New Approaches to Analyze Neurometabolites of <i>Drosophila melanogaster</i> with Capillary Electrophoresis

Open Access
Author:
Kuklinski, Nicholas John
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 18, 2010
Committee Members:
  • Andrew Ewing, Dissertation Advisor
  • Andrew Ewing, Committee Chair
  • Anne M Andrews, Committee Member
  • Peter J Butler, Committee Member
  • Michael Thomas Green, Committee Member
Keywords:
  • fruit fly
  • capillary electrophoresis
  • Drosophila Melanogaster
  • dopamine
  • biogenic amines
  • salsolinol
Abstract:
<p><dd>This thesis describes the development and optimization of capillary electrophoresis (CE) based techniques for neurometabolite analyses of the fruit fly <i>Drosophila melanogaster</i>. Fast separation times (seconds to minutes), minimal sample requirements (nanoliters to femtoliters), and excellent mass detection limits (femtomole to zeptomole) make CE ideally suited for sampling neuromodulators with a high degree of spatial resolution. Significant progress has been made to resolve biogenic amines and to improve sampling techniques used to investigate these complex biological phenomena. </dd></p> <p><dd>Micellar electrokinetic chromatography (MEKC) coupled to amperometric electrochemical detection (EC) was used to resolve and then to quantify biogenic amines and metabolites within <i>Drosophila melanogaster</i>. In Chapter 2, a new separation scheme was devised to allow resolution of 24 compounds of interest. This was accomplished by precisely controlling the amount of base added to the background borate/sodium dodecyl sulfate (SDS) buffer, which optimized the resolution of the separation, and then calculating the pH. Here, I focused on measurements of six of the analytes that are thought to be involved in the response to alcohol: dopamine, salsolinol, norsalsolinol, N-acetyloctopamine, octopamine, and N-acetyldopamine. These were identified and quantified within homogenates of the fly head. The identification of salsolinol and norsalsolinol in the fly brain is novel and may help to elucidate what role this neuromodulator holds in the dopaminergic system. </dd></p> <p><dd>I then used the separation scheme developed in Chapter 2 to quantify biogenic amines within individually, microdissected <i>Drosophila melanogaster</i> brains and brain regions in Chapter 3. The effects of pigment from the relatively large fly eyes on the separation were examined to find that the red pigment from the compound eye masks much of the electrochemical signal from biogenic amines. The brains of <i>white</i> mutant flies, which have characteristically low pigment in the eyes, have a significantly simplified separation profile in comparison to the red-eyed, wild-type, Canton S fly. However, the <i>white</i> mutant flies were also found to have significantly lower amounts of dopamine, l-3,4-dihydroxyphenylalanine (L-DOPA), salsolinol, and N-acetyltyramine in their dissected brains when compared to dissected brains of Canton S flies. In addition, significant variation was observed in the dissected brains between individual flies that might be related to changes in neurotransmitter turnover. The transgenic tyrosine hydroxylase-green fluorescent protein (TH-GFP) fly line, for which the overall profile of biogenic amines is not found to be significantly different from Canton S, was then used to visualize the location of and to dissect dopamine neurons. Biogenic amines were then quantified in three brain regions observed to have dopamine levels: the central brain, optic lobes, and posterior superiormedial protocerebrum (PPM1) region. </dd></p> <p><dd>Microdissection techniques were expanded in Chapter 4 to analyze homogenates of fly-brain populations through the process of freeze-drying, a dehydration process where a material is quickly frozen, has its surrounding pressure reduced, and is then heated to allow sublimation of the water from the sample. Extraction and drying times were quantitatively explored and optimized. Drying for too long produced low signals possibly due to sample loss, while long freezer storage times led to samples with red pigment from the eyes being extracted into the brain. Freeze-drying fly heads makes the outer walls of the cuticles hard and brittle while also freeing the brain from the inner walls of the cuticle. This significantly decreased the time needed to dissect a fly brain and enabled larger numbers of brains to be homogenized to make a sample. The number of brains in a homogenate was increased, effectively concentrating the sample, which helped to increase the signal and resolution. Biogenic amines were then quantified in samples containing fifteen freeze-dried brains. N-acetylserotonin, N-acetyltyramine, N-acetyldopamine, L-DOPA, and tyramine were discovered to all correspond well with previous dissected studies. </dd></p> <p><dd>CE-EC is a powerful technique for the analysis of neuromodulators offering low limits of detection and high selectivity against background signals in biological samples. Yet, this selectivity of electroactive metabolites and transmitters prevents the analysis of many other chemical analytes of interest. To begin to overcome this, Chapter 5 presents the development of separations using CE electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry (MS) for the analysis of neurotransmitters, metabolites, and drugs within the brains of <i>Drosophila</i>. Optimizing a volatile buffer at a specific pH ensured that a unique m/z ratio was available for each analyte for detection. </dd></p>