratas , Ratas Sprague-Dawley , Retina/ultraestructura , Proteína G de Unión al Calcio S100/metabolismo , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2 , Proteína delta-6 de Union ComunicanteRESUMEN
The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and heterologous oligodendrocyte gap junctions provide essential pathways for intra- and intercellular ionic homeostasis.
Asunto(s)
Sistema Nervioso Central/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/ultraestructura , Conexina 43/metabolismo , Citoplasma/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Uniones Comunicantes/ultraestructura , Homeostasis , Inmunohistoquímica , Iones , Masculino , Ratones , Ratones Endogámicos , Microscopía Electrónica , Vaina de Mielina/ultraestructura , Oligodendroglía/ultraestructura , Potasio/metabolismo , Nódulos de Ranvier/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismo , Distribución Tisular , Proteína beta1 de Unión ComunicanteRESUMEN
Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular cross-bridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4.
Asunto(s)
Acuaporinas/química , Astrocitos/ultraestructura , Membrana Celular/ultraestructura , Epéndimo/ultraestructura , Técnica de Fractura por Congelación , Empalme Alternativo/genética , Animales , Acuaporina 4 , Acuaporinas/genética , Astrocitos/química , Células CHO , Membrana Celular/química , Cricetinae , Epéndimo/química , Femenino , Aumento de la Imagen , Inmunohistoquímica , Sustancias Macromoleculares/química , Masculino , Modelos Moleculares , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 co-localization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1. Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions.
Asunto(s)
Comunicación Celular/fisiología , Conexinas/biosíntesis , Oligodendroglía/metabolismo , Uniones Estrechas/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Western Blotting , Encéfalo/metabolismo , Conexina 26 , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Confocal , Oligodendroglía/ultraestructura , Fosfoproteínas/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/metabolismo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1RESUMEN
Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.
Asunto(s)
Mapeo Encefálico/métodos , Conexinas/análisis , Proteínas del Ojo/análisis , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Proteómica/métodos , Receptores de N-Metil-D-Aspartato/análisis , Animales , Conexinas/biosíntesis , Proteínas del Ojo/biosíntesis , Carpa Dorada , Inmunohistoquímica , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Noqueados , Fosfoproteínas/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biosíntesis , Proteína de la Zonula Occludens-1 , Proteína delta-6 de Union ComunicanteRESUMEN
Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.
Asunto(s)
Conexinas/fisiología , Proteínas del Ojo/fisiología , Uniones Comunicantes/fisiología , Carpa Dorada/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiología , Animales , Conexinas/genética , Conductividad Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Proteínas del Ojo/genética , Técnica de Fractura por Congelación , Terminales Presinápticos/fisiología , Proteína delta-6 de Union ComunicanteRESUMEN
Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30-positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47-positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.
Asunto(s)
Astrocitos/fisiología , Conexinas/genética , Conexinas/metabolismo , Oligodendroglía/fisiología , Animales , Astrocitos/citología , Comunicación Celular/fisiología , Conexina 26 , Conexina 30 , Conexina 43/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas del Tejido Nervioso , Oligodendroglía/citología , Proteína beta1 de Unión ComunicanteRESUMEN
Auditory afferents terminating as "large myelinated club endings" on goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that offer the unique opportunity to correlate physiological properties with biochemical composition and specific ultrastructural features of individual synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we demonstrate that gap junctions at these synapses contain connexin35 (Cx35). This connexin is the fish ortholog of the neuron-specific human and mouse connexin36 that is reported to be widely distributed in mammalian brain and to be responsible for electrical coupling between many types of neurons. Similarly, connexin35 was found at gap junctions between neurons in other brain regions, suggesting that connexin35-mediated electrical transmission is common in goldfish brain. Conductance of gap junction channels at large myelinated club endings is known to be dynamically modulated by the activity of their colocalized glutamatergic synapses. We show evidence by confocal microscopy for the presence of the NR1 subunit of the NMDA glutamate receptor subtype, proposed to be a key regulatory element, at these large endings. Furthermore, we also show evidence by FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor is present at postsynaptic densities closely associated with gap junction plaques containing Cx35 at mixed synapses across the goldfish hindbrain. Given the widespread distribution of electrical synapses and glutamate receptors, our results suggest that the plastic properties observed at these identifiable junctions may apply to other electrical synapses, including those in mammalian brain.
Asunto(s)
Conexinas/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Transmisión Sináptica , Animales , Especificidad de Anticuerpos , Astrocitos/química , Astrocitos/ultraestructura , Vías Auditivas , Sistema Nervioso Central/fisiología , Conexinas/análisis , Conexinas/inmunología , Conductividad Eléctrica , Proteínas del Ojo/fisiología , Uniones Comunicantes/química , Uniones Comunicantes/ultraestructura , Carpa Dorada , Inmunohistoquímica , Microscopía Confocal , Terminaciones Nerviosas/química , Terminaciones Nerviosas/citología , Plasticidad Neuronal , Neuronas/química , Neuronas/citología , Terminales Presinápticos/química , Receptores de N-Metil-D-Aspartato/análisis , Rombencéfalo/fisiología , Sinapsis/química , Sinapsis/ultraestructura , Proteína delta-6 de Union ComunicanteRESUMEN
The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.
Asunto(s)
Conexinas/análisis , Técnica de Fractura por Congelación , Inmunohistoquímica , Nervio Ciático/química , Animales , Western Blotting , Conexinas/inmunología , Uniones Comunicantes/química , Ratones , Ratones Noqueados , Vaina de Mielina/química , Proteínas del Tejido Nervioso , Células de Schwann/química , Proteína beta1 de Unión ComunicanteRESUMEN
The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions.