Capillary Electrophoresis Coupled to Fluorescence Detection for the Determination of in Vivo Release of Multiple Neuropeptides from the Ewe Median Eminence

Abstract

The link between brain and reproduction is the release of the decapeptide luteinizing hormone-releasing hormone (LHRH) from the brain median eminence (ME), a release modulated by other neuropeptides such as β-endorphine (BEND) and neuropeptide Y (NPY) impinging on the LHRH neuron. Analysis of brain perfusates is the metnod of choice to determine in vivo release from discrete brain areas. For this, push-pull cannula (PPC) and microdialysis sampling are the most prominent technologies. However, sample volume is a limiting issue when attempting to measure by radioimmunoassays (RIA) in vivo release of multiple neuropeptides from these sources, especially when neuropeptide concentration is low and the whole sample (100 μl PPC perfusate/10 min) has to be dedicated to measure only one neuropeptide. We developed a capillary electrophoresis (CE)-based assay for the simultaneous determination of LHRH, BEND and NPY in PPC samples from the ewe ME. This assay uses CE for the separation of these neuropeptides and their derivatization with the fluorogenic chromophore fluorescamine to amplify their signal. The CE-based assay (120 nl of PPC perfusate injected into the capillary column) allows the simultaneous electrophoretic assessment of endogenous NPY and BEND (found at levels between 10–100 pg/100 μl PPC perfusate/10 min as resolved by RIA). However, simultaneous detection of endogenous LHRH (present at levels between 0.1-1 pg/100 μI PPC perfusate/10 min) is only marginal. A working assay for LHRH might only be achieved when laser-induced fluorescence detection of derivatized neuropeptides is coupled to additional sensitivity enhancers already in existence. An appealing aspect of this technology is the simplicity of the methodology, its efficiency coupled to the need of low volume samples, and most important, its ability to separate and to detect multiple neuropeptide components, simultaneously, in a single sample.