bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats
Authors: Ikeshima H. , Yuasa S. , Matsuo K. , Kawamura K. , Hata J. , Takano T.
Published: January 2005

By Northern blot analysis with the digoxigenin‐labeled antisense RNA probes of the noncoding regions, the transcripts of three calmodulin (CaM) genes, CaMI, CaMII, and CaMIII, were separately detected in 12 different tissues of adult Wistar albino rats, without any cross‐hybridization. The mRNAs of all three CaM genes were abundant in the central nervous system (CNS) as well as in the testis, although ubiquitous expression was detected at low levels in the other tissues. There were subtle but significant differences in the tissue‐specific distribution of the three CaM gene RNAs. By in situ hybridization, strong hybridization of the three CaM gene probes was observed in common in large projection neurons of the CNS: the hippocampal pyramidal cells, the cerebellar Purkinje cells, and the large neurons of the cerebral neocortex, the pyriform cortex, the mesencephalon, the pons, and the spinal cord. The expression of the three CaM genes was at lower levels in small interneurons of the CNS. These profiles of expression were almost the same among the three CaM genes. Thus, all three CaM genes were coordinately expressed in neurons of the adult rat CNS. Certain reulatory mechanisms of the three CaM genes seemed to mediate similar tissue‐ and cell type‐specific expression in the CNS. © 1993 Wiley‐Liss, Inc.
Outstations of the golgi complex are present in the processes of cultured rat oligodendrocytes
Authors: de Vries H. , Schrage C. , Hoekstra K. , Kok J. W. , van der Haar M. E. , Kalicharan D. , Liem R. S. B. , Copray J. C. V. M. , Hoekstra D.
Published: January 2005

Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6‐[N‐(7‐nitrobenz‐2‐oxa‐1,3,‐diazol‐4‐yl)amino]hexanoylceramide (C6NBD‐ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37°C most of the C6NBD‐ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microspcopy. Interestingly, C6NBD‐ceramide was found to acculmulate also in smaller, oval‐shaped structures in many of the processes, at distances up to 30 μm from the nucleus. This implies the possibility that these structures are Golgi (‐derived) complexes. Indeed, after incubation of oligodendrocytes with C6‐ceramide and rhodamine‐labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structres in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryor, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes. © 1993 Wiley‐Liss, Inc.
AMPA‐Selective glutamate receptor subunits in astroglial cultures
Authors: Condorelli D. F. , Dell'Albani P. , Corsaro M. , Barresi V. , Stella A. M. Giuffrida
Published: January 2005

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR‐1 to ‐4) and at the protein level (GluR‐1 and GluR‐2/3/4C) in “primary astroglial cultures” (non‐neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocamps, and striatum and in “brain non‐neuronal cell cultures” (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined ampa subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (glur‐1,‐2,‐3,‐4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti‐GluR‐1 and anti GluR‐2/3/4C polyclonal antibodies confirmed the presence of low leve of immunoreactive proteins of the same size of those identifice in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR‐1, ‐2, and ‐3 were mailly expressed in cortical cultures, while GluR‐4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR‐4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutmate ionotropic receptors of the AMPA type in primary astroglial cultrues and suggest that GluR‐4 is the main AMPA receptor subunit expressed in non neuronal cells of the central nervous system. © 1993 Wiley‐Liss, Inc.
Myelin gene activation: A glucose sensitive critical period in development
Authors: Royland J. E. , Konat G. W. , Wiggins R. C.
Published: January 2005

Glucose deprivation was employed to model caloric undernutrition in newborn rat mixed glial cultures. Six day‐old cultures were placed in serum‐free media containing glucose concentrations from 0.55 mg/ml to 10 mg/ml. The expression of the myelin PLP, BP, and MAG genes was determined by Northern blot analysis. The activation of the myelin genes began at approximately 6 day in vitro (DIV), and a period of rapid upregulation followed through 14 DIV. The gene activity was directly related to the glucose concentrat.

The increase in glucose concentration from 0.55 to 1.5 mg/ml (which spans the physiological range) resulted in 2–3 fold increases in expression of the myelin genes, whereas further increases in glucose (2–10 mg/ml) produced only slight additional elevation in the gene activity. We used high glucose (5–6 mg/ml) as control, or low glucose (0.55 mg/ml) as deprived, to delineate possible critical periods of oligodendrocytic differentiation. Cultures were deprived for 4‐day intervals, staggered from 6 to 22 DIV. Deprivation from 6 to 10 DIV produced an 80–90% suppression of the myelin gene upregulation at 22 DIV; deprivation from 10 to 14 DIV produced 60–70% suppression; whereas deprivation from 14 to 18 DIV was fully recoverable by 22 DIV. These results show that mixed glial cultures model the developmental pattern of myelin gene expression, as well as their vulnerability. Furthermore, the period of rapid upregulation of the myelin genes appears to be a critical period in development, during which glucose deprivation irreversibly alters oligodendrocyte differentiation. © 1993 Wiley‐Liss, Inc.
Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia
Authors: Manzerra P. , Rush S. J. , Brown I. R.
Published: January 2005

