RESUMO
Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to â¼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGlu f ) and ultrafast (iGlu u ) variants with comparable brightness but increased Kd for glutamate (137 µM and 600 µM, respectively). Compared with iGluSnFR, iGlu u has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGlu u is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGlu u responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.
Assuntos
Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Masculino , Plasticidade Neuronal , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s-1, compared with 109 s-1 for f-RGECO1 and f-RGECO2 (37 °C). Thus, the CaM-peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM-peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.
Assuntos
Antraquinonas/metabolismo , Sinalização do Cálcio , Cálcio/análise , Cálcio/metabolismo , Antraquinonas/química , Células HEK293 , Humanos , Cinética , Modelos MolecularesRESUMO
Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic guanylate kinase-associated protein (GKAP) controls the bidirectional synaptic scaling of AMPA receptors (AMPARs); however, mechanisms by which chronic activity triggers cytoskeletal remodeling to downscale synaptic transmission are barely understood. Here, we report that the microtubule-dependent kinesin motor Kif21b binds GKAP and likewise is located in dendritic spines in a myosin Va- and neuronal-activity-dependent manner. Kif21b depletion unexpectedly alters actin dynamics in spines, and adaptation of actin turnover following chronic activity is lost in Kif21b-knockout neurons. Consistent with a role of the kinesin in regulating actin dynamics, Kif21b overexpression promotes actin polymerization. Moreover, Kif21b controls GKAP removal from spines and the decrease of GluA2-containing AMPARs from the neuronal surface, thereby inducing homeostatic synaptic downscaling. Our data highlight a critical role of Kif21b at the synaptic actin cytoskeleton underlying homeostatic scaling of neuronal firing.
Assuntos
Actinas , Cinesinas , Actinas/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Miosinas/metabolismo , Espinhas Dendríticas/metabolismoRESUMO
The strength of an excitatory synapse relies on the amount of glutamate it releases and on the amount of postsynaptic receptors responding to the released glutamate. Here we describe a strategy to investigate presynaptic release independently of postsynaptic receptors, using a genetically encoded glutamate indicator (GEGI) such as iGluSnFR to measure synaptic transmission in rodent organotypic slice cultures. We express the iGluSnFR in CA3 pyramidal cells and perform two-photon glutamate imaging on individual Schaffer collateral boutons in CA1. Sparse labeling is achieved via transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning microscopy. A spiral scan path over an individual presynaptic bouton allows to sample at high temporal resolution the local release site in order to capture the peak of iGluSnFR transients.
Assuntos
Ácido Glutâmico , Transmissão Sináptica , Hipocampo , Microscopia Confocal , Células Piramidais/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.
Assuntos
Cálcio , Vesículas Sinápticas , Ácido Glutâmico , Hipocampo/fisiologia , Neurotransmissores , Terminações Pré-Sinápticas/fisiologia , Probabilidade , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologiaRESUMO
The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.
RESUMO
The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.