(75f) Hybrid Electro-Plasmonic Neural Stimulation and Its Implications for Prosthetic Devices

Electrical stimulation tends to spread to surrounding tissues, making it difficult to stimulate discrete neural sites with traditional neural prosthetic devices, such as cochlear implants, cardiac pacemakers, electromyography stimulators, sciatic nerve stimulators, etc. used for neural stimulation. To facilitate specific point stimulation, various nanomaterial-assisted neural stimulation approaches have been reported in recent years^1-12 where different localized fields are activated (electric, magnetic, thermal) employing different nanomaterials for stimulation. Infrared stimulation has been shown to excite neurons, but with limitation of heating surrounding tissue^13-14. In a previous study¹⁵ we demonstrated the use of visible light (532 nm) laser pulses to trigger a localized surface plasmon resonance (LSPR) effect from the gold nanoparticles coated microelectrodes for stimulating plasmonic modulation of SH-SY5Y neuroblastoma cells response, but with a limited success rate and detrimental effects on the cell membrane with higher levels of pure optical stimulation. To address the limitations, we developed a novel platform for spatially and temporally precise neural excitation, using hybrid electro-plasmonic stimulation technique in a whole-cell patch-clamp configuration to elicit electrical responses in primary trigeminal neurons.

Experimental Design/Methods:

Borosilicate glass micropipettes were silanized with 10 % solution of γ-aminopropyl triethoxy silane in ethanol, following the Turkevich method¹⁶. Then, the tip of the micropipette was dip-coated with 20 nm-diameter colloidal gold nanoparticles (Au NPs) for making the gold-coated micropipettes¹⁵. These micropipettes were used for producing the needed plasmonic stimulation effect for the in-vitro neuron stimulations. 5-7 weeks C57B1/6 mice were used for the study. Mouse was decapitated, and the trigeminal ganglia nerve was removed. The nerve tissue was dissociated enzymatically, with HBSS containing collagenase type I and dispase II, and finally neurons were cultured in L15 media containing 10 % FBS and left to incubate in an incubator maintained at 37 C, 5 % CO₂ over night after dissociation. All cells were used within 36 hours timeframe. All the electrophysiology experiments were done using whole-cell patch-clamp methods. Microelectrode was placed next to a single patched neuron cell and a 532 nm green laser beam was focused onto the gold coated electrode tip using an optical fiber. Whole cell patch-clamp techniques were used in conjunction with an Axopatch 700B amplifier, Digidata 1440 interface, and pCLAMP-9 software (Axon Instruments, Union City, CA, USA). Extracellular solution was used to flood the cells in the petri dish. For the hybrid stimulation, electrical stimulus was added, in addition to the optical plasmonic stimulation. Neural responses were recorded using the patch electrode.

Major Findings/Results:

Electrical stimulation was used before and after the optical stimulation to verify that cells are healthy and can fire action potentials. Initially, experiments were done with pure optical stimulations (1-5 ms pulse, 100-120 mW power). Plasmonic stimulations were more detrimental to the cells as compared to pure electrical stimulation, with success rate of firing action potential less than 30 % of stimulated cells. Also, pure optical stimulations led to detrimental effects on the cell membrane. To overcome these issues, we combined electrical and optical stimulation to add the advantages from both stimulation methods, plasmonic and electrical, while decreasing the disadvantages from both methods when used alone. The percentage reduction in input current with hybrid stimulation, as compared to the current required to fire action potential with pure electrical stimulation, was approximately 40%. Also, electrical action potentials were recorded at a higher success rate with hybrid stimulation as compared to pure plasmonic stimulation (83% vs. 26%). Neuron cells survival and viability after hybrid stimulation was superior to that of pure optical stimulation (72% vs. 13%). We were also able to achieve multiple consecutive action potentials firings when applying consecutive hybrid stimulation on the cells. Further optimization of the lead and lag time of the electrical vs. plasmonic stimulus was also studied. The most optimal output is achieved when electrical stimulus leads before optical by 0.4 ms and not more than 1.4 ms, as well as when optical stimulus leads electrical by 0.6ms or less. The best repeatability of the action potential firing was achieved when electrical leads before optical by less than 1 ms.

Summary:

We demonstrated that with our hybrid stimulation platform, a reduction of up to ~40% of the input current threshold is achieved when optical stimulation is added to the sub-threshold electrical stimuli. Nanomaterials, specifically gold, maximize the utility of thermal stimulation via the LSPR phenomena in the triggering action potentials. Our findings show the applicability of short duration pulses (1-5 ms) when applied repeatedly, for sub-threshold electrical and LSPR visible light stimulation pulses, in combination with AuNPs-coated substrates (nanoelectrodes), can reliably trigger a train of action potentials, for obtaining repeatable multiple trains of APs from neurons. Neural cell survival rates and viability after hybrid stimulation was superior to that of pure optical stimulation. The input current sufficient to trigger APs with multiple hybrid stimulation was 35-40% lower, matching what was observed earlier with single AP recordings. This reinforces the previous findings above, supporting the effectiveness of the proposed platform for hybrid electro-plasmonic stimulation of neurons, which opens doors of opportunities to develop a new generation of high-acuity neural modulation prosthetic devices, tunable for the individual patient’s needs. Our long-term goal is to develop nanoparticle-light based cochlear implants for the hearing-impaired population. The reduction of current required to trigger action potentials, and the evidence that cells stay healthy after repeated exposure to hybrid stimulation are ground breaking results for a new generation tunable cochlear implants that can offer more precise frequency modulation enabled by more selective activation of auditory neurons.

Reference:

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