1

Nerve Degeneration and Regeneration Rowena’s & Nia’s Notes Rowena’s Notes 1) Is activity necessary for synaptic elimination?

Experimental data supports two opposing views of the role of activity in SE. A large proportion of literature suggests that active axons have a competitive advantage over the less active. Preventing activity with TTX in all motor neurons delays SE, whilst increasing activity accelerates it. As SE is only delayed by the absence of activity & not abolished, this suggests that activity is not the only factor for SE & has other requirements. This view is contradicted by Callaway et.al., (1987, 1989) who concluded that complete presynaptic inactivity improves the chances of survival of the axon. Barber & Lichtman (1999) formulated a mathematical model of activity-mediated SE to determine activities influence on SE. The model shows that axons with a high frequency of activity have a competitive advantage at early stages in development. The advantage is shifted in the later stages of development to axons with greatest synaptic efficacy and a lower firing frequency. The reasoning why it is thought that active axons have a competitive advantage is controversial, but there are hypothesises:

1) Axon terminals may compete for a finite amount of tropic factor produced by muscle fiber. Inactive m. fibers produce a large quantity of this tropic factor; activity in the fiber suppresses tropic production so that only a single synaptic terminal can be maintained.

~ supporting this several tropics factor have been shown to delay elimination, support axon branching & regulate motor unit size. 2) Another hypothesis is that in proportion to activity the axon generates a

punishing signal (possibly protease or locally generated thrombin) which could damage or repulse its neighbours.

Asynchronous or uncorrelated activity of the pre & postsynapse within a narrow time window is particularly successful in inducing SE. This can be shown using ∝-Bungarotoxin (∝BTx) to cause a partial or entire block of AChR in the adult NMJ. Partial blockade initiated SE in the blocked region & an entire block showed no change in SE compared to controls. (Balice-Gordon & Lichtman, 2002) The protective effect of synchronous activity is now widely accepted. It is possible to measure motorneuron activity using single-unit electromyography to study the synchrony of firing during postnatal development. It has been shown in both rats & mice that synchronous activity predominates in early stages of development & that this changes to asynchronous activity in parallel with SE. (Buffelli et.al., 2002) Costanzo et.al., (2000) showed that SE could occur in reinnervated adult muscle in total absence of activity. This suggests that activity acts as a modulator or elimination rather than a direct requirement.

2

References on activities involvement in SE: Balice-Gordon R.J., Lichtman J.W. Long-term synapse loss induced by focal blockade of postsynaptlc receptors. (2002). Nature, 372; 519 – 524. Barber M.J., Lichtman J.W.. Activity-Driven Synapse Elimination Leads Paradoxically to Domination by Inactive Neurons. (1999), The Journal of Neuroscience, 19(22): 9975-9985 Buffelli M., Busetto G., Cangiano L., Cangiano A.. Perinatal switch from synchronous to asynchronous activity of motoneurons: Link with synapse elimination. (2002) Proceedings of the National Academy of Sciences, 99(20); 13200-13205. Callaway E.M., Soha J.M., Van Essen D.C.. Differential loss of neuromuscular connections according to activity level and spinal position of neonatal rabbit soleus motor neurons. (1989). Journal of Neuroscience, 9; 1806-1824. Costanzo E.M., Barry J.A., Ribchester R.R., Competition at silent synapse in reinnervated skeletal muscle. (2000). Nature Neuroscience. 3(7); 694-700. Herrera A.A., Zeng Y., Activity-dependent switch from synapse formation to synapse elimination during development of neuromuscular junctions. (2003), J. Neurocytology, 32; 817-833.

3

2) What is the role of the immune system in degeneration?

See Rosie’s fantastic presentation & handout on the complement system. T-cells & macrophages invade causing axonal injury – macrophages lead to clearance of axonal debris. Macrophages ∝ TNF IFN Induce MHC II activation ⊕ IL-8 CD4 T-cell activation Demyelination & nerve injury Tumour necrosis factor-alpha (∝TNF ); Interlekin –8 (IL-8); Interferon (IFN); Major Histocompatibility Complex (MHC) ∝ TNF is produced by macrophages & appears to be involved in both immune-mediated demyelination & non-immune myelin degradation after axotomy.

Disintergration Axonal Schwann cells Genes differentially Proteins expressed breakdown differentiate regulated (cytokines) Degenerating axons themselves trigger cytokine response normally. References on the role of immune system in degeneration: I didn’t really use references for this bit as I couldn’t find any suitable. So the information is what was compiled in the lecture. Renno T., Krakowski M., Piccirillo C., Lin J.Y., Owens T.. TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines. (1995) The Journal of Immunology, 154(2); 944-953,

4

5) How are nerve injuries assessed clinically?

To understand hoe nerve injury is assessed I thought it would be best to briefly go over what happens to nerves when they are injured. The following paragraph from Evans (2001) provides a nice summary. (& a good picture!): ‘But what happens when a nerve is cut or injured? Waller (1850) examined the hypoglossal nerve in the frog and demonstrated discrete, reproducible pathologic changes following injury. Following injury, chromatolysis of the cell bodies occurs, as demonstrated by an increase in the size, and a migration of the nucleus to the periphery with an increase in protein and RNA metabolism. In the distal segment, one sees Schwann cell proliferation, myelin phagocytosis by both neuronal and non-neuronal cells, formation of bands of Bünger and axonal sprouting. Axon sprouting occurs at a rate of 1–4 mm per day and initially unmyelinated fibers soon are incorporated with Schwann cells and become myelinated.’

Fig. In the distal segment, one sees Schwann cell proliferation (tan cells), myelin (blue material wrapping the axons) phagocytosis by both neuronal and non-neuronal cells, formation of bands of Bünger (tubes formed by the bundling of Schwann cells), and axonal sprouting. Axon sprouting occurs at a rate of 1–4 mm per day and initial unmyelinated fibers soon are incorporated with Schwann cells and become myelinated. Although we are aware of the pathologic changes seen in nerve injury, the exact underlying molecular mechanisms of Schwann cell/axon interactions are unknown. Shen & Zhu (1996) suggest that assessment of nerve injury should consist of motor, sensory, and functional assessment autonomic nerves. The method for evaluating motor function should be selected according to location of nerve injury. Two methods of evaluation are widely used; Lovett's method & British Medical Research Council (BMRC) method although I haven’t been able to access any of the articles which descried what each method entails.

5

Electrophysiological & clinical examination can localise damage and assess its magnitude. Injured nerves show a decreased size in compound muscle action potential in response to distil stimulation. BUT… a complete absence of c. muscle AP does not mean complete transaction. (Bari et.al., 1999) Examine electromyoneurographically (EMNG) Motor & sensory fibres studies by surface electrode stimulation & results recorded.

EMNG records: - Spontaneous denervation activity - Decrease in no of motor unit potentials (MUPs) - Changes in c. muscle AP. (suggestive of denervation/reinnervation) - Sensory nerve action potential aplitude

References for assessment of nerve injury:

Bari N., Perovi D., Mitrovi Z., Jureni D., Zagar M.. Assessment of war and accidental nerve injuries in children. (1999), Pediatric Neurology; 21(1); 450-455.

Evans G.R.D. Peripheral Nerve Injury: A Review and Approach to Tissue Engineered Constructs. (2001), The Anatomical Record, 263: 396–404 . Shen N., Zhu J.. Functional assessment of peripheral nerve injury and repair. (1996), Journal of reconstructive microsurgery. 12(3); 153-157. Nia’s Notes 3. Define the following terms: Gangliosides

Found in the brain, liver, spleen and red blood cells (particularly abundant in nerve cell membranes)

Compounds composed of glycosphingolipid, with one or more sialic acids linked on the sugar chain

Component of cell membranes Functional ligands for myelin stability and control of nerve regeneration by

binding to a specific myelin associated glycoprotein Intact – inhibits growth of nerve

Removing a side group – stimulates growth Involved in cell-to-cell and cell-to-ECM interactions Found to be highly important in immunology - Have receptors for interferon

(IFN) α-latrotoxin

A neurotoxin Enhances both “spontaneous” and “depolarization-evoked” quantal sectretion

from neurons Causes formation of Ca2+ pores, which increases the secretion of

neurotransmitter

6

If enough pores are produced, the cell dies Inefficient endocytosis Presynaptic nerve terminal swells

Ethidium

Used as an intercalating agent (a molecule that inserts in the space between two adjacent DNA or RNA base pairs)

A dye that stains red/orange when intercalated into DNA or RNA Normally impermeable, therefore fluorescence indicates that the cell

membrane is damaged Very strong mutagen, and may possibly be a carcinogen or a teratogen

Disialoside

Targets sialic acid in gangliosides Forms part of the epitope that antibodies bind to Known to play an important role in various events of cellular communication

4. What is the role of the perisynaptic Schwann cells at the neuromuscular junction? What happens when they are damaged and what happens to them when axons are damaged? Useful reference: “Perisynaptic Schwann cells at the NMJ: Nerve- and activity-dependent contributions to synaptic efficacy, plasticity and reinnervation.” By Daniel Auld. The Neuroscientist 2003 Vol 9(2) 144-157

In amphibians: PSC processes

envelop the nerve terminal and occasionally intrude into the synaptic cleft

In mammals: PSC processes do not traverse the synaptic cleft

PSCs distinguished from myelinating Schwann cells by ACh receptors (muscarinic) and ion channels

PSCs are active partners in synaptic

transmission because of the following: 1. PSCs detect synaptic activity

Neurotransmitter release from the nerve terminal causes an increase in PSC [Ca2+]i due to mobilization of Ca2+ from intracellular stores

2. PSC acitivity is changed by synaptic activity 3. PSC response to synaptic activity subsequently changes synaptic output

7

Receptor coupled G-proteins are likely to activate pathways in PSCs that enhance (effected by Ca2+) and reduce (unidentified effector) neurotransmission

Any changes in PSC modulation of transmitter release make contributions to the observed long-term changes in synaptic efficacy

Following denervation,

PSCs invade the synaptic cleft PSCs can release ACh PSCs extend elaborate process networks PSCs express new neurotransmitter receptors

Following partial denervation,

PSC processes from denervated endplates find innervated endplates, where they then induce a terminal sprout and guide it to the denervated endplate

In studies where PSC s are specifically ablated:

- Very few NMJs showed terminal growth - Nearly half of the NMJs underwent retraction

In demyelinating diseases (e.g. Miller Fisher syndrome and Guillain Barre), retraction of terminal Schwann cells is seen, which subsequently causes degeneration of the nerve terminal. NB Nicotinic AChRs - found on postsynaptic membrane of NMJ - depolarizing - opens pores which allow Na+ and K+ ions to pass through Muscarinic AChRs - found on perisynaptic Schwann cells and possibly on

presynaptic membranes - G-protein coupled activation