Abstract
BackgroundOpioid 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.MethodsNeonatal 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.ResultsIn 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.ConclusionsThese 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.
Highlights
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
Alcohol effects on microglia and proopiomelanocortin (POMC) neuron interactions in the hypothalamus during the developmental period 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 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
Summary
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. Macrophage-like cells of the brain, are a type of innate immune cell in the central nervous system (CNS). Microglia are known to secrete neurotrophins and protective cytokines to promote neuronal development and survival. Upon CNS insult or injury, microglia can acquire complex phenotypes in order to participate in the cytotoxic response, immune regulation, and injury resolution. It has been shown that chronic alcohol drinking activates microglia to an M1 phenotype and promotes inflammatory response [2]. Activation of microglia to the M1 phenotype is detrimental during the developmental period, as this may lead to neurotoxicity and developmental disorders [3]
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