This mouse study investigated the effects of N,N-dipropyltryptamine (DPT), a psychedelic tryptamine, on audiogenic seizures in a mouse model of fragile X syndrome (genetic cause of autism). DPT was found to prevent seizures at a 10 mg/kg dose completely but not at lower doses (3 or 5.6 mg/kg). Despite being a serotonin receptor agonist, the antiepileptic effects of DPT were not mediated through specific serotonin receptor subtypes (5-HT2A, 5-HT1B, or 5-HT1A), nor through sigma1 receptors.
The serotonergic psychedelic psilocybin shows efficacy in treating neuropsychiatric disorders, though the mechanism(s) underlying its therapeutic effects remain unclear. We show that a similar psychedelic tryptamine, N,N-dipropyltryptamine (DPT), completely prevents audiogenic seizures (AGS) in an Fmr1 knockout mouse model of fragile X syndrome at a 10 mg/kg dose but not at lower doses (3 or 5.6 mg/kg). Despite showing in vitro that DPT is a serotonin 5-HT2A, 5-HT1B, and 5-HT1A receptor agonist (with that rank order of functional potency, determined with TRUPATH Gα/βγ biosensors), pretreatment with selective inhibitors of 5-HT2A/2C, 5-HT1B, or 5-HT1A receptors did not block DPT’s antiepileptic effects; a pan-serotonin receptor antagonist was also ineffective. Because 5-HT1A receptor activation blocks AGS in Fmr1 knockout mice, we performed a dose-response experiment to evaluate DPT’s engagement of 5-HT1A receptors in vivo. DPT elicited 5-HT1A-dependent effects only at doses greater than 10 mg/kg, further supporting that DPT’s antiepileptic effects were not 5-HT1A-mediated. We also observed that the selective sigma1 receptor antagonist, NE-100, did not impact DPT’s antiepileptic effects, suggesting DPT engagement of sigma1 receptors was not a crucial mechanism. Separately, we observed that DPT and NE-100 at high doses caused convulsions on their own that were qualitatively distinct from AGS. In conclusion, DPT dose-dependently blocked AGS in Fmr1 knockout mice, but neither serotonin nor sigma1 receptor antagonists prevented this action. Thus, DPT might have neurotherapeutic effects independent of its serotonergic psychedelic properties. However, DPT also caused seizures at high doses, showing that DPT has complex dose-dependent in vivo polypharmacology.
Papers cited by this study that are also in Blossom
Johnson, M. W., Garcia-Romeu, A., Cosimano, M. P. et al. · Journal of Psychopharmacology (2014)
Bogenschutz, M. P., Ross, S., Bhatt, S. R. et al. · JAMA Psychiatry (2022)
Daws, R. E., Timmermann, C., Giribaldi, B. et al. · Nature Medicine (2022)
Carhart-Harris, R. L., Bolstridge, M., Rucker, J. et al. · Lancet Psychiatry (2016)
Earlier research has explored serotonergic psychedelics such as psilocybin and related tryptamines for neuropsychiatric indications including major depressive disorder, substance-use disorders, autism spectrum disorder and fragile X syndrome (FXS). Psychedelic tryptamines are known 5-HT2A/2B/2C receptor agonists but also interact with other serotonin receptor subtypes (for example 5-HT1A and 5-HT1B) and non-serotonergic targets such as sigma1 receptors. Because different 5-HT receptor subtypes and sigma1Rs have been implicated in seizure modulation, the specific receptor mechanisms that might underlie any antiseizure or broader therapeutic effects of tryptamines remain uncertain. Tyagi and colleagues set out to test whether the psychedelic N,N-dipropyltryptamine (DPT) prevents audiogenic seizures (AGS) in the juvenile Fmr1 knockout (KO) mouse model of FXS and, if so, whether its effects are mediated by serotonergic receptors or sigma1 receptors. The study combines in vivo AGS assays and behavioural pharmacology with in vitro radioligand binding and TRUPATH functional assays to characterise DPT's receptor pharmacology and to probe mechanism using selective antagonists. The work aims to determine whether DPT's antiepileptic action is linked to its serotonergic psychedelic activity or to alternative targets.
Experimental subjects were juvenile Fmr1 KO mice (postnatal day 23–25), both sexes, bred at Mercer University; sighted FVB wild-type (WT) mice were used for some in vivo pharmacology. All in vivo compounds (including DPT hydrochloride, antagonists and controls) were administered intraperitoneally (i.p.) at 1 mL/100 g, with vehicle consisting of Milli-Q water except for SB-224289 (dissolved in 2% DMSO, 4% Tween-80, 4% PEG-20). Pretreatment intervals for antagonists were 10 min before DPT; mice were tested 5 min after DPT injection. AGS testing used a 5 min alarm stimulus presented in a clear box while video recording behaviour; primary outcomes included prevalence of AGS (defined by tonic-clonic seizures), wild-running and jumping (WRJ), seizure duration and lethality. Sound levels were monitored to ensure consistency (alarm ~105 ± 4 dB; baseline ~55 ± 9 dB). Behavioural scoring was performed from video and analysed with Fisher's exact test for categorical outcomes. For in vitro pharmacology, human 5-HT2A, 5-HT1A and 5-HT1B receptor cDNAs were expressed in HEK293T cells. Radioligand competition binding used [3H]LSD for 5-HT2A and [3H]5-CT for 5-HT1A/1B receptors, with nonspecific binding defined by high concentrations of established competitors. Functional signalling was assessed using the TRUPATH bioluminescence resonance energy transfer (BRET2) platform to measure G-protein activation by ligands (Rluc8-Gα q for 5-HT2A, Rluc8-Gα i3 for 5-HT1A/1B), with nonlinear regression applied to concentration–response data (two-site model used for 5-HT2A binding where appropriate). In vivo receptor engagement assays included the head-twitch response (HTR) as a 5-HT2A-dependent behavioural readout and observation of 5-HT1A-dependent signs (for example hind-limb abduction, flat body posture, Straub tail). Doses tested for DPT spanned 3, 5.6, 10, 15 and 20 mg/kg. Antagonists tested in AGS experiments were pimavanserin (5-HT2A/2C; 3 and 10 mg/kg), WAY-100635 (5-HT1A; 0.1 and 1 mg/kg), SB-224289 (5-HT1B; 5 mg/kg), methiothepin (pan-5-HTR; 2 and 4 mg/kg) and NE-100 (sigma1R; 30 and 50 mg/kg). Statistical analyses used Fisher's exact test for seizure prevalence, one-way ANOVA with multiple comparisons for HTR data, Student's t test for control comparisons, and nonlinear regression for in vitro assays.
DPT prevented AGS in juvenile Fmr1 KO mice in a dose-dependent manner, with a complete block of AGS at 10 mg/kg. Vehicle-treated Fmr1 KO mice had an AGS prevalence of 72%. At 10 mg/kg DPT, seizure prevalence dropped to 0% (p < 0.0001). Among mice given 10 mg/kg DPT, 65% exhibited normal behaviour during the auditory alarm while 35% showed WRJ; WRJ duration in DPT-treated mice (mean ± SEM 78 ± 20.7 s) was significantly longer than WRJ in vehicle-treated mice (22 ± 6.26 s; p < 0.01), which the authors interpret as prevention of progression to tonic-clonic seizure (TCS). Lower DPT doses (3 and 5.6 mg/kg) did not alter AGS prevalence, latency, duration or lethality (p values ≥ 0.46; comparisons versus vehicle gave p = 0.69 for prevalence at each lower dose). In vitro and in vivo pharmacology at serotonergic receptors showed that DPT is a moderate-potency full agonist at human 5-HT2A receptors (Emax 106% ± 1.13 relative to 5-HT) and a low-potency partial agonist at 5-HT1A receptors (Emax 53% ± 3.67). At 5-HT1B receptors DPT behaved as a very low-potency agonist with Emax 83% ± 1.95. In vivo, DPT elicited a dose-dependent head-twitch response (HTR), a 5-HT2A-dependent behaviour, with maximal HTR at 10 mg/kg in both juvenile and adult WT mice. Juvenile HTR counts (mean ± SEM) were vehicle 3 ± 0.3; DPT 3 mg/kg 13 ± 0.7 (p = 0.047); 5.6 mg/kg 18 ± 3.9 (p = 0.007); 10 mg/kg 18 ± 2.9 (p = 0.007); 15 mg/kg 14 ± 3.1 (p = 0.046); and 20 mg/kg 7 ± 1.5 (p = 0.287). Adult mice showed a similar dose–response profile with significant HTRs at 5.6–20 mg/kg. Despite evidence that DPT engages 5-HT2A receptors at the antiepileptic dose, pharmacological blockade of serotonin receptors did not reverse its anti-AGS action. Pretreatment with the 5-HT2A/2C antagonist pimavanserin (3 and 10 mg/kg) did not significantly restore AGS (p = 0.10 for pimavanserin 3 mg/kg + DPT versus DPT alone, and p > 0.99 for pimavanserin 10 mg/kg). Antagonism of 5-HT1A receptors with WAY-100635 (0.1 and 1 mg/kg) also failed to block DPT's antiseizure effect (0.1 mg/kg: 13% vs 0% prevalence, p = 0.33; 1 mg/kg: 0% vs 0%, p > 0.99). SB-224289 (5-HT1B antagonist) produced a non-significant rise in seizure prevalence (25% vs 0%, p = 0.10). A pan-5-HTR antagonist, methiothepin (4 mg/kg), had no effect on DPT's efficacy (0% vs 0%, p > 0.99), although methiothepin produced pronounced sedation during observation. Because tryptamines can interact with sigma1Rs, the authors tested NE-100, a selective sigma1 antagonist. NE-100 at 30 and 50 mg/kg did not reverse the anti-AGS effect of DPT (all mice remained free of AGS; p > 0.99). High doses of DPT and NE-100 produced seizures when administered alone, but these drug-induced convulsions were qualitatively distinct from AGS. Specifically, DPT at 20 mg/kg induced convulsions in 90% of tested mice (N = 10), characterised by myoclonic tremors progressing to brief tonic convulsions with opisthotonos and vocalisation, lasting less than ~20 s; mice recovered and later displayed 5-HT1A-dependent behaviours. NE-100 at 50 mg/kg induced convulsions in 69% of mice (N = 13), with longer events lasting 150–200 s and recovery to normal behaviour within 20–30 min. Cotreatment with NE-100 50 mg/kg and DPT 10 mg/kg caused seizures within ~2 min, with characteristics matching NE-100-induced seizures and not AGS. Collectively, these results indicate that DPT blocks AGS at a specific dose but that this effect is not reversed by antagonising major serotonin receptor subtypes or sigma1Rs, and that DPT has dose-dependent proconvulsant effects at higher doses.
Tyagi and colleagues interpret their findings as evidence that DPT dose-dependently prevents audiogenic seizures in juvenile Fmr1 KO mice, with full protection at 10 mg/kg. They note that this antiepileptic dose coincided with in vivo engagement of 5-HT2A receptors (maximal head-twitch response), whereas engagement of 5-HT1A receptors required higher doses. In vitro data mirrored the in vivo observations: DPT behaved as a moderate-potency full agonist at 5-HT2A receptors and as a low-potency partial agonist at 5-HT1A receptors, with low potency at 5-HT1B receptors. Despite evidence of 5-HT2A engagement at the effective dose, co-administration of selective antagonists for 5-HT2A/2C, 5-HT1A, 5-HT1B, a pan-5-HTR antagonist, or a selective sigma1R antagonist did not meaningfully reverse DPT's antiepileptic effect. From these negative antagonist challenges the investigators conclude that DPT's blockade of AGS may be mediated by mechanisms independent of the serotonin receptors and sigma1Rs tested, although they remain cautious about the conclusion. The authors highlight the dose-dependent polypharmacology of DPT: at higher doses (for example 20 mg/kg) it became proconvulsant, producing brief tonic seizures that differed in phenomenology and time course from AGS; similar dose-dependent reversals have been reported for other serotonergic ligands. The discussion presents several possible explanations and limitations. One hypothesis is that DPT may modulate auditory processing directly, reducing auditory hypersensitivity in Fmr1 KO mice; the authors note anecdotal human reports that related tryptamines alter sound perception, and suggest this as an avenue for future work. A key limitation acknowledged is that some antagonists (pimavanserin, methiothepin) caused sedation, which could non-specifically alter auditory input and confound interpretation of antagonist challenge experiments. The authors also point out gaps in knowledge about DPT's pharmacokinetics and the complexity of GPCR signalling: TRUPATH readouts probe specific Gα subunits and DPT could have different potencies across other G-protein pathways or intracellular signalling cascades not examined here. Finally, because drug-elicited seizures and AGS arise from different anatomical loci, the distinct seizure phenomenology observed with DPT and NE-100 supports the view that different mechanisms produce those convulsions. Overall, the investigators remain parsimonious: their data show that DPT can prevent AGS in this FXS mouse model, but the antiepileptic mechanism does not appear to depend on the major serotonin receptor subtypes or sigma1Rs tested, and further work is needed to delineate the relevant targets, local circuit actions and dose-dependent effects of DPT.
