Introduction

Functional connectivity (FC) estimated with resting-state fMRI offers a unique window into the brain for characterizing the effects of pharmacological interventions. Many pharmacological agents used to treat psychiatric disorders, such as antidepressants, affect synaptic activity at or binding to receptors related to neurotransmitter systems projecting from midbrain nuclei such as the serotonergic system in the dorsal raphe nucleus (DRN) and dopaminergic systems in the ventral tegmental area (VTA). This is the case for the hallucinogenic drug psilocybin, a 5-HT2A agonist with potential antidepressant effects. Yet, assessing the contribution of individual neurotransmitter systems to resting-state networks (RSNs) remains challenging. RSNs generally do not include midbrain nuclei, and placing seeds on these regions fails to capture functionally coupled distal regions. In this study, we propose a novel approach for assessing the functional connectivity changes induced by psilocybin injection in mice by assessing contributions of individual neurotransmitter systems to resting-state signal using gene expression and projection fields within the dual-regression framework.

Method

Mice were anesthetized using medetomidine / isoflurane (Grandjean et al. 2014). Psilocybin (1mg/kg n=13 or 2mg/kg n=12) or vehicle (n=15) was injected i.v. 20 min before acquisition of mutli-echo EPI scans (800 volumes, TR=1500ms, TE=[11,17,24]ms) at 9.4T with a 2x2 cryoprobe. Images were processed with meica.py script (Kundu et al. 2012). Standard analysis included dual-regression (Filippini et al. 2009) using 17 reference RSNs identified previously (Zerbi et al. 2015). Structural connectivity maps for midbrain nuclei derived from anterograde viral tracer injection in the DRN and VTA, as well as gene expression maps for serotonin 5HT2a and dopamine D1 receptors, were obtained from the Allen Brain Institute database (http://mouse.brain-map.org/) and transformed to reference space. Injection sites and white matter fibers were masked from tracer injection maps. Tracer and gene expression maps were thresholded to the 90th percentile and used as spatial reference maps in dual-regression analysis. Statistical analysis comparing psilocybin 1mg/kg or 2mg/kg to vehicle, and combined psilocybin effect (1 and 2 mg/kg) to vehicle was carried using nonparametric permutation testing with threshold-free cluster enhancement inference with Bonferroni correction.

Results

Dual-regression analysis identified robust psilocybin effects relative to vehicle in the ventral striatum network, indicating decreased FC in psilocybin treated animals (Figure 1). The highest significant cluster from this analysis was used as a reference in seed-based analysis (Figure 2): one-sample t-tests carried out on the seed-based maps revealed FC relative to a seed in the ventral striatum that were confined to the striatum in the vehicle group, whereas FC extended to prefrontal cortex and midbrain in the two psilocybin groups. The seed coordinate was converted to Allen atlas space and used to determine gene expression clusters using the Anatomic Gene Expression Atlas (AGEA). The AGEA cluster revealed spatial distribution comparable to the seed-based map. In particular, both dopaminergic D1 and D2 receptors were expressed in this cluster (1.75 and 2.06 fold change in expression in the AGEA cluster relative to the remaining voxels, respectively). To further investigate the role of dopamine and serotonin on the resting-state signal, in situ hybridization maps for D1 and 5HT2a receptor genes were used as spatial reference maps in dual-regression analysis (Figure 3). With respect to D1 gene expressing regions, psilocybin decreased FC within the striatum, prefrontal cortex, hippocampus and midbrain. With respect to 5HT2a gene expression map, psilocybin increased FC in the striatum. Comparable results were obtained when the viral tracer maps from the VTA and DRN were used as spatial references in the dual regression analysis (Figure 4).

Discussion

Gene expression and structural connectivity maps related to the dopaminergic and serotonergic system were used as spatial reference maps in dual-regression analysis in order to discriminate two opposing effects of psilocybin on FC, a) decreased in FC within the ventral striatum related to dopaminergic activity and b) increased striatum coupling to 5HT2a receptor expressing regions and projection field from the DRN. Combining FC analysis with online databases offers a new perspective to gain insights into whole-brain mechanisms of pharmacological agents and to capture different dynamics in neurotransmitter systems.

Acknowledgements

No acknowledgement found.

References

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