High temporal resolution for in vivo monitoring of neurotransmitters in awake epileptic rats using brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection

Abstract

A method for high temporal resolution monitoring of five neurotransmitters, dopamine (DA), noradrenaline (NA), gamma-aminobutyric acid (GABA), glutamate (Glu), l-aspartate (L-Asp), in freely-moving rats using microdialysis and capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) was developed. An on-line device, including microdialysis and derivatization with naphthalene-2,3-dicarboxaldehyde, mixes the dialysate with derivatization reagents directly in the collection tube, i.e. with no reactor. Thereafter, collected derivatized samples are analyzed off-line with an automated CE system coupled to a LIFD using a 442 nm excitation. The sampling time was limited by the minimal volume required for the analysis by the automated CE system used: neurotransmitters could be determined in 667 nl dialysates (940 nl after derivatization), i.e. in samples collected every 20 s with a flow rate of 2 μl/min. The detection limits at the dialysis probe were 3 × 10 −9, 1 × 10 −9, 1.9 × 10 −8, 4.2 × 10 −7, 2.1 × 10 −7 mol/l for DA, NA, GABA, Glu and L-Asp, respectively. The protocol was validated using in vitro/in vivo tests and the performances—repeatability, linearity, characteristics of the probes—were determined. Finally, the high temporal resolution allowed the simultaneous monitoring of these neurotransmitters in rats with genetic absence epilepsy and revealed, for the first time, increases in GABA concentrations concomitantly with the seizures, detected when our new microdialysis method was combined to electroencephalographic recordings.