Light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy of cerebellar basket cells.

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.

Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain.

Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.

Further observations on cerebellar climbing fibers. A study by means of light microscopy, confocal laser scanning microscopy and scanning and transmission electron microscopy

The intracortical pathways of climbing fibers were traced in several vertebrate cerebella using light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. They were identified as fine fibers up to 1(micron thick, with a characteristic crossing-over bifurcation pattern. Climbing fiber collaterals were tridimensionally visualized forming thin climbing fiber glomeruli in the granular layer. Confocal laser scanning microscopy revealed three types of collateral processes at the interface between granular and Purkinje cell layers. Scanning electron microscopy showed climbing fiber retrograde collaterals in the molecular layer. Asymmetric synaptic contacts of climbing fibers with Purkinje dendritic spines and stellate neuron dendrites were characterized by transmission electron microscopy. Correlative microscopy allowed us to obtain the basic three-dimensional morphological features of climbing fibers in several vertebrates and to show with more accuracy a higher degree of lateral collateralization of these fibers within the cerebellar cortex. The correlative microscopy approach provides new views in the cerebellar cortex information processing.(AU)

Further observations on cerebellar climbing fibers. A study by means of light microscopy, confocal laser scanning microscopy and scanning and transmission electron microscopy

The intracortical pathways of climbing fibers were traced in several vertebrate cerebella using light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. They were identified as fine fibers up to 1(micron thick, with a characteristic crossing-over bifurcation pattern. Climbing fiber collaterals were tridimensionally visualized forming thin climbing fiber glomeruli in the granular layer. Confocal laser scanning microscopy revealed three types of collateral processes at the interface between granular and Purkinje cell layers. Scanning electron microscopy showed climbing fiber retrograde collaterals in the molecular layer. Asymmetric synaptic contacts of climbing fibers with Purkinje dendritic spines and stellate neuron dendrites were characterized by transmission electron microscopy. Correlative microscopy allowed us to obtain the basic three-dimensional morphological features of climbing fibers in several vertebrates and to show with more accuracy a higher degree of lateral collateralization of these fibers within the cerebellar cortex. The correlative microscopy approach provides new views in the cerebellar cortex information processing.

Three-dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy.

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.

Correlative microscopy of cerebellar Golgi cells.

The cerebellar Golgi cells of mouse, teleost fish, primate and human species have been studied by means of light and Golgi light microscopic techniques, confocal laser scanning microscopy, slicing technique, ethanol-cryofracturing and freeze-fracture methods for scanning electron microscopy and ultrathin sectioning and freeze-etching replicas for transmission electron microscopy. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform macroneurons surrounded by the granule cell groups. They exhibited ascending dendrites toward the molecular layer and horizontal dendrites and a short beaded axonal plexus confined to the granular layer. Scanning electron microscopy revealed their three-dimensional neuronal geometry and smooth outer surfaces. Freeze-fracture method for SEM showed the stereospatial cytoplasmic arrangement of endoplasmic reticulum, cell organelles and nuclear envelope. By means of transmission electron microscopy the asymmetric synaptic connections of Golgi cell horizontal dendrites--with mossy fiber rosettes at the cerebellar glomerulus--and of Golgi cell axons--with granule cell dendrites at the periphery of glomerular region--were identified. At the molecular layer, Golgi cell ascending dendrites exhibited short neckless spines establishing asymmetric contacts with granule cell axons or parallel fibers. Shaft asymmetric axodendritic and axospinodendritic contacts between Golgi cell dendrites and climbing fibers were also found in the molecular layer.

Correlative microscopy of cerebellar Golgi cells

The cerebellar Golgi cells of mouse, teleost fish, primate and human species have been studied by means of light and Golgi light microscopic techniques, confocal laser scanning microscopy, slicing technique, ethanol-cryofracturing and freeze-fracture methods for scanning electron microscopy and ultrathin sectioning and freeze-etching replicas for transmission electron microscopy. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform macroneurons surrounded by the granule cell groups. They exhibited ascending dendrites toward the molecular layer and horizontal dendrites and a short beaded axonal plexus confined to the granular layer. Scanning electron microscopy revealed their three-dimensional neuronal geometry and smooth outer surfaces. Freeze-fracture method for SEM showed the stereospatial cytoplasmic arrangement of endoplasmic reticulum, cell organelles and nuclear envelope. By means of transmission electron microscopy the asymmetric synaptic connections of Golgi cell horizontal dendrites--with mossy fiber rosettes at the cerebellar glomerulus--and of Golgi cell axons--with granule cell dendrites at the periphery of glomerular region--were identified. At the molecular layer, Golgi cell ascending dendrites exhibited short neckless spines establishing asymmetric contacts with granule cell axons or parallel fibers. Shaft asymmetric axodendritic and axospinodendritic contacts between Golgi cell dendrites and climbing fibers were also found in the molecular layer.(AU)

