452. Primary neuronal cultures to study neuroplastic effects of hallucinogen drugs

Abstract Background Chronic stress is a key risk factor for major depressive disorder (MDD), one of the leading causes of disability worldwide1. Despite available pharmacological therapies, treatment-resistant depression (TRD) remains a clinical challenge. Hallucinogens such as ketamine and serotonergic psychedelics including psilocybin and lysergic acid diethylamide (LSD)2 show rapid antidepressant effects involving modulation of synaptic plasticity3,4. However, their neuronal mechanisms are not yet fully characterized. p11 (S100A10) is a multifunctional protein, which has been shown to interact with serotonergic receptors and to modulate the antidepressant response by regulating synaptic function and receptor trafficking. Interestingly, hippocampal p11 was implicated in the sustained antidepressant effect of ketamine and peripheral p11 was proposed as a predictor of ketamine response. Aims & Objectives In this study, we aimed to characterize molecular, synaptic and functional changes induced by ketamine, psilocybin and LSD in primary neuronal cultures exposed chronically to corticosterone (as an in vitro model of chronic stress exposure) and to test whether ketamine effects are modulated by p11 (S100A10) using primary neuronal cultures from p11 KO mice.

Method: To establish the in vitro stress model, mature primary neuronal cultures from C57BL/6 mouse embryos (E16.5) were chronically exposed to corticosterone (CORT, 200 nM) and vehicle (VEH: DMSO) twice daily (9 am and 5 pm) from DIV12 to DIV15. After the final CORT exposure, cultures were incubated with ketamine (1 μM), psilocybin (1 μM), or LSD (100 nM) for 4 hours. The same in vitro stress protocol followed by acute ketamine was also applied to primary neuronal cultures derived from p11 WT and p11 KO mice. Changes in the protein expression of molecular effectors of synaptic plasticity were quantified by western blotting. In parallel, neuronal activity was evaluated through calcium imaging, focusing on intracellular calcium dynamics in response to the stress hormone and drug treatments. Alterations in the number of synaptic spines were assessed through immunostaining for vGLUT1, vGAT and synaptophysin and puncta-based quantification of excitatory and inhibitory presynaptic markers across conditions.

Results: In primary cultures from C57BL/6 mouse embryos exposed to the stress protocol, chronic CORT decreased neuronal dendritic length and reduced glutamatergic presynaptic markers (vGLUT1) compared to VEH controls. Acute ketamine increased the levels of CaMKII, pSer880-GluA2, and GluA1 compared with CORT controls suggesting the activation of plasticity mechanisms. Similarly, calcium imaging showed an enhanced cellular response after acute ketamine indicating rapid functional changes at the network level. Immunostaining experiments revealed a reduction of the density of excitatory contacts in primary cultures exposed to chronic CORT while hallucinogens largely rescued this alteration. Experiments in the p11 WT/KO cultures treated with ketamine are currently underway. Discussion & Conclusions These preliminary findings suggest that the in vitro stress protocol induces structural and synaptic alterations, and that ketamine rapidly affects expression of several proteins involved in synaptic plasticity. Ongoing analyses will clarify whether psilocybin and LSD show similar effects and whether p11 modulates ketamine-induced molecular and functional effects.