Sodium oligomannate activates the enteroendocrine-vagal afferent pathways in APP/PS1 mice.

Enteroendocrine cells (EECs) and vagal afferent neurons constitute functional sensory units of the gut, which have been implicated in bottom-up modulation of brain functions. Sodium oligomannate (GV-971) has been shown to improve cognitive functions in murine models of Alzheimer's disease (AD) and recently approved for the treatment of AD patients in China. In this study, we explored whether activation of the EECs-vagal afferent pathways was involved in the therapeutic effects of GV-971. We found that an enteroendocrine cell line RIN-14B displayed spontaneous calcium oscillations due to TRPA1-mediated calcium entry; perfusion of GV-971 (50, 100 mg/L) concentration-dependently enhanced the calcium oscillations in EECs. In ex vivo murine jejunum preparation, intraluminal infusion of GV-971 (500 mg/L) significantly increased the spontaneous and distension-induced discharge rate of the vagal afferent nerves. In wild-type mice, administration of GV-971 (100 mg· kg-1 ·d-1, i.g. for 7 days) significantly elevated serum serotonin and CCK levels and increased jejunal afferent nerve activity. In 7-month-old APP/PS1 mice, administration of GV-971 for 12 weeks significantly increased jejunal afferent nerve activity and improved the cognitive deficits in behavioral tests. Sweet taste receptor inhibitor Lactisole (0.5 mM) and the TRPA1 channel blocker HC-030031 (10 µM) negated the effects of GV-971 on calcium oscillations in RIN-14B cells as well as on jejunal afferent nerve activity. In APP/PS1 mice, co-administration of Lactisole (30 mg ·kg-1 ·d-1, i.g. for 12 weeks) attenuated the effects of GV-971 on serum serotonin and CCK levels, vagal afferent firing, and cognitive behaviors. We conclude that GV-971 activates sweet taste receptors and TRPA1, either directly or indirectly, to enhance calcium entry in enteroendocrine cells, resulting in increased CCK and 5-HT release and consequent increase of vagal afferent activity. GV-971 might activate the EECs-vagal afferent pathways to modulate cognitive functions.

5-Hydroxytryptamine 4 Receptor Agonist Attenuates Diabetic Enteric Neuropathy through Inhibition of the Receptor-Interacting Protein Kinase 3 Pathway.

Necroptosis, considered as a form of programmed cell death, contributes to neural loss. The 5-hydroxytryptamine 4 receptor (5-HT4R) is involved in neurogenesis in the enteric nervous system. However, whether the activation of 5-HT4R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is unclear. This study aimed to explore the beneficial effects of 5-HT4R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT4Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT4R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3, and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly up-regulated in the colon of diabetic mice. Treatment with 5-HT4R agonist appeared to inhibit diabetes-induced elevation of RIPK3, phosphorylated RIPK3, and MLKL in the colon of mice. Diabetes-induced up-regulation of MLKL in both the mucosa and the muscularis of the colon was prevented by Ripk3 deletion. Moreover, diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by Ripk3 removal. These findings suggest that activation of 5-HT4Rs could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

G protein-coupled estrogen receptor 1 deficiency impairs adult hippocampal neurogenesis in mice with schizophrenia.

