Psychedelic-like Properties of Quipazine and Its Structural Analogues in Mice

Abstract

Known classic psychedelic serotonin 2A receptor (5-HT_2AR) agonists retain a tryptamine or phenethylamine at their structural core. However, activation of the 5-HT_2AR can be elicited by drugs lacking these fundamental scaffolds. Such is the case of the N-substituted piperazine quipazine. Here, we show that quipazine bound to and activated 5-HT_2AR as measured by [³H]ketanserin binding displacement, Ca²⁺ mobilization, and accumulation of the canonical G_q/11 signaling pathway mediator inositol monophosphate (IP₁) in vitro and in vivo. Additionally, quipazine induced via 5-HT_2AR an expression pattern of immediate early genes (IEG) in the mouse somatosensory cortex consistent with that of classic psychedelics. In the mouse head-twitch response (HTR) model of psychedelic-like action, quipazine produced a lasting effect with high maximal responses during the peak effect that were successfully blocked by the 5-HT_2AR antagonist M100907 and absent in 5-HT_2AR knockout (KO) mice. The acute effect of quipazine on HTR appeared to be unaffected by serotonin depletion and was independent from 5-HT₃R activation. Interestingly, some of these features were shared by its deaza bioisostere 2-NP, but not by other closely related piperazine congeners, suggesting that quipazine might represent a distinct cluster within the family of psychoactive piperazines. Together, our results add to the mounting evidence that quipazine’s profile matches that of classic psychedelic 5-HT_2AR agonists at cellular signaling and behavioral pharmacology levels.

Graphical Abstract

INTRODUCTION

The chemical structures of classic psychedelics, such as lysergic acid diethylamide (LSD), psilocybin, and mescaline, contain either a tryptamine or phenethylamine scaffold with different substituents.^1,2 Modifications to these core structures can involve minor short-chained substitutions, like in the case of N,N-dimethyltryptamine (DMT) or 2,5-dimethoxy-4-methylamphetamine (DOM) series, but can also be rather extensive to the point of configuring separate subfamilies like in the case of ergolines or NBOMEs. These core structures are present among a wide variety of bioamines and drugs that are not necessarily psychedelic.^3,4 However, within the pharmacological class of psychedelic serotonin (or 5-hydroxytryptamine) 2A receptor (5-HT_2AR) agonists, efavirenz—a retroviral drug chemically unrelated to classic psychedelics—is the only known representative lacking a phenethylamine or tryptamine core embedded on its structure.⁵ Quipazine may belong to such group of psychedelics with unconventional scaffolds, but decades since its discovery it still remains an open case of study as the available data on the potential psychedelic effects in humans of this N-substituted piperazine and well-known 5-HT_2AR agonist are both scant and fragmentary, when not contradictory.^6–8

Although devoid of the canonical phenethylamine or tryptamine skeleton of classic psychedelic 5-HT_2AR agonists, quipazine does retain the essential features of these scaffolds as it pertains to molecular recognition of 5-HT_2AR;^9,10 namely, a protonatable nitrogen attached by a short aliphatic chain to an aromatic ring (Figure 1). While these basic pharmacophoric traits are common to a large number of psychoactive drugs and endogenous chemicals, quipazine displays—in vitro and in animal models—an activity profile comparable to classic psychedelics.^11–13 For this reason, we sought to also explore the in vitro pharmacology of quipazine and a small series of congeners, evaluate their effect in predictor models of psychedelic activity, and determine the differential contribution of receptors other than 5-HT_2AR to the observed behavioral output. In light of the renewed interest of the scientific community on psychedelic research fueled by its therapeutic potential as fast-acting and long-lasting antidepressants,¹⁴ unveiling novel structure–activity relationships for 5-HT_2AR-binding ligands out of the classic phenethylamine and tryptamine core structures can aid to better understand the intertwining of structural requirements, pharmacodynamics, and potential manifestation of clinically relevant effects.

Figure 1.

Structures of quipazine, the position isomer isoquipazine, and the corresponding deaza isosteres 2-NP and 1-NP. The piperazine positions N_1′ and N_4′ are indicated in the structure of quipazine.

RESULTS

Psychedelic-like Effect of Quipazine on Mouse Head-Twitch Response.

First, we evaluated the effect of different doses of quipazine on head-twitch response (HTR) as a behavior-based model of psychedelic activity.^15,16 While the effect of quipazine on HTR in rats has been known for decades,^11,17–19 to the best of our knowledge, the extent and duration of its effect in mice have not been reported. Quipazine produced a robust and long-lasting increase on HTR counts that peaked during the first 30 min post injection and lasted for over 90 min at the highest doses evaluated (Figure 2A) (two-way repeated measures ANOVA treatment effect: F[4,24] = 56.12, P < 0.001). We then collapsed HTR counts during the first 30 min for each dose (Figure 2B) (one-way ANOVA F[4,24] = 35.18, P < 0.001), and calculated the ED₅₀ for quipazine (Figure 2B). On a separate experiment, we recorded HTR events for a longer period of time (180 min, data not shown) to capture the tail of the drug effect on its entirety and determine the half-life of the effect of quipazine (5 mg/kg) on HTR (t_1/2 = 31.48 ± 1.62 min).

Figure 2.

(A) Time-course showing HTR counts in 15 min blocks corresponding to different doses of quipazine (n = 5–6). Black arrow shows the administration time point (t = 0). (B) HTR counts corresponding to the first 30 min after injection of quipazine. Two-way (A) and one-way (B) ANOVA Bonferroni’s post hoc test vs vehicle. *P < 0.05, **P < 0.01, ***P < 0.001.

To evaluate the involvement of 5-HT_2AR on the HTR induced by quipazine, mice were pretreated with the selective 5-HT_2AR antagonist M100907 (also known as volinanserin) prior to the administration of quipazine (Figure 3A). As expected, pretreatment with the antagonist completely blocked the effect of quipazine on HTR over the time course (two-way repeated measures ANOVA treatment effect: F[2,13] = 340.0, P < 0.001). This observation was further corroborated by the comparison of HTR during the first 30 min post drug(s) or vehicle administration (Figure 3A, right panel, one-way ANOVA: F[2,13] = 240.9, P < 0.001). It had previously been shown that pretreatment with the 5-HT_2AR/5-HT_2CR antagonist ketanserin partially blocked quipazine-elicited HTR.²⁰ In our case, full blockade of an equivalent dose of quipazine was attained with a 40-fold lower dose of antagonist. However, it should be noted that M100907 is a particularly efficacious 5-HT_2AR antagonist at blocking HTR induced by psychedelics in rodents.^21,22

Figure 3.

