Elsevier

Neuroscience

Volume 81, Issue 2, 8 September 1997, Pages 535-551
Neuroscience

Changes in immunoreactivity for growth associated protein-43 suggest reorganization of synapses on spinal sympathetic neurons after cord transection

https://doi.org/10.1016/S0306-4522(97)00151-6Get rights and content

Abstract

Cervical or high thoracic spinal cord injury often results in autonomic dysreflexia, a condition characterized by exaggerated spinal reflexes and episodic hypertension, that may be caused by reorganization of synapses on sympathetic preganglionic neurons after loss of supraspinal input. To assess remodelling of synaptic input to identified preganglionic neurons, immunoreactivity for growth associated protein-43 was examined by fluorescent and electron microscopy in control rats with intact spinal cords and in rats seven to 30 days after midthoracic cord transection. This protein is found in mature bulbospinal axons that supply spinal sympathetic nuclei and it is also known to be up-regulated in growing or sprouting axons.

In the thoracic cord of control rats, fibres containing growth associated protein-43 surrounded histochemically- or retrogradely-labelled preganglionic neurons and formed a ladder-like pattern in the gray matter. Fibres travelled rostrocaudally along the lateral horn and, at approximately regular intervals, they coursed mediolaterally to form the “rungs” of a ladder. Electron microscopy revealed concentrated growth associated protein-43 in many intervaricose axon segments in the intermediolateral cell column. Less frequently, faint immunoreactivity for this protein was found in varicosities, some of which synapsed on retrogradely-labelled sympathoadrenal preganglionic neurons. Electron microscopy of conventionally processed tissue was used to determine the time-course of degeneration of severed axon terminals in the intermediolateral cell column. In spinal rats, terminals with ultrastructural signs of degeneration were numerous in the intermediolateral cell column three days after transection, but were rare at seven days and absent at 14 days. Degenerating terminals were never found in this region in control rats. Thus virtually all supraspinal inputs to preganglionic neurons had been eliminated by seven days after transection. At longer times after injury, terminals containing immunoreactivity for growth associated protein-43 must therefore arise from intraspinal neurons. The distribution of fibres immunoreactive for growth associated protein-43 changed markedly in the first 30 days after cord transection. By 14 days, the ladder-like pattern was distorted rostral to the transection by enlarged masses of immunoreactive fibres surrounding preganglionic neurons, suggesting sprouting of bulbospinal or intraspinal axons or accumulation of this protein in their terminals after the parent axon had been severed. Caudal to the transection, the ladder-like arrangement of fibres was completely replaced by a reticular network of immunoreactive fibres that extended throughout the intermediate gray matter and increased in density between 14 and 30 days. In the intermediolateral cell column, at fourteen days after transection, axons with the ultrastructural features of growth cones contained intense growth associated protein-43 immunoreactivity. Although varicosities of bulbospinal axons containing this protein had degenerated by 14 days, weak immunoreactivity was still found in varicosities that synapsed on labelled sympathoadrenal neurons. Furthermore, immunoreactivity appeared in numerous somata of presumed interneurons throughout the intermediate gray matter by 14 days and the number of somata increased by 30 days. These interneurons may be the source of this protein in the reticular network, and in growth cones and synapses.

The loss of supraspinal inputs by seven days after cord transection, and the new intraspinal network of immunoreactive fibres, synapses and cells are consistent with new synapse formation on preganglionic neurons. New synapses on preganglionic neurons may be crucial for the development of autonomic dysreflexia.

Section snippets

Experimental procedures

All protocols for these experiments were approved by the University of Western Ontario Animal Care Committee in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care. Fifty-four adult male Wistar rats (Charles River, St Constance, Quebec, Canada), weighing approximately 250–350 g, were prepared for surgical interventions by sedation with 2.5 mg/kg Diazepam (i.p.) 10 min prior to anaesthesia with sodium

Light microscopy

GAP-43 immunoreactivity was found in fibres in all thoracic spinal segments in rats with intact spinal cords. In horizontal sections of spinal cord, clearly defined immunoreactive fibres extended rostrocaudally along the intermediolateral gray matter, forming several dense clusters in each cord segment (Fig. 1). These clusters usually surrounded FluoroGold- or NADPH-diaphorase-labelled SPN in the IML (Fig. 1B). The fibres immunoreactive for GAP-43 formed a ladder-like pattern in the gray

Discussion

We have demonstrated a major change in the distribution of GAP-43 immunoreactivity in the IML and in the intermediate gray matter as a result of complete transection of the upper thoracic spinal cord. In the intact cord, GAP-43-immunoreactive fibres were organized in a regular, ladder-like pattern in autonomic areas of the spinal gray matter and these GAP-43-containing fibres synapsed on retrogradely labelled SPN. Within 30 days of transection, this orderly arrangement of GAP-43-immunoreactive

Conclusions

We have demonstrated a striking change in the pattern of GAP-43 immunoreactivity in the intermediolateral gray matter caudal to a cord transection that is consistent with remodelling of synaptic inputs to SPN. The ladder-like arrangement of GAP-43 immunoreactivity in the intact cord was replaced by a reticular network of GAP-43-immunoreactive fibres coinciding with the appearance of GAP-43 immunoreactivity in the somata of spinal neurons. These findings suggest that intraspinal neurons with

Acknowledgements

This research was supported by grants from the Heart and Stroke Foundation of Ontario (T2679), the Medical Research Council of Canada and the National Health and Medical Research Council of Australia. L. C. Weaver is the recipient of a Career Investigator award from the Heart and Stroke Foundation of Ontario; I. J. Llewellyn-Smith is a Senior Research Fellow of the National Health and Medical Research Council of Australia; A. V. Krassioukov held a Fellowship from the Medical Research Council of

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