Correction to: Piezo proteins: incidence and abundance in the enteric nervous system. Is there a link with mechanosensitivity?

The Authors regret forgetting in the original version of this article to mention that this work was also supported by the US National Institute of Health (NIH) (1OT2OD024899-01).

Piezo proteins: incidence and abundance in the enteric nervous system. Is there a link with mechanosensitivity?

Piezo channels play fundamental roles in many physiological processes. Their presence and functional role in the enteric nervous system is still not known. We hypothesize that they play a role in mechanotransduction in enteric neurons. Our aims are to quantify the presence of both Piezo1 and 2 in enteric neurons throughout the gastrointestinal tract using immunohistochemistry and analyze their function(s) using neuroimaging techniques and pharmacological investigations. In order to perform a systematic and comparative study, we performed our experiments in gastrointestinal tissue from guinea pigs, mice and humans. Piezo1 (20-70%) is expressed by both enteric neuronal cell bodies and fibers in the myenteric and submucosal plexi of all the species investigated. Generally, Piezo1 expressing somata are more numerous in the submucosal plexus (50-80%) than in the myenteric plexus (15-35%) apart from the stomach where Piezo1 is expressed in up to 60% of cell bodies. Myenteric Piezo1 neurons mainly (60-100%) but not exclusively, also express nitric oxide synthase, a minority express choline acetyltransferase. In the submucosal plexus, Piezo1 neurons co-express vasoactive intestinal peptide (40-90%). Conversely, expression of Piezo2 is extremely rare in the somata of enteric neurons and is present in few neurites. In functional experiments, 38-76% of the mechanosensitive neurons expressed Piezo1 channels. Statistical analysis showed a positive significant correlation between mechanosensitive and Piezo1 positive neurons. However, pharmacological experiments using an activator and an inhibitor of Piezo channels did not demonstrate changes in mechanotransduction. A major role of Piezo1 in the mechanosensitivity of enteric neurons can be excluded.

Dronabinol Is a Safe Long-Term Treatment Option for Neuropathic Pain Patients.

Treatment of neuropathic pain (NP) symptoms associated with multiple sclerosis (MS) is frequently insufficient. Yet, cannabis is still rarely offered for treatment of pain. This clinical trial aimed at showing the positive benefit-risk ratio of dronabinol. Two hundred forty MS patients with central NP entered a 16-weeks placebo-controlled phase-III study followed by a 32-weeks open-label period. One hundred patients continued therapy for overall up to 119 weeks. Primary endpoint was change of pain intensity on the 11-point Numerical Rating Scale over a 16-weeks treatment period. Safety was assessed on the basis of adverse reactions (ARs), signs of dependency and abuse. Pain intensity during 16-weeks dronabinol and placebo treatment was reduced by 1.92 and 1.81 points without significant difference in between (p = 0.676). Although the proportion of patients with ARs was higher under dronabinol compared to placebo (50.0 vs. 25.9%), it decreased during long-term use of dronabinol (26%). No signs of drug abuse and only one possible case of dependency occurred. The trial results demonstrate that dronabinol is a safe long-term treatment option.

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity.

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca(2+) dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca(2+) transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca(2+)]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca(2+)]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatants.

Sensitivity to Strain and Shear Stress of Isolated Mechanosensitive Enteric Neurons.

Within the enteric nervous system, the neurons in charge to control motility of the gastrointestinal tract reside in a particular location nestled between two perpendicular muscle layers which contract and relax. We used primary cultured myenteric neurons of male guinea pigs to study mechanosensitivity of enteric neurons in isolation. Ultrafast Neuroimaging with a voltage-sensitive dye technique was used to record neuronal activity in response to shear stress and strain. Strain was induced by locally deforming the elastic cell culture substrate next to a neuron. Measurements showed that substrate strain was mostly elongating cells. Shear stress was exerted by hydrodynamic forces in a microchannel. Both stimuli induced excitatory responses. Strain activated 14% of the stimulated myenteric neurons that responded with a spike frequency of 1.9 (0.7/3.2) Hz, whereas shear stress excited only a few neurons (5.6%) with a very low spike frequency of 0 (0/0.6) Hz. Thus, shear stress does not seem to be an adequate stimulus for mechanosensitive enteric neurons (MEN) while strain activates enteric neurons in a relevant manner. Analyzing the adaptation behavior of MEN showed that shear stress activated rapidly/slowly/ultraslowly adapting MEN (2/62/36%) whereas strain only slowly (46%) and ultraslowly (54%) MEN. Paired experiments with strain and normal stress revealed three mechanosensitive enteric neuronal populations: one strain-sensitive (37%), one normal stress-sensitive (17%) and one strain- and stress-sensitive (46%). These results indicate that shear stress does not play a role in the neuronal control of motility but normal stress and strain.

The mast cell degranulator compound 48/80 directly activates neurons.

BACKGROUND: Compound 48/80 is widely used in animal and tissue models as a "selective" mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. METHODOLOGY/PRINCIPAL FINDINGS: We used in vivo recordings from extrinsic intestinal afferents together with Ca(++) imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca(++) and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca(++) transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H(1) and H(2) antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca(++) transients in mast cell-free enteric neuron cultures. CONCLUSIONS/SIGNIFICANCE: The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.