Shared and Distinct Brain Regions Targeted for Immediate Early Gene Expression by Ketamine and Psilocybin

Abstract

Psilocybin is a psychedelic with therapeutic potential. While there is growing evidence that psilocybin exerts its beneficial effects through enhancing neural plasticity, the exact brain regions involved are not completely understood. Determining the impact of psilocybin on plasticity-related gene expression throughout the brain can broaden our understanding of the neural circuits involved in psychedelic-evoked neural plasticity. In this study, whole-brain serial two-photon microscopy and light sheet microscopy were employed to map the expression of the immediate early gene, c-Fos, in male and female mice. The drug-induced c-Fos expression following psilocybin administration was compared to that of subanesthetic ketamine and saline control. Psilocybin and ketamine produced acutely comparable elevations in c-Fos expression in numerous brain regions, including anterior cingulate cortex, locus coeruleus, primary visual cortex, central and basolateral amygdala, medial and lateral habenula, and claustrum. Select regions exhibited drug-preferential differences, such as dorsal raphe and insular cortex for psilocybin and the CA1 subfield of hippocampus for ketamine. To gain insights into the contributions of receptors and cell types, the c-Fos expression maps were related to brain-wide in situ hybridization data. The transcript analyses showed that the endogenous levels of Grin2a and Grin2b predict whether a cortical region is sensitive to drug-evoked neural plasticity for both ketamine and psilocybin. Collectively, the systematic mapping approach produced an unbiased list of brain regions impacted by psilocybin and ketamine. The data are a resource that highlights previously underappreciated regions for future investigations. Furthermore, the robust relationships between drug-evoked c-Fos expression and endogenous transcript distributions suggest glutamatergic receptors as a potential convergent target for how psilocybin and ketamine produce their rapid-acting and long-lasting therapeutic effects.

Keywords: activity-dependent transcription; antidepressant; c-Fos; immediate early gene; neural plasticity; psychedelics.

Figure 1.. Whole-brain mapping of drug-induced c-Fos expression.

(A) Transgenic c-Fos-GFP mice were injected with either saline, ketamine (10 mg/kg), or psilocybin (1 mg/kg) at 3.5 hours before sacrifice and collection of brains (n = 4 per condition). (B) Schematic of the serial two-photon microscope setup. (C) Left: tiled image of a coronal block-face of a brain from c-Fos-GFP mouse. Right: zoomed in view demonstrating expression of c-Fos puncta in neurons. (D) C57/BL6 mice were injected with either saline, ketamine (10 mg/kg), or psilocybin (1 mg/kg) at 2 hours before sacrifice and collection of brains (n = 4 per condition). Brains were cleared and then immunolabeled with antibody against c-Fos protein. (E) Schematic of the light sheet microscope setup. (F) Left: image of a horizontal plane of a cleared mouse brain labeled with c-Fos antibody. Right: zoomed in view demonstrating expression of c-Fos puncta in neurons of the cortex. (G) Total number of c-Fos+ cells detected in the entire brain across different drug conditions using serial two-photon microscopy. Symbol, individual animal. Box plot shows the median, 25^th and 75^th percentiles. (H) Total number of c-Fos+ cells detected in the entire brain across different drug conditions using light sheet microscopy. Symbol, individual animal. Box plot shows the median, 25^th and 75^th percentiles. (I) Top: formula to calculate c-Fos+ cell density for a region. Middle: formula to calculate the change in c-Fos+ cell density due to drug compared to saline. Bottom: formula to calculate difference in drug-evoked change in c-Fos+ cell density between the two imaging modalities. S2P, serial two-photon microscopy. LS, light sheet microscopy. (J) Difference in psilocybin-evoked change in c-Fos+ cell density between the two imaging modalities, plotted by brain region. Region indices are listed in Supporting Table 1. Blue lines, threshold for exclusion. Dashed line, zero percent difference. (K) Difference in psilocybin-evoked change in c-Fos+ cell density between the two imaging modalities, plotted as a function of the volume of brain region as assessed by serial two-photon microscopy. Dashed line, zero percent difference.

Figure 2.. Effects of psilocybin and ketamine on regional c-Fos expression.

Drug-evoked percent change in c-Fos+ cell density for psilocybin (red) and ketamine (blue). Circle, mean. Line, bootstrapped 95% confidence intervals assuming normal distribution.

Figure 3.. Common and distinct regions targeted for c-Fos expression by psilocybin and ketamine.

(A) Scatter plot of mean drug-evoked percent change in c-Fos+ density for psilocybin (x-axis) versus ketamine (y-axis). For list of abbreviations, see Supporting Table 3. (B) Example images from light sheet microscopy for select cortical and subcortical brain areas. Due to background intensity, for visualization purposes, we performed gamma correction on the magnified images, using the same adjustment for each row of images.

Figure 4.. Potential receptors and cell types contributing to drug-evoked c-Fos expression.

(A) Schematic illustrating the analysis procedure. The mRNA transcript levels of a particular gene (e.g., Htr2a) (left, interpolated from sagittal sections to yield 3D rendering using Brainrender) was compared with drug-evoked percent change in c-Fos+ density (middle), on a region-by-region basis to calculate a correlation coefficient (right). (B) Correlation coefficients computed for psilocybin condition using regions across the entire brain. Colored lines, correlation coefficients for select serotonin and dopamine receptor genes, with percentile indicated within the parenthesis. Grey line, histogram of correlation coefficients for all 19,413 genes in the mouse genome. (C) Similar to (B) for ketamine. (D) Similar to (B) for glutamate receptors. (E) Similar to (D) for ketamine. (F - I) Similar to (B - E) except using only regions within the cortex. (J, K) Similar to (F, G) for major cell-type marker genes.