DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

The overall goal of this procedure is to visualize the branching patterns of individual axons of DRG neurons in a whole mount preparation of mouse embryonic spinal cord. In order to identify molecules involved in axonal branching, the projections of DRG neurons into the mouse spinal cord offer a simple, easy, accessible experimental system to study axonal branching. When ENT axons of DRG neurons reach the dorsal root entry zone of the cord, they display a stereotype pattern of T or Y shaped bifurcation.

The two resulting daughter axons then proceed in rostral or cordal directions, respectively at the dorsal lateral margin of the cord. Only after a waiting period collateral sprout from these stem axons to penetrate the gray matter and project a relay neurons in specific laminate of the spinal cord where terminal arborization occurs. This video demonstrates first how to dissect the spinal cord.

With its attached DRG from an embryonic day 12.5 mouse following fixation of the specimen minute mounts of dii are applied to the DRG using glass needles pulled from capillary tubes After an incubation step, the labeled spinal cord is mounted as an inverted open book preparation to analyze individual axons using fluorescence microscopy. The branching of axons is an important aspect in the development of neural connectivity. The method presented here allows a comparison of branching patterns of Y type with those of mutant mice, and thereby enables the identification of molecules that regulate axonal branching.

First, set up the dissecting microscope and lay out the surgical instruments needed for dissection. Place a sheet of filter paper in a 100 millimeter silk guard coated Petri dish. Next, pour cold PBS In the wells of a 12 well plate a 100 millimeter Petri dish and a 12 milliliter tube and leave on ice.

Pipette two milliliters of 4%paraldehyde fixation buffer in each well of a second 12 well plate and place on ice. After isolating the bilateral uterine horns from a mouse, transfer them into the 100 millimeter dish with ice cold PBS. After decapitating the embryos transfer the toci of the embryos to the 12.

Well plate wet the filter paper in the S guard dish with a few drops of PBS and position an embryonic torso with its dorsal side up on the paper for better stability. Straighten its tail and extremities away from the body working under dissecting microscope with a magnification of approximately 16 times. Carefully pinch the skin of the embryo above the spinal cord with two pairs of fine tipped forceps beginning in the middle.

Gently tear the skin first towards the tail, and then resuming from the middle towards the anterior side. Wet the embryo from time to time with two or three drops of PBS to prevent it from drying to ensure that the dorsal root ganglia or DRG are not torn from the spinal cord. When it is removed, detach the DRG and the spinal cord from the surrounding cartilaginous vertebral column by starting in the middle of the embryo's right side and working towards the tail, and then to the anterior end to completely detach the loosened spinal cord from the embryo.

Pick up its cervical part with fine forceps and pull it out in one piece towards its cordal.End. Submerge the isolated spinal cord with attached DRG in a well. Of the 12 well plate filled with fixation buffer, and proceed with the preparation of spinal cords from the remaining embryos.

Fix the spinal cords for at least two hours on ice for each vinyl cord to be labeled, prepare two glass needles using a micro pipette puller under microscopic control. Dip the tip of each glass needle three to five times in a 5%solution of dye in ethanol to create a thin layer of dai crystals on the needle, working under a dissecting microscope with a magnification of approximately 40 times. Place a spinal cord with its ventral side up on a slide using spring scissors.

Open the cord on the ventral side at its whole length by cutting through the floor plate to allow for inverted open book mounting. Position the spinal cord with the dorsal side up on the slide. Then manually approach the cord with the dai covered tip of the glass needle and carefully pierce every second DRG on one side of the spinal cord.

Almost no traces of dai should be visible in the pierce DRG to ensure the labeling of only a small number of neurons using the second needle. Repeat for the other side of the cord, aiming only every second. DRG avoids an overlap of labeled axonal projections from neighboring, which might complicate the differentiation of individual axons.

Return the spinal cord to the fixation buffer and incubate in the dark for at least six hours at ambient temperature or overnight at four degrees Celsius to give time for the diet to diffuse along the axon within the plasma membrane. Following diet diffusion position a single spinal cord with its dorsal side down on a cover slip in a drop of PBS aspirate excess fluid, and flatten out the cord in an inverted open book mode. Using forceps.

Take care that the attached DRG are correctly oriented laterally and do not intermingle mount the preparation on a microscope slide using PBS to prevent the increase of unwanted background staining. Fluorescence microscopic analysis of the labeled axonal projections should be carried out on the day of mounting. Until then, keep the slides in the dark at four degrees Celsius.

Here, dorsal views of Dai labeled DRG from wild type mice at increasing magnification are shown as described every second. DRG has been labeled by Dai. Here, a small number of axons are labeled and at a higher magnification, the presence of T like branches can be identified in the dorsal root entry zone of the spinal cord.

T-shaped branches. Single rostral R or cordal C turns are quantified here at different vertebral levels in both wild type and C type natriuretic peptide CMP deficient mice. At E 13.5.

The total number of neurons counted is shown in bracket and the percentage of trajectory type is shown below. In contrast to the wild type axons of DRG neurons in CMP signaling mutants turn only in one direction instead of bifurcating at the dorsal root entry zone of the spinal cord. This method helped us to identify a CGP signaling cascade that is essential for the bifurcation of sensory accident as the dorsal root entry zone.

In contrast to the wild type accents of cgm, P signaling movement only turn in one direction instead of bifurcating at the dors root entry zone. These studies also revealed that co formation is not affected in CG P signaling mutants, indicating that the second type of axonal branching observed in DGen neurons is differently regulated.