Anne Stiene-Martin

Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media (controls), 1 microM met-enkephalin, 1 microM met-enkephalin plus the opioid antagonist naloxone (3 microM), or naloxone alone (3 microM). Absolute numbers of neural cells were counted in unstained preparations, while combined [3H]-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in [3H]-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture.

μ-Opioid receptor-induced Ca2+ mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca2+-dependent mechanism, few studies have examined whether Ca2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca2+ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca2+ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca2+-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca2+ ([Ca2+]o), or in unmodified media containing Ca2+ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca2+ ([Ca2+]i) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphines actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca2+]i in developing astroglia. At normal [Ca2+]o, morphine attenuated DNA synthesis by increasing [Ca2+]i; low [Ca2+]o (0.3 mM) blocked this effect, while treatment with Ca2+ ionophore or glutamate mimicked morphines actions. At extremely low [Ca2+]o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca2+]i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca2+ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP3-sensitive Ca2+ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca2+-dependent Ca2+ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca2+-dependent Ca2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: Impact on gliogenesis in vivo

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2–5‐day‐old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express μ‐, δ‐, and/or κ‐receptor immunoreactivity in a cell type‐specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential μ‐agonist) significantly decreased the number of BrdU‐labeled GFAP+ cells compared with controls or mice co‐treated with naltrexone plus morphine. Alternatively, in S100β+ cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP−/S100β−. When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP−/S100β− cells is tonically suppressed by endogenous opioids. Assuming that S100β and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors. GLIA 36:78–88, 2001.

Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes.

To identify the possible cellular sites of opioid gene expression during ontogeny, proenkephalin mRNA and enkephalin peptide expression were examined, respectively, by in situ hybridization and immunocytochemistry in organotypic explants of rat cerebellum and in astrocyte-enriched cultures of murine cerebral hemispheres. High levels of proenkephalin mRNA and enkephalin immunoreactivity were detected in immature cells identified as astrocytes. Double-labeling studies combining in situ hybridization and immunocytochemical localization of the astrocytic marker, glial fibrillary acidic protein, provided direct evidence that proenkephalin mRNA is expressed by astrocytes in culture. Based on previous studies that Met-enkephalin can inhibit astrocyte growth in vitro, the present results suggest that proenkephalin gene expression by astrocytes is important during central nervous system maturation.

Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation

To determine whether exogenous opiate drugs with abuse liability directly modify neural growth, the present study investigated the effects of morphine on astrocyte proliferation and differentiation in primary cultures of murine glial cells. The results indicate that morphine decreases glial cell production in a dose-dependent, naloxone-reversible manner. Most notably, gliogenesis virtually ceased in the presence of 10(-6) M morphine during the first week in culture, whereas 10(-8) M or 10(-10) M morphine caused an intermediate suppression of growth compared to control or 10(-6) M morphine treated cultures. Moreover, morphine treatment inhibited [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes, suggesting that the decrease in glial cell production was due in part to an inhibition of astrocyte proliferation. Morphine also caused significant increases in both cytoplasmic area and process elaboration in flat (type 1) astrocytes indicating greater morphologic differentiation. In the above experiments, morphine-dependent alterations in astrocyte growth were antagonized by naloxone, indicating that morphine action was mediated by specific opioid receptors. These observations suggest that opiate drugs can directly modify neural growth by influencing two critical developmental events in astrocytes, i.e., inhibiting proliferation and inducing morphologic differentiation.

HIV-1 neuropathogenesis: glial mechanisms revealed through substance abuse.

Neuronal dysfunction and degeneration are ultimately responsible for the neurocognitive impairment and dementia manifest in neuroAIDS. Despite overt neuronal pathology, HIV‐1 does not directly infect neurons; rather, neuronal dysfunction or death is largely an indirect consequence of disrupted glial function and the cellular and viral toxins released by infected glia. A role for glia in HIV‐1 neuropathogenesis is revealed in experimental and clinical studies examining substance abuse–HIV‐1 interactions. Current evidence suggests that glia are direct targets of substance abuse and that glia contribute markedly to the accelerated neurodegeneration seen with substance abuse in HIV‐1 infected individuals. Moreover, maladaptive neuroplastic responses to chronic drug abuse might create a latent susceptibility to CNS disorders such as HIV‐1. In this review, we consider astroglial and microglial interactions and dysfunction in the pathogenesis of HIV‐1 infection and examine how drug actions in glia contribute to neuroAIDS.

κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: relationship to Ca2+ mobilization, development, and the antiproliferative effect of opioids

To assess the role of kappa-opioid receptors in astrocyte development, the effect of kappa-agonists on the growth of astroglia derived from 1-2-day-old mouse cerebra was examined in vitro. kappa-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of kappa-receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6-7, as many as 50% of the astrocytes displayed kappa-receptor (KA8) immunoreactivity or exhibited increases in [Ca2+]i in response to kappa-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the kappa-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were kappa-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca2+]i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that kappa-receptor activation increases astroglial [Ca2+]i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express kappa-opioid receptors in vitro, and suggest that the activation of kappa-receptors mobilizes [Ca2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing kappa-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Opiates selectively increase intracellular calcium in developing type-1 astrocytes: role of calcium in morphine-induced morphologic differentiation

