title: Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon.
abstract: OBJECTIVE
  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 circular muscle 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 (ChAT), 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, ChAT, 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 ChAT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near ChAT-IR neurons, and some of these fibers were even distributed around ChAT-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 ChAT, 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.
  
  
  CONCLUSIONS
  EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Jun-Ping
authors.initials: JP
authors.lastname: Li
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Gao
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Zhen-Zhen
authors.initials: ZZ
authors.lastname: Kou
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Xu-Wen
authors.initials: XW
authors.lastname: Jiao
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Lian-Xiang
authors.initials: LX
authors.lastname: Zhang
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Wu
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Zhong-Yi
authors.initials: ZY
authors.lastname: He
authors.affiliation: Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
authors.forename: Yun-Qing
authors.initials: YQ
authors.lastname: Li
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D003106:Colon / Q000294:innervation*; D005455:Fluorescent Antibody Technique; D007413:Intestinal Mucosa / Q000294:innervation*; D008297:Male; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000294:innervation*; D009197:Myenteric Plexus / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D009924:Organ Culture Techniques; D010641:Phenotype; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D013373:Substance P / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
chemical_list: D015415:Biomarkers; D009842:Oligopeptides; D013373:Substance P; D014660:Vasoactive Intestinal Peptide; C105294:endomorphin 2; D019001:Nitric Oxide Synthase; D002795:Choline O-Acetyltransferase; D010751:Phosphopyruvate Hydratase; D015740:Calcitonin Gene-Related Peptide
keywords: Colon; Endomorphin-2; Enteric neuron; Submucosal plexus; μ-opioid receptor