This article describes a method for tracing sensory neurons in rats using a retrograde tracer. The procedure involves surgical exposure of the dura mater, application of the tracer, and subsequent analysis of neuronal cell bodies.
Take an anesthetized rat with an exposed skull that is covered with a drape.
Drill a hole to expose the dura mater, the brain’s outermost layer, innervated by pain-sensitive sensory neurons originating from the trigeminal ganglion and cervical dorsal root ganglia.
Construct a barrier to prevent leakage, introduce a retrograde tracer, and seal the hole.
Suture the wound and allow the rat to recover.
The tracer enters the sensory neuronal axons, travels retrogradely to their ganglia, and enters the neuronal cell bodies.
Euthanize the rat and dissect the ganglia.
Prepare sections of the ganglia and mount them onto slides.
Add a solution to permeabilize cellular membranes and mask non-specific binding sites.
Incubate with primary antibodies targeting the tracer and sensory neuron-specific neuropeptides.
Add fluorescently labeled secondary antibodies specific to the primary antibodies.
Using fluorescence microscopy, observe the colocalization of neuropeptides and tracers within the cell bodies, confirming the ganglionic origin of the sensory neurons.
Shave the rat's head with an electric razor. Then put blunt ear bars to the rat and place it on the stereotaxic device. Position the mouth holder and apply ophthalmic ointment on the eyes. Clean the surgical site of the head skin using 75% ethanol, and make an incision along the midline of the scalp.
Bluntly remove the periosteum and muscle tissues away from the skull using sterile cotton tipped applicators. Drill a small hole using a burr drill with a round tip bit on the left parietal and temporal bones above the middle meningeal artery. Build a bank around the hole with dental silicate cement, and add 2 microliters of fluoro gold into the hole around the middle meningeal artery with a 10-microliter micro syringe.
Cover the hole with a small piece of hemostatic sponge. Put a piece of paraffin film on the hole and seal the edges with bone wax. Suture the wound with sterile thread. Keep the rats in a warm area until they have fully recovered, then return the rats back to their cages.
After seven survival days, perfuse these rats with 0.9% normal saline, followed by 4% paraformaldehyde in 0.1 molar phosphate buffer. Dissect out the TG and cervical 1 to 4 DRGs, then cut them at the thickness of 30 micrometers on a cryostat microtome system in the sagittal direction. Mount the sections on silane-coated glass slides.
Circle the sections with a histochemical pen, and incubate them for 30 minutes in blocking solution. Transfer the samples into the solution of rabbit anti-fluorogold and mouse anti-CGRP antibody, and incubate at 4 degrees Celsius overnight.
On the next day, wash the sections three times in 0.1 molar phosphate buffer. Incubate the washed sections in a mixed solution of donkey anti-rabbit Alexa Fluor 594 and Donkey anti-mouse Alexa Fluor 488 secondary antibody. Then, wash them three times in 0.1 molar phosphate buffer. And apply coverslips with 50% glycerin for observation. Take images of the fluorogold-labeled neurons in TG and DRGs under UV illumination using a fluorescent microscope. Then capture images of the fluorogold and CGRP-labeled Neurons in TG and DRGs.
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