title: Expression of the P2X₂ receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse.
abstract: OBJECTIVE
  To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility.
  
  
  METHODS
  The study examined the distribution of the P2X₂ receptor (P2X₂R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X₂R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm²) and area profile (μm²) of P2X₂R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area.
  
  
  RESULTS
  In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X₂R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X₂R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm²) of P2X₂R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (μm²) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups.
  
  
  CONCLUSIONS
  We demonstrate increases in P2X₂R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.
authors.affiliation: Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, CEP São Paulo 05508-900, Brazil.
authors.forename: Márcia Sanae
authors.initials: MS
authors.lastname: Mizuno
authors.forename: Amanda Rabello
authors.initials: AR
authors.lastname: Crisma
authors.forename: Primavera
authors.initials: P
authors.lastname: Borelli
authors.forename: Patricia
authors.initials: P
authors.lastname: Castelucci
mesh_terms: D000818:Animals; D064032:Calbindin 2; D002795:Choline O-Acetyltransferase / Q000032:analysis; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D005260:Female; D007082:Ileum / Q000294:innervation*; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D009197:Myenteric Plexus / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I / Q000032:analysis; D058476:Receptors, Purinergic P2X2 / Q000032:analysis*; D064030:S100 Calcium Binding Protein G / Q000032:analysis
chemical_list: C576511:Calb2 protein, mouse; D064032:Calbindin 2; D058476:Receptors, Purinergic P2X2; D064030:S100 Calcium Binding Protein G; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D008058:Dihydrolipoamide Dehydrogenase; D002795:Choline O-Acetyltransferase