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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

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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.


Related in: MedlinePlus

Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α (a), mRNA level of cytokine MCP1 (b), cytokine receptors CSFR1 (c), and TLR4 (d) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells (e) and histograms representing the mean ± SEM number of IBA-1-positive cells (f) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified (g); partially amoeboid (h); fully amoeboid (i)) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting (j).Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons (k). Representative images of POMC-stained neurons in the arcuate nucleus (l) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 (m). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus (n) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 (o). Scale bars are 200 μm/each. Data are represented as mean ± SEM (n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. *p < 0.05, **p < 0.01, ***p < 0.001, AF vs PF and AD, ap < 0.05, AF vs AD, #p < 0.05, AF vs PF, ##p < 0.01, AF vs PF, ###p < 0.001, AF vs PF
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Fig1: Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α (a), mRNA level of cytokine MCP1 (b), cytokine receptors CSFR1 (c), and TLR4 (d) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells (e) and histograms representing the mean ± SEM number of IBA-1-positive cells (f) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified (g); partially amoeboid (h); fully amoeboid (i)) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting (j).Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons (k). Representative images of POMC-stained neurons in the arcuate nucleus (l) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 (m). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus (n) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 (o). Scale bars are 200 μm/each. Data are represented as mean ± SEM (n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. *p < 0.05, **p < 0.01, ***p < 0.001, AF vs PF and AD, ap < 0.05, AF vs AD, #p < 0.05, AF vs PF, ##p < 0.01, AF vs PF, ###p < 0.001, AF vs PF

Mentions: Previously, we have shown that alcohol exposure during the developmental period reduces POMC/ß-EP cell number by increasing this neuronal apoptotic death in the hypothalamus [8, 10]. Using the rat model of neonatal alcohol feeding (equivalent to third trimester binge human alcohol use) which elevates blood level of alcohol about 150–200 mg/dl and increases POMC/ß-EP neuronal apoptotic death [10], we determined the changes in the levels of pro-inflammatory signal molecules in the mediobasal hypothalamus where most of the POMC neurons are distributed. We measured some of the important pro-inflammatory signal molecules (TNF-α, MCP1, CSFR1, and TLR4), which are known to promote neuronal death [24]. As shown in Fig. 1, protein levels of TNF-α (Fig. 1a) and mRNA levels of MCP1 (Fig. 1b) were elevated in the hypothalamus of alcohol-fed (AF) animals as compared to control-fed animals. Similarly, mRNA levels of TLR4, an innate immune system activating receptor, and CSFR1, a cytokine receptor, were elevated in the hypothalamus of AF rats as compared to controls (Fig. 1c, d).Fig. 1


Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol
Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α (a), mRNA level of cytokine MCP1 (b), cytokine receptors CSFR1 (c), and TLR4 (d) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells (e) and histograms representing the mean ± SEM number of IBA-1-positive cells (f) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified (g); partially amoeboid (h); fully amoeboid (i)) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting (j).Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons (k). Representative images of POMC-stained neurons in the arcuate nucleus (l) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 (m). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus (n) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 (o). Scale bars are 200 μm/each. Data are represented as mean ± SEM (n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. *p < 0.05, **p < 0.01, ***p < 0.001, AF vs PF and AD, ap < 0.05, AF vs AD, #p < 0.05, AF vs PF, ##p < 0.01, AF vs PF, ###p < 0.001, AF vs PF
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Fig1: Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α (a), mRNA level of cytokine MCP1 (b), cytokine receptors CSFR1 (c), and TLR4 (d) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells (e) and histograms representing the mean ± SEM number of IBA-1-positive cells (f) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified (g); partially amoeboid (h); fully amoeboid (i)) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting (j).Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons (k). Representative images of POMC-stained neurons in the arcuate nucleus (l) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 (m). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus (n) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 (o). Scale bars are 200 μm/each. Data are represented as mean ± SEM (n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. *p < 0.05, **p < 0.01, ***p < 0.001, AF vs PF and AD, ap < 0.05, AF vs AD, #p < 0.05, AF vs PF, ##p < 0.01, AF vs PF, ###p < 0.001, AF vs PF
Mentions: Previously, we have shown that alcohol exposure during the developmental period reduces POMC/ß-EP cell number by increasing this neuronal apoptotic death in the hypothalamus [8, 10]. Using the rat model of neonatal alcohol feeding (equivalent to third trimester binge human alcohol use) which elevates blood level of alcohol about 150–200 mg/dl and increases POMC/ß-EP neuronal apoptotic death [10], we determined the changes in the levels of pro-inflammatory signal molecules in the mediobasal hypothalamus where most of the POMC neurons are distributed. We measured some of the important pro-inflammatory signal molecules (TNF-α, MCP1, CSFR1, and TLR4), which are known to promote neuronal death [24]. As shown in Fig. 1, protein levels of TNF-α (Fig. 1a) and mRNA levels of MCP1 (Fig. 1b) were elevated in the hypothalamus of alcohol-fed (AF) animals as compared to control-fed animals. Similarly, mRNA levels of TLR4, an innate immune system activating receptor, and CSFR1, a cytokine receptor, were elevated in the hypothalamus of AF rats as compared to controls (Fig. 1c, d).Fig. 1

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&ndash;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&rsquo;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&rsquo;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.


Related in: MedlinePlus