Enhanced Phosphorylation of NMDA Receptor 1 Subunits in Spinal Cord Dorsal Horn and Spinothalamic Tract Neurons after Intradermal Injection of Capsaicin in Rats

Abstract

The functional enhancement of NMDA receptors after peripheral tissue injury is proposed to contribute to the sensitization of spinothalamic tract (STT) cells and hyperalgesia. Protein phosphorylation is a major mechanism for the regulation of NMDA receptor function. In this study, Western blots, immunofluorescence double labeling, and the retrograde tracing method were used to examine whether phosphorylation of NMDA receptor 1 (NR1) subunits increases in spinal cord tissue and spinal dorsal horn neurons, especially in STT cells, after injection of capsaicin (CAP) into the glabrous skin of one hindpaw of anesthetized rats. Western blots showed that phosphorylated NR1 protein in spinal cord tissue was increased 30 min after CAP injection. Immunofluorescence double-labeling staining showed no significant difference in the number of the NR1-like immunoreactive neurons in laminae I-VII in the lumbosacral segments (L 4 -S 1 ) on the ipsilateral and the contralateral sides 30 min after CAP or vehicle injection. However, the numbers of phospho-NR1-like immunoreactive neurons were significantly increased on the ipsilateral side compared with the vehicle injection group. STT cells were labeled by bilateral microinjections of the retrograde tracer fluorogold into the lateral thalamus, including the ventral-posterior lateral nucleus. Immunofluorescence staining was performed at 30, 60, and 120 min after CAP injection or at 30 min after vehicle injection. There was a significant increase in the proportion of STT cells with phosphorylated NR1 subunits compared either with the contralateral side 30 and 60 min after CAP injection or either side of animals after intradermal injection of vehicle. These results provide direct evidence that NMDA receptors in STT cells are phosphorylated after CAP injection. Key words: NMDA receptor subunit; phosphorylation; STT cell; retrograde tracing; dorsal horn; nociceptionSpinothalamic tract (STT) cells transmit nociceptive information from the spinal cord to the thalamus (Willis, 1985;Gybels and Sweet 1989;Willis and Coggeshall, 1991). Extracellular glutamate and aspartate concentrations increase significantly in the spinal cord dorsal horn after peripheral inflammation (Sluka and Westlund, 1992, 1993a;Sorkin et al., 1992;Sorkin and McAdoo, 1993;Sluka and Willis, 1998), and this increase is believed to underlie hyperalgesia and allodynia (Sluka and Westlund, 1993b) by enhancing the excitability of nociceptive dorsal horn neurons, including STT cells (Aanonsen et al., 1990; Willis, 1991, 1992; Dougherty et al., 1992a,b). Intrathecal administration of glutamate agonists such as NMDA elicits nociceptive behavior (Aanonsen and Wilcox, 1987), and spinal cord administration of an NMDA receptor antagonist attenuates hyperalgesia and allodynia (Haley et al., 1990; Ren et al., 1992a,b;Ren and Dubner, 1993;Sluka et al., 1994). At a cellular level, central sensitization of STT cells can be initiated by iontophoretic co-release of NMDA and substance P (cf. Randic et al., 1990;Dougherty and Willis, 1991...

Electroacupuncture Inhibits Spinal Interleukin-17A to Alleviate Inflammatory Pain in a Rat Model

“…It is well known that p-NR1 modulates NMDAR activity and facilitates nociceptive input transmission in inflammatory pain models [3][4][5]. This suggests that inflammation-enhanced IL-17A might facilitate pain through NR1 phosphorylation, and EA might decrease spinal …”

“…These cells produces a number of algesic substances such as the proinflammatory cytokines interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 to promote spinal transmission and processing of noxious signals [1,2]. It is reported that IL-1β is up-regulated in spinal astrocytes to increase NMDA NR1 phosphorylation, an essential subunit of the N-methyl Daspartate receptor (NMDAR), which in turn promote pain in an inflammatory pain rat model [3][4][5][6] . IL-17A, recently found in astrocytes of patients with multiple sclerosis [7], increase in concentration in the spinal cord of rats with nerve injury [8].…”

Enhanced NMDA receptor NR1 phosphorylation and neuronal activity in the arcuate nucleus of hypothalamus following peripheral inflammation

“…Peripheral tissue injury dramatically enhances the function of spinal NMDA receptor that is involved in the initiation and maintenance of central sensitization, a persistent increase in the excitability of nociceptive neurons [2,3,28,29] . The hyperfunction of the NMDA receptor could result from phosphorylation, upregulation, or a combination of both [30,31] . Our western blot analysis indicated that the NMDA receptor NR1 subunit was phosphorylated but not upregulated in the ARC, and this effect was enhanced in inflamed rats.…”

“…Phosphorylation of NR1 has been shown to induce NMDA receptor trafficking from storage sites in the endoplasmic reticulum to the synaptic plasma membrane, leading to a hyperactivation state of NMDA receptor in nociceptive transmis sion [35,36] . Furthermore, NR1 phosphorylation has been correlated with hyperalgesia and allodynia, which are characteristic behavioral manifestations of central sensitization [31,37] . Previous investigations have demonstrated that the blockage of NR1 phosphorylation could reverse allodynia [37] .…”

Substance P inhibits progesterone conversion to neuroactive metabolites in spinal sensory circuit: A potential component of nociception

“…Consequently, involvement of 5␣-DHP and 3␣,5␣-THP biosynthetic pathways in the regulation of nociception needs to be considered in regard to various neurobiological mechanisms occurring in both acute and chronic phases of pain, even though in vivo studies have shown that 3␣,5␣-THP and its synthetic analogs induce antinociceptive effects in rats and humans (40-43). It is also important to recall that rNK1-mediated hyperalgesia seems to be an extremely complex mechanism involving the release of prostanoids (44,45) and glutamate (46,47), and the phosphorylation of NMDA receptor through a protein kinase C transduction process (48,49). Therefore, we suggest here an hypothetical model to recapitulate and clarify neurochemical events that may occur in spinal Values are the mean Ϯ SEM of four independent experiments.…”