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T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Background & Aims YAP (Yap1) and TAZ (Wwtr1) are transcriptional co-activators and downstream effectors of the Hippo pathway, which play crucial roles in organ size control and cancer pathogenesis. Genetic deletion of YAP/TAZ has shown their critical importance for embryonic development of the heart, vasculature, and gastrointestinal mesenchyme. The aim of this study was to determine the functional role of YAP/TAZ in adult smooth muscle cells in vivo. Methods Because YAP and TAZ are mutually redundant, we used YAP/TAZ double-floxed mice crossed with mice that express tamoxifen-inducible CreERT2 recombinase driven by the smooth muscle–specific myosin heavy chain promoter. Results Double-knockout of YAP/TAZ in adult smooth muscle causes lethality within 2 weeks, mainly owing to colonic pseudo-obstruction, characterized by severe distension and fecal impaction. RNA sequencing in colon and urinary bladder showed that smooth muscle markers and muscarinic receptors were down-regulated in the YAP/TAZ knockout. The same transcripts also correlated with YAP/TAZ in the human colon. Myograph experiments showed reduced contractility to depolarization by potassium chloride and a nearly abolished muscarinic contraction and spontaneous activity in colon rings of YAP/TAZ knockout. Conclusions YAP and TAZ in smooth muscle are guardians of colonic contractility and control expression of contractile proteins and muscarinic receptors. The knockout model has features of human chronic intestinal pseudo-obstruction and may be useful for studying this disease., Graphical abstract

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.

Trihexyphenidyl, a nonselective muscarinic receptor antagonist, is the small molecule drug of choice for the treatment of DYT1 dystonia, but it is poorly tolerated due to significant side effects. A better understanding of the mechanism of action of trihexyphenidyl is needed for the development of improved treatments. Because DTY1 dystonia is associated with both abnormal cholinergic neurotransmission and abnormal dopamine regulation, we tested the hypothesis that trihexyphenidyl normalizes striatal dopamine release in a mouse model of DYT1 dystonia using ex vivo fast scan cyclic voltammetry and in vivo microdialysis. Trihexyphenidyl increased striatal dopamine release and efflux as assessed by ex vivo voltammetry and in vivo microdialysis respectively. In contrast, ʟ-DOPA, which is not usually effective for the treatment of DYT1 dystonia, did not increase dopamine release in either Dyt1 or control mice. Trihexyphenidyl was less effective at enhancing dopamine release in Dyt1 mice relative to controls ex vivo (mean increase WT: 65% vs Dyt1: 35%). Trihexyphenidyl required nicotinic receptors but not glutamate receptors to increase dopamine release. Dyt1 mice were more sensitive to the dopamine release decreasing effects of nicotinic acetylcholine receptor antagonism (IC50: WT = 29.46 nM, Dyt1 = 12.26 nM) and less sensitive to acetylcholinesterase inhibitors suggesting that nicotinic acetylcholine receptor neurotransmission is altered in Dyt1 mice, that nicotinic receptors indirectly mediate the differential effects of trihexyphenidyl in Dyt1 mice, and that nicotinic receptors may be suitable therapeutic targets for DYT1 dystonia.

Funder: Sosei Heptares Therapeutics Ltd, AIMS: HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. METHODS: This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. RESULTS: Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2-16.3], 35-mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9-18.9], 15-mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. CONCLUSION: HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired hippocampus-dependent memory, but the underlying mechanism remains elusive. Given that hippocampal cholinergic systems modulate memory and rapid development of hippocampal cholinergic systems occurs during nicotine exposure, here we investigated its impacts on cholinergic function. Both nicotinic and muscarinic activation produce transient or long-lasting depression of excitatory synaptic transmission in the hippocampal CA1 region. We found that postnatal nicotine exposure impairs both the induction and nicotinic modulation of NMDAR-dependent long-term depression (LTD). Activation of muscarinic receptors decreases excitatory synaptic transmission and CA1 network activity in both wild-type and α2 knockout mice. These muscarinic effects are still observed in nicotine-exposed mice. M1 muscarinic receptor activity is required for mGluR-dependent LTD. Early postnatal nicotine exposure has no effect on mGluR-dependent LTD induction, suggesting that it has no effect on the function of m1 muscarinic receptors involved in this form of LTD. Our results demonstrate that early postnatal nicotine exposure has more pronounced effects on nicotinic function than muscarinic function in the hippocampal CA1 region. Thus, impaired hippocampus-dependent memory may arise from the developmental disruption of nicotinic cholinergic systems in the hippocampal CA1 region.

Although data on the prevalence of anticholinergic misuse is scarce, it has been reported among psychiatric patients. Anticholinergic drugs can act as potent indirect dopamine agonists in the limbic system, a mechanism that has been hypothesized to explain their misuse potential among patients. In psychiatric practice settings, the use of typical antipsychotics in conjunction with anticholinergics is common, with the latter mainly used to manage extrapyramidal side effects of the former. Haloperidol is a first-generation (typical) antipsychotic with weak anticholinergic properties that may sometimes be potentiated when it is used in combination with other anticholinergic medications. This combination can induce significant gastrointestinal hypomotility, constipation, and rarely even paralytic ileus. We present the case of a 67-year-old African American male with a history of schizophrenia, benign prostatic hyperplasia, and essential hypertension, who abruptly started misusing benztropine, without any prior history of a substance use disorder. This case highlights the importance of obtaining a detailed history when previously stable psychiatric patients develop acute physical symptoms. It also illustrates the importance of care coordination among care providers and the central role of the psychiatrist in the care of patients with medical comorbidities.

Ripples are brief high-frequency electrographic events with important roles in episodic memory. However, the in vivo circuit mechanisms coordinating ripple-related activity among local and distant neuronal ensembles are not well understood. Here, we define key characteristics of a long-distance projecting GABAergic cell group in the mouse hippocampus that selectively exhibits high-frequency firing during ripples while staying largely silent during theta-associated states when most other GABAergic cells are active. The high ripple-associated firing commenced before ripple onset and reached its maximum before ripple peak, with the signature theta-OFF, ripple-ON firing pattern being preserved across awake and sleep states. Controlled by septal GABAergic, cholinergic, and CA3 glutamatergic inputs, these ripple-selective cells innervate parvalbumin and cholecystokinin-expressing local interneurons while also targeting a variety of extra-hippocampal regions. These results demonstrate the existence of a hippocampal GABAergic circuit element that is uniquely positioned to coordinate ripple-related neuronal dynamics across neuronal assemblies.

