Regenerating supernumerary axons are cholinergic and emerge from both autonomic and motor neurons in the rat spinal cord

doi:10.1016/j.neuroscience.2005.08.022

Neuroscience

Volume 136, Issue 2, 2005, Pages 417-423

https://doi.org/10.1016/j.neuroscience.2005.08.022 Get rights and content

Abstract

Multipolar neurons in the mammalian nervous system normally exhibit one axon and several dendrites. However, in response to an axonal injury, adult motoneurons may regenerate supernumerary axons. Supernumerary axons emerge from the cell body or dendritic trees in addition to the stem motor axon. It is not known whether these regenerating axons contain neurotransmitters for synaptic transmission at their terminals. Here, using immunohistochemistry for choline acetyltransferase, an enzyme that synthesizes acetylcholine, we demonstrate the emergence of cholinergic supernumerary axons at 6 weeks after a unilateral L5–S2 ventral root avulsion and acute implantation of the avulsed L6 ventral root into the adult rat spinal cord. Light microscopic serial reconstruction of choline acetyltransferase immunoreactive arbors shows that these supernumerary axons originate from both autonomic and motor neurons. The supernumerary axons emerge from the cell body or dendrites, exhibit an abnormal projection pattern within the intramedullary gray and white matters, make frequent abrupt turns in direction, and form bouton-like swellings as well as growth cone-like terminals. Double labeling immunohistochemistry studies show that the choline acetyltransferase immunoreactive supernumerary axons co-localized with two proteins associated with axonal growth and elongation, growth-associated protein 43 and p75, the low affinity neurotrophic factor receptor. Our findings suggest that regenerating supernumerary axons selectively transport and store choline acetyltransferase, supporting the notion that supernumerary axons may develop functional and active synaptic transmission. Therefore, regenerating supernumerary axons may contribute to the plasticity in neural circuits following injury in the adult nervous system.

Section snippets

Experimental procedures

All animal procedures followed the guidelines of the NIH guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996) and were approved by the Chancellor’s Animal Research Committee at UCLA. Adult male Sprague–Dawley rats (n=14, 175–225g, Charles River Laboratories, Raleigh, NC, USA) were included in the studies. The animals were housed in a room with a 12-h light/dark cycle with food and water access ad libitum. Efforts were made to minimize the number of animals

Autonomic and motor neurons develop cholinergic supernumerary axons

Using IHC for ChAT, we studied the morphological features of autonomic and motor neurons in the L6 and S1 segments at 6 weeks after an L5–S2 ventral root avulsion and implantation of the L6 avulsed root into the spinal cord. ChAT IR neurons were detected in the ventral horn, intermediolateral nucleus, adjacent to the central canal, and occasionally in the dorsal horn, corresponding to previously described populations of motoneurons, PPNs, central canal cluster cells, and interneurons,

Discussion

We demonstrated that axotomized motoneurons and PPNs in the adult rat spinal cord developed ChAT IR supernumerary axons following a lumbosacral ventral root avulsion injury and acute implantation of an avulsed ventral root into the spinal cord. The supernumerary axons originated from either the soma or dendrites, exhibited an aberrant projection pattern, commonly extending into the lateral funiculus toward the site of the implanted root, and formed both bouton-like swellings and growth

Conclusion

We show that supernumerary axons originating from spinal cord neurons may develop in adult rats following a lumbosacral ventral root avulsion and subsequent implantation of an avulsed ventral root into the spinal cord. In addition, supernumerary axons can originate not only from motoneurons, but also autonomic neurons in the spinal cord. Furthermore, the presence of ChAT IR in these supernumerary axons and their terminal arbors with bouton-like swellings raises the possibility that

Acknowledgments

Supported by: NIH/NINDS NS042719, the Roman Reed Funds for Spinal Cord Injury Research of California, the Paralysis Project of America, and the ARCS Foundation. We would like to thank Dr. Matthew Schibler for excellent assistance with confocal microscopy at the Brain Research Institute/Carol Moss Spivak Cell Imaging Facility, UCLA.

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Citation Excerpt :
For instance, the axotomized neurons show early and sustained cell body atrophy in combination with altered metabolism, which includes a markedly reduced synthesis of choline acetyl transferase during the first month after injury (Hoang et al., 2003). When preganglionic parasympathetic neurons survive following a root avulsion injury, i.e. supported by the reimplantation of lesioned roots into the conus medullaris, they may develop supernumerary axons, which emerge from the cell body or proximal dendrites, show a cholinergic phenotype, and express both GAP-43 and the low-affinity neurotrophic factor receptor p75 (Hoang et al., 2005). Such regenerative axons share many features of supernumerary axonal growth and altered neuronal polarity, encountered also in axotomized spinal motor neurons (Havtorn and Kellerth, 1987; Rose et al., 2001; MacDermid et al., 2002), and represent an interesting target for neurotrophic treatment strategies.
Spinal nerve root avulsion injury interrupts the transverse segmental spinal cord nerve fibers. There is degeneration of sensory, motor, and autonomic axons, loss of synapses, deterioration of local segmental connections, nerve cell death, and reactions among non neuronal cells with central nerve system (CNS) scar formation, i.e., a cascade of events similar to those known to occur in any injury to the spinal cord. This is the longitudinal spinal cord injury (SCI). For function to be restored, nerve cells must survive and there must be regrowth of new nerve fibers along a trajectory consisting of CNS growth-inhibitory tissue in the spinal cord as well as peripheral nervous system (PNS) growth-promoting tissue in nerves. Basic science results have been translated into a successful surgical strategy to treat root avulsion injuries in man. In humans, this technique is currently the most promising treatment of any spinal cord injury, with return of useful muscle function together with pain alleviation. Experimental studies have also identified potential candidates for adjunctive therapies that, together with surgical replantation of avulsed roots after brachial plexus and cauda equina injuries, can restore not only motor but also autonomic and sensory trajectories to augment the recovery of neurological function. This is the first example of a spinal cord lesion that can be treated surgically, leading to restoration of somatic and autonomic activity and alleviation of pain.
A spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression after lumbar ventral root avulsion and implantation
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Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves.
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Cellular neuroscienceRegenerating supernumerary axons are cholinergic and emerge from both autonomic and motor neurons in the rat spinal cord

Abstract

Section snippets

Experimental procedures

Autonomic and motor neurons develop cholinergic supernumerary axons

Discussion

Conclusion

Acknowledgments

Trends Neurosci

Neuroscience

Curr Opin Neurobiol

Prog Brain Res

Neuroscience

Brain Res

Prog Neurobiol

Neuroscience

Neurobiol Dis

Neuron

The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cordan immunocytochemical study

J Comp Neurol

Neuronal polarity

Annu Rev Neurosci

Development of neuronal polarityGAP-43 distinguishes axonal from dendritic growth cones

Nature

Regeneration by supernumerary axons with synaptic terminals in spinal motoneurons of cats

Nature

Transformation of synaptic vesicle phenotype in the intramedullary axonal arbors of cat spinal motoneurons following peripheral nerve injury

Exp Brain Res

Autonomic and motor neuron death is progressive and parallel in a lumbosacral ventral root avulsion model of cauda equina injury

J Comp Neurol

Cellular neuroscience
Regenerating supernumerary axons are cholinergic and emerge from both autonomic and motor neurons in the rat spinal cord