This work was supported by American Heart Association postdoctoral fellowship (AY), Defense Advanced Research Projects Agency (DARPA) Reorganization and Plasticity to Accelerate Injury Recovery (REPAIR; N66001-10-C-2010), R01.NS073940, and by the UCSF Neuroscience Imaging Center. This work was also supported by the Eunice Kennedy Shiver National Institute of Child Health &#38; Human Development of the National Institutes of Health under Award Number K12HD073945, the Washington National Primate Research Center (WaNPCR, P51 OD010425), and the Center for Neurotechnology (CNT, a National Science Foundation Engineering Research Center under Grant EEC-1028725). We thank Camilo Diaz-Botia, Tim Hanson, Viktor Kharazia, Daniel Silversmith, Karen J. MacLeod, Juliana Milani, and Blakely Andrews for their help with the experiments and Nan Tian, Jiwei He, Peter Ledochowitsch, Michel Maharbiz, and Toni Haun for technical help.

All procedures have been approved by the University of California, San Francisco Institutional Animal Care and Use Committee (IACUC) and are compliant with the Guide for the Care and Use of Laboratory Animals. The following procedure was performed using two adult male rhesus macaques of 8 and 7 years of age, weighing 17.5 kg and 16.5 kg (monkey G and monkey J), respectively.
NOTE: Use standard aseptic techniques for all surgical procedures.
1. Baseline Imaging
<ol...

<ol>
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H., 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=Convection-enhanced+delivery+of+macromolecules+in+the+brain.">Convection-enhanced delivery of macromolecules in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2076-2080 (1994).</li><li>Lieberman, D. M., Laske, D. W., Morrison, P. F., Bankiewicz, K. S., Oldfield, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+distribution+of+large+molecules+in+gray+matter+during+interstitial+drug+infusion.">Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion.</a> Journal of Neurosurgery. 82 (6), 1021-1029 (1995).</li><li>Lonser, R. R., Gogate, N., Morrison, P. F., Wood, J. D., Oldfield, E. 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PNS has financial interest in Neuralink Corp., a company that is developing clinical therapies using brain stimulation.

Here, we outline a feasible and efficient technique for achieving large-scale optogenetic expression in NHP primary somatosensory and motor cortex by MR-guided CED. The use of MR-guided CED presents significant advantages over traditional methods of viral infusion in the NHP brain. One such advantage is the ability to attain expression over large areas with fewer required infusions. For instance, with conventional methods, multiple injections of 1-2 &#181;L of the vector yield expression in a 2-3 mm diameter region<sup c...

Optogenetics is a powerful tool that allows for the manipulation of neural activity and the study of network connections in the brain. Implementing this technique in non-human primates (NHPs) has the potential to enhance our understanding of large-scale neural computation, cognition, and behavior in the primate brain. Although optogenetics has been successfully implemented in NHPs in recent years1,2,3,4,5,6,7, a challenge that researchers face is achieving high levels of expression across large brain areas in these animals. Here, we are providing an efficient and simplified approach to achieve high levels of optogenetic expression across large areas of the cortex in macaques. This technique has great potential to improve current optogenetic studies in these animals in combination with state-of-the-art recording8,9 and optical stimulation10 technologies.
Convection enhanced delivery (CED) is an established method of delivery of pharmacological agents and other large molecules, including viral vectors, to the central nervous system11,12,13. Whereas conventional delivery methods involve multiple low volume infusions distributed across small regions of the brain, CED can achieve broader and more even agent distribution with fewer infusions. Pressure-driven bulk fluid flow (convection) during infusion allows for more widely and uniformly distributed transduction of the target tissue when delivering viral vectors with CED. In recent studies, we demonstrated the transduction and subsequent optogenetic expression of large areas of primary motor (M1) and somatosensory (S1) cortices9 and thalamus14 using magnetic resonance (MR)-guided CED.
Here, we outline the use of CED to achieve optogenetic expression across large cortical areas with only a few cortical injections.

<table>
	<tbody>
		<tr>
			<td>0.2 mL High Pressure IV Tubing</td>
			<td>Smiths Medical Inc., Dublin, OH, USA</td>
			<td>533640</td>
			<td></td>
		</tr>
		<tr>
			<td>0.32 mm ID, 0.43 mm OD Silica Tubing</td>
			<td>Polymicro Technologies</td>
			<td>1068150027</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm ID, 0.76 mm OD Silica Tubing</td>
			<td>Polymicro Technologies</td>
			<td>1068150625</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2.5-CamKII-C1V1-EYFP</td>
			<td>Penn Vector Core, University of Pennsylvania</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABS plastic</td>
			<td>Stratasys, MN, USA</td>
			<td>ABSplus-P430</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimicrobial incise drape</td>
			<td>3M</td>
			<td>6650EZ</td>
			<td>Ioban Drape</td>
		</tr>
		<tr>
			<td>Dental Acrylic</td>
			<td>Henry Schein, Inc.</td>
			<td>1013117</td>
			<td>Acrylic Bonding Agent</td>
		</tr>
		<tr>
			<td>Elevators</td>
			<td>VWR International, LLC.</td>
			<td>10196-564</td>
			<td>Langenbeck Elevator, Wide Tip</td>
		</tr>
		<tr>
			<td>Fine suture</td>
			<td>McKesson Medical-Surgical Inc.</td>
			<td>1034505</td>
			<td></td>
		</tr>
		<tr>
			<td>Gadoteridol</td>
			<td>Prohance, Bracco Diagnostics, Princeton, NJ</td>
			<td>0270-1111-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser for light stimulation</td>
			<td>Omicron-Laserage, Germany</td>
			<td>PhoxX 488-60</td>
			<td></td>
		</tr>
		<tr>
			<td>MR compatible 3cc syringe</td>
			<td>Harvard apparatus, Holliston, MA, USA</td>
			<td>59-8377</td>
			<td></td>
		</tr>
		<tr>
			<td>MR Imaging Software</td>
			<td>Pixmeo</td>
			<td>OsiriX MD 10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MR-Compatible Pump</td>
			<td>Harvard apparatus, Holliston, MA, USA</td>
			<td>Harvard PHD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>MR-compatible stereotaxic frame</td>
			<td>KOPF</td>
			<td>1430M MRI</td>
			<td></td>
		</tr>
		<tr>
			<td>Perifix Clamp Style Catheter Connector</td>
			<td>B-Braun, Bethlehem, PA, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Screws</td>
			<td>Plastics 1</td>
			<td>0-80 x 1/8N</td>
			<td>Nylon screws</td>
		</tr>
		<tr>
			<td>Titanium screws</td>
			<td>Crist Instrument Co., Inc.</td>
			<td>6-YCX-0312</td>
			<td>Self-tapping bone screws</td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>GerMedUSA Inc,</td>
			<td>SKU:GV70-42</td>
			<td></td>
		</tr>
		<tr>
			<td>uPrinter SE 3D printer</td>
			<td>Stratasys, MN, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin E Capsule</td>
			<td>Pure Encapsulations, LLC.</td>
			<td>DE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet sterile absorbable gelatin</td>
			<td>Pfizer Inc.</td>
			<td>AZL0009034201</td>
			<td>Gelfoam</td>
		</tr>
	</tbody>
</table>

convection enhanced delivery of optogenetic adeno-associated viral vector to the cortex of rhesus macaque under guidance of online mri images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Convection Enhanced Delivery (CED) under MRI Guidance
The spread of the viral vector was monitored during CED infusion under the guidance of online MR images (Figure 3A). In this study, S1 and M1 of two monkeys were targeted (Figure 3B). The three-dimensional distribution volumes were estimated in a post-hoc analysis of the MR images (<strong class="xfi...

Watch this Scientific Journal Video about Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images at JoVE.com