Quipazine induced HTR time course (A–C, left panel) and sum of totals during the first 30 min (A–C, right panels) after different pretreatments. Quipazine was administered at t = 0 min (as indicated by the black arrow). (A) Effect of pretreatment with M100907 (0.1 mg/kg) or vehicle 5 min prior to the administration of quipazine (5 mg/kg) or vehicle (n = 3–10). (B) Effect of treatment with PCPA (100 mg/kg) or vehicle daily during the 4 previous days on quipazine (5 mg/kg) induced HTR on day 5 (n = 6). (C) Effect of pretreatment with ondansetron (1 mg/kg) or vehicle 5 min prior to the administration of quipazine (5 mg/kg, n = 6). (D) Feces count during the first 30 min after quipazine (5 mg/kg), ondansetron (1 mg/kg), or vehicle administration (n = 9). Two-way (A, left and D) and one-way ANOVA (A, right) Bonferroni’s post hoc. Studentťs t-test (B and C, right). ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Quipazine binds and blocks the serotonin transporter.^23,24 Serotonin and its precursor 5-hydroxytryptophan (5-HTP) when administered directly at high doses have been shown to induce HTR in rodents.^19,25 To exclude the possibility that the effect of quipazine on HTR could result from an acute increase of serotonin levels, we induced depletion of the neurotransmitter by administering para-chlorophenylalanine (PCPA) for 4 days.^26,27 Approximately 24 h after the last dose of PCPA, mice were then treated with quipazine and their HTR monitored for 90 min (Figure 3B). No effect of the PCPA treatment on quipazine-induced HTR was observed (two-way repeated measures ANOVA treatment effect F[1,10] = 3.070, P > 0.05; right panel, t₁₀ = 1.636, P > 0.05), indicating that serotonin depletion induced by PCPA did not reduce the ability of quipazine to elicit HTR. This further verifies that the effect of quipazine on HTR is independent of its ability to acutely increase synaptic levels of serotonin.

Considering the high affinity of quipazine for the 5-HT₃R,²⁸ and the purported role of this receptor in the gastrointestinal (GI) discomfort experienced by human volunteers that received quipazine,⁶ we next evaluated the contribution of 5-HT₃R to the effect of quipazine on HTR following pretreatment with the 5-HT₃R antagonist ondansetron prior to the administration of quipazine (Figure 3C). The HTR counts between vehicle and ondansetron pretreated mice were statistically comparable (two-way repeated measures ANOVA treatment effect F[1,10] = 0.663, P > 0.05; right panel, t₁₀ = 0.219, P > 0.05), suggesting that the action of quipazine on HTR is independent of its 5-HT₃R agonism. Additionally, we counted the number of feces following treatment with quipazine with and without ondansetron to assess the effect of each treatment on GI motility (Figure 3D). Two-way ANOVA analysis showed an effect for quipazine and a trend for ondansetron on the count of feces (two-way ANOVA, quipazine effect: F[1,32] = 10.82, P < 0.01; ondansetron effect F[1,32] = 2.14, P = 0.057). Post hoc analysis revealed that ondansetron successfully reduced the effect of quipazine on feces count increase during the 30 min following cotreatment (Figure 3D).

Quipazine Effects on the Mouse Frontal Cortex Consistent with That of Classic Psychedelics.

Mounting evidence demonstrates the involvement of 5-HT_2AR activation on the mechanism of action of psychedelics, a receptor that engages canonical G_q/11 proteins as its primary signaling mechanism.¹⁶ To further demonstrate the agonist action of quipazine on 5-HT_2AR, we aimed to measure the relative accumulation of IP₁, a downstream effector of the G_q/11 signaling pathway, in vivo. The procedure required the adaptation of a homogeneous time-resolved fluorescence (HTRF) protocol for the ratiometric quantification of IP₁ and the pretreatment of the animals with LiCl to prevent the degradation of the metabolite on the brain tissue (Figure 4A).^29,30 Compared to vehicle, quipazine-treated animals showed greater accumulation of IP₁ in the frontal lobe of the cortex (expressed as a IP₁ signal fold-change in the cortex relative to the cerebellum for each animal); this effect was successfully blocked by the same dose of the 5-HT_2AR antagonist M100907 that blocked quipazine-induced HTR (Figure 4A) (one-way ANOVA F[2,6], P < 0.01).

Figure 4.

(A) In vivo IP1 accumulation by quipazine (5 mg/kg) and in the presence of the antagonist M100907 (0.1 mg/kg). Upper panel represents the timeline of the experiment: administration of LiCl (200 mg/kg, all animals), administration of the corresponding treatments or vehicle (Tx, n = 3 per group), and harvesting of tissue. (B) IEGs and housekeeping gene expression in the somatosensory cortices of 129S6/SvEv (A) wild-type (n = 12) and (B) 5-HT_2AR-KO (n = 5) mice 60 min after the administration of quipazine (5 mg/kg) or vehicle. One-way ANOVA Bonferroni’s post hoc vs vehicle (A). Multiple t-test with Bonferroni’s correction test for multiple comparisons (B). *P < 0.05, **P < 0.01, ***P < 0.001.

Classic psychedelics induce, via 5-HT_2AR, a characteristic pattern of immediate early genes (IEG) expression in the mouse somatosensory cortex not paralleled by nonpsychedelic 5-HT_2AR agonists.^16,31 Wild-type male mice treated with quipazine exhibited changes in the cortical expression of c-fos, egr-1, and egr-2 (Figure 4B) consistent with those of other phenethylamine and tryptamine psychedelic drugs [wild-type (per gene vehicle vs quipazine multiple t-test): c-fos (t₁₅₄ = 11.34, P < 0.001), egr-1 (t₁₅₄ = 3.15, P = 0.01), egr-2 (t₁₅₄ = 9.23, P < 0.001), GAPDH (t₁₅₄ = 0.043, P > 0.05)]. Importantly, such changes were not observed in the somatosensory cortex of 5-HT_2AR-KO littermates treated with quipazine (Figure 4C), suggesting that 5-HT_2AR was ultimately responsible for the fingerprint of quipazine on c-fos, egr-1 and egr-2 expression [5-HT_2AR-KO (per gene vehicle vs quipazine multiple t-test): c-fos (t₅₄ = 0.152, P > 0.05), egr-1 (t₅₄ = 0.157, P > 0.05), egr-2 (t₅₄ = 0.328, P > 0.05), GAPDH (t₅₄ = 0.623, P > 0.05)]. Additional housekeeping genes were evaluated for both genotypes as controls: wild-type β-actin (t₁₅₄ = 0.73, P > 0.05), rps3 (t₁₅₄ = 0.135, P > 0.05), Mapkapk5 (t₁₅₄ = 0.098, P > 0.05); 5-HT_2A-KO β-actin (t₅₄ = 0.188, P > 0.05), rps3 (t₅₄ = 0.101, P > 0.05), Mapkapk5 (t₅₄ = 0.130, P > 0.05).

[³H]Ketanserin Displacement by Quipazine Is Comparable across Species.

In light of the consistency of quipazine’s psychedelic-like effects on rodent-models and its elusive effect in humans, we aimed to determine whether species-related 5-HT_2AR molecular idiosyncrasies could potentially drive divergences in drug-mediated interactions between mouse and human 5-HT_2ARs. For this purpose, we sought to perform [³H]ketanserin displacement by quipazine in the mouse frontal cortex and human postmortem prefrontal cortex as a surrogate of binding affinity for the 5-HT_2AR. Our data showed comparable magnitude of binding affinities across both species (Figure 5A) (post-mortem human prefrontal cortex pK_i = 5.290 ± 0.16, mouse frontal cortex pK_i = 5.601 ± 0.16, F-test F[12,12] = 1.474, P > 0.05).

Figure 5.

(A) [³H]Ketanserin binding displacement in membrane preparations from human post-mortem brain (n = 3) and mouse frontal cortices (n = 4). (B) Quipazine (hot pink carbon atoms) bound in the in the orthosteric binding site of the active (PDB: 6WHA) conformation of the 5-HT_2AR (green carbon atoms). Ligand and residues (shown within 5 Å from ligand) are rendered as capped sticks. Salt bridge and H-bond interactions (i.e., D155^3.32–N_4′, S159^3.36–N) are indicated by yellow dashed lines. (C) Schematic representation of the residues in the binding site relative to quipazine. F-test (A).

To gain insight on how quipazine (Figure 1) interacts with 5-HT_2ARs, we generated a model of the h5-HT_2AR-based on the recent X-ray crystal structures of the active state receptor.³² The highest scoring solution of quipazine docked into the 5-HT_2AR revealed that the aromatic moiety of quipazine is situated in a hydrophobic pocket comprised of residues I152^3.29, D155^3.32 V156^3.33, S159^3.36, V366^7.39, G369^7.42, and Y370^7.43 (superscript indicates the Ballesteros–Weinstein numbering system³³) (Figure 5B and C). As expected, quipazine formed a salt bridge interaction between the N_4′ atom and the conserved D155^3.32 (2.6 Å), an interaction shown to be essential to the binding of most 5-HT_2AR ligands.³⁴ GOLD and HINT analysis also revealed a hydrogen bond between the quinoline N atom and S159^3.36. Importantly, this hydrogen bond-forming S159^3.36 has been shown to be essential for the activity of several 5-HT_2AR ligands, including agonists and antagonists.^35–37 Modeling data suggested possible interactions between S159^3.36 risperidone and arylpiperazines,^9,35,36 and mutagenesis studies showed that point mutations of S159 resulted in the reduced activity of 5-HT and N,N-dimethyl-5-HT(bufotenine) at 5-HT_2ARs.³⁷ These molecular modeling and computational ligand docking studies indicate that quipazine interacts with the h5-HT_2AR in a manner that is comparable to what has been previously reported for other 5-HT_2AR ligands.

Although the primary sequence of the 5-HT_2AR is highly conserved between human and rodent species,³⁸ we aimed to evaluate whether the residues involved in the putative binding pose of quipazine was evolutionarily conserved. For this purpose, we performed a sequence alignment of the human, mouse, and rat 5-HT_2AR that showed that the amino acids in the sequence of the 5-HT_2AR that partake in the highest scoring pose of quipazine bound to the h5-HT_2AR are preserved in the two main rodent species used to investigate the effects of quipazine (Supplementary Figure 1).

Quipazine and Congeners Exhibit Different Profiles on Mouse HTR.

The N-substituted piperazine scaffold is common to many pharmacologically active drugs with various activity profiles.³⁹ To gain additional insight on the pharmacology of quipazine, we screened a small set of the commercially available positional isomer and deaza analogues of quipazine (Figure 1). Other than quipazine, only 2-NP was able to produce an increase in HTR that differed statistically from vehicle, though to a much lesser extent than the same dose of quipazine (Figure 6A) (one-way ANOVA: F[4,31] = 89.68, P < 0.001). Neither isoquipazine nor 1-NP produced changes in HTR counts as compared to vehicle-treated animals. The increase in HTR counts by 2-NP is consistent with a previous report on DOM stimulus-generalization studies. In this model, 2-NP was able to produce full generalization for the DOM stimulus in rats trained to distinguish the drug from vehicle whereas 1-NP did not.⁴⁰ Additionally, and further supporting the involvement of 5-HT_2AR on this behavior (see Figure 3A, above), HTR did not differ between vehicle and quipazine or 2-NP treated 5-HT_2AR-KO mice (Figure 6B) (one-way ANOVA: F[2,20] = 1.196, P > 0.05).

Figure 6.

(A) HTR-induced by different structural analogues of quipazine shown in Figure 1 (5 mg/kg all) during the first 30 min postinjection (n = 6–12) in wild-type and (B) 5-HT_2A-KO mice (n = 5–12). One-way ANOVA Bonferroni’s post hoc vs vehicle (A, B). *P < 0.05, ***P < 0.001.

