Ultrastructural studies of severed medial giant and other CNS axons in crayfish

Summary The distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.This work was supported by NIH research grant #NS-14412 and an RCDA#00070 to GDB     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Calmodulin Acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons

Summary Lateral axons from the abdominal nerve cord of cray-fish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa>7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca²⁺-CaM) the junctional resistance between the axons increases from control values of about 60 to 500–600 k in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused.The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca²⁺-CaM there are regions where the synaptic gap between the membranes decreases from a control 4–6 to 2–3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.     

Differential survival of isolated portions of crayfish axons

Summary Electron microscopic studies show that transplanted segments of sensory axons of varying lengths degenerate within 7–14 days whereas transplanted segments of crustacean motor axons survive morphologically intact for 20–30 days. The middle portion of an isolated motor axon segment degenerates less rapidly than portions of the same axon located nearer the periphery or nearer the ventral nerve cord. One week after transplantation, glial cells appear to phagocytize sensory axons whereas glial cells around motor axons appear to hypertrophy and to have more rough endoplasmic reticulum. After three weeks, motor axons also appear to be phagocytized by glial cells.These data suggest that the glia surrounding isolated motor axons can change from a supportive to a destructive function, whereas glial cells surrounding severed sensory axons primarily have a destructive function. These and other data also indicate that crustacean motor axons receive significant trophic inputs from their own perikaryon, from post-synaptic contacts, and from adjacent glial cells. The possibility that adjacent healthy cells may supply metabolically deficient cells with needed substances could be a significant adaptive advantage for the evolution of multicellular organisms.Supported in part by an NIH grant (NS-1186101) to Dr. BittnerThe authors wish to thank Mr. Martis Ballinger and Mr. Robert Riess for their valuable assistance in all stages of this research     

Gap junctions and intercellular communication in the hepatopancreas of the crayfish (Orconectes propinquus) during molt

Summary The crustacean hepatopancreas is a major metabolic center intimately involved in molting and vitellogenesis. Cells of the hepatopancreas exhibit one of the richest endowments of gap junctions known and are thus presumed to be linked for intercellular communication. In order to monitor hepatopancreatic activity during the molt cycle of crayfish (Orconectes propinquus), the electrical coupling between cells of the hepatopancreatic tubules was measured during postmolt, intermolt and premolt. Samples of hepatopancreas from each of these stages were fixed and freeze-fractured to correlate morphologic features of gap junctions with electrophysiological data. Analysis of the data revealed that ionic coupling was greater in postmolt and premolt tubule cells than in cells of intermolt animals. Platinum replicas of hepatopancreatocyte plasmalemmata revealed that in postmolt, gap junction plaques were smaller and more numerous than those in intermolt and premolt; however, the total area of gap junction plaques per unit membrane area analyzed was approximately the same for hepatopancreatocytes from all molt stages. Although the hepatopancreatic gap junctions exhibited no quantitative differences, those from post- and premolt animals were rounded with tightly packed particles, while plaques from intermolt animals were generally pleomorphic with loosely packed particles. Results of this study suggest that cells of the crayfish hepatopancreas are more coupled in pre- and postmolt, with macular plaques of tightly packed particles, perhaps as a response to the increased metabolic demands of molt, and less well coupled, with irregular plaques of loosely packed junctional particles, during intermolt. The only recognizable morphological correlates of increased cell coupling were tight packing of junctional particles into rounded plaques, while decreased coupling corresponded to junctions with loosely packed irregular aggregates of particles.Supported by the Natural Sciences and Engineering Research Council of Canada (RRS)     

The stage-specific function of gap junctions during tumourigenesis

Tumour development is a process resulting from the disturbance of various cellular functions including cell proliferation, adhesion and motility. While the role of these cell parameters in tumour promotion and progression has been widely recognized, the mechanisms that influence gap junctional coupling during tumorigenesis remain elusive. Neoplastic cells usually display decreased levels of connexin expression and/or gap junctional coupling. Thus, impaired intercellular communication via gap junctions may facilitate the release of a potentially neoplastic cell from the controlling regime of the surrounding tissue, leading to tumour promotion. However, recent data indicates that metastatic tumour cell lines are often characterized by relatively high levels of connexin expression and gap junctional coupling. This review outlines current knowledge on the role of connexins in tumorigenesis and the possible mechanisms of the interference of gap junctional coupling with the processes of tumour invasion and metastasis. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication costs were covered by the organisers of this meeting.     

Electron-microscopic studies on the presence of gap junctions between corneal fibroblasts in rabbits

Summary Corneal fibroblasts, major cellular components of the corneal stroma, are loosely arrayed between collagen lamellae. They play an important role in the metabolic and physiological homeostasis mechanisms by which the cornea is kept transparent. This paper deals with the demonstration of the gap junctions between the corneal fibroblasts of rabbits by transmission electron microscopy of thin sections and of freeze-fracture specimens. Under the transmission electron microscope, the corneal fibroblasts are seen between the lamellae of collagen fibers of the corneal stroma. Their long cytoplasmic processes are in contact with those of neighboring fibroblasts. Typical gap junctions are found between these cytoplasmic processes. In the freeze-fracture images, intramembrane particles with a diameter of 10.3 nm form polygonal aggregates on P faces. These findings suggest that corneal fibroblasts, coupled with each other, might function synchronously through gap junctions responsible for metabolic activities essential for the maintenance of corneal transparency.A part of this study was published in Kinki Daigaku Igaku Zasshi in Japanese as the thesis for Atsuko Ueda, M.D. This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, from Osaka Eye Bank, Osaka, Japan, and from an intramural research fund of Kinki University     

Passage of 17 kDa calmodulin through gap junctions of three vertebrate species

Gap junctions of some vertebrates are capable of passing the elongate molecule, calmodulin, with a molecular weight 8-17 times greater than the previously recognized size limits. Fluorescently labeled calmodulin (FCaM) (17.34 kDa) microinjected into oocytes of ovarian follicles from an amphibian, Xenopus laevis, and from two species of teleost fish, Danio rerio (Zebrafish) and Oryzias latipes (Medaka), is shown to transit their gap junctions and enter the surrounding epithelial cells. Passage of FCaM was terminated when follicles were first treated with 1 mM octanol, a molecule known to down-regulate gap junctions. There was no FCaM detected in the surrounding medium, nor did epithelial cells become fluorescent when follicles were incubated in medium containing dye. Calmodulin is well known to modulate many cytoplasmic reactions; thus, its passage through gap junctions opens possibilities of additional means by which cells may be supplied with this signaling molecule, and by which their supply may be regulated.     

Structural changes in cardiac gap junctions after hypoxia and reoxygenation: a quantitative freeze-fracture analysis

Summary Isolated rat hearts were subjected to increasing periods of hypoxia with or without subsequent reoxygenation and the gap-junctional particle configuration was followed quantitatively. Irregular contractions were prevented by K⁺-arrest; glucose, counteracting the effects of hypoxia, was omitted. Hyperkalemia alone and a maximum of 20 min of hypoxia do not produce reorganization of the gap-junctional particles normally forming multiple hexagonally packed arrays separated by smooth aisles. After 30 min of hypoxia, the aisles disappear in a proportion of the junctions, thereby increasing the particle density from 9400±800/m² to 10200±900/m². After 40 min of hypoxia, the normal configuration is no longer found and numerous junctions are arranged as uninterrupted hexagonal lattices. The particles are further condensed to 11600±900/m². Following reoxygenation after both 30 and 40 min of hypoxia, the proportion of crystalline gap junctions dramatically augments and the mean particle density has further increased significantly. Corresponding thin sections show irreversible cell damage. When reoxygenation is performed with a control solution containing normal levels of K⁺ and glucose, the particle density does not increase substantially in comparison to the respective 30- and 40-min hypoxic periods. In both groups, the gap junctions display either a normal, a crystalline or an intermediate configuration with crystalline margins and loose centers. The gap-junctional reorganization during hypoxia essentially represents a particle condensation, while the mean center-to-center distances between the particles and pits remain constant. Furthermore, the reappearance of normal gap junctions after reoxygenation appears to depend on glucose availability.     

Reformation of gap and tight junctions in regenerating liver after cholestasis

Summary Morphometric analysis of the alterations in interhepatocyte junctions induced by bile duct ligation revealed that after 48 h, during which time the serum bilirubin increased 6 to 8 fold, the membrane area occupied by gap junctions on the apico-lateral and medio-lateral sides decreased from 3.6% in controls to 0.02% in the ligated group. The strands of the zonulae occludentes were reduced in number and showed increased discontinuities.Within 45 min of recanalization of the common bile duct, clusters of particles appeared within and adjacent to the tight junctional areas or in the lateral hepatocyte membrane. Subsequently, the particle aggregations localized in the apico-lateral membrane areas increased in number and size becoming finally indistinguishable from those of controls within 96 h after the onset of recanalization. The zonulae occludentes also rearranged and reestablished their original structure during this period. The serum bilirubin fell to normal within 24 h of recanalization. It is concluded that metabolic and ultrastructural restitution associated with the recanalization of the ligated bile duct have no strict temporal correlation to one another.These studies provide further evidence that alterations in gap and tight junctions induced by pathological processes, e.g. during bile duct ligation, are completely reversible when regeneration occurs.Summer student from Harvard Medical School, Boston (USA)     

Unusual neuromuscular junctions in the heart of the crayfish (Procambarus clarkii)

Summary The neuromuscular junctions in the crayfish heart were studied with the electron microscope and were classified into two types based on the characteristics of the post-synaptic side. Type I junction was characterized by a mazy post-synaptic apparatus which has been referred to in this work as the junctional envelope, consisting of the cytoplasmic processes and/or lamellae of the muscle cell. Type II junction on the other hand, lacked the junctional envelope. The nerve terminals in both Type I and Type II junctions contained two types of synaptic vesicles: large granular and small agranular vesicles, which were about 1000 Å and 450 Å in diameter respectively. The physiological significance of these neuromuscular junctions and the nature of their synaptic vesicles are discussed.Acknowledgement. The author wishes to express sincere gratitude to Prof. T.Yamamoto for the kind encouragement and guidance during the course of this study.The presence of this unusual neuromuscular junction, coupled with the histological characteristics of heart muscles themselves (Komuro, 1968), may be involved in the different physiological properties of the crustacean heart. This subject will be discussed in a later publication by the author.     

Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

Gap junctions between pinealocytes

Summary The intercellular junctions between the pinealocytes of male rats were investigated by freeze-fracture and conventional electron microscopy.Our findings reveal that the intercellular contacts between pineal cells, formerly described as zonulae adhaerentes or zonulae occludentes, are in fact gap junctions which are difficult to characterize in thin sections due to their peculiar geometrical arrangement, which is in the form of fenestrated communicating zonules.The arrangement of these communicating zonules around rudimentary lumina of pineal clusters and rare transitions between tight and gap junctions may point to phylogenetic transformations of occluding into communicating zonules, corresponding with the change of the pineal gland from a sensory to a secretory organ. Alternatively, these tight-to-gap junctional transitions may reflect the periodic (circadian or seasonal) activity of the pineal gland.These Studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Gap junctions between guinea-pig pinealocytes

Summary In accordance with previous results in rats, belt-like arrangements of fenestrated gap junctions have been found around the collicular segments of pineal cells in the guinea pig. In addition, macular interpinealocyte gap junctions have been observed in this species.S.-K. Huang was a recipient of a Humboldt Foundation fellowship.     

The role of gap junctions in trophoblastic cell fusion in the guinea-pig placenta

Summary Fusion of cytotrophoblast cells in the guinea-pig placenta occurs at regions of plasma membrane interdigitation where the cells are attached to one another by complex arrays of gap junctions and desmosomes. Fusion begins at the gap junctions, which are lost in this process. The desmosomes play no obvious part in the fusion mechanism and remain after fusion as sites of attachment of syncytiotrophoblast membrane to itself. It is proposed that a major role of gap junctions in placental development is to bring trophoblast plasma membranes into a close relationship which may act as a starting point for cell fusion.     

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

Passage through vertebrate gap junctions of 17/18 kDa molecules is primarily dependent upon molecular configuration

In fish, amphibians and mammals, gap junctions of some cells allow passage of elongate molecules as large as 18 kDa, while excluding smaller, less elongate molecules. Fluorescently labeled Calmodulin (17 kDa) and fluorescently labeled Troponin-C (18 kDa), when microinjected into oocytes of Danio rerio, Xenopus laevis or Mus domestica, were able to transit the gap junctions between these oocytes and the granulosa cells which surrounded them. Co-microinjected with these Ca²⁺-binding proteins, Texas-red-labeled dextran (10 kDa) remained in the microinjected cell. Osteocalcin (6 kDa), also a Ca²⁺-binding protein, but with a wide “V” shape proved unable to transit these gap junctions. Calmodulin, but not Troponin-C, was able to transit gap junctions of gonadotropin treated WB cells in culture. We show evidence that molecules as large as 18 kDa can pass through some vertebrate gap junctions, both homologous and heterologous, and that it is primarily molecular configuration which governs gap junctional permeability.     

Closing and opening of gap junction pores between two- and threedimensionally cultured tumor cells

Intercellular signal transfer via gap junction pores in cultured multicell spheroids of BICR/M1R-K cells decreases with increasing spheroid age. In two days old spheroids the pores allow passage of Lucifer yellow molecules. Two days later, this fluorescent dye is retained in the injected cell even though the cells are still electrically coupled. Gap junction plaques of considerable size are still found in 9 days old spheroids, when the cells are completely uncoupled. The same cells growing as monolayer cultures do not exhibit such a gradual closing of their gap junction pores: Their coupling is established at first cell contact, probably by a gradual opening of the pores, which remain open even up to 9 days in culture.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.