Repair of the transected spinal cord at different stages of development in the North American opossum, Didelphis virginiana.

When the thoracic spinal cord is transected on postnatal day (PD) 5 in the North American opossum, descending and ascending axons grow through the lesion site. When the lesion is made on PD20, comparable growth is limited to a subset of descending axons. To better understand the mechanisms underlying these differences, we analyzed the transection site at different times after lesioning at both ages. Axons which crossed the lesion site could be identified using silver impregnation and immunostaining for neurofilament. Nissl stains revealed that abnormal appearing grey matter was also present in some of the PD5 cases. In many PD5 cases, however, and in all of the animals transected at PD20, grey matter was not present at the lesion site. Immunostaining with a neuron specific antibody supported that conclusion. However, immunostaining with phenotypic specific antibodies revealed that glial cells were present in all cases. Immunostaining for Schwann cells was negative. Fibronectin-positive cells were also present at the lesion site after transection of the thoracic cord at PD20, but their identity was uncertain. When injections of bromodeoxyuridine (BrdU), a thymidine analog, were made at different times after lesioning and the pups were sacrificed for BrdU immunohistochemistry up to 40 days later, labeled cells were found in the tissue which bridged the lesion site indicating that cell proliferation contributed to reconstruction at the lesion site.

Regeneration of descending spinal axons after transection of the thoracic spinal cord during early development in the North American opossum, Didelphis virginiana.

Opossums are born in an immature, fetal-like state, making it possible to lesion their spinal cord early in development without intrauterine surgery. When the thoracic spinal cord of the North American opossum, Didelphis virginiana, is transected on postnatal day 5, and injections of Fast Blue (FB) are made caudal to the lesion site 30-40 days or 6 months later, neurons are labeled in all of the spinal and supraspinal areas that are labeled after comparable injections in age-matched, unlesioned controls. Double-labeling studies document that regeneration of cut axons contributes to growth of axons through the lesion site and behavioral studies show that animals lesioned on postnatal day 5 use their hindlimbs in normal appearing locomotion as adults. The critical period for developmental plasticity of descending spinal axons extends to postnatal day 26, although axons which grow through the lesion site become fewer in number and more restricted as to origin with increasing age. Animals lesioned between postnatal day 12 and 26 use the hindlimbs better than animals lesioned as adults, but hindlimb function is markedly abnormal and uncoordinated with that of the forelimbs. We conclude that restoration of anatomical continuity occurs after transection of the spinal cord in developing opossums, that descending axons grow through the lesion site, that regeneration of cut axons contributes to such growth, and that animals lesioned early enough in development have relatively normal motor function as adults.

Rescue of axotomized rubrospinal neurons by brain-derived neurotrophic factor (BDNF) in the developing opossum, Didelphis virginiana.

Many rubrospinal neurons die in developing opossums when their axon is cut at thoracic levels of the spinal cord and in the present study we asked whether they can be rescued by brain-derived neurotrophic factor (BDNF). Bilateral injections of Fast Blue (FB) were made into the rostral lumbar cord to prelabel rubrospinal neurons and 5 days later the rubrospinal tract was cut unilaterally by hemisecting the thoracic cord. Immediately after hemisection, BDNF-soaked gelfoam was placed into the lesion cavity. Since pilot data indicated that one application of BDNF was not sufficient to produce a rescue effect, a second application was made 7 days later. Seven days after the second application the pups were killed by an overdose of anesthetic so that the red nucleus contralateral and ipsilateral to the lesion site could be examined for labeled neurons. The rubrospinal tract is almost entirely crossed, so the red nucleus contralateral to the lesion contained many axotomized neurons, whereas the red nucleus ipsilateral to it did not. Age-matched controls were subjected to the same procedures, but the gelfoam applied to the lesion site in the experimental animals was soaked only in the vehicle used to deliver BDNF. In all cases, labeled neurons were fewer in number in the red nucleus contralateral to the lesion than ipsilateral to it. It was of particular interest, however, that labeled neurons contralateral to the lesion were more numerous in the animals treated with BDNF than in the controls. We conclude that BDNF rescues at least some rubrospinal neurons from axotomy-induced cell death in developing opossums suggesting that loss of access to BDNF, and perhaps other neurotrophins, contributes to failure of rubrospinal neurons to survive axotomy.

