Mo1872 Enhanced Serotonergic Innervation of DRG During Colitis and Its Role in CaMKII-Mediated CREB Activation

Abstract

The transcription factor CREB (cAMP response element-binding) is implicated to function as a molecular switch underlying neural plasticity, and is activated in the primary sensory neurons in dorsal root ganglia (DRG) during experimental colitis. Activation of CREB is regulated by a list of kinases including Ca2+/calmodulin-dependent protein kinases (CaMK). The neuronal mediators in the sensory pathways contributing to CREB activation in the DRG during colitis are not identified. Recent studies have suggested a role of 5-hydroxytryptamine (5-HT) receptors in mediating sensory activity and visceral hypersensitivity. The AIMS of this study are 1) to examine serotonergic innervation of the DRG during colitis; 2) to examine the expression of metabotropic 5-HT receptor 5-HT4 and its association with phospho (p) -CREB expression in the DRG; and 3) to examine the effects of 5-HT on CREB activation and 5-HT-induced signaling pathways in the DRG. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 7 following induction of colitis. The DRG explants were incubated with 5-HT in the presence or absence of various inhibitors. Western blot and immunohistochemistry techniques were used for examination of protein expression. RESULTS: At 7 days of colitis, we have observed robust serotonergic innervation of the L1-L2 DRG where CREB was also activated. The serotonergic fibers contained small and regularly spaced varicosities. The expression level of 5-HT4 was significantly increased in these DRGs, and was co-localized with p-CREB. Application of 5-HT (10 μM) to DRG culture in the presence of Pargyline (1 mM) increased the expression level of p-CREB. 5-HT-elicited CREB activation was blocked by KN-62 (5 μM) and KN-93 (5 μM), the specific inhibitors of CaMKII. During colitis, the phosphorylation level of CaMKII was also increased in DRG neurons expressing p-CREB. CONCLUSION: TNBS colitis-induced CREB activation in primary sensory neurons is likely mediated by 5-HT-induced Ca2+/calmodulin-dependent pathways.