Name: Using Affordable LED Arrays for Photo-Stimulation of Neurons (Video) | JoVE Journal
Uploaded: 2011-11-15T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Learn to use affordable LED arrays for precise optogenetic control of adult-born neurons in brain slice preparations. This cost-effective method enables accurate measurement of activation thresholds and studies of neuronal integration.

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This work was supported by the life insurance company "AG2R-La-Mondiale", Ecole des Neurosciences de Paris (ENP), the Agence Nationale de la Recherche "ANR-09-NEUR-004" in the frame of "ERA-NET NEURON" of FP7 program by the European Commission, and the Pasteur Foundation. Sebastien Wagner was supported by the Letten Foundation.

1. Optical Calibration: Measuring LED Power
<ol>
	<li>Attach a LED array to a heatsink actively cooled by a fan and affix this LED/heatsink aparatus to a collimating lens.</li>
	<li>Replace lamp used in brightfield illumination with LED/heatsink/fan/lens apparatus. This apparatus must be carefully positioned so that the collimated LED beam travels along a straight optical path towards the condenser lens. Make sure the heatsink/fan is properly grounded to the system's common ground.</li>
	<li>Drive the LED array with a power supply that can give fast and square pulses of current. This power supply may be controlled by a 5V TTL pulse originating from a pulse generator.</li>
	<li>Center the collimated beam along the light path defined between the field diaphragm and the condensor lens. Ideally, the LED beam will slightly overfill the fully opened field diaphragm. Typically, a more tightly collimated LED beam will fill this aperture less, and will produce more power at the expense of uniformity. In our setup we increased light uniformity by choosing a collimating lens that projected a slightly expanded image of the LED array at its conjugate plane at the consenser diaphragm.</li>
	<li>Achieve Kohler illumination by focusing the condenser so that the image of the field diaphragm (the diaphragm closest to the light source) is focused on the slice chamber (fig. 1). A thin tissue of lens paper can act as a projection screen to visualize the focused image of the field diaphragm at other depths.</li>
	<li>Drill a series of pinholes of known diameter in an opaque material. Place one of these small pinholes over the sensor of an optical power-meter. Place the power meter on the specimen stage and center the power meter over the focused image of the field diaphragm by simply moving the power meter until it gives a maximum reading. Affix the power meter in this position.</li>
	<li>Fully open all apertures (aperature diaphragm and field diaphragm). Systematically move the attached slice chamber/power meter relative to the optical lightpath and calculate the uniformity of optical power within the illuminated area. Build the uniformity plot for your system. If the microscope is properly setup with Kohler illumination centered at the objective's focus, maximum power should be directly underneath the objective, and regions outside of this focus should now receive a known amount of power according to the uniformity plot.</li>
	<li>For each size of pinhole, build a standard curve of optical power vs. pinhole surface area. By adjusting the input current to the LED array, produce this curve at multiple power levels and from each curve calculate the power per mm2. If the LED array is to be used for patching optics, make sure to introduce optical elements necessary for patching (condensers, pinholes and filters) to know how much light is transmitted under patching illumination. </li>
	<li>Flipping the power meter to face the objective, calculate the intensity of illumination at 470nm when the mercury lamp is turned on.</li>
	<li>Add a live slice to the chamber, and rebuild standard curves to determine the light power transmitted through the scattering brain tissue.</li>
</ol>
2.	Slicing Procedure and Electrophysiology
Part A: Slice Preparation
<ol>
	<li>Anesthetize (60 mg/kg Ketamine and 2mg/kg Xylazine) and decapitate the mouse. Dissect the brain in artifical cerebral spinal fluid (ACSF, in mM: 124 NaCl, 3 KCl, 1.3 MgSO4, 26 NaHCO3, 1.25 NaHPO4, 20 glucose, 2 CaCl2; ~310mOsm, pH 7.4 when bubbled with a mixture of 95% O2 and 5% CO25,1), taking care not to damage the olfactory bulbs. Separate the two hemispheres and place on agar with the ventral surface even with one edge (for horizontal sections).</li>
	<li>Glue the agar and dorsal surface of each cortical hemisphere to the vibratome chuck, and slowly fill the bath with ice-cold ACSF. Slice from ventral surface in 300 μm sections, transferring each section to warmed (34-36°C) and oxygenated ACSF, allowing them to recover for 30-45 minutes.</li>
</ol>
Part B: Loose Patch Measurement of the Threshold to Spike
<ol start="3">
	<li>After bringing the slices to room temperature for 30 minutes, gently place a slice in the recording chamber of the microscope chamber under constant perfusion of oxygenated ACSF. 
	At risk of excessively stimulating ChR2 infected neurons, the presence of EYFP-ChR2 can be confirmed under epifluorescence (fig. 2a)</li>
	<li>Pull glass electrodes on a pipette puller (Sutter P-97). Fill this electrode with ACSF. When placed in the ACSF bath the tip resistance should be between 7-10 MOhms.</li>
	<li>Under fluorescent illumination, locate in the slice a healthy ChR2-EYFP neuron with mature morphology. Also locate this neuron's soma under patching optics.</li>
	<li>With light positive pressure passed through the electrode tip, lower the patch electrode towards the identified fluorescent neuron. When membrane contact is made, quickly release positive pressure and apply a small and brief amount of suction through the tip. A giga-ohm seal should be made between the plasma membrane and the walls of the patch electrode.</li>
	<li>Even if a giga-seal is not formed, if the electrode is sufficiently close to a fluorescent neuron spiking activity should produce a measurable local field potential. Activate ChR2 in this neuron by flashing different doses of light. Because light-dose is a function of both LED power and duration, calculate how much light is necessary to evoke an action potential at multiple powers and durations (fig. 2b). Also observe how much spiking occurs under mercury lamp illumination.</li>
</ol>
3. Representative Results: 
On our microscope (Olympus BX51WI), our LED is in line with 2 apertures and a condenser lens, thus maintaining the original lightpath of the factory-installed arc lamp. By closing both the field diaphragm and the aperature diaphragm, we can achieve brightfield contrast sufficient for patch clamp recordings (fig. 1b). With all diaphragms fully open we expose the slice to maximum light power for channelrhodopsin activation (fig. 1a). On our microscope, this patching configuration produces light-density that is approximately three orders of magnitude lower than the maximum full field density (4.1 μW/mm2 versus 6.88 mW/mm2).
We see robust labeling of adult-born olfactory bulb granule and periglomerular neurons weeks after lentiviral infection of migrating neuroblasts in the rostral migratory stream (fig. 2a) A loose-patch recording from a single adult-born ChR2-EYFP expressing granule cell indicates that a 5 ms stimulation at maximum power (6.88 mW/mm2) is sufficient to evoke spiking (fig. 2b). Since expression level varies between cells, the amount of light that passes the threshold to spike will vary and should be described statistically for each cell type of interest. 
<img src="/files/ftp_upload/3379/3379fig1.jpg" alt="Microscopy setup diagram showing light path through lens system for optical analysis and comparison." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. LED array setup for full-field photostimulation and patch-clamp slice electrophysiology. To activate channelrhodopsin (ChR2) we project a collimated beam through open back apertures and condenser optics (a). This configuration can be changed into high-contrast patching optics by fully closing the field diaphragm and modulating the width of the field diaphragm (b). Abbreviations: a. fan, b. heatsink, c. LED array, d. collimating lens, e. mirror, f. field diaphragm, g. aperture diaphragm, h. condenser lens, i. sample stage, j. objective.
<img src="/files/ftp_upload/3379/3379fig2.jpg" alt="Confocal microscopy image, electrophysiology results; neural response, fluorescence analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Image of a 300μm horizontal slice of olfactory bulb for patch clamp and whole-field photostimulation (a). Lentivirally infected adult-born granule cells expressing ChR2-EYFP can be seen radiating from the core of the olfactory bulb. The light-dose required to evoke spiking can be found by increasing the duration of the LED flash (b). The threshold for this granule cell was 5ms at full LED intensity (2.43mW/mm2). Scale in (a) = 500μm, scale in (b) = 50ms.

<ol><li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li>
<li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li>
<li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((genetically+targeted+optical+control+of+neural+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li>
<li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li>
<li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+maturation+of+the+first+synapse+in+olfaction%3A+development+and+adult+neurogenesis%5BTitle%5D)+AND+%22The+Journal+of+neuroscience%22%5BJournal%5D)">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li>
<li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circuit-breakers%3A+optical+technologies+for+probing+neural+signals+and+systems%5BTitle%5D)+AND+%22Nature+reviews.+Neuroscience%22%5BJournal%5D)">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li>
<li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-site+optical+excitation+using+ChR2+and+micro-LED+array%5BTitle%5D)+AND+%22Journal+of+neural+engineering%22%5BJournal%5D)">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li>
<li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse%5BTitle%5D)+AND+%22Nature+neuroscience%22%5BJournal%5D)">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li>
<li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting%5BTitle%5D)+AND+%22Stem+cells%22%5BJournal%5D)">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li>
<li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li>
<li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 Forthcoming.</li>
<li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li>
</ol>

No conflicts of interest declared.

Recent years have seen an explosion in the popularity of optogenetic tools for neuroscience research6. As a result, it is increasingly important to lower the barrier of entry for labs wishing to begin using these new tools. Here we describe how to conduct a simple and low cost retrofitting and calibration of a conventional patch-clamp rig so that it can do full-field optical stimulation of channelrhodopsin-expressing neurons. In particular, we apply this technique to the study of olfactory bulb adult neuroge...

<table><tbody><tr><td>Ketamine</td><td>Imalg&amp;#232;ne 1000</td><td></td><td>100 mg/ml</td></tr><tr><td>Xylazine</td><td>Rompun</td><td></td><td>2%</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td>&amp;amp;nbps;</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M1880</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>NaHPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5011</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>Pipette Puller</td><td>Sutter Instrument Co.</td><td></td><td>P-97</td></tr><tr><td>Glass Capillaries</td><td>Harvard Apparatus</td><td>GC150T-10</td><td>1.5 mm O.D./1.17 mm I.D.</td></tr><tr><td>LED array</td><td>Bridgelux</td><td>BXRA-C2000</td><td></td></tr><tr><td>Collimating lens</td><td>Thorlabs Inc.</td><td>LEDC1</td><td>40 mm beam diameter</td></tr><tr><td>Power supply</td><td>A1W Electronik</td><td>HKO2800</td><td>2.8 amp</td></tr><tr><td>Optical power meter</td><td>Thorlabs Inc.</td><td>PM 100</td><td></td></tr><tr><td>Heatsink </td><td>Thermaltake</td><td>A1838</td><td>Silent Boost K8</td></tr><tr><td>Fan</td><td>Thermaltake</td><td>A1838</td><td>Silent Boost K8</td></tr><tr><td>Vibratome</td><td>Leica Microsystems</td><td>VT1200S</td><td></td></tr></tbody></table>

using affordable led arrays for photo-stimulation of neurons

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 ...

