Discovery of 2-Methyl-5-(1H-pyrazol-4-yl)pyridines and Related Heterocycles as Promising M4 mAChR Positive Allosteric Modulators for the Treatment of Neurocognitive Disorders.

The M4 muscarinic acetylcholine receptor (mAChR) is a biological target for neurocognitive disorders. Compound 1 is an ago-PAM for the M4 mAChR. Herein, we report the design, synthesis, and evaluation of novel putative M4 mAChR PAMs based on 1. These analogs were screened and then fully characterized in two functional assays (GoB protein activation and CAMYEL activation) to quantify their allosteric and ago-PAM properties against ACh. A selection of 7 M4 PAMs were assessed for their ability to modulate ACh-mediated ß-arrestin recruitment and revealed 4 distinct clusters of M4 PAM activity: (1) analogs similar to 1 (24d), (2) analogs demonstrating only allosteric agonism (23d), (3) analogs with increased allosteric properties in CAMYEL activation (23b/23f and 24a/24b), and (4) analogs with a biased modulatory effect toward ß-arrestin recruitment (23i). These novel M4 chemical tools disclose discrete molecular determinants, allowing further interrogation of the therapeutic roles of cAMP and ß-arrestin pathways in neurocognitive disorders.

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.

Design and Synthesis of Clinical Candidate PF-06852231 (CVL-231): A Brain Penetrant, Selective, Positive Allosteric Modulator of the M4 Muscarinic Acetylcholine Receptor.

Selective activation of the M4 muscarinic acetylcholine receptor subtype offers a novel strategy for the treatment of psychosis in multiple neurological disorders. Although the development of traditional muscarinic activators has been stymied due to pan-receptor activation, muscarinic receptor subtype selectivity can be achieved through the utilization of a subtype of a unique allosteric site. A major challenge in capitalizing on this allosteric site to date has been achieving a balance of suitable potency and brain penetration. Herein, we describe the design of a brain penetrant series of M4 selective positive allosteric modulators (PAMs), ultimately culminating in the identification of 21 (PF-06852231, now CVL-231/emraclidine), which is under active clinical development as a novel mechanism and approach for the treatment of schizophrenia.

Muscarinic Modulation of Synaptic Transmission and Short-Term Plasticity in the Dorsal and Ventral Hippocampus.

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Activation of M4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

BACKGROUND AND PURPOSE: Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. EXPERIMENTAL APPROACH: Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. KEY RESULTS: Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. CONCLUSION AND IMPLICATIONS: Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.

M1/M4 receptors as potential therapeutic treatments for schizophrenia: A comprehensive study.

Muscarinic acetylcholine receptors (mAChRs) play a significant role in the pathophysiology of schizophrenia. Although activating mAChRs holds potential in addressing the full range of schizophrenia symptoms, clinical application of many non-selective mAChR agonists in cognitive deficits, positive and negative symptoms is hindered by peripheral side effects (gastrointestinal disturbances and cardiovascular effects) and dosage restrictions. Ligands binding to the allosteric sites of mAChRs, particularly the M1 and M4 subtypes, demonstrate activity in improving cognitive function and amelioration of positive and negative symptoms associated with schizophrenia, enhancing our understanding of schizophrenia. The article aims to critically examine current design concepts and clinical advancements in synthesizing and designing small molecules targeting M1/M4, providing theoretical insights and empirical support for future research in this field.

PET imaging of M4 muscarinic acetylcholine receptors in rhesus macaques using [11C]MK-6884: Quantification with kinetic modeling and receptor occupancy by CVL-231 (emraclidine), a novel positive allosteric modulator.

Stimulation of the M4 muscarinic acetylcholine receptor reduces striatal hyperdopaminergia, suggesting its potential as a therapeutic target for schizophrenia. Emraclidine (CVL-231) is a novel, highly selective, positive allosteric modulator (PAM) of M4 muscarinic acetylcholine receptors i.e. acts as a modulator that increases the response of these receptors. First, we aimed to further characterize the positron emission tomography (PET) imaging and quantification performance of a recently developed M4 PAM radiotracer, [11C]MK-6884, in non-human primates (NHPs). Second, we applied these results to determine the receptor occupancy of CVL-231 as a function of dose. Using paired baseline-blocking PET scans, we quantified total volume of distribution, binding potential, and receptor occupancy. Both blood-based and reference region-based methods quantified M4 receptor levels across brain regions. The 2-tissue 4-parameter kinetic model best fitted regional [11C]MK-6884-time activity curves. Only the caudate nucleus and putamen displayed statistically significant [11C]MK-6884 uptake and dose-dependent blocking by CVL-231. For binding potential and receptor occupancy quantification, the simplified reference tissue model using the grey cerebellum as a reference region was employed. CVL-231 demonstrated dose-dependent M4 receptor occupancy in the striatum of the NHP brain and shows promise for further development in clinical trials.

Stimulation of the muscarinic receptor M4 regulates neural precursor cell proliferation and promotes adult hippocampal neurogenesis.

Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction.

[Muscarinic M1 and/or M4 receptor agonists as potential novel treatments for psychoses]. / Muscarine 1- en/of 4-receptoragonisten als potentieel nieuwe behandeling voor psychosen.

BACKGROUND: Research suggests that cholinergic muscarinic 1 (M1) and/or muscarinic 4 (M4) receptors may be involved in the pathophysiology of psychotic disorders. Agonistic modulation of these receptors can offer new treatment options. AIM: To provide an overview of current research on the role of cholinergic M1 and M4 receptors in the development and treatment of psychoses, with special attention to the development of new drugs such as xanomeline and emraclidine. METHOD: To obtain an overview, we searched for English-language studies published in PubMed, Embase, and PsycInfo up until June 1, 2023. We examined the role and effects of M1 and/or M4 agonists in schizophrenia. Additionally, we consulted clinical trial registers. RESULTS: Our search strategy resulted in nine published articles on five clinical studies. These studies revealed that reduced presence of M1 receptors, primarily in the frontal cortex, and M4 receptors, primarily in the basal ganglia, are associated with psychoses. M1 and M4 receptors modulate dopaminergic activity in the ventral tegmentum and striatum through various pathways. Several M1 and/or M4 agonists, partial agonists, and positive allosteric modulators (PAMs) have been developed. Drugs exhibiting agonistic activity on M1 and/or M4 receptors, such as xanomeline-trospium (phase 2 and 3 studies) and emraclidine (phase 1b studies), have shown positive effects on cognitive and potentially negative symptoms in patients with schizophrenia. CONCLUSION: M1 and/or M4 receptor agonists show potential as new treatment strategies for individuals with psychotic disorders. Although initial studies with xanomeline-trospium and emraclidine have shown positive results, further research is needed to assess their long-term efficacy, safety, and tolerability before these new medications can be evaluated.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

M1/M4-Preferring Muscarinic Cholinergic Receptor Agonist Xanomeline Reverses Wake and Arousal Deficits in Nonpathologically Aged Mice.

Degeneration of the cholinergic basal forebrain is implicated in the development of cognitive deficits and sleep/wake architecture disturbances in mild cognitive impairment (MCI) and Alzheimer's disease (AD). Indirect-acting muscarinic cholinergic receptor agonists, such as acetylcholinesterase inhibitors (AChEIs), remain the only FDA-approved treatments for the cognitive impairments observed in AD that target the cholinergic system. Novel direct-acting muscarinic cholinergic receptor agonists also improve cognitive performance in young and aged preclinical species and are currently under clinical development for AD. However, little is known about the effects of direct-acting muscarinic cholinergic receptor agonists on disruptions of sleep/wake architecture and arousal observed in nonpathologically aged rodents, nonhuman primates, and clinical populations. The purpose of the present study was to provide the first assessment of the effects of the direct-acting M1/M4-preferring muscarinic cholinergic receptor agonist xanomeline on sleep/wake architecture and arousal in young and nonpathologically aged mice, in comparison with the AChEI donepezil, when dosed in either the active or inactive phase of the circadian cycle. Xanomeline produced a robust reversal of both wake fragmentation and disruptions in arousal when dosed in the active phase of nonpathologically aged mice. In contrast, donepezil had no effect on either age-related wake fragmentation or arousal deficits when dosed during the active phase. When dosed in the inactive phase, both xanomeline and donepezil produced increases in wake and arousal and decreases in nonrapid eye movement sleep quality and quantity in nonpathologically aged mice. Collectively, these novel findings suggest that direct-acting muscarinic cholinergic agonists such as xanomeline may provide enhanced wakefulness and arousal in nonpathological aging, MCI, and AD patient populations.

Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

Muscarinic Receptors Expression in the Peripheral Blood Cells Differentiate Dementia with Lewy Bodies from Alzheimer's Disease.

BACKGROUND: Central nervous system disruption of cholinergic (ACh) signaling, which plays a major role in cognitive processes, is well documented in dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). The expression of muscarinic ACh receptors type 1 and 4 (CHRM1 and CHRM4) has been reported to be altered in the brain of DLB patients. OBJECTIVE: We aim to assess the peripheral gene expression of CHRM1 and 4 in DLB as a possible marker as compared to AD and healthy control (HC) subjects. METHODS: Peripheral blood mononuclear cells were collected from 21 DLB, 13 AD, and 8 HC matched subjects. RT-PCR was performed to estimate gene expression of CHRM1 and CHRM4. RESULTS: Peripheral CHRM1 expression was higher and CHRM4 was lower in DLB and AD compared to HC, whereas both CHRM1 and CHRM4 levels were higher in AD compared to DLB patients. Receiver operating characteristics curves, with logistic regression analysis, showed that combining peripheral CHRM1 and CHRM4 levels, DLB and AD subjects were classified with an accuracy of 76.0%. CONCLUSION: Alterations of peripheral CHRM1 and CHRM4 was found in both AD and DLB patients as compared to HC. CHRM1 and CHRM4 gene expression resulted to be lower in DLB patients compared to AD. In the future, peripheral CHRM expression could be studied as a possible marker of neurodegenerative conditions associated with cholinergic deficit and a possible marker of response to acetylcholinesterase inhibitors.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes - Part 2.

This Letter details our efforts to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core which lead to the discovery of two novel tricyclic cores: a 7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine core and 2,4-dimethylthieno[2,3-b:5,4-c']dipyridine core. Both tricyclic cores displayed low nanomolar potency against the human M4 receptor.

Monoamine Oxidase Inhibition by Major Tanshinones from Salvia miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I.

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.

Muscarinic M1, but not M4, receptor antagonism impairs divided attention in male rats.

Divided attention may be more important than ever to comprehend, given ubiquitous distractors in modern living. In humans, concern has been expressed about the negative impact of distraction in education, the home, and the workplace. While acetylcholine supports divided attention, in part via muscarinic receptors, little is known about the specific muscarinic subtypes that may contribute. We designed a novel, high-response rate test of auditory sustained attention, in which rats complete variable-ratio runs on one of two levers, rather than emitting a single response. By doing this, we can present a secondary visual distractor task during some trials, for which a correct nosepoke response is reinforced with a more palatable food pellet. The nonspecific muscarinic antagonist scopolamine impaired performance, and slowed and reduced lever press activity. We then explored antagonists that preferentially block the M1 and M4 subtypes, because these receptors are potential therapeutic targets for cognitive enhancers. Telenzepine, an M1-preferring antagonist, impaired divided attention performance, but not performance of the attention task without distraction. Telenzepine also had fewer nonspecific effects than scopolamine. In contrast, the M4-preferring antagonist tropicamide had no effects. Analysis of overall behavior also indicated that accuracy in the main attention task decreased as a function of engagement with the distractor task. These results implicate the M1 receptor in divided attention.

Nerve growth factor interacts with CHRM4 and promotes neuroendocrine differentiation of prostate cancer and castration resistance.

Nerve growth factor (NGF) contributes to the progression of malignancy. However, the functional role and regulatory mechanisms of NGF in the development of neuroendocrine prostate cancer (NEPC) are unclear. Here, we show that an androgen-deprivation therapy (ADT)-stimulated transcription factor, ZBTB46, upregulated NGF via ZBTB46 mediated-transcriptional activation of NGF. NGF regulates NEPC differentiation by physically interacting with a G-protein-coupled receptor, cholinergic receptor muscarinic 4 (CHRM4), after ADT. Pharmacologic NGF blockade and NGF knockdown markedly inhibited CHRM4-mediated NEPC differentiation and AKT-MYCN signaling activation. CHRM4 stimulation was associated with ADT resistance and was significantly correlated with increased NGF in high-grade and small-cell neuroendocrine prostate cancer (SCNC) patient samples. Our results reveal a role of the NGF in the development of NEPC that is linked to ZBTB46 upregulation and CHRM4 accumulation. Our study provides evidence that the NGF-CHRM4 axis has potential to be considered as a therapeutic target to impair NEPC progression.

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

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

The M1/M4 preferring muscarinic agonist xanomeline modulates functional connectivity and NMDAR antagonist-induced changes in the mouse brain.

Cholinergic drugs acting at M1/M4 muscarinic receptors hold promise for the treatment of symptoms associated with brain disorders characterized by cognitive impairment, mood disturbances, or psychosis, such as Alzheimer's disease or schizophrenia. However, the brain-wide functional substrates engaged by muscarinic agonists remain poorly understood. Here we used a combination of pharmacological fMRI (phMRI), resting-state fMRI (rsfMRI), and resting-state quantitative EEG (qEEG) to investigate the effects of a behaviorally active dose of the M1/M4-preferring muscarinic agonist xanomeline on brain functional activity in the rodent brain. We investigated both the effects of xanomeline per se and its modulatory effects on signals elicited by the NMDA-receptor antagonists phencyclidine (PCP) and ketamine. We found that xanomeline induces robust and widespread BOLD signal phMRI amplitude increases and decreased high-frequency qEEG spectral activity. rsfMRI mapping in the mouse revealed that xanomeline robustly decreased neocortical and striatal connectivity but induces focal increases in functional connectivity within the nucleus accumbens and basal forebrain. Notably, xanomeline pre-administration robustly attenuated both the cortico-limbic phMRI response and the fronto-hippocampal hyper-connectivity induced by PCP, enhanced PCP-modulated functional connectivity locally within the nucleus accumbens and basal forebrain, and reversed the gamma and high-frequency qEEG power increases induced by ketamine. Collectively, these results show that xanomeline robustly induces both cholinergic-like neocortical activation and desynchronization of functional networks in the mammalian brain. These effects could serve as a translatable biomarker for future clinical investigations of muscarinic agents, and bear mechanistic relevance for the putative therapeutic effect of these class of compounds in brain disorders.