bluuebunny/crossref_metadata_2025 huggingface dataset:
SNARE Complex Proteins, Including the Cognate Pair VAMP‐2and Syntaxin‐4, Are Expressed in Cultured Oligodendrocytes
Authors: Madison Dana L. , Krueger Winfried H. , Cheng David , Trapp Bruce D. , Pfeiffer S. E.
Published: March 2003

Abstract : Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane‐directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP‐2/synaptobrevin‐2, syntaxin‐2 and ‐4, nsec‐1/munc‐18‐1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP‐2, the small GTP‐binding protein Rab3a, and the target SNARE syntaxin‐4 were up‐regulated. VAMP‐2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network ; in situ anti‐VAMP‐2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane‐directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.
Altered Calcium Homeostasis and Mitochondrial Dysfunction in Cortical Synaptic Compartments of Presenilin‐1 Mutant Mice
Authors: Begley James G. , Duan Wenzhen , Chan Sic , Duff Karen , Mattson Mark P.
Published: March 2003

Abstract : Alzheimer's disease is characterized by amyloid β‐peptide deposition, synapse loss, and neuronal death, which are correlated with cognitive impairments. Mutations in the presenilin‐1 gene on chromosome 14 are causally linked to many cases of early‐onset inherited Alzheimer's disease. We report that synaptosomes prepared from transgenic mice harboring presenilin‐1 mutations exhibit enhanced elevations of cytoplasmic calcium levels following exposure to depolarizing agents, amyloid β‐peptide, and a mitochondrial toxin compared with synaptosomes from nontransgenic mice and mice overexpressing wild‐type presenilin‐1. Mitochondrial dysfunction and caspase activation following exposures to amyloid β‐peptide and metabolic insults were exacerbated in synaptosomes from presenilin‐1 mutant mice. Agents that buffer cytoplasmic calcium or that prevent calcium release from the endoplasmic reticulum protected synaptosomes against the adverse effect of presenilin‐1 mutations on mitochondrial function. Abnormal synaptic calcium homeostasis and mitochondrial dysfunction may contribute to the pathogenic mechanism of presenilin‐1 mutations.
Rat Brain Thioltransferase : Regional Distribution, Immunological Characterization, and Localization by Fluorescent In Situ Hybridization
Authors: Balijepalli Sadguna , Tirumalai P. S. , Swamy K. V. , Boyd M. R. , Mieyal J. J. , Ravindranath Vijayalakshmi
Published: March 2003

Abstract : Thioltransferase (TTase) is a member of the family of thiol‐disulfide oxidoreductases that are involved in the maintenance of sulfhydryl homeostasis in cells by catalyzing thiol‐disulfide interchange reactions. One of the major consequences of oxidative stress in brain is the formation of protein‐glutathione mixed disulfides (through oxidation of protein thiols), which can be reversed by TTase during the recovery of brain from oxidative stress. We therefore examined the presence of TTase in brain regions from rat. In the rat, TTase activity in the whole brain was comparable with the corresponding activity in liver, but significantly higher in hippocampus. The enzyme activity was significantly lower in striatum and cerebellum compared with activity in whole brain. Rat brain TTase shared immunological similarity with the human red blood cell enzyme, but not with the pig liver enzyme. The constitutive expression of the mRNA to TTase was demonstrable by northern blotting. Localization of the TTase mRNA in rat brain by fluorescent in situ hybridization showed the presence of high amounts of mRNA in the olfactory bulb, cortex, and hippocampus and its predominant localization in the neurons. TTase mRNA was also present in Purkinje cells in the cerebellum, in giant reticular neurons in the midbrain, and in the striatal and thalamic neurons. This study demonstrates the constitutive presence of a functional TTase system in brain and delineates the regional and cellular localization of the enzyme in rat brain.
Subtype‐Specific Enhancement of NMDA Receptor Currents by Mutant Huntingtin
Authors: Chen Nansheng , Luo Tao , Wellington Cheryl , Metzler Martina , McCutcheon Krista , Hayden Michael R. , Raymond Lynn A.
Published: March 2003

Abstract: Evidence suggests that NMDA receptor‐mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch‐clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B‐type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.
Endogenous Bax Translocation in SH‐SY5Y Human Neuroblastoma Cells and Cerebellar Granule Neurons Undergoing Apoptosis
Authors: McGinnis Kim M. , Gnegy Margaret E. , Wang Kevin K. W.
Published: March 2003

