Antibodies specifically produced against pain transduction proteins
can be used to visualize nociceptors and to examine the expression
patterns of proteins involved in the generation of the pain signal.
The sensory endings of
nociceptors are thin fibers with diameters near 1 µm. The image below shows such a fiber in the skin, growing along the interface
between epidermis (the outer skin) and dermis (the true skin). The
epidermis is composed of several layers of cells (here the cell nuclei
are stained blue) and topped by a layer of dead cells which forms the
body surface. The epidermis, which is composed of various cell types
and connective tissue, is the black area below the red pain fiber. The
pain fiber was stained
with an antibody against TRPV1,
heat-activated ion channel that mediates the perception of noxious
heat. The antibody was visualized by a second antibody which carried a
red fluorescent label.
Immunohistochemistry can be
used to identify distinct populations of pain cells within dorsal root
ganglia. The fluorescence image below shows cells expressing
the heat-gated ion channel TRPV1, visualized using TRPV1-specific
antibodies (red). The blue DAPI stain of cell nuclei in this 10
µm-thick section of a DRG results mostly from non-neuronal satellite
cells which form a
protective cell layer around each neuron. The
unstained neurons themselves leave elliptic, black patches on the
image – some are marked by white circles. TRPV1-positive cells are
small-to-medium diameter neurons. The green stain results from the
binding of the lectin IB4 – labeled with a green fluorophore – to a
subpopulation of DRG neurons. Neurons expressing both TRPV1 and IB4
show yellow fluorescence.
Pain cells form synaptic
connection in the dorsal horn of the spinal cord. The dorsal horn
receives sensory input from somatosensory neurons, whereas the ventral
horn is the origin of the motoroutput that controls muscle movements.
To understand the neural circuitry in the spinal cord one first has to
find out in which layer of the dorsal horn each subpopulation of pain
cells forms their synapses. The image on the right shows that most
TRPV1-expressing pain cells terminate in the most superficial layer of
the dorsal horn (layer 1), while IB4-expressing pain cells form their
synapses within layer 2, the substantia gelatinosa. In the
outer part of layer 2, yellow immunofluorescence indicates that both
antigens are localized in the same synapses.