Effects of Acute and Chronic Phenytoin on the Electrolyte Content and the Activities of Na+, K+-, Ca2+, Mg2+ -, and HCO3--ATPases and Carbonic Anhydrase of Neonatal and Adult Rat Cerebral Cortex

Various parameters of anion and cation transport were measured in the cerebral cortex of neonatal (3-day-old) and adult rats following acute and chronic treatment with phenytoin (PHT). Acutely, PHT significantly inhibited the enzyme Na+, K+-ATPase in both neonatal and adult rats. This effect was accompanied by a significant increase in cerebral cortical Na+ content and a decrease in K+ content only in neonatal animals. Chronic treatment (two and four times a day for 7 days) of adult rats with PHT significantly reduced Na+ content without affecting whole homogenate Na+, K+-ATPase activity. The activity of this enzyme was markedly increased in the myelin- (glial product) and slightly decreased in the synaptosomal- (neuronal) fractions following chronic (four times a day for 7 days) PHT treatment. These results suggest that PHT differentially affects the two forms (neuronal and glial) of the enzyme Na+, K+-ATPase. The possible relevance of this hypothesis in relationship to the anticonvulsant and excitatory properties of PHT is discussed. Chronic (two and four times a day for 7 days) PHT treatment increased both DNA content and activity of the glial marker enzyme carbonic anhydrase. Activity of the mitochondrial enzyme HCO3- -ATPase was also increased following chronic PHT treatment. These two enzymes are intimately involved in the regulation of HCO3- -Cl- transport across glial cell and mitochondrial membranes, and these results suggest that PHT is able to affect beneficially glial regulatory processes. The ability to enhance glial regulation of anions and cations in extracellular fluid provides new and important insights into the mechanism of the anticonvulsant action of PHT.     

Effects of methadone on ornithine decarboxylase and cyclic nucleotide phosphohydrolase in neuronal and glial cell cultures

Mixed neuronal and nonneuronal cell cultures were obtained from 8-day-old chick embryos cerebral hemispheres and glial-enriched cultures were obtained from fifteen-day-old chick embryo cerebral hemispheres. Cultures were exposed to methadone, a narcotic drug, from days four to six. The activity of ornithine decarboxylase (ODC) was determined at day eight and the activity of cyclic nucleotide phosphohydrolase (CNP) was determined at day fifteen. Both ODC and CNP activity were higher in mixed neuronal-nonneuronal cell cultures treated with methadone as compared to control. No effect was observed in the neuronal-enriched or glial-enriched cultures. These findings are interpreted to reflect that neuronal-glial interaction is important in the response of primary neural cells to methadone.     

Role for glial cells in regulating the functional expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from dissociated fetal brain cells

A series of studies from our laboratory have established an aggregate culture system of fetal rat brain cells expressing neuropeptide Y (NPY) which can serve as a model to study the role of glia-neuron paracrine interactions in the developmental expression of NPY neurons. In this system, NPY production increases progressively with culture-age and it is induced by forskolin (FOR) and phorbol 12-myristate 13-acetate (PMA). We addressed the following question: Is the functional expression of the NPY neurons impaired in the absence of glial cells (particularly astrocytes) and if so, can secretory products of aggregates composed of the full complement of brain cells ( intact aggregates) restore the function of the impaired NPY neurons? Aggregates were generated from 17-day-old fetal rat cortex and maintained in serum-free medium for 13–15 days. Cytosine arabinoside (CA; doses of 0.5–8 μM) was added to the cultures on day 1 and the effectiveness in elimination of glial cells was verified on day 15 by measuring the incorporation of ³H thymidine into DNA and by immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Basal NPY production and FOR (10 μM) + PMA (20 nM) stimulated production of NPY on days 13–15 were taken as functional criteria. FOR + PMA induced ≈2-fold increase in NPY production in control cultures (no CA). CA inhibited both basal and FOR + PMA induced production of NPY and DNA synthesis in a dose-dependent manner: at 6 μM CA, basal NPY production was reduced by about 50%, FOR + PMA stimulated production of NPY and DNA synthesis were completely inhibited, and astrocytes were essentially eliminated. A competing dose of 100 μM deoxycytidine completely reversed the effects of 6 μM CA on DNA synthesis and basal and FOR + PMA stimulated production of NPY. Conditioned media derived from intact aggregates completely restored basal and partially (≈50%) restored FOR + PMA stimulated production of NPY in CA-treated aggregates. These results implicate secretory products of glial cells (astrocytes?) and/or glial-neuron paracrine interactions in regulating the developmental expression of NPY neurons in culture such that activation of the cAMP and protein kinase C pathways leads to increased production of NPY. © 1994 Wiley-Liss, Inc.     

