Adult neurogenesis in the short-lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging

We studied adult neurogenesis in the short‐lived annual fish Nothobranchius furzeri and quantified the effects of aging on the mitotic activity of the neuronal progenitors and the expression of glial fibrillary acid protein (GFAP) in the radial glia. The distribution of neurogenic niches is substantially similar to that of zebrafish and adult stem cells generate neurons, which persist in the adult brain. As opposed to zebrafish, however, the N. furzeri genome contains a doublecortin (DCX) gene. Doublecortin is transiently expressed by newly generated neurons in the telencephalon and optic tectum (OT). We also analyzed the expression of the microRNA miR‐9 and miR‐124 and found that they have complementary expression domains: miR‐9 is expressed in the neurogenic niches of the telencephalon and the radial glia of the OT, while miR‐124 is expressed in differentiated neurons. The main finding of this paper is the demonstration of an age‐dependent decay in adult neurogenesis. Using unbiased stereological estimates of cell numbers, we detected an almost fivefold decrease in the number of mitotically active cells in the OT between young and old age. This reduced mitotic activity is paralleled by a reduction in DCX labeling. Finally, we detected a dramatic up‐regulation of GFAP in the radial glia of the aged brain. This up‐regulation is not paralleled by a similar up‐regulation of S100B and Musashi‐1, two other markers of the radial glia. In summary, the brain of N. furzeri replicates two typical hallmarks of mammalian aging: gliosis and reduced adult neurogenesis.     

Neuronal or glial progeny: regional differences in radial glia fate

The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS.     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

Colchicine-induced tubular, vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. II.--Endocytosis studies

Administration of the antimicrotubular agent colchicine to adult rats (0.5 mg/100 g of body weight for 6 hr) induces formation of extended aggregates of tubular, vesicular, and cisternal organelles in the absorptive cells of the small intestine. The phosphatase reaction pattern (thiamine pyrophosphatase, acid phosphatase, acid trimetaphosphatase) suggests that the majority of them belongs to the lysosomal system (Ellinger and Pavelka, 1984). The present study extends these findings and examines the uptake and fate of intravenously injected horseradish peroxidase (HRP) at the basal and lateral cell surfaces and of intraluminally applied HRP at the apical cell surface. HRP, applied to control animals and animals pretreated with colchicine, was internalized at both apical and basolateral cell surfaces of the absorptive cells, and delivered into endosome-like vesicles, multivesiculated bodies (mvbs), dense bodies (dbs), and in several instances into Golgi cisternae. Following intraluminal application, evidence was obtained for the transport of HRP across the cell; in contrast, intravenously applied HRP was never detected at the apical cell surface. Colchicine pretreatment did not stop the uptake of HRP, which was rapidly sequestered to the clustered tubules, vesicles, and cisternae, as well as to the mvbs and dbs. After longer intervals, the portion of HRP-reactive tubules, vesicles, and cisternae within the clusters increased: 60 min after HRP-administration all of them contained HRP-activity. These results indicate that the colchicine-induced clustered organelles are recipients of endocytic materials internalized at the apical as well as at the basolateral cell surface.     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1^-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.     

Generation of a radial-like glial cell line

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase β, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (∼60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (∼30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 291–304, 1998     

Initial stages of radial glia astrocytic transformation in the early postnatal anterior subventricular zone

In the early postnatal subventricular zone (SVZ), two seemingly unrelated events occur simultaneously: a massive tangential migration of neuroblasts towards the olfactory bulb, known as the rostral migratory stream (RMS), and the outward movement of radial glia (RG) undergoing astrocytic transformation. Because of the orthogonal arrangement between these two sets of cells, little, if any, relevance has been ascribed for their possible interactions. By depositing DiI at the pial surface we have studied RG transformation within the SVZ/RMS, from birth up to the end of the first postnatal week. While still within the SVZ/RMS, RG morphology changed from simple bipolar to highly complex branched profiles, attaining their highest degree of complexity at the interface of the SVZ with the overlying white matter. At this interface cell bodies of radial glia accumulate and their processes run tangentially, surrounding the SVZ/RMS. Processes of RG surrounding the SVZ/RMS could also be observed by immunostaining for vimentin, GFAP, and nestin. In contrast, in the white matter all DiI-labeled RG presented a simple bipolar profile. These results indicate that the outward radial migration of the transforming RG does not occur uniformly. Instead, the different morphologies and cell densities that RG assume when they cross the SVZ/RMS and overlying white matter imply different migratory behaviors. Finally, our data suggest that RG provide a cellular scaffold to the early postnatal SVZ/RMS, much in the same way as astrocytes in the adult RMS.     

Distribution of immunoglobulin G receptors in the small intestine of the young rat

Conjugates of horseradish peroxidase (HRP) and immunoglobulin G (IgG) were used to map the distribution of cell surface receptors that can bind IgG at 0 degrees C within the small intestine of 10-12-d-old rats. Luminal receptors are present only within the duodenum and proximal jejunum. In these locations, receptors are limited to absorptive cells that line the upper portion of individual villi. Near villus tips, receptors are relatively evenly distributed over the entire luminal plasmalemma. In the midregion of villi, receptors are unevenly distributed over the luminal surface. Receptors (a) specifically bind rat and rabbit IgG, (b) recognize the Fc portion of the immunoglobulins, and (c) bind at pH 6.0 but not pH 7.4. To determine whether IgG receptors are confined to the luminal portion of the plasmalemma, intact epithelial cells were isolated from the proximal intestine of 10-12-d-old rats and incubated with HRP conjugates at 0 degree C. The specific binding of rat IgG-HRP to cells at pH 6.0 indicates that IgG receptors, which are functionally similar to those found on the luminal surface, are also present over the entire abluminal surface of absorptive cells. These results are consistent with the transport of IgG to the abluminal plasma membrane in the form of IgG-receptor complexes on the surface of vesicles. Exposure of these complexes to the serosal plasma, which is presumably at pH 7.4, would cause release of IgG from the receptors. To assess possible inward movement of vesicles from the abluminal surface after discharge of IgG, intravenously injected HRP was used as a space-filling tracer in the serosal plasma. HRP could be visualized within the coated and tubular vesicles responsible for transport of IgG in the opposite direction. These vesicles may, therefore, provide a pathway whereby receptors shuttle between the luminal and abluminal surfaces of cells.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division

Neurogenesis in the adult avian brain is restricted to the telencephalon. New neurons originate in the ventricular zone (VZ) from cells that have not been identified. We mapped the position of [3H]thymidine-labeled cells in the walls of the ventricles of the adult canary brain. Labeled VZ cells were restricted to the telencephalon (lateral ventricles) and concentrated in "hot spots". The coincidence of these hot spots with regions rich in radial cells suggested that radial cells may be the cells undergoing mitosis. We used smears prepared from fragments of the VZ containing the hot spots to show directly that radial cells accumulate [3H]thymidine. In addition, grain counts at different survival times demonstrated that these cells divide. Hot spots of VZ cell division also coincided with sites of neuronal origin. We suggest that radial cell division may give rise to new neurons.     

Adult neurogenesis: From canaries to the clinic

Neuronal precursor cells persist in the adult vertebrate forebrain, residing primarily in the ventricular/subventricular zone (SZ). In vivo, SZ precursors yield progeny which may die or give rise to glia. Yet they may also generate neurons, which are recruited to restricted regions such as the avian telencephalon and mammalian olfactory bulb. The survival of neurons arising from adult progenitors is dictated by both the availability of a permissive pathway for migration and the environment into which migration occurs. In the songbird higher vocal center (HVC), both humoral and contact-mediated signals modulate the migration and survival of new neurons, through an orchestrated set of hormonally regulated paracrine interactions. New neurons of the songbird brain depart the SZ to enter the brain parenchyma by migrating upon radial guide fibers, which emanate from cell bodies in the ventricular epithelium. The radial guide cells coderive with new neurons from a common progenitor, which is widespread throughout the songbird SZ. Neural precursors are also widely distributed in the adult mammalian SZ, although it is unclear whether avian and mammalian progenitor cells are homologous: Whereas neuronal recruitment persists throughout much of the songbird forebrain, in mammals it is limited to the olfactory bulb. In humans, the adult SZ appears to largely cease neurogenesis in vivo, although it, too, can produce neurons in vitro. In both rats and humans, the differentiation and survival of neurons arising from the postnatal SZ may be regulated by access to postmitotic trophic factors. Indeed, serial application of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) has allowed the generation and maintenance of neurons from the adult human SZ. This suggests the feasibility of inducing neurogenesis in the human brain, both in situ and through implanted progenitors. In this regard, using cell-specific neural promoters coupled to fluorescent reporters, defined progenitor phenotypes may now be isolated by fluorescence-activated cell sorting. Together, these findings give hope that structural brain repair through induced neurogenesis and neurogenic implants will soon be a clinical reality. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 267–286, 1998     

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.     

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of the African lungfish, Protopterus annectens (Dipnoi: Lepidosirenidae)

The distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin, in the brain and spinal cord of the African lungfish, Protopterus annectens, was examined by light microscopy immunoperoxidase cytochemistry. Glial fibrillary acidic protein immunoreactivity is clear and is evident in a radial glial system. It consists of fibers of different lengths and thicknesses that are arranged in a regular radial pattern throughout the central nervous system (CNS). They emerge from generally immunopositive radial ependymoglia (tanycytes), lining the ventricular surface, and are directed from the ventricular wall to the meningeal surface. These fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls forming the glia limitans externa and the perivascular glial layer, respectively. GFAP-immunopositive star-shaped astrocytes were not found in P. annectens CNS. In the gray matter of the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependymal layer. Vimentin-immunopositive structures are represented by thin fibers mostly localized in the peripheral zones of the brain and the spinal cord. While a few stained fibers appear in the gray matter, the ependymal layer shows no antivimentin immunostaining. In P. annectens the immunocytochemical response of the astroglial intermediate filaments is typical of a mature astroglia cell lineage, since they primarily express GFAP immunoreactivity. This immunocytochemical study shows that the glial pattern of the African lungfish resembles that found in tetrapods such as urodeles and reptiles. The glial pattern of lungfishes is comparable to that of urodeles and reptiles but is not as complex as that of teleosts, birds, and mammals.     

Characterization of glial fibrillary acidic protein and astroglial architecture in the brain of a continuously growing fish, the rainbow trout

Unlike mammals, some fish, including carp and trout, have a continuously growing brain. The glial architecture of teleost brain has been intensively studied in the carp and few data exist on trout brain. In this study, using immunoblotting we characterized the topographic distribution of glial fibrillary acidic protein (GFAP) in larval and adult rainbow trout brain and studied by immunohistochemistry the distribution and morphology of GFAP-immunoreactive cell systems in the rainbow trout hindbrain and spinal cord. Immunoblotting yielded a double band with an apparent molecular weight of 50-52 kDa in the spinal cord homogenate in the trout larval and adult stages. In the adult hindbrain and forebrain, our antibody cross reacted also with a second band at a higher molecular weight (90 kDa). Because the forebrain contained this band alone the two brain regions might contain two distinct isoforms. Conversely, the larval total brain homogenate contained the heavy 90 kDa band alone. Hence the heavy band might be a GFAP protein dimer or vimentin/GFAP copolymer reflecting nerve fiber growth and elongation, or the two isoforms might indicate two distinct astroglial cell types as recently proposed in the zebrafish. In sections from trout hindbrain and spinal cord the antibody detected a GFAP-immunoreactive glial fiber system observed in the raphe and in the glial septa separating the nerve tracts. These radial glia fibers thickened toward the pial surface, where they formed glial end feet. The antibody also labeled perivascular glia around blood vessels in the white matter, and the ependymoglial plexus surrounding the ventricular surface in the grey matter. Last, it labeled round astrocytes. The GFAP-immunoreactive glial systems had similar distribution patterns in the adult and larval spinal cord suggesting early differentiation.     

Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells

In parotid acinar cells, horseradish peroxidase (HRP) administered via the main excretory duct is endocytosed from the apical cell surface in smooth C- or ring-shaped vesicles (Oliver, C. and A. R. Hand. 1979. J. Cell Biol. 76:207). These vesicles ultimately fuse with lysosomes adjacent to the Golgi apparatus. The present investigation extends these findings and examines the uptake and fate of intravenously injected HRP from the lateral and basal cell surfaces of resting and stimulated parotid and pancreatic acinar cells from rats and mice. Isoproterenol and pilocarpine were used to stimulate the parotid gland and the pancreas, respectively. HRP was internalized in smooth and coated vesicles primarily in areas of membrane infoldings. Both the number of coated vesicles and the amount of tracer internalized increased markedly following secretagogue administration. In both resting and stimulated cells, the HRP was rapidly sequestered in a unique system of basally located lysosomes that possess trimetaphosphatase activity, but not acid phosphatase activity. At 1-3 h after HRP administration, reaction product was also found in multivesicular bodies, vesicles, and lysosomes adjacent to the Golgi apparatus. With time, more HRP was localized in Golgi-associated lysosomes. By 6-7 h, tubules in the apical cytoplasm of stimulated cells contained HRP reaction product. When native ferritin was administered retrogradely and HRP injected intravenously, both tracers could be localized in the same lysosome after 4-5 h, indicating that material taken in from all cell surfaces mixes in Golgi-associated lysosomes. The results of this study suggest that two separate and distinct endocytic pathways exist in exocrine acinar cells: one involves membrane retrieval from the apical cell surface; and the other is a stimulation-dependent process at the lateral and basal cell surfaces.     

Radial astrocytes and ependymocytes in the spinal cord of the adult toad (Bufo bufo L.)

Summary Immunohistochemical and ultrastructural techniques have been used to demonstrate glial fibrillary acidic protein (GFAP) immuno-positive cells in the adult toad spinal cord. Two types of GFAP-immunoreactive cells were observed: ependymocytes and radial astrocytes. GFAP-positive ependymocytes were scarce and contained the immunoreactive product in their processes. They showed intermediate filaments in the basal pole and in their processes when studied with the electron microscope. These immuno-positive ependymocytes represent the tanycytic form of ependymal cells because their processes ended at the subpial zone. The radial astrocytes showed a more intensive immunoreactive product in somata and processes when they were located far away from the ependymal layer. Cell bodies and processes were also associated with blood vessels, but most of the processes ended at the subpial zone forming a continuous subpial glia limitans. The GFAP-positive processes, which form this subpial glia limitans in the toad spinal cord, belong to both tanycytic ependymocytes and radial astrocytes, whose somata are located in the grey matter. These findings lead us to suggest that both types of GFAP-immunopositive cells might be the functional equivalents of mammalian astrocytes.     

Uptake of exogenous protein tracer in cells of the rat renal papilla

In order to study the phagocytic potential of different cell types of the rat renal papilla with special emphasis on interstitial cells, horseradish peroxidase (HRP) (8 mg/100 g body weight) was injected intravenously into adult rats. The distribution of peroxidase was studied in animals perfusion-fixed 60 and 180 min after injection and was found to be similar after both time intervals. The epithelial cells of the collecting ducts took up the largest amounts of the tracer. HRP was mainly located in large lysosome-like bodies in the basal part of the cytoplasm, suggesting peritubular uptake from the interstitial space. However, small amounts of the tracer were also seen in apical vesicles close to the luminal plasma membrane. The interstitial cells of peroxidase-injected animals were ultrastructurally altered and had large irregular invaginations of the cell membrane. The cells had taken up only small amounts of the tracer which were located in small round lysosome-like bodies. Thus, the interstitial cells displays no macrophage characteristics, either in the native state or when challenged with an extracellular protein.