Development of a capillary electrophoresis based assay for the characterization of multiple neuropeptides from the brain median eminence

The biochemical determination of communication signals, such as the in vivo release of multiple neuropeptides from specific brain areas, is important to understand the operation of integration sites involved in defined physiological processes. However, in most cases the concentration of the in vivo neuropeptide release is found in the subnanomolar range and the amount of the sample volume available for such studies is extremely low (μL level). With the advent of capillary electrophoresis (CE), a high-resolution analytical technique of great value for the identification and characterization of a wide range of compounds, it is possible to identify a large number of neuropeptides simultaneously. When CE is coupled to adequate detection system, such as laser induced fluorescence detection (LIF) an enhanced sensitivity is achieved to identify multiple neuropeptides released at specific brain integration sites. Thus the objective of this thesis was to develop such a CE based assay. For this, it was used a CE separation condition employing borate buffer and the detection of the separated neuropeptides by absorbance based UV light and laser induced fluorescence. In preliminary experiments, pools of exogenous (synthetic) neuropeptides were employed to develop the CE based system. However, the limits of sensitivity were tested using the endogenous neuropeptides content of in in vivo push-pull cannula (PPC) samples from the median eminence (ME), a specific brain integration site. The neuropeptide content of the test samples used were previously measured by specific radio-immunoassays (RIA) aimed at the quantification of a single neuropeptide per assay.; The CE-LIF based assay is consisted of Beckman CE unit coupled to an Argon ion laser (ex. 488 nm, em. 520 nm). The separation buffers were 25 mM sodium phosphate and 50 mM sodium borate buffer. The CE method enabled the detection of several neuropeptides simultaneously in a single analysis. The results obtained using the CE-LIF based assay on pooled PPC samples, are correlated well with those obtained using RIA. In addition to separate and quantify neuropeptides, the CE-LIF method was able to detect the effect of endopeptidase inhibitor in neuropeptides secretion in ME. Furthermore, the CE-LIF method enabled the identification of several neuropeptides in progesterone blocked ewe model. Moreover, the CE-LIF method permitted the detection of the effect of ethanol on expression of preovulatory GnRH. One major drawback, however, was that the CE-LIF method was not sensitive enough to determine multiple neuropeptides in samples derived from in vitro studies performed under the laboratory experimental conditions employed during the course of this thesis.