THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE. Biomedical Seminar Room 7, Mondays 9:30-11:30

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1 THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE Biomedical Seminar Room 7, 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 tens of millions (about 10 5 /µm 2 ) of ligand-gated acetylcholine receptors and voltagegated sodium ion channels. High-fidelity synaptic transmission and homeostatic, synaptic strength-regulating mechanisms empower NMJ s, enabling them to activate skeletal muscles with an extraordinary degree of reliability; thus enabling a myriad of delicate-tointense voluntary movements, thereby linking cognition and intention to behaviour. When these highly-tuned, impedance-matched nerve-muscle 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 myasthenia gravis or botulism, respectively; 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 Central Nervous System (CNS), which further adds to the value of studying mechanisms of peripheral nerve repair: since a deeper understanding of these mechanisms could have an impact on the quest to find ways to repair damaged axons more effectively in the brain and spinal cord. The structure of neuromuscular synapses, as one might expect, is intimately entwined with their function. 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. Homeostatic mechanisms regulate the size and strength of neuromuscular connections and ensure a high margin of safety for neuromuscular transmission, ensuring reliability of movement and behaviour. During development, the exquisite interplay of the different molecular and cellular components of neuromuscular synapses lies somewhere between and akin to the co-operativity of an intimate love-affair and the competitive struggle of all-out war. These mechanisms are at least partly activity-dependent, implying the presence of Hebbian use-it-lose-it mechanisms, with respect to strength and maintenance of synaptic connections. 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. Finally, in addition to the synaptic connections between motor neurone terminals and the motor end-plates of muscle fibres, 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. 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 four or five members and each group will be responsible for delivering one of the mini-symposia. Four members of the group ( Motor Nerve Terminals ) 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. The Group identifies, defines and clarifies any difficult terms or terminology 3. The 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. Mini-symposium topics : I. Structure and function of neuromuscular junctions II. Physiology and pathophysiology of neuromuscular transmission III. Development, Degeneration and Repair of the NMJ IV. Activity-dependent plasticity of NMJ V. The NMJ in Motor Neurone Disease 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. Quantal analysis and the safety-factor for neuromuscular transmission 5. Neuromuscular synapse formation, elimination and regeneration 8. Activity-dependent plasticity of the NMJ (*Rosalind Brown) 10. Involvement of the NMJ in Motor Neurone Disease 2

3 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: RRR s Top Ten NMJ Papers 1: Fatt P, Katz B. Spontaneous subthreshold activity at motor nerve endings. J Physiol May;117(1): PMID: : Boyd IA, Martin AR. The end-plate potential in mammalian muscle. J Physiol Apr 27;132(1): 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: : Betz WJ, Bewick GS. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science Jan 10;255(5041): 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: : 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: : Walsh MK, Lichtman JW. In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron Jan 9;37(1): PMID:

4 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 4

5 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 5

6 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 6

7 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 7

8 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. 8

9 4. In a study by Wood & Slater (1997) it was found that the action potential firing threshold in rat soleus muscle fibres is reached by an endplate potential (EPP) with a mean quantal content of about 15. Quantal size did not vary substantially between muscle fibres in either soleus or extensor digitorum longus muscles in this study. Answer the following (weighting of percentage marks in brackets): a. Explain what is meant by the terms quantal content and quantal size b. Explain briefly how recordings of endplate currents could be made from this fibre, why endplate currents sum linearly with increasing quantal content but EPP s sum non-linearly, and how EPP-amplitude measurements may be corrected for non-linear summation. c. Predict the number of failures in a train of 100 EPC s if the mean quantal content became reduced to 2 quanta. d. Use the Failures Method to calculate the probability of failures in a train of EPC s evoked by repetitive stimulation when the mean quantal content is 15. Calculate the predicted variance and standard deviation of quantal contents in a train of EPPs of the same mean quantal content (ie 15). e. Discuss briefly what experimental or clinical conditions lead to a reduction of quantal content and the mechanisms that can enhance the safety factor for neuromuscular transmission. 5. Intracellular recordings of endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were obtained from a muscle fibre in an isolated mouse skeletal muscle bathed in normal physiological saline but in which the Ca 2+ concentration was reduced from 2mM to 1mM and the Mg 2+ concentration was increased from 1mM to 4 mm. The preparation was then stimulated with 100 trains-of-four nerve stimuli, delivered at 50Hz with intervals of 10s between each stimulus train. Each stimulus train evoked a corresponding train of four EPPs (EPP1-EPP4). The acetylcholinesterase inhibitor neostigmine (final concentration 3 µm) was then added to the physiological saline. After 20 minutes, recordings were resumed from the same muscle fibre and a further 100 trains-of-four supramaximal nerve stimuli were applied to the nerve. Other constituents of the bathing solution were unaltered from that of normal physiological saline. The resting membrane potential of the muscle fibre remained steady throughout at -72 mv. The following data were obtained Before and After adding neostigmine. Before adding neostigmine Mean MEPP amplitude: 1.2 mv Stimulus trials: 100 EPP1 EPP2 EPP3 EPP4 Number of EPP Failures Mean EPP Amplitude (mv) Rise time (ms) Half -Decay time (ms) After adding neostigmine Mean MEPP amplitude: 1.5 mv Stimulus trials: 100 EPP1 EPP2 EPP3 EPP4 Number of EPP Failures Mean EPP Amplitude (mv) Rise time (ms) Half-Decay time (ms) A. Describe and explain the responses to nerve stimulation and the effects of neostigmine on amplitude and time course of the EPPs. Sketch their appearance in the trains-of-four before and after the change in bathing solution (25 % of marks) B. What would you conclude from a quantal analysis of these data and should correction for non-linear summation of EPPs be considered? (25%) 9

10 C. Anticholinesterases may have additional direct effects on properties of presynaptic or post-synaptic acetylcholine receptors. Formulate hypotheses to explain the effects of neostigmine on the amplitude and time course of the synaptic potentials, with justification of your reasoning (25%). D. Suggest how the hypotheses you have proposed might be tested experimentally and what alternative outcomes of these experiments would imply. (25%) Note: The Poisson equation is P(x)=m x.exp(-m)/x! where P(x) is the probability of occurrence of an EPP with a quantal content of x, given a mean quantal content equal to m. 10

11 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). Massoulié J, Millard CB. Cholinesterases and the basal lamina at vertebrate neuromuscular junctions. Curr Opin Pharmacol Jun;9(2): PMID: (PDF here). 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: 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: Nagwaney S, Harlow ML, Jung JH, Szule JA, Ress D, Xu J, Marshall RM, McMahan UJ. Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. J Comp Neurol Apr 10;513(5): PubMed PMID: Gaffield MA, Tabares L, Betz WJ. Preferred sites of exocytosis and endocytosis colocalize during high- but not lower-frequency stimulation in mouse motor nerve terminals. J Neurosci Dec 2;29(48): PubMed PMID:

12 Week 4 MINI-SYMPOSIUM II Physiology and Pathophysiology of Neuromuscular Transmission Background reading: Slater, CR Reliability of neuromuscular transmission and how it is maintained.handbook of Neurology. 2008: 91, (PDF available see course website) Schwarz,T. (2005) Transmitter Release at the Neuromuscular Junction. Int Rev Neurobiol 75, (PDF available see course website) Spillane J, Beeson DJ, Kullmann DM. Myasthenia and related disorders of the neuromuscular junction. J Neurol Neurosurg Psychiatry Aug;81(8): PMID: For presentation: 1. Ruiz R, Cano R, Casañas JJ, Gaffield MA, Betz WJ, Tabares L. Active zones and the readily releasable pool of synaptic vesicles at the neuromuscular junction of the mouse. J Neurosci Feb 9;31(6): PMID: Kaja S, van de Ven RC, van Dijk JG, Verschuuren JJ, Arahata K, Frants RR, Ferrari MD, van den Maagdenberg AM, Plomp JJ. Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a-mutant mouse rolling Nagoya. Eur J Neurosci Apr;25(7): 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: 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: Additional Reading: Costanzo EM, Barry JA, Ribchester RR. Co-regulation of synaptic efficacy at stable polyneuronally innervated neuromuscular junctions in reinnervated rat muscle. J Physiol Dec 1;521 Pt 2: PMID:

13 Week 6 MINI-SYMPOSIUM III Development, degeneration and repair of the NMJ Background articles: 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: 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: 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: Conforti L, Wilbrey A, Morreale G, Janeckova L, Beirowski B, Adalbert R, Mazzola F, Di Stefano M, Hartley R, Babetto E, Smith T, Gilley J, Billington RA, Genazzani AA, Ribchester RR, Magni G, Coleman M. WldS protein requires Nmnat activity and a short N- terminal sequence to protect axons in mice. J Cell Biol Feb 23;184(4): PubMed 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:

14 MINI-SYMPOSIUM IV Activity-dependent regulation of structure and function at the NMJ Background articles: Dorlöchter M, Irintchev A, Brinkers M, Wernig A. Effects of enhanced activity on synaptic transmission in mouse extensor digitorum longus muscle. J Physiol May;436: PMID: Barry JA, Ribchester RR. Persistent polyneuronal innervation in partially denervated rat muscle after reinnervation and recovery from prolonged nerve conduction block. J Neurosci Oct;15(10): PMID: Davis GW. Homeostatic control of neural activity: from phenomenology to molecular design. Annu Rev Neurosci. 2006;29: PMID: For presentation: 1. Valdez G, Tapia JC, Kang H, Clemenson GD Jr, Gage FH, Lichtman JW, Sanes JR. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci U S A Aug 17;107(33): PMID: Tsujimoto T, Umemiya M, Kuno M. Terminal sprouting is not responsible for enhanced transmitter release at disused neuromuscular junctions of the rat. J Neurosci Jul;10(7): PMID: Costanzo EM, Barry JA, Ribchester RR. Competition at silent synapses in reinnervated skeletal muscle. Nat Neurosci Jul;3(7): PMID: Favero M, Buffelli M, Cangiano A, Busetto G. The timing of impulse activity shapes the process of synaptic competition at the neuromuscular junction. Neuroscience May 5;167(2): PMID:

15 Week 8 MINI-SYMPOSIUM V 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: 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: 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: For presentation: 1. 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: 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 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): PMID:

16 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 kinds 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

17 ABSTRACTS FOR MINI-SYMPOSIUM I Structure and Function of Neuromuscular junctions (PDF s of the full articles can be downloaded from the course website: or via the PubMed PMID number (if accessing from a University computer). PLoS Biol Feb 10;7(2):e32. The interscutularis muscle connectome. Lu J, Tapia JC, White OL, Lichtman JW Source Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA Erratum in PLoS Biol Apr;7(4):e Abstract The complete connectional map (connectome) of a neural circuit is essential for understanding its structure and function. Such maps have only been obtained in Caenorhabditis elegans. As an attempt at solving mammalian circuits, we reconstructed the connectomes of six interscutularis muscles from adult transgenic mice expressing fluorescent proteins in all motor axons. The reconstruction revealed several organizational principles of the neuromuscular circuit. First, the connectomes demonstrate the anatomical basis of the graded tensions in the size principle. Second, they reveal a robust quantitative relationship between axonal caliber, length, and synapse number. Third, they permit a direct comparison of the same neuron on the left and right sides of the same vertebrate animal, and reveal significant structural variations among such neurons, which contrast with the stereotypy of identified neurons in invertebrates. Finally, the wiring length of axons is often longer than necessary, contrary to the widely held view that neural wiring length should be minimized. These results show that mammalian muscle function is implemented with a variety of wiring diagrams that share certain global features but differ substantially in anatomical form. This variability may arise from the dominant role of synaptic competition in establishing the final circuit Comment in PLoS Biol Feb;7(2):e PMID:

18 J Neurocytol Feb;10(1): The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. Desaki J, Uehara Y Abstract Skeletal neuromuscular junctions (NMJs) of vertebrates were examined by scanning electron microscopy after removal of connective tissue components by HCl hydrolysis. In addition to the surface texture of NMJs, the subsynaptic organization of the sarcolemma was visualized in specimens in which nerve endings were detached from the muscle surface. A remarkable morphological variability between animal species was observed. The NMJs in the frog sartorius muscle consisted of longitudinal ribbon-like endings which fitted into a shallow synaptic gutter containing highly ordered cross-bands of junctional folds. The NMJs of the posterior latissimus dorsi muscle of the zebra finch were characterized by varicose swellings of the nerve endings which fitted into a round pit of the sarcolemma. NMJs in the sternothyroid muscle of the Chinese hamster consisted of thin ramified endings which were confined to an oval area on the muscle surface. The labyrinthine synaptic groove contained well-developed junctional folds without preferential spatial arrangement. The procedure used for the present study illustrates in great detail the terminal arborization of the motor nerve ending and the surface features of the subsynaptic sarcolemma. It may also allow quantitative study of the synaptic morphology of NMJs. PMID:

19 J Comp Neurol Apr 10;513(5): Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. Nagwaney S, Harlow ML, Jung JH, Szule JA, Ress D, Xu J, Marshall RM, McMahan UJ Source Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305, USA Abstract Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the presynaptic membrane, and to synaptic vesicles docked on the membrane prior to fusing with it during synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked vesicles may help account for a greater vesicle-presynaptic membrane contact area during docking and a greater probability of fusion during synaptic transmission in mouse. Certain AZM macromolecules in mouse were connected to synaptic vesicles contacting the presynaptic membrane at sites where fusion does not occur. These secondary docked vesicles had a different relationship to the membrane and AZM macromolecules than primary docked vesicles, consistent with their having a different AZM-regulated behavior. (c) 2009 Wiley-Liss, Inc. PMID:

20 J Neurosci Dec 2;29(48): Preferred sites of exocytosis and endocytosis colocalize during high- but not lower-frequency stimulation in mouse motor nerve terminals. Gaffield MA, Tabares L, Betz WJ. Department of Physiology and Biophysics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA Abstract The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptophluorin (sph) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of sph fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the sph hot spots, but their rate of disappearance after stimulation was much slower (t(1/2) approximately 9 min vs approximately 10 s for sph hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of sph and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation. PMID: