Hepatitis C virus infection among paediatric patients attending University of Assiut Hospital, Egypt.

Few studies have evaluated the epidemiology and risk factors of hepatitis C virus (HCV) infection in children in Egypt. This study of 465 children attending Assiut University Hospital measured the rates of anti-HCV positivity by 3rd-generation ELISA test and of HCV-RNA positivity by PCR, with analysis of some relevant risk factors. The rate of HCV-RNA positivity among ELISA-positive cases (n = 121) was 72.2% overall: 100% in the subgroup with hepatitis, 70.8% in those with a history of multiple transfusions and 58.3% in those without hepatitis or multiple transfusions. History of blood transfusions, frequent injections, hospitalization or surgical procedures were significant risk factors for anti-HCV positivity by ELISA.

Regrowth of severed axons in the neonatal central nervous system: establishment of normal connections.

When pyramidal tract axons are cut in the adult hamster, fibers degenerate in both anterograde and retrograde directions from the lesion. If the same operation is performed on infant hamsters, however, there is massive regrowth of the severed axons via a new brainstem pathway to their appropriate terminal sites in the medulla and spinal cord. In contrast to previous studies, these results suggest that axons in the mammalian central nervous system damaged early in life may regenerate in a functionally useful way.

Axon branching requires interactions between dynamic microtubules and actin filaments.

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence time-lapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth.

Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches.

Local changes in microtubule organization and distribution are required for the axon to grow and navigate appropriately; however, little is known about how microtubules (MTs) reorganize during directed axon outgrowth. We have used time-lapse digital imaging of developing cortical neurons microinjected with fluorescently labeled tubulin to follow the movements of individual MTs in two regions of the axon where directed growth occurs: the terminal growth cone and the developing interstitial branch. In both regions, transitions from quiescent to growth states were accompanied by reorganization of MTs from looped or bundled arrays to dispersed arrays and fragmentation of long MTs into short MTs. We also found that long-term redistribution of MTs accompanied the withdrawal of some axonal processes and the growth and stabilization of others. Individual MTs moved independently in both anterograde and retrograde directions to explore developing processes. Their velocities were inversely proportional to their lengths. Our results demonstrate directly that MTs move within axonal growth cones and developing interstitial branches. Our findings also provide the first direct evidence that similar reorganization and movement of individual MTs occur in the two regions of the axon where directed outgrowth occurs. These results suggest a model whereby short exploratory MTs could direct axonal growth cones and interstitial branches toward appropriate locations.

Fibroblast growth factor-2 promotes axon branching of cortical neurons by influencing morphology and behavior of the primary growth cone.

Interstitial branching is an important mechanism for target innervation in the developing CNS. A previous study of cortical neurons in vitro showed that the terminal growth cone pauses and enlarges in regions from which interstitial axon branches later develop (Szebenyi et al., 1998). In the present study, we investigated how target-derived signals affect the morphology and behaviors of growth cones leading to development of axon branches. We used bath and local application of a target-derived growth factor, FGF-2, on embryonic pyramidal neurons from the sensorimotor cortex and used time-lapse digital imaging to monitor effects of FGF-2 on axon branching. Observations of developing neurons over periods of several days showed that bath-applied FGF-2 significantly increased growth cone size and slowed growth cone advance, leading to a threefold increase in axon branching. FGF-2 also had acute effects on growth cone morphology, promoting rapid growth of filopodia within minutes. Application of FGF-2-coated beads promoted local axon branching in close proximity to the beads. Branching was more likely to occur when the FGF-2 bead was on or near the growth cone, suggesting that distal regions of the axon are more responsive to FGF-2 than other regions of the axon shaft. Together, these results show that interstitial axon branches can be induced locally through the action of a target-derived growth factor that preferentially exerts effects on the growth cone. We suggest that, in target regions, growth factors such as FGF-2 and other branching factors may induce formation of collateral axon branches by enhancing the pausing and enlargement of primary growth cones that determine future branch points.

Projections of the cerebellar and dorsal column nuclei upon the inferior olive in the rhesus monkey: an autoradiographic study.

Projections from the cerebellar and dorsal column nuclei to the inferior olive of the rhesus monkey were traced with anterograde autoradiographic methods. The cerebellar nuclei give rise to a massive projection which reaches the contralateral inferior olivary complex by way of the descending limb of the superior cerebellar peduncle. Dentato-olivary fibers project exclusively upon the principal olivary nucleus (PO) and observe a strict topography. The dorsal, lateral, and ventral dentate project respectively to the dorsal, lateral, and ventral lamellae of the PO. Within the lamellae, the dentato-olivary fibers are related point for point in the medio-lateral axis. By contrast, the rostro-caudal topography is reversed so that the rostral pole of the dentate projects to the caudal PO and the caudal dentate to the rostral PO. These connections are predominantly crossed but a small ipsilateral component recrosses the midline at the olivary commissure and mirrors the topography on the opposite side. The anterior interpositus projects only to the medial half of the DAO and the posterior interpositus projects only to the rostral two thirds of the MAO. The ipsilateral component is minor in comparison with the contralateral projection, but appears to be more substantial than the ipsilateral projection to the PO arising from the dentate nucleus. The fastigial nucleus does not project upon the olivary complex. The dorsal column nuclei project topographically upon the contralateral accessory nuclei with the gracile nucleus sending fibers primarily to the lateral half of the DAO and the cuneate nucleus projecting to rostral cell groups of the MAO. The present results when compared with other olivary connections described by previous studies in a variety of species suggest that regions of the MAO and DAO receiving sensory information from the periphery may lie outside the influence of cerebellar feedback loops.

Projections of the cerebellar and dorsal column nuclei upon the thalamus of the rhesus monkey.

Projections from the cerebellar and dorsal column nuclei to the midbrain and thalamus of the rhesus monkey were traced with anterograde autoradiographic techniques, or, in a few cases, with the Fink-Heimer method. The cerebellar nuclei give rise to a massive projection to the contralateral midbrain and thalamus via the ascending limb of the superior cerebellar peduncle. Cerebellar efferent fibers terminate contralaterally in both divisions of the red nucleus, and bilaterally in the interstitial nucleus of Cajal, the nucleus of Darkschewitsch, the oculomotor nucleus, and the central gray. All the deep cerebellar nuclei project upon a broad area of the contralateral ventral thalamus as well as certain intralaminar nuclei. Corresponding ipsilateral thalamic terminations are sparse. The topographic organization of cerebellothalamic fibers does not correspond to individual cerebellar nuclei or to cytoarchitectonic divisions of the ventral thalamic nuclei. Rather there are longitudinally oriented strips of terminal labeling which extend through all divisions of the ventral lateral nucleus, i.e., the VLps, the VLc, the VLo, as well as nucleus X, the oral division of the ventral posterolateral nucleus (VPLo), the central lateral nucleus (CL), and the most caudal region of the ventral anterior nucleus (VA). The topography of the cerebellothalamic fibers is arranged in a mediolateral pattern with fibers originating from anterior zones of the dentate and interpositus ending most laterally and those from posterior dentate and interpositus terminating most medially. The fastigial contribution is relatively sparse. The longitudinal strips of terminal labeling in the ventral thalamic nuclei are made up of still smaller terminal units consisting of disk-like aggregates of silver grains separated from one another by grain-free spaces. The dorsal column nuclei terminate primarily in the contralateral caudal division of the VPL (VPLc) and never extend rostrally into VPLo. These results demonstrate a segregation of cerebellar and dorsal columnar inputs to motor and sensory regions of the thalamus, respectively. Since these regions are separate and discrete in their cortical associations as well (Kalil, '76), it seems unlikely that fast afferent pathways relaying to motor cortex (Lemon and Porter, '76) could arise from the dorsal column nuclei.

Development of the pyramidal tract in the hamster. II. An electron microscopic study.

We undertook a qualitative and quantitative electron microscopic study of the growth and development of the pyramidal tract in the hamster to investigate the mode of growth of the axons, the possibility of fiber degeneration during development, and the process of myelination. By calculating the total fiber number as the product of axon density and tract area for several postnatal ages, we found that the pyramidal tract grows through the medulla as a compact bundle containing nearly twice the number of fibers as the mature tract. During the second postnatal week there is a substantial loss of axons followed in the third and fourth weeks by a more gradual loss such that by 34 days after birth the total number of axons reaches the adult value. Myelination in the hamster pyramidal tract begins at 7 days and continues at a very slow rate until the third postnatal week, when a dramatic increase in myelin formation occurs. By 34 days after birth the number of myelinated axons is approximately 80% that of the adult. as has been reported for other CNS tracts, there does not seem to be a "critical diameter" of an axon that absolutely determines the presence or absence of myelin on a fiber. However, all axons above 0.5 micron in diameter are myelinated at approximately the same rate, while those under this diameter are myelinated much more slowly and even in the adult make up only a small percentage of the total myelinated fibers.

Development of the pyramidal tract in the hamster. I. A light microscopic study.

The development of the pyramidal tract and other projections from the sensorimotor cortex was studied in the postnatal hamster with both (3H) proline and horseradish peroxidase (HRP) as anterograde tracers. In the 1-day-old animal labeled axons extend as far as the pons. Other corticofugal fibers have penetrated into the corpus striatum and the thalamus. By 2 days postnatally, the pyramidal tract has grown to midmedullary levels and there is substantial retrograde (HRP) and anterograde labeling in the thalamus. The pyramidal decussation is formed at 3 days of age and by 4 days the pyramidal tract has descended in the dorsal funiculus as far as midcervical spinal cord. Corticofugal fibers invade the pontine nuclei at 4 days and both the dorsal column nuclei and the superior colliculus at 6 days of age. At 6 days the pyramidal tract can be traced to mid-thoracic levels of the spinal cord, by 8 days the tract reaches lumbar levels, and by 14 days it has completed its caudal growth to the coccygeal spinal cord. Fibers first penetrate the gray matter of a given spinal cord level approximately 2 days after the tract has grown past that level in the dorsal funiculus. Pyramidal fibers continue their lateral growth into the dorsal horn at all levels of the cord throughout the third postnatal week such that by 21 days of age the pyramidal tract appears similar to that of the adult. The projections from sensorimotor cortex to the pontine nuclei, the superior colliculus, and the dorsal column nuclei appear to have a pattern similar to that of the adult soon after the fibers grown into these structures. There is a consistent delay of 2 to 3 days between the arrival of the pyramidal tract axons in the white matter adjacent to target structures and their innervation of a given terminal field. The pyramidal tract grows more quickly through the dorsal funiculus of the spinal cord than it does along the ventral surface of the medulla. Extensive elongation of pyramidal tract axons is achieved long before the growth and differentiation of the sensorimotor cortical neurons from which they originate. Finally, the pyramidal tract appears to grow as a compact bundle and not by the addition of temporally staggered groups of fibers. The relatively protracted period of innervation of the spinal cord by the pyramidal tract coupled with the immaturity of the cortical neurons at birth may be factors contributing to the significant regrowth of pyramidal tract axons severed early in development.

Cell death of corticospinal neurons is induced by axotomy before but not after innervation of spinal targets.

The response of corticospinal neurons to axotomy at postnatal ages from 5 days to adulthood was studied in the golden hamster (Mesocricetus auratus). Corticospinal neurons were retrogradely labeled with fluorescent rhodamine latex beads injected into the cervical or lumbar spinal cord. A unilateral lesion of the medullary pyramidal tract was made 1-2 days later and the brains fixed 1-30 days after axotomy. Comparisons of labeled axotomized corticospinal neurons with labeled normal corticospinal neurons in the contralateral cortex showed that axotomy at 14 days or later caused cell shrinkage but not cell death. Axotomy prior to 14 days caused cell death of corticospinal neurons. More neurons died the earlier the lesion was made, culminating in virtual complete cell death of corticospinal neurons following axotomy at 5 days. Axotomy at a given age did not affect all corticospinal neurons uniformly. Lumbar projection neurons underwent cell death ranging from slight to complete following axotomy at 13 and 9 days, respectively. Cervical projection neurons, in contrast, survived axotomy after a lesion at 9 days but underwent complete cell death if the lesion occurred at 5 days. Since corticospinal axons innervate the cervical cord from postnatal days 4-8 and the lumbar cord from 10-14 days (Reh and Kalil, '81; J. Comp. Neurol. 200:55-67), the ability of corticospinal neurons to survive axotomy appears to be temporally well correlated with their innervation of spinal targets. These neurons die if their axons are cut prior to target innervation but are able to survive if axotomy occurs after their axons innervate spinal targets. The results show that plasticity in the corticospinal pathway documented in previous reports cannot take the form of regrowth of severed axons, since early lesions cause extensive corticospinal cell death. Aberrant corticospinal pathways resulting from early lesions must therefore arise from undamaged axons. Additional retrograde labeling experiments showed that the opposite cortex responded to contralateral pyramidotomy by sprouting into denervated areas of the spinal cord. Thus another source of plasticity after early pyramidal tract lesions is sprouting from corticospinal axons arising from the intact cortex.