We sought to explore further the effect of the two deaza analogues 1-NP and 2-NP in the HTR model. HTR counts elicited by 2-NP were dose-dependent (Figure 7A) (one-way ANOVA F[5,28] = 5.897, P < 0.001) and time-dependent (Figure 7B) (two-way ANOVA repeated measures time effect: F[5,80] = 9.787, P < 0.001; treatment effect: F[1,16] = 37.11, P < 0.001). Even if the maximal effect was modest, the ED₅₀ value of 2-NP was approximately 1 order of magnitude superior to that of quipazine (ED₅₀ = 0.42 ± 0.2 mg/kg). On the other hand, 1-NP produced an apparent suppression of HTR counts at all tested doses (Figure 7A) (one-way ANOVA F[4,25] = 5.382, P = 0.01). In the case of 1-NP there was time-dependent trend (Figure 7B) (two-way ANOVA repeated measures time effect: F[1.97,31.47] = 2.78, P = 0.08; treatment effect: F[1,16] = 18.91, P < 0.001). The effect of 1-NP on locomotor activity as a potential confounding factor HTR was not evaluated, however, based on previous reports motor suppression appears unlikely.¹⁹

Figure 7.

(A) HTR dose–response for 2-NP and 1-NP (n = 6). (B) Time-course of the effect of 2-NP and 1-NP on HTR (n = 6). (C) Effect of pretreatment with 5-HT_1AR antagonist WAY100635 (n = 3–6) on HTR induced by 2-NP or suppressed by 1-NP. (D) Effect of pretreatment with 5-HT_2CR antagonist SB242084 on HTR induced by 2-NP or suppressed by 1-NP (n = 6–12). One-way (A and C) and two-way ANOVA (B and D), Bonferroni’s post hoc vs vehicle (A and B), or vehicle pretreatment (C and D). *P < 0.05, **P < 0.01, ***P < 0.001.

The prominent role of 5-HT_2AR activation in the mechanism of action of psychedelics is well established.^2,16,41 However, classic psychedelics target a wide array of receptors in the serotonergic and other monoaminergic systems.⁴² While the interaction between these different receptors can be synergistic, other receptors commonly activated by classic psychedelics such as psilocybin and LSD such as 5-HT_1AR and 5-HT_2CR have been proposed to attenuate both the subjective effects in humans and HTR in rodents.^43–45 Both 1-NP and 2-NP have been shown to displace [³H]-OH-DPAT and [³H]ketanserin binding in rat brain membrane preparations.⁴⁰ Considering this, we next aimed to evaluate whether pretreatment with the 5-HT_1AR antagonist WAY100635 (Figure 7C) or the 5-HT_2CR antagonist SB-242084 (Figure 7D) modulated the effect of 1-NP or 2-NP on HTR counts. Pretreatment with SB-242084 or WAY100635 did not significantly affect HTR in mice administered with 1-NP or 2-NP (Figure 7C, two-way ANOVA WAY100 pretreatment effect: F[2,23] = 0.675, P > 0.05; Figure 7D, two-way ANOVA SB24 pretreatment effect: F[1,25] = 0.432, P > 0.05).

Quipazine and Congeners Exhibit Matching Activity Profiles on 5-HT_2AR and HTR.

Next, we sought to determine the affinity of the compounds against 5-HT_2AR by displacement of [³H]ketanserin binding in membrane preparations from HEK293 cells stably expressing the human receptor (Figure 8A) (1-NP pK_i = 7.00 ± 0.11; 2-NP pK_i = 6.14 ± 0.11; isoquipazine pK_i = 5.42 ± 0.16; quipazine pK_i = 4.7 ± 0.14). The relative affinities for the human 5-HT_2AR is consistent with those previously reported in rat brain membrane preparations in which 1-NP and 2-NP showed greater affinity for [³H]ketanserin binding sites than quipazine.⁴⁰

Figure 8.

(A) [³H]ketanserin binding displacement (n = 4–6; 2–3 independent experiments performed in duplicate) on membrane preparation from HEK293 cells stably expressing 5-HT_2AR. (B) In vitro IP₁ accumulation screening in HEK293 cells stably expressing 5-HT_2AR by different drugs (n = 2). Ca²⁺-mobilization on HEK293 cells with Fluo-4: (C) concentration-dependent Ca²⁺ mobilization for serotonin, quipazine P (n = 6–12; 3–4 independent experiments performed in duplicate or quadruplicate). (D) Antagonist concentration-dependent response of isoquipazine and 1-NP on serotonin (1 μM)-induced Ca²⁺ mobilization (n = 6–12; 3–4 independent experiments performed in duplicate or quadruplicate). (E) 5-HT_2AR antagonist M100907 blocking effect on Ca²⁺ release induced by 5-HT, quipazine, and 2-NP (n = 8–12; 2–3 independent experiments performed in duplicate or quadruplicate). (B) One-way ANOVA Bonferroni’s post hoc vs M100907 (B), M100907, ***P < 0.001. (E) Two-way ANOVA Bonferroni’s post hoc vs vehicle. *P < 0.05, ***P < 0.001.

In light of the 5-HT_2AR-agonism inferred from HTR studies (Figure 6), we sought to characterize the activity profile of its congeners in vitro in HEK293 stably expressing 5-HT_2AR. We screened the ability of the different compounds to stimulate the production of inositol monophosphate (IP₁) accumulation by HTRF as a proximal downstream effector of the G_q/11 protein pathway. Consistent with our previous findings in vivo (Figure 4A), quipazine produced a robust increase of IP₁ accumulation (Figure 8B) comparable in magnitude to that of the psychedelic 5-HT_2AR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) and slightly below the maximal effect of serotonin (100% on y-axis), followed by a more modest response by 2-NP (~50%) (Figure 8B) (one-way ANOVA F[6,7] = 86.28, P < 0.001). The reference antagonist/inverse agonist M100907 resulted in a slightly negative shift in IP₁ accumulation; in contrast, 1-NP and isoquipazine showed a residual stimulation effect (~10%) close enough to the baseline to be considered physiologically irrelevant.⁸

