My thesis investigates the role of brain-resident immune cells, particularly microglia, in angiogenesis during brain development and their impact on germinal matrix hemorrhage (GMH) in premature infants. While microglia were postulated to promote cerebrovascular formation, the exact mechanism remains unclear. To address this question, I developed a multidisciplinary approach to examine the distribution, transcriptomics, and functions of immune cell subtypes in distinct brain regions and ages. My findings showed that prenatal microglia age-dependently interact with nascent vasculature and that ablation of these cells reduces angiogenesis in the ganglionic eminences (GE), which correspond to the human germinal matrix. Furthermore, single-cell transcriptomics and flow cytometry showed that distinct subsets of immune cells employ diverse signaling mechanisms to promote vascular formation in the GE. In contrast, immune cells from preterm infants with GMH included neutrophils/monocytes that produce bactericidal factors and chemokine, which can disrupt vascular integrity and lead to GMH. Finally, my results showed that changes in microglia states adversely affect neural progenitor density and positioning. Taken together, this project offers insights into the complex tripartite interplay among immune, vascular, and neuronal cells during brain development, shedding light on their implications for GMH pathogenesis.

Finely tuned arousal control is required for animal survival. To catch food, a predatory animal is keenly attentive on a hunt, and to survive, prey is acutely vigilant. These critical attentional dynamics require sufficient sleep, which promotes optimal neuronal function by facilitating waste clearance, metabolic homeostasis, and immune regulation1,2. In humans, disordered sleep is associated with attentional and cognitive deficits, increased risk of accident, disease, and psychiatric disease, underscoring the importance of unravelling the cellular mechanisms underlying the balance of sleep and wake3–5. That said, our understanding of arousal control is mostly limited to neuronal studies, leaving the role of non-neuronal brain cells largely unknown. Classical neuromodulators regulate arousal states, spanning deep sleep to vigilant wakefulness, primarily by activating cortical neurons1,6,7. However, cortical astrocytes also express neuromodulatory G-protein coupled receptors (GPCRs)8,9. While astrocytic noradrenergic receptors can modulate two critical regulators of arousal—cortical synchrony10 and extracellular adenosine levels11–14—how other neuromodulatory signaling pathways similarly shape arousal remains unclear. Astrocytes in mammalian cortex express particularly high levels of the wake-promoting histamine-1-receptor (H1R) GPCR8, yet little is known about how astrocytic H1R contributes to regulation of arousal. To address this gap, we used pharmacological and genetic approaches in murine cortex to test how astrocytic H1R signaling affects astrocyte Ca2+, cortical activity across sleep/wake, and release of adenosine—an output of astrocytic activity known to promote sleep. Using ex vivo two-photon Ca2+ imaging in acute cortical slices, we show that H1R activation drives cell-autonomous astrocyte Ca2+ elevations and stimulates release of ATP, which is extracellularly metabolized to adenosine. In a parallel project published by Silvia Pittolo et al.15, we show that DA also triggers ATP release in the cortex, suggesting that a critical function of astrocyte-neuromodulatory signaling may be to increase cortical ATP/adenosine levels. Next, in vivo fiber photometry and electrophysiology results show that astrocyte-specific H1R deletion in cortex disrupts local astrocyte Ca2+ and extracellular adenosine dynamics specifically around rapid eye movement (REM) sleep transitions, when HA release is minimal. We observe concurrent changes in cortical oscillations during REM, suggesting that H1R activation, during wake when HA is released, induces lasting changes in astrocyte physiology to modulate extracellular adenosine and cortical dynamics in REM sleep. These findings contribute to an emerging model in which astrocytes integrate neuromodulators on a minutes-long timescale to regulate sleep/wake neuronal dynamics via adenosine signaling.

Between 1.5 – 3.8 million people experience a traumatic brain injury each year in the United States and at least 75% of these injuries are a mild TBI (concussive injuries without loss of consciousness). TBI remains a growing health concern, as it’s a leading cause of long-term neurological disability in the world, induces accelerated cognitive aging, and is the strongest environmental risk factor for the development of dementia. Even mild TBI (concussive injury) is associated with a >2-fold increase in the risk of dementia diagnosis. The pathophysiology of TBI is very complex, resulting in chronic behavioral and cognitive impairments that affect the quality of life of millions of individuals. Little is known about the cellular mechanisms responsible for the development of cognitive deficits after injury, thus with no identified targetable mechanisms, there are no treatments to prevent or mitigate deficits resulting from TBI. This dissertation investigates mechanisms contributing to TBI-induced cognitive decline and broadens the scope of TBI research to understand the effect biological sex, aging, and therapeutic whole-brain irradiation.In Chapter 2, I investigated the effect of concussive injury and ISR inhibition on cortical spine dynamics and density and short-term memory function. Concussive injury induced aberrant changes in spine dynamics and density and short-term memory dysfunction. Targeting the ISR using the small-molecule inhibitor ISRIB reversed the maladaptive changes in cortical spine dynamics and the short-term memory dysfunction. Importantly these restorative effects were maintained weeks after treatment ended. In Chapter 3, we investigated the effect of mild repetitive traumatic brain injury, ISR inhibition, and biological sex on cognitive and behavioral function and neuronal function. Mild repetitive injury resulted in increased risk-taking behavior only in male mice and this behavior phenotype was associated with increased activation of the ISR and cell-specific synaptic alterations in type A layer V pyramidal neurons of the medial prefrontal cortex. Importantly, brief pharmacological inhibition of the ISR with ISRIB reversed injury-induced risk-taking behavior and the associated cell-specific deficits in synaptic function. In Chapter 4, we investigated the role of the ISR in healthy age-related cognitive decline. Temporary treatment with ISRIB leads to improvement in spatial, working, and episodic memory. At a cellular level the cognitive enhancement was paralleled by i) improved intrinsic neuron excitability, ii) increased dendritic spine density, iii) reversal of age-induced changes in IFN and T cell responses in the hippocampus and blood, and iv) reversal of ISR activation. In Chapter 5, we investigated the effect whole-brain therapeutic irradiation and concussive injury on the functional role of brain engrafted macrophages. Replacement of microglia with monocyte derived brain engrafted macrophages (BEMs) prevented loss of synapses and consequent memory deficits. Importantly, BEMs replacing microglia were also protective against a second injury to the brain. These chapters collectively add to our understanding of the pathophysiology of TBI with the aim that we are closer to providing effective treatment for a major health crisis in the future.