Expression Of Muscarinic Acetylcholine Receptors Research Articles

Neuroanatomical substrates of circuit specific cholinergic modulation across the primate anterior cingulate cortex.

Acetylcholine is a robust neuromodulator of the limbic system and a critical regulator of arousal, emotions, and stress. The anterior cingulate cortex (ACC) and the amygdala (AMY) are key limbic structures that are both densely innervated by cholinergic afferents and interact with each other for emotional regulation. The ACC is comprised of functionally-distinct dorsal (A24), rostral (A32), and ventral (A25) areas that differ in their connections with the AMY. The structural substrates of cholinergic modulation of distinct ACC microcircuits and outputs to AMY are thought to depend on the laminar and subcellular localization of cholinergic receptors. The present study examines the distribution of muscarinic acetylcholine receptors, m1 and m2, on distinct excitatory and inhibitory neuronal classes and on AMY-targeting projection neurons within ACC areas, via immunohistochemistry and in vivo injections of neural tracers into the basolateral AMY in adult rhesus monkeys of both sexes. We found that laminar densities of m1+ and m2+ expressing excitatory and inhibitory neurons depended on area and cell-type. Among the ACC areas, ventral subgenual ACC A25 exhibited greater m2+ localization on presynaptic inhibitory axon terminals and greater density of m1+ and m2+ expressing AMY-targeting (tracer+) pyramidal neurons. These patterns suggest robust cholinergic dis-inhibition and potentiation of amygdalar outputs from the limbic ventral ACC, which may be linked to the hyperexcitability of this subgenual ACC area in depression. These findings reveal the anatomical substrate of diverse cholinergic modulation of specific ACC microcircuits and amygdalar outputs that mediate cognitive-emotional integration and dysfunctions underlying stress and affective disorders.Significance Statement Cholinergic neuromodulation of anterior cingulate (ACC) cortico-limbic networks are consequential for cognitive-emotional integration. This study explored muscarinic acetylcholine receptor (m1 and m2) expression on distinct excitatory and inhibitory neurons across functionally-distinct ACC subregions and their outputs to the amygdala, a key limbic structure for emotional regulation. Our findings reveal that muscarinic receptor expression is most robust in ventral subgenual ACC area 25, in patterns that suggest cholinergic dis-inhibition and potentiation of area 25 outputs to the amygdala. These neuromodulatory interactions may be linked to the hyperexcitability of subgenual ACC found in depression. Our findings yield novel insight on how cholinergic modulation of specific ACC microcircuits and amygdalar outputs can contribute to cognitive-emotional processing and dysfunction in stress and affective disorders.

A comprehensive assessment of the cholinergic-supporting and cognitive-enhancing effects of Rosa damascena Mill. (Damask rose) essential oil on scopolamine-induced amnestic rats.

Alzheimer's disease (AD) is a neurodegenerative condition characterized by gradual loss of cognitive abilities (dementia) and is a major public health problem. Here, we aimed at investigating the effects of Rosa damascena essential oil (RDEO) on learning and memory functions in a rat model of amnesia induced by scopolamine, as well as on changes in acetylcholinesterase (AChE) activity, M1 muscarinic acetylcholine receptor (mAChR) expression, and brain-derived neurotrophic factor (BDNF) levels in the extracted brain tissues. The control, amnesia (scopolamine, 1mg/kg/i.p.) and treatment (RDEO, 100μL/kg/p.o. or galantamine, 1.5mg/kg/i.p.) groups were subjected to Morris water maze and new object recognition tests. AChE activity was assayed by ELISA, and M1 mAChR and BDNF concentration changes were determined by western blotting. Also, using computational tools, human M1 mAChR was modeled in an active conformation, and the major components of RDEO were docked onto this receptor. According to our behavioral tests, RDEO was able to mitigate the learning and memory impairments caused by scopolamine in vivo. Our in vitro assays showed that the observed positive effects correlated well with a decrease in AChE activity and an increase in M1 mAChR and BDNF levels in amnestic rat brains. We also demonstrated in an in silico setting that the major components of RDEO, specifically -citronellol, geraniol, and nerol, could be accommodated favorably within the allosteric binding pocket of active-state human M1 mAChR and anchored here chiefly by hydrogen-bonding and alkyl-π interactions. Our findings offer a solid experimental foundation for future RDEO-based medicinal product development for patients suffering from AD.