M any studies are investigating the therapeutic potential of serotonergic psychedelics, including psilocybin and related psychedelic tryptamines, for various psychiatric conditions. Indications under study include but are not limited to major depressive disorder (MDD) and substance-use disorders, whereas other indications under consideration include autism spectrum disorder (ASD) and fragile X syndrome (FXS).Despite the proliferation of clinical studies, the specific pharmacodynamic properties that contribute to the therapeutic efficacies of psychedelics are not well understood. Psychedelic tryptamines are serotonin (5-HT) 5-HT 2A , 5-HT 2B , and 5-HT 2C receptor (5-HT 2 R) agonists, but they bind various other targets.For example, they are 5-HT 1A and 5-HT 1B R agonists, and some, including N,Ndimethyltryptamine have direct modulatory effects in vivo on non-serotonergic receptors, including sigma1Rs.This poses the question of whether targets in addition to 5-HT 2 Rs contribute to the pharmacotherapeutic effects of psychedelics.We have been researching 5-HTRs as targets for treating FXS and ASD.FXS is a monogenic neurodevelopmental disorder that is the leading cause of intellectual disability and ASD.In addition to various other neurobehavioral issues, individuals with FXS present with auditory hypersensitivities and seizures.Seizures affect approximately 12% of FXS patients,with three times higher risk in comorbid ASD patients.5-HT and its receptors were under investigation as novel antiepileptics decades ago, and there has been a resurgence of interest in this area, owing to the recently proven antiepileptic effects of the 5-HT releaser and lowpotency, direct 5-HT 2 R agonist, fenfluramine, in Dravet syndrome and Lennox-Gastaut syndrome.Clinical trials are now underway assessing the efficacy of selective 5-HT 2C R agonists for treating seizures in Dravet syndrome,based partly on historical studies showing susceptibility in 5-HT 2C R knockout (KO) mice to audiogenic seizures (AGS).In some preclinical studies, nonselective activation of 5-HT 2 Rs attenuates generalized tonic-clonic and myoclonic seizures.A classical study showed that the serotonergic psychedelic 5-methoxy-N,N-dimethyltryptamine inhibited myoclonic seizures caused by photic stimulation in lateral geniculate-kindled felines.However, these observations are controversial, as some studies show no seizure-modulating effects of nonselective 5-HT 2 R activation.Still, others show that nonselective 5-HT 2 R antagonists block psychostimulant-induced convulsions,and there are clinical reports of seizures induced by certain, full-efficacy 5-HT 2A agonist psychedelics.Thus, the functions of distinct 5-HT 2 R subtypes in modulating distinct types of epilepsy remain unsolved. With few exceptions, e.g., in absence seizures,in preclinical models, 5-HT 1A R activation is antiepileptic. For example, it inhibits hippocampal focal seizures in felines and prevents AGS in the Fmr1 KO mouse model of FXS.WAY-100635, a selective 5-HT 1A R antagonist, inhibits the anti-AGS effect in Fmr1 KO mice of the selective 5-HT 1A agonist, NLX-112.WAY-100635 also inhibits the anticonvulsant effects of the 5-HT 1A/1B R agonist RU24969 on pentylenetetrazol-induced seizures and inhibits the anticonvulsant effects of the 5-HT 1A R agonist 8-OH-DPAT on picrotoxin-induced seizures in mice,demonstrating that activation of 5-HT 1A Rs is antiepileptic in various seizure models. Several other preclinical studies show that selective serotonin reuptake inhibitors (SSRIs) decrease the occurrence and increase the threshold of various types of induced seizures.This provides ample evidence that targeting the central 5-HT system may be a fruitful approach for treating epilepsies. Sigma1Rs are another target of tryptamines that can modulate epileptiform activity.Also, fenfluramine was shown to act as a positive allosteric modulator at sigma1Rs in mice and zebrafish models,and sigma1R modulation prevents seizures in models of Dravet syndrome, amphetamine-induced seizures, and epileptic encephalopathies.We tested the hypothesis that the serotonergic psychedelic N,N-dipropyltryptamine (DPT) would prevent AGS in juvenile Fmr1 KO mice and that it would be effective via a serotonergic or sigma1R mechanism. DPT is a short-acting psychedelic tryptamine, but there is limited knowledge about its pharmacology and behavioral effects; in vivo, it possesses agonist activity at 5-HT 1A and 5-HT 2A Rs,and in vitro it has been shown to be a substrate of the 5-HT transporter.DPT is not restricted as a Schedule 1 controlled substance and hence is accessible for laboratory research without possessing a Drug Enforcement Agency controlled substances license. Here, we report observations that DPT prevents AGS in Fmr1 KO mice, but our in vitro and in vivo pharmacology experiments did not provide evidence that its antiepileptic effects were 5-HTR-or sigma1R-mediated.
DPT Is an Antiepileptic in Fmr1 KO Mice. Compounds that target the central 5-HT system, such as fenfluramine, have antiepileptic effects in individuals with neurodevelopmental disorders.Hence, we evaluated the antiepileptic effects of DPT in juvenile Fmr1 KO mice using the AGS assay. As shown in Figure, vehicle-treated male and female Fmr1 KO mice showed a prevalence of AGS of 72%, and DPT completely prevented AGS at 10 mg/kg (p < 0.0001). Sixtyfive percent of mice treated with 10 mg/kg DPT showed normal behavior during the presentation of the seizure-eliciting alarm (akin to wild-type (WT) mice), whereas 35% showed a wild-running and jumping (WRJ) response. The duration of WRJ in these mice was significantly longer than WRJ in vehicle-treated mice (vehicle (mean ± SEM), 22 ± 6.26 s vs 10 mg/kg DPT (mean ± SEM), 78 ± 20.7 s; p < 0.01) which could indicate that DPT treatment prevented the transition to the tonic-clonic seizure (TCS) stage of AGS in these mice.Relative to vehicle, DPT did not significantly affect the prevalence of AGS at 3 (p = 0.69) or 5.6 mg/kg (p = 0.69). In addition, 3 and 5.6 mg/kg DPT did not impact latency to seizure onset, seizure duration, or lethality caused by AGS (p values ≥ 0.46, Supplemental Figure). In Vitro, DPT Is a Modest Potency 5-HT 2A R Agonist, and In Vivo, DPT Elicits Peak 5-HT 2A R-Dependent Head-Twitch Responses at a Dose Equivalent to Its Antiepileptic Dose. We next tested DPT's in vitro and in vivo pharmacology at 5-HT 2A Rs to explore whether 5-HT 2A R activation could be mediating its antiepileptic effects. In vitro, DPT was a moderate potency full agonist at 5-HT 2A Rs with E max of 106% ± 1.13 (mean ± SEM), relative to 5-HT, the positive control. EC 50 and K i values are reported in Table. See Figure,B for affinity and function nonlinear regression curves. In vivo, DPT produced a dose-dependent head-twitch response (HTR), with maximal effects at 10 mg/kg (Figure). In juvenile WT mice (P23-25), we observed a main effect of treatment (F(5, 23) = 4.25, p = 0.007). Relative to vehicle treatment, DPT increased the number of HTRs at 3, 5.6, 10, and 15 mg/kg doses (mean ± SEM HTRs vehicle = 3 ± 0.3; DPT 3 mg/kg = 13 ± 0.7, p = 0.047; DPT 5.6 mg/kg = 18 ± 3.9, p = 0.007; DPT 10 mg/kg = 18 ± 2.9, p = 0.007; DPT 15 mg/kg = 14 ± 3.1, p = 0.046). HTRs decreased at the 20 mg/kg dose, generating the classic bi-phasic dose-effect Figure. Dose-effect study of DPT (structure shown) on AGS in juvenile Fmr1 KO mice. DPT at 10 mg/kg, but not 3 or 5.6 mg/kg prevented AGS. The filled square denotes the statistically significant effect of DPT compared to vehicle (Veh, p < 0.0001). D3, D5.6, D10: DPT 3, 5.6 and 10 mg/kg. N = number of subjects per group. The vehicle group includes new (N = 14) and historical (N = 53) data collected in our laboratory, to increase statistical power.curve (mean ± SEM HTRs elicited by DPT 20 mg/kg = 7 ± 1.5, p = 0.287, compared to vehicle). In adult WT mice (∼2 months old), we observed a similar main effect of treatment (F(5, 22) = 5.18, p = 0.027). Relative to vehicle, 3 mg/kg DPT was not sufficient to elicit the HTR (mean ± SEM HTRs vehicle = 1 ± 0.6 and DPT 3 mg/kg = 4 ± 1.0, p = 0.391). DPT elicited a significant HTR at all other doses tested, i.e., 5.6, 10, 15, and 20 mg/kg (mean ± SEM HTRs DPT 5.6 mg/kg = 12 ± 2.7, p = 0.013; DPT 10 mg/kg = 14 ± 1.4, p = 0.004; DPT 15 mg/kg = 11 ± 2.6, p = 0.016; DPT 20 mg/kg = 12 ± 2.8, p = 0.013, compared to vehicle). (±)-2,5-Dimethoxy-4-iodoamphetamine (DOI) at 1 mg/kg� used as a positive control�elicited a very high number of HTRs in both juvenile (3 vehicle vs 35 DOI, p = 0.001) and adult (1 vehicle vs 28 DOI, p < 0.001) WT mice, thus validating our assay (Supplemental Figure). Additionally, DPT at all five test doses did not affect locomotor activity in adult WT mice, but in juvenile WT mice, at 20 mg/kg, DPT reduced the distance traveled (Supplemental Figure). The dose of DPT that elicited the highest number of HTRs (10 mg/kg) was the DPT dose that effectively blocked AGS, suggesting 5-HT 2A R activation might mediate DPT's antiepileptic effects. Antagonism of 5-HT 2A/2C Rs Does Not Block DPT's Antiepileptic Effects. As DPT engaged 5-HT 2A Rs in vivo at 10 mg/kg, which matched its effective dose to block AGS, we tested whether selective antagonism of 5-HT 2A/2C Rs blocks DPT's anti-AGS effects (Figure). Pretreatment with 3 mg/ kg pimavanserin slightly reversed the antiepileptic effects of DPT 10 mg/kg (AGS prevalence 25% for pimavanserin 3 mg/ kg plus DPT 10 mg/kg vs AGS prevalence of 0% for DPT 10 mg/kg alone), but this increase in the seizure prevalence was not significant (p = 0.10). Pretreatment with pimavanserin 10 mg/kg did not impact DPT's anti-AGS effects (AGS prevalence 0 vs 0%; p > 0.99). Mice treated with pimavanserin 3 mg/kg behaved normally, i.e., like vehicle-treated mice. Mice treated with pimavanserin 10 mg/kg showed signs of mild sedation which included partial ptosis, low sensory responses, hypolocomotion, and immobility that took effect within 2 min of injection and which lasted ∼30 min. These observations suggest that the anti-AGS effects of DPT are not mediated by 5-HT 2A / 2C R activation. These data align with our previous observations that the 5-HT 2C R-preferring agonist, lorcaserin, does not prevent AGS in juvenile Fmr1 KO mice.In Vitro, DPT Is a Low-Potency 5-HT 1A R Agonist, and In Vivo, DPT Elicits 5-HT 1A R-Dependent Behavioral Effects at Doses Higher than Its Antiepileptic Dose. As we and others recently showed that 5-HT 1A R activation blocks AGS in Fmr1 KO mice,we next tested whether DPT activation of 5-HT 1A Rs could be mediating its anti-AGS effects. We tested its pharmacology at 5-HT 1A Rs in vitro and in vivo. In vitro, we observed that DPT is a low potency partial agonist at 5-HT 1A Rs, with an E max of 53% ± 3.67 (mean ± SEM) relative to 5-HT, the positive control. These observations are similar to a previous study of DPT at 5-HT 1A Rs.EC 50 and K i values are reported in Table. See Figureand 3B for affinity and function curves. As shown in Figure, relative to vehicle, juvenile and adult mice treated with DPT at 20 mg/kg, but not lower doses, showed distinct behavioral symptoms, which included flat body posture (0 vs 90%, p = 0.0001), hind limb abduction (0 vs 90%, p = 0.0001), tremors (0 vs 70%, p = 0.003), and Straub tail (0 vs 90%, p = 0.0001). These behaviors are 5-HT 1A R-dependent,providing evidence that DPT does not sufficiently engage 5-HT 1A R at its antiepileptic dose of 10 mg/kg. Antagonism of 5-HT 1A Rs Does Not Block DPT's Antiepileptic Effects. To further evaluate whether DPT's antiepileptic effects in the AGS model were 5-HT 1A Rdependent, we tested whether selective antagonism of 5-HT 1A Rs blocks DPT's effects. Pretreatment with WAY-100635 0.1 and 1 mg/kg did not significantly reverse DPT's anti-AGS effects (13% vs 0% p = 0.33, and 0% vs 0% p > 0.99, respectively) (Figure). Importantly, we previously showed that WAY-100635 at a 0.1 mg/kg dose blocks the antiepileptic effects of the highly selective 