Correlative microscopy of cerebellar Golgi cells

The cerebellar Golgi cells of mouse, teleost fish, primate and human species have been studied by means of light and Golgi light microscopic techniques, confocal laser scanning microscopy, slicing technique, ethanol-cryofracturing and freeze-fracture methods for scanning electron microscopy and ultrathin sectioning and freeze-etching replicas for transmission electron microscopy. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform macroneurons surrounded by the granule cell groups. They exhibited ascending dendrites toward the molecular layer and horizontal dendrites and a short beaded axonal plexus confined to the granular layer. Scanning electron microscopy revealed their three-dimensional neuronal geometry and smooth outer surfaces. Freeze-fracture method for SEM showed the stereospatial cytoplasmic arrangement of endoplasmic reticulum, cell organelles and nuclear envelope. By means of transmission electron microscopy the asymmetric synaptic connections of Golgi cell horizontal dendrites--with mossy fiber rosettes at the cerebellar glomerulus--and of Golgi cell axons--with granule cell dendrites at the periphery of glomerular region--were identified. At the molecular layer, Golgi cell ascending dendrites exhibited short neckless spines establishing asymmetric contacts with granule cell axons or parallel fibers. Shaft asymmetric axodendritic and axospinodendritic contacts between Golgi cell dendrites and climbing fibers were also found in the molecular layer.

Further observations on cerebellar climbing fibers. A study by means of light microscopy, confocal laser scanning microscopy and scanning and transmission electron microscopy.

The intracortical pathways of climbing fibers were traced in several vertebrate cerebella using light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. They were identified as fine fibers up to 1(micron thick, with a characteristic crossing-over bifurcation pattern. Climbing fiber collaterals were tridimensionally visualized forming thin climbing fiber glomeruli in the granular layer. Confocal laser scanning microscopy revealed three types of collateral processes at the interface between granular and Purkinje cell layers. Scanning electron microscopy showed climbing fiber retrograde collaterals in the molecular layer. Asymmetric synaptic contacts of climbing fibers with Purkinje dendritic spines and stellate neuron dendrites were characterized by transmission electron microscopy. Correlative microscopy allowed us to obtain the basic three-dimensional morphological features of climbing fibers in several vertebrates and to show with more accuracy a higher degree of lateral collateralization of these fibers within the cerebellar cortex. The correlative microscopy approach provides new views in the cerebellar cortex information processing.

Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining.

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.

High resolution (SE-I) scanning electron microscopy features of primate cerebellar cortex.

This paper provides an exploration into the outer and inner surfaces of primate cerebellar neurons using secondary electron-I (SE-I) topographic contrast. SE-I enriched, chromium coated, cryofractured cerebellum staged within the condenser/objective lens stage of SEMs, equipped with high brightness LaB6 and field emission emitter, generated quality images of intact and fractured nerve cells studied at intermediate and high magnifications. Granule and Golgi cell surfaces revealed smooth, accurately delineated profiles of the true cell surface features, which lacked the SE-III dominated brilliance of conventional gold or gold-palladium decorated images. Fractured non synaptic segments of parallel fibers in the molecular layer showed interconnected anastomotic networks of ER tubules, vesicles and cisterns, whereas cross fractured presynaptic "en passant" endings of these fibers exhibited spheroidal synaptic vesicles and SE-I edge brightness contrast delineated their limiting plasma membranes. Parallel fiber fractured synaptic endings showed a homogeneous extravesicular material surrounding the synaptic vesicles. The neuroglial cytoplasm ensheathing nerve processes exhibited a smooth discontinuous surface. The high mass density surface of the myelin sheath showed a mixed population of globular structures, 10-30 nm, corresponding to protein and phospholipid microdomains.

Microfilament-associated growth cone component depends upon Tau for its intracellular localization.

