bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, culture, and characterization of adult rat oligodendrocytes
Authors: Vick R. S. , Chen S.‐J. , DeVries G. H.
Published: January 2005

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSAZ). The pellet was resuspended and placed in an untreated 6‐well culture dish overnight to allowthe astrocytes to attach. The non‐adherent cells were replated on poly‐l‐lysine‐treated coverslips. Approximately 8.25 × 105 cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2–5 days in vitro (DIV), the oligodendrocytes extended 6–10 thick processes. A membrance sheath between these processes was immunostainable with either anti‐galactocerebroside (GC), anti‐04, anti‐myelin basic protein (MBP), or anti‐2′3′ cyclic nucleotide 3′ phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up nto 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 AS with up to 11 concentric layers of membrance. Immunologically, the adult oligodendrocytes are GC+, 04+, MBP+, CNPase+, and GFAP−. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.
Method for isolation of kappa‐opioid binding sites by dynorphin affinity chromatography
Authors: Simon J. , Benyhe S. , Hepp J. , Varga E. , Medzihradszky K. , Borsodi A. , Wollemann M.
Published: January 2005

A kappa‐opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1–10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600‐fold purification over the membrane‐bound receptor) with a Kd of 9.1 nM. The addition of cholesterol‐phosphatidylethanolamine (2:1) enhanced 3.6‐fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa‐selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.
Conditioned medium from activated splenocytes increases substance P in sympathetic ganglia
Authors: Jonakait G. Miller , Schotland S.
Published: January 2005

Following removal of the presynaptic input to the superior cervical ganglion (SCG) of the neonatal rat, there is an increase in substance P (Kessler et al.:Science 214:335–336, 1981; Kessler and Black: Brain Res 234:182–187, 1982) and the mRNA coding for its prohormone precursor (Roach et al.: Proc Natl Acad Sci USA 84:5078–5081, 1987). However, the functional significance of this increase has been unclear. We report here that SP increases dramatically in cultures of SCG grown in the presence of conditioned medium from con‐A‐stimulated splenocytes. The effect is mimicked by growing SCG explants in the presence of human recombinant interleukin‐1 (hrIL‐1) but not hrIL‐2. Nerve growth factor (NGF) is not involved in mediating this effect since antibodies to NGF included in the culture fail to alter the lympho‐kine‐induced increase in SP. Moreover, the effect is somewhat specific for SP since the activities of tyrosine hydroxylase, tryptophan hydroxylase, and choline acetyltransferase (enzymes in the biosynthetic pathways for norepinephrine, serotonin, and acetylcholine) are not similarly elevated. Dorsal root ganglia respond with only modest increases in SP. The action of lymphokines in stimulating SP may, therefore, be a ganglion‐specific action in promoting recovery following injury.
Method for the identification of brain macrophages/phagocytic cells in vitro
Authors: Jordan F. L. , Wynder H. J. , Booth P. L. , Thomas W. E.
Published: January 2005

The use of labelled latex beads, with acute incubation conditions, for the identification of active macrophages in mixed cell cultures based on their phagocytic activity is described. Fluorescent beads provided the best results and selectively labelled active macrophage cell in cultures of blood monocytes. When this technique was applied to primary cultures of rat cerebral cortex, a specific cell type was significantly labelled. This was a small round cell, previously uncharacterized, which in addition to phagocytic activity indicated by the ingestion of beads also possessed macrophage biochemical markers. Thus, the procedure appears useful for phagocytic macrophage identification in vitro, and should be generally applicable to any tissue culture system. Additionally, this procedure can be rendered compatible with cell viability and, therefore, be utilized for long‐term monitoring of macrophages.
Axonal modulation of myelin gene expression in the peripheral nerve
Authors: LeBlanc A. C. , Poduslo J. F.
Published: January 2005

Myelin gene expre(P0, MBP, P2, and MAG) was investigated during Wallerian degeneration and in the presence or absence of subsequent axonal regeneration and remyelination. The steady state levels of mRNA and protein were assessed in the crushed or permanently transected rat sciatic nerve at 0, 1, 4, 7, 10, 12, 14, 21, and 35 days after injury. The mRNA and protein steady state levels of the myelin specific genes, P0 and the MBPs, decreased to low yet detectable levels during Wallerian degeneration and returned to normal levels with subsequent axonal regeneration. The steady state level of P2 protein also followed a similar pattern of expression. The steady state level of MAG mRNA decreased to unddectable levels by 4 days of injury in the permanently transected nerve. After crush injury, re‐expression of MAG to levels comparable to those of normal nerves preceded that of P2 by 2 days and that of P0 and the MBPs by 3 weeks during axonal regeneration and remyelination. These results support the proposed roles for MAG in the formation of initial Schwann cell‐axonal contact required for myelin assembly, for P2 in fatty acid transport during myelination, and for P0 and the MBPs in the maintenance of the integrity and compactness of the myelin sheath. In addition, these results indicate that the expression of the myelin specific genes, P0 and MBP, is constitutive and that the level of myelin specific mRNAs is modulated by axonal contact and myelin assembly.
Gene transfer into brain tumor cell lines: Reporter gene expression using various cellular and viral promoters
Authors: Oellig C. , Seliger B.
Published: January 2005

