RESUMEN
Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.
Asunto(s)
Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Sinapsis Eléctricas/metabolismo , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Transmisión Sináptica/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Conexinas/genética , Conexinas/metabolismo , Sinapsis Eléctricas/ultraestructura , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen Molecular , Neuronas Motoras/citología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Periodicidad , Terminales Presinápticos/ultraestructura , Proteína Fluorescente RojaRESUMEN
Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor-encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an "ur-" bHLH gene.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Nociceptores/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Biología Computacional , Sinapsis Eléctricas/metabolismo , Sinapsis Eléctricas/ultraestructura , Embrión no Mamífero , Ontología de Genes , Canales Iónicos/genética , Canales Iónicos/metabolismo , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Anotación de Secuencia Molecular , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Nociceptores/citología , Fenotipo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Transcripción GenéticaRESUMEN
Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.
Asunto(s)
Dopamina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Animales , Antipsicóticos/farmacología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Sinapsis Eléctricas/genética , Sinapsis Eléctricas/ultraestructura , Femenino , Genotipo , Humanos , Masculino , Ratones , Polimorfismo de Nucleótido SimpleRESUMEN
Parvalbumin (PV)-positive interneurons form dendritic gap junctions with one another, but the connectivity among gap junction-coupled dendrites remains uninvestigated in most neocortical areas. We visualized gap junctions in layer 4 of the mouse barrel cortex and examined their structural details. PV neurons were divided into 4 types based on the location of soma and dendrites within or outside barrels. Type 1 neurons that had soma and all dendrites inside a barrel, considered most specific to single vibrissa-derived signals, unexpectedly formed gap junctions only with other types but never with each other. Type 2 neurons inside a barrel elongated dendrites outward, forming gap junctions within a column that contained the home barrel. Type 3 neurons located outside barrels established connections with all types including Type 4 neurons that were confined inside the inter-barrel septa. The majority (33/38, 86.8%) of dendritic gap junctions were within 75 µm from at least 1 of 2 paired somata. All types received vesicular glutamate transporter 2-positive axon terminals preferentially on somata and proximal dendrites, indicating the involvement of all types in thalamocortical feedforward regulation in which proximal gap junctions may also participate. These structural organizations provide a new morphological basis for regulatory mechanisms in barrel cortex.
Asunto(s)
Dendritas/ultraestructura , Sinapsis Eléctricas/ultraestructura , Interneuronas/ultraestructura , Corteza Somatosensorial/ultraestructura , Animales , Interneuronas/química , Masculino , Ratones Endogámicos C57BL , Parvalbúminas/análisis , Terminales Presinápticos/ultraestructura , Corteza Somatosensorial/químicaRESUMEN
Alpha-type retinal ganglion cells (alpha cells) of the same class in mammalian retina are connected by gap junctions. Electrical synapses between alpha cells were examined using combined techniques of dual patch-clamp recordings, intracellular labeling and electron microscopy in the albino rat retina. In simultaneous dual whole-cell recordings from pairs of neighboring alpha cells, bidirectional electrical synapses with symmetrical junction conductance were observed in pairs with cells of the same morphological type. Regulatory domains of gap junction protein subunit connexins in electrical synapses between alpha cells by extracellular and intracellular ligands investigated by dual whole-patch clamp recordings. I examined how passage currents through electrical synapses between alpha cells are modulated by specific antibodies against connexin36 proteins, and extracellular or intracellular application of ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance (Gj) (1.35[Formula: see text][Formula: see text][Formula: see text]0.51[Formula: see text]nS). Experimental results show that high level of intracellular cyclic AMP within examined cells suppress electrical synapses between the neighboring cells. Gj between examined cells reduced to 0.15[Formula: see text][Formula: see text][Formula: see text]0.04[Formula: see text]nS. Under application of dopamine (1.25[Formula: see text][Formula: see text][Formula: see text]0.06[Formula: see text]nS) or intracellular cyclic GMP (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS), however, Gj also remains as in the control level. Intracellular application of an antibody against the cytoplasmic loop of connexin36 reduced Gj (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS). Cocktail of the antibody against cytoplasmic connexin36 and intracellular cyclic AMP leaves Gj as in the level by single involvement of the cytoplasmic antibody. The elimination of Gj by the cytoplasmic antibody was in a dose-dependent manner. These results suggest that binding domains against cyclic AMP may be present in the cytoplasmic sites of connexin proteins to regulate channel opening of gap junctions between mammalian retinal alpha ganglion cells.
Asunto(s)
Conexinas/metabolismo , AMP Cíclico/metabolismo , Sinapsis Eléctricas/metabolismo , Espacio Intracelular/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Anticuerpos , Conexinas/antagonistas & inhibidores , Conexinas/inmunología , Relación Dosis-Respuesta a Droga , Sinapsis Eléctricas/efectos de los fármacos , Sinapsis Eléctricas/ultraestructura , Femenino , Inmunohistoquímica , Espacio Intracelular/efectos de los fármacos , Microscopía Electrónica , Técnicas de Placa-Clamp , Ratas Wistar , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/ultraestructura , Proteína delta-6 de Union ComunicanteRESUMEN
In vertebrate retinas, wide-field amacrine cells represent a diverse class of interneurons, important for the extraction of selective features, like motion or objects, from the visual scene. Most types of wide-field amacrine cells lack dedicated output processes, whereas some types spatially segregate outputs from inputs. In the tyrosine hydroxylase (TH)::green fluorescent protein (GFP) mouse line, two types of GFP-expressing wide-field amacrine cells have been described: dopaminergic type 1 and γ-aminobutyric acid-ergic type 2 cells (TH2). TH2 cells possess short and long radial processes stratifying in the middle of the inner plexiform layer, where they collect excitatory and inhibitory inputs from bipolar cells and other amacrine cells, respectively. Although it was shown that these inputs lead to ON-OFF light responses, their spatial distribution along TH2 cell processes is unknown. Also, the postsynaptic targets of TH2 cells have not been identified so far. Here, we analysed the synapse distribution of these cells in TH::GFP mice and show that they form a weakly coupled network. Electrical synapses (made of connexin36) and chemical (excitatory and inhibitory) synapses are uniformly distributed along TH2 dendrites, independent of dendrite length or distance from soma. Moreover, we reveal that TH2 cells contact at least two types of small ganglion cells; one of them is the W3 cell, a ganglion cell sensitive to object motion. Contacts were often associated with markers of inhibitory synapses. Thus, TH2 wide-field amacrine cells likely provide postsynaptic inhibition to W3 ganglion cells and may contribute to object-motion detection in the mouse retina.
Asunto(s)
Células Amacrinas/ultraestructura , Células Ganglionares de la Retina/ultraestructura , Sinapsis/ultraestructura , Animales , Sinapsis Eléctricas/ultraestructura , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Bipolares de la Retina/ultraestructura , Tirosina 3-MonooxigenasaRESUMEN
Trafficking and turnover of transmitter receptors required to maintain and modify the strength of chemical synapses have been characterized extensively. In contrast, little is known regarding trafficking of gap junction components at electrical synapses. By combining ultrastructural and in vivo physiological analysis at identified mixed (electrical and chemical) synapses on the goldfish Mauthner cell, we show here that gap junction hemichannels are added at the edges of GJ plaques where they dock with hemichannels in the apposed membrane to form cell-cell channels and, simultaneously, that intact junctional regions are removed from centers of these plaques into either presynaptic axon or postsynaptic dendrite. Moreover, electrical coupling is readily modified by intradendritic application of peptides that interfere with endocytosis or exocytosis, suggesting that the strength of electrical synapses at these terminals is sustained, at least in part, by fast (in minutes) turnover of gap junction channels. A peptide corresponding to a region of the carboxy terminus that is conserved in Cx36 and its two teleost homologs appears to interfere with formation of new gap junction channels, presumably by reducing insertion of hemichannels on the dendritic side. Thus, our data indicate that electrical synapses are dynamic structures and that their channels are turned over actively, suggesting that regulated trafficking of connexons may contribute to the modification of gap junctional conductance.
Asunto(s)
Sinapsis Eléctricas/fisiología , Canales Iónicos/fisiología , Transmisión Sináptica/fisiología , Animales , Transporte Biológico , Comunicación Celular , Conexinas/química , Conexinas/fisiología , Sinapsis Eléctricas/efectos de los fármacos , Sinapsis Eléctricas/ultraestructura , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Técnica de Fractura por Congelación , Carpa Dorada , Inmunohistoquímica , Canales Iónicos/efectos de los fármacos , Canales Iónicos/ultraestructura , Fusión de Membrana , Plasticidad Neuronal , Fragmentos de Péptidos/farmacología , Transporte de Proteínas , Proteínas SNARE/metabolismo , Proteína 25 Asociada a Sinaptosomas/química , Proteína delta-6 de Union ComunicanteRESUMEN
The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.
Asunto(s)
Microscopía Electrónica de Transmisión , Animales , Microscopía Electrónica de Transmisión/métodos , Vesículas Sinápticas/ultraestructura , Sinapsis Eléctricas/ultraestructura , Sinapsis Eléctricas/fisiología , Sinapsis/ultraestructura , Membranas Sinápticas/ultraestructura , Imagenología Tridimensional/métodosRESUMEN
Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA(A) receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2(SH3+) and CB2(SH3-), and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2(SH3+) and CB2(SH3-) into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2(SH3-), but not CB2(SH3+), can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.
Asunto(s)
Sinapsis Eléctricas/metabolismo , Neuronas GABAérgicas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Discapacidad Intelectual/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Línea Celular , Sinapsis Eléctricas/genética , Sinapsis Eléctricas/ultraestructura , Neuronas GABAérgicas/ultraestructura , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Mutación/genética , Unión Proteica/genética , Ingeniería de Proteínas , Isoformas de Proteínas/genética , Multimerización de Proteína/genética , Factores de Intercambio de Guanina Nucleótido Rho , Transgenes/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Electrical activity may cause observable changes in a cell's structure in the absence of exogenous reporter molecules. In this work, we report a low-coherence interferometric microscopy technique that can detect an optical signal correlated with the membrane potential changes in individual mammalian cells without exogenous labels. By measuring milliradian-scale phase shifts in the transmitted light, we can detect changes in the cells' membrane potential. We find that the observed optical signals are due to membrane electromotility, which causes the cells to deform in response to the membrane potential changes. We demonstrate wide-field imaging of the propagation of electrical stimuli in gap-junction-coupled cell networks. Membrane electromotility-induced cell deformation may be useful as a reporter of electrical activity.
Asunto(s)
Potenciales de Acción , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Sinapsis Eléctricas/fisiología , Sinapsis Eléctricas/ultraestructura , Células HEK293 , Humanos , Microscopía de Interferencia , Imagen Molecular , Fenómenos Ópticos , Técnicas de Placa-ClampRESUMEN
Electron microscopic investigation of gap junctions (GJ) on serial sections of rat barrel cortex has shown that GJ were in contact with one or both processes that formed chemical synapses, however, these connections could not traced in single sections. In the serial sections, it was possible to observe two GJ in the immediate proximity to one another, in a single field of vision, thus, each GJ was traced in two or three successive sections in a series. Considering the described variants of GJ arrangement in the cortex, it is suggested that GJ could be a structural basis for local synchronization of the bioelectrical activity not only at postsynaptic, but also at presynaptic level, and the formation of GJ occurs both before, and after the development of chemical synapses.
Asunto(s)
Sinapsis Eléctricas/ultraestructura , Neuronas/ultraestructura , Sinapsis/ultraestructura , Animales , Corteza Cerebral/ultraestructura , Dendritas/ultraestructura , Masculino , Microscopía Electrónica , RatasRESUMEN
Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes.
Asunto(s)
Proteínas ADAM/metabolismo , Axones/metabolismo , Sinapsis Eléctricas/metabolismo , Guanilato-Quinasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas ADAM/genética , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Homólogo 4 de la Proteína Discs Large , Sinapsis Eléctricas/ultraestructura , Proteínas Fluorescentes Verdes/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Fibras Nerviosas Mielínicas/fisiología , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-Dawley , Canales de Potasio de la Superfamilia Shaker/metabolismo , Tinción con Nitrato de Plata/métodosRESUMEN
Innexin-gap junctions in crayfish lateral giant fibers (LGFs) have an important role in escape behavior as a key component of rapid signal transduction. Knowledge of the structure and function of characteristic vesicles on the both sides of the gap junction, however, is limited. We used electron tomography to analyze the three-dimensional structure of crayfish gap junctions and gap junctional vesicles (GJVs). Tomographic analyses showed that some vesicles were anchored to innexons and almost all vesicles were connected by thin filaments. High densities inside the GJVs and projecting densities on the GJV membranes were observed in fixed and stained samples. Because the densities inside synaptic vesicles were dependent on the fixative conditions, different fixative conditions were used to elucidate the molecules included in the GJVs. The projecting densities on the GJVs were studied by immunoelectron microscopy with anti-vesicular monoamine transporter (anti-VMAT) and anti-vesicular nucleotide transporter (anti-VNUT) antibodies. Some of the projecting densities were labeled by anti-VNUT, but not anti-VMAT. Three-dimensional analyses of GJVs and excitatory chemical synaptic vesicles (CSVs) revealed clear differences in their sizes and central densities. Furthermore, the imaging data obtained under different fixative conditions and the immunolabeling results, in which GJVs were positively labeled for anti-VNUT but excitatory CSVs were not, support our model that GJVs contain nucleotides and excitatory CSVs do not. We propose a model in which characteristic GJVs containing nucleotides play an important role in the signal processing in gap junctions of crayfish LGFs.
Asunto(s)
Aletas de Animales/inervación , Astacoidea/fisiología , Sinapsis Eléctricas/ultraestructura , Fibras Nerviosas Mielínicas/ultraestructura , Animales , Sinapsis Eléctricas/metabolismo , Tomografía con Microscopio Electrónico , Reacción de Fuga/fisiología , Microscopía Inmunoelectrónica , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Fibras Nerviosas Mielínicas/metabolismo , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Sinapsis Eléctricas/fisiología , Uniones Comunicantes/fisiología , Neuronas/fisiología , Optogenética , Factores de Edad , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Conexina 26/genética , Conexina 26/metabolismo , Conexinas/genética , Conexinas/metabolismo , Conductividad Eléctrica , Sinapsis Eléctricas/metabolismo , Sinapsis Eléctricas/ultraestructura , Femenino , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Edad Gestacional , Células HEK293 , Células HeLa , Humanos , Ratones Endogámicos ICR , Ratones Noqueados , Microscopía Confocal , Neuronas/metabolismo , Neuronas/ultraestructura , Embarazo , Potenciales Sinápticos , Proteína delta-6 de Union ComunicanteRESUMEN
Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90-100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36.
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Conexinas/metabolismo , Sinapsis Eléctricas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Retina/citología , Uniones Estrechas/metabolismo , Animales , Conexinas/deficiencia , Conexinas/genética , Sinapsis Eléctricas/ultraestructura , Femenino , Técnica de Fractura por Congelación/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo/métodos , Ratas , Ratas Wistar , Transfección/métodos , Proteína de la Zonula Occludens-1 , Proteína delta-6 de Union ComunicanteRESUMEN
Stretchable and conformable synapse memristors that can emulate the behaviour of the biological neural system and well adhere onto the curved surfaces simultaneously are desirable for the development of imperceptible wearable and implantable neuromorphic computing systems. Previous synapse memristors have been mainly limited to rigid substrates. Herein, a stretchable and conformable memristor with fundamental synaptic functions including potentiation/depression characteristics, long/short-term plasticity (STP and LTP), "learning-forgetting-relearning" behaviour, and spike-rate-dependent and spike-amplitude-dependent plasticity is demonstrated based on highly elastic Ag nanoparticle-doped thermoplastic polyurethanes (TPU : Ag NPs) and polydimethylsiloxane (PDMS). The memristor can be well operated even at 60% strain and can be well conformed onto the curved surfaces. The formed conductive filament (CF) obtained from the movement of Ag nanoparticle clusters under the locally enhanced electric field gives rise to resistance switching of our memristor. These results indicate a feasible strategy to realize stretchable and conformable synaptic devices for the development of new-generation artificial intelligence computers.