Abstract: Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH‐SY5Y human neuroblastoma cells undergoing staurosporine‐ or EGTA‐mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA‐treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase‐3‐like activity. An increase in caspase‐3‐like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase‐3‐like activity in staurosporine‐treated SH‐SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K+‐mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.
Nerve Growth Factor Determines Survival and Death of PC12 Cells by Regulation of the bcl‐x, bax, and caspase‐3 Genes
Authors: Rong Pei , Bennie Antoinette M. , Epa W. Ruwan , Barrett Graham L.
Published: March 2003

Abstract: We investigated the effects of nerve growth factor (NGF)
and NGF withdrawal on expression of members of the bcl‐2 family of
genes and caspase‐3 in PC12 cells. NGF regulated several members of
the bcl‐2 family and caspase‐3 in a manner consistent with
its effect on apoptosis in PC12 cells. Levels of bcl‐xl, bcl‐xs, and
caspase‐3 mRNAs were increased by NGF treatment. The increases in
caspase‐3 and bcl‐xs levels should have disposed the cells
toward apoptosis but were opposed by the simultaneous increase in
bcl‐xl level. NGF withdrawal resulted in abrupt down‐regulation of
bcl‐xl and up‐regulation of bax, favoring apoptosis. Forced
expression of bcl‐xl after NGF withdrawal was sufficient to prevent
cell death. Cell death was rapid when NGF was withdrawn after 5 days of
treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of
treatment. This was consistent with the reduced accumulation of
caspase‐3 mRNA with shorter NGF treatments. These results indicate that Bcl‐xl, Bcl‐xs, Bax, and caspase‐3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.
The Lipid Peroxidation Product 4‐Hydroxynonenal Inhibits Neurite Outgrowth, Disrupts Neuronal Microtubules, and Modifies Cellular Tubulin
Authors: Neely M. D. , Sidell K. R. , Graham D. G. , Montine T. J.
Published: March 2003

Abstract: Oxidative stress is believed to be an important factor in
the development of age‐related neurodegenerative diseases such as Alzheimer's
disease (AD). The CNS is enriched in polyunsaturated fatty acids and is
therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation
of lipid peroxidation products has been demonstrated in affected regions in
brains of AD patients. Another feature of AD is a change in neuronal
microtubule organization. A possible causal relationship between lipid
peroxidation products and changes in neuronal cell motility and cytoskeleton
has not been investigated. We show here that 4‐hydroxy‐2(E)‐nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4‐Oxononanal, and HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE‐induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.
Mazindol Attenuates the 3,4‐Methylenedioxymethamphetamine‐Induced Formation of Hydroxyl Radicals and Long‐Term Depletion of Serotonin in the Striatum
Authors: Shankaran Mahalakshmi , Yamamoto Bryan K. , Gudelsky Gary A.
Published: March 2003

Abstract: The formation of hydroxyl radicals following the systemic
administration of 3,4‐methylenedioxymethamphetamine (MDMA) was studied in the
striatum of the rat by quantifying the stable adducts of salicylic acid and
D‐phenylalanine, namely, 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and
p‐tyrosine, respectively. The repeated administration of MDMA
produced a sustained increase in the extracellular concentration of 2,3‐DHBA
and p‐tyrosine, as well as dopamine. The MDMA‐induced increase in the extracellular concentration of both dopamine and 2,3‐DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long‐term depletion of serotonin (5‐HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine‐dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long‐term depletion of brain 5‐HT content.
Evidence for Phosphatidylinositol 4‐Kinase and Actin Involvement in the Regulation of 125I‐β‐Nerve Growth Factor Retrograde Axonal Transport
Authors: Reynolds Anna J. , Heydon Katharina , Bartlett Selena E. , Hendry Ian A.
Published: March 2003

Abstract: The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4‐kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I‐labeled βNGF was inhibited by PAO (0.5‐2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3‐dimercaptopropanol with PAO reduced its ability to inhibit 125I‐βNGF retrograde transport. PAO (20 nM to 200 μM) also inhibited NGF‐dependent survival of both sympathetic and sensory neuronal populations. F‐actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F‐actin staining in vitro and reduced 125I‐βNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4‐kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I‐βNGF retrograde axonal transport in vivo.
Biochemical and Neurotoxicological Effects of L‐2‐Chloropropionic Acid on Rodent Brain
Authors: Williams R. E. , Jones P. , Lock E. A. , Bachelard H. S.
Published: March 2003

