Herpes Simplex Virus 1 (HSV-1), a member of the alpha herpesvirus subfamily, is among the very few viruses that naturally and productively exploit the mammalian nervous system, and, accordingly have evolved neuron- specific mechanisms to interact with highly-specialized neuronal cell biology. During the typical course of disease, alpha herpesviruses infect and establish latency in the peripheral nervous system (PNS). During lytic replication, such as following reactivation from latency, progeny virus particles spread back to peripheral tissues, causing recurrent herpetic lesions, and can also spread into the central nervous system (CNS). HSV-1 invasion of the CNS can cause severe and debilitating herpes encephalitis, or can be asymptomatic/sub- clinical. Once in the CNS, HSV-1 may trigger neuroinflammation and contribute to the development of neurodegenerative disease. To better understand the molecular and cellular mechanisms that underlie HSV-1 spread to the CNS, we propose an innovative method development/bioengineering goal: to develop methods to directly image virus particle exocytosis at or near neuronal synapses. This will involve culturing primary neurons on patterned substrates in order to induce synapse formation at defined locations and orientations. We will combine this method with our established live-cell virus exocytosis method to be able to image and study cell biological factors involved in viral exocytosis at or near synapses. Elucidating the basic cell biological processes that HSV-1 uses in neurons will increase our understanding of how and why herpesviruses spread in the nervous system, lead to the identification of druggable targets and development of better therapies for viral neuropathology, and may provide fundamental insights into the cell biology of neurons.