Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture

Abstract

S, Mittal RK. Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture. Am J Physiol Gastrointest Liver Physiol 305: G129-G138, 2013. First published May 9, 2013 doi:10.1152/ajpgi.00040.2013The enteric nervous system of the esophagus plays an important role in its sensory and motor functions. Although the esophagus contains enteric neurons, they have never been isolated and characterized in primary culture. We isolated and cultured enteric neurons of the rat esophagus and determined their morphological appearance, chemical coding for neurotransmitters, and functional characteristics. After primary culture for 2 wk, dendrites and axons appeared in the enteric neurons, which usually have one axon and several dendrites. Although the size of neuronal bodies varied from Dogiel type I to type II, their average size was 39 Ϯ 1.8 m in length and 23 Ϯ 1.4 m in width. Immmunocytochemical studies revealed that over 95% of these cells were positively stained for two general neuronal markers, PGP 9.5 or Milli-Mark Fluoro. Chemical coding showed that the neurons were positively stained for choline acetyltransferease (53 Ϯ 6%) or nNOS (66 Ϯ 13%). In functional studies, membrane depolarization and stimulation of several G protein-coupled receptors (GPCRs) induced Ca 2ϩ signaling in the esophageal enteric neurons. The GPCR stimulation was found to induce both intracellular Ca 2ϩ release and extracellular Ca 2ϩ entry. The functional expressions of Ca 2ϩ channels (voltage-gated Ca 2ϩ channels and store-operated channels) and Ca 2ϩ pump (sarcoplasmic reticulum Ca 2ϩ -ATPase) were also demonstrated on these neurons. We have grown, for the first time, esophageal enteric neurons in primary culture, and these contain excitatory and inhibitory neurotransmitters. The functional integrity of GPCRs, Ca 2ϩ channels, and Ca 2ϩ pump in these neurons makes them a useful cell model for further studies. choline acetyltransferease; nitric oxide synthase; cytoplasmic free calcium ion; G protein-coupled receptors; calcium ion channels; calcium ion pump THE ENTERIC NERVOUS SYSTEM (ENS) plays an essential role in the regulation of gastrointestinal motility (4,19,35). Enteric neurons have many different functions in the ENS, and broadly they are divided into three major types, 1) sensory neurons (intrinsic primary afferent neurons), 2) interneurons, and 3) motor neurons (4, 19). Motor neurons are further divided into excitatory and inhibitory types based on their regulatory functions on the gastrointestinal motility (4, 18). Excitatory motor neurons contain excitatory neurotransmitters, such as acetylcholine (ACh), substance P, and N-methyl-D-aspartate (NMDA) (19). On the other hand, inhibitory motor neurons predominantly contain inhibitory neurotransmitters, such as NO, ATP, and VIP (19,35).Enteric neurons located in the wall of esophagus play an important role in its sensory and motor functions (18,19,42). The esophageal enteric motor neurons have their cell bodies in the myenteric ganglia, ...

Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive

“…The Institutional Animal Care and Use Committee at the Veterans Affairs San Diego Healthcare Systems and University of California, San Diego approved the study protocol, and all experiments were conducted in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." Isolation and primary culture of enteric neurons from the rat esophagus were conducted as described previously (9). To isolate fresh enteric neurons from the rat esophagus, we used magnetic bead immunoselection of cells expressing p75 neurotrophin receptor (a neural marker).…”

“…Neurons were immunostained with anti-protein gene product (PGP) 9.5 and anti-neuronal NO synthase (nNOS) antibodies as reported previously (9). Primary culture of the neurons was fixed by FormalFixx (Thermo Scientific, Kalamazoo, MI) for 30 min, washed with PBS for 5 min, treated twice with PBS ϩ 0.05% Triton X-100 ϩ 1% bovine serum albumin (BSA) for 30 min, and processed for immunostaining.…”

“…The change in F360 is an indicator of cell stretch induced by changes in the osmolality of extracellular solution. [Ca 2ϩ ]cyt in the neurons was measured using fura 2-AM at 340-and 380-nm excitation and 510-nm emission, as described previously (2,9 cyt) production in the neurons was measured as described previously (5,38). Briefly, after neurons were cultured on 10-mm glass coverslips for 2 wk, they were loaded with 1 M 4-amino-5-methylamino-2=,7=-difluorofluorescein (DAF-FM) diacetate in PSS at room temperature (22°C) for 30 min and washed in PSS for Ն20 min.…”

“…Moreover, although a growing body of literature suggests that enteric neurons respond directly to mechanical stretch in the small intestine and colon (6,28,36,41), whether isolated enteric neurons are mechanosensitive has not been studied. Recently, we successfully cultured myenteric neurons from the rat esophagus (9). We characterized morphology and chemical coding of these neurons and identified functional expression of G protein-coupled receptors and ion channels (9).…”

“…Recently, we successfully cultured myenteric neurons from the rat esophagus (9). We characterized morphology and chemical coding of these neurons and identified functional expression of G protein-coupled receptors and ion channels (9). Because of the integrity and high quality of these isolated neurons in primary culture, they represent a useful cell model for further studies of myenteric neurons.…”