Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis

Schroeder, Timothy; Jankowski, Jeffrey A.; Kawagoe, Kirk T.; Wightman, R. Mark; Lefrou, Christine; Amatore, Christian

doi:10.1021/ac00048a003

Cited by 151 publications

(170 citation statements)

References 12 publications

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“…Exocytotic spikes were identified, and area (pC), t 1/2 (ms), and i max (pA) values for each spike were determined using a multipass algorithm described previously (Schroeder et al, 1992). Signals were designated as spikes if their i max values were eight times the SD (typically 0.6 pA) of a 1-s portion of stable baseline recorded before the first stimulation.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells

Colliver

Hess

Pothos

et al. 2000

Journal of Neurochemistry

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Previously used methods of comparing amperometric spike characteristics from two separate groups of cells have entailed pooling all the values for a spike characteristic from each group of cells and then statistically comparing the two samples. Although this approach has indicated that there are significant differences between the spike characteristics from coloboma and control mouse chromaffin cells, the results are not consistent between experiments. We have reexamined the assumptions of the statistical tests used as well as the variability inherent in amperometric data measured from two groups of cells. Our findings indicate that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values. This method consistently indicates that there is no difference between coloboma and control amperometric spikes. These results have been validated by using samples of mean spike characteristics to detect changes in the shape of amperometric spikes from both mouse chromaffin cells at 37°C and PC12 cells previously exposed to 50 M L-3,4-dihydroxyphenylalanine and by the use of an additional analysis method, the nested ANOVA. Together, these results indicate that pooled samples of amperometric spike characteristics can give results that may confound the interpretation of amperometric data.

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Section: Discussionmentioning

confidence: 99%

Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells

Colliver

Hess

Pothos

et al. 2000

Journal of Neurochemistry

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“…Data were displayed in real time (Axoscope 1.1.1.14, Axon Instruments) and stored to a computer with no subsequent filtering. E xocytotic spikes were identified, and the spike characteristics-area (pC), t 1/2 (msec), and I max (pA)-were determined by using a multi-pass algorithm described previously (Schroeder et al, 1992). Signals were designated as spikes if their I max values were six times the SD (typically 0.7 pA) of a 1 sec portion of stable baseline recorded before the first stimulation.…”

Section: Methodsmentioning

confidence: 99%

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

et al. 2000

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It has been well established that the volume of secretory vesicles can be modulated. However, we present the first data demonstrating that the amount of transmitter in a vesicle can regulate its volume. Amperometry and transmission electron microscopy have been used to determine that L-3,4-dihydroxyphenylalanine and reserpine increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles as well as their catecholamine content. Because changes in vesicular catecholamine content are tracked by changes in vesicle volume, our results indicate that when quantal size is altered via the vesicular monoamine transporter the concentration of catecholamines within the vesicles remains relatively constant. This previously unidentified cellular response provides new insight into how catecholamines can be packaged in and released from secretory vesicles.

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“…Event analysis addresses quantal analysis, vesicular concentrations, considerations for patch capacitance measurements and fusion pore analysis 2,3,10-14 , as well as amperometric spike analysis 6,[19][20][21][22][23][24] and the relationship between cell electrical activity and fusion. An example of the type of data obtained with the method is shown in Figure 4.…”

Section: Example Of Applicationmentioning

confidence: 99%

Patch amperometry: high-resolution measurements of single-vesicle fusion and release

et al. 2005

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Patch amperometry is a new technique for the observation of single-vesicle exocytosis. Exocytosis of single vesicles as small as 50 nm in diameter can be detected by cell-attached patch-clamp admittance measurements 1-4 indicating fusion of vesicles with the plasma membrane or by amperometry with a carbon fiber electrode (CFE) 5-9 indicating release of oxidizable molecules such as catecholamines. The admittance measurement provides the membrane capacitance that increases in proportion to the membrane area because of the incorporation of the vesicle into the patch membrane. It also reveals the fusion pore conductance during an exocytotic event, giving an estimate of fusion pore dimensions. Amperometry provides the amount and time course of release of molecules that are readily oxidizable such as dopamine, norepinephrine or serotonin. This technique is capable of detecting as little as a few thousand molecules 8,9 . It also resolves the flux of catecholamines through a narrow fusion pore in a so-called foot signal that precedes rapid release indicated by an amperometric spike 6 . Patch amperometry combines high-resolution patch capacitance measurements with amperometry by placing the amperometric detector inside the patch pipet 10 . The method provides precise information on single-vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines 10-14 . Thus, it is a unique tool to investigate the mechanisms that modulate quantal size and the effect of molecular manipulations affecting the properties of the fusion pore. Here we provide step-by-step instructions for the application of this method.

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Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis

Cited by 151 publications

References 12 publications

Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells

Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

Patch amperometry: high-resolution measurements of single-vesicle fusion and release

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