Aims HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. Methods This randomized, double‐blind, placebo‐controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1–35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross‐over design in 14 healthy subjects. Results Pharmacokinetics of HTL0018318 were well‐characterized showing dose proportional increases in exposure from 1–35 mg. Single doses of HTL0018318 were associated with mild dose‐related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2–16.3], 35‐mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9–18.9], 15‐mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. Conclusion HTL0018318 showed well‐characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Tylosema fassoglense (TFG) is used as an antidiarrheal traditional medicine in Western Kenya. This study aimed to investigate the antidiarrheal activity of its aqueous extracts in-vivo and the putative mechanism (s) of action ex-vivo using Sprague-Dawley rats and New Zealand white rabbits respectively. The in-vivo antidiarrheal effects of the extract were evaluated in castor oil-induced diarrhea, the castor oil-induced enteropooling, and phenol red gastric motility tests. On the other hand, isolated rabbit’s jejunal segments were used to evaluate the spasmolytic effect of TFG on spontaneous contraction, in acetylcholine-induced contraction, in presence of 80mMK+, calcium chloride-induced contraction as well as in presence of the following antagonists: naloxone, methylene blue, L-NAME, prazosin, and propranolol in the ex-vivo studies. The data were express as Mean ± S.E.M and analyzed by one-way ANOVA and Tukey’s post hoc test in cases of significance which was set at p < 0.05. The extract was phytochemically characterized using Liquid chromatography Mass spectroscopy (LC-MS).The extract possessed significant inhibitory effect in the in-vivo experiments. The extract exhibited significant spasmolytic effect on both spontaneous contraction and in jejunal segment pre-contracted acetylcholine as well as in presence of 80mMK+ solution. It also attenuated the spasmogenic effect of various concentration of calcium chloride. The extract’s spasmolytic effect was, however, significantly attenuated in presence of several antagonists (methylene blue and L-NAME) but the adrenergic blockers (prazosin and propranolol) had no significant effect in the ex-vivo studies. LC-MS identified thirty compounds where Proathocyanidin (11.54%), Syringic acid (7.30%), and 4-Hydroxybenzoic acid (6.19%) had the highest percentage abundance. In conclusion, the results obtained in this study partially validate the traditional uses of the tubers of this plant species as an antidiarrheal. These antidiarrheal effects are probably mediated via modulation of nitrous oxide pathway, voltage gated calcium channels, and muscarinic receptors.

© 2018, The Author(s). Rationale: Muscarinic acetylcholine receptors are known to play key roles in mediating cognitive processes, and impaired muscarinic cholinergic neurotransmission is associated with normal ageing processes and Alzheimer’s disease. However, the specific contributions of the individual muscarinic receptor subtypes (M1–M5) to cognition are presently not well understood. Objectives: The aim of this study was to investigate the contribution of M2-type muscarinic receptor signalling to sustained attention, executive control and learning and memory. Methods: M2 receptor-deficient (M2−/−) mice were tested on a touchscreen-operated task battery testing visual discrimination, behavioural flexibility, object-location associative learning, attention and response control. Spontaneous recognition memory for real-world objects was also assessed. Results: We found that M2−/− mice showed an enhancement of attentional performance, but significant deficits on some tests of learning and memory. Executive control and visual discrimination were unaffected by M2-depletion. Conclusions: These findings suggest that M2 activation has heterogeneous effects across cognitive domains, and provide insights into how acetylcholine may support multiple specific cognitive processes through functionally distinct cholinergic receptor subtypes. They also suggest that therapeutics involving M2 receptor-active compounds should be assessed across a broad range of cognitive domains, as they may enhance some cognitive functions, but impair others.

Central cholinergic systems regulate the hypothalamic-pituitary-adrenal (HPA) axis differentially in males and females (sexual diergism). We previously investigated the role of muscarinic receptors in this regulation by administering physostigmine (PHYSO), an acetylcholinesterase inhibitor, to male and female rats pretreated with scopolamine (SCOP), a nonselective muscarinic antagonist. SCOP pretreatment enhanced adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses in both sexes; males had greater ACTH responses while females had greater CORT responses. In the present study, we further explored the role of muscarinic receptor subtypes in HPA axis regulation by administering PHYSO to male and female rats following SCOP or various doses of either the M1 or the M2 selective muscarinic receptor antagonists, pirenzepine (PIREN) or methoctramine (METHO), respectively. Blood sampling occurred before and at multiple times after PHYSO. ACTH and CORT were determined by highly specific immunoassays. PIREN + PHYSO resulted in sustained, dose-dependent increases in ACTH and CORT: ACTH responses were similar in both sexes, CORT responses were greater in females, and percent changes from baseline for both hormones were greater in males. METHO + PHYSO resulted in overall decreases in ACTH and CORT: ACTH and CORT responses were higher in females but lower than those caused by PIREN or SCOP in both sexes, and percent changes from baseline were lower in males. Area under the curve analyses further supported these sexually diergic effects. These results suggest that specific muscarinic receptor subtypes differentially influence the HPA axis in a sexually diergic manner.

Introduction We evaluated the selective M1 muscarinic positive allosteric modulator, MK-7622, as adjunctive cognitive enhancing therapy in individuals with Alzheimer's disease. Methods A randomized, double-blind, proof-of-concept trial was performed. Participants with mild-to-moderate Alzheimer's disease, being treated with an acetylcholinesterase inhibitor, were randomized 1:1 to 45 mg of MK-7622 or placebo for 24 weeks. Endpoints included the mean change from baseline in Alzheimer's Disease Assessment Scale–Cognitive Subscale (ADAS-Cog11) at 12 weeks and Alzheimer's Disease Cooperative Study–Activities of Daily Living Inventory at 24 weeks. Results Two hundred forty participants were randomized. The trial was stopped for futility after meeting prospectively defined stopping criteria. MK-7622 did not improve cognition at 12 weeks (group difference in ADAS-Cog11: 0.18 [95% confidence interval: −1.0 to 1.3]) or function at 24 weeks (group difference in Alzheimer's Disease Cooperative Study–Activities of Daily Living Inventory: 0.06 [95% confidence interval: −2.4 to 2.5]). More participants taking MK-7622 discontinued study medication because of adverse events than those taking placebo (16% vs 6%) and who experienced cholinergically related adverse events (21% vs 8%). Discussion MK-7622 (45 mg) does not improve cognition or function when used as adjunctive therapy in mild-to-moderate Alzheimer's disease., Highlights • MK-7622 is a positive allosteric modulator of the M1 receptor. • MK-7622 did not improve cognition or function in Alzheimer's disease patients. • MK-7622 increased cholinergically related side effects in Alzheimer's disease patients. • M1 positive allosteric modulation is not useful for treating Alzheimer's disease.

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER–PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER–PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface–conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P(2) hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER–PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P(2) recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P(2) or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER–PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER–PM junctions formed by Kv2 channel–VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

The olfactory bulb (OB) receives significant cholinergic innervation and widely expresses cholinergic receptors. While acetylcholine (ACh) is essential for olfactory learning, the exact mechanisms by which ACh modulates olfactory learning and whether it is specifically required in the OB remains unknown. Using behavioral pharmacology and optogenetics, we investigated the role of OB ACh in a simple olfactory fear learning paradigm. We find that antagonizing muscarinic ACh receptors (mAChRs) in the OB during fear conditioning but not testing significantly reduces freezing to the conditioned odor, without altering olfactory abilities. Additionally, we demonstrate that m1 mAChRs, rather than m2, are required for acquisition of olfactory fear. Finally, using mice expressing channelrhodopsin in cholinergic neurons, we show that stimulating ACh release specifically in the OB during odor-shock pairing can strengthen olfactory fear learning. Together these results define a role for ACh in olfactory associative learning and OB glomerular plasticity.

Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections., Acetylcholine modulates the virulence of Candida albicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans. The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections. IMPORTANCE Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Considerable evidence indicates that adenosine and dopamine systems interact in the regulation of basal ganglia function. Nonselective adenosine antagonists such as the methylxanthine caffeine as well as selective adenosine A2A antagonists have been shown to produce antiparkinsonian and antidepressant effects in animal models. The present studies were conducted to assess if another methylxantine, theophylline, can reverse motor and motivational impairments induced by dopamine antagonism in rats. Results Theophylline (3.75–30.0 mg/kg, IP) reversed tremulous jaw movements (TJMs), catalepsy, and locomotor suppression induced by the dopamine D2 antagonist pimozide. It also reversed TJMs induced by the muscarinic receptor agonist pilocarpine, which is a well-known tremorogenic agent. Parallel studies assessed the ability of theophylline (5.0–20.0 mg/kg, IP) to reverse the changes in effort-related choice behavior induced by the dopamine D1 antagonist ecopipam (0.2 mg/kg, IP) and the D2 antagonist haloperidol (0.1 mg/kg, IP). Rats were tested on two different operant choice tasks which assess the tendency to work for a preferred reinforcer by lever pressing (for palatable pellets or a high 5% sucrose solution) vs. approaching and consuming a less preferred reinforcer (freely available lab chow or a less concentrated 0.3% sucrose solution). Theophylline restored food and sucrose-reinforced lever pressing in animals treated with the D2 antagonist. However, it was unable to reverse the effects of the D1 antagonist. Overall, the effects of theophylline resembled those previously reported for adenosine A2A antagonists, and suggest that theophylline could be clinically useful for the treatment of motor and motivational symptoms in humans.

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

Cholinergic system is an important system and a branch of the autonomic nervous system which plays an important role in memory, digestion, control of heart beat, blood pressure, movement and many other functions. This article serves as both structural and functional sources of information regarding cholinergic receptors and provides a detailed understanding of the determinants governing specificity of muscarinic and nicotinic receptor to researchers. The study helps to give overall information about the fundamentals of the cholinergic system, its receptors and ongoing research in this field.

Ethnopharmacological relevance The infusion or decoction of Mirabilis jalapa leaves is used in traditional medicine in Brazil to treat inflammatory and painful diseases. Thus, the present study was designed to investigate whether the leaf ethyl acetate (Eta) fraction from Mirabilis jalapa exhibits antinociceptive effect in clinically relevant pain models in mice. Furthermore, we have investigated the role of cholinergic system in the antinociceptive action produced by Eta in mice. Materials and methods The effect of Eta administered orally (10 mg/kg, p.o.) in mice was verified on the painful hypersensitivity (mechanical allodynia) in models of chronic inflammation (subcutaneous injection of complete Freund’s Adjuvant—CFA in the plantar surface of the right hind paw), postoperative (paw surgical incision) and neuropathic (partial sciatic nerve ligation) pain. In the chronic inflammation model, we further verified the effect of Eta treatment on paw edema and interleukin-1β (IL-1β) levels. We also investigated the role of muscarinic and nicotinic receptors in the antiallodynic action produced by Eta as well as the possible action of Eta on in vitro and ex vivo acetylcholinesterase activity in CFA treated animals. Furthermore, we verified the effect of Eta treatment on the parameters of liver and kidney lesion (level of urea, and activity of aspartate aminotransferase and alanine aminotransferase). Results Eta produced marked reduction in the allodynia caused by CFA, surgical incision and partial sciatic nerve ligation. However, Eta did not alter the paw edema or the increase of IL-1β levels produced by CFA. The antinociceptive effect of Eta was reversed by the pre-treatment of animals with the antagonists of muscarinic (atropine, 5 mg/kg, s.c) or nicotinic (mecamylamine, 0.001 mg/kg, s.c.) receptors. Eta did not alter in vitro acetylcholinesterase activity in blood or spinal cord samples, but it reversed the increase in the acetylcholinesterase activity observed in the spinal cord samples from mice injected with CFA. Moreover, Eta did not alter the indicators of liver or kidney lesion. Conclusions Based on its use in traditional medicine, the results of the present study confirmed the antinociceptive properties of Eta in clinically relevant pain models. Also its effect on the CFA-induced chronic inflammation seems to be related to acetylcholinesterase inhibition and cholinergic system.

As perturbations in auditory filtering appear to be a candidate trait marker of schizophrenia, there has been considerable interest in the development of translational rat models to elucidate the underlying neural and neurochemical mechanisms involved in sensory gating. This is the first study to investigate the effects of the non-selective muscarinic antagonist scopolamine, the muscarinic M1 antagonist biperiden and the cholinesterase inhibitor donepezil (also in combination with scopolamine and biperiden) on auditory evoked potentials (AEPs) and sensory gating. In the saline condition, only the N50 peak displayed sensory gating. Scopolamine and biperiden both disrupted sensory gating by increasing N50 amplitude for the S2 click. Donepezil was able to fully reverse the effects of biperiden on N50 sensory gating, but had residual effects when combined with scopolamine; i.e., it enhanced sensory gating by increasing N50 amplitude of the S1 stimulus. Donepezil by itself improved sensory gating by enhancing N50 amplitude of S1, and reducing N50 amplitude of the S2 click. In conclusion, due to its relatively more selective effects biperiden is to be preferred over scopolamine as a means for pharmacologically inducing cholinergic impairments in auditory processing in healthy rats. Changes in auditory processing and sensory gating induced by cholinergic drugs may serve as a translational model for aging instead of schizophrenia.

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening.

Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.

Controversy over the meaning of pharmacological parameters often arises because of a lack of appreciation of different hierarchical levels of analysis. In a recent letter in Trends in Pharmacological Sciences, Zhang and Kavana [1] concluded that my two-state model for allosterism lacks cooperativity, even though Figures 5 and 6 in my review [2] illustrate examples of how the two-state model yields specific cooperativity values. Here, I explain how the two-state model (receptor-state analysis) gives rise to the cooperativity parameter (α) of the allosteric ternary complex model (receptor-population analysis).

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery., This study is funded by the Medical Research Council (MRC) through funding of program leaders provided by the MRC Toxicology Unit (to A.B.T.).

Tremulous jaw movements (TJMs) are rapid vertical deflections of the lower jaw that resemble chewing but are not directed at any particular stimulus. In rodents, TJMs are induced by neurochemical conditions that parallel those seen in human Parkinsonism, including neurotoxic or pharmacological depletion of striatal dopamine (DA), DA antagonism, and cholinomimetic administration. Moreover, TJMs in rodents can be attenuated by antiparkinsonian agents, including levodopa (L-DOPA), DA agonists, muscarinic antagonists, and adenosine A2A antagonists. In human Parkinsonian patients, exaggerated physiological synchrony is seen in the beta frequency band in various parts of the cortical/basal ganglia/thalamic circuitry, and activity in the tremor frequency range (3–7 Hz) also has been recorded. The present studies were undertaken to determine if tremor-related local field potential (LFP) activity could be recorded from motor cortex (M1) or subthalamic nucleus (STN) during the TJMs induced by the muscarinic agonist pilocarpine, which is a well-known tremorogenic agent. Pilocarpine induced a robust TJM response that was marked by rhythmic electromyographic (EMG) activity in the temporalis muscle. Compared to periods with no tremor activity, TJM epochs were characterized by increased LFP activity in the tremor frequency range in both neocortex and STN. Tremor activity was not associated with increased synchrony in the beta frequency band. These studies identified tremor-related LFP activity in parts of the cortical/basal ganglia circuitry that are involved in the pathophysiology of Parkinsonism. This research may ultimately lead to identification of the oscillatory neural mechanisms involved in the generation of tremulous activity, and promote development of novel treatments for tremor disorders.

The striatum is one of the major forebrain regions that strongly expresses muscarinic and nicotinic cholinergic receptors. This article reviews the current knowledge and our new findings about the striatal cholinoceptive organization and its role in a variety of cognitive functions. Pharmacological and genetic manipulations have indicated that the cholinergic and dopaminergic system in the striatum modulate each other's function. In addition to modulating the dopaminergic system, nicotinic cholinergic receptors facilitate GABA release, whereas muscarinic receptors attenuate GABA release. The striatal cholinergic system has also been implicated in various cognitive functions including procedural learning and intradimensional set shifting. Together, these data indicate that the cholinergic system in the striatum is involved in a diverse set of cognitive functions through interactions with other neurotransmitter systems including the dopaminergic and GABAergic systems. (C) 2010 Elsevier B.V. All rights reserved.

Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD), the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs) are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional) and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

We tested the hypothesis that the de-endothelialized artery rings from the left anterior descending (LAD) coronary artery and its left ventricular branch (LVB) differ in their contractile responses to Na(+)-Ca(2+)-exchanger (NCX) mediated Ca(2+)-entry, muscarinic receptor activation with carbachol, and sarco/endoplasmic reticulum Ca(2+) pump (SERCA) inhibition with thapsigargin. In LVB, the force of contraction (in N/g tissue) produced by the NCX mediated Ca(2+)-entry (17.5 +/- 1.4) and carbachol (18 +/- 1.5) was only slightly smaller than that due to membrane depolarization with KCl (24.0 +/- 1.0). In contrast, in LAD the force of contraction produced with NCX (8.7 +/- 0.7) and carbachol (6.1 +/- 1.1) was much smaller than with KCl (15.7 +/- 0.7). Thapsigargin also contracted LVB with greater force than LAD. When isolated microsomes were used, the binding to the muscarinic receptor antagonist quinuclidinyl benzilate was greater in LVB than in LAD. Microsomes were also used for Western blots. The intensities of signals for both SERCA and NCX were greater in LVB than in LAD. These biochemical observations were consistent with the contractile experiments. Thus, it appears that the differences between LAD and the resistance arteries may begin as early as LVB.

Despite being a favorite delicacy, only 200–300 of the 5,000 known mushroom species have been clearly established to be safe for consumption. Cases of mushroom poisoning have been reported with diverse clinical syndromes. A syndromic classification of mushroom poisoning has recently been developed to facilitate early interventions. We present a series of five cases of mushroom poisoning with muscarinic manifestations to highlight the difficulties we faced with exact species and toxin identification and the importance of this syndromic classification. The common symptoms in our case series are blurred vision, diarrhea, vomiting, and abdominal cramps.

Maintenance of context is necessary for execution of appropriate responses to diverse environmental stimuli. The dorsolateral prefrontal cortex (DLPFC) plays a pivotal role in executive function, including working memory and representation of abstract rules. DLPFC activity is modulated by the ascending cholinergic system through nicotinic and muscarinic receptors. Although muscarinic receptors have been implicated in executive performance and gating of synaptic signals, their effect on local primate DLPFC neuronal activityin vivoduring cognitive tasks remains poorly understood. Here, we examined the effects of muscarinic receptor blockade on rule-related activity in the macaque prefrontal cortex by combining iontophoretic application of the general muscarinic receptor antagonist scopolamine with single-cell recordings while monkeys performed a mnemonic rule-guided saccade task. We found that scopolamine reduced overall neuronal firing rate and impaired rule discriminability of task-selective cells. Saccade and visual direction selectivity measures were also reduced by muscarinic antagonism. These results demonstrate that blockade of muscarinic receptors in DLPFC creates deficits in working memory representation of rules in primates.SIGNIFICANCE STATEMENTAcetylcholine plays a pivotal role in higher-order cognitive functions, including planning, reasoning, impulse-control, and making decisions based on contingencies or rules. Disruption of acetylcholine function is central to many psychiatric disorders manifesting cognitive impairments, including Alzheimer's disease. Although much is known about the involvement of acetylcholine and its receptors in arousal and attention, its involvement in working memory, an essential short-term memory component of cognition dependent on the integrity of prefrontal cortex, remains poorly understood. Herein, we explored the impact of suppressing acetylcholine signaling on neurons encoding memorized rules while macaque monkeys made responses based on those rules. Our findings provide insights into the neural mechanisms by which a disruption in acetylcholine function impairs working memory in the prefrontal cortex.

The medial prefrontal cortex (mPFC) is a key brain region for the control of consummatory behavior. Neuronal activity in this area is modulated when rats initiate consummatory licking and reversible inactivations eliminate reward contrast effects and reduce a measure of palatability, the duration of licking bouts. Together, these data suggest the hypothesis that rhythmic neuronal activity in the mPFC is crucial for the control of consummatory behavior. The muscarinic cholinergic system is known to regulate membrane excitability and control low-frequency rhythmic activity in the mPFC. Muscarinic receptors (mAChRs) act through KCNQ (Kv7) potassium channels, which have recently been linked to the orexigenic peptide ghrelin. To understand if drugs that act on KCNQ channels within the mPFC have effects on consummatory behavior, we made infusions of several muscarinic drugs (scopolamine, oxotremorine, physostigmine), the KCNQ channel blocker XE-991, and ghrelin into the mPFC and evaluated their effects on consummatory behavior. A consistent finding across all drugs was an effect on the duration of licking bouts when animals consume solutions with a relatively high concentration of sucrose. The muscarinic antagonist scopolamine reduced bout durations, both systemically and intra-cortically. By contrast, the muscarinic agonist oxotremorine, the cholinesterase inhibitor physostigmine, the KCNQ channel blocker XE-991, and ghrelin all increased the durations of licking bouts when infused into the mPFC. Our findings suggest that cholinergic and ghrelinergic signaling in the mPFC, acting through KCNQ channels, regulates the expression of palatability.

While historically acetylcholine (ACh) holds a central place in the discovery of chemical transmission in the nervous system, progress in our knowledge of the mechanistic underpinnings of cholinergic transmission in the central nervous system (CNS) has lagged behind its sister transmitters. For example, unlike what we know about glutamatergic transmission, neither the prevalence of fast cholinergic transmission or postsynaptic specializations at cholinergic synapses, is well understood. Every level of inquiry into the cholinergic system reveals a bewildering complexity in signaling and transduction mechanisms. Receptor localizations within and among neurons, along with transmitter source and access to receptors, provide for a system that is capable of neuromodulation at multiple time scales that allow for short- and long-term regulation of circuit output in the brain. The compilation of reviews and primary papers in this research topic provides a sampling of findings at different levels of integration that highlight both the current status of our understanding of CNS cholinergic mechanisms as well as reveals the gaps in our knowledge that need to be filled. How ACh mediates signaling in the brain is still an unresolved issue at the level of synaptic physiology. Cholinergic innervation in the brain arises from two main loci- the basal forebrain and the pendunculo-pontine area of the hindbrain. In both instances a relatively small cluster of neurons send diffuse projections into various target areas. The diffuse nature of the projections, as well as the non-planar relationship between the cholinergic neurons and their targets precludes traditional slice electrophysiology approaches to examining signaling. Recent advances in optogenetic techniques offer a potential solution to this problem and some of the advances made thus far are reviewed in this research topic (Luchicchi et al., 2014). Some of the relevant issues that need to be resolved are the prevalence of synaptic vs. non-synaptic modes of activation, the role of transmitter diffusion, relationship between ACh release sites and the distribution of acetylcholine esterases, and the potential co-release of other transmitters from cholinergic terminals. In addition to the centrifugal cholinergic inputs, areas of the cortex also express their own, resident, cholinergic interneurons. Well studied among these are the cholinergic interneurons of the striatum, relevant to disorders like Parkinson's disease. These are large aspiny neurons that are small in number (about 1% of the total neuronal population) but send dense projections throughout the area. Recent findings that are summarized here (Lim et al., 2014) show that these neurons themselves are subject to complex regulation, both excitatory and inhibitory, from a vast area of other transmitter systems. Another area, where cholinergic interneurons have been described is the hippocampus (Frotscher et al., 2000). Thus, far, these neurons have remained an oddity, with no functional attribution. In this research topic, work done by the group of Josh Lawrence (Yi et al., 2015) has begun the examination of these neurons using transgenic mice where GFP or YFP is expressed driven by the choline acetyltransferase promoter. Interestingly, these neurons might themselves be subject to cholinergic modulation, presumably from basal forebrain innervation. We have a long way to go before we understand the complex variables of cholinergic signaling. Differential expression of various subtypes of the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs), their expression in multiple neuronal types within a region, and varying locations within a neuron (i.e., somatic, dendritic, synaptic etc.) all orchestrate a complex symphony of neuromodulation. A review of receptor localization and function within hippocampal CA 1 interneurons (McQuiston, 2014) illustrates how complex expression of the two classes of receptors can allow for differential control of principal cell activity under varying concentrations of the transmitter. These local control mechanisms can regulate circuit outputs in various regions of the brain, potentially mediating the attention and learning-specific behaviors ascribed to ACh-driven modulation. In the olfactory system, nAChR activation can filter odor signals such that weak inputs are eliminated while strong ones are allowed through, thus providing a mechanism for altering the gain function of a circuit (D'souza and Vijayaraghavan, 2014) and, perhaps, regulating the effects of these receptors on odor discrimination (Hellier et al., 2010). In the visual cortex, differential functional expression of mAChRs might explain neuronal synchrony and gamma oscillations allowing for the tuning of network output during tasks involving perceptual learning (Groleau et al., 2015). In the hippocampus, a potential role for mAChR-regulated release of endocannabinoid in circuit oscillation highlight the complex mechanisms in play that underlie the effects of the cholinergic system in regulation of behavior (Alger et al., 2014). Cholinergic receptors are a potential gold mine as targets for therapies and pharmaceutical companies have not been diffident about exploring various receptor ligands as potential therapies. Emerging studies implicate cholinergic receptors in various addictive mechanisms: the direct interaction between cocaine and nAChRs reported in this research topic (Acevedo-Rodriguez et al., 2014) illustrates how these receptors could play an important role in the reinforcement properties of cocaine. The cholinergic system appears to be involved in specific behavioral endophenotypes of a number of diseases such as Alzheimer's disease (Oddo and LaFerla, 2006), schizophrenia (Wallace and Bertrand, 2013), and Parkinson's disease (Bohnen et al., 2010). More recent animal model studies also implicate the transmitter system in autism (Amodeo et al., 2014). This is prime time for the development of clinical therapies that target the cholinergic system for a host of brain disorders. At the same time, as the collection of studies in this research topic illustrates, much remains to be understood regarding the physiology of cholinergic transmission and modulation. There is a risk of pharmacological advances outpacing our knowledge of cholinergic signaling mechanisms in the brain. History tells us that such a disconnect between therapeutic advances and our knowledge of physiology can often lead to unintended complications from novel therapies, a classic example being the aggressive marketing of heroin as a cough remedy at the turn of the twentieth century. There needs to be significant investment in examining the basic biology of cholinergic modulation of brain circuits in order to develop more rational and safe therapeutic targets that the cholinergic system offers.

Mammalian circadian control is determined by a central clock in the brain suprachiasmatic nucleus (SCN) and synchronized peripheral clocks in other tissues. Increasing evidence suggests that SCN-independent regulation of peripheral clocks also occurs. We examined how activation of excitatory receptors influences the clock protein PERIOD 2 (PER2) in a contractile organ, the urinary bladder. PERIOD2::LUCIFERASE-knock-in mice were used to report real-time PER2 circadian dynamics in the bladder tissue. Rhythmic PER2 activities occurred in the bladder wall with a cycle of ∼24 h and peak at ∼12 h. Activation of the muscarinic and purinergic receptors by agonists shifted the peak to an earlier time (7.2±2.0 and 7.2±0.9 h, respectively). PER2 expression was also sensitive to mechanical stimulation. Aging significantly dampened PER2 expression and its response to the agonists. Finally, muscarinic agonist-induced smooth muscle contraction also exhibited circadian rhythm. These data identified novel regulators, endogenous receptors, in determining local clock activity, in addition to mediating the central control. Furthermore, the local clock appears to reciprocally align receptor activity to circadian rhythm for muscle contraction. The interaction between receptors and peripheral clock represents an important mechanism for maintaining physiological functions and its dysregulation may contribute to age-related organ disorders.—Wu, C., Sui, G., Archer, S. N., Sassone-Corsi, P., Aitken, K., Bagli, D., Chen, Y. Local receptors as novel regulators for peripheral clock expression.

We studied the role of endogenous acetylcholine (Ach) in corticostriatal long-term potentiation (LTP). The muscarinic receptor antagonists scopolamine and pirenzepine fully prevented the induction of LTP suggesting that the activation of M1-like muscarinic receptors is a crucial event in the conditioning phase of this form of synaptic plasticity.

These experiments examined the interaction of muscarinic and dopaminergic systems in influencing memory for one-trial inhibitory avoidance training in mice of the C57BL/6 and DBA/2 strains. In both strains, immediate post-training systemic administration of the muscarinic cholinergic agonist oxotremorine enhanced retention and the cholinergic antagonist atropine impaired retention. No effects were seen with injections 2 h post-training. Furthermore, the drugs did not affect retention performance of animals that received no footshock on the training trial. These results confirm previous findings indicating that muscarinic cholinergic drugs affect memory by influencing memory consolidation. In C57 mice, pretreatment with selective D1 or D2 dopamine (DA) receptor agonists (SKF 38393 or LY 171555, respectively) in otherwise non-effective doses (5 and 0.25 mg/kg, respectively) potentiated the effects of oxotremorine (0.04 mg/kg). Furthermore, in C57 mice pretreatment with selective D1 or D2 receptor antagonists (SCH 23390 or (−)-sulpiride) in otherwise non-effective doses (0.025 and 6 mg/kg, respectively) blocked the memory enhancing effects of oxotremorine. The memory impairing effects of atropine (3 mg/kg) were blocked by the D1 and D2 selective agonists and potentiated by the selective D1 or D2 antagonists. In contrast, in DBA mice, the D1 and D2 selective agonists antagonised the memory enhancing effects of oxotremorine (0.02 mg/kg) and potentiated the effects of atropine (2 mg/kg). Furthermore, the D1 and D2 antagonists potentiated the effects of oxotremorine and antagonised those of atropine. These findings indicate that although muscarinic cholinergic influences on memory storage are comparable in mice of these two strains, the cholinergic-dopaminergic interactions are opposite in the two strains. These results have implications for hypotheses of cholinergic and dopaminergic regulation of memory storage.

1. Intracellular recordings were made from neurones in slice of rat striatum in vitro. 2. The forty-nine neurones studied were immunoreactive for choline acetyltransferase and had the electrophysiological characteristics typical of large aspiny interneurones. 3. Focal stimulation of the slice elicited a hyperpolarizing inhibitory postsynaptic potential in thirty-five neurones. This IPSP lasted 0.5-1 s and reversed polarity at a membrane potential which was dependent on the logarithm of the extracellular potassium concentration. 4. The IPSP was reversibly blocked by scopolamine and methoctramine, which has some selectivity for M2 subtype of muscarinic receptor. It was unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), DL-2-amino-phosphonovaleric acid (30 microM) and bicuculline (30 microM). 5. Exogenous acetylcholine and muscarine also hyperpolarized the neurones, and this was blocked by methoctramine by not by pirenzepine, which is an M1 receptor-selective antagonist. 6. The findings demonstrate that muscarinic IPSPs occur in the central nervous system. The IPSP may mediate an 'autoinhibition' of striatal cholinergic neurone activity.

Purpose To assess the response of rat urinary bladder regenerated by the homologous bladder acellular matrix graft (BAMG) to in vitro electrical and pharmacologic stimuli. Materials and Methods In Sprague-Dawley rats, partial cystectomy (>50%) was performed, followed by BAMG augmentation cystoplasty. After 4 months, organ bath studies of tissue strips in 10 were used to compare the contractility of the BAMG regenerates and the corresponding host detrusor smooth muscle. Results The BAMG regenerates exhibited contractile activity to electrical field stimulation and a qualitatively identical pattern of response to muscarinic, purinergic, alpha- and beta-adrenergic drug administration and nitric oxide. At 4 months after surgery, the maximum forces of contraction of the BAMG regenerates to carbachol stimulation amounted to close to 80% of the host bladder response. With electrical field stimulation, they equaled 44% and 62% of the host bladder response after 2.5 and 4 months, respectively. Histological and immunohistochemical studies confirmed the presence of receptors for neurotransmitters that these functional in vitro studies implied. Conclusions The present study provides further evidence that augmentation cystoplasty with the BAMG leads to functional regeneration of the rat bladder detrusor smooth muscle.

Kylie J MansfieldGraduate School of Medicine, University of Wollongong, NSW, AustraliaAbstract: Muscarinic receptors have long been the target receptors for treatment of patients with overactive bladder (OAB). These patients experience symptoms of urgency, urinary frequency and nocturia, with or without urge incontinence (the involuntary leakage of urine associated with urge). Fesoterodine, a pro-drug, structurally and functionally related to tolterodine, is the newest agent developed for the treatment of OAB. Fesoterodine is broken down to the active metabolite, 5-hydroxy-methyl-tolterodine (5-HMT) by non-specific esterases. This metabolism results in the complete breakdown of the parent compound and is responsible for dose related improvements in clinical efficacy and health related quality of life. Like other antimuscarinic agents including tolterodine, fesoterodine is associated with improvements in clinical variables related both to bladder filling (decreasing micturition frequency and increasing mean voided volume) and urgency (urgency and urge incontinence episodes). Improvements in health related quality of life following treatment with fesoterodine is indicated by improvements in 7 of the 9 variables measured by the King’s Health Questionnaire. Also like other antimuscarinic agents, fesoterodine use is associated with adverse events including dry mouth. However the incidence of dry mouth is reduced with fesoterodine, compared to oxybutynin, due to the improved bladder selectivity of 5-HMT.Keywords: fesoterodine, 5-hydroxymethyl-tolterodine, muscarinic, overactive bladder, urgency, incontinence

Background: AZD9164 has demonstrated potential as an inhaled, long-acting, muscarinic antagonist (LAMA) bronchodilator. However, in patients with COPD, but not in healthy subjects, a transient initial drop in FEV1 was observed following inhalation of nebulised doses of AZD9164 in citrate buffer. Two additional studies were conducted to further assess the safety and tolerability of multiple ascending doses of AZD9164 in 27 white and 18 Japanese healthy subjects and in 4 patients with COPD. In these studies, AZD9164 was inhaled via Turbuhaler™. Methods: These were Phase I, randomised, double-blind, placebo-controlled, multiple ascending dose (MAD) studies conducted in Sweden and UK. Healthy subjects (mean age 25.9 yrs) and patients with COPD (mean age 66 yrs, mean post-bronchodilator FEV1 60.1% predicted normal value) were randomised 2:1 to active treatment (400, 1000 or 2800 μg delivered doses of AZD9164) or placebo. Results: No safety or tolerability concerns were identified in the healthy subjects at doses up to and including 2800 μg and both studies confirmed the bronchodilator effect of AZD9164. However, the first 3 patients in the COPD cohort who received AZD9164 (1000 μg) experienced a transient fall in FEV1 5t o 15 minutes after inhalation of AZD9164 while the patient receiving placebo did not. The study safety review process then resulted in cessation of further activities on AZD9164. Retrospective analysis showed that two healthy subjects had also had transient falls in FEV1 shortly after inhalation of AZD9164 400 and 2800 μg respectively, although neither reported any related respiratory symptoms or other AEs. Conclusions: These results show that transient paradoxical bronchoconstriction ca no ccur in some healthy subjects, in addition to patients with COPD, following inhalation of AZD9164 and that the citrate buffer used in the nebulised formulation cannot have been the only cause of the drop in FEV1 in previous studies. As preclinical data do not provide an explanation, the reasons for this brief post-dose drop in FEV1 remain unclear. However, these results highlight the importance of monitoring lung function immediately post-dose when investigating novel inhaled treatments, even when a rapid onset of effect is not expected. Trial registration: Clinicaltrials.gov NCT01016951 and NCT01096563.

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening., Graphical abstract Individual M2 muscarinic receptors were visualized and tracked in live cardiac tissue slices to establish their abundance, distribution, mobility and oligomeric state in situ. Highlights ► The first observation of single molecules moving on the surface of living tissue ► Only ca. 10% of murine cardiomyocytes expressed M2 receptors at appreciable levels. ► M2 density at birth was 3-fold higher than that in embryonic and adult myocytes. ► M2 receptors were primarily monomeric but formed reversible dimers. ► M2 receptors diffused 4-times faster in heart slices than in cultured myocytes.

While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

Acetylcholine (ACh) is a neurotransmitter acting via muscarinic and nicotinic receptors that is implicated in several cognitive functions and impairments, such as Alzheimer’s disease. It is believed to especially affect the acquisition of new information, which is particularly important when behaviour needs to be adapted to new situations and to novel sensory events. Categorisation, the process of assigning stimuli to a category, is a cognitive function that also involves information acquisition. The role of ACh on categorisation has not been previously studied. We have examined the effects of scopolamine, an antagonist of muscarinic ACh receptors, on visual categorisation in macaque monkeys using familiar and novel stimuli. When the peripheral effects of scopolamine on the parasympathetic nervous system were controlled for, categorisation performance was disrupted following systemic injections of scopolamine. This impairment was observed only when the stimuli that needed to be categorised had not been seen before. In other words, the monkeys were not impaired by the central action of scopolamine in categorising a set of familiar stimuli (stimuli which they had categorised successfully in previous sessions). Categorisation performance also deteriorated as the stimulus became less salient by an increase in the level of visual noise. However, scopolamine did not cause additional performance disruptions for difficult categorisation judgements at lower coherence levels. Scopolamine, therefore, specifically affects the assignment of new exemplars to established cognitive categories, presumably by impairing the processing of novel information. Since we did not find an effect of scopolamine in the categorisation of familiar stimuli, scopolamine had no significant central action on other cognitive functions such as perception, attention, memory or executive control within the context of our categorisation task.

Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision due to glaucoma is caused by the selective death of retinal ganglion cells (RGCs). Treatments for glaucoma, limited to drugs or surgery to lower intraocular pressure (IOP), are insufficient. Therefore, a pressing medical need exists for more effective therapies to prevent vision loss in glaucoma patients. In this in vivo study, we demonstrate that systemic administration of galantamine, an acetylcholinesterase inhibitor, promotes protection of RGC soma and axons in a rat glaucoma model. Functional deficits caused by high IOP, assessed by recording visual evoked potentials from the superior colliculus, were improved by galantamine. These effects were not related to a reduction in IOP because galantamine did not change the pressure in glaucomatous eyes and it promoted neuronal survival after optic nerve axotomy, a pressure-independent model of RGC death. Importantly, we demonstrate that galantamine-induced ganglion cell survival occurred by activation of types M1 and M4 muscarinic acetylcholine receptors, while nicotinic receptors were not involved. These data provide the first evidence of the clinical potential of galantamine as neuroprotectant for glaucoma and other optic neuropathies, and identify muscarinic receptors as potential therapeutic targets for preventing vision loss in these blinding diseases.

Rat and mouse models are widely used for studies in cognition and pathophysiology, among others. Here, we sought to determine to what extent these two model species differ for cholinergic and cholinoceptive features. For this purpose, we focused on cholinergic innervation patterns based on choline acetyltransferase (ChAT) immunostaining, and the expression of muscarinic acetylcholine receptors (mAChRs) detected immunocytochemically. In this brief review we first place cholinergic and cholinoceptive markers in a historic perspective, and then provide an overview of recent publications on cholinergic studies and techniques to provide a literature survey of current research. Next, we compare mouse (C57B1/J6) and rat (Wistar) cholinergic and cholinoceptive systems simultaneously stained, respectively, for ChAT (analyzed qualitatively) and mAChRs (analyzed qualitatively and quantitatively). In general, the topographic cholinergic innervation patterns of both rodent species are highly comparable, with only considerable (but region specific) differences in number of detectable cholinergic interneurons, which are more numerous in rat. In contrast, immunolabeling for mAChRs, detected by the monoclonal antibody M35, differs markedly in the forebrain between the two species. In mouse brain, basal levels of activated and/or internalized mAChRs (as a consequence of cholinergic neurotransmission) are significantly higher. This suggests a higher cholinergic tone in mouse than rat, and hence the animal model of choice may have consequences for cholinergic drug testing experiments. (C) 2010 Elsevier B.V. All rights reserved.

G-protein coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This super-family of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In the current study we demonstrate that a classical Gq/11-coupled GPCR, the M3-muscarinic receptor, was able to regulate apoptosis via receptors that are endogenously expressed in the human neuroblastoma cell line SH-SY5Y and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M3-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, sub-cellular trafficking and modification of p53 in a manner that is in part dependent on the cell type.

Purpose The human physiological bladder contraction is largely mediated by acetylcholine acting on muscarinic receptors, but in pathophysiological settings the relative role of non-cholinergic stimuli gains importance. beta-Adrenoceptor agonists are currently in clinical development as treatments for the overactive bladder syndrome. Therefore, we have explored the ability of the beta-adrenoceptor agonist isoprenaline to induce rat isolated bladder strip relaxation on pre-contraction with the muscarinic agonist carbachol as compared to bladder tone induced by several non-cholinergic stimuli. Methods Bladder tone was induced by passive tension, receptor independently by KCl, carbachol, bradykinin or serotonin. Concentration-response curves were generated for relaxation by isoprenaline, and a single concentration of the receptor-independent relaxant forskolin was also tested. Results The various contractile stimuli induced different degrees of bladder tone, but the ability of isoprenaline or forskolin to relax rat bladder was not correlated with the degree of tone. Isoprenaline was significantly less potent and effective in causing relaxation against carbachol-induced tone than against any other stimulus, whereas no such relationship was observed for forskolin. Conclusions We conclude that beta-adrenoceptor agonists can induce rat bladder relaxation against a wide range of contractile stimuli and are more potent and/or effective against non-cholinergic stimuli than against muscarinic agonism. This profile appears desirable for agents intended for the treatment of overactive bladder

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (infinity). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors., The original publication is available at www.springerlink.com, Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology, 195(4), pp.351-363; 2009

1. In the present study we investigated desensitization phenomena of guinea-pig jejunal longitudinal smooth muscle responses after stimulation with 100 microM histamine or methacholine, using a superfusion method. 2. Histamine H1-receptor-mediated contractions appear to be rapidly reduced after application of 100 microM histamine. Muscarinic responses were not affected following desensitization with 100 microM histamine, indicating a homologous desensitization. 3. Initial contractions to 0.3 microM histamine were reduced by 90%, recovered quickly, but did not reach control levels within 1 h. Desensitization of histamine responses could be separated into two phases; a rapid, but transient, desensitization and a more sustained desensitization. As a consequence of this sustained effect the pD2 for histamine shifted from 6.7 +/- 0.1 (control) to 6.1 +/- 0.1 (desensitized). 4. Desensitization with 100 microM methacholine caused a heterologous desensitization, reflected by the development of a refractory period, in which neither histamine nor methacholine was able to elicit a contraction. After a few minutes responses to both agents recovered to control levels. 5. During the refractory period after methacholine desensitization, muscle strips were still responsive to 40 mM KCl but did not contract in response to 10 mM caffeine, suggesting that the heterologous desensitization is caused by a modification of an intracellular Ca2(+)-store, which is used by both histamine and methacholine. 6. The recovery of the responses after methacholine desensitization was not dependent on extracellular Ca2+, suggesting that the recovery is not dependent on refilling of the intracellular Ca2+ store with extracellular Ca2+. 7. The protein kinase C activator, phorbol-12,13-dibutyrate, concentration-dependently inhibited histamine- and methacholine-induced contractions. Protein kinase C seems therefore not to be implicated in the observed homologous H,-receptor desensitization. 8. These data suggest that different forms of desensitization can be distinguished in this model, each with a different time course and dependent on the applied stimulus.

Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB(1) cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M(3) muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB(1) antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB(1) receptor agonist, mimics M(3) activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. 2-Arachidonoylglycerol (2-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by 20 min of 1-Hz stimulation.