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In order to analyze some aspects of the spatial organization in the primary somatosensory cortex of the rat, we have reconstructed the positions of bundles of apical dendrites and neurons in a cortical prisms measuring 0.5 mm x 0.4 mm x cortical thickness, with special reference to a hypothetical columnar organization. Complete series of semithin (0.65 microm) sections were cut, tangentially from the pial surface down to the white matter, stained and digitizalized into a computer and represented as a stack of 2D images. The mean neuron density (N(V)-value) was (60 x 10(3) +/- 15 x 10(3)) neurons/mm3. The mean number of neurons beneath 1 mm2 of cortical surface (NC-value) was (113 x 10(3) +/- 8 x 10(3)) neurons/mm2. Well-defined bundles of apical dendrites emanating from layer V pyramidal cells were observed. The bundles consisted of 3-12 (mean 5 +/- 2) dendrites. The dendrites within a bundle converged while ascending towards the pial surface and reached a maximal close packing in layer IV. Superficially, the packing density decreased again. The mutual positions of the dendrites within the bundles shifted only slightly along their course towards the pial surface. The occurrence of bundles in tangential sections through layer IV was about 190 bundles/mm2 and the average number of neurons per bundle was estimated at approximately 600. However, when calculating Voronoi-diagrams, the number of neurons, which with this mathematical technique, is ascribed to each of the reconstructed dendritic bundles, varied between 200 and 1000. The possibility that the dendritic bundles are centers in cortical cell modules is discussed.

A single fourth lumbar dorsal rootlet was transected at the entry point into the spinal cord. The nerve fibres were labelled with biotin dextran injected into the rootlet. An endogenous matrix containing olfactory-ensheathing cells (OECs) labelled with green fluorescent protein was applied to the opposing cut surfaces of the rootlet and the spinal cord, which were then brought into apposition and held in place by fibrin glue. Two weeks later, a ladderlike bridging structure has been formed by astrocytic processes growing out for about 200-300 microm from the spinal cord. The transplanted cells remained largely confined to this area. They were elongated along the nerve axis but did not enter the spinal cord itself. Labelled dorsal root axons crossed the repaired dorsal root entry zone in alignment with the bridging astrocytic processes and the transplanted cells and then proceeded beyond the transplant to enter the grey matter of the dorsal horn and send axons both rostrally and caudally for at least 10 mm in the white matter of the ascending dorsal columns.

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

The nodes of Ranvier are sites of specific interaction between Schwann cells and axons. Besides their crucial role in transmission of action potentials, the nodes of Ranvier and in particular the paranodal axon-Schwann cell networks (ASNs) are thought to function as local centers in large motor axons for removal, degradation, and disposal of organelles. In order to test whether ciliary neurotrophic factor (CNTF), which is present at high levels in the Schwann cell cytoplasm, is involved in the maintenance of these structures, we have examined lumbar ventral root nerve fibers of alpha-motor neurons by electron microscopy in 3- and 9-month-old Cntf null ((-/-)) mutant mice. Nerve fibers and nodes of Ranvier in 3-month-old Cntf(-/-) mutants appeared morphologically normal, except that ASNs were more voluminous in the mutants than in wild-type control animals at this age. In 9-month-old Cntf(-/-) animals, morphological changes, such as reduction in nerve fiber and axon diameter, myelin sheath disruption, and loss of ASNs at nodes of Ranvier, were observed. These findings suggest that endogenous CNTF, in addition to its role in promoting motor neuron survival and regeneration, is needed for long-term maintenance of alpha-motor nerve fibers. The premature loss of paranodal ASNs in animals lacking CNTF, which seems to be a defect related to a disturbed interaction in the nodal region between the axon and its myelinating Schwann cells, could impede the maintenance of a normal milieu in the motor axon, preceding more general neuronal damage., (Copyright 2003 Wiley-Liss, Inc.)

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti-sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I-positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered., (Copyright 2002 Wiley-Liss, Inc.)

We have studied the perinatal development of large myelinated axons (adult D > 10 microm) in cat ventral and dorsal lumbosacral spinal roots using autoradiography and electron microscopy (serial section analysis). These axons acquire their first myelin sheaths 2-3 weeks before birth and show nearly mature functional properties first at a diameter of 4-5 microm, i.e. 3-4 weeks after birth. The most conspicuous event during this development takes place around birth, when a transient primary myelin sheath degeneration strikes already well myelinated although short 'aberrant' Schwann cells. The aberrant Schwann cells become completely demyelinated, then measuring about 10 microm in length, and are subsequently eliminated from their parent axons. Morphometry indicates that on average 50% of the Schwann cells originally present along a prospective large spinal root axon suffer elimination. Here it should be noted that in cat lumbo-sacral spinal roots, the longitudinal growth of myelinated Schwann cells that belong to the group containing what will be the largest fibers is on average twice that of their parent axons. The elimination phenomenon is particularly striking in the dorsal roots close to the spinal cord where CNS tissue invades the root for several hundred micrometres. Our observations suggest that, once demyelinated and then eliminated, Schwann cells (i.e. aberrant Schwann cells) colonize neighbouring axons, future myelinated as well as future unmyelinated ones. In the former case the immigrant Schwann cells appear to start myelin production, possibly risking a second demyelination and elimination. We take our observations to indicate that Schwann cells in the cat, during normal development, may switch iteratively between a 'myelin-producing' and a 'non-myelin-producing' phenotype. From a functional point of view the transient presence along a myelinated axon of intercalated unmyelinated segments approximately 10 microm long, due to aberrant Schwann cells, would mean a slowing down of the action potential. The rapid disappearance of aberrant Schwann cells during the two first postnatal weeks could then explain the progressing normalization of the leg-length conduction time.

Here we describe a computer-assisted method which, based on conventional transmission electron microscopy, renders simulated high-voltage electron micrographs. We perform arithmetic minimum filtering on stacks of aligned serial transmission electron microscopic images. In this way, the structural information of the separate images is fused into one compound image that highlights organization patterns otherwise easily overlooked or impossible to comprehend. Our method, like high-voltage electron microscopy, offers the possibility to build stereo-pairs for high-resolution three-dimensional analysis of tissue layers 1-2 microm thick. The use of background elimination and the development of a depth enhancement routine improved the three-dimensional effect and facilitated the analysis of the interior of objects. As an example, we use the method to display the distribution of axonal organelles at nodes of Ranvier and the shape and contents of a highly branched hippocampal dendritic spine.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

We have studied the cellular distribution of gangliosides GD1b, GD3 and GM1 in rat cerebellum by immunostaining, using monoclonal antibodies and confocal microscopy. Antibodies against astroglial, neuronal and synaptic vesicle associated molecules were used for colocalization analyses. In the gray matter, the anti-GD1b antibody stained thin strands in the molecular layer (ML), interpreted as Bergman glia fibers based on colocalized staining with anti-glial fibrillary acidic protein (GFAP). The neuropil in the granule (GL) and Purkinje (PL) cell layers was also anti-GD1b positive. The anti-GD3 antibody stained the ML, the neuropil in the GL and PL and also the granule and Purkinje cell bodies, appearing intracytoplasmically and vesicle associated. Anti-GD1b and anti-GD3 staining in the GL glomeruli were colocalized with anti-synaptophysin staining. The anti-GM1 antibody stained cell bodies in the ML but they could not be characterized in colocalization experiments. The GL and PL were not stained with the anti-GM1 antibody. In the white matter, different staining patterns were seen for the gangliosides, the anti-GM1 staining being the most intense. This study shows cellular layer and cell type specific associations of the investigated gangliosides and localization of GD1b and GD3 at synaptic sites, warranting further studies on their role in synaptic mechanisms., (Copyright 2000 Wiley-Liss, Inc.)

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.

In order to test our hypothesis that myelin-forming Schwann cells early during development, after having been eliminated from their parent axons, colonize neighbouring unmyelinated axons, we studied the distribution of Schwann cells at the PNS-CNS border in the feline S1 dorsal spinal root during pre- and postnatal development using electron microscopy and autoradiography. Myelination of axons peripheral to the PNS-CNS border began about 1.5 weeks before birth. The adult distribution of one-third myelinated and two-thirds unmyelinated axons was noted 3 weeks after birth. Analysis based on to-scale reconstructions of axon and Schwann cell samples from the first 6 postnatal weeks gave the following results. (1) CNS tissue appeared in the proximal part of the root around birth and expanded peripherally during the first three postnatal weeks. (2) The number of Schwann cells associated with myelinated axons decreased. (3) The number of Schwann cells associated with unmyelinated axons increased. (4) The mitotic activity of the Schwann cells was low at birth and nil after the first postnatal weak. (5) Apoptotic cell units were virtually absent. (6) Aberrant Schwann cells, i.e. short and very short Schwann cells with distorted and degenerating myelin sheaths, were common. (7) The endoneurial space contained numerous Schwannoid cells i.e. solitary cells surrounded by a basal lamina. (8) Cytoplasmic contacts between unmyelinated axons and aberrant Schwann cells or Schwannoid cells were observed. We take these results to support our hypothesis.

We have calculated the number of paranodal Schwann cell mitochondria in adult feline ventral and dorsal lumbar spinal roots using ultrastructural serial section analysis. Distinct accumulations of paranodal mitochondria were noted in nerve fibres more than 4-5 microm in diameter. The calculated number of paranodal mitochondria increased linearly with fibre diameter from a few hundred up to 20,000-30,000 per node. A linear increase in the number of paranodal mitochondria per node also appeared as a function of nodal variables such as 'nodal axon membrane area', 'nodal Schwann cell membrane area', and 'node gap extracellular volume'. In large fibres (D = 15-18 microm), a calculated number of about 20,000 paranodal Schwann cell mitochondria were accumulated at each node of Ranvier and related to nodal axon membrane area of about 20 microm2. Our calculations indicate that, on the average, 1000 paranodal Schwann cell mitochondria with a total volume of 6.7 microm3, a total outer membrane area of 250 microm2 and a total inner membrane area of 580 microm2 projected to each microm2 of the nodal axon membrane via the nodal Schwann cell brush border.

The aim of this study was to investigate the potential ability of Schwann cells to sequester axonally transported material via so called axon-Schwann cell networks (ASNs). These are entities consisting of sheets of Schwann cell adaxonal plasma membrane that invade the axon and segregate portions of axoplasm in paranodes of large myelinated mammalian nerve fibres. Rat hindlimb alpha-motor axons were examined in the L4-S1 ventral roots using light/fluorescence, confocal laser, and electron microscopy for detection of retrogradely transported red-fluorescent latex nanospheres taken up at a sciatic nerve crush, and intramuscularly injected horseradish peroxidase endocytosed by intact synaptic terminals. Survival times after tracer administration ranged from 27 hours to 4 weeks. During their retrograde transport toward the motor neuron perikarya, organelles carrying nanospheres/peroxidase accumulated at nodes of Ranvier, where they often appeared in close association with the paranodal myelin sheath. Serial section electron microscopy showed that many of the tracer-containing bodies were situated within ASN complexes, thereby being segregated from the main axon. Four weeks after nanosphere administration, several node-paranode regions still showed ASN-associated aggregations of spheres, some of which were situated in the adaxonal Schwann cell cytoplasm. The data establish the ability of Schwann cells to segregate material from motor axons with intact myelin sheaths, using the ASN as mediator. Taken together with our earlier observations that ASNs in alpha-motor axons are also rich in lysosomes, this process would allow a local elimination and secluded degradation of retrogradely transported foreign substances and degenerate organelles before reaching the motor neuron perikarya. In addition, ASNs may serve as sites for disposal of indigestable material.

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an alpha 2-3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman's incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.

We address three problems concerning the quantitative analysis of nerve cell distribution in the cerebral cortex: (i) preparatory tissue deformation (shrinkage); (ii) difficulties in differentiating between small neurons and astroglia; and (iii) the bias introduced by the counting method. We found that staining with Richardson's solution led to no shrinkage in Vibratome-cut sections of aldehyde-fixed rat brains, but did result in staining of the neurons and left the glial cells unstained. This was in striking contrast to Nissl staining which introduced a linear shrinkage of 20-30% and stained all kinds of cortical cells indiscriminately. A computer-based unbiased counting method was implemented by taking advantage of the stereological procedure referred to as the 'optical disector' (Gundersen, H.J.G. (1986) Stereology of arbitrary particles, J. Microsc., 143: 3-45).

The ultrastructure of developing nodes of Ranvier and adjacent paranodes of future large myelinated fibers in feline lumbar spinal roots is described. The development starts before birth concurrent with myelination and is finished at the end of the first postnatal month when the nodal regions of future large fibers, now 4-5 microns of diameter, for the first time appear like miniatures of those of their 4 times thicker and fully mature counterparts. At this stage the fibers also begin to show mature functional properties. The latent maturation process is denoted "nodalization" and includes two major events: (1) the formation of a narrow node gap bordered by compact myelin segments and filled with Schwann cell microvilli that interconnect an undercoated nodal axolemma with rapidly increasing accumulations of mitochondria lodging in the longitudinal cords of Schwann cell cytoplasm that is distributed outside a more and more crenated paranodal myelin sheath; (2) the setting of a fixed number of nodes along the axons; an event that includes segmental axonal and myelin sheath degeneration and is concluded by the elimination of supernumerary Schwann cells.

We have investigated the number of neurons in three neocortical areas of the rat brain. Our results challenge the uniformity concept proposed by Rockel et al. [Brain, 103 (1980) 221-244]. Area Fr1, HL and Oc2 (primary motor, primary somatosensory and secondary visual cortex) from Sprague-Dawley rats were examined. The brains were glutaraldehyde fixed, sectioned in 50 mu m thick sagittal slices and stained in Richardson's solution. The counting was carried out using a computerized system based on the optical disector. The cortical thickness was measured to be 1.9 mm, 1.9 mm, and 1.4 mm in area Fr1, HL, and Oc2, respectively. The number of neurons under 1 mm2 cortical surface was calculated to be 91 100 in Fr1, 133 500 in HL and 106 100 in Oc2. The number of neurons in a volume of tissue 30 x 25 mu m through the depth of the cortex was calculated to be 68 in Fr1, 100 in HL and 80 in Oc2. The density of neurons was calculated to be 48 500 neurons/mm3 in Fr1, 69 400 neurons/mm3 in HL and 76,900 neurons/mm3 in Oc2. There were significant (P < 0.01) differences between all areas regarding both the number of neurons under a certain area of surface as well as the neuron density. The results indicate that there is no basic uniformity in the number of neurons under a certain area of cortical surface.

Horseradish peroxidase (HRP) was injected into the left dorsal column nuclei of adult cats. Large dorsal funiculi axons of the C3, C5, C8 and L7 segments were searched for HRP-activity after 12, 24, 36 and 48h using light and electron microscopy. Accumulations of intra-axonal HRP-positive bodies occurred at nodes of Ranvier in the C3-C8 segments at 12, 24 and 36h and in the L7 segments at 24, 36, and 48h. The accumulations of HRP in three spatio-temporally different consecutive patterns, noted earlier at nodes of Ranvier in the peripheral nervous system (PNS) portion of feline alpha motor axons for more than 70h after an intramuscular injection of the enzyme, were not observed in the present material. We suggest that the differences in the modes in which large PNS and CNS axons interact with retrogradely transported HRP are due to differences in the organization of the respective nodal regions. We also emphasize that endocytosis via axon terminals in the CNS normally represents uptake of material from an extracellular space which is controlled and protected by the blood-brain barrier. This is in contrast to endocytosis via axon terminals in a muscle, which represents uptake of material from an extracellular space openly exposed to influx of different substances from the blood stream.

In order to study the organization and distribution of dendrites and axons in the cerebral cortex, we have developed a computer-assisted method for 3D reconstruction of neuronal processes based on serial light microscopic images displayed as a continuous sequence. A series of tangential sections (0.65 micron thick) through rat parietal cortex was aligned, digitized into the computer and then used to build a sequence (stack) of images which was stored to a digital real-time video disk. Apical dendrites located in dendritic bundles in laminae III and IV were traced through the sequence. Two tracing modes were tested: (1) cinematographic mode, in which the image stack was displayed continuously and automatically by the computer at various preset speeds (max. speed: 25 images/s) and (2) stepping mode, in which the interval between each image was varied manually according to the choice of the operator. Coordinates were stored in a database and used to build a 3D reconstruction where apical dendrites were displayed as wires or tubes. Tracing in cinematographic mode was about 3 times faster than tracing in stepping mode. We believe that the former mode exploits the built in 'filtering' capacity of the visual system to perform temporal averaging.

We have studied the ratio between the nodal and the internodal diameter (the dn/d(in) ratio) of large myelinated axons in the L7 ventral spinal root of the cat during pre- and postnatal development using light and electron microscopy. A substantial nodal constriction, dn/d(in) = 0.6, was found at the beginning of myelination, about 2 weeks before birth. The ratio decreased during the subsequent 10 weeks and approached the adult value of 0.47 (SE 0.01, N = 45) in the 8 weeks old kitten. The observations are discussed with respect to the maturation of the nodal region and to our earlier idea that the constricted nodal axon segments of large peripheral myelinated nerve fibres of adult cats and kittens 2 months and more of age are sites capable of interacting with and perhaps even controlling the passage of axonally transported materials.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.

A technique for 2D reconstruction of intracellularly labeled neurons from sequential sections is described. The system consists of a Charged Coupled Device-camera mounted on a microscope, a videomixer and a IBM-compatible PC with a framegrabber. The neurons (interneurons from the spinal cord of the cat) were labeled iontophoretically by horshradish peroxidase and subsequently cut in 60 microns sections. The sections were aligned using the video mixer by fitting the cut dendrites and axon from one section with their counterparts in the following section. The images were then digitized in the PC where they were fused to create a superimposed picture of the aligned parts of the neuron; a 2D reconstruction was created.

Using light and electron microscopy, axon diameter, myelin sheath thickness (measured as number of myelin lamellae) and internodal length of alpha and gamma motor axons of the L7 ventral root and spinal cord segment were investigated in serial cross-sections. The CNS internodes of the alpha motor fibres had, on average, an axon diameter of 8.6 microns, 105 myelin lamellae and a length of about 560 microns. The CNS internodes of the gamma motor fibres had, on average, an axon diameter of 3.4 microns, 66 myelin lamellae and a length of about 440 microns. Axon diameter at the nodes of Ranvier was 30-40% of the internodal axon diameter. Axon diameter, number of myelin lamellae and internodal length varied considerably between consecutive internodes. Statistical analysis showed no systematic increases or decreases. Regression analyses of the scatter plots of the number of myelin lamellae and internodal length against axon diameter showed large variations and correlation coefficients of r < 0.50. In conformity with ventral root (PNS) internodes (Nilsson & Berthold, 1988) the plotting of intrafunicular (CNS) internodal myelin volume against internodal axon mantle area showed linear correlations with correlation coefficients of r > 0.90. The mean axon diameter of the investigated CNS internodes was similar to, the mean number of myelin lamellae somewhat lower than, and the mean internodal length considerably shorter than that of internodes of axons of the L7 ventral root (Nilsson & Berthold, 1988). In contrast to the ventral root, the intrafunicular alpha motor fibres had higher g values (axon diameter/fibre diameter value) and lower il/d ratios (internodal length/axon diameter ratio) than is considered optimal for conduction. We consider that these deviations from the theoretical optimum are not large enough to impair the conduction properties of the CNS parts of the motor axons in a significant way.

The occurrence and distribution of axoplasmic organelles in large myelinated axons of the ventral, the lateral and the dorsal funiculi of L7 spinal cord segments of the cat have been studied using electron microscopy (EM). Most organelles were found to be concentrated to the paranode-node-paranode (pnp)-regions and they showed their highest relative concentration in the constricted part of these regions, i.e. at the nodes of Ranvier. In the paranode-node-paranode-regions of the lateral and dorsal funiculi, large dense bodies predominated distal to the nodal mid-level and vesiculo-tubular membranous organelles proximal to it. This pattern of organelle distribution, a proximo-distal (with reference to the neuron soma) segregation of the organelles, was only faintly indicated in the paranode-node-paranode-regions of the alpha motor axons of the ventral funiculus. These paranode-node-paranode-regions were, apart from a weak proximo-distal segregation of a few organelles, characterized by deposits of electron dense granules and clusters of large round mitochondria. We conclude that there are two types of organelle accumulation and distribution in the paranode-node-paranode-regions of large spinal cord nerve fibres of the cat. One type is found in the lateral and dorsal funiculi, i.e. in axons with terminal (synaptic) fields inside the blood-brain-barrier. The other type is found in the alpha motor axons of the ventral funiculus, i.e. in axons with their terminal field in the PNS and thus outside the blood-brain barrier. It should be noted that retrogradely transported material in the alpha motor axons has passed through a long sequence of paranode-node-paranode-regions equipped with Schwann cells before it reaches the CNS, while material transported retrogradely in the axons of the dorsal and lateral funiculi has not. The following discussion includes a comparison of the organelle accumulation and distribution in these two types of CNS paranode-node-paranode-regions with the organelle accumulation and distribution observed in the paranode-node-paranode-regions of PNS axons.

Using light microscopy (LM) and electron microscopy (EM) we have examined the occurrence and distribution of axoplasmic organelles in large myelinated nerve fibres of the L7 ventral and dorsal spinal roots of the cat with special reference to the paranode-node-paranode (pnp)-regions. Ninety-eight percent of the 550 Toluidine Blue-stained paranode-node-paranode-regions examined in the light microscope contained dark-blue bodies accumulated distal to the midlevel of the paranode-node-paranode-region. Further, a veil of Toluidine Blue positive material was observed in about 50% of the paranode-node paranode-regions. In about 25% of these paranode-node-paranode-regions the veil lay distal to the midlevel of the paranode-node-paranode-region and in the remainder it lay proximally. Electron microscopy suggested that the ultrastructural equivalents of the dark-blue bodies and of the veil were dense lamellar bodies and a diffuse granular material, respectively. Our calculations indicate that from 70% to more than 90% of some organelles (dense lamellar bodies, multivesicular bodies and vesiculo-tubular membranous organelles) present in an axon are accumulated in the paranode-node-paranode-regions. The occurrence of these organelles in the individual paranode-node-paranode-regions varied within wide limits also in adjacent fibres. The dense lamellar and multivesicular bodies dominated the distal part of the paranode-node-paranode-regions while the vesiculo-tubular membranous organelles dominated the proximal part, i.e. the organelles showed a mutual proximo-distal segregation with reference to the midlevel of the paranode-node-paranode-region. Of seventeen paranode-node-paranode-regions analyzed ultrastructurally, seven were classified as 'fully segregated', that is 67% or more of the lamellar and multivescular bodies, present in the whole paranode-node-paranode-region, lay distal to the mid-level, and 67% or more of the vesiculo-tubular membranous organelles lay proximal to it.

This paper describes software routines that (a) visualizes a stack of several thousands of aligned sequential photographic two-dimensional (2D) images stored in an image processing system; (b) creates a data base containing information about objects identified sequentially from the 2D images; (c) transfers the data base to a graphical terminal; (d) reconstructs a three-dimensional (3D) object space; and (e) supports on-line interaction between the image processing system and the graphical terminal. As an application example, the cell content of a prism of motor cerebral cortex of the cat is reconstructed. Preliminary results from reconstructing human epileptic temporal cortex (cortical microdysgenesia) are also reported.

A prism of cat cerebral cortex was reconstructed with a method for three-dimensional (3D) representation of biological objects. A series of 918 semithin sections were digitized into an image analysis system. The images were aligned and analyzed, and a data base with the coordinates and a classification of the cells was created. The data base (i.e., the cortical prism) was visualized in a 3D graphic terminal, and parameters such as columnar and lamellar organization, clustering, and cell density were analyzed. A neuronal perikaryon and its neurites was reconstructed and shown together with the cortical prism.

The "close-to-node" regions of myelinated nerve fibres, i.e., the paranodal end segments, are generally thought to be sites of high metabolic activity and myelin sheath turnover. Data on turnover rates of individual myelin constituents are conflicting but there exists a common belief that myelin is metabolized as independent molecules rather than as a unit. The occurrence of paranodal Marchi-positive bodies, with morphological and biochemical properties consistent with partially degraded myelin, prompted us to examine the temporal dynamics of the incorporation of radioactive precursor label in the major proteins of myelin and the Marchi-positive bodies. 3H-leucine was administered intrathecally in adult rabbits. After various survival times, the spinal cord was subfractionated by ultracentrifugation in a discontinuous two-step 0.32 M/0.85 M sucrose gradient. Myelin was collected from the interface and a floating fraction, heavily enriched in Marchi-positive bodies, was recovered on top of the 0.32 M sucrose. By scintillation counting and by gel fluorography combined with immunoblotting, a gradual appearance with time of partially degraded peptides of myelin-associated protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase was seen in the floating fraction but not in myelin. The temporal dynamics of the specific activities of these two proteins and myelin-basic protein and proteolipid protein were consistent with a typical source-product relationship between myelin and the material in the floating fraction. In conjunction with earlier morphological and biochemical findings, these data may suggest that Marchi-positive bodies appear as a consequence of myelin catabolism.

Two computer- and image-analysis-based procedures for realignment of images of consecutive light micrographs of nerve tissue (cat motor cortex) have been developed. One procedure (CENT) was interactive, employing the subjective overall 'best fit' of two images to each other and the other (AUTOCENT) was based on an automatic comparison of two images (image thresholding and binary comparison). Images of light micrographs of several hundred consecutive sections were realigned using the interactive and the automatic methods. The interactive procedure was easy to use; realignment of an image took only a few minutes in the hands of an experienced operator and should be easy to implement on commercially available smaller computers like PCs and workstations, but the realignment was possibly disturbed by the operator's subjective expectancy of regular forms. The automatic procedure realigned 1-4 images per hour, might be implemented on smaller computers if the programming is made more efficient, was probably objective and restored the 'true position of images'.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e. lysosomes, in dorsal column and dorsal root axons of the spinal cord segments C8 and L7 in adult cats was analyzed by light and electron cytochemical methods after injection of horseradish peroxidase (HRP) in the dorsal column nuclei. Axonal lysosomes were, with few exceptions, concentrated at the nodes of Ranvier. We found no changes in nodal occurrence and distribution of lysosomes in axons of the HRP-injected sides, as compared to axons of the uninjected sides or of animals not exposed to HRP. Axonal lysosomes were very rare in the dorsal columns, where the frequency of nodes containing light microscopically detectable AcPase-positive bodies was 0-5% at the HRP-injected sides, 0-6% at the contralateral sides, and 0-3% in control animals. The corresponding values in the cervical and lumbar dorsal roots were 6-23%, 9-20%, 10-12% and 19-37%, 21-40%, 26-43%, respectively. In view of our recent observations in alpha-motor neurons, the results point at a noteworthy difference in local degradative ability between dorsal column axons and alpha-motor axons, the latter being able to accumulate intramuscularly injected and retrogradely transported HRP at their PNS nodes of Ranvier for 48-60 h, during which period the axoplasmic AcPase activity/concentration increases at some nodes. Such a degradative activity, which could protect the motor neurons by restricting axoplasmic transport of exogenous materials imbibed by their axon terminals outside the CNS, may not be of the same significance for neurons, e.g. dorsal root ganglion neurons, the axon terminals of which are located within the CNS.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha-motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha-motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon.

The paranodal occurrence of axon-Schwann cell networks (ASNs), which are entities assumed to take part in the removal of degenerate axonal material, was examined quantitatively by electron microscopical serial section analysis in normal cat ventral and dorsal spinal roots. In nerve fibres greater than or equal to 10 microns in diameter 88% of the nodal regions in the ventral roots and 97% in the dorsal roots showed ASN complexes, which especially in the ventral roots often consisted of many segregated axoplasmic portions. The corresponding frequencies in fibres less than 10 microns were 28% and 62% in the ventral and the dorsal roots, respectively. ASN complexes were rare in fibres less than 5 microns. The results show that the ASN is a part of the normal paranodal architecture in large myelinated nerve fibres. The ASN occurrence seems to differ with neurone type.

Histochemical and electron microscopical studies have shown that Marchi-positive bodies in the normal mammalian CNS are associated with paranodal regions and it has been proposed that the formation of Marchi-positive bodies represents a step in the catabolic events of normal myelin turnover. After a two-step density gradient ultracentrifugation a light 'floating fraction' highly enriched in these structures can be collected and in the present study two monoclonal antibodies, FC4 and 3B5, were produced against proteins present in the floating fraction but absent from the myelin fraction. Immunohistochemical studies showed that these antibodies bound preferentially to the PNS-CNS transitional region and the glia limitans. Double-staining experiments demonstrated an extensive overlap in these regions with cells stained by antibodies against the astrocyte marker glial fibrillary acidic protein. Centrally in the white matter, FC4 and 3B5 mainly stained cells which also stained with the microglia lectin marker Bandeiraea simplicifolia isolectin B4. Three-dimensional reconstructions made from confocal microscopic scans showed that FC4/3B5-positive cells in the white matter extend processes enveloping paranodal Marchi-positive bodies and nodes of Ranvier. It is suggested that astrocyte-like and microglia-like cells both are participants in paranodal myelin turnover and that a division of labour with respect to, for example, protein degradation and immunological functions, may be present between the two cell types.

Lysosomal activity at nodes of Ranvier of feline hindlimb alpha-motor neurons was examined by light and electron microscopical acid phosphatase (AcPase) histochemistry during retrograde axonal transport of intramuscularly injected horseradish peroxidase (HRP). Several nodes along the PNS parts of the alpha-motor axons of the HRP-injected side showed accumulations of AcPase-positive bodies in the constricted nodal axon segment and the adjacent paranodal axoplasm. Such lysosomal accumulations were most prominent in the ventral root and differed in number and intensity depending on survival time after the HRP injection. At nodes showing high AcPase activity the axoplasm proximal to the nodal midlevel was occupied by many small, AcPase-positive, vesiculotubular profiles. Larger AcPase-positive bodies were mainly situated distal to the nodal midlevel. Double incubation for demonstration of both HRP and AcPase activity showed similar accumulations of AcPase-positive bodies at some of the HRP-transporting nodes. The AcPase activity differed considerably between nodes exhibiting comparable levels of HRP-positivity. Many of the AcPase-positive bodies also contained HRP reaction product. At some HRP-positive nodes the number of AcPase-positive bodies situated in the paranodal axon-Schwann cell network was elevated when compared to nodes of the contralateral, control side. In contrast to the PNS nodes, the nodal occurrence and distribution of lysosomes in the CNS part of alpha-motor axons seemed not to be affected by HRP transport. These observations support our previous proposal that nodes of Ranvier in the PNS parts of alpha-motor axons, in contrast to their CNS nodes, possess an ability to control passage of and initiate lysosomal degradation of axonally transported substances. Such an ability may provide a protective function to the motor neuron by restricting the intraneuronal transport of materials imbibed by the axon terminals outside the CNS.

An ultrastructural and morphometric study was performed on sural nerve biopsies of four industrial spray painters (35-59 years) and 11 controls (6-64 years). No difference could be shown in spray painters and age-matched controls as to the number of myelinated nerve fibres per area, their size distribution, variation of internodal length along single nerve fibres or the ratio between the number of myelin lamellae and the axon circumference. There was marked scattering of the two latter parameters in older exposed and and control individuals. The distribution of NADH2-tetrazolium reductase activity was similar in exposed and control cases. The general ultrastructural appearance of nodal-paranodal regions in controls conformed with that noted in experimental animals. The overall ultrastructural organization and age-related changes of nerves of exposed cases were similar to those of control cases except for a presence of paranodal axonal mitochondria which contained glycogen-like particles in exposed cases. In one exposed case abundant dispersed or clustered glycogen-like particles were seen in the paranodal axoplasm. These findings are suggested to be an effect of chronic exposure to organic solvent vapours. Ageing seems, however, to have a much greater impact on the morphology of the sural nerve fibre than occupational exposure to organic solvent.

The general ultrastructural organization of nodes of Ranvier in peripheral nerve fibres from 2 to 20 microns in diameter (D) was investigated in the adult cat using serially sectioned ventral and dorsal spinal roots. The study was performed in order to collect and systematize information considered necessary for a morphometric analysis of the node of Ranvier. In all cases a node of Ranvier could be divided into a central nodal axon segment and a surrounding nodal Schwann cell compartment. The latter included a nodal gap matrix substance, more or less overlapping nodal Schwann cell collars and, as a rule, also a Schwann cell brush-border emanating from the nodal Schwann cell collars and occupying the nodal gap. The relative size and the organization level of the nodal Schwann cell compartment increased with increasing fibre size up to a fibre diameter of 8-10 microns. At this fibre size the nodal gap was of a fairly even height (1 micron) all around the nodal axon and contained a thick brush-border of densely packed, more or less radially arranged Schwann cell microvilli. In very small fibres (D less than 3 microns) the nodal gap was low (less than 0.1 microns) and contained no or few microvilli. In fibres greater than 10 microns in diameter the relative size and the degree of structural order of the nodal Schwann cell compartment decreased with increasing fibre size. Drastic sectorial variations in nodal gap height and local thinning-out of the brush-border became prominent features in the largest fibres. The possible in vivo organization of the nodal Schwann cell compartment is discussed. Preliminary calculations indicate that the extracellular space directly surrounding the nodal axon might be quite small and that the area open for free communication between this extracellular space and the endoneurial space might be very much restricted, measuring as little as 2% of the area of the nodal axolemma. Algorithms for calculating various nodal structural parameters are discussed.

Samples of S1 dorsal root nerve fibers from cats of different pre- and postnatal ages were examined electron microscopically with regard to axon caliber and number of myelin lamellae. Each root was examined at four different cross-sectional levels. Two levels were situation close to the spinal cord entrance on each side of the peripheral (PNS) and central nervous system (CNS) border. The third and fourth levels were located more distally. The first compact myelin lamella was observed in the CNS part of the root in a 47-day-old fetus. In the 53-day-old fetus the degree of myelination was the same in the CNS as distal in the PNS part of the root. Surprisingly, all axons appeared unmyelinated close to the PNS-CNS border and remained so for a further 10-day period. After this time lag, this part of the root became myelinated and showed a rapid increase in myelin sheath thickness. Calculations of axonal growth, mesaxonal length, and myelin volume indicated a maturation process that progressed discontinuously. Myelination did not proceed in a strict somatofugal direction, but was a regionally differentiated process. The maximal myelin production, expressed as the increase in myelin volume per Schwann cell, was found during the second to fourth postnatal months, i.e., very late in development.