| dc.description.abstract | Fast-scan cyclic voltammetry at carbon-fiber microelectrodes (FSCV) is a commonly used electrochemical technique to measure sub-second concentration changes in electroactive neurotransmitters, including dopamine, and neuromodulators both in vivo and in vitro. FSCV provides several advantages, including good chemical selectivity and temporal resolution. Our research group is currently interested in studying rapid interactions between neurotransmitters in the context of neurodegenerative disease and toxic neurological events. Many research groups including ours use rat or mouse animal models to study neurotransmission; however, these animal models have some drawbacks, including low throughput analysis and the need to use invasive treatment methods, such as intravenous tail injections. In addition, developing transgenic rodent models for neurodegenerative disease is expensive and time consuming. Therefore, we seek to develop the use of an alternative animal model that has potential for high-throughput analyses and easy genetic manipulation. Zebrafish (Danio rerio) have recently been established as popular animal model for the study of neuronal functions due to their fast-life cycle, ease of genetic manipulation and drug treatment, and central nervous system similarities with humans. Unfortunately, real time measurement methods to study sub-second neurotransmitter release and uptake events in zebrafish have not yet been well developed, representing a serious roadblock to the use of this organism for elucidating brain function. In this study, FSCV was used to measure locally-evoked dopamine release and uptake in zebrafish whole brain preparations as well as brain slices. Our results indicate that dopamine release is easily measured in the whole brain. Moreover, peak dopamine concentration ([DA]release) was similar to that of sagittal brain slice preparations (0.49 ± 0.13 µM in whole brain and 0.59 ± 0.28 µM in brain slices, p = 0.41, t-test). Several pharmacological studies using α-methyl-p-tyrosine methyl ester (αMPT), an inhibitor of tyrosine hydroxylase, sulpiride, a D2 dopamine autoreceptor antagonist, and nomifensisne, a dopamine reuptake inhibitor, were conducted to confirm the presence of dopamine and to determine if zebrafish dopamine receptor and transporter are similar to that of rodents. In addition to quantifying dopamine release and stimulation optimization, we established zebrafish as a chemobrain animal model. We investigated the changes in dopamine release and uptake in zebrafish that were treated with chemotherapy drugs, carboplatin and 5-fluorouracil using either habitat water treatment or food treatment. The data here suggest that the chemotherapy drugs have an effect on dopamine release and uptake in zebrafish similar to that observed in the rodent animal models. Moreover, zebrafish treated orally with chemotherapy drugs shown greater effect on evoked dopamine release compared to zebrafish exposed to the treated habitat water system. Last, our group has been interested in studying the relationship between glutamate and dopamine. However, glutamate is neurotoxic and cannot be applied to a harvested brain as was the other pharmacological agents. To overcome this challenge, we employed p-hydroxyphenylacyl (pHP) based caged compounds to deliver glutamate to the target location while not interfering with glutamate’s known biological response. However, direct electrochemical quantification of how much glutamate is photo-released is impossible since glutamate is not electroactive. Therefore, instead of measuring the amount of glutamate photoreleased, we decided to measure 4-hydroxyphenylacetic acid (4HPAA) instead. 4HPAA is an electroactive by-product generated from the photostimulation of pHP based caged glutamate. Due to the electroactivity of 4HPAA, the amount of photoreleased glutamate can be quantified using FSCV. In addition, our group has successfully demonstrated using both FSCV and high performance liquid chromatograpy (HPLC) that the quantification of 4HPAA indirectly allow for the quantification of glutamate generated due to the photoactivation. Therefore, we studied 4HPAA’s electrochemistry in order to optimize a waveform for the simultaneous detection of dopamine and 4HPAA in a single voltammetry measurement. | |
