We thank Bakhos A. Tannous, Ph.D., Director of Vector Development and Production in the Neuroscience Center of Massachusetts General Hospital, Charlestown, Massachusetts, for providing us with the rAAV-eGfp vector, and John Whang for technical assistance. This work was supported by grant R01 NS050408 (to J.S.) from the National Institute of Neurological Disorders and Stroke.

The surgical procedure described has been approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
1. Preparation of Equipment and Virus Particle Suspension
<ol>
	<li>Clean and disinfect the equipment, sterilize the surgical instruments and the V notch spikes that will be used to fix vertebra L1.</li>
	<li>Pull and bevel glass pipettes. We use pipettes that have a tip diameter of 40 &mu;m and are beveled at an angle of 20&deg;. Sterilize the glass pipettes.</li>
	<li>Set up the stereotaxic frame, mount the microsyringe injector onto the manipulator and connect the injector to the controller.</li>
	<li>Attach one of the glass pipettes to the microsyringe using the compression fitting kit.</li>
	<li>Remove the plunger from the microsyringe and fill the syringe with mineral oil. Oil Red O (1-(2,5-dimethyl-4-(2,5-dimethylphenylazo)phenylazo)-2-naphthol) may be added to the mineral oil to increase its visibility. Reinsert the plunger and push it to all the way to the tip. Carefully avoid creating an air bubble.</li>
	<li>Prepare the virus particle suspension in a biosafety cabinet. Thaw the frozen virus on ice and, just before use, dilute it with sterile phosphate-buffered saline to the desired particle concentration.</li>
	<li>Insert the microsyringe into the holder on the injector.</li>
	<li>Pipette 5 &mu;l of the virus suspension onto a small plastic sheet, e.g. Parafilm. Lower the glass pipette tip into the drop and pull the plunger to fill the microsyringe. Create a small air bubble at the pipette tip to prevent it from clogging.</li>
</ol>
2. Laminectomy
<ol>
	<li>Prepare the surgery area by wiping bench and heating pad with disinfectant.</li>
	<li>Anesthetize the mouse. We use inhalation anesthesia with isoflurane (3% during induction, 2%-3% during maintenance).</li>
	<li>Place lubricant on each eye to protect the eyes from drying during the operation.</li>
	<li>Shave the fur from the lower back to the neck of the mouse and disinfect the skin with alternating wipes of a topical antiseptic such as chlorhexidine or povidone-iodine and 70% ethanol. Isolate the aseptically prepared site with surgical drape and infiltrate the incision site with bupivacaine (0.25%, diluted 1:10 with physiological saline).</li>
	<li>Incise the skin at the caudal end of the rib cage along the midline (2-3 cm) and separate the fascia covering the spine.</li>
	<li>Because the spinal cord stops growing earlier during postnatal development than the vertebral column, spinal segment L4 lies underneath the first lumbar vertebra (L1). Vertebra L1 is located caudal to the vertebra that holds the last pair of ribs. Identify and expose vertebra L1 by removing the small spinal muscles and ligaments attached to its dorsal surface.</li>
	<li>Slightly lift and hold vertebra L1 with an Adson forceps. Use a dedicated laminectomy forceps to remove the dorsal portion of the vertebra (spine and lamina) and expose the spinal cord. Avoid damaging the spinal cord.</li>
	<li>Transfer the mouse onto the heating plate in the stereotaxic frame. Monitor the temperature of the mouse during the subsequent operation.</li>
</ol>
3. Injection
<ol>
	<li>Fix vertebra L1 with V notch spikes. The spikes must stabilize the spine so that the vertebra does not move during breathing.</li>
	<li>Bring the microsyringe closer so that the pipette tip is above the laminectomy site. Lower the plunger until you see the virus suspension exiting the pipette. Remove the droplet with a sterile cotton tip.</li>
	<li>Position the pipette tip at the rostral-most portion of the exposed spinal cord. Center the pipette above the posterior median sulcus, then move the tip 500 &mu;m laterally. Lower the tip to the surface of the cord and puncture the dura mater or, if you are working with an unbeveled glass pipette, use a beveled steel cannula to puncture the dura. Lower the tip of the glass pipette 300 &mu;m into the spinal cord.</li>
	<li>Inject 1 &mu;l of virus suspension at a rate of 200 nl/min.</li>
	<li>At the end of the injection, wait at least 2 min before slowly retracting the pipette.</li>
	<li>Repeat steps 3.3 to 3.5 at the caudal-most portion of the exposed spinal cord to achieve complete distribution of the viral vector in spinal segment L4. The two injection sites are located rostral and caudal of the L4 segment to avoid tissue damage in the target region.</li>
</ol>
4. Wound Closure
<ol>
	<li>Release vertebra L1 from the V notch clamps and remove the mouse from the stereotaxic frame.</li>
	<li>Suture the fascia with 5.0 vicryl. The closed fascia provides cover for the laminectomy site.</li>
	<li>Close the skin with nylon sutures or surgical staples.</li>
</ol>
5. Postoperative Care
<ol>
	<li>Transfer the mouse to a recovery cage with soft, nonparticular bedding. Place it on the side for comfortable breathing. Monitor the animal until it is fully alert, ambulant and starts drinking.</li>
	<li>We provide postsurgical analgesia for 72 hr with daily subcutaneous injections of carprofen (5.0 mg/kg).</li>
	<li>Monitor postsurgical recovery by daily inspection for the first 3 days, then every other day or 3 days per week until the experiment is complete.</li>
	<li>Remove the skin sutures or staples 7 to 10 days after the operation, when wound healing is complete.</li>
	<li>Euthanize the animal after completion of the experiment.</li>
</ol>

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The authors declare that they have no competing financial interests.

Stereotaxic vector injection allows targeting spinal cord neurons for applications such as neuronal network mapping based on transsynaptic virus spreading 6,7 or optogenetic dissection 8, axon guidance during regeneration from injury 9,10, or gene therapy for the prevention or treatment of neurodegeneration 11,12. Viral vectors have been used for gene manipulation in the spinal cord to study somatosensory, motor and autonomic pathways 9,10,13-15. The mouse is the mod...

Advanced technologies of conditional gene manipulation in the mouse enable multifaceted approaches to the exploration of synaptic pathways and functional connections in the central nervous system. Transgenes may be regulated by small-molecule effectors such as doxycycline acting on a tetracycline-controlled transactivator, which can be designed to function as a repressor or an activator of gene transcription, or tamoxifen recognizing a mutated ligand-binding domain of the estrogen receptor 1. Irreversible transgene modification is commonly achieved by deoxyribonucleic acid (DNA) recombinases. Cre (causes recombination) and Flp (flippase recombination enzyme) catalyze the excision, inversion or translocation of DNA fragments that are flanked by loxP (locus of crossing x over, P1) or Frt (flippase recognition target) sites, respectively 1. Applications include gene activation or silencing and inducible ribonucleic acid (RNA) interference 2. Conditional expression of fluorescent or enzymatic reporters such as &beta;-galactosidase or alkaline phosphatase can be used to label neurons and examine their topical organization and connectivity 3. Large-scale mutagenesis projects in North America (<a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a>) and Europe (<a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a>) are producing libraries of mouse embryonic stem cell clones with conditional gene targets and traps that will eventually cover the entire mouse genome. Mice generated from these clones may be crossed with an expanding number of mouse lines that express DNA recombinases under promoters or loci specific to a particular population of neurons for selective gene manipulation (<a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index.php</a>).
However, restricting gene manipulation to distinct populations of neurons or particular regions of interest may not be achieved by genetic targeting alone if a promoter specific to the neuron population of interest is not known or not expressed by all neurons in the region of interest. In the spinal cord, experimental designs may require spatial restriction of the gene manipulation to one or two craniocaudal segments. Stereotaxic injection of a viral vector that expresses Cre or Flp allows limiting gene recombination to regions in the spinal cord of mice in which DNA fragments are flanked by loxP or Frt sites, so-called floxed or flrted alleles. Unlike constitutive DNA rearrangement, which would result from crossbreeding the animals with recombinase expressing mice, this strategy also provides temporal control over gene activation or silencing. Viral vectors encoding floxed or flirted transgenes offer a reverse option of gene manipulation in mice expressing the corresponding recombinase downstream of a neuron-specific promoter. Several recombinant vectors with affinity to neurons are available 4. High-capacity (gutless) adenovirus, adeno-associated virus, herpes simplex virus and lentivirus are commonly used neurotropic vectors. Selecting the appropriate virus for a research question is a crucial part of the experimental design. Size of the transgene, delivery route, specificity of the infection to neurons as opposed to glial cells, infection efficacy, inflammatory and toxic side effects need to be considered 4.
Here we describe the stereotaxic injection of a viral vector into the dorsal horn of the spinal cord, a technique that we employ for conditional gene regulation in our research on the neurobiology of pain. The dorsal horn receives afferent input from primary somatosensory neurons including nociceptive neurons. Local interneurons process the information before projection neurons convey it from the dorsal horn to the brain 5. We demonstrate the infection of dorsal horn neurons at spinal segmental level L4 with a neurotropic recombinant adeno-associated virus (rAAV) that expresses enhanced green fluorescent protein (eGfp) under a constitutively active cytomegalovirus promoter.