Endogenous opioids and opiate drugs inhibit nervous system maturation, in part, by affecting the growth of astrocytes. Opiates inhibit astrocyte proliferation and cause premature differentiation. The emerging importance of Ca2+ in astrocyte function prompted us to explore whether opiates might affect astrocyte development by altering Ca2+ homeostasis. Astrocyte-enriched cultures were derived from newborn ICR mouse cerebra. Quantitative fluorescent measurements of intracellular free Ca2+ ([Ca2+]i) using Fura-2 as well as fluo-3 and computer-aided image analysis showed that 1 microM morphine significantly increased [Ca2+]i in flat, polyhedral, glial fibrillary acidic protein (GFAP) immunoreactive astrocytes at 2 and 6 min, and at 72 h. Co-administration of 3 microM naloxone blocked morphine-dependent increases in [Ca2+]i. Treatment with 1 microM concentrations of the kappa-opioid receptor agonist, U69,593, but not equimolar amounts of mu ([D-Ala2,MePhe4,Gly(ol)5]enkephalin)- or delta ([D-Pen2,D-Pen5]enkephalin)-opioid receptor agonists, significantly increased [Ca2+]i in astrocytes. To assess the role of Ca2+ in morphine-induced astrocyte differentiation, untreated and 1 microM morphine-treated astrocyte cultures were incubated for 5 days in < 0.01, 0.3, 1.0, or 3.0 mM extracellular Ca2+ ([Ca2+]o), or incubated with 1.0 mM [Ca2+]o in the presence of 1 microM of the Ca2+ ionophore, A23187. The areas of single astrocytes were measured and there was a positive correlation between astrocyte area and [Ca2+]o. Morphine had an additive effect on area and form factor measures when [Ca2+]o was 1.0 mM. High [Ca2+]o (3.0 mM) alone mimicked the action of morphine. Morphine alone had no effect on astrocyte area in the presence of 3.0 mM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of opioid-dependent glial development in dissociated and organotypic cultures of mouse central nervous system: critical periods and target specificity

Opioid-dependent changes in glial growth were characterized in primary dissociated and organotypic explant cultures of the developing mouse central nervous system (CNS) continuously grown in the presence of an endogenous opioid, [Met5]enkephalin, or an opiate drug, morphine. The glia in dissociated, astrocyte-enriched cultures derived from the cerebra of postnatal day 1, 3, or 5 mice, respectively, displayed age-dependent reductions in glial numbers that occurred at 3, 7, or 9 days in vitro (DIV) in response to continuous [Met5]enkephalin (10(-6) M) exposure. In contrast, in cultures derived from gestational day 19 mice, glial numbers were not reduced following continuous exposure to 10(-6) M [Met5]enkephalin during the first 7 days in vitro. An examination of [3H]thymidine incorporation by glial fibrillary acidic protein-(GFAP) immunoreactive astrocytes with flat (type 1) morphology in dissociated cultures derived from postnatal day 1 mice revealed that the reduction in glial numbers at 3 DIV was not immediately preceded by a reduction in the rate of [3H]thymidine incorporation at 2 DIV, although previous studies have shown that opioids inhibit the rate of [3H]thymidine incorporation by more mature astrocytes at 4 or 6 DIV. Early (i.e., at 2 to 3 DIV) changes in glial numbers may result from an inhibition of the proliferative rate of non-GFAP-containing glia or astrocyte precursors, or an enhanced rate of glial death. The rate of [3H]thymidine incorporation by GFAP-immunoreactive astrocytes with process-bearing (type 2) morphology was unchanged by opioid treatment. In separate experiments, a comparison of the area of growth of GFAP-immunoreactive astrocytes in paired symmetrical (right vs left) organotypic explant cultures demonstrated that opiates (i.e., 10(-5) M morphine) can inhibit astrocyte growth when the normal histiotypic organization of neurons and glia are maintained, and that there are regional differences in astrocyte responsiveness. Opioid-dependent alterations in astrocyte growth were mediated through specific opioid receptors since they were prevented by simultaneous treatment with (-)naloxone. The results suggest that the ability of opioids to modify glial growth is highly selective and varies depending on astrocyte type, as well as temporal and regional factors. Spatial and temporal differences in the response of developing glia to opioids may determine critical periods of CNS vulnerability to opioids in the maturing brain.

Morphine suppresses DNA synthesis in cultured murine astrocytes from cortex, hippocampus and striatum

To determine whether there are regional differences in the ability of opiates to affect astrocyte proliferation, the effects of morphine were examined in astrocyte-enriched cultures from striatum, hippocampus and cerebral cortex derived from newborn mouse brains. Cultures from each region were continuously incubated in media alone (controls), or in media treated with 1 microM morphine, 1 microM morphine plus 3 microM naloxone, or 3 microM naloxone alone. Before harvesting at 6 days in vitro, cultures were exposed to [3H]thymidine (0.24 mu CI/ml for 16 h). Thymidine-labeling index was determined autoradiographically in flat, polyhedral (type 1) glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Morphine significantly inhibited [3H]thymidine incorporation in astrocytes from all three brain regions, although regional differences in labeling indices were noted. The results show that opiates can intrinsically affect the proliferative rate of astrocytes from diverse brain regions.