Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors

Abstract

Decreased dendritic spine density in the cortex is a hallmark of several neuropsychiatric diseases, and the ability to promote cortical neuron growth has been hypothesized to underlie the rapid and sustained therapeutic effects of psychedelics. Activation of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) is essential for psychedelic-induced cortical plasticity, but it is currently unclear why some 5-HT2AR agonists promote neuroplasticity, whereas others do not. We used molecular and genetic tools to demonstrate that intracellular 5-HT2ARs mediate the plasticity-promoting properties of psychedelics; these results explain why serotonin does not engage similar plasticity mechanisms. This work emphasizes the role of location bias in 5-HT2AR signaling, identifies intracellular 5-HT2ARs as a therapeutic target, and raises the intriguing possibility that serotonin might not be the endogenous ligand for intracellular 5-HT2ARs in the cortex.

Fig. 1.. Compound-Induced Neuronal Growth Correlates with Ligand Lipophilicity.

(A) Chemical structures of serotonin, tryptamine, and 5-MeO-tryptamine as well as their corresponding N-methyl and N,N-dimethyl analogs. (B) Sholl analysis demonstrates that increasing N-methylation leads to a concomitant increase in dendritic arbor complexity. The shaded area represents 95% confidence intervals. Sholl plots were generated from rat embryonic cortical neurons (DIV6) treated with compounds (10 μM). (C) Maximum numbers of crossings (N_max) of the Sholl plots in B (N = 45–64 neurons per treatment). (D) Images of rat embryonic cortical neurons (DIV6) treated with compounds (10 μM). (E–J) Correlation plots of Sholl analysis % efficacy (N_max values relative to 10 μM ketamine as the positive control) vs [³H]-IP accumulation (E), activation of psychLight2 (F), G_q activation (G), β-arrestin recruitment (H), or calculated LogP (J). PsychLight2 activation correlates well with both G_q activation and β-arrestin recruitment (I). Compounds treated at 10 μM. Data for [³H]-IP accumulation was obtained from literature values (22). VEH = vehicle, KET = ketamine, TRY = tryptamine, NMT = N-methyltryptamine, DMT = N,N-dimethyltryptamine, 5-HT = serotonin, N-Me-5-HT = N-methylserotonin, BUF = bufotenin, 5-MeO-TRY = 5-methoxytryptamine, 5-MeO-NMT = 5-methoxy-N-methyltryptamine, 5-MeO-DMT = 5-methoxy-N,N-dimethyltryptamine. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to VEH controls (one-way ANOVA followed by Dunnett’s multiple comparisons test). R² values calculated via simple liner regression.

Fig. 2.. Cortical neurons express intracellular 5-HT2A receptors.

(A) Live cell images of HEK293T cells and rat embryonic cortical neurons (DIV6) expressing Myc-5-HT2AR-CFP, β2AR-ECFP, or ECFP. Signals from the fluorescent protein and fluorescent plasma membrane marker (Cellbrite^® Steady) are shown in cyan and white, respectively. The expression patterns of 5-HT2ARs and β2ARs are comparable in HEK293T cells. However, in neurons, β2ARs exhibit higher expression on the plasma membrane than 5-HT2ARs. The expression of ECFP is diffuse in both HEK293T cells and cortical neurons. (B) Pearson’s Correlation Coefficient (PCC) and Manders’ Colocalization Coefficients (MCC) quantify the extent of colocalization between MYC-5-HT2AR-CFP, β2AR-ECFP, or ECFP and the fluorescent plasma membrane marker (N = 20–43 cells per group). ECFP = enhanced cyan fluorescent protein. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, bars indicate comparisons between data (one-way ANOVA followed by Tukey’s multiple comparisons test).

Fig. 3.. Intracellular 5-HT2ARs mediate structural plasticity induced by serotonergic psychoplastogens.

