This work was made possible by grants from the NIH (EY012977), a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, the Whitehall Foundation, and the Sloan Foundation (A.K.M.).

1. Virus Handling and Storage
<ol>
<li>Proper protection and handling techniques should be chosen based on the biosafety level of the virus to be used. These practices can be found in Biosafety in Microbiological and Biomedical Laboratories 5th edition, available on the CDC website (<a href="http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm">http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm</a>). The use of AAV vectors is approved for Biosafety Level 1 (BSL-1). For the experiment shown here, a lab coat and gloves will be worn when handling the virus, in accordance with procedures for handling BSL-1 agents.</li>
<li>To preserve the activity of the virus it is best to divide it into small aliquots to avoid repeated freezing and thawing.</li>
<li>Prepare a Biosafety Cabinet (BSC) Class II for aliquoting virus by clearing the BSC of any unnecessary objects and sterilizing the surface with 70% EtOH. Place a beaker containing 10% bleach solution in the hood for collecting AAV-contaminated waste. Also placed in the BSC are sterile 0.5mL tubes and a container of dry ice.</li>
<li>Thaw viral stock on ice outside the BSC.</li>
<li>Vortex virus and open the tube inside the BSC.</li>
<li>Pipette the desired aliquot volume (e.g. 5 &mu;L) into a 0.5mL tube, close the tube and place it in the dry ice to flash-freeze the virus.</li>
<li>When all virus has been aliquoted, dispose of the virus tube and pipette tips in the waste container containing 10% bleach.</li>
<li>Remove the waste container from the hood and add additional 10% bleach, then pour the bleach down the sink. Dispose of plastic waste in a biohazard container as instructed by your institute's biosafety officer.</li>
<li>Clean any equipment or surfaces that came in contact with the virus with 10% bleach. Discard gloves.</li>
<li>Store aliquots in -80&deg;C freezer.</li>
</ol>
2. Surgery
<ol>
<li>Cover the surgical area with absorbent lab bench paper. Surgical tools should be sterilized and surgery done under aseptic conditions in accordance with your institution's biosafety and animal use guidelines.</li>
<li>Choose an area adjacent to the surgical area that will be dedicated to loading the micropipettes with virus, and cover it with absorbent lab bench paper. Place an aliquot of virus in a container of ice in this area, and allow the virus to thaw on ice as the surgery is being performed.</li>
<li>Setup a waste container of 10% bleach in the dedicated virus handling area for disposing of pipette tips, etc. that come in contact with the virus.</li>
<li>Pull a glass Wiretrol micropipette to a tip diameter of approximately 20 microns. Place a small drop of mineral oil on the blunt end of the micropipette and insert the wire plunger provided with the micropipettes.</li>
<li>Secure the micropipette in the clamp of the micropump arm.</li>
<li>Inject a mouse subcutaneously with buprenorphine at a dose of 0.5 mg/kg. Anesthetize the mouse with Avertin via intraperitoneal injection with a dose of 200 mg/kg and remove the hair from the top of the head with surgical scissors, making sure to leave margins large enough to prevent hair from entering the incision.</li>
<li>Attach a heating blanket to the base of a stereotax to maintain a body temperature of 37&deg;C throughout the surgery and secure the mouse in the stereotax.</li>
<li>Bathe the head with three alternating scrubs of ethanol and betadine to sterilize the area.</li>
<li>Place a drop of Tobradex eye ointment on each eye to keep the eyes moist during surgery.</li>
<li>Make an incision down the midline of the head and pull the skin back to expose the skull.</li>
<li>Carefully remove the fascia from the skull using fine-tipped forceps.</li>
<li>Locate the area to be injected using stereotaxic coordinates and mark the skull with a surgical pen.</li>
<li>Using a dental drill with a 1.4mm burr, thin an area of the skull approximately 2mm in diameter until the skull cracks, dividing the thinned area into several segments.</li>
<li>Throughout this procedure, keep the skull moist with applications of sterile saline.</li>
<li>Perform a craniotomy by gently removing the thinned skull segments using extra-fine tipped forceps.</li>
</ol>
3. Injection Preparation
<ol>
<li>With a KimWipe placed over the lid, open the tube of virus and pipette 1.5uL (for a 1uL injection) of virus onto a small piece of parafilm.</li>
<li>Place the tip of the micropipette into the viral stock and manually withdraw the plunger. If difficulty is experienced drawing viral stock into the micropipette, the tip can be enlarged slightly be piercing a KimWipe with the micropipette to break a small portion of the glass.</li>
<li>Lower micropump arm onto plunger until a small amount of virus is dispelled from the tip of the micropipette. Remove this small drop with a cotton tip applicator and discard applicator in waste container.</li>
<li>Apply a drop of mineral oil to the tip of the micropipette to prevent clogging as the micropipette is lowered into the brain.</li>
</ol>
4. Virus Injection
<ol>
<li>Using X and Y stereotaxic coordinates, position the micropipette over the area to be injected. In this experiment, the stereotaxic coordinates used to locate primary visual cortex are 2.7mm posterior to bregma and 2.5mm lateral to the midline. Very slowly lower the micropipette (at an approximate rate of 1mm/1minute) to the proper Z position.</li>
<li>Enter the desired injection parameters into the SYS Micro4 micropump controller box and initiate the injection. For this experiment, 1 microliter over 10 minutes will be used as a rate of injection.</li>
<li>When the injection is finished, leave the pipette to rest for one-two minutes to prevent efflux of virus during removal. After this period, very slowly remove the micropipette from the brain (same rate as above).</li>
<li>Suture the scalp and seal it with tissue glue. Inject the animal subcutaneously with buprenorphine at a dose of 0.1 mg/kg every 8-12 hours over the next 72 hours, or as long as the animal is exhibiting signs of pain. Allow the animal to recover under a heat lamp until it is ambulating and ready to be returned to its cage. Imaging experiments can be initiated after the desired incubation time (days to weeks after viral injection).</li>
</ol>
5. Cleanup
<ol>
<li>Rinse the micropipette with 10% bleach and discard it in a sharps container.</li>
<li>Dispose of waste container in the same manner as Section 1.</li>
<li>Discard lab bench paper into biohazard bin and wipe down all surfaces and instruments that may have come in contact with the virus with 10% bleach.</li>
<li>Unused virus may be frozen again, keeping in mind that repeated freeze/thaw cycles cause degradation of the virus.</li>
</ol>

