Neurofibrillary tangles composed of hyperphosphorylated tau are a major hallmark of Alzheimer's Disease. This phosphorylated tau may be a root cause of the disorder and therefore understanding its regulation is important for therapeutic intervention. To model this pathology, Okadaic acid (OA) has been used in primary cultured hippocampal neurons to investigate effects on tau, and the role of the JNK pathway in tau phosphorylation. The use of high content screening has allowed us to quantitatively assess the profound spatiotemporal profile of these proteins, finding dramatic and inhibitable changes. Furthermore, in vitro phosphorylation experiments show that the JNK3 isoform, which is predominantly expressed in the brain, can strongly autophosphorylate itself. This has profound implications on the importance of JNK3 in the CNS and its ability to sustain signaling both towards tau and other apoptotic targets. Together these data provide novel insights into the JNK pathway and a high resolution perspective on how this pathway influences tau phosphorylation and dynamics in neurons.