This research was supported by the Robert A. and Rene E. Belfer Family Foundation, NIA R21 AG038856 (JLJ), BrightFocus Foundation Alzheimer&#8217;s Disease research grant A2010097 (JLJ), and NIA Biology of Aging Training grant T32 AG000183 (support for SDG).

Perform all procedures and protocols involving animals in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The procedures described here were reviewed and approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.
Replication-incompetent adeno-associated virus (AAV) vectors for transgene delivery in the rodent brain are approved for Biosafety Level 1 use. Refer to the CDC website for the US Government publication &#8220;Biosafety in Microbiology and Biomedical Laboratories (BMBL)&#8221; which details specific requirements regarding proper protection and virus handling procedures. Check with local veterinary and environmental safety staff to learn institutional specific requirements for procedures using viruses. Regulations regarding procedure rooms, quarantine, and identification of biohazard cages vary across institutions.
1. Prepare P0 Pups and Foster Mothers
<ol>
	<li>Provide high fat chow for all pregnant and nursing females to support the energy demands of breeding and lactation.</li>
	<li>In the evening, set up a breeding cage by adding one or two healthy females to the cage of a single resident male. The following morning, check the females for a mating plug and separate the females from the males if a plug is present.
	<ol>
		<li>Use foster mothers to raise pups from a genetic background with poor maternal characteristics (for example, C57BL/6 or C3HeJ). Foster mothers accept pups from another litter if the transferred pups are born within four days of the foster mother&#8217;s own litter.</li>
		<li>Set up a separate mating cage using ICR or FVB females alongside the experimental breeders so that the two sets of females deliver at approximately the same time.</li>
	</ol>
	</li>
	<li>Females will deliver in 19 &#177;1 days from the plug date, depending on the background strain. Three days prior to delivery (16 days after the plug date), place the pregnant female into a clean cage with fresh nesting material and a covered shelter.</li>
	<li>Check for newborn pups twice daily starting 2 days prior to the expected delivery date (17 days after the plug date). Examine the cage with minimum stress to the mothers by peeking through the bottom for the presence of pink newborns. Mice generally deliver pups in the morning but on rare occasions will deliver in afternoon.</li>
	<li>Plan to perform injections as soon as the pups are nursing (which will be evident by visible milk spots), or within 6 hr of birth, whichever comes first.</li>
</ol>
2. Prepare Equipment for Injection
<ol>
	<li>Prepare a 10 &#181;l injection syringe with a 32 G&#160;needle for general P0 neonates. If performing stereotaxic injection, mount the syringe onto the manipulator arm.</li>
	<li>If using a stereotaxic frame for injections, cool the neonatal stage to 4-8 &#176;C by adding 100% ethanol and dry ice to the reservoir at the front end of the block. Maintain the temperature above 1 &#176;C to avoid frostbite of the pups.</li>
</ol>
3. Prepare Viral Dilutions for Injection
<ol>
	<li>Prepare a 1% trypan blue dye solution (20x stock).</li>
	<li>Prepare 5-25 &#181;l aliquots of adeno-associated virus (AAV) stock inside a Class 2 biosafety cabinet. Place these aliquots at -80 &#176;C for future use. Avoid additional freeze-thaw cycles as this causes the virus to lose transduction efficacy.</li>
	<li>Remove an aliquot of AAV from the -80 &#176;C freezer and place on ice to thaw.</li>
	<li>Prepare the viral solution for injection by diluting the virus into ice-cold 1x phosphate buffered saline (PBS). Start with a ten-fold serial dilution (1010 to 108 viral particles/hemisphere) for initial optimization of the transduction pattern.</li>
	<li>Add 20x trypan blue to the AAV solution to a final concentration of 0.05%.</li>
</ol>
4. Staging the Newborn Pups for Injection
<ol>
	<li>Place a small aluminum plate on ice to cool. Place a dry task wipe on top of the plate to protect the pup&#8217;s skin from the cold metal. This serves as a flat cold surface for anesthetizing and injecting the pups.</li>
	<li>Prepare a warming pad suitable for keeping neonatal mice warm before and after injection.</li>
	<li>Once the newborn pups have begun to nurse, remove approximately half of them from the cage and leave the other half with their mother. Place the collected pups on the warming pad while awaiting injection. 
