Psychedelics Produce Complex And Heterogeneous Transcriptional Responses In Diverse Cortical Cell Types In The Brain Of Rats As Determined By New Flow Cytometric Methods Allowing For Sorting Of Distinct Cell And Neuronal Populations From Whole Brain
Authors: Martin David , Porretta Connie , Nichols Charles D 
Published: November 2023

Psychedelics, also known as classic serotonin hallucinogens, are currently being investigated by several groups for their potential as therapeutic agents for a variety of psychiatric indications. Although the neural circuitry underlying the effects of psychedelics is being established by others, the role of specific cell types and populations within the brain that respond to these agents remain largely uninvestigated. We conducted a series of experiments examining the transcriptional effects of the psychedelic and selective 5‐HT2 receptor agonist, (R)‐DOI on a variety of cell types in the brain. Pre‐existing flow cytometry methods were initially used to sort and to examine the transcriptional effects of (R)‐DOI on activated and non‐activated neurons, as well as on specific non‐neuronal populations, including astrocytes, in the medial prefronal cortex (mPFC) and somatosensory cortex (SSC). QRTPCR analysis of mRNA extracted from sorted cortical cells indicated that a variety of cell types produce cfos and other immediate early genes (IEGs) in response to (R)‐DOI, including parvalbumin and somatostatin expressing interneurons, HTR2A expressing neurons, and astrocytes. The induction of cFos protein in a subset of somatostatin‐expressing and 5‐HT2A receptor‐expressing neurons was confirmed using immunohistochemistry. Additionally, a pronounced redistribution of 5‐HT2A receptors to the peri‐nuclear region of neurons in cortical neurons was observed following (R)‐DOI administration. To separate neurons by cell type, we developed a new methodology using flow cytometry. This involved developing methods that isolated cells with cytoplasm and extracellular membranes intact, rather than only nuclei, from whole brain. Pure populations of somatostatin and parvalbumin expressing interneurons were each isolated from whole cortex in the SSC and mPFC. Further separation of these particular cell types into activated and non‐activated populations using antibodies directed against cFos allowed us to analyze IEG induction within specific subpopulations of cortical cells. QRTPCR analyses of sorted neurons from distinct cell types revealed that different IEGs (such as egr2) are not induced uniformly in all activated (cFos+) cells by (R)‐DOI. Our results highlight the diversity of cells that are recruited by psychedelic drugs (e.g. excitatory glutamatergic neurons, GABA interneurons, astrocytes), and the differential transcriptional responses of these cells. Importantly, we demonstrate the utility of our new flow cytometric methods for investigating cell‐type‐specific transcriptional and translational effects of drug administration in the brain. Significantly, our new technique can be generally applied to study the effects of any manipulation (ie., drug‐induced, genetic, or environmental) within specific neural populations of whole brain.

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LSU Health Science Center. Heffter Research Institute.