Development of assays for neuronal peptide content and release

This thesis describes the development of neuropeptide assays for neuropeptides in individual neurons and in release from a cellular cluster. These assays are performed to elucidate chemical signaling between neurons. A review of methodologies for studying neuronal signaling is presented. Included in the review is a description of capillary electrophoresis (CE).; The assay methods described in this thesis employ a common molluscan neuronal model, Aplysia californica. Rationale is offered for using Aplysia in numerous studies such as this one. Also, there is a review of the bag cell literature.; Assays of the neuropeptide content of individual neurons were performed with CE. Development of a detector, a UV laser-induced fluorescence system, is outlined. The 354-nm line from a HeCd laser is used to excited fluorescamine derivatives (CPP) of amino acids and peptides. The linear, concentration dependent signal for CPP derivatives stretches over four orders of magnitude and affords a 2.3 nM limit of detection for CPP-egg-laying hormone (ELH). Trace level peptide assay is demonstrated for concentrations down to 10 nM ELH prior to derivatization. Also, assays for different neuron types gives reproducible and unique electropherograms.; Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF MS) was used to explore neuropeptide content and release. The reproducibility of profiling cellular neuropeptide content from identified neurons is high and rinsing cellular samples in the MALDI matrix before analysis provides sensitive detection. All predicted neuropeptides from the ELH prohormone are seen including novel peptides.; Neuropeptide release is also studied with MALDI TOF MS. In this method, a small chamber (10 {dollar}mu{dollar}l) is used to hold an isolated bag cell cluster in a small volume of physiological saline. The chamber consists of a standard pipette tip with the narrow end melted around two sections of fused silica capillaries. Physiological saline flows into and from the chamber via capillaries and gravimetric pressure flow. Following electrical stimulation of a bag cell cluster, 50-100 nl samples are collected from the outlet end of the sampling capillary for analysis. MALDI MS is performed on the sample for peptide detection. Even in high salt concentrations peptides are detected by MALDI MS.