Extended Data Fig. 3: Effects of psilocybin on structural plasticity of IT neurons residing in layer 2/3 or layer 5.

a, Volumetric reconstruction from z-stack images of GFP-expressing IT neurons. IT neurons can be divided into two groups based on the laminar position of their cell body (200–400 μm or 450–600 μm). For the deep-lying IT neurons, sometimes the cell body could not be imaged due to the depth limitation of two-photon microscopy, but nonetheless the apical trunk was observed at >450 μm. b, Density of dendritic spines (left), spine head width (middle), and spine protrusion length (right) in apical tuft of IT neurons residing in layer 2/3 (top row) or layer 5 (bottom row) after psilocybin (1 mg/kg, i.p.) or saline across days, expressed as fold-change from baseline in first imaging session (day -3). The same mice were used for both depth ranges (200–400 μm and 450–600 μm). c, Left: spine formation rate determined by number of new and existing spines in consecutive imaging sessions across two-day interval, expressed as difference from baseline in first interval (day -3 to day -1). Right: similar to left for elimination rate. d-e, Similar to (b) for density of dendritic spines and spine head width but further divided the data based on the sex of the animal. n = 8 mice (IT neurons, saline, 5 females, 3 males), n = 8 (IT neurons, psilocybin, 5 females, 3 males) in (b-e). The analysis was motivated by the question: is psilocybin-evoked increase in spine size specific to cell type (IT versus PT), or specific to laminar position (layer 2/3 versus layer 5)? This is because IT neurons can be both superficial and deep, but PT is only found in deep layer. We detected no significant depth dependence for spine size for layer 2/3 and deep layer 5 IT neurons (interaction effect of treatment × soma depth, indicated in plots, mixed effects model). Data are mean and s.e.m. across dendrites. Detailed sample size n values are provided in Methods. Statistical analyses are provided in Supplementary Table 1.

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