	NOTE: If the biological mother does not care for the pups, remove the entire litter from the cage immediately and place the pups onto a warming pad to await injection. This situation may arise with the first litter born to C57BL/6 females or with other inbred strains that have poor breeding characteristics. In this situation, milk spots will not be visible on newborn pups.</li>
	<li>Transfer one pup from the warming pad onto the cold metal plate to induce hypothermia anesthesia. Wait 2-3 min for the pup to become fully anesthetized. Confirm anesthesia by very gently squeezing a paw and monitor for lack of movement or respiration.</li>
</ol>
5. Injection of AAV into Neonatal Mice
<ol>
	<li>Free-hand intracranial injection of AAV into neonatal mice:
	<ol>
		<li>Load 5 &#181;l of diluted AAV with 0.05% trypan blue into the injection syringe.</li>
		<li>Gently wipe the head of the anesthetized pup with a cotton swab soaked in 70% ethanol.</li>
		<li>Identify the injection sites at 2/5 of the distance from the lambda suture to each eye. An alternative injection site is located approximately 0.8-1 mm lateral from the sagittal suture, halfway between lambda and bregma. These landmarks are visible through the skin at P0.</li>
		<li>Mark the injection site with a non-toxic laboratory pen.</li>
		<li>Hold the syringe with the scale visible for monitoring the volume of solution dispensed.</li>
		<li>Ensure that the thumb can reach the top of the plunger. Then remove the thumb from the plunger while positioning the needle to avoid accidentally dispensing virus.</li>
		<li>Find a comfortable position to brace the arm for injection by putting the elbow on the bench and leaning the arm on the ice bucket.</li>
		<li>Lay the pup on its side with its head directly under the syringe. Turn the pup&#8217;s head so that the marked injection site is facing up, and gently but firmly hold this position with an open hand.</li>
		<li>Hold the syringe perpendicular to the surface of the skull and insert the needle at the marked injection site to a depth of approximately 3 mm. Inject the needle until the resistance decreases slightly, indicating that the needle has penetrated into the lateral ventricle. If a reference is needed, mark 3 mm from the tip of needle with non-toxic maker. Adjust the injection depth for pup size and strain based on dye targeting of the ventricles.</li>
		<li>Hold the syringe rigidly so that the plunger can be depressed without moving the needle farther into the brain. If the injection is displaced into the thalamus, viral spread will be severely limited.</li>
		<li>Begin slowly injecting virus while monitoring the volume dispensed from the syringe. Administer a maximum volume of 2 &#181;l into each ventricle. If the needle is in the correct position, the dye will spread to fill the ventricle.</li>
		<li>Slowly withdraw the needle.</li>
		<li>Allow the first injection site to close before injecting the other hemisphere. 
		NOTE: Do not inject the same site more than once. Reinserting the needle forces virus that has already been injected to leak out and causes injury or death of the pup.</li>
		<li>Inject the contralateral ventricle using the same procedure.</li>
	</ol>
	</li>
	<li>Stereotaxic injection of AAV into neonatal mice:
	<ol>
		<li>Load 5 &#181;l of diluted AAV with 0.05% trypan blue into the injection syringe held by the stereotaxic manipulator.</li>
		<li>Gently place the pup&#8217;s head between the ear bars of the neonatal frame. Make sure the head is level in the Y-axis (front to back) by checking that the line between lambda and bregma is parallel to the stage. Make sure the head is level in the X-axis (side to side) by checking that an imagined line between the ears, or a line between the eyes, is parallel to the stage.</li>
		<li>Gently wipe the anesthetized pup&#8217;s head with a cotton swab soaked in 70% ethanol.</li>
		<li>Use the stereotaxic manipulator to position the syringe above lambda and then zero the X and Y coordinates. Move the stereotaxic arms to (X, Y) = (0.8, 1.5) mm for standard P0 pups. 
		NOTE: Pup size and stereotaxic coordinates may vary by strain and age. Adjust coordinates based on dye injections as needed to target the lateral ventricles.</li>
		<li>Slowly lower the needle into the injection site. The surface of the skull will indent and then release once the needle has penetrated the skin. Retract the needle until the skull recovers its normal concave shape, but keep the bevel of the needle under the skin. Zero the Z coordinate at this point.</li>
		<li>Insert the needle until Z = -1.7 mm and then retract to -1.5 mm.</li>
		<li>Slowly inject the virus. Each hemisphere can accept up to 2 &#181;l of solution.</li>
		<li>Keep the syringe in place for 30-60 sec after completing the injection and then slowly retract the needle over 1-2 min.</li>
