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Notes: Objective In obstetrics, both area and hospital variation has previously been documented for operative procedures, but not for other treatments or diagnostic procedures. This study investigated the extent of variation in care during pregnancy, at birth and after birth.Design and methods The variation was studied by hospitals (n= 52) by hospital level. To account for differences in women's background characteristics, logistic regression analyses were done. The data came from the 1991 Finnish Birth Register which records data on all births (n= 64 171).Results All interventions and care practices studied varied notably from one hospital to another, with the variation usually being greater within hospitals of the same level than between hospitals of different levels. Each of the seven non-operative interventions showed a larger variation than did each of the four operative interventions. Standardisation for mothers’ background characteristics did not eliminate this variation. Some interventions, but not all, were clustered in the same hospitals.Conclusions Such large variation indicates a need for further studies on the benefits of obstetric interventions and of care practices, and on better application of research results to obstetric care.

Notes: Objective To establish the reliability of fetal magnetocardiography as a method of measuring the time intervals of the fetal heart during the antenatal period.Design A prospective study.Setting Wellcome Biomagnetism Unit, Southern General Hospital.Subject One hundred and six low risk pregnant women at 20 to 42 weeks gestation.Main outcome measures Success in obtaining QRS complexes, P waves and T waves. Correlation of time intervals with fetal outcome.Results The technique was acceptable to pregnant women. A QRS complex was successfully demonstrated in 68 (67%) of the unaveraged traces. Using off-line averaging techniques on these 68 cases, P waves were obtained in 75% and T waves in 72%. Although good quality traces were obtained throughout the range of gestational ages, in general it was more difficult below 28 weeks. QRS duration (R2= 7%, P= 0.02) demonstrated a positive linear correlation with increasing gestation. Of the 35 (51%) cases with umbilical vein pH analysis available, only one result was less than 7.2. No significant relation was found between measurements of the fetal waveforms and the pH results.Conclusion The technique of fetal magnetocardiography provides a significant advance in the technological field for the demonstration of QRS complexes and the full PQRST waveforms in gestations from 20 weeks onwards. With further technical improvements the clinical impact of this technique can be assessed more fully.

Notes: Objective To assess the effects of dihydralazine, labetalol and magnesium sulphate on the vascular tone in the isolated, perfused human placental cotyledon.Methods In vitro perfusion of the fetal compartment of isolated, human placental cotyledons.Results None of the drugs affected basal vascular tone. The thromboxane A2-mimic U46619 and endothelin-1 induced a concentration-dependent increment in perfusion pressure, while 5- hydroxytryptamine induced a variable increase, and norepinephrine induced a small, transient increase in perfusion pressure. After preconstriction with U46619, magnesium sulphate (1.5×10−3 to 6.10−3 mol/1) induced a decrease in perfusion pressure, while dihydralazine (10−6to 10−4 mol/1) or labetalol (10−7 to 10−4 mol/1) enhanced the perfusion pressure. These effects of dihydralazine and labetalol were unaffected by treatment with indomethacin 10−6 mol/1, but could be reversed by addition of magnesium sulphate 6.10−3 mol/1. Labetalol 10−6 to 10−4 mol/1 also caused an increase in the perfusion pressure induced by endothelin-1, but showed no effects after preconstriction with 5-hydroxytryptamine. Pretreatment with labetalol 10−4 mol/1 inhibited the transient increase in perfusion pressure induced by norepinephrine 3.10−5 mol/1.Conclusions The present data demonstrated that the commonly used vasodilating agents labetalol and dihydralazine do not produce vasodilatation in the human perfused cotyledon after vasoconstriction induced by agents of suggested importance for maintenance of fetal placental vascular tone, and that high concentrations of these drugs may even enhance vasoconstriction induced by thromboxane and endothelin-1 in this area. Magnesium sulphate may show the potential to reverse such unwanted effects of dihydralazine and labetalol.

Notes: Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µM) and pirenzepine (10 nM), but not by methoctramine (25 nM). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.

Notes: Abstract: Fura-2 digital imaging microfluorimetry was used to evaluate the Ca2+ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca2+i) but of different magnitudes (ET-1 = ATP〉 NE). The Ca2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca2+ influx. The Ca2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca2+ signal, which was characteristic of the response to Oxo-M, was observed in 〈40% of cells stimulated with ET-1 and absent from Ca2+ signals elicited after NE addition. Removal of extracellular Ca2+ enhanced the rate of decay of Ca2+ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca2+ influx. In the absence of extracellular Ca2+, NE elicited asynchronous multiple Ca2+ transients. In either the absence or presence of extracellular Ca2+,〉94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were ∼74 and ∼48%. Sequential addition of agonists to cells maintained in a Ca2+-free buffer indicated that each ligand mobilized Ca2+ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four- to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET-1 or ATP resulted in larger increases in the net formation of inositol 1,4,5-trisphosphate than did either Oxo-M or NE. These results indicate that, in SK-N-MCIXC cells, the characteristics of both Ca2+ signaling and inositol phosphate production are agonist specific.

Notes: Abstract: The interaction of triethyltin (TET) and trimethyltin (TMT) with rat brain astrocytes in vitro was investigated. Both compounds are highly neurotoxic after in vivo application, cause neurobehavioral changes, and elicit neuronal and glial responses in the CNS. In this study, 5-week-old cultures were exposed to TMT or TET (0.1–2.5 µM) for 24 h. A concentration-dependent cytotoxicity was observed for both agents by vital dye uptake assay using neutral red (NR). The order of potency for half-maximal cytotoxicity (NR-50) was TET (0.7 µM)〉 TMT (2.5 µM), in agreement with results found after in vivo administration. TET and TMT caused similar morphological changes: large holes extending through the plasma membrane appeared initially in the flattened cell bodies, cytoplasmic extensions were retracted, and long cellular processes formed. Later, the cell bodies rounded up and had only a few extremely long and thin processes. Indirect immunofluorescence staining using anti-vimentin and anti-glial fibrillary acidic protein (GFAP) antibodies revealed that the orderly array of the intermediate filament system was severely disturbed. At lower concentrations, an increased bundling was observed, and at higher concentrations the disassembly of the intracellular framework was seen, and cellular staining appeared rather diffuse. Western blot analysis of cellular extracts was carried out to determine the protein levels of GFAP and vimentin. In this culture system, TET and TMT caused an almost two-fold increase in the levels of GFAP at concentrations around and below NR-50, indicating that astrocytes react to organotics independently of neuronal signals. Concomitantly, TET (0.7 µM) and TMT (2.5 µM) led to a 63% decrease in the activity of ecto-5′-nucleotidase, which in addition to its enzymatic function, represents a transmembrane cell surface protein and has been implicated in cellular adhesion and cell communication processes. Thus, triorganotins directly affect astrocytes in culture and alter their functional stage even in the absence of neurons.

Notes: Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.

Notes: Abstract: The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of τ were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow-mobility (66- and 68-kDa) forms of τ. The percentage of immunoreactive τ in faster-mobility pools (42–54 kDa) increased in proportion to the decrease in content of 66–68-kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal-appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimer's disease are formed.

Notes: Abstract: The regulation of expression of two different types of neuronal intermediate filament proteins, ON1/ON2 and plasticin, was studied during optic nerve regeneration in the goldfish. During regenerative growth of optic axons, there is a rapid and dramatically increased expression of plasticin, a recently cloned, novel type III intermediate filament protein, in the retinal ganglion cells. At the time when the growing axons reinnervate the optic tectum, expression of plasticin declines and there is an increased expression of ON1 and ON2. This time course suggests that the target tissue participates in the regulation of these proteins. The aim of this study was to characterize the regulatory role played by the optic tectum. To address this issue, a repeated-crush paradigm was used whereby growing axons were hindered from reaching their target. It was found that in absence of tectal contact, the increased expression of ON1 and ON2 normally seen during regeneration was not induced. In contrast, expression of plasticin increased both in the presence and in the absence of tectal contact.

Notes: Abstract: Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar-type H+-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 nM baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.

Notes: Abstract: We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3′ untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3′ untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.

Notes: Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first-line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to “therapeutic” concentrations (0.6 mM) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme-selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or 3H-phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross-talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.

Notes: Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP3) over extended incubation periods, and consequently to affect the level of InsP3 receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 mM) evoked a biphasic accumulation of InsP3, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP3 receptor, it was demonstrated that 〉50% down-regulation of type I InsP3 receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 mM CCh. Over 24 h, 1 mM CCh caused an ∼85% decrease in type I InsP3 receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP3 receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1S,3R-dicarboxylic acid (200 µM), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP3 receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP3 production in cerebellar granule cells is the down-regulation of InsP3 receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.

Notes: Abstract: We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2L receptor prelabeled with 32Pi or [3H]-palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.

Notes: Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5-min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5- or 20-min ischemia was induced. When 5-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15-fold) was observed in the histologically normal CA1 field. When 20-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20-min ischemia, but remarkable ischemia-induced increases in glutamate were observed during the last 10 min of 20-min ischemia in both areas. An excessive increase in glutamate (100-fold) was observed during 20-min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5-min ischemia was induced, a significant increase in glutamate was observed during the last half of 5-min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia-induced glutamate accumulation in the CA1 field of the hippocampus during 5-min ischemia but contributes to the ischemia-induced increase in glutamate level during ischemia with a longer duration and that ischemia-induced efflux of glutamate in the CA1 field during 5-min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.

Notes: Abstract: Paralytic tremor (pt) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents ∼30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T → A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Notes: Abstract: Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down-regulation of β1-adrenergic receptors (β1AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of β1AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered β1AR mRNA levels in a biphasic manner; i.e., short-term exposure (30–60 min) increased by 50%, whereas longer exposure (2–6 h) decreased by 50% the levels of β1AR mRNA. The extent of both the up- and down-regulation was dependent on agonist concentration. Similar regulation of β1AR mRNA was observed in forskolin-treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol- and forskolin-mediated down-regulation of β1AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6-h time course. The mechanisms underlying β1AR mRNA down-regulation were examined. The half-life of β1AR mRNA was slightly increased (from 61 to 77 min) after a 2-h exposure of the cells to either isoproterenol or forskolin. Nuclear run-on analysis demonstrated that the rate of β1AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90–240 min, consistent with the time course for up- and down-regulation of β1AR mRNA. Isoproterenol treatment (120 min) also decreased the level of β1AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4-thiouridine-pulsed cells. The results demonstrate that β1AR mRNA has a relatively short half-life and that agonist regulation of β1AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of β1AR mRNA occurs at the level of β1AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that β1AR mRNA down-regulation may be mediated by the induction of a repressor of the β1AR gene.

Notes: Abstract: Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle-shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P0 glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin-related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin-related components.

Notes: Abstract: Prior exposure to a mild thermal stress can protect neuronal cells from a subsequent more severe stress including high temperature, ischemia, glutamate toxicity, or stimuli inducing apoptosis. Although the protective effect of thermal stress correlates with the elevated expression of the heat shock proteins (hsps), the protective effect of individual hsps has never been directly demonstrated in neuronal cells. Here we show that the constitutive overexpression of either of the major hsps, hsp90 or hsp70, can protect neuronal cells from thermal stress but not from stimuli that induce apoptosis. The possible mechanisms by which thermal stress can protect neuronal cells from apoptosis are discussed.

Notes: Abstract: The membrane lipid composition of human frontal and temporal cortices and white matter has been studied in 118 subjects, age 20–100 years. The brain specimens were selected from subjects who lived a normal social life and died suddenly and unexpectedly with no history of neurologic or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The sudden death eliminated all risk of changes over a long agonal stage. The data for total solids and major lipids are summarized in graphic form. Total solids, phospholipids, and cholesterol diminished linearly from 20 years of age in frontal and temporal cortices, whereas total solids phospholipids, cholesterol, cerebroside, and sulfatide showed a curvilinear diminution in frontal and temporal white matter. Gangliosides differed from the other lipids, showing an almost constant concentration between 20 and 70 years of age with a slight peak around 50 years of age. The ganglioside pattern showed continuous change with aging, with decreasing proportions of GM1 and GD1a and increasing proportions of GD1b, GM3, and GD3. Equations are given that can be used to calculate the lipid composition of normal human frontal and temporal cortices and white matter at any age between 20 and 100 years of age. These data can be used where data by direct analysis are not available for comparison with values for various pathological states.

Notes: Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose-dependent Ca2+ influx, which was reduced to 14% by the absence of external Ca2+. Dantrolene inhibited the C48/80-induced increase in Ca2+ permeability of plasma membranes in a concentration-dependent manner (0.33–10 µM, IC50 value was 5 µM). We next examined C48/80-induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h0/h−1 was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration-dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µM) itself did not change the membrane fluidity, but it significantly reduced the C48/80-induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80-induced increase in fluidity was dependent on extracellular Ca2+. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80-induced membrane perturbation, which causes Ca2+ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.

