(K-425) Deep Brain Stimulation Waveform Parameter Exploration in C. elegans

Introduction::

Parkinson's disease is a neurological disorder severely affecting muscle control in about 1% of the American population over age 60 [1]. The resulting tremors can be debilitating and lead to poor quality of life. The most effective oral medication for symptom relief is Levodopa, but over time its benefits often lessen. Deep brain stimulation (DBS) is effective for altering patients' responses to Levodopa and reducing tremors. DBS involves an implanted electrical stimulation device with electrodes to deliver varying waveforms to the thalamus and have been shown to increase muscle control long term. While DBS has proven effective for minimizing tremors, little is known of the biological mechanism behind this improvement. We have explored the effect of varying stimulation voltage, frequency, and duty cycle on neural firing. These selections of parameters are currently done manually by trial and error in vivo which is far from optimal. The long term consequences of these different parameters are also unknown. This study aimed to explore how variations in the duty cycle and frequency of non-invasive electrical stimulation waveforms modulate the excitability of C. elegans AWA neurons. We chose to use a model organism to investigate the physiological impacts of DBS because of their neurochemical and genetic homology to mammalian brains as well as their noninvasively observable neural activity across many animals at once.. By tracking the neural output and watching for changes in the strength of the signal when stimulation is applied, the impact each parameter imparts on the animal's neural excitability was assessed. 

Materials and Methods:: Microfluidic arenas shaped as parallel trap arrays were created to efficiently trap the C. elegans in place for experiments. The arenas were designed in CAD and molds were fabricated using photolithography on silicon wafers. Polydimethylsiloxane (PDMS) was poured onto the molds to form the devices. The designs were then individually cut out and a 1 mm dermal punch was used to create holes for the tubing to connect. The devices were plasma bonded to a glass slide and flow induced using hydrostatic pressure. The animals were genetically encoded with GCaMP, a calcium sensor to report neural activity by fluorescence brightness. The animals fed on OP50 lawns covered in 50 µM All-Trans-Retinal for 12-18 hours prior to experimentation. This allows for non-invasive depolarization of neurons expressing the Chrimson cation channel using a red LED (𝝀=617 nm). One hour prior to experimentation the animals expressing Chrimson and GCaMP in the AWA chemosensory neurons were placed in a 1 mM Tetramisole hydrochloride paralytic solution for immobilization. Images were acquired on a 4X (0.28 NA) wide-field inverted fluorescence microscope at 10 fps. The experiments were coded using ImageJ and a custom-built Arduino controller automated experiment acquisition and the application of the optical and electrical stimulation. The data was analyzed using NeuroTracker [3], an ImageJ/Fiji plugin, where brightness of the neuron signals were measured. MATLAB was then used for data aggregation and visualization.

Results, Conclusions, and Discussions::

Using red light stimulation, we can establish a baseline for what typical neural activity looks like. Variation from the baseline pattern is the result of neuromodulation from electrical stimulation. To investigate the effects of variation in the applied voltage we ran a voltage sweep ranging from 0-15V. This data indicates that the directionality of the animal causes inverse responses. While the response of animals facing the positive electrode showcased increased excitability with the applied charge, a decrease in excitability was seen in those facing the negative electrode. We also assessed that a range of neuromodulation is possible, found to be between 6-9V, varying from animal to animal. Before this range, animals seem to respond comparably to the controls. After each animal has reached its unique value within this range, it will directly depolarize. With this high-throughput model, we intend to run more studies to get an accurate representation of precisely where this range of neuromodulation falls. To investigate how duty cycle impacts neuronal depolarization, a sweep ranging from 0.4-100% at a constant 12V and 80Hz was run. This was aimed to mimic the physiological parameters of in vivo DBS devices ranging from 70-120Hz and 3.0-3.5V, with consideration to limitations in machinery and the model organism (Figure 3). From these experiments, we found that duty cycles below 55% appear to modulate responses, while duty cycles above this inhibit optogenetic depolarization for all animals. We found that duty cycles between 5-55% inhibited the responses of animals facing the grounded electrode, although they exhibited rebounding responses within a minute of removal of the electrical stimulation. A duty cycle of 25% led to consistent response modulation for animals in both orientations, so it was chosen as the new default level. Next, we ran a frequency sweep at 12V ranging from 40-120Hz. The applied levels of stimulation lead to an increase in the amplitude of neural responses, this increase was consistent throughout and did not correlate with the frequency applied. This indicates that frequencies within this range do not play a role in the strength of the neural response. 

Acknowledgements (Optional): : This work was supported by NSF REU Grant EEC2150076
We thank Hamilton White and Fox Avery for their assistance in lab and experimental guidance to the completion of this work.

References (Optional): :

[1] “Parkinson’s Disease.” Mayo Clinic, 26 May 2023, www.mayoclinic.org/diseases-conditions/parkinsons-disease/diagnosis-treatment/drc-20376062. 

[2] “Neurodegenerative Diseases.” National Institute of Environmental Health Sciences, www.niehs.nih.gov/research/supported/health/neurodegenerative/index.cfm#:~:text=Alzheimer’s%20disease%20and%20Parkinson’s%20disease,Alzheimer’s%20Disease%20Association%20in%202022. Accessed 24 July 2023.

[3] Larsch, J., Ventimiglia, D., Bargmann, C. I., Albrecht, D. R. High-throughput imaging of neuronal activity in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 110 (45), E4266-E4273 (2013)

[4] Created with BioRender.com