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Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

View Article: PubMed Central - PubMed

ABSTRACT

Background: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.

Methods: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.

Results: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.

Conclusions: These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0844-3) contains supplementary material, which is available to authorized users.

No MeSH data available.


The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 106/well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone (a); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole (b). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml (c)), antibody to IL-6 (0.5 ng/ml (d)), antibody to IL-4 (1 ng/ml (e)), or antibody to IL-13 (5 ng/ml (f)) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs
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Fig6: The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 106/well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone (a); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole (b). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml (c)), antibody to IL-6 (0.5 ng/ml (d)), antibody to IL-4 (1 ng/ml (e)), or antibody to IL-13 (5 ng/ml (f)) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs

Mentions: In order to evaluate if opioid receptors on microglia participate in ethanol neurotoxic action on POMC neurons, we treated microglial cell cultures with ethanol with or without MOR or DOR agonist or antagonist and then microglial conditioned media were removed and added to POMC cell cultures to conduct the apoptotic studies. Data shown in Fig. 6a indicate that the conditioned medium of microglial cells treated with the MOR agonist DAMGO or with ethanol (EtOH) increased nucleosome levels in POMC neuronal cells in a similar magnitude. However, conditioned medium of microglial cells treated with both DAMGO and ethanol increased nucleosome levels in POMC cells more than those produced by ethanol alone. The conditioned medium from MOR antagonist naltrexone-treated microglia had no effect on nucleosome levels. The conditioned medium of microglia treated with both naltrexone and ethanol increased nucleosome levels in POMC cells much less than those produced by ethanol alone. Naltrexone co-treatment also reduced the ability of ethanol to increase nucleosome levels in POMC cells. Figure 6b shows that DOR agonist DPDPE or antagonist naltrindole alone treated microglia culture media produced no significant effects on the basal level of nucleosomes in POMC cells. However, the conditioned medium of microglia treated with both ethanol and DPDPE, but not with ethanol and naltrindole, had lower levels of nucleosomes in POMC cells than those produced by ethanol alone. Naltrindole co-treatment also reduced the ability of ethanol and DPDPE to increase nucleosome levels in POMC cells. These data support a mediatory role of MOR in ethanol-activated microglia killing of POMC neurons. Also, these data identify a protective effect of the DOR agonist in ethanol-activated microglia killing of POMC neurons.Fig. 6


Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol
The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 106/well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone (a); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole (b). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml (c)), antibody to IL-6 (0.5 ng/ml (d)), antibody to IL-4 (1 ng/ml (e)), or antibody to IL-13 (5 ng/ml (f)) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs
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Fig6: The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 106/well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone (a); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole (b). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml (c)), antibody to IL-6 (0.5 ng/ml (d)), antibody to IL-4 (1 ng/ml (e)), or antibody to IL-13 (5 ng/ml (f)) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs
Mentions: In order to evaluate if opioid receptors on microglia participate in ethanol neurotoxic action on POMC neurons, we treated microglial cell cultures with ethanol with or without MOR or DOR agonist or antagonist and then microglial conditioned media were removed and added to POMC cell cultures to conduct the apoptotic studies. Data shown in Fig. 6a indicate that the conditioned medium of microglial cells treated with the MOR agonist DAMGO or with ethanol (EtOH) increased nucleosome levels in POMC neuronal cells in a similar magnitude. However, conditioned medium of microglial cells treated with both DAMGO and ethanol increased nucleosome levels in POMC cells more than those produced by ethanol alone. The conditioned medium from MOR antagonist naltrexone-treated microglia had no effect on nucleosome levels. The conditioned medium of microglia treated with both naltrexone and ethanol increased nucleosome levels in POMC cells much less than those produced by ethanol alone. Naltrexone co-treatment also reduced the ability of ethanol to increase nucleosome levels in POMC cells. Figure 6b shows that DOR agonist DPDPE or antagonist naltrindole alone treated microglia culture media produced no significant effects on the basal level of nucleosomes in POMC cells. However, the conditioned medium of microglia treated with both ethanol and DPDPE, but not with ethanol and naltrindole, had lower levels of nucleosomes in POMC cells than those produced by ethanol alone. Naltrindole co-treatment also reduced the ability of ethanol and DPDPE to increase nucleosome levels in POMC cells. These data support a mediatory role of MOR in ethanol-activated microglia killing of POMC neurons. Also, these data identify a protective effect of the DOR agonist in ethanol-activated microglia killing of POMC neurons.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.

Methods: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.

Results: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.

Conclusions: These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0844-3) contains supplementary material, which is available to authorized users.

No MeSH data available.