5-methoxy-N,N-dimethyltryptamine induces a rapid multi-level neuroplastic cascade in mouse primary cortical neuron–astrocyte cultures

Introduction

The renaissance of psychedelic research in recent decades has highlighted the promising therapeutic potential of this class of psychoactive drugs in neuropsychiatric disorders. The underlying mechanism appears to involve neuroplasticity mediated by cortical 5-HT_2A receptor activation. However, neuroplastic effects across molecular and structural biological levels remain unclear; particularly for 5‑methoxy‑N,N‑dimethyltryptamine (5‑MeO‑DMT), which, despite emerging clinical evidence of rapid antidepressant effects in treatment‑resistant depression, is still comparatively under‑studied relative to other classical psychedelics. We hypothesise that 5-MeO-DMT induces multi-level neuroplasticity via rapid transcriptional activation of plasticity-related genes, leading to subsequent protein upregulation and dendritic remodelling.

Aims

To identify the molecular and structural neuroplasticity‑promoting effects of 5‑MeO‑DMT in mouse primary cortical neuron–astrocyte co-cultures.

Methods

Primary cortical neuron–astrocyte co-cultures from embryonic C57BL/6J mice were used as an in vitro model to study neuroplasticity. To assess structural neuroplasticity, cells were exposed to 5-MeO-DMT (10 µM) for 24 h at 4 days in vitro (DIV4), followed by a 48h drug-free period. At DIV7, cultures were fixed, immunostained for microtubule‑associated protein 2 (MAP2), and dendritic morphology was quantified using Sholl analysis (maximum number of crossings (Nmax)), number of primary dendrites, number of secondary branches, and total dendritic length). To evaluate early transcriptional responses, cells were exposed to 5‑MeO‑DMT (10 µM) for 45 min at DIV7 and mRNA levels were quantified by qRT-PCR; including immediate early genes FBJ murine osteosarcoma viral oncogene homolog (C-fos), early growth response protein 2 (Egr2), activity-regulated cytoskeleton-associated protein (Arc) and plasticity-related transcripts Brain-derived neurotrophic factor (Bdnf), cAMP response element-binding protein (Creb). To test changes in levels of neuro-plasticity-related proteins, cells were exposed to 5-MeO-DMT (10 µM) for 3 h or 24 h at DIV7, and analysed by western blot to assess ARC, BDNF, CREB (3 h) and synaptic proteins synaptophysin, postsynaptic density protein 95 (PSD-95) (24 h). Statistical significance was determined using unpaired student’s t-test (α = 0.05). N = independent experiments; n neurons analysed per condition. Immunocytochemistry: N = 3, n = 30–40; qRT-PCR: N = 6; western blot N = 7.

Results

5-MeO-DMT significantly increased dendritic arbour complexity, as reflected by Sholl-derived metrics compared with control cultures: Nmax (p=0.0004), number of primary dendrites (p=0.0204), number of secondary branches (p=0.0010), total dendritic length (p=0.0078). At the molecular level, qRT-PCR showed upregulated mRNA levels of immediate early genes (C-fos (p=0.0045),Egr2 (p=0.0005), Arc (p=0.0031), together with plasticity-related transcripts (Bdnf (p=0.0061), and Creb (p=0.0211)). This transcriptional response was followed by increased protein levels of ARC (p=0.0023), BDNF (p=0.0034) and total CREB (p=0.0018) at 3 h. At 24 h, levels of both presynaptic protein synaptophysin (p=0.0253) and postsynaptic protein PSD-95 (p=0.0012), were also augmented.

Conclusions

Our findings provide evidence that 5‑MeO‑DMT promotes a rapid multi-level neuroplastic response in mouse cortical neuron–astrocyte cultures, spanning transcriptional activation, protein upregulation, and structural dendritic remodelling. Overall, this supports convergent molecular and structural plasticity pathways, potentially relevant to 5-MeO-DMT rapid antidepressant-like effects and warrants further mechanistic investigation in translational models of neuropsychiatric disease.

References

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