		<li>Repeat for the contralateral hemisphere, using negative coordinates in the X-axis for the injection site. 
		NOTE: Alternative coordinates for injection are (X, Y, Z) = (0.8, 2.0, -1.5) mm and (1.2, 1.0, -1.5) mm. Perform the first injection at (0.8, 2.0), then move to (0.8, -2.0), (1.2, 1.0) and finally (1.2, -1.0). Inject 1 &#956;l of solution at each site, for a total of 2 &#956;l per hemisphere. Move quickly between injections to complete the procedure in less than 10 min.</li>
	</ol>
	</li>
</ol>
6. Post-injection Care
<ol>
	<li>After completing injections into both hemispheres, place the pup back on the warming pad until its body temperature and skin color return to normal and the pup begins to move.</li>
	<li>If using the biological mother to nurse the injected neonates, return the injected pups to the biological mother after they recover normal movement. Place the remaining uninjected pups on the warming pad. Repeat the procedure until all mice have been injected.</li>
	<li>If using a foster mom to nurse the injected neonates, transfer all injected pups to the foster mother for care after they recover normal movement.
	<ol>
		<li>Remove most or all of the pups belonging to the foster mother from the cage to ensure the success of the injected animals.</li>
		<li>If foster mother&#8217;s pups and injected pups have the same eye and skin color, mark pups from the foster mother with a laboratory pen so they can be distinguished later.</li>
		<li>Place the foster mother&#8217;s pups and some of their bedding together with the injected pups. Ensure that the injected pups acquire the scent of her biological offspring to improve acceptance of the new pups.</li>
		<li>Place only the injected pups back into the foster mother&#8217;s cage. Cull any remaining pups from the foster mother.</li>
	</ol>
	</li>
	<li>Ensure that the mother is attending to and nursing the injected pups within 10 min&#160;of placing them into the cage. If the mother does not collect the pups within this time, transfer pups to another foster mother.</li>
	<li>Label the cage with a biohazard card to show that it contains virally-injected animals. House the cage in an approved area for 72 hr.</li>
	<li>After the 72 hr quarantine period, transfer the mother and pups (but no bedding or other cage items) to a clean cage. Perform this transfer inside a Level 2 biosafety cabinet to avoid exposure to any virally-contaminated bedding. Return the clean cage with the injected animals to the regular mouse facility.</li>
	<li>Place the dirty cage into a biohazard bag, sterilize by autoclaving, and then return the cage to the vivarium.</li>
</ol>
7. Cleanup
<ol>
	<li>Place the remaining virus solution at 4 &#176;C for future use. The virus retains transduction efficiency for at least 2 months when stored at 4 &#176;C8.</li>
	<li>Disinfect the injection needle and syringe with 2% bleach followed by repeated rinses in deionized water.</li>
	<li>Clean the working area with 2% bleach followed by 70% ethanol.</li>
	<li>Collect all disposable items including pipette tips, tubes, mask, and gloves in a plastic biohazard bag. Dispose of waste materials as required by local law and institutional policies (i.e. autoclave or incinerate).</li>
</ol>
8. Prepare Mouse Brains for Imaging
<ol>
	<li>Place an injected mouse into a carbon dioxide euthanasia chamber at 3-4 weeks post-injection. Follow institutional guidelines for proper euthanasia procedures. Do not perfuse animals with paraformaldehyde as this will quench the fluorescence label.</li>
	<li>Remove the whole brains from the skull and fix for 3-12 hr in 4% paraformaldehyde in PBS at 4 &#176;C.</li>
	<li>Transfer the fixed brain to 30% sucrose for cryoprotection. Incubate at 4 &#176;C until the brain sinks to the bottom.</li>
	<li>Collect a bucket of finely crushed dry ice. Cut the brain in half down the midline. Bury the brain halves in the dry ice to freeze.</li>
	<li>Cool the microtome stage by adding dry ice and 100% ethanol.</li>
	<li>Secure the brain to the stage with the midline down using PBS, and allow to freeze.</li>
	<li>Section through the brain at 30-45 &#181;m thickness using a freezing-sliding microtome.</li>
	<li>Place sections into 24-well plates containing antifreeze solution (250 ml glycerol, 300 ml ethylene glycol, 500 ml 0.1 M phosphate buffer, pH 7.4). Cover plates with tin foil and store at -20 &#176;C for future use.</li>
	<li>Collect sections from 1-4 wells and wash 3x in PBS.</li>
	<li>Mount sections onto microscope slides and coverslip. Allow slides to dry in a dark location.</li>
	<li>Image slides using appropriate filters for native YFP or tdTomato to check the virus transduction pattern.</li>
</ol>

<ol><li>De Vry, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation+of+the+central+nervous+system%3A+a+non-viral+approach+for+targeted+gene+delivery%5BTitle%5D)+AND+%22Progress+in+neurobiology%22%5BJournal%5D)">In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.</a> Progress in neurobiology. 92, 227-244 (2010).</li>
<li>Sun, B., Gan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manipulation+of+gene+expression+in+the+central+nervous+system+with+lentiviral+vectors%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Manipulation of gene expression in the central nervous system with lentiviral vectors.</a> Methods Mol Biol. 670, 155-168 (2011).</li>
<li>Puntel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+transfer+into+rat+brain+using+adenoviral+vectors%5BTitle%5D)+AND+%22Curr+Protoc+Neurosci%22%5BJournal%5D)">Gene transfer into rat brain using adenoviral vectors.</a> Curr Protoc Neurosci. 4, (2010).</li>
<li>Passini, M. A., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Widespread+gene+delivery+and+structure-specific+patterns+of+expression+in+the+brain+after+intraventricular+injections+of+neonatal+mice+with+an+adeno-associated+virus+vector%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Widespread gene delivery and structure-specific patterns of expression in the brain after intraventricular injections of neonatal mice with an adeno-associated virus vector.</a> J Virol. 75, 12382-12392 (2001).</li>
<li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraventricular+brain+injection+of+adeno-associated+virus+type+1+%28AAV1%29+in+neonatal+mice+results+in+complementary+patterns+of+neuronal+transduction+to+AAV2+and+total+long-term+correction+of+storage+lesions+in+the+brains+of+beta-glucuronidase-deficient+mice%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice.</a> J Virol. 77, 7034-7040 (2003).</li>
<li>Kim, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viral+transduction+of+the+neonatal+brain+delivers+controllable+genetic+mosaicism+for+visualising+and+manipulating+neuronal+circuits+in+vivo%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo.</a> Eur J Neurosci. 37, 1203-1220 (2013).</li>
<li>Chakrabarty, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capsid+serotype+and+timing+of+injection+determines+AAV+transduction+in+the+neonatal+mice+brain%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain.</a> PloS one. 8, (2013).</li>
<li>Croyle, M. A., Cheng, X., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+formulations+that+enhance+physical+stability+of+viral+vectors+for+gene+therapy%5BTitle%5D)+AND+%22Gene+therapy%22%5BJournal%5D)">Development of formulations that enhance physical stability of viral vectors for gene therapy.</a> Gene therapy. 8, 1281-1290 (2001).</li>
<li>Bauer, H. C., Bauer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+induction+of+the+blood-brain+barrier%3A+still+an+enigma%5BTitle%5D)+AND+%22Cellular+and+molecular+neurobiology%22%5BJournal%5D)">Neural induction of the blood-brain barrier: still an enigma.</a> Cellular and molecular neurobiology. 20, 13-28 (2000).</li>
<li>Cearley, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanded+repertoire+of+AAV+vector+serotypes+mediate+unique+patterns+of+transduction+in+mouse+brain%5BTitle%5D)+AND+%22Molecular+therapy+%3A+the+journal+of+the+American+Society+of+Gene+Therapy%22%5BJournal%5D)">Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 16, 1710-1718 (2008).</li>
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</ol>

Authors declare that they have no competing financial interests.

We have described a versatile procedure for manipulating neuronal gene expression using AAV as a vehicle for widespread delivery into the neonatal mouse brain. Compared with other methods of neuronal transgenesis such as in utero electroporation1 or stereotaxic intracranial injection2,3, neonatal viral injection is relatively easy and simple. The basic procedure can be performed in minutes with only an ice bucket and a microliter syringe. Optimal survival and transgene expression can be attained by attending to a few technical details, the most important being the quality of viral stocks, the timing and accuracy of injection, and the post-natal care.
The quality of viral preparation is critical for successful transduction. Bad viral preparations will significantly diminish neuronal infectivity and produce significant astrocytic transduction, possibly by phagocytosis of non-infectious particles. Many universities have core laboratories on-site that specialize in viral packaging, and large facilities at the University of North Carolina and the University of Pennsylvania offer high quality off-the-shelf reagents in a variety of serotypes at reduced cost. These facilities also provide custom packaging for vectors that are not available as pre-packaged stocks. Once received into the laboratory, remember that AAV particles can be stable for years at -80 &#186;C, but are very sensitive to temperature fluctuations. For this reason, aliquots should be stored at -80 &#176;C and should not be refrozen once thawed.
For highest transduction efficiency, it is critical to inject virus as soon as possible after pups are delivered. Based on our studies with AAV8 and those of our collaborators, delaying the injections not only diminishes the spread of virus from the ventricle, but will also bias the transduction from neurons to astrocytes6,7. Therefore we recommend injecting on the day of birth for optimal neuronal expression. At this age - before the cerebrospinal fluid-brain barrier has matured9 - viral particles injected into the lateral ventricle will diffuse throughout the ventricular system and then follow the flow of cerebrospinal fluid into the brain4. Consequently, precise targeting of the lateral ventricle is critical to maximize viral spread. Accurate targeting also minimizes tissue damage from the rapid injection of a relatively large volume of fluid. Therefore, we strongly recommend repeated practice until targeting the lateral ventricles becomes reliable and reproducible. Note that the coordinates provided in this protocol are appropriate for general P0 pups and should be adjusted for the strain and age of experimental pups. Initially, injections should be performed with dye solutions, such as trypan blue or India ink, so the targeting and spread can be visualized by harvesting the brain immediately after injection. If the lateral ventricles have been successfully targeted, the dye will spread throughout the ventricular chambers and be visible all the way from the olfactory bulb to the cerebellum. A stereotaxic device is recommended if the ventricles cannot be reliably targeted by free-hand injection.
If the injections are performed correctly, the proportion of mice lost to injection will be negligible. Instead, the survival rate after injection is most affected by maternal care. Start with healthy animals. The mother&#8217;s health is indicated by her behavior and her coat. She should be well-groomed and clean. The litter size can also be an indication of well-being. Healthy young females should produce litters of 6 - 8 pups for C57BL/6, or 10 - 16 pups for ICR. Healthy pups should be pink and wiggly. If pups do not look well or have no milk spot on their belly, they should be transferred to a new foster female immediately. We recommend ICR or FVB for fostering because they produce ample milk and readily accept new pups into their litter. It is imperative that disruptions to the mother are minimized before and after the injections as stress may cause her to reject or cannibalize the pups. Reduce stressors by limiting noise, light, and other disturbances and keeping the cage in a quiet place. Open the cage as little as possible when checking for newborn pups and in the first few days after injection, although it is essential to monitor the mother&#8217;s attentiveness to the pups once they are replaced. The mother should display innate behaviors such as bunching the pups in one place and sitting atop the pile of offspring. If the mother does not respond immediately when the pups are returned to the cage, try again to mix the pups with dirty bedding and nesting material from her cage ensure they have acquired her scent. Check again later in the day that the pups still have milk spots on their bellies. If possible, do this by looking into the cage from underneath rather than opening the cage from above. The first days are most critical for survival, but also when the female will be most sensitive to disruption.
When done carefully, intraventricular AAV injection provides a fast and easy means of manipulating neuronal gene expression in vivo without the cost and time of traditional germline transgenesis. The native mosaicism of viral transduction can be harnessed to control the density of expression, making it an ideal approach for experiments to separate cell-intrinsic and cell-extrinsic consequences of transgenesis. In addition, two viruses can be co-injected making it possible to express multiple proteins in either overlapping or distinct neuronal populations. These manipulations highlight the flexibility of the approach, but we believe we&#8217;ve only just scratched the surface of its potential. The growing availability of off-the-shelf reagents will make it easier to develop viral injection for new experimental needs, while the emergence of hybrid serotypes with distinct tropisms may expand the cellular repertoire that can be targeted10,11.

Traditional methods for modifying neural gene expression require time-consuming and expensive germline manipulations. Alternative de novo approaches such as in utero electroporation or stereotaxic lentiviral injection yield faster results and are less costly but have the disadvantage of requiring complex surgical intervention1-3. Furthermore, transgene expression has a limited spatial range with these methods. Herein, we describe a fast, easy, and economical method for widespread neuronal manipulation via intraventricular injection of adeno-associated virus (AAV) into the neonatal mouse brain. The method was first described by John Wolfe and Marco Passini in 2001, where they suggested small particle size of AAV allowed it to diffuse within the cerebral spinal fluid as it passes from the lateral ventricles through the immature ependymal barrier and into the brain parenchyma4,5. Intraventricular injection of AAV within the first 24 hr after birth yields widespread viral transduction of neural subsets spanning every region of the brain, from the olfactory bulbs to the brain stem6,7. Virally-delivered transgenes are expressed and active within days of injection and persist for up to a year after transduction. Thus, this versatile manipulation enables studies ranging from early postnatal brain development to aging and degeneration in the adult.
In adapting the technique to our specific experimental needs, we have focused primarily on AAV8 serotype because it is the most efficient at transducing neurons6. We show that viral titer can be diluted to control the density of transduced neurons for experiments testing cell-intrinsic consequences of genetic manipulation. In addition, we demonstrate that two viruses could be co-injected to produce expression patterns that are biased towards distinct or overlapping sets of neurons, depending on the serotypes chosen for viral packaging. Our work expands the versatility of this technique for use in a broad range of experimental neuroscience settings.

<table><tbody><tr><td>ICR outbred mice</td><td>Harlan</td><td>Hsd:ICR (CD-1)</td><td>This strain is also known as CD-1</td></tr><tr><td>FVB inbred mice</td><td>The Jackson Laboratory</td><td>1800</td><td>5-6 weeks of age</td></tr><tr><td>Nestlets</td><td>Lab Supply</td><td>NESTLETS</td><td></td></tr><tr><td>Shepherd shacks</td><td>Lab Supply</td><td>SS-mouse</td><td></td></tr><tr><td>High fat rodent chow</td><td>Purina Mills</td><td>PicoLab Mouse diet 20, #5058</td><td>This is our standard breeder chow</td></tr><tr><td>High fat rodent chow (alternative)</td><td>Harlan Laboratories</td><td>Teklad Global 19% protein rodent diet #2019S</td><td>If low phytoestrogen, autoclavable diet is needed</td></tr><tr><td>Injection syringe</td><td>Hamilton</td><td>7653-01</td><td>10 ml syringe</td></tr><tr><td>Injection needles</td><td>Hamilton</td><td>7803-04, RN 6PK PT4, 0.375&quot;</td><td>32 G, for standard P0 injections</td></tr><tr><td>Metal plate for cryoanesthesia</td><td>McMaster Carr</td><td>8975K439</td><td>Raw aluminum plate, 6&amp;rdquo; x 12&amp;rdquo;, 0.25&amp;rdquo; thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop</td></tr><tr><td>Small animal stereotaxic device with digital readout</td><td>David Kopf Instruments</td><td>Model 940</td><td></td></tr><tr><td>Universal syringe holder with needle support foot</td><td>David Kopf Instruments</td><td>Model 1772-F1</td><td></td></tr><tr><td>Neonatal frame</td><td>Stoelting</td><td>51625</td><td>Officially called a mouse and neonatal rat adaptor</td></tr><tr><td>Biohazard disposal bags with sterile indicator</td><td>VWR</td><td>14220-030</td><td>Important! &amp;ndash; Check with local veterinary and environmental safety staff to learn your institute&amp;rsquo;s protocol for biohazard waste disposal</td></tr></tbody></table>

intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Successful intraventricular viral injection yields widespread and robust neuronal expression. Here we evaluated viral transduction using YFP or tdTomato fluorescent genes under the control of chicken beta actin promoter (CBA promoter). These constructs were packaged into AAV8 and injected into the lateral ventricles of neonatal (P0) ICR mice. High viral titers (1010 particles per hemisphere) resulted in dense labeling of the olfactory bulb, striatum, cerebral cortex, hippocampus and cerebellum (Figure 1A, left). Labeling was also apparent in cerebellar Purkinje neurons, but notably absent from cerebellar granule neurons which are not yet present in large numbers at P0. Injection of fewer viral particles (107) resulted in sparse labeling and lower intensity expression (Figure 1A, right). We commonly use AAV8 within a concentration range between 107 and 1010 particles per hemisphere as an easy and reliable way to control the degree of transgene mosaicism produced by injection (Figure 1B). We have also adapted the technique to express multiple transgenes by co-injecting two or more viruses. Co-injection of two viruses packaged into the same serotype (i.e. both AAV8) biases the resulting transduction to overlapping neuronal populations, so that many cells express both transgenes (Figure 2A). At lower concentrations, the expression patterns become more independent, and the percentage of co-labeled cells is decreased (Figure 2B). Non-overlapping expression can also be achieved by co-injecting different AAV serotypes. Co-injection of AAV1 and AAV8 encoding distinct fluorescent reporters produces a largely independent pattern of dual-mosaicism (Figure 2C).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51863/51863fig1highres.jpg" width="600" /> 
Figure 1. Viral titer can be used to control the density of viral transduction. (A) A broad range of transgene mosaicism can be achieved by diluting the viral solution from 1010 down to 107 particles/hemisphere. Representative images of sagittal sections show two distinct transduction patterns based on viral titer. Images were taken from mice harvested 3 weeks after injection with AAV8-YFP to deliver 2.0 x 1010 (left) or 5.0 x 107 (right) particles/hemisphere. (B) Higher magnification images of cortex (upper row) and cerebellum (lower row) show the spectrum of transgene mosaicism achieved by serial dilution of AAV8. Transduction is visualized by native YFP fluorescence. Exposure times for whole brain images were determined by the cortex and hippocampus, which fluoresce most brightly, while exposure times for the magnified panels were adjusted to show patterns within each region. <a href="https://www.jove.com/files/ftp_upload/51863/51863fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51863/51863fig2highres.jpg" width="600" /> 
Figure 2. Titer and serotype independently control the degree of viral co-expression. Representative images show the transduction pattern in cerebral cortex (A, B) and cerebellum (C) 3 weeks after viral injection. (A) Co-injection of two AAV8 viruses containing different fluorescent reporters (tdTomato or YFP) produced extensive transduction throughout the brain. The majority of transduced cells expressed both transgenes. (B) Co-injection of the same viruses at lower titers produced sparse expression of each protein, in which fewer cells were transduced by both viruses. (C) Co-injection of AAV packaged into different serotypes (AAV8 and AAV1), even at moderately high titers, produced a largely non-overlapping pattern of viral expression. The pattern of each fluorescent protein was nearly identical to its expression when injected alone, indicating that transduction of one AAV is independent of the other. TdTomato fluorescence shown in red, YFP in green. Transduction is visualized by native fluorescence. <a href="https://www.jove.com/files/ftp_upload/51863/51863fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction at JoVE.com

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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Universidad Científica del Sur

Research

JoVE Journal

Neuroscience

59.0K Views.  Baylor College of Medicine. Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Video: Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

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 ...

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Immunology and Infection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Biology

A Comprehensive Protocol for Delivering Intrathecal Injections in Neonatal Mice

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

Author Spotlight: Intrathecal Injection &#8211; An Efficient and Reliable Delivery Method to Test the Efficacy of Gene Editing in Neonatal Mouse Brains

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

Widespread Transduction of Mouse Neocortical Neurons by Subarachnoid Injection of AAV2

Recombinant adeno-associated viruses are a flexible and powerful tool for the delivery and expression of various genes of interest in many areas of experimental biology, particularly in neuroscience. The most popular method to drive the expression of a desired transgene in a particular brain area is to inject an AAV vector directly into the brain parenchyma. However, this method does not allow widespread neuronal transduction that is required for some in vivo experiments. In this article, we present a new technique for widespread gene expression in the mouse neocortex based on viral infusion into the subarachnoid space of the brain. This neuronal labeling method not only ensures widespread transduction of neurons in adult mouse superficial neocortical layers but also results in expression of the transgene in a large population of layer five pyramidal neurons with high specificity even when using a strong non-selective promoter such as CAG. Moreover, because cell transduction takes place at a significant distance from the injection site, this method can help preserve brain tissue for subsequent optical or electrophysiological recordings of neuronal activity.

A new technique for widespread delivery of adeno-associated virus that uses subarachnoid virus infusion is described. This method not only ensures widespread transduction of mouse neocortical neurons in superficial layers but also results in selective expression of the transgene in layer five pyramidal neurons, even when using a non-selective promoter.

Recombinant adeno-associated viruses are a flexible and powerful tool for the delivery and expression of various genes of interest in many areas of ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Summary

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

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

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