OBJECTIVE: This study aimed to confirm that G protein-coupled estrogen receptor 1 (GPER1) deficiency affects cognitive function by reducing hippocampal neurogenesis via the PKA/ERK/IGF-I signaling pathway in mice with schizophrenia (SZ). METHODS: Mice were divided into four groups, namely, KO Con, WT Con, KO Con, and WT SZ (n = 12 in each group). All mice were accustomed to the behavioral equipment overnight in the testing service room. The experimental conditions were consistent with those in the animal house. Forced swimming test and Y-maze test were conducted. Neuronal differentiation and maturation were detected using immunofluorescence and confocal imaging. The protein in the PKA/ERK/IGF-I signaling pathway was tested using Western blot analysis. RESULTS: GPER1 KO aggravated depression during forced swimming test and decreased cognitive ability during Y-maze test in the mouse model of dizocilpine maleate (MK-801)-induced SZ. Immunofluorescence and confocal imaging results demonstrated that GPER1 knockout reduced adult hippocampal dentate gyrus neurogenesis. Furthermore, GPER1-KO aggravated the hippocampal damage induced by MK-801 in mice through the PKA/ERK/IGF-I signaling pathway. CONCLUSIONS: GPER1 deficiency reduced adult hippocampal neurogenesis and neuron survival by regulating the PKA/ERK/IGF-I signaling pathway in the MK-801-induced mouse model of SZ.

Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance.

The rostral ventromedial medulla (RVM) exerts bidirectional descending modulation of pain attributable to the activity of electrophysiologically identified pronociceptive ON and antinociceptive OFF neurons. Here, we report that GABAergic ON neurons specifically express G protein-coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, but their ablation abrogated, pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain, and ameliorated morphine analgesia through desensitizing µ-type opioid receptor-mediated (MOR-mediated) activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia, and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly indicate that GPER is a marker for GABAergic ON cells and illuminate the mechanisms underlying hormonal regulation of pain and analgesia, thus highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

Selective activation of the hypothalamic orexinergic but not melanin-concentrating hormone neurons following pilocarpine-induced seizures in rats.

Introduction: Sleep disorders are common comorbidities in patients with temporal lobe epilepsy (TLE), but the underlying mechanisms remain poorly understood. Since the lateral hypothalamic (LH) and the perifornical orexinergic (ORX) and melanin-concentrating hormone (MCH) neurons are known to play opposing roles in the regulation of sleep and arousal, dysregulation of ORX and MCH neurons might contribute to the disturbance of sleep-wakefulness following epileptic seizures. Methods: To test this hypothesis, rats were treated with lithium chloride and pilocarpine to induce status epilepticus (SE). Electroencephalogram (EEG) and electromyograph (EMG) were recorded for analysis of sleep-wake states before and 24 h after SE. Double-labeling immunohistochemistry of c-Fos and ORX or MCH was performed on brain sections from the epileptic and control rats. In addition, anterograde and retrograde tracers in combination with c-Fos immunohistochemistry were used to analyze the possible activation of the amygdala to ORX neural pathways following seizures. Results: It was found that epileptic rats displayed prolonged wake phase and decreased non-rapid eye movement (NREM) and rapid eye movement (REM) phase compared to the control rats. Prominent neuronal activation was observed in the amygdala and the hypothalamus following seizures. Interestingly, in the LH and the perifornical nucleus, ORX but not MCH neurons were significantly activated (c-Fos+). Neural tracing showed that seizure-activated (c-Fos+) ORX neurons were closely contacted by axon terminals originating from neurons in the medial amygdala. Discussion: These findings suggest that the spread of epileptic activity from amygdala to the hypothalamus causes selective activation of the wake-promoting ORX neurons but not sleep-promoting MCH neurons, which might contribute to the disturbance of sleep-wakefulness in TLE.

Activation of uroepithelial 5-HT4R inhibits mechanosensory activity of murine bladder afferent nerves.

