The authors declare that they have no competing financial interests.

All endoscopic procedures including animal subjects have been approved at the Institute of Animal Physiology and Genetics, Academy of Science Czech Republic (Biomedical Center PIGMOD), Libechov, Czech Republic (project Experiments in implantation of battery-less and battery devices into submucosa of the esophagus and stomach &#8212; experimental study). All experiments are done in compliance with Czech law 246/1992 Sb. &#34;On the protection of animals against maltreatment, as amended&#34;.&#160;Transmitter device is not required to be sterilized, because it is an external device that is not in direct contact with the animal.
1. Implantable Device Design
<ol>
	<li>Prepare the PCB using a third-party PCB manufacturing service. The complete printed circuit board design is provided in the supplementary file &#34;gerber_implant.7z&#34;. The schematic diagram is provided in Figure 1.</li>
	<li>Place the PCBs on a flat surface (Figure 2a). Use a solder paste dispenser with 0.6 mm needle and 60 psi pressure to manually dispense the soldering paste onto every metallic pad on the PCB. Begin with the top side of the PCB (Figure 2b). The total amount of soldering paste for both sides of the PCB should not exceed 15 &#956;L.</li>
	<li>With a pair of antistatic tweezers, place all components on the top layer of the PCB (Figure 2e). Use Figure 3 for component position and supplementary file &#34;bom_implantabledevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>Use a PCB hot air gun station at 260 &#176;C to solder all components (Figure 4a). Wait until all the solder paste melts, then put away the hot air gun and allow the board cool to room temperature.</li>
	<li>Turn the PCB over and dispense solder paste on the other side. Use the same needle and pressure as stated in 1.2 (Figure 2d).</li>
	<li>As in step 1.3., place all components of the bottom layer of the PCB. Refer to Figure 3 for component position and the supplementary file &#34;bom_implantabledevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>Repeat the heating of the PCB with a hot air gun to solder all components on the bottom side. Use the same process as in step 1.4.</li>
	<li>Check the PCB for any short circuits visually. If any short circuit is found, remove it with a soldering iron.</li>
	<li>Manufacture the wireless charging/communication coil. Use 17 turns of AWG42 wire. The size of the coil is 26 x 13.5 mm2 (Figure 4d). Twist the two output wires.</li>
	<li>Design and manufacture the electrode. The electrode design is provided in the supplementary file &#34;gerber_electrodes.7z&#34;. Use the same manufacturing process as in step 1.1. This PCB is fully completed after manufacturing, and no components are required to be soldered onto it. Solder two AWG42 wires to the small rectangular contacts (Figure 4f)</li>
	<li>Prepare the antenna by using 7 cm of enameled wire and scraping off 3 mm of the enamel from one end (Figure 4e)</li>
	<li>Connect the PICkit 3 programmer to the PCB (Figure 4b-c)
	<ol>
		<li>Connect pads 6 and 7, according to Figure 5, to pins 2 and 3 of the PICkit programmer, respectively.</li>
		<li>Connect pads TP1, TP2 and TP3 (refer to Figure 3) to pins 1, 5 and 4 of the PICkit programmer, respectively</li>
	</ol>
	</li>
	<li>Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.</li>
	<li>Run the MPLAB IPE software and program the firmware into the microcontroller.
	<ol>
		<li>Run the MPLAB IPE v3.61. Select &#34;Settings | Advanced Mode&#34;</li>
		<li>Into the Password field, enter the default password which is &#39;microchip&#39;. Click &#34;Log on&#34;. A tab with different panels on the left will appear.</li>
		<li>On the top left, click &#34;Operate&#34;, then in top middle part of the screen, click &#34;Device field&#34; and type in &#34;PIC16LF1783&#34;. Click &#34;Apply&#34;.</li>
		<li>Select panel &#34;Power&#34; on the left (Figure 6).</li>
		<li>Change the VDD voltage value to 2.55. This step is critical. 
		Caution: Setting this value above 2.8 V will damage the board (Figure 7).</li>
		<li>Click the checkbox &#34;Power Target Circuit&#34; from &#34;Tool&#34; (Figure 7).</li>
		<li>Click the &#34;Operate&#34; tab on the left (Figure 6).</li>
		<li>Click &#34;Connect&#34;.</li>
		<li>Download the supplementary file &#34;IMPLANTABLE_V2.X.production.hex&#34; and note its location on the hard drive. In the IPE software, find the Source line and click the &#34;Browse&#34; button near it (Figure 8).</li>
		<li>Click Program. Wait till the software says that the software has been successfully downloaded to the microcontroller (Figure 9).</li>
	</ol>
	</li>
	<li>Desolder the wires soldered to pads TP1, TP2 and TP3 (Figure 3) as well as wires soldered to pads 6 and 7 (Figure 5).</li>
	<li>Connect the PCB to all other electrical components except for the battery (Figure 10a).
	<ol>
		<li>Solder the wireless charging/communication coil to pads 2 and 3 according to Figure 8. The polarity is not important.</li>
		<li>Connect the antenna to pad 1 according to Figure 5. Connect the PCB electrodes to pads number 4 and 5 according to Figure 5. The polarity is not important.</li>
	</ol>
	</li>
	<li>Solder the CG-320 battery to pads 6 and 7 (Figure 5). The negative terminal of the battery must be soldered onto pad 7. Be careful while performing the next steps. The device is now powered and is sensitive to short circuits and contact with metallic objects.</li>
	<li>To test the functionality of the wireless charging circuitry, all steps in part 2 need to be completed. After that, place the wireless charger/transmitter in close proximity of the device. Use a multimeter to measure the voltage of the battery. If the battery voltage is slowly rising (several mV per min), the charging function is working.</li>
	<li>Wind the antenna around the device in a spiral (Figure 10b)</li>
	<li>Cut a 32 mm long piece of a heat-shrinkable tubing with an internal diameter of 9.5 mm.</li>
	<li>Place the coil on the PCB. Refer to Figure 7b for the correct placement.</li>
	<li>Put the heat-shrinkable tubing over the device, coil and antenna. Only the electrodes should protrude from the tubing. Refer to Figure 7c for correct placement.</li>
	<li>Heat the tubing with a hot air gun to 150 &#176;C to shrink and then allow it to cool (Figure 10d).</li>
	<li>Apply epoxy glue to the left end to seal one side of the tubing (Figure 10e).</li>
	<li>Glue the electrode to the back side of the PCB with tubing. Also glue the other end of the tubing. Refer to Figure 10f for correct placement.</li>
	<li>Wait for at least 24 h for the glue to harden and fully cure.</li>
	<li>After the completion of the wireless charger/transmitter device, test the implantable device for water leaks by placing it into a 30 cm high column of saturated saline solution for 1 h. Any major leak can be spotted as a sudden drop of the battery voltage or the malfunction of the device caused by the saline solution shorting the electronics. After the test, the device is fully prepared to be implanted.</li>
	<li>Test the stimulation function of the implant using an oscilloscope. Connect two measurement electrodes of the oscilloscope to the tin metal plated contact pads of the electrode on the implantable device. Observe the stimulation pattern on the oscilloscope screen. The correct stimulation pattern is given in Figure 11.</li>
</ol>
2. Wireless Charger/Transmitter Design
<ol>
	<li>The PCB design is provided in the supplementary file &#34;gerber_transmitter.7z&#34;. Use the same manufacturing process as for the implantable device. The schematic diagram is provided in Figure 12.</li>
	<li>Place the PCBs on a flat surface. Use a solder paste dispenser with 0.6 mm needle and 60 psi pressure to manually dispense the soldering paste onto every metallic pad on the PCB. The total amount of solder paste dispensed on the PCB should not exceed 50 &#956;L.</li>
	<li>With a pair of antistatic tweezers, place all components on the top layer of the PCB. Consult Figure 13 for the component position and the supplementary file &#34;bom_transmitterdevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>Use a PCB hot air gun station preset to 260 &#176;C to solder all components. Wait until all the solder paste melts, put away the hot air gun and allow the board to cool to room temperature.</li>
	<li>Repeat the steps 2.3&#8211;2.4&#160;for the bottom side of the device. Follow a similar procedure as during the manufacturing of the implantable device.</li>
	<li>Create a coil with 3 turns of AWG18 enameled wire (Figure 14c) and connect it to pads COIL1 and COIL2 (Figure 13).</li>
	<li>Make an aluminum heatsink for the power transistors (Figure 13, Q1 and Q2). The exact shape of the heatsink is not critical. One of the possible embodiments is shown in Figure 9d. In this case, the heatsink also forms an enclosure for the device.</li>
	<li>Connect the PICkit 3 programmer to the assembled PCB. Connect pads TP1 to TP5 (Figure 13) with pins 1 to 5 of the PICkit programmer, respectively.</li>
	<li>Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.</li>
	<li>Run the MPLAB IPE software and program the firmware into the microcontroller. The process is the same as for the implantable device, except for the VDD voltage and file uploaded.
	<ol>
		<li>Run the MPLAB IPE v3.61. Select &#34;Settings | Advanced Mode&#34;.</li>
