This procedure describes a versatile in vivo tracing protocol that exploits wheat germa glutenin conjugated to Alexa fluorophores to trace neuronal circuits. First, an incision is made into the lower thoracic region, upper lumbar region, and the fascia and superficial spinal muscles are cut. The second step is to perform a dorsal laminectomy.
Next, the micro electrode is set up and attached to the micro manipulator. This is followed by pressure injection of neural traces into the spinal cord. The final step of this procedure is to provide proper post-surgical care during recovery and give ample time for the tracers to be transported throughout the targeted tracts.
Ultimately, results can be obtained that show the organization and connectivity of topographic maps in the cerebellum cortex, brainstem, and spinal cord through immunofluorescence microscopy. The main advantage of this tracer over existing tracers like BDA or W-G-A-H-R-P, is that WGA Alexa tracers are bright enough for all mount imaging directly after tracing. This method could help answer key questions in systems neuroscience field, such as what is the logic underlining the global patterning of multiple topographic projections within and between cortical, cerebellum, brainstem, and spinal systems Before beginning the procedure.
Ensure that all manipulations are in compliance with iacuc guidelines, that a sterile field is available for the surgery, and that all instruments to be used have been sterilized. Sterile surgical techniques should be used throughout the surgery after administering an approved anesthetic to the animal, ensure that a suitable plane of anesthesia has been achieved by performing a toe pinch. The pedal reflex should be absent once the animal is completely anesthetized.
Shave the fur over the area to be operated on with hair clippers. Next, lay the animal dorsal side up on a sterile gauze pad using an inside to outside circular pattern. Wipe the shaved region with two sets of alcohol wipes, then 70%alcohol Betadine.
Then once more with alcohol wipes, then clearly demarcate the surgical field by placing a sterile gauze pad with a small hole cut into it over the animal after the animal is properly prepared. In this way, proceed to the surgery and maintain proper sterile surgical technique. Lift the skin using forceps and make an incision with scissors at the midline directly above the lower thoracic upper lumbar region.
Cut the fascia and superficial spinal muscles if necessary, stop any bleeding by cauterizing the blood vessels. Then identify the midway point between the articular process and the dorsal spinal process, and cut the lamina here on both sides of the vertebral column. Next, perform a dorsal laminectomy by removing the spinous processes of one or two segments of the lower thoracic upper lumbar spinal cord.
Using a micrometer syringe, fill a pooled glass pipette with the tracer. Then tighten the glass micro electrode into place on a micro manipulator. Next, fill the needle with reagents.
Insert the pooled pipette 0.1 to 0.5 millimeters into the lower thoracic upper lumbar region of the spinal cord. Halfway between the midline and the lateral edge of the spinal cord pressure inject approximately 0.5 microliters of a 2%solution of wheat agglutinin LOR conjugate wheat gem agglutinin, horse radish peroxidase conjugate, or a 10%biotinylated dextran amine in PBS supplemented with 0.5%fast green over a period of three to five minutes. If the tracer does not easily eject from the pipette, slowly retract the tip to create a small pocket in the tissue.
Then try injecting again following completion of the microinjection. Gently close the skin with wound clips and tissue glue. Then place the animal in a recovery chamber on a heating pad.
Monitor the breathing rate and check the signs that the animal is attempting to move. After the animal has recovered from anesthesia, return it to the home cage and provide water and food ad libitum and analgesics as required. Routinely check for signs of pain, discomfort, or infection, and for adequate eating and drinking animals injected with wheat germ gluten and conjugate tracers are sacrificed.
24 hours after microinjection. BDA typically requires longer survival times to fully trace neural projections, and therefore, the complete mapping of spino cerebellar projections. These animals are sacrificed after approximately 11 days.
Wheat germa Glutenin Alexa tracers were injected into the lower thoracic upper lumbar spinal cord. This schematic illustrates the origin and termination of wheat germa glutenin. Alexa 5 55 labeled spino cerebellar neurons.
This figure shows the injection site after delivery of wheat germ agglutinin Alexa 5 55 into the lower thoracic upper lumbar region of the adult spinal cord. This whole mount image shows the anterior loles following the injection of wheat. Gemma Glutenin.
Alexa 5 55 into the lower thoracic upper lumbar region of the spinal cord. Loles are indicated by Roman numerals. Here we see that wheat germa glutenin Alexa 5 55 antegrade tracing of the spin cerebellar tract reveals defined bands of mossy fibers as seen on this coronal tissue section.
This schematic of the mouse cerebellum summarizes the overall pattern of spinal cerebellum. Mossy fiber bands wheatgermagluten in Alexa. 5 55 was injected into the right side of the lumbar spinal cord and wheatgermagluten in Alexa, 4 88 into the same segment on the left side, as seen on a coronal tissue section cut through the posterior LOEs.
Both Weger agglutinin Alexa tracers were transported to the cerebellum and successfully labeled distinct subsets of mossy fiber terminals, mainly in the granule cell layer, which is labeled GCL Weger Agglutinin Alexa tracers can be used in combination with immunohistochemistry and histology for triple labeling of neurons and projections. Here, Weger Agglutinin Alexa 5 55 is deposited in mossy fiber terminals in labial three. This higher power image of the boxed region illustrates clear labeling of the mossy fiber terminals.
This image of zein two staining reveals an array of bikini cell stripes in Lole three. This higher power image of the boxed region illustrates clear labeling of the bikini cells in the bikini cell layer and molecular layer. Here DPI staining reveals neuronal and glial nuclei.
This higher power image of the boxed region illustrates intense labeling of the nuclei rich granule cell layer and bikini cell layer, and less dense labeling in the molecular layer. Here emerged, image shows triple labeling by Wheatgerm Agglutinin, Alexa 5 55 serin two, and DPI of afferent bands, bikini cell stripes, and general cyto architecture. Finally, this higher power image of the boxed region clearly illustrates the power of this technique to reveal the cyto architecture of brain structures.
Once mastered, this technique can be performed in 20 minutes per animal if done properly. While attempting this procedure, it's important to remember to image the tissue immediately after cutting. Although imaging within two days of cutting still allows labeled fibers and terminals to be successfully imaged following this procedure.
Other methods like genetic lineage tracing can performed in order to answer additional questions like, what are the projection patterns and termination patterns of genetically defined populations of cells?