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Materiales Biomiméticos/síntesis química , Sinapsis Eléctricas/química , Electrónica/instrumentación , Plasticidad Neuronal , Dispositivos Electrónicos Vestibles , Inteligencia Artificial , Dimetilpolisiloxanos/química , Sinapsis Eléctricas/fisiología , Sinapsis Eléctricas/ultraestructura , Potenciación a Largo Plazo , Conformación Molecular , Nanopartículas/química , Nanopartículas/ultraestructura , Poliuretanos/química , Plata/químicaRESUMEN
The endosomal system is proposed as a mediator of synaptic vesicle recycling, but the molecular recycling mechanism remains largely unknown. Retromer is a key protein complex which mediates endosomal recycling in eukaryotic cells, including neurons. Retromer is important for brain function and mutations in retromer genes are linked to neurodegenerative diseases. In this study, we aimed to determine the role of retromer in presynaptic structure and function. We assessed the role of retromer by knocking down VPS35, the core subunit of retromer, in primary hippocampal mouse neurons. VPS35 depletion led to retromer dysfunction, measured as a decrease in GluA1 at the plasma membrane, and bypassed morphological defects previously described in chronic retromer depletion models. We found that retromer is localized at the mammalian presynaptic terminal. However, VPS35 depletion did not alter the presynaptic ultrastructure, synaptic vesicle release or retrieval. Hence, we conclude that retromer is present in the presynaptic terminal but it is not essential for the synaptic vesicle cycle. Nonetheless, the presynaptic localization of VPS35 suggests that retromer-dependent endosome sorting could take place for other presynaptic cargo.
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Membrana Celular/metabolismo , Sinapsis Eléctricas/metabolismo , Hipocampo/patología , Neuronas/fisiología , Enfermedad de Parkinson/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Sinapsis Eléctricas/ultraestructura , Humanos , Ratones , Transporte de Proteínas , ARN Interferente Pequeño/genética , Receptores AMPA/sangre , Proteínas de Transporte Vesicular/genéticaRESUMEN
Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and "low-tech"-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses. This pre-embedding approach does not require specialized embedding devices but only commonly available equipment. The cryostat section-based procedure allows optimization of the pre-embedding immunolabelling conditions at the less laborious and time-consuming light microscopic (LM) level before the ultrastructural analyses of the immunolabelled structures can be performed on the same sample after ultrathin sectioning without further modification. The same photoreceptor synapse that has been first studied at the light microscopic level can be subsequently analyzed with this approach at the electron microscopic level at individual ultrathin sections or serial ultrathin sections from individual, identical synapses. Higher resolution EM analyses of the immunolabelled synapses can be performed with only minor modifications of the combined LM/EM procedure. The detergent-free procedure is applicable even for weakly fixed cryostat sections which is a relevant aspect for many antibodies that do not work with more strongly fixed biological samples.
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Sinapsis Eléctricas/inmunología , Sinapsis Eléctricas/ultraestructura , Interpretación de Imagen Asistida por Computador/métodos , Inmunohistoquímica/métodos , Microscopía Electrónica de Transmisión/métodos , Células Fotorreceptoras Retinianas Bastones/inmunología , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Adhesión del Tejido/métodos , Animales , Bovinos , Proteínas del Ojo/inmunología , Interpretación de Imagen Asistida por Computador/instrumentación , Inmunohistoquímica/instrumentación , Microscopía Electrónica de Transmisión/instrumentación , Microtomía , Fijación del TejidoRESUMEN
The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.
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Giro Dentado/metabolismo , Sinapsis Eléctricas/metabolismo , Potenciales Postsinápticos Excitadores , Vía Perforante/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Giro Dentado/citología , Giro Dentado/ultraestructura , Sinapsis Eléctricas/ultraestructura , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Vía Perforante/citología , Vía Perforante/ultraestructura , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genéticaRESUMEN
Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labeling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harbored Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labeling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons.