Addressing the Neurochemical Problem: Sensitive and Selective Measurements of Neurotransmitters, Neuropeptides, and Synaptic Vesicles

Abstract

The neurochemical problem (1) and the directive of the neuroanalytical chemist (2) can be stated as follows: (1) The chemical space of the nervous system is populated by hundreds of neuroactive species linked through extensive biological circuits which are dynamically changing in time and space in response to myriad inputs. (2) Neurochemical analysis techniques should therefore have the appropriate temporal, spatial, and chemical resolution to study these systems, while perturbing them so minimally as to allow unfettered in vivo measurements. New tools and concepts for analytical measurements of neurotransmitters, neuropeptides, and synaptic vesicles are developed and presented in this dissertation on analytical measurements for addressing the neurochemical problem. The introduction gives a broad overview of chemical neuroscience and introduces quantitative visualization of the multidimensional resolution paradigm for analytical chemists seeking to design effective experiments. Chapters two through four detail advancements in data processing and instrument design which decrease detection limits and allow for improved spatial, temporal, chemical resolution in capillary electrophoresis measurements of neurotransmitters, metabolites, and synaptic vesicles. Chapter five discusses the development of fast-scan controlled-adsorption voltammetry which has dramatically increases the spatial and temporal resolution of basal dopamine measurement in vivo. Chapter six introduces online-preservation microdialysis as a way to overcome enzymatic degradation of endogenous opioid neuropeptides during in vivo sample collection. Because of this discovery of the secretory behavior these neuropeptides is reported in the anterior cingulate cortex (ACC), a region of the brain deeply associated with pain signaling. The advancement of peptide drugs particularly glycosylated neuropeptide analogs through new methods of mass spectrometry analysis for rapid feedback in drug development are presented in chapter 7. Chapter eight concludes this work with future directions pointing towards single-cell electrochemical and mass spectrometry measurements, shotgun-microdialysis for high-throughput screening of neurotherapeutics, preliminary data on the effect of chronic pain of endogenous opioids in the ACC, and the beginnings of in vivo neuroproteomics analysis in rodent pain models.