6. Representative Results
<img src="/files/ftp_upload/2140/2140fig1.jpg" alt="Figure 1" /> 
Figure 1. Transduced neuron after injection of double-stranded adeno-associated virus serotype 1 (dsAAV S1) carrying green fluorescent protein (GFP) under control of the CMV promotor. The cell body as well as proximal and distal dendrites are clearly visible in fixed sections imaged using an epifluorescent microscope. Scale bar= 100 &mu;m.
<img src="/files/ftp_upload/2140/2140fig2.jpg" alt="Figure 2" /> 
Figure 2. Labeling typical of an intracranial virus injection in primary visual cortex using dsAAV S1 showing the extent of viral spread as well as labeled neurons, glia and processes. Scale bar= 250 &mu;m.
<img src="/files/ftp_upload/2140/2140fig3.jpg" alt="Figure 3" /> 
Figure 3. Labeling of cells in hippocampus using dsAAV S1. Scale bar= 250 &mu;m. These figures are adapted from Lowery et al. 20091

<ol>
	<li>Kaplitt, M. G., Leone, P., Samulski, R. J., Xiao, X., Pfaff, D. W., O'Malley, K. L., During, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nat Genet. 8, 148-154 (1994).</li><li>Harding, T. C., Dickinson, P. J., Roberts, B. N., Yendluri, S., Gonzalez-Edick, M., Lecouteur, R. A., Jooss, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+gene+transfer+efficiency+in+the+murine+striatum+and+an+orthotopic+glioblastoma+tumor+model,+using+AAV-7-+and+AAV-8-pseudotyped+vectors.">Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8-pseudotyped vectors.</a> Hum Gene Ther. 17, 807-820 (2006).</li><li>Lo, W. D., Qu, G., Sferra, T. J., Clark, R., Chen, R., Johnson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+the+brain:+duration+and+modulation+of+expression.">Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression.</a> Hum Gene Ther. 10, 201-213 (1999).</li><li>Lowery, R. L., Zhang, Y., Kelly, E. A., Lamantia, C. E., Harvey, B. K., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+long-term+labeling+of+cells+in+the+developing+and+adult+rodent+visual+cortex+using+double-stranded+adeno-associated+viral+vectors.">Rapid long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.</a> Dev Neurobiol. 69, 674-688 (2009).</li></ol>

The animal and experimental protocols were approved by the University of Rochester University Committee on Animal Resources (UCAR) in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals.

Virally-mediated gene delivery holds great potential for the study of neurological processes and treatment of brain disorders1,2,3. The great versatility of this technique can also be exploited to fluorescently label cells for imaging both in vitro and in vivo4. Here we demonstrate a detailed procedure for the transduction of neurons and glia in mouse visual cortex using a double-stranded adeno-association virus expressing enhanced green fluorescent protein.
<p class="jove_con...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stoelting Mouse and Neonatal Rat Adaptor</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51625</td>
<td>Regular stereotax for securing animals for surgery may be substituted</td>
</tr>
<tr>
<td>Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14084-08</td>
<td>&nbsp;</td>
<tr>
<td>Eye Dressing Forceps, 10cm, tip width 0.5mm, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5/45 Forceps- Dumoxel Standard Tip, 11cm, angled</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11251-35</td>
<td>Extra-fine tipped forceps for performing craniotomy</td>
</tr>
<tr>
<td>Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>Microtorque Control Box and Tech2000 handpiece</td>
<td>&nbsp;</td>
<td>Ram Products, Inc.</td>
<td>TECH2000ON/OFF</td>
<td>Dental drill</td>
</tr>
<tr>
<td>Micro Drill Stainless Steel Burrs 1.4mm tip diameter</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>19008-14</td>
<td>&nbsp;</td>
<tr>
<td>Wiretrol micropipettes, to deliver 1-5 Ul</td>
<td>&nbsp;</td>
<td>VWR International/ Drummond</td>
<td>5-000-1001 or 53480-287</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>29447-338</td>
<td>&nbsp;</td>
<tr>
<td>Manual Micromanipulator and Tilting Base (right-handed)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>M3301-M3-R</td>
<td>Used for determining stereotaxic co-ordinates</td>
</tr>
<tr>
<td>UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>UMP3-1</td>
<td>&nbsp;</td>
<tr>
<td>Sutures</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>95056-952</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Tobradex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Available from your institution&#x2019;s veterinary services</td>
</tr>		</tbody>
		</table>
		</div>

intracranial injection of adeno-associated viral vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Watch this Scientific Journal Video about Intracranial Injection of Adeno-associated Viral Vectors at JoVE.com

Intracranial Injection of Adeno-associated Viral Vectors

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

intracranial-injection-of-adeno-associated-viral-vectors

Research

JoVE Journal

Neuroscience

47.2K ビュー.  University of Rochester. ここでは、視覚野の神経細胞とグリア細胞の蛍光標識用AAVベクターの頭蓋内注射を提示する。

ビデオ: Intracranial Injection of Adeno-associated Viral Vectors

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

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;

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 ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

Analyzing the Parkinson&#039;s Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &amp;#945;-Synuclein

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.