The Psychedelic N,N-Dipropyltryptamine Prevents Seizures in a Mouse Model of Fragile X Syndrome via a Mechanism that Appears Independent of Serotonin and Sigma1 Receptors

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.