Constipation is a common symptom in patients with Parkinson\'s disease (PD) and is often associated with depression. Enteric glial cells (EGCs) are crucial for regulating intestinal inflammation and colon motility, and their activation can lead to the death of intestinal neurons. Glial cell line-derived neurotrophic factor (GDNF) has been recognized for its neuroprotective properties in various neurological disorders, including PD. This study explores the potential of GDNF in alleviating intestinal reactive gliosis and inflammation, thereby improving constipation and depressive behavior in a rat model of PD. A PD model was established via unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA). Five weeks post-injury, AAV5-GDNF (2 ~ 5 × 10^11) was intraperitoneally injected into experimental and control rats. Fecal moisture percentage (FMP) and colonic propulsion rate (CPPR) were used to evaluate colon motility. Colon-related inflammation and colonic epithelial morphology were assessed, and depressive behavior was analyzed one week before sampling. PD rats exhibited reduced colonic motility and GDNF expression, along with increased EGC reactivity and elevated levels of pro-inflammatory cytokines IL-1, IL-6, and TNF-α. Additionally, there was an up-regulation of CX43 and a decrease in PGP 9.5 expression. The intraperitoneal injection of AAV-GDNF significantly protected colonic neurons by inhibiting EGC activation and down-regulating CX43. This treatment also led to a notable reduction in depressive-like symptoms in PD rats with constipation. GDNF effectively reduces markers of reactive gliosis and inflammation, and promotes the survival of colonic neurons, and improves colonic motility in PD rats by regulating CX43 activity. Furthermore, GDNF treatment alleviates depressive behavior, suggesting that GDNF or its agonists could be promising therapeutic agents for managing gastrointestinal and neuropsychiatric symptoms associated with PD.

Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disorder characterized by visceral pain and gut dysmotility. However, the specific mechanisms by which Lactobacillus strains relieve IBS remain unclear. Here, we screened Lactobacillus strains from traditional Chinese fermented foods with potential IBS-alleviating properties through in vitro and in vivo experiments. We demonstrated that Lactiplantibacillus plantarum D266 (Lp D266) administration effectively modulates intestinal peristalsis, enteric neurons, visceral hypersensitivity, colonic inflammation, gut barrier function, and mast cell activation. Additionally, Lp D266 shapes gut microbiota and enhances tryptophan (Trp) metabolism, thus activating the aryl hydrocarbon receptor (AhR) and subsequently enhancing IL-22 production to maintain gut homeostasis. Mechanistically, Lp D266 potentially modulates colonic physiology and enteric neurons by microbial tryptophan metabolites. Further, our study indicates that combining Lp D266 with Trp synergistically ameliorates IBS symptoms. Together, our experiments identify the therapeutic efficacy of tryptophan-catabolizing Lp D266 in regulating gut physiology and enteric neurons, providing new insights into the development of probiotic-mediated nutritional intervention for IBS management.

UNASSIGNED: Sacral nerve stimulation (SNS) has been employed for treating constipation. However, its mechanisms involving enteric nervous system (ENS) and motility are largely unknown. In this study, we investigated the possible ENS involvement of SNS in treating Loperamide-induced constipation in rats.
UNASSIGNED: Experiment-1 was designed to study the effects of acute SNS on whole colon transit time (CTT). In experiment-2, we induced constipation by Loperamide and then applied daily SNS or sham-SNS for 1 week. Choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and PGP9.5 in colon tissue were examined at the end of the study. Moreover, survival factors such as phosphorylated AKT (p-AKT) and Glial cell-derived neurotrophic factor (GDNF) were measures by immunohistochemistry (IHC) and western blot (WB).
UNASSIGNED: (1) SNS with one set of parameters shortened CTT starting at 90 min after phenol red administration (p < 0.05). (2) While Loperamide induced slow transit constipation with a significant reduction in fecal pellet number and feces wet weight, daily SNS for a week resolved constipation. (3) Moreover, SNS was able to shorten whole gut transit time comparing to sham-SNS (p = 0.01). (4) Loperamide reduced the number of PGP9.5 and ChAT positive cells, and downregulated ChAT protein expression and upregulated nNOS protein expression, whereas these detrimental effects were significantly reversed by SNS. (5) Furthermore, SNS increased expressions of both GDNF and p-AKT in colon tissue. (6) Vagal activity was reduced following Loperamide (p < 0.01); yet SNS normalized vagal activity.
UNASSIGNED: SNS with appropriate parameters improves opioid-induced constipation and reversed the detrimental effects of Loperamide on enteric neurons possibly via the GDNF-PI3K/Akt pathway.GRAPHICAL ABSTRACT.

Glucagon-like peptide-1 (GLP-1) is a signal peptide released from enteroendocrine cells of the lower intestine. GLP-1 exerts anorectic and antimotility actions that protect the body against nutrient malabsorption. However, little is known about how intestinal GLP-1 affects distant organs despite rapid enzymatic inactivation. We show that intestinal GLP-1 inhibits gastric emptying and eating via intestinofugal neurons, a subclass of myenteric neurons that project to abdominal sympathetic ganglia. Remarkably, cell-specific ablation of intestinofugal neurons eliminated intestinal GLP-1 effects, and their chemical activation functioned as a GLP-1 mimetic. GLP-1 sensing by intestinofugal neurons then engaged a sympatho-gastro-spinal-reticular-hypothalamic pathway that links abnormal stomach distension to craniofacial programs for food rejection. Within this pathway, cell-specific activation of discrete neuronal populations caused systemic GLP-1-like effects. These molecularly identified, delimited enteric circuits may be targeted to ameliorate the abdominal bloating and loss of appetite typical of gastric motility disorders.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

In the enteric nervous system, there exist a huge number of local intrinsic neurons, which control the gastrointestinal functions. Culture of enteric neurons provides a good model system for physiological, electrophysiological, and pharmacological studies. Here, we describe two methods to obtain sufficient enteric neurons from mouse myenteric plexuses by directly culturing primary neurons or inducing neuronal differentiation of enteric neural stem/progenitor cells.

