This research was supported by Convergent Technology R&#38;D Program for Human Augmentation through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (NRF-2019M3C1B8090805), and supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (no. 2019R1A2C1088909). We thank Seung-Hee Lee&#39;s laboratory at the Department of Biological Sciences, KAIST, Daejeon, Korea, for kindly providing the transgenic mice.

The animal care and surgical procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Ewha Womans University (no. 20-029).
1. Preparation of an optrode array (Figure 1 and Figure 2)
<ol>
	<li>Attach optical fibers with the microlens array.
	<ol>
		<li>Remove the passivation coating of the optical fiber and cut it into 5 mm-long pieces using a precis...

The feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording was verified (Figure 6). The big spikes during light stimulation are photoelectric artifacts occurring at the same time as the light stimulation (Figure 6A). This is clear in the zoomed view of the waveform in the red dashed rectangle (Figure 6A). As shown in Figure 6A, the photoelectrical artifacts...

Recording and controlling neural activity is essential for understanding how the brain functions in a neural network and at cellular levels. Conventional electrophysiological recording methods include the patch clamp1,2,3,4 using a micropipette and extracellular recording using microneural electrodes5,6,7,8. As a neuromodulation method, electrical stimulation has been frequently used to directly stimulate a focal brain region through direct or indirect depolarization of neuronal cells. However, the electrical method cannot distinguish neuronal cell types for recording or stimulation because the electrical currents spread in all directions.
As an emerging technology, optogenetics has ushered in a new era in understanding how the nervous system works9,10,11,12,13,14,15,16. The essence of optogenetic techniques is to use light to control the activity of light-sensitive opsin proteins expressed by genetically modified cells. Thus, optogenetics enables the sophisticated modulation or monitoring of genetically selected cells in complicated neural circuits14,17. The wider use of the optogenetic approach has necessitated simultaneous neural recording to directly confirm optical neuromodulation. Therefore, an integrated device with light control and recording functions would be extremely valuable16,18,19,20,21,22,23,24,25.
There are limitations of conventional, laser-based optogenetic stimulation, which requires a bulky and expensive light delivery system26,27,28,29,30. Therefore, some research groups employed &#181;LED-based silicon probes to minimize the size of the light delivery system31,32,33,34. However, there is a risk of thermal brain damage caused by direct contact with &#181;LEDs due to the low energy conversion efficiency of LEDs. Light waveguides, such as optical fibers, SU-8, and silicon oxynitride (SiON), have been applied to avoid thermal damage30,35,36,37,38,39. However, this strategy also has a drawback due to its low coupling efficiency between light sources and the waveguides.
The microlens array was previously introduced to enhance the light coupling efficiency between LEDs and optical fibers40. An optrode system was developed based on microelectromechanical systems (MEMS) technologies for optical stimulation and electrical recording on a microscale40. The microlens array between an LED and optical fibers increased the light efficiency by 3.13 dB. As shown in Figure 1, a 2x2 optical fiber array is aligned on the 4x4 microlens array, and the LED is positioned below the microlens array. The 2x2 optical fibers are mounted instead of 4x4 to reduce brain damage. A tungsten electrode array is positioned adjacent to the optrode array using silicon via holes for electrophysiological recording (Figure 1B).
The system consists of a top disposable part and detachable bottom parts. The top disposable part, which includes the optical fiber array, microlens array, and the tungsten electrode array, is designed to be permanently implanted into the brain for in vivo experiments. The bottom part includes an LED light source and an external power supply line, which is easily removable and reusable for another animal experiment. An attachable plastic cover protects the disposable part when the detachable part is removed.
The feasibility of the system is verified by implantation into the brains of transgenic mice expressing channelrhodopsin-2 (ChR2) in Ca2+/calmodulin-dependent protein kinase II-positive neurons (CaMKII&#945;::ChR2 mouse). Recording electrodes were used to record the neural activities from individual neurons during optical stimulation of the neurons.

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			<td>5-pin Connector</td>
			<td>NW3</td>
			<td>HD127K</td>
			<td>1.27 mm (.050&#34;) pitch</td>
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			<td>Bovie</td>
			<td>Fine Science Tools(F.S.T)</td>
			<td>18010-00</td>
			<td>High Temperature Cautery Kit</td>
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			<td>Data Acquisition Software</td>
			<td>Intan Technologies, LLC</td>
			<td>USB Interface Board software</td>
			<td>Work with the RHD USB Interface Board</td>
		</tr>
		<tr>
			<td>Dental Cement</td>
			<td>Lang Dental Manufacturing Company, Inc.</td>
			<td>1223CLR</td>
			<td>Use Jet Liquid and powder in jet denture repair package</td>
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			<td>Digital Manipulator Arm</td>
			<td>Stoelting Co.</td>
			<td>51904/51906</td>
			<td>Left, Right each Digital Manipulator Arm, 3-Axes, Add-On</td>
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			<td>Gel Foam</td>
			<td>Cutanplast</td>
			<td>Standard (70*50*10 mm)</td>
			<td>Sterile re-absorbable gelatin sponge with a haemostatic effect</td>
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		<tr>
			<td>Headstage Preamplifier</td>
			<td>Intan Technologies, LLC</td>
			<td>#C3314</td>
			<td>RHD 16-Channel Recording Headstages</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Stoelting Co.</td>
			<td>53800R</td>
			<td>Stoelting Rodent Warmer X1 with Rat Heating Pad</td>
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			<td>LED</td>
			<td>OSLON</td>
			<td>GB CS8PM1.13</td>
			<td>&#955; typ. 470 nm, Viewing angle 80 &#176;, Forward voltage 2.85 V</td>
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			<td>MATLAB</td>
			<td>MathWorks, Inc.</td>
			<td>R2019a</td>
			<td></td>
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			<td>Micro Clamp</td>
			<td>SURGIWAY</td>
			<td>12-1002-04</td>
			<td>Straight type, Serre-fine DIEFFENBACH droite 3.5 cm</td>
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		<tr>
			<td>Optical Fiber</td>
			<td>Thorlabs, Inc.</td>
			<td>FT200UMT</td>
			<td>0.39 NA, &#216; 200 &#181;m Core Multimode Optical Fiber, High OH for 300 - 1200 nm</td>
		</tr>
		<tr>
			<td>PFA-Coated Tungsten Wire</td>
			<td>A-M System</td>
			<td>Custom ordered</td>
			<td>Rod type, &#216; 101.6 &#956;m (.004&#34;)</td>
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		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>S121C</td>
			<td></td>
		</tr>
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			<td>power meter</td>
			<td>Thorlabs Inc.</td>
			<td>PM100D</td>
			<td></td>
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			<td>Precision cleaver</td>
			<td>FITEL</td>
			<td>S326</td>
			<td>Fiber slicer tool</td>
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			<td>Prism</td>
			<td>GraphPad</td>
			<td>5.01 version</td>
			<td></td>
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			<td>Scalpel</td>
			<td>Feather&#8482;</td>
			<td>#20</td>
			<td>Scalpel blade with 100mm long Scalpel Handle</td>
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		<tr>
			<td>screw</td>
			<td>Nasa Korea</td>
			<td>stainless steel</td>
			<td>diameter: 1.2 mm, length: 3 mm</td>
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		<tr>
			<td>Silver Wire</td>
			<td>The Nilaco Corporation</td>
			<td>AG-401265</td>
			<td>&#216; 200 &#181;m</td>
		</tr>
		<tr>
			<td>Stereotaxic Fxrame</td>
			<td>Stoelting Co.</td>
			<td>51500D</td>
			<td>Digital new standard stereotaxic, rat and mouse</td>
		</tr>
		<tr>
			<td>suture</td>
			<td>ETHICON</td>
			<td>W9106</td>
			<td>suture size: 4-0, length:75 cm, wire diameter: 4-0</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Unilever PLC</td>
			<td>Original</td>
			<td>100% pure petroleum jelly</td>
		</tr>
		<tr>
			<td>Wave_Clus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://github.com/csn-le/wave_clus</td>
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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.

The optrode system is successfully fabricated to provide sufficient light power to activate the target neurons. The fine alignment of the tungsten electrodes is achieved through the microfabricated silicon via the holes. The measured light intensity is 3.6 mW/mm2 at the optical fiber tip when 50 mA current is applied. The microlens increased the light efficiency by 3.13 dB. Due to the microlens array, which enhances the light coupling, the applied current is approximately half of the current required to achiev...

Watch this Scientific Journal Video about Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording at JoVE.com

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

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 ...

This protocol includes fabricating on optrode system for simultaneous optogenetic stimulation and electrophysiological recording. The proposed LED-based system enhances the light coupling efficiency through a microlens array. An LED light source has a simple lighting set up than a laser light source so that the system is readily applicable to the wireless system.Optogenetics is a path technique for understanding the pathology of neurological disease and treatment mechanisms. Optogenetics can be applied to treat spinal cord injury, epilepsy, Parkinson's disease and Alzheimer disease. Our LED-based device can be implemented further as a wireless system with several advantages, such as low system complexity, cost effectiveness, and low power consumption.First, connect the 1.27 millimeter pitch female connector to the head stage preamplifier. Place the LED in the 3D printed housing. Measure the light intensity at the end of the optical fiber tip using a photodiode.Immerse the tungsten electrodes and optical fibers in alcohol for 15 minutes and air dry them. Shave the head skin and place the anesthetized mouse in the stereotactic frame. Position the head within the stereotactic frame and insert ear bars into the meatus.Turn on the thermal heating pad embedded in the stereotactic frame and maintain the body temperature at 37 degrees Celsius throughout the surgical procedures. Adjust the incision bar for setting the height of the incision bar and tighten the nose clamp against the snout. Cover the eyes with petroleum jelly to prevent dryness.Lift the skin in the head with forceps, secure an injection space and inject 1%lidocaine under the scalp. Perform a sagittal incision using a scalpel and fine scissors. Hold the incised skin with a microclamp to broaden the visibility of the surgical area.Remove the periosteum using cotton swabs. If there is bleeding, cauterized the skull using a bovie to seal the blood vessels. Clean the skull with saline and mark the craniotomy sites using a manipulator arm.Drill a hole above the cerebellum and insert a screw for the ground. Using a precision screw, put the ground screw 0.5 millimeters deep until it reaches the top of the cerebellum. Drill the marked area and remove a piece of the skull with forceps.Bend a 26-gauge needle tip clockwise at an angle of 120 degrees and expose the brain area by inserting the bevel side of the needle facing upward. Fix the optrode array in the manipulator arm and move close to the exposed area. Slowly insert the optrode array and connect the ground screw to the silver wire attached to the system.Insert gel foam between the exposed brain and the device to protect chemicals from direct contact with the brain. Carefully apply dental cement to the skull to fix the device and cover the gel foam. Before the dental cement is completely hardened, separate the scalp if attached to the dental cement.Pull the incised skin with forceps to cover the hardened dental cement and suture the scalp. Place the anesthetized mouse in the stereotactic frame. Set the light pulse recipe to 4%duty cycle in 10 Hertz frequency.Set up the light stimulation for two seconds during neural recording. Take off the plastic cover and attach the light delivery systems upper part with the reusable LED in circuits. Connect the head stage preamplifier to the implanted five-pin connector.Open the software and set up the software filters. Next, click and set amplifier sampling rate. Then click change bandwidth.Set amplifier lower bandwidth and amplifier upper bandwidth. Check software DAC High-Pass Filter. Open Spike Scope and check neural signals using voltage threshold.Click Stop and record. Load the acquired data using MATLAB and check raw data. Then run spike sorting code which contains wave clust algorithm.Check sorted spikes in raster plot. Now plot counting spikes and check evoked spikes during light stimulation. The figure shows whole recorded wave forms with exact conditions, including two seconds light on period in the middle.The number of evoked individual neural spikes following each light stimulation pulse significantly increased compared with the baseline with different time bins of two seconds and 0.2 seconds. The figure shows a spike histogram for each channel before, during, and after light stimulation. The inset figure indicates the location of the implanted optrode array.Further, the zoomed version shows a spike histogram with a 100 millisecond time bin. The blue bar represents the time period the LED light was on to record the response. To minimize the artifact noise from LED driving current process, the system should be designed to ensure enough distance between the electro signal pathway and the LED circuit.The proposed LED-based optrode system can investigate the various neuro signaling related to animals'behaviors because it can deliver optogenetic stimulation and record the evoked neuro responses. An LED optrode array is compatible with the wireless optogenetics interface, which can be used in freely moving animal research.

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Neuroscience

3.5K vistas.  Ewha Womans University. Este protocolo incluye la fabricación en el sistema optrode para la estimulación optogenética simultánea y el registro electrofisiológico. El sistema basado en LED propuesto mejora la eficiencia del acoplamiento de luz a través de una matriz de microlentes. Una fuente de luz LED tiene una configuración de iluminación simple que una fuente de luz láser para que el sistema sea fácilmente aplicable al sistema inalámbrico.La optogenética es una técnica de camino para comprende...