High-speed imaging of glutamate release with genetically encoded sensors

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

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 that allow investigating 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 of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

Related collections

Most cited references 35

Record: found
Abstract: found
Article: not found

Short-term synaptic plasticity.

Robert S. Zucker, Wade Regehr (2002)

Synaptic transmission is a dynamic process. Postsynaptic responses wax and wane as presynaptic activity evolves. This prominent characteristic of chemical synaptic transmission is a crucial determinant of the response properties of synapses and, in turn, of the stimulus properties selected by neural networks and of the patterns of activity generated by those networks. This review focuses on synaptic changes that result from prior activity in the synapse under study, and is restricted to short-term effects that last for at most a few minutes. Forms of synaptic enhancement, such as facilitation, augmentation, and post-tetanic potentiation, are usually attributed to effects of a residual elevation in presynaptic [Ca(2+)]i, acting on one or more molecular targets that appear to be distinct from the secretory trigger responsible for fast exocytosis and phasic release of transmitter to single action potentials. We discuss the evidence for this hypothesis, and the origins of the different kinetic phases of synaptic enhancement, as well as the interpretation of statistical changes in transmitter release and roles played by other factors such as alterations in presynaptic Ca(2+) influx or postsynaptic levels of [Ca(2+)]i. Synaptic depression dominates enhancement at many synapses. Depression is usually attributed to depletion of some pool of readily releasable vesicles, and various forms of the depletion model are discussed. Depression can also arise from feedback activation of presynaptic receptors and from postsynaptic processes such as receptor desensitization. In addition, glial-neuronal interactions can contribute to short-term synaptic plasticity. Finally, we summarize the recent literature on putative molecular players in synaptic plasticity and the effects of genetic manipulations and other modulatory influences.

0 comments Cited 1024 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

An optimized fluorescent probe for visualizing glutamate neurotransmission

Jonathan S. Marvin, Bart G. Borghuis, Lin Tian … (2013)

We describe an intensity-based glutamate-sensing fluorescent reporter (“iGluSnFR”) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted post-synaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

0 comments Cited 354 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.

Björn Granseth, Benjamin Odermatt, Stephen J Royle … (2006)

The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.