bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosinated and detyrosinated microtubules in axonal processes of cerebellar macroneurons grown in culture
Authors: Arregui C. , Busciglio J. , Caceres A. , Barra H. S.
Published: January 2005

We have used the monoclonal antibody YL 1/2 (Tyr) specific for tyrosinated tubulin, and a polyclonal antibody (Glu) specific for detyrosinated tubulin to visualize the distribution of microtubules and microtubule assembly sites during axonal outgrowth. Cerebellar macroneurons growing in culture initially extend several short and thin neurites which have the potential to differentiate either as axons or dendrites (Ferreira and Caceres: Developmental Brain Research 49:205‐213, 1989). At the onset of axonal outgrowth the Tyr antibody labels the minor neurites, the axon, and its growth cone, while the Glu antibody only shows immunoreactivity in the axonal shaft. After nocodazole treatment, the Tyr staining disappears, whereas that produced by the Glu antibody remains practically unchanged. When nocodazole was removed, tyrosinated microtubules reappeared first at the tip of the axon, in a more distal region than that occupied by detyrosinated microtubules; another focal site of tyrosinated tubulin incorporation was detected in the cell body. Incorporation of tyrosinated tubulin into growing axons was also studied after taxol treatment. After long incubation periods in the presence of taxol, the Tyr staining disappeared from the axon but remained in the cell body; however, immunoreactivity in this site was negative when the cells were preincubated in the presence of protein synthesis inhibitors. Release from taxol results in the reappearance of Tyr immunoreactivity at the distal end of the axon. Taken collectively, the present results indicate (1) that in cerebellar macroneurons axonal differentiation is accompanied by a temporal and spatial differentiation of microtubules fcnd (2) that there is an active site of tyrosinated tubulin assembly at the tip of axonal processes, and they suggest that the highly tyrosinated domain in this region is a consequence of rapid microtubule turnover and tubulin tyrosine ligase activity.
Differential neuronal expression of c‐fos proto‐oncogene following peripheral nerve injury or chemically‐induced seizure
Authors: Jones K. J. , Evinger C.
Published: January 2005

Previous work by others has demonstrated that neocortical injury results in the induction of c‐fos proto‐oncogene both at and distal to the site of injury. However, secondary effects of focal brain injury, such as spreading depression and seizure activity, also have been shown to induce the expression of c‐fos. Thus, it is unclear whether the stimulus inducing c‐fos expression after generalized brain trauma is direct neuronal injury or associated, secondary effects of injury. In this study, we tested the hypothesis that a specific axonal disconnection would induce the expression of c‐fos proto‐oncogene in the injured neurons. The injury paradigm that was used was peripheral axotomy of rodent facial motoneurons. The right facial nerve was severed distal to the stylomastoid foramen, with the left side serving as an internal control. As positive controls, in a separate group of animals, seizures were invoked using bicucullin administered intraperitoneally. At the end of postoperative survival times ranging from 30 min to 24 hr, the animals were sacrificed. For northern blot analysis using a c‐fos cDNA probe, total RNA was isolated from the dissected facial nuclear groups in the injury experiments, or whole brain and neocortex in the seizure experiments. For immunocytochemical analysis using a battery of c‐fos antibodies, the animals were perfused with paraformaldehyde and processed for routine light microscope immunocytochemistry. In the bicucullin‐injected animals, c‐fos mRNA was massively induced in whole brain in a manner proportional to the overall level of gross seizure activity. The localization of the c‐fos expression occurred primarily in neurons within the neocortex. In the axotomized facial motoneurons, no induction of c‐fos mRNA or protein was found to occur at any of the timepoints examined and no basal expression of c‐fos was observed in the uninjured motoneurons. The results suggest that the rapid induction of c‐fos proto‐oncogene product is not a specific component of the nerve cell body response to injury.
Influence of innervation on molecular forms of acetylcholinesterase in regenerating fast and slow skeletal muscles
Authors: Črne N. , Sketelj J. , Brzin M.
Published: January 2005

Nerve‐intact muscle regenerates were prepared by ischemic‐toxic injury of slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of the rat. Rapid innervation of regenerating myotubes modified intrinsic patterns of AChE molecular forms, revealed by velocity sedimentation in linear sucrose gradients. Regarding their onset, the effects of innervation can be classified as early and late. The earliest changes in the SOL regenerates appeared a few clays after inner vation by their motoneurons: The activity of the 13 S AChE form (A 8) increased significantly in compari son to non‐innervated regenerates. The pattern of AChE molecular forms became similar to that in the normal SOL muscle during the 2nd week after in jury. In contrast, no major differences were observed between 8 day‐old innervated and non‐innervated EDL regenerates. Their patterns of AChE molecular forms resembled that in the normal EDL. However, the predominance of the 10 S AChE form (G 4) char acteristic for the 2‐week old non‐innervated regener ates was prevented by innervation. Early effect of innervation observed in the SOL regenerates but not in the EDL may be due to intrinsically different re sponse of the regenerating SOL myotubes to innerva tion. Rather high extrajunctional activity of the asymmetric 16 S (A 12) molecular form of AChE in early regenerates was reduced to adult level in about 3 weeks in the SOL, and nearly completely sup pressed in 5 weeks after innervation in the EDL re generates. This reduction is assumed to be a late ef fect of innervation, as well as a decrease of the activity of the 4 S AChE form (G 1) in the SOL regenerates. A suppressive mechanism is activated in the extra junctional regions of the innervated muscle regener ates during their maturation. This mechanism coun teracts the enhancing influence of contractile activity on the expression of the asymmetric AChE forms in immature muscle cells, and is more efficient in the EDL than in the SOL. Its onset may be apparently delayed by protracted fusion of the satellite cells with muscle fibers.
Analysis of a decreased Na+ conductance by tumor necrosis factor in identified neurons of Aplysia kurodai
Authors: Sawada M. , Hara N. , Maeno T.
Published: January 2005

The ionic mechanism of the effect of extracellularly ejected recombinant human tumor necrosis factor‐alpha (rhTNF‐α) on the membrane of identified neurons R9 and R10 of Aplysia kurodai was investigatedwith conventional voltage‐clamp, micropressure ejection, and ion substitution techniques. Micropressureejected rhTNF caused a marked hyperpolarization in the undamped neuron. Clamping the same neuron at its resting potential level (–60 mV) and reejecting rhTNF‐α with the same doseproduced a slow outward current [Io(TNF)] associated with a decrease in input membrane conductance. Io(TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io}(TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that I}o(TNF) in identified neurons R9 and R10 of A. kurodai is due to a decreased Na+ conductance but not due to an activation of the Na+ ‐K+ Pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the immune system.
Age‐dependent decrease of process formation by cultured oligodendrocytes is augmented by protein kinase C stimulation
Authors: Yong Voon Wee , Cheung J. C. B. , Uhm J. H. , Kim S. U.
Published: January 2005

The proportion of cultured rat oligodendrocytes (OL) that extended processes of over three soma diameter in length is dependent on the age of the animals from which the brains were derived; up to 70% of neonatal OL attained this criterion within 3 days, and this proportion progressively decreased with advancing ages of the animals (1, 3, and 6 months). The lower extent of process formation from older rat OL could he augmented, and indeed to equal neonatal levels, by treatment of cells with phorbol esters that stimulate protein kinase C: 4β‐phorbol‐12,13‐dibutyrate (PDB) and phorbol‐12‐myristate‐13‐acetate (PMA). Enhancement of process formation, by PDB and PMA was also observed for cultured adult human and bovine OL. For adult OL from all three species, a phorbol ester that binds but that does not activate protein kinase C, 4α‐phorbol‐12,13‐didecanoate, did not result in enhancement of process formation. Selectively to biologically active phorbol esters was shown by the inability of a wide range of growth factors to promote process extension. Immunohistochemical analyses indicate that the type III isozyme of protein kinase C predominates in cultured OL; the apparent intensity of immunoreactive PKC was not different between controls or cultures treated for 12 days with PDB, suggesting that the persistent presence of PDB might not have down‐regulated the enzyme, in contrast to other cell types. We propose that stimulation of protein kinase C is critical to the triggering of process formation by cultured OL in vitro.
Quantitation of the growth‐associated protein B‐50/GAP‐43 and neurite outgrowth in PC12 cells
Authors: San E. R. A. Jap Tjoen , Schmidt‐Michels M. H. , Spruijt B. M. , Oestreicher A. B. , Schotman P. , Gispen W. H.
Published: January 2005

A combined assay to measure neurite outgrowth and B‐50/GAP‐43 levels in PC 12 cells is reported. During NGF‐induced neuritogenesis, B‐50/GAP‐43 expression was monitored by enzyme‐linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose–response and time curve of β‐NGF‐induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of β‐NGF ≥5 ng/ml and was accompanied by a linear increase in the amount of B‐50/GAP‐43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.
Olfactory Schwann cells are derived from precursor cells in the olfactory epithelium
Authors: Chuah M. I. , Au C.
Published: January 2005

In this morphological and immunohistochemical study we show that olfactory schwann cells (OSC) are derived from precursor cells residing in the olfactory epithelium. During development, they migrate out of the epithelium and extend processes to ensheath the olfactory axons.

Olfactory mucosa from E14 rat embryos and juvenile rats were treated with trypsin‐pancreatin to remove the underlying connective tissue. The epithelial explant was then maintained for two days in culture, during which cells migrated out from the explant. Among them were spindly bipolar cells which were identified as OSC by their positive immunoreaction for glial fibrillary acidic protein, ultrastructure, and association with growing axons. Axonal growth was significantly more profuse in the embryonic explants, in which the polarity of the OSC was oriented parallel with the axons. Ultrastructural observations showed that ensheathment of the bundles of axons resembled those in vivo.
Oligodendrocytes of the jimpy phenotype can be partially restored by environmental factors in vivo
Authors: Lachapelle F. , Lapie P. , Campagnoni A. T. , Gumpel M.
Published: January 2005

Cross‐transplantations of neural tissue between jimpy (jp) shiverer (shi) and normal mice have been performed under heterochronic conditions. In all series, fragments of E14–E15 embryonic neural tissue from the different donors have been transplanted into newborn host brain in order to study environmental influences by differentiated tissue on transplanted embryonic cell lines. Large patches of proteolipid protein (PLP)‐positive myelin have been observed in the jp brain after transplantation of shi or normal embryonic tissue into the newborn jp brain, suggesting that the jp parenchyma did not inhibit the differentiation of other Oligodendrocytes (ODCs). Jp embryonic tissue had the same mitotic potential as normal tissue, as demonstrated by the larger size of myelin patches observed when jp embryonic tissue was used instead of newborn jp tissue. By contrast, whatever the conditions, jp myelin patches were always obviously smaller than normal or shi myelin patches, suggesting that the myelinating capacity of jp ODCs was not enhanced by environmental factors. Finally, comparison of the ratie of successful outcomes observed following embryonic vs. newborn jp donor tissue, strongly suggests a partial or total normalization of jp embryonic ODCs survival by a more mature shi environment.
Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing wallerian degeneration
Authors: Komiyama A. , Novicki D. L. , Suzuki Kinuko
Published: January 2005

Proliferation of Schwann cells during nerve degeneration or regeneration is well documented in vivo. We investigated whether the proliferative response of Schwann cells to injury is retained in vitro. Using 5‐month‐old male C57BL mice, Schwann cells were isolated from sciatic nerves under 3 experimental conditions: (1) uninjured, (2) after permanent nerve‐transection, or (3) after nerve‐crush, which permits axonal regeneration. Schwann cells rarely attached to polylysine‐coated coverslips when isolated from uninjured or 1 day posttransection/crush nerves. The number of adherent cells increased when Schwann cells were isolated 3 days after nerve‐transection or‐crush. When cells were isolated from transected nerves, cell adhesion reached a peak 2 weeks after the injury and then declined. Maximal attachment of Schwann cells occurred when the cells were isolated 2–4 weeks after nerve‐crush. The percentage of Schwann cells with spreading processes corresponded closely with the number of thymidine‐labeled cells at 1 day in vitro. The in vitro capacity of cells to spread and incorporate thymidine reached maximal levels at 5 days posttransection/crush. Capacity of cells to spread and incorporate thymidine subsequently decreased with time following transection. However, a biphasic elevation in cell spreading and thymidine incorporation was observed in Schwann cells isolated from crushed nerves. Maximal growth of Schwann cells in vitro occurred at 1–2 weeks posttransection and at 1–4 weeks postcrush. Adhesion and spreading of Schwann cells were promoted by coating coverslips with laminin or fibronectin. Preincubation of Schwann cells with soluble laminin or fibronectin prevented the initial cell attachment induced by the corresponding protein. Our results suggest that Schwann cells from injured nerves possess binding sites for laminin and fibronectin, which are, in part, responsible for the enhanced adhesion of Schwann cells in vitro. This study provides a new method for preparation of Schwann cells from peripheral nerves of adult mice.
Autoradiographic study of iron‐binding sites in the rat brain: Distribution and relationship to aging
Authors: Barkai A. I. , Durkin M. , Dwork A. J. , Nelson H. D.
Published: January 2005

Specific binding of 59Fe to various brain structures was investigated in rats using nanomolar concentrations of 59FeCl3 and quantitative aiitoradiography. Saturation studies revealed high affinity binding (kd in the nanomolar range) with binding sites density (Bmax) which varied in different brain regions from 462 fmol per mg tissue in the central thalamic nuclei to over 4 pmol per mg tissue in the cerebral peduncle. Binding was seen in both white and gray matter structures. Bmax values for frontal cortex, dentate gy‐rus, and substantia nigra were significantly lower in older rats. The distribution of 59Fe binding sites was not consistent with the distribution of brain iron as reported by other investigators. 59Fe binding was reduced significantly in the presence of free radicals. These observations suggest that high affinity binding sites for iron are localized differentially in various brain structures and may play an important role in the translocation and storage of potentially harmful ferric cations in brain. The finding that the capacity of the brain tissue to bind iron diminished with age in discrete brain regions suggests that in the aged animal, the removal of “free” iron from the cellular domain may be impaired in such regions, leading to increased susceptibility to iron‐enhanced lipid peroxidation and cell death.
Peripheral nerve regeneration is impeded by interleukin‐1 receptor antagonist released from a polymeric guidance channel
Authors: Guénard V. , Dinarello C. A. , Weston P. J. , Aebischer P.
Published: January 2005

Interleukin‐1 receptor antagonist (IL‐lra), a true antagonist of the interleukin‐1 (IL‐1) receptors, is released by activated macrophages and binds specifically to the IL‐1 receptors without triggering IL‐1 effects. Following peripheral nerve axotomy, activated macrophages release IL‐1, which induces the expression of nerve growth factor (NGF) mRNA in Schwann cells. IL‐lra may therefore impede peripheral nerve regeneration by blocking the NGF‐mediated effect of IL‐1. Peripheral nerve regeneration occurring through polymeric guidance channels releasing IL‐lra was investigated in a 4‐mm gap transected mouse sciatic nerve model. Cohorts of five animals were implanted with tubes releasing either bovine serum albumin (BSA), BSA with IL‐lra, or BSA with deactivated IL‐lra (dlL‐lra) for 4 weeks. In vitro release kinetics indicated that after an initial burst, IL‐lra release was linear for the next 3½ weeks. Following implantation of a polymeric guidance channel, a regenerated cable bridged the nerve gap in all animals. The cables were similar in size and were composed of nerve microfascicles containing both unmyelinated and myelinated axons in association with their Schwann cells. Tissue regenerated in tubes releasing BSA‐IL‐lra contained, however, significantly fewer myelinated and unmyelinated axons and blood vessels than did tubes releasing BSA alone or BSA‐dIL‐lra. We conclude that a naturally occurring antagonist of IL‐1 receptors impedes peripheral nerve regeneration, suggesting that macrophages play an essential role in controlling peripheral nerve regeneration through the release of stimulatory and/ or inhibitory molecules.
Protein kinase C stimulation enhances the process formation of adult oligodendrocytes and induces proliferation
Authors: Althaus H. H. , Schröter J. , Spoerri P. , Schwartz P. , Klöppner S. , Rohmann A. , Neuhoff V.
Published: January 2005

Oligodendrocytes (OL) isolated from adult pig brains regenerate their processes and form a network of fibers after 14–18 days in vitro (DIV). Stimulation of protein kinase C (Pk‐C) by tumour promoters such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) produces at day 7 in vitro a similar network after only 20 hr. H‐7, an inhibitor of Pk‐C, as well as amiloride, which inhibits the subsequent Na+/H+ exchange, reversibly suppress this effect. Activation of the protein kinase A or calmodulin pathway do not result in an increased OL process production. Furthermore, TPA induced proliferation in a subpopulation of OL. We conclude that the stimulation of Pk‐C is of utmost importance for OL regeneration.
Analysis of neurotransmitter receptors mediating changes in LH release induced by electrical stimulation of the dorsal raphe nucleus in the rat
Authors: Johnson J. H. , Kitts C. S.
Published: January 2005

