THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE. Biomedical Seminar Room 5, Mondays 9:30-11:30

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1 THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE Biomedical Seminar Room 5, Mondays 9:30-11:30 a lifetime, a part; neuro-muscular junctions: mind meeting matter. We can t live without our neuromuscular junctions (NMJ s). Thousands of motor neurones each supply an axon branch that delivers hundreds of motor nerve terminals to our skeletal muscle fibres. Each terminal arbor forms synapses on the surface of a single muscle fibre at a single patch, about 400 µm 2 in area and each of these motor endplates is endowed with millions of ligand-gated acetylcholine receptors and voltage-gated sodium ion channels. High-fidelity synaptic transmission empowers NMJ s and activates skeletal muscles, enabling a myriad of delicate-to-intense voluntary movements, thus linking cognition and intention to behaviour. When these highly-tuned, impedance-matched nervemuscle connections fail, as they may do with advancing age or disrepair - or as a result of injury, poisoning or disease affected individuals may suffer symptoms and show signs of severe motor disturbances, ranging from painful seizures or cramps to weakness or complete paralysis. In fact, respiratory paralysis - due to failure of neuromuscular junctions - is a critical feature in illnesses or infections such as botulism or myasthenia gravis; and degeneration of NMJ s in respiratory muscle is a harbinger of death in incurable motor neurone diseases such as amyotrophic lateral sclerosis (ALS). Injuries to peripheral nerves can also be highly debilitating, triggering Wallerian degeneration of axons and motor nerve terminals disconnected from their cell bodies, which leads to partial or complete denervation and paralysis of muscle fibres. Fortunately, injured peripheral nerve axons are capable of successful regeneration, unlike most axons in the CNS; which further adds to the value of studying mechanisms of peripheral nerve repair. Given the crucial importance of NMJ s in our lives, the structure of neuromuscular synapses, as one might expect, is intimately entwined with their function. We now know that two kinds of supporting cells, terminal Schwann cells and kranocytes, co-exist at NMJ s and co-operate in their formation, maintenance and plasticity. Drugs that act selectively on neuromuscular transmission, influencing either the release of acetylcholine from nerve terminals or the action of these molecules on postsynaptic receptors, play important roles both in revealing the normal function of these synapses and in treatment for neuromuscular disorders. During development, the exquisite interplay of the different molecular and cellular components of neuromuscular synapses lies somewhere betwixt and akin the co-operativity of an intimate love-affair and the competitive struggle of all-out war. Similar processes - and perhaps similar molecules - regulate the repair of damaged connectivity and these processes are important targets for developing more effective treatments for neurodegenerative diseases such as ALS. Structure of the NMJiHaD course The NMJiHaD course comprises five mini-symposia, prepared and delivered by student members of the class. Each symposium focuses on a different aspect of the structure, function, development and plasticity of neuromuscular synaptic connections and their relevance to the understanding of disease or injury affecting motor neurones. Thus, the course is not comprehensive and several areas of interest are not covered or touched on in a limited way. (For example, we do not dwell very much on the biochemistry or pharmacology of neuromuscular junctions). However, the topics we do cover will include discussion of cutting-edge research. 1

2 The class will be divided into five groups ( Motor Units ), each with five members, and each group will be responsible for delivering one of the mini-symposia. Four members of the group (the Junctions ) will deliver minute presentations of the research papers that illustrate the topic (one paper per presenting student). The fifth member of the group (the Axon ) will Chair the mini-symposium. As well as steering questions from the audience, the Chairperson should also think of questions to ask each speaker. This is a valuable generic skill for anyone chairing a meeting: it is quite often necessary for the chair to get the ball rolling, or to maintain the momentum of discussion when audience members or other attendees appear reticent. The Chair shall also be a rapporteur, responsible for summarizing their mini-symposium and writing a brief (2-page) overview of all the papers presented, for circulation to the class. The mini-symposia will be held in alternate weeks. In the interleaving weeks, the session will normally begin with an introduction to the topic of the next mini-symposium by the course organiser (RRR: the Soma ), followed by a discussion of the abstracts of the papers to be presented by one of the groups the following week. The format of these group discussions will be structured as follows: 1. Each abstract is read aloud by a member of the group 2. Group identifies, defines and clarifies any difficult terms or terminology 3. Group freely discusses the issues raised by the paper 4. Each group decides on up to four burning questions (BQ s) from the issues discussed 5. In plenary discussion, the class narrows down the number of BQ s to four Big Burning Questions (BBQ s) that the presenting group should endeavour to address in the following week s mini-symposium. General Reading Byrne, JH & Roberts, JL (2009) From Molecules to Networks 2nd edn. Sinauer. Chapters 2, 5, 8,11,13,16, 20 Katz B. Neural transmitter release: from quantal secretion to exocytosis and beyond.. J Neurocytol Jun-Sep;32(5-8): PMID: Sanes JR, Lichtman JW. Development of the vertebrate neuromuscular junction. Annu Rev Neurosci. 1999;22: PMID: Hughes BW, Kusner LL, Kaminski HJ. Molecular architecture of the neuromuscular junction. Muscle Nerve Apr;33(4): PMID: Ribchester RR. Mammalian neuromuscular junctions: modern tools to monitor synaptic form and function. Curr Opin Pharmacol Jun;9(3): PMID: Mini-symposium topics : I. Structure and function of neuromuscular junctions II. Development and remodelling of the NMJ III. Homeostatic regulation of structure and function at the NMJ IV. Neuromuscular Junctions in Motor Neurone Disease V. Neuromuscular synaptic protection in the Wld S mouse mutant Introductory talks (RRR unless otherwise indicated) Week Topic 1. Overview of course structure; MCQ revision of NMJ; review of anatomy and physiology of the NMJ; 3. Neuromuscular synapse formation, elimination and repair 5. Quantal analysis and the safety-factor for neuromuscular transmission 7. Animal models of NMJ in Motor Neurone Disease 9. Prospective cell biology of the NMJ 2

3 Week 2 MINI-SYMPOSIUM I Structure and Function of Neuromuscular junctions Background reading: Ribchester, R.R. (2009) Mammalian neuromuscular junctions: modern tools to monitor synaptic form and function. Curr Opin Pharmacol. 9, PMID: (PDF here). For Presentation: 1. Lu J, Tapia JC, White OL, Lichtman JW. The interscutularis muscle connectome. PLoS Biol Feb 10;7(2):e32. PMID: Desaki J, Uehara Y. The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J Neurocytol Feb;10(1): PMID: Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ.The architecture of active zone material at the frog's neuromuscular junction.nature Jan 25;409(6819): PMID: Court FA, Gillingwater TH, Melrose S, Sherman DL, Greenshields KN, Morton AJ, Harris JB, Willison HJ, Ribchester RR. Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions. J Cell Sci Dec 1;121(Pt 23): PMID: Additional reading Massoulié J, Millard CB. Cholinesterases and the basal lamina at vertebrate neuromuscular junctions. Curr Opin Pharmacol Jun;9(2): PMID:

4 Week 4 MINI-SYMPOSIUM II Development and remodelling of the NMJ Background articles: Brown MC, Jansen JK, Van Essen D. Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation.j Physiol Oct;261(2): PMID: Son YJ, Trachtenberg JT, Thompson WJ. Schwann cells induce and guide sprouting and reinnervation of neuromuscular junctions. Trends Neurosci Jul;19(7): PMID: For presentation: 1. Walsh MK, Lichtman JW. In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron Jan 9;37(1): PMID: Costanzo EM, Barry JA, Ribchester RR. Competition at silent synapses in reinnervated skeletal muscle. Nat Neurosci Jul;3(7): PMID: Murray LM, Comley LH, Thomson D, Parkinson N, Talbot K, Gillingwater TH. Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. Hum Mol Genet Apr 1;17(7): PMID: Liu Z, Chen Y, Wang D, Wang S, Zhang YQ. Distinct presynaptic and postsynaptic dismantling processes of Drosophila neuromuscular junctions during metamorphosis. J Neurosci Sep 1;30(35): PMID: Additional reading Pun S, Sigrist M, Santos AF, Ruegg MA, Sanes JR, Jessell TM, Arber S, Caroni P.An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles. Neuron Apr 25;34(3): PMID:

5 Week 6 MINI-SYMPOSIUM III Homeostatic regulation of structure and function at the NMJ Background articles: Schwarz,T. (2005) Transmitter Release at the Neuromuscular Junction. Int Rev Neurobiol 75, Urbano FJ, Pagani MR, Uchitel OD. Calcium channels, neuromuscular synaptic transmission and neurological diseases. J Neuroimmunol Sep 15; C: PMID: Palace J, Beeson D. The congenital myasthenic syndromes. J Neuroimmunol Sep 15; :2-5. PMID: Lang B, Vincent A. Autoimmune disorders of the neuromuscular junction. Curr Opin Pharmacol Jun;9(3): PMID: Slater, CR Reliability of neuromuscular transmission and how it is maintained.handbook of Neurology. 2008: 91, For presentation: 1. Wood SJ, Slater CR.The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. J Physiol Apr 1;500 ( Pt 1): PMID: DiAntonio A, Haghighi AP, Portman SL, Lee JD, Amaranto AM, Goodman CS. Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature Jul 26;412(6845): PubMed PMID: Collins CA, Wairkar YP, Johnson SL, DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal. Neuron Jul 6;51(1): PMID: Slater CR, Fawcett PR, Walls TJ, Lyons PR, Bailey SJ, Beeson D, Young C, Gardner- Medwin D. Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia'. Brain Aug;129(Pt 8): PMID:

6 Week 8 MINI-SYMPOSIUM IV Neuromuscular Junctions in Motor Neurone Disease Background articles: Bruijn LI, Miller TM, Cleveland DW. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci. 2004;27: PMID: Dupuis L, Loeffler JP. Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models. Curr Opin Pharmacol Jun;9(3): PMID: Veldink JH, Bär PR, Joosten EA, Otten M, Wokke JH, van den Berg LH. Sexual differences in onset of disease and response to exercise in a transgenic model of ALS. Neuromuscul Disord Nov;13(9): PMID: Fischer LR, Culver DG, Tennant P, Davis AA, Wang M, Castellano-Sanchez A, KhanJ, Polak MA, Glass JD. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol Feb;185(2): PMID: For presentation: 1. Fischer LR, Culver DG, Tennant P, Davis AA, Wang M, Castellano-Sanchez A, Khan J, Polak MA, Glass JD. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol Feb;185(2): PMID: Schaefer AM, Sanes JR, Lichtman JW. A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis. J Comp Neurol Sep 26;490(3): PMID: David G, Nguyen K, Barrett EF. Early vulnerability to ischemia/reperfusion injury in motor terminals innervating fast muscles of SOD1-G93A mice. Exp Neurol Mar;204(1): PMID: Wong M, Martin LJ. Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum Mol Genet Jun 1;19(11): PMID:

7 Week 10 MINI-SYMPOSIUM V Neuromuscular synaptic protection in the Wld S mouse mutant Background articles: Perry VH, Lunn ER, Brown MC, Cahusac S, Gordon S. Evidence that the Rate of Wallerian Degeneration is Controlled by a Single Autosomal Dominant Gene. Eur J Neurosci. 1990;2(5): PMID: Coleman MP, Freeman MR. Wallerian degeneration,wld(s), and Nmnat. Annu Rev Neurosci. 2010;33: PMID: For presentation: 1. Mack TG, Reiner M, Beirowski B, Mi W, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L, Fernando FS, Tarlton A, Andressen C, Addicks K, Magni G, Ribchester RR, Perry VH, Coleman MP. Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci Dec;4(12): PubMed PMID: Gillingwater TH, Thomson D, Mack TG, Soffin EM, Mattison RJ, Coleman MP, Ribchester RR. Age-dependent synapse withdrawal at axotomised neuromuscular junctions in Wld(s) mutant and Ube4b/Nmnat transgenic mice. J Physiol Sep 15;543(Pt 3): PMID: Beirowski B, Babetto E, Gilley J, Mazzola F, Conforti L, Janeckova L, Magni G, Ribchester RR, Coleman MP. Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo. J Neurosci Jan 21;29(3): PubMed PMID: Wong F, Fan L, Wells S, Hartley R, Mackenzie FE, Oyebode O, Brown R, Thomson D, Coleman MP, Blanco G, Ribchester RR. Axonal and neuromuscular synaptic phenotypes in Wld(S), SOD1(G93A) and ostes mutant mice identified by fiber-optic confocal microendoscopy. Mol Cell Neurosci Dec;42(4): Epub 2009 Aug 14. PubMed PMID: Additional reading: Ferri A, Sanes JR, Coleman MP, Cunningham JM, Kato AC. Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease. Curr Biol Apr 15;13(8): PMID:

8 Tips on reading and presenting research papers What is the aim of the study? Is there a hypothesis? How has the study been designed to address the aim/hypothesis? What methods/techniques have been used? Are they appropriate for the design/question? Figures contain the most important data: what kind of data were acquired? How have the data been quantitatively analysed? Is the data analysis adequate? Could it be improved and if so how? Are the authors conclusions justified by the quality and quantity of the data? Are there alternative interpretations? What are the strengths and weaknesses of the study? What should be done next? See also - 8

9 RRR s Top Ten NMJ Papers 1: Fatt P, Katz B. Spontaneous subthreshold activity at motor nerve endings. J Physiol May;117(1): No abstract available. PMID: : Boyd IA, Martin AR. The end-plate potential in mammalian muscle. J Physiol Apr 27;132(1): No abstract available. PMID: : Dodge FA Jr, Rahamimoff R.Co-operative action a calcium ions in transmitter release at the neuromuscular junction. J Physiol Nov;193(2): PMID: : Brown MC, Jansen JK, Van Essen D. Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation. J Physiol Oct;261(2): PMID: : McLachlan EM, Martin AR. Non-linear summation of end-plate potentials in the frog and mouse. J Physiol Feb;311: PMID: : Mishina M, Takai T, Imoto K, Noda M, Takahashi T, Numa S, Methfessel C, Sakmann B. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature May 22-28;321(6068): PMID: : Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ. The architecture of active zone material at the frog's neuromuscular junction. Nature Jan 25;409(6819): PMID: : Wood SJ, Slater CR. The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. J Physiol Apr 1;500 ( Pt 1): PMID: : Richards DA, Guatimosim C, Betz WJ. Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron Sep;27(3): PMID: : Walsh MK, Lichtman JW. In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron Jan 9;37(1): PMID:

10 MCQ : Knowledge Review/Revision of the Neuromuscular System 1. The following are part of the descending motor pathway EXCEPT: A. Muscle spindles B. The motor cortex C. Upper motor neurones D. Motor neurone pools E. Lower motor neurones 2. Alpha motor neurones may receive synaptic inputs from the following EXCEPT: A. Afferent fibres from muscle spindles B. Spinal interneurones C. Gamma motor neurones D. Upper motor neurones E. Group Ia afferent fibres 3. In muscle innervation by lower motor neurones the following is true EXCEPT: A. Efferent axons exit the spinal cord via the ventral root B. Alpha motor neurones are responsible for the generation of muscle force C. Axons of lower motor neurones are unmyelinated D. Gamma motor neurones innervate muscle spindles E. The cell body of the alpha motor neurone is located in the ventral horn of the spinal cord 4. The compound action potential in a whole nerve: a) is activated in an all-or-none manner b) is 1-2 s in duration c) is composed of small and large diameter axons with identical conduction velocities d) is mediated by ligand gated ion channels e) exhibits an absolute refractory period 5. Which of the following statements concerning the conduction of action potentials in axons is FALSE a. Group Aα fibres may conduct at a velocity of 60 ms -1 b. Conduction in Group Aβ fibres is saltatory c. Conduction in Group Aδ fibres is faster than in Group C fibres d. Group C fibres conduct at velocities from 1ms -1 to 10 ms -1 e. Conduction in Group C axons is continuous because they are unmyelinated 6. Sodium ionic channels in motor axons are normally blocked by which of the following drugs: a. tetrodotoxin b. µ-conotoxin c. tubocurarine d. 4-aminopyridine e. ω-agotoxin 10

11 7. The following events occur during chemical synaptic transmission EXCEPT: A. The contents of a vesicle are released from the presynaptic terminal B. Calcium ions enter the presynaptic terminal C. A neurotransmitter binds to a neurotransmitter receptor D. Neurotransmitter molecules diffuse across a synaptic cleft E. Magnesium ions in the extracellular fluid enhance transmitter release 8. Indicate which of the following is FALSE. Calcium ions: A. Are pumped out of the synaptic terminal via voltage gated ion channels following neurotransmitter release B. Enter the synaptic terminal as a result of depolarisation C. Are at very low concentrations within the cytoplasm of resting neurones D. Can shape the neuronal action potential E. Enter the synaptic terminal via voltage gated ion channels located at active zones 9. Indicate which of the following is FALSE. Synaptic vesicles: A. Are primed for exocytosis following docking with the presynaptic membrane B. Undergo fusion as a result of increased intracellular calcium C. Undergo fusion following inhibition of synaptotagmin D. Dock with the presynaptic membrane using synaptobrevin E. Are targeted to the active zone 10. With regard to the process that take place during exocytosis of neurotransmitter at synapses, which of the following statements is FALSE: a. v-snare s interact with t-snare s to bring about vesicular fusion with synaptic terminal membranes in response when intracellular Ca ion concentration increases b. the rate of vesicular fusion is transiently increased by application of α-latrotoxin c. acetylcholine diffuses through a fusion pore formed by a synaptic vesicle with the presynaptic membrane d. docked vesicles may be replenished by vesicles from a reserve pool in the synaptic terminal e. a molecular cage of clathrin molecules forms around docked vesicles immediately prior to exocytosis 11. Indicate which of the following is FALSE. Postsynaptic potentials: A. Make communication between neurones possible B. Occur around 1 ms after the presynaptic action potential C. Propagate from sensory receptors D. Result from neurotransmitter molecules binding to postsynaptic receptors E. Can be either excitatory or inhibitory 12. A recording from a neuromuscular junction revealed spontaneous MEPPs of mean amplitude 0.5 mv; and EPPs in response to nerve stimulation (in a low Ca 2+ solution), of mean amplitude 4 mv. What was the quantal content (number of vesicles released by nerve stimulation) at this junction? a) 0.5 mv b) 8 mv c) 2 quanta d) 4 quanta e) 8 quanta 11

12 13. If the magnesium ion concentration in a solution bathing a nerve-muscle preparation is increased to about 5 mm and Ca ionic concentration is reduced to about 0.5 mm, nerve stimulation fails to evoke transmitter release on a significant number of occasions. The average number of synaptic vesicles (quantal content, m) undergoing exocytosis can be calculated under these conditions using the formula : m=ln (trials/failures); where Ln is the Natural Logarithm (Ln x = log 10 x). In a run of 100 test stimuli during such an experiment, there was no endplate-potential response to 10 of the stimuli. This suggests the average quantal content was: a. about 23.0 b. about 10.0 c. about 2.3 d. about 1.0 e. about The rat diaphragm twitch: a) results from release of acetylcholine at the parasympathetic neuroeffector junction b) is blocked by atropine c) is blocked by hexamethonium d) is unaffected by tubocurarine and tetrodotoxin e) is inhibited by suxamethonium 15. Application of the following drugs leads to block of synaptic transmission evoked by nerve stimulation at neuromuscular junctions of isolated nerve-muscle preparations. For which of the following drugs is the above statement FALSE : a. botulinum toxin b. α-bungarotoxin c. atracurium d. 4-aminopyridine e. suxamethonium 16. Atracurium (AtC) is used as a muscle relaxant during surgery. Its effect and mechanism of action are similar to those of tubocurarine, If AtC were applied during a recording from a neuromuscular junction, what would be observed? a) a decrease in the amplitude of MEPPs b) an increase in the amplitude of EPPs c) a decrease in EPP quantal content d) an increase in MEPP quantal size e) repetitive firing due to the inhibitory effect of AtC on acetylcholinesterase 17. Which of the following statements about the development of the motor innervation of skeletal muscle in rodents (rats or mice) is FALSE: a) motor neurones are generated in ventricular germinal zones of the neural tube then migrate and aggregate in the presumptive ventral horns of spinal cord grey matter b) many more motor neurones are normally generated prenatally than survive postnatally c) by birth all or nearly all muscle fibres are polyneuronally innervated by axons of different motor neurones d) postnatal synapse elimination is due mainly to loss of entire motor units by motor neurone death e) acetylcholine receptors at newly formed NMJ s contain γ-subunits rather than ε-subunits 12

13 18. In the developing muscle fibre: a) myoblasts are multinucleated cells b) myotubes are multinucleated syncitia c) muscle fibres are monucleated d) acetylcholine receptors are only expressed once neuromuscular synapses have formed e) sodium channels become concentrated at the crests of the neuromuscular junctional folds 19. Which of the following is a normal regressive event during neuromuscular development: a) neural induction b) outgrowth of motor axons from the neural tube c) prenatal death of motor neurones d) postnatal death of motor neurones e) sprouting of motor nerve terminals following axon degeneration in adults 20. When electrophysiological recordings are made from newborn rat or mouse muscles: a) end-plate potentials (EPP) no longer fluctuate randomly in size b) graded nerve stimulation may produce systematic increments in the size of the EPP c) all motor units give the same percentage of the total muscle tension as in adults d) action potentials are rarely obtained because there are no sodium channels present e) single channel recordings from acetylcholine receptors show the same kinetics as those in adults 21. The following findings may be taken as evidence in support of activity-dependent competitive synapse elimination EXCEPT (i.e. which is FALSE): a) partial denervation at birth inhibits the reduction in the size of intact motor units b) partial denervation at birth leads to shrinkage of intact motor units c) transgenic expression of trophic factors delays synapse elimination d) muscle stimulation accelerates the appearance of mononeuronal innervation e) rats increase their motor activity during the loss of polyneuronal innervation 22. In the disease myasthenia gravis, patients have antibodies in their blood against their own acetylcholine receptors, producing symptoms and signs of muscle weakness. At a cellular level, neuromuscular junctions would be expected to show which of the following characteristics: a. abnormally large end-plate potentials in response to nerve stimulation b. abnormally small spontaneous miniature end-plate potentials c. insensitivity to neostigmine d. insensitivity to tubocurarine e. long-lasting facilitation of end-plate potentials in response to repetitive nerve stimulation at 30 Hz 23. In the motor neurone disease Amyotrophic Lateral Sclerosis (ALS): a. All forms of the disease are caused by mutations in the SOD1 gene b. Motor neurones supplying the legs are nearly always the first to degenerate c. Surviving motor units may be enlarged due to compensatory axonal sprouting d. There is no impairment of glutamate transport by glial cells in the spinal cord e. Riluzole, an antagonist of glutamate release, completely cures some patients 13

14 Neuromuscular Transmission/Quantal Analysis Problems 1. In an experiment on a partially curarised frog neuromuscular junction, acetylcholine (ACh) was applied to the endplate by iontophoresis, using 1 na, 1 ms current pulses at a frequency of 2 Hz. A train of five endplate potentials (EPPs) was then evoked by stimulating the muscle nerve at 50Hz. The iontophoretic pulses were resumed within 20 ms of the end of the stimulus train. The following data were obtained: Mean ACh response before EPP train = mv (mean ± S.D.; n=10) Mean ACh response after EPP train = mv (mean ± S.D.; n=7) EPP number Amplitude (mv) a) calculate the amount of charge delivered by each of the iontophoretic current pulses; b) sketch the characteristic responses to ACh and nerve stimulation indicating the time course of the responses; c) how might the iontophoretic responses to ACh change, if a low concentration of ACh (1 µm) were also continuously present in the medium? d) is the hypothesis that short-term synaptic depression is caused by desensitisation of ACh receptors supported or refuted by these data? Give your reasoning. 2. Intracellular recordings were made from a mouse neuromuscular junction. The nerve supply was stimulated 150 times at 1Hz. The mean size of the EPP evoked was 1.00 mv. Five of the stimuli evoked no response (i.e. there were 5 'failures'). a What was the mean quantal content at this neuromuscular junction? b What do you predict for the quantal size, the amplitude of the uniquantal event (MEPP)? c How many of the EPPs would you predict to have quantal contents of 1,2,3 and 4 quanta? d What do you predict would be the standard deviation of the EPP amplitudes? e If the baseline noise level peak-to-peak was 500 µv, how would this affect the accuracy of your estimates? 3. In an experiment on an isolated flexor digitorum brevis nerve-muscle preparation dissected from a mouse, intracellular microelectrode recordings were made of spontaneous miniature endplate potentials (MEPP). Endplate potentials (EPP) were then evoked by nerve stimulation at a frequency of 1 Hz. In total, 97 of the stimuli applied to the nerve evoked an EPP but 3 stimuli failed to evoke any EPP. The following mean data with their standard deviations were obtained: Mean MEPP amplitude (± SD) : 1.20 ± 0.72 mv Mean EPP amplitude (± SD) : 4.25 ± 2.42 mv A. Speculate on the ratio of Ca 2+ to Mg 2+ ions in the medium bathing this preparation. B. Calculate the mean quantal content of the EPP using the Direct, Variance and Failures Methods. C. What does the standard deviation of the MEPP amplitude (quantal size) indicate and how might this affect the estimation of mean quantal content? D. Give one other possible reason for a low quantal content, in the contexts of health and disease. 14