We would like to thank Ionis Pharmaceuticals for supplying the ASOs described in the article.

All in vivo procedures were performed under Biogen Institutional Animal Use and Care Committee (IACUC) approved protocols which follow the guidelines set forth by the United States National Institutes of Health guide for the care and use of laboratory animals.
1. Material and instrument preparation
<ol>
	<li>Prepare the special guide cannulas.
	<ol>
		<li>Use a rotary tool with cut-off wheel (or a sharp saw) to cut off the two ends of a 19 G needle, resulting in a ~1.5&#8722;2 cm long guide cannula (Figure 1Aiii). Use the grinding wheel of the rotary tool to smooth the two ends. 
		NOTE: Alternatively, premade and sterile guide cannulas can be purchased from a commercial vendor (Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the catheter/wire assembly.
	<ol>
		<li>Cut an 8 cm long piece of PE-10 tubing (polyethylene tubing, diameter 0.011 inch) to serve as the intrathecal catheter. Make a mark 2 cm from one end with an ethanol resistant marker pen. Cut an 11 cm long stylet wire from polytetrafluoroethylene coated stainless steel wire. Insert the stylet wire (Figure 1Aii) into the lumen of the PE-10 catheter (Figure 1Ai). 
		NOTE: One catheter/wire assembly set (Figure 1Av) is needed for each animal. Alternatively, catheters and stylet wires can be purchased from a commercial vendor (Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare delivery catheter assembly.
	<ol>
		<li>Cut a piece of PE-50 catheter (5&#8722;10 cm, polyethylene tubing, diameter 0.023 inch) (Figure 1Bi). To one end of the PE50 catheter insert a 23 G tubing adaptor (Figure 1Biv). This end will be connected to a 100 &#181;L syringe (Table of Materials) during surgery. Insert a modified 30 G needle with hub cut off (Figure 1Biii) into an approximately 0.5 cm piece of PE-10 tubing (Figure 1Bii) and connect the PE-10 tubing to the other end of the catheter.</li>
		<li>The 30 G needle end of the delivery catheter assembly (Figure 1Bv) will be connected to the distal end of the implanted PE-10 catheter in the rat during surgery. 
		NOTE: Alternatively, the delivery catheter assembly (Figure 1Bv) can be purchased from a commercial vendor (Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the guide cannula-needle assembly (Figure 1Avi) by placing a guide cannula (Figure 1Aiii) over the end of a 23 G needle. </li>
	<li>Sterilize all surgical instruments including the guide cannula and the wire/catheter sets using an ethylene oxide sterilizer for 12 h. 
	NOTE: All surgical instruments except the catheter can be autoclaved; catheter will melt at high temperature.</li>
</ol>
2. Surgery preparation
NOTE: This procedure is routinely performed on male and female Sprague Dawley rats with body weights between 200 g and 400 g. Two rats are housed per cage under a 12 h light/dark cycle with free access to food and water.
<ol>
	<li>Weight a rat and place it in an isoflurane chamber to induce anesthesia (1&#8722;5% isoflurane in O2, titrated to effect). 
	NOTE: The rat is then continuously anesthetized with isoflurane to maintain deep anesthesia via a nose cone throughout the procedure. An alternative anesthesia method (e.g., administration of ketamine 100 mg/kg and xylazine 10 mg/kg) can be used as approved by the IACUC.</li>
	<li>When the rat fails to respond to a toe pinch, shave its back from the tail to the caudal thoracic spine and place the shaved rat on the a sterile sheet laid on top of a heating pad.</li>
	<li>Position a 50 ml conical centrifuge tube under the abdomen of the rat to flex the spine in the lumbar region (Figure 2A) and apply ophthalmic ointment to the eyes.</li>
	<li>Inject sustained release buprenorphine (1.0 mg/kg; Table of Materials) subcutaneously in the rat. Clean the exposed skin with alternating povidone and alcohol scrubs and repeat this three times. 
	NOTE: An alternative pain relief can be used as approved by the IACUC protocol.</li>
	<li>Drape the animal with a sterile transparent drape that has been fenestrated over the surgical site.</li>
</ol>
3. Surgery
<ol>
	<li>With the rat supported by the 50 mL conical tube, identify the two natural pits between muscles above the pelvis (arrows in Figure 2A). With one hand holding those pits, use the other hand to gently press and feel the spine from caudal to rostral direction and find the first major indentation between vertebrae and this is the intervertebral space between S1 and L6 vertebrae (Figure 2B).</li>
	<li>Move slightly rostrally to identify the next indentation, the intervertebral space between the L5 and L6 vertebrae and the injection site (* in Figure 2A). Use a scalpel to make an incision no more than 2 cm long in the skin along the midline from rostral to caudal so that the injection site is at the center of the incision (dotted line in Figure 2A).</li>
	<li>Use dissection scissors to dissect away the connective tissue in order to visualize the muscle layer. Then make a 1 cm incision in the muscle capsule immediately lateral to the dorsal spinal process of the L6 lumbar vertebra. 
	NOTE: The bones of the L6 lumbar vertebra could be visualized at this point.</li>
	<li>Position the guide cannula-needle assembly near the anterior aspect of the 6th lumbar vertebra and push it into the intervertebral space along the anterior aspect of the 6th vertebra so that the end of the needle penetrates the spinal canal. Push the guide cannula in place along the needle and remove the 23 G needle leaving only the guide cannula in place. 
