NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity

Abstract

Background: Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing.

Results: CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs.

Conclusion: Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation.

Figure 1

CANs express TTX-resistant sodium channel Na_v1.9. Photomicrographs of DiI labelled neurons in the submucosal plexus of colon showing co-localization with known nociceptive sodium channel Na_v1.9. Scale bars = 20 μm.

Figure 2

Expression of the TRPV1 receptor is eliminated following treatment with RTX. Photomicrographs of DiI and TRPV1 expression in control(a') and RTX(a") lesioned rats in submucosal plexus of the colon. RTX results in a decrease in the expression of TRPV1 labeling in the colon. Photomicrographs that show a decrease in both DiI and TRPV1 expression in lumbosacral DRG neurons(b") as compared to controls(b'). (c) Immunohistochemical characterization of CANs following RTX lesioning showed no change in peptide and receptor expression as compared to controls. RTX results in a decrease in the expression of TRPV1 labeling in thoracolumbar DRG neurons (e). Scale bars = 20 μm(a) and 50 μm(b, e).

Figure 3

Colonic innervation from lumbosacral DRG neurons is reduced following RTX treatment. (a)Application of the retrograde tracer DiI into the colon wall revealed a 70% decrease in the number of DRG neurons that innervate colon as compared to controls (control 87 ± 12;RTX 26 ± 6;n = 5, p < 0.001). DiI neurons no longer co-localized with the TRPV1 receptor(b) (p < 0.0001) or the PAR2 receptor(c) (p < 0.0001) following RTX lesioning. However, there was no change in the expression of the NK1 receptor (d). Scale bars = 50 μm.

Figure 4

TNBS induced visceral but not somatic hypersensitivity is still present following RTX treatment. The presence of inflammation was seen in animals 14 days post-TNBS inflammation as shown by infiltration of neutrophils in the lamina propria, an increase in mast cells and mucosal ulceration (a). Fourteen days post-inflammation resulted in a decrease in balloon threshold for both Non-RTX and RTX animals. However, a decrease in peripheral mechanical threshold was seen in non-RTX animals but not RTX animals (b).

Figure 5

NMDA receptor expression on CANs increases following TNBS-induced inflammation. The NMDA receptor NR1 cassettes N1 (a) and C1(c) are up-regulated in DiI labelled neurons 14 days post-inflammation. N1 co-localization increased 50% (70% ± 3.3) as compared to controls (10% ± 1.145;p < 0.0001) (b). Where as C1 co-localization increased 51% (59.8% ± 1.7) as compared to controls (8% ± 8.6;p < 0.001) (d). Scale bars = 20 μm.

Figure 6

Activation of the PAR2 receptor expressed on CANs following TNBS induced inflammation. Approximately 40% of CANs expressing the PAR2 receptor are activated following inflammation as shown by an antibody that detects the N-terminal end of the PAR2 receptor (a, b) (p < 0.0003). Typical RT-PCR products for PAR2 (598 bp) and GAPDH (720 bp) (c). RT-PCR analysis revealed that mRNA expression of PAR2 does not change due to TNBS induced inflammation as compared to controls (c, d). A 60% decrease in the co-localization of SP expressed on CANs was seen following inflammation as compared to controls (p < 0.0008) (e). Scale bars = 20 μm.