bluuebunny/crossref_metadata_2025 huggingface dataset:
Diffusible factor(s) from adult rat sciatic nerve increases cell number and neurite outgrowth of cultured embryonic ventral mesencephalic tyrosine hydroxylase‐positive neurons
Authors: Collier T. J. , Sladek C. D. , Gallagher M. J. , Gereau R. W. , Springer J. E.
Published: January 2005

Dissociated embryonic rat ventral mesencephalon containing the developing A8‐A10 dopamine (DA) neurons was cultured alone or in the presence of a 10 mm segment of adult rat sciatic nerve that had been explanted and maintained in separate culture for 72 hours prior to introduction to mesencephalic cultures. Nerve segments were contained in a co‐culture basket, so that midbrain cells and nerve shared medium but were not in physical contact. The number and morphology of cultured DA neurons was assessed via immunocytochemistry for tyrosine hydroxylase (TH). Co‐cultures of ventral midbrain tissue and nerve exhibited an increased number of TH‐positive neurons, with larger neuronal perikarya, and increased length and complexity of neurites, than cultures of midbrain tissue alone. Increased number and growth of TH‐positive neurons was obtained with as little as 2 days of exposure to nerve. This evidence suggests that a diffusible, soluble factor(s) from sciatic nerve can enhance the number and development of TH‐positive neurons detected in cultures of embryonic ventral mesencephalon.
Expression of chimeric human transferrin genes in vitro
Authors: Fischbach K. , Lu Y. , Tiffany‐Castiglioni E. , Minter A. , Bowman B. H. , Adrian G. S.
Published: January 2005

Transferrin (TF), a major plasma protein, binds and transports ferric iron. Evidence exists for unique roles for TF in brain in oligodendrocyte differentiation, myelination and neuronal development. In this study, 5' flanking regions of the TF gene important in regulating gene expression were identified by transfected cell studies and a comparison of 5' flanking sequences of the human TF and TF receptor genes. Human glioma cell lines HTB‐16 and HTB‐17 were shown to synthesize TF identical in size and immunological reaction to TF synthesized by liver. The expression of a series of human chimeric TF genes in glioma cells was compared with hepatoma and HeLa cells. A difference in transient expression was observed in hepatoma and glioma cells transfected with TF chimeric genes containing 3.9 kb of the 5' region; hepatoma cells demonstrated significantly more expression than did glioma cells, suggesting that a DNA region present in the 3.9‐kb construct is important either in liver‐specific expression or in repression of brain expression, or in both. Smaller constructs containing ≤ 0.622 kb of the 5' regulatory region of the TF gene failed to demonstrate cell‐specific expression; they were expressed in HeLa cells, a line that does not synthesize TF. High levels of expression of 0.15‐kb TF constructs were also observed in hepatoma and glioma cell lines, but not in transgenic mice. Possible explanations of differences observed in expression of shorter TF constructs in vitro and in vivo are discussed.
Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor
Authors: Clagett‐Dame M. , Chung C. , Chao M. V. , DiStefano P. S.
Published: January 2005

Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF‐R) as well as to a soluble, truncated form of the receptor (NGF‐Rt). The antibodies were generated against partially purified NGF‐Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk−) that expresses large numbers of the low affinity form of the human NGF‐R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan A A, Buck CR, Sehgal A [1986]: Science 232:518–521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross‐linked to [125‐I]‐NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl, k subclass. All monoclonal antibodies bound to both NGF‐R and NGF‐Rt. Two monoclonal antibodies (VIIDl, XIF1) immunoblotted the NGF‐R from E9b cell preparations resolved on non‐reducing sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The antibodies immunoprecipitated NGF‐R from both E9b cells and from SH‐SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF‐Rt, but did not cross‐react with NGF‐R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF‐Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2‐site RISA. The 2‐site RISA can be used to rapidly quantitate NGF‐R and NGF‐Rt in large numbers of biological samples in the absence of added [125‐I]‐labeled NGF.
Differential effect of lithium on fos protooncogene expression mediated by receptor and postreceptor activators of protein kinase C and cyclic adenosine monophosphate: Model for its antimanic action
Authors: Divish M. M. , Sheftel G. , Boyle A. , Kalasapudi V. D. , Papolos D. F. , Lachman Herbert M.
Published: January 2005

