Co-stimulation of muscarinic M1 and M4 acetylcholine receptors prevents later cocaine reinforcement in male and female mice, but not place-conditioning.

Acute stimulation of M1 or M4 muscarinic cholinergic receptors reduces cocaine abuse-related effects in mice and rats. The combined activation of these receptor subtypes produces synergistic effects on some behavioural endpoints in mice. M1 and M1 + M4 receptor stimulation in a cocaine vs. food choice assay in rats and microdialysis in rats showed delayed and lasting "anticocaine effects". Here, we tested whether these putative lasting neuroplastic changes are sufficient to occlude the reinforcing effects of cocaine at the behavioural level in mice. Mice were pre-treated with the M1 receptor partial agonist VU0364572, M4 receptor positive allosteric modulator VU0152100, or VU0364572 + VU0152100 two weeks prior to acquisition of cocaine intravenous self-administration (IVSA). Male C57BL/6JRj mice received vehicle, VU0364572, VU0152100, or VU0364572 + VU0152100. Female mice were tested with two VU0364572 + VU0152100 dose combinations or vehicle. To attribute potential effects to either reduced rewarding effects or increased aversion to cocaine, we tested VU0364572 alone and VU0364572 + VU0152100 in acquisition of cocaine-conditioned place preference (CPP) in male mice using an unbiased design. The acquisition of cocaine IVSA was drastically reduced and/or slowed in male and female mice receiving VU0364572 + VU0152100, but not either drug alone. Food-maintained operant behaviour was unaffected, indicating that the treatment effects were cocaine-specific. No treatment altered the acquisition of cocaine-CPP, neither in the post-test, nor in a challenge 14 days later. The cocaine IVSA findings confirm unusual long-lasting "anticocaine" effects of muscarinic M1 + M4 receptor stimulation. Thus, in mice, simultaneous stimulation of both receptor subtypes seems to produce potential neuroplastic changes that yield lasting effects.

Cadmium-induced neurotoxic effects on rat basal forebrain cholinergic system through thyroid hormones disruption.

Cadmium (Cd) single and repeated exposure produces cognitive dysfunctions. Basal forebrain cholinergic neurons (BFCN) regulate cognitive functions. BFCN loss or cholinergic neurotransmission dysfunction leads to cognitive disabilities. Thyroid hormones (THs) maintain BFCN viability and functions, and Cd disrupts their levels. However, Cd-induced BFCN damages and THs disruption involvement was not studied. To research this we treated male Wistar rats intraperitoneally with Cd once (1 mg/kg) or repetitively for 28 days (0.1 mg/kg) with/without triiodothyronine (T3, 40 µg/kg/day). Cd increased thyroid-stimulating-hormone (TSH) and decreased T3 and tetraiodothyronine (T4). Cd altered cholinergic transmission and induced a more pronounced neurodegeneration on BFCN, mediated partially by THs reduction. Additionally, Cd antagonized muscarinic 1 receptor (M1R), overexpressed acetylcholinesterase S variant (AChE-S), downregulated AChE-R, M2R, M3R and M4R, and reduced AChE and choline acetyltransferase activities through THs disruption. These results may assist to discover cadmium mechanisms that induce cognitive disabilities, revealing a new possible therapeutic tool.

Effects of prenatal ethanol exposure on choline-induced long-term depression in the hippocampus.

Choline is an essential nutrient under evaluation as a cognitive enhancing treatment for fetal alcohol spectrum disorders (FASD) in clinical trials. As a result, there is increased pressure to identify therapeutic mechanism(s) of action. Choline is not only a precursor for several essential cell membrane components and signaling molecules but also has the potential to directly affect synaptic mechanisms that are believed important for cognitive processes. In the current work, we study how the direct application of choline can affect synaptic transmission in the dentate gyrus (DG) of hippocampal slices obtained from adolescent (postnatal days 21-28) Sprague-Dawley rats (Rattus norvegicus). The acute administration of choline chloride (2 mM) reliably induced a long-term depression (LTD) of field excitatory postsynaptic potentials (fEPSPs) in the DG in vitro. The depression required the involvement of M1 receptors, and the magnitude of the effect was similar in slices obtained from male and female animals. To further study the impact of choline in an animal model of FASD, we examined offspring from dams fed an ethanol-containing diet (35.5% ethanol-derived calories) throughout gestation. In slices from the adolescent animals that experienced prenatal ethanol exposure (PNEE), we found that the choline induced an LTD that uniquely involved the activation of N-methyl-d-aspartate (NMDA) and M1 receptors. This study provides a novel insight into how choline can modulate hippocampal transmission at the level of the synapse and that it can have unique effects following PNEE.NEW & NOTEWORTHY Choline supplementation is a nutraceutical therapy with significant potential for a variety of developmental disorders; however, the mechanisms involved in its therapeutic effects remain poorly understood. Our research shows that choline directly impacts synaptic communication in the brain, inducing a long-term depression of synaptic efficacy in brain slices. The depression is equivalent in male and female animals, involves M1 receptors in control animals, but uniquely involves NMDA receptors in a model of FASD.

Long-term effects of early postnatal nicotine exposure on cholinergic function in the mouse hippocampal CA1 region.

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.

The muscarinic M1 receptor modulates associative learning and memory in psychotic disorders.

BACKGROUND: Psychotic disorders are characterized by prominent deficits in associative learning and memory for which there are currently no effective treatments. Functional magnetic resonance imaging (fMRI) studies in psychotic disorders have identified deficits in fronto-temporal activation during associative learning and memory. The underlying pathology of these findings remains unclear. Postmortem data have suggested these deficits may be related to loss of muscarinic M1 receptor mediated signaling. This is supported by an in-vivo study showing improvements in these symptoms after treatment with the experimental M1/4 receptor agonist xanomeline. The current study tests whether reported deficits in fronto-temporal activation could be mediated by loss of M1 receptor signaling in psychotic disorders. METHODS: Twenty-six medication-free subjects diagnosed with a psychotic disorder and 29 age-, gender-, and IQ-matched healthy controls underwent two functional magnetic resonance imaging (fMRI) sessions, one under placebo and one under selective M1 antagonist biperiden, while performing the paired associated learning task. M1 binding potentials (BPND) were measured in the dorsolateral prefrontal cortex (DLPFC) and hippocampus using 123I-IDEX single photon emission computed tomography. RESULTS: In the subjects with psychotic disorders DLPFC hypoactivation was only found in the memory phase of the task. In both learning and memory phases of the task, M1 antagonism by biperiden elicited significantly greater hyperactivation of the parahippocampal gyrus and superior temporal gyrus in subjects with a psychotic disorders compared to controls. Greater hyperactivation of these areas after biperiden was associated with greater hippocampal M1 receptor binding during learning, with no association found with M1 receptor binding in the DLPFC. M1 receptor binding in the DLPFC was related to greater functional sensitivity to biperiden of the cingulate gyrus during the memory phase. CONCLUSION: The current study is the first to show differences in M1 receptor mediated functional sensitivity between subjects with a psychotic disorder and controls during a paired associate learning and memory task. Results point to subjects with psychotic disorders having a loss of M1 receptor reserve in temporal-limbic areas.

Ketamine: The final frontier or another depressing end?

