Abstract
Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1−/− colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1−/− ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies.
Highlights
Neuronal nitric oxide synthase neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns
In diagnosing gut motility dysfunction, damage to the ENS; or loss of neuronal subtypes, is attributed as the causative factor based on histopathological screening for a panel of common neural, glial and interstitial cell markers
We demonstrate that Vasoactive intestinal peptide (Vip) is upregulated, at the transcriptional level, upon global loss of Nos[1]; pointing to compensatory mechanisms within ENS development
Summary
Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1−/− ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Nos[1] (nNOS) deficient mice have been shown to exhibit the clinical phenotype of a number of human gut motility disorders including delayed gastric emptying[8,18,19], and slow transit in the colon[7,20] The use of this global Nos1−/− knockout model provides an opportunity to examine differences in the ENS, after specific loss of nNOS signalling. We propose that disruption of specific neuronal subtypes can have wide ranging; potentially clinically relevant, effects within the ENS which are often undetected
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