BACKGROUND: Injection of glutamate (Glu) in normal animals can cause neuronal c-Fos expression; however, whether Glu can induce spinal neuronal c-Fos expression in pain models is unclear. OBJECTIVE: To examine the effects of intraplantar and intrathecal injection of Glu on c-Fos expression in the L_5 spinal cord dorsal horn Ⅰ/Ⅱ and Ⅲ/Ⅳ layers after spinal nerve ligation, and to study the effects of the N-methyl-D-aspartic acid (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoate (D-AP5), and a selective group I mGluR antagonist, 7-hydroyiminocyclo propan[a]chromen-1a- carboxylic acid ethyl ester (cpccoEt). DESIGN, TIME AND SETTING: A randomized, controlled animal study was performed at the Department of Pharmacology, Oral Anatomy, and Neurobiology, Osaka University Graduate School of Dentistry, from December 2005 to December 2006.MATERIALS: Glu (5 μmol), D-AP5 (50 nmol) and cpccoEt (250 nmol) were provided by Wako Pure Chemical Industries, Osaka, Japan, and diluted in saline (50 μL). The pH of all solutions was adjusted to 7.4.METHODS: Twelve rats were randomly divided into sham operation (n = 6) and spinal nerve ligation (SNL; n = 6) groups for behavioral assessments of neuropathic pain after ligation surgery of the left L_(5-6) nerve segment. Another 60 rats were randomly divided into sham operation, SNL, saline-intraplantar, saline-intrathecal, Glu-intraplantar, Glu-intrathecal, D-AP5-intrathecal, Glu-D-AP5-intrathecal, cpccoEt-intrathecal, and Glu-cpccoEt-intrathecal groups, with 6 rats in each group. All groups except sham operation group received a similar SNL. On day 14, rats received a 50-μL injection of saline, Glu, D-AP5, and/or cpccoEt into the left intraplantar or intrathecal L_(5-6) segments. MAIN OUTCOME MEASURES: The number of c-Fos positive neurons in both Ⅰ/Ⅱ and Ⅲ/Ⅳ spinal layers at L_6 was observed using immunohistochemistry 2 hours after administration. RESULTS: (1) SNL increased the level of c-Fos expression in two sides of the spinal cord, particularly on Ⅲ/Ⅳ spinal layers of the ligated side. (2) Intraplantar or intrathecal administration of saline significantly increased the c-Fos labeled neurons in Ⅰ/Ⅱ spinal layers of the ligated side, compared with SNL alone (P < 0.01). (3) Intraplantar Glu (5 μmol) increased the number of c-Fos positive neurons in Ⅰ/Ⅱ spinal layers compared with intraplantar saline (P < 0.01). (4) The number of c-Fos neurons in Ⅰ/Ⅱ spinal layers on both the ipsilateral and contralateral side after intraplantar Glu was lower than intrathecal Glu (P < 0.01), with a 3-fold higher induction by intrathecal Glu. (5) Co-administration of D-AP5 or cpccoEt reduced the effects of intrathecal Glu (P < 0.01). CONCLUSION: Intrathecal Glu increases c-Fos expression more than intraplantar Glu. Antagonists of NMDA and group I mGluRs block this effect.