<table border="1">
	<tbody>
 	<tr>
 	<td>Material Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td></td>
 	 </tr>
 <tr>
 		<td>Spinal base plate</td>
 <td>David Kopf Instruments</td>
 <td>912</td>
 <td></td>
 </tr>
 <tr>
 		<td>Small animal stereotaxic instrument</td>
 <td>David Kopf Instruments</td>
 <td>900</td>
 <td></td>
 </tr>
 <tr>
 		<td>Mouse gas anesthesia head holder</td>
 <td>David Kopf Instruments</td>
 <td>923-B</td>
 <td></td>
 </tr>
 <tr>
 	<td>Adjustable base mounts</td>
 <td>David Kopf Instruments</td>
 <td>982</td>
 <td></td>
 </tr>
 <tr>
 	<td>V notch spikes</td>
 <td>David Kopf Instruments</td>
 <td>987</td>
 <td></td>
 </tr>
 <tr>
 	<td>Small animal temperature control system</td>
 <td>David Kopf Instruments</td>
 <td>TCAT-2LV</td>
 <td></td>
 </tr>
 <tr>
 	<td>Adson forceps</td>
 <td>Fine Science Tools</td>
 <td>11006-12</td>
 <td></td>
 </tr>
 <tr>
 	<td>Laminectomy forceps</td>
 <td>Fine Science Tools</td>
 <td>11223-20</td>
 <td></td>
 </tr>
 <tr>
 	<td>UltraMicroPump (one) with SYS-Micro4 Controller</td>
 <td>World Precision Instruments</td>
 <td>UMP3-1</td>
 <td></td>
 </tr>
 <tr>
 	<td>Microsyringe, 65RN</td>
 <td>Hamilton</td>
 <td>7633-01</td>
 <td></td>
 </tr>
 <tr>
 	<td>RN compression fitting, 1 mm</td>
 <td>Hamilton</td>
 <td>55750-01</td>
 <td></td>
 </tr>
 <tr>
 	<td>Borosilicate glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td></td>
 </tr>
 <tr>
 	<td>Microgrinder</td>
 <td>Narishige</td>
 <td>EG-44</td>
 <td></td>
 </tr>
 </tbody>
</table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Successful transfection yields robust gene expression in neurons of the injected dorsal horn (Figure 1), sparing the dorsal horn of the contralateral side, the ventral horn and the dorsal root ganglia.
<img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figure 1" /> 
Figure 1. Transfection of dorsal horn neurons. (A) Expression of the fluorescent reporter eGfp (green) in the left dorsal horn of the L4 spinal cord, two weeks after the s...

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Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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Research

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Neuroscience

30.2K Views.  Centre National de la Recherche Scientifique (CNRS). Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Video: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Behavior

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

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.

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 ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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. ...

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, ...

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 Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...