Involvement of AMPA receptor GluR2 and GluR3 trafficking in trigeminal spinal subnucleus caudalis and C1/C2 neurons in acute-facial inflammatory pain

Abstract

To evaluate the involvement of trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR2 and GluR3 subunits in an acute inflammatory orofacial pain, we analyzed nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) and Fos expression in Vi/Vc, Vc and C1/C2 in GluR2 delta7 knock-in (KI), GluR3 delta7 KI mice and wild-type mice. We also studied Vc neuronal activity to address the hypothesis that trafficking of GluR2 and GluR3 subunits plays an important role in Vi/Vc, Vc and C1/C2 neuronal activity associated with orofacial inflammation in these mice. Late nocifensive behavior was significantly depressed in GluR2 delta7 KI and GluR3 delta7 KI mice. In addition, the number of pERK-immunoreactive (IR) cells was significantly decreased bilaterally in the Vi/Vc, Vc and C1/C2 in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice at 40 min after formalin injection, and was also significantly smaller in GluR3 delta7 KI compared to GluR2 delta7 KI mice. The number of Fos protein-IR cells in the ipsilateral Vi/Vc, Vc and C1/C2 was also significantly smaller in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice 40 min after formalin injection. Nociceptive neurons functionally identified as wide dynamic range neurons in the Vc, where pERK- and Fos protein-IR cell expression was prominent, showed significantly lower spontaneous activity in GluR2 delta7 KI and GluR3 delta7 KI mice than wild-type mice following formalin injection. These findings suggest that GluR2 and GluR3 trafficking is involved in the enhancement of Vi/Vc, Vc and C1/C2 nociceptive neuronal excitabilities at 16-60 min following formalin injection, resulting in orofacial inflammatory pain.

Figure 1. Face scratching and mechanical head-withdrawal threshold following subcutaneous formalin injection into the whisker pad.

A, B and C: The duration of face scratching events in wild-type, GluR2 delta7 KI and GluR3 delta7 KI mice. Gray lines in A, B and C represent individual animal values and dotted lines indicate mean values. D: Mean face scratching duration after formalin injection was plotted before, and in the 1^st phase (0–15 min) and 2^nd phase (16–60 min) after formalin injection. E: Mean head-withdrawal threshold before and 40 min after formalin injection into the whisker pad skin in in the wild-type, GluR2 delta7 KI and GluR3 delta7 KI mice.

Figure 2. Expression of pERK-IR cells in Vi/Vc, Vc and C1/C2 after formalin injection.

A: High magnification photomicrographs of pERK-IR cells (Aa), NeuN positive cells (Ab) and NeuN-labeled pERK-IR cells (Ac) at 5 min after formalin injection in the Vc of wild-type mice. B, C and D: low magnification photomicrographs of pERK-IR cells in the dorsal (B), middle (C) and ventral (D) portions of the Vc 5 min after formalin injection in wild-type mice. E: Camera lucida drawings of pERK-IR cells in the wild-type mice at 5 min after subcutaneous formalin injection into the whisker pad skin in sections from −1440 µm to the obex. F and G: Rostro-caudal distribution of pERK-IR cells in the wild-type, GluR2 delta7 KI and GluR3 delta7 KI mice 40 min after formalin injection in the ipsilateral and contralateral whisker pads, respectively. The mean number of pERK-IR cells in the ipsilateral (H) and contralateral (I) Vi/Vc, Vc and C1/C2 in the wild-type, GluR2 delta7 KI and GluR3 delta7 KI mice 40 min after subcutaneous formalin injection into the whisker pad skin. Boxes in the 3^rd panel in E indicate areas in B, C and D. Five min data are presented besides the 40 min ones in H and I.

Figure 3. Expression of Fos protein-IR cells in Vi/Vc, Vc and C1/C2 after formalin injection.

A: High magnification photomicrographs of Fos protein-IR cells (Aa), NeuN positive cells (Ab) and NeuN-labeled pERK-IR cells (Ac) at 40 min after formalin injection in the Vc of wild-type mice. B, C and D: low magnification photomicrographs of Fos protein-IR cells in the dorsal (B), middle (C) and ventral (D) portions of the Vc 40 min after formalin injection in wild-type mice. E: Camera lucida drawings of Fos protein-IR cells in the wild-type mice at 40 min after subcutaneous formalin injection into the whisker pad skin in sections from −1440 µm to the obex. Rostro-caudal distribution of Fos protein-IR cells at 40 min after formalin injection (F: ipsilateral side to injection, G: contralateral side to injection). The mean number of Fos protein-IR cells in the ipsilateral (H) and contralateral (I) Vi/Vc, Vc and C1/C2 in wild-type and GluR2 delta7 KI and GluR3 delta7 KI mice after subcutaneous formalin injection into the whisker pad skin. Boxes in the 3^rd panel in E indicate areas in B, C and D. Both 5 and 40 min data are presented in H and I.

Figure 4. Effect of intrathecal administration of vehicle, CNQX or APV on pERK or Fos expression.

A and B: Mean number of pERK-IR cells in ipsilateral and contralateral Vi/Vc, Vc and C1/C2 regions following i.t. administration of vehicle, CNQX or APV 5 min (A) and 40 min (B) after subcutaneous formalin injection into the whisker pad skin in wild-type mice. C and D: Mean number of Fos protein-IR cells in ipsilateral and contralateral Vi/Vc, Vc and C1/C2 regions following i.t. administration of vehicle, CNQX or APV 5 min (C) and 40 min (D) after subcutaneous formalin injection into the whisker pad skin in wild-type mice.

Figure 5. Reponses of WDR neurons in the Vc.

A: recording sites of WDR neurons in the Vc of the wild-type, GluR2 delta7 KI and GluR3 delta7 KI mice. B: Low- and high-threshold RF areas of WDR neurons in Vc. C: Spontaneous activities of WDR neurons in the Vc. D: Mean spikes of WDR neurons in the Vc following graded mechanical stimulation (brush, press and pinch) of the whisker pad skin. E: Mean afterdischarges of WDR neurons in the Vc followed by noxious mechanical stimulation. The RF size was presented only for untreated animals since spontaneous activity was too high to allow for record accurate delineation of RF size after formalin injection in C.

Figure 6. Responses of Vc WDR neurons following subcutaneous formalin injection into the whisker pad.

Post-stimulus time histograms at the 1^st phase (A) and at the 2^nd phase (B). C: Mean firing frequency of Vc WDR neurons after subcutaneous formalin injection into the whisker pad skin before injection, at 1^st phase and 2^nd phases after subcutaneous formalin injection into the whisker pad skin.