Patterns of neuronal differentiation in developing cultures of neonatal mouse cerebellum: A living and silver impregnation study

Abstract

Organotypic explants of neonatal mouse cerebellum were cultured for periods up to 56 days in vitro. Living cultures were compared to those fixed and stained by the Holmes' reduced silver nitrate method. The explants consist of a heterologous neuronal population comparable to that found in situ. Since the differentiation of the cerebellum is actively in progress during the neonatal and postnatal periods, cultures derived from such tissue demonstrate the developmental phases that are present at the time of explantation.The in vitro maintenance of neonatal mouse cerebellum allows the expression of those aspects of neuronal development which are intrinsic to the cell proper and primarily concerned with type-specific differentiation, e.g., neurofibrillary development; axonal growth; myelination; and the acquiring of basic dendritic patterns. The secondary aspects of differentiation, which determine the adult state and which are characterized primarily by the increasing complexity of dendritic formation and of synaptic interactions, occur to a much lesser extent in vitro.The importance of recognizing transitional forms as normal and not peculiar to the in vitro situation is emphasized.

Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes

“…Undifferentiated astrocytes could be distinguished from other types of undifferentiated cells in cerebellar explants. Undifferentiated astrocytes were of distinct size (11.8 ± 1.4 μm perikaryal diameter); often had multiple small nucleoli within a nucleus that was eccentrically positioned within the perikarya; and were distributed at the periphery of the outgrowth at a position normally occupied almost exclusively by astrocytes in cerebellar explant cultures after 7 days in vitro 1,23 (Hauser, unpublished 35 , and are of distinct size (7.2 ± 0.7 μm), morphology, and distribution (within the explant) from immature astrocytes. Astrocytes were not labeled for proenkephalin mRNA when pretreated with RNase or an excess of unlabeled probe, or when using an [ 35 S]-labeled RNA probe complementary to prodynorphin mRNA with the same hybridization conditions, exposure time, and specific activity as used for the proenkephalin cRNA probe (Fig.…”

An Organotypic Co‐culture of Embryonic Rat Brainstem and Tongue or Skeletal Muscle

“…Sections were made in order to promote cellular and neuritic outgrowth (Allerand, 1971) and to reduce the size of the explant. The final explants were removed to Petri dishes identified according to the motor nucleus explanted.…”

“…Sections containing the oculomotor nucleus, motor trigeminal nucleus, facial and abducens nuclei or hypoglossal nucleus together with the dorsal nucleus of the vagus nerve and nucleus ambiguus, were identified under the stereo microscope and explants were obtained by cutting away unnecessary tissue. Sections were made in order to promote cellular and neuritic outgrowth (Allerand, 1971) and to reduce the size of the explant. The final explants were removed to Petri dishes identified according to the motor nucleus explanted.…”

A Modified Silver Method for Demonstrating Developing Nervous Tissue in Culture

“…Nonetheless, despite its obvious limitations, the Holmes reduced silver nitrate method for neurofibrils has proven to be a practical and reliable method for light microscopic analysis of neuronal de velopment in whole mount cultures of the central nerv ous tissue [Allerand, 1971;Wolf, 1964], The Golgi method, moreover, although of obvious value as dem onstrated in situ, remains a notoriously difficult and quite unreliable technique when applied in vitro [Wolf et al, 1967]. The modification of Bodian' s protargol copper method has been widely used.…”