In the present study we determined the optimal conditions for transferring DNA into rat and human brain tumor cell lines of glial and neuronal origin using electroporation as the transfection method. Gene transfer efficiency was measured in terms of transient chloramphenicol acetyltransferase (CAT) activity and stable neomycin expression. Moreover, the activity of a variety of cellular and viral promoters in brain tumor cell lines of distinct origin was characterized. The results revealed various expression patterns, including glial as well as neuronal specific promoter activity.
Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo
Authors: Kidd G. J. , Hauer P. E. , Trapp B. D.
Published: January 2005

Expression of myelin protein genes by myelinating Schwann cells in vivo is dependent on axonal influences. This report investigated the effect of axons on myelin protein mRNA levels in the central nervous system (CNS). In situ hybridization studies of rat spinal cord sections localized mRNAs encoding proteolipid protein (PLP) and myelin basic protein (MBP) 20 and 40 days after unilateral rhizotomy. Compared with control tissue, hybridization intensity was reduced in transected tissue, but there was little change in the number of oligodendrocytes labeled. Cellular RNA was extracted from transected and age‐matched control optic nerves 5, 10, 20, and 40 days after surgery, and levels of the following mRNAs were determined by slot blot procedures: PLP, MBP, myelin‐associated glycoprotein (MAG), and 2′,3′ cyclic nucleotide 3′‐phosphodiesterase (CNP). In transected nerves, PLP and MBP mRNA levels were approximately 85%, 45%, and 25% of control values at 5, 20 and 40 days posttransection, respectively. Axonal transection had a lesser effect on CNP and MAG mRNA levels, which declined to approximately 60% of control levels at 40 days. Immunocytochemical studies indicated that the number of oligodendrocytes was not decreased 40 days after optic nerve transection. These data demonstrate that axons modulate myelin protein mRNA levels in oligodendrocytes. In contrast to Schwann cells, however, oligodendrocytes continue to express significant levels of myelin protein mRNA in vivo following loss of axonal contact.
Effects of short‐ and long‐term ganglioside treatment on the recovery of neurochemical markers in the ibotenic acid‐lesioned rat striatum
Authors: Contestabile A. , Virgili M. , Migani P. , Barnabei O.
Published: January 2005

The striatum of adult rats was bilaterally lesioned with stereotaxic injections of ibotenic acid in a dose (16 nmoles) that resulted in subtotal lesions. Some rats received systemic ganglioside treatment starting the day before operation and lasting for 6 or 24 days after operation; they were compared with lesioned rat receiving systemic saline injections as well as with corresponding groups of sham‐operated animals. Specific neurochemical markers for cholinergic neurons (choline acetyltransferase, ChAT), GABAergic neurons (glutamate decarboxylase; GAD), and astrocytes (glutamine synthetase; GS) were assayed to asses the neurochemical recovery promoted by ganglioside treatment. Twenty‐four, but not six, days after operation a significant increase of ChAT and GAD was measured in the striatum of lesioned rats treated with gangliosides in comparison with the saline group. Furthermore, a significant increase of both enzymes occurred in the striatum of lesioned rats receiving ganglioside treatment for 24 days in comparison with rats receiving gangliosde treatment for 6 days only. A small but significant increase of ChAT was measured in the striatum of sham‐operated rats after 24 days of ganglioside treatment in comparison with the corresponding saline group. Finally, the increase of GS caused by the glial reaction to the ibotenic acid lesion was not affected by ganglioside treatment. The results indicate that a relatively long‐lasting ganglioside treatment stimulates the recovery of specific neuronal transmitter markers and that some effect is, in addition, exerted on unlesioned cholinergic striatal neurons.
Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system
Authors: Ernfors P. , Lönnerberg P. , Ayer‐LeLievre C. , Persson H.
Published: January 2005

A cDNA clone encoding rat basic fibroblast growth factor (bFGF) was used as a hybridization probe to determine the level of bFGF mRNA during rat brain development as well as in different adult rat brain regions. In the rat brain, a 3.7kb bFGF RUNA was detected together with lower levels of two minor bFGF mRNA species of 1.8kb and 1.5kb, respectively. The 3.7kb bFGF mRNA was detected in the rat brain already at embryonic day 16, the earliest time point tested. The embryonic brain contained 1.5 to 2 times higher levels of the 3.7kb bFGF mRNA than the adult brain. The amount of the 3.7kb bFGF mRNA in the adult rat brain was approximately 50 times higher than the level of β‐nerve growth factor mRNA in the rat brain. bFGF mRNA was found in all 12 brain regions tested in the adult rat brain with the highest level in colliculi, cerebral cortex, thalamus, and olfactory bulb. The lowest levels were found in pons and medulla oblongata. All three bFGF mRNA species showed the same regional distribution in the brain. In contrast to nerve growth factor mRNA, the level of bFGF mRNA in the neonatal hippocampus was slightly decreased 10 days after a cholinergic denervation by transection of the fimbria‐fornix.
Basic fibroblast growth factor in neuronal cultures of human fetal brain
Authors: Torelli S. , Dell'Era P. , Ennas M. G. , Sogos V. , Gremo F. , Ragnotti G. , Presta M.
Published: January 2005

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week‐old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA‐stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross‐reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell‐associated protein experiments. All the neurons showed bFGF immunoreactivity as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.
Damage to bulbospinal serotonin‐, tyrosine hydroxylase‐, and TRH‐containing axons occurs early in the development of experimental allergic encephalomyelitis in rats
Authors: White S. R. , Samathanam G. K. , Bowker R. M. , Wessendorf M. W.
Published: January 2005

Spinal cord monoaminergic and peptidergic axonal damage occurring during the development of experimental allergic encephalomyelitis (EAE) was assessed using immunohistochemistry. Spinal cord axons immunoreactive for serotonin, catecholamines, or a thyrotropin‐releasing hormone marker peptide were found to be markedly swollen and distorted by the earliest stage of detectable paralysis during EAE development (the flaccid tail stage). As clinical signs progressed to complete hindlimb paralysis, axonal damage became increasingly extensive. Axonal damage was equally pronounced whether EAE was induced by inoculation with purified myelin basic protein or with whole spinal cord homogenate, suggesting that the damage did not result from an immune attack directed against specific monoaminergic and/or peptidergic antigens present in the inoculant. However, two observations suggested that mechanical or chemical factors associated with the inflammatory foci contribute to the axonal damage: first, distorted axons were nearly always located adjacent to blood vessels or the pial surface, sites at which inflammation occurs during EAE. Second, the severity of axonal damage correlated with the severity of the inflammation. The early onset of axonal damage during development of EAE and the close correlation that was found between the severity of axonal damage and the severity of clinical signs suggested that the axonal damage may contribute to the clinical signs of the disease.
Changes in PC12 cell morphology induced by transfection with 42C cDNA, coding for a member of the S‐100 protein family
Authors: Masiakowski P. , Shooter E. M.
Published: January 2005

The cloned DNA coding for 42C protein (light chain of calpactin I), whose mRNA is induced in PC12 cells by treatment with nerve growth factor (NGF), was reintroduced into these cells. A cell line was obtained in which the outgrowth of processes in the absence of added NGF, similar to that induced in the parental PC12 cells by the factor, was accompanied by high levels of 42C RNA. The apparent reason for this constitutive overexpression of 42C is the stable integration of multiple copies of the 42C DNA into the cell genome. These results further support the notion that S‐100 protein family, of which 42C is a member, may play an important role in development.
Immunohistochemical studies on cross‐transplantations between jimpy, shiverer, and normal newborn mice
Authors: Lachapelle F. , Lapie P. , Nussbaum J. L. , Gumpel M.
Published: January 2005

Cross‐transplantations of neural tissue have been performed between jimpy (jp), shiverer (shi), and normal mice. Taking advantage of the absence of immunodetectable myelin basic protein (MBP) in the shi brain, jp myelin has been identified in the shi recipient by using an anti‐MBP antiserum. By contrast, shi as well as normal myelin have been identified in the jp brain by using an anti‐C‐terminal hexapeptide of the proteolipid protein (PLP) (this PLP hexapeptide being absent in the jp PLP). When transplanted under homochronic conditions (newborn into newborn), jp oligodendrocytes (ODC) express their usual phenotype in a normal or a shi environment, suggesting that at birth the jp ODCs phenotype is strictly established and cannot be modified by environmental conditions. The reverse transplantations (newborn shi or normal into newborn jp brain) demonstrate that the jp environment does not modify the phenotype of normal or shi ODCs. Finally, these experiments demonstrated a normal timing of differentiation of jp axons and of jp ODCs.
Nicotinic acetylcholine receptor subtypes in human frontal cortex: Changes in Alzheimer's disease
Authors: Sugaya K. , Giacobini E. , Chiappinelli V. A.
Published: January 2005

