bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microtubule dynamics in axons and dendrites
Authors: Baas P. W. , Slaughter T. , Brown A. , Black M. M. 
Published: January 2005

AbstractWe have investigated the stability, α‐tubulin composition, and polarity orientation of microtubules (MTs) in the axons and dendrites of cultured sympathetic neurons. MT stability was evaluated in terms of sensitivity to nocodazole, a potent anti‐MT drug. Nocodazole sensitivity was assayed by quantifying the loss of MT polymer as a function of time in 2 μg/ml of the drug. MTs in the axon and the dendrite exhibit striking similarities in their drug sensitivity. In both types of neurites, the kinetics of MT loss are biphasic, and are consistent with the existence of two types of MT polymer that depolymerize with half‐times of ≈︁3.5 min and ≈︁130 min. We define the more rapidly depolymerizing polymer as drug‐labile and the more slowly depolymerizing polymer as drug‐stable. The proportion of MT polymer that is drug‐stable is greater in axons (58%) than in dendrites (25%). On the basis of current understanding of the mechanism of action of nocodazole, we suggest that the drug‐labile and drug‐stable polymer observed in both axons and dendrites correspond to two distinct types of polymer that differ in their relative rates of turnover in vivo. In a previous study, we established that in the axon, these drug‐stable and drug‐labile types of MT polymer exist in the form of distinct domains on individual MTs, with the labile domain situated at the plus end of the stable domain (Baas and Black, J Cell Biol 111:495–509, 1990).Because of the great difference in drug sensitivity between the drug‐labile and drug‐stable MT polymer, we were able to dissect them apart by appropriate treatments with nocodazole. This permitted us to evaluate the drug‐labile and drug‐stable polymer in terms of polarity orientation and relative content of α‐tubulin variants generated by posttranslational detyrosination or acetylation. In both the axon and the dendrite, the modified as well as unmodified α‐tubulins are present in both drug‐labile and drug‐stable polymer, but at different levels. Specifically, the modified forms of α‐tubulin are enriched in the drug‐stable MT polymer compared to the drug‐labile MT polymer. In studies on MT polarity orientation, we demonstrate that in axons, MTs are uniformly plus‐end‐distal, whereas in dendrites, MTs are non‐uniform in their polarity orientation, with roughly equal levels of the MTs having each orientation. We also demonstrate that both drug‐stable and drug‐labile polymer are incorporated into MTs of both polarity orientations in the dendrite.
Involvement of mature tau isoforms in the stabilization of neurites in PC12 cells
Authors: Hanemaaijer R. , Ginzburg I. 
Published: January 2005

AbstractTau microtubule—associated proteins are believed to play a role in regulation of the growth of neuronal processes. In order to study the function of tau protein in vivo, we examined the inhibition of tau expression in PC12 cells by exposing the cells to tau antisense oligodeoxynucleotides. A speceific retraction of neurites was observed after 3–4 days of incubation with nerve growth factor (NGF) and the antisense oligodeoxynucleotides. This is different from the previously described retraction of neurites at the initiation step following exposure to tubulin antisense oligodeoxynucleotides, indicating that tau proteins are involved at later stages of neurite outgrowth. Analysis of tau protein isoforms in NGF‐induced PC12 cells showed a transition from immature to mature tau isoforms, thus relating the appearance of the latter with the stabilization step of neurite outgrowth. Use of an RNase‐protection assay demonstrated a similar switch from immature to mature tau mRNA species. The transition to stable microtubules was verified by the appearance of microtubule bundles and their stability to colchicine treatment. Both phenomena occurred between 2 and 4 days of NGF induction. These results indicate that in vivo only mature tau isoforms are involved in the transition from unstable to stable neurites, which is a key step in neuronal development.
Structure and organization of membrane organelles along distal microtubule segments in growth cones
Authors: Dailey M. E. , Bridgman P. C. 
Published: January 2005

AbstractAdvance and stabilization of organelle‐rich cytoplasm within the neuronal growth cone is coupled to axon elongation (Goldberg and Burmeister, 1986; Aletta and Greene, 1988), and this involves forward movement of organelles from the growth cone base along distinct tracks toward the leading edge. Membrane‐bound organelles that advance first within the growth cone often make transient excursions toward the leading edge, and at the light microscope level these leading organelles appear to colocalize with distal microtubule (MX) segments (Dailey and Bridgman, 1989).We have used electron microscopy (EM) to identify the membranous organelles adjacent to distal MT segments, and to examine their structural interactions with MTs. In both glutaraldehyde‐fixed and rapid frozen whole‐mount growth cones, attenuated endoplasmic reticulum (ER)‐like membrane elements were the most common organelle type located adjacent to distal MX segments. These ER‐like membrane elements coursed roughly parallel to MTs and frequently terminated within an electron‐dense bulb at the MX tip. Blind‐ended membrane tubes, dense‐core vesicles, clear vesicles, and vacuoles were also found adjacent to distal MX segments. Quantitative analyses of organelle‐MT associations suggest that elements of the ER‐like membrane system may frequently advance ahead of other membrane‐bound organelles. Freeze‐etch EM revealed crossbridging structures between MTs and membranous organelles, which is consistent with the idea that advance of leading membrane organelles into the growth cone periphery is mediated by microtubule‐based motor transport mechanisms.The results suggest that distal microtubule segments serve as transport elements for advance of membrane organelles into more peripheral growth cone regions, and together MTs and ER‐like membrane organelles may initiate the conversion of dynamic F‐actin‐rich cytoplasm to more stable organelle‐rich cytoplasm (i.e., axoplasm).
Two types of Na +‐currents in cultured rat optic nerve astrocytes: Changes with time in culture and with age of culture derivation
Authors: Sontheimer H. , Minturn J. E. , Black J. A. , Ransom B. R. , Waxman S. G. 
Published: January 2005

AbstractNa+ channel expression was studied in cultures of rat optic nerve astrocytes using whole‐cell voltage‐clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h∞ curves. Stellate, GFAP +/A2B5 + astrocytes showed currents with h∞ curve midpoints close to−65 mV, similar to Na + currents in most neurons. In contrast, flat fibroblast‐like GFAP +/A2B5− astrocytes showed Na+ currents with h∞ midpoints around −85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5 + astrocytes. Interestingly, Na + current expression was maintained in A2B5 Na+ astrocytes but began to decrease in A2B5− astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP +/A2B5‐. These cells did not display measurable Na + currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5− astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na + channels; in P0‐derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.
Mitogenic response of rat schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels
Authors: Chen J.‐K. , Yao L.‐L. , Jenq C.‐B. 
Published: January 2005

AbstractRat sciatic nerve Schwann cells either do not proliferate, or proliferate very slowly, in medium containing 10% fetal bovine serum (FBS). They were previously shown to respond only to a limited number of mitogens associated with cells of central and peripheral nervous systems, which appeared to be distinct from FGFs and PDGF, and to agents that raise intracellular cAMP levels. In a basal medium consisting of 75% DMEM, 25% Ham's F‐12, 5 nM sodium selenite, 50 μM 2‐amino ethanol, and 2 mM histidine, supplemented with 5% FBS, we showed that aFGF, bFGF, and PDGF were all capable of stimulating Schwann cell growth and the stimulation was greatly potentiated by forskolin and dibutyryl‐cAMP. In addition, pretreating culture surface with purified matrix proteins such as laminin, fibronectin, or type 1 collagen, was necessary for obtaining a better cellular response to the mitogenesis of these growth factors even in 10% FBS. Our results clearly indicated that providing a suitable medium and substratum, aFGF, bFGF and PDGF are mitogens for rat sciatic nerve Schwann cells in medium with and without forskolin or dibutyryl‐cAMP.
EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture
Authors: Casper D. , Mytilineou C. , Blum M. 
Published: January 2005

AbstractEpidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia. In this study, we investigated the effect of EGF on cells cultured from 16‐day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo. EGF receptors were detected in untreated cultures by [125I]‐EGF binding. Treatment of the cultures with EGF resulted in up to 50‐fold increases in neuronal high‐affinity dopamine uptake. Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase‐immunore‐active cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons. By contrast, the high‐affinity uptake for serotonin was increased only threefold over its controls. There was no significant effect on high‐affinity γ‐aminobutyric acid (GABA) uptake. These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures. Immunocytochemistry for glial flbrillary acidic protein demonstrated an increase in astroglia with EGF treatment. Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.
Saturable transport of Ca into CSF in chronic hypo‐ and hypercalcemia
Authors: Murphy V. A. , Smith Q. R. , Rapoport S. I. 
Published: January 2005

AbstractTo further characterize possible saturable transport of Ca into CSF during chronic plasma [Ca] changes, weanling rats were fed diets differing in Ca for 10 weeks. Transfer coefficients for unidirectional uptake of 45Ca and 36Cl into CSF (Kcsf) were determined 3 or 10 min after intravenous tracer injection in unanesthetized animals. In rats fed low Ca diet, KcsfS for 45Ca and 36Cl were elevated above control, but the 45Ca/36Cl ratio for Kcsf, a more specific measure of Ca transport, was also increased. In animals fed high Ca diet, KcsfS both radiotracers were not statistically different from control, but the 45Ca/36Cl ratio was decreased. Injection of CaCl2 into hypocalcemic rats elevated plasma [Ca], depressed 45Ca Kcsf, and returned the 45Ca/36Cl ratio to the control value. The inverse relationship between plasma ionized [Ca] and 45Ca/ Kcsf was fitted to saturation kinetics with Km ≤ 0.53 μmol/ml, maximal Ca influx for the saturable component between 27 and 67 × 10−5 μmol·g−1·s−1, and the passive component of Kcsf ≤ 15 × 10−5 ml·g−1·s−1. We conclude that Ca transport into CSF is saturable and this transport is important is the regulation of CSF [Ca].
Molecular modeling of the weak glycine antagonist iso‐THAO
Authors: Aprison M. H. , Lipkowit K. B. 
Published: January 2005

AbstractWhen compared to strychnine, a potent glycine antagonist, iso‐THAO, a bicyclic 5‐isoxazolol zwitterion, has been reported to be a weak glycine antagonist. Since there are so few glycine antagonists, and there is a striking lack of similarity between the structures of these two antagonists, iso‐THAO was studied using current molecular modeling techniques and quantum mechanical calculations in order to compare the structural features and charge distributions of iso‐THAO with glycine. The results of this study confirm our earlier hypothesis that an antagonist to inhibitory neurotransmitters like glycine and GABA has at least three binding sites to the natural receptor that are very similar to three such binding sites in the transmitter and its agonists, and each antagonist has an additional negative binding site. We speculate that the latter negative binding site can attach to the top of the chloride channel within the receptor complex. The diminished inhibitory activity of iso‐THAO is attributed to its poor structural congruence with the three atom attachment sites used by glycine at its natural recognition site.
Differentiation and heterogeneity in T‐antigen immortalized precursor cell lines from mouse cerebellum
Authors: Redies C. , Lendahl U. , McKay R. D. G. 
Published: January 2005

AbstractRecently, various techniques have been developed to transfer oncogenes into brain cells in order to generate immortalized neural cell lines. It is of interest to establish how well such cell lines reflect their cellular origin. Here we report the characterization of sixteen cell lines from mouse cerebellum and, as a control, six cell lines from skin. Lines were established by immortalizing postnatal primary cell cultures with a retro‐virus carrying a modified temperature‐sensitive variant of SV40 large T antigen. The cell lines reflect many properties of the cell type from which they were derived. All of the sixteen cerebellar lines expressed one or more markers of the neural precursor cells, namely, nestin and epitopes for NG2 and A2B5. In contrast, none of the six skin lines expressed neural precursor markers. Both types of cell lines expressed vimentin and fibronectin. Differentiation occurred in some of the cerebellar lines and was enhanced in defined medium. A small percentage of cerebellar cells, usually less than 5%, was positive for a marker of differentiation, e.g., glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), or L1, Expression of GFAP colocalized with that of nestin at varying levels of intensity, indicating a gradual replacement of nestin by GFAP in the cytoskeleton. Both the cells positive for precursor markers and those positive for differentiation markers tended to be located in clusters, suggesting that stochastic processes or cell‐cell interactions are important for the determination of the fate of cells within a clonal cell line in vitro. The degree of differentiation seemed to correlate with a shift from serum‐containing to defined medium, but not with a shift from the permissive to the nonpermissive temperature for T antigen expression.The immortalization approach described here thus allows the establishment of cell lines which are “captured” in the precursor state of the developing mouse neuroepithelium.
Induction of anti‐myelin antibodies in EAE and their possible role in demyelination
Authors: Sadler R. H. , Sommer M. A. , Forno L. S. , Smith M. E. 
Published: January 2005

AbstractExperimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7–95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance pf antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10–95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26–27 days after immunization but remained significantly elevated up to 95 days post‐immunization compared to the activity of serum from the Freund's adjuvant‐injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol‐fast blue stained spinal cords up to 60 days post‐immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.
Rescue of dying neurons: A new action for deprenyl in MPTP parkinsonism
Authors: Tatton W. G. , Greenwood C. E. 
Published: January 2005

AbstractDeprenyl slows the progression of disabling symptoms in Parkinson's disease (PD) by an unknown mechanism. It can block the action of MPTP on substantia nigra compacta (SNc) neurons by inhibiting monoamine oxidase B necessary to mediate the conversion of MPTP to MPP+, its active metabolite, in astroglia. Mice were pretreated with saline or the PD‐producing toxin, MPTP (30 mg/kg) daily for 5 days and then after a further 3 days (to allow for the metabolism and excretion of the MPTP) were treated with deprenyl (0.25 or 10 mg/kg ) or saline 3 times weekly for 20 days. In three series of mice treated with MPTP alone or MPTP‐saline, serial sections through the SNc showed that averages of 37–42% of tyrosine hydroxylase (TH) immunoreactive neurons were lost gradually over 20 days. Joint counts of the numbers of TH‐immunoreactive and Nissl‐stained SNc somata from immediately adjacent sections established that the reductions in the numbers of TH immunoreactive somata at 20 days after MPTP treatment represented neuronal death. Deprenyl treatment reduced the loss of TH‐immunoreactive SNc neurons to averages of 14–16% for the 10‐mg/kg and 0.25‐mg/kg doses, respectively, and joint Nissl/ TH counts for adjacent sections showed that reduction in the loss of TH‐immunoreactive soma represented the rescue of SNc neurons that would have died by 20 days. The gradual loss of SNc neurons over the 20 days following MPTP exposure may reflect the toxin's axotomy‐like effects on SNc neurons or the prolonged action of sequestered MPP+. In either case, the research shows that deprenyl can increase SNc neuronal survival by a mechanism that is independent of the blockade of MPTP's conversion to MPP+ and may be responsible for slowing the progressioon of PD.
Receptor‐mediated endocytosis and degradation of insulin‐like growth factor I and II in neonatal rat astrocytes
Authors: Auletta M. , Nielsen F. C. , Gammeltoft S. 
Published: January 2005

AbstractReceptor‐mediated internalization and degradation of insulin‐like growth factors, IGF‐I and IGF‐II, were studied in primary cultures of neonatal rat astrocytes. Surface‐bound IGF‐II was rapidly internalized and 80% of cell‐associated radioactivity was located intracellularly after 30 min. IGF‐I was internalized at a slower rate, and only 40% of cell‐associated radioactivity was inside the cell utter 30 min. A pulse‐chase experiment demonstrated that 55% and 70% of internalized IGF‐I and IGF‐II, respectively, was degraded to free amino acids after a 3‐hr chase. Lysosomal and protease inhibitors had different effects on the binding, internalization, and processing of IGF‐I and IGF‐II. Inhibition of lysosomal acidification by chloroquine increased the amounts of surface‐bound IGF‐II and intracellular IGF‐I and reduced the degradation of IGF‐I. The chelating agent phenanthroline increased the surface binding of IGF‐I and IGF‐II and internalization of IGF‐II and reduced the degradation of IGF‐I and IGF‐II. Finally, receptor‐bound IGF‐II on the cell surface was decreased with increasing cell density, whereas IGF‐I binding was unaltered. Our data suggest that cell‐surface expression of IGF‐I receptors and IGF‐II receptors is regulated by different mechanisms and that receptor‐bound IGF‐I and IGF‐II are trafficked and processed by different intracellular pathways in neonatal rat astrocytes.
Long‐term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization
Authors: Pollack I. F. , Lee L. H.‐C. , Zhou H. F. , Lund R. D. 
Published: January 2005

AbstractPrevious studies have suggested that the incidence of spontaneous rejection among immunogenetically mis matched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron‐rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4–6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non‐neuronal graft material composed predominantly of xenogeneic glial cells (post natal day 3, PD3, CD‐1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague‐Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2‐positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no his‐tological signs of rejection or major histocompatabil‐ity complex antigen expression in and around the transplant‐derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well‐integrated 1‐month‐old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2‐positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 “long‐term” animals showed evidence of graft rejection.These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune‐mediated process is unlikely to be responsible for the time‐dependent elimination of the donor astrocytes. We conclude that the presence of donor astrocytes does not itself predispose a graft to rejection. Accordingly, these cells are unlikely to be responsible for the high incidence of spontaneous rejection seen with cortical grafts.