Abstract
BackgroundPaw carrageenan induces activation of phosphatidylinositol 3-kinase (PI-3K) and Akt in dorsal horn neurons in addition to induction of pain behavior. Spinal PI-3K activation is also thought to be required for inflammation-induced trafficking of GluA1, AMPA receptor subunits, into plasma membranes from cytosol. Phosphorylation of Akt has a unique time course. It occurs first in the superficial dorsal horn (0.75 h), then soon dissipates and is followed an hour later by Akt phosphorylation in deeper dorsal horn laminae, primarily lamina V. Initially, we wished to determine if Akt phosphorylation in the deeper laminae were dependent on the presence of lamina I, neurokinin receptor bearing projection neurons. As the study progressed, our aims grew to include the question, whether carrageenan-induced GluA1 subunit trafficking was downstream of Akt phosphorylation.ResultsRats pretreated with spinal saporin conjugated to a stabilized form of substance P had substantial loss of neurons with neurokinin 1 receptors throughout their superficial, but not deep dorsal horns. Animals pre-treated with substance P-saporin exhibited no change in locomotor ability and a small, but significant decrease in carrageenan-induced mechanical allodynia when compared to animals pre-treated with spinal saporin alone. Importantly, carrageenan-induced phosphorylation of Akt was blocked, in the substance P-saporin treated group, throughout the spinal cord grey matter. In marked contrast, carrageenan induced-trafficking of the GluA1 receptor subunit increased equivalently in both treatment groups.ConclusionsWe infer from these data that 1) phosphorylation of Akt in the deep dorsal horn is dependent on prior activation of NK1 receptor bearing cells in superficial dorsal horn, and 2) there are parallel spinal intracellular cascades initiated by the carrageenan injection downstream of PI-3K activation, including one containing Akt and another involving GluA1 trafficking into neuronal plasma membranes that separately lead to enhanced pain behavior. These results imply that the two pathways downstream of PI-3K can be activated separately and therefore should be able to be inhibited independently.
Highlights
Paw carrageenan induces activation of phosphatidylinositol 3-kinase (PI-3K) and Akt in dorsal horn neurons in addition to induction of pain behavior
Parallel experiments were conducted to determine the effects of SSPSap pretreatment on paw carrageenan-induced pain behavior and increases in plasma membrane-associated GluA1 in dorsal horn, an index of increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptor trafficking, which is thought to contribute to spinal long- term potentiation and sensitized pain states [15]
Few large neurokinin 1 (NK1) receptor positive neurons were seen in laminae I-III and the remaining receptor appeared to be located in small cells or scattered in the neuropil
Summary
Paw carrageenan induces activation of phosphatidylinositol 3-kinase (PI-3K) and Akt in dorsal horn neurons in addition to induction of pain behavior. The present study re-examined this laminar specific time course in the presence and absence of spinal pre-treatment with stabilized substance P-conjugated to saporin (SSP-Sap) This agent, when used at the appropriate dose and duration after administration, is a specific neurotoxin, which kills neurons with neurokinin 1 receptors in the superficial but not the deeper laminae of the dorsal horn [7,8]. The purpose of these experiments was to ascertain if loss of these superficial neurons was sufficient to prevent phosphorylation of Akt induced by paw carrageenan in laminae V and IX. Parallel experiments were conducted to determine the effects of SSPSap pretreatment (loss of superficial dorsal horn NK1 receptor bearing neurons) on paw carrageenan-induced pain behavior and increases in plasma membrane-associated GluA1 in dorsal horn, an index of increased AMPA receptor trafficking, which is thought to contribute to spinal long- term potentiation and sensitized pain states [15]
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