The authors would like to acknowledge NanoVentures Australia for travel funding support and Prof. John Haycock for having partially hosted this research at the University of Sheffield and Ms. Jaimee Mayne for her help during the filming.

1. Au NRs Preparation
Note: Au NRs can be synthesized by a number of recipes16, or purchased from commercial vendors.
<ol>
	<li>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.</li>
	<li>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 provided by the vendor.</li>
	<li>Prepare a 5 ml stock solution by diluting the initial Au NR sample to reach an OD = 1. For the best repeatability, keep the OD constant for all of the tested samples. Follow the commercial vendor&#8217;s protocol for the composition of the diluting solvent. If unsure, use deionized water.</li>
	<li>Centrifuge 1 ml of the Au NR solution twice for 15 min at 7,800 x g to remove any chemical excess from the solution. Centrifugation cycles can vary in force and time (e.g. 20 min at 5,600 &#215; g)18.</li>
	<li>Remove the supernatants and re-suspend the NRs in deionized water. As re-suspended particles might form aggregates in solution, prepare them daily to have the best results. Alternatively, store in a fridge for no longer than 1 week. Do not freeze.</li>
	<li>Before using under cell culture conditions, sonicate the Au NR solution for 5 min and then sterilize with UV light for 30 min (UV radiation intensity not less than 400 mW&#8729;m-2 at 254 nm).</li>
</ol>
2. NG108-15 Neuronal Cell Line Culture and Differentiation
<ol>
	<li>For the cell culture medium, prepare 500 ml of sterile Dulbecco&#39;s modified Eagle medium (DMEM) containing 10% (w/v) fetal calf serum (FCS), 1% (w/v) L-glutamine, 1% (w/v) penicillin/streptomycin and 0.5% (w/v) amphotericin B. 
	Note: Supplements can be aliquoted, stored at -20 &#176;C and added to the media on the day required. Cell culture medium can be refrigerated in a sterile condition for a maximum of 1 month.</li>
	<li>For the cell differentiation medium, prepare 50 ml of sterile DMEM containing 1% (w/v) L-glutamine, 1% (w/v) penicillin/streptomycin and 0.5% (w/v) amphotericin B.</li>
	<li>Grow NG108-15 neuronal cells in 10 ml of cell culture medium in T75 flasks made of polystyrene in an incubator with humidified atmosphere (5% CO2 at 37 &#176;C). Normally, seed 1.5-2 &#215; 105 cells in each flask to be ready in 3-4 days. Change cell culture medium every two days. 
	Note: To prevent genetic drifts or variation, do not use cells older than passage 21 for experiments.</li>
	<li>When 70-80% confluent in culture, change the medium with warm fresh cell culture medium. Mechanically detach the cells by gently knocking the bottom of the confluent flask. Do not use trypsin.</li>
	<li>Centrifuge the cell suspension for 5 min&#160;at 600 x g and re-suspend the cell pellet in 2 ml of warm cell differentiation medium.</li>
	<li>Seed 2 &#215; 104 cells/cm2 in a tissue culture polystyrene 96 well plate with 200 &#181;l of cell differentiation medium. Incubate the experiment for 1 day at 5% CO2/37 &#176;C.</li>
	<li>Add between 3.2 &#215; 109-4.2 &#215;1 010 particles/ml of Au NR solution on day 2 and incubate it for additional 24 hr. Do not add the particles for the control experiments. 
	Note: As an alternative control, Au NPs with a well-differentiated peak absorption wavelength can be used for comparison purposes14. For a more consistent cell behavior, keep the cell density constant and do not modify the well&#160;surface prior to cell seeding.</li>
</ol>
3. Neurite Outgrowth Enhancement
<ol>
	<li>Couple the laser with a single mode optical fiber (numerical aperture = 0.13) and terminate it with a fiber connector (FC connectors are convenient and commonly available). Measure the output laser power with a standard power meter. To obtain the most effective results, match the peak wavelength of the laser to the plasmon resonance peak of the NRs.</li>
	<li>On day 3 following NR incubation, fix the FC connector to the well. Irradiate samples and controls at RT for 1 min&#160;in continuous wave, for different laser powers. Allow the culture to proceed for 3 additional days at 5% CO2/37 &#176;C. Repeat the laser irradiation for a minimum of 3 independent measurements. 
	Note: Different irradiation times and pulse frequencies may be selected, depending on the application.</li>
	<li>Characterize the laser in terms of beam diameter and laser irradiance (W&#8729;cm-2).
	<ol>
		<li>For a standard single mode fiber, use NA = n&#8729;sin&#952;, where n is the refractive index of the medium in use and &#952; is the half-angle of the cone of light exiting the fiber (see Figure 1A). From trigonometry, r = tan&#952;&#8729;d, where r is the beam radius and d is the distance between the fiber and the sample. The FC connector matches the diameter of the well, therefore illuminating the sample at a distance d = 2.70 &#177; 0.20 mm. Light exits the fiber in water (n = 1.33), giving r = tan(sin-1(NA/n))&#8729;d.</li>
		<li>Using the latter equation, calculate the laser beam radius, the corresponding beam area and the average laser irradiance (laser power divided by beam area) at the target (see the example graph in Figure 1B). These values represent the average irradiance over the illuminated area.</li>
		<li>Evaluate the errors for the measurements with the general theory of error propagation. 
		Note: The distance between the FC connector and the sample can be measured by photographs of the experimental arrangement and post-processing of the image using appropriate software (e.g. ImageJ).</li>
	</ol>
	</li>
	<li>At day 5, remove cell differentiation medium from the experiments and fix the samples with 3.7% (v/v) formaldehyde solution for 10 min, then permeabilize the cells with 0.1% (v/v) Triton X-100 for 20 min.</li>
	<li>Add 3% (w/v) bovine serum albumin (BSA) to the samples for 60 min to block the unreacted protein binding sites. Label the samples for anti-&#946;III-tubulin overnight (5 &#181;g/ml in PBS supplemented with 1% of BSA) at 4 &#176;C.</li>
	<li>Incubate the cells for 90 min in the dark with an appropriate secondary antibody (e.g. TRITC-conjugated anti-mouse IgG antibody) using a concentration of 0.4-2 &#181;g/ml in 1% BSA in PBS. Label the cell nuclei with DAPI (0.1 &#181;g/ml in de-ionized water) for 10 min. 
	Note: Antibody conjugation and concentration might vary according to the company protocol. Wash samples with PBS twice for 5 min&#160;after each staining stage. 
	<img alt="Figure 1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li>
	<li>Image samples by epifluorescence or confocal microscopy using at least a 20&#215; objective. Choose the microscope filters accordingly to the secondary antibody. Select a DAPI filter (&#955;EX = 358 nm; &#955;EM = 488 nm) to visualize the cell nuclei.</li>
	<li>Analyze the pictures by assessing: i) the maximum neurite length (record the length from the tip of the neurite to the beginning of the cell body), ii) the number of neurites per neuronal cell (sum up all of the neurites per cell) and iii) the percentage of cells with neurites (divide the total number of cells expressing &#946;III-tubulin by the total number of cells with a positively stained nucleus)19.</li>
</ol>
4. Laser-induced Intracellular Ca2+&#160;Imaging
<ol>
	<li>Prepare a 20% (w/v) stock solution of Pluronic F-127 by dissolving 2 g of solute in 10 ml of anhydrous dimethyl sulfoxide (DMSO). Heat the solution of Pluronic F-127 at 40 &#176;C for about 20 min&#160;to increase the solubility. Prepare it in advance if stored at RT.</li>
	<li>On day 3 following NR incubation, prepare a balanced salt solution (BSS; 135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2 , 0.5 mM MgCl2 , 5.6 mM glucose, and 10 mM HEPES, pH 7.4) and supplemented it with 5 &#181;M of Fluo-4 AM in DMSO and 0.1% (w/v) of Pluronic F-127 solution. 
	Note: BSS solution can be prepared in advance and refrigerated for a maximum of 1 month. Add the supplements (Fluo-4 AM and Pluronic F-127) on the day of the experiment.</li>
	<li>Remove cell differentiation medium and replace it with the supplemented BSS solution. To obtain the best results with an inverted confocal microscope, incubate the cells in a micro-slide well.</li>
	<li>Load NG108-15 cells with Fluo-4 AM for 30 min&#160;in the dark at 5% CO2/37 &#176;C. Following the incubation time, wash samples twice with BSS to remove any extracellular unloaded fluorophore.</li>
	<li>Couple the laser with a single mode optical fiber and cleave the tip using standard techniques. Observe the resulting tip under an optical microscope, to ensure a high-quality surface (i.e.&#160;the tip should be perpendicular to the fiber axis and flat upon microscopy inspection).
	<ol>
		<li>Measure the output laser power with a standard power meter. Insert the light delivery fiber into an optical fiber holder and affix it to a micropositioner. Refer to Brown et al.20 for more details on optical fiber preparation and positioning. 
		Caution: Follow the general rules for laser safety during the measurement of the laser power (e.g.&#160;do not look directly into laser beam, wear laser safety goggles during handling, prevent stray light exposure to other lab users).</li>
	</ol>
	</li>
	<li>Connect an oscilloscope to the portable laser to monitor the optical modulation. Use a binary signal with variable frequencies (0.5-2 Hz) and pulse lengths (20-100 msec). Connect the laser using the modulation signal as transistor-transistor logic (TTL) input for the microscope, following the setup shown in Paviolo et al.13.
	<ol>
		<li>Place the cells under an inverted confocal microscope and position the light delivery fiber 250 &#177; 50 &#181;m away from the target cell in transmission illumination mode. At this distance, use the equations presented in 3.3.1 to calculate the beam radius at the target.</li>
	</ol>
	</li>
	<li>Use an argon-ion laser (488 nm) to excite the internalized Fluo-4 AM dye (&#955;EX = 494 nm; &#955;EM = 516 nm) and the synchronized LD for the excitation of the endocytosed NRs. Perform the imaging at RT of samples and controls using at least a 40&#215; objective by collecting time-series scans with 256 &#215; 256 pixels/frame resolution in roundtrip mode with a frequency of at least 4 Hz. Perform a recording without any LD excitation to identify any argon-ion laser interference (baseline noise).</li>
	<li>Remove the background from the data using adaptively weighted penalized least-squares algorithms21. Plot the induced Ca2+ variations as a function of time. Analyze the transient peaks in the fluorescence intensity using image post-processing software by thresholding at a level three times the standard deviation of the baseline noise (3&#963;).</li>
</ol>

