RESUMO
Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.
Assuntos
Caenorhabditis elegans , Canal de Liberação de Cálcio do Receptor de Rianodina , Vesículas Sinápticas , Animais , Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Gap junctions mediate intercellular communication and are critical for development and nervous system function. Initially thought to function solely as stand-alone molecules, it has now been shown that a stomatin-like protein regulates a gap junction channel in Caenorhabditis elegans.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Junções Comunicantes/metabolismo , Proteínas de Membrana/fisiologia , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos BiológicosRESUMO
Nicotinic (cholinergic) neurotransmission plays a critical role in the vertebrate nervous system, underlies nicotine addiction, and nicotinic receptor dysfunction leads to neurological disorders. The C. elegans neuromuscular junction (NMJ) shares many characteristics with neuronal synapses, including multiple classes of postsynaptic currents. Here, we identify two genes required for the major excitatory current found at the C. elegans NMJ: acr-16, which encodes a nicotinic AChR subunit homologous to the vertebrate alpha7 subunit, and cam-1, which encodes a Ror receptor tyrosine kinase. acr-16 mutants lack fast cholinergic current at the NMJ and exhibit synthetic behavioral deficits with other known AChR mutants. In cam-1 mutants, ACR-16 is mislocalized and ACR-16-dependent currents are disrupted. The postsynaptic deficit in cam-1 mutants is accompanied by alterations in the distribution of cholinergic vesicles and associated synaptic proteins. We hypothesize that CAM-1 contributes to the localization or stabilization of postsynaptic ACR-16 receptors and presynaptic release sites.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Nicotínicos/fisiologia , Transmissão Sináptica/fisiologia , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Comportamento Animal , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Colina/farmacologia , Antagonistas Colinérgicos/farmacologia , Diagnóstico por Imagem , Di-Hidro-beta-Eritroidina/farmacologia , Estimulação Elétrica/métodos , Regulação da Expressão Gênica/genética , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Levamisol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Microscopia Eletrônica de Transmissão/métodos , Biologia Molecular , Movimento/fisiologia , Músculos/efeitos dos fármacos , Músculos/fisiologia , Mutagênese , Mutação , Junção Neuromuscular/efeitos dos fármacos , Neurônios/fisiologia , Nicotina/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Interferente Pequeno , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Fatores de Tempo , Transativadores/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The nematode Caenorhabditis elegans has long been popular with researchers interested in fundamental issues of neural development, sensory processing and behavior. Recently, advances in applying electrophysiological techniques to C. elegans have made this genetically tractable organism considerably more attractive to neurobiologists studying the molecular mechanisms of synaptic organization and function. The development of techniques that involve voltage-clamp of specific neurons and muscles has allowed the coupling of genetic perturbation techniques with electrophysiological analyses of nervous system function. Recent studies combining these biophysical and genetic techniques have provided novel insights into the mechanisms of presynaptic neurotransmitter release, postsynaptic responses to neurotransmitters and information processing by neural circuits.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Eletrofisiologia/métodos , Locomoção/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Expressão Gênica , Genoma de Protozoário , Interneurônios/fisiologia , Neurônios Motores/fisiologia , Músculo Esquelético/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Junção Neuromuscular/fisiologia , Neurônios/fisiologia , Neurônios Aferentes/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
The nematode Caenorhabditis elegans provides numerous experimental advantages for the identification and characterization of genes required for the function of the nervous system. These advantages include forward and reverse genetic tractability, a relatively simple body plan with an invariant cellular lineage, and a fully sequenced and well-annotated genome. However, one limitation of C. elegans is the relative scarcity of electrophysiological data from excitable cells. To address this limitation, high-resolution cellular techniques for probing the roles of specific gene products in the C. elegans nervous system have been recently developed. This chapter will provide an overview of the technical requirements for patch-clamp electrophysiological analysis of C. elegans neurons and muscle cells, as well as provide some illustrative examples of insights gained from the pairing of electrophysiological techniques with molecular and genetic analysis.
Assuntos
Caenorhabditis elegans/fisiologia , Músculos/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Eletrofisiologia/métodos , Técnicas de Patch-Clamp/métodosRESUMO
Ionotropic glutamate receptors (iGluRs) in Caenorhabditis elegans are predicted to have high permeability for Ca2+ because of glutamine (Q) residues in the pore loop. This contrasts to the low Ca2+ permeability of similar iGluRs in principal neurons of mammals, because of an edited arginine (R) at the critical pore position in at least one channel subunit. Here, we introduced the R residue into the pore loop of a glutamate receptor subunit, GLR-2, in C. elegans. GLR-2(R) participated in channel formation, as revealed by decreased rectification of kainate-evoked currents in electrophysiological recordings when GLR-2(R) and the wild-type GLR-2(Q) were coexpressed in worms. Notably, the transgenic worms exhibited, at low penetrance, strong phenotypic impairments including uncoordination, neuronal degeneration, developmental arrest, and lethality. Penetrance of adverse phenotypes could be enhanced by transgenic expression of an optimal GLR-2(Q)/(R) ratio, implicating channel activity as the cause. In direct support, a mutation in eat-4, which prevents glutamatergic transmission, suppressed adverse phenotypes. Suppression was also achieved by mutation in calreticulin, which is necessary for maintainance of intracellular Ca2+ stores in the endoplasmic reticulum. Thus, synaptically activated GLR-2(R)-containing iGluR channels appear to trigger inappropriate, neurotoxic Ca2+ release from intracellular stores.
Assuntos
Substituição de Aminoácidos , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Cálcio/metabolismo , Degeneração Neural/genética , Receptores de AMPA/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Arginina/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Calreticulina/genética , Calreticulina/fisiologia , Anormalidades Congênitas/genética , Retículo Endoplasmático/metabolismo , Genótipo , Glutamina/química , Transporte de Íons/genética , Ácido Caínico/farmacologia , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Dados de Sequência Molecular , Necrose , Neurônios/patologia , Fenótipo , Edição de RNA , Receptor Cross-Talk , Receptores de AMPA/química , Receptores de AMPA/deficiência , Receptores de AMPA/genética , Receptores de Glutamato/genética , Receptores de Glutamato/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Transmissão Sináptica , Tato , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
A current challenge in neuroscience is to bridge the gaps between genes, proteins, neurons, neural circuits, and behavior in a single animal model. The nematode Caenorhabditis elegans has unique features that facilitate this synthesis. Its nervous system includes exactly 302 neurons, and their pattern of synaptic connectivity is known. With only five olfactory neurons, C. elegans can dynamically respond to dozens of attractive and repellent odors. Thermosensory neurons enable the nematode to remember its cultivation temperature and to track narrow isotherms. Polymodal sensory neurons detect a wide range of nociceptive cues and signal robust escape responses. Pairing of sensory stimuli leads to long-lived changes in behavior consistent with associative learning. Worms exhibit social behaviors and complex ultradian rhythms driven by Ca(2+) oscillators with clock-like properties. Genetic analysis has identified gene products required for nervous system function and elucidated the molecular and neural bases of behaviors.