IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment.

Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/ß-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.

Loss of Rsph9 causes neonatal hydrocephalus with abnormal development of motile cilia in mice.

Hydrocephalus is a brain disorder triggered by cerebrospinal fluid accumulation in brain cavities. Even though cerebrospinal fluid flow is known to be driven by the orchestrated beating of the bundled motile cilia of ependymal cells, little is known about the mechanism of ciliary motility. RSPH9 is increasingly becoming recognized as a vital component of radial spokes in ciliary "9 + 2" ultrastructure organization. Here, we show that deletion of the Rsph9 gene leads to the development of hydrocephalus in the early postnatal period. However, the neurodevelopment and astrocyte development are normal in embryonic Rsph9-/- mice. The tubular structure of the central aqueduct was comparable in Rsph9-/- mice. Using high-speed video microscopy, we visualized lower beating amplitude and irregular rotation beating pattern of cilia bundles in Rsph9-/- mice compared with that of wild-type mice. And the centriolar patch size was significantly increased in Rsph9-/- cells. TEM results showed that deletion of Rsph9 causes little impact in ciliary axonemal organization but the Rsph9-/- cilia frequently had abnormal ectopic ciliary membrane inclusions. In addition, hydrocephalus in Rsph9-/- mice results in the development of astrogliosis, microgliosis and cerebrovascular abnormalities. Eventually, the ependymal cells sloughed off of the lateral wall. Our results collectively suggested that RSPH9 is essential for ciliary structure and motility of mouse ependymal cilia, and its deletion causes the pathogenesis of hydrocephalus.

Autoradiography and inmmunolabeling suggests that lizard blastema contains arginase-positive M2-like macrophages that may support tail regeneration.

BACKGROUND: The regenerating blastema of the tail in the lizard Podarcis muralis contains numerous macrophages among the prevalent mesenchymal cells. Some macrophages are phagocytic but others are devoid of phagosomes suggesting that they have other roles aside phagocytosis. METHODS: The presence of healing macrophages (M2-like) has been tested using autoradiographic, immunohistochemical and ultrastructural studies. RESULTS: Autoradiography shows an uptake of tritiated arginine in sparse cells of the blastema and in the regenerating epidermis. Bioinformatics analysis suggests that epitopes for arginase-1 and -2, recognized by the employed antibody, are present in lizards. Immunofluorescence shows sparse arginase immunopositive macrophages in the blastema and few macrophages also in the apical wound epidermis. The ultrastructural study shows that macrophages contain dense secretory granules, most likely inactive lysosomes, and small cytoplasmic pale vesicles. Some of the small vesicles are arginase-positive while immunolabeling is very diffuse in the macrophage cytoplasm. CONCLUSIONS: The presence of cells incorporating arginine and of arginase 1-positive cells suggests that M2-like macrophages are present among mesenchymal and epidermal cells of the regenerative tail blastema. M2-like macrophages may promote tail regeneration differently from the numerous pro-inflammatory macrophages previously detected in the scarring limb. The presence of M2-like macrophages in addition to hyaluronate, support the hypothesis that the regenerative blastema of the tail in lizards is an immuno-privileged organ where cell proliferation and growth occur without degenerating in a tumorigenic outgrowth.

Regional differences in the ependyma of the optic tectal ventricle of adult zebrafish with structures referring to brain hydrodynamics.

Classical electron microscopic morphological studies provide detailed ultrastructural information, which may lend insights into cellular functions. As a follow-up to our morphological investigation of the adult zebrafish (Danio rerio) optic tectum, in this study, we have analyzed the ependymal structures lining the surfaces of the tectal ventricle: the torus, tegmental surface of the valvula cerebelli and the periventricular gray zone of the optic tectal cortex. We used toluidine blue stained plastic (semithin) sections for light microscopy and scanning electron microscopy. Our morphological findings of gated entrances and/or egresses indicate that, at least in the adult zebrafish brain, there may be a bidirectional direct flow communication between the ventricular cerebrospinal fluid and the parenchymal interstitial fluid.

Calaxin is required for cilia-driven determination of vertebrate laterality.

Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.

Ependymal cilia beating induces an actin network to protect centrioles against shear stress.

Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.

Methionine Sulfoxide Reductase-B3 (MsrB3) Protein Associates with Synaptic Vesicles and its Expression Changes in the Hippocampi of Alzheimer's Disease Patients.

Genome-wide association studies (GWAS) identified susceptibility loci associated with decreased hippocampal volume, and found hippocampal subfield-specific effects at MSRB3 (methionine sulfoxide reductase-B3). The MSRB3 locus was also linked to increased risk for late onset Alzheimer's disease (AD). In this study, we uncovered novel sites of MsrB3 expression in CA pyramidal layer and arteriolar walls by using automated immunohistochemistry on hippocampal sections from 23 individuals accompanied by neuropathology reports and clinical dementia rating scores. Controls, cognitively intact subjects with no hippocampal neurofibrillary tangles, exhibited MsrB3 signal as distinct but rare puncta in CA1 pyramidal neuronal somata. In CA3, however, MsrB3-immunoreactivity was strongest in the neuropil of the pyramidal layer. These patterns were replicated in rodent hippocampi where ultrastructural and immunohistofluorescence analysis revealed MsrB3 signal associated with synaptic vesicles and colocalized with mossy fiber terminals. In AD subjects, the number of CA1 pyramidal neurons with frequent, rather than rare, MsrB3-immunoreactive somatic puncta increased in comparison to controls. This change in CA1 phenotype correlated with the occurrence of AD pathological hallmarks. Moreover, the intensity of MsrB3 signal in the neuropil of CA3 pyramidal layer correlated with the signal pattern in neurons of CA1 pyramidal layer that was characteristic of cognitively intact individuals. Finally, MsrB3 signal in the arteriolar walls in the hippocampal white matter decreased in AD patients. This characterization of GWAS-implicated MSRB3 protein expression in human hippocampus suggests that patterns of neuronal and vascular MsrB3 protein expression reflect or underlie pathology associated with AD.

Simvastatin Promotes Hematoma Absorption and Reduces Hydrocephalus Following Intraventricular Hemorrhage in Part by Upregulating CD36.

We previously found that hematoma worsens hydrocephalus after intraventricular hemorrhage (IVH) via increasing iron deposition and aggravating ependymal cilia injury; therefore, promoting hematoma absorption may be a promising strategy for IVH. Recently, some investigations imply that simvastatin has the ability of accelerating hematoma absorption. Thus, this study was designed to examine the efficacy of simvastatin for IVH in rats. Intracerebral hemorrhage with ventricular extension was induced in adult male Sprague-Dawley rats after autologous blood injection. Simvastatin or vehicle was administered orally at 1 day after IVH and then daily for 1 week. MRI studies were performed to measure the volumes of intracranial hematoma and lateral ventricle at days 1, 3, 7, 14, and 28 after IVH. Motor and neurocognitive functions were assessed at days 1 to 7 and 23 to 28, respectively. Iron deposition, iron-related protein expression, ependymal damage, and histology were detected at day 28. Expression of CD36 scavenger receptor (facilitating phagocytosis) was examined at day 3 after IVH using western blotting and immunofluorescence. Simvastatin significantly increased hematoma absorption ratio, reduced ventricular volume, and attenuated neurological dysfunction post-IVH. In addition, less iron accumulation and more cilia survival was observed in the simvastatin group when compared with the control. What's more, higher expression of CD36 was detected around the hematoma after simvastatin administration. Simvastatin significantly enhanced brain hematoma absorption, alleviated hydrocephalus, and improved neurological recovery after experimental IVH, which may in part by upregulating CD36 expression. Our data suggest that early simvastatin use may be a novel therapy for IVH patients.

Bi- and uniciliated ependymal cells define continuous floor-plate-derived tanycytic territories.

Multiciliated ependymal (E1) cells line the brain ventricles and are essential for brain homeostasis. We previously identified in the lateral ventricles a rare ependymal subpopulation (E2) with only two cilia and unique basal bodies. Here we show that E2 cells form a distinct biciliated epithelium extending along the ventral third into the fourth ventricle. In the third ventricle floor, apical profiles with only primary cilia define an additional uniciliated (E3) epithelium. E2 and E3 cells' ultrastructure, marker expression and basal processes indicate that they correspond to subtypes of tanycytes. Using sonic hedgehog lineage tracing, we show that the third and fourth ventricle E2 and E3 epithelia originate from the anterior floor plate. E2 and E3 cells complete their differentiation 2-3 weeks after birth, suggesting a link to postnatal maturation. These data reveal discrete bands of E2 and E3 cells that may relay information from the CSF to underlying neural circuits along the ventral midline.

Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier.

Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier.

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.

Intracerebral Hematoma Contributes to Hydrocephalus After Intraventricular Hemorrhage via Aggravating Iron Accumulation.

BACKGROUND AND PURPOSE: The intraventricular hemorrhage (IVH) secondary to intracerebral hemorrhage (ICH) was reported to be relevant to a higher incidence of hydrocephalus, which would result in poorer outcomes for patients with ICH. However, the mechanisms responsible for this relationship remain poorly characterized. Thus, this study was designed to further explore the development and progression of hydrocephalus after secondary IVH. METHODS: Autologous blood injection model was induced to mimic ICH with ventricular extension (ICH/IVH) or primary IVH in Sprague-Dawley rats. Magnetic resonance imaging, Morris water maze, brain water content, Evans blue extravasation, immunohistochemistry staining, Western blot, iron determination, and electron microscopy were used in these rats. Then, deferoxamine treatment was used to clarify the involvement of iron in the development of hydrocephalus. RESULTS: Despite the injection of equivalent blood volumes, ICH/IVH resulted in more significant ventricular dilation, ependymal cilia damage, and iron overload, as well as more severe early brain injury and neurological deficits compared with IVH alone. Systemic deferoxamine treatment more effectively reduced ventricular enlargement in ICH/IVH compared with primary IVH. CONCLUSIONS: Our results show that ICH/IVH caused more significant chronic hydrocephalus and iron accumulation than primary IVH alone. Intracerebral hematoma plays a vital role in persistent iron overload and aggravated hydrocephalus after ICH/IVH.

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo's Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain's slices in a customized glass-bottom plate containing Dulbecco's Modified Eagle's Medium (DMEM)/High-Glucose at 37 °C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Clustering of Kir4.1 at specialized compartments of the lateral membrane in ependymal cells of rat brain.

Brain ependymal cells, which form an epithelial layer covering the cerebral ventricles, have been shown to play a role in the regulation of cerebrospinal and interstitial fluids. The machinery underlying this, however, remains largely unknown. Here, we report the specific localization of an inwardly rectifying K(+) channel, Kir4.1, on the ependymal cell membrane suggesting involvement of the channel in this function. Immunohistochemical study with confocal microscopy identified Kir4.1 labeling on the lateral but not apical membrane of ependymal cells. Ultrastructural analysis revealed that Kir4.1-immunogold particles were specifically localized and clustered on adjacent membranes at puncta adherens type junctions, whereas an aquaporin water channel, AQP4, that was also detected on the lateral membrane only occurred at components other than adherens junctions. Therefore, in ependymal cells, Kir4.1 and AQP4 are partitioned into distinct membrane compartments that might respectively transport either K(+) or water. Kir4.1 was also expressed in a specialized form of ependymal cell, namely the tanycyte, being abundant in tanycyte processes wrapping neuropils and blood vessels. These specific localizations suggest that Kir4.1 mediates intercellular K(+) exchange between ependymal cells and also K(+)-buffering transport via tanycytes that can interconnect neurons and vessels/ventricles. We propose that ependymal cells and tanycytes differentially operate Kir4.1 and AQP4 actively to control the property of fluids at local areas in the brain.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.

Defective motile cilia in Prickle2-deficient mice.

Motile cilia play diverse roles across phyla and cell types, and abnormalities in motile cilia lead to numerous disease states, including hydrocephalus. Although motile ciliary abnormalities in Prickle2 mutants have not yet been described, the planar cell polarity genes, including Prickle2, are implicated in the development and function of motile cilia. This report evaluates Prickle2-deficient mice for dysfunction in processes known to depend on functioning motile cilia. Prickle2-deficient mice do not develop hydrocephalus, but do display abnormal morphology and motility in the motile cilia of the ependyma. The morphology of tracheal motile cilia is also abnormal. Taken together, these results demonstrate that Prickle2 is required for normal ependymal motile cilia development and function.

Age-related changes in astrocytic and ependymal cells of the subventricular zone.

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.

Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning and juvenile hydrocephalus.

Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1000 children born today, making it one of the most common human developmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animal models suggest a broad genetic program underlying the regulation of CSF balance. In this study, the random integration of a transgene into the mouse genome led to the development of an early onset and rapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (Jhy(lacZ)) inherit communicating hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed as early as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4-8 weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reduced in number in Jhy(lacZ/lacZ) brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of Jhy(lacZ/lacZ) cilia develop axonemes with 9+0 or 8+2 microtubule structures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie the hydrocephalus of Jhy(lacZ/lacZ) mice, and the Jhy transcript and protein are decreased in Jhy(lacZ/lacZ) mice. Partial phenotypic rescue was achieved in Jhy(lacZ/lacZ) mice by the introduction of a bacterial artificial chromosome (BAC) carrying 60-70% of the JHY protein coding sequence. Jhy is evolutionarily conserved from humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains. Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSF homeostasis.

Disruption of CDH2/N-cadherin-based adherens junctions leads to apoptosis of ependymal cells and denudation of brain ventricular walls.

Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.

Ependymal cells variations in the central canal of the rat spinal cord filum terminale: an ultrastructural investigation.

OBJECTIVE: The ependymal cells, considered today as an active participant in neuroendocrine functions, were investigated by electron microscopy in the central canal of the lowest spinal cord, the filum terminale (FT), in adult rats. In this area of the spinal cord, the central canal is covered by a heterogeneous population of ependymal cells. The aim of the present work was to compare the regional features of the ependymal cells in two different parts of the FT with a special regard to their ultrastructure. METHODS: Two parts of the FT were selected for the ultrastructural observations: the rostral (rFT) and the caudal (cFT) ones. The rTF was removed at the level of the immediate continuation of the conus medullaris, while the cFT 30 mm further caudally. After formaldehyde fixation, the spinal cord was removed and cut into small blocks for electron microscopic processing. The material was embedded into durcupan, contrasted with uranyl acetate, lead citrate as well as osmium tetroxide, and investigated under JEOL 1200 EX electron microscope. RESULTS: In the rFT, the ependymal lining is pseudostratified and one-layered in the cFT, whereas the shape of the ependymal cells may vary from cuboidal to flatten in the rostro-caudal direction. The basal membrane of many ependymal cells possesses deep invaginations, so called "filum terminale labyrinths". Many neuronal processes occur in the pericanalicular neuropil. In contrast to the rFT, the cFT is less rich in the neuropil particles. Some of the ependymal cells concurrently reach both the intracanalicular and extracanalicular cerebrospinal fluid (CSF), thus they may represent a new variant of the ependymal cells designated as "bridge cells of the FT". CONCLUSIONS: The present data indicate that the FT ependymal cells exhibit clear differences in anatomy as well as ultrastructure that may reflect their distinct functional activity. Therefore, observations presented here may serve for the better understanding of the physiological role of the individual ependymal areas in this special portion of the rat spinal cord.