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The excitability of spinal neurons that transmit pain is modulated by glutamate and substance P (SP). Glutamate is an excitatory neurotransmitter in the dorsal horn, and its effects are enhanced by SP acting on neurokinin 1 receptors (NK1Rs). We assessed activation of NK1Rs by studying their internalization in spinal cord slices. NK1Rs were localized in sections from the slices by using immunohistochemistry combined with fluorescence and confocal microscopy. Incubating the slices with SP induced internalization in most NK1R-positive neurons in laminae I, IIo, and X and in half of NK1R-positive neurons in laminae III–V. SP-induced internalization was abolished by the specific NK1R antagonist L-703,606 (1 μm). Stimulating the dorsal root with long-duration (0.4 msec) pulses evoked EPSPs in dorsal horn neurons with latencies consistent with the conduction speed of A∂- and C-fibers. High-frequency (100 Hz) stimulation of the dorsal root with these pulses induced NK1R internalization in neurons in laminae I–IIo of the stimulated side of the slice but not in the contralateral side or in other laminae. Stimulation at lower frequencies (1 and 10 Hz) failed to elicit significant internalization, suggesting that the release of SP is frequency-dependent. Internalization produced by the 100 Hz tetanus was mimicked by NMDA and blocked by an NMDA antagonist, 2-amino-5-phosphonopentanoic acid, but not by the AMPA and kainate antagonist CNQX. The NK1R antagonist L-703,606 abolished the internalization produced by 100 Hz stimulation or NMDA. Therefore, the release of SP in the dorsal horn appears to be controlled by NMDA receptors.

Neurokinin 1 Receptor Internalization in Spinal Cord Slices Induced by Dorsal Root Stimulation Is Mediated by NMDA Receptors