title: Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep.
abstract: We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20-25% of myenteric and 65-75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80+/-13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77+/-7% of CALB-IR cells were NeuN-positive. In mature sheep, 73+/-10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.
authors.affiliation: Department of Veterinary Morphophysiology and Animal Production, University of Bologna, 40064 Ozzano Emilia, Bologna, Italy. chiocchetti@vet.unibo.it
authors.forename: Roberto
authors.initials: R
authors.lastname: Chiocchetti
authors.forename: Annamaria
authors.initials: A
authors.lastname: Grandis
authors.forename: Cristiano
authors.initials: C
authors.lastname: Bombardi
authors.forename: Paolo
authors.initials: P
authors.lastname: Clavenzani
authors.forename: Giovanna Lalatta
authors.initials: GL
authors.lastname: Costerbosa
authors.forename: Maria L
authors.initials: ML
authors.lastname: Lucchi
authors.forename: John B
authors.initials: JB
authors.lastname: Furness
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D064092:Calbindin 1; D064026:Calbindins; D017615:Enteric Nervous System / Q000166:cytology* / Q000378:metabolism; D005260:Female; D007082:Ileum / Q000294:innervation*; D007774:Lactation / Q000502:physiology; D008297:Male; D009197:Myenteric Plexus / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D012756:Sheep / Q000254:growth & development*; D013368:Submucous Plexus / Q000166:cytology / Q000378:metabolism
chemical_list: D015415:Biomarkers; C576498:CALB1 protein, human; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G