Snapshot IP₁ Detection Following 5-HT₂A Receptor Stimulation in the Mouse Brain

The distinct subjective effects that define psychedelics such as lysergic acid diethylamide (LSD), psilocybin, or 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) as drug class are causally linked to the activation of the serotonin 2A receptor (5-HT_2AR). However, some aspects of 5-HT_2AR pharmacology remain elusive, such as what molecular drivers differentiate psychedelic from nonpsychedelic 5-HT_2AR agonists. We developed an ex vivo platform to obtain snapshots of drug-mediated 5-HT_2AR engagement of the canonical G_q/11 pathway in native tissue. This nonradioactive methodology captures the pharmacokinetic and pharmacodynamic events leading up to changes in inositol monophosphate (IP₁) in the mouse brain. The specificity of this method was assessed in homogenates from the frontal cortex in DOI-treated wild-type and 5-HT_2AR knockout (5-HT_2AR-KO) animals compared to other brain regions, namely, striatum and cerebellum. The effect of DOI on mouse frontal cortex IP₁ was time-bound, dose-dependent, and was correlated to head twitch response counts. We observed that IP₁ levels in frontal cortex homogenates from mice treated with LSD and lisuride varying in magnitude, consistent with LSD's 5-HT_2AR agonism and psychedelic nature and lisuride's lack thereof. 3,4-Methylenedioxymethamphetamine (MDMA) evoked an increase in the IP₁ signal in the frontal cortex that was not matched by the serotonin precursor 5-hydroxytryptophan or the serotonin reuptake inhibitor fluoxetine. We attribute the differences in the readout primarily to the indirect stimulation of 5-HT_2AR by MDMA via the release of serotonin from its presynaptic terminals. This methodology enables one to capture a snapshot of IP₁ turnover in the mouse brain that can provide mechanistic insights into the study of psychedelics and G_q/11-coupled receptors.