Transplantation of neural stem cells encapsulated in hydrogels improve functional recovery in a cauda equina lesion model.

This study explored the therapeutic effects of transplantation of neural stem cells (NSCs) encapsulated in hydrogels in a cauda equina lesion model. NSCs were isolated from neonatal dorsal root ganglion (nDRG) and cultured in three-dimensional porous hydrogel scaffolds. Immunohistochemistry, transmission electron microscopy and TUNEL assay were performed to detect the differentiation capability, ultrastructural and pathological changes, and apoptosis of NSCs. Furthermore, the functional recovery of sensorimotor reflexes was determined using the tail-flick test. NSCs derived from DRG were able to proliferate to form neurospheres and mainly differentiate into oligodendrocytes in the three-dimensional hydrogel culture system. After transplantation of NSCs encapsulated in hydrogels, NSCs differentiated into oligodendrocytes, neurons or astrocytes in vivo. Moreover, NSCs engrafted on the hydrogels decreased apoptosis and alleviated the ultrastructural and pathological changes of injured cauda equina. Behavioral analysis showed that transplanted hydrogel-encapsulated NSCs decreased the tail-flick latency and showed a neuroprotective role on injured cauda equina. Our results indicate transplantation of hydrogel-encapsulated NSCs promotes stem cell differentiation into oligodendrocytes, neurons or astrocytes and contributes to the functional recovery of injured cauda equina, suggesting that NSCs encapsulated in hydrogels may be applied for the treatment of cauda equina injury.

Acellular Cauda Equina Allograft as Main Material Combined with Biodegradable Chitin Conduit for Regeneration of Long-Distance Sciatic Nerve Defect in Rats.

Autologous nerve grafting (ANG), the gold standard treatment for peripheral nerve defects, still has many restrictions. In this study, the acellular cauda equina allograft (ACEA), which consists of biodegradable chitin conduit and acellular cauda equina, is developed. The cauda equina is able to complete decellularization more quickly and efficiently than sciatic nerves under the same conditions, and it is able to reserve more basal lamina tube. In vitro, ACEA shows superior guidance capacity for the regeneration of axons and migration of Schwann cells compared to acellular sciatic nerve allograft (ASNA) in dorsal root ganglion culture. In vivo, ACEA is used to bridge 15 mm long-distance defects in rat sciatic nerves. On day 21 after transplantation, the regenerative distance of neurofilaments in the grafting segment is not significantly different between the ACEA and ANG groups. At week 12, ACEA group shows better sciatic nerve repair than chitin conduit only and ASNA groups, and the effect is similar to that in the ANG group as determined by gait analysis, neural electrophysiological, and histological analyses. The above results suggest that the ACEA has the potential to become a new biological material as a replacement for autografting in the treatment of long-distance nerve defects.

The brain of the tree pangolin (Manis tricuspis). I. General appearance of the central nervous system.

Here, we describe the superficial appearance of the brain of the rarely studied tree pangolin. Phylogenetic analyses have placed the pangolins, order Pholidota, as a sister group to the order Carnivora. The majority of features visible on the surface of the tree pangolin brain, and its overall appearance can be described as typically mammalian. The pattern of sulci and gyri, while simple, appears very similar to that observed in carnivores. Two derived features of the Pholidota were observed, the first being the rostral decussation of the pyramidal tract, which instead of occurring at the spinomedullary junction, decussates at the level of the caudal pole of the facial nerve nucleus in the rostral medulla oblongata. This appears to be related to the need for voluntary control of the tongue, with a potentially enlarged corticobulbar tract ending in the hypoglossal nucleus. The second derived feature is the very short spinal cord, which terminates midway along the thoracic vertebrae before giving rise to a long and extensive cauda equina. This foreshortened spinal cord appears to be related to anisotropic growth of the somatic and neural elements following early development of the central nervous system. The olfactory system appears to be generally enlarged and is likely the predominant sense used in foraging. Vision and hearing do not appear specialized based on the relative size of the superior and inferior colliculi, but potential somatic specializations indicate that the somatosensory system is heavily relied upon for food consumption and prehensile tail usage.

Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans.

Neural stem cells (NSCs) reside in a unique microenvironment within the central nervous system (CNS) called the NSC niche. Although they are relatively rare, niches have been previously characterized in both the brain and spinal cord of adult animals. Recently, another potential NSC niche has been identified in the filum terminale (FT), which is a thin band of tissue at the caudal end of the spinal cord. While previous studies have demonstrated that NSCs can be isolated from the FT, the in vivo architecture of this tissue and its relation to other NSC niches in the CNS has not yet been established. In this article we report a histological analysis of the FT NSC niche in postnatal rats and humans. Immunohistochemical characterization reveals that the FT is mitotically active and its cells express similar markers to those in other CNS niches. In addition, the organization of the FT most closely resembles that of the adult spinal cord niche. J. Comp. Neurol. 525:661-675, 2017. © 2016 Wiley Periodicals, Inc.

The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties.

BACKGROUND: The filum terminale is a fibrous band, consisting of the filum terminale internum (FTI), connecting the conus medullaris (CM) with the dural sac (DS), and the filum terminale externum (FTE), connecting the DS with the coccyx. Despite its importance in tethered cord syndrome, published anatomic and physiologic data on the filum terminale remain scarce. We describe 1) the dimensions and position of the FTI and FTE; 2) the histology of the FTI-DS-FTE transition zone; and 3) the extensibility and elastic properties of the FTI and the CM. METHODS: Anatomic measurements were performed on 10 fresh and 10 embalmed human cadavers. Four other fresh cadavers were used for strain and elasticity measurements. RESULTS: The mean FTI and FTE lengths were 158.75 and 69.33 mm, respectively. From cranially to caudally, the FTI diameter decreased from 1.93 to 0.69 mm. The most frequent vertebral level of the CM-FTI and the FTI-DS-FTE junction were L1 and S2, respectively. FTE length correlates with body length (r = 0.54; P = 0.014) and with FTI-DS-FTE junction vertebral level (ρ =-0.76; P < 0.001). Histologically, the FTI fuses with DS fibers and continues as FTE. The FTI and the CM show an exponential loaded weight-strain relationship, with the FTI showing higher strain than the CM and almost perfect elastic properties. The CM strain is increased when the dentate ligaments are cut. CONCLUSIONS: The FTI is an overturned oblate cone-shaped structure, showing bigger strain under weight loading compared with the CM, thereby protecting the CM from traction, together with the dentate ligaments.

The isolation, differentiation, and survival in vivo of multipotent cells from the postnatal rat filum terminale.

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

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.

The postnatal human filum terminale is a source of autologous multipotent neurospheres capable of generating motor neurons.

BACKGROUND: Neural progenitor cells (NPCs) are undifferentiated and mitotic and can be induced to differentiate into neurons and glia, the building blocks of the nervous system. NPCs have great therapeutic potential for nervous system trauma and degenerative disorders. They have been identified in the mammalian central nervous system, but current sources are difficult to access surgically and come from regions that are critical for normal brain function. OBJECTIVE: To identify and characterize in detail a novel source of human NPCs in the filum terminale (FT), a vestigial structure at the caudal end of the spinal cord, which is easily accessed and plays no functional role in the postnatal nervous system. METHODS: Cells were isolated and cultured in vitro from the FT of terminated fetuses and from children and adolescents who had undergone surgical resections for tethered spinal cords. Cell culture techniques, immunohistochemistry, and immunocytochemistry were applied to examine FT cells. RESULTS: : FT cells gave rise to neurospheres that proliferated over extended periods of time in culture. These neurospheres were positive for neural stem/progenitor cell markers by immunocytochemical staining. The neurospheres were able to be induced to differentiate in vitro into neurons and glial cells, which were confirmed by the use of antibodies against the cell type-specific markers. Moreover, they have been induced to form motor neurons capable of innervating striated muscle in vitro. CONCLUSION: Multipotent NPC cells from the FT are both accessible and expendable. They may allow autologous cell-based transplantation therapy that circumvents immunological rejection.

Distribution and characterization of progenitor cells within the human filum terminale.

BACKGROUND: Filum terminale (FT) is a structure that is intimately associated with conus medullaris, the most caudal part of the spinal cord. It is well documented that certain regions of the adult human central nervous system contains undifferentiated, progenitor cells or multipotent precursors. The primary objective of this study was to describe the distribution and progenitor features of this cell population in humans, and to confirm their ability to differentiate within the neuroectodermal lineage. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that neural stem/progenitor cells are present in FT obtained from patients treated for tethered cord. When human or rat FT-derived cells were cultured in defined medium, they proliferated and formed neurospheres in 13 out of 21 individuals. Cells expressing Sox2 and Musashi-1 were found to outline the central canal, and also to be distributed in islets throughout the whole FT. Following plating, the cells developed antigen profiles characteristic of astrocytes (GFAP) and neurons (ß-III-tubulin). Addition of PDGF-BB directed the cells towards a neuronal fate. Moreover, the cells obtained from young donors shows higher capacity for proliferation and are easier to expand than cells derived from older donors. CONCLUSION/SIGNIFICANCE: The identification of bona fide neural progenitor cells in FT suggests a possible role for progenitor cells in this extension of conus medullaris and may provide an additional source of such cells for possible therapeutic purposes. Filum terminale, human, progenitor cells, neuron, astrocytes, spinal cord.

Exploring atypical locations of mammalian neural stem cells: the human filum terminale.

Neurogenesis is a multifactorial event determined by local environmental cues, inherent cellular program as well as cellular milieu and may not necessarily be restricted to the SVZ and SGZ. NSCs have been isolated from or neurogenesis has been demonstrated in traditionally non neurogenic regions. This more permissive view of neurogenesis, however, is not widely accepted due to concerns regarding the methodologies used. Furthermore, it is compounded by the fact that the basal levels of increased neurogenesis in such regions has not been completely confirmed and thus precludes a paradigm shift. Were this non limited view of neurogenesis to be generally accepted after thorough investigation, it would open new avenues for regenerative medicine and stem cell therapy.

Isolation of human multipotent neural progenitors from adult filum terminale.

Stem cells have been isolated from several CNS regions, including the spinal cord. However, the terminal end of the spinal cord, filum terminale, has been referred to as a fibrovascular tag without neurogenic potential and of no clinical significance. Recently, we were fortunate to acquire some samples of this tissue. We show for the first time that progenitor cells exhibiting the hallmarks of stem cells can be isolated from adult human filum terminale (FTNPs). More specifically, FTNPs self-renew and proliferate to form neurospheres, and exhibit tripotent differentiation into neurons, astrocytes, and oligodendrocytes. Equally important, FTNPs develop the electrophysiological profile of neurons and glia. Whole-cell patch-clamp recordings show beta-III-tubulin(+) neurons exhibiting overshooting action potentials, displaying both the fast inactivating TTX-sensitive sodium current as well as 4-AP and TEA sensitive potassium currents. To assess potency in vivo, FTNPs were transplanted into the posterior periventricular region of control or ischemic rat brains. Despite a vigorous immune response against the xenograft, FTNPs survived and were found not only in the graft area but had also migrated to the lesioned CA1 region. Notwithstanding the immune response, FTNPs differentiated into astrocytes, but no neuronal differentiation was observed in the transplant milieu tested. However, neuronal differentiation in vivo cannot be ruled out and assessment of the conditions necessary to promote neurogenesis in vivo requires more research. Significantly, no tumor formation or aberrant cell morphology was seen in or adjacent to the graft area. Thus, filum terminale provides a novel source of adult human neural progenitor cells that develop into functional neurons with possible clinical applications.

ISSLS prize winner: integrating theoretical and experimental methods for functional tissue engineering of the annulus fibrosus.

STUDY DESIGN: Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering. OBJECTIVE: Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios. SUMMARY OF BACKGROUND DATA: There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy. METHODS: Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model). RESULTS: The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading. CONCLUSION: AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction. Further, the model suggested that the native AF fiber architecture is uniquely designed to support shear stresses encountered under multiple loading configurations.

Neurochemical architecture of the filum terminale in the rat.

Contrary to the widespread assumption, the filum terminale in the rat possesses a precise glial and neuronal organization. The processes of glial fibrillary acidic protein-stained astrocytes form a rich, three dimensional array. The crescent shaped white matter could be outlined with antibody detecting oligodendrocytes. The neurons in the filum terminale, labeled with neuron-specific nuclear protein, are distributed in a small midline group (dorsal nucleus) dorsal to and in two symmetrical clusters at both sides of the central canal (lateral nuclei). Nitric oxide synthase-, calretinin-, choline acetyltransferase-, substance P- and neurokinin receptor-1-immunoreactive neurons were detected in the lateral nuclei. Axons were classified based on their course and termination. Small number of calcitonin gene-related peptide-immunoreactive fibers was found exclusively in the dorsal nucleus. Nitric oxide synthase-, substance P-, and neurokinin receptor-1-stained axon arborizations were detected mainly in the lateral nucleus. A dense array of extremely fine vesicular glutamate transporter 2- and fine, synaptophysin-immunoreactive varicosities covered densely the lateral nuclei. Fine glycine-transporter 2-immunoreactive axon arborization like structures were seen also in the lateral nucleus. Vesicular glutamate transporter 1- and choline acetyltransferase-immunoreactive axons arborized in the entire gray matter. Serotonin- and enkephalin-immunoreactive fibers congregated in the dorsolateral portion of the white matter, called "shoulder region", while calretinin- and thick, varicose neurokinin receptor-1-stained axons were also seen in the same area of the white matter. Synaptophysin-immunoreactive fine varicosities colocalized only with vesicular glutamate transporter 2 immunoreaction. Substance P and glycine-transporter 2-immunoreactive puncta were found in close contact with neurokinin receptor-1-immunostained perikarya and dendrites.

The filum terminale externum.

OBJECT: The filum terminale externum (FTE) is the extradural component of the filum terminale internum and little attention has been dedicated to this structure in the literature. The authors theorized that the rare intrasacral ependymomas may originate from ependymal cell collections within the FTE. METHODS: To address this hypothesis, the FTE was dissected and analyzed histologically in 15 adult cadavers. None of the specimens was found to harbor ependymal or other glial cell collections. CONCLUSIONS: The authors found previously undescribed smooth-muscle cells within the FTE. Furthermore, histological analysis identified adipose, nerve, bone, and cartilage cells.

Segmental variation in the in vitro cell metabolism of nucleus pulposus cells isolated from a series of bovine caudal intervertebral discs.

STUDY DESIGN: This study focuses on the association between cell metabolism and molecular matrix composition of nucleus pulposus (NP) tissue with spine level in sequential bovine caudal intervertebral discs. OBJECTIVE: To explore the hypothesis that the molecular composition of NP tissue and corresponding cell metabolism varies with caudal spine. A secondary hypothesis is tested that potential cellular differences are maintained after monolayer culture. SUMMARY OF BACKGROUND DATA: In articular cartilage, cell metabolism and molecular matrix composition are influenced by loading history. This may also be a feature of intervertebral discs in series. METHODS: NP cells (nonpooled or level pooled) were isolated from four sequential bovine caudal intervertebral discs (levels 3-4, 4-5, 5-6, and 6-7) and cultured in alginate beads immediately or following monolayer culture. Levels of 3H-TdR (proliferation) and 35SO4 (GAG synthesis) incorporation were determined from 14 animals. In a separate set of 6 animals, total content of water, DNA, collagen (and type), GAG (and type) were also determined. RESULTS: The rate of 3H-TdR and 35SO4 incorporation in freshly isolated NP cells increased nonlinearly from level 3-4 to 6-7 (P < 0.05). Monolayer cultured cells retained level-specific differences for 35SO4 and 3H-TdR incorporation similar to that of freshly isolated cells. GAG content and chondroitin sulfate proportion decreased distally (P < 0.05); however, total collagen and Type I proportion increased distally (P < 0.05). No significant differences in water or DNA content could be determined. CONCLUSIONS: The results support the hypothesis that level-specific differences in NP cell metabolism and molecular composition are dependent on spine level potentially reflecting subtle mechanical differences between levels. Retention of level-specific differences in monolayer may suggest a certain level of cell "programming." This may be important for cellular strategies to repairspecific sites of degeneration.

Tapering of human nerve fibres.

To determine the tapering of human nerve fibres, rostral and caudal root pieces of cauda equina nerve roots were removed and nerve fibre diameter distributions were constructed for 4 myelin sheath thickness ranges for the two sites, and compared with each other. The reduction of the group diameter in the different alpha-motoneuron groups was 0.2 % per 13 cm. Accounting for systematic errors, there may be even less tapering. An identified single nerve fibre showed no tapering. Further, there is indication that gamma-motoneurons, preganglionic sympathetic and parasympathetic fibres and skin afferents also reduce their fibre diameter by 0.2 % per 13 cm or less. Consequently, a nerve fibre with a diameter of 10 microm would be reduced to approximately 9.8 microm at 1m from the cell soma. Preganglionic parasympathetic fibres were found to be represented in roots S1 to S5. At similar distances from the spinal cord, the mean diameter of ventral root alpha1-motoneuron (FF) axons increased from the thoracic towards the lumbo-sacral region before decreasing again in the lower sacral region. Usually no alpha1-motoneuron axons were found in S5 roots. The diameter distribution of unmyelinated nerve fibres of a ventral S5 root showed three peaks at 0.25, 0.95 and 1.2 microm. The unmyelinated fibres with diameters around 0.25 microm may represent parasympathetic fibres. In six selected areas of the ventral S5 root, 6.6 times more unmyelinated nerve fibres than myelinated fibres were found on the average.

Development of the posterior neural tube in human embryos.

Development of the posterior neural tube (PNT) in human embryos is a complicated process that involves both primary and secondary neurulation. Because normal development of the PNT is not fully understood, pathogenesis of spinal neural tube defects remains elusive. To clarify the mechanism of PNT development, we histologically examined 20 human embryos around the stage of posterior neuropore closure and found that the developing PNT can be divided into three parts: 1) the most rostral region, which corresponds to the posterior part of the primary neural tube, 2) the junctional region of the primary and secondary neural tubes, and 3) the caudal region, which emerges from the neural cord. In the junctional region, the axially-condensed mesenchyme (AM) intervened between the neural plate/tube and the notochord at the stage of posterior neuropore closure, while the notochord was directly attached to the neural plate/tube in the most rostral region. A single cavity was found to be formed in the AM as the presumptive luminal surface cells were radially aligned in the junctional region prior to the formation of the neural cord. The single cavity was continuous with the central cavity of the primary neural tube. In contrast, multiple or isolated cavities were frequently observed in the caudal region of the PNT. Our observation suggests that the junctional region of the PNT is distinct from other regions in terms of the relationship with the notochord and the mode of cavitation during secondary neurulation.

Intraoperative urodynamics in spinal cord surgery: a study of feasibility.

Intraoperative monitoring (IOM) of bladder function in spinal cord surgery is a challenging task due to vegetative influences, multilevel innervation and numerous supraspinal modulating factors. Despite routine use of urodynamics in neurosurgery for implantation of bladder stimulators or denervation of nerve fibres in spastic reflex bladders, application of IOM in patients with spinal cord tumours or tethered-cord syndrome is not widespread. Combining urodynamics with sphincter electromyography (EMG) in IOM enables identification of bladder efferents responsible for contraction and continence. We monitored four patients with ependymoma of the Cauda equina, one patient with tethered-cord syndrome and two patients with cervical intramedullary tumours. In all patients undergoing operations of the Cauda equina, identification of bladder efferents responsible for detrusor contraction was possible. There was good correlation between preoperative bladder dysfunction, preoperative urodynamics and intraoperative pressure increase by bladder contraction or latency between stimulation and contraction. This method proved unsuitable for intramedullary tumours where no contraction of the bladder could be observed while stimulating the spinal cord. Intraoperative monitoring of urodynamics is an effective tool for identifying bladder efferents in the Cauda equina. Intraoperative conclusions on bladder dysfunction through registration of pressure increase and latency are possible.

Membrane currents and morphological properties of neurons and glial cells in the spinal cord and filum terminale of the frog.

Using the patch-clamp technique in the whole-cell configuration combined with intracellular dialysis of the fluorescent dye Lucifer yellow (LY), the membrane properties of cells in slices of the lumbar portion of the frog spinal cord (n=64) and the filum terminale (FT, n=48) have been characterized and correlated with their morphology. Four types of cells were found in lumbar spinal cord and FT with membrane and morphological properties similar to those of cells that were previously identified in the rat spinal cord (Chvátal, A., Pastor, A., Mauch, M., Syková, E., Kettenmann, H., 1995. Distinct populations of identified glial cells in the developing rat spinal cord: Ion channel properties and cell morphology. Eur. J. Neurosci. 7, 129-142). Neurons, in response to a series of symmetrical voltage steps, displayed large repetitive voltage-dependent Na(+) inward currents and K(+) delayed rectifying outward currents. Three distinct types of non-neuronal cells were found. First, cells that exhibited passive symmetrical non-decaying currents were identified as astrocytes. These cells immunostained for GFAP and typically had at least one thick process and a number of fine processes. Second, cells with the characteristic properties of rat spinal cord oligodendrocytes, with passive symmetrical decaying currents and large tail currents after the end of the voltage step. These cells exhibited either long parallel or short hairy processes. Third, cells that expressed small brief inward currents in response to depolarizing steps, delayed rectifier outward currents and small sustained inward currents identical to rat glial precursor cells. Morphologically, they were characterized by round cell bodies with a number of finely branched processes. LY dye-coupling in the frog spinal cord gray matter and FT was observed in neurons and in all glial populations. All four cell types were found in both the spinal cord gray matter and FT. The glia/neuron ratio in the spinal cord was 0.78, while in FT it was 2.0. Moreover, the overall cell density was less in the FT than in the spinal cord. The present study shows that the membrane and morphological properties of glial cells in the frog and rat spinal cords are similar. Such striking phylogenetic similarity suggests a significant contribution from distinct glial cell populations to various spinal cord functions, particularly ionic and volume homeostasis in both mammals and amphibians.

Extracellular diffusion parameters in spinal cord and filum terminale of the frog.

Extracellular space (ECS) diffusion parameters were studied in isolated frog spinal cord grey matter and filum terminale (FT), that is predominantly composed of glial cells and axons. We compared the cell swelling induced by K(+) application, hypotonic stress and tetanic stimulation of afferent input. The ECS diffusion parameters, volume fraction alpha (alpha = ECS volume/total tissue volume), tortuosity lambda (lambda(2) = free/apparent diffusion coefficient in the tissue) and non-specific cellular uptake k', were determined by the real-time iontophoretic method using TMA(+)-selective microelectrodes. Stimulation-evoked changes in extracellular K(+) concentration ([K(+)](e)) were measured by K(+)-selective microelectrodes. Histological analysis revealed that in the central region of the FT, the cell density was lower than in SC, neurons and oligodendrocytes were scarce, GFAP-positive astrocytes were abundant, and they showed thicker and more densely stained processes than in spinal cord. In the FT, alpha was 58% higher and lambda significantly lower than in the spinal cord. In 50 mM K(+), alpha in spinal cord decreased from about 0.19 to 0.09, i.e., by 53%, whereas in FT from about 0.32 to 0.20, i.e., by only 38%; lambda increased significantly more in FT than in spinal cord. Hypotonic solution (175 mmol/kg(-1)) resulted in similar decreases in alpha, and there were no changes in lambda in either spinal cord or FT. Stimulation of VIII or IX dorsal root (DR) by 30 Hz evoked an increase in [K(+)](e) from 3 to 11-12 mM in spinal cord, but to only 4-5 mM in FT. In the spinal cord this stimulation led to a 30% decrease in alpha and a small increase in lambda whereas in the FT the decrease in alpha was only about 10% and no increase in lambda was found. We conclude that in spinal cord, a complex tissue with a higher density of cellular elements than the FT, 50 mM K(+), hypotonic stress as well as DR stimulation evoked a greater decrease in ECS volume than in FT. Nevertheless, the K(+)-induced increase in tortuosity was higher in FT, suggesting that a substantial part of the K(+)-evoked increase in lambda was due to astrocytic swelling.