Combined methods elucidate the multi-organ toxicity of cylindrospermopsin (CYN) on Daphnia magna

Global water bodies are now at risk from inevitable cyanobacterial blooms and their production of multiple cyanotoxins, in particular cylindrospermopsin (CYN). However, research on the CYN toxicity and its molecular mechanisms is still limited, whilst the responses of aquatic species against CYN are uncovered. By integrating behavioral observations, chemical detections and transcriptome analysis, this study demonstrated that CYN exerted multi-organ toxicity to model species, Daphnia magna. The present study confirmed that CYN could cause protein inhibition by undermining total protein contents, and altered the gene expression related to proteolysis. Meantime, CYN induced oxidative stress by increasing reactive oxygen species (ROS) level, decreasing the glutathione (GSH) concentration, and interfered with protoheme formation process molecularly. Neurotoxicity led by CYN was solidly determined by abnormal swimming patterns, reduced acetylcholinesterase (AChE), and downward expression of muscarinic acetylcholine receptor (CHRM). Importantly, for the first time, this research determined CYN directly interfered with energy metabolism in cladocerans. CYN distinctively reduced filtration and ingestion rate by targeting on heart and thoracic limbs, which declined the energy intake, and could be further displayed by the reduction of motional strength and the trypsin concentration. These phenotypic alterations were supported by transcriptomic profile, including the down-regulation of oxidative phosphorylation and ATP synthesis. Moreover, CYN was speculated to trigger the self-defense responses of D. magna, known as “abandon-ship” by moderating lipid metabolism and distribution. This study, overall, comprehensively demonstrated the CYN toxicity and the responses of D. magna against it, which is of great significance to the advancements of CYN toxicity knowledge.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation.Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method.Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level.Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia.

The effect of combination pretreatment of donepezil and environmental enrichment on memory deficits in amyloid-beta-induced Alzheimer-like rat model

Alzheimer's disease (AD) is progressive neurodegeneration known as the most common cause of dementia, and it is the sixth leading cause of death in older people. Given the promising data on the additive effect of combination therapy with donepezil (Aricept), an acetylcholinesterase inhibitor (AChEI), and regarding the similar neuronal mechanisms through which donepezil (DON) and environmental enrichment (EE) exert their enhancing effects on cognition; we asked whether simultaneous treatment with two paradigms in amyloid-beta-induced AD rats may lead to greater protection against the cognitive impairments than either treatment individually. The experimental groups consisted of Alz, sham-operated, Alz + DON, Alz + EE, and Alz + DON + EE. AD was induced by intrahippocampal injection of amyloid-beta (1–42, 6 μg), and DON was orally administrated (4 mg/kg) for 21 days. Environmental enrichment consisted of housing animals in large cages (50 × 50 × 50 cm) containing a running wheel and differently shaped objects for 21 days. Spatial learning and memory were assessed in the Morris water maze (MWM) and Real-time PCR was performed to assess the expression of brain-derived neurotrophic factor (BDNF) and M1 muscarinic acetylcholine receptor (AchM1R) within the hippocampus. Spatial memory was impaired in Alz animals, and while neither pretreatment with DON nor EE alone could significantly restore spatial memory scores in Alz rats, combination therapy was effective. BDNF expression was suppressed in Alz rats and pretreatment with DON plus EE could increase it to the saline levels. The data suggest that a cholinesterase inhibitor and cognitive stimulation can be used effectively in combination to protect cognitive loss in an AD rat model. This additive protective effect may be in part due to the augmented influence of this combination on BDNF levels and cholinergic neuronal system within the hippocampus.

