Cobalt-coupled neurons of a giant fibre system in Diptera

Certain intact nerve cells in flies can be filled with cobalt from presynaptic or postsynaptic neurons. This cobalt coupling is best demonstrated in giant fibre systems where the phenomenon was originally termed 'transsynaptic staining'. Fine structural analysis of silver-intensified, cobalt-coupled neurons indicates that the passage of cobalt ions occurs at gap junctions that are accompanied by conventional chemical synapses. Cobalt-coupled systems in dipterous insects are uniquely identifiable and can always be detected between the same kinds of neurons. The visualization of cobalt-coupled neurons allows the identification of functional pathways linking the brain to motor neuropils.     

Morphology of spinal electromotor neurons and presynaptic coupling in the gymnotid Sternarchus albifrons.

Spinal electromotor neurons in the gymnotid Sternarchus albitrons were studied by light and electron microscopy. In this species, the electric organ discharge, which is of high and relatively constant frequency, is generated by specialized axons which arise from the spinal electromotor neurons. The cell bodies are located in medial regions of the spinal cord. They are round to ellipsoid in outline and dendrites are not peresent. The initial myelin segment often extends partially over the cell body. Fine glial lamellae are interposed between closely adjacent cells, and somato-somatic gap junctions are not observed. The large majority of axosomatic synapses are characterized by gap junctions. Single axons are commonly found to establish gap junctions with two adjacent neurons. Only a few synapses have the chracteristics associated with chemically mediated transmission. The morphological data provide evidence for electrotonic soupling between electromotor neurons by way of presynaptic fibres. The absence of dendrites in these neurons may provide a morphological correlate for their simple relay function.     

Neuro-epitheliomuscular cell and neuro-neuronal gap junctions inHydra

Summary Gap junctions have been described ultrastructurally between neurons and epitheliomuscular cells and between neurons and their processes in the hypostome peduncle and basal disc ofHydra. All gap junctions examined inHydra exhibit two apposed plasma membranes having a 2–4 nm gap continuous with the extracellular space. The gap junctions are variable in length from 0.1–1.6 m and appear linear or V-shaped in section. Neuronal gap junctions inHydra occur infrequently as compared to chemical synapses. Electron microscopy of serial sections has demonstrated the presence of adjacent electrical and chemical synapses (neuromuscular junctions) formed by the same neuron. In addition multiple gap junctions were present between two neurons. This is the first ultrastructural demonstration of electrical synapses in the nervous system ofHydra. Such synapses occur in neurons previously characterized as sensory-motor-interneurons on the basis of their chemical synapses; these neurons appear to represent a type of stem cell characterized by having both electrical and chemical synapses.     

Membrane ultrastructure of the giant synapse of the squidLoligo pealei

The ultrastructure of the ‘giant synapse’ of the stellate ganglion of the squid was studied with freeze-fracture and thin-sectioning techniques. A sheath of glial cells separates the pre- and post-synaptic axons. At intervals, round-topped processes of the postsynaptic axon pierce the sheath to contact the presynaptic axon. This area of synaptic contact is marked by a widened intercellular cleft containing electron-dense material and by a cluster of synaptic vesicles within the presynaptic cytoplasm. The number of synaptic vesicles in such clusters was greatly reduced by electrical stimulation of the synapse during fixation. Freeze-fracture reveals a roughly circular patch (0.3 μm diameter) of 10 nm particles on the cytoplasmic leaflet of the presynaptic membrane. A similar patch of particles lies on the external leaflet of the apposed postsynaptic membrane.The squid giant synapse thus consists of multiple small pre- and postsynaptic active zones where neurotransmitter is released from the presynaptic terminal and sensed by postsynaptic receptors. Comparison of the structure of these postsynaptic active zones with those at synapses where the transmitter or transmitter action is known suggests that the excitatory transmitter at this synapse is an amino acid.Presumptive gap junctions, marked by particles in the cytoplasmic leaflet, are found between small-diameter axons in the stellate ganglion but not at the giant synapse. Glial-cell membranes contain aggregates of particles and pits suggestive of gap junctions. The aggregates of pits are embedded within linear arrays of particles which somewhat resemble tight junctions.     