Functional role of regrowing pyramidal tract fibers.

When pyramidal tract axons are severed in the infant hamster, the damaged fibers regrow via a new pathway to their normal terminal sites in the medulla and spinal cord and there form synaptic connections (Kalil and Reh, '79, '82). We studied the behavior of animals with infant and adult lesions of the medullary pyramid to determine the functional significance of the new pathway in maintaining normal motor behavior. Examination of behaviors normally mediated by the pyramidal tract, particularly the manipulation of sunflower seeds during feeding, revealed a correlation between the presence of the new tract and the preservation of function. Furthermore, in the adult animal with an infant lesion, the spared behaviors were lost when the new pathway was destroyed.

A light and electron microscopic study of regrowing pyramidal tract fibers.

Autoradiographic and EM techniques were used to study the regenerative capacity of severed axons in the mammalian CNS. In infant and adult hamsters the pyramidal tract was severed unilaterally in the medulla several millimeters rostral to the decussation. After survival to adulthood, the animals received injections of [3H] proline in the sensorimotor cortex ipsilateral to the lesion. Autoradiography showed that labeled pyramidal tract axons in the medulla did not cross the lesion site. Instead, in animals with infant lesions there was massive new axonal growth arising from the severed pyramidal tract several millimeters rostral to the cut. Most of these labeled fibers crossed to the contralateral brainstem, coalesced into a compact bundle, descended just medial to the spinal trigeminal nucleus, and grew caudally for 6-7 mm. Although the trajectory of the regrowing axons was completely abnormal, their pattern of termination in the dorsal column nuclei and dorsal horn of the cervical spinal cord was normal. Synapse formation by the anomalous regrowing pyramidal tract axons in their appropriate terminal areas was confirmed by electron microscopy of terminal degeneration in animals with infant pyramidotomies followed by adult cortical lesions. Autoradiographic labeling of the new pathway at short postlesion survival times showed that the fibers grew out rapidly at about 1 mm/day, a rate somewhat slower than normal (2-4 mm/day). There was a dramatic difference in the capacity of the pyramidal axons to regrow in animals operated as infants vs. those operated as adults. The regrowth was maximal with lesions at 4-8 days of age. Capacity for new growth declined sharply thereafter such that after 20 days of age, pyramidal tract lesions elicited no new growth but instead a progressive axon degeneration retrograde to the lesion. These results, in contrast to many previous findings, show that significant regrowth of severed axons can occur in the neonatal CNS. Most importantly pyramidal tract fibers regrowing by anomalous routes can nevertheless establish synaptic connections in appropriate terminal areas and thus, as we show in the following paper, play a functional role in maintaining normal motor behavior.

Development of specificity in corticospinal connections by axon collaterals branching selectively into appropriate spinal targets.