	NOTE: It is helpful to use blunt forceps to locate the dorsal process of the L6 lumbar vertebra before inserting the needle. Generally, CSF fluid can be seen entering the needle hub (this fluid may be tinged with a hint of blood, but this does not indicate that harm has been done or that the needle is not placed correctly). The authors have not seen large amount of blood or severe bleeding during this procedure. If either occurs, a veterinarian should be contacted to determine the appropriate treatment and if animals should be euthanized.</li>
	<li>Insert the catheter-wire assembly into the guide cannula. Angle down the catheter-wire assembly at approximately a 45&#176; angle to the spinal canal and force the end approximately 0.3 cm into the spinal canal.</li>
	<li>Remove the guide cannula, leaving the catheter with the stylet wire in place. Remove the stylet wire approximately 2.5 cm from the intrathecal tip of the catheter and advance the catheter into the spinal canal until the 2 cm mark is at the entrance of the canal (just visible below the muscle) as shown in Figure 1Bvi. 
	NOTE: The inserted catheter should extend rostrally into the subarachnoid space. Successful placement should allow for free movement of the catheter in that space.</li>
	<li>Completely withdraw the stylet wire, and CSF may be seen entering the implanted catheter.</li>
	<li>Connect the delivery catheter assembly to the distal end of the implanted catheter via the 30 G needle end (Figure 1Bv,vi).</li>
	<li>Load 60 &#181;L of sterile saline into a 100 &#181;L syringe (flushing syringe). Load a bolus of 30 &#181;L of the test compound (e.g., ASO solution) into a second syringe (injection syringe).</li>
	<li>Connect the flushing syringe (loaded with saline) to the tubing adaptor end of the delivery catheter assembly (Figure 1Bv). Inject 20 &#181;L of sterile saline into the intrathecal space (pre-injection flushing).</li>
	<li>Connect the injection syringe (loaded with test compound) to the tubing adaptor end of the delivery catheter assembly (Figure 1Bv). Inject 30 &#181;L of test compound into the intrathecal space over 30 s. 
	NOTE: The routine injection volume of ASO is 30 &#181;L to achieve good knockdown in the spinal cord and in the cortex. It has been reported that the injection volumes may impact compound distribution12, though different injection volumes have not been tested. If other compound or volume is used, the safety and effectiveness need to be empirically determined.</li>
	<li>Repeat step 3.10 and flush the catheter with another 40 &#181;L of sterile saline (post-injection flushing). Then detach the delivery catheter assembly from the implanted catheter. 
	NOTE: The pre and post-injection flush is thought to reduce local sequestration of the compounds and improve their distribution to the rostral structures12.</li>
	<li>Aseptically cut and heat seal the implanted catheter: Place a pair of sterile dissection forceps in a bead sterilizer until they are very hot, then clamp down on the tubing with the hot end of the forceps. 
	NOTE: This action melts the catheter. Thus, the hole in the tube is collapsed and all sides stick to each other, sealing the tubing in an aseptic fashion and then it is placed into the subcutaneous space.</li>
	<li>Use absorbable monofilament sutures to secure the remaining heat-sealed catheter to the connective tissue. Then use non-absorbable monofilament sutures to close the skin over the secured heat-sealed catheter. 
	NOTE: Wound clips can also be used as approved by the IACUC protocol. The technique is compatible with repeated injections though it has only been used for one-time injections in our hands. The feasibility of repeated injections should be empirically evaluated with the approval of the IACUC.</li>
	<li>Use gauze and saline to wash any blood from the skin and allow the animal to recover from the anesthesia in a heated incubator until mobile, at which point it is returned to its home cage (two rats per cage). 
	NOTE: When performing surgeries on multiple rats on the same day, clean tools using water to remove blood and re-sterilize using a heated dry bead sterilizer (for at least 20 s, with time to cool) between animals. A new set of instruments is used every 5 animals.</li>
	<li>Monitor the animals daily for at least 3 days after surgery and continue to monitor the animals weekly after recovering from surgery according to the IACUC protocol. 
	NOTE: If any complications occur (urine retention, incision infections, neurological disorder such as seizure or paralysis), a veterinarian should be contacted to determine the appropriate treatment and if animals should be euthanized. If sustained release buprenorphine is not used, pain relief should be given daily after surgery according to the IACUC protocol.</li>
</ol>
4. Evaluation of tissue specific knockdown after IT injection
<ol>
	<li>Two weeks after IT bolus injection of ASOs, collect different regions of the brain (i.e., cerebral cortex, striatum and cerebellum) as well as different segments of the spinal cord (i.e., cervical, thoracic, and lumbar). Extract total tissue RNA using a commercial RNA extraction kit and perform cDNA synthesis reaction as described previously13. 
	NOTE: Standard reagents were used for qPCR with the following assays: rat Malat1 and rat GAPDH. The relative transcript levels were calculated using the 2-&#916;&#916;CT method (CT = threshold cycle).</li>
</ol>

<ol>
	<li>Abbott, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+CNS+barriers:+evolution,+differentiation,+and+modulation.">Dynamics of CNS barriers: evolution, differentiation, and modulation.</a> Cellular and Molecular Neurobiology. 25 (1), 5-23 (2005).</li><li>Greene, C., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+modulation+of+the+blood+brain+barrier:+CNS+delivery+of+small+molecules.">Tight junction modulation of the blood brain barrier: CNS delivery of small molecules.</a> Tissue Barriers. 4 (1), e1138017 (2016).</li><li>Daneman, R., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers+in+health+and+disease.">Brain barriers in health and disease.</a> Neurobiology of Disease. 107, 1-3 (2017).</li><li>Ballabh, P., Braun, A., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+an+overview:+structure,+regulation,+and+clinical+implications.">The blood-brain barrier: an overview: structure, regulation, and clinical implications.</a> Neurobiology of Disease. 16 (1), 1-13 (2004).</li><li>Cardoso, F. L., Brites, D., Brito, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+the+blood-brain+barrier:+molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches.</a> Brain Research Reviews. 64 (2), 328-363 (2010).</li><li>Larsen, J. M., Martin, D. R., Byrne, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+delivery+through+the+blood-brain+barrier.">Recent advances in delivery through the blood-brain barrier.</a> Current Topics in Medicinal Chemistry. 14 (9), 1148-1160 (2014).</li><li>Brinker, T., Stopa, E., Morrison, J., Klinge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+cerebrospinal+fluid+circulation.">A new look at cerebrospinal fluid circulation.</a> Fluids Barriers CNS. 11, 10 (2014).</li><li>Standifer, K. M., Chien, C. C., Wahlestedt, C., Brown, G. P., Pasternak, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+delta+opioid+analgesia+and+binding+by+antisense+oligodeoxynucleotides+to+a+delta+opioid+receptor.">Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor.</a> Neuron. 12 (4), 805-810 (1994).</li><li>Wahlestedt, C., 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=Antisense+oligodeoxynucleotides+to+NMDA-R1+receptor+channel+protect+cortical+neurons+from+excitotoxicity+and+reduce+focal+ischaemic+infarctions.">Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.</a> Nature. 363 (6426), 260-263 (1993).</li><li>Wahlestedt, C., Pich, E. M., Koob, G. F., Yee, F., Heilig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+anxiety+and+neuropeptide+Y-Y1+receptors+by+antisense+oligodeoxynucleotides.">Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides.</a> Science. 259 (5094), 528-531 (1993).</li><li>Mazur, C., 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=Development+of+a+simple,+rapid,+and+robust+intrathecal+catheterization+method+in+the+rat.">Development of a simple, rapid, and robust intrathecal catheterization method in the rat.</a> Journal of Neuroscience Methods. 280, 36-46 (2017).</li><li>Wolf, D. 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=Dynamic+dual-isotope+molecular+imaging+elucidates+principles+for+optimizing+intrathecal+drug+delivery.">Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery.</a> Journal of Clinical Investigation Insight. 1 (2), e85311 (2016).</li><li>Becker, L. 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=Therapeutic+reduction+of+ataxin-2+extends+lifespan+and+reduces+pathology+in+TDP-43+mice.">Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.</a> Nature. 544 (7650), 367-371 (2017).</li><li>Zhang, X., Hamblin, M. H., Yin, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+noncoding+RNA+Malat1:+Its+physiological+and+pathophysiological+functions.">The long noncoding RNA Malat1: Its physiological and pathophysiological functions.</a> RNA Biology. 14 (12), 1705-1714 (2017).</li><li>Crooke, S. T., Witztum, J. L., Bennett, C. F., Baker, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Targeted+Therapeutics.">RNA-Targeted Therapeutics.</a> Cell Metabolism. 27 (4), 714-739 (2018).</li><li>DeVos, S. L., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intraventricular+delivery+of+drugs+to+the+rodent+central+nervous+system.">Direct intraventricular delivery of drugs to the rodent central nervous system.</a> Journal of Visualized Experiments. (75), e50326 (2013).</li><li>McCampbell, 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=Antisense+oligonucleotides+extend+survival+and+reverse+decrement+in+muscle+response+in+ALS+models.">Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.</a> Journal of Clinical Investigation. 128 (8), 3558-3567 (2018).</li><li>Schoch, K. M., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+Oligonucleotides:+Translation+from+Mouse+Models+to+Human+Neurodegenerative+Diseases.">Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases.</a> Neuron. 94 (6), 1056-1070 (2017).</li><li>Lane, R. 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=Translating+Antisense+Technology+into+a+Treatment+for+Huntington's+Disease.">Translating Antisense Technology into a Treatment for Huntington's Disease.</a> Methods in Molecular Biology. 1780, 497-523 (2018).</li><li>Wurster, C. D., Ludolph, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotides+in+neurological+disorders.">Antisense oligonucleotides in neurological disorders.</a> Therapeutic Advances in Neurological Disorders. 11, (2018).</li><li>Hach&#233;, 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=Intrathecal+Injections+in+Children+With+Spinal+Muscular+Atrophy:+Nusinersen+Clinical+Trial+Experience.">Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience.</a> Journal of Child Neurology. 31 (7), 899-906 (2016).</li><li>Goodkey, K., Aslesh, T., Maruyama, R., Yokota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nusinersen+in+the+Treatment+of+Spinal+Muscular+Atrophy.">Nusinersen in the Treatment of Spinal Muscular Atrophy.</a> Methods in Molecular Biology. 1828, 69-76 (2018).</li><li>Wurster, C. 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The authors are all employees of Biogen, Inc. or Ionis Pharmaceuticals. The authors receive the antisense oligonucleotides described in the article from Ionis Pharmaceuticals.

