Many human viral pathogens, such as the widely prevalent herpes simplex virus (HSV), exhibit neurotropism. The recurring latency and reactivation of HSV throughout a lifelong infection period make it a fascinating subject for study. However, investigating virus neurotropism and interactions with the nervous system requires accessible models. This chapter offers a detailed account of various in vitro models employing primary mouse neuronal cultures, encompassing ganglia explants and dissociated neurons from superior cervical ganglia, trigeminal ganglia, and dorsal root ganglia. These models include two-dimensional (2D) and 3D cultures, as well as microfluidic chambers (MFCs) capable of isolating neurites and neuronal somata. These setups enable the study of different infection pathways and processes, including infection routes (i.e., through neurite ends or neuronal somata) as well as virus transport within neurites. Additionally, they can also be useful for addressing the interaction between pathogens and neuronal cells, such as the regulation of neuroelectrophysiological activity and neurite outgrowth. Furthermore, these protocols could be easily adapted for isolating and culturing neurons from the central nervous system. Overall, this chapter provides a versatile toolkit for obtaining large amounts of mouse primary neurons, which are essential for studies requiring neuronal models in the field of life sciences.