Microglial regulation of intermittent hypoxia induced phrenic motor plasticity

About This Grant

ABSTRACT A well-studied model of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic motor output following moderate acute intermittent hypoxia (mAIH). Over 2 decades, we developed a nuanced understanding of intra-cellular mechanisms giving rise to pLTF, and factors regulating its expression in the rest/light phase. With mAIH, serotonin-driven pLTF is constrained by modest adenosine 2A (A2A) receptor activation. When extracellular adenosine levels are high, greater A2A receptor activation initiates an alternate adenosine-dependent mechanism of plasticity. Although the serotonin driving plasticity arises from raphe serotonergic neurons, the cellular source of adenosine was unknown. Thus, in the first 4 years of this grant, we asked the question: what is the source of hypoxia-evoked adenosine? We developed an inter-cellular model whereby phrenic motor neuron to microglia signaling evokes spinal adenosine formation and regulates pLTF. During hypoxia, phrenic motor neurons release Fractalkine (Fkn) and activate receptors on the lone CNS cell- type expressing Fkn receptors—microglia. Microglia respond by converting extracellular ATP to adenosine, restraining serotonin-driven pLTF. These experiments were performed during the daily rest/light phase; our discovery that mAIH delivered in the active (vs rest) phase profoundly impacts the mechanism driving pLTF due to diurnal shifts in basal adenosine levels lead us to realize we must update our model to account for rest/active phase differences. Two fundamental hypotheses guide this proposal: 1) shifts in microglial state across the rest/active cycle alter phrenic motor neuron/microglia interactions and, thus, mAIH-induced pLTF; and 2) microglial (and pLTF) responses to pro-inflammatory stimuli reverse in the active/dark phase. Using a multi-disciplinary approach, we will interrogate a revised inter-cellular model by testing 5 hypotheses: 1) Phrenic motor neuron-microglia fractalkine signaling inhibits pLTF less in the active/dark phase; even though 2) Fkn receptor activation evokes greater active phase spinal adenosine formation; 3) Microglial expression of key molecules for adenosine formation increases in the active (dark) phase; 4) Unlike the rest/light phase, mild systemic inflammation has minimal impact, or even enhances active phase pLTF; and 5) Due to differences in microglial reactivity, ventilatory LTF exhibits sexual dimorphism during the rest (not active) phase. We know little concerning the impact of “biological clocks” in any aspect of ventilatory control, let alone respiratory plasticity. Thus, we will expand on our discovery that time-of-day has powerful effects on AIH-induced pLTF by considering the complex rest/active phase effects on phrenic motor neuron/microglia interactions. Regardless of outcome, these studies will greatly advance our understanding of diurnal rhythms in respiratory plasticity and accelerate ongoing efforts to develop AIH as a therapeutic modality to treat devastating human clinical disorders that compromise breathing, threatening life itself. Greater understanding of diurnal rhythms is sorely needed since nocturnal rodents and diurnal humans are typically studied in opposite phases of the rest/active cycle.