Abstract Synaptic failure is an important feature of HIV infection of the brain, and a likely key contributor to HIV- associated neurocognitive disorders (HAND). Yet current animal models have proven of limited utility in defining the mechanisms of this process, in part because of the species-specific nature of HIV infection, but also because of the greater complexity of human astrocytes relative to those of mice. To address this issue, we will utilize mice chimeric for both human microglia and human astrocytes, to assess the effects of HIV infection on central neurons. To that end, we will engraft mice with both human glial progenitor cells (hGPCs) and microglia, each derived from embryonic stem cells (hESCs). We have established the methods of generating these human glial chimeras, by the neonatal implantation of hGPCs, which outcompete and ultimately replace the host mouse GPCs, yielding adult chimeras broadly colonized with human astroglia1-5. This process is especially robust in the neostriatum, allowing the glial humanization of regions critically involved in striatal reward and addiction circuits. We have recently extended this approach to include chimerization with hESC-derived microglia, paired with the use of CSF1R null mice lacking host microglia, crossed to NSG SGM3 mice to allow the stable xenograft of hGPCs. The mice are thus chimeric for hGPC-derived astrocytes as well as microglia, in a T- and B-cell deficient background that allows the effects of glial HIV infection on neurons to be isolated, following intracerebral inoculation with HIV-infected microglia. These chimeric human astroglial-microglial (CHAM) mice are especially attractive, since they incorporate the hominid-specific features of human astroglia, which are themselves key components of central synapses. Using this model, we will test the postulate that astrocytes become both structurally and functionally impaired by microglial HIV infection, resulting in the loss of synaptic engagement by affected astrocytes, with consequent dendritic involution and network disruption. By infecting CHAM mice with HIV, and using rabies viral-EGFP to trace striatal dendrites, we will assess the effects of astrocytic HIV infection on the dendritic architecture and synaptic structure of resident medium spiny neurons. In parallel, we will study the effects in CHAM mice of HIV infection complicated by methamphetamine use – a common and disabling comorbidity that suppresses dopaminergic input to the striatum – focusing on the structural and transcriptional responses of human glia to the combination of infection and addiction, as well as on the behavioral effects of that combination. To that end, we will use single cell RNA-Seq to assess the changes in gene expression by human astrocytes and their partnered mouse neurons caused by HIV infection, both alone and together with chronic methamphetamine use, to identify those changes that contribute to the striatal synaptic disruption and behavioral...