This research was supported by the National Natural Science Foundation of China (31871085), the Natural Science Foundation of Shanghai (21ZR1407300), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX01), ZJ Lab, and Shanghai Center for Brain Science and Brain-Inspired Technology.

This protocol received approval from the Animal Care and Use Committee at Fudan University and was conducted following the guidelines and regulations designed by the National Institutes of Health Guide for Care and Use of Laboratory Animals. All possible measures were implemented to minimize the number of animals utilized in this study. The time required to perform each step is included in the respective steps.
1. Preparation of electrodes (Figure 1)
<ol>
	<li>Cut an appropriate length of tungsten wire (~20 mm) and burn off the insulation layer at one end to expose the tungsten wire (1.6-2 mm). Bend the other end of the wire into an &#34;L&#34; shape (see Figure 1A, ~1 min)</li>
	<li>Carefully place the &#34;L&#34; end of the tungsten wire onto the optical fiber (see Figure 1B, ~1 min). 
	NOTE: The tungsten wire should be 0.2-0.4 mm longer than the tip of optical fiber.</li>
	<li>Dip a drop of adhesive and apply it to the interface of the optical fiber and the tungsten wire (see Figure 1C, ~1 min). Due to the surface tension of the liquid, the tungsten wire will attach to the optical fiber.</li>
	<li>Apply the adhesive once and allow it to dry. Then, apply a second coat. This will further tighten the bond between the tungsten wire and the optical fiber (see Figure 1C, ~1 min). 
	NOTE: During the application process, care should be taken to prevent the adhesive from flowing to the tip of the optical fiber, as this may affect its transmittance.</li>
	<li>At the corner of the &#34;L&#34; shape, use adhesive to closely bond the base of the optical fiber and the tungsten wire together (~1 min). 
	NOTE: The increased contact area and the spherical solidification of the adhesive droplet ensure a firm bond between the optical fiber and the tungsten wire.</li>
	<li>Use soldering to establish a connection between the tungsten wire and the pin of the female connector (see Figure 1D, ~3 min for each) 
	NOTE: Since tungsten wire is not sensitive to heat, the success rate of soldering can be increased to nearly 100% by a metal sleeve fashioned by cutting the pin slot location to physically unite the two components. Subsequently, solder filling the sleeve ensures a stable connection between the inserted tungsten wire and the pin.</li>
	<li>According to the experimental design, solder the required number of optrodes (see Figure 1E, ~10 min) 
	NOTE: Three optrodes were utilized in this study.</li>
	<li>Cut the enameled wire to the appropriate length (~30 mm) and burn off the insulation on both ends. Wrap one end of the enameled wire around the base of the screw and solder it. Connect the other end of the enameled wire to the pins of the female connector via a soldering iron, serving as the EEG recording and ground connection for the skull electrode (see Figure 1F, ~5 min) 
	NOTE: Due to the flexibility of the enameled wire, it is convenient to connect the skull and the pins of the female connector. Soldering after winding the enameled wire around a screw is more reliable than surface-mount soldering.</li>
	<li>Apply hot-melt adhesive to the female connector&#39;s pin array to encapsulate the female connector and ensure the solder joints are fully enveloped. During the solidification of the hot-melt adhesive, shape the adhesive using a plastic board to minimize space occupancy during implantation (see Figure 1F, ~3 min) 
	NOTE: Due to the potential burn risks associated with using hot-melt glue, AB adhesive is considered a safer alternative.</li>
	<li>Next, use a light intensity meter and digital multimeter to measure luminousness and conductivity. Upon passing these tests, the setup is ready for use (see Figure 1G, ~2 min) 
	NOTE: When working with lasers, always wear protective eyewear suitable for the specific wavelength and power of the laser light. High-quality fibers are required to transmit at least 80% of the light from the end of the patch cord to the tip of the implantable fiber. Additionally, the resistance from the tip of the tungsten wire to the pin header slot was measured with an acceptable range of 1-5 &#937;.</li>
	<li>Before use, immerse the prepared electrodes in 75% alcohol for 15 min, then transfer them to sterile saline (~16 min). 
	NOTE: The total weight of the encapsulated electrode device (0.6701 &#177; 0.01123 g, n = 4) does not affect the activity of the mice. The electrodes are sterilized in advance using plasma sterilization.</li>
</ol>
2. Protocol for electrode implantation (Figure 2)
<ol>
	<li>Anesthetize the mice with isoflurane (2%-4%) and secure them in a stereotaxic apparatus (see Figure 2A, ~2 min). Use eye ointment on mouse eyes to prevent dryness while under anesthesia. Apply lidocaine to the skin around the perimeter of the cranial incision and a subcutaneous injection of carprofen at a dose of 5 mg/kg. 
	NOTE: Proper anesthetization is confirmed through the absence of the righting reflex and the loss of response to toe pinch. For prolonged surgical procedures, using an injectable&#160;anesthetic is more appropriate.</li>
	<li>After securing the animal in the stereotaxic apparatus, trim away the scalp along the edge of the skull and wipe the skull surface with 75% alcohol to expose the positions of the bregma and lambda (see Figure 2B, ~3 min). 
	NOTE: The pre-surgical procedure includes clipping the hair, disinfecting the surgical site, pre-sterilizing the instruments and surgical tools.</li>
	<li>Level the brain and drill holes (~0.8 mm) above the regions of interest (ROI) using a microdrill. Inject the relevant viruses with a syringe pump (see Figure 2C - F, ~20 min). 
	NOTE: Detailed information about the viruses and ROIs in this study is shown in the Table of Materials and Figure 3.</li>
	<li>Hold the optrodes with a holder and move their tip to the bregma point, setting this position as &#39;zero&#39;. Using the bregma point as the reference coordinate, move the optrodes to the target regions (see Figure 2G, ~3 min). Position the optrodes 0.3 mm above the virus injection site. 
	NOTE: In the vertical implantation strategy, the minimum&#160;distance between the two electrodes is approximately 2 mm, depending on the thickness of the fiber optic sleeve and the outer diameter of the ferrule. An angled insertion method can be used for simultaneous recording from closely located regions.</li>
	<li>After inserting the optic fibers into the corresponding brain areas, apply a small amount of tissue adhesive around them to cover the exposed tissue. Then, sequentially apply additional adhesive and a little dental cement. After the dental cement solidifies, release the holder carefully (see Figure 2H, ~5 min).</li>
	<li>Before each new optrode implantation, perform recalibration, which involves repeating step 2.4 (see Figure 2I). 
	NOTE: For each implantation, position the optrode at bregma as the &#39;zero&#39; point.</li>
	<li>After implanting all optrodes, drill the holes behind the lambda point for &#39;Ground&#39; and above the neocortex for &#39;EEG&#39; using a microdrill (~1 min). 
	NOTE: The size of the drilled hole (~1.1 mm) needs to match the diameter of the screw.</li>
	<li>Insert skull screws into the hole above the cortex and behind the lambda point (see Figure 2J, ~5 min). 
	NOTE: Skull screws are used for EEG recording and grounding, respectively.</li>
	<li>Organize the tungsten wires and enameled wires so they are positioned above the implantation area and not exposed outside the skull. Secure the position of the female connector using a holder (see Figure 2K, ~2 min).</li>
	<li>Fill the implantation site with dental cement to ensure a stable connection between the electrode device and the skull. Encapsulate the internal wires, leaving the female connector&#39;s insertion holes exposed for connection to the headstage (see Figure 2L, ~6 min).</li>
	<li>After dental cement hardening, apply iodine to the skin around the perimeter of the exposed scalp and then place the animal on a warming pad (~1 min). 
	NOTE: Ensure that the animal is not left unattended until it has regained sufficient consciousness to maintain sternal recumbency. Sterile conditions should be maintained throughout the survival surgery.</li>
	<li>Administer antibiotics and analgesics to support post-surgical recovery. 
	NOTE: Animals that have undergone surgery are not reintroduced to the company of other animals until they have fully recovered.Animals were administered intraperitoneal injections of ceftriaxone sodium (180 mg/kg),&#160;and subcutaneous injections of carprofen (5 mg/kg),&#160;and lidocaine gel was applied to the surgical wounds daily for 3 days. The animals are required to survive for a designated period to complete the behavioral testing. Subsequently, they will be euthanized using 4%-5% isoflurane, followed by perfusion and tissue sectioning to assess viral infection and the sites of electrode implantation.</li>
</ol>
3. Protocol for recording in vivo brain
<ol>
	<li>Before conducting signal recording and behavioral testing (open field test)15, ensure that the mouse undergoes a 3-day period of gentle handling to acclimatize them to the environment. 
	NOTE: In this study, the mice were placed in an open field (42 cm &#215; 42 cm) to move around freely, allowing us to observe their seizure behavior.</li>
	<li>After anesthetizing the mice with isoflurane (2%-4%), connect the optical fibers sequentially in the order of the recording site before inserting the headstage.</li>
	<li>Set the 635 nm light pulse with a 10% duty cycle at 20 Hz. Set up the light stimulation for 10 s (200 pulses) during recording. Set the intensity at the optical fiber tips at 3 mW.</li>
	<li>Use a fiber photometry system to record Gcamp6m signals behavioral testing period at a sampling rate of 30 Hz by 470 nm LED channel.</li>
	<li>Simultaneously record calcium signals, LFPs, and EEG under optogenetic stimulation, along with corresponding behavioral performances (see Figure 3).</li>
	<li>Employ transistor-transistor logic (TTL) signal synchronization tagging to ensure temporal consistency in recorded data.</li>
</ol>
4. Data processing
<ol>
	<li>Load photometry data to MATLAB software. Then, use custom coding (Supplementary File 1) to detrend the calcium signal.</li>
	<li>Define the changes in fluorescence intensity with equation (1): 
	&#916;F/F = (Ft&#160;-&#160;F0)/ F0&#160; &#160; (1) 
	Where Ft represents the real-time calcium signal fluorescence values, and F0 denotes the averaged baseline fluorescence intensity before the event.</li>
	<li>Import the electrophysiological data, sampled rate of 1000 Hz, into MATLAB (Supplementary File 2).</li>
	<li>Coordinate the display of calcium signaling and electrophysiological data in alignment with synchronized markers.</li>
</ol>

Flexible Multi-Region Neural Recording Device for Epilepsy Research

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The authors declare no competing financial interests to disclose.

Here, we employed a self-made optrode device for in vivo neural signal recording across multiple regions. The feasibility of this system for simultaneous optogenetic stimulation, calcium signal recording, and electrophysiological recording has been validated. The electrode preparation method described herein is efficient and cost-effective. According to the experimental design, we could record signals from relevant brain regions. The strategic arrangement of optrodes allows for the potential extraction of abunda...

Epilepsy is a common neurological condition characterized by recurrent seizures, which manifest as convulsions, sensory disturbances, and loss of consciousness1. The pathophysiological mechanisms underlying epilepsy are complex and involve multiple interconnected brain regions2,3. Recent advances in neuroimaging have shed light on the large-scale networks involved in epilepsy4,5. However, understanding of the intricate circuitry and network mechanisms underlying the generation and propagation of epilepsy remains limited, partly due to the insufficient application of multi-region neural recording techniques6. Therefore, developing a flexible, integrated method capable of simultaneously monitoring neural activity across disparate brain regions is imperative.
Electrophysiological recordings are conducted to capture seizures and determine the presence of epilepsy7. Except for recording electrophysiological activity, there is a growing emphasis on the precise calcium activity of specific&#160;neural populations in epilepsy studies8,9. Advancements in calcium indicator synthesis and various probe designs have prompted researchers to adopt fiber photometry for capturing changes in neuronal activity and neural substances within the brain10,11. The two independent methods of detecting neuronal activity, namely electrophysiology and fiber photometry recording, complement each other, facilitating a more comprehensive understanding of dynamic neuronal processes.
Additionally, synchronous recording and modulating neural activity are pivotal for gaining insights into the brain functioning at both network and cellular levels. This approach enables researchers to observe and manipulate the brain&#39;s complex processes in real-time. Optogenetics has emerged as an indispensable tool for probing neural signaling, owing to its distinctive ability for selective stimulation or inhibition12. Despite widespread applications of multi-site electrophysiological recording in neuroscience13, the integration of multi-region electrophysiological recording with fiber photometry and optogenetic manipulation remains limited. More importantly, current methods for fabricating and implanting multi-region electrodes lack flexibility14. These limitations hinder our capacity to dissect specific circuit functions and interactions across multiple regions. Here, we present a cost-effective and convenient multi-region in vivo recording approach that sheds light on neuronal processes across regions in kindling-induced seizures and other neuropsychiatric disorders.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-32 adapter</td>
			<td>Plexon</td>
			<td>Custom ordered</td>
			<td>Connect the female connector and headstage</td>
		</tr>
		<tr>
			<td>AAV-CaMKII&#945;-ChrimsonR-mcherry</td>
			<td>Taitool Bioscience</td>
			<td>S0371-9</td>
			<td>4 x 1012 VG/mL&#160;</td>
		</tr>
		<tr>
			<td>AAV-hsyn-Gcamp6m</td>
			<td>Taitool Bioscience</td>
			<td>S0471-9</td>
			<td>4 x 1012 VG/mL&#160;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>236276</td>
			<td>Titered 1:500</td>
		</tr>
		<tr>
			<td>Dental Cement</td>
			<td>New Century Dental</td>
			<td>430205</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophysiological recordings system</td>
			<td>Plexon</td>
			<td>Omniplex</td>
			<td></td>
		</tr>
		<tr>
			<td>Enameled wire</td>
			<td>N/A</td>
			<td>Custom ordered</td>
			<td>Diameter = 0.2 mm</td>
		</tr>
		<tr>
			<td>Female connector</td>
			<td>N/A</td>
			<td>Custom ordered</td>
			<td>1.25 mm pitch</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Loctite</td>
			<td>45282</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Changchun New Industries</td>
			<td>BH81563</td>
			<td>635 nm&#160;</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2021b</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel fiber photometry</td>
			<td>ThinkerTech</td>
			<td>FPS-SS-MC-LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Xi&#39;an Bogao</td>
			<td>L-200UM</td>
			<td>Select the appropriate fiber length based on the depth of the targeted brain regions.</td>
		</tr>
		<tr>
			<td>PFA-Coated Tungsten wire</td>
			<td>A-M System</td>
			<td>795500</td>
			<td>Bare 0.002&#34;; Coated 0.0040&#34;</td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic Fxrame</td>
			<td>RWD</td>
			<td>68807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

an integrated method for crafting flexible and convenient electrophysiological optrodes for multi-region in vivo recording

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the propagation of epileptic signals through associated neural circuits. Therefore, in vivo recording of local field potential (LFP) from the critical brain regions is&#160;essential for deciphering the circuits involved in seizure propagation. However, current methods for electrode fabrication and implantation lack flexibility. Here, we present a handy device designed for electrophysiological recordings (LFPs and electroencephalography [EEG]) across multiple regions. Additionally, we seamlessly integrated optogenetic manipulation and calcium signaling recording with LFP recording. Robust after-discharges were observed in several separate regions during epileptic seizures, accompanied by increasing&#160;calcium signaling. The approach used in this study offers a convenient and flexible strategy for synchronous neural recordings across diverse regions of the brain. It holds the potential for advancing research on neurological disorders by providing insights into the neural profiles of multiple regions involved in these disorders.

Author Spotlight: Unraveling Neural Communication and Circuit Interactions in Health and Disease

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

We investigate brain networks and neural circuits using multimodal approaches which encompass techniques such as multichannel electrophysiological recording, fiber photometry, neuro modulation, and functional magnetic resonance imagery. We are trying to answer how different brain regions communicate and interact in vivo both in healthy and diseased states. We use methods for natural replication and implantation to connect the molecular cellular cycles and network layers.This comprehensive approach allowed us to study neurological disorders more effectively and a deep understanding of their underlying mechanisms. Our results pave the way for new scientific question about how flexible multiregion brain recordings can be, how different brain circuits interact in epilepsy, and how this could apply to other neurological disorders. It offers fringe insights into your neural activities across various brain regions.

We combined optogenetics with multiregional electrophysiological recording and calcium imaging to observe neuronal activity across various brain regions during optogenetic seizures. For this purpose, an adeno-associated virus (AAV) expressing ChrimsonR under the control of the CaMKII&#945; promoter (AAV-CaMKII&#945;-ChrimsonR-mcherry)16 was injected into a classical epileptogenic site, the piriform cortex (ROI 1)17, in rodents. Additionally, AAV-hsyn-Gcamp6m<sup class="xref...

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the ...

an-integrated-method-for-crafting-flexible-convenient

Research

JoVE Journal

Neuroscience

Designing Flexible Optrode for Electrophysiological Recordings Across Multiple Regions in Freely Moving Mice

To begin, cut an appropriate length of tungsten wire and burn off the insulation layer at one end to expose the tungsten wire. Then, fold the other end of the wire into an L shape. Carefully place the L end of the tungsten wire onto the optical fiber.Dip a drop of adhesive and apply it at the interface of the optical fiber and the tungsten wire. At the corner of the L shape, use adhesive to closely bond the base of the optical fiber and the tungsten wire together. Then, use soldering to establish a connection between the tungsten wire and the pin of the female connector.According to the experimental design, solder the required number of optrodes. Next, cut the enameled wire to the appropriate length and burn off the insulation on both ends. Wrap one end of the enameled wire around the base of the screw and solder it.After that, connect the other end of the enameled wire to the pins of the female connector via soldering iron. Then, apply hot melt adhesive to the female connector's pin array to encapsulate the connector and ensure the solder joints are fully enveloped. Shape the adhesive using a plastic board during its solidification to minimize space occupancy during implantation.Next, use a light intensity meter and digital multimeter to measure luminousness and conductivity. Once these tests are passed, the setup is ready for use. Before use, immerse the prepared electrodes in 75%alcohol for 15 minutes.Then, transfer them to sterile saline.

Procedure for Electrode Implantation Surgery in Mice

After securing the anesthetized animal in the stereotaxic apparatus, trim away the scalp along the edge of the skull. Then wipe the skull surface with 75%alcohol to expose the positions of the bregma and lambda. Using a micro drill, level the mouse and drill holes above the regions of interest.Then inject the relevant viruses with a syringe pump. Now hold the optrodes with a holder and move their tip to the bregma point, setting this position as zero. Using the bregma point as the reference coordinate, move the optrodes to the target regions.Position the optrodes 0.3 millimeters above the virus injection site. After inserting the optic fibers into the corresponding brain areas, apply a small amount of tissue adhesive around them to cover the exposed tissue. Sequentially apply additional adhesive and a little dental cement.After the dental cement solidifies, release the holder carefully. Once all erodes are implanted, drill the holes behind the lambda point for ground and above the neocortex for EEG using a micro drill. Insert skull screws into the hole above the cortex and behind the lambda point.Then organize the tungsten and enameled wires so they are positioned above the implantation area and not exposed outside the skull. Secure the position of the female connector using a holder. Now fill the implantation site with dental cement to ensure a stable connection between the electrode device and the skull.Encapsulate the internal wires, leaving the female connector's insertion holes exposed for connection to the head stage. Once the dental cement hardens, apply iodine to the wound and place the animal on a warming pad.

Recording In Vivo Synchronous Electrophysiological and Calcium Activity in Mice

To begin, implant the recording electrodes and inject the relevant viruses in the mouse skull. After anesthetizing the mice, connect the optical fibers sequentially in the order of the recording site before inserting the head stage. Then, set the 635 nanometer light pulse with a 10%duty cycle at 20 hertz, and set up the light stimulation for 10 seconds during recording.Set the intensity at the optical fiber tips at three milliwatts. Next, use a fiber photometry system to record Gcamp6m signals throughout the behavior testing. Simultaneously, record calcium signals, local field potentials, and electroencephalogram under optogenetic stimulation, along with corresponding behavioral performances.Finally, employ transistor-transistor logic signal synchronization tagging to ensure temporal consistency in recorded data. Brief optogenetic stimulation elicited robust after discharges, along with remarkable increases in calcium activities in the target brain regions. A 30 hertz response was observed in the opto-stimulating regio, induced by the activation of optogenetic proteins via blue light LED.

384 Views.  Fudan University.  We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

Flexible Multi-Region Neural Recording Device for Epilepsy Research

Summary