Analysis of small molecule neurotransmitters using capillary electrophoresis-mass spectrometry: applications for studying development and disease

Murphy, Shannon Elaine

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https://hdl.handle.net/2142/115728 Copy

Description

Title

Analysis of small molecule neurotransmitters using capillary electrophoresis-mass spectrometry: applications for studying development and disease

Author(s)

Murphy, Shannon Elaine

Issue Date

2022-04-20

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan V

Doctoral Committee Chair(s)

Sweedler, Jonathan V

Committee Member(s)

• Gillette, Martha U
• Han, Hee-Sun
• Rodriguez-Lopez, Joaquin

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Mass spectrometry
• Neurotransmitters
• Capillary electrophoresis

Abstract

Neurotransmitters play a key role in neuronal growth and differentiation during central nervous system (CNS) development. The disruption of neurotransmitter systems during development has been linked to various neurological disorders. A better understanding of how neurotransmitters develop normally in the CNS and how their disruption leads to neurological disorders is needed to diagnose and treat these disorders. However, there are several analytical challenges with measuring the suite of neurotransmitters from a defined location in the CNS. Sample volumes ranging from picoliters in single cells to microliters in whole tissues provide analytical challenges of limited sample volume and the need to measure a wide concentration range to analyze neurotransmitters during development. Capillary electrophoresis-mass spectrometry (CE-MS) has advantages such as low sample volumes (which allows for single cell analysis), low limits of detection, and a wide linear dynamic concentration range that make it well suited for analyzing neurotransmitters during development. Additionally, CE-MS can measure many electroactive and non-electroactive compounds simultaneously, offering advantages over microelectrodes or biosensor techniques which can only measure a few electroactive compounds in a single experiment. Animal and in vitro models are commonly used to study human development and disease. However, in order for these models to be useful, they need to accurately represent the system they are trying to mimic. One of the goals here is to use CE-MS to analyze neurotransmitters in in vitro models to ensure that these models have the correct chemical contents and chemical interplay, which provides insight into their functionality and therefore their accuracy. Similarities were found in the levels of acetylcholine during development for the spinal cord organoid models and the spinal cord samples, though more data measuring the levels of other neurotransmitters is needed to fully determine how well these organoids neurochemically model spinal cords. In evaluating the functionality of an engineered in vitro model of hepatic encephalopathy, neurotransmitters in cortical neuron constructs cultured on collagen and collagen-HA hydrogels showed opposite trends with increasing ammonia concentration, demonstrating how the composition of the collagen hydrogel effects the interaction between neurons and ammonia under hyperammonemia conditions. Additionally, the neurotransmitter levels in the cortical neurons cultured on collagen-HA hydrogels decreased with increasing ammonia concentrations (instead of increasing like expected) and the levels of GABA and glutamate at the high concentration (200 μmol/L) were lower in this condition than the control treatment for both collagen hydrogels, suggesting that this experiment should be repeated with adjusted hyperammonemia conditions to minimize the loss of neuron functionality and verify the neurotransmitter levels measured in this model. The use of CE-MS to measure neurotransmitters in these biological systems advances the development of these in vitro biological models to model development and disease states. Microfluidic CE-MS has the same advantages of conventional CE-MS that enable it to be a powerful technique for the analysis of neurotransmitters with the additional benefits of faster analysis times and higher sample throughput. 908 Devices recently developed a microfluidic CE device that’s compatible with the mass spectrometers present in the Sweedler lab. Additional goals of this work are to determine the ability of this microfluidic CE-MS system to analyze neurotransmitters in small and complex biological samples in a high throughput manner and to compare the performance of microfluidic and conventional CE system using the same samples. Results demonstrate that the performance of the two CE systems is similar for analyzing a combined analytical standard of monoamine neurotransmitters. However, results comparing the systems with spinal cord samples show inconsistent results in the measured neurotransmitter levels between two developmental timepoints for most neurotransmitters, which could be due to biological variability or batch effects. Microfluidic CE-MS was also used to build a library of monoamine neurotransmitters found in planarians (where dopamine, octopamine, and serotonin were detected in all samples) and to analyze planarians with altered monoamine neurotransmitter levels (RNAi aadc knockdown planarians) to see which neurotransmitters were altered. Relative serotonin levels measured in the RNAi samples trended towards being decreased compared to the levels measured in the control planarians, suggesting that serotonin could be involved in regulating factors of germ cell development. The work presented here shows the utility of conventional and microfluidic CE-MS to measure neurotransmitters in various biological systems for the applications of studying development and disease.

Graduation Semester

2022-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Shannon Murphy

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