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P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), (2012).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J. Biomed. Opt. 4 (1), 36-46 (1999).</li><li>Byrnes, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers Surg. Med. 36 (3), 171-185 (2005).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=810+nm+wavelength+light:+an+effective+therapy+for+transected+or+contused+rat+spinal+cord.">810 nm wavelength light: an effective therapy for transected or contused rat spinal cord.</a> Lasers Surg. Med. 41 (1), 36-41 (2009).</li><li>Grossman, N., Schneid, N., Reuveni, H., Halevy, S., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=780+nm+low+power+diode+laser+irradiation+stimulates+proliferation+of+keratinocyte+cultures:+involvement+of+reactive+oxygen+species.">780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.</a> Lasers Surg. Med. 22 (4), 212-218 (1998).</li><li>Wong-Riley, M. T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+directly+benefits+primary+neurons+functionally+inactivated+by+toxins+-+Role+of+cytochrome+c+oxidase.">Photobiomodulation directly benefits primary neurons functionally inactivated by toxins - Role of cytochrome c oxidase.</a> J. Biol. Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

The authors have no competing interests to disclose.

The protocols outlined in this presentation describe how to culture, differentiate and optically stimulate neuronal cells using extrinsic absorbers. The NR characteristics (e.g.&#160;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 ...

Recent studies have demonstrated that the transient heating associated with the absorption of infrared light by water (wavelength &#62;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 models suggested that the target tissue/cells may be affected by cumulative heating effects, when multiple stimulus sites and/or high repetition rates are used3,4.
In response to these challenges, researchers have recognized the potential to use extrinsic absorbers for nerve stimulation to produce more localized heating effects in the tissue. Huang et al. demonstrated this principle by using superparamagnetic ferrite nanoparticles to remotely activate the temperature-sensitive TRPV1 channels in HEK 293 cells with a radio-frequency magnetic field5. Although this technique may allow for deeper penetration (magnetic fields interact relatively weakly with tissue), the responses were only recorded over periods of seconds, rather than the millisecond durations required in bionic devices5. Similarly, Farah et al. demonstrated electrical stimulation of rat cortical neurons with black micro-particles in vitro. They showed cell-level precision in stimulation using pulse durations on the order of hundreds of &#181;s and energies in the range of &#181;J, potentially allowing for faster repetition rates6.
The use of extrinsic absorbers has also been applied to induce morphological changes in vitro. Ciofani et al. showed a ~40% increase in neuronal cell outgrowth using piezoelectric boron nitride nanotubes excited by ultrasound7. Similarly, endocytosed iron oxide nanoparticles in PC12 cells have been reported to enhance neurite differentiation in a dose-dependent manner, due to the activation of cell adhesion molecules with the iron oxide8.
Recently, the interest in extrinsic absorbers to assist neural stimulation has also focused on the use of gold nanoparticles (Au NPs). Au NPs have the ability to efficiently absorb laser light at the plasmonic peak and to dissipate it into the surrounding environment in the form of heat9. Amongst all of the available particle shapes, the optical absorption of gold nanorods (Au NRs) conveniently matches the therapeutic window of biological tissues (near infrared - NIR, wavelength between 750-1,400 nm)10. Moreover, in the context of neural stimulation, the use of Au NRs provides relatively favorable biocompatibility and a wide range of surface functionalization options11. Recent studies have shown that a stimulatory effect on differentiation can be induced after continuous laser exposures of Au NRs in NG108-15 neuronal cells12. Similarly, intracellular calcium transients were recorded in neuronal cells cultured with Au NRs after laser irradiation modulated with variable frequencies and pulse lengths13. Cell membrane depolarization was also recorded after NIR laser illumination of Au NRs in primary cultures of spiral ganglion neurons14. The first in vivo application with irradiated Au NRs has been demonstrated just recently. Eom and coworkers exposed Au NRs at their plasmonic peak and recorded a six-fold increase in the amplitude of compound nerve action potentials (CNAPs) and a three-fold decrease in the stimulation threshold in rat sciatic nerves. The enhanced response was attributed to local heating effects resulting from the excitation of the NR plasmonic peak15.
In the present paper, protocols for investigating the effects of laser stimulation in NG108-15 neuronal cells cultured with Au NRs are specified. These methods provide a simple, yet powerful, way to irradiate cell populations in vitro using standard biological techniques and materials. The protocol is based on a fiber-coupled laser diode (LD) that allows safe operation and repeatable alignment. The Au NR sample preparation and laser irradiation methods can be further extended to different particle shapes and neuronal cell cultures, providing that the specific synthesis and culture protocols are known, respectively.

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

gold nanorod-assisted optical stimulation of neuronal cells

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.

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&#183;cm&#8722;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 ...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

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.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K Views.  Swinburne University of Technology. 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.

Video: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Chemistry

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Bioengineering

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

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Using Affordable LED Arrays for Photo-Stimulation of Neurons

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetic Stimulation of the Auditory Nerve

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording