Volume 136, Issue 5, Pages 1732-1740.e3 (May 2009) Up-Regulation of Anterior Cingulate Cortex NR2B Receptors Contributes to Visceral Pain Responses in Rats  Jing Fan, Xiaoyin Wu, Zhijun Cao, Shengliang Chen, Chung Owyang, Ying Li  Gastroenterology  Volume 136, Issue 5, Pages 1732-1740.e3 (May 2009) DOI: 10.1053/j.gastro.2009.01.069 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Spike firing rates of CRD-excited pACC neurons during infusion of NR2A- or NR2B-specific antagonists in normal and VH rats. Results were collected from 6 and 9 CRD-excited pACC neurons per group in normal and VH rats, respectively. A total of 50 mm Hg CRD but not 30 mm Hg CRD evoked an excitatory response. pACC neurons of EA rats had high spontaneous activities. There were marked increases in response to 30 mm Hg CRD- and 50 mm Hg CRD-evoked pACC neuronal responses in EA rats. (A) Application of NR2A antagonist NVP-AAM077 at a dose of 500 μmol/L had no effect on either basal (CRD 0 mm Hg) or CRD-evoked pACC neuronal activity in both normal and EA rats. (B) Administration of NR2B antagonist Ro25-6981 at doses of 100 μmol/L and 500 μmol/L had no effect on either basal or CRD-evoked pACC neuronal activity in normal rats. In contrast, Ro25-6981 dose-dependently (100 μmol/L, 500 μmol/L, and 2 mmol/L) decreased basal pACC neuronal firing and suppressed or abolished CRD-evoked pACC spike firing in EA rats. These observations suggest that NR2B subtypes of NMDA glutamate receptors are responsible for the mediation of pACC neuronal activities evoked by noxious visceral stimulation in VH rats. Data are presented as means ± SEM. (A) *P < .05 compared with 0 mm Hg CRD; (B) *P < .05 compared with EA vehicle. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Recording of CRD-excited pACC neurons during infusion of NR2A or NR2B antagonists in VH rats. CRD (50 mm Hg) markedly increased pACC neuronal firing during microdialysis of vehicle (artificial cerebrospinal fluid) in VH rats. (A) Microdialysis of NR2A antagonist NVP-AAM077 (500 μmol/L) did not change basal or CRD-evoked pACC neuronal firing. (B) Application of NR2B-receptor antagonist Ro25-6981 (500 μmol/L) significantly decreased basal pACC neuronal activities and suppressed CRD-induced pACC neuronal responses. (C–E) Microphotograph of neurons in the pACC labeled by neurobiotin. Thionine-stained coronal sections show the laminar distribution of neurons located in layer II/III. (C′–E′) Higher magnification of neurobiotin-labeled pACC neurons. Scale bars: C–E, 250 μm; C′–E′, 50 μm. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Effects of bilateral injection of selective NMDA NR2B- or NR2A-receptor antagonists into the pACC on the VMR induced by graded-pressure CRD in normal and VH rats. Results were obtained from 10 normal rats and 12 EA rats. Mean amplitude of the abdominal muscle contraction is expressed as the AUC after baseline subtraction. (A) Summary of data is expressed as AUC after vehicle or NR2B antagonist Ro25-6981 were microinjected bilaterally into the pACC. Infusion of Ro25-6981 at a dose higher than 10 mmol/L had no effect on the VMR to CRD in normal rats; however, it produced marked decreases in the VMR to 20, 40, and 60 mm Hg CRD in EA rats. Injection of a lower dose of Ro25-6981 (1 mmol/L) into the pACC also produced significant decreases in the number of muscle contractions in EA rats. The response was dose dependent. Data are presented as means ± SEM. *P < .05 compared with EA vehicle. (B) Administration of NR2A-receptor antagonist NVP-AAM077 (10 mmol/L) did not change the numbers of spontaneous muscle contractions in either normal or EA rats. Data are presented as means ± SEM. *P < .05 compared with normal vehicle-treated rats. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Microphotographs of thionine-stained coronal sections show injection sites in the pACC of (A) 1 normal rat and (B and C) 2 VH rats. Scale bars: 250 μm. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Expression of NR2A and NR2B subtypes of NMDA receptors in the pACC of normal and VH rats. (A–C) Representative immunoblots from the pACC show the expression of NMDA-receptor subunits NR1, NR2A, NR2B, and actin, with an indication of size (kilodaltons). One control lane is shown along with a lane from EA rats. (C) Western blot showed that the expression level of NR2B, but not NR1 or NR2A, was increased significantly in the pACC of EA rats 10 days after the induction of visceral hypersensitivity. (D) Relative intensity of NMDA-receptor protein from immunoblots was measured by densitometric analysis. Quantification of protein expression in the pACC of EA rats is expressed as the percentage of controls. Each column represents the means ± SEM (n = 3–5 rats for each group). *P < .05, significantly different from control group. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Western blot analysis of pACC protein homogenates from animals receiving bilateral microinfusion of control siRNA or NR2B-siRNA. (A) Representative lanes from each group are shown. pACC proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a membrane. The membrane was probed sequentially with antibodies to NR2B. Treatment with NR2B siRNA resulted in a significant reduction in NR2B protein level, whereas there was no change in protein level of the nontargeted NR2B. (B) Quantification of protein expression in the pACC with NR2B-siRNA microinfusion is expressed as the percentage of controls. Each column represents the means ± SEM (n = 3/condition). **P < .01, significantly different from control group. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 GFP and NR2B immunoreactivity in pACC neurons 3 days after electroporation. (Aa) Coronal section showing GFP-expressing neurons 3 days after electroporation. (Ab) Higher magnification illustrates GFP expression of the inset in Aa. (Ac) With terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling costaining, few cells underwent apoptosis in the targeted pACC. (Ba–c) Microinfusion of a mixture of control siRNA and pEGFP-N1 vector. (Bd–f) Microinfusion of a mixture of pEGFP-N1 vector and NR2B-specific siRNA. (Ba and Bd) Expression of GFP staining in pACC neurons in both control and siRNA-treated groups. (Bb) Immunoreactivity for NB2B receptor in control rats. (Be) Lack of NR2B expression in pACC neurons after treatment with NR2B-siRNA. (Bc) pACC neurons expressing both GFP and NR2B (arrows) appear yellow in the merged image. (C–E) Microphotographs of thionine-stained coronal sections show electroporation sites in 3 rats. Scale bars: Aa, 100 μm; Ab and Ac, 10 μm; Ba–f, 20 μm; C–E, 250 μm. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Effects of bilateral injection of NR2B-specific siRNA into the pACC on the VMR induced by graded-pressure CRD in normal and VH rats. (A) Original electromyograms show that microinjection of NR2B-specific siRNA (1 μg/μL) into the pACC of a normal rat (upper panel) did not change the number of muscle contractions in response to CRD. In the VH rat (lower panel), infusion of NR2B-specific siRNA (1 μg/μL) into the pACC produced significant decreases in response to all CRD pressures (20, 40, 60 mm Hg). (B) Summary of data is expressed as the AUC of the VMR in response to CRD. Results were obtained from 15 normal rats and 16 EA rats. NR2B-siRNA injection had no effect on the VMR in normal rats. In the pACC of NR2B-siRNA injected EA rats, significant inhibition of VMR compared with control siRNA-injected EA rats was observed. Data are presented as means ± SEM. *P < .05 compared with control siRNA-injected EA rats. Gastroenterology 2009 136, 1732-1740.e3DOI: (10.1053/j.gastro.2009.01.069) Copyright © 2009 AGA Institute Terms and Conditions