Diabetes can result in pathological changes to enteric nervous system. Our aim was to test the dynamic changes of enteric neurons and identify the role of enteric glial cells (EGCs) in regulating enteric neuron expression in diabetic rats.
A single injection of streptozotocin (STZ) was used to establish diabetic rats. Animals were randomly distributed into diabetic 1-, 4-, 8-, and 16-week groups, as well as age-matched control groups. The PGP9.5- and glial fibrillary acidic protein (GFAP)-immunopositive cells were quantified by immunohistochemistry. The protein levels of PGP9.5, ChAT, nNOS, S-100β, and c-fos were determined by western blotting. The levels of nerve growth factor (NGF), neurotrophin 3 (NT-3), and glial cell-derived neurotrophic factor (GDNF) were tested by ELISA.
An increase in blood glucose and a decrease in body weight were observed following STZ administration. PGP9.5 expression did not change in the diabetic ileum. However, ChAT increased after 16 weeks, and nNOS decreased after 8 and 16 weeks in the ilea of diabetic rats. The absence of degeneration of enteric neurons during the acute stage of the disease could be the consequence of the up-regulation of GFAP, S-100β, and c-fos. Moreover, the content of NGF, NT-3, and GDNF in the ileum increased by varying degrees after 1 and/or 4 weeks of diabetes. Using 2 co-culture models of EGCs and SH-SY5Y cells in a high glucose condition, the supportive role of EGCs was further confirmed.
Enteric glial cell activation can protect enteric neurons from damage due to diabetes in the acute stage of the disease, in part via the promotion of neurotrophin release.

On the basis of the importance of the enteric nervous system (ENS) in gastrointestinal motility, we hypothesized that the ENS may mediate the therapeutic efficacy of electro-acupuncture (EA) in constipation by regulating the mechanisms underlying the effects of EA on gastrointestinal function.
Model mice with constipation were generated by gastric instillation of 0-4°C normal saline. Defecation time and stool (form and wet and dry weight) were assessed. The effect of EA at ST37 or ST25 on colorectal motility and proximal colonic motility was assessed using a water-filled balloon. The expression of protein gene product 9.5 (PGP9.5), the cholinergic neuron marker acetyltransferase (ChAT) and the anticholinergic neuron marker nitric oxide synthase (nNOS) was detected by immunohistochemistry, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis.
ST37 and ST25 improved colorectal pressure; however, ST37 but not ST25 improved proximal colonic pressure. In the proximal colon, the expression of PGP9.5 returned to normal after EA at ST 37, while EA at ST25 did not have this effect. In addition, qPCR and western blot analysis showed that ST37 could downregulate the expression of nNOS and upregulate the expression of ChAT to normal levels, while ST25 could only downregulate the expression of nNOS to normal levels.
Electro-acupuncture at specific acupoints can improve intestinal motility in constipation by altering the ENS and differentially affecting excitatory and inhibitory neurons, restoring the coordination between contraction and relaxation muscles, and working in concert with the central nervous system and peripheral neural pathways.

Optogenetics has emerged as an important tool in neuroscience, especially in central nervous system research. It allows for the study of the brain\'s highly complex network with high temporal and spatial resolution. The enteric nervous system (ENS), the brain in the gut, plays critical roles for life. Although advanced progress has been made, the neural circuits of the ENS remain only partly understood because the appropriate research tools are lacking. In this review, I highlight the potential application of optogenetics in ENS research. Firstly, I describe the development of optogenetics with focusing on its three main components. I discuss the applications in vitro and in vivo, and summarize current findings in the ENS research field obtained by optogenetics. Finally, the challenges for the application of optogenetics to the ENS research will be discussed.

BACKGROUND: Intestinal inflammation is well known to cause gut dysmotility through the effects on the enteric nervous system. Glial-derived neurotrophic factor (GDNF) has been demonstrated to have anti-inflammatory effects and neuronal protective actions. The aim of this study was to investigate whether the GDNF could improve inflammation-induced gut dysmotility.
METHODS: Recombinant adenoviral vectors encoding GDNF (Ad-GDNF) were administered intracolonically in experimental colitis induced by dextran sulfate sodium (DSS). The disease activity index (DAI) and histological score were measured. Colonic transit was measured by using phenol red and assessed with the geometric center. PGP 9.5 immunostaining was used to examine the number and distribution of enteric neurons. The expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and myeloperoxidase (MPO) activity were measured by ELISA assay. The expression of Akt, caspase-3, bcl-2 and PGP 9.5 was analyzed by western blot assay.
RESULTS: A significant neuronal cell loss and a significant delay in colonic transit accompanied with the neuronal loss following inflammation were observed. GDNF prevented partially the loss of enteric neurons and ameliorated significantly experimental colitis and delayed colonic transit by, at least in part, down-regulation of TNF-α and IL-1β expression, decrease of infiltration of leukocytes, and inhibition of neuronal cell apoptosis.
CONCLUSIONS: GDNF reduces inflammation and improves delayed colonic transit in DSS-induced colitis. GDNF may be a useful therapeutic agent for the treatment of gut dysmotility in patients with UC.