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PubMed1989

Effect of cytosine arabinoside on rat cerebral explants: fibroblasts and reactive microglial cells as the primary cellular barrier to neurite growth.

Rat cerebral explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside (AraC) to investigate whether reports of increased neurite outgrowth may be primarily related to a decreased incidence of fibroblastic-reactive microglial cells. Treated explants were exposed to AraC from 7 to 10 days in vitro (DIV) and processed for transmission electron microscopy at 18 DIV. Morphometric analysis of the outgrowth zone revealed a statistically significant decrease in the incidence of fibroblastic-reactive microglial cells (from 12 to 0%), and a significant increase in the incidence of protoplasmic astrocytic-epithelial cells (from 82 to 96%), for AraC-treated explants compared to controls. In contrast, the incidence of fibrous astrocytes was similar to that of control explants (4 and 6%, respectively). Thus, it appears that AraC may primarily enhance neurite growth by curtailing the proliferation of fibroblastic-reactive microglial cells. These results suggest that fibroblastic-reactive microglial cells, rather than fibrous astrocytes, may constitute the primary cellular barrier to neurite growth.

Animals↗

PubMed1993

Tight junction complexity in the retinal pigment epithelium of the chicken during development.

In the avascular retina of birds, the pigment epithelium (RPE) is the main site of the blood-retina barrier. Tight junctions (TJs) connect the pigment epithelial cells and represent the structural substrate of the barrier function. We investigated, by means of the quantitative freeze-fracturing technique, the TJs of the chicken RPE during development and compared them with the TJs of choroid capillary endothelial cells which are known to be fenestrated. The association of TJs with the protoplasmic membrane leaflet (P-face) is more pronounced in the RPE than in the choroid vessels. Between embryonic day 15 (E15) and E19, we observed a significant increase in the TJ complexity in the RPE, but not in the choroid vessels. The increase coincides with the morphological and functional maturation of the chicken retina suggesting that complex P-face-associated TJs in the RPE are necessary for the formation of an effective blood-retina barrier.

Aging↗

PubMed1979

Selective modification of positive chemotaxis in the true slime mold Physarum polycephalum by ethylenediaminetetraacetic acid treatment.

The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.

Chemotaxis↗

PubMed1976

Freeze-fracture properties of central myelin in the bullfrog.

The freeze-fractured membrane of the central myelin sheath has three classes of particulate components: (i) Particles inherent to the compact myelin lamellae. These are distributed at random and cleave predominantly with the P (protoplasmic) face. (ii) Particles which comprise the intramyelinic tight junctions. These are arranged in strands and are located at the inner and outer mesaxon, the paranodal loops, the cytoplasmic incisures, and occasionally within the compact regions of the myelin sheath. (iii) Particles localized exclusively at the portion of the paranodal loop membrane involved in the septate-like junction with the axolemma. These are regularly spaced and are organized in parallel rows. In the central myelin sheaths of bullfrogs fixed by perfusion with aldehydes and cryoprotected in 30% glycerol, the randomly distributed particles differ in size and shape from those of the axolemma. They possess a reasonably well defined bimodal distribution with respect to particle shape--most can be described either as globules or as ellipsoids. The globular particles range in diameter from 60 to 150 A. The ellipsoidal particles are 100-200 A long and 15-50 A wide. The total number of particles per square micron on the P face is approximately 1500. About half of these are of the globular type and half of the ellipsoidal type. In poorly fixed specimens, loss of interlamellar adhesion and loss of randomly distributed particles seem to coincide. Evidence is presented against the hypothesis that the tight junctions between compact myelin lamellae represent the radial component of the myelin. The possible relation between the types of particulate components seen in freeze-fracture and the classes of protein isolated from central myelin fractions is briefly discussed.

Animals↗

PubMed1986

Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction.

Small pieces of Torpedo electric organ were treated with 4-aminopyridine, a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120-150 ms after the stimulus, that is, a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also carried out in a modified solution containing no added calcium, 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro-transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗

PubMed1992

Choline blockage of currents through Ca(2+)-activated K+ channels in bovine chromaffin cells.

The action of choline on "maxi" Ca(2+)-activated K+ channels was studied in excised patches of bovine chromaffin cell membranes. Choline (20-70 mM) applied to the internal surface of the membrane reduced the single channel current amplitudes, which can be explained by a fast channel block. The block is concentration- and voltage-dependent and is rapidly and completely reversed upon washout. The block becomes progressively greater with depolarization. The estimates of blocking parameters vary from channel to channel but appear to fall in two groups. A larger group (two-thirds of cases) with moderate affinity [KD(0) = 88.5 mM] and low voltage dependence (delta = 0.26) and a smaller group (one-third of cases) with very low affinity (KD = 306 mM) and moderate voltage dependence (delta = 0.59). The open state probability appears not to be affected at any choline concentration (up to 70 mM) or membrane potential (from -20 to +60 mV) studied, suggesting that choline does not affect the channel gating kinetics. Since the affinity of the choline block is low to moderate, the intracellular choline is not expected to alter the current flow through "maxi" Ca(2+)-activated K+ channels unless the choline concentration close to the protoplasmic membrane is much higher than the mean cellular concentration.

Adrenal Medulla↗

PubMed1994

Structural and molecular heterogeneity of astrocytes and oligodendrocytes in the gerbil lateral superior olive.

The goal of this study was to determine the distribution and diversity of astrocytes and oligodendrocytes within the lateral superior olive of the gerbil. We used morphometric analyses and several immunocytochemical markers to assess differences in glial cell composition between the lateral (low-frequency projection) and the medial (high-frequency projection) limb of the lateral superior olive. Cell counts from Toluidine-stained semithin sections revealed a similar density of total astrocytes in both the lateral and the medial limbs. However, based on cytologic features, there was a prevalence of fibrous-like astrocytes in the lateral limb and protoplasmic-like astrocytes in the medial limb. In a similar manner, glial fibrillary acidic protein staining of astrocytes was intense in the lateral limb, but was largely restricted to the nucleus borders in the medial limb of the lateral superior olive. While glial fibrillary acidic protein was largely restricted to astrocytic processes, glutamine synthetase and S100 protein staining occurred, for the most part, in glial cell bodies. The density of glutamine synthetase positive cell bodies was homogeneous between the two limbs, while the density of S100-positive somata was significantly greater in the lateral limb. Cell counts obtained from semithin sections demonstrated a greater density of oligodendrocytes in the lateral limb than in the medial limb of the lateral superior olive. In a similar manner, there was a 40% greater density of carbonic anhydrase-positive somata in the lateral limb compared to the medial limb. Transferrin immunostaining was restricted to oligodendrocytes, but the density of labeled somata was identical in the lateral and medial limbs. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein were also localized to the somata of oligodendrocytes, labeling both perisomatic and interfascicular cells. At the ultrastructural level, specialized contacts were found between pairs or clusters of oligodendrocytes. These results suggest that more than one type of astrocyte and oligodendrocyte is present within the gerbil lateral superior olive. Furthermore, glial cells were unevenly distributed, such that a greater density of oligodendrocytes and fibrous-like astrocytes were found in the low-frequency projection region. This heterogeneity is well correlated with known differences in the neuronal morphology within the lateral superior olive.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1994

Response of striatal astrocytes to neuronal deafferentation: an immunocytochemical and ultrastructural study.

This ultrastructural and light microscopic immunocytochemical study describes the time course of anatomical changes that occur in striatal astrocytes in response to neuronal deafferentation in young adult rats and the coordinate distribution of two astrocytic proteins involved in reactive synaptogenesis, glial fibrillary acidic protein and clusterin. We found that following a unilateral lesion of the cerebral cortex, striatal astrocytes undergo a rapid ultrastructural transformation from a protoplasmic to a reactive type of astroglia and are the primary cells involved in the removal of degenerating axon terminals, but not axons of passage, from the neuropil. In addition, at 10 and 27 days postlesion, processes of reactive astrocytes are also seen to occupy vacant postsynaptic spines after degenerating presynaptic terminals are removed, suggesting that they may also participate in the reinnervation of the deafferented neurons. By immunocytochemistry, reactive astrocytes were characterized by a significant increase in the intensity of glial fibrillary acidic protein staining beginning at three days postlesion and lasting for at least 27 days postlesion. Reactive astrocytes were characterized by cellular hypertrophy and an increase in the density of immunoreactive processes distributed throughout the deafferented striatum. However, our analysis of astrocyte cell number found no evidence of astrocyte proliferation in response to the deafferentation lesion. Although previous in situ hybridization studies have reported elevated clusterin messenger RNA in reactive astrocytes after decortication, clusterin immunoreactivity was not seen in the cell soma of reactive astrocytes but was distributed as punctate deposits, ranging from 1 to 2 microns in diameter, within the neuropil of the deafferented striatum. At 10 days postlesion, the distribution of clusterin staining appeared as large aggregates of immunoreactive deposits adjacent to neurons. However, by 27 days postlesion, the aggregates of clusterin reaction product were replaced by a fine scattering of individual punctate deposits distributed evenly over the dorsal part of the deafferented striatum. These data support the notion that reactive astrocytes serve multiple, time-dependent roles in response to brain injury and are involved in both the removal of degenerative debris from the lesion site as well as in reforming the synaptic circuitry of the damaged brain. Our data suggest that, in response to decortication, reactive astrocytes are the primary cells responsible for removing degenerating axon terminals, but not axons of passage, from the deafferented striatum and that the coordinate increase in glial fibrillary acidic protein may serve to stabilize the extension of reactive astrocytic processes during phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed1994

Endothelins are extracellular signals modulating cytoskeletal actin organization in rat cultured astrocytes.

Effects of endothelin-3 on rapid morphological changes and cytoskeletal actin organization of rat cortical cultured astrocytes were examined. In serum-free medium, treatments with 1 mM dibutyryl cAMP and 5 microM cytochalasin B, an inhibitor of actin polymerization, caused astrocytic morphological changes with cytoplasmic retraction (stellation). Concurrent addition of 1 nM endothelin-3 prevented astrocytic stellation by dibutyryl cAMP and cytochalasin B. The inhibition of endothelin-3 on the astrocytic stellation was dose-dependent, where IC50 and maximal effective dose were about 50 pM and 0.1 nM, respectively. Endothelin-1 and sarafotoxin S6b prevented the cytochalasin B-induced stellation with similar potencies to endothelin-3. Endothelin-3 reversed the stellate morphology of cytochalasin B-treated cells. Sixty minutes after addition of endothelin-3, most cytochalasin B-treated astrocytes lost their apparent distinction between cell body and processes. Treatment with dibutyryl cAMP and cytochalasin B decreased actin content in a 0.5% Triton X-100-insoluble fraction (cytoskeletal fraction) of cultured astrocytes. Subsequent treatments with endothelin-3 for 2 h restored the decreased cytoskeletal actin to that of non-treated cells. Rhodamine-phalloidin staining showed that a prominent structure of organized filamentous actin in protoplasmic astrocytes is stress fibers. The astrocytic stress fibers disappeared after treatment with dibutyryl cAMP and cytochalasin B. Endothelin-3 stimulated reorganization of stress fibers both in the dibutyryl cAMP- and the cytochalasin B-treated astrocytes. These results suggest that endothelins are extracellular signals to regulate cytoskeletal actin organization of astrocytes.

Actins↗

PubMed1980

The role of the brain cell nuclei in the function of memory.

The neurophysiological basis of memory may lie in the significance of eccentric cell nuclei found in neurons in certain parts of the brain. Embryonic nerve cells are spherical with central nuclei, whereas fully developed neurons in specific parts of the adult brain are non-spherical with eccentric nuclei. The hypothesis proposes that the mechanism of the sodium pump of the cell membrane is affected by the proximity of the nucleus. Molecules from the nucleus travel through the protoplasm of the cell to the deep surface of the cell membrane. The polarity of the individual cell is determined by that part of the cell membrane which is nearest the nucleus. This "Favoured Spot" is always re-energised first after the sodium pump has been discharged by a stimulus from a synaptic knob. After depolarisation, stimuli arriving at the favoured spot have priority over stimuli arriving simultaneously at any other parts of the cell, for when the latter are still in a refractory phase, the favoured spot is already re-energised to its exitatory state.

Adult↗

PubMed1981

The pathway of photosensory transduction in Physarum polycephalum.

Irradiation of the plasmodia of Physarum with blue and white light results in a transient change of theie oscillatory contraction frequency. This reaction to light decreases with increasing distance from the illuminated area (Block and Wohlfarth-Bottermann, 1981). The first local appearance of light response in non-illuminated parts of the plasmodia was used to analyse the sensory pathway of the light stimulus modulating the contractile apparatus. Different experimental assays revealed that the direction of photosensory transduction is determined by the momentary direction of protoplasmic shuttle streaming. The endoplasmic flow carriers the signal responsible for photosensory transduction and light reaction to the force generating ectoplasmic tube.

Cytoplasm↗

PubMed1984

Fine structural cytology of the rat subfornical organ during ontogenesis.

The main developmental events in the subfornical organ take place between 17 fetal days (fd) and 5 days post natum (dpn) at which time it possesses most of its mature fine structural characteristics. The surface regional characteristics of ependymal cells differentiate primarily during this time as well, while the ependymal cellular fine structure, shape and relationship with neurons and the vascularity are well established prior to birth. Undifferentiated neurons contain glycogen prior to 19 fd and then differentiate by developing processes and organelles characteristic of neurons. By 5 dpn, the various types of neurons found in the mature subfornical organ are all present, except for giant vacuolated cells. Synapses containing only electron-lucent vesicles are first present at 20 fd, those containing additional electron-dense vesicles at 3 dpn. Microglial cells are first identifiable at 17 fd, and the first protoplasmic astrocytes are recognizable at 21 fd, while fibrous astrocytes are not detectable prior to 7 dpn. By 5 dpn, the cytological elements of the subfornical organ are all in place, and further developmental changes leading to adult fine structural characteristics by 30 dpn are essentially quantitative in nature.

Animals↗

PubMed1996

Serum modulates cyclic AMP-dependent morphological changes in cultured neurohypophysial astrocytes.

The effects of serum on the morphological plasticity exhibited by pituicytes in explant cultures of the neurohypophysis of adult rats have been examined. Cultured pituicytes are normally nonstellate, protoplasmic, amorphous cells (< 25% are stellate with a distinct cell body and phase bright processes). After incubation (90 min) of pituicyte cultures in a HEPES buffered salt solution (HBSS) supplemented with isoproterenol or forskolin, the fraction of stellate pituicytes significantly increased. The increase in the fraction of stellate cells induced by isoproterenol was not reversed by subsequent incubation in isoproterenol-free HBSS for 90 min. In contrast, after stellation was induced in cultures by exposure to forskolin (90 min), the fraction of stellate cells was significantly reduced if these cultures were incubated in forskolin-free, serum (0.5%) supplemented HBSS for the same duration. Serum also blocked the increase in the fraction of stellate pituicytes induced by forskolin. These experiments suggest that serum components may have a significant role in controlling the plasticity of neuroglial relations in the neurohypophysis previously demonstrated in vivo.

Adrenergic beta-Agonists↗

PubMed1987

Three-dimensional visualizations of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membranous organelles.

The fine structure of the filamentous and membranous organelles in the stereocilia and in the cuticular plate of sensory hair cells from the guinea pig was examined using a rapid-freeze, deep-etch method. In fixed and unfixed tissue the outer surface of the plasma membrane of the stereocilia had numerous surface protrusions of various sizes and shapes, while the protoplasmic fractured face of the membrane had rather sparse intramembrane particles. Many tiny cross links were present between the adjacent actin filaments and between actin filaments and the plasma membrane of the stereocilia. Numerous fibrils radiating from the hair rootlet were attached to the peripheral actin filaments in the cuticular plate. The radiating fibrils differed from the tiny cross links which interconnected the adjacent, randomly-oriented actin filaments in the cuticular plate. These complex structures consisting of actin filaments in the hair rootlets, radiating fibrils, and peripheral actin filaments may play an important role in regulating stereociliary bending.

Actin Cytoskeleton↗

PubMed1987

Melanin storing cells in anuran gut.

1. The location, distribution and morphological characteristics of the pigment cells found in the frog gut are described. 2. The pigment cells show long and large protoplasmic projections. At the ultrastructural level, the nucleus is elongated with prominent nucleolus and dense marginal chromatin. The cytoplasm is full with pigment granules (2500-7500 A) and typical premelanosome structures have been observed. 3. The pigment cells number is higher in the esophagus and large intestine than in the stomach or small intestine and the pigment cells are always located in close contact with blood vessels and nervous structures (ganglia and fibres). 4. We have observed that the pigment content depends upon seasonal variations, increasing during the cold months. 5. We have demonstrated by histological methods that the cells pigment content is melanin. 6. According to their morphological and tinctorial characteristics the anuran gut melanin storing cells are similar to the skin epidermal melanocytes.

Animals↗

PubMed1992

A comparative fractal analysis of various mammalian astroglial cell types.

Camera-lucida drawings of Golgi-impregnated astroglial cells and their processes are described by the fractal dimension of their borders, which is an objective, quantitative measure of morphological complexity. Protoplasmic astrocytes from human neocortex have fractal dimensions (D) that are larger than those of fibrous astrocytes from the cat optic nerve. Marginal astrocytes from monkey cerebropontile angle have two kinds of processes: (1) short, thick processes with endfeet abutting the pial surface, with relatively high D's, and (2) very long, thin processes extending into the neuronal tissue, with very low D's. These data indicate that short astrocytic processes may have a complex surface (and have a high D), whereas long processes are rather smooth (and have a low D). A comparison between transmission electron microscopy morphometry and measures of D at the light microscopic level, performed on different parts of rabbit retinal Müller glial cells, suggests that D is strongly correlated to the surface-to-volume ratio which, in part, determines the length constant of a cable for core-conductance of currents. We provide data supporting the hypothesis that astroglial cell geometry is adjusted to allow for sufficient spatial buffering K+ currents, even through very long processes.

Animals↗

PubMed1992

A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae.

Scanning electron microscopy (SEM) was used to quantify the intercellular relationships between osteoblasts and osteocytes on the growing endosteal surfaces of the medullary canal of the tibia in four rabbits of different ages. The area of each osteoblast was measured on the SEM micrographs by means of an Image Analyzer. The number of osteocyte cytoplasmic processes was indirectly evaluated by counting the canalicular openings present on the same microscopic fields after the removal of the osteoblasts. The metabolic activity of the osteoblasts was indirectly evaluated from their shape, and the structure was analyzed by transmission electron microscope (TEM) in sections taken from the samples studied by SEM. In all four animals, the surface area of the osteoblasts (OA) was found to vary a great deal, whereas the density of canalicular openings was fairly uniform. Moreover, although the OA mean value increases significantly with the age of the animals, the density of canalicular openings does not; it would therefore appear that the older the animal and the more flattened the osteoblasts, the greater the number of canaliculi beneath them. Since osteoblast activity has previously been shown to be inversely proportional to the area of the protoplasm in contact with the bone surface, it appears that the less active osteoblasts should contact a greater number of osteocyte cytoplasmic processes. These findings suggest that osteocytes might play an important role in modulating osteoblast activity and in recruiting osteoblasts that differentiate into osteocytes, possibly by means of inhibitory signals transmitted via gap junctions.

Animals↗

PubMed2004

Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1.

Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extracytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.

4-Chloro-7-nitrobenzofurazan↗