Morphological correlates of electrotonic coupling in the magnocellular mesencephalic nucleus of the weakly electric fish Gymnotus carapo.

The magnocellular mesencephalic nucleus (MMN) of Gymnotus carapo was studied by electron microscopy. This particular nucleus, characteristic of weakly electric fish, contains two principal classes of neuron. (1) Large neurons (25-35 mum): these are rounded unipolar cells, with the perikaryon partially covered by a sheath of compact myelin. The axon leaves the neuron as a short thick unmyelinated process not resembling the initial segment of multipolar neurons. The axon branches profusely and becomes myelinated very close to its origin. The perikaryal surface not covered by the myelin sheath receives abundant club endings. The synaptic interface between club endings and large neurons is characterized by alternating gap junctions and attachment plaques. In addition, at the periphery of the club endings, "active" zones are generally present, and this synapse is therefore a "mixed" synapse. (2) Small neurons (5-12 mum): these are uni- or bipolar cells, scattered throughout the nucleus, and occasionally, grouped in small clusters. Gap junctions were not observed between neuronal perikarya in such clusters. The synaptic investment of small neurons is formed by long cup endings which almost completely encircle the perikarya. The synaptic interface between cup endings and the perikarya of small neurons is characterized by large areas of gap junctions. A single cup ending establishing gap junctions with two small neurons within the plane of the section was frequently observed and this arrangement provides a morphological basis for electrotonic coupling between small neurons by way of presynaptic fibres. In the neuropil of the MMN, there are abundant synaptic islands constituted by a large axon terminal in synaptic contact with small unidentified profiles; both synaptic elements are surrounded by numerous thin glial lamellae. At the synaptic interface, in the islands, both gap junctions and "active" zones are present. The synaptic islands must also be considered as "mixed" synapses. The morphological results presented here correlate with electrophysiological data (Szabo et al., 1975). The very short delay (0.8-1.3 ms) of the MMS response to the fish's own electric organ discharge can only be explained by the existence of electrotonic transmission along the neuronal chain of the electrosensory pathway. The presence of gap junctions between club endings and large neurons provides a morphological basis for electrotonic transmission at the mesencephalic level of the electrosensory pathway.     

Distribution of electrotonic synapses on identified lamprey neurons: a comparison of a model prediction with an electron microscopic analysis

1. Intracellular recordings of the monosynaptic excitatory postsynaptic potential (EPSP) were made from an identified giant interneuron in the lamprey spinal cord following stimulation of a single identified presynaptic giant axon. The EPSP had both electrotonic and chemical components. 2. The giant interneuron and sometimes the giant axon were labeled with horseradish peroxidase (HRP). Using combined light and electron microscopy (EM), gap junctions formed by the axon on the HRP-labeled dendritic trees of seven interneurons were identified and their position marked on dendritic diagrams made from tracings of the cells. 3. The total number of gap junctions on a giant interneuron varied from a minimum of 9 to a maximum of 23. The number of gap junctions on a branch of a dendritic tree varied from 1 to 9. For all seven neurons the closest gap junction was located approximately 65 microns from the soma and the furthest 280 microns from the soma. 4. When gap junction position was plotted as a function of electrotonic distance from the soma, the variability in their distribution decreased significantly. Synaptic location was found to be on the average about 0.5 space constants from the soma, and this value compared favorably with that determined from an analysis of half-width and rise time of the electrotonic synaptic potential for these neurons. 5. These results suggest that the presynaptic axon tends to make, on the average, synaptic contact at the same electrical distance from the soma.     

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.     

