Effects of Arginine Vasopressin and Oxytocin on Neurite Outgrowth and Cellular Signaling in SH‐SY5Y Human Neuroblastoma Cell Line

Abstract

IntroductionIn the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G‐protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross‐reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression.MethodsThe human neuroblastoma derived SH‐SY5Y cell line demonstrates morphological characteristics of neurons including neurites and formation of functional synapses, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8‐AM to measure ligand‐induced Ca2+ mobilization and FLIPR Membrane Potential (FMP) assays were performed to assess ligand‐induced hyperpolarization. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of AVP and OT for 24 hours, fixed and stained with phalloidin and DAPI.ResultsIn SH‐SY5Y cells, OT was more potent than AVP at both ligand‐induced Ca2+ mobilization and membrane hyperpolarization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L‐371,257. These data are consistent with previous reports that vasopressin 1a receptors are absent in SH‐SY5Y cells. Additionally, OT demonstrated a dose‐dependent increase in neurite outgrowth, which is inhibited by L‐371,257.ConclusionsTogether, these data demonstrate that OTR mediated signaling pathways lead to neurite outgrowth in SH‐SY5Y cells. Additional studies are needed to assess the specific molecular mechanisms that contribute to neurite outgrowth, and whether functional synapses are formed. Integrative studies of behavior, genetics and ligand‐receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.