We have previously investigated the expression of hsp70 genes in the hyperthermic rabbit brain at the mRNA level by Northern blot and in situ hybridization procedures. Our studies have now been extended to the protein level utilizing Western blot and immunocytochemistry. Using an antibody which is specific to inducible hsp70, a prominent induction of hsp70 protein in glial cells of hyperthermic animals was noted. In particular, Bergmann glial cells in the cerebellum are strongly immunoreactive while adjacent Purkinje neurons are immunonegative. Extension of our in situ hybridization studies to a time course analysis revealed that the initial glial induction events were followed by a delayed accumulation of inducible hsp70 mRNA in Purkinje neurons at 10 hr post‐heat shock. In control animals, high levels of constitutively expressed hsc70 mRNA and protein were observed in Purkinje neurons. Similar hsc70 and hsp70 mRNA observations were also made in neurons of the deep cerebellar nuclei and in motor neurons of the spinal cord. Our results suggest that these neuronal cell types accmulate hsp70 mRNA in response to hyperthermic treatment; however, the response is delayed when compared to the rapid response seen in glial cells. The high constitutive levels of hsc70 in certain neuronal cell types may play a role in the initial dampening of the hsp70 induction response in these cells. © 1993 Wiley‐Liss, Inc.
Transferrin in the central nervous system of the shiverer mouse myelin mutant
Authors: Connor J. R. , Roskams A. J. , Menzies S. L. , Williams M. E.
Published: January 2005

Transferrin, the iron mobilization protein, and its mRNA are normally present in oligodendrocytes. Previous reports using myelin mutants have shown both a decrease in transferrin protein and mRNA when the oligodendrocyte population is compromised. In this study the shiverer mouse mutant in which the oligodendrocyte population is numerically normal, but has both quantitatively diminished and qualitatively abnormal myelin was used. This animal model was chosen to address the question whether expression of the transferrin message and/or protein correlated more closely to the number of oligodendrocytes (normal) or the amount of myelin (abnormally low). A 1/2 to 2/3 decrease in transferrin protein occurred in all brain regions examined except for the spinal cord in the shiverer group compared to both heterozygous littermates and wild type controls. Levels of transferrin transcripts in the brain are not affected by the shiverer mutation. These results taken with previous reports from this laboratory indicate that the presence of oligodendrocytes is a requirement for normal expression of transferrin mRNA in brain but is not sufficient for normal values of the protein. The level of Tf protein correlates more closely with the amount of myelin present than it does with the numbers of oligodendrocytes present. These data are consistent with previous reports from our laboratory that transferrin accumulation by oligodendrocytes is associated with myelin production by these cells. These data further suggest transferrin mRNA may be constitutively expressed by oligodendrocytes and that the protein expression is regulated at the level of translation. © 1993 Wiley‐Liss, Inc.
Oxygen radical‐induced neurotoxicity in spinal cord neuron cultures
Authors: Michikawa M. , Lim K. T. , McLarnon J. G. , Kim S. U.
Published: January 2005

The neurotoxic effects of oxygen radical on spinal cord neuron cultures derived from fetal mouse have been studies. Oxygen radicals, superoxide radical and hydrogen peroxide, were generated by adding xanthine oxidase and hypoxanthine in the culture medium. Exposure of neurons to this oxygen radical generating system resulted in a significant cell death and decrease of choline acetyltransferase (ChAT) activity in a time‐dependent manner in spinal cord neuron cultures. The decrease in cell viability and ChAT enzyme activity induced by the oxygen radicals was blocked by scavengers such as superoxide dismutase (SOD), catalase, and tetrakis (2‐pyridylmethyl) ethylenediamine (TPEN), a metal chelator. Antagonista of the N‐methyl‐D‐aspartate (NMDA) receptor, including MK801 (a noncompetitive NMDA antagonist), D‐2‐amino‐5‐phosphonovaleric acid (APV) (a competitive NMDA antagonist), and 7‐chlorokynurenic acid (an antagonist at the glycine site associated with the NMDA receptor), similarly blocked oxygen radical‐induced decrease in cell viability and ChAT activity in spinal cord neuron cultures. These results indicate that both oxygen radicals and excitotoxic amino acids were involved in the oxidant‐ititiated neurotoxicity of spinal cord neurons. © 1994 Wiley‐Liss, Inc.
Autoradiographic characterization of the non‐N‐methyl‐D‐aspartate binding sites in human cerebellum using the antagonist [3H]6‐cyano‐7‐nitroquinoxaline‐2,3‐dione
Authors: Hatziefthimiou A. , Mitsacos A. , Kouvelas E. D.
Published: January 2005

Using quantitative autoradiography, we have characterized the binding properties of the non‐N‐methyl‐D‐aspartate (NMDA) glutamate receptor antagonist [3H]6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) in adult human cerebellum. Saturation experiments revealed [3H]CNQX binding to a single class of sites with similar affinity in the molecular and granule cell layer (Kd = 89.0 ± 6.4 and 83.3 ± 9.9nM, respectively). The maximum number of [3H]CNQX binding sites was much higher in the molecular compared to the granule cell layer (Bmax = 16.2 ± 1.1 and 2.8 ± 0.5 pmol/mg protein, respectively). Inhibition experiments were performed in order to examine the pharmacological profile of [3H]CNQX binding in the molecular layer. [3H]CNQX labeled sites with high affinity for both non‐NMDA agonists, (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) and kainate. Dose‐response curves for inhibition of [3H]CNQX by AMPA and Kainate were biphasic. The potency of AMPA for displacement of [3H]CNQX binding (Ki © 1994 Wiley‐Liss, Inc.:2.8 ± 0.8 nM and 12.5 ± 0.8 μM) was 4‐ to 6‐fold greater than the corresponding potency of kainate (Ki:18.1 ± 5.7 nm and 48.7 ± 9.3 μM). In conclusion, the pharmacological analysis of [3H]CNQX binding in the human cerebellar molecular layer reflects the existence of multiple binding sites of the non‐NMDA receptor that have different affinities for both AMPA and kainate. © 1994 Wiley‐Liss, Inc.
Isoforms of ferritin have a specific cellular distribution in the brain
Authors: Connor J. R. , Boeshore K. L. , Benkovic S. A. , Menzies S. L.
Published: January 2005

Ferritin is the major iron storage protein and accounts for the majority of the iron in the brain. Thus, ferritin is a key component in protecting the brain from iron induced oxidative damage. The high lipid content, high rate of oxidative metabolism, and high iron content combine to make the brain the organ most susceptible to oxidative stress. The role of oxidative damage and disruption of brain iron homeostasis is considered clinically important to normal aging and a potential pathogenic component of a number of neurologic disorders including Alzheimer's disease and Parkinson's disease. Little is known, however, of the mechanism by which the brain maintains iron homeostasis at either the whole organ or cellular level. In this study we report the cellular distribution of the two isoforms of ferritin in the brain of adult subhuman primates. A subset of neurons immunolabel specifically for the H‐chain ferritin protein, whereas cells resembling microglia are immunolabeled only after exposure to the L‐chain ferritin antibody. Only one cell type immunostains for both H‐and L‐chain ferritin; these cells are morphologically similar and have the same distribution pattern as oligodendrocytes. Neither ferritin isoform is usually detected in astrocytes. These data indicate considerable differences in iron sequestration and use between neurons and glia and among neuronal and glial subtypes. This information will be essential in determining the role of each of these cells in maintaining general brain iron homeostasis and the relative abilities of these cells to withstand oxidative stress. © 1994 Wiley‐Liss, Inc.
Phosphorylation of neurofilament H subunit as related to arrangement of neurofilaments
Authors: Gotow T. , Tanaka J.
Published: January 2005

To find out what causes differences in phosphorylation states in neurofilaments (NF), we selected two types of dendrite, one provided with very few NFs (Purkinje cell) and the other with relatively many (anterior horn cell). We examined these with four monoclonal antibodies selected by the Western blot analysis, two (NE14 and SMI31) recongnizing only phosphorylated, SMI32 recognizing only nonphosphorylated, and N52 recognizing phosphorylation‐independent epitopes of NF‐H. The immunoperoxidase labeling of dendrites, and also of perikarya, in both neurons was detectable with all four antibodies. After the tissue was treated with Triton X‐100, the labeling was still detectable with SMI32 or N52, but undetectable with NE14 and SMI31. The brain homogenate Triton‐extracted supernatant after centrifugation at 100,000g for 1 hr showed the staining of NE14, SMI31, and N52 but not that of SMI32. In Purkinje cell dendrite and perikaryon, NFs always appeared singly. In the immunogold labeling, they were labeled only with SMI32 or N52. Labeling by NE14 or SMI31 was distributed throughout the cytoplasm and hardly associated with NFs. In the anterior horn cell dendrite and perikaryon, NFs appeared both single and in bundles. They were predominantly labeled with SMI31 or N52 when they were single, and with NE14, SMI31, or N52 when they were bundled. Even in one NF, portions that appeared single were labeled mostly with SMI32 or N52, while the remainder, to which other NFs approached closely, were labeled mostly with NE14, SMI31, or N52. Thus, when NFs appear singly, NF‐H in their projections or cross‐bridges with other organelles is not phosphorylated, while when NFs are bundled, NF‐H is phosphorylated in crossbridges between NF core filaments. These data may explain why the NF‐H is heavily phosphorylated in axons, where NFs are abundant, and not in dendrites and perikarya, where NFs are sparse. Wiley‐Liss, Inc.
Induction of heat shock (stress) protein 70 and its mRNA in the normal and light‐damaged rat retina after whole body hyperthermia
Authors: Tytell M. , Barbe M. F. , Brown I. R.
Published: January 2005

