bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural localization of phenylethanolamine N‐methyltransferase in sensory and motor nuclei of the vagus nerve
Authors: Pickel V. M. , Chan J. , Park D. H. , Joh T. H. , Milner T. A.
Published: January 2005

The ultrastructural localization of phenylethanolamine N‐methyltransferase (PNMT), the enzyme used in the final step in the synthesis of adrenaline, was examined in the medial nuclei of the solitary tracts (m‐NTS) and in the dorsal motor nuclei of the vagus. Adult rats were anesthetized with Nembutal (50 mg/kg intraperitoneally), and the brains were fixed by vascular perfusion with a solution containing 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer. Coronal Vibratome sections were collected through the intermediate portions of the m‐NTS at the level of the area postrema. These sections were immunocyto‐chemically labeled employing a rabbit polyclonal antiserum against PNMT and the peroxidase‐antiperoxidase method. Immunoreactivity was detected in perikarya, dendrites, and axon terminals in the intermediate portion of the m‐NTS. The labeled perikarya were either small (10–15 μm diameter) and oval or large 20–30 μm) with two or more proximal processes. The PNMT‐containing dendrites received synaptic input from unlabeled, small (0.5–1.0 μm) and large (2–3 μm) vagal‐like afferents as well as from a few terminals, which also showed PNMT immunoreactivity.

Axons and axon terminals containing immunoreactive PNMT were more frequently observed than the perikarya or dendrites in the m‐NTS and were the only labeled profiles in the dorsal motor nuclei. In both regions the PNMT‐labeled terminals formed principally symmetric synapses with unlabeled dendrites. However, a few asymmetric axodendritic and symmetric axosomatic synapses also were detected. These findings indicate that the adrenergic neurons may have multiple, but principally inhibitory, actions on other neurons within cardiovagal portions of baroreflex pathways.
Ependyma formation in adult rat spinal cord after transplantation of fetal cerebral cortex homografts
Authors: Bernstein J. J.
Published: January 2005

The ependymal cells in rat host spinal cord were studied after transplantation of fetal cerebral cortex homografts. Adult Sprague‐Dawley male rats had pieces of E14 fetal rat cortex pressure‐injected into the spinal cord at T6. The tissue was prepared for light and electron microscopy and studied for over 2 months postimplantation. At 7 and 14 days postimplantation (DPI), there were dividing ventricular‐ependymal cells lining cysts in the host spinal cord. After 21 days, cell division was no longer observed in these cells, and only mature ependymal cells lined these cysts. Mature ependymal cells were either: columnar, pseudocolumnar, cuboidal, squamous, or pseudomorphic; had cilia and villi on only one surface of the cell and shared tight junctions when contiguous. These data show that ependymal cells were mature cellular components of adult spinal cord after transplantation of fetal CNS grafts. In addition, ventricular‐ependymal cells continued to divide within the parenchyma of the graft, indicating continued growth of transplanted fetal cerebral cortex in host spinal cord.
Isolation and sequence of cDNA clones coding for the precursor to the γ subunit of mouse muscle nicotinic acetylcholine receptor
Authors: Boulter J. , Evans K. , Martin G. , Mason P. , Stengelin S. , Goldman D. , Heinemann S. , Patrick J.
Published: January 2005

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage (λgt10) vectors with poly (A +) RNA isolated from the nonfusing mouse muscle cell line BC3H‐1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor γ subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor γ subunits from Torpedo californica, chick, calf, and human. One isolate, λBMG419, has 88 nucleotides of 5′‐untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor γ subunit, and 144 nucleotides of 3′‐untranslated sequence. Alignment of the λBMG419‐deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse γ subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled λBMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A +) RNA prepared from BC3H‐1 cells and denervated mouse limb muscle. γ Subunit‐coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of theγ subunit is exerted through regulation of the amount of its mRNA.
Extensive postmortem stability of RNA from rat and human brain
Authors: Johnson S.A. , Morgan D.G. , Finch C.E.
Published: January 2005

The postmortem stability of brain RNA was measured in rat and human samples for up to 48hr. Whole rat brains, cooled at a rate approximating that of human brains awaiting autopsy, were collected at intervals from 0 to 48 hr after death and frozen. These samples were compared with freshly obtained, unfrozen rat brains. Two potentially independent characteristics of these RNA populations were measured: (1) the recovery or yield of RNA/gram tissue (quantity) and (2) the integrity or extent of degradation (quality). Total RNA yields were similar after all postmortem delays. Hybridization of [32P]‐labeled cDNA probes to nitrocellulose filter blots of electrophoretically separated rat brain RNA failed to reveal degradation of the specific rat brain mRNAs during the postmortem period. Similarly, in vitro translation of these same rat total RNA samples produced high molecular weight translation products with no differences between long and short postmortem times.

