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Publisher Summary The chapter discusses the Axon decussation and midline glia in the developing ferret auditory hindbrain. The timing of trapezoid body fiber out growth in relation to the timing of medial superior olive (MSO) and lateral superior olive (LSO) cell migration to the ventral hindbrain, the apparently changing distribution of glycoconjugates in relation to trapezoid body formation, and the distribution of midline glia in the hindbrain. Pioneer fibers from the cochlear nucleus reach the midline before the cells of the superior olivary complex complete migration to the ventral hindbrain. These fibers may be guided to their site of decussation, as are spinal cord commissural neurons, by netrin diffusing from the floor plate. Subsequently, the superior olivary complex begins to release a local chemotropic factor, resulting in the collateralization of cochlear nucleus fibers and projection of those fibers into their target nuclei. As the cochlear nucleus projection in the trapezoid body continues to increase, contralaterally projecting fibers are guided through the midline by a combination of extracellular matrix and glial guidance cues. Inappropriate fibers are prevented from crossing the midline by both the molecular and structural characteristics of the midline glia.

The distribution of glycine-immunopositive cells and axonal endings was studied in the adult and early postnatal ferret superior olive. As in other species, the most prominent glycine-immunopositive cell group in the adult ferret superior olive was the medial nucleus of the trapezoid body. Other darkly immunostained cells were present, although more scattered, in most periolivary regions, including the lateral and ventral trapezoid body nuclei. In the lateral superior olivary nuclei, glycine-immunopositive cells were intermingled with immunonegative cells. A comparable population of cells in the ipsilateral lateral superior olivary nucleus was retrogradely labeled in cases with unilateral injections of tritiated glycine in the inferior colliculus. Glycine-immunopositive puncta were widely distributed in the neuropil in most periolivary regions, including dense accumulations in the dorsomedial periolivary region and ventral and lateral nuclei of the trapezoid body. In the lateral and medial superior olivary nuclei, immunopositive puncta were distributed around the principal cells in characteristic perisomatic halos. In postnatal ferrets, immunopositive cell bodies were first observed by postnatal day 7 and were distributed in regions comparable to regions in the adult, with the exception that immunopositive cells in the lateral superior olivary nucleus did not appear until about postnatal day 28. There was diffuse staining in the neuropil in principal and periolivary nuclei by postnatal day 7. During the third postnatal week, the immunostaining in the neuropil began to take on a more granular appearance and immunopositive puncta could be seen by postnatal day 35. In the lateral and medial superior olivary nuclei, the earliest distribution of immunostaining in the neuropil was nonuniform, being greater in the high-frequency, medial, and ventral regions, respectively. The density gradient in these areas was gradually eliminated over the next 2 postnatal weeks as immunostained processes and endings appeared over greater portions of the nuclei. © 1995 Wiley-Liss, Inc.

The present study examined the fate and migration of transplanted astrocytes in different host ages. Additionally, the effect of donor cell age was examined in relation to cell migration. Cultured astrocytes from 5,12 and 30 days in vitro were transplanted into young (postnatal day 5 and 21), adult (4.5 month), and aged (21 month) animals. The transplanted cells were labeled with Fast Blue, Fluorogold or DiI. The results confirmed previous studies demonstrating that transplanted cells were able to migrate successfully through host central nervous system and extended those findings to show that the age of the host significantly influenced donor cell migration distance. Migration was most extensive in young animals, as conditions supporting cell migration appeared to be lacking in older animals. Donor cells preferentially migrated on myelinated fiber tracts, rather than on unmyelinated fiber tracts or gray matter. The donor cells were not glial fibrillary acidic protein positive, indicating that either the cultured type 1 astrocytes did not survive transplantation or underwent significant remodeling of the intermediate filament network. It is also possible that a subpopulation of cells, possibly immature astrocytes which are present in the transplanted cell suspensions, flourished and subsequently migrated in the host brains.

The laterality of projections from the lateral superior olivary nucleus (LSO) to the inferior colliculus was studied in adult and immature postnatal ferrets. In the adult ferret, large unilateral injections of horseradish peroxidase (HRP) in the inferior colliculus labeled about equal proportions of cells in the ipsilateral and contralateral lateral superior olivary nuclei. The contralateral labeled cells consistently were more densely labeled than those on the ipsilateral side. Double labeling experiments using fluorescent dyes indicated that only about 3% of LSO cells in the adult give rise to collaterals ending in the inferior colliculus on both sides. As expected, the distribution of labeled cells varied topographically in the LSO as a function of the injection site in the inferior colliculus. Dorsolateral inferior collicular injections labeled cells in the lateral limb of the LSO, whereas ventromedial injections labeled cells in the medial limb of the LSO. The proportion of ipsilateral and contralateral labeled cells also varied across the lateral-medial axis of the LSO in some cases. A gradient in laterality was observed in these cases with the lateral limb of the LSO containing the highest proportion of contralateral labeled cells, and the medial limb, the highest proportion of ipsilateral labeled cells. Larger inferior collicular injections resulted in greater proportions of ipsilateral labeling in LSO than smaller injections. Finally, ipsilateral labeled cells tended to be in the marginal region of the LSO, whereas contralateral labeled cells were more common within the core region of the LSO, irrespective of the location along the lateral-medial axis of LSO. The contralateral predominance of labeled cells, greater density of labeling in contralateral cells, different topographic distribution, and regional segregation of ipsilateral and contralateral labeled cells were typical of the LSO in ferret kits by birth, one month before the onset of hearing. Nevertheless, the relative proportion of ipsilateral and contralateral projection cells appears to change during postnatal development. © 1993 Wiley-Liss, Inc.

In recent years, the important role of the extracellular matrix in neural development has been increasingly recognized. In order to begin to examine what role might be played by the extracellular matrix in the developing dorsal lateral geniculate nucleus (dLGN), the present study used immunocytochemistry to assess the distribution of a recognized extracellular matrix molecule, fibronectin (FN), during postnatal development of the ferret dLGN. Prior to the segregation of cell layers, no clear pattern of FN distribution can be distinguished within the dLGN. By P16, when layers A and A1 are separated by an interlaminar space, FN is localized in that space. By P24, FN is present not only between layers A and A1, but also within layers A and A1 as bands parallel to the laminar borders. These bands appear to correspond spatially and temporally to the development of sublaminar boundaries in the ferret (Hahm and Sur, Neurosci Abstr 14:460, 1988). By the end of the first postnatal month, immunoreactivity is diminished but still present. FN is no longer present at P44 or in the adult. Adjacent sections were incubated with antiserum to glial fibrillary acid protein (GFAP). When a laminar pattern of GFAP can be distinguished, it coincides with the interlaminar distribution of FN. The findings described here are consistent with a role for FN in the development of a laminar organization in the dLGN.

The dendritic morphology of cells in the lateral superior olivary nucleus was studied with the Golgi method in adult and postnatal ferrets. The lateral superior olivary nucleus in the adult ferret is a convoluted structure with an M-shape in frontal sections. The major cell type appears to have disk-shaped dendritic trees. Most dendritic trees appear to be approximately orthogonal to the curved medial-lateral axis of the nucleus. Depending on their position in the limb and on the plane of section with respect to the dendritic tree, the disk-shaped cells are either bipolar or radiate in orientation. One subclass of disk-shaped cells has secondary dendritic branches that end as tufts of tendril-like processes. In a second subclass of cells, the dendrites exhibit several orders of dichotomous branching and lack obvious tufts of terminal processes. Marginal cells are observed at the border of the nucleus and have dendrites restricted to the margins of the cell plate. The bipolar orientation of disk-shaped cells orthogonal to the axis of the limbs is already apparent by the time of birth. Transient spines and other appendages are abundant on somata and dendrites during the first postnatal week. By the end of the first postnatal month only distal appendages are found. Tufts of fine tendril-like processes appear at the ends of dendrites between postnatal days 28 and 56.

The development of neurons in the dorsal lateral geniculate nucleus (dLGN) of pigmented ferrets was studied by using the Golgi-Hortega technique. In adult ferrets, four dLGN cell classes were defined on the basis of somatic and dendritic morphology. Classes 1 and 2 were divided into stellate and oriented subtypes. Class 1 and 4 cells are characterized by filiform appendages, class 2 cells by club-like appendages, and class 3 cells by stalked appendages. At birth, dLGN neurons have simple dendritic arbors. During the first postnatal week, dendritic length and proximal branching density increase markedly. By postnatal day 21 (P21), dendritic morphology begins to take on mature characteristics and by the time of eye opening (P30-P35), most neurons can be classified. Also by that time, dLGN cells are covered with abundant filiform appendages. Developmental changes in appendage density were quantified for class 1 stellate cells. These data reveal that appendage density reaches a peak at P56, decreases sharply until P90, and then gradually declines to mature levels by P180. Elaboration and elimination of transient appendages occurs centrifugally; at maturity appendage density remains greater distally.

In normal development, cell layers in the dorsal lateral geniculate nucleus (dLGN) segregate from a relatively homogeneous cell group. If all retinal input is removed prior to this segregation, the layers fail to form. In the present study, we used ultrastructural and morphometric analyses to study dLGN development in the tree shrew following neonatal removal of retinal input. The goal of the present study was to determine whether there are differences between normal animals and enucleates in the development of dLGN cells and their interrelationships with each other and/or with the surrounding glia, which might explain the failure of cellular lamination in enucleated animals. The results indicate that although the development in enucleated animals may take place somewhat more slowly, by P90 cell size and density are not significantly different from normal. These results, coupled with the observation that the dLGN in enucleates is smaller than in normals, suggest that the removal of retinal input results in dLGN cell loss. At both the light and electron microscopic level, cells in the developing normal dLGN are arranged in bands of immediately adjacent cells. In enucleates, dLGN cells are less frequently in immediate contact and are arranged in small groups or clumps which may be separated by degenerating cells. The present data suggest that the presence of retinal input may be necessary to allow dLGN cells to maintain the intercellular relationships necessary for laminar segregation to take place.

Dendritic morphology and development in the medial superior olivary nucleus of the ferret were studied using the Golgi method. In the adult ferret most medial superior olivary neurons had disk-shaped dendritic fields. These dendritic fields were oriented such that cells in a coronal plane of section appeared bipolar with major lateral and medial dendritic axes. In the horizontal plane the dendrites radiated about the soma. Dendrites of principal cells branched distally into tufts of numerous, tertiary processes that were beaded and thin. Peripheral cells in the fiber mantle encircling the nucleus were generally spindle-shaped or tripolar and lacked the tufted dendrites of principal cells. The dendrites of these peripheral cells coursed parallel to the nucleus both dorsoventrally and rostrocaudally. Horizontally oriented dendrites were observed even at birth for some cells in the medial superior olivary nucleus and bipolar dendritic fields were typical of most cells by the end of the second postnatal week. Dendrites of immature cells varied in caliber and radiated in all directions from the soma. Around postnatal days 8-10 transient appendages appeared on the soma and dendrites, first proximally and then more distally. These appendages persisted until the first postnatal month. Cell size and dendritic radius increased markedly during this same period. Postnatal days 28-30 were marked by the first appearance of tufts of tertiary dendritic branches. The tendril-like processes continued to increase in length until about the end of the second postnatal month.

In the spinal ganglia of the chick embryo, two neuronal populations can be distinguished: large, early differentiating ventrolateral (VL) cells and small, late differentiating dorsomedial (DM) cells. It was found that, beginning with stage 25, the DM cells originate from a narrow band of small, immature cells at the medial border of the ganglion, extending to the dorsolateral border. We have designated this band as the inner and outer marginal zone. Neuronal death was investigated in thoracic ganglion 18 and brachial ganglion 15 by counting degenerating cells, separately for the VL and DM populations, at every stage from stage 24 (4% days) to stage 38 (12 days). In both ganglia, separate degeneration periods were found for the VL and DM populations which do not overlap. The peaks of degeneration are: stage 27 (5% days) for the VL population in ganglion 18, stage 30 (6% to 7 days) for VL in ganglion 16, and stage 35 (8% days) for DM in both ganglia. Daily injections of 6 pg of nerve growth factor, (NGF) in 6 to 12 ~1 of 0.9% sterile salt solution into the yolk sac from stage 21 (3% days) to the day of sacrifice resulted in a significant reduction of neuronal death in the VL population of ganglion 18 and in the rescue of practically all VL neurons in ganglion 15 and all DM neurons in both ganglia, which normally would have died. This is the first demonstration of an NGF effect on VL neurons. In the analysis of the multiple effects of nerve growth factor (NGF), the sympathetic ganglia have played the dominant role; the sensory ganglia which are the other target of NGF have attracted much less attention. Yet, if one entertains the notion that NGF might be the natural trophic maintenance agent for these two neuron types (i.e., that NGF is actually produced by their target organs), then one approach to the testing of this hypothesis would be to engage in in viva experiments. For this

In the present study, we examined the colliculogeniculate projection in normal adult tree shrews and in adults that were bilaterally enucleated at birth. We injected lectin-conjugated HRP into superficial superior colliculus and then mapped the pattern of anterogradely transported enzyme in the ipsilateral dLGN. In normal adult tree shrews, the results confirm that the colliculogeniculate projection is laminated and terminates predominantly in small-celled layers 3 and 6 and in the interlaminar space between layers 4 and 5 (Fitzpatrick et al., 1980); we report an additional sparse projection to layer 4. In bilaterally enucleated animals, the colliculogeniculate projection is unlaminated and tends to terminate in the lateral two-thirds of the dLGN even though synaptic sites are vacated throughout the nucleus. We suggest that this preference may be due to a specificity of the colliculogeniculate fibers for the lateral two-thirds of the dLGN, which, in normal adult tree shrews, contains cells with similar physiological characteristics. We further suggest that the normal lamination of the colliculogeniculate projection in the lateral two- thirds of the dLGN may be due to competition with retinogeniculate fibers so that colliculogeniculate fibers terminate predominantly in layers containing small, W-like cells.

We examined the ultrastructural development of the lateral geniculate nucleus (LGN) in postnatal tree shrews to distinguish which features, if any, show a correlation with the development of cell layers. Our data indicate that synaptogenesis has begun at birth (P0) which is prior to the development of cell layers. At postnatal day 8 (P8), when laminar borders can be distinguished, the majority of synaptic profiles are still immature. Although some mature synaptic profiles can be identified at P8 and especially at P15, complex synaptic arrangements characteristic of adults are absent at that time. Growth cones are present at all 3 ages but are less prevalent at P8 and P15 than at birth and appear to be present in slightly higher concentrations in the interlaminar spaces. These results suggest that LGN cell layer formation does not correlate with the beginning of synaptogenesis but that it may correlate with an increase in growth cones in the interlaminar spaces.

We have demonstrated retrograde transport of nerve growth factor to the dorsal root ganglia in the chicken embryo. Polyacrylamide gel pellets impregnated with 125I-labeled nerve growth factor were implanted subcutaneously in the leg of stage 36 (10-day) chicken embryos. The embryos were killed 8 hr after implantation and were processed for routine autoradiography. Examination revealed significant labeling of all lumbar dorsal root ganglia on the side of the implanted pellet. The lateral motor columns and ventral roots as well as the dorsal roots on both sides were unlabeled. These observations show that in the chicken embryo, during the period of sensory cell sensitivity to nerve growth factor, there is a selective uptake and retrograde transport of nerve growth factor from the target area to the dorsal root ganglia. These observations supplement reports from other laboratories in which selective retrograde transport of nerve growth factor in neonatal and adult rodents has been demonstrated.

Publisher Summary This chapter discusses the features of lamination in the mature dorsal lateral geniculate nucleus (LGN), development of layers in LGN, and the role of retinogeniculate fibers in the development of individual features of lamination. In the absence of cytoarchitectural and cytological features of lamination, most mammals exhibit laminar segregation of retinal input to the LGN. The evaluation of the effect of bilateral enucleation on cytological development is based solely on Nissl-stained material. The formation of spaces between cell layers in the LGN can be compared to the formation of other cytoarchitectural features in different systems. The retinogeniculate fibers have a “trophic” effect on the LGN cells, stimulating dendritic growth, and perhaps other features of differentiation. The combination of dendritic growth of cells in adjacent layers and the dense projection of the corticogeniculate fibers on the distal LGN cell dendrites thus result in the formation of the interlaminar spaces and the laminar projection pattern of the corticogeniculate fibers. The development of cytological features characteristic of individual layers, in the absence of retinogeniculate fibers, is presumably due to a combination of intrinsic cellular differentiation independent of afferent input and, perhaps to some degree of “trophic” stimulation by intact afferent systems such as the late-arriving corticogeniculate projection.

The role of retinogeniculate afferents in the development of patterns of connections between visual cortex and the lateral geniculate nucleus (LGN) was addressed by studying the effect of bilateral enucleation at birth on those patterns of connections in tree shrew. In normal adult tree shrews there are six LGN cell layers separated by cell-sparse interlaminar spaces. The reciprocal connections between the LGN and visual cortex are restricted to a column running across all six LGN layers; the geniculocortical projection arises from the cell layers while the corticogeniculate projection terminates primarily in the interlaminar spaces. At birth, when the experimental animals were bilaterally enucleated, the retinogeniculate fibers have begun to segregate by eye but neither the cytological characteristics of individual layers nor the interlaminar spaces have yet formed, and the corticogeniculate fibers have not entered the nucleus. Bilateral enucleation does not prevent the development of the cytological characteristics of individual layers but the interlaminar spaces do not develop. The results of [3H]proline/HRP injections into visual cortex in animals bilaterally enucleated at birth indicate that in the absence of retinogeniculate fibers, and thus interlaminar spaces, the corticogeniculate fibers do not concentrate at the laminar borders but instead spread across all six LGN cell layers. Despite the failure of this projection to concentrate at laminar borders, the corticogeniculate fibers do terminate within a restricted projection column.

In the present study, we quantified the distribution of growth cones and synapses in 2 developing layers, as well as in the intervening interlaminar space of the dorsal lateral geniculate nucleus (dLGN) in tree shrews. Our goal was to gain insight into mechanisms involved in the segregation of dLGN cells into layers during development. We sacrificed tree shrews before (P0), during (P4 and P7), and after (P15) laminar segregation as well as at maturity (P90). The dLGN from each animal was sectioned horizontally, and all tissue for analysis was blocked from the middle third of the nucleus along the dorsoventral axis. Each micrograph was coded and blindly scored for the number of growth cones and synapses in layers 4 and 5 and the intervening interlaminar space. We also measured each growth cone and classified synapse type. Statistical analyses of these data reveal that neither growth cones nor synapses are significantly more common in the interlaminar space early in the period of laminar segregation (P4). By nearly a week after the interlaminar space can first be distinguished (P7), there are more growth cones in the interlaminar space than in the layers, but this difference is no longer present at (P15). These results suggest that, although neuropil development at the laminar borders may not play a role in the onset of laminar segregation, it may contribute to the widening of the interlaminar spaces once this process has begun. In addition, growth cones continuously decrease in number and become less bulbous and more linear in shape with development. Synapses, on the other hand, continuously increase in number with age and pass through a transient period characterized by heavy spinous terminations.

In order to gain insight into the mechanisms involved in the formation of groupings of functionally similar cells in the developing nervous system, we have studied the formation of cell layers in the developing dorsal lateral geniculate nucleus (dLGN). To examine the possibility that a higher affinity or adhesion between cells in individual layers may play a role in laminar segregation, we studied cellular interrelationships in the dLGN of tree shrews before (P0), during (P4 and P7), and just after (P15) laminar segregation has taken place. We compared our observations at these stages of development with similar observations in the adult. In none of the cases do we see evidence of gap junctions either between adjacent neurons or between neurons and processes in the surrounding neuropil. However, we frequently observe the presence of puncta adherentes between adjacent neurons at all stages of development. These profiles are also present between neurons and cellular processes in the neuropil. We also see subsurface cisternae in all of our cases, although these are more pronounced before and during interlaminar space formation. As with the puncta adherentes, these are found both between adjacent neurons as well as between neurons and other elements in the neuropil. We also see some evidence of what appear to be cytoplasmic bridges between adjacent neurons; these are quite rare but appear to be present only before and during laminar segregation. Finally, we frequently see cytoplasmic processes interdigitated between otherwise immediately adjacent cells. These processes also are often found oriented along other portions of the neuronal plasmalemma. Whether these processes are portions of neuronal growth cones or glial processes is impossible to determine at this time. Because of the potential role glial processes may play in the formation and maintenance of laminar cell groupings during layer formation, we have also made a preliminary survey of whether glial cells can be distinguished ultrastructurally at the stages we have studied.