The present article shows a powerful method to deliver therapeutic agents directly into the rat CNS. In theory, a similar technique can be also performed in mice, though due to the smaller size, the method can be more challenging. Therefore, our group performs intracerebroventricular (ICV) injections in mice for CNS drug delivery, which reach the same goals through a different route of administration. That method has been described in another study16.
The advantage of t...

The vascular system of the central nervous system (CNS) has evolved as a critical regulator of homeostasis, controlling traffic of molecules, supplying nutrients and getting rid of waste. This system is also the first line of defense from attacks of external pathogens, thanks to a dense distribution of tight junctions along the walls of the endothelial cells. These tight junctions make up one aspect of the blood brain barrier (BBB). While the BBB allows the transport of molecules required to fulfill nutrient and energy demands (e.g., ions, glucose), it also selectively limits the passage of pathogens as well as toxic chemicals1,2,3.
Ironically, the same protective function of the BBB that limits passage of pathogens and toxic chemicals also is the major obstacle to our ability to easily access the CNS with therapeutic treatments after systemic administration to the organism2,4,5. This role of the BBB has prompted the development of a plethora of new drug distribution technologies and approaches6.
One way to overcome this obstacle is to inject the drugs directly into the cerebrospinal fluid (CSF) that continuously perfuses both the brain and spinal cord7,8,9,10. In this article, we describe a method to successfully deliver agents into the lumbar intrathecal space by placing the internal end of the catheter completely in the cauda equina space of the rat spine. A description of this procedure was previously published by Mazur et al. elsewhere11.
The protocol is very effective and produces a greater than 90% success rate of antisense oligonucleotide (ASO) delivery to the CNS when assessed by quantitative polymerase chain reaction (qPCR) analysis of target gene knockdown8. The procedure causes minimal discomfort to the animals, as 100% of the rats survive the surgery and show minimal swelling around the surgical wound and no signs of distress (e.g., hyperactivity, dehydration, circling, loss of balance, decreased food intake, and dehydration) during post-op observation. Another advantage of the method described here is that it does not require expensive equipment, nor any special tools.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Steri-Drape Small Drape with Adhesive Aperture</td>
			<td>3M</td>
			<td></td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Laboratories, Inc</td>
			<td></td>
			<td>8416-160Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swab sticks</td>
			<td>Dynarex</td>
			<td></td>
			<td>NO 1204</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://punchout.fishersci.com/shop/products/bd-general-use-syringes-5/22124967?crossRef=BD%20302830#?keyword=BD+302830">BD General Use Syringes 1 mL Luer-Lok tip</a></td>
			<td>BD</td>
			<td>1ml TB Luer-Lok tip</td>
			<td>BD 302830</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE50 Diameter 0.023 in</td>
			<td>BD 427400 (10ft, Fischer Scientific 22-204008) or 427401 (100ft, Fischer Scientific 14-170-12P)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE10 Diameter 0.011 in</td>
			<td>BD 427410 (10ft, Fischer Scientific 14-170-11B) or 4274011 (100ft, Fischer Scientific 14-170-12B)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing Adapters</td>
			<td>BD</td>
			<td>23 gauge intramedic luer stub adaper</td>
			<td>BD 427565 or Fisher Scientific 14-826-19E</td>
			<td>120V 1.2A</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Single-use Needles 30G</td>
			<td>BD</td>
			<td></td>
			<td>BD 305128</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine Sustained Release-lab</td>
			<td>ZooPharm</td>