Developmental plasticity of ascending spinal axons studies using the North American opossum, Didelphis virginiana.

The objectives of the present study were to determine if axons of all ascending tracts grow through the lesion after transection of the thoracic spinal cord during development in the North American opossum, and if so, whether they reach regions of the brain they normally innervate. Opossum pups were subjected to transection of the mid-thoracic cord at PD5, PD8, PD12, PD20, or PD26 and injections of Fast Blue (FB) into the lower thoracic or upper lumbar cord 30-40 days or 6 months later. In the PD5 transected cases, labeled axons were present in all of the supraspinal areas labeled by comparable injections in unlesioned, age-matched controls. In the experimental cases, however, labeled axons appeared to be fewer in number and in some areas more restricted in location than in the controls. When lesions were made at PD8, labeled axons were present in the brain of animals allowed to survive 30-40 days prior to FB injections but they were not observed in those allowed to survive 6 months. When lesions were made at PD12 or later, labeled axons were never found rostral to the lesion. It appears, therefore, that axons of all ascending spinal pathways grow though the lesion after transection of the thoracic cord in developing opossums and that they innervate appropriate areas of the brain. Interestingly, the critical period for such growth is shorter than that for most descending axons, suggesting that factors which influence loss of developmental plasticity are not the same for all axons.

Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana.

Spinocerebellar axons have been studied extensively in placental mammals, but there have been no full reports on their origin, laterality, or spinal course in any marsupial. We have used the North American opossum (Didelphis virginiana) to obtain such information and to ask whether any spinocerebellar neurons innervate both the anterior and posterior lobes of the cerebellum through axonal collaterals. To identify spinal neurons that project to the cerebellum, we employed the retrograde transport of Fluoro-Gold (FG) from the anterior lobe, the main target of spinocerebellar axons. In some cases, cerebellar injections of FG were combined with hemisections of the rostral cervical or midthoracic spinal cord, so that laterality of spinocerebellar connections could be established. To determine whether single neurons project to both the anterior lobe and the posterior lobe, injections of Fast Blue (FB) into the anterior lobe were combined with injections of Diamidino yellow (DY) or rhodamine B dextran (RBD) into the posterior lobe, or vice versa. Following injections of FG into the anterior lobe, neurons were labeled throughout the length of the spinal cord, which differed in laminar distribution and laterality of their projections. Among other areas, neurons were labeled in the central cervical nucleus, the nucleus centrobasalis, Clarke's nucleus, the dorsal horn dorsal spinocerebellar tract area, the spinal border region, and Stilling's nucleus. When anterior lobe injections of FB were combined with injections of RBD or DY into the posterior lobe, or vice versa, some double-labeled neurons were present in all major spinocerebellar groups. Cerebellar injections of FG also retrogradely labeled spinocerebellar axons, allowing us to document their locations in the gray matter as well as within the periphery of the lateral and ventral funiculi at all spinal levels. A few spinocerebellar axons also were found in the dorsal funiculus (a dorsal column-spinocerebellar tract), which appeared to originate from neurons in the dorsal part of Clarke's nucleus from the ninth thoracic segment to the first lumbar segment. Our results indicate that spinocerebellar axons in the marsupial opossum are generally comparable in origin, course, and laterality to the same axons in the placental mammals studied to date.

Regeneration of supraspinal axons after transection of the thoracic spinal cord in the developing opossum, Didelphis virginiana.

