Investigating the Origins of Sensory Neurons in the Dura Mater Using Fluorescence Microscopy

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.