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post‐hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley‐Liss, Inc.
Dibutyryl cAMP, interleukin‐1β, and macrophage conditioned medium enhance the ability of astrocytes to promote neurite growth
Authors: Miller C. , Tsatas O. , David S.
Published: January 2005

Astrocytes purified from the neonatal rat brain were cultured for up to 3 weeks prior to being treated with agents that can induce reactive changes in astrocytes. These agents included dibutyryl cAMP, interleukin‐1β, and macrophage conditioned media. After treating astrocytes for 3 days, the agents were removed and the ability of the astrocytes to support neurite growth was assessed by plating neonatal rat cerebellar cortical neurons. All the agents increased the ability of astrocytes to support the growth of long neurites. This was particularly evident with astrocytes treated with dibutyryl cAMP and LPS macrophageconditioned medium. These results provide direct evidence that the reactive changes induced in astrocytes in this in vitro model might aid the growth of neurites. Similar changes may underlie the injury‐induced axonal sprouting that occurs in vivo. © 1994 Wiley‐Liss, Inc.
Actin‐Binding protein, drebrin, accumulates in submembranous regions in parallel with neuronal differentiation
Authors: Asada H. , Uyemura K. , Shirao T.
Published: January 2005

Drebrins are developmentally regulated actin‐binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SHSY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F‐actin was more stable against cytochalasin D than F‐actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F‐actin. © 1994 Wiley‐Liss, Inc.
Effect of L‐glutamic acid on acid secretion and immunohistochemical localization of glutamatergic neurons in the rat stomach
Authors: Tsai L. H. , Tsai W. , Wu J.‐Y.
Published: January 2005

Glutamatergic neurons in the rat stomach were localized immunohistochemically using antibodies against L‐glutamate (L‐Glu) as well as glutamate synthesizing enzyme, glutaminase (GLNase). Myenteric ganglia and nerve bundles in the circular muscle and the longitudinal muscle were found to contain GLU‐ and GLNase‐positive nerve fibers, while submucosa and mucosa were devoid of glutamatergic innervation. The distribution of glutamatergic neurons and their processes in both myenteric ganglia and circular muscle is heterogeneous within the stomach. The effect of L‐Glu on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. L‐Glu at 10−7 and 10−8 M alone had no effect on acid secretion. It was found that the oxotremorine‐, histamine‐, or gastrin‐stimulated acid secretion was markedly reduced by L‐Glu at 10−8 M, whereas L‐Glu had little effect on the acid secretion stimulated by dimethyl‐phenylpiperazinium (DMPP) at this concentration. However, at higher concentration, e.g., 10−7 M, L‐Glu also markedly reduced DMPP‐induced acid secretion. Among L‐Glu receptor agonists tested, quisqualic acid (QA) is most potent, followed by kainic acid (KA) and N‐methyl‐D‐aspartic acid (NMDA) in inhibiting oxotremorine‐stimulated acid secretion. Furthermore, this inhibitory effect of L‐Glu on oxotremorine‐stimulated acid secretion is blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a specific non‐NMDA receptor antagonist.

All these results suggest that glutamatergic neurons are involved in the modulation of gastric acid secretion via ionotropic QA/KA receptors, probably through openings of Ca2+ channels. © 1994 Wiley‐Liss, Inc.
Monoamine‐activated α2‐macroglobulin inhibits choline acetyltransferase of embryonic basal forebrain neurons and reversal of the inhibition by NGF and BDNF but not NT‐3
Authors: Liebl D. J. , Koo P. H.
Published: January 2005

