RESUMO
The location of GABAergic synapses on dendrites is likely key for neuronal integration. In particular, inhibitory inputs on dendritic spines could serve to selectively veto or modulate individual excitatory inputs, greatly expanding the computational power of individual neurons. To investigate this, we have undertaken a combined functional, molecular, and ultrastructural mapping of the location of GABAergic inputs onto dendrites of pyramidal neurons from upper layers of juvenile mouse somatosensory cortex. Using two-photon uncaging of GABA, intracellular labeling with gerphyrin intrabodies, and focused ion beam milling with scanning electron microscopy, we find that most (96-98%) spines lack GABAergic synapses, although they still display GABAergic responses, potentially due to extrasynaptic GABA receptors. We conclude that GABAergic inputs, in practice, contact dendritic shafts and likely control clusters of excitatory inputs, defining functional zones on dendrites.
Assuntos
Espinhas Dendríticas/ultraestrutura , Neurônios GABAérgicos/ultraestrutura , Córtex Somatossensorial/ultraestrutura , Sinapses/ultraestrutura , Animais , Espinhas Dendríticas/fisiologia , Neurônios GABAérgicos/fisiologia , Camundongos , Células Piramidais/fisiologia , Células Piramidais/ultraestrutura , Córtex Somatossensorial/fisiologia , Sinapses/fisiologiaRESUMO
Arc/Arg3.1 is an immediate-early gene whose expression levels are increased by strong synaptic activation, including synapse-strengthening activity patterns. Arc/Arg3.1 mRNA is transported to activated dendritic regions, conferring the distribution of Arc/Arg3.1 protein both temporal correlation with the inducing stimulus and spatial specificity. Here, we investigate the effect of increased Arc/Arg3.1 levels on synaptic transmission. Surprisingly, Arc/Arg3.1 reduces the amplitude of synaptic currents mediated by AMPA-type glutamate receptors (AMPARs). This effect is prevented by RNAi knockdown of Arc/Arg3.1, by deleting a region of Arc/Arg3.1 known to interact with endophilin 3 or by blocking clathrin-coated endocytosis of AMPARs. In the hippocampal slice, Arc/Arg3.1 results in removal of AMPARs composed of GluR2 and GluR3 subunits (GluR2/3). Finally, Arc/Arg3.1 expression occludes NMDAR-dependent long-term depression. Our results demonstrate that Arc/Arg3.1 reduces the number of GluR2/3 receptors leading to a decrease in AMPAR-mediated synaptic currents, consistent with a role in the homeostatic regulation of synaptic strength.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de AMPA/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Biotinilação/métodos , Western Blotting/métodos , Proteínas do Citoesqueleto/genética , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Técnicas In Vitro , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/fisiologia , Depressão Sináptica de Longo Prazo/efeitos da radiação , Modelos Biológicos , Mutagênese/fisiologia , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/efeitos da radiação , Ácido Okadáico/farmacologia , Técnicas de Patch-Clamp/métodos , Interferência de RNA/fisiologia , Ratos , Estatísticas não Paramétricas , Fatores de Tempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its ß-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.
RESUMO
We describe the selective photorelease of gamma-amino butyric acid (GABA) with a novel caged-GABA compound that uses a ruthenium complex as photosensor. This compound ("RuBi-GABA") can be excited with visible wavelengths, providing greater tissue penetration, less photo-toxicity, and faster photorelease kinetics than currently used UV light-sensitive caged compounds. Using pyramidal neurons from neocortical brain slices, we show that RuBi-GABA uncaging induces GABA-A receptor-mediated responses, has no detectable side effects on endogenous GABAergic and glutamatergic receptors and generates responses with kinetics and spatial resolution comparable to the best caged GABA compounds presently available. Finally, we illustrate two potential applications of RuBi-GABA uncaging: GABA receptor mapping, and optical silencing of neuronal firing.