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>

<ol>
	<li>Lewandoski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+control+of+gene+expression+in+the+mouse.">Conditional control of gene expression in the mouse.</a> Nature Reviews Genetics. 2, 743-755 (2001).</li><li>Couto, L. B., High, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector-mediated+RNA+interference.">Viral vector-mediated RNA interference.</a> Curr. Opin. Pharmacol. 10, 534-542 (2010).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nature Reviews Neuroscience. 4, 353-364 (2003).</li><li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11, 823-836 (2010).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 21848-21853 (2010).</li><li>Lo, L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72, 938-950 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8, 745-752 (2011).</li><li>Tang, X. Q., Heron, P., Mashburn, C., Smith, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sensory+axon+regeneration+in+adult+spinal+cord.">Targeting sensory axon regeneration in adult spinal cord.</a> J. Neurosci. 27, 6068-6078 (2007).</li><li>Cameron, A. A., Smith, G. M., Randall, D. C., Brown, D. R., Rabchevsky, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+intraspinal+plasticity+after+spinal+cord+injury+alters+the+severity+of+autonomic+dysreflexia.">Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.</a> J. Neurosci. 26, 2923-2932 (2006).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> The Journal of Clinical Investigation. 120, 1253-1264 (2010).</li><li>Lutz, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsymptomatic+restoration+of+SMN+rescues+the+disease+phenotype+in+a+mouse+model+of+severe+spinal+muscular+atrophy.">Postsymptomatic restoration of SMN rescues the disease phenotype in a mouse model of severe spinal muscular atrophy.</a> The Journal of Clinical Investigation. 121, 3029-3041 (2011).</li><li>Chen, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsAAV+type+2-mediated+gene+transfer+of+MORS196A-EGFP+into+spinal+cord+as+a+pain+management+paradigm.">dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 20096-20101 (2007).</li><li>South, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+deletion+of+the+NR1+subunit+of+the+NMDA+receptor+in+adult+spinal+cord+dorsal+horn+reduces+NMDA+currents+and+injury-induced+pain.">A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.</a> J. Neurosci. 23, 5031-5040 (2003).</li><li>Tappe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+scaffolding+protein+Homer1a+protects+against+chronic+inflammatory+pain.">Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.</a> Nat. Med. 12, 677-681 (2006).</li><li>Colle, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+intracerebral+delivery+of+AAV5+vector+encoding+human+ARSA+in+non-human+primate.">Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.</a> Human Molecular Genetics. 19, 147-158 (2010).</li><li>Carbajal, K. S., Weinger, J. G., Whitman, L. M., Schaumburg, C. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Transplantation+of+Mouse+Neural+Stem+Cells+into+the+Spinal+Cords+of+Mice+Infected+with+Neurotropic+Mouse+Hepatitis+Virus.">Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus.</a> J. Vis. Exp. (53), e2834 (2011).</li><li>Snyder, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+adeno-associated+viral+vector+serotypes+for+spinal+cord+and+motor+neuron+gene+delivery.">Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.</a> Hum. Gene Ther. 22, 1129-1135 (2011).</li><li>Towne, C., Pertin, M., Beggah, A. T., Aebischer, P., Decosterd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+serotype+6+(rAAV2/6)-mediated+gene+transfer+to+nociceptive+neurons+through+different+routes+of+delivery.">Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.</a> Mol. Pain. 5, 52 (2009).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian.</a> 8, 148-154 (1994).</li></ol>

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...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

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 ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.2K vistas.  Centre National de la Recherche Scientifique (CNRS). Los vectores virales permiten la manipulación dirigida de genes. Se demuestra un método para la expresión génica condicional o ablación en la médula espinal de ratón, usando inyección estereotáxica de un vector viral en el asta dorsal, un sitio prominente de contacto sináptico entre las vías aferentes somatosensoriales y las neuronas del sistema nervioso central.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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 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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...