Serotonin (5-HT) is known to act via multiple 5-HT receptors at spinal and supraspinal levels to regulate micturition. However, the contribution of peripheral 5-HT and its receptors in bladder physiology and pathology is not very well understood, despite evidence showing expression of multiple 5-HT receptors in the bladder wall and 5-HT may activate bladder afferent nerves. The current study was designed to investigate the possible role of 5-HT4R in modulation of the sensitivity of bladder afferents to bladder filling. Immunofluorescent staining showed abundant 5-HT4R immunoreactivity largely confined to the uroepithelium in wild type (WT) but not 5-HT4R-/- mice. In the ex vivo bladder-pelvic nerve preparation, intravesical application of the 5-HT4R agonist RS67333 (1-30 µm) caused concentration-dependent decreases of the pelvic nerve response to bladder filling. Such effect was not observed in the presence of 5-HT4R antagonist GR125487 or in 5-HT4R-/- preparations. A cohort of 5-HT4R-/- and WT control mice were treated with intraperitoneal injections of cyclophosphamide (CYP) (75 mg/kg, three times at 2 days interval) to induce chronic cystitis. Void spot analysis showed that CYP-treated 5-HT4R-/- mice urinated more frequently than their CYP-treated WT counterparts. Concomitantly, bladder afferents of CYP-treated 5-HT4R-/- mice displayed exaggerated sensitivity to bladder filling in comparison with the CYP-treated WT controls. These data suggest that 5-HT4R expressed on uroepithelial cells plays an inhibitory role in mechanosensory transduction in the bladder. Loss of 5-HT4R-mediated inhibition may enhance bladder afferent sensitivity and exacerbate bladder overactivity in pathological conditions. We propose that 5-HT4R agonists might be exploited for the treatment of overactive and painful bladder symptoms.

G-Protein-Coupled Estrogen Receptor (GPER) in the Rostral Ventromedial Medulla Is Essential for Mobilizing Descending Inhibition of Itch.

Chronic itch is a troublesome condition and often difficult to cure. Emerging evidence suggests that the periaqueductal gray (PAG)-rostral ventromedial medulla (RVM) pathway may play an important role in the regulation of itch, but the cellular organization and molecular mechanisms remain incompletely understood. Here, we report that a group of RVM neurons distinctively express the G-protein-coupled estrogen receptor (GPER), which mediates descending inhibition of itch. We found that GPER+ neurons in the RVM were activated in chronic itch conditions in rats and mice. Selective ablation or chemogenetic suppression of RVM GPER+ neurons resulted in mechanical alloknesis and increased scratching in response to pruritogens, whereas chemogenetic activation of GPER+ neurons abrogated itch responses, indicating that GPER+ neurons are antipruritic. Moreover, GPER-deficient mice and rats of either sex exhibited hypersensitivity to mechanical and chemical itch, a phenotype reversible by the µ type opioid receptor (MOR) antagonism. Additionally, significant MOR phosphorylation in the RVM was detected in chronic itch models in wild-type but not in GPER-/- rats. Therefore, GPER not only identifies a population of medullary antipruritic neurons but may also determine the descending antipruritic tone through regulating µ opioid signaling.SIGNIFICANCE STATEMENT Therapeutic options for itch are limited because of an as yet incomplete understanding of the mechanisms of itch processing. Our data have provided novel insights into the cellular organization and molecular mechanisms of descending regulation of itch in normal and pathologic conditions. GPER+ neurons (largely GABAergic) in the RVM are antipruritic neurons under tonic opioidergic inhibition, activation of GPER promotes phosphorylation of MOR and disinhibition of the antipruritic GPER+ neurons from inhibitory opioidergic inputs, and failure to mobilize GPER+ neurons may result in the exacerbation of itch. Our data also illuminate on some of the outstanding questions in the field, such as the mechanisms underlying sex bias in itch, pain, and opioid analgesia and the paradoxical effects of morphine on pain and itch.

Emerging roles of the TRPV4 channel in bladder physiology and dysfunction.

The transient receptor potential vanilloid type 4, TRPV4, is a polymodal cation channel which can be activated by diverse stimuli including mechanical, thermal and chemical cues. In the urinary bladder, TRPV4 is not only abundantly expressed in the urothelium but may also be localized in subepithelium, detrusor smooth muscles and afferent neurons. Emerging evidence indicates that the TRPV4 channel plays a sensory role in the uroepithelium, where it may regulate the release of sensory mediators such as ATP, which in turn modulates afferent nerve activity in response to bladder filling during the urination cycle. TRPV4 may also directly regulate detrusor contractility and the urothelial barrier function. Altered TRPV4 expression has been detected in various pathological bladder conditions. As such, TRPV4 may be a promising therapeutic target for bladder dysfunctions.

GPER-Deficient Rats Exhibit Lower Serum Corticosterone Level and Increased Anxiety-Like Behavior.

Ample evidence suggests that estrogens have strong influences on the occurrence of stress-related mood disorders, but the underlying mechanisms remain poorly understood. Through multiple approaches, we demonstrate that the G protein-coupled estrogen receptor (GPER) is widely distributed along the HPA axis and in brain structures critically involved in mood control. Genetic ablation of GPER in the rat resulted in significantly lower basal serum corticosterone level but enhanced ACTH release in response to acute restraint stress, especially in the female. GPER-/- rats of either sex displayed increased anxiety-like behaviors and deficits in learning and memory. Additionally, GPER deficiency led to aggravation of anxiety-like behaviors following single-prolonged stress (SPS). SPS caused significant decreases in serum corticosterone in WT but not in GPER-deficient rats. The results highlight an important role of GPER at multiple sites in regulation of the HPA axis and mood.

[Perinatal antibiotics exposure causes increase in serum 5-hydroxytryptamine level as well as changes in behavior and gastrointestinal motility in the male offspring in mice].

The current study was aimed to investigate the potential effects of perinatal exposure to therapeutic dose of penicillin and cefixime on the cognitive behaviors, gastrointestinal (GI) motility and serum 5-hydroxytryptamine (5-HT) level in the offspring. Pregnant rats were continuously treated with cefixime or penicillin in the period between 1 week before and 1 week after labor. Behavior tests, including social preference, self-grooming and elevated plus maze tests, and intestinal motility tests were carried out on the offspring at age of 4 to 10 weeks. Serum 5-HT levels were detected with ELISA, and potassium/sodium hyperpolarization activated cyclic nucleotide-gated channel 2 (HCN2) and tryptophan hydroxylase 1 (TPH1) expression levels in colon epithelium of offspring were detected by Western blot and RT-qPCR. The results showed that, compared with the naive group, cefixime increased social behavior in the female offspring, but did not affect the male offspring. Compared with the naive group, cefixime significantly decreased colonic and intestinal transits, and increased cecum net weight and standardized cecum net weight in the male offspring, but did not affect the female offspring. The serum 5-HT levels in the male offspring, rather than the female offspring, in cefixime and penicillin groups were significantly increased compared with that in the naive group. The protein expression level of HCN2 in colon epithelium of the offspring in cefixime group was significantly down-regulated, and the TPH1 expression level was not significantly changed, compared with that in the naive group. These results suggest that perinatal antibiotics exposure may affect neural development and GI functions of the offspring, and the mechanism may involve peripheral 5-HT and gender-dependent factor.

[Effects and pathophysiological significance of intestinal flora on the enteric neuro-endocrine-immune system].

Interactions among the nervous, the endocrine and the immune systems enable the gut to respond to the dietary products, pathogens and microbiota, which maintains the homeostasis of the body. However, dysbiosis may induce or aggravate the gastrointestinal (GI) and extra-GI diseases through changing the activities of enteric nervous system (ENS), enteroendocrine cells and enteric immune cells. Here we review recent advances in the understandings on how intestinal flora may impact the enteric neuro-endocrine-immune system in the gut, thereby contributing to the regulation of pathophysiological processes.

The novel estrogen receptor GPER1 decreases epilepsy severity and susceptivity in the hippocampus after status epilepticus.

The steroid hormone 17ß-estradiol (estrogen) exerts neuroprotective effects in several types of neurological disorders including epilepsy. The novel G protein-coupled estrogen receptor 1 (GPER1), also called GPR30, mediates the non-genomic effects of 17ß-estradiol. However, the specific role of GPER1 in status epilepticus (SE) remains unclear. In this report, we evaluated the effects of GPER1 on the hippocampus during SE and the underlying mechanism was studied. Our results revealed that pilocarpine-induced GPER1-KD epileptic rats exhibited a shorter latency to generalized convulsions and strikingly elevated seizure severity. Additionally, the electroencephalographic seizure activity also corresponded to these results. Fast-Fourier analysis indicated an enhancement of power in the theta and alpha bands during SE in GPER1-KD rats. In addition, epilepsy-induced pathological changes were dramatically exacerbated in GPER1-KD rats, including neuron damage and neuroinflammation in hippocampus. GPER1 might be associated with the susceptibility to and severity of epileptic seizures. In summary, our results suggested that GPER1 plays a neuroprotective role in SE, and might be a candidate target for epilepsy therapy.

Activation of PGC-1α and Mitochondrial Biogenesis Protects Against Prenatal Hypoxic-ischemic Brain Injury.

Survivals after prenatal hypoxia-ischemia (HI) usually suffer long-lasting cognitive defects. Reduced blood-oxygen supplies and the following reperfusion cause mitochondrial injury. Damaged mitochondria could be replaced by mitochondrial biogenesis program and peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) is the specific up-regulator. The objective of this study was to determine whether PGC-1α and mitochondrial biogenesis participate in the resistant responses of an immature brain to prenatal HI. We used a pregnant rat model of transient occlusion of uterine perfusion to induce intrauterine HI associated brain injury. SH-SY5Y cells exposed to oxygen-glucose deprivation was used to investigate the HI induced reactions in vitro. PGC-1α and its downstream signaling pathway (NRF-1 and TFAM) were examined by Western blot and quantitative Real-time PCR. Mitochondrial respiratory enzyme COX-IV was investigated by Western blot and immunohistochemistry. Mitochondrial density and morphology was detected by transmission electron microscopy. The hippocampal injury and cognitive function were examined. We found that the intrauterine HI triggered PGC-1α-NRF-1-TFAM pathway in both protein and mRNA levels. COX-IV expression significantly increased after HI injury. Intrauterine HI induced both mitochondrial impairment and mitochondrial biogenesis. Postnatal administration of pioglitazone further promoted PGC-1α and mitochondrial biogenesis, alleviated hippocampal injury, and improved performance in the behavioral tasks after intrauterine HI. Our investigation implicated activation of PGC-1α, and mitochondrial biogenesis is a neuroprotective mechanism against brain injury caused by systemic prenatal HI. Promotion of PGC-1α by pioglitazone might be a potential treatment for protecting against hippocampal injury and cognitive defects after intrauterine HI.

Genetic knockout of the G protein-coupled estrogen receptor 1 facilitates the acquisition of morphine-induced conditioned place preference and aversion in mice.

Drug addiction is considered the pathological usurpation of normal learning and memory. G protein-coupled estrogen receptor 1 (GPER1) plays an important role in normal learning and memory, but the effect of GPER1 on addiction-related pathological memory has not been reported. Our study used GPER1 knockout (GPER1 KO) and wild-type (WT) mice to compare the sensitivity differences of morphine- and sucrose-induced conditioned place preference (CPP) and naloxone-induced conditioned place aversion (CPA), and differences in dopamine (DA) content in the nucleus accumbens (NAc) were determined by high performance liquid chromatography (HPLC). The results showed that GPER1 KO mice showed higher sensitivity to morphine-induced CPP and naloxone-induced CPA, and corresponding to the behavioral effect, the DA content in the NAc of GPER1 KO mice was significantly higher than that of WT mice. Interestingly, the sensitivity of GPER1 KO mice to sucrose-induced CPP did not differ from that of the WT mice, and there was no significant difference in the DA content in the NAc between the two genotypes of mice. GPER1 knockout promoted the formation of morphine addiction-related positive and aversive memory, and its molecular biological mechanism may be associated with increased DA content in the NAc. Therefore, GPER1 plays an important role in the formation of addiction-related pathological memory and may become a potential molecular target for drug addiction therapy.

TRPV4 receptor as a functional sensory molecule in bladder urothelium: Stretch-independent, tissue-specific actions and pathological implications.

The newly recognized sensory role of bladder urothelium has generated intense interest in identifying its novel sensory molecules. Sensory receptor TRPV4 may serve such function. However, specific and physiologically relevant tissue actions of TRPV4, stretch-independent responses, and underlying mechanisms are unknown and its role in human conditions has not been examined. Here we showed TRPV4 expression in guinea-pig urothelium, suburothelium, and bladder smooth muscle, with urothelial predominance. Selective TRPV4 activation without stretch evoked significant ATP release-key urothelial sensory process, from live mucosa tissue, full-thickness bladder but not smooth muscle, and sustained muscle contractions. ATP release was mediated by Ca2+-dependent, pannexin/connexin-conductive pathway involving protein tyrosine kinase, but independent from vesicular transport and chloride channels. TRPV4 activation generated greater Ca2+ rise than purinergic activation in urothelial cells. There was intrinsic TRPV4 activity without exogeneous stimulus, causing ATP release. TRPV4 contributed to 50% stretch-induced ATP release. TRPV4 activation also triggered superoxide release. TRPV4 expression was increased with aging. Human bladder mucosa presented similarities to guinea pigs. Overactive bladders exhibited greater TRPV4-induced ATP release with age dependence. These data provide the first evidence in humans for the key functional role of TRPV4 in urothelium with specific mechanisms and identify TRPV4 up-regulation in aging and overactive bladders.

Oral Administration of Penicillin or Streptomycin May Alter Serum Serotonin Level and Intestinal Motility via Different Mechanisms.

BACKGROUND/AIMS: Enterochromaffin cells (EC cells) constitute the largest population of enteroendocrine cells and release serotonin (5-HT) in response to mechanical and chemical cues of the gastrointestinal tract (GIT). How EC cells respond to altered microbiota such as due to antibiotic treatments remain poorly understood. We hypothesized that the pacemaker channel HCN2 might contribute to the regulation of EC cells functions and their responses to antibiotics-induced changes in intestinal flora. METHODS: Mice were given either penicillin or streptomycin or both in drinking water for 10 consecutive days. The changes in the profile of short chain fatty acids (SCFAs) in the cecum following penicillin or streptomycin treatments were tested by GC-MS. Serum 5-HT content, whole intestinal transit time, fecal water content, cecum weight and expression of HCN2 and TPH1 in cecal mucosa were measured. Ivabradine (a HCN channels blocker) was used to explore the role of HCN2 in penicillin-induced changes in 5-HT availability and intestinal motility. RESULTS: HCN2 immunofluorescence was detected on intestinal EC cells. Both penicillin and streptomycin caused significant reduction in total SCFAs in the cecum, with the penicillin-treated group showing greater reductions in butyrate, isobutyrate and isovalerate levels than the streptomycin group. The expression of HCN2 was increased in the mice treated with penicillin, whereas TPH1 expression was increased in the mice treated with streptomycin. Mice treated with antibiotics all had larger and heavier cecum, elevated serum 5-HT level and increased fecal water content. Besides, mice treated with penicillin had prolonged intestinal transit time. Intraperitoneal injection of Ivabradine attenuated the effect of penicillin on serum 5-HT level, cecum size and weight, intestinal motility, and fecal water content. CONCLUSION: Disruptions of the intestinal flora structure due to oral administration of penicillin may significantly increase serum 5-HT level and inhibit intestinal motility, at least partially through up-regulating the expression of HCN2. Oral administration of streptomycin may alter 5-HT availability by up-regulating TPH1 expression thus increasing synthesis of 5-HT. Alterations of intestinal flora composition due to exposure to different antibiotics may regulate 5-HT availability and intestinal motility through different mechanisms.

TLR4 mediates upregulation and sensitization of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate-induced colitis.

Elevated excitability of primary afferent neurons underlies chronic pain in patients with functional or inflammatory bowel diseases. Recent studies have established an essential role for an enhanced transient receptor potential vanilloid subtype 1 (TRPV1) signaling in mediating peripheral hyperalgesia in inflammatory conditions. Since colocalization of Toll-like receptor 4 (TLR4) and TRPV1 has been observed in primary afferents including the trigeminal sensory neurons and the dorsal root ganglion neurons, we test the hypothesis that TLR4 might regulate the expression and function of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis using the TLR4-deficient and the wild-type C57 mice. Despite having a higher disease activity index following administration of 2,4,6-trinitrobenzene sulfate, the TLR4-deficient mice showed less inflammatory infiltration in the colon than the wild-type mice. Increased expression of TLR4 and TRPV1 as well as increased density of capsaicin-induced TRPV1 current was observed in L4-S2 dorsal root ganglion neurons of the wild-type colitis mice till two weeks post 2,4,6-trinitrobenzene sulfate treatment. In comparison, the TLR4-deficient colitis mice had lower TRPV1 expression and TRPV1 current density in dorsal root ganglion neurons with lower abdominal withdrawal response scores during noxious colonic distensions. In the wild type but not in the TLR4-deficient dorsal root ganglion neurons, acute administration of the TLR4 agonist lipopolysaccharide increased the capsaicin-evoked TRPV1 current. In addition, we found that the canonical signaling downstream of TLR4 was activated in 2,4,6-trinitrobenzene sulfate-induced colitis in the wild type but not in the TLR4-deficient mice. These results indicate that TLR4 may play a major role in regulation of TRPV1 signaling and peripheral hyperalgesia in inflammatory conditions.

Activation of the G Protein-Coupled Estrogen Receptor Elicits Store Calcium Release and Phosphorylation of the Mu-Opioid Receptors in the Human Neuroblastoma SH-SY5Y Cells.

Estrogens exert extensive influences on the nervous system besides their well-known roles in regulation of reproduction and metabolism. Estrogens act via the nuclear receptor ERα and ERß to regulate gene transcription (classical genomic effects). In addition, estrogens are also known to cause rapid non-genomic effects on neuronal functions including inducing fast changes in cytosolic calcium level and rapidly desensitizing the µ type opioid receptor (MOR). The receptors responsible for the rapid actions of estrogens remain uncertain, but recent evidence points to the G protein-coupled estrogen receptor (GPER), which has been shown to be expressed widely in the nervous system. In the current study, we test the hypothesis that activation of GPER may mediate rapid calcium signaling, which may promote phosphorylation of MOR through the calcium-dependent protein kinases in neuronal cells. By qPCR and immunocytochemistry, we found that the human neuroblastoma SH-SY5Y cells endogenously express GPER and MOR. Activation of GPER by 17ß-estradiol (E2) and G-1 (GPER selective agonist) evoked a rapid calcium rise in a concentration-dependent manner, which was due to store release rather than calcium entry. The GPER antagonist G15, the PLC inhibitor U73122 and the IP3 receptor inhibitor 2-APB each virtually abolished the calcium responses to E2 or G-1. Activation of GPER stimulated translocation of PKC isoforms (α and ε) to the plasma membrane, which led to MOR phosphorylation. Additionally, E2 and G-1 stimulated c-Fos expression in SH-SY5Y cells in a PLC/IP3-dependent manner. In conclusion, the present study has revealed a novel GPER-mediated estrogenic signaling in neuroblastoma cells in which activation of GPER is followed by rapid calcium mobilization, PKC activation and MOR phosphorylation. GPER-mediated rapid calcium signal may also be transmitted to the nucleus to impact on gene transcription. Such signaling cascade may play important roles in the regulation of opioid signaling in the brain.