5-HT 1A R agonist, NLX-112, in the AGS assay in Fmr1 KO mice.Thus, these data support the conclusion that DPT's antiepileptic effects are not 5-HT 1A R-mediated. In Vitro, DPT Is a Very Low-Potency 5-HT 1B R Agonist, and In Vivo, Antagonism of 5-HT 1B Receptors Does Not Block DPT's Antiepileptic Effects. We next tested DPT's in vitro pharmacology at 5-HT 1B Rs and observed that it is a very low-potency 5-HT 1B R agonist, relative to 5-HT, the positive control. DPT's E max was 83% ± 1.95 (mean ± SEM), relative to 5-HT. EC 50 and K i values are reported in Table. See Figure,B for affinity and function curves. We tested whether selective 5-HT 1B R antagonism would block DPT's anti-AGS effects. As shown in Figure, pretreatment with SB-224289 at 5 mg/kg increased seizure prevalence when compared to treatment with DPT 10 mg/kg alone (25 vs 0%). However, the difference was not statistically significant (p = 0.10). These observations suggest that activation of 5-HT 1B Rs does not mediate DPT's anti-AGS effects. Pan-5-HTR Antagonism Does Not Block DPT's Antiepileptic Effects. Given DPT's structural similarity and shared targets with 5-HT, and our objective to investigate the potential involvement of multiple 5-HTRs in mediating the anti-AGS effects of DPT, we next tested the effects of the pan- 5-HTR antagonist methiothepin. Pretreatment with methiothepin at 4 mg/kg did not influence DPT's anti-AGS effects (seizure prevalence 0 vs 0%; p > 0.99) (Figure). During the observation period, mice pretreated with methiothepin appeared to be highly sedated (partial or complete ptosis) while lying in a prone or medial-lateral position being either slightly responsive or unresponsive to the AGS-eliciting alarm. These effects were more prominent in the methiothepintreated group as compared to the pimavanserin 10 mg/kg group (data not shown). We did a pilot test of pretreatment with methiothepin at 2 mg/kg; it also did not impact the antiepileptic effects of DPT (data not shown). Collectively, our observations suggest that the anti-AGS effects of DPT may be mediated by a mechanism(s) that is independent of 5-HTRs. Antagonism of Sigma1Rs Does Not Block DPT's Antiepileptic Effects. Since no 5-HTR antagonist challenged DPT's antiepileptic efficacy in the AGS assay, we tested whether a nonserotonergic receptor could mediate DPT's antiepileptic effects. DPT and other tryptamines bind sigma1Rs, which studies have shown modulate epileptic activity.NE-100, a selective sigma1R antagonist, is structurally similar to DPT, sharing an N,N-dipropyl moiety(see Figuresand). NE-100 potentiates seizures at a 25 mg/ kg dose and induces seizures at 50 mg/kg and above.To determine suitable doses of NE-100 for evaluation in the AGS assay�doses of NE-100 that straddle the threshold dose for NE-100 causing seizures on its own�we tested NE-100 at six different doses, 5, 10, 15, 20, 30, and 50 mg/kg, in juvenile WT Figure. In vitro and in vivo pharmacology of DPT at 5-HT 2A Rs and examination of the impact of inhibiting 5-HT 2A/2C Rs on the antiepileptic properties of DPT. (A) In vitro radioligand competition binding of DPT and 5-HT at human (h) 5-HT 2A Rs. The 100 μM data point was interpolated, so curves reached asymptote (no specific binding). Data were obtained from two separate experiments in which 5-HT was tested in duplicate and DPT was tested in sextuplicate per concentration. Data best fit to a "two-site, fit K i " model, which is shown. (B) In vitro, functional activity of 5-HT and DPT at h5-HT 2A Rs. Data were obtained from three experiments in which 5-HT and DPT were tested in quadruplicate per concentration. (C) DPT dose-dependently elicited the 5-HT 2A R-dependent HTR, with maximal effects at 10 mg/kg in juvenile and adult WT mice. (We previously found no difference in DOI-elicited HTRs between WT and Fmr1 KO mice.) Note that this dose was equivalent to the effective dose of DPT to prevent seizures, demonstrating that DPT engaged 5-HT 2A Rs while it prevented seizures. *represents p < 0.05 and ## , **represents p < 0.01 compared to vehicle. Despite this, as shown in (D), the selective 5-HT 2A/2C R antagonist, pimavanserin, did not significantly block the anti-AGS effects of DPT in juvenile Fmr1 KO mice. **** represents p < 0.0001 DPT 10 mg/kg compared to vehicle; data reproduced from Figureto show the comparison to the pimavanserin-treated groups. Veh: Vehicle; D3, 5.6, 10, 15 and 20: DPT 3, 5.6, 10, 15 and 20 mg/kg; P3 and 10: pimavanserin 3 and 10 mg/kg. N = number of mice tested. All data with error bars are means and SEMs. and Fmr1 KO mice, and observed them for 30 min following administration. Like a previous report,we observed that 50 mg/kg NE-100 induced generalized TCS (Table), whereas the mice exhibited normal behavior when administered the lower doses. Thus, we tested the impact of 30 mg/kg and 50 mg/kg NE-100 on DPT's anti-AGS effects. Neither 30 mg/kg nor 50 mg/kg NE-100 pretreatment reversed the anti-AGS effects of 10 mg/kg DPT (all mice showed no AGS; 0%, p > 0.99) (Figure). DPT and NE-100 Cause Seizures on Their Own that Are Qualitatively Distinct from AGS. During doseresponse testing of DPT and NE-100, we observed that at high doses, i.e., 20 mg/kg DPT and 50 mg/kg NE-100, both compounds caused seizures on their own. Hence, we compared the behavioral signs of convulsions due to AGS, DPT, and NE-100; each caused a unique time-dependent repertoire of behavioral symptoms (Table). DPT 20 mg/kg induced convulsions in 90% of mice tested (N = 10 total WT mice tested). These began between 5 and 10 min after administration, and the duration was brief. Mice exhibited myoclonic tremors that transitioned to tonic convulsions with prominent opisthotonos, and 50% of the mice vocalized during the episode. The entire seizure episode lasted less than 20 s. All mice recovered, and then showed behavioral signs of 5-HT 1A R activation (see Table). One adult male mouse also showed excessive salivation after the seizure. NE-100 50 mg/kg induced convulsions in 69% of mice tested (N = 13 total tested, including 10 Fmr1 KO and 3 WT mice). Behaviors that preceded seizures and behaviors indicative of seizures were similar to those reported by Vavers et al.NE-100-induced seizures lasted 150-200 s, and mice recovered to normal behavior between 20 and 30 min after treatment. Like DPT, NE-100-induced seizures were not lethal. Importantly, we did not observe a treatment by genotype effect (Supplemental Figure). In the AGS experiments, cotreatment with NE-100 50 mg/kg and DPT 10 mg/kg (N = 8, the same subjects as those in Figure) caused seizures within ∼2 min of administration, i.e., prior to sounding the AGSeliciting alarm. These seizures presented the same symptomology as observed with NE-100 50 mg/kg alone. After sounding the alarm, the characteristics of the seizures observed were like NE-100-induced seizures. In other words, mice did not exhibit AGS (see Methods and Table). In conclusion, DPT and NE-100 caused seizures that were qualitatively distinct from AGS, suggesting different mechanisms.
