Muscarinic Cholinergic Receptors Research Articles

The effects of galangin in prepulse inhibition test and experimental schizophrenia models.

Acetylcholinesterase inhibitors are the focus of interest in the management of schizophrenia. We aimed to investigate the effects of acute galangin administration, a flavonoid compound with acetylcholinesterase inhibiting activity, on schizophrenia-associated cognitive deficits in rats and schizophrenia models in mice. Apomorphine-induced prepulse inhibition (PPI) disruption for cognitive functions, nicotinic, muscarinic, and serotonergic mechanism involvement, and brain acetylcholine levels were investigated in Wistar rats. Apomorphine-induced climbing, MK-801-induced hyperlocomotion, and catalepsy tests were used as schizophrenia models in Swiss albino mice. The effects of galangin were compared with acetylcholinesterase inhibitor donepezil, and typical and atypical antipsychotics haloperidol and olanzapine, respectively. Galangin (50,100 mg/kg) enhanced apomorphine-induced PPI disruption similar to donepezil, haloperidol, and olanzapine (p < 0.05). This effect was not altered in the combination of galangin with the nicotinic receptor antagonist mecamylamine (1 mg/kg), the muscarinic receptor antagonist scopolamine (0.05 mg/kg), or the serotonin-1A receptor antagonist WAY-100635 (1 mg/kg) (p > 0.05). Galangin (50,100 mg/kg) alone increased brain acetylcholine concentrations (p < 0.05), but not in apomorphine-injected rats (p > 0.05). Galangin (50 mg/kg) decreased apomorphine-induced climbing and MK-801-induced hyperlocomotion similar to haloperidol and olanzapine (p < 0.05), but did not induce catalepsy, unlike them. We suggest that galangin may help enhance schizophrenia-associated cognitive deficits, and nicotinic, muscarinic cholinergic, and serotonin-1A receptors are not involved in this effect. Galangin also exerted an antipsychotic-like effect without inducing catalepsy and may be considered as an advantageous antipsychotic agent.

Sexually dimorphic muscarinic acetylcholine receptor modulation of contextual fear learning in the dentate gyrus

Contextual fear conditioning, where the prevailing situational cues become associated with an aversive unconditional stimulus such as electric shock, is sexually dimorphic. Males typically show higher levels of fear than females. There are two components to contextual fear conditioning. First the multiple cues that encompass the context must be integrated into a coherent representation, a process that requires the hippocampus. The second is that representation must be communicated to the basolateral amygdala where it can be associated with shock. If there is inadequate time for forming the representation prior to shock poor conditioning results and this is called the immediate shock deficit. One can isolate the contextual processing component, as well as alleviate the deficit, by providing an opportunity to explore the context without shock prior to the conditioning session. The purpose of the present study was to determine the extent to which cholinergic processes within the dentate gyrus of the hippocampus during contextual processing contribute to the sexual dimorphism. Clozapine-n-oxide (CNO) is a putatively inactive compound that acts only upon synthetic genetically engineered receptors. However, we found that CNO infused into the dentate gyrus prior to exploration eliminated the sexual dimorphism by selectively decreasing freezing in males to the level of females. Biological activity of CNO is usually attributed to metabolism of CNO to clozapine and we found that clozapine, and the muscarinic cholinergic antagonist, scopolamine, produced results similar to CNO, preferentially affecting males. On the other hand, the muscarinic agonist oxotremorine selectively impaired conditioning in females. Overall, the current experiments reveal significant off-target effects of CNO and implicate muscarinic cholinergic receptors in the dentate gyrus as a significant mediator of the sexual dimorphism in contextual fear conditioning.

Neuron-specific responses to acetylcholine within the spinal dorsal horn circuits of rodent and primate

Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord.

Muscarinic regulation of the neuronal Na+/K+‐ATPase in rat hippocampus

Stimulation of postsynaptic muscarinic receptors was shown to excite principal hippocampal neurons by modulating several membrane ion conductances. We show here that activation of postsynaptic muscarinic receptors also causes neuronal excitation by inhibiting Na+ /K+ -ATPase activity. Muscarinic Na+ /K+ -ATPase inhibition is mediated by two separate signalling pathways that lead downstream to enhanced Na+ /K+ -ATPase phosphorylation by activating protein kinase C and protein kinase G. Muscarinic excitation through Na+ /K+ -ATPase inhibition is probably involved in cholinergic modulation of hippocampal activity and may turn out to be a widespread mechanism of neuronal excitation in the brain. Stimulation of muscarinic cholinergic receptors on principal hippocampal neurons enhances intrinsic neuronal excitability by modulating several membrane ion conductances. The electrogenic Na+ /K+ -ATPase (NKA; the 'Na+ pump') is a ubiquitous regulator of intrinsic neuronal excitability, generating a hyperpolarizing current to thwart excessive neuronal firing. Using electrophysiological and pharmacological methodologies in rat hippocampal slices, we show that neuronal NKA pumping activity is also subjected to cholinergic regulation. Stimulation of postsynaptic muscarinic, but not nicotinic, cholinergic receptors activates membrane-bound phospholipase C and hydrolysis of membrane-integral phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3 ). Along one signalling pathway, DAG activates protein kinase C (PKC). Along a second signalling pathway, IP3 causes Ca2+ release from the endoplasmic reticulum, facilitating nitric oxide (NO) production. The rise in NO levels stimulates cGMP synthesis by guanylate-cyclase, activating protein kinase G (PKG). The two pathways converge to cause partial NKA inhibition through enzyme phosphorylation by PKC and PKG, leading to a marked increase in intrinsic neuronal excitability. This novel mechanism of neuronal NKA regulation probably contributes to the cholinergic modulation of hippocampal activity in spatial navigation, learning and memory.

Pulsed Infrared Stimulation of Vertical Semicircular Canals Evokes Cardiovascular Changes in the Rat.

A variety of stimuli activating vestibular end organs, including sinusoidal galvanic vestibular stimulation, whole body rotation and tilt, and head flexion have been shown to evoke significant changes in blood pressure (BP) and heart rate (HR). While a role for the vertical semicircular canals in altering autonomic activity has been hypothesized, studies to-date attribute the evoked BP and HR responses to the otolith organs. The present study determined whether unilateral activation of the posterior (PC) or anterior (AC) semicircular canal is sufficient to elicit changes in BP and/or HR. The study employed frequency-modulated pulsed infrared radiation (IR: 1,863 nm) directed via optical fibers to PC or AC of adult male Long-Evans rats. BP and HR changes were detected using a small-animal single pressure telemetry device implanted in the femoral artery. Eye movements evoked during IR of the vestibular endorgans were used to confirm the stimulation site. We found that sinusoidal IR delivered to either PC or AC elicited a rapid decrease in BP and HR followed by a stimulation frequency-matched modulation. The magnitude of the initial decrements in HR and BP did not correlate with the energy of the suprathreshold stimulus. This response pattern was consistent across multiple trials within an experimental session, replicable, and in most animals showed no evidence of habituation or an additive effect. Frequency modulated electrical current delivered to the PC and IR stimulation of the AC, caused decrements in HR and BP that resembled those evoked by IR of the PC. Frequency domain heart rate variability assessment revealed that, in most subjects, IR stimulation increased the low frequency (LF) component and decreased the high frequency (HF) component, resulting in an increase in the LF/HF ratio. This ratio estimates the relative contributions of sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) activities. An injection of atropine, a muscarinic cholinergic receptor antagonist, diminished the IR evoked changes in HR, while the non-selective beta blocker propranolol eliminated changes in both HR and BP. This study provides direct evidence that activation of a single vertical semicircular canal is sufficient to activate and modulate central pathways that control HR and BP.

Effect of solvent fractions of crude extract of Liushenqu on gastrointestinal motility in guinea pigs, and the underlying mechanism(s)

Purpose: To study the effect of solvent fractions of the crude extract of liushenqu on gastrointestinal motility in guinea pigs, and the mechanism of action.&#x0D; Methods: The effects of solvent fractions of crude extract of liushenqu (LSQ) on receptors in guinea pig isolated small intestinal cells were determined by treatment with different receptor blockers, including diphenhydramine (0.067 mg/mL), atropine sulfate (0.064 mg/mL), propranolol hydrochloride (0.033mg/mL), phentolamine mesylate (0.04mg/mL) and ondansetron hydrochloride (0.048mg/mL), to investigate the possible pharmacological mechanism of action.&#x0D; Results: There was no significant change in the maximum amplitude of muscle tension before and after administration in the control group, petroleum ether fraction group, and dichlormethane fraction group, while muscle tension in the 95 % ethanol and n-butanol fractions significantly increased (p &lt; 0.01). The mean changes in tension were significantly different from that of control group (p &lt; 0.01), but ethyl acetate fraction showed significant intestinal muscle inhibition (p &lt; 0.01). Addition of LSQ did not alleviate the inhibition caused by diphenhydramine, but it significantly reversed the inhibition caused by blockers of cholinergic muscarinic receptor, adrenergic alpha- and beta- receptors, and 5-HT receptor (p &lt; 0.01).&#x0D; Conclusion: These results indicate that n-butanol fraction is the most effective bioactive fraction of LSQ, while ethyl acetate fraction has the opposite effect. In addition, its mechanism of action is related to increase in the amplitude of small intestine smooth muscle contraction and acceleration of small intestine peristalsis.

The Non‐Neuronal Cholinergic System is Causal for Cardioprotection by Hypoxic Preconditioning in Isolated Adult Rat Cardiomyocytes

Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although this phenomenon is known for more than 3 decades, the underlying signaling mechanisms are unclear in their details. Among various cardioprotective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is certainly involved. However, a non-neuronal cardiac cholinergic system (NNCCS) has been recently identified, suggesting a nervous system-independent, cardiomyocyte-derived ACh signaling with signal amplification by autocrine cholinergic receptor activation. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of cardioprotection. We therefore in a reductionist approach used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Hearts from male Lewis rats were harvested, and adult ventricular cardiomyocytes were isolated. In suspended cardiomyocytes HPC was induced by one cycle of 10 min brief hypoxia and 20 min reoxygenation. Thirty min normoxia served as control. Afterwards, cardiomyocytes were exposed to 30 min sustained hypoxia and 5 min reoxygenation (H/R) or to 35 min normoxia as time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod-shaped, unstained (0.5% trypan blue) cells over all cells. In parallel experiments, physostigmine (0.2 mmol/L) was added to cardiomyocyte suspensions to block the ubiquitous ACh esterase. Intracellular ACh was quantified with and without HPC via liquid chromatography-coupled tandem mass spectrometry. In subsets, the nicotinic cholinergic receptor antagonists hexamethonium (1.0 µmol/L) and α-bungarotoxine (0.1 µmol/L) or the muscarinic cholinergic receptor antagonist atropine (0.1 µmol/L) were added to cardiomyocyte suspensions with and without HPC, respectively, and exposure to H/R or TC, respectively, afterwards. Again, cardiomyocyte viability was quantified. Results With HPC, the viability of cardiomyocytes subjected to H/R was better preserved than without (24±1% vs. 7±1%, figure A). Also, intracellular ACh was slightly (n.s.) increased after sustained hypoxia (6±2 pmol/mg protein), but significantly with HPC (13±3 pmol/mg protein) in comparison to baseline (3±1 pmol/mg protein) or TC (3±1 pmol/mg protein, figure B). However, this increase was only detectable in the presence of physostigmine (figure B). Addition of nicotinic and muscarinic cholinergic receptor antagonists attenuated the protection by HPC to the same extent (hexamethonium: 12±2%, α-bungarotoxine: 11±1%, atropine: 10±1%, figure A). Conclusion The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular ACh, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an autocrine signal amplification on the cardiomyocyte level.

Gender dimorphic effect of dopamine D2 and muscarinic cholinergic receptors on memory retrieval.

Episodic memory retrieval is fundamental for daily activities of humans and animals. Muscarinic cholinergic signaling is important for memory functioning and shows gender-dependent response in episodic memory retrieval. Dopamine D2 receptors influence memory formation and retrieval by influencing cholinergic signaling in the brain. This study aimed to determine the gender-dependent effects of D2 and muscarinic activity on memory retrieval. Male and female mice were trained for Morris water maze test and contextual fear conditioning. Memory retrieval was assessed following sub-chronic treatment (for 5days) with D2 antagonist (risperidone 2.5mg/kg) alone or in combination with scopolamine (1mg/kg) or donepezil (1mg/kg). Open field test was performed prior to the retrieval test to evaluate effects of risperidone treatment on locomotor activity and exploratory behavior. Risperidone co-treatment with donepezil impaired spatial memory retrieval in males only. Muscarinic and D2 simultaneous antagonism tend to impair fear retrieval in males but significantly enhanced retrieval of fear memories in female mice. These results suggest that D2 signaling influence muscarinic receptor activity during memory retrieval in gender-dependent manner.

Neuromodulation of Persistent Activity and Working Memory Circuitry in Primate Prefrontal Cortex by Muscarinic Receptors

Neuromodulation by acetylcholine plays a vital role in shaping the physiology and functions of cerebral cortex. Cholinergic neuromodulation influences brain-state transitions, controls the gating of cortical sensory stimulus responses, and has been shown to influence the generation and maintenance of persistent activity in prefrontal cortex. Here we review our current understanding of the role of muscarinic cholinergic receptors in primate prefrontal cortex during its engagement in the performance of working memory tasks. We summarize the localization of muscarinic receptors in prefrontal cortex, review the effects of muscarinic neuromodulation on arousal, working memory and cognitive control tasks, and describe the effects of muscarinic M1 receptor stimulation and blockade on the generation and maintenance of persistent activity of prefrontal neurons encoding working memory representations. Recent studies describing the pharmacological effects of M1 receptors on prefrontal persistent activity demonstrate the heterogeneity of muscarinic actions and delineate unexpected modulatory effects discovered in primate prefrontal cortex when compared with studies in rodents. Understanding the underlying mechanisms by which muscarinic receptors regulate prefrontal cognitive control circuitry will inform the search of muscarinic-based therapeutic targets in the treatment of neuropsychiatric disorders.

Muscarinic Cholinergic Receptor Agonist and Peripheral Antagonist for Schizophrenia

BackgroundThe muscarinic receptor agonist xanomeline has antipsychotic properties and is devoid of dopamine receptor–blocking activity but causes cholinergic adverse events. Trospium is a peripherally restricted muscarinic receptor antagonist that reduces peripheral cholinergic effects of xanomeline. The efficacy and safety of combined xanomeline and trospium in patients with schizophrenia are unknown.MethodsIn this double-blind, phase 2 trial, we randomly assigned patients with schizophrenia in a 1:1 ratio to receive twice-daily xanomeline–trospium (increased to a maximum of 125 mg of xanomeline and 30 mg of trospium per dose) or placebo for 5 weeks. The primary end point was the change from baseline to week 5 in the total score on the Positive and Negative Syndrome Scale (PANSS; range, 30 to 210, with higher scores indicating more severe symptoms of schizophrenia). Secondary end points were the change in the PANSS positive symptom subscore, the score on the Clinical Global Impression–Severity (CGI-S) scale (range, 1 to 7, with higher scores indicating greater severity of illness), the change in the PANSS negative symptom subscore, the change in the PANSS Marder negative symptom subscore, and the percentage of patients with a response according to a CGI-S score of 1 or 2.ResultsA total of 182 patients were enrolled, with 90 assigned to receive xanomeline–trospium and 92 to receive placebo. The PANSS total score at baseline was 97.7 in the xanomeline–trospium group and 96.6 in the placebo group. The change from baseline to week 5 was −17.4 points with xanomeline–trospium and −5.9 points with placebo (least-squares mean difference, −11.6 points; 95% confidence interval, −16.1 to −7.1; P<0.001). The results for the secondary end points were significantly better in the xanomeline–trospium group than in the placebo group, with the exception of the percentage of patients with a CGI-S response. The most common adverse events in the xanomeline–trospium group were constipation, nausea, dry mouth, dyspepsia, and vomiting. The incidences of somnolence, weight gain, restlessness, and extrapyramidal symptoms were similar in the two groups.ConclusionsIn a 5-week trial, xanomeline–trospium resulted in a greater decrease in the PANSS total score than placebo but was associated with cholinergic and anticholinergic adverse events. Larger and longer trials are required to determine the efficacy and safety of xanomeline–trospium in patients with schizophrenia. (Funded by Karuna Therapeutics and the Wellcome Trust; ClinicalTrials.gov number, NCT03697252.)

