Biomedical subjects

L Rönnbäck

Publications and source records attributed to L Rönnbäck.

At least 19 recordsLinked to original sources

PubMed2006

Corticosterone inhibits expression of the microglial glutamate transporter GLT-1 in vitro.

The present study investigates the effect of the glucocorticoid corticosterone on microglial glutamate transporters in vitro. Microglial cultures obtained from rat cerebral cortex were found to express the excitatory amino acid transporter GLT-1, but not GLAST, and this expression was increased by 1 ng/ml lipopolysaccharide after 12 h of stimulation. This increase has previously been shown to be mediated by tumor necrosis factor-alpha, a cytokine released by microglia during pathological conditions. Furthermore, lipopolysaccharide increased the microglial release of tumor necrosis factor-alpha and 1 microM corticosterone inhibited this effect. Corticosterone also inhibited the lipopolysaccharide-induced increase of the GLT-1 expression as well as the expression in non-activated cells. The effect of corticosterone on the GLT-1 expression was dose dependent and accompanied by similar effects on the microglial glutamate uptake capacity. Additionally, exogenous tumor necrosis factor-alpha was found to counteract the effect of corticosterone on microglial GLT-1 expression. The effect of corticosterone appeared to be glucocorticoid receptor specific since 10 microM of the glucocorticoid receptor antagonist mifepristone inhibited the effect. Thus, corticosterone decreased the microglial uptake of glutamate by decreasing the expression of glutamate transporters, probably due to the inhibited microglial tumor necrosis factor-alpha release. These results provide insights into the mechanisms behind microglial glutamate transporter expression during pathological conditions, and contribute to the debate about the beneficial or harmful effects of glucocorticoids.

Analysis of Variance↗

PubMed2001

Signaling and gene expression in the neuron-glia unit during brain function and dysfunction: Holger Hydén in memoriam.

Holger Hydén demonstrated almost 40 years ago that learning changes the base composition of nuclear RNA, i.e. induces an alteration in gene expression. An equally revolutionary observation at that time was that a base change occurred in both neurons and glia. From these findings, Holger Hydén concluded that establishment of memory is correlated with protein synthesis, and he demonstrated de novo synthesis of several high-molecular protein species after learning. Moreover, the protein, S-100, which is mainly found in glial cells, was increased during learning, and antibodies towards this protein inhibited memory consolidation. S-100 belongs to a family of Ca(2+)-binding proteins, and Holger Hydén at an early point realized the huge importance of Ca(2+) in brain function. He established that glial cells show more marked and earlier changes in RNA composition in Parkinson's disease than neurons. Holger Hydén also had the vision and courage to suggest that "mental diseases could as well be thought to depend upon a disturbance of processes in glia cells as in the nerve cells", and he showed that antidepressant drugs cause profound changes in glial RNA. The importance of Holger Hydén's findings and visions can only now be fully appreciated. His visionary concepts of the involvement of glia in neurological and mental illness, of learning being associated with changes in gene expression, and of the functional importance of Ca(2+)-binding proteins and Ca(2+) are presently being confirmed and expanded by others. This review briefly summarizes highlights of Holger Hydén's work in these areas, followed by a discussion of recent research, confirming his findings and expanding his visions. This includes strong evidence that glial dysfunction is involved in the development of Parkinson's disease, that drugs effective in mood disorders alter gene expression and exert profound effects on astrocytes, and that neuronal-astrocytic interactions in glutamate signaling, NO synthesis, Ca(2+) signaling, beta-adrenergic activity, second messenger production, protein kinase activities, and transcription factor phosphorylation control the highly programmed events that carry the memory trace through the initial, signal-mediated short-term and intermediate memory stages to protein synthesis-dependent long-term memory.

Animals↗

PubMed2001

Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V(max) and the K(m) of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca(2+) concentration, whereas the intracellular Ca(2+) antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca(2+).

Amino Acid Sequence↗

PubMed2000

Differential expression of delta opioid receptors and mRNA in proliferating astrocytes during the cell cycle.

Previous immunohistochemical and radioligand binding studies have shown a cell cycle-dependent regulation of the delta opioid receptor (DOR). The relationship between DOR expression and mitosis in primary astroglial cultures of rat cerebral cortex was investigated in this study. The cultures were arrested during the G(1)/S transition or during mitosis. The DOR protein level increased twofold (P = 0.009) during mitosis and DOR mRNA level increased threefold (P = 0.002) during the G(1)/S transition compared to nonsynchronized cultures. DOR mRNA was also elevated (1.6-fold, P = 0.008) during the G(1)/S transition compared with mitotic cells. A premitotic increase in DOR mRNA suggests that elevated DOR protein levels during mitosis might be regulated during transcription.

Animals↗

PubMed2000

Astrocyte beta1-adrenergic receptor immunoreactivity and agonist induced increases in [Ca2+]i: differential results indicative of a modified membrane receptor.

Antibodies against the C-terminus of the beta1-adrenergic receptor were used for staining cultured astrocytes from the rat cerebral cortex. Immunoreactivity was found to be localized exclusively to an intracellular organelle structure similar to the Golgi complex, with no staining of the plasma membrane. The astrocytes stained positive with BODIPY CGP 12177, a FITC-conjugated beta-adrenergic receptor agonist, and this staining was blocked by the beta1-adrenergic antagonist atenolol, indicating that these receptors are expressed on the surface of the astrocytes. The presence of functional plasma membrane beta1-adrenergic receptors was further verified using microspectrofluorometry for measurements of intracellular calcium changes upon beta-adrenergic agonist stimulation. Intracellular immunoreactivity confined to the organelles was also found in astrocytes from mixed astroglial-neuronal cultures. In contrast, the neurons in these cultures showed a strong labeling of the cell bodies by the beta1-adrenergic receptor antibodies. Thus, the beta1-adrenergic receptor antibody, which stains the cell bodies of the neurons, recognizes the astroglial receptors only intracellularly, although functional beta1-adrenergic receptors are present on the astroglial surface. Taken together, these data suggest that the beta1-adrenergic receptors observed intracellularly might be processed on their passage to the surface to a modified form of the final plasma membrane receptor, which is not recognized by the antibodies.

Adrenergic alpha-Agonists↗

PubMed2000

[Astrocytes--anonymous cells in the brain, participating in neurasthenic symptoms].

During rehabilitation after brain damage due to stroke or trauma, or after meningitis, patients often suffer from mental fatigue and decreased capacity for concentration and learning. We hypothesize that one pathogenic cause of these symptoms could be a failure on the part of astroglia to remove glutamate and K+ from synaptic regions during glutamatergic transmission. As astroglial support of neuronal glutamate transmission has been shown to be of utmost importance, such an astroglial failing might lead to a decreased signal-to-noise ratio in glutamatergic neurotransmission, especially when the transmission is intense for a considerable time period. Three aspects of astroglial failing are focused upon: decreased capacity of the astroglial glutamate transporters, depolarization of the astroglial network, and uncoupling of the gap junction coupled astroglial network. It has been shown that several neuroactive substances, present within the nervous system even long after damage or infection, influence upon these three astroglial properties.

Animals↗

PubMed2000

Growth hormone increases connexin-43 expression in the cerebral cortex and hypothalamus.

Several studies indicate that systemic GH influences various brain functions. Connexin-43 forms gap junctions that mediate intercellular communication and establish the astroglial syncytium. We investigated the effects of peripheral administration of bovine GH (bGH) and recombinant human insulin-like growth factor I (rhIGF-I) on the expression of connexin-43 in the rat brain. Hypophysectomized female Sprague Dawley rats were substituted with cortisol (400 microg/kg x day) and L-T4 (10 microg/kg x day) and treated with either bGH (1 mg/kg x day) or rhIGF-I (0.85 mg/kg x day) for 19 days. The abundance of connexin-43 messenger RNA (mRNA) and protein in the brainstem, cerebral cortex, hippocampus, and hypothalamus was quantified by means of ribonuclease protection assays and Western blots. Treatment with bGH increased the amounts of connexin-43 mRNA and protein in the cerebral cortex and hypothalamus. No changes were found in the brainstem or hippocampus. Infusion of rhIGF-I did not affect connexin-43 mRNA or protein levels in any of the brain regions studied. These results show that administration of bGH increases the abundance of cx43 in specific brain regions, suggesting that GH may influence gap junction formation and thereby intercellular communication in the brain.

Animals↗

PubMed1999

Connexin43 mRNA and protein expression during postnatal development of defined brain regions.

Connexin43 (cx43) mRNA and protein were quantified in seven brain regions using RNase protection assays and Western blotting from postnatal day 3, 10, and 17, and adult Sprague-Dawley rats. Increases in cx43 mRNA abundance preceded strong increases in cx43 protein in the cerebral cortex, hippocampus, hypothalamus, and striatum, whereas smaller postnatal increases were seen in the cerebellum, brain stem, and olfactory bulbs.

Animals↗

PubMed1999

Delta-opioid receptor immunoreactivity on astrocytes is upregulated during mitosis.

Endogenous opioid peptides and opioid receptors are expressed by brain cells early during normal development, and exogenous opiate exposure in this period is known to affect brain cell proliferation and maturation. Despite the abundant evidence that opioids affect brain development, little is known about the mechanisms involved. In this study cortical astrocytes in primary culture were examined immunohistochemically by using antibodies against the opioid receptors. The immunoreactivity for delta-opioid receptors was strongly upregulated during mitosis with an increase in immunostaining that started in early prophase and lasted through the M-phase to cytokinesis. Similar effects could not be observed when antibodies against the mu- or kappa-opioid receptor subtypes were used. Cultured neurons and microglia presented a strong and homogenous immunostaining for the delta-opioid receptor and no further upregulation of immunoreactivity could be detected in these cells. The presence of functional delta-opioid receptors on the mitotic astrocytes was verified by using microspectrofluorometry for detection of delta-opioid agonist induced changes in intracellular free calcium concentrations ([Ca2+]i). In these experiments fluo-3/AM incubated cells showed a rapidly induced delta-opioid agonist (DPDPE, 10(-6) M) evoked increase in [Ca2+]i. These results suggest an upregulation of the delta-opioid receptors that could represent a mechanism involved in the response to opioids in the developing brain.

Animals↗

PubMed1999

5-Hydroxytryptamine and glutamate modulate velocity and extent of intercellular calcium signalling in hippocampal astroglial cells in primary cultures.

The effects of 5-hydroxytryptamine or glutamate treatment on mechanically induced intercellular calcium waves were studied in gap junction-coupled astroglial cells using rat astroglial-neuronal primary cultures from hippocampus. Imaging software was developed to study amplitude, velocity and extent of wave propagation. Velocity software was designed to find the cell contours automatically and to calculate travelled distance and time-delay of the calcium wave as it propagates from the stimulated cell to all other cells. Propagation analyses were performed to calculate the area of wave propagation. Mechanical stimulation of a single astroglial cell induced an intercellular calcium wave spreading from cell to cell in the astroglial syncytium. When registering the appearances of calcium signals in individual cells along the wave path upon re-stimulation of the same cell, 44.7% of the cells responded with similar calcium signal appearances the second time as the first time. A second wave from the opposite direction resulted in similar calcium signal appearances in 27.3% of the studied cells. Both amplitude and velocity of the calcium signal decreased most prominently in the first part and showed a later flattening out. Treatment with 5-hydroxytryptamine or glutamate for 20-30 s before mechanical stimulation increased the velocity of the calcium waves. 5-Hydroxytryptamine treatment for varying times decreased the propagation area of the calcium waves. In contrast, glutamate treatment increased the propagation area.

Animals↗

PubMed1999

Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions.

Astrocytes are coupled via gap junctions, predominantly formed by connexin-43 proteins, into cellular networks. This coupling is important for the propagation of intercellular calcium waves and for the spatial buffering of K+. Using the scrape-loading/dye transfer technique, we studied gap junction permeability in rat astrocytes cultured from four different brain regions. The cultures were shown to display regional heterogeneity with the following ranking of the gap junction coupling strengths: hippocampus = hypothalamus > cerebral cortex = brain stem. Similar relative patterns were found in connexin-43 messenger RNA and protein levels using solution hybridization/RNase protection assay and western blots, respectively. The percentages of the propagation area of mechanically induced intercellular calcium waves for cortical, brain stem and hypothalamic astrocytes compared with hippocampal astrocytes were approximately 77, 42, and 52, respectively. Thus, the extent of calcium wave propagation was due to more than just gap junctional permeability as highly coupled hypothalamic astrocytes displayed relatively small calcium wave propagation areas. Incubation with 5-hydroxytryptamine decreased and incubation with glutamate increased the calcium wave propagation area in hippocampal (67% and 170% of the control, respectively) and in cortical astrocytes (82% and 163% of the control, respectively). Contrary to hippocampal and cortical astrocytes, the calcium wave propagation in brain stem astrocytes was increased by 5-hydroxytryptamine incubation (158% of control), while in hypothalamic astrocytes, no significant effects were seen. Similar effects from 5-hydroxytryptamine or glutamate treatments were observed on dye transfer, indicating an effect on the junctional coupling strength. These results demonstrate a strong relationship between connexin-43 messenger RNA levels, protein expression, and gap junction permeability among astroglial cells. Furthermore, our results suggest heterogeneity among astroglial cells from different brain regions in intercellular calcium signaling and in its differential modulation by neurotransmitters, probably reflecting functional requirements in various brain regions.

Animals↗

PubMed1999

Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca(2+) signaling.

Astrocytes represent a major target for endothelins (ETs), a family of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in the brain. In the present study we have investigated the involvement of different ET receptor subtypes on intercellular dye diffusion, intracellular Ca(2+) homeostasis, and intercellular Ca(2+) signaling in cultured rat astrocytes from hippocampus and striatum. Depending on the ET concentration and the receptor involved, ET-1- and ET-3-induced intracellular Ca(2+) increases with different response patterns. Both ET-1 and ET-3 are powerful inhibitors of gap junctional permeability and intercellular Ca(2+) signaling. The nonselective ET receptor agonist sarafotoxin S6b and the ET(B) receptor-selective agonist IRL 1620 mimicked these inhibitions. The ET-3 effects were only marginally affected by an ET(A) receptor antagonist but completely blocked by an ET(B) receptor antagonist. However, the ET-1-induced inhibition of gap junctional dye transfer and intercellular Ca(2+) signaling was only marginally blocked by ET(A) or ET(B) receptor-selective antagonists but fully prevented when these antagonists were applied together. The ET-induced inhibition of gap junction permeability and intercellular Ca(2+) signaling indicates that important changes in the function of astroglial communication might occur when the level of ETs in the brain is increased.

Animals↗

PubMed1998

Receptor-activated Ca2+ increases in vibrodissociated cortical astrocytes: a nonenzymatic method for acute isolation of astrocytes.

A new nonenzymatic method for the acute isolation of astrocytes from rat cerebral cortex is described. A vibratory device was used to dissociate the cells from thin brain slices, and the method yielded fresh and relatively well-preserved astrocytes without previous enzyme incubation. These cells were examined in a microspectrofluorometric system for measurement of changes in intracellular free calcium concentrations ([Ca2+]i), and their expression of various neurotransmitter receptors was determined. Acutely isolated glial fibrillary acidic protein (GFAP)-positive astrocytes (p7-p18) were seen to respond to the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10(-4) M) with increases in [Ca2+]i, and this response was blocked by (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA, 10(-3) M), an antagonist to group 1 metabotropic glutamate receptors. The delta-opioid receptor agonist D-Pen2, D-Pen5-enkephalin (DPDPE, 10(-6) M) evoked [Ca2+]i increases that were blocked by the delta-opioid antagonist ICI 174.388 (10(-5) M). The astrocytes failed to respond to 5-hydroxytryptamine (5-HT, 10(-5) M), although the same cells subsequently were found to respond to other agonists. Furthermore, [Ca2+]i responses evoked by phenylephrine (10(-5) M) were blocked by prazosin (0.2x10(-6) M), suggesting the expression of alpha1-adrenergic receptors on the acutely isolated astrocytes. The cells were also shown to react with [Ca2+]i increases in response to depolarization with high extracellular potassium concentrations (50x10(-3) M). The signals induced by depolarization were not seen in Ca2+-free buffer, indicating the presence of voltage-activated calcium channels in these cells. Thus, the present study confirms some of the results earlier obtained in cell cultures, suggesting that cortical astrocytes in vivo express glutamate, opiate, and adrenergic receptors, coupled to increases in [Ca2+]i, whereas no receptors for 5-HT could be detected.

Animals↗

PubMed1998

Intracellular sulfatide expression in a subpopulation of astrocytes in primary cultures.

A highly specific antibody against sulfatide, a myelin-associated glycolipid, has been investigated using indirect double immunocytochemistry in rat primary astroglial cultures from cerebral cortex. Sulfatide was expressed in a selected subpopulation of astrocytes (2-3%) and was found to be exclusively located intracellularly. The sulfatide-positive cells appeared in two different morphologies: flat and stellate. Immunolabeling of the astroglial cultures showed that sulfatide always co-existed with GFAP or S-100, and in some cells with GD3 (flat 90% and stellate 50%) or A2B5 (1%) antibody. The sulfatide-positive cells did not bind the O1 antibody, which is used as a marker for oligodendrocytes. Glial cultures from other regions and mixed cultures, with both neurons and glial cells, were examined and showed similar results. Biochemical analysis by TLC-ELISA verified the presence of sulfatide in the astroglial culture and showed decreasing amounts of sulfatide with days in vitro; 0.05 nmol/mg protein at day 10 and 0.01 nmol/mg protein at day 17. This analysis also showed that neither sulpholactosylceramide nor seminolipid was present, each of which also has affinity for the sulfatide antibody. This selective and intracellular expression encourages further identification of the astrocytes expressing sulfatide and the biological role of sulfatide in these cells.

Animals↗

PubMed1998

Modulation of mechanically induced calcium waves in hippocampal astroglial cells. Inhibitory effects of alpha 1-adrenergic stimulation.

The effects of different adrenoceptor agonists were investigated on mechanically induced Ca2+ waves in astroglial cells in astroglial-neuronal mixed cultures from rat hippocampus. In the initial part of the study some properties of the waves were characterized. The results show that the initiation of the Ca2+ waves was not critically dependent on extracellular Ca2+ but both the calcium signal and the propagation area of the calcium wave were significantly reduced when the experiments were performed in Ca2+-free buffer. In addition, using the phospholipase C (PLC) inhibitor U-73122 (1 microM) and the gap junction uncoupler octanol (1 mM), the results showed that the Ca2+ wave propagation required PLC activation and functional gap junctions. Further, the data also showed that the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA 150 nM) reduced the spreading of the waves. The adrenoceptor agonists isoproterenol (iso; beta), phenylephrine (phe; alpha1) and clonidine (clon; alpha2) were evaluated for their short-term (<30 s) effects on the wave propagation. The propagation area was persistently decreased 1, 3 and 5 min after removal of phe. No effects were observed after incubation with iso or clon. Furthermore, using U-73122 or PMA together with phe, shortly incubated, the experiments showed that PLC was a central regulator in the initial phase of the initiation procedure of wave propagation. However, under these conditions PKC was shown not to be involved. Instead it appeared that PKC exerted its inhibitory action on the Ca2+ waves in a latter phase, after prolonged phe exposure. Taken together, the results show that the propagation of Ca2+ waves between astroglial cells in primary cultures can be inhibited/regulated in two principally different ways which involve a pronounced time component. The results also further point out the adrenergic signaling system as an important mediator of dynamic neuron-astroglial information exchange.

1-Octanol↗

PubMed1998

Regulation of the glial glutamate transporter GLT-1 by glutamate and delta-opioid receptor stimulation.

The excitatory effect of presynaptically released glutamate is tightly regulated and terminated by high affinity sodium-dependent glutamate transporters. The regulation of the glial glutamate transporter GLT-1 is potentially important in synaptic modulation. Using astroglial cultures prepared from the rat cerebral cortex, we found that the delta-opioid receptor agonist [D-pen2,D-pen5]-enkephalin decreases and glutamate increases the expression of the GLT-1 transporter mRNA. Corresponding changes in the uptake kinetics were found after incubation for 48 h with the respective agonists when glial glutamate uptake was measured in primary astroglial cultures. The data suggest that long-term receptor activation induces alterations in glial glutamate uptake properties.

ATP-Binding Cassette Transporters↗

PubMed1998

Delta-opioid receptors on astroglial cells in primary culture: mobilization of intracellular free calcium via a pertussis sensitive G protein.

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.

Animals↗

PubMed1997

Glutamate induced astroglial swelling--methods and mechanisms.

Glutamate (Glu) plays an important role in the early development of brain injuries caused by ischemia, i.e. stroke, or brain trauma. Glu induces a rapid astroglial swelling which, in turn, deranges the composition of neuroactive substances in the extracellular space. We report that Glu can induce astroglial cell swelling by interaction with metabotropic Glu receptors (mGluRs). Furthermore, the Na(+)-K(+)-2Cl- cotransporter, a Na(+)-K(+) ATPase, and the Na(+)-dependent electrogenic Glu carrier seem to be involved in this Glu-induced astroglial cell swelling. Two methods for studying cell swelling arc described. One is based on variations in the signal emitted by the fluorescent probe fura-2/AM when excited at its isosbestic point. These variations were shown to be directly proportional to variations in intracellular volume. Relative changes in cell volume and intracellular calcium concentration could be detected simultaneously in single astroglial cells. The other method used permits the cell volume to be calculated in relative terms with the aid of image processing techniques.

Animals↗