We discovered that DPT prevents AGS in juvenile Fmr1 KO mice, a genetic model of FXS. We also report DPT's affinity and function at 5-HT 2A , 5-HT 1A , and 5-HT 1B receptors. DPT was effective at preventing AGS only at a 10 mg/kg dose, whereas at lower doses AGS persisted. In separate studies of in vivo receptor engagement, DPT activated 5-HT 2A receptors, i.e., elicited the HTR,at its antiepileptic dose. It took a higher dose to engage 5-HT 1A Rs, e.g., to elicit hind-limb abduction and flat body posture, which are elicited by 5-HT 1A R activation.These results corroborated our in vitro data which showed DPT was a moderate-potency full agonist at 5-HT 2A Rs but a low-potency partial agonist at 5-HT 1A Rs. We also showed that DPT was a low-potency 5-HT 1B R agonist in vitro. The in vitro and in vivo receptor pharmacology studies suggested that DPT engaged 5-HT 2A Rs but not 5-HT 1A or 5-HT 1B Rs at its antiepileptic dose. As an additional approach to investigate if these receptors contributed to DPT's antiepileptic properties, we tested if co-administration of selective 5-HT 2A/2C , 5-HT 1A , or 5-HT 1B R antagonists could block DPT's antiepileptic effects. None did nor did a pan 5-HTR antagonist. Interestingly, we observed that at high doses, DPT switched from an antiepileptic (in the AGS assay) to a proconvulsant, eliciting tonic seizures when administered on its own. The proconvulsant effects of DPT that we observed at 20 mg/kg align with a study in rats that showed DPT was proconvulsant at 30 mg/kg.Such dose-dependent switches in effects were also described with the 5-HT 1 R agonist, sumatriptan. Sumatriptan increases pentylenetetrazol-induced seizure thresholds in mice at 1 mg/kg but reduces the threshold at 20 mg/kg, suggesting the engagement of different targets or neural circuits at different doses.Similarly, 5-methoxy-N,Ndimethyltryptamine, mentioned in the Introduction section as having antiseizure effects in lateral geniculate kindled felines, has also been reported anecdotally to induce convulsions� akin to seizures�in humans when administered at strong doses (see experience ID:39420 and 76059 at Erowid.org). Based on the available evidence, the effects of DPT are dosedependent, consistent with DPT having polypharmacology like other tryptamines. Sigma1Rs are targets of several tryptaminesand modulate epileptiform activity,which provided us the rationale to investigate them as antiepileptic targets of DPT. We used the sigma1R antagonist, NE-100, based on its structural similarity to DPT. Two observations lead us to conclude that DPT's anti-AGS effects were not caused by activation or inactivation of sigma1Rs. NE-100 failed to reverse DPT's effects, and DPT failed to impact (either suppress or potentiate) NE-100elicited seizures. Also, NE-100 caused characteristically distinct convulsions at a 50 mg/kg dose. These seizures differed from AGS and DPT-induced seizures in terms of the behavioral sequelae and duration. Furthermore, drug-elicited seizures differ in underlying anatomical loci than AGS. Drug-elicited seizures affect various neural systems,whereas in Fmr1 KO mice, AGS have a localized origin, being dependent on altered activity in the inferior colliculus, an auditory pathway structure in the midbrain. One possible mechanism for why DPT was antiepileptic in the AGS assay is that it directly modulates auditory processing, reducing auditory hypersensitivity in Fmr1 KO mice. In humans, a closely related tryptamine N,N-diisopropyltryptamine reduces sound pitch and causes harmonic distortion while keeping the relationship between tones intact; subjects report that sounds from music are an octave lower than usual, i.e., as if they are listening to music underwater.The possibility that DPT or related tryptamines can target auditory processing is worth exploring in the future, as it may help improve understanding of auditory hypersensitivity in FXS and other neurodevelopmental disorders. A limitation of the in vivo pharmacological antagonism studies is the side-effect of sedation (no locomotion, flaccid bodies, and eyes closed or partially closed) caused by pimavanserin and methiothepin. The sedation caused by brain-wide inhibition of 5-HT 2A/2C Rs in the case of pimavanserin and brain-wide inhibition of 5-HT 1 , 5-HT 2 , 5-HT 3 , 5-HT 5 , 5-HT 6 , 5-HT 7 Rs (and other receptors) in the case of methiothepin may have been sufficient to block auditory signals from reaching the inferior colliculus to cause AGS. For example, 5-HT 2A R blockade in the frontal cortex may have diminished auditory processing, and potentiated the anti-AGS effects of DPT, i.e., could have had anti-AGS effects independent of DPT. However, we previously showed that the selective 5-HT 2A R antagonist/inverse agonist, M100907, which causes sedation in mice, does not block AGS in juvenile Fmr1 KO mice.Another possibility for the inefficacy of pimavanserin to block the anti-AGS effects of DPT is that DPT's effects were due to precise, localized modulation of 5-HT 2A Rs. 5-HT 2A R modulation�5-HT 2A R biased signaling, antagonism or agonism of distinct 5-HT 2A R signal transduction pathways�in auditory neural pathways might have underlied the antiepileptic effects of DPT, and we were unable to detect this because of brain-wide inhibition of 5-HT 2A Rs that obfuscated this effect. It is yet to be determined whether local blockade or inactivation of 5-HT 2A Rs in auditory neural pathways would block DPT's anti-AGS effects, and conversely, whether local activation of 5-HT 2A Rs by DPT would be sufficient to block AGS. DPT has not been studied extensively. A PubMed search of articles with "dipropyltryptamine" in their abstracts produced only 23 results. Little is known about DPT's pharmacodynamics. We investigated DPT's functional effects at 