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Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.

ABSTRACT: Cholinergic neurons have been identified with the acetylcholine synthetic enzyme choline acetyltransferase (ChAT). However, ChAT is difficult to localize in newly differentiated peripheral neurons making the study of cholinergic neuronal development problematic. Consequently, researchers have used mouse reporter lines to indicate the presence of ChAT.Our objective was to determine which ChAT reporter line was the most sensitive indicator of ChAT expression. We utilized two different fluorescent ChAT reporter lines (ChAT-GFP and ChAT-Cre;R26R:floxSTOP:tdTomato) together with immunolocalization of ChAT protein (ChAT-IR) to characterize the spatial and temporal expression of ChAT in myenteric neurons throughout enteric nervous system (ENS) development.ChAT-IR cells were first seen in the intestine at E10.5, even within the migration wavefront of neural precursors. Myenteric neurons within the distal small intestine (dSI) and proximal colon were first labeled by ChAT-IR, then ChAT-GFP, and finally ChAT-Cre tdTomato. The percentage of ChAT-IR neurons is equivalent to adult levels in the dSI by E13.5 and proximal colon by P0. After these stages, the percentages remained relatively constant throughout development despite dramatic changes in neuronal density.These observations indicate that neurotransmitter expression occurs early and there is only a brief gap between neurogenesis and neurotransmitter expression. Our finding that the proportion of ChAT myenteric neurons reached adult levels during embryonic development suggests that the fate of cholinergic neurons is tightly regulated and that their differentiation might influence further neuronal development. ChAT-GFP is a more accurate indicator of early ENS cholinergic neuronal differentiation than the ChAT-Cre;R26R:floxSTOP:tdTomato reporter mouse.

SUBMITTER: Erickson CS

PROVIDER: S-EPMC4037379 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.

Erickson C S CS Lee S J SJ Barlow-Anacker A J AJ Druckenbrod N R NR Epstein M L ML Gosain A A

Neurogastroenterology and motility 20140408 6

<h4>Background</h4>Cholinergic neurons have been identified with the acetylcholine synthetic enzyme choline acetyltransferase (ChAT). However, ChAT is difficult to localize in newly differentiated peripheral neurons making the study of cholinergic neuronal development problematic. Consequently, researchers have used mouse reporter lines to indicate the presence of ChAT.<h4>Methods</h4>Our objective was to determine which ChAT reporter line was the most sensitive indicator of ChAT expression. We ...[more]

PMID: 24712519

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Project description:BACKGROUND: Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions. METHODOLOGY/PRINCIPAL FINDINGS: We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca(++)/high Mg(++). Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin. CONCLUSIONS/SIGNIFICANCE: We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.

Project description:The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca2+ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca2+ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f+/ChAT+) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT3Rs). Cholinergic MG neurons also display small, spontaneous Ca2+ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na+ channel dependent impulses, presynaptic Ca2+ channels and postsynaptic receptor function reveal that the Ca2+ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT3Rs. Electrical stimulation of axonal connectives to MG evoked Ca2+ responses in the neurons that similarly depended on nAChRs or/and 5-HT3Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca2+ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca2+ transients and stimulus evoked Ca2+ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT3Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca2+ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.

Dataset Information

Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.

Publications

Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.

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