We report studies of neurotransmitter and receptor species mediating the inhibition of LH release in ovariectomized (OVX) rats and stimulation in OVX estrogen‐primed rats induced by electrical stimulation of the dorsal raphe nucleus (DRN). Attempts were made to block effects of stimulation of the DRN in groups of four to nine ovariectomized rats with or without priming with estradiol benzoate (EB) by pre‐treatment with the 5HT receptor antagonists ke‐tanserin, methysergide, or metergoline; the α‐adrenergic antagonist phenoxybenzamine; or the opiate antagonist naltrexone. Blood samples were collected via jugular cannulae from the unanesthetized rats at 10‐min intervals before and during electrical stimulation of the DRN, and assayed by double antibody radioimmunoassay for LH. To test the effect of stimulation, data from animals in each treatment group were subjected to analysis of variance to obtain the contrast matrix including prestimulation and stimulation time points of interest. The contrast matrix was then used to construct an F ratio and thereby to evaluate the level of significance. In unprimed OVX rats the sustained decrease in plasma LH concentration during stimulation was prevented in rats pretreated with ketanserin or phenoxybenzamine. Methysergide pretreatment delayed the inhibitory effect of DRN electrical stimulation for 30 min, whereas metergoline and naltrexone were ineffective. In EB‐primed animals the increase in plasma LH observed during stimulation was prevented in rats pretreated with metergoline, and reversed in those receiving naltrexone. Ketanserin limited the duration of the increase to 10 min, while phenoxybenzamine and methysergide had no significant effect. These results suggest that the inhibitory effect of dorsal raphe stimulation on release of LH in OVX rats may be mediated by 5HT2 and α‐adrenergic systems, whereas the stimulatory effects (OVX + EB) may involve primarily 5HT1 and opiate receptors.
Taurine‐deficient cultured cerebellar astrocytes and granule neurons obtained by treatment with guanidinoethane sulfonate
Authors: Morán J. , Pasantes‐Morales H.
Published: January 2005

Mouse cerebellar granule neurons and astrocytes grown in the presence of 2 mM guanidinoethane sul‐fonate (GES) exhibited a progressive and rapid decrease in taurine concentration. A reduction of 20% was observed as early as 1 hr after exposure to GES and the loss of cell taurine continued until the taurine pool was reduced by about 90%. This remaining taurine persisted without further decrease even after 3 weeks of exposure to GES. Taurine reduction caused by GES was similar in both types of cells. The effect of GES was dose‐dependent, with significant decreases in taurine levels already detected at 100 μM. It was selective for taurine, since none of the other free amino acids were affected. Taurine depletion induced by GES was totally reversible. Intracellular taurine was not mobilized by GES. Taurine uptake in both astrocytes and granule neurons, examined at the taurine concentration present in the culture medium, was practically abolished by 2 mM GES. This approach represents an in vitro model of taurine depletion that may be useful to investigate the cell abnormalities responsible for the failure of differentiation and migration of granule cells and astrocytes observed in taurine‐deficient cats.
Inhibition of GFAP synthesis by antisense RNA in astrocytes
Authors: Yu A. C. H. , Lee Y. L. , Eng L. F.
Published: January 2005

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with LipofectinTM (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H‐antisense GFAP RNA alone and reached an equilibrium of 7–8.8 ηg per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 ηg per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose‐ and time‐dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)‐treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30–40% decrease in GFAP content 2 days after treatment. A similar decrease in GFAP content was obtained in cultures grown in a chemically defined medium, another condition known to induce an increase in GFAP content in cultures. This study has demonstrated that antisense GFAP RNA can inhibit GFAP synthesis in astrocytes and may be useful for regulating astrogliosis immediately following CNS injury.
Microtubule dynamics in axons and dendrites
Authors: Baas P. W. , Slaughter T. , Brown A. , Black M. M.
Published: January 2005

We 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

Tau 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

Advance 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

Na+ 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

Rat 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

Epidermal 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

To 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

When 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

Recently, 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

Experimental 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

Deprenyl 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

Receptor‐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.