ABSTRACT
The cerebellar basket cells of mice, hamsters, teleost fishes and human have been studied by means of Golgi light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. Golgi light microscopy showed ascending dendrites toward the molecular layer and descending and transverse axonal collaterals contributing to the formation of Purkinje cell pericellular basket. Confocal laser scanning microscopy provided z-series of optodigital sections of ascending basket cell dendrites and descending axonal collaterals participating in the Purkinje cell pericellular nest and the pinceaux. Scanning electronmicrographs displayed the three-dimensional relief of Purkinje pericellular nest formed by basket cell descending and transverse axonal collaterals. Transmission electron microscopy, used as a complementary technique, showed the synaptic contacts formed by basket cell axonal collaterals on Purkinje cell soma and the axosomatic contacts on basket cells by parallel and climbing fiber endings, basket cell axonal terminals and/or Purkinje cell recurrent axonal collaterals. Pre- and postsynaptic membrane specializations were mainly found in parallel fiber axosomatic contacts on basket cells, but not in the Purkinje cell pericellular basket and in other axosomatic contacts on basket cells. These latter findings have been adscribed to a phenomenon of cerebellar synaptic plasticity related with motor learning performance. The correlative microscopy approach demonstrates the potential value of these methodologies for studying the three-dimensional aspect of short intracortical circuits in the central nervous system.
Subject(s)
Cerebellar Cortex/ultrastructure , Animals , Catfishes/anatomy & histology , Female , Guinea Pigs , Humans , Mice , Microscopy, Confocal , Microscopy, Electron, Scanning , Species SpecificityABSTRACT
A correlative microscopic study of vertebrate cerebellar mossy fiber glomeruli has been carried out to obtain a three-dimensional view of the multisynaptic contacts formed by afferent mossy fibers with the granule and Golgi cell dendrites and by the monosynaptic relationship of Golgi cell axonal ramifications with granule cell dendrites. Samples of mice, hamsters, teleost fishes and human species were studied by means of one of the following procedures: confocal laser scanning microscopy (CLSM), ethanol-cryofracturing technique and conventional scanning electron microscopy (CSEM) and transmission electron microscopy (TEM) by ultrathin sections and freeze-etching replicas. CLSM, by means of montages of z-series of the cerebellar granular layer, provided a new approach to explore mossy fiber trajectory and branching bifurcation pattern and the quantitative relationship between mossy fibers and granule cell dendrites. CSEM and freeze-fracture method for SEM offered a more detailed in-depth, higher resolution image of outer and inner surface organization of mossy fiber glomeruli. TEM, either by ultrathin sections or freeze-etching replicas, was used as complementary technique for proper orientation, comparative purposes and rational identification of pre- and postsynaptic structures. Freeze-etching replicas showed in addition the real extent of glial cell cytoplasm encapsulating the synaptic glomeruli. The integrated microscopy approach offers a new and more comprehensive view of three-dimensional morphology, organization and quantitative aspects of mossy fiber glomeruli.
Subject(s)
Nerve Fibers/ultrastructure , Animals , Cricetinae , Female , Humans , Microscopy, Electron , Microscopy, Electron, ScanningABSTRACT
The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.
