Automatic analysis for amperometrical recordings of exocytosis

Segura, Fernando; Brioso, Miguel A.; Gómez, José Luís Graña; Machado, José David; Borges, Ricardo

doi:10.1016/s0165-0270(00)00309-5

Cited by 84 publications

(77 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The analysis of individual exocytotic events included the measurement of the following parameters: I max , maximum oxidation current, expressed in picoamperes; t 1/2 , spike width at half height, expressed in milliseconds; Q, spike net charge, expressed in picocoulombs; m, ascending slope of spike, expressed in nanoamperes/second (for details, see Machado et al, 2000;Segura et al, 2000). To analyze the falling part of the spike, two extra measurements were taken: Ft 1 and Ft 2 (falling times 1 and 2), as the time from the spike maximum point to reach the I max /e and the I max /1 Ϫ e points of the spike, respectively.…”

Section: Methodsmentioning

confidence: 99%

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Montesinos¹,

Machado²,

Camacho³

et al. 2008

J. Neurosci.

121

142

View full text Add to dashboard Cite

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited ϳ30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with L-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that L-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

show abstract

Section: Methodsmentioning

confidence: 99%

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Montesinos¹,

Machado²,

Camacho³

et al. 2008

J. Neurosci.

121

142

View full text Add to dashboard Cite

show abstract

“…An ADInstrument MacLab/4e interface controlled by a Macintosh G4 running the MacLab Chart application was used to record, display, and analyze simultaneously the [Ca 2ϩ ] c and electrochemical data. Individual spike characteristics were analyzed using the automatic analysis program IGOR (Wavemetrics, Lake Oswego, OR) and the macros described by Segura et al (2000).…”

Section: Methodsmentioning

confidence: 99%

Modulatory Mechanism of the Endogenous Peptide Catestatin on Neuronal Nicotinic Acetylcholine Receptors and Exocytosis

et al. 2002

View full text Add to dashboard Cite

The catestatin fragment of chromogranin A is the first known endogenous compound able to inhibit catecholamine release elicited by the activation of neuronal nicotinic acetylcholine receptors (nAChRs) of different animal species and catecholaminergic cell types. However, how catestatin regulates the receptor activity, which subunit combination of the heteropentameric forms of receptor is better blocked by the peptide, or how it affects the different stages of the exocytotic process have not yet been evaluated. To address these questions, we have assayed the effects of catestatin: (first) on the inward currents elicited by ACh (I ACh ) in voltage-clamped oocytes expressing different combinations of nAChR subunits; and (second) on the cytosolic Ca 2ϩ concentration, [Ca 2ϩ ] c , and quantal release of catecholamines simultaneously monitored in single adrenal chromaffin cells stimulated with ACh. Catestatin potently blocks all the subtypes of nAChRs studied. Furthermore, it inhibits the ␣ 3 ␤ 4 current in a reversible, noncompetitive, voltage-, and use-dependent manner, a behavior compatible with open-channel blockade. In fura-2-loaded single chromaffin cells, the peptide reduced the [Ca 2ϩ ] c signal and the total release of catecholamines elicited by ACh; however, catestatin did not modify the kinetics or the last step of the exocytotic process. Our results suggest that catestatin might play an autocrine regulatory role in neuroendocrine secretion through its interaction with different native nAChR subtypes; the extent of receptor blockade by the peptide could be acutely regulated by the intensity and duration of the presynaptic stimulus.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

Automatic analysis for amperometrical recordings of exocytosis

Cited by 84 publications

References 17 publications

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Modulatory Mechanism of the Endogenous Peptide Catestatin on Neuronal Nicotinic Acetylcholine Receptors and Exocytosis

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

Contact Info

Product

Resources

About