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After spinal cord injury at birth, axotomized brainstem-spinal and corticospinal neurons are capable of permanent regenerative axonal growth into and through a fetal spinal cord transplant placed into the site of either a spinal cord hemisection or transection. In contrast, if fetal tissue which is not a normal target of the axotomized neurons (embryonic hippocampus or cortex) is placed into a neonatal spinal cord hemisection, brainstem-spinal serotonergic axons transiently innervate the transplant, but subsequently withdraw. The first set of experiments was designed to test the hypothesis that after spinal cord transection, serotonergic axons would cross the nontarget transplant, reach normal spinal cord targets caudal to the transection, and gain access to requisite target-derived cues, permitting permanent maintenance. Surprisingly, after a complete spinal cord transection, brainstem-spinal axons failed to grow into an inappropriate target even transiently. These observations suggest that the transient axonal ingrowth into nontarget transplants may represent lesion-induced axonal sprouting by contralateral uninjured axons. We have used double-labeling with fluorescent dyes, to test directly whether axonal sprouting of neurons which maintain collaterals to uninjured spinal cord targets (1) provide the transient ingrowth of brainstem-spinal axons into a nontarget transplant and (2) contribute to permanent ingrowth into target-specific transplants. Uninjured red nucleus, raphe nucleus, and locus coeruleus neurons extend axons into the nontarget transplant while maintaining collaterals to the host spinal cord caudal to the transplant. The lesion-induced sprouting by uninjured axons was also observed with a target-specific transplant. Taken together, these studies suggest that sprouting and regenerating axons may differ in their requirements for growth after injury.

Neurons that maintain extensive axon collaterals proximal to the site of axotomy may be better able to survive injury. Early lesions of the rubrospinal tract lead to retrograde cell death of the majority of axotomized immature neurons. Transplants of fetal spinal cord tissue rescue axotomized rubrospinal neurons and promote their axonal regeneration. Rubrospinal neurons develop many of their axon collaterals postnatally. The present study tests the hypothesis that the axotomized rubrospinal neurons that are rescued by transplants and regenerate their axons are those neurons that have established axon collaterals to targets rostral to the lesion. Neonatal rats received a transplant of fetal spinal cord tissue placed into a midthoracic spinal cord hemisection. One month after transplantation, the retrogradely transported fluorescent tracers fast blue (FB) and diamidino yellow (DY) were used to identify rubrospinal neurons with collaterals to particular targets. FB was injected either into the interpositus nucleus of the cerebellum or into the gray matter of the cervical enlargement to identify collaterals to these targets, and DY was injected into the spinal cord approximately 5 mm caudal to the transplant and lesion site to label retrogradely the neurons that regenerated their axons. Double labeling was observed in the axotomized neurons of the red nucleus after tracer injections into the cervical spinal cord but not after injections into the cerebellum. This labeling pattern indicates that axotomized rubrospinal neurons that are rescued and regenerate axons caudal to the transplant maintain axon collaterals at cervical spinal cord levels. Cerebellar collaterals do not appear to play a role in the survival and regrowth of axotomized rubrospinal neurons.

After spinal cord lesions in newborn rats, transplants of fetal spinal cord tissue rescue immature axotomized neurons, support the growth of axons into and through the site of injury and prolong the critical period for developmental plasticity. Both late-developing (uninjured) and regenerating axons contribute to this transplant-induced anatomical plasticity. After lesions in the mature CNS, transplant-induced axonal elongation is spatially restricted. The current study was designed (1) to determine the magnitude of transplant-induced regeneration, (2) to test the hypothesis that the long distance growth beyond the site of injury is mediated by late-developing axonal pathways, whereas axonal elongation by regenerating pathways is spatially restricted as it is in the adult, and (3) to determine if particular nuclei have a greater inherent capacity for regeneration than others. We used temporally spaced retrograde tracing with the fluorescent dyes fast blue and diamidino yellow to address this issue. Fast blue was placed into the site of a spinal cord overhemisection in rat pups < 48 h old to label those neurons which were axotomized by a neonatal lesion. The tracer was removed and a transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. Three to six weeks later a second tracer (diamidino yellow) was injected bilaterally into the host spinal cord caudal to the transplant. We counted the number of double-labeled (regenerated), single diamidino yellow-labeled (late-growing), and single fast blue-labeled (nonbridging) neurons in the cortex, red nucleus, raphe nuclei, and locus coeruleus. By systematically varying the distance of the diamidino yellow injection site caudal to the transplant, we were able to compare the distance which injured axons regenerate with the distance that late-growing axons extend. When the diamidino yellow injection was placed within 5 mm caudal to the transplant 28% of the axotomized neurons in the red nucleus, 32% of the axotomized neurons in the locus coeruleus, and 37% of the axotomized neurons in the raphe nuclei were double-labeled (regenerating). Although the percentage of double-labeled neurons decreased as the distance beyond the transplant increased, a substantial population of regenerating neurons was identified in each of the brain stem nuclei examined following diamidino yellow injections up to 15 mm caudal to the transplant. Thus, after spinal cord lesions and transplants at birth, both regenerating neurons and late-developing neurons extended axons long distances (up to 15 m) caudal to the lesion site. The capacity for regenerative growth was similar in each of the nuclei examined.