When the thoracic spinal cord of the North American opossum is transected early in development, supraspinal axons grow through the lesion. In the experiments reported here, we asked whether regeneration of cut axons contributes to such growth. Fast Blue (FB) was injected into the lumbar cord on postnatal day (PD)5, 8, 15, or 20. Five days later, FB was removed by gentle suction, and the spinal cord was transected at thoracic levels. Fourteen days later, rhodamine B dextran was injected between the site of the FB injection and the lesion. The pups were maintained for an additional 7-10 days before killing and perfusion. We assumed that supraspinal neurons that contained FB survived axotomy and those that contained both FB and rhodamine B dextran supported regenerating axons. In the PD5 group (lesioned at PD10), regenerative growth was documented for axons originating in all of the supraspinal nuclei that innervate the lumbar cord by PD10. When the injections were made at the later ages, however, neurons that supported regenerative growth were fewer in number and regionally restricted. In some cases, they were limited primarily to the red nucleus, the medullary raphe, and the adjacent reticular formation. Our results show that regeneration of cut axons contributes to growth of supraspinal axons through the lesion after transection of the thoracic cord in developing opossums and that the critical period for regenerative growth is not the same for all axons.

Adult opossums (Didelphis virginiana) demonstrate near normal locomotion after spinal cord transection as neonates.

When the thoracic spinal cord of the North American opossum (Didelphis virginiana) is transected on postnatal day (PD) 5, the site of injury becomes bridged by histologically recognizable spinal cord and axons which form major long tracts grow through the lesion. In the present study we asked whether opossums lesioned on PD5 have normal use of the hindlimbs as adults and, if so, whether that use is dependent upon axons which grow through the lesion site. The thoracic spinal cord was transected on PD5 and 6 months later, hindlimb function was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. All animals supported their weight with the hindlimbs and used their hindlimbs normally during overground locomotion. In some cases, the spinal cord was retransected at the original lesion site or just caudal to it 6 months after the original transection and paralysis of the hindlimbs ensued. Surprisingly, however, these animals gradually recovered some ability to support their weight and to step with the hindlimbs. Similar recovery was not seen in animals transected only as adults. In order to verify that descending axons which grew through the lesion during development were still present in the adult animal, opossums subjected to transection of the thoracic cord on PD5 were reoperated and Fast blue was injected several segments caudal to the lesion. In all cases, neurons were labeled rostral to the lesion in each of the spinal and supraspinal nuclei labeled by comparable injections in unlesioned, age-matched controls. The results of orthograde tracing studies indicated that axons which grew through the lesion innervated areas that were appropriate for them.

Developmental plasticity of selected spinocerebellar axons. Studies using the North American opossum, Didelphis virginiana.

When the thoracic spinal cord of the opossum is hemisected at postnatal day 5 or 8, but not at day 12 or later ages, spinocerebellar axons which originate from spinal border cells, the sacral/coccygeal ventrolateral nucleus, and Stilling's nucleus grow through the lesion and reach the cerebellum. The critical period for such growth is comparable to that reported previously for spinocerebellar axons originating within Clarke's nucleus and for axons of the fasciculus gracilis, but shorter than that for most descending spinal axons. It appears, therefore, that differences exist in the ability of ascending and descending axons to traverse a lesion of their spinal pathway during development.

Early development and developmental plasticity of the fasciculus gracilis in the North American opossum (Didelphis virginiana).

The first objective of the present study was to ask when axons of the fasciculus gracilis reach the nucleus gracilis in the North American opossum (Didelphis virginiana). When Fast Blue (FB) was injected into the lumbar cord on postnatal day (PD) 1 and the pups were killed 2 days later, labeled axons were present within a distinct fasciculus gracilis at thoracic and cervical levels of the cord. When comparable injections were made at PD3 or 5 and the pups were allowed to survive for the same time period, a few labeled axons could be followed to the caudal medulla where they were located dorsal to the presumptive nucleus gracilis. In order to verify these observations and to determine if any of the axons which innervate the nucleus gracilis early in development originate within dorsal root ganglia, we also employed cholera toxin conjugated to horseradish peroxidase (CT-HRP) to label dorsal root axons transganglionically. When CT-HRP was injected into the hindlimb on PD1 and the pups were maintained for 1 day prior to death and HRP histochemistry, labeled axons were present within the fasciculus gracilis at thoracic and cervical levels, but they could not be traced into the medulla. When comparable injections were made on PD3, and the pups were maintained for 2 days, labeled axons were present within the caudal medulla. Our second objective was to determine whether axons of the fasciculus gracilis grow through a lesion of their spinal pathway during early development. In one group of animals, the thoracic cord was transected at PD5, 8, 12, 20 and 26 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30-40 days later. After a 3-5 day survival, the pups were killed and perfused so that the spinal cord and brainstem could be removed and sectioned for fluorescence microscopy. In all of the cases lesioned at PD5, axons of the fasciculus gracilis were labeled rostral to the site of transection and they could be followed to the nucleus gracilis. Evidence for growth of fasciculus gracilis axons into the caudal medulla was also seen in cases lesioned at PD8. In contrast, labeled axons were not observed rostral to the lesion when it was made at PD12 or at later stages of development. In order to verify that some of the axons which crossed the lesion originated within dorsal root ganglia, the thoracic cord was transected at PD5 in another group of animals and 7 days later, injections of CT-HRP were made into one of the hindlimbs. After a 3 day survival, labeled axons could be traced through the lesion site and into the caudal medulla. We conclude that axons of the fasciculus gracilis reach the nucleus gracilis by at least PD5 in the opossum and that they grow through a lesion of their spinal pathway when it is made at the same age or shortly thereafter. The critical period for such growth appears to end between PD8 and PD12.

Evidence for growth of supraspinal axons through the lesion after transection of the thoracic spinal cord in the developing opossum Didelphis virginiana.

In the present study, we asked whether supraspinal axons grow through a complete transection of the spinal cord in the developing opossum Didelphis virginiana. When the thoracic cord was transected at postnatal day (PD) 5 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30-40 days later, FB-containing neurons were found in each of the supraspinal nuclei labeled by comparable injections in age-matched unlesioned controls. Continuity between the cut ends of the cord was obviously gross when the animals were killed, and histologically recognizable spinal cord was present at the lesion site. When the same procedure was followed on pups subjected to transection at PD12, FB-containing neurons were still present at supraspinal levels, but they appeared to be fewer in number than in the PD5 cases or the age-matched controls, and none were found within the medial pontine reticular and lateral vestibular nuclei. When the lesion was made at PD20, labeled neurons were even fewer in number, and when it was made at PD26, they were restricted to the medullary raphe and the red nuclei. There was no evidence for growth of supraspinal axons across lesions made at PD33. We conclude that supraspinal axons grow through the lesion after transection of the spinal cord in neonatal opossums and that the critical period for growth of reticulospinal and vestibulospinal axons through the lesion ends earlier than that for comparable growth of raphespinal and rubrospinal axons.

Growth of dorsal spinocerebellar axons through a lesion of their spinal pathway during early development in the North American opossum, Didelphis virginiana.