Molecular genetic and pharmacological studies have suggested that several subtypes of nicotinic acetylcholine receptors exist in the mammalian and avian brain. Combining 3H‐(−)‐nicotine, 125 I‐α‐bungarotoxin, and 125I‐κ‐bungarotoxin as ligands, we report here the first evidence for the existence in human frontal cortex of at least three different subtypes of nicotinic receptors. Autoradiographic analysis shows that specific 125I‐κ‐bungarotoxin binding sites are concentrated mainly in several cortical layers. We also show that κ‐bungarotoxin, but not κ‐bungarotoxin decreases the evoked release of 3H‐acetylcholine in rat cortical slices, indicating a likely presynaptic localization for some of the α‐bungarotoxin‐insensitive κ‐bungarotoxin sites in mammalian brain. The brains of patients with Alzheimer's disease show marked decreases in Bmax values for low‐affinity 125I‐κ‐bungarotoxin sites and both high‐ and low‐affinity 3H‐nicotine sites, whereas 125I‐κ‐bungarotoxin sites are not significantly different in number from age‐matched control brains. We conclude that Alzheimer's disease does not affect all subtypes of nicotinic receptors in the frontal cortex to the same extent.
Characterization of functional nerve growth factor‐receptors in a CNS glial cell line: Monoclonal antibody 217c recognizes the nerve growth factor‐receptor on C6 glioma cells
Authors: Kumar S. , Huber J. , Peña L. A. , Perez‐Polo J. R. , Werrbach‐Perez K. , de Vellis Jean
Published: January 2005

The biological effects of nerve growth factor (NGF) have been shown to be mediated by the high‐affinity form of the nerve growth factor receptor (NGF‐R) in sympathetic and sensory neurons, and in PC12 cells. We report here that the central nervous system C6 rat glioma cell line likewise expresses functional high‐affinity NGF‐Rs. The expression of NGF‐R mRNA in C6 cells can be up‐regulated by cycloheximide and its own ligand, NGF; and it can be rapidly down‐regulated by epidermal growth factor (EGF). Furthermore, C6 cells display NGF responsiveness by expressing c‐fos mRNA within 30 minutes of treatment with NGF; and after 4—5 days of NGF exposure, C6 cells cease dividing as measured by [3H]‐thymidine uptake, change shape, and reveal neurite‐like ‐processes. Scatchard analysis of [125 I]‐labelled bound to solubilized C6 cells confirms the presence of both high‐ and low‐affinity receptor protein. Cross‐linking radiolabeled NGF to its receptor in the presence or absence of excess unlabeled NGF, followed by immunoprecipitation with monoclonal antibody (mAb) 192‐IgG (a known anti‐NGF‐R antibody) and SDS‐PAGE reveals a 100 kD band corresponding to the NGF/NGF‐R complex. An identical band is observed when the immunoprecipitation is carried out with mAb 217c, suggesting that the 217c epitope is related to NGF‐R. The 217c antibody was generated against C6 cells and shown to be a cell surface antibody (Peng et al., Science 215:1102–4, 1982); several investigators have used it subsequently as an immunocytochemical marker for Schwann cells. The significance of NGF‐Rs in a CNS glial cell line is unclear, but association of NGF with the control of proliferation and/or differentiation of primitive glial cells is suggested.
Effect of gender on glucose utilization rates in healthy humans: A positron emission tomography study
Authors: Miura S. A. , Schapiro M. B. , Grady C. L. , Kumar A. , Salerno J. A. , Kozachuk W. E. , Wagner E. , Rapoport S. I. , Horwitz B.
Published: January 2005

Positron emission tomography (PET) was used with 18fluorodeoxyglucose to see if gender differences in resting cerebral glucose utilization could be detected. Thirty‐two healthy subjects (15 women and 17 men; age range: 21–38 yr) were examined using a high‐resolution PET scanner to determine the regional cerebral metabolic rate for glucose (CMRglc) in 65 gray matter regions of interest. Whole brain CMRglc did not differ significantly between the two genders, nor did any of the regional CMRglc values. Only 1 of 65 ratios of regional‐to‐whole brain CMRglc differed significantly between men and women, which is consistent with chance. These results indicate that there are no differences in resting regional cerebral glucose utilization between young men and women.
Effects of light/dark‐ and dark‐rearing on synaptic morphology in the superior colliculus and visual cortex of the postnatal and adult rat
Authors: Bakkum B. W. , Benevento L. A. , Cohen R. S.
Published: January 2005

Serveral synaptic parameters, previously shown to undergo alterations with changes in the internal and external environment, were examined in the visual system of light/dark‐ and dark‐reared postnatal and adult rats. Animals were raised in either 14hr light/10 hr dark (Lt/Dk) or in total darkness (Dk). The specific synaptic parameters in the superficial layers of the superior colliculus (SC), visual cortex (laminae II/III and IV) (VC), and, as contro, the auditory cortex (laminae II/III and IV) (AC), examined during the postnatal period (i.e., postnatal days 7,14,21, and 28) and in the adult (i.e., day 56) included:(1) mean number of synapses, (2) mean synaptic length, (3) percentages of perforated postsynaptic densities, (4) percentages of asymmetric and symmetric synapses, (5) percentages of dendritic, spinous, anmd somatic synapses, and (6) percentages of synapses with positive, negative, or no curvature. Developmental patterns in rats rearded in normal lighting conditions were noted. Specifically, in the SC and VC of Lt/Dk animals, the number of synapses increased up to postnatal day in the mean number of synapses increased; no significant changes in the mean number of synapses between PND =28 and 56 were detected in any of the areas examined. Changes in synaptic length in the SC and VC were not observed during postnatal development or in the adult in any of the aforementioned brain areas. Low percentages of postsynaptic densities (PSDs) were found at all time points and in all brain areas during the postnatal period. Increases in perforated PSDs were seen at PND = 56 compared to PND = 28 in the VC. In the VC and AC, there was decrease in symmetric synapses with age. Asymmetric synapses were prevalent in all brain areas at PND = 28 AND 56. dendritic synapses predominated in the SC, while spinous synapses were the preponderant type in the VC and AC during postnatal development and in the adult, A decrease in the percentage of spinous synapses in the SC was observed at PND = 56 vs. PND = 28. A decrease in the percentage of negatively curved synapses with age and a trend toward a concomitant increase in the percentage of positively curved synapses were seen in all brain areas during development and in the adult. Quantitative analyses of the SC and VC tissues examined from all postnatal animals demonstrated no significant differences between Lt/Dk and Dk animals in all the synaptic parameters measured. On the other hand, similar analyses of adult synapses in the SC and VC showed a significantly smaller mean number of synapses in rats dark‐reared to adulthood as compared to those raised in normal lighting conditions. Dark‐rearing had no effect on synaptic number at any time point in the auditory cortex, indicating that this is a specific light effect. Therefore, the effects of visual deprivation on synaptic numbers occur during early adulthood and not during postnatal development.
Changes in glial fibrillary acidic protein mRNA expression after corticospinal axotomy in the adult hamster
Authors: Kost‐Mikucki S. A. , Oblinger M. M.
Published: January 2005

We examined changes in the expression of glial fibrillary acidic protein (GFAP) mRNA during Wallerian degeneration in the corticospinal system of the adult Golden hamster following axotomy. GFAP is the product of a type III intermediate filament (IF) gene that is expressed specifically in mature astrocytes. A well‐studied component of a complex response termed reactive astrogliosis that occurs after various types of CNS injury is the increased production of astrocytic processes filled with GFAP‐containing IFs. While increased expression of GFAP during reactive astrogliosis has been well established at the protein level, little is known about whether or not changes in GFAP mRNA levels occur after CNS injury. In the present study we used in situ hybridization methods, to examine, this issue. A 35S labeled mouse GFAP cDNA probe was used for in situ hybridizations of sections of the brain stem obtained 2, 7, and 14 days after unilateral transections of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed a dramatic increase in GFAP mRNA labeling associated with the degenerating corticospinal tract. GFAP mRNA levels were already dramatically increased in the injured corticospinal tract by 2 days post axotomy and remained elevated at 14 days. Interestingly, in addition to the robust increase in GFAP mRNA levels specifically associated with the degenerating tract, a diffuse increase in GFAP mRNA labeling was observed throughout the grey matter of the brain stem at 2 days post‐axotomy, but not after this time. Immunoblotting and immunocytochemical experiments verified that the increased GFAP mRNA levels in the degenerating corticospinal system were accompanied by an increased expression of, the protein. These results demonstrate that an increase in GFAP mRNA levels occurs during Wallerian degeneration in the CNS and suggest that increased expression of the GFAP gene is a major contributor to CNS scarring that results after direct traumatic injury.
Verapamil regulation of Na‐K pump levels in rat skeletal myotubes: Role of spontaneous activity and Na channels
Authors: Brodie C. , Sampson S. R.
Published: January 2005

