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Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle.

ABSTRACT: Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal-type AChR with an adult-type AChR preserves myoblast fusion, muscle and end-plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end-plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.

SUBMITTER: Koenen M

PROVIDER: S-EPMC1369094 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle.

Koenen Michael M Peter Christoph C Villarroel Alfredo A Witzemann Veit V Sakmann Bert B

EMBO reports 20050601 6

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscl ...[more]

PMID: 15905852

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Project description:BackgroundCell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF) is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood.ResultsThe cloning and analysis of three additional zebrafish (Danio rerio) BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes) and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR.ConclusionThe zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are expressed in an independently regulated and cell-specific fashion. An initial structural and functional analysis has shown that the regions controlling zebrafish BDNF gene expression have been cloned and identified. They can now be subjected to detailed molecular and genetic analyses to identify the cellular mechanisms by which the transcription factors that act on these regions control BDNF gene expression.

Project description:We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.

Project description:BackgroundThe increased expression of the nicotinic acetylcholine receptor (nAChR) in muscle denervation is thought to be associated with electrophysiological acetylcholine supersensitivity after nerve injury. Hence, we investigated the utility of the 18F-ASEM alpha7-nAChR targeting radiotracer as a new diagnostic method by visualizing skeletal muscle denervation in mouse models of sciatic nerve injury.MethodsTen-week-old C57BL/6 male mice were utilized. The mice were anesthetized, and the left sciatic nerve was resected after splitting the gluteal muscle. One week (n = 11) and three weeks (n = 6) after the denervation, 18F-ASEM positron emission tomography/magnetic resonance imaging (PET/MRI) was acquired. Maximum standardized uptake values (SUVmax) of the tibialis anterior muscle were measured for the denervated side and the control side. Autoradiographic evaluation was performed to measure the mean counts of the denervated and control tibialis anterior muscles at one week. In addition, immunohistochemistry was used to identify alpha7-nAChR-positive areas in denervated and control tibialis anterior muscles at one week (n = 6). Furthermore, a blocking study was conducted with methyllycaconitine (MLA, n = 5).Results18F-ASEM PET/MRI showed significantly increased 18F-ASEM uptake in the denervated tibialis anterior muscle relative to the control side one week and three weeks post-denervation. SUVmax of the denervated muscles at one week and three weeks showed significantly higher uptake than the control (P = 0.0033 and 0.0277, respectively). The relative uptake by autoradiography for the denervated muscle was significantly higher than in the control, and immunohistochemistry revealed significantly greater alpha7-nAChR expression in the denervated muscle (P = 0.0277). In addition, the blocking study showed no significant 18F-ASEM uptake in the denervated side when compared to the control (P = 0.0796).ConclusionsOur results suggest that nAChR imaging with 18F-ASEM has potential as a noninvasive diagnostic method for peripheral nervous system disorders.

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Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle.

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Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle.

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