Dopamine Transporter Genetic Reduction Induces Morpho-Functional Changes in the Enteric Nervous System

Abstract

Antidopaminergic gastrointestinal prokinetics are indeed commonly used to treat gastrointestinal motility disorders, although the precise role of dopaminergic transmission in the gut is still unclear. Since dopamine transporter (DAT) is involved in several brain disorders by modulating extracellular dopamine in the central nervous system, this study evaluated the impact of DAT genetic reduction on the morpho-functional integrity of mouse small intestine enteric nervous system (ENS). In DAT heterozygous (DAT^+/-) and wild-type (DAT^+/+) mice (14 ± 2 weeks) alterations in small intestinal contractility were evaluated by isometrical assessment of neuromuscular responses to receptor and non-receptor-mediated stimuli. Changes in ENS integrity were studied by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (). DAT genetic reduction resulted in a significant increase in dopamine-mediated effects, primarily via D1 receptor activation, as well as in reduced cholinergic response, sustained by tachykininergic and glutamatergic neurotransmission via NMDA receptors. These functional anomalies were associated to architectural changes in the neurochemical coding and S100β immunoreactivity in small intestine myenteric plexus. Our study provides evidence that genetic-driven DAT defective activity determines anomalies in ENS architecture and neurochemical coding together with ileal dysmotility, highlighting the involvement of dopaminergic system in gut disorders, often associated to neurological conditions.

Keywords: confocal microscopy; dopamine transporter; enteric nervous system; neuromuscular contractility; small intestine.

Figure 1

DAT hypofunction affects dopaminergic neuromuscular response. (A) Inhibitory concentration–response curves to dopamine (DA) on spontaneous contractile activity of isolated ileal preparations from WT and DAT^+/− mice. (B) Representative tracings of responses induced by increasing dopamine (DA) concentration of in WT and DAT^+/− preparations. (C) 30 µM dopamine-induced relaxation of CCh-precontracted ileal preparations with or without SCH23390 or sulpiride in isolated ileal segments of WT and DAT^+/− mice. (D) Representative confocal microphotographs showing the distribution of D1R (green, marker for D1 receptor) and HuC/D (red, pan-neuronal marker) and (E) D1R density index in LMMP preparations of WT and DAT^+/− mice. Scale bars = 22 μm. Data are reported as mean ± SEM. * p < 0.05, *** p < 0.001 vs. WT mice; °° p < 0.01, °°° p < 0.001 vs. respective control in absence of antagonist. n = 5 mice/group.

Figure 2

DAT hypofunction affects dopaminergic neuromuscular response. Neuromuscular excitatory response induced by 4 Hz-EFS in absence or presence of 10 µM dopamine after pretreatment with SCH23390, sulpiride, MRS2500 or L-NAME in isolated ileal preparations of WT (A) and DAT^+/− (B) mice. Data are reported as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. respective control mice. n = 5 mice/group.

Figure 3

DAT influences NO-mediated relaxation. (A) 10 Hz EFS-evoked relaxation in NANC conditions with or without or 100 μM L-NAME (pan-NOS inhibitor) in ileal segments of WT and DAT^+/− mice. (B) Representative confocal microphotographs showing the distribution of HuC/D (red) and nNOS (green) and (C) analysis of nNOS⁺ neurons in ileal LMMPs of WT and DAT^+/− mice (bars = 22 μm). Data are reported as mean ± SEM. * p < 0.05 vs. WT; ° p < 0.05 vs. respective control in NANC condition. n = 5 mice/group.

Figure 4

DAT hypofunction influences neuromuscular response. (A) Neuromuscular excitatory response induced by EFS (0–50 Hz) in isolated ileal preparations of WT and DAT^+/− mice. (B) Representative tracings of responses induced by EFS in WT and DAT^+/− preparations. (C) Representative confocal microphotographs showing the distribution of ChAT (green, marker for cholinergic neurons) and HuC/D (red, pan-neuronal marker) and (D) ChAT density index in LMMP preparations of WT and DAT^+/− mice. Scale bars = 22 μm. Data are reported as mean ± SEM. * p < 0.05 vs. WT mice. n = 5 mice/group.

Figure 5

DAT hypofunction influences tachykininergic neurotransmission. (A) Tachykininergic nerve-evoked contractions induced by 10 Hz-EFS, in NANC condition with or without L-NAME or L732138 in isolated ileal preparations of WT and DAT^+/− mice. (B) Representative confocal microphotographs showing the distribution of substance P (green) and HuC/D (red, pan-neuronal marker) and (C) substance P density index in LMMP preparations of WT and DAT^+/− mice. Scale bars = 22 μm. Data are reported as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT mice; °° p < 0.01, °°° p < 0.001 vs. respective control in absence of antagonists; #p < 0.05 vs. respective control in NANC condition; §§ p < 0.01, §§§ p < 0.001 vs. respective control with L-NAME. n = 5 mice/group.

Figure 6

DAT hypofunction influences glutamatergic neurotransmission. (A) 0.1 mM and 1 mM NMDA-induced contractions in isolated ileal preparations of WT and DAT^+/− mice. (B) GluN1 mRNA levels in LMMP preparations of WT and DAT^+/− mice. Data are reported as mean ± SEM. * p < 0.05 vs. WT mice. n = 5 mice/group.

Figure 7

DAT hypofunction affects neuroglial phenotype. (A) Representative confocal microphotographs showing the distribution of HuC/D⁺ neurons (cyan), GFAP⁺ (yellow) and S100β⁺ (magenta) glial cells in WT and DAT^+/− LMMPs preparations (bars = 22 µm). (B,C) Changes in GFAP (B) and S100β (C) density index in WT and DAT^+/− LMMPs preparations. (D) Analysis of HuC/D⁺ neurons in ileal LMMPs of WT and DAT^+/− mice. Data are reported as mean ± SEM. * p < 0.05 vs. WT. n = 5 mice/group.