Neuronal gap junctions in the rat main olfactory bulb,with special reference to intraglomerular gap junctions

The structural features of neuronal gap junction-forming processes in the rat olfactory bulb were analyzed electron microscopically. Gap junctions were present in glomeruli and extraglomerular regions. In extraglomerular regions, mitral/tufted cell somata, dendrites and axon hillock-initial segments made gap junctions and mixed synapses with interneuronal processes, some of which were confirmed to be GABA positive. In glomeruli gap junctions were encountered mainly between mitral/tufted cell dendrites and diverse types of processes; a small population of them were conclusively identified as periglomerular cell dendrites. Gap junction-forming processes frequently received synapses from olfactory nerve terminals, suggesting that they could be type 1 periglomerular cells. However, the majority were GABA negative or only faintly positive and none were tyrosine hydroxylase positive, indicating that they were different from previously reported type 1 periglomerular cells. Furthermore serial sectioning analyses revealed that the majority of those processes forming gap junctions with mitral/tufted dendrites were smooth cylindrical and had few presynaptic sites, indicating that they were different from previously described periglomerular cells. These findings revealed that mitral/tufted cells make gap junctions with diverse types of neurons; and some of these gap junction-forming processes originated from some types of periglomerular cells but others from hitherto uncharacterized neuron type(s).     

Ultrastructural study of gap junctions between dendrites of parvalbumin-containing GABAergic neurons in various neocortical areas of the adult rat

Parvalbumin (PV)-containing GABAergic neurons in the hippocampus form dual networks linked by both dendrodendritic gap junctions and mutual inhibitory synapses. Recent physiological studies have demonstrated similar functional connectivity among cortical GABAergic neurons, but the corresponding structures have not been fully analyzed at the electron microscopic level. In this study we examined detailed ultrastructural features of gap junctions between PV neurons in the mature neocortex. Light microscopic observations and confocal laser scanning microscopy revealed frequent dendrodendritic contacts between PV neurons. Electron microscopic analysis provided direct morphological evidence for the existence of gap junctions between 22 pairs of PV-immunoreactive dendrites in the visual, auditory, and somatosensory cortices. Their ultrastructural features that were characteristic of immunolabeled profiles were consistent with the general structure of gap junctions. In one case a gap junction coexisted with a dendrodendritic chemical synapse, making a mixed synapse. Importantly, we also encountered a gap junction between PV positive and negative, presumptive non-principal cell-derived, dendrites. Quantitative analysis was made in 16 pairs of PV positive dendrites forming gap junctions in the infragranular layers of the somatosensory cortex. Diameters of these dendrites ranged from 0.3 to 2.7 microm, suggesting diverse locations of gap junctions along the proximal-distal axis of dendritic trees, but the majority (81%) were less than 1 microm. The mean size of gap junctions along apposing membranes was 0.22+/-0.09 microm. By using this size, the theoretical value of a junctional conductance was estimated to be 2.1-5.3 nS. Dendrites of PV neurons in the infragranular layers of the somatosensory cortex were reconstructed light microscopically and the sites of contacts with other PV neurons were mapped. Although these contacts do not necessarily imply gap junctional coupling, their number (5.3+/-2.3 per cell, n=11) suggested the degree of connectivity of less than 10 coupling from single PV neurons with others. Sholl analysis revealed that only 38% of their dendrites occurred within 200 microm from the soma. The present study demonstrated detailed ultrastructural features of gap junctions between mature cortical PV neurons. These features will facilitate not only identification of gap junctions in variously labeled neurons but also analysis of their functional aspects by enabling theoretical estimate of their junctional conductances.     

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.     

Gap junctions are required for NMDA receptor dependent cell death in developing neurons

A number of studies have indicated an important role for N-methyl-D-aspartate (NMDA) receptors in cell survival versus cell death decisions during neuronal development, trauma, and ischemia. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all three of these conditions. However, whether neuronal gap junctions contribute to NMDA receptor-regulated cell death is not known. Here we address the role of neuronal gap junction coupling in NMDA receptor-regulated cell death in developing neurons. We report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In contrast, increasing or decreasing NMDA receptor function when gap junction coupling is low has no or greatly reduced impact on cell survival. Pharmacological inactivation of gap junctions or knockout of neuronal connexin 36 prevents the cell death caused by NMDA receptor hypofunction or hyperfunction. The results indicate the critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons. Based on these data, we propose the novel hypothesis that NMDA receptors and gap junctions work in concert to regulate neuronal survival.     

