AIM:To investigate the distribution and neurochemical phenotype of endomorphin-2(EM-2)-containing neurons in the submucosal plexus of the rat colon.METHODS:The mid-colons between the right and left flexures were removed from rats,and transferred into Kreb’s solution. For whole-mount preparations,the mucosal,outer longitudinal muscle and inner circularmuscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides,and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide(CGRP),choline acetyltransferase(Ch AT),nitric oxide synthetase(NOS),neuron-specific enolase(NSE),substance P(SP) and vasoactive intestinal peptide(VIP). After staining,all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP,Ch AT,NOS,NSE,SP and VIP,ganglia,which have a clear boundary and neuronal cell outline,were randomly selected from each specimen for this analysis. RESULTS:In the submucosal plexus of the mid-colon,many EM-2-immunoreactive(IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for Ch AT,SP,VIP or NOS. EM-2-IR nerve fibers coursed near Ch AT-IR neurons,and some of these fibers were even distributed around Ch AT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers,but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for Ch AT,SP,VIP or NOS were approximately 91% ± 2.6%,36% ± 2.4%,44% ± 2.5% and 44% ± 4.7%,respectively,but EM-2 did not co-localize with CGRP. CONCLUSION:EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers. AIM: To investigate the distribution and neurochemical phenotype of endomorphin-2 (EM-2) -containing neurons in the submucosal plexus of the rat colon. METHODS: The mid-colons between the right and left flexures were removed from rats, and transferred into The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin -coated glass slides and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide (CGRP), choline acetyltransferase (Ch AT), nitric oxide synthetase (NOS), neuron-specific enolase After staining, all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGR P, Ch AT, NOS, NSE, SP and VIP, ganglia, which have a clear boundary and neuronal cell outline, were randomly selected from each specimen for this analysis. RESULTS: In the submucosal plexus of the mid-colon, many EM- 2-immunoreactive (IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM- 2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for Ch AT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near Ch AT-IR neurons, and some of these fibers were even distributed around Ch AT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers, but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for Ch AT, SEM, P, VIP or NOS were approximately 91% ± 2.6%, 36% ± 2.4%, 44% ± 2.5% and 44% ± 4.7%, respectively, but EM- 2 did not co-localize with CGRP. CONCLUSION: EM-2 -IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.