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Convection enhanced delivery, or CED, of optogenetic viral vectors in non human primates will enable researchers to manipulate neural activity at a large scale to understand complex neural computations and behaviors. With CED we can achieve high optogenetic expression across large regions of the primate brain with only a few injections in a short amount of time compared to traditional methods. Magnetic resonance or MR compatible chamber implantation is performed using standard practice in non human primate surgeries, the procedure is described in detail in the text protocol and outlined briefly in this video.Following animal sedation, positioning, and initial incision as described in the text protocol, create a circular craniotomy to cover the preplanned trajectories for injections using a trephine. Create an indentation at the center of the planned craniotomy sufficiently deep in the skull to anchor the trephine using the adjustable centering point of the trephine. Once the center has been made, lower the trephine onto the skull and rotate the trephine clockwise and counterclockwise while applying downward pressure until the bone cap can be removed with forceps.After lifting the dura as detailed in the text protocol, cut the dura from the center to the edge of the craniotomy and continue along the edge with fine ophthalmic scissors. Mount the cylindrical custom made convection enhanced delivery MR compatible chamber to the skull on top of the craniotomy to provide cannula support during infusion such that the curvature of the chamber flange aligns well with the curvature of the skull. Secure the implants to the skull using either plastic screws and dental acrylic or a few titanium screws.Soon after implanting the MR compatible chamber and inserting the cannula injection grid into the chamber, fill the grid with 0.9%saline for visualizing the injection locations via MR anatomical images. Also fill the chamber cavities with wet sterile absorbable gel foam to maintain the moisture of the brain. Cover the skin and the cylinder with a sterile antimicrobial incise drape to maintain the sterility of the cylinders during transport and MR infusions.Place a vitamin E capsule to mark the top of the injection grid for positive identification. While the animal remains intubated, detach the endotracheal tube from the anesthesia circuit and reattach it to a portable MR compatible isoflurane machine. Transport the animal to the MRI scanner.Acquire T1 images to calculate the distance from the top of the injection grid and the cortical surface. The vitamin E capsule is clearly visible in T1 images and should be used as a marker for the top of the injection grid. Acquire T2 images to determine the optimal cannula guides for each site based on the targeted site of infusion.The cannula grid is filled with saline which is best visible in T2 images. Using the MR imaging software, scroll through the coronal and sagittal planes to find the target location of infusion. After thawing the viral vector for a few hours at room temperature, mix the viral vector with the MR contrast agent gadoteridol by pipette or vortex mixing.Load the mixed virus into 0.2 milliliter high pressure IV tubing. Using high pressure IV tubing, connect a long extension line to an MR compatible three milliliter syringe and prime with saline. Connect the other end of the long extension tubing to the 0.2 milliliter IV tubing loaded with the virus.Then attach the reflux resistant cannula with a one milliliter stepped tip to the distal end of this assembly with a clamp style catheter connector. Lastly set the syringe in an MR compatible pump. Using the obtained baseline anatomical MR images, select the cannula injection grid location and insertion depth needed to reach the target infusion site.Mark the insertion depth on the cannula using sterile tape. Begin the infusion at a rate of one microliter per minute and visually validate the flow of the fluid in the infusion line by observing the ejection of fluid from the cannula tip. Insert the cannula manually through the injection grid to the target depth while maintaining flow in the infusion line as this will prevent the penetrated tissue from clogging the cannula during insertion.Now acquire fast flash T1-weighted images for online monitoring of the viral vector infusion. After infusing approximately 10 microliters of the vector such that enough virus is infused to detect in the MR scanner, obtain MR images to verify correct cannula placement as evident by the observed spread of the virus. If the depth of the inserted cannula is incorrect, adjust the depth accordingly or slowly remove the cannula and reattempt the insertion.Monitor the infusion via guidance of online MR images. Increase the infusion rate to five microliters per minute by one microliter per minute steps every few minutes. It is important to increase the infusion rate slowly and not to exceed five microliters per minute as high infusion rates could cause tissue damage.After infusing approximately 40 microliters of the viral vector, begin reducing the infusion rate by one microliter per minute steps. Stop the infusion after approximately 50 microliters is injected. Leave the cannula in place for ten minutes.Slowly remove the cannula from the brain and move to the next location. Cover the cylinder with a sterile drape at the end of injections before animal transport, then transport the animal back to the operating room. Histological analyses on serial coronal sections reveal large scale optogenetic expression around the infusion sites.Shown here is the baseline coronal MR image and the spread of the contrast agent after the infusion for the same MR coronal slice. A coronal tissue section is shown from approximately the same. Peroxidase staining reflects expression of the yellow fluorescent protein or EYFP reporter.Good alignment is observed between the area of EYFP expression, measured with surface epifluorescense and with histological staining. These include the region of vector spread estimated from MR images. White dots indicate injection sites and the entire black region represents the area exposed by the craniotomy.Shown here is a low magnification image of the coronal sections stained with anti GFP antibody showing the medial lateral aspect of YFP expression in the somatosensory cortex. The black arrowhead indicates the location of the cannula track. The adjacent tissue is shown at greater magnification to show laminar distribution of the YFP positive cells.Densely populated regions of YFP positive cells are located predominately in layers two through three and five through six. Further enlargement reveals typical pyramidal cells in layers two through three, layer five, and layer six. It is important to leave the cannula in place for 10 minutes after the flow has stopped.This will ensure that the virus has percolated into the surrounding targeted neural tissue. CED is an efficient approach to achieve uniformly high expression levels across large brain regions in mammals. It has the potential to expand our understanding of neural circuits and connectivity.After viral transduction by CED, subsequent optogenetic stimulation experiments can be paired with electrical recording and behavioral essays to reveal the complex circuit dynamics within the brain. This technique has been previously employed by Yazdan-Shahmorad and colleagues in 2018 in which optogenetic neural modulation was used to induce network wide connectivity changes in the sensory motor cortex.

convection-enhanced-delivery-optogenetic-adeno-associated-viral

Research

JoVE Journal

Neuroscience

6.8K Görüntüleme.  University of Washington. Konveksiyon gelişmiş teslimat, ya da CED, insan olmayan primatlar optogenetik viral vektörler, araştırmacılar karmaşık nöral hesaplamalar ve davranışları anlamak için büyük bir ölçekte nöral aktivite işlemek için sağlayacaktır. CED ile primat beyninin büyük bölgelerinde geleneksel yöntemlere göre kısa sürede sadece birkaç enjeksiyon la yüksek optogenetik ekspresyon elde edebiliriz. Manyetik rezonans veya MR uyumlu oda implantasyonu insan olmayan primat ameliyatl...