Effects of the HIV-1 Envelope Glycoprotein gp120 in Cerebellar Cultures. [Ca2+]i Increases in a Glial Cell Subpopulation

The various types of cells present in cultures prepared from the postnatal rat cerebellum, identified by their gross morphology and immunocytochemistry, were loaded with the specific dye fura-2 and analysed individually for [Ca²⁺]_i changes induced by the HIV-1 envelope glycoprotein gp120 and a variety of other treatments. In granule neurons [Ca²⁺]_i increases were induced by high KCl and glutamate (mainly through the NMDA receptor) while in type-1 astrocytes this effect was observed after serotonin, carbachol and also quisqualate. In contrast, administration of gp120 was always without effect in these cells. Type-2 astrocytes (an arborized cell type responsive to agonists targeted to the glutamatergic AMPA and cholinergic receptors) were also most often unresponsive to the viral glycoprotein. However, among the cells exhibiting the arborized phenotype, a subpopulation (-13%) responded to gp120 with conspicuous [Ca²⁺]_i increases sustained by both release from intracellular stores and influx across the plasma membrane. These responses to the viral protein did not involve activation of either voltage-gated Ca²⁺ channels or glutamatergic receptors. Although not yet conclusively identified by specific cytochemical markers, the gp120-responsive cells resemble type-2 astrocytes and differ from neurons and type-1 astrocytes both in gross phenotype and in a number of receptor/channel properties: positivity to AMPA and cholinergic agonists; negativity to NMDA, serotonin and high KCl. From these results it is concluded that a subpopulation of glial cells is affected by gp120. The role of these cells in HIV brain infection and damage requires further studies to be precisely established.     

Effects of Dopamine D1 and D2 Receptor Agonists and Antagonists on Basal and Ethanol-Modulated β-Endorphin Secretion from Hypothalamic Neurons in Primary Cultures

In the present study, we determined the effects of dopamine receptor agonists and antagonists on basal and ethanol-modulated β-endorphin (β-EP) secretion from hypothalamic neurons in primary cultures. Treatment with various concentrations of dopamine D1 agonist SKF 38393 and D1 antagonist SCH 23390 did not affect basal IR-β-EP release. However, dopamine D2 receptor agonist LY 141865 reduced basal immunoreactive (IR)-β-EP release in a concentration dependent manner. D2 receptor antagonist, sulpiride, on the other hand, stimulated basal IR-β-EP release and blocked LY 141865-induced inhibition of IR-β-EP release in a concentration dependent manner. When the actions of these DA receptor agents on ethanol-modulated IR-β-EP release were studied, both D1 and D2 receptor agents failed to affect ethanol-modulated IR-β-EP release. These data suggest that the endogenous secretion of β-EP from hypothalamic neurons is under the influence of an inhibitory dopaminergic system involving the D2 receptor. Furthermore, ethanol's effects on β-EP secretion are not mediated by dopamine.     

Effects of Dibutyryl Cyclic AMP on Na+, K+-ATPase Activity and Intracellular Na+ and K+ in Primary Cultures of Astrocytes from DBA and C57 Mice

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na⁺, K⁺-ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na⁺)_i and (K⁺)_i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na⁺, K⁺-ATPase activity in cell homogenates was greater and (Na⁺)_i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K⁺)_i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na⁺, K⁺-ATPase activity in cell homogenates and decreased (Na⁺)_i, but had no significant effect on (K⁺)_i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K⁺ and Na⁺ concentration in medium altered Na⁺, K⁺-ATPase activity in cell homogenates as well as (Na⁺)_i and (K⁺)_i in cultured astrocytes of both DBA and C57 mice. Changes in (Na⁺)_i and (K⁺)_i at different K⁺ concentrations in medium paralleled those in Na⁺, K⁺-ATPase activity in cell homogenates. Results indicate that the ability to transport Na⁺ across the cell membrane and the response of Na⁺, K⁺-ATPase to db-cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.