Human cerebral cortex RNA was prepared by the same methods as those used for rat brain from a neurosurgical sample and four other donors with postmortem intervals from 7 to 36 hr. Typically, the yield of total RNA from human brain was 40–50% of the yield from rat brain. When analyzed by RNA gel blot hybridization studies, as for rat brain RNA, human cortical RNA appeared slightly degraded. However, the degree of apparent RNA degradation was not related to the postmortem interval. In vitro translation products of human cortical RNA revealed high molecular weight peptides at all postmortem intervals, but slightly less [35S]incorporation into these bands was found at the longer postmortem intervals relative to the shorter times. Together, these results demonstrate an extensive stability of brain RNA that invites aggressive use of molecular genetic techniques for the study of human neurodegenerative diseases.
Lipophilin (PLP) Gene in X‐linked myelin disorders
Authors: Fahim S. , Riordan J.R.
Published: January 2005

There are several X‐linked diseases in animals and at least one in man in which there is a failure of CNS myelination. We have recently cloned cDNAs for lipophilin (PLP) with which PLP sequences were localized to a region of the long arm of the X chromosome (Xq13–q22 in man) close to the jimpy (jp) locus in that mouse mutant. The present communication pursues the postulate that some of this class of diseases may involve mutations at the PLP locus. Blot hybridization analysis of PLP mRNA levels revealed a five‐to tenfold reduction in the brains of hemizygous jp/Y mice. The major PLP mRNA species of those mice was also reduced in size. However, Southern blots of jp DNA digested with many different restriction enzymes failed to detect major deletions or other rearrangements in the PLP gene. A human PLP cDNA was isolated and employed to similarly analyze DNA from four patients diagnosed as having Pelizaeus‐Merzbacher disease. In one of these four a significant rearrangement of the PLP gene was found. These findings suggest that there may be alterations in the PLP gene in both jp mouse and Pelizaeus‐Merzbacher disease.
In vitro cytotoxicity and demyelination induced by theiler viruses in cultures of spinal cord slices
Authors: Shahar A. , Frankel G. , David Y. , Friedmann A.
Published: January 2005

The cytopathic effects caused by Theiler viruses to myelinated organotypic spinal cord cultures was studied by light and electron microscopy. Heavily myelinated cultures, 2–3 weeks in vitro were infected with WW and GD VII viruses. Mock infection served as control. On light microscopy cytopathic effects and demyelination become evident about 16–17 hr after infection. Demyelination observed in WW virus‐infected cultures was much more pronounced than in cultures infected with GD VII viruses. The myelin in mock‐infected cultures remained undamaged.

Electron microscopy revealed that in control cultures cells were intact, exhibiting numerous synapses and a network of axons enwrapped by multilayered myelin sheaths. Virus‐infected spinal cord slices showed that a more severe cytotoxicity was caused by GD VII virus than by WW virus. The cytopathology included accumulation of cytoplasmatic vacuoles, margination of chromatin, synapse and cell disintegration, and various degrees of demyelination. Several GD VII virions were observed, arranged in crystalline arrays, mainly in electron‐opaqe cells, but not within axons. WW virions on the other hand were only occasionally encountered.
Nerve growth factor (NGF) in serum: Evaluation of serum NGF level with a sensitive bioassay employing embryonic sensory neurons
Authors: Stephani U. , Sutter A. , Zimmermann A.
Published: January 2005

Considerable controversy surrounds the question of whether or not nerve growth factor (NGF) or a related nerve growth‐promoting factor is present in serum. Recently, supporting its role as a local neuronotrophic factor, the presence of NGF in glial cells and its production in target tissues of NGF‐responsive neurons were demonstrated [Rush: Nature 312:364–367, 1984; Heumann, Korsching, Scott, Thoenen: EMBOJ 3:3183–3189, 1984; Shelton and Reichardt: Proc Natl Acad Sci USA 81:7952–7955, 1984]. At the same time, the concept that NGF may play a role as a humoral factor has been questioned, since careful analyses of serum with specific and sensitive radioimmunoassays [Suda, Barde, Thoenen: Proc Natl Acad Sci USA 75:4042–4046, 1978; Korsching and Thoenen; Proc Natl Acad Sci USA 80:3513–3516, 1983; Furukawa, Kamo, Furukawa, Akazawa, Satoyoshi, Itoh, Hayashi: J Neurochem 40:734–744, 1983] as well as bioassays [Skaper and Varon: Exp Neurol 76:655–665, 1982] have not confirmed earlier reports [Levi‐Montacini and Booker; Proc Natl Acad Sci USA 46:373–391, 1960; Banks, Banthorpe, Charlwood, Pearce, Vernon: Nature 246:503–504, 1973; Hendry: Biochem J 128:1265–1272, 1972] on NGF's representation in serum.

In this study serum from mouse, rat, and man was analyzed with an in vitro bioassay system which employs sensory neurons from chicken embyro dorsal root ganglia and which allows the measurement of NGF concentrations as low as 0.8 pM. It was found that sera from all these species contained neuronotrophic activity (S‐NGF). The target cell spectrum as well as characteristic parameters of the neuronal growth response of S‐NGF and of NGF were identical. S‐NGF of mouse serum was completely inhibitable by polyclonal and monoclonal antibodies to mouse submandibular gland β NGF. On polyacrylamide isoelectric focussing gels mouse and human S‐NGF could be recovered from the same position as NGF as well as from the region where α2‐macroglobulin and serum albumin focused.

In newborn and adult male and female mice basal S‐NGF levels were equivalent to 10–50 pM NGF. A. fraction of the serum samples of male mice showed elevated S‐NGF levels. The incidence of high S‐NGF levels was more frequent in NMRI and C57BL/6 males than in BALB/c males. Following sialectomy of male mice only basal S‐NGF levels were observed up to 5 weeks after the operation. This indicates that although the submandibular gland may contribute to S‐NGF levels in serum under certain conditions that appeared to be stress related, it cannot be the only source of S‐NGF.
Development of transferrin‐positive oligodendrocytes in the rat central nervous system
Authors: Connor J. R. , Fine R. E.
Published: January 2005

Transferrin is the second most abundant plasma protein and functions to transport iron. It is an essential constituent in culture media for virtually all cells. In a recent study, we reported that transferrin (Tf) is specifically located in oligodendrocytes in the rat nervous system. This investigation examines immunohistochemically the development of Tf in the cerebral cortex, corpus striatum, and spinal cord. Tf is first seen in oligodendrocytes in the spinal cord white matter at 5 days of age. The immunoreactivity is confined to the white matter in the periphery of the spinal cord between 5 and 8 days of age. By 10–12 days of age, the number of immunoreactive oligodendrocytes in the spinal cord white matter increases considerably, corresponding to the onset of myelination. Tf‐positive oligodendrocytes are first found in the gray matter at 15 days of age. By 30 days of age, the number and distribution of Tf‐positive oligodendrocytes in both the brain and spinal cord have reached the adult pattern. The results of this study demonstrate a spatial and temporal association between Tf development and myelinogenesis. This suggests that part of the process of differentiation of oligodendrocytes includes the accumulation of Tf, perhaps in order to support the metabolic demands associated with the production and maintenance of myelin.
Regulation of sterol synthesis and of 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase by lipoproteins in glial cells in primary culture
Authors: Langan T. J. , Iimori Y. , White G. , Volpe J. J.
Published: January 2005

Although plasma lipoproteins have been demonstrated to have a major role in regulating cholesterol biosynthesis in extraneural cells, no data concerning such regulation are available for developing brain, when cholesterol synthesis is especially active. Glial primary cultures derived from neonatal rat brain and by morphological and biochemical criteria essentially exclusively composed of astrocytes were utilized to examine such regulation. When the primary cultures, which had been maintained in 10% fetal calf serum, were placed in 10% lipoprotein‐poor serum on day 7 of culture, an induction of sterol synthesis (1.6–2.2‐fold) and of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase‐specific activity (1.5‐2‐fold) resulted after 24 hr. Addition of low‐density lipopro‐tein (LDL) to the 10% lipoprotein‐poor serum prevented the induction of both sterol synthesis and HMG‐CoA reductase. However, addition of high‐den‐sity lipoprotein (HDL) to the 10% lipoprotein‐poor serum caused a 1.5–2‐fold further induction of sterol synthesis relative to that in cultures containing 10% lipoprotein‐poor serum alone. In contrast to the glial primary cultures, cultures of C‐6 glioma cells responded to replacement of 10% fetal calf serum with 10% lipoprotein‐poor serum with much more marked increases of sterol synthesis and HMG‐CoA reductase. Although, as with the primary cultures, addition of LDL to the C‐6 glioma cell cultures prevented the increases in sterol synthesis and reductase activity, addition of HDL had no effect. Thus, these results indicate that in developing glial cells in primary culture, cholesterol synthesis and HMG‐CoA reductase are capable of responsiveness to both LDL and HDL. Moreover, at least in terms of regulation of sterol synthesis, C‐6 glioma cells and developing glia in primary culture clearly are not identical.
Experimental allergic encephalomyelitis (EAE): Role of B cell and T cell epitopes in the development of EAE in lewis rats
Authors: Hashim G. A. , Day E. D. , Carvalho E. , Abdelaal A.
Published: January 2005

Studies from our laboratory have shown that classical clinical and histological signs of experimental allergic encephalomyelitis (EAE) may be induced in Lewis rats by synthetic peptides S49 or S55. Peptides S49S and S55S are defined by residues 69–84 and 72–84 of the guinea pig myelin basic protein (MBP), respectively. Peptide S53 (residues 75–84 of the guinea pig MBP), six residues shorter than S49S at the N‐terminal end, induced mild clinical signs of disease unaccompanied by hind leg paralysis, incontinence, or central nervous system pathology. In contrast, peptide S67 (residues 69–81 of the guinea pig MBP), three residues shorter than S49S at the C‐terminal end, did not induce either clinical or histological signs of EAE despite the fact that the S67‐sequence houses an epitope known to induce cell‐mediated immunity. Peptides S49S, S55S, and S53 are antigenic and gave rise to antibodies that recognized either of the three peptide sequences. In this report we explore the interrelationship between cellular immunity induced by the S67 sequence and humoral immunity, induced by the S53 sequence and the development of classical clinical and histological signs of EAE. The results show that the nonencephalitogenic sequence of S67 may be rendered encephalitogenic in the presence of antibody directed against the S53 sequence. Lewis rats immunized with S53 developed pathological signs of EAE only after they were challenged with S67. The fact that a simultaneous challenge with S67 and S53 was as effective in inducing EAE pathology as a delayed one (up to 40 days) suggests that the cellular response to S67 is dependent upon the humoral response to S53. The response to both peptides injected into separate sites mimics the response to the encephalitogenic S49S within which the S67 and the S53 sequences are subsumed. Clearly, a causal relationship is established between the humoral and cellular immunities in inducing EAE. This conclusion is supported by the results of the transfer experiments which showed that either anti‐S53 antibodies or S53‐primed lymphocytes may substitute for S53‐priming in potentiating the development of S67‐induced cell‐mediated immunity and therefore EAE pathology.
Arachidonic acid inhibits uptake of glutamate and glutamine but not of GABA in cultured cerebellar granule cells
Authors: Yu A. C. H. , Chan P. H. , Fishman R. A.
Published: January 2005

The effects of arachidonic acid (20:4) on the uptake of glutamate were studied in primary cultures of cerebellar granule cells and were compared to cortical neurons and astrocytes. At a dose of 0.005 mM, the glutamate uptake was significantly inhibited in cerebellar granule cells. This inhibition was dose and time dependent. The uptake of glutamate was equally sensitive to 20:4 in primary cell cultures of cortical neurons, whereas the uptake in astrocytes was much less sensitive to 20:4. Glutamine uptake was inhibited by 20:4 in cultured cerebellar granule cells and cerebral cortical astrocytes but was not affected in cerebral cortical neurons. Furthermore, the uptake of gammaaminobutyric acid was not affected by 20:4 in cerebellar granule cells.
Role of growth hormone in regulating T‐Dependent immune events in aged, nude, and transgenic rodents
Authors: Davila D. R. , Brief S. , Simon J. , Hammer R. E. , Brinster R. L. , Kelley K. W.
Published: January 2005

Growth hormone (GH) appears to play a major role in a reciprocal axis that has been postulated between the thymus and pituitary glands. Our previous studies showed that thymic structure, as well as T‐cell proliferation and IL‐2 synthesis, could be restored in aged female Wistar‐Furth rats by the implantation of GH3 pituitary adenoma cells. These cells secrete GH and some prolactin. We have now used three different approaches to determine whether GH affects a variety of immune events in vivo in both old and young rodents, and whether GH3 cells can directly affect progenitor T‐cells in nude rats that congenitally lack a thymus gland. To test the effects of GH in aged rats, 750 μg of pituitary‐derived ovine GH was injected 2× daily into 26‐month‐old Fischer 344 rats for 5 weeks. This approach demonstrated that GH augments splenocyte proliferation to T‐cell lectins as well as natural killer (NK) activity at low effector: target ratios even though morphologic characteristics of the thymus were not altered. To assess the effect of GH in young rodents, mice were studied that were transgenic for the rat metallothionein‐GH gene. Histologic evaluation of thymus glands revealed that the amount of adipose tissue and the number of epithelial cells and Hassall's corpuscles are augmented in transgenic mice. Splenocyte proliferation at suboptimal mitogen doses is greater in transgenic than in control littermate mice, but neither IL‐2 synthesis nor antibody synthesis to sheep erythrocytes is affected. The role of pituitary hormones on progenitor T‐cells was then explored by implanting GH3 cells into Rowett nude rats. GH3 cells are unable to augment proliferation to T‐cell lectins, IL‐2 synthesis, antibody production to sheep erythrocytes, or NK activity in nude rats. Collectively, these experiments show that T‐cells and NK cells from aged rats are not inherently defective because their proliferative responses to lectins and cytolytic activity can be augmented by exogenous GH. If GH affects progenitor T‐cells, these effects are not apparent in nude rats. Finally, GH can also augment T‐cell proliferative responses in young rodents, but the immunoenhancing effects are not as apparent as thos observed in immunocompromised aged animals.
The influence of nerve growth factor on the in vitro proliferative response of rat spleen lymphocytes
Authors: Thorpe L. W. , Perez‐Polo J. R.
Published: January 2005

Splenic lymphocytes and accessory cells express receptors for nerve growth factor (NGF), a well‐characterized neurotropic peptide that influences the development and survival of neuronal elements in the central and peripheral nervous systems. In the present study, we report that when rat splenic mononuclear cells (MC) are incubated in the presence of NGF, a dose‐dependent increase in DNA synthesis occurs during 96–120 hours of culture as measured by 3H thymidine (3H‐Thd) uptake. The minimal molar concentration of NGF at which the increased proliferative response of the cells (3.7 nM) is seen corresponded to the equilibrium disassociation binding constant of the MC (Kd = 2.5 nM), suggesting that the response was a consequence of receptor‐ligand interaction. In addition, NGF was able to potentiate the lymphoproliferative response to several T‐cell and B‐cell mitogens. Significantly increased 3H‐Thd uptake by NGF‐stimulated cells was noted for concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS), particularly at suboptimal dosages of mitogen. Thus it appears that the NGF receptors on rat splenic MC are physiologically relevant and that NGF can modulate proliferation of T‐ and B‐ cells.
Central action of interleukin‐1 in altering the release of tsh, growth hormone, and prolactin in the male rat
Authors: Rettori V. , Jurcovicova J. , McCann S. M.
Published: January 2005

In order to determine the possible effects of interleukin‐1 on the release of pituitary hormones by direct action on the brian, the peptide was injected into the third brain ventricle of conscious, unrestrained male rats and the effects on hormone release were compared with effects on rectal temperature. The procedure of blood sampling and intraventricular injection resulted in a significant decline in body temperature and a decrease in plasma growth hormone without alteration in the plasma level of thyroid stimulating hormone (TSH) and prolactin. Interleukin‐1 injected intraventricularly at a dose of 5 ng (0.3 pmol) prevented the decline in body temperature that occurred in the saline‐injected controls and resulted in a significant elevation of plasma growth hormone levels that became apparent within 15 min of injection, as well as a highly variable but significant elevation of plasma prolactin and a significant decline in plasma TSH that was observed at 30 min. The results were similar when areas under the release curves for the various hormones were calculated. On the other hand, the higher dose of 25 ng (1.5 pmole) of interleukin‐1, although producing a frank pyrexia, was associated with smaller changes in hormone values, which were no longer significant for any of the three hormones. The results indicate that interleukin‐1 can act in minute doses, presumably on structures near the third ventricle, to stimulate growth homone and prolactin release and to inhibit TSH releas. Apparently when frank febrile responses occur, these hormonal responses are muted for reasons that are yet to be determined. In view of the minute doses injected we favor a hypothalamic site for these effects.
Synthetic peptide analogs to probe the immunological expression of the rat encephalitogenic neuropeptide
Authors: Hashim G. A. , Day E. D.
Published: January 2005

The rationale for designing and synthesizing 24 different peptide analogs and subpepties of residues 69–84 of guinea pig myelin basic protein is presented. The encephalitogenic potential of 19 of these peptides in Lewis rats is also given.
Glial glutamate dehydrogenase: Ultrastructural localization and regional distribution in relation to the mitochondrial enzyme, cytochrome oxidase
Authors: Aoki C. , Milner T. A. , Berger S. B. , Sheu K.‐F. R. , Blass J. P. , Pickel V. M.
Published: January 2005

Glutamate dehydrogenase (GDH) is primarily a mitochondrial enzyme involved in the metabolism of glutamate. We have recently shown by light microscopic immunocytochemistry that, within detergent‐permeabilized brain tissue, GDH is enriched in glial cells, particularly in regions utilizing L‐glutamate as a neurotransmitter. In this study, we used immunogold labeling to quantitatively establish that the forms of the enzyme recognized by the presently used GDH antiserum is associated primarily with a subpopulation of mitochondria in ultrathin, plastic‐embedded sections of the rat cortex and striatum. Permeabilization with detergents was omitted in these studies, so as to preserve the ultrastructure. As expected, labeled mitochondria occurred both in neurons and glia. Furthermore, light microscopic comparisons of the regional distributions of peroxidase immunoreactivity for GDH and a histochemical reaction product for a second mitochondrial enzyme, cytochrome oxidase (CO), were used to demonstrate that high levels of GDH in glia of glutamate‐receptive areas do not necessarily reflect the areas's demand for elevated oxidative metabolism. While all showing intense labeling for glial GDH also exhibited high levels of CO activity, many additional regions showing high levels of CO activity contained no detectable immunoreactivity for glial GDH. These light‐microscopic comparisons reveal that the energy requirments are not the only factors accounting for the regional heterogeneity of the enzyme. We conclude that glial mitochondria are heterogeneous withe respect to their GDH content and that GDH is enriched in areas exhibiting chronically active glutamatergic transmission.
Synthesis of leukotrienes in frog retina and retinal pigment epithelium
Authors: Bazan N. G. , Bazan H. E. P. , Birkle D. L. , Rossowska M.
Published: January 2005

Leukotrienes (LT) were identified in the intact frog (Rana pipiens) retinalpigment epithelium (RPE), retina, isolated rod outersegments (ROS), and ROS‐free (neuronal) retina. Levels of endogenous LTC4 were measured by radioimmunoassay (RIA) in both unpurified by reverse‐phase high‐performance liquid chromatography (HPLC). The purified LTC4 exhibited characteristic ultraviolet absorption spectrum with λmax at 280 nm. The Ca2+ ionophore A23187 (10 μM) increased LTC4 production in intact and ROS‐free retina and RPE but had no effect on LTC4 levels in isolated ROS. This lack of effect suggests that LTC4 is present but notsynthesized in ROS. Synthesis of radiolabeled LTC4 in frog retina and RPE prelabeled in vivo by intravitreal injection of [1‐14C]arachidonic acid (20:4, n‐6) provided an additional verification of the presence of LTC4 in these tissues. The physiological significance of the presence of these biologically active derivatives of arachidonic acid in photoreceptorcells and retinal pigment epitelium may be related to the interactions between these cells, consisting of photoreceptor membrane shedding and phagocytosis.
Chronic nicotine administration incrreases binding of [3H]Domperidone in rat nucleus accumbens
Authors: Reilly M. A. , Lapin E. P. , Maker H. S. , Lajtha A.
Published: January 2005

An apparent inverse relationship between smoking and Parkinson's disease prompted and investigation of the effect of chronic nicotine administration on dopaminergic and serotonergic receptors in rat brain. Nicotine, 0.8 mg/kg, was injected once daily, five times per week, for 6 weeks. In nucleus accumbens the Kd for [3H]domperidone was increased 2–4‐fold, and the Bmax was increased 1.5–2‐fold. No changes were observed in the binding of [3H]domperidone in caudateputamen or in that of [3H]ketanserin in frontal cortex. It is concluded that chronic nicotine administration may have a suppressant effect on central nervous system release of dopamine that in pre‐parkinsonian persons causes an aversion to the effects of smoking.
Dopamine and serotonin inhibition of neurite elongation of different identified neurons
Authors: McCobb D. P. , Haydon P. G. , Kater S. B.
Published: January 2005

This study demonstrates that a second classical neurotransmitter, dopamine, can act to suppress regenerative neurite outgrowth. Single identified neurons were dissected from two central ganglia of the snail Helisoma, and growth cone motility was studied as neurites regenerated in cell culture. Both dopamine and serotonin inhibited growth cone motility and elongation of neurites. Outgrowth inhibition ranged from sustained arrest to a similar but transient response. The effects of dopamine and serotonin are neuron‐selective. Specific neurons affected by dopamine and serotonin represent distinct sets. One neuron was found that response to both agents. The implications of neurotransmitter regulation of the dynamics of neuronal morphology are discussed.
Developmental effect of dimethyl sulfoxide on hypothalamo‐neurohypophysial neurons in vitro
Authors: Schilling K. L. , Pilgrim C.
Published: January 2005

Primary dissociated cultures were established from diencephalic tissue of 14‐day‐old fetal rats. Neurons exhibiting immunocytochemical staining for neurophsin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio‐immunometric quantitation of neuron‐specific enolase. In contrast, in DMSO‐treated cultures the number of neurophysin‐immunoreactive neurons was more than doubled as compared to control cultures. [3H] Thymidine labeling and autoradiography in conjuction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time‐course analysis of the action of DMSO revealed a 6‐day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin‐immunoreactive cells. These findings suggest that DMSO, which has previously been on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.
Phorbol ester enhances morphological differentiation of oligodendrocytes in culture
Authors: Yong V. W. , Sekiguchi S. , Kim M. W. , Kim S. U.
Published: January 2005

The effects of phorbol esters have been attributed to the activation of the enzyme protein kinase C. While much has been described for the actions of phorbol esters on neurons and synaptic transmission, sparse data exist on the effects of phorbol esters on oligodendrocytes, the cells that make and maintain myelin in the central nervous system. In this report, we show that 10 and 100 nM of a phorbol ester, 4β–phorbol–12, 13–dibutyrate, extensively enhanced process formation by cultured bovine oligodendrocytes. This effect was blocked by two inhibitors of protein kinase C, sodium heparin and polymixin B. We propose the hypothesis that activation of protein kinase C is an important process that leads to the differentiation of oligodendrocytes and the formation of myelin in vivo.
A chemically defined medium for the culture of mature oligodendrocytes
Authors: De Los Monteros A. Espinosa , Roussel G. , Neskovic N. M. , Nussbaum J. L.
Published: January 2005

A new chemically defined medium consisting of equal parts of Dulbecco modified Eagle's and Ham's F‐12 media supplemented with insulin, sodium selenite, putrescine, and D+ galactose, which allows the longterm survival of mature oligodendrocyte pure cultures, is described. Immunohistochemical staining has shown that over 90% of the cells become positive for myelin proteins shortly following subculture. Contaminating astrocytes (2%) do not survive in this medium. Biochemical data have indicated that these purified oligodendrocytes express 2′ 3′‐cyclic nucleotide 3′‐phosphohydrolase and UDP‐galactose ceramide galactosyltransferase activities. Electoron microscopical examination revealed that the oligodendrocytes were mostly of medium‐dark type and appeared to be identical to cells cultured in serum‐containing medium. The ability to maintain pure oligodendrocyte cultures in such a defined medium will allow investigations concerning exogenous and endogenous factors involved in oligodendrocyte metabolism.
Effect of laminin and cytoskeletal agents on neurite formation by NG108‐15 cells
Authors: Kleinman H. K. , Luckenbill‐Edds L.
Published: January 2005

Laminin promotes attachment and process formation in the neuroblastoma x glioma hybrid cell line NG108–15. As cells attached to laminin, they flattened and remained dispersed rather than associated in clumps. Process formation was observed within 1 hr after exposure to laminin and was dose dependent. Cycloheximide, an inhibitor of protein synthesis, did not block laminin‐mediated attachment and neurite formation. Addition of drugs that depolymerize the cytoskeleton led to different behaviors for cells grown on plastic compared with those in the presence of laminin. Cells on plastic treated with either vinblastine or cytochalasin neither flattened nor grew processes. Cells plus laminin and vinblastine retracted processes, but remained flat, suggesting that laminin‐induced processes can be destabilized by disrupting microtubules. Cells sequentially treated with laminin and cytochalasin produced processes that were thin and highly branched. Cells in high concentrations of cytochalasin on a laminin substrate formed aberrant processes even when their soma did not flatten. Since laminin counteracted the effect of cytochalasin on process outgrowth but did not alter the effect of cytochalasin on flattening of the cell body, different mechanisms mediated by microfilaments may be involved in cell flattening and in process formation.
Angiotensinogen gene expression in neuronal and glial cells in primary cultures of rat brain
Authors: Kumar A. , Rassoli A. , Raizada M. K.
Published: January 2005

