Dendrodendritic electrical synapses between mammalian retinal ganglion cells.
ABSTRACT: Electrical synapses between alpha-type ganglion cells were detected using combined techniques of dual patch-clamp recordings, intracellular labeling, electron microscopy, and channel subunit connexin immunocytochemistry in the albino rat retina. After intracellular injection of Neurobiotin into alpha-cells of inner (ON-center) and outer (OFF-center) ramifying types, measurement of tracer coupling resulted in a preferentially homologous occurrence among cells of the same morphological type (n = 19 of 24). In high-voltage as well as conventional electron microscopic analysis, direct dendrodendritic gap junctions (average size, 0.86 mum long) were present in contact sites between tracer-coupled alpha-cells. In simultaneous dual whole-cell recordings from pairs of neighboring alpha-cells, these cells generated TTX-sensitive sustained spiking against extrinsic current injection, and bidirectional electrical synapses (maximum coupling coefficient, 0.32) with symmetrical junction conductance (average, 1.35 nS) were observed in pairs with cells of the same morphological type. Precise temporal synchronization of spike activity (average time delay, 2.7 msec) was detected when depolarizing currents were simultaneously injected into the pairs. To address whether physiologically identified electrical synapses constitute gap junctional connectivity between cell pairs, identified neuronal connexin36 immunoreactivity was undertaken in Lucifer yellow-labeled cell pairs after patch-clamp recordings. All alpha-cells expressed connexin36, and confocal laser-scanning imaging demonstrated that connexin36 is primarily located at dendritic crossings between electrically coupled cells (seven sites in a pair, on average). These results give conclusive evidence for electrical synapses via dendrodendritic gap junctions involving connexin36 in alpha retinal ganglion cells of the same physiological type.
Project description:Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36-EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin.
Project description:Neurons in the enteric nervous system utilize numerous neurotransmitters to orchestrate rhythmic gut smooth muscle contractions. We examined whether electrical synapses formed by gap junctions containing connexin36 also contribute to communication between enteric neurons in mouse colon. Spontaneous contractility properties and responses to electrical field stimulation and cholinergic agonist were altered in gut from connexin36 knockout vs. wild-type mice. Immunofluorescence revealed punctate labelling of connexin36 that was localized at appositions between somata of enteric neurons immunopositive for the enzyme nitric oxide synthase. There is indication for a possible functional role of gap junctions between inhibitory nitrergic enteric neurons.
Project description:In the mammalian retina, amacrine cells represent the most diverse cell class and are involved in the spatio-temporal processing of visual signals in the inner plexiform layer. They are connected to bipolar, other amacrine and ganglion cells, forming complex networks via electrical and chemical synapses. The small-field A8 amacrine cell was shown to receive non-selective glutamatergic input from OFF and ON cone bipolar cells at its bistratified dendrites in sublamina 1 and 4 of the inner plexiform layer. Interestingly, it was also shown to form electrical synapses with ON cone bipolar cells, thus resembling the rod pathway-specific AII amacrine cell. In contrast to the AII cell, however, the electrical synapses of A8 cells are poorly understood. Therefore, we made use of the Ier5-GFP mouse line, in which A8 cells are labeled by GFP, to study the gap junction composition and frequency in A8 cells. We found that A8 cells form <20 gap junctions per cell and these gap junctions consist of connexin36. Connexin36 is present at both OFF and ON dendrites of A8 cells, preferentially connecting A8 cells to type 1 OFF and type 6 and 7 ON bipolar cells and presumably other amacrine cells. Additionally, we show that the OFF dendrites of A8 cells co-stratify with the processes of dopaminergic amacrine cells from which they may receive GABAergic input via GABAA receptor subunit ?3. As we found A8 cells to express dopamine receptor D1 (but not D2), we also tested whether A8 cell coupling is modulated by D1 receptor agonists and antagonists as was shown for the coupling of AII cells. However, this was not the case. In summary, our data suggests that A8 coupling is differently regulated than AII cells and may even be independent of ambient light levels and serve signal facilitation rather than providing a separate neuronal pathway.
Project description:A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.
