Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Summary

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Abstract

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Introduction

Recent studies have demonstrated that the transient heating associated with the absorption of infrared light by water (wavelength >1,400 nm) can be used to induce action potentials in nerve tissue¹ and intracellular calcium transients in cardiomyocytes². The use of infrared light has raised great interest for applications in neural prostheses, due to the potential finer spatial resolution, lack of direct contact with the tissue, minimization of stimulation artifacts, and removal of the need to genetically modify the cells prior to stimulation (as required in optogenetics)¹. Despite all of these benefits, recently developed thermal ....

Protocol

1. Au NRs Preparation

Note: Au NRs can be synthesized by a number of recipes¹⁶, or purchased from commercial vendors.

1. Measure the initial optical density (OD) of the Au NR solution via UV-Vis spectroscopy, by recording the absorption values from 300 nm to 1,000 nm with a resolution of 0.5-2 nm. Vary the volume of the solution to be used with the available cuvette.
2. Evaluate the initial NP molar concentration with a suitable technique¹⁷ (e.g. UV-Vis spectroscopy, single particle inductively coupled plasma mass spectrometry, transmission electron microscopy) or use the concentration values p....

Results

By using Protocols 1, 2, and 3 described here, a stimulatory effect on differentiation was observed in NG108-15 neuronal cells cultured with Au NPs (Au NRs, poly(styrenesulfonate)-coated Au NRs and silica-coated Au NRs) after laser exposures between 1.25 and 7.5 W·cm⁻² . Confocal images of rhodamineB-labelled Au NRs demonstrated that the particles were internalized from day 1 of incubation¹². The localization was predominantly observed in the cell cytoplasm, indicating that the preferred mechanism o.......

Discussion

The protocols outlined in this presentation describe how to culture, differentiate and optically stimulate neuronal cells using extrinsic absorbers. The NR characteristics (e.g. dimensions, shape, plasmon resonance wavelength and surface chemistry) and the laser stimulation parameters (such as wavelength, pulse length, repetition rate, etc.) can be varied to match different experimental needs. The effects on cell behavior can be monitored using standard biological assays and materials. Overall the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Au NR	Sigma Aldrich	716812
NG108-15	Sigma Aldrich	8811230
DMEM	Sigma Aldrich	D6546
FCS	Life Technologies	10100147
L-glutamine	Sigma Aldrich	G7513
Penicillin/streptomycin	Life Technologies	15140122
Amphotericin B	Life Technologies	15290018
Formaldehyde	Sigma Aldrich	F8775
Triton X-100	BDH	T8532
BSA	Sigma Aldrich	A2058
Anti-βIII-tubulin	Promega	G7121
TRITC-conjugated anti-mouse IgG antibody	Sigma Aldrich	T5393
DAPI	Invitrogen	D1306
Fluo-4 AM	Invitrogen	F14201
DMSO	Sigma Aldrich	472301
Pluronic F-127	Invitrogen	P6867
UV-Vis spectrometer	Varian Medical Systems Inc.	Cary 50 Bio
Mini centrifuge	Eppendorf	Mini Spin
Sonic bath	Unisonics Australia	FPX 10D
Cell culture incubator	Kendro	Hera Cell 150
Cell culture centrifuge	Hettich	Rotofix 32A
Laser diode	Optotech	780 nm single mode fibre - coupled LD
Optical fiber	Thorlabs	780 HP
Power meter	Coherent	Laser Check
ImageJ	http://rsb.info.nih.gov/ij/index.html
Epifluorescent microscope	Axon Instruments	ImageX-press 5000A
μ-slide well	Ibidi	80826
Inverted confocal microscope	Carl Zeiss Microscopy Ltd.	LSM 510 meta-confocal microscope
Oscilloscope	Tektronix	TDS210