Two decades ago, the observation of a rapid and sustained antidepressant response after ketamine administration provided an exciting new avenue in the search for more effective therapeutics for the treatment of clinical depression. Research elucidating the mechanism(s) underlying ketamine's antidepressant properties has led to the development of several hypotheses, including that of disinhibition of excitatory glutamate neurons via blockade of N-methyl-d-aspartate (NMDA) receptors. Although the prominent understanding has been that ketamine's mode of action is mediated solely via the NMDA receptor, this view has been challenged by reports implicating other glutamate receptors such as AMPA, and other neurotransmitter systems such as serotonin and opioids in the antidepressant response. The recent approval of esketamine (Spravato™) for the treatment of depression has sparked a resurgence of interest for a deeper understanding of the mechanism(s) underlying ketamine's actions and safe therapeutic use. This review aims to present our current knowledge on both NMDA and non-NMDA mechanisms implicated in ketamine's response, and addresses the controversy surrounding the antidepressant role and potency of its stereoisomers and metabolites. There is much that remains to be known about our understanding of ketamine's antidepressant properties; and although the arrival of esketamine has been received with great enthusiasm, it is now more important than ever that its mechanisms of action be fully delineated, and both the short- and long-term neurobiological/functional consequences of its treatment be thoroughly characterized.

Muscarinic and Nicotinic Modulation of Neocortical Layer 6A Synaptic Microcircuits Is Cooperative and Cell-Specific.

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4ß2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4ß2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

Central muscarinic receptor subtypes (M1 and M3) involved in carbacol-induced hypophagia in neonatal broiler chicken.

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist), 4-DAMP (M3 receptor antagonist), and PD102807 (M4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD3) neonatal broiler chicken.Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3-5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection.Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group (P < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake (P < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol (P < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake (P < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol (P > 0.05).Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors in neonatal chicken.

M1 muscarinic receptor is a key target of neuroprotection, neuroregeneration and memory recovery by i-Extract from Withania somnifera.

Memory loss is one of the most tragic symptoms of Alzheimer's disease. Our laboratory has recently demonstrated that 'i-Extract' of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

Effects of novel hexahydropyrimidine derivatives as potential ligands of M1 muscarinic acetylcholine receptor on cognitive function, hypoxia-induced lethality, and oxidative stress in rodents.

The neurodegenerative diseases have a complex pathogenetic mechanism comprising oxidative stress and receptor system dysfunction caused by various damaging factors such as, for example, brain hypoxia. The purpose of this study was to elucidate the influence of hexahydropyrimidine derivatives on learning, memory, and orientation and locomotor activities in the passive avoidance (PA) and open field (OF) tests and to evaluate these compounds for their potential antihypoxic and antioxidant action on normobaric hypercapnic hypoxia and toxic hypoxia models. We demonstrated that compounds 1a and 1e administered as a single 100 mg/kg dose (p.o.) one hour before the tests increased the latency time to enter the dark compartment for the first time and reduced the time spent in the dark compartment on the 2nd, 7th, and 14th days of PAT and increased the number of squares crossed and hole-pokings in the OF test. It was also shown that single administration of compounds 1a and 1e (in 100 mg/kg dose, p.o.) one hour before generation of hypoxia increased the life span of mice under normobaric hypoxia by 30% (P < 0.05) and, after injection of sodium nitroprusside, they decreased the malondialdehyde (MDA) level and increased the catalase level in the brain of mice. According to molecular docking results, compounds 1а and 1е are bound in the orthosteric active site of M1 muscarinic receptor via supramolecular interactions with a number of functional amino acids. The results indicate that hexahydropyrimidine derivatives have a beneficial effect on the memory, learning processes, and orientation and locomotor activities of rats in an unfamiliar environment and exhibit antihypoxic and antioxidant activities under hypoxia in mice. The cognitive enhancement can be mediated by the effect of lead compounds on the M1 muscarinic acetylcholine receptor.

Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor Pharmacology and Photochromic Properties.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra- ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra- ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.

M1 muscarinic receptors facilitate hippocampus-dependent cognitive flexibility via modulating GluA2 subunit of AMPA receptors.

Cognitive flexibility is an important aspect of executive function. The cholinergic system, an important component of cognition, has been shown to modulate cognitive flexibility mainly through the striatum and prefrontal cortex. The role of M1 muscarinic receptors (M1 mAChRs), an important therapeutic target in the cholinergic system, in hippocampus-dependent cognitive flexibility is unclarified. In the present study, we demonstrated that selective activation of M1 mAChRs promoted extinction of initial learned response and facilitated acquisition of reversal learning in the Morris water maze, a behavior test that is mainly dependent on the hippocampus. However, these effects were abolished in GluA2 mutant mice with deficiency in phosphorylation of Ser880 by protein kinase C (PKC). Further long-term depression (LTD) in the hippocampal CA1 area induced by M1 mAChR activation was shown to be dependent on AMPA receptor subunit GluA2 but not GluA1. M1 mAChRs increased GluA2 endocytosis through phosphorylation of Ser880 by PKC. Inhibition of PKC blocked M1 mAChR-mediated LTD, memory switching and reversal learning facilitation. Moreover, the slow memory extinction observed in GluA2 mutant mice and PKC inhibitor-treated mice appeared to affect the consolidation and retrieval of reversal learning. Thus, these results demonstrate that M1 mAChRs mainly facilitate acquisition in spatial reversal learning and further elucidate that such an effect is dependent on the phosphorylation of GluA2 by PKC. The study helps clarify the role of M1 mAChRs in cognitive flexibility and may prompt the earlier prevention of cognitive inflexibility.

Central muscarinic and LPBN mechanisms on sodium intake.

Central cholinergic activation stimulates water intake, but also NaCl intake when the inhibitory mechanisms are blocked with injections of moxonidine (α2 adrenergic/imidazoline agonist) into the lateral parabrachial nucleus (LPBN). In the present study, we investigated the involvement of central M1 and M2 muscarinic receptors on NaCl intake induced by pilocarpine (non-selective muscarinic agonist) intraperitoneally combined with moxonidine into the LPBN or by muscimol (GABAA agonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN and in the lateral ventricle were used. Pirenzepine (M1 muscarinic antagonist, 1 nmol/1 µl) or methoctramine (M2 muscarinic antagonist, 50 nmol/1 µL) injected intracerebroventricularly (i.c.v.) reduced 0.3 M NaCl and water intake in rats treated with pilocarpine (0.1 mg/100 g of body weight) injected intraperitoneally combined with moxonidine (0.5 nmol/0.2 µL) into the LPBN. In rats treated with muscimol (0.5 nmol/0.2 µL) into the LPBN, methoctramine i.c.v. also reduced 0.3 M NaCl and water intake, however, pirenzepine produced no effect. The results suggest that M1 and M2 muscarinic receptors activate central pathways involved in the control of water and sodium intake that are under the influence of the LPBN inhibitory mechanisms.

TAK-071, a novel M1 positive allosteric modulator with low cooperativity, improves cognitive function in rodents with few cholinergic side effects.

The muscarinic M1 receptor (M1R) is a promising target for treating cognitive impairment associated with cholinergic deficits in disorders such as Alzheimer's disease and schizophrenia. We previously reported that cooperativity (α-value) was key to lowering the risk of diarrhea by M1R positive allosteric modulators (M1 PAMs). Based on this, we discovered a low α-value M1 PAM, TAK-071 (α-value: 199), and characterized TAK-071 using T-662 as a reference M1 PAM with high α-value of 1786. Both TAK-071 and T-662 were potent and highly selective M1 PAMs, with inflection points of 2.7 and 0.62 nM, respectively. However, T-662 but not TAK-071 augmented isolated ileum motility. TAK-071 and T-662 increased hippocampal inositol monophosphate production through M1R activation and improved scopolamine-induced cognitive deficits in rats at 0.3 and 0.1 mg/kg, respectively. TAK-071 and T-662 also induced diarrhea at 10 and 0.1 mg/kg, respectively, in rats. Thus, taking into consideration the fourfold lower brain penetration ratio of T-662, TAK-071 had a wider margin between cognitive improvement and diarrhea induction than T-662. Activation of M1R increases neural excitability via membrane depolarization, reduced afterhyperpolarization, and generation of afterdepolarization in prefrontal cortical pyramidal neurons. T-662 induced all three processes, whereas TAK-071 selectively induced afterdepolarization. Combining sub-effective doses of TAK-071, but not T-662, with an acetylcholinesterase inhibitor, significantly ameliorated scopolamine-induced cognitive deficits in rats. TAK-071 may therefore provide therapeutic opportunities for cognitive dysfunction related to cholinergic deficits or reduced M1R expression, while minimizing peripheral cholinergic side effects.

Exposure to far-infrared rays attenuates methamphetamine-induced recognition memory impairment via modulation of the muscarinic M1 receptor, Nrf2, and PKC.

We demonstrated that activation of protein kinase Cδ (PKCδ) and inactivation of the glutathione peroxidase-1 (GPx-1)-dependent systems are critical for methamphetamine (MA)-induced recognition memory impairment. We also demonstrated that exposure to far-infrared rays (FIR) causes induction of the glutathione (GSH)-dependent system, including induction of the GPx-1 gene. Here, we investigated whether exposure to FIR rays affects MA-induced recognition memory impairment and whether it modulates PKC, cholinergic receptors, and the GSH-dependent system. Because the PKC activator bryostatin-1 mainly induces PKCα, PKCε, and PKCδ, we assessed expression of these proteins after MA treatment. MA treatment selectively increased PKCδ expression and its phosphorylation. Exposure to FIR rays significantly attenuated MA-induced increases in PKCδ phosphorylation. Importantly, bryostatin-1 potentiated MA-induced phosphorylation of PKCδ. MA treatment significantly decreased M1, M3, and M4 muscarinic acetylcholine receptors (mAChRs) and ß2 nicotinic acetylcholine receptor expression. Of these, the decrease was most pronounced in M1 mAChR. Exposure to FIR significantly attenuated MA-induced decreases in the M1 mAChR and phospho-ERK1/2, while it facilitated Nrf2-dependent GSH induction. Dicyclomine, an M1 mAChR antagonist, and l-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, counteracted against the protective potentials mediated by FIR. More importantly, the memory-enhancing potential of FIR rays was significantly counteracted by bryostatin-1, dicyclomine, and BSO. Our results suggest that exposure to FIR rays attenuates MA-induced impairment in recognition memory via up-regulation of M1 mAChR, Nrf2-dependent GSH induction, and ERK1/2 phosphorylation by inhibiting PKCδ phosphorylation by bryostatin-1.

L-Satropane Prevents Retinal Neuron Damage by Attenuating Cell Apoptosis and Aß Production via Activation of M1 Muscarinic Acetylcholine Receptor.

Muscarinic acetylcholine receptor (mAChR) agonists have been used to treat glaucoma due to their intraocular pressure-lowering effects. Recently, it has been reported that retinal mAChRs activation can also stimulate neuroprotective pathways. PURPOSE: In our study, we evaluated the potential neuroprotective effect of L-satropane, a novel mAChR agonist, on retinal neuronal injury induced by cobalt chloride (CoCl2) and ischemia/reperfusion (I/R). METHODS: CoCl2-induced hypoxia injury in cultured cell models and I/R-induced retinal neuronal damage in rats in vivo were used to evaluate the abilities of L-satropane. In detail, we measured the occurrence of retinal pathological changes including molecular markers of neuronal apoptosis and Aß expression. RESULTS: Pretreatment with L-satropane protects against CoCl2-induced neurotoxicity in PC12 and primary retinal neuron (PRN) cells in a dose-dependent manner by increasing retinal neuron survival. CoCl2 or I/R-induced cell apoptosis by upregulating Bax expression and downregulating Bcl-2 expression, which resulted in an increased Bax/Bcl-2 ratio, and upregulating caspase-3 expression/activity was significantly reversed by L-satropane treatment. In addition, L-satropane significantly inhibited the upregulation of Aß production in both retinal neurons and tissue. We also found that I/R-induced histopathological retinal changes including cell loss in the retinal ganglion cell layer (GCL) and increased TUNEL positive retinal ganglion cells in GCL and thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) were markedly improved by L-satropane. The effects of L-satropane were largely abolished by the nonselective mAChRs antagonist atropine and M1-selective mAChR antagonist pirenzepine. CONCLUSION: These results demonstrated that L-satropane might be effective in preventing retinal neuron damage caused by CoCl2 or I/R. The neuroprotective effects of L-satropane may be attributed to decreasing cell apoptosis and Aß production through activation of M1 mAChR.

An improved, high-efficiency assay for assessing serum anticholinergic activity using cultured cells stably expressing M1 receptors.

Assessments of total anticholinergic activity (SAA) in serum are of considerable interest for its potential involvement in cognitive impairment associated with polydrug states in the elderly and other populations. Such estimations have been based on the displacement of radioligand binding in rat brain tissues. The validity of such measurements has been questioned, as a potentially distorting effect of large serum proteins was identified. We sought to develop a modified assay that would be more efficient and free of this potential confound. Cultured CHO cells stably expressing M1 receptors M1WT3 were used. Binding of 3H-radioligands was conducted in 96-well plates and tested in serum containing known amounts of anticholinergic medications. Effects of endogenous serum proteins were assessed by pre-assay filtration and also by deproteinization with perchloric acid (PCA). Binding of [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]N-methyl-scopolamine ([3H]NMS) to M1WT3 cells proved reliable and equally sensitive to varying concentrations of anticholinergic agents. In agreement with previous findings (Cox, Kwatra, Shetty, & Kwatra, 2009), filtration of proteins heavier than 50kDa essentially reduced SAA values to zero. In contrast, PCA preserved more than 70% of the binding seen untreated cell membranes. Cell-based assays also showed significant signal increases compared to the conventional rat brain-based protocol. Further advantages of the cell-based protocol described here include increased sensitivity and reliability, smaller amounts of radioligand needed, and higher throughput. PCA pretreatment eliminates potential artifacts attributable to serum proteins. This step, together with improvements in efficiency, should contribute significantly to the usefulness of the assay.

Disease-Modifying Effects of M1 Muscarinic Acetylcholine Receptor Activation in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is the leading cause of dementia worldwide, and currently no disease-modifying therapy is available to slow or prevent AD, underscoring the urgent need for neuroprotective therapies. Selective M1 muscarinic acetylcholine receptor (mAChR) activation is an attractive mechanism for AD therapy since M1 mediates key effects on memory, cognition, and behavior and has potential for disease-modifying effects on Aß formation and tau phosphorylation. To validate M1 as a neuroprotective treatment target for AD, the M1-selective agonist, VU0364572, was chronically dosed to 5XFAD mice from a young age preceding Aß pathology (2 months) to an age where these mice are known to display memory impairments (6 months). Chronic M1 activation prevented mice from becoming memory-impaired, as measured by Morris water maze (MWM) testing at 6 months of age. Additionally, M1 activation significantly reduced levels of soluble and insoluble Aß40,42 in the cortex and hippocampus of these animals, as measured by ELISA and immunohistochemistry. Moreover, soluble hippocampal Aß42 levels were strongly correlated with MWM memory impairments and M1 activation with VU0364572 abolished this correlation. Finally, VU0364572 significantly decreased oligomeric (oAß) levels in the cortex, suggesting one mechanism whereby VU0364572 may be exerting its neuroprotective effects is by reducing the available oAß pool in the brain. These findings suggest that chronic M1 activation has neuroprotective potential for preventing memory impairments and reducing neuropathology in AD. M1 activation therefore represents a promising avenue for preventative treatment, as well as a promising opportunity to combine symptomatic and disease-modifying effects for early AD treatment.

Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs.

Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.

Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3ß, and an increase in Aß and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3ß, AChE splice variants gene expression, and Aß and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity.