Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

The overall goal of this procedure is to discover the central premotor network of distributed cells that impinge on a specific peripheral target and to then confirm the specific connections between those cells in the brain. This is accomplished by first retrograde infecting the premotor circuits with a trans synaptic viral vector that is delivered peripherally through the muscle. The second step of the procedure is to visualize the infected cells with light fluorescent microscopy.

The third step is to confirm specific connections in the brain by injecting monos synaptic tracers targeted at previously identified nodes in a pre-motor circuit. Ultimately, results can show how cells in a distributed pre-motor network are connected to each other through a combination of trans synaptic viral tracing, and traditional monos synaptic neuro anatomical techniques. The main advantage of this technique over existing methods like traditional monos synaptic neuro tracing, is that the nodes of a distributed neuro network can be identified and the specific connections between cells in that network can be resolved On an anesthetized mouse.

Prepare the surgical site by trimming the hair and disinfecting the skin with at least three alternating scrubs of Betadine and 70%alcohol. Now make a skin incision in the throat area and reveal the muscle of interest. For example, to access the cricothyroid laryngeal muscle, it is necessary to first remove the overlying portion of the Sterno hyoid muscle seal any transected muscles with vet bond tissue adhesive.

Next load the 10 microliter nano fill micro syringe system with freshly thawed PR RV solution. Slowly clear the dead space and verify that solution exits the micro syringe tip. Discard the fluid as biohazardous waste and carefully mount it on the stereotaxic device.

Using the micro positioning device, carefully place the needle tip into the muscle of interest. Then slowly fill the muscle until a slight swelling is visible. Now move the syringe to the next muscle of interest, the lateral cricoarytenoid in this case and repeat.

The injection procedure only puncture each muscle. Once the injection rate will vary depending on the size of the muscle and the volume to be injected. For example, five injections of 200 nanoliters at a rate of four nanoliters per second should be made one minute apart at the same site to fill the cricothyroid muscle.

After retracting the micro syringe, seal the break in the fascia using vet bond tissue adhesive. Once all the injections are finished, close the wound using vet bond tissue adhesive. Monitor the animal until it shows sternal recumbent.

Then provide analgesia, food, water, and care as required by your institutional animal. Use guidelines. The procedure for analyzing EGFP expression and PRV infected neurons is covered in detail in the text protocol.

The tissue processing is very similar to that covered later in this video. Begin by anesthetizing an adult mouse protecting its eyes and fixing the position of its head. Then prepare the surgical site as outlined previously with alternating scrubs of Betadine and 70%alcohol.

Now make a scalp incision and retract the scalp over the brain. Region of interest. Perform a small craniotomy with the drill at the appropriate stereotaxic coordinates.

For example, the coordinates for laryngeal connected motor cortex identified by PRV tracing in an adult mouse are 1.2 millimeters lateral and 0.2 millimeters rostral to the bgma. Next, prepare a 7.5%solution of biotinylated dextran amines in sterile water. Then load the nano jet two micro pipette system with enough BDA solution for the planned injections.

Slowly clear the dead space and verify that solution is exiting the micro pipette tip. Using the micro positioning device, carefully lower the micro pipette tip into the brain region of interest and slowly inject the BDA solution. Adjust the rate depending on the size of the region to be labeled.

For example, 12 injections of 4.6 nanoliters should be made at four different locations. 0.2 millimeters apart. Stro coddle to cover the laryngeal connected motor cortex in an adult mouse.

The final injection volume should be adjusted according to the size of the region of interest. Keeping in mind that label may diffuse from the injection core through brain tissue and along the processes of labeled neurons. Clean and wet craniotomy with sterile saline and close the scalp wound using vet bond tissue adhesive.

Monitor the animal until it shows sternal recumbent. Then provide analgesia, food, water, and care as required by your institutional animal. Use guidelines.

Six days later, cholera toxin subunit B can be injected into the animal's muscle. Then in three days, the animal should be sacrificed for analysis. Cut sections at 40 microns on a cryostat and save floating sections in 0.1 molar PBS quench the sections with 0.3%hydrogen peroxide in PBS for 30 minutes with shaking and protected from light.

Wash the sections three times in PBS for five minutes per wash. Then react them for an hour in freshly prepared A b, C solution at room temperature. Now wash the sections three times in phosphate buffer for 10 minutes per wash.

Then develop the sections for eight minutes with agitation in phosphate buffer. pH 7.4 containing DAB hydrogen peroxide and nickel chloride. The DAB reaction product inside the cells should appear black.

Next block the sections while shaking for 30 minutes in PBS containing 0.3%tween 20 with normal rabbit serum from the vector stain elite kit. Next, incubate the blocked sections in goat anti CTB with shaking for two hours at room temperature. Then wash the sections three more times in PBS for five minutes per wash.

Apply the rabbit antigo secondary antibody from the VE kit and incubate the sections while shaking for one hour at room temperature. Wash sections three times in PBS for five minutes per wash and then react them for 30 minutes in freshly prepared A, b, C solution at room temperature. Wash sections three times in phosphate buffer for 10 minutes and incubate the sections for eight minutes.

In the development solution, the DAB reaction product inside the cells should appear brown. Now mount the sections on super frosts plus slides on the slides, dehydrate the tissue through graded alcohol series, followed by two clearing washes in xylene. Then cover slip the slides with paramount and proceed with imaging.

Staining for EGFP should begin showing weak signal in primary motor neurons 72 hours after injecting P RV 1 52 with staining becoming robust after 90 hours. This nucleus ambiguous, which houses the laryngeal motor neurons is seen 94 hours after injection of P RV 1 52 into two of the seven laryngeal muscles. Because the virus entered the brain through neurons that innervate the infected muscles.

Other atory motor neuron pools such as the hypoglossal nucleus do not express EGFP after 90 hours. EGFP can be seen in second order cells. These brainstem neurons were stained subsequent to the primary infection in nucleus ambiguous.

The injection was unilateral and the resulting pattern of label in the brainstem shows infection of the ipsilateral nucleus. Ambiguous and solitary nucleus reticular inter neurons were also infected and stain strongly for EGFP. These inter neurons connect various structures including the atory motor neuron pools and the contralateral nucleus.Ambiguous.

However, their efferent targets remain unlabeled because the virus only spreads in the retrograde direction. Survival time with PRV is limited, but can show EGFP staining in third and higher order cells infected premotor neurons in the motor cortex expressed EGFP contralateral to the peripheral injection site. Presumably only the population of cells that composed the motor cortical representation of the laryngeal musculature were infected.

BDA labeled axons at the level of nucleus ambiguous marked by arrowheads, make contact with the CTB positive motor neurons retrograde labeled by injections into laryngeal muscles seen in brown the axons form varicosities and putative terminal batons near the dendrites of laryngeal motor neurons and near the soma of laryngeal motor neurons. Further experiments using electron microscopy or electrophysiology are required to determine whether these varicosities represent synaptic. A bilateral injection of BDA placed into the laryngeal connected motor cortex is seen in black because BDA is not directionally specific, both afferent and efferent connections may be labeled.

For example, both a projection field of anterograde label and cell bodies labeled by retrograde are seen in the thalamus. Thalamic cells are identified by the arrowhead and the internal capsule is labeled for orientation. Don't forget that working with pseudo rabies virus and DAB can be extremely hazardous and precautions such as sterilizing.

All materials that were in contact with either PRV or DAB using 10%bleach solution. An appropriate disposal of biohazardous solid and liquid waste according to the regulations of your institution should always be taken while performing this procedure.