(A) Structures of membrane permeable (blue) and impermeable (red) compounds. (B) Images of rat embryonic cortical neurons (DIV6) administered compounds with (+) and without (–) electroporation. (C) N_max values obtained from Sholl plots. Membrane impermeable analogs of psychedelics (1 μM) only promote growth when administered with electroporation (N = 35–110 neurons per treatment). Unlike KTSN (10 μM), MKTSN (10 μM) only blocks psychedelic-induced plasticity when administered with electroporation (N = 45–109 neurons per treatment). (D) Membrane impermeable agonists of 5-HT2ARs (1 μM) cannot promote spinogenesis in cultured embryonic rat cortical neurons (DIV15) (N = 30–34 neurons per treatment). (E) Images of treated cortical neurons (DIV15). (F) HEK293T cells expressing psychLight1 were pretreated with VEH or MKTSN (10 μM) prior to the administration of 5-HT (10 μM) or 5-MeO-DMT (10 μM). (N = 41–54 cells per treatment). p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to VEH controls in (D) and (C) (one-way ANOVA followed by Dunnett’s multiple comparisons test), or between indicated pairs of data in (F) (two-way ANOVA followed by Šídák’s multiple comparisons test). For box-and-whisker plots, center line = median; box limits = upper and lower quartiles; whiskers = min and max values.

Fig. 4.. Cellular uptake of 5-HT induces structural plasticity in vitro.

(A) Rat embryonic cortical neurons were administered compounds (1 μM) with (+) and without (–) electroporation. N_max values obtained from Sholl plots (DIV6) demonstrates that unlike ketamine, serotonin only promotes growth when administered with electroporation (N = 49–59 neurons per treatment). (B) Images of embryonic rat cortical cultures (DIV6) sparsely transfected with CaMKII-SERT-EYFP and treated with compounds. (C) N_max values obtained from Sholl plots demonstrate that 5-HT (10 μM) can only increase the dendritic arbor complexity of SERT+ neurons, while DMT (10 μM) can promote the growth of both SERT+ and SERT– neurons (N = 69–93 neurons per treatment). (D) KTSN pretreatment (10 μM) blocks the plasticity-promoting effects of 5-HT (1 μM) in SERT+ neurons and DMT (1 μM) in both SERT+ and SERT– neurons. CIT (10 μM) only blocks the plasticity-promoting effects of 5-HT (1 μM) in SERT+ neurons (N = 59–100 neurons per treatment). (E) Images of CaMKII-SERT-EYFP positive and negative dendrites (DIV15) treated with compounds (10 μM). (F) 5-HT only promotes spine growth in SERT+ neurons (N = 11–35 neurons per treatment). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to VEH controls in (A) (one-way ANOVA followed by Dunnett’s multiple comparisons test), or compared to VEH controls from the same genotype in (C), (D), and (F) (two-way ANOVA followed by Tukey’s multiple comparisons test). For box-and-whisker plots, center line = median; box limits = upper and lower quartiles; whiskers = min and max values.

Fig. 5.. Cellular uptake of 5-HT produces antidepressant-like effects in vivo.

(A) Schematic displaying experimental design for measuring spine density in Thy1-EGFP mice following administration of a serotonin-releasing agent. (B) Histology images of Thy1-EGFP mice expressing CaMKII-mCherry or CaMKII-SERT-mCherry in the mPFC. (C) Images of dendritic spines in the mPFC of mice treated with PCA (5 mg/kg, IP). (D) Mice expressing CaMKII-SERT-mCherry display increased dendritic spine density following PCA treatment. (E) Schematic displaying experimental design for determining the sustained antidepressant-like effects of serotonin in mice expressing SERT in the mPFC. (F) NIL demonstrates no difference between CaMKII-SERT-EYFP and CaMKII-GFP expressing mice. (G) CaMKII-SERT-EYFP and CaMKII-GFP expressing mice exhibit no differences in the FST. After PCA (5 mg/kg, IP) administration, CaMKII-SERT-EYFP expressing mice display a sustained antidepressant-like effect. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared between indicated pairs of data in (D) and (F) (two-tailed unpaired Student’s t test), or (G) (two-way repeated measures ANOVA followed by Šídák’s multiple comparisons test. For box-and-whisker plots, center line = median; box limits = upper and lower quartiles; whiskers = min and max values.