			<td>Prescription required</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene oxide sterilizer</td>
			<td>Andersen Sterilizer INC.</td>
			<td>AN 74i, gas sterilizer</td>
			<td>AN 74i</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>BD</td>
			<td>19G x 1 WT (1.1 mm x 25mm) needle</td>
			<td>BD 305186</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 100ul</td>
			<td>Hamilton company</td>
			<td>Hamilton syringe 100ul</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>STERILIZER MODELNO FST 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra veterranery product</td>
			<td></td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Pocket Pro Pet Trimmer</td>
			<td>Braintree Scientific</td>
			<td></td>
			<td>CLP-9931 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone scrub</td>
			<td>PDI</td>
			<td></td>
			<td>S48050</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>Sodium Chloride 0.9% Intravenous Infusion BP 50ml</td>
			<td>FE1306G</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>disposable scalpel</td>
			<td>No. 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal heating pad</td>
			<td>K&#38;H Manufacturing</td>
			<td></td>
			<td>Model # 1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>McMaster-Carr</td>
			<td>1749T14</td>
			<td>LH-36233780</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery Towel drape</td>
			<td>Dynarex</td>
			<td></td>
			<td>4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors and forceps</td>
			<td>FST and Fisher Scientific</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td>4-0 or 5-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool to make the Guide cannular</td>
			<td>Grainger</td>
			<td>Rotary tool (Dremel)</td>
			<td>14H446 (Mfr: EZ456)</td>
			<td>1.5&#8221; diameter, Pk5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock cut off Wheel</td>
			<td>1PKX5 (Mfr: 3000-1/24)</td>
			<td>1.5&#8221;, Pk2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Grinding Wheel, Aluminum Oxide</td>
			<td>38EY44 (Mfr: EZ541GR)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock Mandrel</td>
			<td>1PKX8 (Mfr: EZ402-01)</td>
			<td>1.5&#8221; diameter</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Diamond wheel floor Tile</td>
			<td>3DRN4 (Mfr: EZ545)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alternative source for pre-made and sterilized materials for this procedure</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dosing catheter system</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-GCA</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Catheters</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-INC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-STY</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrathecal delivery of antisense oligonucleotides in the rat central nervous system

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Using the method described here, we injected two groups of adult female rats (250&#8722;300 g; n = 10/group) with either a single bolus of phosphate-buffered saline PBS or 300 &#181;g of ASO targeting the long non-coding (linc) RNA Malat1; in our lab we routinely use the Malat1 ASO as a tool compound, because Malat1 is expressed ubiquitously and at high levels in all tissues14, including brain and spinal cord. The Malat1 ASO works via an RNaseH1-mediated mechanism<...

Watch this Scientific Journal Video about Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System at JoVE.com

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

intrathecal-delivery-antisense-oligonucleotides-rat-central-nervous

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JoVE Journal

Neuroscience

18.2K Views.  Biogen, Inc.. Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Video: Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

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Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

In vivo Optogenetic Stimulation of the Rodent Central Nervous System