Fetal spinal cord transplants prevent the retrograde cell death of immature axotomized central nervous system (CNS) neurons and provide a terrain which supports axonal elongation in the injured immature spinal cord. The current experiments were designed to determine whether the axons which grow across the site of the neonatal lesion and transplant are derived from axotomized neurons and are therefore regenerating or whether the axons which grow across the transplant are late-growing axons that have not been axotomized directly. We have used an experimental paradigm of midthoracic spinal cord lesion plus transplant at birth and temporally spaced retrograde tracing with the fluorescent tracers fast blue (FB) and diamidino yellow (DY) to address this issue. Fast blue was placed into the site of a spinal cord hemisection in rat pups less than 48 h old. After 3-6 h to allow uptake and transport of the tracer, the source of fast blue was removed by aspiration and the lesion was enlarged to an "over-hemisection." A transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. The animals survived 3-6 weeks prior to the injection of the second tracer (DY) bilaterally into the host spinal cord caudal to the lesion plus transplant. Neurons with late-developing axons would not be exposed to the first dye (FB), but could only be exposed to the second tracer, diamidino yellow. Thus, neurons with a diamidino yellow-labeled nucleus are interpreted as "late-developing" neurons. Neurons axotomized by midthoracic spinal cord lesion at birth could be exposed to the first tracer, fast blue. If after axotomy they regrew caudal to the transplant, they could be labeled by the second tracer as well. We interpret these double-labeled neurons as regenerating neurons. If neurons labeled with fast blue and axotomized by the spinal cord hemisection either failed to regenerate or grew into the transplant but not caudal to it, they would be labeled only by the first dye. We have examined the pattern and distribution of single (FB or DY)- and double (FB + DY)-labeled neurons in the sensorimotor cortex, red nucleus, locus coeruleus, and raphe nuclei. The sensorimotor cortex contains only DY-labeled neurons. The red nucleus contains both FB- and FB + DY-labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

We are using neural tissue transplantation after spinal cord injury to identify the rules which determine the response of young neurons to injury, to identify the mechanisms underlying anatomical plasticity and recovery of function following spinal cord injury, and to determine the conditions which change during development, leading to the more restricted growth capacity of mature neurons following injury. Spinal cord lesions at birth interrupt different pathways at different relative stages in their development. Neural tissue transplants modify the response of the immature central nervous system neurons to injury. In the current studies, we have used neuroanatomical and behavioral methods to compare the response of the late-developing corticospinal pathway with that of brainstem-spinal pathways which are intermediate in their development and that of the relatively mature dorsal root pathway. We find that both late-developing and regenerating neuronal populations contribute to the transplant-induced anatomical plasticity, and suggest that this anatomical plasticity underlies the transplant-mediated sparing and recovery of function.

Expression and development of specific markers of the adrenergic phenotype were studied in central neurons grown in transplant system. Medulla oblongata from embryonic day 12.5 (E12.5) or E18 rat was grafted into the anterior chamber of the eye of adult rat hosts. After two months, grafts were examined for the presence of immunoreactivity (IR) and catalytic activity to the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28), a specific adrenergic marker. In addition, grafts were examined for immunoreactivity to neuropeptide Y (NPY) and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. In E12.5 grafts, PNMT was expressed de novo, enzyme activity developed to levels similar to those in adult rat brainstem and PNMT-IR neurons were observed. TH-IR and NPY-IR neurons were also observed. In contrast, PNMT-IR was not observed in E18 grafts even though these already contained PNMT-IR neurons at the time of grafting. This was not due to poor growth of E18 grafts, in general, since TH-IR neurons were present and the protein content of the grafts was similar to that of E12.5 grafts. These studies suggest that adrenergic neurons survive well in oculo if they are transplanted prior to the age when neuroblasts have initially expressed the adrenergic phenotype, migrated to their final positions and elaborated processes. In addition, these studies establish a transplant system in which factors required for the development of central adrenergic neurons can be more easily studied than in situ.