THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE. Biomedical Seminar Room 3, Thursdays 9:30-11:30

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1 THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE Biomedical Seminar Room 3, Thursdays 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-tointense voluntary movements, thus 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 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, coexist 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.

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 minisymposium 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 minisymposium by the course organiser (RRR: the Soma ), followed by a discussion in the mini-groups of the abstracts of the papers to be presented by one of the groups the following week. As a result of these discussion, each mini-group will be asked to come up with a single burning question, which the presenting group should incorporate into their mini-symposium. General Reading 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, plasticity and repair of the NMJ III. Cellular and molecular biology of normal and abnormal NMJ IV. Homeostatic regulation of synaptic transmission at NMJ in health and disease V. The involvement of 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. Synapse formation, elimination, degeneration and regeneration 5. Cell biology and molecular genetics of normal and mutant NMJ (GP) 7. Quantal analysis and the safety-factor for neuromuscular transmission. 9. Animal models of Motor Neurone Disease

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: Massoulié J, Millard CB. Cholinesterases and the basal lamina at vertebrate neuromuscular junctions. Curr Opin Pharmacol Jun;9(2): PubMed PMID: (PDF here). For Presentation: 1. Desaki J, Uehara Y. The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J Neurocytol Feb;10(1): PMID: Lu J, Tapia JC, White OL, Lichtman JW. The interscutularis muscle connectome. PLoS Biol Feb 10;7(2):e32. 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: Gaffield MA, Tabares L, Betz WJ. The spatial pattern of exocytosis and postexocytic mobility of synaptophluorin in mouse motor nerve terminals. J Physiol Mar 15;587(Pt 6): PMID:

4 Week 4 MINI-SYMPOSIUM II Development, plasticity 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: Papers for presentation: 1. 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: 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: Costanzo EM, Barry JA, Ribchester RR. Competition at silent synapses in reinnervated skeletal muscle. Nat Neurosci Jul;3(7): PMID:

5 Week 6 MINI-SYMPOSIUM III Cellular and molecular biology of normal and abnormal NMJ Background review articles: Schwarz,T. (2005) Transmitter Release at the Neuromuscular Junction. Int Rev Neurobiol 75, (see course website for pdf) Dolly JO, Lawrence GW, Meng J, Wang J, Ovsepian SV. Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics. Curr Opin Pharmacol Jun;9(3): PMID: Papers for presentation: 1. Eaton BA, Fetter RD, Davis GW. Dynactin is necessary for synapse stabilization. Neuron May 30;34(5): 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: Miech C, Pauer HU, He X, Schwarz TL. Presynaptic local signaling by a canonical wingless pathway regulates development of the Drosophila neuromuscular junction. J Neurosci Oct 22;28(43): PMID: ; 4. Chai A, Withers J, Koh YH, Parry K, Bao H, Zhang B, Budnik V, Pennetta G. hvapb, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet Jan 15;17(2): PMID:

6 Week 8 MINI-SYMPOSIUM IV Homeostatic regulation of normal and diseased NMJ Background review articles: Urbano FJ, Pagani MR, Uchitel OD. Calcium channels, neuromuscular synaptic transmission and neurological diseases. J Neuroimmunol Sep 15; C: PMID: Dolly JO, Lawrence GW, Meng J, Wang J, Ovsepian SV. Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics. Curr Opin Pharmacol Jun;9(3): 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, (See course website for PDF). Papers 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: Frank CA, Kennedy MJ, Goold CP, Marek KW, Davis GW. Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis. Neuron Nov 22;52(4): PMID: 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: 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:

7 Week 10 MINI-SYMPOSIUM V Involvement of NMJ in the SOD1 mouse model of 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: Papers for presentation: 1. 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: Fischer LR, Culver DG, Davis AA, Tennant P, Wang M, Coleman M, Asress S, Adalbert R, Alexander GM, Glass JD. The WldS gene modestly prolongs survival in the SOD1G93A fals mouse. Neurobiol Dis Jun-Jul;19(1-2): PMID: Scott S, Kranz JE, Cole J, Lincecum JM, Thompson K, Kelly N, Bostrom A, Theodoss J, Al-Nakhala BM, Vieira FG, Ramasubbu J, Heywood JA. Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler. 2008;9(1):4-15. PMID: (See course website for PDF)

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 -

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 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 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 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 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 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 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

11 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 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 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 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 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 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 endplatepotential 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 0.1 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 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 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

13 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 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 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 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

14 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 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 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

15 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.

16 Hons Neuroscience Professor R.R. Ribchester QUANTAL ANALYSIS AT THE NEUROMUSCULAR JUNCTION Our present understanding of the fundamental physiological mechanism of transmitter release at synapses is mainly due to the work of B.Katz and his colleagues, based on their studies of transmission at the frog neuromuscular junction made during the 1950 s-1970 s at University College London. (Katz was awarded a Nobel Prize for his work in 1970). They showed that neurotransmitter is released in multi-molecular packets ( quanta ). Electron microscopy established that these corresponded with synaptic vesicles in the motor nerve terminals. The evidence for quantized release of transmitter was based on Katz s statistical analysis of electrophysiological recordings made from neuromuscular junctions during stimulation and at rest. Refinements of this quantal analysis are still widely used in cellular electrophysiology to establish the amount of transmitter released at synapses during activity, in a variety of tissues: including the neuromuscular junction, autonomic ganglia, the hippocampus, and the cerebral cortex. At the resting neuromuscular junction, miniature end-plate potentials (MEPPs) are generated spontaneously at the endplate. An evoked end-plate potential (EPP) is produced in the muscle fibre following nerve stimulation. Normally this response triggers a muscle fibre action potential and contraction. When transmission is weakened either by blocking receptors with a nicotinic antagonist (e.g. tubocurarine) or by suppressing transmitter release with solutions containing reduced Ca 2+ ions, the EPP becomes too small to trigger an active response: the EPP is said to be subthreshold. The essence of Katz s quantal hypothesis of synaptic transmission was threefold: 1. The quantum of transmitter underlying the smallest nerve-evoked EPP and the spontaneous MEPP are one and the same; 2. The release of each quantum of neurotransmitter is independent of the release of other quanta and occurs with a very low statistical probability (i.e. random); 3. The evoked EPP is caused by the synchronous release of several quanta, due to a transient and large increase in the probability of release of individual quanta. Evidence supporting the hypothesis was obtained by recording intracellular EPPs and MEPPs and ascertaining the relationship between their amplitudes. In particular it was noted that EPPs are variable in amplitude from stimulus to stimulus, whereas the MEPPs are roughly constant in amplitude. The variability could be accounted for on the basis of point (2) above, by showing that the distribution of EPP amplitudes conformed to a binomial distribution, which simplified under conditions of low release probability to a poisson distribution. Binomial model of synaptic transmission Consider a nerve terminal containing a number (n) of quanta /synaptic vesicles. Suppose each has a small chance (p) of fusing with the plasma membrane and releasing transmitter across the synapse. If the synapse is stimulated repetitively, say 100 times, then the mean number (m) of quanta released will be : m = n.p (1) By analogy, imagine tossing a coin 100 times. The probability of each toss coming up heads is 0.5. The average number of times the coin will come up heads is therefore 100 times 0.5: i.e., 50. 1

17 In matters of transmitter release, however, the probability of a vesicle fusing with the presynaptic membrane is normally considerably less than 0.5, but for the sake of argument let s suppose that n=3 and p=0.1. On average, a stimulus will evoke 0.3 quanta. In other words, some of the time there will be no release (a failure ). On the other occasions, release will consist of 1, 2 or 3 quanta. Thus the quantal content of the EPPs will vary between zero and three. How are the quantal amplitudes distributed? How often would one or two quanta be released in response to a stimulus? And how often would no quantal release occur? We make the assumption that all the quanta released following a stimulus are recycled, so that the number available on each occasion remains constant. Under this condition, the overall probability that all 3 quanta will be released (P) is simply the product of their individual release probabilities: P(3) = p.p.p = p 3 (= 0.001) Similarly, the probability that no quanta are released can be stated formally. By the rules of probability, either something or nothing must happen and certainty has the value of 1.0. So the probability for each vesicle not being released (q) is 1-p. Therefore the overall probability of a stimulus failing to release any of the three quanta in our imaginary synaptic terminal is: P(0) = (1-p).(1-p).(1- p) = q.q.q = q 3 (= 0.729) How about the overall probability of release of one quantum? By similar reasoning, for each quantum in the store this is p.q.q. The rules of probability require us to apply this condition to each of the three quanta in the store, thus: P(1) = p.q.q + p.q.q + p.q.q = 3 p.q 2 (= 0.243) Likewise, P(2) = p.p.q + p.p.q + p.p.q = 3 p 2.q (=0.027) 2

18 The overall distribution of transmitter release is obtained by adding together all four probability terms, and these must all add up to one (i.e. there are no other possibilities): P(0) + P(1) + P(2) + P(3) = p 3 + 3p 2 q + 3 pq 2 + q 3 = 1 This simplifies to : (p+q) 3 = 1 The above is a binomial expression : it contains two terms, p and q. Mathematical theory shows that in general we can predict from such an expression that for any number of quanta n with release probability p, that a particular nerve stimulus will release x quanta (x n) from the formula: P(x) = n!. p x. q (n-x) The Binomial Distribution (2) (n-x)!x! Try this out on the example we have used above with n=3 and p=0.1 ( m=0.3): P(0) = P(1) = P(2) = P(3) = The Poisson model The problem with applying binomial analysis to real synapses is that there is rarely any independent way of estimating n or p. We can only estimate the mean quantal content, m, by dividing the mean EPP amplitude by the mean MEPP amplitude. It turns out that we can still nonetheless predict the distribution of amplitudes if we assume that n is very large (n>>p) and p is very small (p<<1). Under these conditions x<<n. Based on these assumptions we can make a number of simplifications to the binomial distribution (equation 2, above). For example, we may write: n! n x (e.g. try this with n=10,000 and x=3) (n-x)! and q (n-x) q n recalling that q = (1-p), we can now substitute these terms in the binomial distribution (equation 2): P(x) = n x. p x. (1-p) n x! 3

19 since m = n.p (equation 1) this immediately simplifies to : P(x) = m x. (1-p) n (3) x! A little mathematical tinkering further simplifies the expression (1-p) n. First we apply natural logarithms in the identity: Ln (1-p) n = n. Ln (1-p) A mathematical formulation called McLaurin s theorem can be used to express Ln (1-p): Ln (1-p) = -p - p 2 - p 3 - p p 2! 3! 4! But since p<<1 by our assumption in the present analysis, then all the terms after the first one in the McLaurin series must be very small and we can ignore them. Thus: Ln(1-p) -p and therefore n.ln (1-p) -n.p Taking the antilogarithm of both sides: (1-p) n = exp (-n.p) = exp (-m) [Note: Theory of logarithms - if y=ln(x), then x=exp(y)] Substituting back in equation (3) we obtain: P(x) = m x. exp(-m) The Poisson Distribution (4) x! Once again, calculate what the distribution of probability of occurrence of EPPs containing 0,1,2,3 quanta are when the mean quantal content is 0.3 P(0) = P(1) = P(2) = P(3) = 4

20 Application of the Poisson Distribution to estimating quantal contents Experimentally, what does P(x) mean? It is simply the fraction of occasions on which the evoked postsynaptic potential (EPP, or in the case of CNS synapses, the EPSP) has a quantal content of x. To evaluate the quantal hypothesis and to use the Poisson distribution, we must compare the predicted variability in the amplitudes of EPPs with the actual variability observed experimentally. One of the most elegant demonstrations of the coincidence of the model and the data was obtained in a study by Boyd & Martin in a study of synaptic transmission in cat muscle: The data in the figure below were obtained from intracellular microelectrode recordings at a single neuromuscular junction in an isolated preparation in which neuromuscular transmission was depressed using a low Ca ion-high Mg ion bathing medium. The upper right of the figure shows the histogram of MEPP amplitudes as a bar chart and the superimposed graph is a fit of a normal (gaussian) distribution to the amplitudes. The lower graph shows the distribution of EPP amplitudes as a bar chart (including failures ) and a fit of the Poisson distribution, taking account of the gaussian variation in MEPP amplitudes. Note that the number of failures is accurately predicted, as well as the distribution of the peaks. The mean and variance of each peak is a unit multiple of the first, which has the same mean and variance as the MEPP amplitude distribution. Data such as these provide confirmation of the quantal hypothesis. 5

21 ESTIMATING QUANTAL CONTENT There are three principal methods. Other methods are based on complex analysis of EPP amplitudes. 1. Direct method: Under favourable conditions, both MEPPs and EPPs can be recorded in sufficient numbers to allow cross checking of the quantal content of EPPs, comparing Poisson statistics with direct estimation of the mean quantal content.m = (mean EPP amplitude) / (mean MEPP amplitude) This methos is not always possible for various technical reasons, e.g the MEPPs might be very infrequent; or they may be too small, buried in the noise of the recording system; or the mean quantal content may be large, resulting in non-linear summation of EPPs (see below). Applying the Poisson equation alone is sometimes sufficient in such cases. There are two methods of estimating quantal content based on the Poisson distribution: the Method of Failures and the Variance Method. 2. Method of Failures If the mean quantal content is low enough (as in the examples above), a significant fraction of stimuli will fail to evoke a response. This represents the P(0) expression in the Poisson distribution: P(0) = exp (-m). m 0 /0! since, by mathematical definintion, both m 0 and 0! are equal to 1 : P(0) = exp (-m) Taking natural logartithms : Ln (P0) = -m Substituting for P(0)=(Number of Failures)/(Number of Stimuli) and rearranging: m = Ln (Stimuli/Failures) 3. Variance method Another property of the Poisson distribution is that its variance equals its mean. From this it can be derived that: m = (mean EPP amplitude) 2 (variance of EPP amplitudes) This is often expressed in terms of the coefficient of variation (standard deviation/mean = σ/µ) : m = C.V. -2 6

22 Non-linear summation of synaptic potentials As the amount of transmitter released onto the postsynaptic membrane increases - e.g. as the quantal content increases - the effectiveness of each quantal packet declines. This is because transmitter molecules at excitatory synapses like the neuromuscular junction act on receptors coupled to ion channels. When the channels open, positive ionic current flows into the postsynaptic cell. The electromotive driving force is determined by the ion gradient and the membrane potential. For example, at neuromuscular junctions the receptor/ion channel is the nicotinic ACh receptor which gates permeability to Na and K ions about equally. The reversal potential is about -5 mv. This means that as the membrane potential approaches -5 mv, the ionic current flowing through the open channels becomes vanishingly small; and if membrane potential becomes more negative than -5 mv, an outward ionic current is produced instead and the EPP reverses in sign. Each quantal component of the EPP depolarises the membrane potential towards the reversal potential by a small amount, but as the amount of overall depolarisation becomes greater each successive quantal component has a weaker and weaker effect on further depolarisation. For instance, if the mean MEPP amplitude is 1 mv, then an EPP comprising 5 quanta may well produce a depolarisation of 5 mv. But an EPP comprising 20 quanta may only produce about 15 mv of depolarisation. This is called non-linear summation of synaptic potentials. It means that under normal conditions of synaptic transmission when quantal contents can be quite high, the direct method of quantal analysis will underestimate mean quantal content and the variance method will overestimate mean quantal content. (Note: The failures method cannot usually be applied when mean quantal content is greater than about 5 because there are so few failures; P(0)=exp(-5) = 0.007; i.e. less than 1 in 100 stimuli would be expected to result in failure of transmission). The relationship between membrane potential, synaptic current and synaptic potential were investigated by McLachlan & Martin (1981), by alternately voltage- and current-clamping of the endplate. They showed that the relationship between the observed amplitude of the EPP, and the amplitude which would be obtained if transmitter quanta produced linear summation is: V = V / (1-f.V/E) where V is the predicted amplitude, V the observed amplitude; E is the difference between the resting membrane potential and the reversal potential and f - critically - is a factor which varies from muscle fibre to muscle fibre depending on its length, diameter and specific membrane and cytoplasmic electrical resistance. Normally it is not possible to measure this fudge factor directly for every muscle fibre (it requires alternate voltage and current clamping of the endplate to do this). But there are rules of thumb: long muscle fibres mostly have an f-factor of 0.8; short muscle fibres have factors of about 0.3. Applying the correction for non linear summation to each EPP before calculating mean quantal content results in more accurate estimates by either the direct or variance methods. References Katz,B.(1969) The release of neural transmitter substances. Liverpool University Press. McLachlan EM. Martin AR. (1081) Non-linear summation of end-plate potentials in the frog and mouse. Journal of Physiology. 311: Katz,B.(1996) Neural transmitter release: from quantal secretion to exocytosis and beyond. The Fenn Lecture. Journal of Neurocytology. 25, JH Byrne & JL Roberts (2009) From molecules to networks. 2 nd edn. Sinauer (Chapter 8) 7