bluuebunny/crossref_metadata_2025 huggingface dataset:
Regional distribution of messenger RNAs in postmortem human brain
Authors: Wood T.L. , Frantz G.D. , Menkes J.H. , Tobin A.J.
Published: January 2005

The ability to isolate intact RNAs from postmortem human brain permits analysis of gene expression and may help uncover the nature of the molecular lesions in neurological diseases. Starting with poly(A) RNA from postmortem brain of neurologically normal patients, we have constructed two complementary DNA libraries in the plasmid vector pBR322. Each of these libraries contains 2–3 × 104 recombinants. One library represents RNA species from the cerebellar cortex, the other from the neostriatum.

Using differential colony hybridization, we identified more than 100 relatively abundant RNA species that appeared to be expressed in brain but not in liver. We then used 16 of these clones to analyze brain and liver RNAs by RNA blot hybridization. Thirteen of the 16 clones hybridized to RNAs of both liver and brain. One clone hybridized only to brain RNA, while seven hybridized to RNA species that were present at higher concentrations in brain than in liver. Eleven of the 16 clones hybridized to more than one species of RNA.

None of the RNA species examined by RNA blot hybridization was limited to a single brain region, though seven of the cDNA clones hybridized to RNAs that were present at different concentrations in different regions. We have also examined the regional distribution of the RNA encoding glutamic acid decarboxylase, which catalyzes the production of gamma‐aminobutyric acid (GABA). GAD RNA showed differential expression among brain regions and was not detectable in liver or kidney. Our data support a model of gene regulation that is based on cell identity, rather than regional specificity.
Growth patterns of primary cultures dissociated from 3‐day‐old chick embryos: Morphological and biochemical comparisons
Authors: Vernadakis A. , Sakellaridis N. , Mangoura D.
Published: January 2005

Cultures were prepared by dissociating 3‐day‐old whole chick embryos and plating the dispersed cells on poly‐L‐Iysine‐coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts bwtween neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell poopulation was characterized histochemically by the method of Karnovsky and Roots and biochemical by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase‐positive staining; however, with days in culture, several aggregates had no staining, and some positive‐stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.
Chloride‐dependent binding sites for L‐[3H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb
Authors: Quinn M.R. , Spraguer P.A.
Published: January 2005

Dendrodendritic synapses occur between granule cell dendrites and secondary dendrites of mitral cells within the olfactory bulb and are attainable in a subcellular fraction (DDS). Since the mitral cells are thought to utilize an excitatory amino acid as a neurotransmitter, we determined the pharmacologic specificity of Na+‐independent L‐[3H]glutamate binding to fresh membrances of DDS in 50 mM Tris‐HCI, pH 7.1. Binding of L‐glutamate to membrances of DDS was specific, CI− dependent, and saturable. Scatchard plots were analyzed by nonlinear regression analysee using the computer program LIGAND, and the data was best‐fitted to a one‐site model with KD of 0.56 ± 0.04 μM and an apparent Bmax of 48 ± 5 pmol/mg protein. Hill plots also indicated the presence of one site and no cooperativity (nH = 0.99 ± 0.03). However, the relative effictiveness of several compounds in inhibiting L‐glutamate binding to membranes of DDS clearly demonstrated the presence of more than one site. Electrophysiological studies suggest that 2‐amino‐4‐phosphonobutyrate (APB) is a potent antagonist of evoked responses elicited by simulation of mitral cell axons and that quisqualate is a potent agonist; both of these compounds were highly effective inhibitors of L‐glutamate binding to DDS membrances. APB displaced about 70% of tke sites labeled with 200 nM L‐glutamate with a K1 of 1.6 μM, whereas quisqualate inhibition of L‐glutamate binding yielded a line that was curvilinear in the Scatchard plot and was resolved into two sites of relatively high affinity (K1 values of 0.02 and 0.65 μM). Several other compounds, including N‐methyl‐D‐aspartate and kainate, were relatively ineffective inhibitors. Although a small amount of CI−‐independent L‐glutamate binding was demonstrable in the preparation, CI−‐dependent sites predominated and were stimulated by clacium. These results suggest an important role of L‐glutamate, or a closely related compound, at dendrodendritic synapses of the olfactory bulb.
Putative glutamate receptors in membranes obtained from heads of Drosophila melanogaster
Authors: Fiedler J.L. , Inestrosa N.C. , Bustos G.
Published: January 2005

The specific binding of L‐[3H]‐glutamic acid (GLU) was examined in membrance preparations derived from heads of Drosophila melanogaster. L‐[3H]‐GLU bound to the membrane preparation with biphasic in a reversible and saturable way. The specific binding was inhibited by Cl− and Ca2+ ions. Saturation analysis of the data fit a model of two independent binding sites with dissociation constants (KD) of 29 nM and 249 nM and corresponding binding site densities (Bmax) of 5.7 and 24.6 pmol/mg protein. A series of excitatory amino acid receptor agonists and antagonists were tested as inhibitors of L‐[3H]‐GLU specific binding. Some compounds almost totally abolished the specific binding, whereas others reduced the binding to 45% over the concentration range used. Among the first type of inhibitors, L‐GLU was the most potent, D‐aspartate (D‐ASP) and D‐GLU were 10 times less effective than L‐GLU, whereas quisqualic acid had the lowest potency. Among the second type of inhibitors, glutamate diethyl ester, D‐α‐amino adipate, and N‐methyl‐D‐aspartate showed the highest potency, and DL‐2‐amino‐4‐phosphonobutyrate was the less effective. Kainic acid and γ‐amino butyric acid were not able to modify at any concentration used the specific binding of L‐[3H]‐GLU. These data suggest the presence of putative GLU receptors in the brain of Drosophila.
Modifications of light responses in the superior colliculus following cortical cryoblockade
Authors: Molotchnikoff S. , Lamothe L. , Cérat A. , Lachapelle P.
Published: January 2005

Responses to light stimuli of cells in the superior colliculus (SC) were studied in anesthetized and paralyzed rabbits after reversible inactivation of corticocollicular impulses by cooling of the visual cortex. The recording of flash‐evoked responses and EEG allowed us to monitor the time course of the inactivation and its recovery. The cell's responses were measured prior to, during, and after cortical blockade. In about half of the collicular cells, disruption of cortical functions resulted in a sharp decline of collicular responsivity. All types of response were affected equally. Hence, the cortex has a global tonic excitatory impact.
Androgen and estrogen receptors in adult zebra finch brain
Authors: Siegel L.I. , Akutagawa E. , Fox T.O. , Konishi M. , Politch J.A.
Published: January 2005

Androgens and estrogens have been implicated in the activation of a variety of sexually‐dimorphic, hormone‐dependent behaviors in the adult zebra finch. In the present report, several biochemical characteristics of two putative sex steroid receptors, androgen‐ and estrogen‐binding activities, were determined by DNA‐cellulose chromatography in brain tissues from these birds. High‐affinity, limited‐capacity receptors for androgens and estrogens were found in cytosolic extracts of telencephalon, diencephalon‐mesencephalon, and metencephalon‐myelencephalon from adult male and female zebra finches. Androgen‐binding activity reproducibly adhered to DNA‐cellulose and was eluted within the 110‐150 mM NaCl region of a linear salt concentration gradient. Estrogen receptors adhered to DNA‐cellulose and exhibited elution maxima between 170 mM and 210 mM NaCl. Collectively these data indicate that brain regions of zebra finches contain steroid receptors that are similar to those found in the brains of other vertebrates and that their biochemical properties are similar in males and females. Quantitatively, males consistently exhibited higher androgen binding than females in both anterior and posterior telencephalic areas. Females treated with estradiol immediately after hatching exhibited adult levels of androgen binding corresponding to those detected in males. Masculinization of the androgen receptor system by early steroid exposure is discussed.
Myelin‐associated glycoprotein (MAG) in the CNS of adult shiverer (Shi/Shi) mice
Authors: Sheedlo H.J. , Siegel G.J.
Published: January 2005

Brain fractions of adult control (+ / +) and shiverer (Shi/Shi) mice were investigated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. Immunostaining with specific antisera against rat brain myelin‐associated glycoprotein (MAG) was detected at about the 96‐kD region of gels in electrophoresed samples of the total homogenate, low‐speed supernatant fraction, and low‐ and high‐speed sedimentable portions of brain from +/+ mice. Reduced immunostaining was observed in the corresponding samples of brain fractions from Shi/Shi mice. The cerebrum, cerebellum, and medulla of +/+ and Shi/Shi mice were examined immunocytochemically for MAG on paraffin‐embedded sections. Periaxonal immunostaining for MAG was observed in all the regions and the highest concentrations were in the corpus callosum, in the central cores of cerebellar folia, and in the medulla. Patterns of distribution were similar in +/+ and Shi/Shi mice, although the density of immunostaining around individual axons and the number of immunostained axons were significantly reduced in Shi/Shi mice. In addition, the three brain regions of Shi/Shi mice exhibited oligodendrocyte‐like cells that contained immunostaining for MAG in the cytoplasm and periphery of their perikarya. This type of immunostained cell was not observed in +/+ mice. In this study, immunoblotting with brain fractions and immunocytochemistry revealed strong evidence for reduced concentrations of MAG in the CNS of Shi/Shi mice compared to control mice. In addition, there is immunocytochemical evidence for abnormal accumulation of MAG in perikarya of oligodendroglial‐like cells, suggesting the possibility of a transport block for myelin proteins in the shiverer mutant.
Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligodeoxyribonucleotide probe
Authors: Han V. K. M. , Snouweart J. , Towle A. C. , Lund P. K. , Lauder J. M.
Published: January 2005

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the synthesis of catecholamines in neural tissues and adrenal medulla. To study the expression of the TH gene and its regulation in adult and developing neural tissues, we have synthesized an oligodeoxyribonucleotide probe (oligomer) that is specific for TH mRNA. Using Northern blot hybridization of polyadenylated RNAs from adrenal gland, brain stem, liver, and cerebral cortex with the 32P‐labeled oligomer, a single TH mRNA of 1.9 kb was detected in adrenal gland and brain stem but not in liver and cerebral cortex. Using this TH‐specific oligomer, TH mRNAs were localized to the chromaffin cells in the adrenal medulla and to catecholaminergic neurons in locus coeruleus and substantia nigra by in situ hybridization histochemistry. After reserpine administration, the intensity of hybridization signal was increased to threefold that of normal in sections of adrenal medulla and twofold that of normal in locus ceruleus. No difference in hybridization signal intensity was observed in the substantia nigra of normal and reserpine‐treated animals. Use of this specific TH probe in in situ hybridization procedures represents a powerful approach to the study of regulation of TH gene expression at the cellular level.
Ganglioside‐induced alterations in hippocampal cholinergic enzymes and Na, K–ATPase after fimbria–fornix transection
Authors: Fass B. , Ramirez J. J. , Stein D. G. , Mahadik S. P. , Karpiak S. E.
Published: January 2005

Previous biochemical findings suggest that exogenous gangliosides enhance cholinergic sprouting in the hippocampus after partial lesions of the septohippocampal pathway. To assess whether GM1 ganglioside accelerates the onset of this sprouting after complete lesions, we measured cholinergic enzymes and Na, K‐ATPase activity in the hippocampus of rats with unilateral fimbria‐fornix transection. At 14 and 18 days postlesion, histochemical staining showed that acetylcholinesterase (AChE) was almost completely eliminated in the hippocampus ipsilateral to the transection in untreated and GM1‐treated rats. Biochemical assays confirmed that GM1 treaments did not increase AChE activity in the denervated hippocampus. Rather, there were significant reductions of AChE and choline acetyltransferase activities in the ipsilateral hippocampus relative to the contralateral value (P < .001); and the reductions were greater in GMI‐treated rats than in untreated controls (P < .001). Na, K‐ATPase activity in the ipsilateral hippocampus increased by 10.1% in GM1‐treated rats, whereas it decreased by 21.7% in untreated controls (P < .05). Since Na, K‐ATPase is enriched in synaptic membranes, the increased activity of this enzyme may indicate that GM1 treatments stabilize surviving synaptic membranes and/or accelerate the onset of sprouting in the denervated hippocampus. The reductions in cholinergic enyzymes, however, imply that the sprouting pathway must be noncholinergic.
Identification of two cell surface proteins of rat brain oligodrocytes in culture
Authors: Pari F. M. , de los Monteros A. Espinosa , Roussel G. , Nussbaum J. L.
Published: January 2005

Antibodies specific for the surface of oligodendrocytes were prepared by incubating living cultures of pure oligodendrocytes with a crude anti‐oligodendrocyte antiserum. These specific antibodies, when used in the technique of immunoelectroblotting, led to the characterization of at least two major plasma membrane proteins of 43 kilodaltons (kDa) and 53 kDa, respectively, as accessible at the external surface of the oligodendrocytes. The 53‐kDa protein was also found in oligodendrocyte‐conditioned medium in significant amounts. Additional oligodendrcyte surface proteins were also detected in the Wolfgram protein fraction.
Recovery of cholinesterases in soman‐injected superior cervical ganglion of the rat in the presence and absence of innervation
Authors: Klinar B. , Brzin M.
Published: January 2005

We have previously described the procedure for quantitative separation of extracellular and intracellular ChEs using mild treatment of rat superior cervical ganglion with papain. Here, this procedure was used in order to investigate the recovery of ChEs in the two pools after irreversible inhibition by soman which was directly injected into the ganglion. After such treatment only ganglion ChEs were totally inhibited, whereas the activity of ChEs in preganglionic neurons and their axons remained unaffected. Comparing in innervated and decentralized ganglia the pattern of recovery rate and ultrastructural reappearance of ChEs after local inhibition, it was possibleto distinguish between the indirect effects of innervation on the recovery rate and pattern of ChEs of ganglion origin and the direct contribution to the total ganglion enzyme activity of ChEs originating in the preganglionic elements.

The absence of nerve contracts affects mostly extracellular activity, particularly AChE, whereas the intracellular activity of AChE was only slightly decreased and the activity of nsChE was somewhat increased. This increase coincides with the enhanced cytochemical reaction of nsChE in some nonneuronal cells in the ganglion.

Actinomycyn D decreased the rate of initial rapid phase of recovery of intracellular ChEs when injected in the ganglion daily for three days, whereas the recovery of extracellular ChEs was already decreased the first day of Actinomycyn D application. This indicates that the externalization of the enzyme is more affected than its synthesis by inhibition the translation step.

The results further suggest that functional innervation of the ganglion down‐regulate the rate of reappearance of the new enzyme at the extracellular and intracellular locations and changes the ratio between AChE and nsChE. ChEs synthesized in preganglionic neurons begins to contribute directly to the total ganglion ChEs activity in the late stages of enzyme recovery in spite of the presence of the preganglionic ChEs which was not inhibited after intraganglionic injection of soman. This finding suggests that the externalization and extracellular attachment of ChEs and not their preganglionic resynthesis may be the cause for delayed reappearance of ChEs affiliated with nerve terminals in the ganglion.
Neurite‐promoting activity from fetal skeletal muscle: Immunological comparison with laminin
Authors: Steele J. G. , Dalton B. A.
Published: January 2005

Polycation‐binding neurite‐promoting factor (PNPF) from skeletal muscle of bovine fetuses has been compared to mouse tumor laminin by an immunological approach. Antiserum prepared against PNPF blocked the neurite‐promoting activity in a dose‐dependent manner. The anti‐PNPF antiserum bound to Mr = 200,000‐;195,000 bands as well as several components of higher molecular weight, as detected in immunoblots of reduced PNPF. The anti‐PNPF antiserum did not inhibit the neurite‐promoting activity of mouse laminin, although it did bind to native laminin. Antilaminin antiserum bound to PNFP and identified the same Mr = 200,000−195,000 bands and two other higher‐molecular‐weight bands, as did the anti‐PNPF antiserum. The activity of PNPF was confirmed to be sensitive to treatment with heparitinase. It is concluded that PNPF from bovine fetal muscle contains a complex of heparan sulfate and high‐molecular‐weight components, particularly of Mr = 200,000‐195,000, which share antigenic determinants with mouse laminin. However antilaminin antiserum fails to inhibit the activity of PNPF, suggesting that the active site, and/or adjacent regions, of PNPF are antigenically different from the neurite‐promoting site of purified mouse tumor laminin.
Receptors for nerve growth factor on rat spleen mononuclear cells
Authors: Thorpe L. W. , Stach R. W. , Hashim G. A. , Marchetti D. , Perez‐Polo J. R.
Published: January 2005

Considerable evidence is mounting to support the concept of a modulatory role for the brain and neuroendocrine system on the immune response. This neuroimmunomodulation occurs in part through the interaction of specific neurosubstances with receptors on lymphocytes and monocytes. Nerve growth factor (NGF) is a neuronotrophic factor necessary for the development and maintenance of sympathetic and embryonic sensory neurons. This trophic effect is initiated through binding of NGF at specific cell surface receptor sites on NGF‐responsive cells. Several recent studies suggest that NGF may interact with cells of the immune system and may play a role in the regulation of some immunologic reactions. In this study we report on the presence of specific receptors for NGF on the surface of mononuclear cells from rat spleens. The NGF‐binding sites are of the low‐affinity type with Kd's in the 10−9 M range. These receptors migrate on SDS‐PAGE as two molecular species of approximately 190 and 125 kilodaltons. Our findings of receptors for NGF on lymphocytes and accessory cells support other evidence that NGF may influence immunoreactivity in vivo.
Is the muscarinic receptor that mediates potentiation of dopamine release negatively coupled to the cyclic GMP system?
Authors: Marchi M. , Paudice P. , Gemignani A. , Raiteri M.
Published: January 2005

Dopamine (DA) terminals in rat corpus stratum and frontal cortex possess muscarinic receptors that mediate enhancement of the depolarization‐evoked release of the catecholamine. The effects of the membrane‐permeating cyclic guanosine monophosphate (cyclic GMP) analog 8‐Br‐cyclic GMP and of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) on the muscarinic‐induced increase of DA release were investigated in striatal synaptosomes prelabeled with [3H]DA and exposed in superfusion to 15 mM KCl and to acetylcholine (ACh). Preincubation of synaptosomes with 8‐Br‐cyclic GMP (10‐200 μM) or with IBMX (200 μM) prevented the ACh‐induced enhancement of [3H]DA release, without affecting the K+ ‐evoked release of the [3H]amine. No significant decrease of the ACh effect was observed when 8‐Brcyclic GMP or IBMX were added concomitantly with ACh to the superfusion medium. The data suggest that stimulation of presynaptic muscarinic receptors on DA terminals may produce enhancement of 3HDA release through a decrease of the intraterminal cyclic GMP content.
Glial fibrillary acidic protein immunohistochemistry of spinal cord astrocytes after induction of ischemia or anoxia in culture
Authors: Goldberg W. J. , Connor J. R. , Bernstein J. J.
Published: January 2005

The effects of ischemia (removal of oxygen and glucose for 4 h) and anoxia (removal of oxygen alone) on astrocytes were studied in dissociated cultures of E14 spinal cord containing both neurons and astrocytes. In addition, a group of cultures was treated with a low Na+, low Ca2+, and high K+ medium during the 4‐h ischemic period (ischemia‐protected group), a process that protects neurons from ischemic damage under identical conditions. Astrocytes were examined immunohistochemically using glial fibrillary acidic protein (GFAP) antiserum 24 h after insult. Densitometry and statistical analysis (1‐way analysis of variance [ANOVA], a priori; 2‐tailed Tukey‐t, a posteriori) of the digitized images of the somata and processes of astrocytes in the anti‐GFAP reacted cultures showed significant differences between the groups; a significant increase (P<0.01) in the GFAP‐positive reaction in the somata of ischemic astrocytes and a significant decrease (P<0.01) in the GFAP‐positive reaction in the processes of ischemic, ischemia‐protected, and anoxic astrocytes. There were no significant differences in the GFAP immunoreactivity of somata between control, ischemia‐protected, and anoxic astrocytes or of processes from ischemic, ischemia‐protected, and anoxic astrocytes. These data show that following ischemia cultured astrocytes increase somatic GFAP immunoreactivity compared to all other groups tested whereas the staining intensity for GFAP was decreased in the processes of all three experimental groups compared to controls. Ischemia protection resulted in the absence of the enhancement of somatic GFAP immunoreactivity. The relationship of the astrocytic response and the type of cellular stress is discussed.
The macroglial cells of the leech are molecularly heterogeneous
Authors: Flaster M. S. , Zipser B.
Published: January 2005

Monoclonal antibodies derived from fusions employing either whole leech nerve cords or frationated proteins (gel bands) bind to the macroglial cells of the nerve cord. Three different antibodies bind to either one, two, or three of the four macroglial cell types in the leech CNS, serving as markers and showing that these four cell types, which differ primarily by anatomical position, all differ molecularly as well. Conventional microscopy confirms the existence of a novel macroglial cell type first noted because it binds antibody. Western (immunoblot) blot analyses demonstrate a polypeptide antigen of 77 kD in the macroglial cells of the connectives and a 130‐kD polypeptide antigen associated with the macroglial cells of the connectives, of the root nerves, and of the ganglionic neuropil. The extensive molecular heterogeneity of leech neurons demonstrated by monoclonal antibody techniques is shared by macroglia.
Dehydroepiandrosterone and its sulfated derivative reduce neuronal death and enhance astrocytic differentiation in brain cell cultures
Authors: Bologa L. , Sharma J. , Roberts E.
Published: January 2005

Human studies of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA‐S) have shown age‐related changes in serum levels of these two sex hormone precursors. The levels of both DHEA and DHEA‐S are characterized by monotonic decreases after puberty in females and after 20–24 yr of age in males. Further studies have shown that DHEA and DHEA‐S levels are significantly low or close to minimal at ages when the incidence of senile dementia of Alzeimer's type (SDAT) begins to increase. We propose that DHEA and DHEA‐S play a significant role in normal function of neuronal cells and that supplementation with them may prevent neuronal loss and/or damage. In the present study, using methods of immunocytochemistry, autoradiography, and scanning electron microscopy, we show that a supplement of as little as 10−8 M DHEA or DHEA‐S greatly increases neuronal survival and differentiation and reduces astroglial proliferation rates in mouse brain cells in cultures. These results suggest that correcting the DHEA and the DHEA‐S deficit may prevent and/or improve the SDAT condition in humans.
Effect of purkinje cell loss on cerebellar gangliosides in nervous mutant mice
Authors: Seyfried T. N. , Bernard D. J. , Yu R. K.
Published: January 2005

The distribution of cerebellar gangliosides was studied in adult (73 ± 2 days) nervous (nr/nr) mutant mice which lose 50–90% of their Purkinje cells. This neuronal loss is associated with significant reductions in cerebellar weight and ganglioside concentration. The cerebellar dry weights (mg) and the ganglioside concentrations (μg N‐acetylneuraminic acid per 100 mg dry weight) in nr/nr mice and age‐matched normal littermates (+/?) are 7.4 ± 0.3 mg and 13.2 ± 0.4 mg; and 411.7 ± 4.8 μg and 438.5 ± 2.1 μg, respectively. Abnormalities were also observed for the concentration of certain ganglioside species. Most notably, GT1a is significantly reduced by 42%, and GD3 is significantly increased by 29% in the nr/nr mice compared to the +/? mice. The nr/nr mice also express a slight but significant reduction in GT1b. No ganglioside abnormalities were observed between the nr/nr and +/? mice in cerebral cortex. We previously found reduced cerebellar GT1a content in other mutants that also lose Purkinje cells, i.e., sg/sg, pcd/pcd, and Lc/+. GT1a is not reduced, however, in wv/wv mice that lose mostly granule cells. The findings in nr/nr mice are therefore consistent with our hypothesis that GT1a is enriched in Purkinje cells. GD1a, which is enriched in mature granule cells, is not reduced in the nr/nr mice. Since we previously found that GD3 is a good marker for reactive glia in neurological disease, the elevated GD3 concentration in the nr/nr mice indicates a mild gliosis. Our findings with nr/nr and the other neurological mutants indicate that gangliosides can be useful as cell‐surface markers for monitoring changes in the cytoarchitecture of the mouse cerebellum.
Effect of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]Dopamine, and [14C]GABA
Authors: Sershen H. , Debler E. A. , Lajtha A.
Published: January 2005

The effects of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA were examined in attempts to understand the mechanism of ascorbic acid attenuation of MPTP neurotoxicity. [3H]Dopamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of ascorbic acid. Ascorbic acid inhibited [3H]MPP+ uptake (IC50 = 0.1 mM) and [14C]GABA uptake (IC50 = 10 mM). Washout of ascorbic acid restored uptake of [3H]dopamine and [3H]MPP+, suggesting that ascorbic‐acid‐induced lipid peroxidation was not involved in the effect on uptake. In addition to the possible involvement of antioxidant mechanisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct effect of ascorbic acid in inhibiting the uptake of MPP+ into dopaminergic nerve terminals.
Myelin‐specific domain on the plasmalemma of oligodendroglia: Differential expression in the rat and hypomyelinating mouse mutants jimpy and quaking
Authors: Gard A. L. , Dutton G. R.
Published: January 2005

Monoclonal antibody, 1A9, prepared against bovine white matter, recognizes a proteinaceous, myelin‐specific domain in the CNS that is restricted to the surface of oligodendroglia in primary dissociated cell cultures. The antigen is not detected in the PNS or non‐neural tissues. Antibody binding is abolished by heating, exposure to SDS and delipidation, indicating that a conformationally sensitive epitope is recognized. The antigen is present in tracts of developing white matter in rat cerebellum beginning at 5 days postnatally. In developing cultures of fetal rat brain the period of rapid onset for the phenotypic expression of 1A9 antigen is similar to that of galactocerebroside, corresponding to 2–4 postnatal days of age. The 1A9 antigen is not observed in white matter or cultured oligodendroglia of the hypomyelinating jimpy mutant mouse, but its expression is qualitatively normal in the quaking mutant. The possibility is raised that 1A9 may be the primary target of the jimpy mutation.
Response of rat encephalitogenic T lymphocyte lines to synthetic peptides of myelin basic protein
Authors: Offner H. , Hashim G. , Vandenbark A. A.
Published: January 2005

T lymphocyte lines and clones selected from Lewis rats immunized with guinea pig basic protein (GP‐BP) proliferate and acquire the ability to transfer experimental autoimmune encephalomyelitis (EAE) after activation by the 68–88 peptide of GP‐BP in concert with autologous I‐A major histocompatibility antigens. In order to evaluate the amino acid sequence required for activation, encephalitogenic T lymphocytes were stimulated with synthetic peptides representing the 69–89, 69–84, 72–84, and 75–84 sequence of GP‐BP. The three longest peptides, but not the 75–84 peptide, induced encephalitogenic lines and clones to proliferate and to transfer clinical EAE; none of the peptides, however, could activate T cell lines of a different epitope specificity. The 69–89 sequence was the most efficient of the synthetic peptides, inducing optimal stimulation comparable to GP‐BP at 10 μg/ml. The 69–84 and 72–84 sequences induced comparable levels of stimulation at 250 μg/ml, but the 75–84 sequence was not active at any concentration. These results show that the 11 amino acids representing the 72–84 sequence of GP‐BP are sufficient to trigger encephalitogenic T cell activity, and suggest that the 85–89 sequence may stabilize the conformation of the encephalitogenic epitope. The close association observed between proliferation and EAE transfer activities, induced in highly purified T cell populations using synthetic peptides, suggests that these two functional properties of T cells result from a common activation pathway involving a single T cell receptor specificity.
Maturation of a large neurofilament protein (NF 150K) in rat postnatal development
Authors: Dahl D.
Published: January 2005

The mammalian neurofilament is made of three neu‐ron‐specific proteins with approximate molecular weights of 70 kilodaltons (kDa) (NF 70K), 150 kDa (NF 15OK), and 200 kDa (NF 200K) by SDS‐PAGE. As previously reported in the rat by Strocchi et al (J Neurochem 39:1132–1141,1982) and Nixon et al (J Cell Biol) 94:150–58, 19821, NF 150K comprises three molecular weight variants with the same isoelectric point. A fourth lower molecular weight and slightly less acidic variant was identified by monoclonal and polyclonal antibodies reacting with the a‐helical middle domain of NF 150K. With few exceptions, this lower molecular weight variant did not stain with monoclonal antibodies reacting with the peripheral carboxyterminal domain. Staining with these antibodies was abolished or markedly reduced following neurofilament dephosphorylation. The distribution of the NF 150K variants varied in different regions of the nervous system. The higher molecular weight variant (component a) was less prominent in brain compared to spinal cord, optic nerve, and sciatic nerve. Further‐more, the lower molecular weight variant (component d) was not identified in optic nerve and sciatic nerve. All four variants were identified in brain and spinal cord extracts of newborn rats with monoclonal and polyclonal antibodies reacting with the a‐helical middle domain of NF 150K. As a general rule (see Results for exceptions) monoclonal antibodies reacting with the carboxy‐terminal region of NF 150K did not stain the variants in newborn rat brain extracts until day 10 when immunoreactivity of component a first appeared. The adult pattern was first observed on postnatal day 15. With the same antibodies component a was already stained at birth in spinal cord extracts while the adult pattern first appeared on day 10. The delayed appearance of immunoreactivity of the NF 150K variants in development suggests the occurrence of post‐translational modifications probably related to axonal maturation.
Gangliosides fail to enhance behavioral recovery after bilateral ablation of the visual cortex
Authors: Butler W. M. , Griesbach E. , Labbe R. , Stein D. G.
Published: January 2005

Postsurgical injections of GM1 gangliosides (30 mg/kg IP) reduced neither behavioral deficits in rats following bilateral ablation of the visual cortex nor the extent of retrograde degeneration of neurons in the dorsolateral geniculate nucleus that typically accompanies large lesions of the visual cortex. Our findings are in contrast to previous research, in which ganglioside treatments have been shown to enhance the rate of functional recovery after lesions in other parts of the central nervous system. The negative findings in the present experiment may be due to the disruption of normal circadian rhythms caused by occipital cortex injury.
The polyclonal antibody responses of lewis rats to the synthetic encephalitogenic neuropeptide S55S (residues 72–84 of guinea pig myelin basic protein) and its analogs
Authors: Day E. D. , Hashim G. A. , Ireland D. J.
Published: January 2005

Immunochemical analysis of polyclonal antisera from Lewis rats immunized with peptide analogs and sub‐peptides of residues 72–84 of guinea pig myelin basic protein (MBP) indicated that there were four main epitopic specificities involved: one represented by residues 76–80, one by residues 72–74, one by 81–84‐Gly, and one involving 74–76 in a heteroclitic way even when the immunogen contained a tetraleucine spacer between residues 74 and 75. The affinities were all relatively low, ranging from 3 × 104M−1 to 7.2 × 106M−1, ad each affinity population was very restricted with respect to heterogeneity. The result were commensurate with out hypothesis that autoimmune responses to homologous encephalitogenic neuropeptide determinants reflect an evolutionarily imposed degeneration of the overall response, and were also i agreement with our hypothesis that the affinities of autoantibodies against epitopes in the molecular neighbourhood of an encephalitogen would be necessarily low. I t was also found that peptide 69–81‐Gly(S67) in solution would not interact in liquid phase radioimmunoassays with any of the above antibody populations, thus indicating that its preferred conformation did not allow the expression of autoreactive epitopes. There was no evidence that S67 in solution, even in the presence of S53, would express any epitopes reactive with autoantibodies. How this finding for a B cell product correlates with T cell‐mediated immunologic responses is not totally understood, but it will be recalled that S67 by itself does not induce T cell‐mediated experimental allergic encephalomyelitis (EAE), that S53 (residues 75–84‐Gly) induces only clinical symptoms of EAE without attendant pathological disease, but that the two together induce classical EAE. Thus, it is hypothesized that residues 76–80 (Gln‐Arg‐Ser‐Gln), modulated by Asn‐84, form the B cell determinant essential for subsequent activation of T cell responses to 72–77 to produce the full expression of EAE disease.
Asymmetrical involvement of the cerebral neocortex on the response to an immunopotentiator, sodium diethyldithiocarbamate
Authors: Renoux G. , Bizière K.
Published: January 2005

We have previously shown that the neocortex in mice has a lateralized influence on the immune system. A partial left or bilateral neocortical lesion selectively decreases spleen T‐cell numbers and function, natural killer and (NK) activity, but a right neocortical lesion do not affect NK activity, and increases T‐cell numbers and T‐cell‐mediated events. Here we report that the immunopotentiating activity of sodium diethyldi‐thiocarbamate (Imuthiol®), a compound that selectively increases T‐cell numbers and activities, is dependent on an intact neocortex. The effects of Imuthiol were examined in female C3H/HeJ mice 10 seeks after partial neocortical lesions. In animals with right or bilateral neocortical lesions, Imuthiol failed to increase the percentage of spleen T cells, did not influence the expression of class I MHC antigen on these cells, no longer induced the release in serum of specific T‐cell‐inducing factors, and failed to enhance T‐cell numbers and activities in a fashion that was comparable to that observed in unlesioned controls, but did not enhance NK activity. It is concluded that Imuthiol may affect immune responsiveness by acting directly on the neocortex and/or by interacting at subcortical levels with signals emitted by the neocortex. Moreover, this study reveals a major hemispheric asymmetry in the response to a drug.
Carrier‐mediated transport of vasorpressin across the blood‐brain barrier of the mouse
Authors: Banks W. A. , Kastin A. J. , Horvath A. , Michals E. A.
Published: January 2005

A brain to blood carrier‐mediated transport system for agrinine vasopressin (AVP) was investigated in mice after intraventricular injection of iodinated AVP and varying amounts of unlabeled material or candidate inhibitors. Residual activity in the brain detected after decaptiation was used as the main determinant ot transport activity. The half‐time disappearance of iodinated AVP from the brain was 12.4 min, the Vmax was 1.41 nmol/g min, and the apparent Km was 28.7 nmol/g. A 30‐nmol dose of AVP, mestocin, arginine vasotocin, pressionoic, amide, pressinoic acid, tocinoic acid, and lysine vasotocin, but not oxytocin, lysine vasopressin, AVP free acid, tocinoic amide, Tyr‐MIF‐1, or cyclo Leu‐Gly, significantly (P<0.05) inhibited the transport of iodinated AVP out of the brain. The 30 nmol dose of AVP had no effect on the tranport of iodide or iodotyrosine out of the brain. High‐preformance liquid chromatography showed that 59.2% of the radioactivity found in the blood 2 min after an i.c.v. injection of labeld AVP eluted at same postiton as labeled AVP compared with 68.8% of radioactivity eluting at that postition after material was infused i.v. for 2 min. This indicates that intact peptide is transported across the blood‐brain barrier and that most of the degradation of AVP occurs during circulation in the blood. Calculations based on the appearance of radioactivity in the periphery showed that 56.2% of the material injected centrally would have been transported into the periphery by 10 min. This appearance of material in the periphery was inhibited by the simulataneous injection of an excess of unlabeled peptide. Water loading significantly decreased the brain to blood transport rate of AVP by 40%. It is concluded that a saturable system exists for brain to blood transport of AVP and some structurally similar peptides.
Dopamine and noradrenaline content in fish retina: Modulation by serotonin
Authors: Jaffé E. H. , Urbina M. , Ayala C. , Drujan Y. , Drujan B. D.
Published: January 2005

The presence of noradrenaline (NA) and the possible interaction of serotonin (5‐HT) with dopaminergic and noradrenergic neuronal elements was studied in the retina of the teleost Eugerres plumieri. By Means of a histofluorescent technique, paired amacrine cells can be visualized after intravitreal injection of NA or 5,6‐dihydroxytriptamine, suggesting their probable catecholaminergic and indoleaminergic nature. With a high‐performance liquid chromatographic (HPLC) method and after pargyline treatment of the animal, 6.86 ng/mg protein ofdopamine (DA) was detected, while NA content was 0.50 ng/mg protein. The NA levels of the retina increased, whereas the DA levels decreased in a significant manner after in vivo treatment with 5‐HT. 5‐methoxy‐N, N‐demethyltryptamine, a 5‐HT agonist, was able to mimic this effect partially, while the agonists tryptamine and quipazine did not affect the levels of DA and NA. The antagonissts methysergide, metergoline, and cyproheptadine significantly blocked the 5‐HT‐induced NA increase, whereas only the first two antagonists were effective in inhibiting the5‐HT‐induced DA decrease. The 5‐HT modulation of NA and DA is apparently receptor mediated and is not due to a hetero‐exchange, since mipramine was not able to block the 5‐HT effect.

These findings support the suggsted interaction between serotoninergic and dopaminergic cells [Negishi et al: J Neurosci Res 5:621–635, 1980; Neurosci Lett 25:1‐5, 1981]. Furthermore, they demonstrate the possible modulation which 5‐HT exerts on the endogenous livels of NA.
Heterogeneity of opioid receptor binding in brain slices
Authors: Barchfeld‐Rothschild C. C. , Medzihradsky F.
Published: January 2005

A methodological approach was established for the study of ligand binding to multiple opioid receptors in slices from rat brain striatum. Specific binding of radiolabeled opiates was resolved from total binding with enantiomers or excess unlabeled ligand. Equilibrium binding of triated etorphine, dihydromorphine, and ethylketocyclazocine, and competitive displacement of [3H]dihydromorphine by the unlabeled opiates were used to assess both high and low affinity receptor sites. The high‐affenity binding components of the radiolabeled opiates were characterized by linear Scatchard plots, Kd values of 2.8–3.7 nM, and binding site densities of 180‐297 fmol/mg protein. The displacement of [3H]etorphine by morphine and ethylketocyclazocine displayed Hill coefficients of 0.62 and 0.47, respectively, and revealed receptor sites with much lower affinities than those described by the direct binding of these opiates. On the other hand, both morphine and ethylketocyclazocine displaced [3H]dihydromorphine with similar high potencies (apparent Kds, 3‐4 nM). The results support the feasibility of using brain slices as a cellular preparation to study opioid receptor mechanisms.
Hypothalamo‐neurohypophysial neurons in vitro: Developmental potentials depend on the donor rat stock
Authors: Schilling K. , Pilgrim C.
Published: January 2005

Primary dispersed cell cultures were established from fetal diencephalic tissue derived from two stocks of outbred rats. The overall growth pattern, as followed by phase‐contrast microscopy and visualized by immunocytochemistry for neurofilament proteins and neuron specific enolase, depended on the age of the donor animal. Stock differences were not revealed by these methods. In contrast, when the cultures were immunostained in order to assess the yield of a specific type of cell, i.e., magnocellular hypothalamic neurons, striking differences were observed between the two stocks of rats. Whereas neurophysin‐immunoreactive cells could be grown in culture established from fetal Chbb: Thom rats at embryonic days 14, 15, and 18, no such cells could be detected in cultures derived from CrI: CD (SD) BR rats older than embryonic day 14. These findings may reflect differences in the differentiation schedule and/or requirements for certain epigenietic and trophic factors between magnocellular neuroblasts derived from the two stock of rats.
A major epitope of synthetic rabbit encephalitogen S24 disclosed through the use of a tritiated peptide probe
Authors: Tyrey S. J. , Potter N. T. , Day E. D. , Hashim G. A.
Published: January 2005

The antibody recognition of synthetic rabbit encephalitogen S24 (TTHYGSLPQKG), which had been inactivated by extensive iodination, was fully restored by tritium‐iodine exchange during boron hydride reduction. A highly active form of 3H‐S24 was obtained by reverse‐phase high‐performance liquid chromatography (HPLC) purification of the crude product and could be further separted into three continuous subfractions, the latter two of which were fully active from an immunochemical point of view. A major epitope was diclosed through use of the 3H‐S24 probe, which required the intact S24 peptide for its expression. Confirmation of two linear epitopes previously encountered, GSLPQK and THYGSL, was also obtained, the latter through the use of the third HPLC subfraction of 3H‐S24.
Age‐dependent differences in 125I‐nerve growth factor binding properties of rat adrenal chromaffin cells
Authors: Hofmann H.‐D. , Ebener C. , Unsicker K.
Published: January 2005

Nerve growth factor (NGF) has ben shown to influence survival, morphology, and transmitter phenotype of young postnatal rat chromaffin cells in vitro. Significant differences in NGF responses of chromaffin cells from newborn rats compared to 8–10‐day‐old ones have been reported. For this reason we studied equilibrium binding and dissociation kinetics of 125I‐NGF on newborn (D1) and 10‐day‐old (D10) rats. Under equilibrium conditions no differences were found between the two cell types, with respect to dessociation constant (∼2.5 × 10−9 M) and receptor number (10–22,000 per cell). In dissociation experiments D10 chromaffin cells exhibited two classes of NGF receptors, similar to those found in other NGF‐responsive cells. Fromfast receptors 125I‐NGF wasreleased rapidly both at 4°C and at 37°C, whereas dissociation from “slow” receptors was observed only at 37°C. The slow receptor class was not found on D1 cells. Instead, more than 50% of specifically bound 125I‐NGF did not dissociate in the presence of excess unlabeled NGF at 37°C. These age‐dependent differences seem to indicate regulatory developmental changes in NGF‐binding properties of rat chromaffin cells.
48‐kilodalton intermediate‐filament‐associated protein in astrocytes
Authors: Abd‐El‐Basset E. M. , Kalnins V. I. , Subrahmanyam L. , Ahmed I. , Fedoroff S.
Published: January 2005

We provide evidence that a protein of 48 kilodaltons (KD), recognized by a normal rebbit serum (F2N), is associated with intermediate filaments (IF) of astrocytes both in cell cultures and in situ. Immunofluorescence staining shows that the F2N serum gives a fibrous staining pattern similar to that seen with antiserum to glial filament protein (GFP), a protein sepecific for IF of astrocytes, and that both proteins are present in the perinuclear fibrous aggregates of IF produced by treating the cells with colchicine. At the ultrastructural level the gold particles decorating the 48‐KD protein are localized on the IF in a linear patterns. This difference in distribution and the fact that the two proteins have different electrophoretic mobilities on SDS gels indicates that the 48‐KD protein although associated with IF is different from GFP.

The 48‐KD protein appears to be a distinct, developmentally regulated intermediate‐filament‐associated protein (IFAP), different from other IFAP'S reported to date and the first IFAP described in astrocytes. Its appearance in late developmental stages when motile astroblasts are changing into nonmotile setllate cells suggests that the 48‐KD protein may be involved in cross‐linking the GFP‐containing IF to provide more tensile stregth to the cytoplasm at the expense of flexibility.