Psychedelic compounds directly excite 5-HT2A layer V medial prefrontal cortex neurons through 5-HT2A Gq activation

Abstract

Psilocybin, and its active metabolite psilocin, have seen renewed interest due to studies suggesting potential therapeutic utility. 5-Hydroxytryptamine2A receptors (5-HT_2ARs) are primary mediators of the psychoactive effects of psychedelics in animals and humans, but the underlying neurobiological mechanisms remain poorly understood. Functional magnetic resonance imaging identified significant psilocin-induced increases in medial prefrontal cortex (mPFC) activity, a site of enriched 5-HT_2AR expression. We identified a population of 5-HT_2AR neurons in the prelimbic/anterior cingulate mPFC. Psilocin and the 5-HT_2AR-selective compound 25-CN-NBOH increased excitability, and stimulated firing across a range of current injections in these neurons that was both 5-HT_2AR and Gα_q dependent. Similar effects were observed with a novel, non-hallucinogenic psychedelic compound. These findings provide valuable insight into the specific role of 5-HT_2AR-containing neurons in psychedelic-associated plasticity in mPFC regions that are likely implicated in the clinical effects of psychedelics and further identify membrane-bound 5-HT_2ARs and subsequent intracellular Gα_q signaling as therapeutic targets.

Fig. 1. Psilocin effects in medial prefrontal cortex and on layer 5 pyramidal neurons from C57B6/J mice.

A A representative 3D plot of the three selected anatomical regions within the prefrontal cortex: prelimbic (PrL), infralimbic (IL), and anterior cingulate cortex (ACC). B The fMRI signal normalized to the baseline (Error bars represent the standard error) in experiment and control groups. C Averaged fMRI signal changes in PrL, ACC, and IL during the psilocin period in the experimental group and the vehicle period in the control group (F-value = 8.10, p < 0.001). PrL exhibited a trend toward higher fMRI activity compared to IL (β = 2.36, ^#p = 0.059). Additionally, fMRI responses in ACC and PrL were significantly elevated in the experimental group relative to the control group (ACC: β = 3.13, ***p < 0.005; PrL: β = 2.43, ***p < 0.005). D Representative PFC Layer 5 pyramidal neuron location within slice. Representative recordings of neuronal firing after psilocin (10 µM) showing increase (E), decrease (F), and no change (G). H Graphical depiction of differential neuronal responses to psilocin (10 µM). (I) Averaged psilocin effects on firing in L5P (mean ± SEM, n = 19). J Membrane characteristics of layer V pyramidal neurons (mean ± SEM).

Fig. 2. Psilocin effects on excitability and synaptic transmission in 5-HT2A neurons in medial prefrontal cortex.

A Visualization of tamoxifen-induced expression (red) and 5-HT_2AR (green) in the PFC of 5-HT_2AR-eGFP-CreERT2xAi9 model. B Representative 5-HT_2A neuron and its location within PFC slice. C Representative recording of 5-HT_2A neuron firing after psilocin (10 µM). D Averaged psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 13 cells/N = 10 mice; paired t-test **p < 0.01). E Normalized psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 13 cells/N = 10 mice. One sample t-test ***p < 0.001). F Representative recordings of 5-HT_2A neuron firing before and after psilocin (10 µM) in response to 400 ms injected currents of 20, 60 and 100 pA with averaged psilocin effect on number of action potentials evoked (mean ± SEM, n = 10–25 cells/N = 12 mice; paired t-test *p < 0.05, **p < 0.01, ***p < 0.001). G Averaged psilocin effect on threshold to fire (mean ± SEM, n = 25 cells/N = 12 mice; paired t-test *p < 0.05). H Averaged psilocin effect on rheobase (mean ± SEM, n = 25 cells/N = 12 mice; paired t-test ***p < 0.001). I Representative recording of 5-HT_2A neuron spontaneous excitatory post synaptic currents (sEPSCs) after psilocin (10 µM). J Averaged psilocin effect on sEPSC frequency (mean ± SEM, n = 12 cells/N = 8 mice). K Normalized psilocin effect on sEPSC frequency (mean ± SEM, n = 12 cells/N = 8 mice). L Average of psilocin effect on sEPSC amplitude (mean ± SEM, n = 12 cells/N = 8 mice). M Normalized psilocin effect on sEPSC amplitude (mean ± SEM, n = 12 cells/N = 8 mice).

Fig. 3. 5-HT2A-selective agonist NBOH-2C-CN effects on excitability and synaptic transmission in 5-HT2A neurons.

A Gq dissociation concentration-response curves at mouse 5-HT_2AR for psychedelic drugs psilocin and NBOH-2C-CN. B Representative recording of 5-HT_2A neuron firing after NBOH-2C-CN (200 nM). C Averaged NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 8 cells/N = 4 mice; paired t-test *p < 0.05). D Normalized NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 8 cells/N = 4 mice; one sample t-test **p < 0.01). E Representative recordings of 5-HT_2A neuron firing before and after NBOH-2C-CN (200 nM) in response to 400 ms injected currents of 20 pA, 60 pA, and 100 pA with averaged NBOH-2C-CN effect on number of action potentials evoked (mean ± SEM, n = 10–12 cells/N = 8 mice). F Averaged NBOH-2C-CN effect on threshold to fire (mean ± SEM, n = 11 cells/N = 7 mice; paired t-test *p < 0.05). G Averaged NBOH-2C-CN effect on rheobase (mean ± SEM, n = 11 cells/N = 7 mice; paired t-test *p < 0.05). H Representative recording of 5-HT_2A sEPSCs after NBOH-2C-CN (200 nM). I Averaged NBOH-2C-CN effect on sEPSC frequency (mean ± SEM, n = 7 cells/N = 5 mice). J Normalized NBOH-2C-CN effect on sEPSC frequency (mean ± SEM, n = 7 cells/N = 5 mice). K Averaged NBOH-2C-CN effect on sEPSC amplitude (mean ± SEM, n = 7 cells/N = 5 mice). L Normalized NBOH-2C-CN effect on sEPSC amplitude (mean ± SEM, n = 7 cells/N = 5 mice).

Fig. 4. The role of 5-HT2A receptors in the effects of psilocin and NBOH-2C-CN on 5-HT2A neurons.

A Representative recording of 5-HT_2A neuron firing after M100907 (200 nM) and psilocin (10 µM). B Averaged M100907 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 6 mice). C Normalized M100907 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 6mice; one sample t-test **p < 0.01). D Averaged M100907 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 6 mice). E Normalized M100907 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 6 mice). F Representative recording of 5-HT_2A neuron firing after M100907 (200 nM) and NBOH-2C-CN (200 nM). G Averaged M100907 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cell/N = 5 mice; paired t-test *p < 0.05). H Normalized M100907 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cells/N = 5 mice; one sample t-test *p < 0.01). I Averaged M100907 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 11 cells/N=5mice). J Normalized M100907 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10/N = 5 mice).

Fig. 5. The role of 5-HT2C receptors in the effects of psilocin and NBOH-2C-CN on 5-HT2A neurons.

A Representative recording of 5-HT_2A neuron firing after RS102221 (5 µM) and psilocin (10 µM). B Averaged RS102221 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 11 cells/N = 6 mice; paired t-test **p < 0.005). C Normalized RS102221 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 11 cells/N = 6 mice; one sample t-test ****p < 0.0001). D Averaged RS102221 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 11 cells/N = 6 mice; paired t-test *p < 0.05). E Normalized RS102221 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 6 mice ; one sample t-test *p < 0.05). F Representative recording of 5-HT_2A neuron firing after RS102221 (5 µM) and NBOH-2C-CN (200 nM). G Averaged RS102221 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cells/N = 5 mice; paired t-test **p < 0.01). H Normalized RS102221 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cells/N = 5 mice; one sample t-test ***p < 0.001). I Averaged RS102221 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 5 mice; paired t-test **p < 0.01). J Normalized RS102221 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 5 mice; one sample t-test *p < 0.05).

Fig. 6. The role of Gαq signaling in the effects of psilocin and NBOH-2C-CN on 5-HT2A neurons.

A Cartoon depicting extracellular application of FR900359 drug onto recorded neurons. B Representative recording of 5-HT_2A neuron firing after FR900359 (1 µM) and psilocin (10 µM). C Averaged FR900359 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 4 mice; paired t-test **p < 0.01). D Normalized FR900359 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 4 mice; one sample t-test **p < 0.01). E Averaged FR900359 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 4 mice). F Normalized FR900359 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 10 cells/N = 4 mice). G Representative recording of 5-HT_2A neuron firing after FR900359 (1 µM) and NBOH-2C-CN (200 nM). H Averaged FR900359 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 6 cells/N = 3 mice; paired t-test **p < 0.01). I Normalized FR900359 effects on firing in 5-HT_2A neurons (mean ± SEM, n = 6 cells/N = 3 mice; one sample t-test **p < 0.01). J Averaged FR900359 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 7 cells/N = 3 mice). K Normalized FR900359 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 7 cells/N = 3 mice). L Cartoon depicting intracellular application of FR900359 drug onto recorded neurons. M Representative recording of 5-HT_2A neuron firing with FR900359 (1 µM) in internal solution and psilocin (10 µM). N Averaged FR900359 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cells/N = 5 mice; paired t-test *p < 0.05). O Normalized FR900359 + psilocin effects on firing in 5-HT_2A neurons (mean ± SEM, n = 9 cells/N = 5 mice). P Representative recording of 5-HT_2A neuron firing with FR900359 (1 µM) in internal solution and NBOH-2C-CN (200 nM). Q Averaged FR900359 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 8 cells/N = 4 mice.). R Normalized FR900359 + NBOH-2C-CN effects on firing in 5-HT_2A neurons (mean ± SEM, n = 8 cells/N = 4 mice).