Rebecca Oliphant and Scott Steffensen, Department of Psychology
Introduction
Dopamine (DA) neurons are at the core of many highly-researched diseases. Dopaminergic neuronal degeneration has been linked to oxidative stress, a state that occurs when high levels of normally-occurring reactive oxidative species (ROS) are formed. Depending on the location of such degradation, the result could be either Parkinson’s disease or the very common state of addiction and withdrawal.
Neuromelanin is a dark polymer pigment found on some catecholaminergic neurons and contains a stable radical that is able to inactivate ROS and possibly protect DA neurons from degradation. The mechanism for spontaneous melanization is unknown, but an understanding of this process and how it can combat sources of oxidative stress could help researchers learn how to protect the body against diseases that result in loss of DA neurons.
Methodology
L-DOPA has been formerly presented to induce melanization in cell culture (Sulzer et al., 2000), based off of early formation of ROS. Our lab has previously shown that methamphetamine (METH) generates ROS (Jang et al., 2016). In order to understand the mechanism of melanization, our research team proposed to use L-DOPA and METH to induce ROS and melanization in DA tissue brain slices.
Our proposed procedure was to dissociate neurons from the ventral tegmental area (VTA) of brain slices of GAD GFP mice. Because this strain of mouse has a knock-in GFP that is expressed only in GABA neurons, and the VTA only contains GABA and DA neurons, DA neurons would be able to be differentiated and experimented on.
Once the DA neurons were discriminated, the plan was to bathe them in either L-DOPA or different concentrations of METH, with a further experiment to add TEMPOL (a superoxide dismutase mimetic that has been shown to decrease the effectiveness of METH on creating ROS) to the METH solutions should there be a melanization response.
Results and Discussion
Unfortunately, this project failed. After trying the procedure outlined above multiple times as well as several variants, we had to conclude that we couldn’t keep the neurons alive long enough to observe any type of melanization. Therefore, we brainstormed other formats we could use to induce and observe this phenomenon.
We tried the following other methods: cultured slices in media, chronic injections in vivo, and even growing dopamine-expressing cell culture (PC-12 cells) with the help of our collaborator at the University of Hawaii and then measuring the lysate by spectrophotometry, background subtracting all but the 480 nm peak. We have yet to have any positive results.
Ultimately, we are confused as to our inability to achieve a viable experimental protocol. In the near future, we will be having part of our lab certified as a Biosafety Level 2 (BSL 2) facility, which will enable us to use established cell lines. Several lines exist which express dopamine. We plan on using one of these cultures to replicate our original proposed experiment and following up with a silver stain that is specific to melanins. We are anxious to start this experiment, but are currently waiting on the approval and finalization of the BSL 2 facility.
Sources
Jang, E. Y., Yang, C. H., Hedges, D. M., Kim, S. P., Lee, J. Y., Ekins, T. G., Garcia, B. T., Kim, H. Y., Nelson, A. C., Kim, N. J., and Steffensen, S. C. (2016) The role of reactive oxygen species in methamphetamine self-administration and dopamine release in the nucleus accumbens. Addiction Biology, doi: 10.1111/adb.12419.
Sulzer D, Bogulavsky J, Larsen KE, Behr G, Karatekin E, Kleinman MH, Turro N, Krantz D, Edwards RH, Greene LA, Zecca L (2000) Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles. Proc Natl Acad Sci U S A 97:11869-11874.