Efficacy of iv infusion of magnesium sulphate and dromperidol on the management of neuropathic and somatosensory chronic pain - a two pain center study

Introduction: Droperidol as a medicine has unique properties and it has been successfully used as analgesic, antipsychotic, antiemetic and as a sedative [1]. It is an antagonist for d2-dopamine, serotonin, histamine, and nicotinic and muscarinic cholinergic receptors. It is also a dose-dependent agonist/antagonist of GABA receptors and an agonist of a2 adrenergic receptors. Moreover, is a sodium channel blocker such as lidocaine and potentiate μ opioid receptors. Its multi-receptor action justifies its use as analgesic, antiemetic and antiphycotic medication [2]. Magnesium seems to function as an antagonist for NMDA receptors and it has been used for relief of acute and chronic pain [3, 4]. The aim of the current study is the evaluation of the efficacy of droperidol and magnesium co-administration on the patients with chronic pain. Method: All patients that attended the pain clinic for chronic pain and aged over 18 years old were included in the study. Patients with atrioventricular block any grade and patients with chronic renal failure were excluded from the study. All included patients received droperidol to the dose 2.5 mg maximum and 1 gr magnesium sulphate iv. DN4 questionnaire was used to assess the presence of neuropathic pain. VAS scale and NPS scale were used to assess the intensity of pain before and after the administration. Change in the pain scales calculated as a percentage. Results: 48 patients included in the study. 23 of them had neuropathic pain. There was a 40–50% reduction of the intensity of pain after iv administration of droperidol and magnesium. There weren’t any complications. Conclusions: The co-administration of magnesium and droperidol can relieve neuropathic and somatosensory pain. Long-term evaluation is needed.

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

Effects of biperiden (cholinergic muscarinic m1/m4 receptor antagonist) on ethanol conditioned place preference in mice

BackgroundPrevious studies suggest that muscarinic cholinergic receptors might act upon the dopamine release in the mesolimbic system and alter drug-reinforcing values related to drug craving. AimsWe examined the effects of systemic biperiden administration, a muscarinic cholinergic (M1/M4) receptor antagonist, on ethanol (dose of 2 g/Kg) conditioned place preference (CPP), neuronal activation, dopamine and its metabolites levels in the nucleus accumbens. MethodsThirty minutes before the ethanol-induced CPP test, mice received saline or biperiden at doses of 1.0, 5.0, or 10.0 mg/kg. The time spent in each compartment was recorded for 15 min. After the CPP protocol, animals were euthanized, and we investigated the activation of the nucleus accumbens by immunohistochemistry for Fos. We also quantified dopamine, homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC) levels in the nucleus accumbens by high-performance liquid chromatography (HPLC). Additionally, the rotarod was employed to evaluate the effects of biperiden on motor coordination. ResultsBiperiden at different doses (1.0, 5.0, and 10.0 mg/kg) blocked the expression of ethanol-induced CPP. These biperiden doses increased the number of Fos-positive cells and the dopamine turnover in the nucleus accumbens. None of the doses affected the motor coordination evaluated by the rotarod. ConclusionsOur results show that biperiden can modulate the effect of alcohol reward, and its mechanism of action may involve a change in dopamine and cholinergic mesolimbic neurotransmission.

Castanea sativa Mill. bark extract cardiovascular effects in a rat model of high-fat diet.

Ellagitannins may have a beneficial impact in cardiovascular diseases. The aim of the study was to evaluate the effect of high-fat diet (HFD) and the efficacy of Castanea sativa Mill. bark extract (ENC) on cardiac and vascular parameters. Rats were fed with regular diet, (RD, n = 15), HFD (n = 15), RD + ENC (20 mg/kg/day by gavage, n = 15), and HFD + ENC (same dose, n = 15) and the effects on body weight, biochemical serum parameters, and inflammatory cytokines determined. Cardiac functional parameters and aorta contractility were also assessed on isolated atria and aorta. Results showed that ENC reduced weight gain and serum lipids induced by HFD. In in vitro assays, HFD decreased the contraction force of left atrium, increased right atrium chronotropy, and decreased aorta K+ -induced contraction; ENC induced transient positive inotropic and negative chronotropic effects on isolated atria from RD and HFD rats and a spasmolytic effect on aorta. In ex vivo experiments, ENC reverted inotropic and chronotropic changes induced by HFD and enhanced Nifedipine effect more on aorta than on heart. In conclusion, ENC restores metabolic dysfunction and cardiac cholinergic muscarinic receptor function, and exerts spasmolytic effect on aorta in HFD rats, highlighting its potential as nutraceutical tool in obesity.