Monoamine‐activated α2‐macroglobulin (α2M) has recently been shown to inhibit the growth and survival of cholinergic neurons of the basal forebrain (Liebl and Koo: J Neurosci Res 35:170–182, 1993). The mechanism of this inhibitory effect is believed to involve the regulation of growth factor activities by α2M. The objectives of this study are to determine whether monoamine‐activated α2M can inhibit choline acetyltransferase (ChAT) activity of cholinergic basal forebrain neurons, and whether some common neurotrophins in the CNS can reverse the inhibition. This study demonstrates that both methylamine‐activated α2M (MA‐α2M) and serotonin‐activated α2M (5HT‐α2M) can dose‐dependently suppress the expression of normal basal levels of ChAT activity in embryonic rat basal forebrain cells in vitro, while normal α2M has little or no effect. As little as 0.35 μM monoamine‐activated α2M can suppress the ChAT activity, whereas either nerve growth factor (NGF) or brain‐derived neurotrophic factor (BDNF), but not neurotrophin‐3 (NT‐3), stimulates ChAT expression of these cells. The addition of either NGF or BDNF to the α2M‐suppressed cells can increase ChAT activity back to its normal levels, while NT‐3 can not. These results demonstrate that (1) monoamine‐activated α2M is a potent non‐cytotoxic inhibitor of the ChAT activity in cholinergic basal forebrain neurons, and (2) NGF and BDNF are capable of not only stimulating the ChAT activity but can also specifically reverse the α2M inhibition. The potential physiological role of monoamine‐activated α2M and neurotrophins in the degeneration and regeneration of cholinergic neurons is discussed. In addition, we propose that α2M may serve as an important tool for evaluating the roles of growth factors in the nervous system. © 1994 Wiley‐Liss, Inc.
Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex
Authors: Takács J. , Hámori J.
Published: January 2005

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm2) and less rapidly between PN days 30 to 48 (up to 9,300 μm2), followed by a moderate decrease at PN day 90 (about 6,600 μm2). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm3 at PN day 6,93,000 μm3 at PN day 25, and 100,000 μm3 at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 104 spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 105 spines/Purkinje cell), which then declined to 6.3 × 104 spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley‐Liss, Inc.
Neurological mouse mutants and the genes of myelin
Authors: Nave K.‐A.
Published: January 2005

The prospect to create mouse mutants of virtually any cloned gene has renewed interest in the genetic analysis of mammalian brain development. A diverse group of spontaneous and engineered mouse mutants, characterized by a defect of myelin formation, has been intensively studied from the morphological to the molecular level. In this system, genetics has been successfully applied to analyze a corresponding set of membrane proteins which help to elaborate a defined structural entity, compact myelin. Shiverer, jimpy, Trembler, and protein zero (P0)‐deficient mice demonstrate the overall function of myelination and have become models for human neurological diseases. They also illustrate some of the problems encountered in defining protein functions from complex mutant phenotypes. © 1994 Wiley‐Liss, Inc.
Fluid‐phase endocytosis of horseradish peroxidase by cerebral endothelial cells in primary culture: Characterization and kinetic analysis
Authors: Noble L. J. , Kalinyak J. E. , Pitts L. H. , Hall J. J.
Published: January 2005

Endocytosis of horseradish peroxidase (HRP) was studied in primary cultures of cerebral endothelial cells prepared from 2‐week‐old rats. These cultures were considered “endothelial‐like” on the basis of their ability to internalize acetylated low density lipoprotein. Cellular localization of HRP protein was examined at the light and ultrastructural levels and endocytosis of the protein was evaluated by a colorimetric assay. HRP was localized in discrete cytoplasmic granules by light microscopy. At the ultrastructural level these granules corresponded to pleomorphic membrane‐bound structures that were present throughout the cytoplam. The amount of internalized HRP was directly related to the concentration of the protein in the medium, was not saturable at high concentrations of HRP, and increased with time. Endocytosis proceeded at 37°C, but was abolished at 4°C. In pulse‐chase experiments, the quantity of internalized protein in the cells did not significantly change during the 2 hr chase period. Taken together, these findings suggest that internalization of HRP occurs by fluid‐phase endocytosis, a non‐receptor‐mediated process, and that the protein is stable within an intracellular compartment for at least several hours. © 1994 Wiley‐Liss, Inc.
Presence of axonal paried helical filament‐tau in Alzheimer's disease: Submicroscopic localization
Authors: Bondareff W. , Harrington C. R. , McDaniel S. W. , Wischik C. M. , Roth M.
Published: January 2005

While normal adult tau protein is found typically in axons, paired helical filament (PHF) tau has been shown immunohistochemically to be in the somatodendritic compartment in Alzheimer's disease (AD). The small amount of PHF‐tau (8‐‐11%) found in white matter with immunochemical methods is shown here by immunoelectron microscopy to be located in axons. It is localized to straight filament variants of PHFs. The small amount of PHF‐tau in white matter, therefore, appears not to result from the contamination of immunochemical preparations but to be an integral constituent of affected axons in AD. © 1994 Wiley‐Liss, Inc.
Solubilization of full‐length amyloid precursor proteins from PC12 cell membranes
Authors: Ripellino J. A. , Vassilacopoulou D. , Robakis N. K.
Published: January 2005

The amyloid β protein (Aβ) of Alzheimer disease (AD) is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane proteins. Production of Aβ from a tranmembrane precursor predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Here we show that incubation of a membrane fraction of PC12 cells at 37°C results in the solubilization of an APP species which migrates on SDS‐PAGE as full‐length APP. The release of this full‐length APP was pH‐dependent with a peak of activity of pH 9.0. At this pH about 19% of the membrane APP was released from the active subcellular fraction. Under the same conditions other transmembrane proteins remained insoluble. Very little APP was solubilized at 4°C. APP solubilization was specifically inhibited by the serine protease inhibitors aprotinin and pefabloc. Other protease inhibitors, including leupeptin and α1‐antitrypsin, had no effect. Several metal cations, including Ca+ + and Zn+ +, also inhibited release of soluble full‐length APP. Low levels of full‐length APP were also detected in both the soluble fraction of PC12 cell extracts and in the media of PC12 cell cultures. These data suggest the involvement of serine protease in the solubilization of membrane, full‐length APP. The release of this APP could provide a soluble substrate for the proteolytic enzymes involved in the production of Aβ.Copyright © 1994 Wiley‐Liss, Inc.
Neurotrophin‐3 enhances neurite outgrowth in cultured hippocampal pyramidal neurons
Authors: Morfini G. , Ditella M. C. , Feiguin F. , Carri N. , Cáceres A.
Published: January 2005

Low density dissociated cultures of embryonic rat hippocampal cells were used to study the effects of neurotrophin‐3 (NT‐3) on neuronal morphogenesis. The results obtained indicate that NT‐3 enhances neurite outgrowth and branching; this is a dose‐dependent effect, detected in approximately 50% of the neurons, and prevented by K‐252a, an inhibitor of the trk family of receptor protein kinases. NT‐3 also accelerates the development of neuronal polarity, a phenomenon preceded by a dramatic accumulation of bundles of looped microtubules within axonal growth cones; these microtubule bundles contain tyrosinated, detyrosinated, and acetylated α‐tubulin. Taken collectively, our data suggest that even though the basic shape of hippocampal neurons may be endogenously determined, critical aspects of their morphological development may be modulated by trophic factors such as NT‐3. In addition, our observations suggest that at least some of the neuritogenic effects of NT‐3 involve a stimulation of microtubule assembly and/or transport. Copyright © 1994 Wiley‐Liss, Inc.
Differentiation‐specific demethylation of myelin associated glycoprotein gene in cultured oligodendrocytes
Authors: Grubinska B. , Laszkiewicz I. , Royland J. , Wiggins R. C. , Konat G. W.
Published: January 2005

The methylation status of a 4.4‐kb 5′ end of the myelin‐associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O‐2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation‐sensitive restriction enzymes, and the cuts were mapped by the indirect end‐labeling technique. The restriction sites within the 4.4‐kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at −1836 and at −39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear‐cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the −1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin.

The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure. © 1994 Wiley‐Liss, Inc.
Proliferation and differentiation of fetal human oligodendrocytes in culture
Authors: Satoh J. , Kim S. U.
Published: January 2005

Phenotypic expression and proliferative capacity of the cells of oligodendrocyte lineage were investigated in primary cultures isolated from fetal human brains of 12–15 weeks' gestation using double immunolabeling with Ranscht‐monoclonal antibody (R‐mAb) or O4 and antibromodeoxyuridine (BrdU) antibody. Cultured cells of oligodendrocyte lineage consisted of a major population of R‐mAb+O4− cells and minor populations of R‐mAb−O4+ and R‐mAb+O4+ cells. Most of the R‐mAb+O4− cells exhibited a uni‐, bi‐, or tripolar immature morphology, while the majority of the R‐mAb+O4+ cells exhibited a multipolar mature morphology. R‐mAb−O4+ cells contained a mixture of immature and mature cell types. When incubated in serum‐free culture medium containing BrdU for 4 days, 42% of total oligodendrocytes expressed nuclear BrdU immunolabeling. R‐mAb+ cells exhibited a higher degree of BrdU immunolabeling, indicating that they have greater capacities for proliferation than O4+ cells. The large majority of BrdU+ cells exhibited an immature morphology. Inclusion of insulin, insulin‐like growth factor (IGF)‐I, basic fibroblast growth factor (bFGF), or fetal bovine serum in culture medium did not stimulate proliferation of oligodendrocytes, while platelet‐derived growth factor (PDGF) of PDGF plus bFGF increased the number of R‐mAb+BrdU+ and O4+BrdU+ cells over control, even though the results were not statistically significant. In addition, insulin and IGF‐I induced a 3‐fold increase in the number of R‐mAb+O4+ cells, indicating that they promoted differentiation of oligodendrocytes. The present study indicates that fetal human oligodendrocytes in culture exhibit a considerable degree of proliferative capacity without requirement of exogenous growth factors and that both insulin and IGF‐I promote their differentiation. © 1994 Wiley‐Liss, Inc.
Prolonged alkylcatechol‐induced expression of c‐jun proto‐oncogene followed by elevation of NGF mRNA in cultured astroglial cells
Authors: Omae F. , Katsumata T. , Sakuma M. , Furukawa Y. , Furukawa S.
Published: January 2005

We have already shown that alkylcatechol markedly enhances synthesis/secretion of nerve growth factor (NGF) in cultured mouse fibroblasts and astroglial cells through immediate accumulation of NGF mRNA and that the stimulatory effect of alkylcatechol on NGF synthesis/secretion is synergistically enhanced by the coadministration of phorbol 12‐myristate 13‐acetate (PMA). The stimulatory effect on NGF mRNA expression of astroglial cells in culture by 4‐methylcatechol (MC), an alkylcatechol, and/or PMA was blocked by treatment of the cells with cycloheximide, suggesting de novo synthesis of some cellular protein(s) is essential for the observed increase in the NGF mRNA level. The exposure to MC and/or PMA caused a rapid increase in c‐fos mRNA content, which was immediately followed by an increase in c‐jun mRNA, prior to NGF mRNA elevation. The expression of c‐fos mRNA was transiently enhanced in all cases of the treatment with MC and/or PMA. The c‐jun mRNA expression was also observed transiently when the cells were treated with PMA alone, while the expression of c‐jun mRNA was pronounced and long‐lasting after the treatment with MC, which was much further enhanced by the coadministration of PMA. The result that the profile of the change in c‐jun mRNA expression resembled that in NGF mRNA expression suggests that the increase in c‐jun mRNA is responsible for the subsequent increase in NGF mRNA after MC treatment. The cotransfection of mouse astroglial cells with expression plasmids of c‐fos and/or c‐jun and NGF promoter gene showed that simultaneous expression of both c‐fos and c‐jun genes was necessary to enhance NGF promoter activity. These results suggest that alkylcatechol induces NGF mRNA by means of transient induction of c‐fos mRNA and long‐lasting induction of c‐jun mRNA. © 1994 Wiley‐Liss, Inc.
Regional CNS uptake of blood‐borne nerve growth factor
Authors: Loy R. , Taglialatela G. , Angelucci L. , Heyer D. , Perez‐Polo R.
Published: January 2005

Nerve growth factor (NGF), in addition to being a neurotrophic substance, has effects on the endocrine and immune systems. For example, intravenous injection of NGF results in a cascade of events leading to an increase in glucocorticoid secretion. While this response appears to be mediated centrally, there has been no evidence that circulating NGF has access to the CNS. Using intravenous injections of 125I‐NGF, we find specific uptake at 1 hr but none at 6 hr, into homogenates of the basal forebrain, cerebellum, frontal cortex, hippocampus, and olfactory bulb. By autoradiography, uptake is localized to circumventricular organs, deep layers of the cerebellum, and all layers of the hippocampal region CA1, but not the dentate gyrus. Thus, uptake of blood‐borne NGF could affect the hypothalamic‐pituitary‐adrenal axis via binding to NGF receptors present in the hippocampus. However, the sources of endogenous NGF, the mechanism of access through the blood‐brain barrier, the eventual fate of NGF entering from the blood, and the physiological significance of this uptake remain to be elucidated. © 1994 Wiley‐Liss, Inc.
Calpain inhibitors block Ca2+‐induced suppression of neurite outgrowth in isolated hippocampal pyramidal neurons
Authors: Song D.‐K. , Malmstrom T. , Kater S. B. , Mykles D. L.
Published: January 2005

Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca2+‐dependent effects is nuclear. Since cytoskeletal proteins are rapidly degraded by Ca2+‐dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca2+‐induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal‐like neurons were cultured and the ability of the membrane‐permeable calpain inhibitors ethyl(+)‐(2S,3S)‐3‐[(S)‐methyl‐1‐(3‐methylbutylcarbamoyl)‐butylcarbamoyl]‐2‐ oxiranecarboxylate (EST) and carbobenzoxyl‐valyl‐phenylalanyl‐H (MDL 28170) to block the Ca2+ ionophore A23187‐induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 m̈M) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 m̈M) showed the same effect as EST in preventing ionophore‐induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 m̈M) did not block the A23187‐induced rise of [Ca2+]i as measured with fura‐2. These results suggest that calpains play a role in Ca2+‐induced pruning of neurites in isolated hippocampal pyramidal neurons. © 1994 Wiley‐Liss, Inc.
Tau‐like immunoreactivity in Alzheimer and control skin fibroblasts
Authors: Matsuyama S. S. , Bondareff W.
Published: January 2005