Dysregulation of the Enteric Nervous System in the Mid Colon of Complement Component 3 Knockout Mice with Constipation Phenotypes.

Complement component 3 (C3) contributes to neurogenesis, neural migration, and synaptic elimination under normal and disease conditions of the brain, even though it has not been studied in the enteric nervous system (ENS). To determine the role of C3 in the regulatory mechanism of ENS during C3 deficiency-induced constipation, the changes in the markers of neuronal and interstitial cells of Cajal (ICCs), the markers for excitatory and inhibitory transmission of ENS, and expression of C3 receptors were analyzed in the mid colon of C3 knockout (KO) mice at 16 weeks of age. Prominent constipation phenotypes, including the decrease in stool parameters, changes in the histological structure, and suppression of mucin secretion, were detected in C3 KO mice compared to wildtype (WT) mice. The expression levels of the neuron specific enolase (NSE), protein gene product 9.5 (PGP9.5), and C-kit markers for myenteric neurons and ICCs were lower in the mid colon of C3 KO mice than WT mice. Excitatory transmission analysis revealed similar suppression of the 5-hydroxytryptamine (5-HT) concentration, expression of 5-HT receptors, acetylcholine (ACh) concentration, ACh esterase (AChE) activity, and expression of muscarinic ACh receptors (mAChRs), despite the mAChRs downstream signaling pathway being activated in the mid colon of C3 KO mice. In inhibitory transmission analysis, C3 KO mice showed an increase in the nitric oxide (NO) concentration and inducible nitric oxide synthase (iNOS) expression, while neuronal NOS (nNOS) expression, cholecystokinin (CCK), and gastrin concentration were decreased in the same mice. Furthermore, the levels of C3a receptor (C3aR) and C3bR expression were enhanced in the mid colon of C3 KO mice compared to the WT mice during C3 deficiency-induced constipation. Overall, these results indicate that a dysregulation of the ENS may play an important role in C3 deficiency-induced constipation in the mid colon of C3 KO mice.

Autophagy Induced by Muscarinic Acetylcholine Receptor 1 Mediates Migration and Invasion Targeting Atg5 via AMPK/mTOR Pathway in Prostate Cancer.

Increasing numbers of researchers discovered the expression of muscarinic acetylcholine receptor 1 in human cancers, while its function in human prostate cancer is still unclear. Our present study focused on CHRM1 to clarify its role in mediating autophagy in prostate cancer. We used immunohistochemistry, western blotting, and immunofluorescence experiments to observe the expression of muscarinic acetylcholine receptor 1 both in nude mice with subcutaneous tumors and in prostate cancer cells. The autophagy was observed through transmission electron microscopy, western blotting, quantitative real-time PCR, and immunofluorescence. After that, we used lentivirus to establish CHRM1 and Atg5 knockdown models. Then, the migration and invasion abilities after knocking down muscarinic acetylcholine receptor 1 and Atg5 were detected by transwell assays. In addition, the AMPK/mTOR pathway-related targets were detected by western blotting. We found that muscarinic acetylcholine receptor 1 was abundantly expressed both in vitro and in vivo in prostate cancer. The overexpression of muscarinic acetylcholine receptor 1 positively regulated migration and invasion in tumor cells as well as the activation of autophagy. Muscarinic acetylcholine receptor 1 was highly correlated with Atg5 and activated the AMPK/mTOR signaling pathway. Downregulation of Atg5 inhibited cell autophagy in prostate cancer cells and the migration and invasion of prostate cancer cells. Meanwhile, abnormal expressions of AMPK/mTOR pathway-related proteins were found. In conclusion, the present findings indicated that muscarinic acetylcholine receptor 1 is highly expressed in prostate cancer cells and promotes cell invasion and migration of prostate cancer. Autophagy is activated in prostate cancer cells and the activation of muscarinic acetylcholine receptor 1 positively regulates autophagy in prostate cancer cells. Moreover, muscarinic acetylcholine receptor 1 induces autophagy-mediated cell migration and invasion by targeting Atg5 in prostate cancer cells via AMPK/mTOR pathway, which uncovered that regulating muscarinic acetylcholine receptor 1, identified in this study, can be a promising solution for treating prostate cancer.

Expression of M3 muscarinic acetylcholine receptors in gastric cancer.

Introduction: Gastric cancer represents a real public health problem as far as incidence, aggressiveness and unfavorable prognosis are concerned. The autonomous nervous system might be one of the major factors involved in the onset, progression, and metastasis, both sympathetically and parasympathetically. The increased activation of the M3 muscarinic acetylcholine receptors (mAChRs) triggers pro-oncogenic mechanisms, especially at a gastric level, through the activation of the Hippo signaling pathway and the increase of the nerve growth factor. Patients, Materials and Methods: In this study, biopsy or postoperative gastric resection pieces have been evaluated by histopathological (HP) and immunohistochemical (IHC) examination in a group of 77 gastric patients and 23 patients without an oncological diagnosis. To quantify the IHC signal, also considering the HP aspect, light microscopy images were obtained. Results: The M3 mAChR expression analysis has been correlated with the different gastric adenocarcinoma differentiation degrees (G1–G3). M3 mAChR presence has been observed also in the non-malignant gastric tissue, but it was significantly increased in the tumor tissue. The highest receptor expression was recorded in patients with a poorly-differentiated (G3) adenocarcinoma, these expressions decreasing with the increase of the differentiation degree towards moderately-differentiated (G2) and well-differentiated (G1). Conclusions: Surgical or pharmacological parasympathetic activity inhibition could decrease the development and progression of gastric tumors and could improve the gastric cancer patient’s prognosis.

Laxative Effects of Phlorotannins Derived from Ecklonia cava on Loperamide-Induced Constipation in SD Rats.

This study investigated the laxative effects of phlorotannins (Pt) derived from Ecklonia cava (E. cave) on chronic constipation by evaluating alterations in stool parameters, gastrointestinal motility, histopathological structure, mucin secretion, gastrointestinal hormones, muscarinic cholinergic regulation, and fecal microbiota in SD rats with loperamide (Lop)-induced constipation subjected to Pt treatment. Stool-related parameters (including stool number, weight, and water contents), gastrointestinal motility, and length of intestine were significantly enhanced in the Lop+Pt-treated group as compared to the Lop+Vehicle-treated group. A similar recovery was detected in the histopathological and cytological structure of the mid-colon of Lop+Pt-treated rats, although the level of mucin secretion remained constant. Moreover, rats with Lop-induced constipation subjected to Pt treatment showed significant improvements in water channel expression, gastrointestinal hormone secretions, and expression of muscarinic acetylcholine receptors M2/M3 (mAChRs M2/M3) and their mediators of muscarinic cholinergic regulation. Furthermore, the Lop+Pt-treated group showed a significant recovery of Bifidobacteriaceae, Muribaculaceae, Clostridiaceae, and Eubacteriaceae families in fecal microbiota. Taken together, these results provide the first evidence that exposure of SD rats with Lop-induced constipation to Pt improves the constipation phenotype through the regulation of membrane water channel expression, GI hormones, the mAChR signaling pathway, and fecal microbiota.

Anisodine hydrobromide alleviates hypoxia/reoxygenation (H/R)‐induced brain microvascular endothelial cell injury via muscarinic acetylcholine receptor 4

Anisodine hydrobromide (Ah) could delivery to the brain across the blood-brain barrier, exerting its protective effect in cerebral ischemia/reperfusion injury. Metabolic alterations under hypoxic conditions lead to endothelial dysfunction. In the present study, we tested that whether Ah regulates the production of nitric oxide and reactive oxygen species (ROS) and the glycolytic pathway via muscarinic receptors under hypoxia/reoxygenation (H/R) conditions. Under H/R conditions, brain microvascular endothelial cells were treated with Ah. Then, the expression of muscarinic acetylcholine receptors was detected by western blot. The production of nitric oxide was detected by Greiss reaction. ROS was measured by DCFH-DA probes. The expression of hypoxia-inducible transcription factor 1 (HIF-1α) was detected. Specific inhibitor of M2 and M4 muscarinic acetylcholine receptors were used to assess the role of muscarinic receptors in Ah protects against endothelial cell dysfunction. Results showed that H/R induced the expression of M2- and M4-AchRs, but not M1-, M3-, and M5-AchRs. Ah significantly suppressed the H/R-upregulated M2- and M4-AchRs. H/R induced production of NO and ROS. Ah and M4-AchRs inhibitor significantly inhibited the H/R-induced production of NO and ROS. HIF-1α was significantly induced by H/R, whereas it was significantly suppressed by Ah. Therefore, Ah protects against H/R injury in brain microvascular endothelial cells via activating HIF-1α, producing NO and ROS, which is dependent on M4-AchR.

Expression of muscarinic acetylcholine receptors in turkey cardiac chambers

The aim of the present study was to characterize the specific binding sites for [N-methyl-3H]-scopolamine ([3H]-NMS), a radioligand for labeling muscarinic acetylcholine receptors (mAChRs), in membranes of four heart chambers obtained from adult male British United Turkey (BUT) Big 6 (“meat-type”) and Cröllwitzer (“wild-type”) turkeys. MAChR subtypes were examined by inhibiting [3H]-NMS binding with subtype selective non-labelled receptor antagonists. In all left and right atria as well as left and right ventricles of both turkey breeds, the specific [3H]-NMS binding was saturable, reversible and of high affinity (KD range: 0.5–1.0 nM). The maximum receptor density (Bmax) was not significantly different between the four cardiac chambers of BUT Big 6 turkeys, but a significant difference was found between atria and ventricles of Cröllwitzer turkeys. Moreover, significant lower Bmax was found in the atria of Cröllwitzer turkeys than in the atria of BUT Big 6, while the ventricular Bmax was significantly higher. In all cardiac chambers, unlabeled mAChR antagonists competed for specific [3H]-NMS binding sites in a concentration-dependent manner, suggesting the presence of the M3 and M2 receptor subtypes, whereby the latter was the predominant subtype. The presence of the M1 subtype could not be excluded. In conclusion, there was a difference between BUT Big 6 (“meat-type”) and Cröllwitzer (“wild-type”) turkeys with regard to receptor density in heart chambers with dominant M2 and M3 receptor subtypes.

Molecular and clinical aspects of the effect of cytidyndiphosphocholine on cognitive functions

Systematization of the array of publications on cytidyldiphosphocholine (CDP-choline). Systematic computer analysis of all currently available publications on CDP-choline (1750 publications in PUBMED) using the topological theory of big data analysis. CDP-choline is essential for acetylcholine biosynthesis, phospholipid metabolism, and DNA methylation. The article describes the effects of CDP-choline on acetylcholinergic and other types of neurotransmission, anti-inflammatory, neuroprotective and neurotrophic effects of CDP-choline. Also, the paper presents the effects of the molecule on lipid metabolism and gene expression within the post-genomic paradigm (in particular, an increase in the expression of nicotinic and muscarinic acetylcholine receptors). The results of fundamental and clinical studies of CDP-choline in the treatment of cognitive impairments associated with cerebral ischemia and neurodegeneration are presented. The pharmacological effects of CDP-choline are mediated through multiple molecular mechanisms that contribute to the nootropic action of this molecule.

Topical bendazol inhibits experimental myopia progression and decreases the ocular accumulation of HIF-1α protein in young rabbits.

