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-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.

The formation of the cerebellum was studied during the first 6 months of the tadpole stage of the bullfrog by using standard histological methods and reconstructions from serial horizontal sections. Three major developmental phases were noted in the formation of the cerebellum. (1) During the first 5 weeks of development, the neuroepithelium proliferated and the dorsal mesencephalic plates increased in size. (2) Starting in the sixth week, a patch of neuroepithelium began to differentiate and gave rise to a small population of Purkinje cells. In subsequent weeks, the area of differentiation continued to spread and a Purkinje cell layer became established along the dorsal margin of the cerebellar plate. (3) In the 12th week, the ventrolateral part of the cerebellar plate began to increase in size and generate two populations of small cells. The lateralmost part of the neuroepithelium in this area generated a group of cells that formed an external granular layer that was one cell deep. Cells of this external granular layer migrated inward into the primitive molecular layer, and by the 26th week only a remnant of an external granular layer remained in the cerebellum. The more medially situated part of the neuroepithelium gave rise to another population of small cells that formed a column, which appeared to be continuous with the Purkinje cells, but differed from them in size. It should be noted that full maturation of the cerebellum occurs during metamorphosis, which in this species remains some 2 years away.

The cerebellum of the premetamorphic bullfrog tadpole differs from the cerebellum of other frog species in its morphology and maturational state. The cell mass beneath the floor of the lateral recess and bordering its lateral wall that has been reported to form the auricular lobe in other species is absent, the auricular lobe abutting the medial wall of the lateral recess instead and is continuous with the corpus cerebelli. The corpus cerebelli, although immature and yet to acquire an external granular layer, is already massive and displays an incipient molecular, Purkinje cell and granular layers. Cytodifferentiation in the auricular lobe and corpus cerebelli is similar, their constituent cells being in various stages of development. Fully mature cells are absent, but a small population of Purkinje cells and glia in the auricular lobe and along the marginal zone of the corpus cerebelli show advanced development. The orientation of these Purkinje cells is parallel to the pia and appears to approximate the course of the vestibulo-lateral commissural fibers. In the ventral part of the corpus cerebelli, developing climbing fibers are present but Purkinje cells are poorly developed.

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.