Abstract
Glial cells, despite comprising approximately 50% of cells in the central nervous system, were previously believed to be mere passive bystanders to neuronal function. Emerging research over the past two decades has demonstrated that this characterization is entirely erroneous; rather, glia have essential roles in the brain under homeostatic and disease conditions. In particular, glia have been identified as essential components of proper brain development, facilitating necessary processes including neurogenesis, neural cell maturation, required cell death, axon guidance, and synaptic refinement. Microglia, the innate immune cells of the central nervous system, and astrocytes, macroglia constituting the majority of glial cells, are critical mediators of these processes. However, the extent to which these cells interact with one another to coordinate postnatal circuit formation and nervous system assembly remains relatively unexplored.
Moreover, glial cells have gained appreciation in recent years for their sex differences in function, morphology, and transcriptional identity. Previously, our lab characterized sex-dependent activity of microglial receptors in impacting circuit development and mouse behavior. Microglia in particular have been characterized as sexually dimorphic cells during brain development, playing critical roles in masculinization of the male brain, regulating dendritic structure, neuronal density, and other factors essential for circuit function. Sex differences in the activity of astrocytes have not been as thoroughly explored, though studies have documented sex differences in their rate of maturation. Disruptions in the activity of microglia and astrocytes have been identified as risk factors for the incidence of neurodevelopmental disorders such as autism spectrum disorder, which is also characterized by a significant sex bias in diagnosis and presentation. In order to better understand how these disorders arise, a greater scientific understanding of sex differences in glial activity and neural circuit development is warranted. In Chapter 1, this dissertation provides a thorough synthesis of current literature detailing the roles of microglia and astrocytes in postnatal neural circuit development, as well as what is known regarding sex differences in the activity of these cells.
In Chapter 2 of this dissertation, I build upon our understanding of sex-specific activity of microglia and astrocytes in neural circuit development. In interrogating the developing somatosensory cortex, we discovered that microglia exhibit sex differences in phagocytic activity during a discrete window from P12 to P17, driven by gonadal hormones. During this period, microglia phagocytose astrocytes in a sex-dependent manner, resulting in a reduced density of cortical astrocytes in females relative to males. This also results in a reduced density of a critical astrocyte-derived synaptogenic factor, hevin, which is necessary for excitatory synapse production and maturation. We observe that during development and in the adult brain, these sex differences in microglia and astrocyte activity result in a sex difference in thalamocortical synapse density and neuronal activity. These data indicate that microglia indirectly regulate synapses through the regulation of astrocytes, and they do so in a sex-specific manner. These findings provide insight not only into physiological sex-specific cortical development, but also could inform our understanding of sex-biased susceptibility to neurodevelopmental disorders.
In Chapter 3 of this thesis, I characterize the transcriptomes of microglia and astrocytes in males and females across cortical development. To better understand the sex differences that are present in microglia and astrocytes and how these sex-specific functions may change across development, we conducted single-nuclei RNA sequencing of somatosensory and motor cortices from wildtype mice at P7, P14, and P40. We demonstrate that both microglia and astrocytes exhibit sex differences in their expression of genes relating to neurogenesis and cell cycle maintenance at P7, while shifting to differentially express genes involved in synaptic regulation at P14. At P40, fewer sex differences exist in the transcriptomes of both of these glial cells, highlighting a critical role for glial sex differences in shaping neural circuit development during earlier stages of postnatal development. We also demonstrate that there are sex differences in the expression of autism-associated genes at P14, but not other time points, in both microglia and astrocytes. Ongoing and future work should further define how these transcriptomic sex differences in microglia and astrocytes correspond to functional differences in nervous system assembly.
In summary, this dissertation stresses the critical role of glial cell sex differences and their interactions for proper brain development and lifelong function. Our work contributes to an improved understanding of the sex differences that exist in the activity of microglia and astrocytes in the developing cortex, and how these sex-specific functions drive male and female-specific neural circuit development.
