RESUMO
Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.
Assuntos
Inativação Luminosa Assistida por Cromóforo/métodos , Inibição Neural/fisiologia , Optogenética/métodos , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Caenorhabditis elegans , Células Cultivadas , Hipocampo/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Compartimento Celular , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Endocitose , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Genes de Helmintos/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Mutação/genética , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Axons and dendrites of developing neurons establish distributed innervation patterns enabling precise discrimination in sensory systems. We describe the role of the extracellular matrix molecule, laminin beta2, interacting with the Ca(V)2.2 calcium channel in establishing appropriate sensory innervation. In vivo, Ca(V)2.2 is expressed on the growth cones of Xenopus laevis sensory neurites and laminin beta2 is expressed in the skin. Culturing neurons on a laminin beta2 substrate inhibits neurite outgrowth in a specific and calcium-dependent manner. Blocking signaling between laminin beta2 and Ca(V)2.2 leads to increased numbers of sensory terminals in vivo. These findings suggest that interactions between extracellular matrix molecules and calcium channels regulate connectivity in the developing nervous system.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Códon de Terminação/fisiologia , Inibidores do Crescimento/fisiologia , Laminina/genética , Neuritos/fisiologia , Neurônios Aferentes/fisiologia , Transdução de Sinais/genética , Animais , Canais de Cálcio Tipo N/genética , Proliferação de Células , Células Cultivadas , Códon de Terminação/genética , Feminino , Laminina/antagonistas & inibidores , Laminina/biossíntese , Inibição Neural/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Transdução de Sinais/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevisRESUMO
Neurotransmitters are essential for interneuronal signalling, and the specification of appropriate transmitters in differentiating neurons has been related to intrinsic neuronal identity and to extrinsic signalling proteins. Here we show that altering the distinct patterns of Ca2+ spike activity spontaneously generated by different classes of embryonic spinal neurons in vivo changes the transmitter that neurons express without affecting the expression of markers of cell identity. Regulation seems to be homeostatic: suppression of activity leads to an increased number of neurons expressing excitatory transmitters and a decreased number of neurons expressing inhibitory transmitters; the reverse occurs when activity is enhanced. The imposition of specific spike frequencies in vitro does not affect labels of cell identity but again specifies the expression of transmitters that are inappropriate for the markers they express, during an early critical period. The results identify a new role of patterned activity in development of the central nervous system.