A Novel Genotyping Method for Detection of the Muscarinic Receptor M1 Gene rs2067477 Polymorphism and Its Genotype/Allele Frequencies in a Turkish Population.

Gene variation in the cholinergic muscarinic receptor 1 (CHRM1) has potential to become a candidate biomarker in the development of several disorders as well as drug response. In this study, a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to determine the C to A single nucleotide polymorphism at position 267 in the CHRM1 gene. A new reverse primer and a mismatched forward primer were designed to obtain 125 bp PCR products. The PCR products were then digested with the Hae III restriction enzyme to detect the rs2067477 polymorphism that comprises a C to A base change. The novel assay developed was tested in 51 Turkish schizophrenia patients. The genotyping assay was successfully performed in patients with schizophrenia in order to confirm the accuracy and validity of this method. The frequency of CC, CA, and AA genotypes was 72.5%, 25.5%, and 2%, respectively. On the basis of these findings, the allele frequency of C was 0.85 and the allele frequency of A was 0.15. This genotyping assay is practical for screening the CHRM1 C267A polymorphism in pharmacogenetic studies. The present polymorphism may be used as a candidate biomarker to determine genetic susceptibility to related diseases and may contribute to the implementation of individualized drug therapy for M1-related diseases.

Changes in the expression and function of the PDE5 pathway in the obstructed urinary bladder.

Our study aims to explore changes in bladder contractility and the phosphodiesterase type 5 (PDE5) signalling pathway in response to partial bladder outlet obstruction (PBOO). A surgically induced male rat PBOO model and human obstructed bladder tissues were used. Histological changes were examined by H&E and Masson's trichrome staining. Bladder strip contractility was measured via organ bath. The expressions of nitric oxide synthase (NOS) isoforms, PDE5, muscarinic cholinergic receptor (CHRM) isoforms and PDE4 isoforms in bladder were detected by RT‐PCR and Western blotting. The immunolocalization of the PDE5 protein and its functional activity were also determined. PBOO bladder tissue exhibited significant SM hypertrophy and elevated responsiveness to KCl depolarization and the muscarinic receptor agonist carbachol. NOS isoforms, PDE5, CHRM2, CHRM3 and PDE4A were up‐regulated in obstructed bladder tissue, whereas no change in PDE4B and PDE4D isoform expression was observed. With regard to PDE5, it was expressed in the SM bundles of bladder. Interestingly, obstructed bladder exhibited less relaxation responsiveness to sodium nitroprusside (SNP), but an exaggerated PDE5 inhibition effect. The up‐regulation of PDE5 could contribute to the lack of effect on Qmax for benign prostatic hyperplasia/lower urinary tract symptom (BPH/LUTS) patients treated with PDE5 inhibitors. Moreover, PDE5 (with presence of NO) and PDE4 may serve as new therapeutic targets for bladder diseases such as BPH‐induced LUTS and overactive bladder (OAB).

Manual acupuncture stimulation of acupoints at the same and adjacent spinal segments promotes uterine contractility

To investigate the effect of manual acupuncture stimulation of acupoints at different spinal nerve segments on uterine motility and the complicated adrenergic and cholinergic receptors. Eighteen adult non-pregnant SD rats were used in the present study. The contractive activities of the uterus were measured by using a pressure transducer which was connected to an inserted water-filled balloon in the uterus via a PE tube at one end and an amplifier at the other end. Manual acupuncture needle was applied to "Zigong"(EX-CA1),"Huiyin" (CV1), "Xuehai "(SP10) and "Taichong "(LR3) acupoints located at the same or adjacent spinal nerve segments of the uterus, and to "Neiguan" (PC6) situated at the distant spinal nerve segment at about 2 Hz for 1 min, followed by observing changes of the uterine contractility. After acupuncture, α-adrenoceptor antagonist phentolamine (0.5 mg/kg, n=9) or cholinergic muscarinic (M) receptor antagonist atropine (0.5 mg/kg，n=9) was given to the rats of different acupoints respectively through tail vein, followed by observing changes of the uterine automatic systolic pressure difference (value of systolic pressure peak minuses the trough value) and frequency after manual acupuncture stimulation as well as after blocking the activities of α-adrenoceptors and M receptors, separately. After acupuncture stimulation of EX-CA1, CV1, SP10 and LR3, but not PC6, the systolic pressure difference and frequency of the uterus were signi-ficantly increased (P<0.05,P<0.001, P<0.01). Following intravenous injection of phentolamine, both the systolic pressure difference and frequency had no marked changes after acupuncture at the 5 acupoints (P>0.05). After intravenous injection of atropine, the uterine systolic pressure difference and frequency were markedly decreased compared with the basic values before acupuncture stimulation (P<0.001), but had no obvious changes after acupuncture at the 5 acupoints at both the same and distant spinal segments to the uterus (P>0.05). Manual acupuncture stimulation of acupoints at the same and adjacent spinal segments can promote the contractility of uterus in normal rats, which is realized by activating both α-adrenoceptor and cholinergic M receptors.

