Hi everyone, my name is Aaron Rasheen, I'm an MD-PhD candidate at the Mayo Clinic in the Neural Engineering Laboratory under the mentorship of Dr. Kendall Lee. I will be presenting my work on the potential therapeutic mechanism of central median paraphysicular complex stimulation for Tourette syndrome. I would like to thank the organizers of this virtual conference for giving me this opportunity. I have no disclosures. Tourette syndrome is a neurologic disorder characterized by chronic motor and vocal tics. For most patients, tics are successfully treated with behavioral and pharmacotherapy. However, for a small subset of patients, approximately 10%, more intensive therapy is warranted, and DBS has demonstrated considerable efficacy in this regard. There are two primary targets for stimulation. The first is the globus pallidus internus. The second is the central median paraphysicular complex of the intralaminar thalamus. My research focuses on central median paraphysicular complex stimulation. We know that central median paraphysicular complex stimulation is therapeutically beneficial. However, the neurobiologic mechanism by which CMPF-DBS alleviates symptoms is not quite understood. There is increasing evidence showing the potential role of dopamine in the pathophysiology of Tourette syndrome. This includes a positive therapeutic benefit of dopamine receptor antagonists like risperidone, increased dopamine transporter and vesicular monoamine transporter 2-binding as seen with PET imaging, and finally, there's increased dopamine release following amphetamine stimulation. We wanted to understand how brain activity levels change in response to central median paraphysicular complex stimulation for Tourette syndrome patients. To do so, we used functional MRI imaging, and the results from the study are in the figure below. Areas of hypoactivity are in blue, and areas of hyperactivity are in red and yellow. An area of note, as indicated by the arrow, is the caudate nucleus accumbens and butamen. This area is collectively known as the striatum. In response to stimulation, these areas have hypoactivation. This is notable because it is known that the paraphysicular nucleus sends a dense glutamatergic projection to the striatum, and these glutamatergic neurons synapse onto cholinergic interneurons, which in turn are known to modulate striatal dopamine release. This leads us to our hypothesis. CMPF-DBS modulates striatal dopamine, leading to alleviation of tics. To test this hypothesis, we performed paraphysicular nucleus stimulation in anesthetized rats. Stimulation was performed with DBS-like parameters using a bipolar stimulation electrode. We recorded striatal dopamine release in the dorsal striatum, both phasic release and tonic levels using carbon fiber microelectrodes. Fast gain cyclic voltometry is a technique capable of measuring phasic dopamine release from the synapse. In this technique, a negative potential is quickly ramped up to a positive potential and then back down. When this occurs, dopamine is oxidized and reduced, generating a current that is directly related to the concentration of dopamine. To measure tonic dopamine levels, we use a new technique developed by our laboratory called multiple cyclic square wave voltometry. As you can see in this figure, square waveforms are overlaid onto a step-like pattern, generating this forward and reverse sweep, and this technique can measure absolute dopamine values. The first step was to understand where the optimal stimulation location was in the paraphysicular nucleus to evoke striatal dopamine release. We began by stimulating the dorsal portion of the paraphysicular nucleus at negative 5.0 and progressing downward until an optimal location was found. This was typically between negative 5.6 and negative 5.8. The data from these experiments are represented in the two figures to the right. The first figure on the top right, we plot depth on the x-axis, and dopamine release on the y-axis as measured by area under the curve. As we increase depth from 5 to 5.8, we find that dopamine release does increase as we increase our depth. In addition, on the graph on the bottom right, we plot depth on the x-axis and release rate on the y-axis, and we find as we increase our depth, the release rate does indeed increase. We perform linear regression analysis of the data to understand if the release rate is correlated with depth, and our model has an R-squared value of 0.5 and a p-value of 0.02, suggesting that it is significant. Once an optimal stimulation location was found, we performed DBS-like stimulation of the paraphysicular nucleus while recording phasic dopamine release using fast-scan circuit voltmetry. Our stimulation parameters were 130 Hz, 0.4 ms pulse width, and 400 uA. We used biphasic charge-balanced pulses for the stimulation. On the graph on the left, you can see this is a color plot. On the x-axis, we have time. On the y-axis, we have voltage from negative 0.4 V to 1.3 V and back down to negative 0.4 V. Additionally, the intensity of the color plot demonstrates the amount of current being generated. You can see at 0.6 V, as indicated with the line, this is the oxidation voltage for dopamine, and we can see a large current being generated at this voltage, signifying that when we do stimulate, we do get a large current response, which correlates with dopamine release. On the right side of the screen, you can see a line plot with the plotted dopamine versus stimulation, and you can see after we stimulate, we see approximately 100 nm increase in dopamine. We also wanted to understand if dopamine release might vary by frequency and amplitude. To do this, we performed 5 experiments where we varied the frequency from 50 to 150 Hz and the amplitude from 10 to 400 uA. What we found was as we increased amplitude and as we increased frequency, dopamine release did increase as well. We performed multiple linear regression analysis of the data collected during these experiments, and we found, as you can see on the plot on the right, where we plot predicted versus actual dopamine, that our model did indeed predict dopamine response. This model had a p-value of 0.0001 and an R-squared of 0.44, suggesting that it is statistically significant. In addition, both covariates, amplitude and frequency, were also significant in our model. Finally, we wanted to understand if stimulation evoked dopamine release decreases with administration of a dopamine synthesis blocker. We used alpha-methyl p-tyrosine, or AMPT, which blocks tyrosine hydroxylase, to do so. On the graph on the bottom left, you can see the orange and yellow graphs. Orange is our control group, yellow is our AMPT group, and you can see with AMPT, stimulation evoked dopamine doesn't decrease. We've quantified this data on the right in this bar plot. You can see the control versus animal-stimulated 60 minutes after AMPT infusion, and we find that this difference is significant. Next, we'd like to understand how tonic extracellular dopamine levels change in response to direct current deep brain stimulation of the paraphysicular nucleus. Our stimulation parameters were 130 Hz, 0.4 ms pulse width, and a 400 μA amplitude. On the figure on the left, you can see two plots. In the first subplot on the top left, you can see a color plot. On the x-axis, you can see staircase potential. On the y-axis, you can see square wave potential. The intensity of the color plot signifies the amount of current being released. On the bottom plot, you can see on the x-axis time. On that left axis, on the y-axis, you can see dopamine normalized to baseline. When we play this video, you can see the blue line indicating the baseline levels. When stimulation is turned on, you can see this blue line will change to red. In response to stimulation, you can see tonic extracellular levels increase. When stimulation is stopped, this increase remains elevated and eventually returns down to baseline. On the right side, you can see quantification of this data with a control group, which is sham stimulation and the stimulation group receiving direct current DBS. We find this elevation in tonic extracellular dopamine levels to be significant. Next, we wanted to understand how direct voltage DBS might elevate tonic dopamine levels as well. In this experiment, we performed DBS with 130 Hz, 0.4 ms pulse width, and 4 V. As we begin this experiment, we can see the baseline extracellular dopamine levels in the blue. When stimulation is begun, this turns to red, and we can see elevation of these extracellular tonic dopamine levels. When stimulation is turned off, these levels return down to baseline. We quantified the data from these experiments on the right bar plot. You can see control sham stimulation versus stimulation with direct voltage, and we do find a significant increase. In conclusion, for our phasic release experiments, we find that periphysicular nucleus deep brain stimulation evokes synaptic dopamine release. This synaptic dopamine release is dependent on stimulation location and scales with frequency and amplitude. For our tonic experiments, we find that tonic extracellular dopamine levels are elevated during deep brain stimulation. Tonic levels remain elevated for a significant period of time after stimulation and slowly return to baseline. I would like to thank the members of the Neural Engineering Laboratory for their support and guidance throughout this project. I would like to thank my mentor, Dr. Kenda Lee, as well as my mentors, Dr. Kevin Bennett, Dr. Charles Blaha, and Dr. Yim Beiou. I would like to especially thank Juan Rojas Cabrera, a very talented post-bac student, for his critical help during these experiments. I would like to thank my sources of funding, including the NIH F31 grant, as well as the NIH R01 grant, as well as the Grainger Foundation for their continued support. Thank you for your time.

CMPF-DBS

Tourette syndrome

dopamine release

stimulation location

therapeutic potential