Origin of the climbing fibers activated by glossopharyngeal nerve stimulation in the frog

The origin of climbing fibers activated by electrical stimulation of the frog's glossopharyngeal (IXth) nerve was investigated using histological and electrophysiological technique. At the molecular layer near the Purkinje cell layer, where the maximum negative cerebellar field potential could be recorded following electrical stimulation of the IXth nerve, horseradish peroxidase (HRP) was iontophoretically injected through the tip of the recording micropipette. The HRP labeled cells were seen in the contralateral inferior olive (IO). In some cases, a small number of HRP-labeled cells were seen in the ipsilateral IO. Labeled cells were not found in the other areas of the brain stem. After electrolytic lesion of the contralateral IO, the negative cerebellar field potential which would be recorded in the molecular layer following electrical stimulation of the IXth nerve had almost ceased. These results demonstrate that the climbing fibers activated by the IXth nerve stimulation have their origin in the contralateral IO.     

Role of gap junctions in synchronized neuronal oscillations in the inferior olive

Inferior olivary (IO) neurons are electrically coupled through gap junctions and generate synchronous subthreshold oscillations of their membrane potential at a frequency of 1-10 Hz. Whereas the ionic mechanisms of these oscillatory responses are well understood, their origin and ensemble properties remain controversial. Here, the role of gap junctions in generating and synchronizing IO oscillations was examined by combining intracellular recordings with high-speed voltage-sensitive dye imaging in rat brain stem slices. Single cell responses and ensemble synchronized responses of IO neurons were compared in control conditions and in the presence of 18beta-glycyrrhetinic acid (18beta-GA), a pharmacological gap junction blocker. Under our experimental conditions, 18beta-GA had no adverse effects on intrinsic electroresponsive properties of IO neurons, other than the block of gap junction-dependent dye coupling and the resulting change in cells' passive properties. Application of 18beta-GA did not abolish single cell oscillations. Pharmacologically uncoupled IO neurons continued to oscillate with a frequency and amplitude that were similar to those recorded in control conditions. However, these oscillations were no longer synchronized across a population of IO neurons. Our optical recordings did not detect any clusters of synchronous oscillatory activity in the presence of the blocker. These results indicate that gap junctions are not necessary for generating subthreshold oscillations, rather, they are required for clustering of coherent oscillatory activity in the IO. The findings support the view that oscillatory properties of single IO neurons endow the system with important reset dynamics, while gap junctions are mainly required for synchronized neuronal ensemble activity.     

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.     

Structural differences between the desmosome-like contacts in the chemical and afferent synapses of Mauthner neurons in the goldfish

Comparative ultrastructural investigation of the desmosome-like contacts at chemical and mixed afferent synapses of the goldfish Mauthner neurons was carried out. It was revealed that these contacts at mixed synapses differed from those at chemical ones by thin transverse fibrillar bridges which cross the gap and connect two adjoining membranes of the junction. We suppose that these crossbridges together with gap junctional connexons may serve as a substrate for electronic coupling at mixed synapses demonstrated earlier.     

Multiple climbing fibers signal to molecular layer interneurons exclusively via glutamate spillover

Spillover of glutamate under physiological conditions has only been established as an adjunct to conventional synaptic transmission. Here we describe a pure spillover connection between the climbing fiber and molecular layer interneurons in the rat cerebellar cortex. We show that, instead of acting via conventional synapses, multiple climbing fibers activate AMPA- and NMDA-type glutamate receptors on interneurons exclusively via spillover. Spillover from the climbing fiber represents a form of glutamatergic volume transmission that could be triggered in a regionalized manner by experimentally observed synchronous climbing fiber activity. Climbing fibers are known to direct parallel fiber synaptic plasticity in interneurons, so one function of this spillover is likely to involve controlling synaptic plasticity.     

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1)

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.     

Effect of electrical coupling on ionic current and synaptic potential measurements

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.     

Prionic neuroinvasion and cerebellar lesion in Creutzfeldt-Jakob disease

Examining the histological brain sections of patients with Creutzfeldt-Jakob disease (CJD) showed the accumulation of PrP(CJD) in the cerebellar cortical layers. In nCJD, the prion accumulated mainly in the granular layer in the area of synapses of moss fibers with granular cell dendrites in the cerebellar glomeruli and in the molecular layer in the area of the synapses formed by granular cell axons and great stellate neurons of the granular layer with the dendrites of Purkinje's piriform cells of busket cells and Golgi's cells. PrP(CJD) amyloid plaques were formed in these regions. In sporadic CJD, PrP(CJD) accumulated only in the cerebellar molecular layer in the area of the synapses formed by climbing fibers with the dendrites of Purkinje cells. The findings lead to the conclusion that prion spreads along different nerve fibers through the synapses. The preserved Purkinje cells contacting the plaques suggest that prion is not itself highly neurotoxic.     

Electrotonic coupling in the inferior olivary nucleus revealed by simultaneous double patch recordings

Electrotonic coupling in the inferior olivary (IO) nucleus is assumed to play a crucial role in generating the subthreshold membrane potential oscillations in olivary neurons and in synchronizing climbing fiber input into the cerebellar cortex. We studied the strength and spatial distribution of the coupling by simultaneous double patch recordings from olivary neurons in the brain slice preparation. Electrotonic coupling was observed in 50% of the cell pairs. The coupling coefficient (CC), defined as the ratio between voltage responses of the post- and the prejunctional cell, varied between 0.002 and 0.17; most of the pairs were weakly coupled. In more than 75% of the pairs, the CC was <0.05. The coupling resistance varied between 0.7 to 19.8 G(Omega), and 68% of the values fell between 0.7 to 8 G(Omega). The difference between the coupling coefficient measured on stimulation of cell 1 or cell 2 of a coupled pair was 27 +/- 16%. Direct calculation of the coupling resistance revealed an asymmetry of 24 +/- 12%, suggesting a directional preference of coupling. The coupling was voltage independent, although depolarization of either the pre- or the postjunctional neuron reduced the CC. The chance of a cell pair being coupled was 80% in immediate neighboring cells, but dropped to about 30% at a distance of 40 microm. No coupled pairs were observed at distances larger than 70 microm. In 52% of staining experiments neurobiotin injection into an olivary neuron produced indirect labeling of 1-11 nearby cells with an average of 3.8 +/- 2.9. All indirectly labeled cells were found in, or immediately adjacent, to the dendritic field of the directly stained neuron. Two distinct morphological types of olivary neurons, "curly" and "straight" cells, were found. In each case all neurons stained indirectly by dye passage through gap junctions belonged to the same type. Using the physiological data we estimated that each olivary neuron is directly coupled to about 50 neurons. Since somatic recordings may not reveal coupling through remote dendrites, we conclude that each neuron is directly connected to > or =50 neurons forming two distinct networks of curly and straight cells.     

Gap junctions between accessory medulla neurons appear to synchronize circadian clock cells of the cockroach Leucophaea maderae

The temporal organization of physiological and behavioral states is controlled by circadian clocks in apparently all eukaryotic organisms. In the cockroach Leucophaea maderae lesion and transplantation studies located the circadian pacemaker in the accessory medulla (AMe). The AMe is densely innervated by gamma-aminobutyric acid (GABA)-immunoreactive and peptidergic neurons, among them the pigment-dispersing factor immunoreactive circadian pacemaker candidates. The large majority of cells of the cockroach AMe spike regularly and synchronously in the gamma frequency range of 25-70 Hz as a result of synaptic and nonsynaptic coupling. Although GABAergic coupling forms assemblies of phase-locked cells, in the absence of synaptic release the cells remain synchronized but fire now at a stable phase difference. To determine whether these coupling mechanisms of AMe neurons, which are independent of synaptic release, are based on electrical synapses between the circadian pacemaker cells the gap-junction blockers halothane, octanol, and carbenoxolone were used in the presence and absence of synaptic transmission. Here, we show that different populations of AMe neurons appear to be coupled by gap junctions to maintain synchrony at a stable phase difference. This synchronization by gap junctions is a prerequisite to phase-locked assembly formation by synaptic interactions and to synchronous gamma-type action potential oscillations within the circadian clock.     

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.     

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.     

Artificial electrotonic coupling affects neuronal firing patterns depending upon cellular characteristics

While there have been numerous theoretical studies indicating that electrotonic coupling via gap junctions interacts with the intrinsic characteristics of the coupled neurons to modify their electrical behaviour, little experimental evidence has been provided in coupled mammalian neurons. Using an artificial electrotonic junction, two distant uncoupled neurons were coupled through the computer, and the coupling conductance was varied. Tonically firing CA1 hippocampal pyramidal neurons reduced their spike firing frequency when coupled to thalamic or pyramidal cells, showing that the electrical coupling can be considered as a low-pass filter. The strength of coupling needed to entrain spike bursts of pyramidal neurons was considerably lower than the coupling needed to synchronize two neurons with different cellular characteristics (thalamic and pyramidal cells). Coupling promoted burst firing in a non-bursting cell if it was coupled to a spontaneously bursting neuron.These results support modelling studies that indicate a role for gap-junctional coupling in the synchronization of neuronal firing and the expression of low-frequency bursting.     

Involvement of actin in electrotonic conductivity in the mixed synapses of goldfish Mauthner neurons

The effect of highly specific and selective actin-polymerizing and labelling agent, phalloidin, on electrotonic conductivity and structure of the mixed synapses of goldfish Mauthner neurons (MN) was studied. It was shown that the paired subthreshold electrostimulation of afferent input against a background of phalloidin application resulted in the average 80% increase of the amplitude of MN response to the second stimulus. In control group it increased by only 10% and was observed only after suprathreshold stimulation, while subthreshold stimuli were ineffective. We interpret these data as the manifestation of increased conductivity of the mixed synapses, induced by actin polymerization. At the ultrastructural level, phalloidin application at MN and their mixed synapses increased the size and number of actin-containing desmosome-like junctions, as well as the number of fibrillar bridges crossing their cleft. Using the phalloidin-colloid gold marker, the actin nature of these bridges was demonstrated. Interdependent morpho-functional changes found in the mixed synapses, provide the indication of actin involvement in the conduction of electrotonic signal through the mixed synapse. The bridges crossing the cleft of desmosome-like junction could be the structural substrate of this process.     

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.     

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.     

Clustering of cell bodies, bundling of dendrites, and gap junctions: morphological substrate for electrical coupling in the prepacemaker nucleus

The possible morphological basis for electrical coupling between neurons of the prepacemaker nucleus was studied in weakly electric gymnotiform fish at the ultrastructural level. Three structural characteristics were found: Extremely dense clustering of cell bodies; 'bundling' of dendrites; and gap junctions between neurons. Electrical coupling may take place through gap junctions and the spatial arrangement of elements in the prepacemaker nucleus, which could enable ephaptic interactions. Such mechanisms may also be used for averaging the responses of individual neurons in the whole assembly in order to render more predictable behavioral reactions.     

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.     

Calcium ions distribution in mixed synapses of the mauthner neurons of goldfish in the norm, fatigue, and adaptation state

The aim of this investigation was to study the structure of giant myelinated club-shaped terminals (afferent mixed synapses) of goldfish Mauthner (M-) cells in different functional states and to demonstrate calcium ion localization in them using modified pyroantimonate method. It was shown that in intact preparations calcium pyroantimonate precipitate was detected neither in gap junctions (GJ) nor in desmosome-like junctions (DLJ). The fibrillar bridges within DLJ cleft were not contrasted. After natural stimulation, which elaborated a long-term adaptation of M-cells, electron dense precipitate was found in GJ, lining all the cleft. Simultaneously fine granules and aggregates of precipitate appeared in DLJ gap and were intensely deposited over the bridges. It is known that the increase of calcium ion concentration up to and above the level demonstrable by pyroantimonate method blocks the electrotonic coupling and that filamentous actin is able to conduct electrotonic signal as a cationic current. Therefore calcium pyroantimonate staining of DLJ bridges, which were earlier shown to contain actin, indicates the association of calcium ions with filamentous actin, i.e. the functioning of bridges as transsynaptic electrotonic shunts at a moment of fixation. The data obtained allow to make a conclusion that DLJ in mixed synapses have not only a known adhesive function, but also a communicative one. The latter is manifested in extreme conditions, thus permitting synapse to maintain or change their conductivity in accordance with environmental demands.