Cholinergic Receptor Antagonist Research Articles

The Influence of CB2-Receptor Ligands on the Memory-Related Responses in Connection with Cholinergic Pathways in Mice in the Passive Avoidance Test.

Background: Dysfunction of the cholinergic system is associated with the development of Alzheimer’s disease (AD). One of the new possible strategies for the pharmacological modulation of memory-related problems typical of AD, is connected with the endocannabinoid system (ECS) and the cannabinoid (CB: CB1 and CB2) receptors. Methods: The aim of the study was to determine the influence of the selective CB2 receptor ligands: agonist (JWH 133) and antagonist (AM 630) on different stages of memory and learning in mice, in the context of their interaction with cholinergic pathways. To assess and understand the memory-related effects in mice we used the passive avoidance (PA) test. Results: We revealed that co-administration of non-effective dose of JWH 133 (0.25 mg) or AM 630 (0.25 mg/kg) with the non-effective dose of cholinergic receptor agonist - nicotine (0.05 mg/kg) enhanced cognition in the PA test in mice; however, an acute injection of JWH 133 (0.25 mg/kg) or AM 630 (0.25 mg/kg) had no influence on memory enhancement induced by the effective dose of nicotine (0.1 mg/kg). Co-administration of JWH 133 (0.25 mg) or AM 630 (0.25 mg/kg) with the effective dose of the cholinergic receptor antagonist scopolamine (1 mg/kg) attenuated the scopolamine-induced memory impairment in the PA test in mice. Conclusion: Our experiments have shown that CB2 receptors participate in the modulation of memory-related responses, especially those in which cholinergic pathways are implicated.

Involvement of monoaminergic systems / pathways in the mechanism of action of the antidepressant effect of Leptadenia hastata (Pers.) decne methanol leaf extract

Depression is a mental disorder that is highly prevalent in many parts of the globe and a source of burden to patients and their families. Existing treatments for depression are far from satisfactory because many patients with depression do not respond effectively and have significant adverse effects on standard antidepressant drugs. It is vital to develop novel antidepressant from medicinal plants, which are considered a good source of drugs with fewer adverse effects. An earlier study has established the in-vivo antidepressant effect of methanol leaf extract of Leptadenia hastata. However, no scientific data on its involvement in the monoaminergic mechanism of action. This study aimed to evaluate the involvement of monoaminergic systems in the antidepressant potential of methanol leaf extract of Leptadenia hastata. The acute toxicity studies of methanol leaf extract of Leptadenia hastata (MELH) was determined using Lorke's Method. The antidepressant effect of the MELH was evaluated using the Tail Suspension Test (TST) and Open Field Test (OFT) for psychostimulant effect. The possible mechanism of antidepressant actions of the MELH was also investigated by intraperitoneal pre-treatment with adrenergic, serotonergic, dopaminergic, opioidergic and muscarinic cholinergic receptor antagonists. The intraperitonial (i.p.) median lethal dose (LD50) values were estimated to be >5000 mg/kg. Administration of standard drug, Imipramine (10 mg/kg) and MELH (250, 500, and1000 mg/kg) significantly (p<0.01) and dose-dependently decreased the duration of immobility time. However, the anti-immobility effect of the MELH was significantly (p<0.05) reversed by prazosin, cyproheptadine and naloxone, suggesting the involvement of adrenergic, serotonergic and opioidergic pathways. However, no significant interactions with dopaminergic and muscarinic cholinergic receptor antagonists were observed. This study concluded that the observed antidepressant effect in MELH is likely mediated through their interactions with adrenergic, serotonergic and opioidergic systems.

In vitro effect of cholinergic agonists and antagonists, activators and blockers of voltage-sensitive Ca++-channels and Na+-channels on arylalkylamine N-acetyltransferase 2 mRNA levels in pineal organ of the catfish, Clarias gariepinus

The fish pineal organ is innervated by cholinergic nerve fibres. But there is no information on cholinergic regulation of aanat2 gene in the fish pineal organ. Therefore, we investigated in vitro effect of specific agonists and antagonists of cholinergic receptors on aanat2 mRNA levels in the pineal organ of catfish Clarias gariepinus. Acclimatized mature male fish was decapitated and the pineal organ was rapidly removed and placed in culture medium and pre-incubated for 1h. After pre-incubation, the culture medium was replaced with the culture medium containing desired concentrations of specific ago-nists and antagonists of cholinergic receptors, blockers and activators of volt-age-sensitive Ca++ channels and Na+ channels for 1h, after which the pineal organs were collected and processed for measuring relative expression of aanat2 gene by measuring AANAT2 mRNA with the help of Real Time RT-PCR. Acetylcholine and nicotine produced dose-dependent inhibitory and stimulatory effect on aanat2 transcript levels, while carbachol increased aa-nat2 transcript levels in a dose-dependent manner. In vitro treatment of the pineal organ with atropine, hexamethonium, the blockers of voltage-sensitive Ca++ channels and Na+ channels significantly blocked the stimulato-ry effect of carbachol and nicotine on aanat2 transcript levels, respectively. Based on these findings, we suggest that cholinergic mechanism plays an important and direct role in regulation of aanat2 gene expression in the fish pineal organ, and stimulates the gene transcription via both muscarinic and nicotinic receptors. Further, muscarinic and nicotinic receptors seem to in-crease aanat2 transcript levels by affecting voltage-sensitive Ca++ channels and Na+ channels, respectively.

Metabolic improvement during short-term treatment with a GLP-1 receptor agonist does not improve cardiac diastolic function in patients type 2 diabetes: a randomized double-blind placebo-controlled trial

Abstract Background/Introduction The cardioprotective effect of glucagon-like peptide-1 (GLP-1) receptor agonists in patients with type 2 diabetes is still not well explained. In these patients, diastolic dysfunction is significant and linked to outcome, and the cardioprotective effect of GLP-1 receptor agonists may be by improving diastolic function. Purpose To investigate if short-term treatment of liraglutide a GLP-1 receptor agonist improves left ventricular diastolic function. Methods In an investigator-initiated double-blind randomized placebo-controlled trial the effect of 18 weeks of treatment with liraglutide on diastolic function was assessed in type 2 diabetes patients and echocardiographic signs of diastolic dysfunction (echo-Doppler determined E/e'≥9 or/and lateral e'≤10 cm/sec). Primary outcomes were improved left ventricle filling (the early peak filling rate, ePFR) and left atrium ease of emptying (the passive emptying fraction, LAPEF), assessed by cardiac magnetic resonance imaging at rest and during chronotropic stress (glycopyrrolate 4 mg/kg; a cholinergic receptor antagonist increasing heart rate, and thereby inducing chronotropic stress without also affecting contractility). Secondary outcomes included left ventricular and left atrial volumes and systolic function, measures of aortic stiffness, and echocardiographic diastolic parameters. Results Forty patients were randomized to liraglutide s.c. 1.8 mg/day (n=20) or placebo (n=20). Liraglutide reduced HbA1c (−0.47% 95% CI (−0.88 to −0.06)) and weight (−2.9kg 95% CI (−4.6 to −1.2)), both p<0.03). Liraglutide did not change ePFR at rest −24±60 vs. −6±46 ml/sec, during stress 2±58 vs. −2±38 ml/sec, or the changes from rest and stress 12.9±72.5 vs. 4.7±104.0, all p>0.05. LAPEF decreased with liraglutide during stress (median (Q1, Q3)) −3.1 (−9.0, 1.1) vs. 1.0 (−2.9, 6.1) %, p=0.049, but no changes were evident at rest −4.3 (−7.9, 1.9) vs. −0.6 (−3.1, 2.2) %, p=0.19, or for the changes from rest to stress −1.7±8.4 vs. 0.8±8.2, p=0.4. All secondary outcomes were unchanged by liraglutide. Conclusions Short-term treatment with liraglutide did not improve diastolic function in patients with type 2 diabetes and echocardiographic signs of diastolic dysfunction. This suggests that the cardioprotective effect seen in long-term studies of liraglutide is not related to the improvement of left ventricular diastolic function. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ASB has received funding from the Danish Heart Association [16-R107-A6790-22002, and 18-R125-A8444-22110]. Novo Nordisk supported the study by an unrestricted grant covering the costs of CMR scans and blood analyses. Novo Nordisk provided free study medication and matching placebo pens. None of the funding sources played any role in the process of conduction, interpretation of results or publishing the study Primary OutcomesSecondary Outcomes

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.

M2-AChR Mediates Rapid Antidepressant Effects of Scopolamine Through Activating the mTORC1-BDNF Signaling Pathway in the Medial Prefrontal Cortex.

Background: Scopolamine, a non-selective muscarinic acetylcholine receptor (M1~5-AChR) antagonist, has rapid and robust antidepressant effects in humans and other species. However, which of the five M-AChRs mediates these therapeutic effects has not been fully identified. Several studies implicate M2-AChR as a potential antidepressant target of scopolamine. This study aimed to explore the role of M2-AChR in scopolamine's antidepressant-like effects and determine the underlying mechanisms.Methods: We used the classic novelty suppressed feeding test (NSFT), open field test (OFT) and forced swim test (FST) to observe antidepressant-related behaviors of normal rats, medial prefrontal cortex (mPFC) neuron silenced rats and M2-AChR knockdown rats treated with scopolamine. In a further experiment, the M2 cholinergic receptor antagonist methoctramine (MCT) was injected intracerebroventricularly into normal rats. Levels of mTORC1 and brain-derived neurotrophic factor (BDNF) in the mPFC of animals were analyzed by Western blotting.Results: Consistent with previous studies, mPFC was required for the antidepressant-like effects of scopolamine, and intracerebroventricular injection of MCT into rats could produce similar antidepressant-like effects. Use of AAV-shRNA to knock down M2-AChR in the mPFC resulted in the antidepressant-like effects of scopolamine being blunted. Furthermore, Western blotting demonstrated increased expression of mTORC1 signaling and BDNF in MCT-treated rats.Conclusion: Our results indicate that M2-AChR in the mPFC mediates the antidepressant-like effects of scopolamine by increasing the expression of BDNF and activating the mTORC1 signaling pathway.

Evaluation of the role of monoaminergic and nonmonoaminergic systems in the psychotropic effects of morin in mice: an interaction study with receptor blockers

Treatment-resistance depression and memory impairment disease, burdened with high levels of serious adverse effects of currently existing antidepressants and memory enhancers, have necessitated the search for alternative agents. Although previous preclinical studies have revealed the antidepressant-like and memory-enhancing potentials of morin, a compound isolated from Morus alba, the neurotransmitter receptor interaction mechanisms of action remain unknown. Hence, the role of monoaminergic and nonmonoaminergic neurotransmitter receptors in the antidepressant-like and memory-enhancing effects in naive mice were evaluated in this study. Male Swiss mice were pretreated intraperitoneally with D2-dopaminergic receptor antagonists (haloperidol), 5-HT1- and 5-HT2-receptor antagonist (metergoline), 5-HT synthesis inhibitor (para-chlorophenylalanine), α1-noradrenoceptor antagonist (prazosin), α2-noradrenoceptor antagonist (yohimbine), or cholinergic receptor antagonist (atropine) prior to the administration of morin (100 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.). The memory-enhancing and antidepressant-like effects were then evaluated 30 min later using the open-field test (OFT), Y-maze test (YMT), and forced swim test (FST). Morin reduced locomotor activity however, it was reversed by metergoline, pCPA, prazosin, yohimbine, haloperidol, and atropine independent of their actions. Morin exhibited antidepressant property in the FST when compared with control (p < 0.05). Metergoline, para-chlorophenylalanine, prazosin, and haloperidol decreased the antidepressant-like activity of morin. Morin showed memory-enhancing effect in the YMT relative to control. Pretreatment with metergoline, para-chlorophenylalanine, or atropine, however, decreased the memory-promoting effect of morin. These findings suggest that the antidepressant-like activity exhibited by morin might be mediated via interactions with 5-HTergic, noradrenergic, and dopaminergic receptors, while the memory-enhancing effect might involve interaction with both 5-HTergic and cholinergic receptors.

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.

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.

Aristolochia trilobata: Identification of the Anti-Inflammatory and Antinociceptive Effects

Aristolochia trilobata, popularly known as “mil-homens,” is widely used for treatment of stomach aches, colic, asthma, pulmonary diseases, diabetes, and skin affection. We evaluated the antinociceptive and anti-inflammatory activities of the essential oil (EO) and the main constituent, 6-methyl-5-hepten-2-yl acetate (sulcatyl acetate, SA). EO and SA (1, 10, and 100 mg/kg, p.o.) were evaluated using chemical (formalin-induced licking) and thermal (hot-plate) models of nociception or inflammation (carrageenan-induced cell migration into the subcutaneous air pouch, SAP). The mechanism of antinociceptive activity was evaluated using opioid, cholinergic receptor antagonists (naloxone and atropine), or nitric oxide synthase inhibitor (L-NAME). EO and SA presented a central antinociceptive effect (the hot-plate model). In formalin-induced licking response, higher doses of EO and SA also reduced 1st and 2nd phases. None of the antagonists and enzyme inhibitor reversed antinociceptive effects. EO and SA reduced the leukocyte migration into the SAP, and the cytokines tumor necrosis factor and interleukin-1 (TNF-α and IL-1β, respectively) produced in the exudate. Our results are indicative that EO and SA present peripheral and central antinociceptive and anti-inflammatory effects.

Pain and stress: functional evidence that supra-spinal mechanisms involved in pain-induced analgesia mediate stress-induced analgesia.

Analgesia induced by stressful and painful stimuli is an adaptive response during life-threatening situations. There is no evidence linking the mechanisms underlying them, while the former depends on the activation of stress-related brain pathways, the second depends on opioidergic mechanisms in the nucleus accumbens and on nicotinic cholinergic mechanisms in the rostral ventromedial medulla. In this study, we hypothesized that stress-induced analgesia is also dependent on opioidergic mechanisms in the nucleus accumbens and on nicotinic cholinergic mechanisms in the rostral ventromedial medulla. We used immobilization, a classical procedure to induce acute stress, and evaluated its ability to decrease the nociceptive responses induced either by carrageenan or by formalin in rats. Immobilization stress significantly decreased either carrageenan-induced hyperalgesia or formalin-induced tonic nociception in a time-dependent manner. This stress-induced analgesia is similar to pain-induced analgesia, as revealed by contrasting the antinociceptive effect induced by immobilization and by a forepaw injection of capsaicin. The administration of a µ-opioid receptor antagonist (CTOP, 0.5 µg) into the nucleus accumbens, as well as that of a nicotinic cholinergic receptor antagonist (mecamylamine, 0.6 µg) into the rostral ventromedial medulla, blocked immobilization stress-induced analgesia in both pain models. These results demonstrate that supraspinal mechanisms which are known to mediate pain-induced analgesia also mediate stress-induced analgesia. Therefore both forms of analgesia have overlapping mechanisms, probably recruited in response to the perception of danger.

Neuropeptide S Displays as a Key Neuromodulator in Olfactory Spatial Memory.

Neuropeptide S (NPS) is an endogenous peptide recently recognized to be presented in the brainstem and believed to play an important role in maintaining memory. The deletion of NPS or NPS receptor (NPSR) in mice shows a deficit in memory formation. Our recent studies have demonstrated that central administration of NPS facilitates olfactory function and ameliorates olfactory spatial memory impairment induced by muscarinic cholinergic receptor antagonist and N-methyl-D-aspartate receptor antagonist. However, it remains to be determined if endogenous NPS is an indispensable neuromodulator in the control of the olfactory spatial memory. In this study, we examined the effects of NPSR peptidergic antagonist [D-Val5]NPS (10 and 20 nmol, intracerebroventricular) and nonpeptidergic antagonist SHA 68 (10 and 50 mg/kg, intraperitoneal) on the olfactory spatial memory using computer-assisted 4-hole-board olfactory spatial memory test in mice. Furthermore, immunofluorescence was employed to identify the distributions of c-Fos and NPSR immunoreactive (-ir) neurons in olfactory system and hippocampal formation known to closely relate to the olfactory spatial memory. [D-Val5]NPS dosing at 20 nmol and SHA 68 dosing at 50 mg/kg significantly decreased the number of visits to the 2 odorants interchanged spatially, switched odorants, in recall trial, and simultaneously reduced the percentage of Fos-ir in NPSR-ir neurons, which were densely distributed in the anterior olfactory nucleus, piriform cortex, subiculum, presubiculum, and parasubiculum. These findings suggest that endogenous NPS is a key neuromodulator in olfactory spatial memory.

Modulation of hypercapnic respiratory response by cholinergic transmission in the commissural nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is an important area of the brainstem that receives and integrates afferent cardiorespiratory sensorial information, including those from arterial chemoreceptors and baroreceptors. It was described that acetylcholine (ACh) in the commissural subnucleus of the NTS (cNTS) promotes an increase in the phrenic nerve activity (PNA) and antagonism of nicotinic receptors in the same region reduces the magnitude of tachypneic response to peripheral chemoreceptor stimulation, suggesting a functional role of cholinergic transmission within the cNTS in the chemosensory control of respiratory activity. In the present study, we investigated whether cholinergic receptor antagonism in the cNTS modifies the sympathetic and respiratory reflex responses to hypercapnia. Using an arterially perfused in situ preparation of juvenile male Holtzman rats, we found that the nicotinic antagonist (mecamylamine, 5mM), but not the muscarinic antagonist (atropine, 5mM), into the cNTS attenuated the hypercapnia-induced increase of hypoglossal activity. Furthermore, mecamylamine in the cNTS potentiated the generation of late-expiratory (late-E) activity in abdominal nerve induced by hypercapnia. None of the cholinergic antagonists microinjected in the cNTS changed either the sympathetic or the phrenic nerve responses to hypercapnia. Our data provide evidence for the role of cholinergic transmission in the cNTS, acting on nicotinic receptors, modulating the hypoglossal and abdominal responses to hypercapnia.

Loss of endogenous analgesia leads to delayed recovery from incisional pain in a rat model of chronic neuropathic pain

BackgroundPreoperative pain and impaired endogenous analgesia are risk factors of chronic postsurgical persistent pain (CPSP). A Chronic neuropathic pain model induced by spinal nerve ligation (SNL6W) shows impaired endogenous analgesia and delayed recovery from incisional pain. Repeated amitriptyline treatment can restore the endogenous analgesia, but its effects on delayed recovery are not clear. MethodsA plantar incision was made on the side contralateral to the nerve ligation in SNL6W rats. Withdrawal thresholds were measured by von Frey filament test until 28 d after surgery. Amitriptyline (10 mg·kg−1·d−1) or vehicle was administered for 13 d perioperatively. To examine the roles of noradrenergic and cholinergic signals in the spinal dorsal horn, pharmacological antagonism, measurement of each neurotransmitter concentration, and immunohistochemistry were conducted. ResultsRecovery of the withdrawal threshold of SNL6W animals to pre-incision values required 28 d after surgery, while naive animals recovered within 14 d. Intrathecal injection of alpha2 adrenoceptor antagonist (idazoxan) or muscarinic cholinergic receptor antagonist (atropine) decreased the withdrawal threshold on POD14 and 21 in naive animals, but not in SNL6W rats. Repeated amitriptyline treatment attenuated the delayed recovery in SNL6W rats, and the effect was antagonized by muscarinic cholinergic receptor antagonist. Beside the concentration of acetylcholine and its synthetic enzyme were not altered by the treatment. ConclusionsNoradrenergic and cholinergic analgesia, which is necessary for normal recovery, is lost in the SNL6W rats. A strategy to enhance endogenous analgesia using antidepressants, rather than simple analgesia, may help to prevent CPSP in chronic pain patients.

Rewarding effects of M4 but not M3 muscarinic cholinergic receptor antagonism in the rostromedial tegmental nucleus

The rostromedial tegmental nucleus (RMTg) receives inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic nuclei. We tested the effects of RMTg M3 and M4 muscarinic cholinergic receptor antagonism in a conditioned place preference (CPP) paradigm in mice. RMTg infusions of the M3 muscarinic cholinergic receptor antagonist 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) do not result in the acquisition of CPP but increase locomotor activation. By contrast, RMTg infusions of the M4 muscarinic cholinergic receptor antagonist Tropicamide result in the acquisition of CPP but do not increase locomotor activation. The rewarding effects of RMTg Tropicamide infusions are dopamine-dependent as systemic pre-treatment with the broad-spectrum dopamine receptor antagonist flupenthixol prevents the acquisition of CPP induced by RMTg Tropicamide infusions. Under conditions of systemic dopamine receptor blockade, RMTg Tropicamide infusions significantly increase locomotor activation. These data provide further support for an important role of endogenous cholinergic input to the RMTg in reward function and suggest that the contributions of RMTg cholinergic input to rewarding and locomotor-activating effects involve differential contributions of RMTg M4 and M3 muscarinic receptors, respectively.

Strain differences in muscarinic cholinergic receptor antagonism of fat intake and acquisition and expression of fat-conditioned flavor preferences in male BALB/c, C57BL/6 and SWR mice

Murine strain differences occur for both intakes of and preferences for sugars and fats. Previous studies demonstrated that the muscarinic cholinergic receptor antagonist, scopolamine (SCOP) more potently reduced sucrose and saccharin intakes in inbred C57BL/6 and BALB/c than SWR mice, sucrose-conditioned flavor preferences (CFP) expression in BALB/c, but not C57BL/6 or SWR mice, and sucrose-CFP acquisition in BALB/c relative to SWR and C57BL/6 mice. Although fat intake and fat-CFP are observed in all three strains, strain-specific effects were previously observed following dopamine D1, opiate and NMDA receptor antagonism of sweet and fat intake and CFP. The present study investigated whether muscarinic receptor antagonism differentially affected fat (Intralipid) intake and preferences in these strains by examining whether SCOP altered fat (Intralipid) intake and fat-CFP expression and acquisition in BALB/c, C57BL/6 and SWR mice. SCOP (0.1–10 mg/kg) significantly reduced Intralipid (5%) intake in all three strains across 2 h. In fat-CFP expression experiments, food-restricted mice consumed one flavored (conditioned stimulus (CS)+, 5 sessions) Intralipid (5%) solution and a differently-flavored (CS−, 5 sessions) Intralipid (0.5%) solution. Two-bottle CS choice tests with the two flavors mixed in 0.5% Intralipid occurred following vehicle and two SCOP doses (1, 5 mg/kg). SCOP elicited small, but significant reductions in fat-CFP expression in BALB/c and C57BL/6, but not SWR mice. In fat-CFP acquisition experiments, separate groups of BALB/c, C57BL/6 and SWR mice were treated prior to the ten acquisition training sessions with vehicle or two SCOP (2.5, 5 mg/kg) doses followed by six two-bottle choice tests without injections. SCOP eliminated fat-CFP acquisition in all three strains. Thus, muscarinic receptor signaling mediates learning, and to a lesser degree maintenance of fat-CFP while maximally inhibiting fat intake in the three strains.

Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.

Optogenetic analysis of neuromuscular transmission in the colon of ChAT-ChR2-YFP BAC transgenic mice.

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 µM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 µM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-l-arginine (100 µM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 µM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ~88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle.NEW & NOTEWORTHY Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.

Cholinergic system is involved in the therapeutic effect of madecassoside on collagen-induced arthritis in rats

Our previous studies demonstrated that oral administration of madecassoside could markedly attenuate collagen-induced arthritis in rats, a rodent model of rheumatoid arthritis. As the autonomic nervous system is critically involved in the modulation of peripheral inflammation and immune response, the present study aims to explore the possible involvement of adrenergic and cholinergic nerves in the effect of madecassoside on rheumatoid arthritis. Arthritis was induced by chicken collagen in rats, and madecassoside was orally administered daily for two weeks from day 14 after the primary immunization. The antagonists of adrenoceptor and cholinergic receptors were co-administered with madecassoside, respectively. Unilateral cervical vagotomy was performed four days before the arthritis induction. The results showed that madecassoside (30 mg/kg) treatment markedly ameliorated arthritis symptoms in rats, mainly evidenced by the reduction of paw swelling and arthritis index scores. Co-administration of madecassoside with atropine (an antagonist of the muscarinic acetylcholine receptor) or hexamethonium (an antagonist of the nicotinic acetylcholine receptor) markedly diminished the therapeutic effects of madecassoside in arthritis. However, co-administration with phentolamine (an antagonist of the α-adrenoceptor) or propranolol (an antagonist of the β-adrenoceptor) did not alter the effect of madecassoside on arthritis. Furthermore, unilateral cervical vagotomy significantly reduced the anti-arthritis efficacy of madecassoside, including the amelioration of clinical symptoms, as well as the inhibition of the production of pro-inflammatory cytokines except T lymphocytes-related cytokines. These findings suggest that madecassoside exerts inhibitory effects on collagen-induced arthritis through, at least partially, the peripheral cholinergic system.

Scopolamine and Medial Frontal Stimulus-Processing during Interval Timing

Neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and Alzheimer's disease (AD) involve loss of cholinergic neurons in the basal forebrain. Here, we investigate how cholinergic dysfunction impacts the frontal cortex during interval timing, a process that can be impaired in PD and AD patients. Interval timing requires participants to estimate an interval of several seconds by making a motor response, and depends on the medial frontal cortex (MFC), which is richly innervated by basal forebrain cholinergic projections. Past work has shown that scopolamine, a muscarinic cholinergic receptor antagonist, reliably impairs interval timing. We tested the hypothesis that scopolamine would attenuate time-related ramping, a key form of temporal processing in the MFC. We recorded neuronal ensembles from eight mice during performance of a 12-s fixed-interval timing task, which was impaired by the administration of scopolamine. Consistent with past work, scopolamine impaired timing. To our surprise, we found that time-related ramping was unchanged, but stimulus-related activity was enhanced in the MFC. Principal component analyses revealed no consistent changes in time-related ramping components, but did reveal changes in higher components. Taken together, these data indicate that scopolamine changes stimulus processing rather than temporal processing in the MFC. These data could help understand how cholinergic dysfunction affects cortical circuits in diseases such as PD, DLB, and AD.