Calbindin immunoreactivity in the auricular lobe and interauricular granular band of the cerebellum in bullfrogs.

Calcium binding protein (CaBP) immunoreactivity in the cerebellum of bullfrogs was examined, concentrating on cells associated with the auricular lobe. While anti-calretinin and anti-parvalbumin also immunoreacted with the same cell populations, anti-calbindin exhibited the most robust and typical pattern of immunostaining. Calbindin immunoreactivity was observed in various populations of cells in the auricular lobe and interauricular granular band of the cerebellum, in the cerebellar peduncle, and in a bundle of interauricular commissural fibers which course through the dorsal, marginal, part of the molecular layer. Cells in the granular layer of the ventral part (i.e., corpus cerebelli) of the cerebellar plate were not CaBP-immunoreactive, nor were any fibers in the molecular layer of this cerebellar region. We believe that axons of CaBP-immunoreactive granule-like cells of the auricular lobes contribute to the formation of the interauricular fiber bundle, which corresponds to the lateral commissure of urodele amphibians. The pattern of calbindin immunoreactivity in the auricular lobes and marginal part of the cerebellar plate provides additional evidence that this cerebellar compartment, which is already present in tadpoles, has a distinct origin, biochemical characterization and connectivity and is separate from the compartment that forms the corpus cerebelli of frogs during metamorphosis.

Calbindin immunoreactivity in Purkinje cells of the bullfrog cerebellum during thyroxine-induced metamorphosis.

Calbindin-immunoreactive Purkinje cells were identified in the cerebella of frog tadpoles that had been treated with thyroxine to accelerate metamorphosis. The dorsal part of the cerebellar plate contained the full complement of Purkinje cells which were all CaBP-immunoreactive, while in the ventral part of the cerebellum Purkinje cells acquired CaBP-immunoreactivity only after several days of thyroxine treatment. The ventral group of Purkinje cells was separated from the dorsal group by a distinct gap, which is the site of a shallow sulcus in adult frogs. Additionally, following thyroxine treatment, the numbers of CaBP-immunoreactive Purkinje cells in the ventral group were only half the numbers seen in frogs that metamorphosed spontaneously. We suggest that the variation in the CaBP-immunoreactivity of the dorsal and ventral groups of Purkinje cells, along with the gap in the Purkinje cell layer between the two groups, may be indicative of two distinct populations of Purkinje cells, with distinct patterns of generation, maturation, and perhaps, origin and connectivity, in the cerebellum of frogs.

Effects of 60 Hz electric and magnetic fields on the development of the rat cerebellum.

The effects of extremely low frequency (ELF) electromagnetic (EM) fields on the maturation of the rat cerebellum were studied. Newborn rats were exposed to 60 Hz electric and magnetic fields under three different combinations in a specially constructed apparatus. The pups were irradiated for 7-8 h daily, with a 30-min interruption for nursing. Pups were kept with their mothers for the remainder of the time. After approximately 1, 2, or 3 weeks of exposure, the pups were killed. Control pups were sham exposed. The somatic growth of the irradiated rats did not show any significant difference from sham-exposed controls. At 1 kV/m and 10 gauss exposure, there was a small but statistically significant decrease in cerebellar mass. In rats exposed at 1 kV/m and 10 gauss, DNA and RNA levels were significantly higher than those in sham-exposed controls at 6 and 13 days of age, but at 20 days, these two biochemical constituents were similar in both groups of rats. The ELF-EM treatment had no effect on protein and cerebroside concentrations. In terms of age effects, DNA and RNA exhibited increases from 6 to 13 days of age, and declined from 13 to 20 days. Protein and cerebroside levels exhibited increases during the 6-20-day periods. In rats exposed at 100 kV/m and 1 gauss, the DNA levels were initially less than those of sham-exposed controls at 8 days of age, reached approximately the same levels at 14 days, and then were higher than those of controls at 22 days. There was, therefore, a significant ELF-EM effect as well as a significant interaction between age and ELF-EM exposure. In terms of age effects, DNA levels for both control and exposed animals increased from 8 to 14 days. From 14 to 22 days, DNA levels of exposed rats continued to increase while those of the controls decreased. This age effect was significant. RNA levels in both groups of animals showed increases from 8 to 14 days of age, but the increase was less for the irradiated animals than for the controls. From days 14 to 22, RNA levels for both groups showed a reduction, but the decrease was greater in the irradiated than in control rats. ELF-EM treatment significantly reduced protein levels at 8 days of age, but at 14 to 22 days, protein levels of exposed rats were higher than those of controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of 60 Hz electric and magnetic fields on maturation of the rat neopallium.

This study was undertaken to determine the effects of extremely low frequency (ELF; 60 Hz) electromagnetic (EM) fields on somatic growth and cortical development, as well as biochemical and morphological maturation, of the rat neopallium. On the fifth day of pregnancy, female rats were put in pairs into plastic cages that were housed in a specially constructed apparatus for irradiation under three separate sets of combination and intensity: 1) 1 kV/m and 10 gauss; 2) 100 kV/m and 1 gauss; and 3) 100 kV/m and 10 gauss. The dams were exposed for 23 h daily, from days 5 through 19 postconception, after which they were returned to cages outside the exposure apparatus until they littered. The neonates were culled to eight pups per litter. At 0 (birth), 5, 12, and 19 days postnatally, they were killed for biochemical and morphological studies. Another group of pregnant rats was sham-exposed in an identical apparatus, which was not energized, and the pups were used as controls. The irradiated rats exhibited no physical abnormalities, nor did they show brain deformities such as swelling or herniation following exposure to ELF-EM fields. There was no difference in somatic growth between control and exposed rats, but a small reduction in cortical weight was observed in rats exposed at 1 kV/m and 10 gauss, and 100 kV/m and 1 gauss, respectively. Biochemical measurements of DNA, RNA, protein, and cerebroside concentrations indicated that among the three separate exposures, only the neopallium of rats exposed at 1 kV/m and 10 gauss showed a small reduction in DNA level, as well as small reductions in RNA and protein levels. No changes were noticed in cerebroside levels in any exposed animals, and there were no differences in protein/DNA and cerebroside/DNA ratios between control and exposed rats. Morphological observations did not reveal any detectable alterations in the irradiated rats. These results indicate that exposure to ELF-EM fields caused minimal or no changes in somatic growth and cerebral development of the rat.

In vitro tendon cell growth rates on a synthetic fiber scaffold material and on standard culture plates.

Growth rates of rat tendon fibroblasts cultured in a three-dimensional carbon fiber matrix were compared with those of cells cultured on standard flat culture plates. The carbon fiber has been used as a tissue scaffold for tendon and ligament repair in animal and clinical studies. While cell growth on the culture plates appears to follow a growth curve containing a lag phase, a log phase, and plateau phase of growth, cell growth in the fiber matrix was characterized by a suppressed log phase of growth. SEM and cytotoxicity studies indicated that this effect was not caused by growth-inhibiting or cytotoxic substances from the carbon fiber. While we cannot rule out the possibility that cell growth was influenced by the surface chemistry of the carbon substrate, evidence from this and other studies suggests that the observed effect was caused by a lack of readily available surface area for cell attachment and growth on the small fibers. Because cell colonies growing on individual fibers are limited (at least in theory) to growing in two directions only, they enjoy limited opportunities for cell migration and growth--in contrast with cell colonies on flat culture plates. These results suggest fundamental differences in the mechanisms controlling cell growth on planar vs. three-dimensional fiber substrates.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: II. Formation of the interauricular granular band.

This study examines the origin of cells in the interauricular granular band (iagb) in the cerebellum of the frog tadpole during early stages of development by means of histological and autoradiographic methods. Premetamorphic bullfrog tadpoles were exposed to multiple doses of 3H-thymidine (10 microCi/g body weight per exposure) at developmental stages ranging from 1 week to 1 year and were killed at either 6 or 12 months of age. The autoradiographic data were examined to determine the time when cells of the iagb were generated. Our findings show that initial generation of iagb cells begins at week 3 and that a peak in the formation of postmitotic neurons is reached at the age of 10 weeks. This is followed by other peaks of cell generation at the ages of 16 weeks, 10 months, and 11.5 months. The generation cycles of iagb cells are interrupted by periods of quiescence when label cannot be detected in any of the cells. These quiescent periods occur at the ages of 20-26 weeks, 7 months, and 12 months. These findings indicate that cells of the iagb are generated in a cyclical manner over the entire 1-year period which was studied. Comparison of our present data on iagb cell formation with the generation of cells in the EGL shows that the production of these two groups of cells is overlapping, but cells of the iagb begin and cease production before those of the EGL. On the basis of our findings we propose that the cells of the iagb and the EGL belong in separate cell groups which are generated by distinct subpopulations of germinal cells in the neuroepithelial cap.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: I. Generation of the external granular layer.

This study examines the time of origin of cells in the external granular layer (EGL) in the frog cerebellum during early stages of development. Premetamorphic bullfrog tadpoles were given multiple intraperitoneal injections of 3H-thymidine (10 microCi/g body weight per injection) at developmental stages ranging from 4 weeks to 1 year and were killed at either 6 or 12 months of age. Autoradiograms were analyzed to determine the time when cells of the EGL were generated by an examination of the labeling pattern in the neuroepithelial cap where EGL cells were presumably formed and in the EGL into which they migrated. The developmental stage of the cerebellum in the 6-month-old tadpole was essentially the same as that of the 12-month-old animal except for an increased size in the older tadpole. The cerebellum in both age groups contained a distinct neuroepithelial cap and an EGL, which was somewhat better formed in the 12-month-old tadpole. Some heavily labeled cells were found in the neuroepithelial caps of 6-month-old tadpoles from injection times of 6 weeks to 6 months. In the cerebella of 12-month-old tadpoles, however, heavily labeled cells were found in the neuroepithelial cap only with the injection time of 12 months; with injection times from 7 to 11 months, the cells were labeled lightly. Labeled EGL cells were found in the cerebella of 6-month-old tadpoles from an injection time of 6 weeks on; with injection times from 10 weeks to 6 months some EGL cells contained heavy amounts of label. In the cerebella of 12-month-old tadpoles, labeling of EGL cells was not detectable with injection times of 7-9 months; they contained light to medium labeling with injection times of 10 and 11 months and heavy labeling when injected at 12 months. These results indicate that EGL cells are generated continuously in premetamorphic tadpoles from the age of 6 weeks to 12 months. Furthermore, these results suggest that the rate of EGL cell formation is faster during the second half-year of development than during the first.

Granule cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Granule cell maturation in the cerebellum of bullfrog tadpoles was studied during both spontaneous and thyroxine-induced metamorphosis by using electron microscopy and Golgi-impregnated preparations. The production of cerebellar microneurons, a majority of which are granule cell precursors, was quantitatively compared during spontaneous and thyroxine-induced metamorphosis by using stereological methods and biochemical measurements of DNA. Granule cell migration and differentiation appeared morphologically similar during spontaneous and thyroxine-induced metamorphosis. In both instances, granule cells migrated tangentially along the pial surface, migrated into the internal granular layer, developed dendritic arbors, and formed synaptic contacts with the processes of Golgi cells and with mossy fibers. These events are similar to developmental processes that have been described in detail in other animals. Quantitative stereological measurements demonstrated similar overall patterns of change during spontaneous and thyroxine-induced metamorphosis. Most notably, increases in the volume of the external granule layer correlated with increases in the relative and total amounts of DNA. However, measurements of total DNA were consistently reduced during the period of accelerated change that occurs in thyroxine-induced metamorphosis, although external granular layer volume was greater in these tadpoles after 2 and 3 weeks of thyroxine treatment than in spontaneously metamorphosing tadpoles. While granule cell development in the frog is largely dependent on thyroid hormone, differences between thyroid-hormone-induced and spontaneously metamorphosing tadpoles suggest that normal patterns of cerebellar development are also dependent on events that occur in premetamorphic tadpoles in the absence of thyroid hormone.

Stellate cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Stellate cell development was studied in the bullfrog cerebellum during spontaneous and thyroxine-induced metamorphosis using the Golgi-Kopsch method and electron microscopy. Cells that possessed axosomatic synapses and resembled stellate cells were present even in the incipient molecular layer of the cerebellum in the premetamorphic tadpole. These cells may have originated from the early, transient wave of external granule cells that have been reported in the cerebellum of premetamorphic tadpoles in the first 6 months of development, and may constitute the variant population of stellate cells that are present later during development or the degenerating cells that have been observed during metamorphosis as scattered dying cells in the molecular layer. Typical stellate cells, whose development resembled the genesis and differentiation of stellate cells in birds and mammals, were initially observed at the outer border of the molecular layer that is adjacent to the external granular layer during the onset of metamorphosis. These stellate cells were bipolar with processes extending in a plane perpendicular to elongating parallel fibers, and with progressive development, became multipolar with dendrites oriented in various directions with respect to the pia. Stellate cell axons innervate the dendrites and somata of Purkinje cells and other stellate cells, and can be categorized into two types: (1) axons with extensive branching near the soma of origin, and (2) long axons with few branches that occasionally terminate in the Purkinje cell layer. Atypical neurons that did not resemble typical stellate cells were also present in the molecular layer; these might be classified as a stellate cell variant. The generation and differentiation of stellate cells can be induced 1 to 2 years prematurely by administering thyroid hormone to premetamorphic tadpoles. Like most events of cerebellar histogenesis in the frog, stellate cell development also appears to be largely a thyroid-dependent phenomenon.

Immunocytochemical localization and immunological characterization of vitamin D-dependent calcium-binding protein in the bullfrog cerebellum.

Antiserum prepared against rat renal calcium-binding protein (CaBP) was used with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique to localize the 28,000 molecular weight CaBP in the cerebellum of the bullfrog, Rana catesbeiana. Whole brains of premetamorphic tadpoles and adults were fixed in Bouin's solution for 2 or 24 h and embedded in paraffin. 8-microns parasagittal sections were prepared and treated by the PAP method. Purkinje cells of the cerebellum in tadpoles and adults were specifically stained for CaBP. In the premetamorphic corpus cerebelli, the stained Purkinje cells corresponded to the precociously developed Purkinje cells described previously. In the auricular lobe region of the cerebellum mature Purkinje cells were stained. In addition, smaller stained cells were seen. The latter were presumed to be immature Purkinje cells that would mature at the time of metamorphosis. Immunoblot procedure demonstrated cross-reactivity for the ranid brains in the 28,000 molecular weight region. This immunoreactive band comigrated with the immunoreactive band observed with purified rat renal CaBP. Although the exact functional significance of CaBP is unknown at this time, our immunocytochemical and immunological findings indicate that CaBP is an excellent marker for studies of Purkinje cell maturation.

Morphological characteristics of tendon cells cultured on synthetic fibers.

Filamentous carbon is currently being used as an implant material for tendon and ligament repair in humans. This material acts as a scaffold for the organization of new fibrous tissue growth. Primary cultures of rat tendon fibroblast cells (1 degrees RTF cells) were grown on carbon, Dacron, polyethylene and Nylon fibers in vitro. The morphological characteristics of these cells were examined. The process of cell migration from tendon explant to fiber substrate was similar for all four materials. Three morphological categories of cells were observed on these materials. (1) spherical dividing cells, (2) spindle-shaped migrating cells, (3) sheath-like migrating or stationary cells. The morphological characteristics and orientational behavior of cultured fibroblasts on these fiber materials were strongly influenced by the diameters of the fibers and by fiber surface characteristics such as longitudinal striations. The possible mechanisms of cell response to substrate geometric configuration are discussed along with the clinical significance of these experiments.

Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis.

Purkinje cell maturation during thyroxine-induced metamorphosis in premetamorphic bullfrog tadpoles was studied using electron microscopy and Golgi (silver-impregnated) preparations. Cerebella from tadpoles were examined following 1, 2, or 3 weeks of thyroxine treatment. Particular attention was paid to possible differences between the two populations of Purkinje cells previously described, i.e. (i) the smaller population located in the dorsal part of the cerebellum, where the Purkinje cells show dendritic arborization long before the appearance of the external granular layer, and (ii) the larger population located in the middle and ventral regions of the cerebellum, where the Purkinje cells begin to undergo maturation during metamorphosis when the external granular layer is established. Following thyroxine treatment, both populations of Purkinje cells showed rapid maturational change. In the mature (dorsal) group, dendritic growth resumed in the presence of an external granular layer increasing the complexity of their dendritic arbors. Moreover, climbing fiber synapses translocated from contacts on the soma to the thorns of growing dendrites, and somatic processes often disappeared. The immature (ventral) group showed dramatic differentiation of the perikaryon including polarization of cytoplasm with subsequent dendritic outgrowth and formation of somatic processes in the presence of climbing fibers. Stellate cell contacts appeared on the smooth portion of the soma of many Purkinje cells. Dendritic growth during thyroxine-induced metamorphosis was characterized by growth (elongation) with minimal branching, which is initially observed during spontaneous metamorphosis. Typically, these growing dendrites ended in growth cones, some with one or several filopodia. Developing Purkinje cell dendritic spines formed synapses with parallel fibers. The present study has provided an example of the dramatic nature of thyroxine's action in inducing the complex series of detailed maturational changes in the cerebellum 1-2 yr ahead of schedule. In addition, the results show that thyroxine-induced Purkinje cell maturation is more rapid and synchronous than that seen during spontaneous metamorphosis. It is concluded that Purkinje cell maturation during metamorphosis is largely dependent on thyroid hormone.

Effects of thyroidectomy and season on the external granular layer of the cerebellum in metamorphosing bullfrog tadpoles (Rana catesbeiana).

Transient formation of the cerebellar external granular layer (EGL) occurs during metamorphosis in frog tadpoles and is dependent on thyroid hormone. Late prometamorphic bullfrog tadpoles at similar stages of development were thyroidectomized and then killed after 1 month and 2 months during the fall and winter. The cerebellum was studied by qualitative and quantitative light microscopy. Thyroidectomy resulted in an inhibition of external metamorphic changes such as hind limb growth. Examination of the cerebellum after thyroidectomy showed the EGL greatly decreased in thickness after 1 month and in some cases was altogether absent after 2 months. Because the disappearance of EGL cells was a result of their inward migration into the IGL, we conclude that thyroid hormone was not necessary for granule cell migration. In addition, metamorphic change normally occurs in the late spring and summer in bullfrog tadpoles, and is inhibited during the fall and winter (metamorphic stasis). During the winter, the volume of the EGL was significantly less compared with tadpoles undergoing active metamorphosis. Although lowered temperature appears to contribute to the large decline in EGL thickness, significant seasonal differences in the EGL volume ratio were still observed in tadpoles maintained under conditions of constant temperature and light. Thyroxine administered to thyroidectomized tadpoles during the winter increased the thickness of the EGL. Therefore, the seasonal decline in the EGL thickness reflects a decrease in thyroid hormone activity, which in turn appears controlled by both internal (hypothalamic) and environmental (e.g., temperature) factors.

Golgi studies on Purkinje cell development in the frog during spontaneous metamorphosis. III. Axonal development.

The development and organization of Purkinje cell axons and their collaterals was studied in the bullfrog using the Golgi-Kopsch method. In the tadpole, axonal collaterals are few and usually unbranched. In the adult, however, intracortical axonal collaterals of Purkinje cells are more numerous, and they form a meager supraganglionic plexus and a more extensive infraganglionic plexus. In contrast to the pattern seen in higher vertebrates, these plexuses have a tendency to be distributed along the length of the cerebellar plate in both tadpoles and froglets. In addition, collateral branches that form intracortical plexuses apparently increase throughout the course of cerebellar development in this species.

Ultrastructural studies on Purkinje cell maturation in the cerebellum of the frog tadpole during spontaneous and thyroxine-induced metamorphosis.

The maturation of Purkinje cells in the cerebella of both thyroxine (T4)-induced and normally metamorphosing tadpoles was studied by transmission electron microscopy, with particular reference to the perikaryal changes. During the latter part of the prometamorphic phase, many Purkinje cells showed hypertrophied apical cones filled with mitochondria, Golgi elements and rosettes of ribosomes. In early metamorphic climax, the perikaryal cytoplasm displayed stratification, with an inner zone of perinuclear Nissl bodies and an outer region of neurotubules. At the onset of metamorphic climax, there was an abrupt appearance of numerous somatic processes, as well as climbing fiber boutons which synapsed with them on some cells. However, many Purkinje cells did not display somatic processes. Stellate cell synapses also were seen in considerable numbers. As metamorphic climax progressed to completion, the somatic processes steadily grew scarce with a concomitant increase in climbing fiber synapses on the major dendrites. Glial ensheathment of the Purkinje cell soma was also rapidly accomplished during metamorphic climax. In addition, premetamorphic bullfrog tadpoles were induced to metamorphose prematurely following treatment with T4 and then compared to normally metamorphosing tadpoles. Following 3 weeks of T4 treatment, large numbers of Purkinje cells displayed somatic processes. These processes were observed to be postsynaptic to climbing fibers and similar to those seen normally. Additionally, Purkinje cell hypertrophied apical cones were observed in treated tadpoles. These observations indicate that some aspects of Purkinje cell maturation during metamorphosis, especially the interaction of climbing fibers and somatic contacts, are T4-dependent. In both normal and induced metamorphosis, changes in frog Purkinje cells thus proceed at a tempo comparable to that of such gross morphological transformation as hindlimb growth.

Ultrastructural studies on the ventricular surface of the frog cerebellum.

Ultrastructural studies of the ventricular surface of the frog cerebellum showed regional differences. In the midline region of the adult cerebellum was found a band of profusely ciliated squamous ependymal cells. In the rest of the cerebellum the ependymal cells were columnar and each had a single cilium. In the cerebellum of the premetamorphic tadpole, the squamous ependymal cells of the midline region also were monociliated. During metamorphosis they gradually became multiciliated. Additionally, supraependymal cells and synaptic elements were present on the ventricular surface of the cerebellum of adult frogs as well as in late metamorphic tadpoles. In contrast, supraependymal cells were rarely observed in premetamorphic tadpoles, and it was concluded that the supraependymal system develops during metamorphosis. It is postulated that the band of cilia may be associated with the circulation of cerebrospinal fluid, and supraependymal synaptic elements function in neuroendocrine regulation.

Ultrastructural studies on Purkinje cells of the frog tadpole cerebellum.

The Purkinje cells of the premetamorphic frog tadpole cerebellum were studied with the transmission electron microscope. At this stage of histogenesis, when the external granular layer is yet to be formed, their maturational state varied greatly from randomly oriented cells with a thin rim of cytoplasm to well-formed cells with an abundance of organelle-rich cytoplasm and well-developed dendrites. The well-developed cells were seen in the apical (marginal) region of the cerebellar plate and the poorly developed cells in the basal region. Climbing fibers and other unidentified processes formed synapses on the well-developed Purkinje cell somata and somatic dendrites and on the spines and smooth surfaces of the primary dendrites.

Golgi studies on Purkinje cell development in the frog during spontaneous metamorphosis. II. Details of dendritic development.

The development of Purkinje cell dendrites was studied in the bullfrog from premetamorphic tadpoles to 10-week-old postmetamorphic frog-lets by the Golgi-Kopsch method. In this species two distinct patterns of arbor formation may be seen, which appear to be related to differences in the timing of initial dendritic development. In Purkinje cells that begin development in early tadpole stages, the dendritic tree is elaborated by continuous and concomitant growth and branching, a process by which the developing arbor expands in both height and width. Arbor formation in Purkinje cells that begin development in metamorphosing tadpoles proceeds in two separate steps. Initially, dendrites of such cells elongate, but form only a few poorly developed branches; only when the arbor reaches near-adult height does branching become extensive. Additional differences present in Purkinje cells are reflected in the paucity of growth cones and filopodia in the tadpole, and numerous filopodia and growth cones in the metamorphic period. An interesting feature of dendritic development in this species is a tendency to alter the arboreal domain by the formation of extra-arboreal dendrites, and possibly by the occasional resorbtion of other partially formed dendrites. The pattern of dendritic development in the frog is different than in mammals and is difficult to interpret. Such unusual development may be due to disturbances in the timing of the formation of Purkinje cell dendrites and of the establishment of the external granular layer (EGL).

Ultrastructural studies on cerebellar histogenesis in the frog: the external granular layer and the molecular layer.

Maturational changes of the cerebellum of frog tadpoles were studied with the electron miscroscope. In the premetamorphic tadpole, parallel fiber-like processes (PFP) were present in the incipient molecular layer, long before the appearance of the external granular layer (EGL). These PFP showed synaptic contacts with the precociously developed Purkinje cell dendrites. It appears that these PFP may be responsible for inducing the precocious elaboration of the Purkinje cell dendritic arborization. In the metamorphosing tadpoles, the EGL cells migrating into the internal granular layer were frequently seen in close association with the ependymoglial cell processes, which extend from the pia down toward the ependymal surface. This observation lends support to the hypothesis that glial processes guide the migrating EGL cells.