Lithium salts are the most effective agents used in treating manic‐depressive illness. It has been suggested that lithium's therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have investigated the effect of lithium on these two signal transduction pathways in PC12 pheochromocytoma cells by studying a common effector target, expression of the fos protooncogene. We find that lithium, at therapeutic doses, has an augmenting effect on phosphatidylinositol (PI)‐mediated fos expression induced by activating a muscarinic cholinergic pathway, whereas it has no effect, at tenfold the therapeutic dose, on fos expression induced by receptor or postreceptor activators of cyclic adenosine monophosphate (cAMP). The lithium augmenting effect is also observed when the cells are treated with phorbol esters, which directly activate protein kinase C (PKC), suggesting that the level of lithium's interaction with the IP pathway is at the postreceptor level. We also show that phorbol esters induce extensive down regulation of subsequent cholinergic and phorbol ester responsiveness as well as heterologous down regulation of cAMP responses. Treatment of down‐regulated cells with lithium leads to an enhanced responsiveness when cells are rechallenged with agonists that activate PKC but not by agonists that stimulate cAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on cAMP‐mediated fos but no effect on the IP pathway. The opposite effects of lithium and carbamazepine on two critical transducing systems suggest a model for the antimanic action of these agents.
Serial passage of embryonic human astrocytes in serum‐free, hormone‐supplemented medium
Authors: Loo D. T. , Sakai Y. , Rawson C. L. , Barnes D. W.
Published: January 2005

We applied serum‐free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassagc culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibreblast growth factor, heparin, high‐density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage culture.
Nerve growth factor‐induced increase in calcium uptake by PC12 cells
Authors: Nikodijevic B. , Guroff G.
Published: January 2005

Treatment of PC 12 cells with nerve growth factor (NGF) produces a rapid and transient increase in calcium uptake into the cells. The increased uptake is maximal after 5 minutes of NGF treatment, but after 15 minutes of NGF treatment, no such increase can be observed. The effect of NGF is partially inhibited by blockers of L‐type calcium channels. K‐252a, an alkaloid‐like kinase inhibitor that usually is found to inhibit the actions of NGF on PC 12 cells, produces an increase in calcium uptake similar to, but smaller than, that seen with NGF. NGF had no effect on calcium release under these conditions.
K+ channel properties in cultured mouse Schwann cells: Dependence on extracellular K+
Authors: Verkhratsky A. , Hoppe D. , Kettenmann H.
Published: January 2005

In cultured Schwann cells, single‐channel and whole‐cell K+ currents can be activated by depolarizing the membrane to values more negative than −50 mV. In elevated extracellular K+ concentration ([K+]o), however, single‐channel activity and whole‐cell currents could be recorded at more negative potentials. Thus, the threshold of current activation was shifted to more negative potentials. This shift in the activation threshold was only observed with normal (50‐60 mM) intracellular [K+] levels; it was not appparent when [K+]j was elevated to 145 mM. The control of [K+ ]0 on the gating properties of K+ channels may serve to en lance the capability of the Schwann cell to take up [K+ ]o and thus may serve for [K+] homeostasis in the peripheral nerve.
Multidrug transport in human glioblastoma cells is inhibited by transforming growth factors type β1, β2, and β1.2
Authors: Schluesener H. J.
Published: January 2005

The transforming growth factors type β1, β2 and β1.2 suppress multidrug transport in human pat‐1 glioblastoma cells and even in cells that strongly over‐express mdr genes and are resistant to inhibition of multidrug transport by chemosensitizers. Thus, inhibition of multidrug transport by cytokines might be a new approach to increase cellular accumulation of chemotherapeutic agents in multidrug resistant glial tumor cells. Interestingly, a member of the more distantly related decapentaplegic subgroup of transforming growth factors, the bone morphogenetic protein BMP 2, did not inhibit multidrug transport.
Effect of nerve growth factor on C‐1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice
Authors: Fink D. W. , Mirkin B. L.
Published: January 2005

