The kinetic mechanisms of fast-decay red-fluorescent genetically encoded calcium indicators.

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

Vesicular release probability sets the strength of individual Schaffer collateral synapses.

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

ScanImage: Flexible software for operating laser scanning microscopes

BACKGROUND: Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. RESULTS: We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. CONCLUSIONS: We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design

Central synapses release a resource-efficient amount of glutamate.

Why synapses release a certain amount of neurotransmitter is poorly understood. We combined patch-clamp electrophysiology with computer simulations to estimate how much glutamate is discharged at two distinct central synapses of the rat. We found that, regardless of some uncertainty over synaptic microenvironment, synapses generate the maximal current per released glutamate molecule while maximizing signal information content. Our result suggests that synapses operate on a principle of resource optimization

Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

Localized direction selective responses in the dendrites of visual interneurons of the fly

<p>Abstract</p> <p>Background</p> <p>The various tasks of visual systems, including course control, collision avoidance and the detection of small objects, require at the neuronal level the dendritic integration and subsequent processing of many spatially distributed visual motion inputs. While much is known about the pooled output in these systems, as in the medial superior temporal cortex of monkeys or in the lobula plate of the insect visual system, the motion tuning of the elements that provide the input has yet received little attention. In order to visualize the motion tuning of these inputs we examined the dendritic activation patterns of neurons that are selective for the characteristic patterns of wide-field motion, the lobula-plate tangential cells (LPTCs) of the blowfly. These neurons are known to sample direction-selective motion information from large parts of the visual field and combine these signals into axonal and dendro-dendritic outputs.</p> <p>Results</p> <p>Fluorescence imaging of intracellular calcium concentration allowed us to take a direct look at the local dendritic activity and the resulting local preferred directions in LPTC dendrites during activation by wide-field motion in different directions. These 'calcium response fields' resembled a retinotopic dendritic map of local preferred directions in the receptive field, the layout of which is a distinguishing feature of different LPTCs.</p> <p>Conclusions</p> <p>Our study reveals how neurons acquire selectivity for distinct visual motion patterns by dendritic integration of the local inputs with different preferred directions. With their spatial layout of directional responses, the dendrites of the LPTCs we investigated thus served as matched filters for wide-field motion patterns.</p

Two-way communication with neural networks in vivo using focused light

Neuronal networks process information in a distributed, spatially heterogeneous manner that transcends the layout of electrodes. In contrast, directed and steerable light offers the potential to engage specific cells on demand. We present a unified framework for adapting microscopes to use light for simultaneous in vivo stimulation and recording of cells at fine spatiotemporal resolutions. We use straightforward optics to lock onto networks in vivo, to steer light to activate circuit elements and to simultaneously record from other cells. We then actualize this 'free' augmentation on both an 'open' two-photon microscope and a leading commercial one. By following this protocol, setup of the system takes a few days, and the result is a noninvasive interface to brain dynamics based on directed light, at a network resolution that was not previously possible and which will further improve with the rapid advance in development of optical reporters and effectors. This protocol is for physiologists who are competent with computers and wish to extend hardware and software to interface more fluidly with neuronal networks.National Institutes of Health (U.S.) (Postdoctoral Fellowship)Simons Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Predoctoral Fellowship)National Institutes of Health (U.S.)Simons Foundatio

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Dendritic spines compartmentalize synaptically-evoked biochemical signals. The authors show that electrical compartmentalization provided by a spine endows the associated synapse with additional modes of calcium signaling by shaping the kinetics of synaptic calcium currents

Modelling Vesicular Release at Hippocampal Synapses

We study local calcium dynamics leading to a vesicle fusion in a stochastic, and spatially explicit, biophysical model of the CA3-CA1 presynaptic bouton. The kinetic model for vesicle release has two calcium sensors, a sensor for fast synchronous release that lasts a few tens of milliseconds and a separate sensor for slow asynchronous release that lasts a few hundred milliseconds. A wide range of data can be accounted for consistently only when a refractory period lasting a few milliseconds between releases is included. The inclusion of a second sensor for asynchronous release with a slow unbinding site, and thereby a long memory, affects short-term plasticity by facilitating release. Our simulations also reveal a third time scale of vesicle release that is correlated with the stimulus and is distinct from the fast and the slow releases. In these detailed Monte Carlo simulations all three time scales of vesicle release are insensitive to the spatial details of the synaptic ultrastructure. Furthermore, our simulations allow us to identify features of synaptic transmission that are universal and those that are modulated by structure

The Effects of NR2 Subunit-Dependent NMDA Receptor Kinetics on Synaptic Transmission and CaMKII Activation

N-Methyl-d-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function