Stimulation of G_q/11 proteins elicits a transient increase in the concentration of intracellular Ca²⁺ from the endoplasmic reticulum, which can be detected with fluorescent calcium-sensitive dyes. Preliminary attempts with the ratiometric dye Fura-2 to measure Ca²⁺ release in HEK293 stably expressing 5-HT_2AR were unsuccessful due to the interference of some of the compounds’ emission on the fluorometric reading (data not shown). This issue was overcome with the use of the Ca²⁺-sensitive dye with red-shift excitation wavelength Fluo-4 for the characterization of the compounds’ 5-HT_2AR activity in vitro. Consistent with their initial screening on IP₁ production, the intracellular Ca²⁺ mobilization assay in HEK293 cells stably expressing 5-HT_2AR revealed that serotonin (5-HT), quipazine, and 2-NP produced a concentration-dependent increase of signal (Figure 8C) (5-HT pEC₅₀ = 7.356 ± 0.25, E_max = 107.0; 2-NP pEC₅₀ = 6.823 ± 0.68, E_max = 48.9; quipazine pEC₅₀ = 5.139 ± 0.36, E_max = 44.3), whereas isoquipazine and 1-NP blocked the Ca²⁺ signal corresponding to 5-HT 1 μM (Figure 8D) (2-NP pIC₅₀ = 6.402 ± 0.230; isoquipazine pIC₅₀ = 4.608 ± 0.19). The Ca²⁺ signal induced by the agonists was blocked by the selective 5-HT_2AR antagonist M100907 (Figure 8E) (two-way ANOVA, antagonist effect F[1,41] = 65.93, P < 0.001). The specificity of the signal output was corroborated by incubating untransfected HEK293 cells with the different compounds (data not shown).

In light of the 5-HT_2AR antagonism of isoquipazine and 1-NP, we evaluated the effect of these two piperazines on quipazine-induced HTR (Figure 9). As expected,¹⁹ pretreatment with either isoquipazine or 1-NP fully blocked quipazine-induced HTR (one-way ANOVA F[2,6] = 89.05, P < 0.001). In agreement with previous reports, these results further highlight the functional antagonism of 1-NP on 5-HT_2AR-induced HTR.¹⁹

Figure 9.

Effect of pretreatment with isoquipazine and 1-NP on quipazine induced HTR (n = 3). One-way ANOVA Bonferroni’s post hoc vs vehicle pretreatment. ***P < 0.001.

DISCUSSION

The coinage of the term “classical hallucinogens,” herein referred to as classic psychedelics, revolved around their characteristic 5-HT_2AR agonism and the presence of a tryptamine or phenethylamine embedded in their structure.^1,2 To this day, almost no exceptions exist to this structural classification of drugs portending psychedelic effects in humans.⁴⁶ Even in more ambiguous cases, a phenethylamine core is concealed in the structure; such is the case of fenfluramine or lorcaserin in which the psychedelic nature of the drug only becomes apparent at high doses.^47,48 However, the tryptamine or phenethylamine scaffold as a quintessential psychedelic structural requirement might be artifactual. Popular classical psychedelics such as LSD, psilocybin, DMT, and synthetic phenethylamines likely satisfy the demand of psychedelics for recreational use to an extent that leaves little to no incentive to clandestine chemists to venture into the exploration of structural alternatives. On the opposite side of the spectrum, the pharmaceutical industry selection bias would systematically flag an unaffordable risk for potential adverse events to any compound remotely portending psychedelic activity.² Thus, it is plausible that the apparent structural exclusivity of classic psychedelic chemical families remains unchallenged by a historical lack of interest and selection bias rather than some fundamental structural privilege. In other words, it should be unsurprising to encounter psychedelics among 5-HT_2AR agonists, even if they belong in unconventional chemical spaces. Along with efavirenz,⁵ as discussed below, quipazine might be a good example of a psychedelic structural outlier.

The inclusion of quipazine as a classic psychedelic structurally unrelated to classic psychedelics bears reasonable doubt based on the very limited available data on its psychoactive properties in humans.⁶ On the contrary, the observations from animal models are compelling. In monkeys, quipazine induced a symptomatology consistent with LSD-like effects as well as emesis.⁶ Similarly, in rodents—which are devoid of the vomiting reflex—quipazine has been shown to produce stimulus generalization in rats trained with LSD⁴⁹ or DOM.¹³ In addition to these observations,^11,17–19 we have verified that quipazine produced a robust and lasting effect on HTR in mice.

Considering the high HTR numbers and sustained effect of quipazine, an analogy can be drawn in this model between quipazine and the phenethylamines DOI and DOM, two drugs that characteristically produce high maximal counts and long-lasting increases of HTR frequency.^50,51 However, in terms of potency, quipazine’s ED₅₀ was approximately 1 order of magnitude weaker than those of these reference compounds.⁵¹ This contrast in potency was also observed in previous drug discrimination studies: quipazine would attain full-generalization for LSD at a dose 1 order of magnitude greater than that needed for DOM.^46,52 A recent study found a correlation between active dose range in humans, ED₅₀ in mouse HTR, and rat drug discrimination for different psychedelic drugs.⁵¹ While the shifts in potency observed in rodent models might not be directly extrapolated, these observations suggest that full occupation of the 5-HT_2AR by quipazine and effects in human comparable to other well-known psychedelics might be attained at greater doses of quipazine than previously reported.⁶

As previously suggested, GI discomfort could have been a limiting factor in escalating doses in previous efforts aimed to determine the quality of the subjective effects of quipazine in humans.⁶ The GI effects of quipazine have been observed in different occasions and attributed to its activity on 5-HT₃R.⁶ In agreement with previous accounts,²¹ we showed that 5-HT₃R blockade by the selective antagonist ondansetron did not affect HTR induced by quipazine. These results further suggest that this receptor potentially involved in the GI effects of quipazine does not appear to contribute to the behavioral effects mediated by 5-HT_2AR stimulation. One anecdotal report points toward a full psychedelic effect when quipazine was combined with a 5-HT₃R antagonist.⁵³ Based on our findings, it is likely that the effect of such a combination had a neutral effect on the quality of the potential subjective effect of quipazine, other than overcoming emesis. Additionally, while direct interaction with 5-HT₃R can be responsible for the GI effects of quipazine, it should be noted that similar undesirable effects can occur with selective serotonin reuptake inhibitors (SSRIs) used for the treatment of depression.⁵⁴ Quipazine, initially developed as an antidepressant,⁵⁵ has been shown to potently bind to SERT and increase synaptic levels of serotonin.^24,56 Under the consideration that 5-HT₃R antagonists are indicated to overcome the GI adverse effects of SSRIs, it is reasonable to suggest that the GI effects of quipazine might result from a combination of its 5-HT₃R agonist activity and increases of gut serotonin levels following SERT inhibition. Further supporting this idea, our current data suggest that administration of the serotonin precursor 5-HTP (i.p.) produced diarrhea in mice (data not shown).