The presence of the microtubule‐associated protein tau in skin fibroblasts derived from Alzheimer patients and normal controls was investigated using a panel of well‐characterized anti‐tau antibodies against epitopes spanning the tau protein from the amino to the carboxyl end. The antibodies immunolabeled a fine, fibrillar cytoplasmic network in all skin fibroblasts. Disruption of the microtubule network with colchicine did not affect the immunolabeling of the fibrillar network nor did treatment with cytochalasin B known to disrupt the microfilament network. Immunoelectron microscopy with the antitau antibodies revealed colocalization of the label with the 10 nm intermediate filaments. Furthermore, immunoblots found no reactivity against purified vimentin, suggesting that the antibodies recognize an intermediate filament‐associated protein. The findings indicate the presence of tau or a protein with considerable homology to tau in fibroblasts associated with intermediate filaments and not microtubules. © 1994 Wiley‐Liss, Inc.
Retardation of neuritic outgrowth and cytoskeletal changes accompany acetylcholinesterase inhibitor treatment in cultured rat dorsal root ganglion neurons
Authors: Dupree J. L. , Bigbee J. W.
Published: January 2005

Over the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non‐cholinergic and non‐cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non‐cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum MatrigelTM and the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE‐specific inhibitor 1,5‐bis‐(4‐allyldimethylammoniumphenyl) pentan‐3‐one dibromide (BW284c51) ranging from 10−4 to 10−7 M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on MatrigelTM at 10−5 and 10−4 M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10−6, 10−5, and 10−4 M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51‐treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseaes such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body. These data further suggest a novel, non‐cholinergic role for AChE in neural development and regeneration. © 1994 Wiley‐Liss, Inc.
Dopamine and serotonin turnover rate in the retina of rabbit, rat, goldfish, and Eugerres plumieri: Light effects in goldfish and rat
Authors: Lima L. , Urbina M.
Published: January 2005

The concentration of dopamine, and its metabolites 3,4‐dihydroxyphenylacetic and homovanillic acids, as well as serotonin and its metabolite 5‐hydroxyindoleacetic acid, were determined in the retina of two teleosts, C. auratus (goldfish) and E. plumieri (mojarra), and two mammals, R. norvegicus (rat) and O. cuniculus (rabbit). The turnover rate of these monoamines were investigated in the four species by the calculation of the ratio monoamine/metabolite as an indirect index, and in goldfish and rat by the inhibition of the synthesis with α‐methyl‐p‐tyrosine or p‐chlorophenylalanine, by the increase in dopamine or serotonin by the corresponding precursors, 3,4‐dihydroxyphenylalanine or 5‐hydroxytryptophan, and by inhibition of monoaminooxidase with pargyline. The modulation by light and dark stimulation was studied in the goldfish and the rat. Differences in the concentration and turnover rate were observed among the species. Serotonin concentration was higher in the teleosts. The administration of inhibitors of dopamine and serotonin synthesis differentially decreased the levels of the monoamines in the retina of goldfish and rat. The rate of formation of dopamine and serotonin by the corresponding precursors was much higher in the goldfish than in the rat. Pargyline administration decreased 3,4‐dihydroxyphenylacetic and 5‐hydroxyindoleacetic acids at different rates and time dependency in the retina of goldfish and rat. Dopamine and serotonin concentration did not exhibit high modifications by the inhibitor, suggesting the function of regulatory mechanisms or additional effect of pargyline at other sites different from monoaminooxidase. Light stimulation decreased the ratios of dopamine and its metabolites in the retina of goldfish, but not in the rat, probably by a greater release of dopamine with lower uptake capacity in the latter. Goldfish or rat retinal serotonin did not change by light exposure. Homovanillic acid was not detected in either type of retina after the dark period. In the goldfish, there was an increase in dopamine/3,4‐dihydroxyphenylacetic acid ratio by the darkness; in the rat, this ratio decreased, which might be related to the release of dopamine. Retinal serotonin turnover rate was accelerated in the goldfish by dark stimulation. The present comparative results support a differential regulation of monoamine levels among species, and will be useful for further characterization of the regulatory mechanisms related to light and dark exposure. © 1994 Wiley‐Liss, Inc.