Corticospinal projections in adult rodents arise exclusively from layer V neurons in the sensorimotor cortex. These neurons are topographically organized in their connections to spinal cord targets. Previous studies in rodents have shown that the mature distribution pattern of corticospinal neurons develops during the first 2 weeks postnatal from an initial widespread pattern that includes the visual cortex to a distribution restricted to the sensorimotor cortex. To determine whether specificity in corticospinal connections also emerges from an initially diffuse set of projections, we have studied the outgrowth of corticospinal axons and the formation of terminal arbors in developing hamsters. The sensitive fluorescent tracer 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label corticospinal axons from the visual cortex or from small regions of the forelimb or hindlimb sensorimotor cortex in living animals at 4-17 days postnatal. Initially axon outgrowth was imprecise. Some visual cortical axons extended transiently beyond their permanent targets in the pontine nuclei, by growing through the pyramidal decussation and in some cases extending as far caudally as the lumbar enlargement. Forelimb sensorimotor axons also extended past their targets in the cervical enlargement, in many cases growing in the corticospinal tract to lumbar levels of the cord. By about 17 days postnatal these misdirected axons or axon segments were withdrawn from the tract. Despite these errors in axon trajectories within the corticospinal tract, terminal arbors branching into targets in the spinal gray matter were topographically appropriate from the earliest stages of innervation. Thus visual cortical axons never formed connections in the spinal cord, forelimb sensorimotor axons arborized only in the cervical enlargement, and hindlimb cortical axons terminated only in the lumbar cord at all stages of development examined. Corticospinal arbors formed from collaterals that extended at right angles from the shafts of primary axons, most likely by the process of interstitial branching after the primary growth cone had extended past the target. Once collaterals extended into the spinal gray matter, highly branched terminal arbors formed within 2-4 days, beginning at about 4 and 8 days postnatal for the cervical and lumbar enlargements, respectively. These results show that specificity in corticospinal connectivity is achieved by selective growth of axon collaterals into appropriate spinal targets from the beginning and not by the later remodeling of initially diffuse connections. In contrast, errors occur in the initial outgrowth of axons in the corticospinal tract, which are subsequently corrected.

Selective neurite outgrowth of cultured cortical neurons on specific regions of brain cryostat sections.

During development, axons of the mammalian cerebral cortex show a high degree of selectivity in their growth into specific regions of the central nervous system (CNS). A number of studies have shown that growing axons are guided by permissive or inhibitory membrane-bound molecules. Cryostat sections of the developing brain provide a useful assay to investigate possible membrane-bound guidance cues because such cues are retained in their normal in situ locations in specific regions of the CNS. Moreover, cryostat sections can also be subjected to various treatments that affect membrane-bound molecules. Therefore, to determine the ability of such cues to regulate the growth and guidance of cortical neurites into specific brain regions at different stages of development, we used an in vitro assay system in which explants from newborn hamster cortex were plated onto various regions of cryostat sections from developing and adult hamster brain. Neurite outgrowth from cortical explants onto the cryostat sections was visualized with a fluorescent vital dye. Results showed first that cortical neurites grew robustly on neonatal cryostat sections but only sparsely on sections from adult hamster. Second, cortical neurites grew preferentially on regions of the neonatal sections such as the cortex, basal ganglia, brainstem, thalamus, and colliculus, which are either pathways or targets for cortical axons in vivo. In contrast, cortical neurites avoided growing on the cerebellum and olfactory bulb, which are neither targets nor pathways for cortical neurites in vivo. Results also showed that cortical neurites extending onto cortical regions of neonatal sections preferred to grow along the radial axis of the cortex. Finally, heat treatment of the neonatal sections drastically reduced cortical neurite outgrowth. Taken together, these results suggest that the growth and guidance of cortical neurites is influenced by substrate-bound, developmentally regulated, heat-sensitive guidance cues preserved in the cryostat sections.

Critical stages for growth in the development of cortical neurons.

In order to study the role of efferent connectivity in the development of CNS neurons, the growth of pyramidal tract neurons within the hamster sensorimotor cortex was studied during normal development and after early postnatal lesions of the pyramidal tract. We first determined, by a combination of Nissl and retrograde HRP techniques, that within the lumbar representation of cortical layer 5B in adult animals two cell populations exist: a large-celled population (40% of the total) projecting to the spinal cord and a small-celled population (60% of the total) projecting intracortically and to targets rostral to the medulla. We could not determine whether large layer 5B cells in the infant sensorimotor cortex also represent the corticospinal population. Nevertheless, measurements of the growth in cross-sectional area of the large cells from 7 days postnatal to adulthood showed that these cells continue to grow until 51 days of age. The most rapid rate of growth occurs between 7 and 14 days, during which time the cross-sectional area of the cell bodies triples, coincident with the arrival of corticospinal axons in the lumbar cord and the beginning of target innervation (Reh and Kalil, '81). The growth of the large neurons in layer 5B was then charted after the pyramidal tract was cut ipsilaterally in the medulla at various postnatal ages. Early lesions of the tract (4-8 days postnatal) interrupt lumbar projection fibers before they establish synapses in the cord. Nevertheless, cortical cell bodies in the lumbar representation continue to grow normally after axotomy until 11 days after birth. At this time, large cells are arrested in development and their cell size remains in the 11-day stage (50% of normal adult large cell size) indefinitely. In contrast, adult lesions of the tract cause a 60% shrinkage of large cells, which in the adult represent corticospinal neurons. No evidence for cortical cell death was found after pyramidal tract lesions at any age. The results of axotomy reveal a turning point in the development of layer 5B cortical neurons. Before the age of 11 days the large cells have an independent program of cell growth that proceeds despite axotomy. After this time, the large cortical neurons appear to require intact axons for further growth and, in the absence of normal connectivity, are arrested in development.

Organization of motoneuronal pools in the rostral spinal cord of the sea robin, Prionotus carolinus.

The functional organization of the motoneurons in the spinal cord of the sea robin, Prionotus carolinus, was studied by means of retrograde transport of horseradish peroxidase (HRP). This species has a complex pectoral apparatus which includes not only a webbed fin, but also three independently mobile fin rays. The motoneurons in the rostral spinal cord fall into two longitudinal columns: dorsal and ventral. The motoneurons of the ventral column innervate the appendicular musculature of the pectoral apparatus. Within the ventral motor column of the rostral spinal cord, four distinct motoneuronal pools were found. The largest pool is situated at the rostral-most end of the spinal cord and contains the motoneurons that innervate the musculature of the webbed pectoral fin. The motoneurons that innervate the fin rays are located in sequentially more posterior pools so that the anteroventral fin ray is controlled by motoneurons situated farthest caudally. The somatotopic arrangement exactly corresponds to the sensory somatotopy determined previously. Furthermore, each fin ray has its sensory representation in a unique accessory spinal lobe which is connected in a reflex fashion to the motoneuronal pool that provides motor output to the same fin ray.

Specificity of corticospinal axon arbors sprouting into denervated contralateral spinal cord.

Previous studies have reported considerable plasticity in the rodent corticospinal pathway in response to injury. This includes sprouting of intact axons from the normal pathway into the contralateral spinal cord denervated by an early corticospinal lesion. We carried out the present study to obtain detailed information about the time course, origin, and degree of specificity of corticospinal axons sprouting in response to denervation. Hamsters (Mesocricetus auratus) ranging in age from 5 to 23 days received unilateral lesions of the left medullary pyramidal tract. Two weeks after the lesion, small regions of the right sensorimotor cortex opposite the lesion were injected with the plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After a further 2 week survival period, immunohistochemistry was carried out on frozen sections of the fixed brains and spinal cords. Detailed morphological analysis of PHA-L labeled corticospinal axons revealed that sprouting from the intact corticospinal pathway into the contralateral denervated spinal cord occurred only at local spinal levels and not at the pyramidal decussation. Arbors sprouting into the denervated cord frequently arose from corticospinal axons that branched into the normal side of the cord as well. Sprouting was maximal after early lesions (5 days) and declined with lesions at later ages up to 19 days. Sprouting corticospinal axons arborized with the same degree of functional and topographic specificity as previously reported for normal corticospinal arbors (Kuang and Kalil: J. Comp. Neurol. 292:585-598, '90), such that axons arising from somatosensory cortex projected only to the dorsal horn, those from motor cortex innervated only the ventral horn, and normal forelimb and hindlimb topography was preserved. Sprouting fibers also had normal branching patterns. Parallel studies of developing corticospinal arbors showed that sprouting could not be attributed to maintenance or expansion of early bilateral connections. These results suggest that local signals, most likely similar to those governing normal corticospinal development, elicit corticospinal sprouting and determine specificity of axon arbors.

Branching patterns of corticospinal axon arbors in the rodent.

