Inflammation causes a long-term hyperexcitability in the nociceptive sensory neurons of Aplysia

Abstract

Nerve injury, tissue damage, and inflammation all cause hyperalgesia. A factor contributing to this increased sensitivity is a long-term (>24 hr) hyperexcitability (LTH) in the sensory neurons that mediate the responses. Using the cluster of nociceptive sensory neurons in Aplysia californica as a model, we are examining how inflammation induces LTH. A general inflammatory response was induced by inserting a gauze pad into the animal Within 4 days, the gauze is enmeshed in an amorphous material that contains hemocytes, which comprise a cellular immune system. Concurrently, LTH appears in both ipsilateral and contralateral sensory neurons. The LTH is manifest as increased action potential discharge to a normalized stimulus. Immunocytochemistry revealed that hemocytes have antigens recognized by antibodies to TGFbeta1, IL-6, and 5HT. When a localized inflammation was elicited on a nerve, hemocytes containing the TGFbeta1 antigen were present near axons within the nerve and those containing the IL-6 were on the surface. Western blots of hemocytes, or of gauze that had induced a foreign body response, contained a 28-kD polypeptide recognized by the anti-TGFbeta1 antibody. Exposure of the nervous system to recombinant human TGFbeta1 elicited increased firing of the nociceptive neurons and a decrease in threshold. The TGFbeta1 also caused an activation of protein kinase C (PKC) in axons but did not affect a kinase that is activated in axons after injury. Our findings, in conjunction with previous results, indicate that a TGFbeta1-homolog can modulate the activity of neurons that respond to noxious stimuli. This system could also contribute to interactions between the immune and nervous systems via regulation of PKC.

Figure 1

Diagram of the ventral surface of the left pleural and pedal ganglion showing pedal (p) nerves p9 and p8. p9, which is the longest nerve in the animal, extends caudally to innervate the tail. The other pedal nerves have been severed close to the ganglion.

Figure 2

Immunocytochemistry of hemocytes migrating on polylysine-coated plastic dishes. The cells were fixed, permeabilized, and exposed to the primary antibody indicated. Antigen-antibody complexes were subsequently detected with HRP-coupled secondary antibody. Hemocytes were stained by antibodies to vertebrate TGFβ1 and IL-6, but not to IL-1. Positive staining was also obtained with an antibody to 5-HT. The staining was most intense in large granules (arrows). Hemocytes that were recognized by the anti-TGFβ1 antibody were larger than most of the other cells and contained especially prominent lamellipodia (Lam). Bars, 2 μm.

Figure 3

Immunocytochemical localization of cells containing TGFβ1 and IL-6. An inflammation was elicited by tying a loose ligation around nerve p9. Four days later the nerve segment immediately below the ligation was fixed, frozen, and sectioned in the longitudinal plane. A segment of a control nerve from the same animal was treated similarly. Sections were exposed to the primary antibody followed by an HRP-coupled secondary antibody. Images were captured using an Argus 20 system. Groups of TGFβ1 immunopositive cells were detected under the connective tissue sheath and adjacent to the axon bundle (arrow). This section was taken near the end of the nerve segment. In contrast, large accumulations of IL-6-positive cells were found attached to the outer border of the sheath. There was no staining of control nerves. Bars, 30 μm.

Figure 4

Recognition of a TGFβ1-like polypeptide on Western blots. (A) A blot, probed with the anti-TGFβ-1 antibody, showing the prominent 28-kD polypeptide (arrow) in an extract of hemocytes, and authentic recombinant vertebrate TGFβ1 (arrowhead). (B) Blot of hemocytes and a detergent extract of gauze that had been implanted in an animal for 4 days. The anti-TGFβ1 antibody detected a prominent 28-kD polypeptide in the gauze extract (arrow). Much of the antigen in the hemocyte sample was found in high molecular weight polypeptides.

Figure 5

Interaction between hemocytes and neurons in vitro. The motile, phase-dense hemocytes (arrowheads) preferentially attach to the neurites and not the cell body (CB). Here the hemocytes formed clusters near the growing tips of the neurites. Bar, 15 μm.