This work was co-led by post-doc Delong Zhou and grad student Heike Schuler- both yet to land in Bsky. Big thank you to some of the funders that supported this work @ludmercentre.bsky.social and @hbhlmcgill.bsky.social

Taken together, this work points to L5/6NP neurons as a key mediator of psilocybin’s neuroplastic effects. Our findings suggest that integrated 5-HTR signaling mediates acute cell-type specific responses to psilocybin that lead to sustained remodeling of mPFC neuronal network activity.

Finally, we examined differential gene expression. We find that psilocybin induces cell-type specific transcriptional programs associated with synaptic plasticity, upregulating synaptic genes in L5/6 NP and GABAergic neurons while increasing activity associated genes in L2-6 IT and L6 CT neurons.

We then examined cell-cell communication and find that psilocybin predominantly alters communication between neurons, suppressing GABAergic neuron signaling to all excitatory neurons.

This predicts that, psilocybin will primarily activate L5/6 NP neurons while suppressing all other excitatory neurons. We also saw interesting patterns in GABAergic sub-types that predict overall reduction of GABAergic suppression of excitatory neurons.

We find that L5/6 NP neurons are unique among excitatory neurons in that a large number of these neurons only express genes for excitatory 5-HT receptors, predominantly 5-HT2C. In contrast, all other excitatory neuron types predominantly express uniquely inhibitory 5-HT receptor genes.

Single-cell sequencing methods are prone to ‘technical drop-outs’ so not optimal for definitively mapping co-expression identity of at the single-cell level. We used spatial transcriptomics data sets to rigorously map cell-type specific expression and co-expression of a wide range of 5-HT receptors.

Neurons express many 5-HT receptors and psilocin binds these too. We hypothesized that the integrated expression patterns of excitatory and inhibitory 5-HT receptor genes might explain the specific sensitivity of L5/6 NP neurons.

Using an algorithm for cell-type prioritization, we show that a type of deep layer near projecting neuron – L5/6 NP - is most robustly regulated 24h after psilocybin. Interestingly, these neurons are not enriched for the gene encoding 5-HT2A. So why are they especially sensitive to psilocybin?

We used single-cell RNA sequencing to discover which mPFC cell types psilocybin impacts in female mice. We chose single cell over single nucleus RNAseq, which although more technically demanding in brain, can more accurately detect cellular state changes and differential transcriptional regulation.

This differential impact of sex on acute and delayed behavior supports findings suggesting distinct mechanisms mediate the hallucinogenic and neuroplastic effects of psilocybin.

First, we show that, while acutely, psilocybin similarly induced head twitch response in male and female mice, 24h later, psilocybin only reduced anxiety-like behavior in female mice.