Project description:Electrical synaptic transmission via gap junctions has become an accepted feature of neuronal communication in the mammalian brain, and occurs often between dendrites of interneurons in major brain structures, including the hippocampus. Electrical and dye-coupling has also been reported to occur between pyramidal cells in the hippocampus, but ultrastructurally-identified gap junctions between these cells have so far eluded detection. Gap junctions can be formed by nerve terminals, where they contribute the electrical component of mixed chemical/electrical synaptic transmission, but mixed synapses have only rarely been described in mammalian CNS. Here, we used immunofluorescence localization of the major gap junction forming protein connexin36 to examine its possible association with hippocampal pyramidal cells. In addition to labeling associated with gap junctions between dendrites of parvalbumin-positive interneurons, a high density of fine, punctate immunolabeling for Cx36, non-overlapping with parvalbumin, was found in subregions of the stratum lucidum in the ventral hippocampus of rat brain. A high percentage of Cx36-positive puncta in the stratum lucidum was localized to mossy fiber terminals, as indicated by co-localization of Cx36-puncta with the mossy terminal marker vesicular glutamate transporter-1, as well as with other proteins that are highly concentrated in, and diagnostic markers of, these terminals. These results suggest that mossy fiber terminals abundantly form mixed chemical/electrical synapses with pyramidal cells, where they may serve as intermediaries for the reported electrical and dye-coupling between ensembles of these principal cells. This article is part of a Special Issue entitled Electrical Synapses.
Project description:Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14-3-3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.
Project description:The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells.
Project description:The synchronization of neuronal activity is thought to enhance information processing. There is much evidence supporting rhythmically bursting external tufted cells (ETCs) of the rodent olfactory bulb glomeruli coordinating the activation of glomerular interneurons and mitral cells via dendrodendritic excitation. However, as bursting has variable significance at axodendritic cortical synapses, it is not clear if ETC bursting imparts a specific functional advantage over the preliminary spike in dendrodendritic synaptic networks. To answer this question, we investigated the influence of single ETC bursts and spikes with the in vitro rat olfactory bulb preparation at different levels of processing, via calcium imaging of presynaptic ETC dendrites, dual electrical recording of ETC -interneuron synaptic pairs, and multicellular calcium imaging of ETC-induced population activity. Our findings supported single ETC bursts, versus single spikes, driving robust presynaptic calcium signaling, which in turn was associated with profound extension of the initial monosynaptic spike-driven dendrodendritic excitatory postsynaptic potential. This extension could be driven by either the spike-dependent or spike-independent components of the burst. At the population level, burst-induced excitation was more widespread and reliable compared with single spikes. This further supports the ETC network, in part due to a functional advantage of bursting at excitatory dendrodendritic synapses, coordinating synchronous activity at behaviorally relevant frequencies related to odor processing in vivo.
Project description:Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36-both expressed in AII amacrine cells-are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.
Project description:We combined the Hodgkin-Huxley equations and a 36-state model of gap junction channel gating to simulate electrical signal transfer through electrical synapses. Differently from most previous studies, our model can account for dynamic modulation of junctional conductance during the spread of electrical signal between coupled neurons. The model of electrical synapse is based on electrical properties of the gap junction channel encompassing two fast and two slow gates triggered by the transjunctional voltage. We quantified the influence of a difference in input resistances of electrically coupled neurons and instantaneous conductance-voltage rectification of gap junctions on an asymmetry of cell-to-cell signaling. We demonstrated that such asymmetry strongly depends on junctional conductance and can lead to the unidirectional transfer of action potentials. The simulation results also revealed that voltage spikes, which develop between neighboring cells during the spread of action potentials, can induce a rapid decay of junctional conductance, thus demonstrating spiking activity-dependent short-term plasticity of electrical synapses. This conclusion was supported by experimental data obtained in HeLa cells transfected with connexin45, which is among connexin isoforms expressed in neurons. Moreover, the model allowed us to replicate the kinetics of junctional conductance under different levels of intracellular concentration of free magnesium ([Mg2+]i), which was experimentally recorded in cells expressing connexin36, a major neuronal connexin. We demonstrated that such [Mg2+]i-dependent long-term plasticity of the electrical synapse can be adequately reproduced through the changes of slow gate parameters of the 36-state model. This suggests that some types of chemical modulation of gap junctions can be executed through the underlying mechanisms of voltage gating. Overall, the developed model accounts for direction-dependent asymmetry, as well as for short- and long-term plasticity of electrical synapses. Our modeling results demonstrate that such complex behavior of the electrical synapse is important in shaping the response of coupled neurons.