bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon γ, interleukin 4 and transforming growth factor β in experimental autoimmune encephalomyelitis in lewis rats: Dynamics of cellular mrna expression in the central nervous system and lymphoid cells
Authors: Issazadeh S. , Mustafa M. , Ljungdahl Å. , Höjeberg B. , Dagerlind Å. , Elde R. , Olsson Tomas
Published: January 2005

The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon ‐γ (IFN‐γ), interleukin 4 (IL‐4) and transforming growth factor β (TGF‐β) both in sections of spinal cords and the antigen‐induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63–88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats.

In spinal cords, the target organ in EAE, cells expressing mRNA for IFN‐γ, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.), and then gradually decreased concomitant with recovery. Very few IL‐4 mRNA‐expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF‐β did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN‐γ, while TGF‐β may act to limit central nervous system (CNS) inflammation.

In lymphoid organs, primed MBP 63–88 reactive T cells showed an interesting time‐dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63–88‐induced production of both IFN‐γ and IL‐4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63–88 induced lymphoid cells to TGF‐β production. Thus, an autoantigen‐specific production of TGF‐β occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically. © 1995 Wiley‐Liss, Inc.
Kainic acid‐induced seizures and brain damage in the rat: Role of calcium homeostasis
Authors: Berg M. , Bruhn T. , Frandsen Aa. , Schousboe A. , Diemer N. H.
Published: January 2005

Seizure activity induced by kainic acid (KA) and subsequent neuronal death are thought to be associated with an increase in cytoplasmic free calcium ([Ca2+]i) and can be prevented by N‐methyl‐D‐aspartate (NMDA) antagonists. In addition to influx through receptor operated Ca2+ channels the increase in [Ca2+]i may be the result of an increased influx through voltage‐operated calcium channels and/or release from intracellular deposits. It was therefore investigated whether compounds other than NMDA antagonists with known actions on the intracellular Ca2+ homeostasis had any protective effect against KA‐induced neuronal death. Voltage‐operated calcium channels in the cell membrane were blocked with the L‐type ion channel antagonist, Nimodipine (1.0 mg/kg), and release of Ca2+ from internal stores was prevented with Dantrolene (10 mg/kg). Animals from two control groups injected with kainate (8 mg/kg) exhibited a survival rate of 67 and 53%, respectively. Countings of neurons in dorsal hippocampus showed subtotal or total loss in the CA1 and CA3 subregions. There were no significant differences concerning seizure and survival rates in the groups injected with kainate and treated with Dantrolene or Nimodipine and the control groups. The group treated with Dantrolene showed no neuropathological changes in the hippocampal CA3 region and only slight changes in the CA1 region, while the neuron loss in the Nimodipine group did not differ from that of its control group. The results emphasize the importance of Dantrolene‐sensitive Ca2+ release from intracellular stores for the development of seizureinduced neuronal death. © 1995 Wiley‐Liss, Inc.
N‐methyl‐D‐aspartate‐evoked release of cyclo‐oxygenase products in rabbit hippocampus: An in vivo microdialysis study
Authors: Łazarewicz J. W. , Salińska E.
Published: January 2005

In vivo microdialysis of the rabbit hippocampus was used to study the effects of N‐methyl‐D‐aspartate (NMDA) receptor stimulation on dialysate concentrations of thromboxane B2 (Tx B2)‐ and 6‐keto prostaglandin F1α (6‐keto PGF1α)‐immunoreactive materials that are stable metabolites of biologically active thromboxane A2 and prostacyclin. All pharmacological substances were applied in the dialysis medium. The application of 1 mM NMDA for 20 min resulted in five‐ and eightfold increases in Tx B2 and 6‐keto PGF1α concentrations, respectively. An increase in NMDA concentration to 2.5 mM did not potentiate a peak eicosanoid release, but significantly prolonged this effect. Either 10μM MK‐801 or the extrusion of Ca2+ from the dialysis medium inhibited the release by about 50%. Quinacrine, a phospholipase A2 inhibitor (250 μM), decreased the NMDA‐evoked eicosanoid release by 30%, whereas 10μM indomethacin, a cyclo‐oxygenase inhibitor, completely suppressed the release. One hundred micromolar furegrelate, an inhibitor of thromboxane synthase, reduced by 75% Tx B2 release with concomitant 100% increase in 6‐keto PGFμ formation. Thus, stimulation of NMDA receptors induces calcium‐dependent formation of thromboxane A2 and prostacyclin in the hippocampus, which may have pathophysiological implications. The neuronal site of their formation seems probable, although a transcellular mechanism of their synthesis should be also considered. © 1995 Wiley‐Liss, Inc.
Biosynthesis and regulation of expression of the HNK‐1 epitope on myelin‐associated glycoprotein in a transfected cell model system
Authors: Pedraza L. , Spagnol G. , Latov N. , Salzer James L.
Published: January 2005

The HNK‐1 antibody recognizes a carbohydrate epitope expressed by many cell adhesion molecules in the nervous system that has been proposed to be an important adhesive determinant. This epitope is particularly prominent on the myelin‐associated glycoprotein (MAG) and is related to the antigenic target in an autoimmune mediated demyelinating neuropathy. Elucidation of the mechanisms underlying the biosynthesis and regulation of expression of the HNK‐1 epitope is therefore likely to have important functional and clinical implications. In order to investigate its biosynthesis and the regulation of its expression, we have expressed both human and rat MAG in several different cell lines by retroviral infection. These studies indicate that the cellular milieu determines whether the HNK‐1 epitope is expressed on the MAG polypeptide and provide an explanation for the significant variation in HNK‐1 levels that has been noted in different species. Using a transfected human neuroblastoma line, we have determined that this epitope is present on the fourth and/or fifth immunoglobulin‐like domain of rat MAG and that it is added intracellularly, probably in the trans Golgi. Finally we have found that expression of the HNK‐1 epitope is increased by activation of different second messenger systems, providing direct evidence that its expression can be regulated independently from that of the MAG polypeptide.© 1995 Wiley‐Liss, Inc.
Cyclic adenosine‐3′,5′‐monophosphate potentiates the synaptic potential mediated by NMDA receptors in the amygdala
Authors: Huang C. C. , Gean P. W.
Published: January 2005

An in vitro slice preparation of rat amygdala was used to study the actions of forskolin and cyclic adenosine‐3′,5′‐monophosphate (cAMP) analogues on the N‐methyl‐D‐aspartate (NMDA) receptor‐mediated synaptic potential (EPSPNMDA). Intracellular recordings were made from basolateral amygdala neurons in the presence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10μM) and picrotoxin (50 μM) to pharmacologically isolate the EPSPNMDA. Application of forskolin (25 μM) markedly and persistently potentiated the EPSPNMDA In contrast, the inactive forskolin analogue, 1,9‐dideoxy‐forskolin, failed to affect the EPSPNMDA significantly. Superfusion of dibutyryl‐cAMP (dbcAMP, 200 μM) for 15 min caused a transient depression of the amplitude of EPSPNMDA. The EPSPNMDA amplitude was reduced to 68 ± 3% of control (n = 10) 15 min after the application, restored to its control value within 25 min, and followed by a long‐term potentiation (LTP). Pretreating the slices with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 5 μM), a selective A1 receptor antagonist, blocked the transient depressive phase produced by dbcAMP. This result suggests that the transient depression induced by dbcAMP was likely due to the interaction of dbcAMP or its breakdown products with adenosine A1 receptors.

To determine the site of action, we examined the effect of forskolin on the postsynaptic responses to exogenously applied NMDA. Forskolin potentiated the postsynaptic depolarization induced by NMDA, suggesting that the enhancement is mediated, at least in part, by a persistent upregulation of postsynaptic NMDA receptor‐operated conductances.

Occlusion experiments were performed to examine whether the sustained enhancements of EPSPNMDA produced by tetanic stimulation (TS) and forskolin share a common mechanism. Three episodes of TS were delivered to saturate the LTP and, under this condition, forskolin still caused a further potentiation of the EPSPNMDA. Similarly, TS, delivered after the EPSPNMDA was enhanced by forskolin or dbcAMP, produced LTP. These results suggest that the long‐term enhancements of EPSPNMDA produced by TS and forskolin are different and thus do not support the hypothesis that activation of protein kinase A triggers LTP. © 1995 Wiley‐Liss, Inc.
Purification and characterization of a low molecular weight endogenous glutamate binding inhibitor (LGBI) in porcine brain
Authors: Lee Y.‐H. , Bhattacharyya A. , Tang X. Wen , Seah E.‐C. , Schmidt K. , Deupree D. L. , Wu Jang‐Yen
Published: January 2005

One of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse‐phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (< 2 kD) that is heat‐and acid/base‐stable. The highly purified LGBI has no effect on GABAA and benzodiazepine receptor binding. The LGBI is not L‐glutamate, L‐aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L‐glutamate binding is reversible, and it only changes the Bmax while the Kd remains the same. Since the membrane preparations used for [3H]L‐glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)‐sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined. © 1995 Wiley‐Liss, Inc.
Orphan receptor COUP‐TF I antagonizes retinoic acid‐induced neuronal differentiation
Authors: Neuman K. , Soosaar A. , Nornes H. O. , Neuman Toomas
Published: January 2005

Chicken ovalbumin upstream promoter‐transcription factors (COUP‐TF) are expressed in the developing nervous system and interact with nuclear hormone receptors to regulate expression of different genes. The role of COUP‐TF orphan receptors in neurogenesis is virtually unknown. To study the possible function of COUP‐TF I during neuronal differentiation, we generated COUP‐TF I overexpressing teratocarcinoma PCC7 cell lines and analyzed retinoic acid (RA)‐induced neuronal differentiation of these cells. COUP‐TF I overexpression results in the blockade of morphological differentiation after induction to differentiate. COUP‐TF I represses expression of micro‐tubule‐associated protein 2 (MAP2) gene and delays induction of growth‐associated protein 43 (GAP43) gene expression. In contrast, expression of the neurofilament light subunit (NF‐L) gene is not affected by COUP‐TF I overexpression during neuronal differentiation. Also, cells overexpressing COUP‐TF I do not stop proliferating after RA and dBcAMP treatment and possess suppressed transcriptional activation from different RA response elements. These results suggest that COUP‐TF I plays an important role in regulating RA‐induced neuronal differentiation.© 1995 Wiley‐Liss, Inc.
GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion
Authors: Parramón M. , González M. P. , Herrero M. T. , Oset‐Gasque M. J.
Published: January 2005

The activation of GABAB receptors of adrenal chomaffin cells produces an increase of [Ca2+]i measured by fura‐2 AM techniques. GABAB agonists 3‐aminopropylphosphinic acid or (‐)baclofen, at concentrations of 0.5 mM, increased basal Ca2+, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca2+ buffer.

The GABAB‐induced increase of [Ca2+]i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L‐type voltage‐dependent Ca2+ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω‐conotoxin, which blocks N‐type channels, did not produce any change in the GABAB‐evoked Ca2+ increment. The second component was due to a release of Ca2+ from intracellular pools and was about one‐third of the total GABAB‐induced increase of [Ca2+]i. GABAB receptors stimulated inositol 1,4,5‐trisphosphate‐sensitive and not the caffeine‐sensitive Ca2+ store. In a low Ca2+ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3‐APPA nor baclofen were able to produce an additional increase of [Ca2+]i, whereas 4 mM caffeine had no effect on GABAB response. This intracellular Ca2+ mobilization could be due to inositol 1,4,5‐trisphosphate accumulation produced by the activation of GABAB receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5‐trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3‐APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/106 cells), respectively.

In the absence of extracellular Ca2+, GABAB‐evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca2+ in GABAB mechanisms in adrenal chromaffin cells. © 1995 Wiley‐Liss, Inc.
Hyperammonemia increases serotonin 1A receptor expression in both rat hippocampus and a transfected hippocampal cell line, HN2‐5
Authors: Alexander J. J. , Banerjee P. , Dawson G. , Tonsgard J. H.
Published: January 2005

Hyperammonemia is an important cause of cerebral dysfunction in liver failure. We used two well‐established models to induce hyperammonemia in rats, injection of urease and injection of methionine sulfoximine (MSO). Urease gave a 10‐fold increase in blood ammonia while MSO, a glutamine synthetase inhibitor, gave a 4‐fold increase in blood ammonia with no increase in brain glutamine levels. We observed a 2‐fold increase in 5‐HT1A receptor (5‐HT1A‐R) expression ([3H] 8‐OH‐DPAT binding) in hippocampus, and little change elsewhere, including thalamus in both models, thus eliminating a role for increased glutamine in the receptor induction. In contrast, a 4 to 8‐fold increase in 5‐HT1A‐R mRNA was observed both in hippocampus and thalamus, suggesting some post‐transcriptional regulation. In the absence of glutamine, ammonium acetate treatment of a hippocampal cell line which had been engineered to stably express the 5‐HT1A‐R (HN2‐5) gave a 1.5‐fold increase in [3H] 8‐OH‐DPAT binding and a 4‐fold increase in the mRNA levels for the 5‐HT1A‐R. We conclude that the cell line HN2‐5 is a good model for studying some of the biochemical sequelae of hyperammonemia and that changes in brain function are not only at the metabolic level, as thought earlier, but can also occur at the transcriptional level. © 1995 Wiley‐Liss, Inc.
Chromatographic and immunochemical characterization of rat brain neuropeptide Y‐like immunoreactivity (NPY‐LI) following repeated electroconvulsive, stimuli
Authors: Stenfors Carina , Mathé A. A. , Theodorsson E.
Published: January 2005

The present work investigates the possible existence of molecular heterogeneity of endogenous neuropeptide Y (NPY) in the rat brain and, if existing, whether it is affected by repeated electroconvulsive stimuli (ECS). Different column chromatographic techniques (gel‐permeation chromatography, HPLC, ion‐pair reverse‐phase HPLC) were used combined with immunochemical methods based on antisera directed towards two different epitopes of NPY (antiserum N1, directed towards the midportion of NPY, and antiserum C1, directed towards the C‐terminal end of NPY). Following repeated ECS increased concentrations of NPY were seen in the occipital cortex and hippocampus (P<0.001) when analyzed by direct radioimmunoassays (RIA). Chromatographic characterization showed that the NPY mainly consisted of intact NPY (1‐36) and sulphoxidated form of NPY, no short C‐terminal homologues were found. This is of importance since it has been shown that NPY (1‐36) has different biological properties compared to C‐terminal homologues. © 1995 Wiley‐Liss, Inc.
Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures
Authors: Abd‐El‐Basset E. , Fedoroff S.
Published: January 2005

We examined the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the morphology, cell motility, cytoskeletal organization, and phagocytic activity of microglia in tissue cultures initiated from neopallia of newborn C3H/OuJ mice. Normally, the microglia in our cultures are non‐migratory and Mac‐1 positive, have ameboid cell morphology, no polarity, many short processes that extend into lamellipodia in opposing directions, and undulating cell membrane projections.

When 1–5 μg/ml LPS is added to such cultures, some cells acquire polarity by forming a large lamellipodium and begin to migrate. Two hours later migration ceases; the membrane undulations stop; and the cells become non‐polar, assume a large, round, flat shape, and gradually develop many microspikes all over the cell body. Those cells that do not transform into large, round, flat cells enlarge and extend numerous lamellipodia in opposing directions.

We found that the cytoskeleton of microglia is composed of actin, vimentin‐containing intermediate filaments (IF) and microtubules (MT). Vimentin‐containing IF and MT form dense networks that radiate into the cell periphery, whereas F‐actin is diffusely arranged throughout the cytoplasm. The LPS‐treated cells show changes in the organization of the main components of the cytoskeleton. F‐actin is reorganized by the formation of bundles‐ underneath and parallel to the cell membrane and other bundles projecting into the cores of the microspikes. The vimentin‐containing IF dense network reorganizes into two condensed rings, with fine strands of IF extended between the two rings and the MT networks become less dense and extend throughout the cytoplasm. The LPS treatment potentiates the phagocytic activity of the microglia. However, approximately 30% of microglia lose the expression of MHC class II antigens. © 1995 Wiley‐Liss, Inc.
Functional topography of myelin‐associated glycoprotein. II. Mapping of domains on molecular fragments
Authors: Meyer‐Franke A. , Tropak M. B. , Roder J. C. , Fischer P. , Beyreuther K. , Probstmeier R. , Schachner M.
Published: January 2005

The myelin‐associated glycoprotein (MAG), an adhesion molecule of the immunoglobulin (Ig) superfamily with five Ig‐like domains, was investigated with regard to its binding site(s) for the neuronal cell surface, collagen I, and heparin, using a panel of new monoclonal antibodies and cyanogen bromide cleavage fragments of MAG. All antibodies generated competed with each other for binding to MAG, indicating that they reacted with identical or closely related epitopes. Mapping of the reactive epitopes on recombinant deletion fragments of MAG expressed by Chinese hamster ovary (CHO) fibroblasts showed reactivity of monoclonal antibody 513 with domains I, II, and III, comprising the amino‐terminal end of the extracellular domain. Monoclonal antibody 15 recognized domain III only. Binding of MAG‐containing liposomes to neurons was blocked by antibodies 15 and 513. Cyanogen bromide (CNBr) fragments of domains I, II, and III bound to collagen type I under isotonic buffer conditions. CNBr fragments containing domains I and II were involved in binding to heparin. These observations suggest that domain III may be important for binding to the neuronal cell surface receptor for MAG, while domains I, II, and III interact with collagen type I and domains II and III with heparin. © 1995 Wiley‐Liss, Inc.
NGF‐producing transfected 3T3 cells: Behavioral and histological assessment of transplants in nigral lesioned rats
Authors: Westlund K. N. , Lu Y. , Kadekaro M. , Harmann P. , Terrell M. L. , Pizzo D. P. , Hulsebosch C. E. , Eisenberg H. M. , Perez‐Polo J. R.
Published: January 2005

The rodent fibroblast clonal cell line, 3T3, was retrovirally transfected with the rat nerve growth factor (NGF) gene and selected for NGF synthesis. This study tested the hypothesis that transplanted 3T3 cells, transfected to secrete nerve growth factor (3T3NGF+), change motor behavioral indices created by striatal denervation in a dose‐dependent fashion. 3T3NGF+ cells were transplanted into the lateral ventricle of rats following ipsilateral lesions of the substantia nigra pars compacta by stereotaxic injections of 6‐hydroxydopamine (10 μg), an established lesion model. Control groups included vehicle injections and transplanted untransfected cells. The extent of the lesions was measured by determining rotational behavior before and two weeks after transplantation. Immediately prior to transplantation, cells were incubated with the fluorescent dye marker, Dil. To assess cell viability, whole brains were cryosectioned and examined for Dil‐labeled 3T3 cells using fluorescent microscopy. The number Uf Dil‐labeled profiles in five animals per group were counted in at least five noncontiguous sections per animal. From these data a statistically derived estimate of viable, transplanted 3T3 cells was obtained. The number of surviving transplanted cells correlated with the behavioral changes measured. The 3T3NGF + transplants reduced rotational behavior, while control 3T3 transplants exacerbated rotational behavior. Thus, while NGF delivery was found to be beneficial, it was apparent that naive 3T3 had detrimental effects. These results underscore the importance of making doseresponse measurements when attempting transplantbased modifications of CNS behavior. © 1995 Wiley‐Liss, Inc.
Stimulation of choline acetyl transferase activity by l‐ and d‐carnitine in brain areas of neonate rats
Authors: Ayala C. A.
Published: January 2005

Acetyl‐CoA supply to the cytosol and its regulatory influence on acetylcholine biosynthesis is still an unsolved question. Acetylcarnitine through the carnitine acetyl transferase (CarAT) system has been proposed to be the acetyl donor in this process. Carnitine isomers were injected into rat developing brains every day for 14 days after birth. Results showed that carnitine and its associated forms produced a choline acetyl transferase (ChAT) activity increase in the striatum and the hippocampus. Carnitine acetyl transferase activity was stimulated by the treatment of 1‐carnitine in the hippocampus but it remained unchanged in the striatum and the cerebral cortex. These results suggest that ChAT and CarAT activities might be modulated by Acetyl‐CoA derived preferentially from acetylcarnitine. It is suggested that ChAT activity enhancement depends on intrinsic and extrinsic cholinergic afferents to these brain areas. © 1995 Wiley‐Liss, Inc.
Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: Factor secretion, cell differentiation, and proliferation
Authors: Trentin A. G. , Rosenthal D. , Neto V. Moura
Published: January 2005

The effects of triiodothyronine (T3)on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T3‐treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T3. T3 or conditioned medium‐treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T3‐treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells.

We verified that T3 may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism. © 1995 Wiley‐Liss, Inc.
Expression of the nerve growth factor gene is controlled by the microtubule network
Authors: Baudet C. , Naveilhan P. , Jehan F. , Brachet P. , Wion D.
Published: January 2005

Colchicine, nocodazol, and vinblastine, three microtubule‐disrupting drugs were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell‐secreted NGF protein in L929 cells, with levels of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubulestabilizing agent Taxotere®, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down‐regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene. © 1995 Wiley‐Liss, Inc.
Deciphering the native GABAA receptor: Is there hope?
Authors: Yeh H. H. , Grigorenko E. V.
Published: January 2005

The bewildering number of GABAA receptor subunits, their regionally dependent expression in the brain, and their supernumerary expression in single cells present major challenges in studying the function of native GABAA receptors. Which subunit combinations actually exist in native neurons? In this mini‐review, GABAA receptor subunit diversity is considered in light of using the wealth of “structure‐function” information gained from studying recombinant receptor to predict the subunit composition and functional properties of native GABAA receptors. © 1995 Wiley‐Liss, Inc.
Increase in GAP‐43 and GFAP immunoreactivity int the rat hippocampus subsequent to perforant path kindling
Authors: Dalby N. O. , Rondouin G. , Lerner‐Natoli M.
Published: January 2005

Kindling is an animal model of epilepsy which is accompanied by morphological and biochemical changes in the brain, including sprouting of fibers and increased transmitter release. Here we have examined the immunocytochemical expression of (1) GAP‐43, a growth‐associated protein, which is a neuron‐specific PKC substrate, particularly expressed in development and regeneration and (2) glial fibrillary acidic protein (GFAP), part of the astrocytic cytoskeleton, after perforant path kiraling. Subsequent to kindling, GAP‐43 imimunoreacliviiy was increased in CAI stratum lacunosum‐moleculare and the inner and outer molecular layer of the fascia dentata. Other hippocampal subregions showed a lower increase. GFAP immunoreactivity was increased in the entire hippocampus, but especially in stratum lacunosum‐moleculare of the CAI and the hilus of fascia dentata. The difference between the number of GFAP‐positive profiles in the hippocampus of control rats and in fully kindled rats was found to be non‐significant. We interpret these findings as being related to both plastic neuronal changes and possible neuronal degeneration. © 1995 Wiley‐Liss, Inc.
Specificity of nerve growth factor signaling: Differential patterns of early tyrosine phosphorylation events induced by NGF, EGF, and bFGF
Authors: Blumberg D. , Radeke M. J. , Feinstein S. C.
Published: January 2005

The specificity of nerve growth factor (NGF) action was examined by comparing early tyrosine phosphorylation events induced by NGF, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). In PC12 cells, administration of either the differentiation factor NGF or the mitogenic factor EGF led to tyrosine phosphorylation of multiple polypeptides in the 100–110 kDa size range associated with PI‐3 kinase. However, NGF induced a more prolonged phosphorylation, relative to a transient EGF effect. In contrast, the differentiation factor bFGF failed to induce measurable tyrosine phosphorylation of PI‐3 kinase‐associated proteins. Similarly, NGF but not bFGF induced marked tyrosine phosphorylation of PLCγ, another early signaling molecule, suggesting that multiple pathways exist for promoting differentiation, and/or that these signaling molecules are not essential for differentiation. TrkA signaling was also compared between PC 12 cells and NIH‐3T3 cells heterologously expressing trkA, where receptor activation promotes mitogenesis. In this comparison, significant differences were observed in the tyrosine phosphorylation pattern of PI‐3 kinase‐associated polypeptides, suggesting the existence of cell type‐specific molecular interactions influencing trkA signaling. Mechanistically, NGF stimulation of PC12 cells resulted in a weak or possibly indirect association between trkA and PI‐3 kinase. Furthermore, NGF did not appear to activate or substantially alter the overall level of PI‐3 kinase activity, raising the possibility that ligand‐induced phosphorylation may serve instead to relocalize constitutively active PI‐3 kinase molecules within the cell. Taken together, data presented suggest that the temporal pattern of induced phosphorylation, the nature of induced associations with other phosphoproteins, and cell type‐specific components may all contribute to the generation of NGF signaling specificity. © 1995 Wiley‐Liss, Inc.
Differentiation is induced in three‐dimensional cultures of brain cells immortalized by the LAP mammalian regulatory system
Authors: Toth M. , Shenk T. , Madarasz E.
Published: January 2005

Immortalized neuroectodermal precursor cell lines were generated from mouse brain by the SV40 large T antigen expressed under the control of the LAP (lac activating protein) mammalian regulatory system. The LAP system permits the reversible expression of T antigen as a function of the exogenous inducer, isopropyl‐β‐D‐thiogalactopyranoside. Immortalized cells can be stably maintained in an undifferentiated state in monolayer cultures. Cell lines expressed the early neurofilament‐like protein nestin, but not markers characteristic for mature cells such as the neurofilament light protein and glial fibrillary acidic protein. Downregulating the LAP‐controlled T antigen with isopropyl‐β‐D‐thiogalactopyranoside was not sufficient to induce differentiation. However, when cells formed three‐dimensional aggregates, differentiation to a neuronal phenotype occurred, indicating that cell‐cell interaction plays an important role in their differentiation. Cells in aggregates did not proliferate, even in the presence of T antigen, suggesting that an aggregation‐induced signal to cease growth was dominant over the growth signal of T antigen. Further morphological differentiation was induced by basic fibroblast growth factor. These immortalized cells should facilitate molecular and cellular studies concerned with the mechanism of commitment, fate determination, and mitotic arrest of neuronal precursor cells in the developing mammalian CNS. © 1995 Wiley‐Liss, Inc.
Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture
Authors: Satoh J.‐I. , Kim S. U.
Published: January 2005

HSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (>99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (<1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X‐100‐soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43°C90 min), the level of HSP27 exproion ryas not altered in astrocytes but was elevated significantly in neurons (11–21%) and microglia (4–7%) during 8–48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat‐stressed conditions. Following heat shock treatment (45°C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat‐stressed conditions, HSP27 was distributed within the Triton X‐100‐insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and αβ‐crystallin were coexpressed in astrocytes under unstressed and heat‐stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat‐induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous system. © 1995 Wiley‐Liss, Inc.
Distribution of erb‐b2, erb‐b3, and erb‐b4 in the developing avian nervous system
Authors: Francoeur J. R. , Richardson P. M. , Dunn R. J. , Carbonetto S.
Published: January 2005

The neuregulin proteins (GGF/ARIA/NDF/Heregulin) are pleiotrophic growth and differentiation factors whose receptors, erb‐B2, erb‐B3, and erb‐B4, are members of the epidermal growth factor receptor family of receptor tyrosine kinases. With western blots, we have examined the developmental and regional distribution of these receptors within the brain and sciatic nerve of chick embryos. The receptors erb‐B2, erb‐B3, and erb‐B4 are development and spatially regulated within the nervous system. In addition, cultures enriched for neurons or glia indicate that erb‐B2 and erb‐B4 are found in both neurons and glia, whereas erb‐B3 is found in glia alone. © 1995 Wiley‐Liss, Inc.
Excitatory amino acid receptors in glia: Different subtypes for distinct functions?
Authors: Gallo V. , Russell J. T.
Published: January 2005

It is now well established that expression of voltage‐ and ligand‐gated ionic channels, as well as G proteincoupled receptors, is not a property unique to neurons, but is also shared by macroglial cells (astrocytes and oligodendrocytes). These glial cells can receive a variety of signals from neurons at different stages of their development. Activation of membrane receptors may affect glial cell activity, proliferation, maturation, and survival through a complex cascade of intracellular events leading to long‐term changes in glial cell phenotype and functional organization. Here we review the experimental evidence for glutamate receptor expression in glial cells in culture and in situ, and the molecular and functional properties of these receptors. We also describe some experimental models that identify possible functions of glutamate receptors in glia. Now that the existence of glutamate receptors in glia has been unambiguously demonstrated, future research will have to (1) determine which receptor subtypes are expressed in macroglial cells in vivo; (2) analyze, in adequate experimental models, the short and long‐term changes produced by glutamate receptor activation in glia; and (3) establish whether these receptors play a role in neuron‐glia communication in the brain. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Several extracellular domains of the neural cell adhesion molecule L1 are involved in homophilic interactions
Authors: Holm J. , Appel F. , Schachner M.
Published: January 2005

The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. It can interact with itself by a self‐binding, i.e., homophilic adhesion mechanism (Kadmon et al.: J Cell Biol 110: 193‐208, 1990a). To determine the domains of L1 involved in homophilic binding, we have generated protein fragments of Ll in a prokaryotic and a eukaryotic expression system and used these covalently coupled to fluorescent microspheres to quantify aggregation between them by cytofluorometric analysis. Protein fragments containing the first and second Ig‐like domains and the third fibronectin type III homologous repeat showed avid self‐binding. Ig‐like domains III and IV also showed some self‐binding, whereas Ig‐like domains V and VI and fibronectin type III homologous repeats 1 and 2 as well as 4 and 5 were less or not active. Binding between different domains was also observed: fibronectin type III homologous repeats 4 and 5 interacted with Ig‐like domains I and II, and fibronectin type III homologous repeats 3‐5 interacted with all Ig‐like domains. These results were confirmed by experiments testing the binding of fragment‐conjugated microspheres to substrate‐coated L1 or to cell surface‐expressed L1 on cultured neurons. Binding of L1 to itself was interfered with by all protein fragments tested, suggesting that also less avidly binding domains of L1 contribute to homophilic binding. These observations indicate prominent functional roles of both Ig‐like domains and fibronectin type III homologous repeats in homophilic binding of L1. © 1995 Wiley‐Liss, Inc.
Expression of neurotrophins in skeletal muscle: Quantitative comparison and significance for motoneuron survival and maintenance of function
Authors: Griesbeck O. , Parsadanian A. Sh. , Sendtner M. , Thoenen H.
Published: January 2005

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain‐derived neurotrophic factor (BDNF), newrotrophin‐3 (NT‐3) and neurotrophin 4/5 (NT‐4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT‐3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT‐4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT‐3 mRNA essentially remained unchanged, whereas NT‐4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT‐4, whereas BDNF expression seems to play a role mainly after nerve lesion. © 1995 Wiley‐Liss, Inc.
Increased cyclic AMP in in vitro regenerating frog sciatic nerves inhibits Schwann cell proliferation bur has no effect on axonal outgrowth
Authors: Ekström P. A. R.
Published: January 2005

