Amperometric Characterization of Nanosecond Electric Pulse-Evoked Exocytosis in Adrenal Chromaffin Cells: Custom Experimental Setup and Spike Detection Software
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Authors
Balaji, Anithakrithi
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Amperometric spike analysis software , Amperometry , Chromaffin Cells , Custom Amperometry Setup , Exocytosis , Nanosecond electric Pulses
Alternative Title
Abstract
Nanosecond electric pulses (NEPs) have emerged as a promising tool for modulating neurosecretion by bypassing traditional receptor-mediated pathways. This dissertation
explores the use of a single 5 ns, 7–9 MV/m pulse to stimulate catecholamine release from
isolated bovine adrenal chromaffin cells, a well-established model for studying Ca2+
regulated exocytosis. While previous studies using Ca2+ imaging and total internal
reflection fluorescence microscopy (TIRFM) have demonstrated NEP-induced increases
in intracellular calcium concentration ([Ca²⁺]i) and associated exocytotic activity, the goal
of this work was to directly quantify individual granule fusion events in real time using
carbon fiber amperometry and to characterize the kinetic and temporal features of the NEP-evoked amperometric responses.
A major technical challenge in implementing amperometry with high intensity NEP
stimulation is protecting the highly sensitive amplifier circuitry from high-voltage damage
while minimizing the interruption in data acquisition. To address this issue, a customized
switching system was developed using a combination of reed relays and semiconductor
switches controlled by LabVIEW and a National Instruments data acquisition card. This
setup enabled brief disconnection of the carbon fiber electrode (CFE) from the amplifier
during NEP delivery, achieving a data recording gap of less than 5 ms without distorting
either the NEP waveform or amperometric data. This switching platform provides a
broadly applicable solution for studies requiring electrical isolation during stimulation.
To analyze the resulting amperometric data, a custom MATLAB-based tool (ASAT) was
developed for spike detection and quantification. Unlike conventional datasets, NEP stimulated recordings include electrical artifacts that can obscure early spike activity. ASAT was designed to address this challenge by incorporating flexible filtering options, artifact-exclusion regions, and a manual spike review interface to ensure accurate event
detection. The tool extracts key parameters such as spike amplitude, half-width, charge,
and estimated number of molecules released, enabling a detailed characterization of
granule fusion kinetics.
Amperometric recordings revealed that NEP-evoked spikes were comparable to DMPPevoked events in quantal size and kinetic features. However, a notable distinction was the
presence of a delay in the onset of exocytosis following NEP stimulation in a majority of
cells, ranging from 1 to 5 seconds, despite a rapid rise in [Ca²⁺]i. In contrast, DMPP-evoked
exocytosis occurs immediately, suggesting that downstream fusion mechanisms may be
differentially regulated following NEP exposure.
Simultaneous Ca²⁺ imaging and amperometry in cells loaded with the fluorescent Ca²⁺
indicator Calcium Green -1 confirmed that [Ca²⁺]i increased immediately after both stimuli,
but only NEP-exposed cells showed delayed secretion. Additional analyses revealed that
Ca²⁺ response profiles (short-lived vs. long-lived) were reflected in the response pattern of
catecholamine release, with sustained Ca²⁺ elevations supporting prolonged exocytotic
activity. The presence of Calcium Green-1 dye did not significantly alter spike parameters,
validating the dual-mode imaging approach.
These findings represent the first real-time quantification of exocytosis triggered by a
single 5 ns pulse Overall, this work lays a strong technical and analytical foundation for
investigating NEP-induced exocytosis. It also opens the door to future studies aimed at
uncovering the molecular mechanisms behind NEP-specific effects on exocytosis.