The goal of this procedure is to prepare and remotely operate LED arrays to control neural activity in brain slices. This is accomplished using in vitro slices from animals with channel REDSIN two expressing neurons, and using an LED array in conjunction with a microscope to activate those neurons. This video covers how to calibrate an LED and microscope to deliver known intensities of light to the slice chamber.With this setup, the activation thresholds of channel two expressing neurons can be accurately measured in response to light stimulation through widefield stimulation of channel opsin two expressing neurons. This technique allows temporarily precise optical control of a large neuronal population. The main advantage of this technique over existing optogenetic methods, including those that use lasers or shutter based methods, is that it's very inexpensive and can be easily fitted to an existing patch clamp scope.This method can help answer key questions in the field of adult neurogenesis, including how does activity shape the integration of new neurons into the adult brain. The implications of this technique extend toward therapies or diagnoses based on adult neural stem cells as it offers precise temporal control over the activity of adult born neurons integrating into existing neural circuitry. Though this method can provide insight into the functional consequences of adult neurogenesis, it can also be performed on any in vitro preparation.Containing channel adoption expressing neurons Begin by selecting an LED array that produces its peak power at the desired wavelength. In addition, choose the array with a maximum current rating. Integrate the LED array to the microscope.Start by attaching the LED array to a fan called heat sink using thermal paste to increase the effectiveness of the heat sink. Then remove the low power stock LED from its lens assembly and fix the higher power LED to the lens. Now replace the Brightfield lamp with the assembled LED apparatus, and make sure that the fan is securely grounded to the system's common ground.Careful positioning of the LED arrays needed to ensure that the call made light is in line with the objective.Objective. Achieve Kohler illumination by focusing the condenser so that the image of the field diaphragm is focused on the slice chamber and is centered under the objectives optical path. A thin tissue of lens paper can help focus the image of the back aperture at other depths.Now, drill pinholes of known diameters in an opaque material that can act as an aperture for the power meter. Place one of these small pinholes over the sensor of the optical power meter. Then fix the power meter to the microscope to focus the condenser on the pinhole.Manually position the power meter until it gives a maximum reading. Affix the power meter in this position. If the power meter maximum is above the plane of the slice, reestablish Kohler illumination at that plane.Next, fully open all the apertures. Systematically move the power meter relative to the optical light path using the stage manipulators. Once finished, measure the uniformity of optical power within the illuminated area by systematically taking power readings in a grid around the maximum power, which should lie under the focus of the objective, The microscope is properly set up with cooler illumination centered at the objectives focus, the maximum power of the array should be at this focus regions outside of the focus should now receive a known amount of power according to the uniformity plot.Now, measure the maximum power for each size pinhole with knowledge of the drill bit diameter used to make each pinhole plot the optical power as a function of the pinhole's area. Because the drilled pinholes are not perfect, averaging these values will produce a good estimation of optical power density. The LED array is gonna be used for patching optics.Make sure you take into account all the optical elements needed for patching when you build the power plot and the uniformity plot. For instance, many microscopes have a flippable diffuser screen that you can use that will sacrifice power for the sake of uniformity. Prepare brain tissue slices using standard methods, and while allowing the slices to recover for 30 to 45 minutes in warmed A CSF, go and make the patch pipettes.Once the tissues have recovered, gently place a slice into the recording chamber of the microscope under constant perfusion of oxygenated A CSF. Confirm the presence of EYFP channel redsin two channels by epi fluorescence under fluorescent illumination. Locate in the slice a healthy channel two EYFP neuron with mature morphology.Then locate this neuron soma under patching optics and produce a giga ohm seal between the plasma membrane and the walls of the patch electrode. If the electrode is sufficiently closed to a spiking neuron, spontaneous activity should produce a measurable local field potential even without a giga ohm seal. Now activate channel adopts two in this neuron by flashing different doses of light because light doses a function of both LED power and duration calculate how much light is necessary to evoke an action potential at multiple powers and durations.An Olympus BX 51 wi microscope was configured with an LED inline with two apertures and a condenser lens. Sufficient brightfield contrast for patch clamp recording was obtained by closing both the field diaphragm and the aperture diaphragm. A uniformity plot of LED light intensity was constructed in a region around the objective field of view indicated by the dotted circle.With all diaphragms fully opened, slicers were exposed to maximum light power for channel adoptin activation. This configuration produces light three orders of magnitude more powerful than the configuration used for patch clamp visualization. Widespread labeling of adult born olfactory bulb granule and per glomerular neurons were observed four weeks after lentiviral infection of migrating neuroblast in the rostral migratory stream.Loose patch recordings from a single born channel Adoptin two EYFP expressing granule cell indicated that for this neuron, a five millisecond stimulation at maximum power was sufficient to evoke spiking. Once mastered, this technique can be done in an afternoon. It's important to remember after this procedure to always monitor your equipment and your settings for drift over time Since their development, such optogenetic techniques have paved the way for researchers in the field of neuroscience to explore synaptic transmission and integration in a number of model organisms such as worm, fly mouse, and even primate.

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

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The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

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

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Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

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Building a Simple and Versatile Illumination System for Optogenetic Experiments

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.

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

Recent developments in pain research highlight the potential of photoneuromodulation using green light-emitting diodes (GLED) as a non-pharmacological treatment. GLED modulates pain pathways, offering effective pain relief. This article aims to standardize and refine GLED exposure protocols, improving consistency across studies and advancing the clinical application of this therapy.

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Learn to use affordable LED arrays for precise optogenetic control of adult-born neurons in brain slice preparations. This cost-effective method enables accurate measurement of activation thresholds and studies of neuronal integration.

Using Affordable LED Arrays for Photo-Stimulation of Neurons

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