Mini symposium week 1&2 Structure and Function of the NMJ Paper 1 – The interscutularis muscle connectome (Lu J, Tapia JC, White OL, Lichtman JW. The interscutularis muscle connectome. PLoS Biol. 2009 Feb 10;7(2):e32) Overview of Paper

• Paper produced a full connectome (wiring diagram) for the interscutularis (small muscle between ear and skull with 10 motor end plates)

• Used YFP modified mice so that motor neurons were visible • 4 main findings

o Anatomical evidence for size principle Henneman Size Principle - A smaller alpha motor neuron has a lower threshold of excitation, causing it to be recruited first during voluntary contraction. A larger alpha motor neuron has a higher threshold of excitation and is recruited when more motor units are needed to generate greater contractile force. V = IR therefore if resistance (R) is increased due to decreased diameter a smaller current (I) is needed to produce a sufficient voltage change across the membrane for an AP This allows for fine motor control and power based on how many motor units are recruited But – in fact the paper has not proved the size principle as it has shown no physiology along side the anatomy

o Quantitive relationships between – axon cross sectional area, axon arbour and motor unit size; intramuscular

axon length and motor unit size by an inverse square root; cross sectional area of axon just before it splits into muscle is proportional to intramuscular length.

o Axonal branching structure of same neurons different between same species and left and right on same species. Suggests lack of genetic pre-determination of path taken.

This is the key discovery; it was believed that L&R sides would be the same due to c.elegans models. This suggests a different developmental strategy possibly due to the sheer complexity and size of mammalian wiring requiring a huge genome to programme each nerve.

o Axon length suboptimal

• Having discussed the 4 conclusions it was felt that only conclusion 3 required this level of detail and method of viewing the connectome to be reached.

• Even at this level of detail the size of neurons are so small that the authors struggled to differentiate one motor neurone from another and it required manual decisions, to decide if a branch was one new neurone branching or two neurons that had been travelling near each other separating in different directions, as well as the automated computer model therefore it is a slow process.

• A project is planned to produce an entire human connectome map and it is hoped that this will bring the same advances in medicine as the human genome project.

• The Big Questions – Their is limited evidence that an optimal wiring will benefit the functioning motor unit and the variability between individual wiring diagrams could provide an explanation for differences in characters e.g. why some of us are musical, others sporty etc.

Paper 2 - The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. (Desaki J, Uehara Y. The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J Neurocytol. 1981 Feb;10(1):101-10)

• This paper used an electron microscope to view the NMJ in various muscles of various species. • 8M HCL was used to remove the basal lamina (potentially removing some neuronal components as well such as

kranocytes.) • Produced very clear images of the NMJ in a frog, hamster and finch.

Frog • Nerve endings <2µm width • 30-200µm length • Junctional Groove

approximately double width of nerve ending

• Nerve ending runs parallel with the Muscle fibre.

• “Gutter-like” synaptic groove containing junctional folds, ridges 0.4µ diameter

• Obvious schwann cell

Finch • Nerve endings ramify into

branches <2µm diameter • Varicose swellings of 2-3µm • Perpendicular arrangement

with respect to the muscle fibre

• Synaptic pits in which the varicosities of the nerve ending sit , the junctional folds are poorly devleoped

• >20 pits per NMJ

Chinese Hamster • NMJ is oval area of approximately

15 by 30µm • Ramified nerve endings of 2-3µm

(same as with the finch) • Overlapping of nerve endings • Sarcolemma exhibits deep and

irregular synaptic depressions with ridges and folds, there are also elevations of muscle fibre that contain no cross-linked myofibrils.

eminences • Images confirm theories of how the NMJ was structured as provide much greater detail as can reach a much higher

level of resolution. • Huge differences between species suggests a level of non random control over the general NMJ appearance but within

the NMJ detail there is randomness e.g. in the junction folds of the hamster. • Paper: strengths – clarity of images; weaknesses: lack of clear aim and hypothesis, lack of explanation as to choice of

muscle types. • Big question –electron microscopy can be and is a very useful tool for examining the differences between NMJs in

different species Paper 3 – The architecture of active zone material at the frog’s NMJ (Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ. The architecture of active zone material at the frog's neuromuscular junction. Nature. 2001 Jan 25;409(6819):479-84)

• The study aimed to illustrate the arrangement and relationship of active zone material and associated proteins at a model synapse using electron microscope tomography.

o This involves taking lots of images at different angels and piecing them back together to form a 3D image

• The paper reveals an unexpected architecture which has raised many questions and many hypotheses will come from this about the physiology of the active zone and the NMJ. It is thought that this structure of these pegs, beams and ribs explain how vesicles are anchored in the active zone and how calcium channels etc. are positioned in close proximity to these vesicles. It is thought that the calcium channels may constitute part of or even all of a peg extending into the ribs.

• The beams act like a back bone for the structure; ribs run perpendicular and tether vesicles to the back bone and finally pegs tether these ribs to the membrane and are thought to possibly be calcium channels.

• Future work – o Identify the proteins in each of these structures o Confirm if the pegs are calcium channels o Look at other species to see if same structure ( mice have a different structure see - Sharuna Nagwaney, Mark

Lee Harlow, Jae Hoon Jung, Joseph A. Szule, David Ress, Jing Xu, Robert M. Marshall, Uel Jackson McMahan. Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. The Journal of Comparative Neurology. 2009 April 513; 5(457-68))

• Burning Question : Does the architecture of the active zone have specific implication for mechanisms of calcium signalling and exocytosis? - Synaptic vesicle exocytosis requires a large increase in calcium concentration to occur. Therefore by anchoring the calcium channels close to the vesicles it allows for a large increase in local calcium concentration reducing the need for diffusion of calcium through out the presynaptic bulb.

Paper 4 – Identity, developmental restriction and reactivity of extralaminar cells capping mammalian NMJs (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. 2008 Dec 1;121(Pt 23):3901-11)

o Kranocytes are a fourth cell type along with neurons, schwann cells and muscle fibres found at all NMJs o This was discovered incidentally when the antibody 2166 was found to stain these cells (controls were carried out to

check it was not just staining various types of connective tissue) and that when added to a preparation the antibody was found through out the NMJs.

o Function as yet is not confirmed though it is hypothesised due to their increased presence in atrophied muscle suggests that they play a role in NMJ repair and maintenance. As our understanding of their role increases we should gain a better understanding of many neuromuscular diseases.

o Future work will therefore include understanding the role of kranocytes and possibly by observing the effect of ablating them in animal models.

o So far research has been done into their role in the neuromuscular diseases of spinal muscular atrophy and amytrophic lateral sclerosis (Murray LM, Talbot K, Gillingwater TH (2010) Neuromuscular synaptic vulnerability in motor

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. 2008 Dec 1;121(Pt 23):3901-11

neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy. Neuropathol Appl Neurobiol 36:133–156)