Analysis of Amperometric Spike Shapes to Release Vesicles

Watson, Daniel; Gummi, Rohit R.; Papke, Jason B.; Harkins, Amy B.

doi:10.1002/elan.201100441

Cited by 6 publications

(5 citation statements)

References 29 publications

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“…The representative averaged single spike from recorded reduction spikes in mouse ( n = 3297) and rat ( n = 409) brain slices are shown in Figure D,E. Both the current spike shape characteristics and kinetics of glutamate transients with half-time decay on submilliseconds time scale support the sensor’s ability to resolve single synaptic vesicle exocytosis events. − Hence, if commonly used recording speed of 2 Hz was used compared to 10 kHz used here, both the information on neuronal activity and exocytosis dynamics would be missing (Figure F). Summarized temporal dynamics of individual spike kinetics in Figure B,G show a sharp current rise time, a slower decay with half-time decay on submilliseconds time scale.…”

Section: Resultsmentioning

confidence: 95%

Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients

Wang

Mishra

Bergman

et al. 2019

ACS Chem. Neurosci.

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Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.

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Section: Resultsmentioning

confidence: 95%

Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients

Wang

Mishra

Bergman

et al. 2019

ACS Chem. Neurosci.

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“…These data were fitted and analyzed by a believed-novel application of fitting methodologies using previously acquired and published amperometric data (28)(29)(30). Therefore, the methods for the stable syt I knockdown with RNA i , cell cultures, and amperometry are briefly described below with reference to the previously published details.…”

Section: Methodsmentioning

confidence: 99%

“…Stably transfected cells were established as described previously (28,31) with standard stable transfection methods and selection procedures for cells that incorporated plasmids with an insert designed to specifically target syt I (shRNA-syt I) (28). The stable syt I knockdown cells, control knockdown, and untransfected cells were compared throughout the previous work and exhibited no off-target knockdown effects (28)(29)(30)32). Here, all results are reported from 100% syt I knockdown and untransfected control (wild-type, WT) cells (28,30,32).…”

Section: Cell Culture and Stable Knockdownmentioning

confidence: 99%

PC12 Cells that Lack Synaptotagmin I Exhibit Loss of a Subpool of Small Dense Core Vesicles

Adams

Harkins

2014

Biophysical Journal

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Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca(2+) sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.

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“…Furthermore, the analysis of the recorded current traces is critical for understanding the mechanisms of exocytosis. Watson et al have recently proposed a new classification of exocytotic spikes . Some of the characteristics of these peaks were analyzed and were found to be different for each class, showing that these different types of spike may correspond to different biological functions.…”

Section: Electrochemistrymentioning

confidence: 99%