Pala, J. | Search bioRxiv

bioRxiv2024

Effects of serotonergic psychedelics on synaptogenesis and immediate early genes expression - comparison with ketamine, fluoxetine and lithium

BackgroundRecent evidence suggests that psychedelics are able to induce rapid and long-lasting antidepressant effects. The generally acknowledged explanation for these traits is the phenomenon of neuroplasticity, although exact underlying molecular mechanisms remain unclear. AimsThis study investigates selected neuroplastic effects of psilocin, lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) in direct comparison with ketamine, fluoxetine and lithium after acute (1 h) and/or prolonged (24 h) treatment in vitro. MethodsRat primary cortical cultures were treated with 10 {micro}M psilocin, 1 {micro}M lysergic acid diethylamide (LSD), 90 {micro}M N, N-dimethyltryptamine (DMT), 1 {micro}M ketamine, 10 {micro}M fluoxetine and 5 mM lithium. Analysis of synaptic puncta was performed; puncta of presynaptic marker synapsin I/II, postsynaptic density protein 95 (PSD-95), and their co-localization (established synapse) were assessed 24 h after drug treatment. Next, expressions of immediate early genes (IEGs) encoding activity-regulated cytoskeleton-associated protein (Arc), early growth response 1 (Egr1), and neuronal PAS (Per-ArntSim) domain protein 4 (Npas4) were analysed 1 and 24 h after drug treatments. ResultsPsilocin increased synaptic puncta count and induced Arc expression. The effect to promote synaptogenesis was comparable to ketamine and lithium; ketamine additionally increased PSD-95 puncta count. LSD and DMT didnt induce any significant effect. Interestingly, fluoxetine had no effect on synaptic puncta count, but upregulated Egr1 and Npas4. ConclusionsPsilocin demonstrated a significant neuroplastic effect comparable to that of ketamine and lithium, adding another piece of evidence to its profile as a promising therapeutic agent.

neuroscience↗

bioRxiv2021

Cell-Tak coating may cause mis-normalization of Seahorse metabolic flux data

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating may strongly affect the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

biochemistry↗