Electrical behaviour of dendritic spines as revealed by voltage imaging.
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ABSTRACT:
SUBMITTER: Popovic MA
PROVIDER: S-EPMC4594633 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We report sensitive recording of membrane potential in single dendritic spines in cortical neurons within a brain slice using two-photon excitation and a new, fluorinated, intracellularly loaded organic dye, di-2-AN(F)EPPTEA. With a two-photon excitation wavelength of 1060 nm, we achieve voltage sensitivity of >16% change in fluorescence per 100 mV. By targeting single spines in single-voxel recordings, we attain excellent single/noise quality, with back-propagating action potentials (bAPs) visible in single sweeps while recording at 10 kHz. This recording rate allows us to reliably assess fast bAP dynamics on single sweeps including bAP rise times of 0.5 ms. The amplitude and propagation delays of the bAPs are similar among different spines located within the same dendritic region, and this is true despite large differences in spine size. The interregion differences in bAP waveforms in spines vary in relation to their distance from the soma and the caliber of their parent dendrites.
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Project description:Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.
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Project description:Dendritic spines are the primary site of excitatory synaptic input onto neurons, and are biochemically isolated from the parent dendritic shaft by their thin neck. However, due to the lack of direct electrical recordings from spines, the influence that the neck resistance has on synaptic transmission, and the extent to which spines compartmentalize voltage, specifically excitatory postsynaptic potentials, albeit critical, remains controversial. Here, we use quantum-dot-coated nanopipette electrodes (tip diameters ?15-30?nm) to establish the first intracellular recordings from targeted spine heads under two-photon visualization. Using simultaneous somato-spine electrical recordings, we find that back propagating action potentials fully invade spines, that excitatory postsynaptic potentials are large in the spine head (mean 26?mV) but are strongly attenuated at the soma (0.5-1?mV) and that the estimated neck resistance (mean 420?M?) is large enough to generate significant voltage compartmentalization. Nanopipettes can thus be used to electrically probe biological nanostructures.
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Project description:Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.
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Project description:Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.
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Project description:Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments, such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10 ?m. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases. VIDEO ABSTRACT.
| S-EPMC6042854 | biostudies-literature
Project description:Dendritic spines are major loci of excitatory inputs and undergo activity-dependent structural changes that contribute to synaptic plasticity and memory formation. Despite the existence of various classification types of spines, how they arise and which molecular components trigger their structural plasticity remain elusive. microRNAs (miRNAs) have emerged as critical regulators of synapse development and plasticity via their control of gene expression. Brain-specific miR-134s likely regulate the morphological maturation of spines, but their subcellular distributions and functional impacts have rarely been assessed. Here, we exploited atomic force microscopy to visualize in situ miR-134s, which indicated that they are mainly distributed at nearby dendritic shafts and necks of spines. The abundance of miR-134s varied between morphologically and functionally distinct spine types, and their amounts were inversely correlated with their postulated maturation stages. Moreover, spines exhibited reduced contents of miR-134s when selectively stimulated with beads containing brain-derived neurotropic factor (BDNF). Taken together, in situ visualizations of miRNAs provided unprecedented insights into the "inverse synaptic-tagging" roles of miR-134s that are selective to inactive/irrelevant synapses and potentially a molecular means for modifying synaptic connectivity via structural alteration.
| S-EPMC6511049 | biostudies-literature
Project description:Rationale: Monitoring and controlling cardiomyocyte activity with optogenetic tools offers exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. Objective: To test the hypothesis that cardiomyocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electrical activity in isolated cardiomyocytes and the whole heart as well as function and maturity in induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Methods and Results: We first generated mice with cardiomyocyte-restricted expression of VSFP2.3 and demonstrated distinct sarcolemmal localization of VSFP2.3 without any signs for associated pathologies (assessed by echocardiography). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch-clamping. The utility of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiomyocytes. Optical cardiograms (OCGs) could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in iPSC-derived cardiomyocytes. Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof-of-concept for its utility in optogenetic sensing of physiological and pathological excitation in mature and immature cardiomyocytes in vitro and in vivo. Determination of transgene (VSFP2.3) cardiotoxicity
2015-09-02 | E-GEOD-69190 | biostudies-arrayexpress
Project description:The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.
| S-EPMC3022016 | biostudies-literature
Project description:BACKGROUND:Dendritic spines of pyramidal neurons are distributed along the complicated structure of the dendritic branches and possess a variety of morphologies associated with synaptic strength. The location and structure of dendritic spines determine the extent of synaptic input integration in the postsynaptic neuron. However, how spine location or size relates to the position of innervating presynaptic cells is not yet known. This report describes a new method that represents a first step toward addressing this issue. RESULTS:The technique combines two-photon uncaging of glutamate over a broad area (~500 × 250 × 100 ?m) with two-photon calcium imaging in a narrow region (~50 × 10 × 1 ?m). The former was used for systematic activation of layer 2/3 pyramidal cells in the rat motor cortex, while the latter was used to detect the dendritic spines of layer 5 pyramidal cells that were innervated by some of the photoactivated cells. This technique allowed identification of various sizes of innervated spine located <140 ?m laterally from the postsynaptic soma. Spines distal to their parent soma were preferentially innervated by cells on the ipsilateral side. No cluster of neurons innervating the same dendritic branch was detected. CONCLUSIONS:This new method will be a powerful tool for clarifying the microarchitecture of synaptic connections, including the positional and structural characteristics of dendritic spines along the dendrites.
| S-EPMC3269225 | biostudies-literature