Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

doi:10.1016/S0306-4522(99)00512-6

Neuroscience

Volume 95, Issue 4, December 1999, Pages 1101-1109

https://doi.org/10.1016/S0306-4522(99)00512-6 Get rights and content

Abstract

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48±3% (n=5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94±4% (n=5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48±3% (n=5) to 35±4% (n=5). Moreover, only 67±5% (n=5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6±0.7 (n=5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3±0.9 (n=3) motor units were present.

Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.

Section snippets

Surgical procedures

The experiments were carried out in adult female Sprague–Dawley rats, aged five to seven weeks at the time of the initial operation. All experiments conformed to our institutional and British Government guidelines on the ethical use of animals, and all efforts were made to minimize both the number and suffering of the animals used in these experiments. Their body weights ranged between 170 and 190 g. All the operations were carried out using halothane anaesthesia and sterile precautions. In one

Effect of sprouting on the survival of motoneurons to tibialis anterior and extensor digitorum longus muscles

The effect of inducing axonal sprouting by either (i) treatment with BoTx, (ii) PD or (iii) BoTx followed by PD (BoTx/PD) on motoneuron survival in adult rats was tested. The extent of sprouting and paralysis was examined one, two and three weeks after each experimental procedure. The long-term effects on both motoneuron survival and, in some cases, force output and numbers of motor units were examined 10 weeks after the last procedure.

Axonal sprouting

The amount of axonal sprouting was established by

Discussion

The present study examined the effect of inducing extensive growth of motor axons on the survival of motoneurons in adult rats. When axons of motoneurons were induced to grow by either (i) intramuscular injections of BoTx into the TA and EDL muscles or (ii) by PD of TA and EDL muscles, there was no loss of motoneurons. However, when the two procedures were combined so that TA and EDL muscles were initially treated with BoTx and subsequently partially denervated three weeks later (BoTx/PD), many

Conclusion

The results of the present study clearly show that, in adult animals, motoneurons that are sprouting as a result of BoTx die after PD of the affected muscles. Although it is possible that the mechanism for cell death in this case is unrelated to the growth activity of the motoneurons, we favour the explanation that motoneurons that are sprouting are vulnerable when challenged to grow further by a second stimulus, and die as a result.

These results may be relevant to our understanding of the

Acknowledgements

We are grateful to the Motor Neurone Disease Association, the Wellcome Trust and the Leverhulme Trust for their support. C. M. W. would like to thank Professor Di Newham of the Physiotherapy Group, Kings College London for her continued encouragement. L. G. was in receipt of a Wellcome Trust Fellowship.

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Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Abstract

Section snippets

Surgical procedures

Effect of sprouting on the survival of motoneurons to tibialis anterior and extensor digitorum longus muscles

Axonal sprouting

Discussion

Conclusion

Acknowledgements

Expl Neurol.

Trends Neurosci.

Neuroscience

Devl Brain Res.

Brain Res.

Brain Res.

Fedn Eur. biochem. Socs Lett.

Overexpression of the neural growth associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice

Cell

The peripheral action of Clostridium botulinum toxin

J. Physiol.

GAP-43 mRNA in mouse motoneurons undergoing axonal sprouting in response to muscle paralysis or partial denervation

Eur. J. Neurosci.

Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25

Nature

Motor nerve sprouting

A. Rev. Neurosci.

Properties of motor units in fast and slow skeletal muscles of the rat

J. Physiol.