The effects of low and high doses of verapamil on expression of Na‐K pump and Na channels in cultured rat skeletal muscle were studied and compared. Myotubes were treated for 3 Jays with either 20 or 100 μM verapamil, and measurements were made of transmembrane resting potential, spontaneous action potential freguency, and specific binding of [3 H]ouabain and [3 H]saxitoxin upregulated both Na‐K pumps and Na channels, whereas high verapamil down regulated the former and upregulated the latter. Changes in channel levels preceded those in pump levels. Spontaneous contractile activity could be observed during treatment with low but not high verapamil. Simultaneous treatment with verapamil and elevated external K+ reversed the effect of low verapamil on Na‐K pumps, was without effect on high verapamil‐induced changes in pump levels, and potentiated the effects of both concentrations of verapamil on Na channel levels. Scatchard analysis showed that verapamil caused changes in Bmax without altering Kd. The verapamil‐induced changes in both Na‐K pumps and Na channels were blocked by inhibition of protein and RNA synthesis. We conclude that the differences in effects on Na‐K pumps obtained with the two doses are due to the different effects on spontaneous activity and associated changes in intracellular Na concentration.
Endogenous opioid‐immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats
Authors: Akesson T. R. , Micevych P. E.
Published: January 2005

Estrogen stimulates expression of proenkephalin mRNA in neurons of the hypothalamic ventromedial nucleus, and evidence is accumulating that synaptic release of one of the peptide end products, met‐en‐kephalin, influences events that regulate reproductive behavior. To address the question of whether estrogen acts directly on neurons that synthesize met‐en‐kephalin or indirectly through a separate neuronal population, we combined estrogen autoradiography with endogenous opioid peptide (EOP) immunohistochemistry. In agreement with previous studies, the ventrolateral subdivision of the hypothalamic ventromedial nucleus was densely packed with EOP‐immunoreactive cells. In males, 48% of the estrogen‐concentrating cells of the ventrolateral subdivision of the hypothalamic ventromedial nucleus contained EOP, and, in females, 27% of the estrogen‐concentrating cells contained EOP. These findings indicate that estrogen acts directly on neurons that express EOP and suggest a mechanism that underlies sexually differentiated reproductive behavior.
Quantitative autoradiographic study of L‐glutamate binding sites in normal and atrophic human cerebellum
Authors: Hatziefthimiou A. , Mitsacos A. , Mitsaki E. , Plaitakis A. , Kouvelas E. D.
Published: January 2005

In the present work the distribution of L‐glutamate binding sites in the different layers of human cerebellum of normal individuals and of seven patients who died with olivopontocerebellar atrophy (OPCA) was examined with the technique of quantitative autora‐diography. Specific L‐[3H]glutamate binding was higher in the molecular than in the granule cell layer of normal cerebellar tissue. A significant decrease of L‐[3H]glutamate specific binding was observed in me molecular layer of ail OPCA tissues. In the granule cell layer L‐[3H]glumate binding was decreased only in two patients who suffered from late‐onset sporadic OPCA and in one patient who suffered from a form of OPCA inherited in a dominant manner.

Quisqualate‐sensitive binding sites were the most abundant binding sites in the molecular layer of normal cerebella, whereas N‐methyl‐D‐aspartic acid (NMDA)‐sensitive binding sites were the most abundant type in the granule cell layer. A significant decrease of quisqualate‐sensitive and an increase in NMDA‐sensitive binding sites were observed in the molecular layer of OPCA cerebellar tissues. No significant changes were observed in the granule cell layer of these tissues.
Expression and induction of intercellular adhesion molecules (ICAMs) and major histocompatibility complex (MHC) antigens on cultured murine oligodendrocytes and astrocytes
Authors: Satoh J. , Kim S. U. , Kastrukoff L. F. , Takei F.
Published: January 2005

Expression of intercellular adhesion molecule‐1 (ICAM‐1), ICAM‐2‐like molecule (Lgp55), and class I/II major histocompatibility complex (MHC) antigens (H‐2 and Ia) was investigated in cultures of mu‐rine Oligodendrocytes and astrocytes. Under unstim‐ulated conditions, low levels of ICAM‐1 expression were observed on astrocytes (<20%), but not on oligodendrocytes. Lgp55 was expressed intensely on oligodendrocytes (>90%) and to a lesser degree on astrocytes (>70%). A weak class I MHC (H‐2) im‐munoreactivity was identified on both Oligodendrocytes and astrocytes (50‐70%). Class II MHC (Ia) antigen was undetectable on both cell types.

After 48‐hr exposure to immune mediators that include interferon‐γ (IFN‐γ), 500 U/ml, and supernatant from concanavalin A (Con A)‐activated spleen cells, ICAM‐1 expression was markedly increased on astrocytes (>80%), but not on Oligodendrocytes. Lgp55 expression on both cell types was not altered. Induction of H‐2 antigen expression by immune mediators was quite high on both cell types (>95%), while Ia antigen induction was low on astrocytes (<50%) and did not occur on Oligodendrocytes. Cell type‐specific expression and induction of ICAMs and MHC antigens by immune mediators may play roles in lymphocyte‐glial cell interactions at the sites of inflammation in the central nervous system (CNS).
Xenopus Oocytes as immunological vectors to produce monoclonal antibodies to rat brain antigens
Authors: Matute C. , Tigyi G. J. , Miledi Ricardo
Published: January 2005

A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain‐derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA‐injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system.
Phosphorylation of brain (NA+,K+)‐ATPase alpha catalytic subimits in normal and epileptic cerebral cortex: II. Partial seizures in human epilepsy
Authors: Guillaume Daniel , Grisar T. , Delgado‐Escueta A. V. , Laschet J. , Bureau‐Heeren M.
Published: January 2005

We examined the activity and phosphorylation level of (Na+, K+)‐ATPase (E.C. 3.6.1.3) partially puri fied from normal and epileptic human cortices. Con trol patients (n = 11) were operated on for a non‐ epileptogenic deep brain lesion, while epileptic patients (n = 10) were operated on for temporal or frontal originating partial seizures, resistant to med ications or secondary to evolutive brain tumors. No differences in the specific activity of microsomal (Na+, K+)‐ATPase were observed between the two groups of patients. After partial purification of the enzyme followed by SDS‐polyacrylaniide, gel. electrophoresis, (Na+, K+)‐ATPase catalytic subunit had a decreased affinity for K+ in human epileptic cortex and lost its sensitivity to phenytoin dephosphorylation. Indirect evidence suggests that those abnormalities of (Na+, K+)‐ATPase in human epileptic cortex hold preferentially true for the alpha(–) enzymatic subunit. Those result indicate that, in human epileptic cortex, (Na+, K+) glial subtype is altered in its K+ regulation and phenytoin sensitivity and could be responsible for ictal transformation and seizure spread.
Modulation of retinal differentiation by oncogenes: Effect of the v‐src gene on expression of choline acetyltransferase and glutamine synthetase
Authors: Notter M. F. D. , del Cerro M. , Balduzzi P. C.
Published: January 2005

Expression of the protooncogene c‐src in chick neural retina is developmentally regulated and associated with neural differentiation. In the present study, chick neural retina (NR) cell cultures from 7 day embryos were exposed to the exogenous src oncogene, the c‐src counterpart, to establish the effect of expression of v‐src on specific retinal cellular differentiation. NR cells from 7 day chick embryos were placed in monolayer or rotation culture and infected with Rous sarcoma virus (RSV) containing a single transforming gene. Other cultures were infected with a transforming defective mutant of RSV which still possesses mitogenic activity for NR cells. While control cultures showed typical neuronal and Muller cell morphologies at the light and electron microscopic level, NR cells infected with RSV exhibited dramatic morphological alterations in monolayer culture and cell aggregates. However, the mutant src gene induced mitosis without accompanying transforming properties. When aggregate cultures were treated with hydrocortisone to induce glutamine synthetase (GS) expression in Muller cells, control cultures showed the typical immunofluorescence pattern of GS staining, while RSV infected cultures showed no GS fluorescence. Cultures infected with mutant RSV showed some staining for GS. In contrast, choline acetyltransferase activity was shown to increase in both monolayer and aggregate cultures of retinal cells following v‐src expression. These data indicate that the presence of excess v‐src in differentiating cultures of NR inhibits the expression of some neural specific enzymes and enhances the presence of other specific proteins. Moreover, continually growing cultures of oncogene‐altered retinal cells may be useful as models to study gene expression in development of the nervous system.
Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: Myeloperoxidase activity assay and histologic verification
Authors: Barone Frank C. , Hillegass L. M. , Price W. J. , White R. F. , Lee E. V. , Feuerstein G. Z. , Sarau H. M. , Clark R. K. , Griswold D. E.
Published: January 2005

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty‐four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory‐motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin‐and‐eosin‐stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non‐washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non‐perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham‐operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular‐adhering/ infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro‐inflammatory stimuli.
Oligodendrocytes and myelin sheaths in normal, quaking and shiverer brains are enriched in iron
Authors: Levine S. M.
Published: January 2005

