Gap junctions as electrical synapses

Gap junctions are the morphological substrate of one class of electrical synapse. The history of the debate on electrical vs. chemical transmission is instructive. One lesson is that Occams razor sometimes cuts too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. More intriguing are the modulatory actions that chemical synapses can have on electrical synapses. Voltage dependence provides an important window on structure function relations of the connexins, even where the dependence may have no physiological role. The new molecular approaches will greatly advance our knowledge of where gap junctions occur and permit experimental manipulation with high specificity.     

Membrane specializations in the first optic neuropil of the housefly,Musca domestica L. I. Junctions between neurons

Summary Thin section and freeze-fracture replicas of the first optic neuropil (lamina ganglionaris) of the flyMusca were studied to determine the types, extent and location of membrane specializations between neurons. Five junctional types are found, exclusive of chemical synapses. These are gap, tight and septate junctions, close appositions between retinular (R) axons and capitate projections (in which an epithelial glial cell invaginates into an R axon). Junctional types and their cellular associations follow: gap junctions, between lamina (L) interneurons, L1–L2; tight junctions, between L1–L2; L3–L4; L4-epithelial glial cell; and R7–R8. Septate junctions, between L1–L2, L3–L4, L3-, L4-, -, and an unidentified fibre making septate junctions with L1 and L2. Close appositions are found between R axons in the distal portion of the optic cartridges of this neuropil prior to extensive R chemical synapses with L1, L2. These loci (seen in freeze-fracture replicas) have rhomboidal patches of hexagonally arrayed P face particles.Intermembranous clefts between R axons are about 50 Å and are invariably electron lucent. These points of near contact between R terminals are probably the sites of low electrical resistance measured by Shaw (1979). Capitate projections are for the first time revealed in freeze fracture surfaces. Here epithelial glia send many, short, mushroom-shaped processes invaginating into R axons forming a tenacious structural bond. All four membrane leaflets (P and E faces of R axon and glial membrane) in the capitate projection possess particles in higher densities than in the surrounding nonspecialized regions. The known, general functions of each membrane specialization were correlated with the functional capacities of those lamina neurons possessing them in an effort to interpret better the integrative capacity of this neuropil. These data provide some fine structural bases for a putative blood-brain barrier between lamina and haemolymph, between lamina and peripheral retina, and possibly between lamina and second optic neuropil.     

Electron microscopic observations of the mesencephalic nucleus of the fifth nerve in the Selachian brain

Summary The mesencephalic nucleus of the trigeminal nerve (mes V) in the brain of the skate (Raja oscellata) was studied by electron microscopy. Mes V neurons are large (40–80 m diameter) and are located in the periventricular grey matter. Their perikaryal cytoplasm is rich in Golgi apparatus, small mitochondria, rough endoplasmic reticulum, polysomes and bundles of neurofilaments. A striking feature is the presence of masses of glycogen granules, at times surrounded by membrane wrappings and lysosomal bodies.Two types of conventional synaptic contacts were made onto mes V perikarya and dendrites. One had round, agranular vesicles and usually also contained dense-cored vesicles, the other had flattened, pleomorphic, agranular vesicles and usually lacked dense-cored vesicles. Additional membrane complexes consisting of a region of gap junction flanked by sites of desmosomal attachment were observed to link neighbouring mes V neurons. Somato-somatic, dendro-somatic, axo-somatic, and dendro-dendritic junctions were noted. Except for the somato-somatic union, one or more chemical synapses were located close to the sites of gap junctions.     

Synaptic regeneration and glial reactions in the transected spinal cord of the lamprey

Summary We have examined axonal growth and synaptic regeneration in identified giant neurons of the transected lamprey spinal cord using intracellular injection of horseradish peroxidase. Wholemounts together with serial section light and electron microscopy, show that axons from identified Müller and Mauthner reticulospinal neurons grow across the lesion and regenerate new synaptic contacts. Relatively normal swimming returns in these animals by 3–4 weeks after spinal transection. This occurs despite the formation of regenerated synapses in regions of the cord that are not usually occupied by these neurons.The regenerating axons branch profusely in contrast to their unbranched state in the normal animal. In addition to showing the two synaptic configurations found normally, synapses may be formed by slender sprouts from the growing giant axon. These sprout type synaptic contacts appear unique to the regenerating neuron. Only regenerated chemical synapses were seen; the morphologically mixed chemical and electrical (gap junction) synaptic complex common in the normal animal was not observed at regenerated synapses.The site of spinal transection in the functionally recovered animal shows an increase in the number of ependymal and glial cells. Ependymal-like cells appear in regions away from the central canal. The expanded ependymal and glial processes covering the peripheral surface of the injured cord become convoluted, in contrast to their normal smooth configuration. There is no collagen within the cord at the site of transection but a considerable deposition is seen external to the cord surface.Axonal growth across a spinal lesion and subsequent synaptic regeneration can be examined in single identifiable giant interneurons in the spinal cord of the larval lamprey. This preparation may be used as an assay to investigate factors that could contribute to functional recovery following central nervous system injury in the higher vertebrates.     

A second sensory--motor--interneuron with neurosecretory granules in Hydra.

Summary Using serial-sectioning techniques for conventional transmission and high-voltage electron microscopy, we characterized the ultrastructural features and synaptic contacts of the sensory cell in tentacles ofHydra. The sensory cell has an apical specialization characterized by a recessed cilium surrounded by three rodlike stereocilia. This ciliary—stereociliary complex constitutes the receptive or dendritic pole of the sensory cell. The dense filamentous cores of the stereocilia project proximally into a narrow circumciliary cytoplasmic region connected by septate junctions to marginal processes of an enveloping epitheliomuscular cell. The central cilium has a characteristic marginal flare midway along its length and a dense filamentous substructure at its base. Pairs of branched, striated rootlets extend from the axial centriole into a mitochondria-rich region of the cell. Pigment-like granules are present in the cytoplasm around the circumciliary space. The perikaryon is characterized by an elongate nucleus surrounded by a narrow rim of cytoplasm containing prominent Golgi complexes, assorted vacuoles and dense-cored vesicles, free ribosomes, short segments of rough endoplasmic reticulum, microtubules, glycogen particles, and lipid droplets.Generally, one or two thin, naked axons extend laterally from the perikaryon into the nerve net region above the myonemes of the large epitheliomuscular cells. Within the axons are found occasional aggregates of dense-cored vesicles anden passant synapses characterized by the presence of clear or dense-cored vesicles in contact with paramembranous densities and associated intracleft cross filaments. Using these ultrastructural criteria, we demonstrated for the first time that the granule-containing sensory cells have synaptic contacts with other neurons, nematocytes, and epitheliomuscular cells; hence, we considered these cells to be sensory–motor–interneurons with neurosecretory granules. We hypothesize that this unique, apparently multifunctional neuron may be a modern representative of a primitive stem cell that gave rise evolutionarily to the sensory cells, motor neurons, interneurons, and neurosecretory cells of higher animals.     

A giant nerve net with multi-effector synapses underlying epithelial adhesive strips in the mouth ofBero? (Ctenophora)

Summary We present ultrastructural evidence for the first known example of a giant nerve net in the phylum Ctenophora. The giant fibre system inBeroë underlies paired strips of adherent epithelial cells that run inside the lips. Interlocking actin-lined cell junctions between opposing adhesive strips keepBeroë's large mouth closed while the ctenophore searches for prey. The giant neurons, up to 6–8 m in diameter, form a continuous lattice-like plexus rich in vesicles, microtubules, and presynaptic triads. A novel feature is that individual giant axons make synaptic contacts with more than one type of effector, i.e. longitudinal muscle fibres and epithelial adhesive cells. Contact of prey with sensory receptors on the lips ofBeroë induces rapid disappearance of the actin-lined adhesive cell junctions, and muscular opening of the mouth to ingest prey. Electron microscopy of food-opened mouths shows local thickening of longitudinal muscles and widening of the basal ends of epithelial cells in the adhesive strip, correlated with retraction of the adhesive epithelium into the mesoglea. Addition of 1% Triton X-100 to formaldehyde fixative in the absence of prey also elicits regional thickening of longitudinal muscles at the location of the adhesive strips (visualized by rhodamine-phalloidin staining). The giant neuron system may serve as a final common pathway to rapidly signal disassembly of actin-based junctions between adhesive cells as well as contractions of longitudinal muscles underlying the adhesive strips, thereby enablingBeroë to open its mouth rapidly to engulf prey.     

Electron microscopy of synapses in reptile spinal cord

The spinal cord of the reptile Anolis carolinensis was examined by electron microscopy. Motor neurons appear as multipolar cells 30-60 micrometer in diameter. Two types of synaptic endings are endings are present on motor neurons. The first type is characterized by distinct synaptic clefts measuring 15-20 nm between pre- and postsynaptic membranes, and by clear presynaptic vesicles. The second type of synapse, which is less common, is characterized by gap junctions between pre- and postsynaptic membranes. At these synapses, there are also clusters of clear vesicles close to the presynaptic membrane adjacent to the gap junction. These findings indicate that both chemical and electrical synaptic transmission are present in the spinal cord of Anolis.     

Ultrastructure of the Mauthner axon collateral and its synapses in the goldfish spinal cord

Summary The ultrastructure of the synapses formed by the Mauthner axon collateral (MAC) was examined in the goldfish spinal cord. All such collaterals form axo—dendritic chemical synapses with dendrites of motor and interneurons. Besides these chemical synapses, contacts have been found between some MAC and axons of interneurons, which can be characterized as gap junctions with presumed electrical transmission. All cellular processes contacted by the MAC contain dense bands of material in their cytoplasm. These dense bands are absent from other parts of the fish C.N.S.These observations and the peculiar synaptic pattern are discussed and correlated with electrophysiological results.     

Ultrastructure of the rectifying electrotonic synapses between giant fibres and pectoral fin adductor motor neurons in the hatchetfish

Summary Synapses formed by giant fibres on pectoral fin adductor motor neurons were identified by horseradish peroxidase (HRP) injection. The synapses were distributed in clusters on the somata and proximal dendrites of the motor neurons. All of the labelled synapses contained synaptic vesicles and often had clearly defined active zones characteristic of chemical synapses. Some synapses also showed gap junctions with the motor neuron soma, often directly adjacent to an active zone. The gap junctions were asymmetrical, with a thick layer of electron dense material on the postsynaptic side. Previous electrophysiological data indicate that giant fibre inputs to motor neurons are purely electrotonic and that these electrical synapses rectify.     

Giant neurons and associated synapses in the peripheral nervous system of whip spiders

Summary Whip spiders (Amblypygi) are arachnids with a specialized first pair of legs. These legs are unusually long (20–25 cm) and are not used for walking. Instead their lengthy tarsi (7–8 cm) are covered with thousands of sensory hairs (mechano- and chemoreceptors). The legs thus resemble antennae of insects. Each sensory hair is associated with 4–40 neurons whose axons are grouped together to form two large tarsal nerves. The nerves contain about 23 000 sensory axons. Whereas most of the axons measure only 0.1–0.2 m in diameter, a few are exceptionally large (3–20 m). These are giant fibres. Their large somata are located in specific segments of the tarsi. The branched dendrites of the giant neurons receive hundreds of chemical synapses, presumably from the sensory axons of the hair sensilla. Since stimulation of the tarsal tip elicits fast withdrawal reaction (80 ms), it is likely that the giant fibres provide the pathway for the rapid conduction of nerve impulses to the motor centres of the C.N.S. The system is comparable to the giant fibre system of certain insects. In contrast, however, the giant interneurons and associated synapses of whip spiders are not located in the C.N.S., but lie some 20 cm removed in the periphery. Thus, some primary sensory information already becomes processed in the peripheral nervous system, before it reaches the C.N.S.     

Large myelinated club endings on the Mauthner cell in the goldfish

Summary Thin sectioning and freeze-fracturing have revealed the distribution of gap junctions and chemical synapses in the synaptic interface of the large myelinated club endings on the lateral dendrite of the goldfish Mauthner cell. In 12 samples of club endings fractured completely or nearly completely, the apposed synaptic membrane area averaged 39.090 m², of which 16.6% was occupied by gap junctions and about 4 to 5% by the active zones of chemical synapses. The numerical profile density (number per unit area of the synaptic membrane) of gap junctions varied greatly, from 1.78 to 6.30, and was mostly in inverse proportion to their size. The chemical synapses were located mainly in two places: in the circumferential rim of the synaptic membrane next to the widened extracellular space, and in the margins of intraterminal invaginations of the synaptic cleft. The axoplasm of the preterminal axon, just after losing its myelin sheath, was filled with microtubules, among which neurofilaments gathered into many small bundles. The correlation between the areas of gap junctions and the chemical synapses and the amplitude of the excitatory postsynaptic potentials (EPSP) is discussed.     

Altered synaptic and electrical properties of lumbar motoneurons in the neurological glial mutant<Emphasis Type="Italic"> taiep</Emphasis> rat

Maturation and differentiation of electrical properties of neurons and synaptic transmission are modulated by neuronal interaction. In vitro experiments have shown that these processes also seem to be regulated by signals from non-neuronal elements such as glial cells. It is not known, however, whether glial alterations in intact neural networks may also affect the maturation of electrical properties and synaptic transmission during development. We used the taiep rat, a neurological mutant with a progressive demyelination and astrogliosis, as an experimental model to study the postnatal development of motoneurons in an altered glial environment. Using the patch-clamp technique, we made intracellular recording from motoneurons of Rexeds lamina IX in spinal cord slices of neonatal rats (postnatal day P4–P10). The electrical properties of normal motoneurons changed significantly with age, showing decreasing input resistance (R_in) and increasing membrane capacity (C_m). The rheobase increased with age, accompanied by an increase of the amplitude and a decrease of the duration of action potentials (APs). In contrast, mutant neurons showed no age-dependent changes of R_in, C_m, or AP characteristics. After blocking inhibitory transmission, intralaminar bipolar stimulation elicited, in both control and taiep motoneurons, fast glutamatergic excitatory postsynaptic potentials (EPSPs). Two types of taiep motoneurons were identified according to the temporal patterns of synaptic responses; (1) taiep _SYN neurons, which showed no significant differences to control motoneurons, and (2) taiep _ASYN neurons, in which the initial EPSP was followed by a variable number of delayed, asynchronous EPSP responses (for up to 300 ms). All these electrophysiological findings suggest that the mutation in taiep rats interfered with the development of the electrical properties of neurons and with the maturation of synaptic transmission, probably due to alterations in the neuron-glia interactions.This work was presented in part at the Fifth European Meeting on Glial Cell Function in Health and Disease (Euroglia 2002, Rome, Italy)     

The cat locus coeruleus

Summary The neuronal cell bodies of the locus coeruleus (LC) and subcoeruleus (SC) of the cat were investigated using Nissl and Golgi preparations, and electron microscopy. On the basis of morphological criteria — size and shape of cell body, branching pattern of dentrites, distribution of cytoplasmic organelles and number of axosomatic synapses — four types of neuronal perikarya were recognized in each region: medium-sized, small-sized and two groups of intermediate-sized neurons. The medium-sized neurons (30–50 m) had an elongated cell body, thick dendrites with a moderate number of branchings, abundant organelles arranged in concentric rings around the nucleus and a moderate number of axosomatic synapses. They were found throughout the LC and SC and most probably correspond to the larger class of catecholaminergic neurons demonstrated by fluorescence histochemistry. The small neurons (10–25 m) were also seen in both LC and SC and are believed to represent non-monoaminergic local interneurons. They displayed sparsely branching dendrites and a thin rim of cytoplasm containing few organelles. In the SC, some of these small cells occurred in closely associated pairs. Ultrastructural analysis of such pairs revealed a close apposition (80–100 Å) of the cell membranes for long distances (up to 10 m) and a narrowing of the intercellular space (30–40 Å) at some discrete points, perhaps indicative of an electrical interaction. The intermediate-sized neurons exhibited some regional morphological differences, but two distinct subgroups could be distinguished. One was characterized by a low number of axosomatic synapses, while the other exhibited a high number of such contacts. It may be assumed that the two subgroups of intermediate-sized neurons comprise catecholaminergic and indolaminergic neurons.This work was presented at Université Claude Bernard-Lyon as part of a Thèse de 3° Cycle entitled Etude ultrastructurale du Locus Coeruleus du Chat     

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 m (SD ± 0.18; range 0.37–1.29 m) and their mean area 0.26 m² (SD ± 0.145; range 0.075–0.93 m²); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.     

Gap junctions in the liver of parasitic adult lampreys, Petromyzon marinus L

Summary Thin-section and freeze-fracture observations of the plasma membranes of hepatocytes from parasitic adult lampreys, Petromyzon marinus, reveal large (250 nm–4.5 m diameter) gap junctions of highly irregular configuration. The multiformity of these junctions is partially due to the fact that they follow the contours of the undulating cell surface of the irregularly shaped hepatocytes. In addition, junctional membrane is characterized by a slight rippling which is not seen on adjacent non-junctional membrane. Although some annular-shaped junctions are associated to non-junctional membrane, others seem completely internalized and they surround portions of the cytoplasm. In P-face replicas the gap junctions are seen to be composed of closely packed particles of 6.0–6.5 nm diameter. E-face replicas of junctional membrane are relatively smooth, a fact which may be related to the small size of the intramembranous particles. Differences in size and shape of gap junctions in hepatocytes of larval (Peek et al. 1979) and adult lampreys may reflect the absence of bile canaliculi and bile ducts in the adult liver and an increased role of these junctions in co-ordination of an endocrine secretory mechanism.This research was supported by grant #A5945 from the Natural Sciences and Engineering Research Council of Canada to J.H.Y.     

Both electrical and chemical transmission between the ''lobula giant movement detector'' and the ''descending contralateral movement detector'' neurons of locusts are supported by electron microscopy

Summary Conventional electron microscopy combined with cobalt staining techniques has revealed chemical synapses and gap junction-like areas denoting specific regions of contact between two large, uniquely identifiable visual interneurons in the brain of the locustitSchistocerca gregaria. The morphological demonstration of chemical synapses suggests that one of the two neurons, the descending contralateral movement detector, receives a chemically mediated input from its main presynaptic element, the lobula giant movement detector. This observation supports recent electrophysiological studies demonstrating synaptic delays between the two cells, characteristic of chemical synapses. However, regions with the appearance of gap junctions are also observed. This corroborates earlier work which suggested that these two neurons are coupled electrically.Dedicated to Professor Dr B. Rensch, University of Münster, on the occasion of his 85th birthday.     

Ultrastructure and synaptology of neurons immunoreactive for gamma-aminobutyric acid in the myenteric plexus of the guinea pig small intestine

Summary Immunoreactivity for gamma-aminobutyric acid is located in one morphologically-defined class of nerve cell body in the myenteric plexus of the guinea pig small intestine. These are a subgroup of the Dogiel type I nerve cells, characterized by their lamellar dendrites, about 1 m thick and flattened in the plane of the myenteric plexus, and one (or rarely two) long axonal process that extends to either the longitudinal or the circular muscle. At an ultrastructural level the dendrites were characterized by their open cytoplasm in which were scattered granular vesicles, pale mitochondria, Golgi apparatus and endoplasmic reticulum. A large proportion of the dendritic surface was in direct contact with the extra-ganglionic space. In the cell body region, which was away from the ganglion surface, the nucleus was surrounded by a thin rim of cytoplasm. The cytoplasmic features are quite distinct from those of Dogiel type II neurons but they were shared by many other non-immunoreactive neurons. Synaptic inputs, which were all non-immunoreactive, were found on the dendrites, cell bodies, axon hillocks and axons of the gamma-aminobutyric acid-immunoreactive neurons. The predominant vesicle type in the presynaptic elements was the small clear vesicle, 40–60 nm in diameter. Based on two gamma-aminobutyric acid-immunoreactive cells that were examined in serial section, about 40–50% of synapses are dendritic, 20–25% are somatic, and 30–35% are on the axon hillock or first 50–70 m of the axon. No synapses formed by immunoreactive varicosities were found on non-immunoreactive neurons or in the neuropil of the myenteric ganglia. Moreover, the lamellar dendrites or soma of gamma-aminobutyric acid neurons were never presynaptic elements forming relationships with other elements in the ganglia. It is concluded that the gamma-aminobutyric acid reactive Dogiel type I neurons are motor neurons providing inputs to the circular and longitudinal muscle layers.     

Dendritic bottlenecks of crustacean motoneurons

Summary Cobalt-labelled fast flexor motoneurons of the crayfish (Procambarus) were studied by electron microscopy after treatment with diaminobenzidine. The neurons were traced into the abdominal ganglion to locations at which they made contacts with the lateral giant fibres of the nerve cord. Fine secondary dendritic branches extended from the primary dendrites of the fast flexor motoneurons to the lateral giant fibre. These fine branches had bottlenecks at various places along their lengths and also at their junctions with primary dendrites. Chemical synapses occurred at the bottlenecks and at other locations on the fine branches. It is postulated that chemical synapses at dendritic bottlenecks could act to modify the effectiveness of the excitatory drive provided by the lateral giant fibres to the fast flexor motoneurons, most likely by gating electrical signals conveyed by the fine dendrites.     

Regenerated synapses persist in the superior colliculus after the regrowth of retinal ganglion cell axons

Summary Synapse formation by retinal ganglion cell axons was sought in the superior colliculus of four adult rats 16–18 months after the optic nerve was transected and replaced by a peripheral nerve graft that guided regenerating RGC axons from the eye to the superior colliculus. The terminals of retinal ganglion cell axons were labelled by intravitreal injections of tritiated amino acids and studied by light and electron microscopic autoradiography. We found that (i) retinal ganglion cell axons had extended from the tips of the peripheral nerve grafts into the superior colliculus for approximately 350 ,m; (ii) within the superior colliculus, some regenerated retinal ganglion cell axons became ensheathed by CNS myelin; (iii) retinal ganglion cell terminals formed asymmetric synapses with dendrites of neurons in the superficial layers of the superior colliculus, mainly the stratum griseum superficialis.Regenerated (n=418) and normal retinal ganglion cell terminals (n=1775) in the superior colliculus were compared in terms of their size (area, perimeter, and maximum diameter), contacts per terminal, contacts per 10 m terminal perimeter, and post-synaptic structure contacted (dendritic spine, shaft, or soma). No statistically significant differences in the ultrastructural characteristics of the pre-synaptic profiles were apparent between the two groups. The post-synaptic structures contacted by axon terminals were similar in regenerated and control animals, although there were quantitative differences in the distributions of these contacts among dendritic spines and shafts.These results suggest that the regeneration of retinal ganglion cell axons in adult rats can lead to the formation of ultrastructurally normal synapses in the appropriate layers of the superior colliculus. The re-formed connections appear to persist for the life-span of these animals.A short account of this work was presented inSociety for Neurosdence Abstracts 14, 654 (1988).     

Response of auditory system of bats to ultrasonic stimuli

Conclusions Combined electrical responses in the inferior colliculus to short ultrasounds, and responses of single neurons of the inferior colliculus were recorded in the bat,Myotis oxygnathus. The amplitude of the combined electrical response, reflecting the number of excited neurons, is proportional to the intensity of ultrasound within the range from 0 to 100 db. The frequency-threshold curve for this response is Ushaped. The thresholds are relatively low in the zone 10–80 kHz, with a principal minimum at 20–30 kHz, and an additional minimum at 70 kHz. A second, very strong stimulus evokes a minimal electrical response after only 1 msec, and a response of normal amplitude 4 msec after the first stimulus. Singular collicular neurons have different frequency-threshold curves: wide, narrow, and also partly or completely truncated by inhibition arising in the course of supramaximal stimulation.