The in situ C‐1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentra tion in mice Sympathectomized with 6‐hydroxy‐dopamine (6‐OHDA) on postnatal days 4–10. Mice were implanted with 1 × 106 disaggregated MNB cells 3 days after termination of 6‐OHDA administra tion. NGF (12‐15 μg/mouse/day) treatment was ini tiated at the time of MNB cell implantation and con tinued until sacrifice of the animal. The time interval between tumor cell implantation and detection of pal pable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a de crease in myocardial norepinephrine concentration of 88% compared with vehicle‐treated animals as well as a significant reduction in total body and organ weight Average body, brain, heart, and spleen weights were decreased 31% 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was signif icantly increased in neonatally Sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in Sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in Sympathectomized mice receiving exogenous NGF. The norepinephrine (NE) and dopamine (DA) con centrations of tumors removed 8 days after tumor onset were increased 2.8‐ and 3. 6‐fold, respectively, in neonatally Sympathectomized mice. Administration of NGF prevented the increase in tumor NE and DA concentration associated with chemical sympa thectomy. Cell surface receptors for NGF were present on cultured C‐1300 MNB cells. These data suggested the presence of two types of binding sites with KD values of 45.9 pM (high affinity) and 2.4 nM (low affinity). The inhibition of MNB tumor growth and elevation in tumor catecholamine content pro duced by neonatal sympathectomy may be Immorally mediated since they were selectively reversed by the administration of NGF.
Expression of the β‐nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig
Authors: MacGrogan D. , Desprès G. , Romand R. , Dicou E.
Published: January 2005

Steady‐state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGFmRNAs was in the order vas deferens < epididymis ≥ seminal vesicles > testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF‐like and proNGF‐like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis.
Quantitation of tyrosine hydroxylase and neuropeptide Y immunoreactivity in single rat sympathetic neurons: Effects of preganglionic nerve activity
Authors: Gurusinghe C. J. , Harris P. J. , Abbott D. F. , Messina A. , Bell Christopher
Published: January 2005

Using computerised densitometry to measure immunocytochemical reaction product in a model system, we established conditions that produced a linear relationship between the logarithm of antigen concentration and the measured intensity of staining. We then applied the densitometric technique to assess the changes in tyrosine hydroxylase (TH) and neuropep‐tide tyrosine (NPY) within sympathetic neurons of rat superior cervical ganglion following chronic decentralization and following reserpine treatment. One week after surgical or pharmacological decentralization, there was appreciable reduction of neuronal levels of both TH and NPY. However, there remained considerable variation in the immunoreactivities of individual cells. Three days of treatment with reserpine elevated TH levels but substantially reduced NPY. Both these effects were prevented by prior decentralization of the ganglia. No differences were seen between normotensive and the Otago strain of genetically hypertensive rats, either in basal TH or NPY immunoreactivities or in responses to the maneouvres performed. Comparison of our findings with previous biochemical data indicate that densitometric immunocytochemistry provides an accurate index of neuronally localised antigen concentrations but also allows analysis of interneuronal differences that are not otherwise apparent.
Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli
Authors: Negro A. , Corona G. , Bigon E. , Martini I. , Grandi C. , Skaper S. D. , Callegaro L.
Published: January 2005

The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl‐B‐D‐thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse‐phase high performance liquid chromatography. The amino‐terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS‐PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expresson and purification of human CNTF.
Identification of a cDNA clone specific for the oligodendrocyte‐derived repulsive extracellular matrix molecule J1‐160/180
Authors: Fuss B. , Pott U. , Fischer P. , Schwab M. E. , Schachner M.
Published: January 2005

A cDNA clone specific for the oligodendrocyte‐de‐rived extracellular matrix glycoproteins Jl‐160/180 was obtained from a àZAPII expression library using polyclonal antibodies generated against mouse JI‐160. The library was constructed from poly(A) + ‐RNA isolated from O1 antigen‐positive rat oligodendrocytes. The cDNA clone expressed a fusion protein that was recognized by the J1‐160/180‐specific monoclonal antibodies 596, 619, and 620, and, weakly, 597. The fusion protein was not recognized by polyclonal antibodies to mouse J1/tenascin. The cDNA clone with an insert of approximately 5.6 kb in size contained the nucleotide sequence coding for the amino acid sequence of the N‐terminus of a tryptic peptide derived from mouse J1‐160. The developmental and tissue distribution of the mRNA recognized by the cDNA clone is in agreement with the described expression of the J1‐160/180 proteins.
Age‐related acute depletion of cerebral glutathione by peroxidative stress
Authors: Benzi G. , Curti D. , Marzatico F. , Pastoris O.
Published: January 2005

Cyclohexene‐1‐one and cycloheptene‐1‐one cause a severe age‐related depletion of reduced glutathione (GSH) in the forebrain of 5‐ or 15‐ or 25‐month‐old rats. Chronic pretreatment with phosphatidylcholine partially inhibits the GSH depletion in old forebrains by the prooxidants tested, suggesting that in aged animals alterations in mitochondrial inner membrane phospholipid composition and/or cytochrome oxidase activity might play a role in oxygen free‐radical production.
A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF‐M and NF‐H
Authors: Harris J. , Ayyub C. , Shaw G.
Published: January 2005

We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C‐terminal “tail” sequences of the two high molecular weight neurofilament sub‐units, NF‐M and NF‐H. A series of recombinant fusion proteins containing selected NF‐M and NF‐H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS‐PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF‐M and NF‐H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF‐M and NF‐H sequence. These antibody studies showed that the extreme C‐termini of NF‐M and NF‐H are immunologically absolutely distinct from one another and also indicate that the extreme C‐terminus of NF‐M is immunologically much more conserved than the analogous region of NF‐H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C‐terminus of NF‐M. We also show that the unusual immunological properties of the bovine NF‐M tail we have previously observed do not extend to the extreme C‐terminal region, which appears immunologically no different from the analogous region of other NF‐M molecules. The peculiarities of bovine NF‐M could be explained by the presence of a KSP motif that resembles the NF‐H KSP prototype.
Identification of a tektin‐like protein associated with neurofilaments in the developing chick nervous system
Authors: Edson K. J. , Linck R. W. , Letourneau P. C.
Published: January 2005

A 160‐kD polypeptide, which is recognized by an affinity‐purified polyclonal antibody to the 55‐kD tektin‐A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin‐A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti‐tektin‐A in chick brain and spinal cord extracts has been identified as a 160‐kD polypeptide by SDS‐PAGE and has been shown to be distinct from the known neurofilament‐triplet proteins by two‐dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin‐A is a component of the neuronal cytoskeleton and is incorporated into or associated with neurofilaments.
Depletion of acetylated α‐tubulin during microtubule purification from bovine brain gray and white matter regions
Authors: Kim H.
Published: January 2005

We have followed the fate of acetylated α‐tubulin during microtubule (MT) purification from both gray matter‐ and white matter‐enriched bovine brain regions, using quantitative immunoblot assays employing well characterized monoclonal antibodies specific for acetylated α‐tubulin and all α‐tubulins. Our results show that crude homogenates from both gray matter and white matter brain regions contain the same proportion of acetylated α‐ to total tubulin. We have found that the acetylated isoform cycles more efficiently with MTs from gray matter than with MTs from white matter. However, the resultant purified MT preparations from both gray and white matter regions are greatly depleted in the acetylated isoform compared to the tubulin in the initial homogenates because most of the acetylated α‐tubulin from both tissue sources partitions with the cold‐insoluble fraction of the initial brain homogenate. A low percentage of the acetylated α‐tubulin in brain homogenates does become incorporated into MTs initially, but this sub‐population of acetylated α‐tubulin then becomes associated with a cold‐ and calcium‐insoluble fraction of the MT preparation. These results demonstrate that the standard brain MT and tubulin preparations used by most investigators for in vitro studies are greatly depleted in acetylated α‐tubulin, and thus provide poor model systems for the analysis of the function of this tubulin isoform.
Corticospinal neurons exhibit a novel pattern of cytoskeletal gene expression after injury
Authors: Mikucki S. A. , Oblinger M. M.
Published: January 2005

We examined changes in the expression of major cytoskeletal protein mRNAs in adult hamster corticospinal neurons after axotomy. While a number of studies had determined that peripheral neurons exhibit major alterations in cytoskeletal gene expression after axotomy, no previous studies had addressed the question of whether or not intrinsic mammalian CNS neurons, which do not have the ability to successfully regenerate axons after injury, alter their expression of tubulin and neurofilament genes after injury. In the present study we used in situ hybridization methods to examine this issue. 35S‐labeled cDNA probe for the low molecular weight neurofilament protein (NF‐L) mRNA and an α‐tubulin mRNA species (Mα1) were used for in situ hybridizations of sections of the sensorimotor cortex obtained 2, 7, and 14 days after unilateral axotomy of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed dramatic decreases in both α‐tubulin and NF‐L mRNA levels within axotomized neurons in layer Vb of the sensorimotor cortex. Tubulin mRNA levels were decreased as early as 2 days after injury whereas NF‐L mRNA levels were not decreased until later times. Ribosomal RNA (rRNA) levels in axotomized corticospinal neurons were also examined using in situ hybridization with a 35S‐labeled rDNA probe. These studies showed only a slight decrease in rRNA levels in corticospinal neurons at 14 days after axotomy. Immunoblotting experiments of total protein from corticospinal axons in the medulla were performed to assess whether the axonal composition immediately proximal to the injury site reflected changes in cell body gene expression. Both α‐tubulin and NF‐L levels were found to decrease in corticospinal axons by 28 days after injury. These findings, to our knowledge, are the first to demonstrate that a class of mammalian CNS neurons have an intrinsically different cytoskeletal response to axonal injury than do PNS neurons. The failure to up‐regulate tubulin gene expression following injury may contribute to the ineffective regenerative response of these long‐tract CNS neurons.
Biosynthesis and release of tachykinins from rat sensory neurons in culture
Authors: Vedder H. , Otten U.
Published: January 2005

A long‐term culture system of dissociated rat dorsal root (DRG) and trigeminal ganglion cells with high cell density has been developed. Two to 3 weeks after plating, the cultures consist of a nearly pure population of sensory neurons, which can be kept for more than 2 months in culture.

Cultured neurons synthesize and release the tachykinins substance P (SP) and substance K (SK, neurokinin A) with a time course similar to that observed in vivo. High‐pressure liquid chromatography (HPLC) analysis of peptides extracted from neuronal cultures and synthetic tachykinins revealed identical retention characteristics. Northern blot analysis of mRNAs from cultured cells with a specific tachykinin‐probe demonstrated that the preprotachykinin‐gene is expressed in preparations of both DRG and trigeminal ganglia cells.

Depolarizing stimuli such as high potassium (47 mM) evoked a peptide release from cultured neurons in a strictly Ca++‐dependent manner. Capsaicin, a compound known to stimulate nociceptive sensory neurons, dose‐dependently released tachykinins in concentrations as low as 10−9 M. Only total absence of Ca ++ ions from the incubation medium abolished the capsaicin‐induced peptide release. Nifedipine, a blocker of voltage‐dependent L‐type Ca++‐channels, completely blocked the potassium‐induced release of SP but did not reduce the capsaicin‐evoked release.

Mediator substances of pain and inflammation, such as bradykinin, serotonin, and histamine, triggered the release of tachykinins from sensory neurons in vitro. These results clearly demonstrate that the neurons characterized express properties similar to those of sensory neurons in vivo and provide model systems for detailed studies of the biosynthesis and release of neuropeptides as well as the participation of sensory neurons in pain and inflammatory reactions.
Myelin basic protein specific T cell lines and clones derived from the CNS of rats with EAE only recognize encephalitogenic epitopes
Authors: Bourdette D. N. , Vainiene M. , Morrison W. , Jones R. , Turner M. J. , Hashim G. A. , Vandenbark A. A. , Offner H.
Published: January 2005

The epitope specificities of myelin basic protein (BP) specific T cell lines derived from the spinal cords (SC) and lymph nodes (LN) of rats with experimental autoimmune encephalomyelitis (EAE) were compared. To induce EAE, Lewis rats were immunized with guinea pig (GP)‐BP and complete Freund's adjuvant. Mononuclear cells from the SC and LN of these animals proliferated in response to BP and the purified protein derivative (PPD) of mycobacterium. After initially being cultured in growth medium, SC mononuclear cells had an enhanced response to BP and lost their response to PPD. LN cells cultured in identical conditions lost their response to both BP and PPD. LN‐derived BP specific cell lines recognized only two epitopes of GP‐BP: an encephalitogenic epitope in residues 72–89 and a non‐encephalitogenic epitope in residues 43–68. SC‐derived BP specific cell lines and clones recognized the 72–89 epitope and a second encephalitogenic epitope contained in residues 87–99 but not the non‐encephalitogenic 43–68 epitope. Unlike those from LN, BP‐specific T cell lines and clones derived from the CNS appear to recognize only encephalitogenic epitopes, including epitopes not recognized by LN lines.
Hyposmolarity‐induced taurine release in cerebellar granule cells is associated with diffusion and not with high‐affinity transport
Authors: Schousboe A. , Olea R. Sánchez , Morán J. , Pasantes‐Morales H.
Published: January 2005

The effects of hyposmotic conditions on taurine uptake and release were studied in mice cultured cerebellar granule cells. The effect of DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate) and of the divalent cations Mg++ and Mn++ on the hyposmolarity‐induced changes in these parameters was investigated. Mg++ (20 mM) and Mn++ (5mM) inhibited by 25% and 41%, respectively, the release of taurine observed in 30% hyposmolar media. DIDS (100 μM) inhibited this release by 46%. Taurine efflux evoked by 50% hyposmolar solutions was reduced about 40% by Mg++ and 55% by Mn++. Taurine uptake into the granule cells could be resolved into a high‐affinity carrier‐mediated component plus a nonsaturable diffusion component. The kinetic constants (Km and Vmax) for the high‐affinity uptake were unaffected by a 50% decrease in the osmolarity. The diffusion constant for the nonsaturable taurine uptake was increased from 1.5 × 10−4 in isosmotic media to 4.6 × 10−4 ml × min−1 mg−1 in hyposmotic (50%) media. This increase in the diffusional component of taurine uptake elicited by the hyposmotic condition was inhibited approximately 25% by either 100 μM DIDS or 5 mM Mn++. These results strongly suggest that the increase in taurine release induced by swelling under hyposmotic conditions is mediated by a diffusional process and not by a reversal of the high‐affinity taurine carrier.
Increase of macrophage colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor receptors in the regenerating rat facial nucleus
Authors: Raivich G. , Gehrmann J. , Kreutzberg G. W.
Published: January 2005

Proliferation of microglial cells commonly occurs in the response of the central nervous system to injury, but little is known about how this process is regulated in vivo. Here we have studied the expression of receptors to macrophage colony‐stimulating factor (MCSF) and granulocyte‐macrophage colony‐stimulating factor (GMCSF) in the normal and regenerating rat facial motor nucleus using receptor immunocytochemistry and in situ ligand binding methods. Under normal conditions, immocytochemical staining with anti‐MCSF receptor (MCSFR) antibody revealed a moderate but selective labelling of microglia‐like cells of the facial motor nucleus. This immunostaining also colocalized with MUC102, a new monoclonal antibody raised against microglial cells in the rat central nervous system. Axotomy of the facial nerve led to a rapid increase in MCSFR‐staining intensity 1 day after injury, which became maximal 7 days postoperatively and then decreased. A similar but somewhat slower increase was also observed for the specific [125I]MCSF binding wiht a maximum at 7 days Specific [125I]GMCSF binding also increased, peaking at 4 days postoperatively and then rapidly decreasing to normal levels at 21 days after axotomy. In summary, axotomy of the facial nerve led to a rapid increase in receptors for MCSF and GMCSF, which coincided with the pattern of microglial proliferation in the regenerating facial motor nucleus. This apparent up‐regulation of receptors for microglial growth factors may play an important role in preparing the microglia to participate in the cellular response to injury in the regenerating central nervous system.
Galactocerebroside and sulfatide independently mediate Ca2+ responses in oligodendrocytes
Authors: Dyer C. A. , Benjamins J. A.
Published: January 2005

Galactocerebroside (GalC) and sulfated galactocerebroside (sulfatide) are sphingolipids highly enriched in myelin. The binding of antibodies reactive with either sulfatide Or GalC to cultured Oligodendrocytes causes a Ca2+ influx, followed by microtubule depo lymerization; however, antisulfatide is less effective than anti‐GalC in altering cytoskeleton. Typical Ca2+ responses are delayed for both antibodies but are transient for sulfatide‐reactive antibodies in contrast to the sustained responses previously reported for anti‐GalC (Dyer and Benjamins, J Cell Biol 111:625–633, 1990). Approximately one‐half as many oligodendrocytes respond to sulfatide‐reactive antibodies (about 39%) as to anti‐GalC (about 75%). Subpopulations of Oligodendrocytes were identified that responded to neither antibody, only one antibody, or both antibodies, indicating that sulfatide and GalC independently mediate Ca22+ responses. These results suggest that sulfatide and GalC have different physiologic roles in regulating elaboration of myelin membrane by oligodendrocytes in vivo and support the possibility that viral or immune attack via GalC or sulfatide on oligodendrocytes may mimic normal signals in a manner that disrupts the sequence of events that coordinates myelination or maintenance of myelin in vivo.
Cellular immune crossreactivity between myelin basic protein and synapsin in rats with experimental allergic encephalomyelitis
Authors: De Santis M. L. , Roth G. A. , Cumar F. A.
Published: January 2005

We previously demonstrated that antibodies against myelin basic protein (MBP) obtained from animals with experimental allergic encephalomyclitis (EAE), induced with MBP and purified by affinity chromatography, have the property to recognize a neuronal protein, synapsin Ia and Ib. To investigate whether this Crossreactivity also occurs at the cellular level, we purified spleen and lymph node mononuclear cells from rats sensitized with MBP or synapsin using polystyrene plates coated with the respective antigen. We observed that animals injected with MBP have T lymphocytes that bind both antigens. Using the same system, each purified cell population was confronted again to the studied antigens. The anti‐MBP cells recognized once more epitopes of MBP and about 40% of them also recognized synapsin. On the other hand, cells that first were attached to synapsin, in the second exposure to antigens bound to MBP and synapsin in similar amounts. Double immunofluorescent labeling of the mononuclear cells isolated from animals injected with bovine myelin or MBP showed that the same lymphocyte was able to recognize MBP as well as synapsin. In both experimental systems the quantitative results were similar indicating that in bovine myelin‐ or MBP‐sensitized animals practically all the cells that recognize synapsin are anti‐MBP cells, and of the total cells raised against MBP, around 40% of them show this Crossreactivity. On the contrary, animals injected with synapsin have cells that bind to this protein but not to MBP indicating that the described Crossreactivity, as observed at humoral level, is only in one way. These results suggest that the lymphocytes sensitized against MBP that also recognize synapsin could be of relevance in the pathogenesis of EAE, since neuronal structures arc affected by the immunological response to MBP.
Preparation of a protein‐free total brain white matter lipid fraction: Characterization of liposomes
Authors: Díaz R. S. , Monreal J. , Regueiro P. , Lucas M.
Published: January 2005

A method of preparing a total lipid extract (TLE), free of protein, by extracting brain white matter with tetrahydrofuran is presented. The optimal conditions of extraction were found to be 50 ml of THF per gram of lyophilized tissue, though fresh tissue can also be used if larger volumes of solvent are employed. The method allowed, in a short time and in a single step, a yield of TLE of 50% on a dry weight basis. Its analytical characterization revealed a qualitative and quantitative composition very similar to the lipid composition of CNS myelin, including all the phospholipid and galactolipid species, cholesterol and gangliosides, but it contained only traces (0.1%) of protein. TLE has been used to prepare liposomes, either multilamellar (MLVs) or unilamellar (LUVs, SUVs), characterized by freeze ‐fracture electron microscopy. A multilayered, heterogeneous population of liposomes is observed in the MLVs preparation. When these samples were submitted to a freezing and thawing procedure the resulting liposomes were single‐walled, and their intravesicular volume was increased. They were quite impermeable to the mono‐valent cation 86Rb+ and, by contrast rather permeable to 45Ca+ +. Their complex lipid composition, together with their permeability properties and their response to ionophores, make them very useful to study protein‐lipid interactions occurring within the myelin membrane as well as the functional properties of myelin proteins in reconstitution experiments.
Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus
Authors: Silverman R. C. , Gibson M. J. , Silverman A.‐J.
Published: January 2005

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons andgrowth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbo‐cyanine perchlorate (Dil) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With Dil crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most Dil positive cells in the graft contained GnRH. These results indicate that connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.
CG‐4, A new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type‐2 astrocytes
Authors: Louis J. C. , Magal E. , Muir D. , Manthorpe M. , Varon S.
Published: January 2005

We have established a permanent cell line (CG‐4) of rat central nervous system glial precursors from primary cultures of bipotential oligodendrocyte‐type 2‐astrocyte (O‐2A) progenitor cells, which were kept proliferating with the mitogen(s) secreted by the neuronal B104 cell line. The CG‐4 cells have a normal karyotype and display the properties of normal O‐2A cells. CG‐4 cells can be propagated in serum‐free culture medium supplemented with medium conditioned by B104 cells for unrestricted periods of time as O‐2A cells, characterized by the presence of the A2B5 surface marker and the absence of markers specific for Oligodendrocytes (galactocerebroside, myelin basic protein) or type 2‐astrocytes (glial acidic fibrillary protein). bFGF and PDGF are potent mitogens for CG‐4 cells and their combination can substitute for the B104‐derived mitogen(s). CG‐4 cells are capable of differentiating into either Oligodendrocytes or type 2‐astrocytes. Differentiation into Oligodendrocytes occurs after withdrawal of the mitogen. Replacement of the mitogen with fetal calf serum (20%), in contrast, induces 50% of the CG‐4 cells to differentiate into type 2‐astrocytes. Pure cultures of Oligodendrocytes or type 2‐astrocytes can be generated in substantial amounts from CG‐4 cells and maintained for several weeks in medium containing 5% fetal calf serum.
Coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in mouse clonal hybrid neurons derived from the septal region
Authors: Satoh Jun‐ichi , Gallyas F. , Endoh M. , Yamamura T. , Kunishita T. , Tabira T.
Published: January 2005

Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum‐containing medium without extra differentiating agents, one‐third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200‐kDa neurofilament protein, neuron‐specific enolase, microtubule‐associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy‐1.2, saxitoxin‐binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis. © 1992 Wiley‐Liss, Inc.
Dibutyryl cyclic AMP stimulates expression of ependymin mRNA and the synthesis and release of the protein into the culture medium by neuroblastoma cells (NB2a/d1)
Authors: Shashoua Victor E. , Nolan P. M. , Shea T. B. , Milinazzo B.
Published: January 2005

Northern blot, immunoprecipitation, and gel electrophoretic data demonstrate that the mouse neuroblastoma NB2a/d1 cells express ependymin mRNA and synthesize and release into the culture medium a protein with immunoreactivity and electrophoretic mobility properties identical to ependymin. This is a brain extracellular glycoprotein that has been implicated in the consolidation process of memory formation and neuronal regeneration. In labeling experiments with 35S‐methionine, dibutyrylcyclic3′,5′‐adenosine‐monophosphate (dbcAMP) was found to stimulate the expression of ependymin mRNA and the enhanced synthesis and release of ependymin into the culture medium at the same time that dbcAMP stimulation of neurite outgrowth takes place. These results are consistent with the proposed role of the protein in the mechanism of neuronal regeneration and synaptogenesis. The data indicate that the NB2a/d1 cell line is a good model system for studies of the functional properties of ependymin. © 1992 Wiley‐Liss, Inc.
Effect of dark rearing on the volume of visual cortex (areas 17 and 18) and number of visual cortical cells in young kittens
Authors: Takács József , Saillour P. , Imbert M. , Bogner M. , Hámori J.
Published: January 2005

The surface area, total volume, and total number of neurons of areas 17 and 18 in one hemisphere of dark‐reared (DR), dark‐reared and light‐exposed (DRL), and normally reared (NR) kittens were studied at the age of 6 weeks. The thickness of the visual cortex was lower by 13% and 11% (area 17) and by 17% and 16% (area 18) in DR and DRL groups, respectively, when compared with similar cortical areas in NR kittens. The surface area values of area 17 were nearly the same in DR and DRL kittens, both being, however, 37% smaller than in NR animals. The surface area of area 18 was significantly smaller than that of area 17 in each group, and was also lower in DR (by 27%) and DRL (by 21%) groups when compared with the NR group. As a consequence of dark rearing, the numerical density of cortical neurons in area 17 amounted to about double of the value observed in normally reared kittens and was also significantly higher in area 18. The numerical density of nerve cells of DRL kittens fell between the DR and NR groups. The total cortical volume of area 17 was similar in DR and DRL groups but it was by 46% (DR) and by 44% (DRL) smaller than in NR Kittens. In each experimental group, the total volume of area 18 was significantly smaller than that of area 17. The cortical volume of area 18 was also smaller than in the NR group by 39% and 34% in DR and DRL groups, respectively. In DR and NR kittens, the total numbers of neurons in areas 17 (DR =; 26.4 million, NR =; 25.7 million) and 18 (DR =; 8.5 million, NR =; 9.0 million) were essentially similar. In the DRL groups a significantly smaller number of cortical neurons was found both in area 17 (21.5 million) and in area 18 (6.8 million). It is concluded that, in spite of considerable differences in the cortical thickness, surface area, numerical density, and total cortical volume, the absolute numbers of neurons in area 17 and 18 of visually deprived (DR) and NR kittens do not differ at 6 weeks of age. The main deficit in cortical organization following dark rearing, therefore, appears to be confined mainly to the neuropil, as a result of an underdevelopment of neuronal processes and of depressed synaptic organization. The significant decrease of the total cortical cell number both in areas 17 and 18 in the DRL group, in which dark rearing was followed by a brief exposure to light, is apparently the consequence of a rapid cell loss in the visual cortex. © 1992 Wiley‐Liss, Inc.