Early studies employing iron histochemical techniques found a spatially restricted distribution of oli‐godendrocytes and myelin enriched in iron. In particular, Oligodendrocytes and myelin positively stained for iron were found in sparsely myelinated brain regions but not in densely myelinated tracts. Subsequent studies modified the iron histochemical technique and demonstrated that Oligodendrocytes and myelin were stained in densely myelinated brain regions but the staining occurred in patches rather than uniformly throughout densely myelinated tracts. This study further modified the iron histochemistry technique to establish that Oligodendrocytes and myelin are enriched in iron throughout densely myelinated tracts. This finding supports biochemical studies that detected high levels of iron in white matter (Hallgren and Sourander, J Neurochem 3:41–51, 1958) and myelin fractions of brain homogenates (Rajan et al., Life Sci 18:423–432, 1976). Thus, this histochemical study and earlier biochemical studies indicate that white matter is a major site of iron concentration within the brain. The present study also examined the distribution of iron in Oligodendrocytes and myelin from the dysmyelinating mutant mice quaking and shiverer. Results from these studies demonstrate that Oligodendrocytes and myelin are enriched in iron in both quaking and shiverer brains. An unexpected finding was an intense staining of oval structures within the oligodendrocyte cytoplasm. This result indicates a concentration of iron in these structures and may be important for understanding how high concentrations of iron are processed by Oligodendrocytes.
Cell adhesion properties of myelin‐associated glycoprotein in L cell fibroblasts
Authors: Afar D. E. H. , Marius R. M. , Salzer J. L. , Stanners C. P. , Braun P. E. , Bell J. C.
Published: January 2005

Myelin‐associated glycoprotein (MAG) is a cell surface molecule expressed by oligodendrocytes and Schwann cells. In order to determine whether MAG expression can confer adhesive properties to cells which normally do not aggregate in suspension, the cDNA encoding the long form of MAG (L‐MAG) was introduced into L cell fibroblasts by retroviral infection. Clonal L cell lines expressing MAG were then subjected to a cell aggregation assay. Our results indicate that L‐MAG can function as an intercellular adhesion molecule in a heterologous cell system. A critical threshold value of L‐MAG expression was required for cell aggregation to occur. The adhesive properties of these cells were specific to MAG, since monoclonal antibodies directed against its extracellular domain inhibited aggregation. Furthermore, the adhesion was found to be calcium‐ and temperature‐independent. Cell sorting experiments demonstrated that L‐MAG‐expressing cells bind in a heterotypic fashion to parental L cell fibroblasts. These results suggest that L‐MAG can function as a heterotypic cell adhesion molecule recognizing a cell surface mole‐cule(s) expressed by L cells.
Neuronal specific protein NP185 is enriched in nerve endings: Binding characteristics for clathrin light chains, synaptic vesicles, and synaptosomal plasma membrane
Authors: Su B. , Hanson V. , Perry D. , Puszkin Saul
Published: January 2005

The neuronal specific protein NP185, found associated with brain clathrin‐coated vesicles, formed a complex with unphosphorylated, but not with phos‐phorylated, clathrin light chains. The NP185–clathrin light chain complex was associated with casein kinase II activity, which, in the presence of polylysine, phosphorylated clathrin light chain b but not the NP185. The dissociation of this complex with 50% ethylene glycol pH 11.5 suggests that NP185binds to hydrophobic domains of clathrin light chains. When NP185 molecules were retained by monoclonal antibody‐linked Sepharose beads, they bound synaptic vesicles, decoated vesicles and synaptosomal plasma membrane. Immunohistochemistry on mouse cerebellar tissue sections using 8G8, a monoclonal antibody raised against NP185, showed neuronal specific labeling closely following synaptic distribution. In immunoblots, NP185 shares similar epitopes to those detected in another assembly polypeptide, AP‐180, an indication that both proteins are identical. It appears that NP185 plays a specific role in nerve ending functions through its ability to induce clathrin to polymerize into cages, its interaction with synaptic vesicles, with the plasma membrane and with clathrin coat components.
Actin‐depolymerizing factor (ADF) in the cerebellum of the developing rat: A quantitative and immunocytochemical study
Authors: Léna J. Y. , Bamburg J. R. , Rabié A. , Faivre‐Sarrailh C.
Published: January 2005

A specific antiserum against actin‐depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton‐soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.
NF‐L and peripherin immunoreactivities define distinct classes of rat sensory ganglion cells
Authors: Goldstein M. E. , House S. B. , Gainer H.
Published: January 2005

Double immunofluorescence studies using antibodies against NF‐L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin‐positive (NF‐L‐negative), and 48% were large and NF‐L‐positive (peripherin‐negative). About 6% were both peripherin‐ and NF‐L‐positive. All of the DRG neurons reacted with antibodies to NF‐M and nonphosphorylation‐dependent or phosphorylation‐independent antibodies to NF‐H. The neuropeptides were predominantly found in the peripherin‐positive small cell population. Eighty‐seven percent of the peripherin‐positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18–24% of the NF‐L‐positive large‐cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum‐free medium, stably maintained exclusively peripherin‐positive neurons, with about 5% of these containing coexistent NF‐L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture.