Analysis of serotonin release from single neuron soma using capillary electrophoresis and laser-induced fluorescence with a pulsed deep-UV NeCu laser

Abstract

The use of capillary electrophoresis (CE) with laser-induced fluorescence excited by ultraviolet (UV) lasers in the range 200-300 nm has been restricted by the available wavelengths and expense of UV lasers. The integration of a NeCu deep UV laser operating at 248.6 nm with a single channel CE system with post-column sheath flow detection allows detection limits for serotonin and tryptophan of 3.9x10(-8) M and 4.5x10(-8) M respectively. Single cell analysis of serotonergic metacerebral cells from the sea slug Aplysia californica yields a value of 800+/-85 fmol of serotonin in each cell soma. For the first time, serotonin is directly detected in electrically stimulated release from single metacerebral cell soma, with approximately 4% of the serotonin contained in the soma released from a semi-intact preparation with a 2 min electrical stimulation.

Signals from the Brainstem Sleep/Wake Centers Regulate Behavioral Timing via the Circadian Clock

“…For most measurements, the glutamate was analyzed by derivatizing 5 μl of microdialysis sample with 1 μl of 3×10 −6 fluorescein, 2 μl of 10 mM naphthalene-2,3-dicarboxaldehyde (NDA), 1 μl KCN, and 1 μl of 50 mM borate buffer (pH 9.4). NDA forms a stable fluorescent product with glutamate that can be measured by capillary electrophoresis/laser-induced fluorescence (CE-LIF) [53], except that the laser, focusing lens, and optics have been changed to a different excitation wavelength (Ex = 457.9 nm). Briefly, an Argon ion laser (Melles Griot; Carlsbad, CA) was employed to provide the 457.9 nm wavelength laser line and a microscope objective was used to focus the beam to a post-column sheath flow assembly.…”

“…A 488 nm notch filter, a 475 nm long-pass filter (03FCG465/GG475; Melles Griot; Carlsbad, CA), and a 550 nm short-pass filter (03SWP408; Melles Griot; Carlsbad, CA) were used to select the appropriate fluorescence signal. The micro-injector, high voltage power supply, sheath flow assembly, and PMT were the same as previously described [53]. The 50-μm I.D./360-μm O.D.…”

Gasotransmitters: Physiology and Pathophysiology

“…CE-LIF has low detection limits and ultra-small sample need, which is suitable for even single cell analysis (Zhang and Sweedler 2001;Page et al 2002;Miao et al 2003;Stuart and Sweedler 2003;Miao et al 2005;Sheeley et al 2005;Lapainis et al 2007) ). However, these probes often have issues of crossreactivity with other cellular constituents.…”

Bioanalytical Applications of Capillary Electrophoresis with Laser-Induced Native Fluorescence Detection

“…Neurochemicals 257 CZE [20,57] Catecholamines Various CZE [47][48][49]55,56] Serotonin 248.6 CZE [51,54] Profiling, tryptophan 266 CZE/chip [63] …”

“…CE with native fluorescence detection was used to quantify serotonin from individual neurons and in rat peritoneal mast cells [54][55][56]. In these experiments, wavelengthresolved fluorescence detection, which compares fluorescence emission spectra, was used to improve identification of the analyte being separated [57].…”