NMDA receptors regulate developmental gap junction uncoupling via CREB signaling

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.     

Neuronal and synaptic organization of the outer plexiform layer of the pigeon retina

The organization of the outer plexi-form layer (OPL) of the pigeon retina is described by electron microscopy and Golgi impregnation. Six types of photoreceptor, four types of horizontal cell, eight types of bipolar cell, and an interplexiform cell type were found by Golgi impregnation. The OPL was tri-stratified due to the endings of the photoreceptors at three different levels. This stratification was reflected in the laminar arrangement of the dendrites of the horizontal and bipolar cells. Electron microscopy showed that the synaptic endings of the photoreceptors made ribbon synapses, both triads and dyads, and basal junctions with the process of second-order neurons. Horizontal cells formed conventional chemical synapses, while horizontal cell axon terminals were extensively linked by gap junctions.     

The mormyrid brainstem--III. Ultrastructure and synaptic organization of the medullary "pacemaker" nucleus

The ultrastructure and synaptic organization of the presumed medullary pacemaker nucleus, nucleus c of the weakly electric mormyrid fish, Gnathonemus petersii has been investigated. Nucleus c consists of about 12-15 small (20-25 micron) neurones (P-cells), which form a group situated ventrally to the medullary relay nucleus and embedded in a neuropil of myelinated fibres and dendritic processes. The P-cells often exhibit an enhanced electron density of their cytoplasm and dendroplasm. They possess several dendrites of different diameter, a short, thin axon initial segment and a thickly myelinated axon running in dorsal direction. The pacemaker neurons are interconnected by complex electronic coupling, established by somatosomatic, dendrosomatic and dendrodendritic gap junctions. Perikarya and dendrites are frequently interconnected serially by gap junctions; dendrites showed sometimes triadic gap-junction arrangement. It is suggested that this high degree of electrotonic coupling amongst the pacemaker cells represents the first level of the highly ordered synchronization processes which characterize the electric discharge command system of Gnathonemus. Pacemaker cells receive synaptic input from club endings with mixed synapses and from bouton-like terminals with chemical synapses, both of them originating from medium-sized myelinated fibres and contacting mainly neuronal perikarya and dendritic processes. The axon initial segment receives only few synaptic inputs. Bouton-like terminals were found to be of two types according to their vesicle content, namely, boutons with ovoid, clear synaptic vesicles forming Gray type-1 synapses and boutons with pleomorphic clear synaptic vesicles forming Gray type-2 synapses. Different functional roles for the two types of boutons in modulating pacemaker cell activity are suggested.     

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Gap junctions constitute the only form of synaptic communication between neurons in the inferior olive (IO), which gives rise to the climbing fibers innervating the cerebellar cortex. Although its exact functional role remains undetermined, electrical coupling was shown to be necessary for the transient formation of functional compartments of IO neurons and to underlie the precise timing of climbing fibers required for cerebellar learning. So far, most functional considerations assume the existence of a network of permanently and homogeneously coupled IO neurons. Contrasting this notion, our results indicate that coupling within the IO is highly variable. By combining tracer-coupling analysis and paired electrophysiological recordings, we found that individual IO neurons could be coupled to a highly variable number of neighboring neurons. Furthermore, a given neuron could be coupled at remarkably different strengths with each of its partners. Freeze-fracture analysis of IO glomeruli revealed the close proximity of glutamatergic postsynaptic densities to connexin 36-containing gap junctions, at distances comparable to separations between chemical transmitting domains and gap junctions in goldfish mixed contacts, where electrical coupling was shown to be modulated by the activity of glutamatergic synapses. On the basis of structural and molecular similarities with goldfish mixed synapses, we speculate that, rather than being hardwired, variations in coupling could result from glomerulus-specific long-term modulation of gap junctions. This striking heterogeneity of coupling might act to finely influence the synchronization of IO neurons, adding an unexpected degree of complexity to olivary networks.     

The dentato-olivary projection in the rat as a presumptive GABAergic link in the olivo-cerebello-olivary loop. An ultrastructural study

It is here shown that autoradiographically labelled axon terminals of the dentato-olivary projection form a heterogeneous population. However, a majority of them constitute an even class of synapses, characterized by their small axonal size, their content in pleimorphic vesicles, and the establishment of symmetric synapses on small dendrites, about 5% of which are linked through a gap junction. The same material, used for immunocytochemistry of GABA with the postembedding technique, discloses that a majority of boutons with cytological features similar to the dentato-olivary terminals are GABA-immunoreactive, especially those synapsing on dendrites linked by gap junctions. The cerebello-olivary projection, despite its heterogeneity, thus appears as part of the GABAergic system which governs the synaptic modulation of the electrotonic coupling between olivary neurons.     

Neuronal gap junctions in the mouse main olfactory bulb: morphological analyses on transgenic mice

In the present study we analyzed the structural features of extraglomerular gap junction-forming processes in mouse olfactory bulb electron microscopically. This work complements a previous study in which we analyzed the structural features of neuronal gap junction-forming processes within the glomerulus itself. Furthermore we examined connexin 36 expressing cells in the mouse olfactory bulb by analyzing transgenic mice in which the connexin 36 coding sequence was replaced with histological reporters. In extraglomerular regions, the mitral/tufted cell somata, dendrites and axon hillocks made gap junctions and mixed synapses with interneuronal processes. These gap junctions and synapses were associated with various types of interneuronal processes, including a particular type of sheet-like or calyx-like process contacting the somata or large dendrites of mitral/tufted cells. In the olfactory bulbs of the transgenic mice, connexin 36 was expressed in mitral cells, tufted cells, presumed granule cells and periglomerular cells. Multiple immunofluorescent labelings further revealed that presumed interneurons expressing connexin 36 in the periglomerular region rarely expressed calbindin, calretinin or tyrosine hydroxylase and are likely to comprise a chemically uncharacterized class of neurons. Similarly, interneurons expressing connexin 36 in the granule cell layer were rarely positive for calretinin, which was expressed in numerous presumed granule cells in the mouse main olfactory bulb. In summary, these findings revealed that mitral/tufted cells make gap junctions with diverse types of neurons; in the glomeruli gap junction-forming interneuronal processes originated from some types of periglomerular cells but others from a hitherto uncharacterized neuron type(s), and in the extraglomerular region gap-junction forming processes originate mainly from a subset of cells within the granule cell layer.     

High-frequency population oscillations are predicted to occur in hippocampal pyramidal neuronal networks interconnected by axoaxonal gap junctions.

In hippocampal slices, high-frequency (125-333 Hz) synchronized oscillations have been shown to occur amongst populations of pyramidal neurons, in a manner that is independent of chemical synaptic transmission, but which is dependent upon gap junctions. At the intracellular level, high-frequency oscillations are associated with full-sized action potentials and with fast prepotentials. Using simulations of two pyramidal neurons, we previously argued that the submillisecond synchrony, and the rapid time-course of fast prepotentials, could be explained, in principle, if the requisite gap junctions were located between pyramidal cell axons. Here, we use network simulations (3072 pyramidal cells) to explore further the hypothesis that gap junctions occur between axons and could explain high-frequency oscillations. We show that, in randomly connected networks with an average of two gap junctions per cell, or less, synchronized network bursts can arise without chemical synapses, with frequencies in the experimentally observed range (spectral peaks 125-182 Hz). These bursts are associated with fast prepotentials (or partial spikes and spikelets) as observed in physiological recordings. The critical assumptions we must make for the oscillations to occur are: (i) there is a background of ectopic axonal spikes, which can occur at low frequency (one event per 25 s per axon); (ii) the gap junction resistance is small enough that a spike in one axon can induce a spike in the coupled axon at short latency (in the model, a resistance of 273 M omega works, with an associated latency of 0.25 ms). We predict that axoaxonal gap junctions, in combination with recurrent excitatory synapses, can induce the occurrence of high-frequency population spikes superimposed on epileptiform field potentials.