The ability of serotonin and 5-HTP to produce HTR in mice can be regarded as a liability of the HTR model as a predictor of psychedelic activity. However, it should be noted that such effects have been observed at doses orders of magnitude above the ED₅₀s of known psychedelic drugs;^51,57,58 in the case of serotonin in the necessary presence of a monoaminoxidase A inhibitor.⁵⁷ The implications of administering equivalent high doses of serotonin or 5-HTP to humans are unknown, but they could very well result in a full-spectrum serotonin syndrome, which includes delirium and hallucinations.⁵⁹ As it pertains to the behavioral model, the ability of high doses of serotonin to produce HTR unveils the necessity to evaluate the contribution of free-neurotransmitter to HTR in the case of drugs that, like quipazine, can both induce HTR and increase synaptic levels of serotonin. We observed that serotonin depletion induced by subchronic PCPA did not attenuate the HTR elicited by quipazine. These data, together with the ability of the selective 5-HT_2AR antagonist M100907 to block quipazine-induced HTR and our in vitro data (see below), further support the notion that the effect of quipazine on the HTR model is mediated by direct activation of 5-HT_2AR.

Other than behavior-based models such as HTR and generalization of the stimulus, the expression pattern of IEG in the mouse somatosensory cortex may serve as a molecular predictor of psychedelic activity in humans.^16,31 Our results show that quipazine induced a pattern of expression of c-fos, egr-1, and egr-2 in the mouse somatosensory cortex that matched that of classic psychedelics. Consistently, quipazine-mediated expression of these IEGs was dependent on 5-HT_2AR; as shown by the lack of changes in the expression of c-fos, egr-1, and egr-2 in knockout animals for the 5-HT_2AR gene. Taken together, the match on 5-HT_2AR-dependent gene-expression fingerprint adds yet another layer to the parallelism between the action of classic psychedelics and quipazine.

Our structure–activity study focused on analogues bearing minimal variations in the structure of quipazine, namely, positional isomerism and bioisostery in the case of the deaza pair. Although structurally conservative, these changes resulted in notable qualitative changes in pharmacological activity. The in vitro and in vivo results indicated that the effect of quipazine on 5-HT_2AR activation and HTR induction is regiospecific to the heteroaromatic bicycle tethering point of the piperazine N_1′, as illustrated by the antagonist profile of the positional isomer isoquipazine. The analysis of the in vivo data was paralleled by the deaza analogue pair: 2-NP appears to be a less efficacious version of quipazine—in terms of 5-HT_2AR maximal stimulation and HTR counts—while 1-NP exhibits the profile of an antagonist.^8,19

Two different measures related to G_q/11 signaling pathway, intracellular Ca²⁺-mobilization and IP₁ production in HEK293 cells stably expressing 5-HT_2AR, confirmed that the activity profile of the compounds in vivo matched their action on 5-HT_2AR. Additionally, we demonstrated that quipazine was able to increase the relative levels of IP₁ on the 5-HT_2AR-rich frontal cortex of the mouse brain as compared to cerebellum—a region lacking in such receptors.^16,60 Considering the role of the LiCl preventing IP₁ degradation in the mouse brain, the observed increase in IP₁ likely represents a cumulative effect of the drug. To the best of our knowledge, this is the first account measuring IP₁ in vivo following the administration of a drug (suspicious of) portending psychedelic activity. Details on HTRF-measured IP₁ accumulation in the mouse brain by well-known psychedelics and the involvement of 5-HT_2AR in such effects will be addressed in a forthcoming manuscript.

The N-substituted piperazine structure of quipazine is commonly found among compounds with biological activity. Some aryl-N-substituted piperazine analogues like meta-chlorophenylpiperazine (mCPP), 3-trifluoromethylphenylpiperazine (TFMPP), or benzylpiperazine (BZP) have been used recreationally for their stimulant and MDMA-like properties.^61–63 Unlike quipazine, they tend to show a 5-HT_2CR bias over 5-HT_2AR agonism.^7,64 Reciprocal substitution in drug discrimination is not uncommon between N-substituted piperazines, but—with the exception of quipazine and MK-212—they tend to produce a stimulus dissimilar to that of known psychedelics.^65–68 Further highlighting a potential divide in the qualitative effect on HTR between the positional isomers herein studied, 1-NP was previously found to produce full-substitution for TFMPP but not for DOM; the opposite was true for the 2-NP stimulus that, like quipazine, generalized for DOM.⁴⁰ This parallelism further supports the pharmacological similarity of quipazine and 2-NP action, but more importantly suggests that these two compounds’ action in vitro and in vivo is more proximal to that of structurally distant classic psychedelic drugs than most of the best-known psychoactive N-substituted piperazines.

CONCLUSIONS

While no animal model can recapitulate the whole extent of human subjective experience on psychedelics, the combination of different predictor models can offer insights into different domains of the behavioral pharmacology of psychedelics. Our current data suggest that quipazine and its deaza analogue 2-NP exhibit psychedelic-like profiles, making these piperazines exceptional candidates to explore 5-HT_2AR pharmacology outside the classic psychedelic chemical families. However, the similitude between classic psychedelics and quipazine in different models conflicts with some of the limited available data in humans for the latter. Other than the active dose range considerations mentioned above, the consistency of the psychedelic-like effect of quipazine in vitro and in vivo hinders the formulation of any alternative hypothesis to explain the lack of conclusive observations on the nature of the subjective effects of quipazine in humans; leaving no option but questioning whether the available human data might simply be too limited or in the case of self-reports untrustworthy. Our data, herein presented, extend our understanding of the mechanism underlying the psychedelic-like effects induced in mice by quipazine and 2-NP, provide lead chemotypes for the design of new synthetic tools to better understand the therapeutic potential of 5-HT_2AR stimulation, and offer insights toward the potential re-evaluation of the effect of quipazine and related piperazines in human subjects.

METHODS

Animals.

Except for IEG expression studies (see below), experiments were performed on adult (8–16 weeks old) C57BL/6 wild-type or 5-HT_2A-KO male mice rederived from 129sv (F5) from our colony. Animals were housed in groups of up to four littermates with food and water ad libitum in a vivarium with a 12 h light/dark cycle at 23 °C. Experiments were conducted in accordance with NIH guidelines and were approved by the Virginia Commonwealth University Animal Care and Use Committee. All efforts were made to minimize animal suffering and the number of animals used.