Abstract: L‐2‐Chloropropionic acid (L‐CPA) is selectively toxic to cerebellar granule cells; necrosis is first observed in rats 36 h after L‐CPA administration (750 mg/kg p.o.) and becomes marked by 48 h. L‐CPA has also been shown to activate the mitochondrial pyruvate dehydrogenase (PDH) complex in fasted adult rats, resulting in reduced blood glucose and lactate levels. This study aimed to investigate the biochemical and neurotoxicological effects of L‐CPA on the brain. Extracts, prepared from guinea‐pig cerebellar and cerebral cortex slices incubated in the presence of L‐CPA, were analysed using 1H magnetic resonance spectroscopy, 31P magnetic resonance spectroscopy, and amino acid analysis. Glucose metabolism was studied by monitoring the metabolism of [1‐13C]glucose using gas chromatography/mass spectrometry. Increased glucose metabolism and decreases in the pool sizes of lactate and alanine were observed in both tissues, demonstrating activation of the PDH complex. Extracts were also prepared from the forebrain and cerebellum of animals that had been treated in vivo with L‐CPA and analysed as described for the in vitro studies. Similar evidence for PDH activation was demonstrated at 2 and 24 h after dosing in both tissues. At 48 h after dosing, when signs of toxicity are observed, an increase in the lactate concentration and a decrease in N‐acetylaspartate in the cerebellum but not in the forebrain confirmed the selective neurotoxic action of L‐CPA. These results suggest that activation of the PDH complex does not directly lead to the delayed selective neurotoxicity of L‐CPA.
Prenatal Exposure to Aluminum Reduces Expression of Neuronal Nitric Oxide Synthase and of Soluble Guanylate Cyclase and Impairs Glutamatergic Neurotransmission in Rat Cerebellum
Authors: Llansola Marta , Miñana María‐Dolores , Montoliu Carmina , Saez Rosana , Corbalán Regina , Manzo Luigi , Felipo Vicente
Published: March 2003

Abstract : Exposure to aluminum (Al) produces neurotoxic effects in humans. However, the molecular mechanism of Al neurotoxicity remains unknown. Al interferes with glutamatergic neurotransmission and impairs the neuronal glutamate‐nitric oxide‐cyclic GMP (cGMP) pathway, especially in rats prenatally exposed to Al. The aim of this work was to assess whether Al interferes with processes associated with activation of NMDA receptors and to study the molecular basis for the Al‐induced impairment of the glutamate‐nitric oxide‐cGMP pathway. We used primary cultures of cerebellar neurons prepared from control rats or from rats prenatally exposed to Al. Prenatal exposure to Al prevented glutamate‐induced proteolysis of the microtubule‐associated protein‐2, disaggregation of microtubules, and neuronal death, indicating an impairment of NMDA receptor‐associated signal transduction pathways. Prenatal exposure to Al reduced significantly the content of nitric oxide synthase and guanylate cyclase and increased the content of calmodulin both in cultured neurons and in the whole cerebellum. This effect was selective for proteins of the glutamatenitric oxide‐cGMP pathway as the content of mitogen‐activated protein kinase and the synthesis of most proteins were not affected by prenatal exposure to Al. The alterations in the expression of proteins of the glutamate‐nitric oxide‐cGMP pathway could be responsible for some of the neurotoxic effects of Al.
Nicotinic Acetylcholine Receptor Antagonistic Activity of Monoamine Uptake Blockers in Rat Hippocampal Slices
Authors: Hennings Esteban C. P. , Kiss Janos P. , De Oliveira Karine , Toth Peter T. , Vizi E. Sylvester
Published: March 2003

Abstract : The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine‐evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine‐evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 μM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole‐cell patch clamp experiments neither drug blocked Na+ currents at 1 μM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 μM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation‐ and nicotine‐evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine‐induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects.
Mitochondrial Respiratory Function and Cell Death in Focal Cerebral Ischemia
Authors: Anderson Michelle F. , Sims Neil R.
Published: March 2003

Abstract : Pyruvate‐supported oxygen uptake was determined as a measure of the functional capacity of mitochondria obtained from rat brain during unilateral middle cerebral artery occlusion and reperfusion. During ischemia, substantial reductions developed in both ADP‐stimulated and uncoupled respiration in tissue from the focus of the affected area in the striatum and cortex. A similar pattern of change but with lesser reductions was seen in the adjacent perifocal tissue. Succinate‐supported respiration was more affected than that with pyruvate in perifocal tissue at 2 h of ischemia, suggesting additional alterations to mitochondrial components in this tissue. Mitochondrial respiratory activity recovered fully in samples from the cortex, but not the striatum, within the first hour of reperfusion following 2 h of ischemia and remained similar to control values at 3 h of reperfusion. In contrast, impairment of the functional capacity of mitochondria from all three regions was seen in the first 3 h of reperfusion following 3 h of ischemia. Extensive infarction generally affecting the cortical focal tissue with more variable involvement of the perifocal tissue developed following 2 h of focal ischemia. Thus, mitochondrial impairment during the first 3 h of reperfusion was apparently not essential for tissue infarction to develop. Nonetheless, the observed mitochondrial changes could contribute to the damage produced by permanent focal ischemia as well as the larger infarcts produced when reperfusion was initiated following 3 h of ischemia.
Processing of Rat Preprocortistatin in Mouse AtT‐20 Cells
Authors: Puebla L , Mouchantaf R , Sasi R , Khare S , Bennett H.P.J , James S , Patel Y.C.
Published: March 2003

Abstract : Preprocortistatin (PPCST) has been recently identified as a novel somatostatin (SST)‐related gene expressed only in brain. PPCST shares 11 of 14 residues with SST‐14 at its C‐terminal segment, where it features Lys‐Lys and Lys‐Arg basic sites for cleavage to putative cortistatin (CST)‐14 and CST‐29 peptides, respectively. Although synthetic replicates of the two putative CST peptides interact with SST receptors, they also display novel effects suggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslational processing. Here we have cloned rat PPCST cDNA, stably expressed it in AtT‐20 pituitary cells, and characterized the cellular and releasable products of PPCST processing by HPLC and radioimmunoassay using a SST‐14 antibody that recognizes synthetic CST‐14 and CST‐29. Transfected cells released 120 ± 21 pg of total CST‐LI per plate basally, with an increase to 204 ± 33 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST‐14, CST‐29, and unprocessed PPCST (ratio, 41 : 55 : 4.5). CST was released preferentially as CST‐14 (63‐70%) compared with CST‐29 (30‐37%) under basal and forskolin‐stimulated conditions. These studies demonstrate efficient processing of PPCST to both CST‐14 and CST‐29 through putative cleavage at both C‐terminal dibasic sites of PPCST. Although the two peptides are synthesized approximately equally, CST‐14 is released preferentially via the regulated secretory pathway.
Altered Glial Function Causes Neuronal Death and Increases Neuronal Susceptibility to 1‐Methyl‐4‐Phenylpyridinium‐ and 6‐Hydroxydopamine‐Induced Toxicity in Astrocytic/Ventral Mesencephalic Co‐Cultures
Authors: MCNaught Kevin St. P. , Jenner Peter
Published: March 2003

Abstract: Altered glial function in the substantia nigra in Parkinson's disease may lead to the release of toxic substances that cause dopaminergic cell death or increase neuronal vulnerability to neurotoxins. To investigate this concept, we examined the effects of subjecting astrocytes to lipopolysaccharide (LPS)‐induced activation alone or combined withl‐buthionine‐[S,R]‐sulfoximine‐induced glutathione depletion or inhibition of complex I activity by 1‐methyl‐4‐phenylpyridinium (MPP+) on the viability of primary ventral mesencephalic neurones or susceptibility to MPP+and 6‐hydroxydopamine (6‐OHDA) in co‐cultures. LPS‐activated astrocytes caused neuronal death in a time‐dependent manner, but glutathione‐depleted or complex l‐inhibited astrocytes had no effect on neuronal viability. The neurotoxicity of LPS‐activated astrocytes was inhibited by the inducible nitric oxide synthase inhibitor aminoguanidine, by the nitric oxide scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide, and by reduced glutathione (GSH). MPP+‐induced neuronal death was greater in ventral mesencephalic cultures previously cultured with LPS‐activated, glutathione‐depleted, or complex l‐inhibited astrocytes compared with co‐cultures containing normal astrocytes. The increased neuronal susceptibility to MPP+caused by LPS‐activated or complex l‐inhibited astrocytes and glutathione‐depleted astrocytes was inhibited by the NMDA/glutamate antagonist MK‐801 and by GSH, respectively. Neuronal death caused by 6‐OHDA was increased in ventral mesencephalic cultures previously cultured with LPS‐activated and glutathione‐depleted, but not complex l‐inhibited astrocytes, compared with co‐cultures containing normal astrocytes. Treatment of co‐cultures with GSH prevented the increased neuronal susceptibility to OHDA. These findings suggest that glial dysfunction may cause neuronal death or render neurones susceptible to toxic insults via a mechanism involving the release of free radicals and glutamate. Such a mechanism may play role in the development or progression of nigrostriatal degeneration in Parkinson's disease.
Propofol Hemisuccinate Protects Neuronal Cells from Oxidative Injury
Authors: Sagara Yutaka , Hendler Sheldon , Khoh‐Reiter Sue , Gillenwater Gail , Carlo Dennis , Schubert David , Chang Jennie
Published: March 2003