Despite extensive study of corticospinal connections in a variety of species, little is known about the detailed morphology of corticospinal axon arbors. Results in previous studies of primates based on intra-axonal filling with horseradish peroxidase (HRP) staining of a limited sample of fibers suggest that corticospinal arbors branch widely to multiple motoneuronal pools. To determine whether this pattern of corticospinal connectivity is present in nonprimate species as well, we studied the branching patterns of corticospinal axon arbors in a rodent species, the golden hamster. The axons were labeled by iontophoretic injection of Phaseolus vulgaris-leucoagglutinin (PHA-L) into small regions of the forelimb and hindlimb sensorimotor cortex, and immunohistochemistry with the peroxidase-antiperoxidase (PAP) method was used to reveal fine details of terminal arbors within the cervical and lumbar enlargements of the spinal cord. As in higher mammals, corticospinal connections are topographically organized. Moreover, corticospinal axons arising from somatosensory cortex project primarily to the dorsal horn, whereas those from motor cortex terminate most heavily in the ventral horn. This differential projection pattern, not previously demonstrated in rodents, implies functional differences between somatosensory and motor components of the corticospinal pathway. Reconstruction of corticospinal arbors in the ventral horn showed that in both cervical and lumbar spinal cord segments, axons branch widely into interneuronal regions. A surprising number appear to extend into motoneuron cell groups, and some of these axons branch into multiple motoneuronal pools. Widely divergent corticospinal axons that branch to multiple motoneuron pools have been shown to mediate activity in functionally related muscle groups of the primate forearm. The present results suggest that in other species, such as the rodent, a similar divergence of corticospinal arbors may also function to facilitate activity in subsets of muscles.

Morphology and cellular interactions of growth cones in the developing corpus callosum.

Previous studies of growth cones in invertebrates have shown that they become larger and more complex when changing direction in response to cell-specific contacts (Bentley and Caudy, '83; Raper et al., '83b; Caudy and Bentley, '86). In pathways of the vertebrate nervous system, analogous regions, termed "decision regions," have been identified in which axons change direction and their growth cones become more elaborate than when tracking along straight trajectories (Tosney and Landmesser, '85a; Bovolenta and Mason, '87). In order to assess the generality of these principles to the mammalian CNS, we studied the morphology of growth cones and their interactions with the environment in the developing corpus callosum. Given the straight pathway that callosal axons could use to navigate across the callosum, one might predict that later arriving axons would extend on those growing out earlier and that therefore, by analogy with previous studies, many growth cones would have simple tapered morphologies. Surprisingly, however, virtually all growth cones in the callosal white matter, regardless of age or position, were complex with broad lamellipodial veils and/or numerous, often lengthy filopodia. Only growth cones entering the cortical target were consistently smaller. As seen in the EM, the predominant elements in the callosal pathway are other axons and growth cones; we found no evidence for specialized contacts. These results suggest that there is no specific decision region for the fiber population as a whole; rather it is possible that in this mammalian CNS pathway individual growth cones respond independently to molecular cues broadly distributed in the callosum.

Development of callosal connections in the sensorimotor cortex of the hamster.

To investigate the development of corpus callosal connectivity in the hamster sensorimotor cortex, we have used the sensitive axonal tracer 1,1 dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI), which was injected either in vivo or in fixed brains of animals 3-6 days postnatal. First, to study changes in the overall distribution of developing callosal afferents we made large injections of DiI into the corpus callosal tract. We found that the anterogradely labeled callosal axons formed a patchy distribution in the contralateral sensorimotor cortex, which was similar to the pattern of adult connectivity described in earlier studies of the rodent corpus callosum. This result stands in contrast to previous retrograde studies of developing callosal connectivity which showed that the distribution of callosal neurons early in development is homogeneous and that the mature, patchy distribution arises later, primarily as a result of the retraction of exuberant axons. The initial patchy distribution of callosal axon growth into the sensorimotor cortex described in the present study suggests that exuberant axons destined to be eliminated do not enter the cortex. In addition, small injections of DiI into developing cortex resulted in homotopic patterns of callosal topography in which reciprocal regions of sensorimotor cortex are connected, as has been shown in the adult. Second, to study the radial growth of callosal afferents we followed the extension of individual callosal axons into the developing cortex. We found that callosal axons began to invade the contralateral cortex on about postnatal day 3, with little or no waiting period in the callosal tract. Callosal afferents then advanced steadily through the cortex, never actually invading the cortical plate but extending into layers on the first day that they could be distinguished from the cortical plate. The majority of callosal axons grew radially through the cortex and did not exhibit substantial branching until postnatal day 8, the age when the cortical plate disappears and callosal afferents reach the outer layer of cortex. This mode of radial growth through cortex prior to axon branching could serve to align callosal afferents with their radial or columnar targets before arborizing laterally.