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 Ca 2+ -dependent mechanism, few studies have examined whether Ca 2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca 2+ 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 Ca 2+ mobilization in morphineinduced astrocyte development. Morphine (1 μM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca 2+ -free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca 2+ ([Ca 2+ ] o ), or in unmodified media containing Ca 2+ 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 Ca 2+ ([Ca 2+ ] 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 morphine's actions were mimicked by the selective μ agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca 2+ Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca 2+ -dependent Ca 2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.

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.

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.

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 mu-, delta-, and/or kappa-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 mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) 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(-)/S100beta(-). 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(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta 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.

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. KeywordsNeural development; In situ hybridization; Endogenous opioids; Proenkephalin A; Glial fibrillary acidic protein; Astrocyte; In vitroThe growth of neuronal and non-neuronal cells of the postnatal rat cerebral cortex, hippocampus, and cerebellum is modified by experimental manipulation of endogenous opioid systems (i.e, endogenous opioids and opioid receptors). Cellular differentiation 17,18 , cell number and packing density 54,55 , and cortical thickness 54,55 are modifiable by treatment with opioid antagonist drugs in vivo. In the cerebellum, [ 3 H]-thymidine incorporation by neuroblasts of the external granular layer (EGL) in 6-day-old rats is inhibited by systemic administration of Met-enkephalin, while treatment with the opioid antagonist naltrexone, at dosages sufficient to completely block opioid receptors, is reported to increase [ 3 H]-thymidine incorporation compared to untreated controls 56 . These and other experiments suggest that endogenous opioids are normally available to cells in the developing CNS in sufficient quantities to tonically inhibit growth 17,18,41,[54][55][56] . In support of this hypothesis, opioid peptide levels and opioid binding are greatly increased in the rat cerebellum 48,49 28,32,43,52 . In the present study, proenkephalin (proenkephalin A) mRNA and enkephalin peptide expression were examined by in situ hybridization and immunocytochemistry, respectively, in primary cultures of the developing CNS (i) to identify the specific cellular sites of proenkephalin gene and peptide expression, and (ii) to determine whether opioids are expressed by replicating astrocytes prior to their reaching confluence. We were particularly interested in determining whether rapidly dividing cells in astrocyte-enriched cultures express the proenkephalin gene prior to reaching confluence, because we have recently found that the proenkephalin peptide product, Met-enkephalin, selectively inhibits type 1 astrocyte proliferation at this time (i.e., at 4 or 6 days in vitro) 46,47 . Initial studies localized high levels of proenkephalin mRNA in cells with astrocytic morphology irrespective of culture conditi...