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increases in axonal sodium currents in peripheral nerves are some of the earliest excitability changes observed in Amyotrophic Lateral Sclerosis (ALS) patients. Nothing is known, however, about axonal sodium channels more proximally, particularly at the action potential initiating region -the axon initial segment (AIS). Immunohistochemistry for Nav1.6 sodium channels was used to investigate parameters of AISs of spinal motoneurones in the G127X SOD1 mouse model of ALS in adult mice at presymptomatic time points (~190 days old). In vivo intracellular recordings from lumbar spinal motoneurones were used to determine the consequences of any AIS changes. AISs of both alpha and gamma motoneurones were found to be significantly shorter (by 6.6% and 11.8% respectively) in G127X mice as well as being wider by 9.8% (alpha motoneurones). Measurements from 20-23 day old mice confirmed that this represented a change during adulthood. Intracellular recordings from motoneurones in presymptomatic adult mice, however, revealed no differences in individual action potentials or the cells ability to initiate repetitive action potentials. To conclude, despite changes in AIS geometry, no evidence was found for reduced excitability within the functional working range of firing frequencies of motoneurones in this model of ALS.Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease which preferentially affects motoneurones in the brain and spinal cord. Although a number of different mutations have been found to account for a small proportion of cases, the underlying pathophysiology of the disease is not fully understood and there is currently no cure. Riluzole, the only established treatment, has only modest effects on survival time 1-5 and blocks glutamatergic neurotransmission and Na + and Ca 2+ channels, indicating that excitability plays a role in the degenerative process in ALS.Threshold tracking techniques have suggested a hyperexcitability of motoneurone axons in ALS patients consisting of an increase in sodium currents occurring with a decrease in potassium currents [6][7][8][9][10][11][12][13][14] . The magnitude of the ion current changes appears to correlate with disease progression 15 , with excitability changes occurring before detectable axon loss 7,15 . In humans, the cell body and proximal axons of motoneurones, however, are largely inaccessible, preventing direct investigations of sodium and potassium channel activity centrally in ALS patients. This has therefore been investigated in vitro in motoneurones cultured from induced pluripotent stem cells (iPSCs) from ALS patients with genetic forms of the disease including superoxide dismutase 1 (SOD1), C9orf72 repeat expansions, TAR DNA binding proteins (TARDBP) and fused-in-sarcoma (FUS) mutations [16][17][18][19] . Increased action potential firing is observed at early stages after plating 16,19 due to reductions in delayed rectifier potassium currents 19 and increases in peak sodium currents 16 . At later time points post-plating however, motoneurones ex...

intramuscular injections of botulinum toxin block pre-synaptic cholinergic release at neuromuscular junctions producing a temporary paralysis of affected motor units. There is increasing evidence, however, that the effects are not restricted to the periphery and can alter the central excitability of the motoneurones at the spinal level. This includes increases in input resistance, decreases in rheobase currents for action potentials and prolongations of the post-spike after-hyperpolarization. the aim of our experiments was to investigate possible anatomical explanations for these changes. Unilateral injections of Botulinum toxin A mixed with a tracer were made into the gastrocnemius muscle of adult rats and contralateral tracer only injections provided controls. immunohistochemistry for Ankyrin G and the vesicular acetylcholine transporter labelled axon initial segments and cholinergic c-boutons on traced motoneurones at 2 weeks post-injection. Soma size was not affected by the toxin; however, axon initial segments were 5.1% longer and 13.6% further from the soma which could explain reductions in rheobase. Finally, there was a reduction in surface area (18.6%) and volume (12.8%) but not frequency of c-boutons on treated motoneurones potentially explaining prolongations of the afterhyperpolarization. Botulinum Toxin A therefore affects central anatomical structures controlling or modulating motoneurone excitability explaining previously observed excitability changes. Botulinum neurotoxin type-A (BoNT/A) is one of the most dangerous toxins known to man, yet it is being increasingly used, both cosmetically and clinically, to produce long lasting paralysis of specific muscles. After intramuscular injection, the toxin enters the synaptic vesicles of motor axon terminals by endocytosis. From here it silences synaptic transmission via specific proteolytic cleavage of SNAP-25 which prevents the release of acetylcholine from the motoneurone terminal resulting in flaccid paralysis 1. There is increasing evidence, however, that the effects may not be restricted to the periphery. Studies show that BoNT/A, injected intramuscularly, is transported both anterogradely along sensory axons and retrogradely along motoneurone axons to the motoneurone soma in the spinal cord 2-5. Furthermore, there is now also evidence that BoNT/A can spread between networks of cells 6 , although this claim has been questioned by other studies and would appear to be dose dependent 7,8. This raises the question as to what central effects BoNT/A may have on the motoneurone itself or on central cholinergic synapses contacting the motoneurone. Electrophysiological studies suggest changes in spinal motoneurone excitability after intramuscular BoNT/A injections of the gastrocnemius muscle. Increased stretch reflexes are seen at two weeks post-injection in rats 9. This increased reflex was also seen when tested as simple monosynaptic responses to dorsal root stimulation suggesting the enhanced reflexes may be due to an increase in motoneurone excitability 1...

Amyotrophic lateral sclerosis is a neurodegenerative disease preferentially affecting motoneurones. Transgenic mouse models have been used to investigate the role of abnormal motoneurone excitability in this disease. Whilst an increased excitability has repeatedly been demonstrated in vitro in neonatal and embryonic preparations from SOD1 mouse models, the results from the only studies to record in vivo from spinal motoneurones in adult SOD1 models have produced conflicting findings. Deficits in repetitive firing have been reported in G93A SOD1 mice but not in presymptomatic G127X SOD1 mice despite shorter motoneurone axon initial segments (AISs) in these mice.These discrepancies may be due to the earlier disease onset and prolonged disease progression in G93A SOD1 mice with recordings potentially performed at a later sub-clinical stage of the disease in this mouse. To test this, and to explore how the evolution of excitability changes with symptom onset we performed in vivo intracellular recording and AIS labelling in G127X SOD1 mice immediately after symptom onset. No reductions in repetitive firing were observed showing that this is not a common feature across all ALS models. Immunohistochemistry for the Na+ channel Nav1.6 showed that motoneurone AISs increase in length in G127X SOD1 mice at symptom onset. Consistent with this, the rate of rise of AIS components of antidromic action potentials were significantly faster confirming that this increase in length represents an increase in AIS Na+ channels occurring at symptom onset in this model.HighightsIn vivo electrophysiological recordings were made in symptomatic G127X SOD1 mice.There were no deficits in repetitive firing in motoneurones in G127X mice.Increased persistent inward currents were still present in the symptomatic mice.Results suggest increases in Na+ currents at axon initial segments (AISs).Immunohistochemistry showed that motoneurone AISs were longer and thinner.

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