We report here a novel intracellular localization and function of Tau proteins in cultured cerebellar neurons. Immunofluorescence staining of detergent-extracted cytoskeletons with antibodies specific for Tau proteins revealed intense labeling of growth cone microtubules. Besides, suppression of Tau by antisense oligonucleotide treatment results in the complete disappearance of antigen 13H9, a specific growth cone component with properties of microfilament- and microtubule-associated protein [Goslin et al., 1989: J. Cell Biol. 109:1621-1631], from its normal intracellular location. This phenomenon is unique to neurite-bearing cells, is not associated with the disappearance of microtubules from growth cones, and is not reversed by taxol, a microtubule-stabilizing agent. In addition, Tau-suppressed neurons display a significant reduction in growth cone area and fillopodial number; on the contrary, fillopodial length increases significantly. The alterations in growth cone morphology are accompanied by considerable changes in the phalloidin staining of assembled actin. Taken together, the present results suggest that in developing neurons Tau proteins participate in mediating interactions between elements of the growth cone cytoskeleton important for maintaining the normal structural organization of this neuritic domain.

Conventional and high resolution field emission scanning electron microscopy of vertebrate cerebellar parallel fiber-Purkinje spine synapses.

Purkinje dendrite-parallel fiber spine synapses of human, teleost fishes, Rhesus monkey and mouse cerebellar cortex have been studied by means of conventional scanning electron microscopy (SEM) using ethanol-cryofracturing technique and by high resolution field emission scanning electron microscopy (HRFESEM) for studying the outer and inner surface morphology of pre- and postsynaptic endings. Transmission electron microscopy of mouse cerebellar cortex either by means of ultrathin sections and freeze-etching replicas have been complementarily used for proper identification and comparative observations. Normal teleost fishes showed short neck and neckless Purkinje spines with exhibited spread or extended postsynaptic densities. In pathological human cerebellum, the ethanol cryofracturing technique exposed the outer surface of edematous flat and invaginated Purkinje spine synapses. In fractured presynaptic endings HRFESEM showed in Rhesus monkey cerebellar cortex a homogeneous extravesicular material binded to the synaptic vesicles and joining them to the presynaptic membrane. HRFESEM partially resolved the synaptic cleft as currently observed in high magnification transmission electron microscopy. Round subunits, 25-35 nm in diameter, were observed associated to postsynaptic membrane, apparently corresponding to the localization and distribution of E face postsynaptic intramembrane particles, which suggest that such subunits correspond to the domains of neurotransmitter postsynaptic receptors.

Scanning electron microscopy of the granular layer of rat cerebellar cortex.

The rat cerebellar cortex has been studied with respect to its cytoarchitectural arrangement using Scanning Electron Microscope. We have distinguished several types of cells in the granular layer and characterized the granules and the Golgi cells. We also characterized the mossy fiber glomeruli which appears round or ovoid. The mossy fibers revealed their "en passant" nature. The climbing fibers formed tendril collaterals and glomeruli. The Golgi cell axon appeared "beaded" and it takes part in the formation of both types of glomeruli.

Three-dimensional morphology of cerebellar protoplasmic islands and proteoglycan content of mossy fiber glomerulus: a scanning and transmission electron microscope study.

The present review summarizes the outer and inner surface features of mossy fiber glomeruli in vertebrate cerebellar granular layer as seen by conventional scanning electron microscopy (SEM) and SEM freeze-fracture method. The intracortical trajectory of mossy fibers and their synaptic contacts with granule cell dendrites were traced by the slicing and freeze-fracture techniques revealing the radial distribution of granule cell dendrites around the central mossy rosette. The "en passant" nature of mossy fiber synaptic contacts and the participation of Golgi cell axonal ramifications were demonstrated. The results obtained were compared with available light and transmission electron microscopy data. The freeze-etching technique disclosed the true extension of glomerular neuroglial investment. The proteoglycan content of mossy fiber rosette has been also studied by Alcian Blue staining, enzymatic digestion with testicular hyaluronidase and neuraminidase and Os-DMEDA staining method resulting in the presence of an electron dense material at the mossy fiber axoplasmic matrix and some synaptic vesicles, pre-and postsynaptic densities and cleft substance. The axoplasmic material appears to be constituted by proteoglycans with hyaluronic acid or chondroitin sulphate in their composition. The possible role of proteoglycans in synaptic functions is also discussed. Scanning electron microscopy is a promising methodology for analysis of short intracortical circuits and for the study of complex multisynaptic arrangements.

Immunolocalization of cytosolic aspartate aminotransferase (cAAT) in axon terminals that form synapses in the rat cerebellar cortex. A study at the electron microscopic level.

This study was conducted to determine the ultrastructural localization of cytosolic aspartate aminotransferase (cAAT)-like immunoreactivity in the cerebellar cortex in the rat. The isoenzyme was found both in excitatory and inhibitory axon terminals, but not in the climbing fibers of the molecular layer. These findings suggest that cAAT may have a different role in the excitatory and inhibitory synapses, and that climbing fibers of the molecular layer do not appear to use aspartate as neurotransmitter.

Freeze-fracture scanning electron microscopy and comparative freeze-etching study of parallel fiber-Purkinje spine synapses of vertebrate cerebellar cortex.

The three dimensional surface morphology, topographical arrangement and intramembrane features of parallel fiber-Purkinje spine synapses of teleost fishes and Swiss albino mice cerebellar cortex have been complementarily studied with the scanning electron microscope (SEM) using the freeze-fracture method and with transmission electron microscope (TEM) by means of freeze-etching replica and thin-sectioning techniques. The SEM fractographs revealed for the first time the outer surface morphology of non-synaptic segments and synaptic varicosities of parallel fibers, which showed clusters of spheroidal synaptic vesicles joined by an amorphous substance. Freeze-etching preparations showed an 'en face' view of P face parallel fiber varicosity (presynaptic P face) characterized by a depressed synaptic active zone with exocytotic vesicle sites, round IMPs (7.5-15 nm), and rod particles (38.5-53 nm in length). Conversely the presynaptic E face exhibited a smooth surface, randomly distributed pits and very low density distribution of round IMPs. The P face of Purkinje spine membrane (postsynaptic P face) showed high density distribution of round or ovoid IMPs, 8-18.5 nm in diameter, and rod IMPs up to 79.2 nm in length. IMPs were also observed beneath the postsynaptic membrane (subsynaptic IMPs) corresponding to the localization of postsynaptic density. The E face of Purkinje spine membrane (postsynaptic E face) showed aggregation of round IMPs, 5.6-17.4 nm in diameter, at the synaptic active zone. Some of these IMPs, particularly the large ones, may morphologically represent the glutamate-receptor ion channels complex. The subsynaptic IMPs may correspond to protein kinases. Granular material and fine filaments were observed in the postsynaptic density. A better insight into the topographical relationship between pre- and postsynaptic structures was obtained of flat and invaginated spine synapses and beside a more realistic view of their glial ensheathment. The SEM fractographs and freeze-etching images were compared with corresponding TEM thin sections for orientation and proper identification of pre- and postsynaptic structures.

Scanning electron microscopy of vertebrate cerebellar cortex.

In this study, the Golgi method for light microscopy, transmission and conventional scanning electron microscopy, the ethanol-cryofracturing technique, the freeze-fracture method for SEM, and the freeze-etching method have been used in conjuction to analyze the three-dimensional cytoarchitectonic arrangement and intracortical circuits of vertebrate cerebellar cortex. Approximately more than 100 specimens of mice, rat, teleost fishes and human cerebelli were processed by the above mentioned techniques. A chronological review of other methods for studying hidden surfaces of cerebellar nerve cell has been also described. The three-dimensional morphology, outer and inner surfaces of granule, Golgi, Purkinje and stellate cells were reviewed by means of a correlative and comparative study. The cerebellar circuits formed by mossy and climbing fibers with granule cell dendrites and Purkinje cell-granule cell synapses have been traced by ethanol-cryofracturing technique, freeze-fracture method for SEM and freeze-etching technique. These findings have been correlated with previous light and electron microscope findings published in the last century. Some advantages and limitations of each method are pointed out. The review emphasizes the paramount importance of correlating light microscope Golgi method with ethanol-cryofracturing and slicing techniques for SEM. The correlation between freeze-fracture method for SEM and freeze-etching technique provides a new approach for studying three-dimensional morphology of nerve cells at cellular and macromolecular levels. This modern methodology of three-dimensional analysis offers new potential areas for future experimental investigation in embryology and pathology of the central nervous system.