BACKGROUND: Musk, a traditional Chinese medicine, is broadly used in inducing resuscitation and refreshing the mind, activating blood and alleviating pain. It is commonly used for the treatment of ischemic stroke, and muscone is its core medicinal component.
OBJECTIVE: The aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors.
METHODS: The effects of muscone were tested in a rat model of middle cerebral artery occlusion (MCAO) as well as injured neurons induced by oxygen-glucose deprivation (OGD) in PC12 cells. Cell counting kit 8 (CCK8) assay was used to measure the cell viability, and the production of lactate dehydrogenase (LDH) and adenosine-triphosphate (ATP) were examined by kit. 2\',7\'-Dichlorodihydrofluorescein diacetate (DCFH-DA), tetramethylrhodamine ethyl ester (TMRE) and Fluo-4 acetoxymethyl ester (Fluo-4 AM) staining were used to demonstrate effect of muscone on the reactive oxygen species (ROS) level, mitochondria membrane potential (MMP) and intracellular Ca2+ measurement in cells respectively, in which all of those staining was visualized by laser confocal microscope. For in vivo experiments, rats\' cerebral blood flow was measured using laser Doppler blood flowmetry to evaluate the MCAO model, and a modified neurological severity score (mNSS) was used to assess the recovery of neurological function. Calculate infarct rate was measured by 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining. Except DCFH-DA and Fluo-4 AM staining, 5,5\',6,6\'-tetrachloro-1,1\',3,3\'-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) staining was used to observe intracellular Ca2+ measurement in brain cells. Protein levels in cells and tissues were detected by Western blot.
RESULTS: Pretreatment with muscone significantly improved the cell viability, lactic acid production, mitochondrial membrane potential collapse and function, Ca2+ overload, ROS generation, and cell apoptosis in OGD PC12 cells. Muscone also regulated PI3K, ERK and AKT signal pathways by activating cholinergic signaling of muscarinic receptors in PC12 cells induced with OGD. More importantly, the blocking of cholinergic signaling of muscarinic receptors by atropine significantly reduces the neuroprotective effects of muscone, including the cell viability, Ca2+ efflux, and mitochondrial repair. Furthermore, muscone was found to effectively alleviate mitochondrial dysfunction and elevated levels of ROS induced by the MCAO in the brain tissue. Notably, this beneficial effect of muscone was attenuated by atropine but not by (+)-Sparteine.
CONCLUSIONS: Our study indicates that muscone exerts its neuroprotective effects by activating muscarinic receptors of cholinergic signaling, thus providing a promising therapeutic target for the treatment of OGD-induced nerve injury in stroke. The findings suggest that these treatments may hold potential benefits for stroke patients.

Cholinergic nerves are widely distributed throughout the human body and participate in various physiological activities, including sensory, motor, and visceral activities, through cholinergic signaling. Cholinergic signaling plays an important role in pancreatic exocrine secretion. A large number of studies have found that cholinergic signaling overstimulates pancreatic acinar cells through muscarinic receptors, participates in the onset of pancreatic diseases such as acute pancreatitis and chronic pancreatitis, and can also inhibit the progression of pancreatic cancer. However, cholinergic signaling plays a role in reducing pain and inflammation through nicotinic receptors, but enhances the proliferation and invasion of pancreatic tumor cells. This review focuses on the progression of cholinergic signaling and pancreatic diseases in recent years and reveals the role of cholinergic signaling in pancreatic diseases.

Bursting of midbrain dopamine (DA) neurons is believed to represent an important reward signal that instructs and reinforces goal-directed behaviors. In DA neurons, many afferents, including cholinergic and glutamatergic inputs, induce bursting, and it is suggested that a synergy exists between these afferents in bursting induction. However, the underlying mechanisms of the role and the synergy of muscarinic receptors (mAChRs) and NMDA receptors (NMDARs) in bursting induction remain unclear. Present work bestowed analysis using a mathematical model of DA neurons to demonstrate the underlying mechanisms. Activation of mAChRs, leading to rapid translocation of TRPC channels to cell surface, recruited C a 2 + -activated nonspecific (CAN) current ( I CAN ), meanwhile NMDARs excitation triggered C a 2 + influx, which induced the positive feedback loop of C a 2 + and I CAN , respectively, yielded a robust ramping depolarization with a superimposed high-frequency spiking. In some DA cells, neither NMDARs nor mAChRs induced positive feedback loop unless they were activated simultaneously to induce bursting. Our experimental results verified those theoretical findings. These together unveil the underlying mechanisms of the role and synergy of mAChRs and NMDARs in bursting induction emerge from the nonlinear relationship between C a 2 + influx and I CAN . Given the diverse and complex nature of neural circuitry and the DA neuron heterogeneity, our work provides new insights to understand specific afferents, the synergy between those afferents, and the differences in intrinsic excitability to be integrated by the bursting to accurately characterize the dopamine signals in the valances of reward and reinforcement, and a broad spectrum of neuropsychiatric disorders.

Persistent inward currents (PICs) play important roles in regulating neural excitability. Results from our previous studies showed that serotonergic (5-HT) neurons of the brainstem expressed PICs. However, little is known about cholinergic (ACh) modulation of PICs in the 5-HT neurons. The whole-cell patch-clamp recordings were performed in the brainstem slices of ePet-EYFP mice to investigate the electrophysiological properties of PICs with cholinergic modulation. PICs in 5-HT neurons were activated at - 51.4 ± 3.7 mV with the amplitude of - 171.6 ± 48.9 pA (n = 71). Bath application of 20-25 μM ACh increased the amplitude by 79.1 ± 42.5 pA (n = 23, p < 0.001) and hyperpolarized the onset voltage by 2.2 ± 2.7 mV (n = 23, p < 0.01) and half-maximal activation by 3.6 ± 2.7 mV (n = 6, p < 0.01). Muscarine mimicked the effects of ACh on PICs, while bath application of nicotine (15-20 μM) did not induce substantial change in the PICs (n = 9). Muscarine enhanced the amplitude of PICs by 100.0 ± 27.4 pA (n = 28, p < 0.001) and lowered the onset voltage by 2.8 ± 1.2 mV (n = 28, p < 0.001) and the half-maximal activation by 2.9 ± 1.4 mV. ACh-induced increase of amplitude and hyperpolarization of onset voltage were blocked by 3-5 μM atropine. Furthermore, the muscarine-induced enhancement of the PICs was antagonized by 5 μM 4-DAMP, the antagonist of M3 receptor, while the antagonists of M1 (Telenzepine, 5 μM) and M5 (VU6008667, 5 μM) receptors did not significantly affect the PIC enhancement. This study suggested that ACh potentiated PICs in 5-HT neurons of the brainstem by activating muscarinic M3 receptor.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

OBJECTIVE: To investigate the effect of manual acupuncture stimulation of acupoints at different spinal nerve segments on uterine motility and the complicated adrenergic and cholinergic receptors.
METHODS: Eighteen adult non-pregnant SD rats were used in the present study. The contractive activities of the uterus were measured by using a pressure transducer which was connected to an inserted water-filled balloon in the uterus via a PE tube at one end and an amplifier at the other end. Manual acupuncture needle was applied to \"Zigong\"(EX-CA1),\"Huiyin\" (CV1), \"Xuehai \"(SP10) and \"Taichong \"(LR3) acupoints located at the same or adjacent spinal nerve segments of the uterus, and to \"Neiguan\" (PC6) situated at the distant spinal nerve segment at about 2 Hz for 1 min, followed by observing changes of the uterine contractility. After acupuncture, α-adrenoceptor antagonist phentolamine (0.5 mg/kg, n=9) or cholinergic muscarinic (M) receptor antagonist atropine (0.5 mg/kg，n=9) was given to the rats of different acupoints respectively through tail vein, followed by observing changes of the uterine automatic systolic pressure difference (value of systolic pressure peak minuses the trough value) and frequency after manual acupuncture stimulation as well as after blocking the activities of α-adrenoceptors and M receptors, separately.
RESULTS: After acupuncture stimulation of EX-CA1, CV1, SP10 and LR3, but not PC6, the systolic pressure difference and frequency of the uterus were signi-ficantly increased (P<0.05,P<0.001, P<0.01). Following intravenous injection of phentolamine, both the systolic pressure difference and frequency had no marked changes after acupuncture at the 5 acupoints (P>0.05). After intravenous injection of atropine, the uterine systolic pressure difference and frequency were markedly decreased compared with the basic values before acupuncture stimulation (P<0.001), but had no obvious changes after acupuncture at the 5 acupoints at both the same and distant spinal segments to the uterus (P>0.05).
CONCLUSIONS: Manual acupuncture stimulation of acupoints at the same and adjacent spinal segments can promote the contractility of uterus in normal rats, which is realized by activating both α-adrenoceptor and cholinergic M receptors.
目的：观察针刺不同节段穴位(部位)对SD大鼠子宫运动的调节效应及自主神经的相关联系。方法：健康成年未孕SD大鼠18只，采用球囊置入法记录未孕状态时子宫的自发性收缩活动；随后分别在与支配子宫相同/近节段穴位“子宫”“会阴”“血海”“太冲”及异节段穴位“内关”给予手针干预(刺激频率2次/s，持续1 min)，每次干预间隔5 min，比较针刺不同节段穴位对子宫运动的影响；分别经尾静脉注射肾上腺素能α受体阻断剂酚妥拉明(0.5 mg/kg)和胆碱能M受体阻断剂阿托品(0.5 mg/kg)，观察子宫运动及针刺上述穴位调节效应的变化。结果：生理条件下，针刺与子宫同/近节段穴位“子宫”“会阴”“血海”“太冲”后子宫的收缩压力差和收缩频率较基础值显著增加(P<0.05，P<0.001，P<0.01)，而针刺异节段“内关”与基础值比较差异无统计学意义(P>0.05)。与基础值比较，尾静脉注射阿托品后大鼠子宫的收缩压力差和收缩频率显著降低(P<0.001)，而尾静脉注射酚妥拉明后差异无统计学意义(P>0.05)。与注射阿托品或酚妥拉明后比较，针刺与子宫相同节段和异节段的上述穴位后子宫收缩压力差和收缩频率差异均无统计学意义(P>0.05)。结论：副交感胆碱能M受体与子宫节律性收缩运动联系密切，针刺与支配子宫同/近神经节段的穴位可显著促进子宫的收缩运动，这种促进效应可能与肾上腺素α受体和胆碱能M受体激活相关。.

Opioids strongly inhibit GABAergic neurons in the rostromedial tegmental nucleus (RMTg) that expresses μ-opioid receptors to induce rewarding and psychomotor effects. M3 and M4 muscarinic receptors are co-localized with μ-opioid receptors at these GABAergic neurons. This study explored whether RMTg M3 and M4 muscarinic receptors are involved in regulating opioid-induced reward and locomotion via a conditioned place preference (CPP) paradigm. Selective muscarinic receptor agonists and antagonists were both singly and combinatorically injected into the RMTg to examine their effects on the acquisition of systemic morphine-induced CPP and locomotor activity. The M3 muscarinic receptor agonist, pilocarpine, inhibited the acquisition of morphine-induced CPP, whereas its antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP, 1 μg/side), reversed the inhibitory effect of pilocarpine (30 μg/side). Additionally, 4-DAMP increased locomotor activity while pilocarpine (30 μg/side) partially decreased locomotor activity when combined with morphine. In contrast, the M4 muscarinic receptor agonist, LY2033298 (0.1 and 0.2 μg/side), and antagonist, tropicamide (20 and 40 μM/side), did not affect the acquisition of morphine-induced CPP or locomotor activity. Taken together, our findings suggest that RMTg M3 muscarinic receptors are involved in opioid-induced rewarding and psychomotor effects. Therefore, RMTg M3 muscarinic receptors may represent a promising target for the treatment of opioid addiction.

Morin (3,5,7,2\',4\'-pentahydroxyflavone) is a yellow coloured natural flavonoid found in plants of the Moraceae family. This favonoid is easily sources from readily available fruits, vegetables and eve certain beverages. Among the sources that was identified, it is clear that morin is most abundantly found in almond, old fustic, Indian guava, and Osage orange. Multiple studies have suggested that morin has multiple therapeutic actions and possess potential to be a functional potent drug. Previous studies demonstrated that morin is capable of resolving deoxycorticosterone acetate-salt-induced hypertension and possess strong vasorelaxant properties. However, the exact mechanisms remains unknown. Therefore, this study is designed to investigate the in vitro mechanism of morin-induced vasorelaxant effects. The underlying mechanisms of morin\'s vasorelaxant activities were evaluated on thoracic aortic rings isolated from Sprague-Dawley rats. Results from the study demonstrated morin causing vasodilatory reaction in phenylephrine and potassium chloride pre-contracted endothelium-intact aortic rings with the effect being significantly affected in endothelium-denuded aortic rings. Pre-incubation of the aortic rings with ODQ (selective cGMP-independent sGC inhibitor), indomethacin (nonselective COX inhibitor), L-NAME (endothelial nitric oxide inhibitor), propranolol (β2-adrenegic receptors blocker), and atropine (muscarinic receptors blocker) significantly reduced the vasorelaxant effect of morin. It was also found to be able to reduce the intracellular calcium level by blocking VOCC and calcium intake from the extracellular environment and the intracellular release of calcium from the sarcoplasmic reticulum. The present study showed that the vasorelaxant effect of morin potentially involves the NO/sGC, muscarinic receptors, β2-adrenegic receptors, and calcium channels.

The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.

Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M1 -M5 muscarinic receptors were all encoded in HBSMCs. The expression rank order was M2  > M1  > M5  > M3  > M4 . The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M3 -preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M3 receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO.