Mechanisms that underlie axon growth and directional guidance during neural development have been extensively studied, yielding evidence that chemotropic gradients of growth factors exert an important role in axonal target finding, dendritic growth, and terminal synapse formation 25-29
. Studies that examine mechanisms underlying axon growth and potential target-finding after injury in the adult spinal cord have by necessity been far more limited, because until recently there was only limited success in promoting axonal regeneration beyond lesion sites. Taking advantage of combinatorial strategies for promoting bridging regeneration beyond lesion sites 8, 9, 30
together with the capability to control local concentrations and topographies of administered growth factors in vivo in adult animals, we have identified distinct chemotropic mechanisms underlying axonal regeneration in the injured adult spinal cord. Using methods that achieve chemotropic gradients of growth factors in appropriate and inappropriate targets in vivo, we report for the first time the reinnervation of a normal target, the nucleus gracilis, by lesioned, regenerating spinal cord axons, and the chemotropic dependence of this regeneration.
During neural development, regional patterns of neurotrophin expression exert control over axonal guidance and termination in various regions of the nervous system 31-33
, including visual cortex 26, 27, 34
, the peripheral nervous system 35, 36
, central projections of sensory systems 29
, and the vestibular system 37
. In the present experiment, we expressed NT-3 in both an appropriate target for regenerating axons, and an inappropriate target, and found that axons extended in a topographical distribution that precisely matched regions of NT-3 expression. Indeed, axons could be drawn to inappropriate regions based upon ectopic NT-3 expression, even bypassing a correct target to grow into an NT-3 source. In the absence of NT-3 expression, axons did not reinnervate the target nucleus. In contrast, increasing NT-3 expression in the target (by increasing NT-3 vector dose) significantly increased regeneration into the appropriate region. Collectively, these findings indicate that NT-3 guided regenerating axons to specific topographies, and that an absence of NT-3 resulted in elimination of regenerating axons. Thus, chemotropic guidance mechanisms observed during neural development may be critical in supporting target reinnervation by regenerating axons in adulthood.
This study is the first to demonstrate reinnervation of a natural brainstem target by regenerating spinal cord axons. While previous studies in models of axonal injury in the brain demonstrated target reinnervation in the visual system 38
, hippocampus 39
and nigrostriatal projection 40
, mechanisms guiding target reinnervation, including chemotropic gradients established by growth factors, have not previously been addressed. In previous studies, lesions were placed either within
the appropriate target (e.g., striatum), or bridges for regenerating axons ended within an appropriate target (e.g., direct placement of a sciatic nerve bridge in the lateral geniculate 41
), thereby providing unitary targets rather than a choice between appropriate or inappropriate targets for regenerating axons. In some disease models, and spinal cord injury in particular, innumerable incorrect targets become available to regenerating axons, thus guidance
of regenerating axons is of paramount importance. We have demonstrated in the present study that axons can be guided to appropriate targets utilizing chemotropic gradients of growth factors expressed in correct regions.
Further, we have demonstrated that axons regenerating into the target form synapses. These synapses exhibited appropriate excitatory features of the pre-injured projection, including asymmetric synaptic specializations and clear rounded vesicles. Axo-dendritic rather than axo-somatic synapses and the presence of multiple synaptic targets arising from single axonal terminal shafts are features consistent with the pre-injured ultrastructural phenotype of the dorsal column nuclei 20
. Thus, using ultrastructural criteria, the regenerating axon terminals closely resembled the corresponding synaptic boutons in intact animals.
While axons formed synapses when regenerating into the nucleus gracilis, we did not determine whether axons regenerating into an ectopic target, the reticular formation, also formed synapses. Studies in other systems indicate that axons sprouting into ectopic locations modify functional outcomes, suggesting that they likely form synapses in these regions (e.g.42, 43, 44
). For example, chronic constrictive lesions of the sciatic nerve cause spontaneous and ectopic growth of sympathetic axons into dorsal root ganglia, associated with dysesthetic pain that can be prevented by blockade of ectopic axon growth 42
. Thus, axons growing into either appropriate (present study) or ectopic 42
targets are likely capable of forming synapses.
An important consideration in any study reporting axonal regeneration is the possibility that spared axons may be mistaken for regenerating axons 45
. Several observations suggest that regenerating axons observed in our lesioned animals were not spared. First, we employed conservative criteria to eliminate subjects wherein lesion extent was uncertain, based on observations of two experienced, blinded observers. Further, we observed axons emerging from all levels of the rostral host/graft interface, rather than simply at the most dorsal or ventral parts where spared axons might be expected. In addition, we observed axons in ectopic locations beyond the lesion site, where axons are not found in intact animals. In all cases, axons beyond the lesion grew only within regions of NT-3 expression, a finding that would not be expected had axons been spared. Finally, we did not observe axons within the target region in any animals that received control (GFP) lentiviral vector injections. Together, these observations support the presence of regenerated rather than spared axons in the nucleus gracilis.
While lesioned axons in combinatorially-treated animals could be directed into the target and formed synapses, we did not detect electrophysiological responses in the nucleus gracilis following sciatic nerve stimulation. Since we sampled a relatively small volume of the total structure, the absence of target responsiveness may have resulted from limited sampling. However, intact animals exhibited electrophysiological responses in 67% of sampled sites, and NT-3 high+CL treatment restored the number of axonal profiles in lesioned animals to 27% of those in intact subjects. Thus, if reinnervating axons formed fully functional synapses, one would predict detection of electrophysiological responses in approximately 18% of sampled sites (67% of 27%) in combinatorially-treated animals.
Alternatively, lack of detectable activity might occur if the majority of regenerating axons failed to form synapses; however, synapses associated with CTB-labeled boutons were readily identifiable at the ultrastructural level in both intact and lesioned/combinatorially-treated animals. Further, synapses in regenerating animals exhibited ultrastructural features of normal synapses, including abundant rounded synaptic vesicles and synaptic specializations, suggesting the synapses could be functional. Importantly, however, we found that CTB-labeled regenerating dorsal column sensory axons emerging from the lesion site were either unmyelinated or poorly myelinated, in contrast to the intact state of these projections 46
. Demyelination of dorsal column sensory projections leads to conduction failure 23
. While it is possible that remyelination might have occurred after the six week time point at which we assessed electrophysiological activity, a recent report indicated a persistent loss of dorsal column sensory axon myelination in the thoracic spinal cord even six months after regeneration induced by anti-NG2 and conditioning lesions 24
. Further, spontaneous remyelination of injured host axons after spinal cord injury is reportedly unstable, falling over extended time periods post-lesion 47
. Persistent demyelination is therefore a candidate mechanism for the lack of synaptic activity observed in the present study. Thus, despite
the demonstration that conditioning of the injured neuron together with chemotropic guidance can direct regenerating central axons to form synaptic contacts with appropriate gracilar targets, synaptic activity was not restored.
To our knowledge this is the first direct observation of a concept that has been previously advanced but not proven: that comprehensive attempts to restore functional neural circuitry after spinal cord injury must address not only axonal growth and target location, but remyelination. It is not likely that regenerated axons will be functional unless action potentials can be efficiently transmitted through regenerated segments. Our studies highlight the complexities of restoring function, and provide a model system in which these complexities can be further explored.
In summary, we demonstrate a tropic dependency for target reinnervation, and evidence for supraspinal target reinnervation after combinatorial application of therapies to the injured adult CNS. Progress in understanding mechanisms of appropriate axon guidance and termination is of critical importance in the field of CNS regeneration, as experimental strategies that enhance axonal growth after injury in the adult CNS continue to be identified. Axons face far more inappropriate than appropriate targets when extending beyond lesion sites after spinal cord injury, and the elucidation of mechanisms that guide directional growth into appropriate targets is essential. The present study identifies chemotropic guidance as an important mechanism that can guide regenerating axons into appropriate targets and lead to synapse formation.