Notes: Abstract: A procedure was established for determining the calcium content of mitochondria isolated from rat brain subregions based on changes in fura-2 fluorescence after disruption of the organelles with Triton X-100 and sodium dodecyl sulfate. Mitochondria isolated from the forebrain of normal rats contained 2.5 ± 0.9 nmol of calcium/mg of protein. A 30-min ischemic period produced an approximately twofold increase in the calcium content of mitochondria isolated from the dorsolateral striatum, a region in which most neurons die within 24 h after this period of ischemia. The calcium content of mitochondria from the paramedian cortex, a region in which there are few ischemia-susceptible neurons, tended to be similarly increased, although this difference was not statistically significant. Larger increases (to approximately five times control values) were seen in mitochondria isolated from both regions after 10 min of recirculation. By 1 h of recirculation, mitochondrial calcium had returned close to preischemic control values in both regions. Longer recirculation periods produced no further changes in the calcium content of mitochondria from the paramedian cortex. However, mitochondrial calcium was again increased in the dorsolateral striatum after 6 h (6.5 nmol of calcium/mg of protein) and 24 h (8.7 nmol of calcium/mg of protein) of recirculation. This regionally selective increase in calcium in the dorsolateral striatum preceded the period during which the majority of neurons in this region exhibit advanced degenerative changes. Thus, this increase may be an essential step, albeit a late one, in the development of neuronal loss.

Notes: Abstract: Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 µM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol 〉 1-palmitoyl lysophosphatidylcholine 〉 1-oleoyl lysophosphatidylcholine ≫ lysophosphatidylserine 〉 lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.

Notes: Abstract: Levels of immunoreactive β-amyloid precursor protein and interleukin-1α were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa β-amyloid precursor protein isoform were elevated to fourfold (p 〈 0.05) those of controls and those of the 130-kDa isoform to threefold (p 〈 0.05), whereas those of the 120-kDa isoform (p 〉 0.05) were not different from control values. β-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 ± 12 vs. 8 ± 3/mm2; p 〈 0.001). However, neither β-amyloid precursor protein-immunoreactive dystrophic neurites nor β-amyloid deposits were found in this tissue. Interleukin-1α-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 ± 8 vs. 25 ± 5/mm2; p 〈 0.001), and these cells were often found adjacent to β-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of β-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Notes: Abstract: Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the postsynaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43-kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43-kDa protein and to compare them with hybrid muscle nAChRs containing a β subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit α1β1γδ and 43-kDa protein transcripts produced discrete receptor aggregates with a diameter of 1–5 µm whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the β1 subunit by the neuronal β2 protein produced a functioning receptor that did not aggregate in the presence of 43-kDa protein and was substantially blocked by the same concentration of NBT. Hybrid α1β4γδ receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43-kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing β2. These results suggest that the β subunit is an important determinant in receptor localisation and sensitivity to NBT.

Notes: Abstract: LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage-sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid-treated cells. Both high K+- and maitotoxin-induced [Ca2+]i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd3+, an inorganic Ca2+-entry blocker, and by the snail toxin ω-conotoxin GVIA, which interacts with the N sub-type of voltage-sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel sub-type, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K+-induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K+-induced increase of [Ca2+]i was still present. In conclusion, the results of the present study demonstrated that retinoic acid-induced differentiation of LAN-1 cells, which lack a high K+-evoked [Ca2+]i increase in the undifferentiated state, induces the functional expression of an ω-conotoxin GVIA-sensitive, dihydropyridine-insensitive N-type voltage-sensitive Ca2+ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.

Notes: Abstract: Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [3H]muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [3H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.

Notes: Abstract: Exposure of human SK-N-MC neurotumor cells to 4β-phorbol 12-myristate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down-regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein Gi was unlikely. Cholate extracts of membranes from control and PMA-treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc− membranes, which lack the stimulatory G protein subunit Gsα; thus, Gs did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn2+ and Mn2+ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the Kact for isoproterenol but not for dopamine or CGP-12177 (a β3-adrenergic agonist) stimulation. Thus, the β1 but not the D1 or β3 receptors were being desensitized by PKC activation. Analysis of SK-N-MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC-α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC-α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.

Notes: Abstract: Several reports have suggested a characteristic decrease in glucose use in the striatum of patients with Huntington's disease (HD) may contribute to the cellular atrophy of the caudate and putamen. We examined the expression of the two major glucose transporter isoforms of brain, GLUT1 and GLUT3. GLUT1 is found largely in capillary endothelial cells and to a lesser extent in the brain parenchyma, whereas GLUT3 is localized primarily in neurons. Membranes prepared from postmortem samples of HD caudate and cortex and non-HD caudate and cortex were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and probed with antisera to GLUT1 and GLUT3 by western blotting. Compared with controls, GLUT1 and GLUT3 transporter expression in caudate was decreased by three- and fourfold, respectively, in grade 3 of the disease. At earlier stages (grade 1), there was no significant difference in the expression of the two transporter isoforms compared with nondiseased controls. It is surprising that despite a substantial increase in glial fibrillary acidic protein immunoreactivity (an indicator of the extent of gliosis), glucose transporter expression was diminished significantly in HD caudate. The results suggest in the absence of a significant number of neurons, as in grade 3, glial cell GLUT1 and GLUT3 expression is down-regulated, perhaps reflecting the decreased metabolic demand of this brain region in HD.

Notes: Abstract: Previously, we identified protein kinase FA/glycogen synthase kinase-3α (GSK-3α) as a brain microtubule-associated τ kinase that phosphorylates Ser235 and Ser404 of τ and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament-associated pathological τ (PHF-τ) in Alzheimer's disease brains. In this study, we found that the activity of kinase FA/GSK-3α towards phosphorylation of brain τ could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of τ, resulting in a reduced mobility of τ with an apparent molecular mass shift to ∼68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer-τ. Tryptic digestion of 32P-labelled τ, followed by HPLC and two-dimensional separation on TLC cellulose plates, revealed eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and protein sequence analysis further revealed that, in addition to Ser235 and Ser404, heparin generated Thr212, Thr231, Ser262, Ser324, and Ser356, the five extra phosphorylation sites in τ. As Ser235, Ser262, Ser324, Ser356, and Ser404 (particularly the site of Ser262) have been identified as five of the most potent sites in τ responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr231, Ser235, Ser262, and Ser404 are four of the most well documented sites abnormally phosphorylated in Alzheimer-τ, the results provide initial evidence that protein kinase FA/GSK-3α after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-τ in Alzheimer's disease brains.

Notes: Abstract: The pH dependency of the binding of ligands to adenosine A2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5′-N-ethylcarboxamidoadenosine, adenosine, 3′-deoxyadenosine, 5′-deoxyadenosine, inosine, and N6-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2′-deoxyadenosine, although 2′-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2′-hydroxyl group of adenosine agonists.

Notes: Abstract: In the neostriatum, amphetamine and other dopamine agonists elevate the extracellular level of ascorbate, which is known to modulate neostriatal function. Although both D1 and D2 receptors have been linked to neostriatal ascorbate release, ample evidence suggests it is controlled by areas outside the neostriatum. The present series of experiments used selective lesions and intracerebral drug infusions to probe the involvement of the ventromedial thalamus and substantia nigra pars reticulata. Our results implicate both of these sites in amphetamine-induced increases in the release of neostriatal ascorbate. Thus, whereas unilateral electrolytic lesions of the substantia nigra pars reticulata completely abolished the ability of systemic amphetamine (2.5 mg/kg) to increase extracellular ascorbate in ipsilateral neostriatum, intranigral infusions of this drug (10 and 30 µg/µl) elevated neostriatal ascorbate release. This infusion effect, moreover, was blocked by electrolytic lesions of the ipsilateral ventromedial thalamus, which receives input from the substantia nigra pars reticulata and projects to the cerebral cortex. These results, combined with previous evidence implicating cortical projections to neostriatum as the source of extracellular ascorbate, suggest that neostriatal ascorbate release is regulated, at least in part, by a nigro-thalamo-cortico-neostriatal pathway.

Notes: Abstract: The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), α subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to ∼10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X-100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0°C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37°C, by which the content of CaMKIIα in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII α subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKIIβ/β′.

Notes: Abstract: This study examines the proteolysis of the carboxy terminal domain of the full-length (α1212) and truncated (α1190) forms of the rabbit skeletal muscle L-type calcium channel α1 subunit by calpain I and calpain II. Although both forms of the α1 subunit show little sensitivity to proteolysis by calpain II, α1212 is relatively more sensitive than α1190 to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of α1212 in a time- and concentration-dependent manner and proteolysis is independent of the α1212 phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from α1212 and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.

Notes: Abstract: Previous studies have shown that potassium-evoked, calcium-dependent, endogenous aspartate release is greater from hippocampal slices of adult epileptic (EL) mice than from nonepileptic control C57BL/6J (B6) mice. To examine further the association between epilepsy and enhanced aspartate release in EL mice, endogenous neurotransmitter release from hippocampal slices was studied in young, seizure-free EL mice and in two nonseizure control mouse strains, DDY and B6. DDY is the parental strain from which EL arose, and it has a genetic background similar to EL. Released amino acid neurotransmitters were quantitated by HPLC with fluorescent detection and were expressed as picomoles of amino acid released per minute of incubation per slice ± SEM. Aspartate release was significantly higher in EL mice (15.8 ± 0.8) than in either the control B6 or DDY mice (8.5 ± 1.4 and 8.4 ± 1.7, respectively). No significant differences were found among the B6, DDY, and EL mice for the release of glutamate (23.0 ± 2.0, 32.3 ± 5.8, and 25.9 ± 2.6, respectively) or GABA (23.5 ± 0.7, 19.5 ± 3.2, and 21.8 ± 3.2, respectively). Thus, enhanced aspartate release precedes the onset of EL seizures and may be related to the cause rather than to the effects of seizure activity.

Notes: Abstract: The binding of an agonist, p-[125I]iodoclonidine, and an antagonist, [3H]yohimbine, to α2-adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant-free victims of suicide and age-matched controls. Agonist binding to α2-adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α2-adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α2-adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high-affinity state of autoinhibitory α2-adrenoceptors with suicide.

Notes: Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP+) on the oxygen consumption, ATP production, H2O2 production, and mitochondrial NADH-CoQ1 reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µM MPP+ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP+ produced a concentration-dependent decrease in H2O2 production. Incubation of mitochondria with ADP for 60 min after loading with 100 µM MPP+ caused no loss of complex I activity after washing of MPP+ from the mitochondrial membranes. These data are consistent with MPP+ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H2O2 by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H2O2 in the presence of Cu2+ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP+ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.

Notes: Abstract: Mutations in the Caenorhabditis elegans unc-41 gene result in an allele-dependent elevation of acetylcholine content. Eight recessive alleles (cn252, e268, e399, e650, e1175, e1199, e1294, and e870) lead to phenotypes including uncoordinated locomotion, slow growth, a small mature body, and resistance to the acetylcholinesterase inhibitors as well as the elevation of acetylcholine content. The remaining two alleles, e554 and e1162, exhibit normal acetylcholine levels but display the short-body phenotype in a semidominant way. To determine the localization of the elevated acetylcholine content, a method for the isolation of synaptic vesicles from C. elegans was established. The elevation of acetylcholine content in the unc-41 mutants is accompanied by the accumulation of synaptic vesicles. We propose that at least one function of the unc-41 gene relates to the release of neurotransmitters.

Notes: Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine-mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.

Notes: Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d-fenfluramine has been shown to act as a potent releaser of brain serotonin [5-hydroxytryptamine (5-HT)]. Here we have investigated the effects of d-fenfluramine on the release of [3H]5-HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [3H]5-HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d-fenfluramine used. At 5 µM, the release of [3H]5-HT was blocked by the 5-HT transporter inhibitor fluoxetine and was Ca2+ independent and insensitive to the human autoreceptor 5-HT1D agonist sumatriptan. The release of [3H]5-HT elicited by 0.5 µMd-fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca2+ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d-fenfluramine largely diffuses into serotonergic terminals and causes release of 5-HT through the 5-HT carrier working in the inside-outside direction; at relatively low concentrations d-fenfluramine enters the terminals through the 5-HT transporter but elicits release of 5-HT by an exocytotic-like mechanism.

Notes: Abstract: In mammalian brain, ischemic injury could be mediated by delayed glutamate neurotoxicity. We have studied the possibility of altered genetic expression of quiescent NMDA receptor subunits in an in vitro model of hypoxia-hypoglycemia, using the reverse transcription-polymerase chain reaction technique. It was found that mRNA corresponding to the NR2C subunit was present 1 h after a brief hypoxic-hypoglycemic episode in adult rat hippocampus and cortex but absent in control tissue. These findings indicate that the phenotypic characteristics of certain brain cells after an ischemic insult are altered by the expression of genes that are quiescent in those cells under normal physiological conditions.

Notes: Abstract: The mRNA encoding μ-opioid receptors is expressed in neurons of the globus pallidus, a region of the basal ganglia that receives a dense enkephalinergic innervation from the striatum. The regulation of the mRNAs encoding the opioid peptide enkephalin in the striatum and the μ-opioid receptor in the globus pallidus was examined with in situ hybridization histochemistry following short- or long-term haloperidol treatments, which alter striatal enkephalin mRNA levels. Animals were administered haloperidol daily for 3 or 7 days (1 mg/kg, s.c.) or continuously for 8 months (1 mg/kg, depot followed by oral). Enkephalin and μ-opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, the enkephalin mRNA level was increased in the striatum, and μ-opioid receptor mRNA levels were markedly decreased in the globus pallidus after 7 days of haloperidol administration. Similar effects were observed in rats treated with haloperidol for 8 months. The results provide the first evidence of regulation of μ-opioid receptor mRNA in vivo.

Notes: Abstract: In vivo microdialysis was used to assess the hypothesis that the stress-induced increase in dopamine release in the prefrontal cortex is mediated by stress-activated glutamate neurotransmission in this region. Local perfusion of an α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, blocked the stress-induced increase in dopamine levels, whereas an NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid, at the dose tested, was not able to alter this response significantly. These data indicate that the effect of stress on dopamine release in the prefrontal cortex is mediated locally by activation of AMPA/kainate receptors, which modulate the release of dopamine in this region.

Notes: Abstract: A PCR-based strategy was used to clone DNAs encoding Na+- and Cl−-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

Notes: Abstract: The rat Schwannoma cell line D6P2T constitutively expresses the mRNA encoding the major myelin protein, P0, but only expresses the mRNA encoding myelin basic protein (MBP) after exposure to forskolin or other substances that raise the levels of intracellular cyclic AMP. In this study we have investigated the molecular basis for forskolin induction of MBP transcription in D6P2T cells. We have found that a 9-bp sequence element, CACTTGATC, located between nucleotides −85 and −77 in the MBP promoter, is necessary for forskolin induction of chloramphenicol acetyltransferase (CAT) expression after transient transfection of MBP promoter-CAT fusion constructs into D6P2T cells. Although similar DNase I footprints, one of which is located within the above 9-bp sequence element, are produced by nuclear extracts prepared from both forskolin-treated and untreated cells, this same sequence can be shown to interact with a forskolin-inducible protein complex using an electrophoretic mobility shift assay. In addition, mutation of this 9-bp sequence abolishes both formation of this new protein-DNA complex and forskolin-inducible CAT expression from the heterologous SV40 promoter. Finally, we have shown that the appearance of this forskolin-inducible protein-DNA complex precedes that of MBP mRNA. Taken together, these data strongly support the notion that the induction of MBP transcription by forskolin in D6P2T cells is mediated by the binding of a forskolin-inducible protein complex to the MBP promoter sequence CACTTGATC.

Notes: Abstract: The human D4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [35S]GTPγS binding assay. By measuring D4 receptor stimulation of [35S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D4 receptor, dopamine, quinpirole, and dp-2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [35S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [35S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [3H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.

Notes: Abstract: 1-Methyl-4-phenylpyridinium (MPP+), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′-alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4′-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ∼600-fold, versus only a 36-fold, increase in the IC50 of MPP+ versus 4′-pentyl-MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′-decyl-MPP+ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol. In addition, the lipophilic anion TPB− was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.

Notes: Abstract: Aromatic l-amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12-myristate 13-acetate administered intracerebroventricularly transiently increased AAAD activity by 30–50% over control values within ∼30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of Vmax with little change of Km for either l-3,4-dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester-induced increase of AAAD. Moreover, okadaic acid, a serine/threonine-selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C-mediated pathways modulate AAAD activity in vivo.

Notes: Abstract: In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N-terminal residues 253–267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877–889 increased about 1.6-fold following permeabilization. Our data suggest that the N- and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross-reactivities in different species and their effect on ligand binding activity were also established.

Notes: Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [3H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly (t1/2 〈 1 min) to steady-state values that averaged 〈5% of plasma specific activity. Incorporation was rapid, as 〉85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.

Notes: Abstract: Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono-Q steps. The phosphocellulose eluate showed two BBGM-CK II activities. The first minor component (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., α (40 kDa), α′ (38 kDa), and β (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM-CK IIa and b showed chromatographic and biochemical differences including differing Km for ATP and GTP and Ki for heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin β-light chain. The unusual chromatographic behavior and biochemical properties of these BBGM-CK II isozymes, compared with the classical CK II, could be explained at least in part by their subunit ratios.

Notes: Abstract: The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of ∼33–45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is ∼16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at ∼40 pg/ml, or 1 pM, and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.

Notes: Abstract: The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly undissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Notes: Abstract: The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF-M) protein. The tissue-specific expression of goldfish NF-M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF-M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF-M protein is similar to higher vertebrate NF-M proteins. In addition, the organization of the goldfish NF-M gene is identical to the NF-M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF-M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF-M genes.

Notes: Abstract: Leech lectin 35 (LL35) is a calcium-independent galactoside-binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium-independent, galactose-specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity-purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.

Notes: Abstract: Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5-HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5-HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56- and 45-kDa forms of SBP are produced by posttranslational cleavage of a 68-kDa precursor molecule; and (b) that 45-kDa SBP is a constituent of serotonergic secretory vesicles. Pulse-chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with l-[35S]methionine, the cells were incubated in the presence of an excess of unlabeled l-methionine for 0, 30, 60, or 90 min at 37°C. Alternatively, the chase was performed under conditions (20°C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20°C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post-Golgi compartment. When secretion was induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was released to the medium. When antibodies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces after 20 min, at which time antibodies to SBP were found inside the cells. It is suggested that 45-kDa SBP is packaged with 5-HT in secretory vesicles. Some 45-kDa SBP is lost during secretion as a result of exocytosis; however, a fraction of the 45-kDa SBP remains bound to the luminal surface of the membrane of secretory vesicles. This protein is exposed to the ambient medium as a consequence of exocytosis, but is reinternalized when the vesicular membrane is recaptured during vesicle recycling.

Notes: Abstract: This study explores the role of cyclic AMP in electrically evoked [3H]noradrenaline release and in the α2-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α2-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on 3H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [3H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca2+ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca2+ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α2-adrenergic inhibition of noradrenaline release involves voltage-activated Ca2+ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α2-adrenergic reduction, apparently through a disinhibition of Ca2+ channels.

Notes: Abstract: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]Enkephalin and selective δ- and μ-, but not κ-, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb δ- and μ-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.

Notes: Abstract: Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1-[2-[bis(4-fluorphenyl)methoxy]-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6-hydroxydopamine (6 µg/µl) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6-hydroxydopamine-treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6-hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10−5 mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.

Notes: Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [3H]acetylcholine ([3H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A1/A2 agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [3H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [3H]ACh release was prevented by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [3H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [3H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [3H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A1 inhibitory receptors modulate ACh release, whereas in the CA3 area, both A2A excitatory and A1 inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A1 inhibitory and A2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.

Notes: Abstract: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [3H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [3H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N-methyl-d-aspartate-induced [3H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [3H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.

Notes: Abstract: Fetal cerebral metabolism changes during development. The normal fetal metabolic rate must be known to evaluate pathophysiological changes. Therefore, we determined the regional cerebral glucose consumption in the fetal guinea pig. This required the application of the 2-deoxyglucose method to this species. We measured both the transfer coefficients of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the lumped constant in chronically prepared undisturbed guinea pig dams using a three-compartment model. Furthermore, the ratio between the initial clearances of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the ratio between the phosphorylation coefficients of these substrates in the fetal brain were determined. The total cerebral glucose consumption measured by the deoxyglucose method (10 ± 1.2 µmol/100 g/min) was similar to that calculated from the glucose concentration and the phosphorylation coefficient of glucose in the cerebrum (10 ± 0.4 µmol/100 g/min). We conclude that the 2-deoxyglucose method is applicable to the guinea pig, and we further conclude that in the fetal guinea pig cerebral glucose consumption is 10 times lower than that in the adult.

Notes: Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 mM 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 mM 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.

Notes: Abstract: Effects of thyroxine (T4) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T4 caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T4 was effective on the post-natal rats only up to day 11. These results suggest that T4 plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T4 suggest that the effects of T4 on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T4.

Notes: Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.

Notes: Abstract: When isolated myelin membranes were ADP-ribosylated by [32P]NAD+ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg9 and Arg54 by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.

Notes: Abstract: Using a brain microdialysis technique, we have shown in the rat that local infusion of a selective and competitive N-methyl-d-aspartate (NMDA) receptor antagonist, cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS-19755), into the medial frontal cortex via dialysis tubing caused a concentration-related increase in the extracellular release of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the cortical region. Coinfusion of a sodium channel blocker, tetrodotoxin, completely inhibited the ability of the NMDA antagonist to augment frontal dopamine metabolism. These findings suggest that dopamine neurons projecting to the frontal cortex might be under a tonic transsynaptic inhibition exerted by excitatory amino acid neurotransmission via the NMDA receptor at the level of dopamine terminal fields.

Notes: Abstract: Plasma and urinary catecholamines were quantified to assess global sympathoadrenal function in rats with preganglionic lesions caused by antibodies to acetyl-cholinesterase (AChE). Rats were given intravenous injections of normal mouse IgG or murine monoclonal anti-acetylcholinesterase IgG (1.5 mg). Five or 16 days afterward, basal blood samples were taken through indwelling arterial cannulae. A few hours later, the rats were immobilized for 10 min in padded restrainers, and another blood sample was drawn. HPLC determinations showed low basal levels of norepinephrine and epinephrine (〈0.2 ng/ml in all rat plasma samples). In control rats, immobilization stress increased levels of plasma catecholamines up to 35-fold. In rats tested 5 days after injection of antibody, the norepinephrine response was much smaller (15% of control), and (he epinephrine response was nearly abolished (5% of control). There was some recovery at 16 days after antibody treatment, but stress-induced catecholamine release was still markedly impaired. Reduced stress-induced release: was not accompanied by major changes in tissue epinephrine or norepinephrine (heart, spleen, adrenal glands, and brain), although adrenal dopamine content dropped by 60%. Urinary excretion was studied in parallel experiments to gain insight into the effects of AChE anti-bodies on basal sympathoadrenal activity. Epinephrine, norepinephrine, dopamine, and selected metabolites were quantified in 24-h urine samples collected at frequent intervals for 30 days after antibody injection. No statistically gnificant changes were detected in the urinary output of dopamine, 3-methoxytyramine, normetanephrine, or 3-methoixy-4-hydroxyphenylglycol. On the other hand, epinephrine and norepinephrine output increased sharply at the time of antibody injection and then fell significantly below control levels. Norepinephrine output returned to normal after 2 weeks, but epinephrine output remained depressed. These results are consistent with previous evidence of widespread and persistent antibody-mediated βmade to the preganglionic sympathetic system.

Notes: Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [3H]adenine-or [3H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [3H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [3H]cGMP accumulation (forskolin EC50 value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [3H]-cAMP in [3H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [3H]cAMP response or a potentiation of the SNP-induced [3H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. AG-Nitro-l-arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [3H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [3H]cGMP response to either SNP or the combination of SNP plus forskolin. N6,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [3H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [3H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.

Notes: Abstract: A histidine residue (His394) that is likely to be located in the ligand-binding region of the D2 dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394 residue with these compounds.

Notes: Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2-and 3-day-old embryos within the same sub-cellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

Notes: Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D1 agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D2 receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d-Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D1 receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D2 antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D1 receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.

Notes: Abstract: Ten-minute incubation with 1 mMS-nitroso-l-cysteine (NO-CYS), a nitric oxide (NO) generator, induced a 3.5-fold increase in the basal dopamine (DA) efflux from rat striatal slices. Reduced hemoglobin (100 µM), a NO scavenger, blocked this response. Nomifensine (10 µM), an inhibitor of high-affinity DA transport, reduced the NO-CYS effect by 39%. In a synaptosomal preparation, NO-CYS induced a concentration-dependent efflux of [3H]DA that was also significantly inhibited by 10 µM nomifensine. Because these findings indicated that NO could alter the activity of the striatal DA transporter, we tested the effect of NO and NO-CYS on the uptake of [3H]DA into crude striatal synaptosomes. Although both compounds inhibited [3H]DA uptake with similar dose-response characteristics (IC50≈300 µM and ≈400 µM, respectively), the effect of NO was quicker in onset. The presence of superoxide dismutase (30 U/ml) and catalase (50 U/ml) had a small impact on the NO-CYS inhibition of [3H]DA uptake (1.8-fold increase in IC50). NO (1 mM) inhibited striatal [3H]glutamate uptake by 23%, but lower concentrations had no significant effect. The duration of the effect of NO gas on [3H]DA uptake varied from 〈5 min to〉10 min, depending on the concentration used. All concentrations of NO-CYS tested produced an inhibition of [3H]DA uptake that lasted at least 10 min. However, only concentrations ≤1 mM NO-CYS (or NO) were washable. The effect of 3 mM NO-CYS lasted〉20 min and could not be reversed by washing. Reduced hemoglobin (300 µM) prevented the action of 3 mM NO-CYS, whereas methemoglobin had no effect. The action of 3 mM NO-CYS was temperature dependent and took about 2 min to fully develop. Scatchard analysis revealed that NO-CYS diminished the capacity of the DA transporter without having a significant effect on the affinity. NO-CYS had no effect on striatal [3H]-mazindol binding, suggesting that NO-CYS did not interact with the DA recognition site of the transporter. These data suggest that NO may play a role in the regulation of DA and, to a lesser extent, glutamate transport in the striatum.

Notes: Abstract: Previous research has shown that systemically administered antipsychotic drugs enhance dopamine release from the nigrostriatal and mesocortical dopamine pathways. However, the degree of enhancement differs as a function of the drug used (atypical versus typical antipsychotic) and the dopamine pathway examined. The present studies examined whether these differences result from differential actions of these drugs on dopamine terminal regions. Clozapine or haloperidol was infused locally into the caudate-putamen or prefrontal cortex through reverse microdialysis. Although both drugs increased extracellular dopamine levels, clozapine produced greater effects than haloperidol in the prefrontal cortex, whereas haloperidol produced greater effects in the caudate-putamen. These results suggest that neurochemical differences within dopamine terminal regions may explain the differential actions of antipsychotic drugs on striatal and cortical dopamine release.

Notes: Abstract: The carbohydrate structures of β-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N-glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N-acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N-acetylglucosamine residues of nearly all structures were found to be α1,6-fucosylated. Peripheral fucose (about 20%α1,3-linked to N-acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N-acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N-acetylglucosamine only were present. At least 20 different N-glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N-acetylglucosamine, as well as terminal N-acetylglucosamine and galactose residues, and significant amounts of N-acetylneuraminic acid in α2,3 linkage), this protein can be classified as “brain-type” glycosylated.

Notes: Abstract: We have studied the effect of isonicotinic acid hydrazide (INH), a convulsant agent, on the extracellular levels of amino acids in the hippocampus, and the effect of sodium valproate (VPA) administration in INH-treated rats. INH (250 mg/kg) caused a rapid and sustained decrease in basal levels of GABA, and during this period convulsions of increasing severity were observed. Basal levels of glutamine, taurine, aspartate, and glutamate were unchanged by INH. When VPA was coadministered with INH, basal GABA levels were increased and no convulsions were observed. When transmitter release was evoked using 100 mMK+, the increase in dialysate GABA observed in INH-treated animals was less than that seen in controls and convulsions increased in frequency. K+-evoked release of glutamate and aspartate tended to be higher following INH treatment, and in the case of aspartate, this increase was significant. VPA reversed the changes in evoked release of glutamate and aspartate, and release of GABA was considerably greater than that seen in control or INH-treated rats. No drug effect on evoked changes in taurine or glutamine level was seen. These are the first data to show decreased extracellular GABA in conjunction with convulsions in freely moving animals in vivo.

Notes: Abstract: The effect of the Kunitz proteinase inhibitor (KPI) on potential β-amyloid precursor protein (βPP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in βPP. In addition, the level of secretion of KPI-containing βPP751 and KPI-lacking βPP695 from transfected cells was examined to assess the effect of the KPI on βPP secretion. βPP751 and βPP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations βPP751, but not βPP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of βPP or in the amyloidogenic cleavage of βPP. The amounts of βPP695 and βPP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in βPP751 did not inhibit the secretory cleavage in transfected cells.

Notes: Abstract: Abstract: Previous observations have implicated GABA as a neurotransmitter released by the vestibular sensory neurons („hair cells”) of the snail Hermissenda onto visual sensory neurons, the type B cells, whose cell bodies are the sites of biophysical and biochemical changes during and following Pavlovian conditioning. Still other observations demonstrated that light-GABA pairings that simulate stimuli presented during Pavlovian conditioning cause prolonged elevatiori of intracellular Ca2+ and transformation of GABA-induced synaptic inhibition into excitation. Intracellular Ca2+ signals in response to GABA perfused onto the postsynaptic: type B terminal branches are shown here to be prolonged on days after conditioning, but not after control paradigms. These and past results demonstrate two separate sites, i.e., the cell body and the terminal branches, for learning-induced changes after Pavlovian conditioning.

Notes: Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca2+],) was characterized in GH3 cells, a pituitary tumor cell line, which are known to possess CCKB receptor subtype. The CCK-8-induced [Ca2+], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca2+], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 nM). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.

Notes: Abstract: Activation of trophic factor receptors stimulates tyrosine phosphorylation on proteins and supports neuronal survival. We report that in the recovery phase following reversible cerebral ischemia, tyrosine phosphorylation increases in the membrane fraction of the resistant hippocampal CA3/dentate gyrus (DG) region, whereas in the sensitive CA1 region or striatum, tyrosine phosphorylation is less marked or decreases. In the cytosolic fractions, a 42-kDa protein, identified as mitogen-activated protein (MAP) kinase, is markedly phosphorylated and activated immediately following ischemia, in particular in CA3/DG, but not in striatum. In the CA1 region, phosphorylation of MAP kinase is less intense and decreases later during re- perfusion, which could explain the delay of neuronal degeneration in this structure. The data suggest that in ischemia-resistant neurons the growth factor receptor-coupled signaling cascade is stimulated and, through its effects on DNA transcription and mRNA translation, supports neuronal survival.

Notes: Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5-15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]-glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the Km of glutamine synthetase for glutamate, which we determined to be ∼7 mM.

Notes: All known nicotinic receptor α subunits include a conserved disulfide bond that is essential for function and is a site for labeling via biochemical modification. In an effort to develop a universal ligand for all subtypes of nicotinic receptors, we previously studied the effects of arsenylation with two compounds, ρ-aminophenyldichloroarsine (APA) and bromoacetyl-ρ-aminophenylarsenòxide (BAPA) on nicotinic receptors from Torpedo electroplax. Here we apply these reagents to immunoisolated receptors containing α4, β2, and possibly other subunits from chick brain that bind [3H]cytisine with high affinity (KD∼5 nM). These are distinct from another receptor subtype that also binds [3H]cytisine and [3H]nicotine and can be arsenylated with APA, but instead contains α5,β2, and probably other subunits. Reduction of α4 β2 receptors with dithiothreitol blocked [3H]cytisine binding and this effect was reversed upon reoxidation by dithiobisnitrobenzoic acid. APA or BAPA prevented the dithiobisnitrobenzoic acid reactivation of dithiothreitol-treated receptors with IC50 values of 15 and 70 nM, respectively. However, the antiarsenical dimercaptopropanesulfonic acid restored function to APA- or BAPA- “arsenylated” receptors (EC50∼100 μM). APA-treated receptors remained blocked for up to 24 h, but treatment with dimercaptopropanesulfonic acid at any time restored [3H]cytisine binding. APA treatment of reduced receptors protected against irreversible alkylation by Bromoacetyl choline, indicating that arsenylation occurs at least in part in the agonist binding site. Thus, these reagents have similar effects on different nicotinic receptor subtypes from both muscle and nerves.

Notes: Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li+ in serum reached 0.6–1.2 mM, a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [32P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more 32P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.

Notes: Abstract: An analogue of colchicine,β-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. β-Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol-stimulated 36Cr-uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopusoocytes expressing human α1β2γ2S, but not α1β2, GABAA receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of β-Lumicolchicine on the GABAA receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, β-Lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.

Notes: Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18-carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20-and 22-carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo 13C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly 13C-enriched 16-and 18-carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U-13C-polyunsaturated fatty acid mixture, 13C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; 13C enrichment remained high in the phospholipids and cholesterol for 15 days. 13C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in 13C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16-and 18-carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long-chain saturated and monounsaturated fatty acids and cholesterol.

Notes: Abstract: Native horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) conjugated to HRP are protein probes represented in the blood-brain barrier (BBB) literature for elucidating morphological routes of passage between blood and brain. We report the application of established pharmacokinetic methods, e.g., multiple-time regression analysis and capillary depletion technique, to measure and compare bidirectional rates of passage between blood and brain for radioactive iodine-labeled HRP (I-HRP), WGA-HRP (I-WGA-HRP), and the serum protein albumin (I-ALB) following administration of the probes intravenously (i.v.) or by intracerebroventricular (i.c.v.) injection in mice. The pharmacokinetic data are supplemented with light and electron microscopic analyses of HRP and WGA-HRP delivered i.v. or by i.c.v. injection. The rates of bidirectional movement between blood and brain are the same for coinjected I-HRP and I-ALB. Blood-borne HRP, unlike WGA-HRP, has unimpeded access to the CNS extracellularly through sites deficient in a BBB, such as the circumventricular organs and subarachnoid space/pial surface. Nevertheless, blood-borne I-WGA-HRP enters the brain ˜10 times more rapidly than I-HRP and I-ALB. Separation of blood vessels from the neocortical parenchyma confirms the entry of blood-borne I-WGA-HRP to the brain and sequestration of I-WGA-HRP by cerebral endothelial cells. Nearly half the I-WGA-HRP radioactivity associated with cortical vessels is judged to be subcellular. Light microscopic results suggest the extracellular pathways into the brain available to blood-borne native HRP do not represent predominant routes of entry for blood-borne WGA-HRP. Ultrastructural analysis further suggests WGA-HRP is likely to undergo adsorptive transcytosis through cerebral endothelia from blood to brain via specific subcellular compartments within the endothelium. Entry of blood-borne I-WGA-HRP, but not of I-ALB, is stimulated with coinjected unlabeled WGA-HRP, suggesting the latter may enhance the adsorptive endocytosis of blood-borne I-WGA-HRP. With i.c.v. coinjection of I-WGA-HRP and I-ALB, I-WGA-HRP exits the brain more slowly than I-ALB. The brain to blood passage of I-WGA-HRP is nil with inclusion of unlabeled WGA-HRP, which does not alter the exit of I-ALB. Adsorptive endocytosis of i.c.v. injected WGA-HRP appears restricted largely to cells lining the ventricular cavities, e.g., ependymal and choroid plexus epithelia. In summary, the data suggest that the bidirectional rates of passage between brain and blood for native HRP are comparable to those for albumin. Blood-borne WGA-HRP is assessed to enter the brain more rapidly than native HRP and albumin, perhaps by the process of adsorptive transcytosis through BBB endothelia, but has difficulty leaving the CNS; the latter result may be due to avid binding and adsorptive endocytosis of WGA-HRP by exposed CNS cells. Neither native HRP nor WGA-HRP alters the integrity of the BBB to albumin. For this reason, both native HRP and WGA-HRP are suitable probes for investigating the permeability of the BBB to macromolecules in vivo.

Notes: Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O2/5% CO2. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ∼6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca2+ channel, but were completely inhibited in Ca2+-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH4CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca2+ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca2+ release from intracellular stores, which is also induced by the Ca2+ influx.

Notes: In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [3H]dopamine ([3H]DA) release and neurotensin (NT)-induced facilitation of [3H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [3H]DA release and decreased K+-evoked [3H]DA release, whereas apamin was without effect. K+-evoked [3H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [3H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K+-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K+-evoked [3H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K+-evoked [3H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [3H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.

Notes: Abstract: We have investigated the effect of unsaturated free fatty acids (FFAs) on the brain GABA/benzodiazepine receptor chloride channel complex from mammalian, avian, amphibian, and fish species in vitro. Unsaturated FFAs with a carbon chain length between 16 and 22 carbon atoms enhanced [3H]diazepam binding in rat brain membrane preparations, whereas the saturated analogues had no effect. The enhancement of [3H]diazepam binding by oleic acid was independent of the incubation temperature (0-30°C) of the binding assay and not additive to the enhancement by high concentrations of C1. In rat brain preparations, the stimulation of [3H]diazepam binding by oleic acid (10−4M) was independent of the ontogenetic development. Phylogenetically, large differences were found in the effect of unsaturated FFAs on [3H]diazepam and [3H]muscimol binding: In mammals and amphibians, unsaturated FFAs enhanced both [3H]-muscimol and [3H]diazepam binding to 150-250% of control binding. In 17 fish species studied, oleic acid (10−4M) stimulation of [3H]diazepam binding was weak (11 species), absent (four species), or reversed to inhibition (two species), whereas stimulation of [3H]muscimol binding was of the same magnitude as in mammals and amphibians. In 10 bird species studied, only weak enhancement of [3H]muscimol binding (110–130% of control) by oleic acid (10−4M) was found, whereas [3H]diazepam binding enhancement was similar to values in mammal species. Radiation inactivation of the receptor complex in situ from frozen rat cortex showed that the functional target size for oleic acid to stimulate [3H]flunitrazepam binding has a molecular mass of ∼200,000 daltons. Our data show that unsaturated FFAs have distinct effects on membranebound GABA/benzodiazepine receptors in vitro.

Notes: Abstract: Apoptosis, also known as programmed cell death, is a mechanism used by different tissues to regulate their cell content. In the nervous system, this process is supposed to adjust the final number of neurons to the number of the target cells they are innervating. The demonstration that, in several systems in vitro and in vivo, neuronal apoptosis can be prevented by inhibiting RNA or protein synthesis suggests that an activation of gene expression is required in the cells that are going to die. The genes involved and their products, named “killer proteins”, are not known in the superior vertebrates. In order to identify such proteins, we have used and characterized an in vitro model consisting of neurons derived from 8-day-old embryonic chicken ciliary ganglia. RNA and protein synthesis inhibitors can prevent the death of these neurons when they are deprived of trophic support. Comparing the synthesis of proteins in trophic-supported neurons with that in trophic-deprived neurons by the use of two-dimensional polyacrylamide gel electrophoresis, we have observed that several proteins were overexpressed reproducibly in the apoptotic cells. We found that all these proteins are localized in the nucleus, suggesting that they may be transcription regulators.