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Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring’s forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment., Correlative data in humans has hinted at an association between maternal sleep apnea during pregnancy and altered neuronal function in offspring. This study shows that in a rat model of sleep apnea, maternal gestational intermittent hypoxia leads to sex-specific changes in neuronal structure and function in offspring, accompanied by impaired behavioral phenotypes.

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

Hypoxia is a component of multiple disorders, including stroke and sleep-disordered breathing, that often precede or are comorbid with neurodegenerative diseases. However, little is known about how hypoxia affects the ability of microglia, resident CNS macrophages, to respond to subsequent inflammatory challenges that are often present during neurodegenerative processes. We therefore tested the hypothesis that hypoxia would enhance or “prime” microglial pro-inflammatory gene expression in response to a later inflammatory challenge without programmatically increasing basal levels of pro-inflammatory cytokine expression. To test this, we pre-exposed immortalized N9 and primary microglia to hypoxia (1% O2) for 16 hrs and then challenged them with pro-inflammatory lipopolysaccharide (LPS) either immediately or 3-6 days following hypoxic exposure. We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to analyze primed microglial inflammatory gene expression and modifications to histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of primed genes. We found that microglia exhibited enhanced responses to LPS 3 days and 6 days post-hypoxia. Surprisingly however, the majority of primed genes were not enriched for H3K4me3 acutely following hypoxia exposure. Using the bioinformatics tool MAGICTRICKS and reversible pharmacological inhibition, we found that primed genes required the transcriptional activities of NF-ĸB. These findings provide evidence that hypoxia pre-exposure could lead to persistent and aberrant inflammatory responses in the context of CNS disorders.