Subject(s)
Cerebellar Cortex/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , Nerve Fibers/ultrastructure , Animals , Cricetinae , Female , Fishes , Macaca mulatta , Microscopy, Confocal/methods , Microscopy, Electron, Scanning/methods , Purkinje Cells/ultrastructureABSTRACT
The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to study its cytoarchitectonic arrangement and intracortical course of mossy and climbing fibers. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30 and 100 microM solutions of FM4-64 in 0.1 M sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Cellular tomography of cerebellar cortex allowed us to follow the course of mossy and climbing fibers in the cerebellar white and gray substance. They exhibited high fluorescence signal at the level of myelin sheath. Mossy fibers were identified only in the granular layer by their typical rosette formations and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving off collateral branches in the granular and molecular layers in a crossing-over pattern. They ascend to the Purkinje cell layer in their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket and stellate cells) showed optimal intracellular staining of cell soma, axonal and proximal dendritic processes. The montage of z-series of stacked optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship of intrinsic cerebellar neurons with the afferent mossy and climbing fibers
Subject(s)
Animals , Cricetinae , Female , Quaternary Ammonium Compounds , Cell Size/physiology , Cerebellum/cytology , Fluorescent Dyes , Microscopy, Confocal/methods , Nerve Fibers/ultrastructure , Neural Pathways , Pyridinium CompoundsABSTRACT
The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to study its cytoarchitectonic arrangement and intracortical course of mossy and climbing fibers. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30 and 100 microM solutions of FM4-64 in 0.1 M sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Cellular tomography of cerebellar cortex allowed us to follow the course of mossy and climbing fibers in the cerebellar white and gray substance. They exhibited high fluorescence signal at the level of myelin sheath. Mossy fibers were identified only in the granular layer by their typical rosette formations and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving off collateral branches in the granular and molecular layers in a crossing-over pattern. They ascend to the Purkinje cell layer in their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket and stellate cells) showed optimal intracellular staining of cell soma, axonal and proximal dendritic processes. The montage of z-series of stacked optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship of intrinsic cerebellar neurons with the afferent mossy and climbing fibers
Subject(s)
Animals , Cricetinae , Female , Cerebellum , Fluorescent Dyes , Microscopy, Confocal , Nerve Fibers , Pyridinium Compounds , Quaternary Ammonium Compounds , Cell Size/physiology , Neural PathwaysABSTRACT
The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30, and 100 microns solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances. They exhibited a high fluorescence signal at the level of the myelin sheath. Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z-series of stacks of optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship with the afferent fibers.
Subject(s)
Cerebellar Cortex/ultrastructure , Fluorescent Dyes , Pyridinium Compounds , Quaternary Ammonium Compounds , Animals , Cricetinae , Dendrites/ultrastructure , Female , Microscopy, Confocal , Nerve Fibers/ultrastructure , Neurons/ultrastructure , Purkinje Cells/ultrastructure , Staining and Labeling/methodsABSTRACT
The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to study its cytoarchitectonic arrangement and intracortical course of mossy and climbing fibers. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30 and 100 microM solutions of FM4-64 in 0.1 M sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Cellular tomography of cerebellar cortex allowed us to follow the course of mossy and climbing fibers in the cerebellar white and gray substance. They exhibited high fluorescence signal at the level of myelin sheath. Mossy fibers were identified only in the granular layer by their typical rosette formations and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving off collateral branches in the granular and molecular layers in a crossing-over pattern. They ascend to the Purkinje cell layer in their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket and stellate cells) showed optimal intracellular staining of cell soma, axonal and proximal dendritic processes. The montage of z-series of stacked optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship of intrinsic cerebellar neurons with the afferent mossy and climbing fibers.
Subject(s)
Cerebellum/cytology , Microscopy, Confocal/methods , Nerve Fibers/ultrastructure , Animals , Cell Size/physiology , Cricetinae , Female , Fluorescent Dyes , Neural Pathways , Pyridinium Compounds , Quaternary Ammonium CompoundsABSTRACT
The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to study its cytoarchitectonic arrangement and intracortical course of mossy and climbing fibers. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30 and 100 microM solutions of FM4-64 in 0.1 M sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Cellular tomography of cerebellar cortex allowed us to follow the course of mossy and climbing fibers in the cerebellar white and gray substance. They exhibited high fluorescence signal at the level of myelin sheath. Mossy fibers were identified only in the granular layer by their typical rosette formations and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving off collateral branches in the granular and molecular layers in a crossing-over pattern. They ascend to the Purkinje cell layer in their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket and stellate cells) showed optimal intracellular staining of cell soma, axonal and proximal dendritic processes. The montage of z-series of stacked optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship of intrinsic cerebellar neurons with the afferent mossy and climbing fibers.