Noise analysis of acetylcholine induced current at neuromuscular junctions of the mouse diaphragm

Miniature end-plate currents (mepcs) and membrane noise elicited by acetylcholine (ACh) iontophoresis were investigated at neuromuscular junctions of the mouse diaphragm. All the experiments were performed at a holding potential of -70 mV at a temperature of 19 degrees C. The equilibrium potential of the ACh response was estimated to be near zero; the mepcs displayed a peak amplitude of 2.46 +/- 0.13 nA (mean +/- S.E.) and relaxed exponentially with a time constant to 1.63 +/- 0.11 msec. Single ACh-activated channels had a conductance of 26.5 +/- 1.5 pS and a mean life time of 1.69 +/0- 0.13 msec.     

大鼠舌乳头神经结构的乙酰胆碱受体亚型配布

目的:探索味觉的传递机制是否系味蕾细胞所接受的信息直接通过神经递质受体作用于舌乳头的感觉神经来完成。方法:免疫组化SABC技术对大鼠舌粘膜各部位各种乳头内神经结构上乙酰胆碱受体(AchR)的8种亚型(VR1、α2、α3、α4A、α4H、α7、β1及β2)的分布进行观察。结果:除丝状乳头外,其他3种乳头的支配神经束及纤维分支不同程度地分布有AchR的各亚型受体。阳性反应的AchR自乳头中轴上的神经束(干)起,并沿其分支分布,在临近味蕾前即中止,未见受体阳性的神经纤维终末到达味蕾细胞。结论:味觉感受的传递机制并非由AchR阳性感觉神经纤维直接从味蕾细胞“接力”上行。     

Muscarinic M3 acetylcholine receptor immunostaining in paraffin-embedded normal and neoplastic prostatic gland tissue

Recent in vitro studies suggest that muscarinic receptors are important indicators of prostatic epithelial differentiation. Using choline in the mapping of prostatic lesions, radiographic studies have been interpreted as showing malignant transformation when high levels of receptors are found, but to our knowledge no correlation with the grade of prostatic cancer has been reported. To demonstrate the relationship of muscarinic receptors to differentiation, we immunostained normal and neoplastic prostatic tissue with antibodies that recognize receptors of acetylcholine, dividing the neoplastic tissue into two groups according to grade of differentiation. As far as we know, this is the first time that immunostaining of benign and neoplastic prostatic gland tissue with cholinergic receptors has been reported. Thirty-five cases of prostatic carcinoma were retrieved from our files and subdivided into two groups; a well to moderately differentiated group and a poorly differentiated group. Both groups were immunostained with M3 muscarinic receptor, along with a proliferation marker (Ki-67). The proliferating index measured with Ki-67 in the well to moderately differentiated group was low (<5% of the cells) and high in the poorly differentiated group (>20% of the cells). A strong correlation was observed between the strength of expression of the muscarinic M3 receptor and the differentiation of the lesion. The 18 cases of well to moderately differentiated carcinoma showed a mean staining intensity of approximately 1 with no case showing an intensity grade greater than 2. Normal controls along with normal peritumoral prostatic tissue also demonstrated a staining intensity of approximately 1. However, none of the 11 cases of poorly differentiated carcinoma showed staining. Our conclusion is that the presence of muscarinic acetylcholine receptor M3 by immunostaining distinguishes normal glandular epithelium and low-grade carcinomas from poorly differentiated tumors, which suggests that its demonstration in vivo could be a useful tool in distinguishing grade of tumor clinically.     

Potentiation of the nicotinic acetylcholine receptor by aluminum in mammalian neurons

Aluminum (Al³⁺), a known neurotoxic substance, has long been implicated in the pathogenesis of Alzheimer’s disease and other neurodegenerative diseases. Al³⁺ targets many ligand-gated and voltage-gated ion channels and modulates their functions. In the present study, the actions of Al³⁺ on the nicotinic acetylcholine receptor (nAChR) were investigated by whole-cell patch clamp technique in acutely isolated rat trigeminal ganglion neurons. We observed that Al³⁺ potentiated nicotine-evoked inward currents in a concentration-dependent manner (10–1000 μM). The effects of Al³⁺ on nicotine-evoked currents were voltage independent. Al³⁺ appeared to increase the affinity of nicotine to nAChR but not the efficacy. Al³⁺ reduced the agonist concentration producing a half-maximal response (EC₅₀) for nicotine from 74.4±1.9 μM to 32.9±2.6 μM, but did not alter the threshold nor maximal response. On the contrary, another trivalent cation, Ga³⁺, had little effect on nicotine-evoked currents. The present results indicated that Al³⁺ enhanced the function of nAChR and this potentiation might underlie the neurological alteration induced by Al³⁺.     

alpha-Dystrobrevin isoforms differ in their colocalization with and stabilization of agrin-induced acetylcholine receptor clusters

In skeletal muscle, alpha-dystrobrevin (alphaDB) is expressed throughout the sarcolemma with high concentrations at the neuromuscular junction. Mice lacking alphaDB display a mild muscular dystrophy and perturbations at the neuromuscular junction that include disruptions to acetylcholine receptor (AChR) cluster stability and patterning. In adult skeletal muscle, three alternatively spliced isoforms (alphaDB1, alphaDB2, alphaDB3) are expressed, while two other splice variants (alphaDB1(-) and alphaDB2(-)) are expressed only during early development. alphaDB is clearly important in AChR stabilization; however, the degree to which individual alphaDB isoforms and their specific functional domains contribute to AChR cluster stability is not fully understood. To investigate this, we established a primary muscle cell culture system from alphaDB knockout mice and stably expressed individual alphaDB isoforms using retroviral infection. A comparison between wild-type and alphaDB knockout muscle cells showed that in the absence of alphaDB, fewer AChR clusters formed in response to agrin treatment, and these AChR clusters were very unstable. Retroviral expression studies revealed that the largest isoforms (alphaDB1, alphaDB1(-), alphaDB2, alphaDB2(-)) colocalized with agrin-induced AChR clusters and rescued the AChR cluster formation defects back to wild-type levels, while only the first three isoforms fully rescued AChR cluster stability back to wild-type levels. alphaDB2(-) conferred an intermediate level of stability to the AChR clusters. In contrast, alphaDB3 showed no specific colocalization with AChR clusters and little effect on AChR cluster formation or stabilization. Twice as much syntrophin was found associated with alphaDB2 compared with alphaDB2(-) in myotubes suggesting that increased recruitment of syntrophin by alphaDB may enhance the stability of AChR clusters. Taken together, these data demonstrate that different alphaDB isoforms have different functional capabilities in the formation and maintenance of AChR clusters in muscle cells, and that these differences are likely due to the presence of different functional domains in each isoform.     

烟碱型乙酰胆碱受体在认知功能中的作用

烟碱型乙酰胆碱受体（nAChR）是化学（配体）门控的离子通道蛋白，属于半胱氨酸环受体家族，广泛分布于中枢和周围神经系统中。中枢nAChR参与许多复杂的功能，如：注意、学习、觉醒和认知等。越来越多的研究表明，中枢nAChR在认知功能活动中发挥了重要的作用。临床资料提示，nAChR与一些认知功能障碍性疾病的发病机制有关，如：阿尔茨海默病（AD）、帕金森氏病（PD）以及癫痫等。     

Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes

Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.     

A neuronal nicotinic acetylcholine receptor in crayfish neurons

In warm-blooded vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) are distinguished from muscle endplate receptors by their ligand affinities and sensitivity to several toxins. In the crayfish optic lobe, synaptic and acetylcholine (ACh)-elicited responses are blocked by toxins (F-toxin and neosurugatoxin) selective for neuronal nAChRs and are insensitive to the alpha-neurotoxins selective for endplate nAChRs.     

Decreased vesicular acetylcholine transporter and alpha(4)beta(2) nicotinic receptor density in the rat brain following 192 IgG-saporin immunolesioning

Degeneration of cholinergic neurons is a well known characteristic of Alzheimer's disease (AD). Two radioligands were studied in a rat model of cholinergic degeneration to evaluate their potential efficacy for molecular imaging of AD. Following specific cholinergic-cell immunolesioning with 192 IgG-saporin (SAP), ex vivo autoradiography was performed with (123)IBVM, a radioligand which targets the vesicular acetylcholine transporter (VAChT). Following the decay of (123)I, the same animals had in vitro autoradiography performed with (125)I-A-85380, a marker for nicotinic acetylcholine receptors (nAChRs). As expected significant, widespread decreases in (123)IBVM uptake were observed in SAP treated animals. Moderate but significant reductions in (125)I-A-85380 binding in the hippocampus (Hip) and cerebellum (Cbm) were also observed following SAP immunolesioning. The results with (123)IBVM confirm and extend previous work investigating the uptake of radioiodinated IBVM in this animal model. The results with (125)I-A-85380 are unique and are in contrast with work performed in this animal model with other nAChR radioligands, indicating the favourable properties of this radioligand for molecular imaging.     

Spontaneous synchronization by 4-aminopyridine of acetylcholine release in the neuromuscular junction

The effect of 4-aminopyridine (4AP) on the character of acetylcholine release was investigated by intracellular recording of spontaneous synaptic activity in an isolated rate phrenic nervediaphragm preparation. No significant changes were found in the mean frequency and amplitude of miniature end-plate potentials (MEPP) in response to 4AP in concentrations of 1·10^–6 to 1·10^–3M. Meanwhile, 4AP caused the appearance of large spontaneous EPP capable of inducing spreading action potentials. 4AP changed the character of distribution of MEPP amplitudes, converting it to polymodal; in some cases the principal mode was shifted into the region of lower values. It is concluded that 4AP changes the character of acetylcholine release and potentiates spontaneous synchronization.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Department of Pharmacology, Faculty of Internal Medicine and Hygiene, I. M. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 259–262, September, 1979.     

Effect of exogenous acetylcholine on ionic currents of the frog and mouse motor nerve ending

Department of Normal Physiology, Kazan' Medical Institute. (Presented by Academician of the Russian Academy of Medical Sciences A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 8, pp. 124–126, August, 1992.     

5-Hydroxytryptamine blocks the fetal more potently than the adult mouse muscle acetylcholine receptor

 The action of 5-hydroxytryptamine (5-HT) on fetal (γ-AChR) and adult (ɛ-AChR) muscle acetylcholine receptors was studied in transfected BOSC 23 cells expressing mouse AChRs and in acutely dissociated mouse muscle fibres. In transfected cells, coapplication of 5-HT (0.01–10 mM) with ACh reversibly reduced the amplitude and accelerated the decay of the ACh-activated whole-cell currents, in a dose- and voltage-dependent manner. 5-HT blocked faster and with higher potency the γ-AChR than the ɛ-AChR. Cell-attached recordings from transfected BOSC 23 cells and embryonic or neonatal muscle fibres were made. When 5-HT (5 μM) was included in the patch pipette, ACh-evoked unitary events acquired a bursting behaviour, which was more pronounced for γ- than ɛ-AChR, though it was enhanced by membrane hyperpolarization for both AChR species. There was no effect on the single-channel conductance. It is concluded that 5-HT behaves as an open-channel blocker of muscle AChRs, with higher efficacy on γ- than on ɛ-AChR. Received: 5 November 1998 / Received after revision: 12 January 1999 / Accepted: 21 January 1999     

Detection of antibodies directed against the cytoplasmic region of the human acetylcholine receptor in sera from myasthenia gravis patients

The nicotinic acetylcholine receptor (AChR) is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore the determination of their specificities requires the use of native AChR. Antibody competition studies suggest that most MG antibodies are directed against the extracellular part of the molecule, whereas antibodies directed against the cytoplasmic region of the AChR have not been detected. To determine whether even small quantities of such antibodies exist in MG sera, we performed competition experiments based on the inhibition by MG sera of the binding of MoAbs to the human AChR, rather than inhibition by MoAbs of the binding of MG sera performed earlier. When MoAbs directed against cytoplasmic epitopes on the alpha or beta subunits (alpha 373-380 and beta 354-360) were used as test MoAbs, 17% or 9% of MG sera inhibited the binding of the anti-alpha or anti-beta subunit MoAbs, respectively, by > or = 50%. Non-specific inhibition was excluded. These results suggest the presence, in several MG sera, of antibodies directed against cytoplasmic regions of the AChR; yet these antibodies seemed to represent a relatively small proportion of the total anti-AChR antibodies. The corresponding epitopes may be involved in the inducing mechanisms in certain MG cases, and knowledge of the presence of such antibodies may be useful in understanding the autoimmune mechanism involved in MG.