Supraspinal axons grow around or through lesions of their spinal pathway during specific critical periods of mammalian development, but comparable plasticity has not been documented for axons which form ascending tracts. In the present study, we asked whether axons of the dorsal spinocerebellar tract (DSCT) are capable of such growth. The spinal cord of the North American opossum, Didelphis virginiana, was hemisected at mid-thoracic levels between postnatal day (PD) 5 and 68 and after varying survival times, bilateral injections of Fluoro-Gold or Fast Blue were made into the anterior lobe of the cerebellum, the major target of DSCT axons. Seven days later, the pups were sacrificed and their spinal cord processed for fluorescence microscopy. In animals lesioned between PD5 and 9, and allowed to survive for 37-269 days, neurons were labeled bilaterally in Clarke's nucleus (CN) caudal to the lesion, but they were fewest in number and smallest in size on the lesioned side. Since the DSCT originates almost entirely within CN on the ipsilateral side, we conclude that the neurons labeled ipsilateral and caudal to the lesion supported axons which grew around or through it. Histological examination revealed that recognizable spinal cord was present at the lesion site and that labeled spinocerebellar axons were located in their normal position ipsilateral to the lesion. It appears, therefore, that growth occurred through the lesion. In animals lesioned between PD13 and 68, labeled neurons were not found in CN caudal and ipsilateral to the lesion although they were present on the contralateral (control) side. We conclude that DSCT axons, like axons which form descending tracts, grow through a lesion of their spinal pathway if it is made early in development.

Flocculation of Esch. coli with cationic polymers: a model for the dose curve based on charge.

Continuously grown cells from a glucose limited chemostat were flocculated with four different cationic polymers. The polymer was added to the cells either dropwise or as a slug at the start of the flocculation period. Dose curves for each polymer type and using each method of polymer addition were constructed. It was evident that classical overdose was possible with all four polymers if slug addition was used. Continuous addition produced dose curves overdose was possible with all four polymers if slug addition was used. Continuous addition produced dose curves with no overdosing except when low molecular weight, low charge density polymer was used. The dose curves could be combined if they were based on amount of charge added. The dose curve was not linear, but fitted a logarithmic model well. Charge density was much more important than MW of the polymer.

Evidence for GAP-43 within descending spinal axons in the North American opossum, Didelphis virginiana.

We have shown previously that GAP-43, a growth associated protein characteristically present in growing and regenerating axons, is relatively abundant in the spinal cord of adult opossums. In the present study, we combined the orthograde transport of the fluorescent marker Fluoro-Ruby with immunofluorescence for GAP-43 to determine if any of it is present within descending spinal axons. When Fluoro-Ruby was injected into the red nucleus and midbrain tegmentum, the medial pontine or medullary reticular formation, the medullary raphe or the lateral vestibular nucleus, axons were labeled in the expected areas of the spinal cord, but in most cases none showed evidence for GAP-43. In two of the four cases with rubral injections, however, a few labeled axons within the rubrospinal tract showed GAP-43 immunofluorescence, and in one case with an injection of the gigantocellular reticular nucleus and adjacent raphe, labeled axons within lamina IX immunostained for the protein. Since serotoninergic neurons are present within the gigantocellular reticular nucleus and adjacent raphe, and axons of the same phenotype are abundant within lamina IX, we asked whether serotoninergic axons contain GAP-43. When sections of the spinal cord were immunostained for both serotonin and GAP-43, many axons within lamina IX showed evidence for both substances. Such axons appeared to contact presumptive motoneurons. In cases with Fluoro-Ruby injections of the forelimb motor cortex, labeled axons were present within the pyramidal tract, and some of them showed evidence for GAP-43.

The distribution of GAP-43 immunoreactivity in the central nervous system of adult opossums (Didelphis virginiana) with notes on their development.

The distribution of the growth associated protein GAP-43 has been described in the brain and spinal cord of rats and other placental mammals but not marsupials. In order to provide such information, we employed a monoclonal antibody to immunostain for GAP-43 in the central nervous system of adult and developing opossums, Didelphis virginiana. The GAP-43 immunoreactivity was widely distributed in the brain of adult opossums, but it was particularly dense within specific layers of the olfactory bulb and hippocampal formation, layer I of the cerebral cortex, the bed nucleus of the stria terminalis, the nucleus accumbens, the striatum, the amygdala, the septum, the olfactory tubercle, medial parts of the preoptic area and diencephalon, the substantia nigra, the ventral tegmental area, the periaqueductal grey matter, the interpeduncular nucleus, the periventricular grey, the molecular layer of the cerebellum, the superior central nucleus, the basilar pons, the dorsal vagal and solitary nuclei, and laminae I and II of the spinal trigeminal nucleus. Immunoreactivity for GAP-43 was also present within the spinal cord, where it was densest within laminae I, II, IX, and X and within the intermediolateral cell column. In most areas of the brain and some areas of the spinal cord, an inverse correlation existed between the location of GAP-43 and myelin. Immunostaining for GAP-43 was found throughout most of the central nervous system during early development, but it decreased with age in a regionally specific manner until the adult pattern was reached. Our results suggest that the distribution of GAP-43 in opossums is similar in many respects to that reported in rats and that it is developmentally regulated.

Developmental plasticity of reticulospinal and vestibulospinal axons in the north American opossum, Didelphis virginiana.

We have shown previously that rubral axons grow around a lesion of their spinal pathway in the North American opossum if it is made at early stages of development. In the present experiments, we have asked whether reticular and vestibular axons have the same ability. The spinal cord was hemisected at postnatal day 20, 12, or 5, well within the critical period for rubrospinal plasticity, and, approximately 30 days later, bilateral injections of fast blue were made about four segments caudal to the lesion. The pups were killed 4 or 5 days after the injections. In most of the animals lesioned on postnatal day 20, labeled neurons were not found in the medial part of the pontine reticular nucleus or the dorsal part of the lateral vestibular nucleus ipsilateral to the lesion. The spinal projections from both areas are exclusively ipsilateral. When the lesions were made at postnatal day 12 or 5, however, labeled neurons were present in both areas, suggesting that they supported axons that had grown caudal to the lesion. As was expected from previous studies, rubral neurons were labeled contralateral to the lesion in all three groups. In the opossum, as in other species, the red nucleus projects contralaterally. We conclude that reticular and vestibular axons, like axons from the red nucleus, grow around a lesion of their pathway during development and that the critical period for their plasticity ends earlier than that for rubrospinal axons.

The early development of major projections from caudal levels of the spinal cord to the brainstem and cerebellum in the gray short-tailed Brazilian opossum, Monodelphis domestica.

The Brazilian short-tailed opossum, Monodelphis domestica, is born 14-15 days after copulation and is available for experimentation at stages of development corresponding to those which occur in utero in placental mammals. In the present study, we took advantage of the opossum's embryology to study the development of projections from caudal levels of the spinal cord to the brainstem and cerebellum using axonal tracing methods. In all cases, a 2-3 day survival time was used for axonal transport. When injections of Fast blue (FB) were made into caudal levels of the thoracic cord at postnatal day (PD) 1 or 2, axonal labeling could not be identified at supraspinal levels. When injections were made at PD3, however, labeled axons were found in the fasciculus gracilis at caudal medullary levels, within the ventrolateral medulla and pons, within an incipient inferior cerebellar peduncle, and within the cerebellar anlage. The dorsal root origin of at least some of the axons within the fasciculus gracilis was evidenced by the transganglionic transport of cholera toxin conjugated to horseradish peroxidase from the hindlimbs. After FB injections at PD7, a few labeled axons could be traced from the fasciculus gracilis into the nucleus gracilis and from the ventrolateral pathway to the inferior olive. Generally comparable results were obtained using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). In cases injected with FB at PD9, the pattern of brainstem labeling was adult-like. Although labeled axons were present within the cerebellum of animals injected with FB on PD3, they were limited to the marginal zone. Axonal labeling was present within an identifiable internal granular layer in cases injected with either FB or WGA-HRP at PD16, and it appeared to be limited to specific bands which foreshadowed those seen at later stages of development and in the adult animal. In some cases, labeled axons were present within the molecular layer where they were not seen in the adult animal. Our results provide a timetable for the normal development of projections from caudal levels of the spinal cord to the brainstem and cerebellum in Monodelphis and show that such development occurs postnatally rather than prenatally, as in placental mammals.