Neuronal and glial cells in primary culture prepared from the hypothalamic‐brain stem areas of one‐day‐old rat brain were analyzed for the presence of angiotensinogen messenger RNA (mRNA) to further confirm our previous conclusion that the brain contains an exclusive angiotensin system. Angiotensinogen mRNA was quantitated by Northern analysis using nick‐translated angiotensinogen cDNA as the hybirdization probe (Kunapuli and Kumar, 1987). Angiotensinogen mRNA sequences were present in the RNA isolated from both neuronal and glial cultures. Quantitative measurements of the mRNA using dot‐blot analysis revealed that the level of angiotensinogen mRNA was three times higher in neuronal cultures compared with glial cultures. These observations provide the first evidence for the synthesis of angiotensinogen in neuronal as well as in glial cells form the brain.
Comparison of the morphological effects of acidic and basic fibroblast growth factors on rat astroblasts in culture
Authors: Perraud F. , Labourdette G. , Miehe M. , Loret C. , Sensenbrenner M.
Published: January 2005

The effect of acidic and basic fibroblast growth factors (aFGF and bFGF) on the morphology of cultured rat astroblasts and on the expression of glial fibrillary acidic protein (GFAP) were compared. The addition of either aFGF or bFGF affected the morphology of the flat, irregular, polygonal‐shaped astroblasts, which formed processes and acquire a fibrous appearance. Appreciable different morphological aspects were observed between aFGF‐ and bFGF‐induced cells, essentially between 11 and 14 days in culture. In the presence of bFGF the astroglial cells were more fibrous with a more compact perikaryon as compared to aFGF treated cells.

At the ultrastructural level abundant intermediate filaments were observed in astroglial cells as an effect of aFGF and rare filaments but numerous microtubules were seen in bFGF‐treated cells.

The immunoreactivity for GFAP increased with time in culture and was much stronger in aFGF‐treated cells compared to bFGF‐treated cells at day 14. An intense positive staining was observed in the somata of the astroglial cells and their processes in the presence of aFGF, while essentially the processes were stained in the presence of bFGF. After 21 days in culture GFAP immunoreaction was also found in the perikarya of cells treated with bFGF.

These results show that rat astroglial cells respond somewhat differently to aFGF and bFGF.
Localization of ingensin in rat central nervous system and skeletal muscle
Authors: Kamakura Keiko , Ishiura S. , Nonaka I. , Sugita H.
Published: January 2005

A high molecular weight, fatty acid‐ and SDS‐sensitive protease named ingensin was purified from rat brain in this study. The enzyme purified from rat brain has the same biochemical properties as those purified from other tissues, e.g., porcine skeletal muscle, human placenta, and rat liver in our laboratory, and rat skeletal muscle and bovine pituitary gland in other laboratories, independently. Immunoblot bands were detected in the same positions as those in the case of ingensin from rat liver.

In addition, its topographical distribution was studied in rat brain and muscle by means of the immunohistochemical method. The cytoplasm of motor neurons of the spinal cord, pyramidal cells, and granular cells were stained. Axons were stained. The cytoplasm of muscle was also stained, especially that of type 2 fibers.
Spread of Trypanosoma brucei to the nervous system: Early attack on circumventricular organs and sensory ganglia
Authors: Schultzberg M. , Ambatsis M. , Samuelsson E.‐B. , Kristensson K. , van Meirvenne N.
Published: January 2005

The distribution of Trypanosoma brucei brucei in the nervous system of experimentally infected Sprague‐Dawley rats and BALB/c and deer mice was examined with immunohistochemical techniques. The trypanosomes showed an early invasion in areas lacking a so‐called blood‐brain or blood‐nerve barrier, i.e., in sensory ganglia and circumventricular organs including the area postrema, pineal gland, and median eminence. This distribution of trypanosomes may relate to the origin of cardinal symptoms of the disease, e.g., sensory disturbances, nausea, disturbed circadian rhythm, and neuroendocrinological dysfunctions. Trypanosome infections in rodents may provide a model for studies of how an infectious agent or factors released by the immune response may relatively selectively interfere with these functionally defined regions of the nervous system.
Growth and differentiation properties of O‐2A Progenitors purified from rat cerebral hemispheres
Authors: Behar T. , McMorris F. A. , Novotný E. A. , Barker J. L. , Dubois‐Dalcq M.
Published: January 2005

We have used the monoclonal antibody A2B5 (which binds to subclasses of surface gangliosides) to select glial precursor cells from postnatal rat brain and compare their properties in culture with those of the bipotential O‐2A progenitor cells of newborn optic nerve. Two methods, fluorescence‐activated cell sorting (FACS) and differential adhesion, resulted in >90% enrichment in A2B5‐positive bipolar cells and multipolar cells with short processes. These cells expressed vimentin and reacted with yet another antibody (NSP4), which binds to O‐2A progenitor cells of optic nerve. The 2–10% of the remaining cells consisted of type 1 astrocytes and/or microglial cells. When maintained in defined medium for 3 days, 28‐40% of A2B5‐positive cells incorporated thymidine, while most other cells became differentiated into galactocerebroside‐positive oligodendrocytes. In the presence of 10% fetal calf serum for 3 days, over 50% of the cells developed a stellate phenotype and expressed GFAP, characteristic of type 2 astrocytes. This phenotypic plasticity of the A2B5 positive cells was also observed in clones derived from single cells grown on a layer of type 1 astrocytes. Thus, A2B5‐positive cells from cerebrum are O‐2A progenitors that can generate O‐2A lineage cells. The effects of the two growth factors, insulin and platelet derived growth factor (PDGF) (which is synthesized by type 1 astrocytes), were tested on cerebrum O‐2A progenitors. PDGF induced a doubling of the percentage of A2B5‐positive cells incorporating thymidine during a 20‐hr pulse and a large increase (up to 40‐fold) of the progenitor population over 3 days. The largest number of O‐2A lineage cells was obtained when purified progenitors were grown in the presence of PDGF and insulin. Thus, A2B5‐positive glial cells from cerebrum overall behave as the O‐2A progenitors of optic nerve, but they more readily divide than differentiate, as if they were at an earlier stage along the O‐2A lineage pathway.
Ability of developing epithelia to attract neurite outgrowth in culture is not correlated with the appearance of laminin
Authors: Nishi R.
Published: January 2005

Using a new technique for organ explants that facilitates the visualization of developing epithelia, we tested the abilities of salivary gland and lung rudiments to support de novo axonal outgrowth from the embryonic submandibular ganglion. We confirmed that salivary gland epithelia, but not lung epithelia, are able to support axonal outgrowth. This neurite outgrowth is also supported by salivary gland epithelia that have been lightly fixed with paraformaldehyde. When given a choice of both salivary gland and lung epithelia as a substrate for axonal outgrowth, the submandibular ganglion neurons showed an absolute preference for the salivary gland. Immunohistochemical localization of laminin was performed on whole mounts of developing epithelia after growing neurites were localized with a histochemical stain for esterase. Areas of lung epithelium devoid of any neurite outgrowth contained substantial immunoreactivity for laminin. In addition, Western blot analyses of extracts of embryonic lung and salivary gland indicate that the same amount of laminin or more is present on a per protein basis in the lung as in the salivary gland. An antiserum directed against laminin and a monoclonal antibody that blocks axonal regeneration on basal laminae in vitro (INO) were unable to block the outgrowth of axons over fixed epithelia. This suggests that molecules other than laminin are responsible for the preferential growth of axons over the salivary gland epithelia.
Developmental and regional regulation of rab3: A new brain specific “ras‐like” gene
Authors: Ayala J. , Olofsson B. , Tavitian A. , Prochiantz A.
Published: January 2005

Recently, the expression of rab3, a new ras‐like gene, has been shown to be restricted to brain tissues (Olofsson et al., 1988). This finding has prompted us to study the expression of rab3 in different brain regions of the developing mouse. The two transcripts corresponding to rab3 (1.8 and 1.3 kb) were first detected in the brains of E13 mouse embryos and were not randomly distributed. Highest levels were found in the mesencephalon, followed by the cortex, striatum, cerebellum, and brain stem in that order. In vitro, the expression of the 1.8‐kb transcript was neuron specific, whereas the small transcript was present in neurons and astrocytes. This is the first report showing developmental and regional regulation of a nervous system‐restricted ras‐like gene. Based on the homologies found between the rab genes and YPT1 or sec‐4, we suggest that the physiological role of rab3 might be related to the stabilization of the neuronal cytoskeleton or to post‐Golgi vesicle transport and fusion.
Acute effects of lithium on catecholamines, serotonin, and their major metabolites in discrete brain regions
Authors: Gottberg E. , Grondin L. , Reader T. A.
Published: January 2005

The acute effects of lithium on the central catecholamine and serotonin systems were investigated in welldefined cortical areas in the rat: the anterior cingulate cortex (CIN), the piriform‐entorhinal region (PiEn), and the primary visual area (VIS) as well as in the hippocampus (HIP), the neostriatum (CPU; caudateputamen), and the olfactory bulbs (OBs). In these microdissected regions, the catecholamines noradrenaline (NA) and dopamine (DA), the indoleamine 5hydroxytryptamine (5‐HT; serotonin), as well as some of their major metabolites (3‐methoxy‐4‐hydroxyphenylglycol; 3,4‐dihydroxyphenylacetic acid; homovanillic acid; 3‐methoxytyramine; 5‐hydroxy‐ltryptophan; and 5‐hydroxyindole‐3‐acetic acid) were assayed by using high‐performance liquid chromatography (HPLC) with electrochemical detection. One hour after the administration of lithium chloride (2 and 10 mEq/kg; i.p.) the endogenous NA levels increased in the CIN and PiEn cortices, in the HIP, and in the CPU. The DA contents remained unchanged in the CPU, HIP, OB, and VIS cortex but were increased in the CIN and PiEn regions. These increases in cortical DA levels were accompanied by reductions in HVA and DOPAC. The levels of HVA and DOPAC but not 3‐MT were also reduced in the CPU, in spite of a normal DA content. The discrepancies between changes of DA and the levels of its metabolites indicate changes in the turnover rates as well as an action of lithium on DA synthesis and/or storage in the nigrostriatal and mesocortical systems. The 5‐HT contents were also increased by lithium throughout all regions, except for the OB. The 5‐HTP and 5‐HIAA levels remained constant except for slight decreases of 5‐HIAA in the CIN and VIS cortices.