Project Summary/Abstract The human brain has about 100 billion neurons and each neuron is connected to up to 10,000 other neurons, passing information to each other through as many as 1,000 trillion synaptic connections, equivalent to a computer with a 1 trillion bit per second processor. Normal brain function critically depends on the proper function of these neural networks, with synapses being crucial components of neurons to communicate and transmit information through neuronal networks. In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and is associated with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV- associated neurocognitive disorders (HAND). Strong indicators of HAND are dendritic injury and disruption of the synaptic machinery. While the HIV-1 protein transactivator of transcription (Tat) is known to induce synaptic dysfunction and impairments in neurocognition, the endocannabinoid (eCBs) system has been shown to play a protective role in neurodegenerative diseases, such as multiple sclerosis, Parkinson?s disease, and Alzheimer?s disease. Thus, the focus of this proposal is to determine 1) HIV-1 Tat-induced synaptic injury at the macromolecular level via cryo-electron microscopy (cryo-EM), and to define 2) the neuroprotective functional and structural effects of eCBs in Tat-induced synaptic injury using eCB catabolic enzyme inhibitors as a tool. We hypothesize that eCBs will affect Tat-induced functional and structural changes at the synapse level. In Specific Aim 1, we will visualize the macromolecular architecture of control versus Tat-treated primary hippocampal neurons preserved in their close-to-physiological state using cryo-EM. Our recent advances in accessing the internal structure of frozen-hydrated biological material using cryo-focused ion beam milling (cryo-FIB) will enable us to assess structural changes of neuronal networks at and below the synaptic level via 3D cryo-electron tomography (CET). The microstructure of control and Tat-treated hippocampal neurons will be compared with focus on synaptic vesicles on the presynaptic side and the postsynaptic density (PSD) on the postsynaptic side. In Specific Aim 2, we will determine if eCB enzyme inhibitors are naturally neuroprotective in the presence of Tat by focusing on functional and structural changes on the synapse level. Functional information, in particular on communication between neurons by tracking firing patterns of neurons on a millisecond time scale, will be obtained by electrophysiology. At the same time microscopy techniques will be used to reveal changes in the molecular structure of synapses. Fluorescence light microscopy provides functional readout, and the opportunity to visualize and monitor subcellular functional changes in ion homeostasis at the synapse level in real time. Combining CET, electrophysiology and fluorescence techniques will allow us to examine how microscopic changes in the structure of synapses affect synaptic function in the context of neuroAIDS and to assess the role of eCB enzyme inhibitors.