Biomedical subjects

K F Hauser

Publications and source records attributed to K F Hauser.

At least 37 records · Page 2Linked to original sources

PubMed2000

Neurotoxicity and dysfunction of dopaminergic systems associated with AIDS dementia.

Infection with the human immunodeficiency virus (HIV) selectively targets the basal ganglia resulting in loss of dopaminergic neurons. Although frequently asymptomatic, some patients may develop signs of dopamine deficiency de novo. Accordingly, they are highly susceptible to drugs that act on dopaminergic systems. Both neuroleptics and psychostimulants may exacerbate these symptoms. Experimental evidence suggests that viral proteins such as gp120 and Tat can cause toxicity to dopaminergic neurons, and this toxicity is synergistic with compounds such as methamphetamine and cocaine that also act on the dopaminergic system. In addition, other neurotransmitters that modulate dopaminergic function, such as glutamate and opioids, may also modify the susceptibility of the dopamine system to HIV. Therefore, a thorough understanding of the mechanisms that lead to this selective neurotoxicity of dopaminergic neurons would also likely lead to the development of therapeutic modalities for patients with HIV dementia.

AIDS Dementia Complex↗

PubMed1999

Abnormal Ca(2+) regulation in oligodendrocytes from the dysmyelinating jimpy mouse.

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca(2+) ([Ca2+]i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca2+](i) dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca2+](i) in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca2+](i) ranging from 60% to 150% over control values. A higher baseline [Ca2+](i) in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.

Animals↗

PubMed1999

Dynorphin A (1-13) neurotoxicity in vitro: opioid and non-opioid mechanisms in mouse spinal cord neurons.

Dynorphin A is an endogenous opioid peptide that preferentially activates kappa-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1-13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1-13) on intracellular calcium concentration ([Ca2+]i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1-13) elevated [Ca2+]i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates kappa-opioid receptors and suggests that kappa receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1-13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.

Analgesics, Opioid↗

PubMed1999

Opioids modulate cell division in the germinal zone of the late embryonic neocortex.

Opioid effects on cell division in the embryonic cerebral cortex were examined using two experimental approaches: (i) the presence of opioid receptors in the embryonic day 16 mouse neocortex was tested using immunohistochemical techniques; (ii) the values of the indices of [3H]thymidine pulse labelled cells and mitotic indices were estimated in the ventricular zone of the embryonic day 16 mouse neocortex 2.5, 4.5 and 8.5 h after administration to pregnant females of selected opioid receptor agonists or the opioid antagonist naloxone. The immunohistochemical study demonstrated that distinct subpopulations of the ventricular zone cells express mu, delta or kappa opioid receptors. Acute exposure of mouse embryos to mu, delta and kappa opioid receptor agonists or naloxone differentially affects the indices of [3H] thymidine pulse labelled cells and mitotic indices indicating changes in the cell cycle composition. Treatment with the mu opioid receptor agonist D-Ala2-MePhe4, Gly-ol5-enkephalin (DAGO), or the partially selective kappa opioid receptor agonist bremazocine, increased the [3H]thymidine labelling and mitotic indices. In contrast, the delta receptor agonist (D-Ser8)-leucine enkephalin-Thr (DSLET) produced a decrease in the labelled cell indices and mitotic indices. Naloxone provided a biphasic effect: a decrease in the values of labelled cell indices 2.5 h after naloxone administration, followed by an increase in the values of the indices at 4.5 and 8.5 h. These results suggest that the endogenous embryonic/maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the late embryonic cortex.

Animals↗

PubMed1998

Opposing actions of the EGF family and opioids: heparin binding-epidermal growth factor (HB-EGF) protects mouse cerebellar neuroblasts against the antiproliferative effect of morphine.

Endogenous opioids and opiate drugs of abuse inhibit the proliferation of cerebellar external granular layer (EGL) neuroblasts by mechanisms that are incompletely understood. Opioids do not act alone, rather multiple extracellular factors regulate granule cell neurogenesis and these undoubtedly act in concert with opioids to shape developmental outcome. We examined whether, heparin binding-epidermal growth factor-like growth factor (HB-EGF), a recently described member of the epidermal growth factor (EGF) family, might compete with an inhibitory opioid signal. The results confirmed our ongoing studies that morphine inhibited neuroblast proliferation, while HB-EGF enhanced cell replication. HB-EGF not only counteracted the antiproliferative morphine signal, but invariably enhanced DNA synthesis irrespective of morphine treatment. Our findings suggest that regional and temporal differences in the availability of endogenous HB-EGF may serve to limit the response of EGL neuroblasts to opioids, and HB-EGF may be neuroprotective in opiate drug abuse. If similar responses occur in vivo, then the EGF family and the opioid system may represent distinct and contrasting components of an extracellular signaling system serving to coordinate EGL neurogenesis.

Animals↗

PubMed1998

Endogenous opioid system in developing normal and jimpy oligodendrocytes: mu and kappa opioid receptors mediate differential mitogenic and growth responses.

The early development of both neurons and neuroglia may be modulated by signaling through opioid mediated pathways. Neurons and astroglia not only express specific types of opiate receptors, but also respond functionally to opioids with altered rates of proliferation and growth. The present study was undertaken to determine if opioids also modulate development of the other major CNS macroglial cell, the oligodendrocyte (OL). Using well-characterized polyclonal antibodies specific for delta-, kappa-, and mu-opiate receptors, OLs grown in vitro were shown to express mu-receptors at a very immature stage prior to expression of kappa-receptors. This developmentally regulated sequence differs from the pattern of expression in neurons and astroglia. delta-receptors are apparently absent from cultured OLs. OLs also have physiologic responses to selective mu- and kappa-receptor agonists and antagonists. Exposure of relatively immature O4+ OLs to the mu-receptor agonist PL017 [H-Tyr-Pro-Phe(N-Me)-D-Pro-NH2] resulted in a significant enhancement in the rate of DNA synthesis. This effect, which was not observed in more mature MBP+ OLs, was entirely blocked by the antagonist naloxone. Although the kappa-receptor pathway appeared to be uninvolved in controlling proliferation, the kappa-receptor antagonist nor-binaltorphimine significantly increased the size of myelin-like membranes produced by the cultured OLs. Interestingly, OLs derived from the jimpy mouse, a mutant characterized by an almost complete lack of CNS myelin and premature death of OLs, were found to be deficient in kappa-opiate receptors. Our findings clearly show that OLs not only express specific opiate receptors, but also respond to changes in their level of stimulation in ways that could profoundly impact nervous system morphology and function. If opiate receptors are expressed by OLs in vivo, their pharmacological manipulation might provide a novel pathway for modulating OL and myelin production both during development and in demyelinated conditions.

Animals↗

PubMed1998

Opioids disrupt Ca2+ homeostasis and induce carbonyl oxyradical production in mouse astrocytes in vitro: transient increases and adaptation to sustained exposure.

Pharmacologically distinct subpopulations of astroglia express mu, delta, and/or kappa opioid receptors. Activation of mu, delta, or kappa opioid receptors can destabilize intracellular calcium ([Ca2+]i) in astrocytes leading to cellular hypertrophy and reactive injury. To assess whether acute or sustained opioid exposure might adversely affect astroglial function by disrupting Ca2+ homeostasis or by producing reactive oxygen species, fura-2 and a novel fluorescent-tagged biotin-4-amidobenzoic hydrazide reagent, respectively, were used to detect [Ca2+]i and carbonyl oxidation products within individual murine astrocytes. Acute (3 h) exposure to mu; (H-Tyr-Pro-Phe (N-Me) -D-Pro-NH2; PLO17), delta ([D-Pen2, D-Pen5]-enkephalin), and kappa (trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrr olidinyl) cyclohexyl] benzeneacetamide methanesulfonate; U50,488H) opioid agonists caused significant mean increases in [Ca2+]i and in the levels of oxidative products in astrocytes. In contrast, following 72 h of continuous opioid exposure, [Ca2+]i and carbonyl levels returned to normal, irrespective of opioid treatment. These preliminary findings indicate that opioids initially destabilize [Ca2+]i and increase reactive oxygen species in astrocytes; however, astrocytes later recover and adapt to sustained opioid exposure.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1998

Diversity of the endogenous opioid system in development. Novel signal transduction translates multiple extracellular signals into neural cell growth and differentiation.

This review explores the role of individual opioid receptor types and signal transduction pathways on cell growth and differentiation. The findings reviewed herein provide suggestive evidence that while no single opioid receptor or peptide type exclusively regulates growth, depending on the cell type, the activation of all three (mu, delta, or kappa) opioid receptor types can affect maturation in a cell type-dependent manner. Specific developmental responses are determined primarily by how a particular opioid receptor type is coupled to intracellular signaling effectors. Moreover, the coupling of opioid receptors appears to be developmentally regulated, and these protein-protein interactions change during ontogeny. The diversity of opioid receptor types and intracellular effectors may be a mechanism by which individual cells discriminate among different opioid signals, and may permit diverse opioid signals to be translated into a unique developmental logic in distinct neuronal and glial subpopulations.

Animals↗

PubMed1998

Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa-opioid receptor expression among cultured mouse astrocytes.

The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. Mu, delta, and kappa opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca2+]i). Significant spatial and temporal differences were evident in the pattern of expression of mu, delta, and kappa receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed mu-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26-34%) than in the cerebellum or striatum (7-12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed mu-immunoreactivity. Fewer astrocytes possessed delta-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16-22%). The proportion of delta receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. Kappa-opioid receptors were uniformly expressed by 27-34% of astrocytes from all regions, except in cortical cultures, where the proportion of kappa expressing cells was 38% at low-density and decreased to 22% at confluence. Selective mu (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH2, delta ([D-Pen2, D-Pen5] enkephalin), or kappa (U50,488H; trans-(+/-)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca2+]i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G2/M phase of the cell cycle were mu or delta receptor immunofluorescent than at G0/G1. When astrocytes were reversibly arrested in G1, significantly fewer cells expressed delta receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

Opioid-related changes in nociceptive threshold and in tissue levels of enkephalins after target disruption of the gene for neutral endopeptidase (EC 3.4.24.11) in mice.

Neutral endopeptidase EC 3.4.24.11 (NEP) is localized in peptidergic neurons and various colocalized peptides or other humoral mediators may serve as substrates. Target disruption of the NEP gene was reported to enhance the lethal response to endotoxin shock in mice. We examined thermonociceptive thresholds and enkephalin (ENK) tissue levels in transgenic NEP (-/-) and control wild type NEP (+/+) mice. Hot plate (52 degrees C) latency was 13.1 +/- 1.4 s in NEP (+/+) mice (n = 16) while latency increased significantly (P = 0.031) to 17.7 +/- 1.6 s in NEP (-/-) mice. Naloxone (10 mg/kg) had no effect on hot plate latency in NEP (+/+) mice (12.5 s, n = 8), but significantly decreased the latency in NEP (-/-) mice compared to untreated NEP (-/-) deficient mice (10.5 s, n = 8). Morphine (3 or 10 mg/kg) analgesic response was similar in knockout mice and wild type mice. Methionine-ENK (MET-ENK) and leucine-ENK (LEU-ENK) levels were determined in extracts from cortex, brain stem, hypothalamus, striatum, spinal cord, trigeminal ganglion and heart in treated and untreated mice. ENK-levels varied in a regionally-dependent manner and were significantly decreased in hypothalamus and spinal cord. We conclude that deletion of the NEP gene results in an opioid-related increase in thermonociceptive threshold. Regional differences in opioid metabolism indicate that NEP evokes tissue-specific patterns of ENK-regulation. NEP selectively controls opioid biosynthesis in hypothalamus and spinal cord presumably by feedback regulation.

Analgesics, Opioid↗

PubMed1996

mu-Opioid receptor activation enhances DNA synthesis in immature oligodendrocytes.

Opioids disrupt nervous system development by inhibiting the proliferation of neuronal and glial progenitors. These studies explored the hypothesis that mu opioid receptors are expressed by immature oligodendrocytes (OLs) and are functionally related to growth. Antibodies identifying the cloned mu opioid receptor demonstrated that cultured OLs expressed mu opioid receptor immunoreactivity very early during development. Cultures were treated with the selective mu opioid receptor agonist H-Tyr-Pro-Phe (N-Me)-D-Pro-NH2 (PL017; 1 microM), or PL017 (1 microM) plus the antagonist naloxone (3 microM). Opioid-dependent changes in DNA synthesis were assessed by determining the proportion of bromodeoxyuridine (BrdU)-labeled O4-immunoreactive OLs. Treatment with PL017 caused a 311% increase in the proportion of O4-immunoreactive OLs incorporating BrdU compared to untreated controls, and these effects were prevented by co-administering naloxone. These preliminary results indicate that (i) immature OLs express mu opioid receptors and that (ii) the activation of this receptor type is functionally coupled to DNA synthesis and the cell division cycle. The expression of opioid receptors by OLs suggests that the endogenous opioid system is widely distributed among glial types.

Animals↗

PubMed1996

kappa-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.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

mu-Opioid receptor-induced Ca2+ mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca(2+)-dependent mechanism.

Morphine, a preferential mu-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 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 mu-opioid receptors and Ca2+ mobilization in morphine-induced astrocyte development. Morphine (1 microM) 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 Ca2+ ([Ca2+]o), or in unmodified media containing Ca2+ ionophore (A23187), nifedipine (1 microM), dantrolene (10 microM), thapsigargin (100 nM), or L-glutamate (100 microM) for 0-72 h. mu-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 mu-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective mu 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 morphine's 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 Ca(2+)-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 Ca(2+)-dependent Ca2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.

Animals↗

PubMed1994

Morphine inhibits Purkinje cell survival and dendritic differentiation in organotypic cultures of the mouse cerebellum.

The effects of morphine on the morphogenesis and survival of calbindin-D28k-immunoreactive Purkinje cells were studied in organotypic explant cultures isolated from 1- or 7-day-old mouse cerebella. To reduce experimental variability, bilaterally matched pairs of organotypic cultures were used to compare the effects of opiate drug treatment. One explant within each pair was untreated, while the remaining explant was continuously treated for 7 to 10 days with morphine, morphine plus naloxone, or naloxone alone. In explants derived from 1-day-old mice, morphine treatment significantly reduced Purkinje cell dendritic length compared to symmetrically matched untreated control explants. The concentration of morphine estimated to cause a half-maximal reduction (EC50) in dendritic length was 4.9 x 10(-8) M. At higher concentrations (EC50 = 3.6 x 10(-6) M), morphine also significantly decreased the number of Purkinje cells in explants from 1-day-old mice compared to untreated explants. Electron microscopy identified increased numbers of degenerating Purkinje cells in explants derived from 1-day-old mice. This showed that high concentrations (10(-5) M) of morphine reduced Purkinje cell numbers by decreasing their rate of survival. In explants derived from 7-day-old mice, morphine (10(-5) M) neither affected Purkinje cell dendritic length nor cell numbers compared to symmetrically matched untreated (control) explants. Collectively, these findings suggest that morphine per se, through a direct action on the cerebellum, can affect Purkinje cell differentiation and survival. The results additionally suggest that there is a critical period during development when Purkinje cells are especially vulnerable to the effects of morphine.

Animals↗

PubMed1993

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)

Animals↗

PubMed1993

Morphine does not affect astrocyte survival in developing primary mixed-glial cultures.

In mixed-glial cultures, high concentrations of morphine (1 microM) have previously been shown to completely inhibit any increase in glial numbers, although DNA synthesis continues in flat, polyhedral astrocytes (type 1 astrocytes). This suggests that high concentrations of morphine are toxic to glia. Morphine toxicity was assessed in mixed-glial cultures using calcein-AM and ethidium homodimer dyes as viability markers to identify live and dead cells, respectively. At 3, 5, and 7 days in vitro there was no significant difference in the number of dead cells between untreated and opiate-treated groups. Comparable numbers of ethidium homodimer-labeled cells were present in all groups. The greatest amount of cell death (16-19%) occurred at 3 days in vitro, while fewer cells (8-12%) were dying at 7 days in vitro. To further characterize the dying glia, glial fibrillary acidic protein (GFAP) and A2B5 immunocytochemistry were combined with viability markers. Only GFAP immunoreactive process-bearing cells and A2B5 immunoreactive cells (process-bearing cells and possibly some neurons) were dying in culture, whereas the death of flat, polyhedral GFAP-positive cells was not observed. Cell survival was not affected by morphine, but may be affected by culture conditions. Thus, morphine-induced reductions in glial numbers did not result from an increased rate of cell death. Collectively, the present and previous findings suggest that morphine inhibits the production of flat, polyhedral astrocytes solely by decreasing their rate of proliferation.

Animals↗

PubMed1993

Ontogeny of proenkephalin mRNA and enkephalin peptide expression in the cerebellar cortex of the rat: spatial and temporal patterns of expression follow maturational gradients in the external granular layer and in Purkinje cells.

Proenkephalin mRNA and peptide products were examined in developing cells of the postnatal rat cerebellar cortex using in situ hybridization and immunocytochemistry. On day 7, proenkephalin mRNA was first detected as discrete cellular labeling in Golgi cells and as a diffuse hybridization signal over the Purkinje cell layer. On day 14, proenkephalin mRNA and peptide products primarily appeared in distinct subpopulations of Purkinje cells present in the posterior and lateral cerebellum. Similarly, in the external granular layer (EGL), enkephalin immunoreactivity was present only in the posterior and lateral portions of the cerebellum on day 14. However, proenkephalin mRNA was not detected in enkephalin-immunoreactive EGL cells. On day 21, the subset of Purkinje cells that expressed proenkephalin mRNA and peptides were distributed more uniformly throughout the cerebellum. On day 28, a few enkephalin-immunoreactive Purkinje cells were uniformly present throughout the cerebellum, but proenkephalin mRNA was not detected in most of these cells. The spatial gradients in proenkephalin mRNA expression evident in the Purkinje cells of younger rats were no longer present in 28-day-old rats. These findings are important, because endogenous opioids such as enkephalin have been previously shown to inhibit the growth of Purkinje cell dendrites and dendritic spines, and inhibit the rate of mitosis in EGL neuroblasts. Cells do not develop at uniform rates within the cerebellum. There are regional differences in the timing of the formation of the EGL, and in the morphogenesis of Purkinje cells. In conjunction with previous work, the present findings suggest that during development, the pattern of enkephalin immunoreactivity in Purkinje and EGL cells closely follows the spatial and temporal gradients of maturation in both these cell types. The emergence and disappearance of enkephalin immunoreactivity in Purkinje and EGL cells is spatially and temporally related, and coincides with proenkephalin mRNA expression in Purkinje cells. Thus, the transient and coordinated appearance of enkephalin in cerebellar Purkinje and EGL cells may contribute to regional differences in the rate of cerebellar maturation, and may help synchronize the developmental interactions between these two cell types.

Animals↗

PubMed1993

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.

Animals↗