		<li>Into password box, enter the default password which is &#39;microchip&#39;. Click&#160;&#34;Log on&#34;. A tab with different panels on the left will appear.</li>
		<li>On the top left, click &#34;Operate&#34;, then in the top middle part of the screen, click the &#34;Device&#34; and type in &#34;PIC16LF1783&#34;. Click &#34;Apply&#34;.</li>
		<li>Select panel &#34;Power&#34; on the left</li>
		<li>Change the VDD voltage value to 3.3.</li>
		<li>Click the checkbox &#34;Power Target Circuit&#34; from &#34;Tool&#34;.</li>
		<li>Click the &#34;Operate&#34; tab on the left.</li>
		<li>Click &#34;Connect&#34;.</li>
		<li>Download the supplementary file &#34;IMPLANTABLE_V2_TRANSMITTER.X.production.hex&#34; and note its location on the hard drive. In the IPE software, find the Source line and click the &#34;Browse&#34; button near to it.</li>
		<li>Click &#34;Program&#34;. Wait till the software says that the software was downloaded to the microcontroller successfully.</li>
	</ol>
	</li>
	<li>Desolder the wires soldered to pads TP1 to TP5</li>
	<li>Connect a 12 V power supply to the V- and V+ pads (Figure 5). The negative terminal has to be connected to the V- pad.</li>
	<li>Plug in a mini-USB to USB-A cable to the X1 connector (Figure 5) and connect to a computer with PuTTy software pre-installed.</li>
	<li>Open the PuTTY software and set it up (Figure 15).
	<ol>
		<li>Open the PuTTY software. Select &#34;Serial&#34; as connection type.</li>
		<li>Enter COMx as a Serial line, where x is the number of the COM port of the device. If no other COM port device was installed, this number will be 1.</li>
		<li>Enter &#34;38400&#34; as Speed. Click &#34;Open&#34;. The charger/transmitter device is now ready to be used. Press H key for help.</li>
	</ol>
	</li>
</ol>
3. Endoscopic Implantation
<ol>
	<li>Use a live mini pig as an in vivo model, adult (8-36 months), 20-30 kg weight.
	<ol>
		<li>Let the pig fast for 24 h prior to the procedure.</li>
		<li>Allow clear liquids ad libitum.</li>
		<li>Administer intramuscular tiletamine (2 mg/kg), zolazepam (2 mg/kg) and ketamine (11 mg/kg) as a premedication.</li>
		<li>Apply intravenous thiopental ad effectum (5% solution) and inhalation anesthesia with isoflurane, N2O and propofol injection. Proper anesthesia is confirmed by reflexes and muscle tone, eye position, palpebral reflex and pupillary reflex. Circulation, oxygenation, ventilation and body temperature are continuously monitored.</li>
	</ol>
	</li>
	<li>In order to perform the implantation and visualization, use an animal model dedicated endoscope. Insert it using the standard way into the in vivo model.</li>
	<li>Grasp the device externally with a snare. After that, insert it into the stomach, then release it.</li>
	<li>Extract the endoscope, equip it with a dissection cap (15.5 mm), and then re-insert it to the stomach.</li>
	<li>In order to implant the device to submucosa, apply saline solution mixed with methylene blue into the submucosal layer using an injection therapy needle catheter (25 G).</li>
	<li>Make a horizontal incision to create an opening in the submucosa using an electrosurgical knife with a knob-shaped tip.</li>
	<li>Using the affixed cap, insert the cap into the newly created space, and with the use of an electrosurgical knife, continue disrupting, dilating, and dissecting the submucosal layer, creating a sufficiently large-enough pocket to insert the stimulation device.</li>
	<li>Grasp the device which is lying freely inside the stomach with insertion and extraction loops and, using grasping forceps, navigate it into the submucosal pocket. Place the stimulation electrodes in contact with the muscularis propria using grasp forceps.</li>
	<li>Use an over the scope clip to secure the device in place inside the submucosal pocket and prevent any migration or dislodging.</li>
</ol>
4. Experiment &#8212; After Implantation
<ol>
	<li>After successful implantation, place the charger/transmitter coil in proximity of the implanted device.</li>
	<li>Plug in the RTL2832 dongle in the PC.</li>
	<li>Run the HDSDR software and set the center frequency to 432 MHz.
	<ol>
		<li>Open the HDSDR software (Figure 15) for correct settings and PuTTY software (Figure 16). In the HDSDR software, click &#34;Options | Select Input | ExtIO&#34;.</li>
		<li>Select Bandwidth &#8212; &#34;960000&#34;. Select the LO frequency to 431.95 MHz. Select the Tune frequency to 432.00 MHz.</li>
	</ol>
	</li>
	<li>Transmit a Manchester coded sequence from the charger/transmitter by pressing the R key in the PuTTY terminal and receive the OOK modulated reply from the implant by observation of the main HDSDR window ( Figure 17e-f).</li>
</ol>
5. Euthanasia after the Experiment
<ol>
	<li>Use an anesthetic overdose for euthanasia (lethal dose of thiopental and KCl).</li>
</ol>