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs, using new TRUPATH technology, which probes the activity of ligands to stimulate individual Gα subunits coupled to GPCRs, and often, ligands have unique potencies to activate different Gα subunits.Future studies might find, for example, that DPT has different potencies at 5-HT 1A and 5-HT 1B coupled to Gα i/o family subunits other than Gα i3 , which we examined. One study assessed DPT's affinity and function at 5-HT 1A receptors, using [ 3 H]8-OH-DPAT and GTPγS incorporation, respectively; 71 DPT's 5-HT 1A R affinity was substantially higher than the affinity we measured with [ 3 H]5-CT competition binding, but it was a 5-HT 1A R partial agonist, like we observed. Another study reported that DPT was inactive at 5-HT 1A Rs up to 10 μM but used calcium mobilization as the functional readout.The higher affinity of DPT at 5-HT 1A compared to 5-HT 1B that we observed is similar to other psychedelic tryptamines.DPT's functional activity at 5-HT 2A Rs was also measured by Blough et al.,and its potency to stimulate canonical 5-HT 2A -Gα q signaling was higher than what we observed with TRUPATH; still, its full agonist efficacy was consistent with our results. Finally, there is no information to our knowledge about DPT's pharmacokinetics in any species. Thus, we are parsimonious in our conclusion about DPT's in vivo mechanism(s). We conjecture that our observations gel with recent research that concludes that some effects of psychedelics are mediated by nonserotonergic mechanisms.Our observations of an apparent nonserotonergic mechanism underlying the antiepileptic effects of DPT add to the growing literature about the pharmacological mechanisms underlying the potential therapeutic effects of serotonergic psychedelics.
All experimental protocols involving FVB.129P2-Pde6b + Tyr c-ch Fmr1 tm1Cgr /J (Fmr1 KO mice, stock #004624, Jackson Laboratory) and FVB.129P2-Pde6b + Tyr c-ch /AntJ (sighted FVB or WT mice, stock #004828) were approved by the Mercer University Institutional Animal Care and Use Committee and were performed following the Guide for the Care and Use of Laboratory Animals, 8th edition. We used Fmr1 KO juvenile mice (P23-P25), male and female, for tests of AGS. The mice were bred and raised in the vivarium at Mercer University College of Pharmacy as previously described.All tests were performed during the light cycle (7:00-19:00). Compounds. DPT hydrochloride, NE-100 hydrochloride, DOI hydrochloride, and methiothepin maleate were purchased from Cayman Chemical. WAY-100635 maleate was purchased from Tocris. Pimavanserin was obtained from Selleckchem, and SB-224289 hydrochloride was purchased from R&D Systems. 5-HT hydrochloride and mianserin hydrochloride were obtained from Alfa Aesar. For in vivo pharmacology tests, all compounds were dissolved in Milli-Q (Millipore Sigma) water, which served as the vehicle, except for SB-224289, which was dissolved in 2% DMSO, 4% Tween-80, and 4% PEG-20 and subsequently q.s. with Milli-Q. The solutions were made fresh on the day of the experiments. Vehicle and all compounds for in vivo studies were administered intraperitoneally (i.p.) to Fmr1 KO and WT mice at a volume of 1 mL/100 g. Doses of compounds were selected based on studies showing their in vivo efficacy. For in vitro pharmacology studies, 10 mM stocks of test ligands were prepared in DMSO. [ 3 H]Lysergic acid diethylamide (LSD) and [ 3 H]5-Carboxamidotryptamine (5-CT) were purchased from PerkinElmer and were diluted in assay buffer. Cell Growth, Maintenance, and Transfection. Plasmids encoding human 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs were obtained from the cDNA Resource Center. Dulbecco's modified Eagle's medium (DMEM) and OptiMEM were obtained from Gibco. Fetal bovine serum (FBS) and dialyzed FBS (dFBS) were purchased from Corning Life Sciences and Gibco. HEK 293 T cells (CRL-3216, ATCC) were used for in vitro binding and functional assays. Cells were cultured in 10 cm dishes with DMEM medium containing 10% FBS and were maintained in an incubator at 37 °C, 5% CO 2 , and 95% humidity. For radioligand competition binding assays, cells were transfected at ∼80% confluency with 7-10 μg of cDNA and 40 μg of transfection grade polyethyleneimine (PEI, 40,000 molecular weight, Polysciences, Inc., prepared as 1 mg/mL in Milli-Q). The transfection cocktail was prepared by separately mixing PEI and plasmids in two vials containing 2.5 mL of OptiMEM and then subsequently combining them. After incubating the transfection cocktail for 30 min at 37 °C, cells were washed with phosphate-buffered saline then cells were gently covered with the transfection cocktail together with 5 mL DMEM and a final concentration of 5% dFBS (transfection media). For the TRUPATH functional assays, cells at ∼80% confluency were transfected with 5-HT 2A , 5-HT 1A , and 5-HT 1B R cDNA (5-10 μg), Rluc8-Gα q for 5-HT 2A and Rluc8-Gα i3 for 5-HT 1A and 5-HT 1B Rs, untagged Gβ 3 , and GFP2-Gγ 9 plasmids in 1:1:1 ratio (750 ng) using the same transfection protocol described earlier. Radioligand Competition Binding. Cell membranes expressing 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs were collected after 48 h of transfection. Cells were collected and homogenized in ice-cold 50 mM Tris HCl buffer. Homogenate was spun thrice at 12,000 × g for 10 min at 4 °C using an Avanti JXN-26 centrifuge (Beckman Coulter). The supernatant was discarded after each spin and the final pellet was stored at -80 °C for later testing. Competition binding assays with DPT and control compounds were performed in 96 well plates, using ∼0.7 nM [ 3 H]LSD to radiolabel 5-HT 2A Rs, and ∼0.2 and ∼0.3 nM [ 3 H]5-CT to radiolabel 5-HT 1A and 5HT 1B Rs, respectively. Nonspecific binding was determined in the presence of 10 μM mianserin for 5-HT 2A Rs, 10 μM serotonin for 5-HT 1A Rs, and 10 μM SB-224289 for 5-HT 1B Rs. After the addition of assay buffer (50 mM tris-HCl, 10 mM MgCl 2 , and 0.1 mM EDTA, pH = 7.4 at room temperature), test ligands, radioligand, and cell membranes expressing 5-HT 2A , 5-HT 1A , or 5-HT 1B Rs, the plates were covered and incubated on a shaker for 90 min at room temperature. Plate contents were rapidly filtered through Whatman GF/B filter mats using a 96-well cell harvester (PerkinElmer) and then washed with ∼800 mL ice-cold 50 mM Tris•HCl to remove unbound radioligand. Filter mats were dried and saturated with a scintillation cocktail (ScintiVerse Cocktail, Fisher Scientific), and scintillations were counted using a PerkinElmer Microbeta 2 instrument. Bioluminescence Resonance Energy Transfer 2 Assay (TRUPATH). Cells were plated in white opaque 96-well microplates (Perkin Elmer) 48 h after transfection in bioluminescence resonance energy transfer (BRET) buffer at a density of 50,000 cells/well. After 2 h in an incubator, cells were treated with freshly prepared luminescent enzyme substrate coelenterazine (5 μM). After 5 min of the equilibration period, 5-HT (positive control) and DPT were added to the wells. After another 5 min, plates were then read in an LB940 Mithras plate reader (Berthold Technologies, Oak Ridge, TN) with 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters. G-protein activation was measured as BRET2 ratios (the ratio of the GFP2 emission to RLuc8 emission).Audiogenic Seizures. Experiments testing the induction of AGS in Fmr1 KO were conducted as previously described.Juvenile mice were acclimated to the test room for 30-60 min in their home cages. Mice were then administered vehicle or DPT at 3, 5.6, or 10 mg/kg. To determine the potential contribution of 5-HT 2A , 5-HT 1A, 5-HT 1B , other 5-HTRs, and sigma1Rs to the antiepileptic effects of DPT, separate groups of mice were pretreated with the selective 5-HT 2A R antagonist/inverse agonist pimavanserin (3 and 10 mg/kg),the selective 5-HT 1A R antagonist WAY100635 (0.1 and 1 mg/ kg),the selective 5-HT 1B R antagonist SB-224289 (5 mg/ kg),the pan-5-HTR antagonist methiothepin (2 and 4 mg/ kg),and the sigma1R antagonist NE-100 (30 and 50 mg/ kg)10 min before treatment with DPT (10 mg/kg). All mice were placed back in their cages and were tested 5 min after injection with DPT. Pretreatment periods were decided based on prior studies conducted in rodents.Also, we previously showed that 0.1 mg/kg WAY100635 at this pretreatment interval is effective at preventing the anticonvulsant effects of the selective 5-HT 1A R agonist, NLX-112.Mice were placed in a clear, polycarbonate box (46 cm × 20 cm × 20 cm) covered with a perforated, clear, polycarbonate lid 1 min before being exposed to an alarm (RadioShack Kit #49-1010, doorstop alarm). The alarm was held by hand ∼10 cm away from the test box and the duration of exposure was 5 min. A sound-level meter/data logger (REED Model SD-4023) was placed ∼20 cm from the alarm and read during testing to ensure a uniform level of sound pressure in each experiment. Tests were video-recorded using a high-definition camcorder (Vixia HF R800, Canon). A maximum of 4 mice (2 per box) were observed simultaneously by two experimenters.The average (±standard deviation (SD)) baseline sound pressure in the testing room was 55 ± 9 dB, and the average alarm sound pressure was 105 ± 4 dB. Behavioral responses, including normal behavior, WRJ, TCS, and death, were documented during AGS testing. Normal behavior was defined as coordinated locomotion, alertness, exploring, sniffing, sitting, rearing, grooming, socializing, and squinting of eyes. The beginning of AGS was marked by a startle response, squinting of eyes, followed by WRJ phase(s), brief opisthotonos, a clonic phase with the mouse lying on either side of its body with head, neck, trunk, and limbs ventroflexed (muscle jerking and twitching with rigidity), a short (∼5 s) tonic seizure phase with full extension of extremities (muscle stiffening), and finally, respiratory arrest. Seizure was defined by TCS. In the case of recovery from the TCS phase, mice exhibited a second round of WRJ, Straub tail, a full body vibrating shudder, and tremors which finally ended with either freezing or a transition to normal behavior. The frequencies of AGS were documented by visual observations of video recordings.DPT 5-HT 2A R and 5-HT 1A R In Vivo Pharmacology. WT mice were acclimated to a procedure room for ≥30 min before administering test compounds. Juvenile (P23-P25) and adult (>P60) mice were injected (i.p.) with Milli-Q water (vehicle) or DPT (3, 5.6, 10, 15, or 20 mg/kg) and were immediately placed in a clear open-field plexiglass chamber (43 × 43 cm; Med Associates). 5-HT 2A R-dependent HTRs were counted using a hand-held tally counter for 15 min postinjection. Locomotor activity (distance traveled in cm) was video recorded and calculated by Ethovision software (Noldus Information Technology). Observations of 5-HT 1A R-dependent effects (see Figure) were also documented. Statistical Analysis. Statistical tests were performed using GraphPad Prism, version 9. AGS and other behaviors were analyzed using Fisher's exact test (two-sided, α = 0.05). To evaluate the efficacy of various doses of DPT to elicit the HTR compared to vehicle, a one-way ANOVA with Holm-S ̌i ́daḱ's multiple comparisons test was used. Student's t test was used for HTR comparison between DOI and vehicle treatment. Nonlinear regression was used for analyzing in vitro pharmacology results. Of note, for the 5-HT 2A R binding, data fit best to a two-site model. For 5-HT and DPT binding at 5-HT 1A R and 5-HT 1B R, we used a one-site model.The Supporting Information is available free of charge at.
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