Experience enhances certainty about olfactory stimuli under bulbar cholinergic control.

We present evidence that experience and cholinergic modulation in an early sensory network interact to improve certainty about olfactory stimuli. The data we present are in agreement with existing theoretical ideas about the functional role of acetylcholine but highlight the importance of early sensory networks in addition to cortical networks. We use a simple behavioral paradigm in mice which allows us to measure certainty about a stimulus via the response amplitude to a condition and novel stimuli. We conclude that additional learning increases certainty and that the slope of this relationship can be modulated by activation of muscarinic cholinergic receptors in the olfactory bulb.

Functional Characterization of Muscarinic Receptors in Human Schwann Cells.

Functional characterization of muscarinic cholinergic receptors in myelinating glial cells has been well described both in central and peripheral nervous system. Rat Schwann cells (SCs) express different muscarinic receptor subtypes with the prevalence of the M2 subtype. The selective stimulation of this receptor subtype inhibits SC proliferation, improving their differentiation towards myelinating phenotype. In this work, we describe for the first time that human SCs are cholinoceptive as they express several muscarinic receptor subtypes and, as for rat SCs, M2 receptor is one of the most abundant. Human SCs, isolated from adult nerves, were cultured in vitro and stimulated with M2 muscarinic agonist arecaidine propargyl ester (APE). Similarly to that observed in rat, M2 receptor activation causes a decreased cell proliferation and promotes SC differentiation as suggested by increased Egr2 expression with an improved spindle-like shape cell morphology. Conversely, the non-selective stimulation of muscarinic receptors appears to promote cell proliferation with a reduction of SC average cell diameter. The data obtained demonstrate that human SCs are cholinoceptive and that human cultured SCs may represent an interesting tool to understand their physiology and increase the knowledge on how the cholinergic stimulation may contribute to address human SC development in normal and pathological conditions.

Synaptically released acetylcholine evokes Ca2+ elevations in astrocytes in hippocampal slices.

Recent results have demonstrated the existence of bidirectional communication between glial cells and neurons. We investigated in brain slices whether rat hippocampal astrocytes respond to acetylcholine synaptically released by an extrinsic pathway. We stimulated the stratum oriens/alveus, which contains cholinergic afferents from the septum and diagonal band of Broca, and recorded whole-cell membrane currents and intracellular Ca2+ levels of astrocytes located in the hippocampal stratum oriens. Nerve-fiber stimulation evoked a long-lasting inward current and increased the Ca2+ levels in astrocytes. Both astrocytic responses were abolished by tetrodotoxin or Cd2+ and were increased by 4-aminopyridine, indicating that the responses were attributable to synaptically released neurotransmitter. The inward current was inhibited by glutamate transporter antagonists, indicating that it was attributable to the electrogenic glutamate transporter activity. The synaptically evoked intracellular Ca2+ elevations were not affected by glutamate receptor antagonists but were abolished by atropine, indicating that they were mediated by muscarinic cholinergic receptors. Thapsigargin prevented the Ca2+ elevation but did not modify the inward current, indicating that the Ca2+ signal was attributable to intracellular Ca2+ mobilization. These results indicate that hippocampal astrocytes respond to acetylcholine released by synaptic terminals. The synaptically released acetylcholine acts on muscarinic receptors, mobilizing Ca2+ from the intracellular stores. Different regions in the recorded astrocytes showed independent stimulus-induced Ca2+ variations, suggesting the existence of subcellular domains in the astrocytic responses evoked by the synaptic cholinergic activity. Therefore, our results show the existence of cholinergic neuron-astrocyte signaling and suggest that astrocytes are a target of axonal inputs from different brain areas.