Abstract : Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood—brain barrier (BBB). Propofol (2,6‐diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water‐soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non‐receptor‐mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT‐22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT‐22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT‐22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested α‐tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT‐22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water‐soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.
Increased Expression of Rat Synuclein in the Substantia Nigra Pars Compacta Identified by mRNA Differential Display in a Model of Developmental Target Injury
Authors: Kholodilov Nikolai G. , Neystat Michael , Tinmarla F. Oo , Steven E. Lo , Larsen Kristin E. , Sulzer David , Burke Robert E.
Published: March 2003

Abstract : Human α‐synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up‐regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up‐regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up‐regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up‐regulated during periods of natural cell death. We conclude that up‐regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.
Role of High‐Affinity Dopamine Uptake and Impulse Activity in the Appearance of Extracellular Dopamine in Striatum After Administration of Exogenous L‐DOPA
Authors: Miller David W. , Abercrombie Elizabeth D.
Published: March 2003

Abstract: The differential behavioral and neurochemical effects of exogenous L‐DOPA in animals with intact versus dopamine (DA)‐denervated striata raise questions regarding the role of DA terminals in the regulation of dopaminergic neurotransmission after administration of exogenous L‐DOPA. In vivo microdialysis was used to monitor the effect of exogenous L‐DOPA on extracellular DA in intact and DA‐denervated striata of awake rats. In intact striatum, a small increase in extracellular DA was observed after administration of L‐DOPA (50 mg/kg i.p.) but in DA‐denervated striatum a much larger increase in extracellular DA was elicited. Additional experiments assessed the role of high‐affinity DA uptake and impulse‐dependent neurotransmitter release in the effect of exogenous L‐DOPA on extracellular DA in striatum. Pretreatment with GBR‐12909 (20 mg/kg i.p.), a selective DA uptake inhibitor, enhanced the ability of L‐DOPA to increase extracellular DA in intact striatum. However, in DA‐denervated striatum, inhibition of DA uptake did not alter the extracellular DA response to L‐DOPA. Impulse‐dependent neurotransmitter release was blocked by the infusion of tetrodotoxin (TTX; 1 μM), an inhibitor of fast sodium channels, through the dialysis probe. Application of TTX significantly attenuated the L‐DOPA‐induced increase in extracellular DA observed in striatum of intact rats pretreated with GBR‐12909. In a similar manner, TTX infusion significantly attenuated the increase in extracellular DA typically observed in striatum of 6‐OHDA‐lesioned rats after the administration of L‐DOPA. The present results indicate that DA terminals, via high‐affinity uptake, play a crucial role in the clearance of extracellular DA formed from exogenous L‐DOPA in intact striatum. This regulatory mechanism is absent in the DA‐denervated striatum. In addition, this study has shown that DA synthesized from exogenous L‐DOPA primarily is released by an impulse‐dependent mechanism in both intact and DA‐denervated striatum. The latter result suggests an important role for a nondopaminergic neuronal element in striatum that serves as the primary source of extracellular DA formed from exogenous L‐DOPA.
A Nuclear Microscopic Study of Elemental Changes in the Rat Hippocampus After Kainate‐Induced Neuronal Injury
Authors: Ong Wei‐Yi , Ren Min‐Qin , Makjanić Jagoda , Lim Tit‐Meng , Watt Frank
Published: March 2003

Abstract: The effect of intracerebroventricular kainate injection on the elemental composition of the hippocampus was studied in adult Wistar rats, at 1 day and 1, 2, 3, and 4 weeks postinjection, using a nuclear microscope. An increase in calcium concentration was observed on the injected side from 1 day postinjection. The increase peaked at 3 weeks postinjection, reaching a concentration of 18 times normal. Large numbers of glial cells but no neurons were observed in the lesioned CA fields at this time, suggesting that an increased calcium level was present in glial cells. This was confirmed by high‐resolution elemental maps of the lesioned areas, which showed very high intracellular calcium concentrations in almost all glial cells. It is possible that the high intracellular calcium level could activate calcium‐dependent enzymes, including calpain II and cytosolic phospholipase A2, shown to be expressed in reactive glial cells after kainate injections. In addition to calcium, an increase in iron content was also observed at the periphery of the glial scar at 4 weeks postinjection. Because free iron could catalyze the formation of free radicals, the late increase in iron content may be related to oxygen radical formation during neurodegeneration.
Inhibition of Microtubule Formation by Metabotropic Glutamate Receptors
Authors: Huang Xi‐Ping , Hampson David R.
Published: March 2003

Abstract: Activation of glutamate receptors is known to alter the biophysical state of the cytoskeleton of neurons in the developing brain. In this study, we examined the ability of G protein‐coupled metabotropic glutamate receptors (mGluRs) to inhibit the formation of processes induced by the expression of the microtubule‐associated protein MAP2c. The infection of insect MG‐1 cells with a recombinant baculovirus (BV) encoding MAP2c induced the formation of fine filamentous processes. The binding of MAPs to tubulin promotes tubulin polymerization and the formation of microtubules. Co‐infection with BVs for the phosphoinositide (PI)‐linked mGluR1a or mGluR1b receptor subtypes inhibited the formation of processes induced by MAP2c, whereas co‐infection with BVs encoding the mGluR4a or mGluR4b subtypes that couple to adenylyl cyclase did not inhibit the formation of processes. The biochemical pathways responsible for producing the inhibitory effect of mGluR1 were investigated. Inhibitors of protein kinase C, calcium/calmodulin‐dependent kinase, and protein tyrosine kinases did not block the inhibitory effect of mGluR1a. The calcium chelator BAPTA and the calcium depletor thapsigargin also did not affect the ability of mGluR1a to inhibit process formation. In contrast, inhibitors of phospholipase C reversed the effect of mGluR1 on process formation, suggesting that one or more metabolites in the PI pathway were responsible for the inhibitory effect. These findings indicate that PIs generated by activation of mGluRs inhibit the binding of MAPs to tubulin and reduce tubulin polymerization and microtubule stability.
ErbB‐4 Activation Promotes Neurite Outgrowth in PC12 Cells
Authors: Vaskovsky Anna , Lupowitz Zipora , Erlich Shlomit , Pinkas‐Kramarski Ronit
Published: March 2003

Abstract: Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type‐dependent. NDF and its receptor ErbB‐4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB‐4 receptors expressed in PC12 cells mediate NDF‐induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor‐activated Trk receptors. In PC12‐ErbB‐4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF‐induced differentiation in PC12‐ErbB‐4 cells was mimicked by the pan‐ErbB ligand betacellulin but not by other epidermal growth factor‐like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB‐4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB‐4 receptors. Neurite outgrowth induced by NDF in PC12‐ErbB‐4 cells was accompanied by sustained activation of mitogen‐activated protein kinase (MAPK) and induction of the neural differentiation marker GAP‐43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF‐induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB‐4 in PC12 cells requires MAPK and PKC signaling networks.
Expression of T‐Cadherin (CDH13, H‐Cadherin) in Human Brain and Its Characteristics as a Negative Growth Regulator of Epidermal Growth Factor in Neuroblastoma Cells
Authors: Takeuchi Tamotsu , Misaki Akiko , Liang Sheng‐Ben , Tachibana Akiko , Hayashi Nakanobu , Sonobe Hiroshi , Ohtsuki Yuji
Published: March 2003

Abstract: In the present study, we first examined the expression of T‐cadherin in human CNS by northern blot analysis, immunohistochemical staining, and in situ hybridization. Northern blot analysis demonstrated expression of T‐cadherin in human adult cerebral cortex, medulla, thalamus, and midbrain. Immunohistochemical staining with a newly generated monoclonal antibody, designated MA‐511, revealed strong expression of T‐cadherin in neural cell surface membrane and neurites in adult cerebral cortex, medulla oblongata, and nucleus olivaris. Little or no expression of T‐cadherin was found in spinal cord. We further examined T‐cadherin expression in various developing nervous systems, and found that T‐cadherin expression was lower in developing brain than in adult brain. In situ hybridization revealed that neural cells in medulla oblongata and nucleus olivaris, but not in spinal cord, possessed T‐cadherin molecules. We transfected T‐cadherin‐negative TGW and NH‐12 neuroblastoma cells with a T‐cadherin cDNA‐containing expression vector. T‐cadherin‐expressing neuroblastoma cells lost mitogenic proliferative response to epidermal growth factor. Epidermal growth factor is known to be required for proliferation of neural stem cells. This finding, together with those of the present study, suggests that T‐cadherin functions as a negative regulator of neural cell growth.
Modulation of the Neurotoxic Effects of Methamphetamine by the Selective κ‐Opioid Receptor Agonist U69593
Authors: Daly E. El , Chefer V. , Sandill S. , Shippenberg T. S.
Published: March 2003

Abstract:κ‐Opioid receptor agonists prevent alterations in
dopamine neurotransmission that occur in response to repeated cocaine
administration. The present microdialysis study examined whether
administration of the selective κ‐opioid receptor agonist U69593 with
methamphetamine prevents alterations in dopamine levels produced by neurotoxic
doses of methamphetamine. Swiss Webster mice were injected intraperitoneally
with methamphetamine (10.0 mg/kg) or saline, four times in 1 day, at 2‐h
intervals. Prior to the first and third injection, they received U69593 (0.32
mg/kg s.c.) or vehicle. Microdialysis was conducted 3, 7, or 21 days later.
Basal and K+‐evoked (60 and 100 mM) dopamine overflow were
reduced 3 days after methamphetamine administration. These effects were
long‐lasting in that they were still apparent 7 and 21 days after
methamphetamine treatment. Intrastriatal (5.0 and 50 μM) or
systemic (1.0‐10.0 mg/kg) administration of methamphetamine increased dopamine
concentrations in control animals. In mice preexposed to methamphetamine,
methamphetamine‐evoked dopamine overflow was reduced. In animals that had
received methamphetamine with U69593, basal dopamine levels did not differ
from those of vehicle‐treated controls. U69593 treatment attenuated the
decrease in K+‐evoked dopamine produced by prior methamphetamine exposure. The reduction in methamphetamine‐evoked dopamine levels was also attenuated. The administration of U69593 alone did not modify basal or stimulus‐evoked dopamine levels. These data demonstrate that repeated methamphetamine administration reduces presynaptic dopamine neuronal function in mouse striatum and that co‐administration of a selective κ‐opioid receptor agonist with methamphetamine attenuates these effects. U69593 treatment did not modify the hyperthermic effects of methamphetamine, indicating that this κ‐opioid receptor agonist selectively attenuates methamphetamine‐induced alterations in dopamine neurotransmission.
A Late Phase of Exocytosis from Synaptosomes Induced by Elevated [Ca2+]i Is Not Blocked by Clostridial Neurotoxins
Authors: Ashton Anthony C. , Dolly J. Oliver
Published: March 2003

Abstract: Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP‐25, syntaxin 1A/1B, and synaptobrevin and diminished Ca2+‐dependent K+ depolarization‐induced noradrenaline secretion. With botulinum toxin type E, <10% of intact SNAP‐25 remained and K+‐evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore‐induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (>120 s for noradrenaline; >60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4‐kinase inhibitor. Based on these collective findings, we suggest that the ionophore‐induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.
Identification and Cataloging of Genes Induced by Long‐Lasting Long‐Term Potentiation in Awake Rats
Authors: Matsuo Ryota , Murayama Akiko , Saitoh Yoshito , Sakaki Yoshiyuki , Inokuchi Kaoru
Published: March 2003

Abstract: Maintenance of long‐term potentiation (LTP) requires de novo gene expression. Here we report the direct isolation, using PCR‐differential display, of genes whose expression level was altered after induction of long‐lasting LTP in the hippocampus of freely moving awake rats. Differential display using 480 primer combinations revealed 17 cDNA bands that showed a reproducible change in expression level. These cDNAs represented at least 10 different genes (termed RM1‐10), all of which showed up‐regulation at 75 min after LTP induction and a return to basal expression levels within 24 h. Three of these genes were known only from expressed sequence tags (RM1‐3), two were known genes whose up‐regulation by LTP has not been described (GADD153/CHOP and ler5), and five were known genes whose up‐regulation by LTP has already been reported (MAPK phosphatase, NGFI‐A/zif268, vesl‐1S/homer‐1a, Ag2, and krox‐20). We characterized the expression profiles of genes in the two former categories with respect to NMDA receptor dependency, tissue specificity, and developmental regulation using northern blotting and semiquantitative RT‐PCR. The up‐regulation of all five of these genes was NMDA receptor‐dependent and correlated with the persistence of LTP, suggesting that these genes may play functional roles in prolonged LTP maintenance.
Somatostatin Release in the Hippocampus in the Kindling Model of Epilepsy
Authors: Marti Matteo , Bregola Gianni , Morari Michele , Gemignani Anita , Simonato Michele
Published: March 2003

Abstract: Somatostatin biosynthesis in the hippocampus is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon is associated with enhanced somatostatin release in vivo. Experiments have been run in awake, freely moving rats, implanted with a bipolar electrode in the right amygdala (for kindling stimulation), and with a recording electrode and a microdialysis probe in the left hippocampus. Basal somatostatin‐like immunoreactivity (‐LI) release was significantly greater in kindled than naive rats. In naive rats, a 2‐min perfusion with 100 mM K+ did not affect behavior and EEG recordings and nonsignificantly increased somatostatin‐LI release; a 10‐min K+ perfusion evoked numerous wet dog shakes, electrical seizures (class 0; latency ≅8 min, duration ≅8 min), and somatostatin‐LI release (≅350% of basal); and a single kindling after‐discharge (4 ± 3‐s duration in the hippocampus) also evoked somatostatin‐LI release (≅200% of basal). In kindled rats, a 2‐min 100 mM K+ perfusion evoked hippocampal discharges in three of seven animals (latency ≅2 min, mean duration ≅1.5 min) and increased somatostatin‐LI release (≅250% of basal); a 10‐min K+ perfusion evoked behavioral seizures (class 1 to 5, latency ≅4 min, mean duration ≅12 min) with numerous wet dog shakes and robust somatostatin‐LI release (≅350% of basal); and a kindling stimulation evoked generalized seizures (class 4 or 5, 77 ± 15‐s duration in the hippocampus) with remarkable somatostatin‐LI release (≅300% of basal). These data demonstrate that hippocampal somatostatin release is increased in the kindling model in vivo.
Purification and Structure of Neurostatin, an Inhibitor of Astrocyte Division of Mammalian Brain
Authors: Abad‐Rodríguez José , Bernabé Manuel , Romero‐Ramírez Lorenzo , Vallejo‐Cremades Mayte , Fernández‐Mayoralas Alfonso , Nieto‐Sampedro Manuel
Published: March 2003

Abstract: Neurostatin was originally described as an inhibitor of astroblast and astrocytoma division present in rat brain extracts and immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens. It was purified recently from mammalian brain extracts and characterized as a glycosphingolipid, but its precise structure remained unknown. Neurostatin has now been purified to apparent homogeneity from ganglioside extracts of rat, bovine, and porcine brain. It is cytostatic for astroblasts, C6 glioma cells, and various human astrocytomas grades III and IV, with IC50 values ranging from 250 to 450 nM, but does not affect the division of primary or transformed fibroblasts up to concentrations >4 μM. Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry of purified pig neurostatin showed a molecular ion of 1,905 Da and ions of 1,863 and 1,934 Da, compatible with a disialoganglioside. Mono‐ and bidimensional NMR spectra, together with biochemical studies, suggest that neurostatin may be the 9‐O‐monoacetyl ester of GD1b.
Regulatory Effects of D2 Receptors in the Ventral Tegmental Area on the Mesocorticolimbic Dopaminergic Pathway
Authors: Chen Nancy N. H. , Pan Wynn H. T.
Published: March 2003

Abstract: To investigate the regulatory effects of somatodendritic D2 receptors on the terminal’s extracellular dopamine (DA) concentration, a D2 antagonist (eticlopride) was infused directly into the ventral tegmental area via a microdialysis probe in chloral hydrate‐anesthetized rats. Extracellular DA changes in both the nucleus accumbens (N ACC) and the medial prefrontal cortex (mPFC) were monitored. Infusion of 10.0 fM eticlopride had no effect on DA in the mPFC (110.2 ± 10.0% of baseline) but significantly increased DA in the N ACC (150.1 ± 11.7%). Infusion of a higher dose of eticlopride (100.0 or 1,000.0 fM) significantly augmented the DA in the mPFC (121.1 ± 7.6 and 180.7 ± 25.8%, respectively) but surprisingly had no effect on DA in the N ACC (111.5 ± 7.3 and 104.1 ± 8.7%, respectively). To further investigate whether the bluntness of DA increase in the N ACC was due to DA receptor activation in the mPFC, eticlopride or SCH23390 was infused into the mPFC prior to and during intrategmental eticlopride infusion, and the change of DA in the N ACC was simultaneously monitored. During intra‐mPFC 1.0 nM eticlopride infusion but not during 10.0 nM SCH23390 administration (95.5 ± 6.1%), intrategmental 1,000.0 fM eticlopride infusion could further elevate DA in the N ACC (130.0 ± 4.6%). Our results indicated that (1) the mesolimbic and the mesocortical pathways were under tonic inhibition by somatodendritic D2 receptors; (2) the DA concentration in the N ACC first increased and then returned to baseline while the intrategmental infusion dose of eticlopride increased; and (3) the bluntness of DA increase in the N ACC resulted from the D2 receptor activation in the mPFC.