Automatic analysis for amperometrical recordings of exocytosis

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Abstract

Amperometry is a widely used technique for monitoring the secretion of catecholamines (CA) by exocytosis. The use of carbon fibre microelectrodes allows the on-line recording of CA released from a single secretory vesicle. Amperometric signals are generated by oxidation of the quantally released CA close to the electrode tip. Each event of exocytosis is called a secretory spike. Here we describe a program written for IGOR (Wavemetrics, Lake Oswego, OR, USA), which may be used to analyze amperometric signals off-line. The procedures allow, (i) digital filtering and analysis of the current noise, spike identification and calculation of spike kinetic parameters; (ii) spike review; (iii) pooling spikes and data to create galleries, tables and histograms of measured parameters which can be exported to a graphic format or files for further analysis.

Introduction

Chromaffin cells release CA by exocytosis, a calcium-dependent process that entails the fusion of a secretory vesicle and the cell membrane. Single vesicle exocytosis can be monitored using amperometric techniques combined with carbon fibre microelectrodes placed on the cell surface (Wightman et al., 1991). This approach allows the direct observation of the time–course of single secretory events and has been successfully used to study the late phase of exocytosis (Jankowski et al., 1993, Chow et al., 1994, Schroeder et al., 1996).

A carbon fibre microelectrode, acting as an amperometrical working electrode, detects the CA exocytosis of a chromaffin granule. The oxidation of a molecule of CA produces two electrons which are taken up by the positively charged working electrode, thereby creating a current directly proportional to the CA concentration surrounding the electrode tip (Kawagoe et al., 1993). A potentiostat maintains a fixed potential and amplifies the current producing an output voltage signal, which can be acquired on-line.

Amperometrical recording experiments commonly produce over a hundred megabytes of raw data whose analysis requires the help of well-structured programs. Several programming languages like Basic have been used to make locally-written software (Schroeder et al., 1996) although they are very slow to analyze large series of raw data. Commercially available acquisition/analysis software for spike analysis eg AxoScope® (Axon Instruments, Foster City, CA), on the other hand have not been specifically conceived for amperometry (Graham and Burgoyne, 2000). IGOR is a software specially designed to work with data arrays. It offers its own programming environment with extensive libraries of built-in functions, which can be complemented with user-written macros to create procedures for specific purposes. These advantages have been used in the past to create small programs for secretory spike analysis (Chow et al., 1992, Chow et al., 1994, Elhamdani et al., 1998, Elhamdani et al., 1999).

Due to large variability on secretory spikes and characteristics obtained upon cell stimulation, even from a single cell, it becomes necessary to conduct statistical studies on populations of several hundreds of events. One important feature of an analysis program for amperometry should be its versatility. Researchers should be able to choose the type and parameters of filters used and the spike identification criteria. In addition, the program should perform an automatic processing of previously selected data files and manual supervision and correction of results. Furthermore, final results should be exportable to graphic or statistical programs for further analysis.

Here, we present a series of macros written for IGOR for the full analysis of exocytotic events detected by amperometry. All of these macros are freely available (see below).

Section snippets

Chromaffin cell cultures

Bovine adrenal chromaffin cells, enriched in adrenaline through a single-step Urografin® gradient, were prepared as described elsewhere (Moro et al., 1990). Cells were plated on 12 mm-diameter glass coverslips contained in 24-well culture plates at an approximate density of 5×105 cells per well. Cells were maintained at 37°C in a water-saturated and 5% CO2 environment and were used at room temperature between 1 and 5 days after isolation.

Amperometric detection of exocytosis

Carbon fibre microelectrodes were prepared as described (

File location and destination

A flow diagram used for data analysis is illustrated in Fig. 1. Program asks for the folder where raw data were stored. Once this path has been defined, the automatic analysis macro can be run.

File finding and opening

The program loads the file into RAM in the form of a wave.

Data scale adjustment

User introduces calibration scale to convert signal from voltage to current (i.e. 100 pA/V).

Digital filtering

User selects the most suitable filter settings for reduction of line- and high frequency-noise which are present with signal. Program calculates the FFT and

Discussion

Amperometric recording analysis requires the assistance of computer programs capable of dealing with files with a large quantity of data. Several programming languages (basic, fortran, C or pascal) have been used to write analysis procedures (Schroeder et al., 1996, Zhou et al., 1996, Borges et al., 1997, Marszalek et al., 1997). IGOR has become very popular for off-line analysis of biological signals and macros for spike analysis have been made by our group and others (Criado et al., 1999,

Acknowledgements

We thank Dr E. Seward for her revision of the manuscript. JDM is recipient of a fellowship from Instituto Tecnológico de Canarias, JFG is recipient of a fellowship of Consejerı́a de Educación del Gobierno de Canarias. This work was supported in part by a grant from Spanish Ministerio de Educación y Cultura, DGCYT PB97–1483 and FEDER (1FD97-1065-C03-01).

References (18)

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