Characterization of rapid adenosine neuromodulation in mouse brain slices

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Chang, Yuanyu, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Venton, Jill, AS-Chemistry, University of Virginia

Adenosine is an important neuromodulator in the brain, it modulates neurotransmitter release and provides neuroprotection during physiological and pathological conditions. The mechanisms of rapid adenosine release in the brain have been investigated for several years using fast-scan cyclic voltammetry (FSCV). However, the mechanisms of rapid adenosine neuromodulation and formation are still not fully understood. This thesis explored the rapid adenosine modulation and formation by using global knockout mice, and the range of rapid adenosine effects in the brain was also characterized with dual-channel FSCV.
In Chapter 2, spontaneous adenosine and mechanically-stimulated adenosine were separately measured in the caudate-putamen region in a mouse brain. Here, I investigated the regulation of rapid adenosine by adenosine receptors, using global A1 or A2A knockout mice. The results indicate that A1 receptors presynaptically modulate the frequency of spontaneous adenosine but do not modulate the concentration. However, A2A receptors modulate the concentration of spontaneous adenosine but do not significantly influence the frequency of spontaneous adenosine. For the mechanically-stimulated adenosine, adenosine receptors do not significantly influence the concentration of adenosine in vivo, but A1 receptors significantly modulate the concentration of stimulated adenosine in the brain slice model. Understanding the role of adenosine receptors on rapid adenosine release will help for future treatments for different diseases that are related to adenosine neuromodulation. Chapter 3 investigated the mechanisms of rapid adenosine formation in the hippocampus CA1 region using Pannexin 1 knockout mice. There was no significant difference in spontaneous adenosine release between wild-type mice and Pannexin 1 knockout mice. Therefore, Pannexin 1 channels might not be the formation pathways of spontaneous adenosine release. However, the concentration of mechanically-stimulated adenosine decreased around 50% in Panx1KO mice indicating that Pannexin 1 channels partially influence the formation of mechanically-stimulated adenosine due to their mechanosensitive property. In addition, a high dose of a non-selective drug, Carbenoxolone (CBX) did not further influence the rapid adenosine release, indicating that other pannexin channels and connexins are not contributed to the rapid adenosine formation. These results prove that spontaneous adenosine and mechanically-stimulated adenosine has different mechanisms of modulation and formation. Future studies could focus on the impact of exocytosis for rapid adenosine formation.
Chapter 4 describes the range of rapid adenosine effects by using dual channel FSCV. Rapid adenosine was simultaneously detected in two different sites in hippocampus CA1 in the mouse brain slice. By varying the distance of two working electrodes, the spatial range of rapid adenosine was characterized. Mechanically-stimulated adenosine can be detected up to 150 µm away from where it was stimulated, although the signal is smaller and delayed. Spontaneous adenosine was randomly and localized released and could not diffuse to a 50 µm distance. This study shows that spontaneous adenosine events are very localized and thus provide only local neuromodulation. Injury, such as mechanical stimulation, allows adenosine to diffuse farther, but neuroprotective effects are still regional.
Chapter 5 presents a graphene oxide (GO) modified carbon fiber microelectrode (CFME) for dopamine detection in FSCV. GO coating improved the electrode sensitivity by providing more oxygen-functional groups for dopamine adsorption. Different methods, such as drop casting, dip coating, and electrodeposition, were also compared in this chapter. Drop casting was likely to cause a huge GO aggregation which slows down the electron transfer rate of dopamine detection. Dip coating did not sufficiently coat the electrode surface, GO particles tended to form sediments under the solution instead. Electrodeposition showed the best coating on CFME, and the sensitivity of the modified electrode enhanced nearly two folds for dopamine detection. In addition, the modified electrodes were successfully applied to mouse brain slices to monitor the electrically-stimulated dopamine release. Thus, GO is good for improving sensitivity and is stable for complex tissue measurement.
Overall, my thesis demonstrates the new findings of the mechanisms of rapid adenosine modulation and formation by using different types of knockout mouse models, I also characterized the range of rapid adenosine with dual channel FSCV in mouse brain slices. The new information provides more understanding of rapid adenosine release in the brain which will be useful for future treatments of adenosine-related diseases. Furthermore, I also optimized new GO-modified CFMEs to improve the sensitivity of dopamine detection in brain slices. Better method developments, better sensors, and better analytical tools will reveal more understanding of rapid neuromodulation in the future.

PHD (Doctor of Philosophy)
Rapid adenosine, Spontaneous adenosine, Mechanically-stimulated adenosine, Adenosine receptors, Pannexin 1 channels, Caudate-putamen, Hippocampus CA1, Fast-scan cyclic voltammetry, Carbon fiber microelectrode, Graphene oxide, Knockout mice, Brain slice
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