To investigate the inhibitory effect of bendazol on form-deprivation myopia (FDM) in rabbits as well as the underlying biochemical processes. Forty-eight 3-week-old New Zealand white rabbits were randomly assigned to three groups: a control group, a form-deprivation (FD) group and an FD+bendazol group (treated with 1% bendazol in the FD eyes). Refraction, corneal curvature, vitreous chamber depth (VCD) and axial length (AL) were assessed using streak retinoscopy, keratometry, and A-scan ultrasonography, respectively. Eyeballs were enucleated for histological analysis, and ocular tissues were homogenized to determine the mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α) and muscarinic acetylcholine receptors (mAChRs). Bendazol inhibited the progression of FDM and suppressed the upregulation of HIF-1α. At week 6, in the control, FD and FD+bendazol groups, the refraction values were 1.38±0.43, 0.03±0.47 and 1.25±0.35 D, respectively (p<0.001); the ALs were 13.91±0.11, 14.15±0.06 and 13.97±0.10mm, respectively (p<0.001) and the VCDs were 6.56±0.06, 6.69±0.07 and 6.61±0.06mm, respectively (p<0.001). HIF-1α was upregulated in FD eyes but downregulated in FD+bendazol eyes, while the mAChRs were the opposite. In the FD rabbit model, bendazol significantly inhibits the development of myopia and downregulates HIF-1α expression, which may provide a novel therapeutic approach for myopia control.

Anti-Inflammatory Response and Muscarinic Cholinergic Regulation during the Laxative Effect of Asparagus cochinchinensis in Loperamide-Induced Constipation of SD Rats.

Several types of saponins and herbal plants containing saponins have been reported to have anti-inflammatory or laxative activities. To verify the therapeutic effects of saponin-enriched extracts of Asparagus cochinchinensis (SPA) on the anti-inflammatory response and on the cholinergic regulation in the gastrointestinal system, an alteration on the constipation phenotypes, on the inflammatory responses, and on the muscarinic cholinergic regulation were investigated in the transverse colons of Sprague Dawley (SD) rats with loperamide (Lop)-induced constipation after the treatment of SPA. Significant increases were observed on the total number of stools, the gastrointestinal transit, the thickness of the mucosal layer, the flat luminal surface, the number of paneth cells, and the lipid droplets in the Lop + SPA-treated group as compared to the Lop + Vehicle-treated group. SPA treatment induced the recovery of inflammatory cytokines (TNF-α, IL-1β) and IL-6), inflammatory mediators (NF-κB and iNOS), the total number of infiltered mast cells, and mucin secretion. Also, some similar improvements were observed on the levels of acetylcholine esterase (AChE) activity and on the phosphorylation of myosin light chains (MLC) as well as the expression of muscarinic acetylcholine receptors M2/M3 (mAChR M2/M3) and their mediators. The results presented herein provide the first strong evidence that SPA stimulates anti-inflammatory responses and the muscarinic cholinergic regulation when exerting its laxative effects in the chronic constipation of Lop-induced models.

Evaluating Cholinergic Receptor Expression in Guinea Pig Primary Auditory and Rostral Belt Cortices After Noise Damage Using [3H]Scopolamine and [18F]Flubatine Autoradiography.

Noise-induced hearing loss leads to anatomic and physiologic changes in primary auditory cortex (A1) and the adjacent dorsal rostral belt (RB). Since acetylcholine is known to modulate plasticity in other cortical areas, changes in A1 and RB following noise damage may be due to changes in cholinergic receptor expression. We used [3H]scopolamine and [18F]flubatine binding to measure muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) expression, respectively, in guinea pig A1 and RB 3 weeks following unilateral, left ear noise exposure, and a temporary threshold shift in hearing. [3H]Scopolamine binding decreased in right A1 and RB (contralateral to noise) compared to sham controls across all cortical layers. [18F]Flubatine binding showed a nonsignificant upward trend in right A1 following noise but only significantly increased in right RB and 2 layers of left RB (ipsilateral to noise). This selective response may ultimately influence cortical plasticity and function. The mechanism(s) by which cholinergic receptors are altered following noise exposure remain unknown. However, these data demonstrate noise exposure may differentially influence mAChRs that typically populate interneurons in A1 and RB more than nAChRs that are traditionally located on thalamocortical projections and provide motivation for cholinergic imaging in clinical patient populations of temporary or permanent hearing loss.

Analysis of Gene Expression and Neuronal Phenotype in Neuroscreen-1 (NS-1) Cells

Neuroscreen-1 (NS-1) a sub-clone of pheochromocytoma (PC12) cell is gaining broad acceptance as in vitro neuronal model for biochemical and phenotypic assays due to robust growth and differentiation profiles. However, the molecular characteristics of the cell remains to be documented. In this study, we performed comparative analysis for expression of neuronal marker genes in undifferentiated and nerve growth factor (NGF) differentiated NS-1 and PC12 by qPCR and immunoblot assays. We show that differentiation of NS-1 occurred under low concentrations of NGF relative to PC12. Cell growth also occurred more rapidly in NS-1. Transcriptional analysis of neuronal marker genes showed comparable expression of tyrosine receptor kinases (Ntrk1, Ntrk2, NGFR/p75NTR) and muscarinic acetylcholine (Chrm1, Chrm2, Chrm3, Chrm4) receptors in unspecialized cells. Ntrk2, adenosine receptors (Adora1, Adora2A) and choline acetyltransferase (ChAT) were altered in undifferentiated NS-1. In contrast, Ntrk1, Ntrk2, Chrm2 transcripts were vastly increased in NS-1 with NGF exposure, while Ntrk3, Adora1 and Adora2A transcripts were reduced. In differentiated PC12, Chrm4 and ChAT were markedly upregulated. Our data suggests that differences in morphological and phenotypic characteristics that distinguish NS-1 from PC12 is likely the product of altered gene expression. Furthermore, expression of neuron type genes in NS-1 support its use as an alternative model to PC12.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs.

To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor (mAChR) M1 in the eyes of guinea pigs. Thirty guinea pigs (15-20 days old) were randomly divided into three groups (n=10/group). Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks' illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry. There was a remarkable reduction in refractive error and increase in axial length after 8-weeks' green flickering light stimulation (P<0.001). The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III (P<0.01). Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed (P<0.05). However, there was no M1 receptor expression in choroid in 3 groups. Myopia can be induced by 8 weeks' green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

Variation of nicotinic subtype α7 and muscarinic subtype M3 acetylcholine receptor expression in three main types of leukemia.

Cholinergic receptors, such as α7-nicotinic acetylcholine receptor (α7-nAChR) and M3-muscarinic acetylcholine receptor (M3-mAChR), have been demonstrated to serve a significant role in the proliferation, differentiation and apoptosis of leukemic cells. However, the expression of these receptors in samples from patients with leukemia remains unclear. The present study aimed to determine the expression of M3-mAChR and α7-nAChR in the bone marrow or peripheral blood of 51 patients with leukemia, including acute myeloid leukemia (AML; n=33), acute lymphoblastic leukemia (ALL; n=13), and chronic myeloid leukemia (CML; n=5). Peripheral blood mononuclear cells (PBMCs) were also isolated from healthy subjects (n=5) for comparison. Western blot analysis was performed to determine the protein expression profiles, and a pattern of decreased α7-nAChR levels in patients with leukemia was observed. Among the leukemia types, the lowest expression of α7-nAChR and M3-mAChR were identified in patients with T-cell ALL/lymphoma (T-ALL). CML exhibited the highest level of M3-mAChR, which was significantly different from APL and AML-M4, yet not from healthy subjects (P<0.05). Therefore, different expression profiles of α7-nACR and M3-mAChR were detected amongst the leukemia types. Collectively, the present study supports the potential role of cholinergic signaling in mediating leukemogenesis. However, further studies in larger cohorts are required to validate these findings.

Inter- and intra-specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds.

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Correction: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats.

[This corrects the article DOI: 10.1371/journal.pone.0195624.].