Drugs.

Quipazine dimaleate (2-(piperazin-1-yl)quinoline, Tocris), isoquipazine (1-(piperazin-1-yl)isoquinoline, Chem Impex), 2-NP (1-(naphthalen-2-yl)piperazine, Princeton Bio), 1-NP (1-(naphthalen-1-yl)piperazine, Sigma-Aldrich), M100907 ((R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol, National Institute on Drug Abuse), WAY100635 maleate (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate, Tocris), 8-OH-DPAT hydrobromide ((±)-8-hydroxy-2-dipropylaminotetralin hydrobromide, Tocris), SB242084 dihydrochloride (6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride, Tocris), ondansetron hydrochloride (1,2,3,9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one, Tocris), and PCPA (para-chloro-dl-phenylalanine, Alfa-Aesar). All drugs were dissolved in saline 0.9% solution and administered intraperitoneally (5–10 μL/g animal weight). In the case of drugs commercialized in the form of free base, the hydrochloride salt was generated in situ (1–2 equiv). Dissolving PCPA required the addition of 0.5 eq. of NaOH.

Head-Twitch Response Quantification.

Magnetometer-based HTR experiments were performed as previously described.^50,69 Mice were placed in the monitoring chamber for 30 min to get them accustomed to the environment and determine baseline HTR. After this period, the animals were administered with the corresponding treatment (w/o pretreatment 5 min prior) and recorded for additional 90 min (or 180 min for determination of the half-life). HTR were quantified in an automated fashion with minor visual monitoring of the magnetometer processed signal.

Quantification of Feces.

Mice previously habituated to HTR monitoring chambers and to i.p. injections were allowed to get accustomed to the monitoring chamber for 30 min after which they were administered the corresponding drugs or saline vehicle (10 μL/g animal weight). Feces prior or immediately after the i.p. injection were not counted.

Radioligand Binding.

Radioligand binding studies were performed in HEK293 cells that stably expressed human 5-HT_2A receptors, mouse and human postmortem brains, as previously described.^70,71 Using [³H]ketanserin binding saturation curves, we have previously shown that our HEK293 cell line stably expresses ~260 fmol/mg protein of 5-HT_2A receptor.^31,70

Human brain samples (three males ages 34, 33, and 44 years old at the moment of death, postmortem delay 23, 17, and 23 h, respectively) were obtained from subjects who had died by sudden and violent causes (motor-vehicle accidents). Samples from the prefrontal cortex (Brodmann’s area 9) were dissected at the time of autopsy and immediately stored at −70 °C until assay. The collection was performed in accordance with approved protocols of the Instituto Anatómico Forense, Bilbao, Spain for postmortem human studies.

Cell pellets or brain tissue samples were homogenized using a Teflon-glass grinder (50 up-and-down strokes) in 5 mL of binding buffer (50 mM Tris-HCl; pH 7.4) supplemented with 0.25 M sucrose. The volume was made up to 10 mL with binding buffer, and the crude homogenate was centrifuged at 3000 rpm for 5 min at 4 °C. The supernatant was centrifuged at 18 000 rpm for 10 min at 4 °C, and the resultant pellet (P2) was washed with 10 mL of binding buffer and recentrifuged at 18 000 rpm for 15 min. Protein concentration was determined using the BioRad protein estimation assay. Curves were carried out by incubating each drug in binding buffer containing 5 nM [³H]-ketanserin (PerkinElmer Life and Analytical Sciences). The final volume in each well was 200 μL. Methysergide (10 μM, Tocris Bioscience) was used to determine nonspecific binding. The free ligand was separated from bound ligand by rapid filtration under vacuum through GF/C glass fiber filters using a MicroBeta Filtermat-96 harvester (PerkinElmer). These filters were then rinsed with ice-cold incubation buffer, dried at 65 °C for 1 h, and counted for radioactivity using a MicroBeta2 detector (PerkinElmer). Radioligand binding data were analyzed by nonlinear regression by GraphPad PRISM (version 8 for Windows 10, GraphPad Software, La Jolla CA). Data are from two or three independent experiments performed in duplicate and are represented as pK_i ± SEM.

Intracellular Calcium Mobilization.

HEK293 stably expressing 5-HT_2A receptors were plated onto 96-well plates (Greiner Bio-One GmbH) that were coated with poly-d-lysine hydrobromide. On the day of the assay, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and loaded with 3 μM Fluo 4-AM (Thermo Fisher Scientific) in imaging solution (5 mM KCl, 0.4 mM KH₂PO₄, 138 mM NaCl, 0.3 mM Na₂HPO₄, 2 mM CaCl₂, 1 mM MgCl₂, 6 mM glucose, 20 mM HEPES, pH 7.4) supplemented with pluronic acid (10% solution in DMSO). The cells were incubated for 0.5 h, washed thrice with imaging buffer, and placed in the FlexStation 3 instrument. Baselines were recorded for 30 s, test compounds were added at 30 s, and serotonin (1 μM) was added at 180 s. The excitation wavelength was fixed at 494 nm, and the fluo-4 emission signal was acquired at 525 nm. The fluorescence was a measure of intracellular calcium and was normalized to basal fluorescence using Softmax Pro (Molecular Devices, Wokingham, U.K.). Data were further normalized to the responses elicited by 1 μM 5-HT in the same experiment. The functional data were analyzed by nonlinear regression to generate concentration–response curves and EC₅₀/IC₅₀ values by GraphPad PRISM (version 8 for Windows 10, GraphPad Software, La Jolla CA). Data are from two to three independent experiments performed in duplicate and are represented as pIC₅₀/pEC₅₀ ± SEM.

IP₁ Accumulation In Vitro.

Evaluation of the agonist activity of compounds at a single concentration in the human 5-HT_2AR was determined by measuring their effects on IP₁ production on HEK293 cells stably expressing 5-HT_2AR using a Homogeneous Time Resolved Fluorescence (HTRF) assay (IP-one Gq Kit, Cisbio-PerkinElmer) as described by the contractor Eurofins-CEREP (https://www.eurofinsdiscoveryservices.com/). Cells were suspended in a buffer containing 10 mM Hepes/NaOH (pH 7.4), 4.2 mM KCl, 146 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM glucose and 50 mM LiCl and then distributed in microplates at a density of 1.5 × 10⁴ cells/well and incubated for 30 min at 37 °C in the presence of buffer (basal control), test compound, or reference agonist. For stimulated control measurement, separate assay wells contained 10 μM 5-HT. Following incubation, the cells were lysed and the fluorescence acceptor (d2-labeled IP₁) and fluorescence donor (cryptate-labeled anti-IP₁ antibody labeled) were added. After 60 min at room temperature, the fluorescence transfer was measured at an excitation wavelength of 337 nm and emission at 620 and 665 nm using a microplate reader (Envision, PerkinElmer). The ratio between the signals at 665 and 620 nm is an inversely proportional proxy of IP₁ concentration. The results were expressed as a percent of the control response to 10 μM 5-HT, the standard reference agonist.

IP1 Accumulation In Vivo.

The experiment was conducted adapting a previously reported methodology to measure IP₁ in vivo in the mouse brain after stimulation by muscarinic ligands using the IP-One Gq Kit (Cisbio-PerkinElmer) kit.^30,72 All animals (C57BL/6 males, 10 weeks of age) were administered LiCl 200 mg/kg and returned to their home cage for 30 min before receiving the corresponding treatment. Mice administered M100907 and quipazine received both drugs in the same solution. Animals were sacrificed by decapitation 60 min after drug administration, and the heads were maintained on ice until the frontal lobe of the mouse brain cortices and the posterior end of the cerebellum were dissected and frozen at −80 °C.

A 10% solution of the IP-One Gq Kit Lysis and Detection Buffer in 50 mM LiCl (10 μL per 1 mg of tissue) and 0.5 mm glass beads (GS05, NextAdvance) were added to the samples that were subsequently lysed using a tissue homogenizer at speed 6 for 5 min (Bullet Blender, NextAdvance). The homogenates were clarified by centrifugation at 17 000g for 15 min. The whole sample processing procedure was performed at 4 °C. The clarified homogenates were spotted in duplicate (HTRF 96-well low volume white plate, Cisbio-PerkinElmer) by adding sequentially 13 μL of Lysis and Detection Buffer, 1 μL of sample, 3 μL of d2-labeled IP₁, and 3 μL of cryptate-labeled monoclonal anti-IP₁ antibody, in this order. The plates were sealed and incubated at room temperature for 30 min before reading the plate following the kit manufacturer recommendations for terbium cryptate donor/red acceptor readout. Briefly, a VICTOR Nivo (PerkinElmer) plate reader with HTRF capabilities was employed to read the emission at 615 and 665 nm following excitation at 320 nm and a 70 μs delay. Since the concentration of IP₁ is inversely proportional to the 665/615 nm ratiometric signal, we chose to invert the ratio (615/665 nm) so that higher levels of IP₁ in the sample would correlate with a more intuitive increase in signal. Finally, the ratio between the frontal cortex and the cerebellum was calculated to show the relative fold-change of IP₁ signal between both regions. While the actual concentration of IP₁ was not determined, an operative range for sample dilution was defined between the maximum HTRF signal (absence of IP₁) and the signal corresponding to a 1.1 μM IP₁ standard.

Gene Expression.

Gene expression experiments were conducted on adult (10–20 weeks old) 129S6/SvEv wild-type and 5-HT_2AR-KO mice born to heterozygote (Htr2a^±) breeders. Changes in the expression of c-fos, egr-1, and egr-2 were evaluated as previously described.¹⁶

Docking Studies.

The crystal structure of active conformation of the human 5-HT_2A serotonin receptor (PDB: 6WHA)³² was used to dock quipazine (see Figure 1 for structure). Quipazine was sketched using SYBYL-X 2.1.1 (Certara USA, Inc., Princeton, NJ) and was energy-minimized using the Tripos Force Field (TFF) with Gasteiger-Hückel charges, a dielectric constant (ε) of 4.0 D/Å, and an energy-gradient cutoff of 0.05 kcal/(mol·Å). Gold Suite v5.5 5 (Cambridge Crystallographic Data Center, Cambridge, UK) was used to dock quipazine into the 5-HT_2AR orthosteric binding site with a binding radius of 12 Å around the α carbon atom of D155.^3,32 One hundred (100) nondiverse solutions were generated and clustered using a built-in Gold clustering program with an RMSD cutoff of 0.75 Å. The solutions were merged with the receptor and the ligand/receptor complexes were energy-minimized in SYBYL-X 2.1.1 utilizing the TFF with Gasteiger-Hückel charges and a nonbonded interaction cutoff of 8 Å, dielectric constant (ε) of 4.00 D/Å, and termination gradient of 0.05 kcal/(mol·Å). Hydropathic INTeractions (HINT)⁷³ analysis was conducted on the highest scoring Gold solution for quipazine-receptor complex using SYBYL 8.1 (Certara USA, Inc., Princeton, NJ). The PyMOL Molecular Graphics System, version 1.7.4 (Schrödinger, LLC) was used to generate images.

A built-in clustering algorithm was used to cluster the docked solutions with an RMSD cutoff of 0.75 Å. The selection of the docked solution was based on GOLD scoring and clustering. The observed ionic, hydrophobic, and polar interactions between ligands and protein were supported by GOLD (score = 41) and HINT analysis (positive score indicates favorable interactions; Total HINT score = 899; Total H-Bond score = 818; D155–N_4′ score = 860; S159–N score = 28, Total Hydrophobic = 239; Total Acid/Base = 409).

Statistical Analysis.

Statistical analyses and nonlinear regression were performed with GraphPad Prism software version 8. Animals were randomly allocated into the different experimental groups with at least 1 week between HTR testing sessions. Statistical significance of experiments involving different treatment groups were assessed by one-way ANOVA followed by Bonferroni’s post hoc test to compare to the control condition. Statistical significances of experiments involving different treatments, doses, or time points were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test. Statistical significance of experiments involving two experimental conditions was assessed by Student’s t test. The level of significance was chosen at P < 0.05. All data are presented as mean ± standard error of the mean (SEM). In group sizes showing great disparity (i.e., n = 6–12), the larger size corresponds to the control group. The half-life of HTR elicited by quipazine was determined by fitting HTR counts in 15 min intervals to an exponential decay equation (MATLAB, MathWorks R2018a) and calculated as previously described.⁵⁰