Name: targeting the corticospinal tract in neonatal rats with a double-viral vector using combined brain and spine surgery
Uploaded: 2021-06-30T13:30:00
Description: Watch this Scientific Journal Video about Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery at JoVE.com

This work was funded by a fellowship grant from Shriners Hospitals for Children SHC-84706.

All of the following surgical and animal care procedures have been approved by the Animal Care and Use Committee of Temple University. The protocol described is a survival surgery, and the animals were eventually euthanized by intraperitoneal injection of 100 mg/kg sodium pentobarbital at the completion of their time points.
1. Pre-surgical preparation
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	<li>Prepare at least two pulled glass needles for viral injection using 3.5 nL glass capillary pipettes; one needle for the DREADD and ...

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Inducible genetic modulation of brain activity with injectable chemogenetic modifiers is a powerful tool in studying the various mechanisms that underlie the recovery from SCI. The accuracy of the targeting for the inducible G-protein-coupled receptors (DREADDs) is further increased when considering that fluorescence tracing validates the anatomical precision in histology. This paper discusses a reliable method for exploring whether or not inhibiting or stimulating select neuronal pathways (with either excitatory or inhi...

While SCI is a relatively rare occurrence in the pediatric population, it is particularly traumatic and causes a permanent disability requiring immense logistical foresight. Furthermore, a higher proportion of pediatric SCIs is classified as cervical and complete compared to the adult population1,2. A hallmark across mammalian species is that neonates recover notably better from SCI than adults, and this offers an opportunity to assess the driving mechanisms for recovery in younger populations3,4,5. Despite this, there are fewer multimodal studies tackling neonate and infant rodent research, partly due to the added difficulty of accurately targeting select populations of neurons in the much tighter anatomical landmarks of younger animals6. This article focuses on the direct injection of highly efficient anterograde and retrograde adeno-associated vectors into the rat spinal cord to modulate major motor pathways with the application of Cre-dependent-DREADDs, expanding the reach of multimodal regeneration studies.
Viral vectors are important biological tools with a breadth of applications, including the introduction of genetic material to substitute for target genes, upregulate growth proteins, and trace the anatomical landscape of the CNS7,8,9. Many of the anatomical details of spinal motor pathways have been studied using classical tracers, i.e., biotinylated dextran amine. While traditional tracers have been instrumental in unearthing neuroanatomy, they are not without their disadvantages: they indiscriminately label pathways even if correctly injected, and studies have found that they are taken up by damaged axons10,11,12. Consequently, this could lead to incorrect interpretations in regeneration studies where severed axons could be mistaken for regenerating fibers.
The following method utilizes the two-viral vector system recently popularized in modulation studies, with two different viral vectors in two separate areas of the same neuron13,14. The first is a vector that locally infects the cell bodies of projection neurons. The other is a retrograde vector being transported from the axon terminals of the projection neurons (Figure 1). The retrograde vector carries Cre recombinase, and the local vector incorporates the &#34;Cre-On&#34; double-floxed sequence in which a fluorescent protein (mCherry) is encoded. The native transgene expressing both hM3Dq and mCherry is inverted relative to the promoter and is flanked by two LoxP sites (Figure 2). Thus, mCherry is only expressed in the doubly transduced projection neurons where Cre recombinase induces a recombination event between the LoxP sites, flipping the orientation of the transgene into the appropriate reading frame and allowing the expression of both the DREADD and the fluorescent protein. Once the viral transgene is in the correct orientation, and when applicable, the DREADDs can transiently induce neuromodulation through a separately injected ligand, i.e., clozapine-N-oxide. The protocol was designed to authenticate inducible neuromodulation research in neonates, wherein DREADDS are injected to modulate the CSTs selectively. The two-viral system acts as an insurance policy, ensuring that every DREADD-positive&#160;cell is traceable under fluorescence with high fidelity to validate the injections.
This method also helps to bridge the gap in neonatal research. Pediatric SCI presents its challenges, and research analyzing regeneration, sprouting, or plasticity should emphasize the differences between neonates and adults3,15,16,17. By optimizing the surgical procedure and performing prior anatomical studies with Nissl staining, the coordinates for both the cranial and spinal injections were validated. The aim was to provide a method for dual injections into a neonatal rat with increased fidelity and survivability.
For the current model, the anterograde vector was injected into the cell bodies of the somatomotor cortex using bregma as reference18,19. In terms of the spinal injections, the retrograde vector was injected into laminae V-VII, where the CST axon terminals reside20,21. There are many fundamental questions underlying how certain lesion models affect younger animals differently, and how the subsequent recovery diverges from an older animal. This study demonstrates a robust means of studying cervical injuries and the recoverability of forelimb function in neonatal rodents. In contrast, the majority of previous studies have addressed recovery locomotion following lumbar or thoracic injuries5,22,23,24. By pairing the double-viral vector with the novel injection technique described here, this protocol helps mitigate certain issues (i.e., survivability) that may plague neonatal rodent investigations. This method is robust, practical, and versatile: slight variations in the technique will allow for targeting different pathways, i.e., ventral CST, dorsal CST, and the ascending dorsal pathways.
For this system, one locally acting virus (e.g., AAV2) is injected in the region of the neuronal cell bodies of interest. A second retrogradely transported virus that controls the expression of the local virus is injected at the axon terminals for that neuronal population. Thus, by definition, only corticospinal neurons are labeled. The retroAAV-Cre virus was chosen with a constitutively active CMV promoter as the shuttle plasmid is used to generate several AAV serotypes for Cre-dependent expression in several cell types. For cortical injections, AAV2 was chosen with the transgene driven by the synapsin-1 promotor to limit any expression to neurons. Because the 2-viral system relies more on the origin and termination of the neuronal population of interest, several different promoters could be used, if they can drive the expression of the genes of interest within the neuronal population of interest. For example, the excitatory neuronal promoter, CamKII, could be substituted for the synapsin-1. In addition to the use of these AAV serotypes, retrograde transport into immature, and to a much lesser extent, adult corticospinal motor neurons can also be achieved using the high retrograde transportable lentivirus (HiRet)25. HiRet lentiviruses use a chimeric Rabies/VSV glycoprotein to target uptake at the synapse for retrograde transport. Combined with a Tet-On promoter, this 2-viral system supports inducible expression in a retrograde-dependent fashion26,27.
Retrograde viruses insert vectors into the synaptic space of a target neuron, allowing it to be taken up by that cell&#39;s axon and transported to the cell body. While lentiviral vectors have previously had tremendous success, providing long-term expression in gene therapy studies, this method pivoted towards adeno-associated viral vectors for a few simple reasons26,28: AAV is more economical, similarly effective, and presents less of a logistical burden, given that it has a lower biosafety level designation29,30,31,32. While AAV2, the most used serotype, demonstrates robust transfection of CST axons, future researchers may note that AAV1 offers some versatility as it labels transynaptically, thus putting forth several possible iterations in future studies33. The final adaptation is to encode the retrograde virus with Cre-recombinase so that multiple anterograde vectors can be introduced simultaneously, thereby reducing unnecessary in-house virus waste and maximizing the likelihood of the DREADDs expressing in the correct orientation.
Ultimately, this protocol demonstrates simultaneous injection into the cortex and cervical spine, specifically targeting the cell bodies and the axon terminals of the corticospinal tract, respectively. High-fidelity transfection is seen in the cerebral cortex and spinal cord. While the protocol described was perfected for Sprague Dawley rats 5 days of age, it is suitable for postnatal days 4-10 with minor adjustments to anesthesia and&#160;stereotactic coordinates.

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			<td>#11 scalpel blades</td>
			<td>Roboz</td>
			<td>RS-9801-11</td>
			<td>For use with the scalpel.</td>
		</tr>
		<tr>
			<td>#10 Scalpel Blades</td>
			<td>Roboz</td>
			<td>RS-9801-10</td>
			<td>For use with the scalpel.</td>
		</tr>
		<tr>
			<td>1 mL Syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td>For anesthetic SC injection and fluid bolus</td>
		</tr>
		<tr>
			<td>4.0 silk suture</td>
			<td>Ethicon</td>
			<td>771-683G</td>
			<td>For skin closure</td>
		</tr>
		<tr>
			<td>4.0 Chromic Catgut Suture</td>
			<td>DemeTECH</td>
			<td>NN374-16</td>
			<td>To re-bind muscle during closing.</td>
		</tr>
		<tr>
			<td>48000 Micropipette Beveler</td>
			<td>World Precision Instruments</td>
			<td>32416</td>
			<td>Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles.</td>
		</tr>
		<tr>
			<td>5% Iodine Solution</td>
			<td>Purdue Products L.P.</td>
			<td>L01020-08</td>
			<td>For use in sterilzation of the surgical site.</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For sterilization of newly prepared glass needles, animal models during surgical preparation</td>
		</tr>
		<tr>
			<td>Ketamine (Ketaset)</td>
			<td>Zoetis</td>
			<td>240048</td>
			<td>For keeping the animal in the correct plane of consciousness during surgery.</td>
		</tr>
		<tr>
			<td>Bead Sterilizer</td>
			<td>CellPoint</td>
			<td>5-1450</td>
			<td>To heat sterilize surgical instruments.</td>
		</tr>
		<tr>
			<td>Digital Scale</td>
			<td>Okaus</td>
			<td>REV.005</td>
			<td>For weighing the animal during surgical preparation.</td>
		</tr>
		<tr>
			<td>Flexible Needle Attachment</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>For cleaning glass needles and loading red oil into glass needles.</td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes</td>
			<td>World Precision Instruments</td>
			<td>4878</td>
			<td>For pulled glass needles - should be designed for nanoliter injectors.</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Roboz</td>
			<td>RS-7231</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Medium Point Curved Forceps</td>
			<td>Roboz</td>
			<td>RS-5136</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Micromanipulator with a Vernier Scale</td>
			<td>Kanetec</td>
			<td>N/A</td>
			<td>For precise targeting during surgery.</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td>For cutting glass whisps off of freshly pulled glass capillary tubes.</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51600</td>
			<td>To hold the neonatal sterotaxic holder in place</td>
		</tr>
		<tr>
			<td colspan="2">Lab Standard with Mouse &#38; Neonates Adaptor</td>
			<td>51615</td>
			<td>For neonatal skull fixation during cranial surgery and spinal injections</td>
		</tr>
		<tr>
			<td>Microscope with Light and Vernier Scale Ocular</td>
			<td>Leitz Wetzlar</td>
			<td>N/A</td>
			<td>Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles.</td>
		</tr>
		<tr>
			<td>MicroSyringe Pump Controller</td>
			<td>World Precision Instruments</td>
			<td>62403</td>
			<td>To control the rate of injection.</td>
		</tr>
		<tr>
			<td>Nanoliter 2000 Pump Head Injector</td>
			<td>World Precision Instruments</td>
			<td>500150</td>
			<td>To load and inject virus in a controlled fashion.</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td>To heat and pull apart glass capillary tubes to form glass needles.</td>
		</tr>
		<tr>
			<td>pAAV-CMV-scCre</td>
			<td>Wu lab&#160;</td>
			<td></td>
			<td>Cre plasmid</td>
		</tr>
		<tr>
			<td>pAAV-hSyn-DIO-hM3Dq-mCherry (plasmid #44361)</td>
			<td>Bryan Roth&#8217;s lab through Addgene</td>
			<td></td>
			<td>DREADD plasmid</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>To assist with loading virus into the nanoinjector.</td>
		</tr>
		<tr>
			<td>PrecisionGlide Needles (25G x 5/8)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305122</td>
			<td>For use with the 1mL and 10 mL syringes to allow injection of the animal model.</td>
		</tr>
		<tr>
			<td>Rat Tooth Forceps</td>
			<td>Roboz</td>
			<td>RS-5152</td>
			<td>For griping spinous processes.</td>
		</tr>
		<tr>
			<td>Red Oil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To provide a front for visualization of virus entering tissue during injection.</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Roboz</td>
			<td>RS-6510</td>
			<td>To hold open the surgical wound.</td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Roboz</td>
			<td>RS-8300</td>
			<td>To remove muscle from the spinal column during surgery.</td>
		</tr>
		<tr>
			<td>Scalpel Blade Handle</td>
			<td>Roboz</td>
			<td>RS-9843</td>
			<td>To slice open skin and fat pad of animal model during surgery.</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-5980</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Staple Removing Forceps</td>
			<td>Kent Scientific</td>
			<td>INS750347</td>
			<td>To remove the staples, should they be applied incorrectly.</td>
		</tr>
		<tr>
			<td>Sterile Cloth</td>
			<td>Phenix Research Products</td>
			<td>BP-989</td>
			<td>To provide a sterile surface for the operation.</td>
		</tr>
		<tr>
			<td>Sterile Cotton-Tipped Applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td>To soak up blood in the surgical wound while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>Covidien</td>
			<td>2146</td>
			<td>To clean the surgical area and surgical tools while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Baxter Healthcare Corporation</td>
			<td>281324</td>
			<td>For use in blood clearing, and for replacing fluids post-surgery.</td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For use by the surgeon to maintain sterile field during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Heating Pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For maintaining the body temperature of the animal model during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For enhanced visualization of the surgical wound.</td>
		</tr>
		<tr>
			<td>Surgical Stapler</td>
			<td>Kent Scientific</td>
			<td>INS750546</td>
			<td>To apply the staples.</td>
		</tr>
		<tr>
			<td>Water Convection Warming Pad</td>
			<td>Baxter Healthcare Corporation</td>
			<td>L1K018</td>
			<td>For use in the post-operational recovery area to maintain the body temperature of the unconscious animal.</td>
		</tr>
		<tr>
			<td>Weighted Hooks</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold open the surgical wound.</td>
		</tr>
		<tr>
			<td>Liquid bandage</td>
			<td>NewSkin</td>
			<td>985838</td>
			<td>To apply along sutures following surgery and encourage wound healing</td>
		</tr>
		<tr>
			<td>Wire Cage Lamp</td>
			<td>ZooMed</td>
			<td>LF10EC</td>
			<td>To help animals recover from anesthesia and retain warm body temperature naturally</td>
		</tr>
	</tbody>
</table>

targeting the corticospinal tract in neonatal rats with a double-viral vector using combined brain and spine surgery

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

Successful injection and transport of the viral vector should result in the transduction of unilateral neurons in the spinal cord and the motor cortex. Figure 4 demonstrates the labeling of layer V CST neurons in the motor cortex of a brain coronal section expressing Cre-dependent-DREADDs-mCherry co-injected with a contralateral spine injection of rCre. The sections were stained with dsRed antibody.
<img alt="Figure 1" class="xfigi...

Watch this Scientific Journal Video about Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery at JoVE.com

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

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

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

This is the first protocol demonstrating the successful co-infection of the corticospinal tract in neonatal rats. It provides an accurate blueprint to investigate how individual subpopulations of cells in young mammals contribute to the recovery from spinal cord injury. Like other double viral vector techniques, this method allows the user to accurately transduce specific subpopulations of neurons, in turn, allowing researchers to identify the effectiveness of various chemogenetic techniques in promoting recovery.We would expect researchers that are unfamiliar with this technique to struggle with locating the proper spinal anatomy. And so we encourage you to continually count the vertebral segments often and always refer back to your landmarks. After confirming the depth of anesthesia in a neonatal Sprague Dawley pup, identify the area where the incision will be made by pressing the forceps in the midline on the top of the scalp, feeling for the sagittal suture.Holding the skin taut to ensure a clean, straight, and precise incision, make a one-centimeter incision along the sagittal suture plane starting immediately above the I line. Next, identify the bregma by gently probing the forceps along the surface of the skull, paying close attention to the suture lines and an indentation caused by the forceps running along the parietal and frontal bones. Maintain the opening by applying weighted hooks on the side of interest.Clear the aponeurosis using a combination of cotton tips and micro scissors to maximize the exposure of the bregma for increased accuracy. Ensure that the coronal suture is in clear view far enough laterally to provide a rough template for subsequent injections. Using the micro scissors, cut out an approximately three-by-two-millimeter section of the left frontal skull bone immediately adjacent to the bregma.Clear any debris, cerebrospinal fluid, and blood with cotton tips. To inject the virus, place the needle in place and program the injector to inject one microliter at a rate of 250 nanoliters per minute. Upon completion of the injection, allow the needle to rest in the cortex for three minutes.Repeat the injections at the other coordinates for a total of three injections along the cortex in relation to the bregma, all at a depth of 0.6 millimeters. After completing the injections, suture the scalp and transfer the animal to the other operating table for cervical laminectomy. Palpate the base of the skull using fingers to identify the incision site.Hold the skin taut by applying tension with the thumb and index finger. Using a number 11 blade, begin the incision at the midline one to two millimeters posterior to the skull base and extend the incision one centimeter posterior to expose the superficial muscle. Using the sharp point of the blade, gently make a series of cuts along the midline of the superficial muscle to expose the spinal cavity and deep spinal muscles.Then use a pair of forceps to spread open the muscle and visualize the surgical window. Once the deep spinal muscles have been exposed, insert retractors into the surgical window and retract the surgical window to seven to eight millimeters in width, allowing an unobstructed view of the spine. Identify the second cervical vertebra by its prominent spinous process that projects dorsally and encapsulates a large dome-shaped muscle.Using the flat edge of a bone scraper, gently scrape away the deep spinal muscle to the sides to expose the C3 to C7 vertebrae. Start medial and scrape laterally along the direction parallel to the vertebra laminae to ensure proper exposure, allowing clear distinction of the vertebral laminae. Control any bleeding with cotton tips.Using C2 as a guide when counting the vertebral levels, carefully cut the lateral edges of the cartilaginous laminae at C6 and C7 using a pair of micro scissors. Using a pair of fine forceps, carefully remove the dissected portion of the lamina to expose the spinal cord, ensuring that the spinal window is large enough to accommodate the desired injection site. Set the animal up in the stereotaxic cranial holder and place a rolled up piece of gauze under the animal's trunk to elevate its hind quarters.Before beginning injections, clear the spinal window of any blood or cerebrospinal fluid by gently applying cotton tips and Sugi triangles to the area, taking care not to insult the spinal cord. Further, create a barrier around the perimeter of the window by placing a small piece of absorbable cotton in the lateral portions of the spinal window to prevent occlusion of the injection site by continuous bleeding or CSF leakage. For direct injections into the spinal cord, use the tip of the glass injection needle to approximate the midline of the spinal cord by identifying the spinal artery.Next, place the needle just posterior to the C5 lamina at the approximate midline and use this as the reference point. Then, using the micro manipulator, move the needle laterally to the right by 0.3 millimeters and lower the needle until it just touches the surface of the spinal cord. From this depth, plunge the needle.0.6 millimeters into the spinal cord. Inject one microliter of the retro adeno-associated virus two carrying the Cre recombinase gene at 250 nanoliters per minute. Then wait for two minutes for the virus to diffuse into the spinal cord before slowly withdrawing the needle.The labeling of layer five corticospinal tract neurons in the motor cortex of a brain coronal section expressing Cre-dependent DREADDs mCherry co-injected with a contralateral spine injection of retro Cre is shown here. Being able to target specific neuronal populations will help to delineate the nuances driving recovery and provide more accuracy for treating the underlying spinal cord injury in the developing nervous system. This method paved the way for us to investigate the viability of using excitatory DREADDs as a means for treating spinal cord injury in juvenile rats.

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Research

JoVE Journal

Neuroscience

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

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 ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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, ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential for recovery based on clinical assessments alone. The functional integrity of the corticospinal tract is an important prognostic biomarker for recovery of UL function, particularly for those with severe initial UL impairment. This article presents a protocol for evaluating corticospinal tract function within 1 week of stroke. This protocol can be used to select and stratify patients in trials of interventions designed to improve UL motor recovery and outcomes after stroke. The protocol also forms part of the PREP2 algorithm, which predicts UL function for individual patients 3 months poststroke. The algorithm sequentially combines a UL strength assessment, age, transcranial magnetic stimulation, and stroke severity, within a few days of the stroke. The benefits of using PREP2 in clinical practice are described elsewhere. This article focuses on the use of a UL strength assessment and transcranial magnetic stimulation to evaluate corticospinal tract function.

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...