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In JoVE (1)
- Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice
Other Publications (12)
- The Australian Journal of Rural Health
- Australian Family Physician
- Australian Family Physician
- Experimental Neurology
- The Journal of Comparative Neurology
- Paediatrics & Child Health
- Reproduction, Fertility, and Development
- Journal of Chemical Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Neurobiology of Disease
- Chemical Communications (Cambridge, England)
- Nature Protocols
Articles by Megan Wright in JoVE
Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice
Megan Wright1, Amy Kim2, Young-Jin Son3
1Biology Department, Arcadia University, 2Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine
We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.
Other articles by Megan Wright on PubMed
Caring for Depressed Patients in Rural Communities: General Practitioners' Attitudes, Needs and Relationships with Mental Health Services
The Australian Journal of Rural Health. Feb, 2005 | Pubmed ID: 15720311
To examine the needs and practices of rural GPs and their relationships with local acute mental health services, particularly in the provision of care to depressed patients.
Australian Family Physician. Jul, 2005 | Pubmed ID: 15999176
Australian Family Physician. Jan-Feb, 2006 | Pubmed ID: 16489393
Ciliary Neurotrophic Factor is Not Required for Terminal Sprouting and Compensatory Reinnervation of Neuromuscular Synapses: Re-evaluation of CNTF Null Mice
Experimental Neurology. Jun, 2007 | Pubmed ID: 17445802
Loss of synaptic activity or innervation induces sprouting of intact motor nerve terminals that adds or restores nerve-muscle connectivity. Ciliary neurotrophic factor (CNTF) and terminal Schwann cells (tSCs) have been implicated as molecular and cellular mediators of the compensatory process. We wondered if the previously reported lack of terminal sprouting in CNTF null mice was due to abnormal reactivity of tSCs. To this end, we examined nerve terminal and tSC responses in CNTF null mice using experimental systems that elicited extensive sprouting in wildtype mice. Contrary to the previous report, we found that motor nerve terminals in the null mice sprout extensively in response to major sprouting-stimuli such as exogenously applied CNTF per se, botulinum toxin-elicited paralysis, and partial denervation by L4 spinal root transection. In addition, the number, length and growth patterns of terminal sprouts, and the extent of reinnervation by terminal or nodal sprouts, were similar in wildtype and null mice. tSCs in the null mice were also reactive to the sprouting-stimuli, elaborating cellular processes that accompanied terminal sprouts or guided reinnervation of denervated muscle fibers. Lastly, CNTF was absent in quiescent tSCs in intact, wildtype muscles and little if any was detected in reactive tSCs in denervated muscles. Thus, CNTF is not required for induction of nerve terminal sprouting, for reactivation of tSCs, and for compensatory reinnervation after nerve injury. We interpret these results to support the notion that compensatory sprouting in adult muscles is induced primarily by contact-mediated mechanisms, rather than by diffusible factors.
Distinct Patterns of Motor Nerve Terminal Sprouting Induced by Ciliary Neurotrophic Factor Vs. Botulinum Toxin
The Journal of Comparative Neurology. Sep, 2007 | Pubmed ID: 17614103
Both diffusible and surface-bound molecules are thought to induce sprouting of motor nerve terminals in response to paralysis. Here we report that the sprouting induced by ciliary neurotrophic factor (CNTF) is qualitatively different from the sprouting induced by botulinum toxin (BoTX). We show first that subcutaneous application of CNTF to levator auris longus muscles of adult mice evokes sprouting from nearly all nerve terminals. Surprisingly, however, most terminal sprouts remain within the boundaries of the endplate region and rarely grow extrasynaptically even if CNTF is administered chronically. In contrast, terminal sprouts induced by BoTX extend vigorously along the extrasynaptic muscle surface. The different patterns of sprout elongation are attributable in part to different patterns of initiation: whereas CNTF-induced sprouts emerge randomly from the surface of terminal branches, BoTX-induced sprouts emerge exclusively along the perimeter of terminal branches in direct apposition to muscle fiber membranes. Combined treatment with CNTF and BoTX produces exceptionally robust extraterminal sprouting with little if any intrasynaptic growth of terminal sprouts. We interpret these results as showing that paralysis induces sprouting primarily by muscle-associated, surface-bound molecules rather than by diffusible factors. Our findings may be useful in defining the physiological role of the numerous candidate sprouting-inducers and in promoting compensatory sprouting after nerve injury for therapeutic benefit.
Paediatrics & Child Health. May, 2008 | Pubmed ID: 19412363
To reduce the risk of medication errors in paediatric patients, the Canadian Council on Health Services Accreditation endorsed the standardization and limiting of drug concentrations available within an organization.
Replication Asynchrony and Differential Condensation of X Chromosomes in Female Platypus (Ornithorhynchus Anatinus)
Reproduction, Fertility, and Development. 2009 | Pubmed ID: 19874719
A common theme in the evolution of sex chromosomes is the massive loss of genes on the sex-specific chromosome (Y or W), leading to a gene imbalance between males (XY) and females (XX) in a male heterogametic species, or between ZZ and ZW in a female heterogametic species. Different mechanisms have evolved to compensate for this difference in dosage of X-borne genes between sexes. In therian mammals, one of the X chromosomes is inactivated, whereas bird dosage compensation is partial and gene-specific. In therian mammals, hallmarks of the inactive X are monoallelic gene expression, late DNA replication and chromatin condensation. Platypuses have five pairs of X chromosomes in females and five X and five Y chromosomes in males. Gene expression analysis suggests a more bird-like partial and gene-specific dosage compensation mechanism. We investigated replication timing and chromosome condensation of three of the five X chromosomes in female platypus. Our data suggest asynchronous replication of X-specific regions on X(1), X(3) and X(5) but show significantly different condensation between homologues for X(3) only, and not for X(1) or X(5). We discuss these results in relation to recent gene expression analysis of X-linked genes, which together give us insights into possible mechanisms of dosage compensation in platypus.
Journal of Chemical Biology. Nov, 2009 | Pubmed ID: 19898886
N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.
Distinct Muscarinic Acetylcholine Receptor Subtypes Contribute to Stability and Growth, but Not Compensatory Plasticity, of Neuromuscular Synapses
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2009 | Pubmed ID: 19940190
Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M(2) mAChRs were present solely in motor neurons, whereas M(1), M(3), and M(5) mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M(2/4)-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M(2)(-/-) but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M(2)(-/-) NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M(2) or other mAChR subtypes. We also found that many NMJs of M(5)(-/-) mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M(2) and that muscle fiber growth is defective without M(5). Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.
Neurobiology of Disease. Sep, 2010 | Pubmed ID: 20381620
In amyotrophic lateral sclerosis (ALS), the exogenous temporal triggers that result in initial motor neuron death are not understood. Overactivation and consequent accelerated loss of vulnerable motor neurons is one theory of disease initiation. The vulnerability of motor neurons in response to chronic peripheral nerve hyperstimulation was tested in the SOD1(G93A) rat model of ALS. A novel in vivo technique for peripheral phrenic nerve stimulation was developed via intra-diaphragm muscle electrode implantation at the phrenic motor endpoint. Chronic bilateral phrenic nerve hyperstimulation in SOD1(G93A) rats accelerated disease progression, including shortened lifespan, hastened motor neuron loss and increased denervation at diaphragm neuromuscular junctions. Hyperstimulation also resulted in focal decline in adjacent forelimb function. These results show that peripheral phrenic nerve hyperstimulation accelerates cell death of vulnerable spinal motor neurons, modifies both temporal and anatomical onset of disease, and leads to involvement of disease in adjacent anatomical regions in this ALS model.
Chemical Communications (Cambridge, England). Apr, 2011 | Pubmed ID: 21221452
We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins.
Multifunctional Protein Labeling Via Enzymatic N-terminal Tagging and Elaboration by Click Chemistry
Nature Protocols. Jan, 2012 | Pubmed ID: 22193303
A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.