<ol>
	<li>Abell, 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=Gastric+electrical+stimulation+for+medically+refractory+gastroparesis.">Gastric electrical stimulation for medically refractory gastroparesis.</a> Gastroenterology. 125 (2), 421-428 (2003).</li><li>O'Grady, G., Egbuji, J., Du, P., Cheng, L. K., Pullan, A. J., Windsor, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+gastric+electrical+stimulation+for+the+treatment+of+gastroparesis:+a+meta-analysis.">High-frequency gastric electrical stimulation for the treatment of gastroparesis: a meta-analysis.</a> World J Surg. 33 (8), 1693-1701 (2009).</li><li>Chu, H., Lin, Y., Zhong, L., McCallum, R. W., Hou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+high-frequency+gastric+electrical+stimulation+for+gastroparesis.">Treatment of high-frequency gastric electrical stimulation for gastroparesis.</a> J Gastroenterol Hepatol. 27 (6), 1017-1026 (2012).</li><li>Rodr&#237;guez, L., 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=Electrical+stimulation+therapy+of+the+lower+esophageal+sphincter+is+successful+in+treating+GERD:+final+results+of+open-label+prospective+trial.">Electrical stimulation therapy of the lower esophageal sphincter is successful in treating GERD: final results of open-label prospective trial.</a> Surg Endosc. 27 (4), 1083-1092 (2013).</li><li>Ellis, F., Berne, T. V., Settevig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevention+of+experimentally+induced+reflux+by+electrical+stimulation+of+the+distal+esophagus.">The prevention of experimentally induced reflux by electrical stimulation of the distal esophagus.</a> Am J Surg. 115, 482-487 (1968).</li><li>Rinsma, N. F., Bouvy, N. D., Masclee, A. A. M., Conchillo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Stimulation+Therapy+for+Gastroesophageal+Reflux+Disease.">Electrical Stimulation Therapy for Gastroesophageal Reflux Disease.</a> J Neurogastroenterol. 20 (3), 287-293 (2014).</li><li>Medtronic Inc, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enterra Therapy 3116 - Gastric Electrical Stimulation System. , (2016).</li><li>Rodriguez, L., 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=Two-year+results+of+intermittent+electrical+stimulation+of+the+lower+esophageal+sphincter+treatment+of+gastroesophageal+reflux+disease.">Two-year results of intermittent electrical stimulation of the lower esophageal sphincter treatment of gastroesophageal reflux disease.</a> Surgery. 157 (3), 556-567 (2015).</li><li>Hajer, J., Nov&#225;k, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Autonomous+Endoscopically+Implantable+Submucosal+Microdevice+Capable+of+Neurostimulation+in+the+Gastrointestinal+Tract.">Development of an Autonomous Endoscopically Implantable Submucosal Microdevice Capable of Neurostimulation in the Gastrointestinal Tract.</a> Gastroent Res Pract. , 8098067 (2017).</li><li>Deb, S., 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=Development+of+innovative+techniques+for+the+endoscopic+implantation+and+securing+of+a+novel,+wireless,+miniature+gastrostimulator+(with+videos).">Development of innovative techniques for the endoscopic implantation and securing of a novel, wireless, miniature gastrostimulator (with videos).</a> Gastrointest. Endosc. 76 (1), 179-184 (2012).</li><li>Jiang, G., Zhou, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Technology advances and challenges in hermetic packaging for implantable medical devices. , (2017).</li><li>Vonthein, R., Heimerl, T., Schwandner, T., Ziegler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+and+biofeedback+for+the+treatment+of+fecal+incontinence:+a+systematic+review.">Electrical stimulation and biofeedback for the treatment of fecal incontinence: a systematic review.</a> Int J Colorectal Dis. 28 (11), 1567-1577 (2013).</li></ol>

This work was supported by the Research Project PROGRES-Q28, and awarded by Charles University in Prague. The authors thank to Ass. Prof. Jan Mart&#237;nek, Ph.D. and PIGMOD centre.

The design of the implantable device should primarily focus on the overall size of the device, achievable stimulation profiles (maximum voltage, maximum deliverable current, length of pulses and pulse frequency). Main limitation from the hardware perspective is the size and availability of suitable components. To minimize the overall size, surface mount components are preferred because of their compact packaging. The best solution would be to integrate bare chip dies on the substrate. However, this is limited by both the...

Gastric dysmotility can be a sign of several relatively common diseases such as gastroparesis, which is usually characterized by a chronic progression and imposes rather severe consequences on the social, work-related, and physical status of the patient. Most cases of gastroparesis are usually diabetic or idiopathic in origin and are often resistant to available medication1. Patients afflicted with this condition most commonly present with nausea and repeated vomiting. Based on previous research, it is known that the application of high-frequency low-energetic electrical stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility1,2.
Based on previous studies, it is proven that high-frequency gastric electrical stimulation can significantly improve the symptoms and gastric emptying3. It has also been shown that lower esophageal sphincter neurostimulator therapy is safe and effective for the treatment of gastroesophageal reflux disease (GERD), reducing the acid exposure and eliminating daily proton-pump inhibitor (PPI) usage without stimulation related adverse effects4. Before human trials, first studies were performed in animal models (canine models5). Based on these studies, electrical stimulation of the lower esophageal sphincter (LES, 20 Hz, pulse width of 3 ms) caused a prolonged contraction of the LES5. Similar effects of high (20 Hz, pulse width of 200 &#956;s) and low (6 cycles/min, pulse width of 375 ms) frequency electrical stimulation on LES in GERD patients were investigated. Both high and low frequency stimulation were effective6. However, currently, there are only two neurostimulation devices for gastric or esophageal stimulation available on the market7,8. In those devices, the electrodes can be implanted surgically, laparoscopically or robotically. The device itself is implanted subcutaneously. This requires general anaesthesia and have a bulky device fitted, using intramuscular catheters which allow for the stimulation of the gastric or esophageal muscle tissue. So, the option of using a wirelessly communicating device implanted into the gastric submucosal layer would represent a definite advantage and improvement in patient comfort. As stated in the previous research9,10, it was proven that an implantation of a miniature neurostimulator into submucosa is possible. For the endoscopic submucosal implantation, we use a technique called endoscopic submucosal pocketing (ESP), based on endoscopic submucosal tunnel dissection10. The goal of this research is to further improve this concept of an implantable neurostimulator, primarily in the scope of power management (specifically the wireless recharging capability), conformity with respective laws and regulations for wireless communication links in medical implantable devices and possibility of bipolar neurostimulation. Next, the presented microneurostimulator is capable of bidirectional communication and the stimulation parameters can be changed in real-time, even while the device is implanted.
This technique is suitable for teams with a therapeutic endoscopist experienced in endoscopic pocketing or tunnel dissections. Next, a hardware and embedded software designer with experience in building hardware prototypes with microcontrollers and radio frequency circuits using surface mount technology is needed. For building the hardware prototypes, a lab equipped with a reflow soldering station and basic equipment for electrical measurements (at least a digital multimeter, an oscilloscope, a spectrum analyzer and PICkit3 programmer) is required.

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		</tr>
		<tr>
			<td>EIA 0603 inductor 470 nH</td>
			<td>Murata Electronics</td>
			<td>LQW18ANR47G00D</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0402 resistor 47 k&#937;</td>
			<td>Murata Electronics</td>
			<td>CRCW040247K0JNED</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>27.0000 MHz crystal 5032</td>
			<td>AVX / Kyocera</td>
			<td>KC5032A27.0000CMGE00</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0402 capacitor 6.8 pF</td>
			<td>Murata Electronics</td>
			<td>GJM1555C1H6R8CB01D</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0402 inductor 82 nH</td>
			<td>EPCOS / TDK</td>
			<td>B82498F3471J</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>ABS05 32.768 kHz crystal</td>
			<td>ABRACON</td>
			<td>ABS05-32.768KHZ-T</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>CDBU00340-HF schottky diode</td>
			<td>COMCHIP technology</td>
			<td>CDBU00340-HF</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>CG-320S Li-Ion pinpoint battery</td>
			<td>Panasonic</td>
			<td>CG-320S</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>HSMS282P schottky diode rectifier</td>
			<td>Broadcom / Avago</td>
			<td>HSMS-282P-TR1G</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>MAX8570 step-up converter</td>
			<td>Maxim Integrated</td>
			<td>MAX8570EUT+T</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>MICRF113 RF transmitter</td>
			<td>Microchip Technology</td>
			<td>MICRF113YM6-TR</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>4.3 V Zener diode</td>
			<td>ON Semiconductor</td>
			<td>MM3Z4V3ST1G</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>OPA237 operational amplifier</td>
			<td>Texas Instruments</td>
			<td>OPA237N</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>PIC16LF1783 8-bit microcontroller</td>
			<td>Microchip Technology</td>
			<td>PIC16LF1783-I/ML</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>TPS70628 low-drop regulator</td>
			<td>Texas Instruments</td>
			<td>TPS70628DBVT</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 1206 thick film resistor 0 &#937;</td>
			<td>Yageo</td>
			<td>RC1206JR-070RL</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0603 thick film resistor 0 &#937;</td>
			<td>Yageo</td>
			<td>RC0603JR-070RL</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0402 thick film resistor 100 k&#937;</td>
			<td>Yageo</td>
			<td>RC0402FR-07100KL</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0603 thick film resistor 100 k&#937;</td>
			<td>Yageo</td>
			<td>RC0603FR-07100KL</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0805 ceramic capacitor 100 nF</td>
			<td>KEMET</td>
			<td>C0805C104K5RAC7210</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0402 thick film resistor 10 k&#937;</td>
			<td>Yageo</td>
			<td>RC0402JR-0710KL</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 1206 ceramic capacitor 10 nF</td>
			<td>Samsung</td>
			<td>CL31B103KHFSW6E</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0402 thick film resistor 1 k&#937;</td>
			<td>Yageo</td>
			<td>RC0402JR-071KL</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0402 thick film resistor 220 &#937;</td>
			<td>Yageo</td>
			<td>RC0402JR-07220RL</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0402 ceramic capacitor 220 nF</td>
			<td>TDK</td>
			<td>C1005X5R1C224K050BB</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 1206 ceramic capacitor 22 nF</td>
			<td>TDK</td>
			<td>C3216X7R2J223K130AA</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>SMC B tantalum capacitor 22 uF</td>
			<td>AVX</td>
			<td>TPSB226K010T0700&#160;</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>EIA 0402 thick film resistor 27 &#937;</td>
			<td>Yageo</td>
			<td>RC0402FR-0727RL</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 1206 thick film resistor 3.3 &#937;</td>
			<td>Yageo</td>
			<td>RC1206JR-073K3L</td>
			<td>3 pcs</td>
		</tr>
		<tr>
			<td>SOT23 3.3V zener diode</td>
			<td>ON Semiconductor</td>
			<td>BZX84C3V3LT1G</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>SMC A tantalum capacitor 4.7uF</td>
			<td>KEMET</td>
			<td>T491A475M016AT</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 0603 thick film resistor 470 &#937;</td>
			<td>Yageo</td>
			<td>RC0603JR-07470RL</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>EIA 1206 ceramic capacitor 470 nF</td>
			<td>KEMET</td>
			<td>C1206C471J5GACTU</td>
			<td>3 pcs</td>
		</tr>
		<tr>
			<td>Electrolytic capacitor 470 uF</td>
			<td>Panasonic</td>
			<td>EEE-1CA471UP</td>
			<td>3 pcs</td>
		</tr>
		<tr>
			<td>EIA 0402 ceramic capacitor 47 pF</td>
			<td>AVX</td>
			<td>04025A470JAT2A</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>0603 GREEN LED</td>
			<td>Lite-On Inc.</td>
			<td>LTST-C191KGKT</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>0603 RED LED</td>
			<td>Lite-On Inc.</td>
			<td>LTST-C191KRKT</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>16 MHz CX3225 crystal</td>
			<td>EPSON</td>
			<td>FA-238 16.0000MB-C3</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>0805 ferrite bead</td>
			<td>Wurth Electronics Inc.</td>
			<td>742792040</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>IR2110SO FET driver</td>
			<td>Infineon Technologies</td>
			<td>IR2110SPBF</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>FT230XS USB to seri&#225;l converter</td>
			<td>FTDI Ltd.</td>
			<td>FT230XS-R</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Mini USB connector</td>
			<td>EDAC Inc.</td>
			<td>690-005-299-043</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>PIC16F1783 8-bit microcontroller</td>
			<td>Microchip Technology</td>
			<td>PIC16F1783-I/ML</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>REG1117 3.3 V regulator SOT223</td>
			<td>Texas Instruments</td>
			<td>REG1117-3.3/2K5</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Schottky SMB diode rectifier</td>
			<td>STMicroelectronics</td>
			<td>STPS3H100UF</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>SMB package TVS diode</td>
			<td>Littelfuse Inc.</td>
			<td>1KSMBJ6V8</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>IRLZ44NPBF N-channel MOSFET</td>
			<td>Infineon Technologies</td>
			<td>IRLZ44NPBF</td>
			<td>2 pcs</td>
		</tr>
		<tr>
			<td>RTL2832U receiver dongle</td>
			<td>EVOLVEO</td>
			<td>Mars</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>PICkit 3</td>
			<td>Microchip Technology</td>
			<td>PICkit 3</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Mini USB to USB A cable</td>
			<td>OEM</td>
			<td>Mini USB to USB-A</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Printed circuit board, implantable device</td>
			<td>---</td>
			<td>Manufacture with the provided supplementary file</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Printed circuit board, transmitter/receiver device</td>
			<td>---</td>
			<td>Manufacture with the provided supplementary file</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Printed circuit board, implantable device</td>
			<td>---</td>
			<td>Manufacture with the provided supplementary file</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>AWG18 wire</td>
			<td>Alpha Wire</td>
			<td>3055 BK001</td>
			<td>2 m</td>
		</tr>
		<tr>
			<td>AWG42 wire</td>
			<td>Daburn Electronics</td>
			<td>2420/42 BK-100</td>
			<td>1 m</td>
		</tr>
		<tr>
			<td>Olympus GIFQ-160</td>
			<td>Olympus</td>
			<td>N/A (part is obsoleted)</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Single-use electrosurgical knife with knob-shaped tip and integrated jet function</td>
			<td>Olympus</td>
			<td>KD-655L</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Single-use oval electrosurgical snare</td>
			<td>Olympus</td>
			<td>SD-210U-15</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>15.5 mm lens hood</td>
			<td>FujiFilm</td>
			<td>DH-28GR</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Injection therapy needle catheter</td>
			<td>Boston Scientific</td>
			<td>25G</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Alligator law grasping forceps</td>
			<td>Olympus</td>
			<td>FG-6L-1</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Instant Mix 5 min epoxy</td>
			<td>Loctite</td>
			<td>N/A</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>Heat shrinkable tubing, inside diameter 9.5 mm</td>
			<td>TE Connectivity</td>
			<td>RNF-100-3/8-X-STK</td>
			<td>1 pc</td>
		</tr>
		<tr>
			<td>ChipQuik solder paste</td>
			<td>Chip Quik</td>
			<td>SMD4300AX10</td>
			<td>1 pc</td>
		</tr>
	</tbody>
</table>

autonomous and rechargeable microneurostimulator endoscopically implantable into the submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

Figure 17 shows that an endoscopic placement of the gastric neurostimulator into a pocket in submucosa as well as proper placement of the electrodes to the muscular layer was successful. The dimensions of the device (Figure 10) are 35 x 15 x 5 mm3 while the weight is 2.15 g. Figure 17 shows the circuit diagram of the device showing that the device comprises of 6 different modules wh...

Watch this Scientific Journal Video about Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa at JoVE.com

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

autonomous-rechargeable-microneurostimulator-endoscopically

Research

JoVE Journal

Neuroscience

8.4K Views.  Charles University, Prague, Czech Republic. The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Video: Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...