In the present study the role of cAMP for axonal outgrowth and Schwann cell proliferation was studying using the cultured frog sciatic nerve. An intrinsic rise in nerve injury, both in vitro and in vivo. Treatment with 0.1‐1.0μM forskolin, an activator of the cAMP‐generating enzyme adenylyl cyclase, increased the cAMP content up to 13‐fold, but was yet without effect on axonal outgrowth during an 8‐day culturing period. HA‐1004, an inhibitor of cAMP‐dependent protein kinase, also lacked effect on the regeneraton. In contrast, the proliferation of Schwann cells, measured as [3H]thymidine incorporation, was inhibited to about 70% of control by forskolin, whereas HA‐1004 stimulated proliferation to approximately 130% of control. The results suggest that cAMP is involved in the injury‐induced proliferation of Schwann cells of an adult peripheral nerve but that it lacks a central role in the regeneration of sensory axons of such nerves. © 1995 Wiley‐Liss, Inc.
Association of nerve growth factor mRNA levels with MK‐801‐induced explosive behaviors in mice
Authors: Riley L. A. , Hitri A. , Bernstein J. J.
Published: January 2005

MK‐801, a noncompetitive antagonist of the N‐methyl‐D‐aspartate (NMDA) receptor, stimulated an outbred strain of NIH Swiss mice to display discrete episodes of explosive jumping behavior, designated as “popping.” The rapid onset of the MK‐801‐induced “popping” seems to follow the rapid distribution of the drug to the frontal cortex, the area that contains high levels of NMDA receptors. We examined the effect of this drug on the levels of mRNA coding for nerve growth factor (NGF) in the frontal cortex in relation to the exhibited “popping” episodes. Mice treated with 1 mg/kg MK‐801 could be split into two groups based on the total number of “popping” episodes in a 30 min post‐injection period. These groups also differed in the steady‐state levels of frontal cortex NGF mRNA. Animals that exhibited low numbers of “popping” had levels of NGF mRNA significantly higher than saline treated controls or mice that exhibited high numbers of “popping.” Mice treated with 10 mg/kg MK‐801 had a high frequency of “popping” that was impossible to separate into episodes. In addition, these mice had levels of frontal cortex NGF mRNA that were significantly lower than either group of mice treated with 1 mg/kg MK‐801. These data indicated that there was an increased level of NGF mRNA under conditions where MK‐801 induced a low level of “popping” behavior. However, when “popping” intensified, NGF mRNA levels were decreased, suggesting a possible behavioral antagonism of the NGF response. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Differentiation of serum‐free mouse embryo cells into astrocytes is accompanied by induction of glutamine synthetase activity
Authors: Loo D. T. , Althoen M. C. , Cotman C. W.
Published: January 2005

Serum‐free mouse embryo (SFME) cells derived in a defined serum‐free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serumfree medium. Exposure of SFME cells to transforming growth factor beta (TGF‐β) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF‐β or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TGF‐β or serum. The induction of GS activity was dose‐ and time‐dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF‐β or serum. © 1995 Wiley‐Liss, Inc.
Calpain secreted by activated human lymphoid cells degrades myelin
Authors: Deshpande R. V. , Goust J. M. , Hogan E. L. , Banik N. L.
Published: January 2005

Calpain secreted by lymphoid (MOLT‐3, M.R.) or monocytic (U‐937, THP‐1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS‐PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20‐40% of the myelindegradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain‐specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60‐80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune‐mediated demyelination. © 1995 Wiley‐Liss, Inc.
Advantages of using microfabricated extracellular electrodes for in vitro neuronal recording
Authors: Breckenridge L. J. , Wilson R. J. A. , Connolly P. , Curtis A. S. G. , Dow J. A. T. , Blackshaw S. E. , Wilkinson C. D. W.
Published: January 2005

We describe fabrication methods and the characterisation and use of extracellalar microelectrode arrays for the detection of action potentials from neurons in culture. The 100 μm2 platinised gold microelectrodes in the 64 electrode array detect the external current which flows during an action potential with S:N ratios of up to 500:1, giving a maximum recorded signal of several millivolts. The performance of these electrodes is enhanced if good sealing of the cells over the electrodes is obtained and further enhanced if the electrodes and the cells lie in a deep groove in the substratum. The electrodes can be used for both recording and stimulation of activity in cultured neurons and for recording from multiple sites on a single cell. The use of such electrodes to obtain recordings from invertebrate neurons is described. The particular advantages of these electrodes, their long term stability, non‐invasive nature, high packing density, and utility in stimulation, are demonstrated. © 1995 Wiley‐Liss, Inc.