A non-hallucinogenic LSD analog with therapeutic potential for mood disorders
This mice and cell study of the non-hallucinogenic LSD analogue 2-bromo-LSD (2-Br-LSD) found it to be a partial agonist at the 5-HT2A receptor but it doesn't activate the 5-HT2B receptor associated with cardiac valvulopathy (disease of heart valves). It also does not induce tolerance and has been shown to promote neuronal structural plasticity and active coping behaviour in mice. Additionally, 2-Br-LSD reverses the effects of chronic stress. These findings suggest that 2-Br-LSD may have therapeutic potential for mood disorders and other indications.
Abstract
Hallucinations limit widespread therapeutic use of psychedelics as rapidly acting antidepressants. Here we profiled the non-hallucinogenic lysergic acid diethylamide (LSD) analog 2-bromo-LSD (2-Br-LSD) at more than 33 aminergic G protein-coupled receptors (GPCRs). 2-Br-LSD shows partial agonism at several aminergic GPCRs, including 5-HT2A, and does not induce the head-twitch response (HTR) in mice, supporting its classification as a non-hallucinogenic 5-HT2A partial agonist. Unlike LSD, 2-Br-LSD lacks 5-HT2B agonism, an effect linked to cardiac valvulopathy. Additionally, 2-Br-LSD produces weak 5-HT2A β-arrestin recruitment and internalization in vitro and does not induce tolerance in vivo after repeated administration. 2-Br-LSD induces dendritogenesis and spinogenesis in cultured rat cortical neurons and increases active coping behavior in mice, an effect blocked by the 5-HT2A-selective antagonist volinanserin (M100907). 2-Br-LSD also reverses the behavioral effects of chronic stress. Overall, 2-Br-LSD has an improved pharmacological profile compared with LSD and may have profound therapeutic value for mood disorders and other indications.
Research Summary of 'A non-hallucinogenic LSD analog with therapeutic potential for mood disorders'
Introduction
Current pharmacotherapies for major depressive disorder (MDD) and anxiety disorders carry well-recognised drawbacks, including delayed therapeutic onset, the requirement for chronic dosing, and substantial rates of treatment resistance. Growing clinical interest has therefore focused on serotonergic psychedelics such as psilocybin, DMT, and LSD, which have shown durable reductions in depression and anxiety after only one or two doses in placebo-controlled trials. Their therapeutic effects are largely attributed to activation of the serotonin 2A (5-HT2A) receptor, though whether these benefits depend on the subjective hallucinogenic experience or can be achieved independently remains an open question. The present study aimed to characterise 2-bromolysergic acid diethylamide (2-Br-LSD, also known as BOL-148), a structural analogue of LSD first synthesised by Albert Hofmann that does not induce hallucinogenic effects in humans. Preliminary evidence had suggested that 2-Br-LSD may retain therapeutic activity despite lacking psychedelic properties, including findings demonstrating efficacy against cluster headaches. Lewis and colleagues sought to determine whether 2-Br-LSD shares the antidepressant-relevant pharmacological and neuroplasticity-promoting properties of classical psychedelics whilst avoiding the hallucinogenic effects that complicate clinical use.
Methods
A comprehensive pan-aminergic GPCR functional screening campaign was conducted using a bioluminescence resonance energy transfer (BRET)-based G protein dissociation assay platform across 33 human aminergic GPCRs, encompassing serotonin, dopamine, adrenergic, histamine, and muscarinic receptor subtypes. 2-Br-LSD and LSD were tested in parallel under conditions of full receptor occupancy, and relative activity (log Emax/EC50) was calculated to rank receptor targets. Complementary beta-arrestin2 recruitment BRET assays, calcium flux assays, and receptor internalisation measurements were used to characterise biased signalling profiles at key serotonin receptor subtypes. Pharmacodynamic properties of 2-Br-LSD were evaluated in male and female mice following intraperitoneal (i.p.) administration, with plasma and brain concentrations quantified using LSD-d3 as an internal standard across multiple doses and time points. Hallucinogenic potential was assessed in male C57BL/6J mice using the head-twitch response (HTR) assay over a 100-fold dose range (0.1-10 mg/kg i.p.), with additional experiments examining blockade of the psychedelic 5-HT2A agonist DOI and testing interactions with 5-HT1A and D2/3 receptor pharmacology. Tolerance liability was assessed by administering 2-Br-LSD or DOI once daily for seven consecutive days, then challenging with DOI 24 hours after the final injection. Structural neuroplasticity was examined in primary cortical neurons treated with 2-Br-LSD (1-100 nM) for three hours, with dendritic arbor complexity assessed at day in vitro 6 via Sholl analysis and spine density measured at day in vitro 18, using ketamine as an active comparator. In vivo behavioural activity was evaluated using the forced swim test (FST) and open field test (OFT) in male and female mice treated with three doses (0.3, 1.0, and 3.0 mg/kg) 24-25 hours prior to testing. A chronic variable stress (CVS) model over five weeks in female mice was used to assess reversal of stress-induced behavioural changes under either a single acute 3 mg/kg dose or four 1 mg/kg doses of 2-Br-LSD. The 5-HT2A dependence of all key effects was confirmed using the selective antagonist volinanserin in both in vitro and in vivo settings.
Results
Across the 33-receptor GPCR screen, 2-Br-LSD demonstrated agonist activity at 14 aminergic receptors, 10 of which were serotonin GPCRs. Notably, it exhibited potent partial agonism at 5-HT2A (EC50 = 0.81 nM, Emax = 59.8%), a markedly lower efficacy than LSD (Emax = 91.5%), and acted as a potent antagonist at 5-HT2B, in contrast to LSD, which is a robust 5-HT2B agonist. 2-Br-LSD also showed potent pan-agonism at all 5-HT1 receptor subtypes, partial agonism at D2 and D4 receptors, and a broadly cleaner cardiovascular off-target profile than LSD, including very weak hERG channel blockade (EC50 = 31.6 µM) and no significant activity at muscarinic or most adrenergic subtypes. In the HTR assay, 2-Br-LSD did not induce head twitches at any dose tested (0.1-10 mg/kg), whilst LSD at 0.1 mg/kg produced a significant response. Pre-treatment with 2-Br-LSD attenuated DOI-induced HTR by up to 76% (at 3 mg/kg), confirming meaningful 5-HT2A occupancy in the brain. Repeated treatment with 2-Br-LSD for seven consecutive days produced no evidence of tolerance in the HTR assay, whereas DOI caused significant tachyphylaxis (p < 0.001). In vitro, 2-Br-LSD showed substantially weaker beta-arrestin2 recruitment at 5-HT2A (Emax = 36.9%) and reduced receptor internalisation compared with LSD and DOI. In primary cortical neurons, 2-Br-LSD (1-10 µM) produced dose-dependent increases in dendritic arbor complexity and spine density comparable to ketamine (10 µM), with no effect on neuronal viability. In the FST, significant reductions in immobility were observed in female mice (-35.2 ± 10.0 s at 1 mg/kg; p = 0.0089) and in male mice at all doses tested (p = 0.0024). Female mice also showed an 88.2 ± 18.9 second increase in open field centre exploration at 1 mg/kg with no change in locomotion, indicating anxiolytic-like activity. In the CVS model, the repeated low-dose regimen (4 x 1 mg/kg) restored centre exploration in stressed mice to levels matching naive controls and partially reversed deficits in the splash test, with effects on open field behaviour persisting at a 28-day follow-up. Pre-treatment with volinanserin blocked all neuroplasticity and behavioural effects, confirming their 5-HT2A dependence.
Discussion
Lewis and colleagues interpret their findings as demonstrating that 2-Br-LSD has a pharmacological profile consistent with therapeutic utility for mood disorders whilst circumventing the key liabilities of classical psychedelics. The compound's partial 5-HT2A agonism and relatively low Emax are proposed to explain its lack of hallucinogenic activity, consistent with published correlations between 5-HT2A agonist efficacy and peak HTR rates, and supported by historical human data indicating the absence of LSD-like experiences at clinically relevant doses. The potent 5-HT2B antagonism is additionally highlighted as a favourable safety feature, since chronic 5-HT2B activation has been associated with fibrotic cardiac valvulopathy, and the absence of this liability may permit more frequent dosing than is feasible with classical psychedelics. The lack of tolerance after repeated dosing is attributed to 2-Br-LSD's weak beta-arrestin2 recruitment at 5-HT2A, a pathway implicated in receptor downregulation and internalisation. The researchers draw parallels between 2-Br-LSD's neuroplasticity-promoting effects and those reported for psilocybin, LSD, and 5-methoxy-DMT, suggesting a shared mechanism centred on 5-HT2A-mediated reversal of neuronal atrophy in prefrontal cortical circuits. This hypothesis is supported by the volinanserin blockade data, which confirmed 5-HT2A dependence of both the structural plasticity and behavioural effects in the present study. Several limitations are acknowledged. In vitro GPCR assays may not accurately reflect in vivo receptor pharmacology due to cell-type-specific expression of G protein and beta-arrestin subtypes. The HTR model does not fully recapitulate the human psychedelic experience, and the validity of the FST as a depression model has been questioned. The researchers note, however, that concordant results obtained in the CVS paradigm, which carries greater construct validity, strengthen the overall conclusions. Further clinical studies and additional preclinical models, including chronic social defeat and physiological biomarkers such as inflammatory markers and hypothalamic-pituitary-adrenal axis measures, are identified as necessary next steps to confirm the antidepressant potential of 2-Br-LSD in humans.
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IN BRIEF
Lewis et al. perform an extensive pharmacological characterization of 2-Br-LSD, finding distinct aminergic GPCR polypharmacology, including 5-HT 2A partial agonism and lack of psychedelic-like effects in vivo. Further, 2-Br-LSD induces dendritogenesis and spinogenesis in vitro while promoting active coping behavior in vivo, effects dependent on 5-HT 2A activation.
INTRODUCTION
Current pharmacotherapies for major depressive disorder (MDD) and anxiety disorders, which are often comorbid,have drawbacks, including delayed therapeutic onset, the need for chronic dosing, and large numbers of treatment-resistant patients.Recently, there has been growing interest in psychedelics as treatment for a range of psychiatric disorders.Psychedelics such as psilocybin, N,N-dimethyltryptamine (DMT), and (+)-lysergic acid diethylamide (LSD) can induce mystical states and profound alterations of consciousness, effects that are largely mediated by serotonin 2A (5-HT 2A ) receptor activation.In multiple double-blind, placebo-controlled trials, psilocybin, DMT, and LSD produced long-lasting reductions in depression and anxiety after only one or two doses.However, therapeutic use of psychedelics has limitations, including their intense hallucinogenic effects, which require close clinical supervision, as well as anxiety and confusion in some patients.The degree to which the therapeutic effects of serotonergic psychedelics are linked to their subjective effects is not entirely clear. In several clinical trials, the level of symptom reduction produced by psilocybin was significantly correlated with metrics of drug-induced psychedelic phenomenology.Thus, it has been proposed that the subjective effects of psychedelics are required for their therapeutic effects.However, it may be possible to decouple the hallucinogenic effects of psychedelic drugs from their therapeutic effects.The intensity of the psychedelic response induced by psilocybin is closely related to the level of 5-HT 2A occupation,and this correlation could reflect a relationship between therapeutic response and target engagement. Furthermore, the pharmacological mechanism for the antidepressant effects of psilocybin has not been characterized and may include other receptors.An attractive hypothesis for the antidepressant effects of psychedelics involves rapid induction of structural and functional neural plasticity and reversal of neuronal atrophy in cortical regions.Prefrontal pyramidal neurons exert top-down control over activity in regions involved in emotional processing, motivation, and reward, and atrophy of the spines and dendrites of pyramidal neurons could contribute to depression symptomology.Preclinical models show that some psychedelic analogs do not produce behavioral effects associated with hallucinogenic effects but retain the ability to promote cortical neuritogenesis, similar to established antidepressant drugs.Lysergic acid derivatives were a focus of intense research during the 1940s and 1950s. LSD was first synthesized by Dr. Albert Hofmann in 1938and was investigated as a potential treatment for an extensive range of disorders.Hofmann also synthesized (+)-2-bromolysergic acid diethylamide (2-Br-LSD, BOL-148; Figure), which did not induce hallucinogenic effects in humans.Similar to LSD and psilocybin,2-Br-LSD recently showed efficacy against cluster headaches,which is surprising because 2-Br-LSD was initially described as a 5-HT 2A antagonistand could block the psychological response to LSD.Based on these findings, the possibility exists that 2-Br-LSD may also mimic some of LSD's antidepressant and anxiolytic effects. Therefore, this study aimed to investigate the pharmacological profile of 2-Br-LSD, its psychedelic-like effects, and the potential for mood disorder treatment.
RESULTS
Profiling 2-Br-LSD across the serotonergic and aminergic GPCRome Ergolines like LSD have a pronounced aminergic G proteincoupled receptor (GPCR) polypharmacology,making it necessary to interrogate 2-Br-LSD at many targets. Importantly, GPCR functional efficacy needs interrogation because traditional radioligand binding assays (i.e., K i determinations) may not accurately assess agonist molecular efficacy.Therefore, we performed a pan-aminergic-wide GPCR functional screening campaign using a G protein dissociation BRET-based assay platformoptimized for 33 human aminergic GPCRs (including serotonin, dopamine, adrenergic, histamine, and muscarinic subtypes; Figure; Table). We screened 2-Br-LSD and LSD in parallel at each of the 33 aminergic GPCR subtypes, measuring select canonical G protein dissociation activity under conditions necessary for full receptor occupancy (37 C/60 min; supplemental information), which is critical to offset LSD's slow binding kinetics.Next, we ranked the top GPCR targets for 2-Br-LSD and LSD by calculating their relative activity (log E MAX /EC 50 ) using the endogenous control standard and plotted a heatmap of activities (Figure). Calculated potency parameters (EC 50 and K B estimates; Table) at select aminergic GPCRs were similar to affinity (K i ) values determined in radioligand binding studies (Table). Interestingly, all five 5-HT 1 subtypes are within the top 10 targets of 2-Br-LSD, including the known anti-migraine drug targets 5-HT 1B/1D/1F(Figure; Table). In fact, 2-Br-LSD demonstrated potent pan-agonism at all 5-HT 1 G i/o -coupled receptor subtypes with similar G protein efficacies as LSD (Figure). At the other G i/o -coupled serotonin GPCR, 5-HT 5A , however, 2-Br-LSD was a potent antagonist (K B = 4.14 nM; Table), whereas LSD showed partial agonism. Within the top ten targets, 2-Br-LSD and LSD demonstrated sub-nanomolar partial agonism at 5-HT 6 (EC 50 = 0.35 and 0.13 nM, respectively; Figure), which is an emerging target for cognitive deficits.At other G s -coupled serotonin GPCRs, LSD and 2-Br-LSD lack potent agonist or antagonist activity at 5-HT 4 . At the 5-HT 7a subtype, however, 2-Br-LSD and LSD act as antagonists (Table), and both exhibit potent inverse agonism in a cyclic AMP (cAMP) accumulation assay (EC 50 = 5.1 and 17.3 nM, respectively; Figure). Two dopamine receptors, D 2 and D 4 , were also within the top 10 targets activated by 2-Br-LSD (EC 50 = 0.35 and 1.2 nM, respectively; Figures, and S2A; Table). At the D 3 subtype, however, 2-Br-LSD is a weaker partial agonist (E MAX = 32% relative to dopamine) compared with LSD (E MAX = 75%). Interestingly, 2-Br-LSD lacks strong agonism at D 1/5 subtypes, whereas LSD demonstrates agonist activity, as reported previously(Figure; Table). Surprisingly, 5-HT 2A and 5-HT 2C were at the bottom of the top 10 ranking list for 2-Br-LSD (Figure). At 5-HT 2A , 2-Br-LSD demonstrated Gq partial agonism (EC 50 = 0.81 nM, E MAX = 59.8%), whereas LSD was almost a full agonist at this receptor (EC 50 = 0.35 nM, E MAX = 91.5%). At 5-HT 2C , LSD is almost a full agonist, whereas 2-Br-LSD exhibits weaker Gq partial agonism (E MAX = 45.8%). Furthermore, we confirmed many of the top 10 GPCR activities in orthologous assays measuring G protein-dependent second messenger assays (Figure). At the remaining aminergic GPCRs, LSD and 2-Br-LSD show weaker agonist activity in general (Figures). At adrenergic GPCR subtypes, 2-Br-LSD displayed antagonistic activity at a 1A , a 1B , b 1 , and b 2 (K B = 43, 38, 113, and 47 nM, respectively; Table). Interestingly, differences between LSD and 2-Br-LSD were measured at a 2A and a 2B , where LSD shows partial agonism (E MAX = 65% and 62%, respectively; Table), and 2-Br-LSD instead antagonizes these receptor subtypes (K B = 12 and 79 nM, respectively). By contrast, LSD and 2-Br-LSD show partial agonism at the a 2C subtype (E MAX = 80.2% and 40.5%, respectively; Table), but LSD is more efficacious and potent than 2-Br-LSD (EC 50 = 0.56 and 10.4 nM, respectively). At histaminergic receptors, weak partial agonism of 2-Br-LSD was detected at H 2 that was only slightly greater than that of LSD. Importantly, neither 2-Br-LSD nor LSD possessed weak agonism or antagonism at the rest of the histamine and muscarinic GPCRs (Table). In summary, LSD exhibited agonist activity at 20 of 33 aminergic GPCR targets). (D) Top 10 targets of 2-Br-LSD agonist activity in the BRET aminergic GPCRome activity assays comparing 2-Br-LSD (blue) with LSD (red) and the positive control (black; 5-HT for serotonin receptors and DA for dopamine receptors). Data represent mean ± SEM from at least 3 independent experiments performed in triplicate, and are all normalized to their respective positive control. Related to Figuresand; Tables. tested, but 2-Br-LSD was only active as an agonist at 14 of these GPCRs; notably, 10 of the 14 were serotonin GPCRs. 2-Br-LSD is a 5-HT 2A partial agonist and competitive partial antagonist The activated 5-HT 2A receptor is a primary mediator of the psychedelic state and is responsible for the hallucinogenic effects of LSD.While LSD acts as a highly efficacious Gq agonist at 5-HT 2A , 2-Br-LSD produces only partial 5-HT 2A activation (E MAX = 59.8%) but maintains high potency (EC 50 = 0.81 nM; Table), similar to LSD. Partial agonists can also act as partial antagonists, given the basal levels of endogenous neurotransmitters in the brain,as noted for 5-HT 2A receptors.Therefore, to assess partial antagonism of the receptor, 2-Br-LSD was tested as an antagonist in 5-HT 2A Gq dissociation and b-ar-restin2 recruitment assays (Figuresand), where 5-HT and 2-Br-LSD were added simultaneously and incubated for 60 min to detect partial inhibition. Here, 2-Br-LSD potently and partially antagonized 5-HT 2A Gq and b-arrestin2 agonism by 5-HT (K B = 0.18 and 0.07 nM, respectively; Table).
-BR-LSD PHARMACOKINETICS IN THE PLASMA AND BRAIN
The pharmacokinetics of 2-Br-LSD were evaluated in mice to confirm bioavailability and brain penetrance (Figures 2C and S3A-S3C; Table). After intraperitoneal (i.p.) administration, plasma levels of 2-Br-LSD, quantified using LSD-d 3 as an internal standard, increased in a dose-and time-dependent manner. 2-Br-LSD was detected in the plasma 10 min post injection in all mice (Figuresand), with a time to maximum concentration (T max ) of 0.2 h, except for male mice treated with 0.75 mg/kg (T max = 0.5 h). Plasma concentrations were 2-5 times higher in male compared with female mice; for example, the mean maximum plasma concentration (C max ) in mice treated with 6.75 mg/kg 2-Br-LSD was 1,558.74 ng/mL (3.9 mM) in males and 826.06 ng/mL (2.1 mM) in females. As was the case for C max , the mean terminal half-life (T 1/2 ) was dependent on sex and dose, with a range of 1.2-1.4 h for male mice and 0.9-2.6 h for females. 2-Br-LSD rapidly crossed the blood-brain barrier with a mean T max of 0.17 h in male (Figure) and female mice (Figure). The mean T 1/2 of 2-Br-LSD in the brain ranged from 0.7-1.0 h for males and 0.4-1.3 h for females (Table). The level of 2-Br-LSD in the brain was below the lower limit of detection (LLOD) 4 h post dosing. The mean brain/plasma ratios for 2-Br-LSD were dose and time dependent and ranged from 0.27-0.75 at the 10-min point post injection.
EFFECT OF 2-BR-LSD ON THE HEAD-TWITCH RESPONSE IN MICE
The head-twitch response (HTR) is a rapid side-to-side rotational head shaking induced by psychedelic drugs in mice via 5-HT 2A receptor activationand serves as a behavioral proxy in mice for human hallucinogen effects because non-hallucinogenic 5-HT 2A receptor agonists do not induce head twitches.2-Br-LSD was tested in male C57BL/6J mice over a 100-fold range of doses (0.1-10 mg/kg i.p.) but did not induce the HTR (Figure; F 5,25 = 1.91, p = 0.1282). Administration of 0.1 mg/kg LSD, by contrast, produced a significant increase in HTR counts (t 9 = 8.35, p < 0.001). Thus, 2-Br-LSD acts as a non-hallucinogenic 5-HT 2A agonist in mice, consistent with reports in humans. Although 2-Br-LSD is brain penetrant (Figure), we tested whether pre-treatment with 2-Br-LSD can block the HTR induced by the psychedelic 5-HT 2A agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) to confirm that lack of the HTR was not due to a low level of 5-HT 2A receptor occupation in the brain. As expected, 2-Br-LSD attenuated the response to DOI in a dose-dependent manner (F 4,26 = 17.96, p < 0.0001; Figure) and produced a high level of blockade (76% attenuation at 3 mg/kg 2-Br-LSD). Additionally, we examined the time course of the interaction between 2-Br-LSD and DOI (Figure). In mice pre-treated with 1 mg/kg 2-Br-LSD, the response to DOI was almost completely blocked during the first 10 min and then gradually returned to control levels after about 40-60 min, yielding a significant drug 3 time interaction (F 47,658 = 12.45, p < 0.0001). These results indicate that 2-Br-LSD produces significant occupation of 5-HT 2A receptors in the brain for at least 30 min after i.p. administration, matching the pharmacokinetics of 2-Br-LSD in the mouse brain. 2-Br-LSD antagonized the effect of DOI on 5-HT 2A activation, providing further validation in vitro (Figure; K B = 0.17 nM). There are countervailing interactions between 5-HT 1A and 5-HT 2A receptors,and activation of 5-HT 1A can block the HTR induced by psychedelic drugs.Because 2-Br-LSD acts as a potent full agonist at the 5-HT 1A receptor, it is possible that 2-Br-LSD's 5-HT 1A agonism masks the HTR.We therefore tested whether 2-Br-LSD can induce the HTR in the presence of WAY-100635. Pre-treatment with 1 mg/kg WAY-100635 had no effect on the response to 2-Br-LSD in the HTR assay (Figure; WAY-100635 3 2-Br-LSD interaction: F 1,20 = 0.12, p = 0.735). These results confirm that 2-Br-LSD's 5-HT 1A agonism is not suppressing the HTR. We also tested whether 2-Br-LSD's D 2 agonism could explain the lack of HTR. 2-Br-LSD did not induce the HTR after pre-treatment with the selective D 2/3 antagonist S-(À)-raclopride (pretreatment 3 treatment: F 1,16 = 3.32, p = 0.0874; Figure). In addition, although co-administration with the D 2/3 agonist (À)-quinpirole attenuated the response to DOI (F 2,13 = 6.02, p = 0.0141), the inhibitory effect was feeble (an $20% reduction; Figure). Another study reported that quinpirole does not alter the HTR induced by LSD.Therefore, the lack of HTR activity is not likely due to D 2 receptor activation. 2-Br-LSD has a safer cardiovascular profile compared with LSD Chronic 5-HT 2B receptor activation can cause fibrotic cardiac valvulopathy,which necessitated withdrawal of multiple US Food and Drug Administration (FDA)-approved drugs,making 5-HT 2B off-target activity a critical liability.While LSD has robust agonist activity at 5-HT 2B , 2-Br-LSD failed to induce 5-HT 2B Gq dissociation, b-arrestin2 recruitment, or Gq-mediated calcium flux and instead demonstrated potent antagonist activity (Figures). We showed above that 2-Br-LSD shows weak activity at aminergic GPCRs known to affect blood pressure, heart rate, and other autonomic functions. Next, 2-Br-LSD was also tested in a Eurofins panel of 44 known off-targets (Table) and other transporters (Table) and was mostly inactive at all targets at concentrations up to 10 mM (including serotonin 5-HT 3 ).. Exceptions were that 2-Br-LSD showed low micromolar activity at Nav 1.5 sodium channels (EC 50 = 1.1 mM) and submicromolar activity at OCT2 (half-maximal inhibition [IC 50 ] = 0.5 mM). Importantly, 2-Br-LSD produced a weak blockade (Figure; EC 50 = 31.6 mM) of the hERG (K V 11.1) channel, an effect known to cause cardiac arrhythmias. Despite the detected off-target activities in the sub-micromolar range, 2-Br-LSD possesses a greater than 100-fold preference for indicated serotonin and dopamine GPCRs over these off targets, demonstrating a safer cardiovascular toxicity profile. 2-Br-LSD produces weak 5-HT 2A b-arrestin recruitment and has reduced potential to induce tolerance in vivo b-Arrestin recruitment is an important signaling pathway for GPCR internalization and downregulation,and GPCR biased agonism is an essential parameter for on-target efficacy and drug development.Experiments were conducted using b-ar-restin2 recruitment BRET assays to determine biased signaling differences at every 5-HT receptor except 5-HT 7 (Figuresand). We calculated the relative activity (log E MAX /EC 50 ) and plotted a heatmap comparing 5-HT, LSD, and 2-Br-LSD activities (Figure; Table). At 5-HT 1A and 5-HT 2C receptors, where 2-Br-LSD's G protein agonism was robust, we observed no detectable b-arrestin2 recruitment agonism despite LSD and 5-HT showing similar or comparable b-arrestin2 recruitment at these receptor subtypes, indicative of G protein bias (Figuresand). Notably, we observed very little difference in 5-HT 2A -biased agonism for 2-Br-LSD when comparing Gq with b-arrestin2 recruitment activities (Figure). Similar to Gq dissociation efficacy, 2-Br-LSD was a partial agonist for 5-HT 2A b-arrestin recruitment (E MAX = 36.9%, Figure). To confirm the weak b-arrestin efficacy in an orthologous assay, we measured loss of surface of expression using a NanoBit N-terminal HiBiT-fused 5-HT 2A receptor. After 60 min of treatment, 2-Br-LSD exhibited a partial agonist effect on 5-HT 2A internalization (Figure), consistent with our assessment of b-arrestin2 recruitment using BRET-based assays. Because 2-Br-LSD produces a much weaker level of b-arrestin recruitment and internalization compared with psychedelics such as DOI and LSD, studies were conducted to test whether 2-Br-LSD can induce receptor downregulation and tolerance in vivo. Mice received i.p. injections of vehicle, DOI (10 mg/kg/day), or 2-Br-LSD (3 mg/kg/day) once daily for 7 consecutive days and were then challenged with DOI (1 mg/kg) 24 h later (Figure). While repeated treatment with DOI inducedand. Article a significant degree of tachyphylaxis (p < 0.001 versus control, Dunnett's test), no tolerance was observed on the HTR after repeated treatment with 2-Br-LSD (Figure).
-BR-LSD PROMOTES NEURONAL STRUCTURAL PLASTICITY
Loss of neurites, synaptic spines, and contacts in cortical neurons are distinctive aspects of depression pathology,and neural plasticity is thought to underlie the therapeutic response to antidepressant drugs.To determine whether 2-Br-LSD can induce structural plasticity and increase arbor complexity, cultured primary cortical neurons were treated with 2-Br-LSD (1-100 nM) for 3 h on day in vitro 3 (DIV3), and then morphological changes in dendritic arbor complexity were measured on DIV6. As an active comparator, we tested ketamine (10 mM), which induces dendritogenesis and synaptic plasticity, effects potentially underlying its antidepressant activity.2-Br-LSD produced a dose-dependent increase in the number of dendrites crossing the Sholl radii, reaching a maximal effect at the two highest concentrations (1 and 10 mM) (Figuresand; 1-way ANOVA: F 6,35 = 3.287, p = 0.0114). At these two concentrations, the effect of 2-Br-LSD was similar to the effect of ketamine (Figuresand; p = 0.0096, control versus ketamine, Bonferroni's test). Accordingly, 1 and 10 mM 2-Br-LSD increased the total length of the dendritic arbor compared with controls (Figuresand; 1-way ANOVA: F 6,35 = 4.49, p = 0.0018). Next, we analyzed effects of 2-Br-LSD treatment (3 h) on primary cortical neuron spine density at DIV18. An overall significant treatment effect was found 24 h after incubation onset (Figuresand, 1-way ANOVA: F 6,63 = 22.12, p < 0.0001). Specifically, spine density increased after a 3-h incubation with 2-Br-LSD (1 and 10 mM) or ketamine (10 mM) compared with vehicle-treated neurons. The increase in spine density induced by 10 mM 2-Br-LSD was comparable with the effect of ketamine (Figuresand). We also tested the effect 2-Br-LSD on the viability of cultured primary rat cortical neurons and determined that it was not different from control neurons at every tested concentration (Figure; 1-way ANOVA: F 6,14 = 0.8030, p = 0.5838). This indicates that dendritic complexity and dendritic spine density increase at 2-Br-LSD concentrations that do not affect neuronal viability. 2-Br-LSD promotes exploration of stressogenic environments, active coping behaviors, and cortical spinogenesis in vivo LSD and other psychedelics have been shown to relieve depressive symptoms in treatment-resistant MDDand induce behavioral effects in rodents comparable with first-line antidepressants and rapidly acting treatments such as ketamine.To test the potential therapeutic activity of 2-Br-LSD for mood disorders, we evaluated the effects of 2-Br-LSD on activity in the forced swim test (FST) and open field test (OFT), which have been used to screen antidepressant and anxiolytic drugs, respectively.Male and female mice were treated with three doses of 2-Br-LSD (0.3, 1.0, and 3.0 mg/kg) and were evaluated 24 h (OFT) and 25 h (FST) later (Figure), when 2-Br-LSD had cleared from the brain (Figuresand). In the OFT, neither females nor males showed a significant increase in locomotion (distance traveled) following 2-Br-LSD treatment (Figuresand; Brown-Forsythe ANOVA: F 3.00,29.02 = 0.1320, p = 0.9403 and 1-way ANOVA: F 3,39 = 1.824, p = 0.1587, respectively). Importantly, female mice showed increased exploration of the arena center after the 1 and 3 mg/kg treatments (Figuresand; 1-way ANOVA: F 3,40 = 7.431, p = 0.0005), with maximal effects (an increase of 88.18 ± 18.89 s) at the 1 mg/kg dose. Despite a similar trend, the increased exploration of the open field stressogenic area with 2-Br-LSD was not significant in male mice (Figuresand, 1-way ANOVA: F 3,39 = 2.005, p = 0.1291). These results indicate potential anxiolytic effects of 2-Br-LSD in female mice because it increased the exploration of stressogenic environments at doses with no effect on locomotor activity. In the FST, we observed a 35.18 ± 10.03 s decrease in immobility in females at the 1 mg/kg dose (Figure; 1-way ANOVA: F 3,39 = 4.438, p = 0.0089). A similar effect was observed in males at all doses tested (Figure; 1-way ANOVA: F 3,39 = 5.739, p = 0.0024). Decreases in immobility induced by the 0. Following FST testing, brains were collected for synaptic spine analysis $26 h after treatment. We focused on the prefrontal cortex (PFC), given its central role in the response to rapidly acting antidepressantsand controlling active stress-coping behaviors.We found a significant increase in average spine density following 2-Br-LSD treatment in both sexes compared with controls (Figuresand; females: t 20 = 2.142, p = 0.0447; males: t 21 = 3.382, p = 0.0028).
-BR-LSD REVERSES THE BEHAVIORAL EFFECTS OF CHRONIC STRESS IN MICE
Chronic stress is a risk factor for many mood disorders, including MDD.Exposure to chronic stress in rodents leads to behavioral, structural, and molecular adaptations relevant to MDD and other psychiatric disorders.These alterations can be reversed by antidepressant treatments such as ketamine and serotonergic hallucinogens, including LSD.To investigate whether 2-Br-LSD can reverse the dysregulated behavioral effect of chronic stress, female mice were subjected to a chronic variable stress (CVS) regimen over 5 weeks,which co-terminated with two different 2-Br-LSD treatment regimens: 1 dose (D) Graphs of loss of surface expression of 2-Br-LSD (blue) to LSD (red), DOI (green), and 5-HT (black) as measured in the NanoBit internalization assay. Data represent mean ± SEM from at least 3 independent experiments performed in triplicate, normalized to percent 5-HT remaining surface expression. (E and F) Lack of tolerance to a 5-HT 2A agonist after repeated treatment with 2-Br-LSD. Mice were injected i.p. once per day with vehicle (n = 7), 2-Br-LSD (3 mg/kg, n = 7), or DOI (10 mg/kg, n = 7) for 7 consecutive days and then challenged with DOI (1 mg/kg i.p.) 24 h after the last injection. Data are presented as group means ± SEM over the entire 30-min test session. ****p < 0.0001, significant difference between groups (Dunnett's test). Related to Figure. after the last day of stress (3 mg/kg i.p., CVS 2-Br-LSD 1 3 3 mg/kg group) or 4 lower doses (1 mg/kg i.p., CVS 2-Br-LSD 4 3 1 mg/kg group) administered every 48 h, starting on day 28 of CVS (Figure). In the OFT, CVS induced a 55.95 ± 19.28 s decrease in the time mice spent exploring the center of the chamber (Figure; 1-way ANOVA: F 3,43 = 4.649, p = 0.0067) without changing the total distance traveled (Figure; 1-way ANOVA: F 3,43 = 1.074, p = 0.3702). Exploration of the arena center was increased by the repeated 2-Br-LSD treatment regimen in CVS mice to levels matching the control (naive-saline) group (4 3 1 mg/kg; Figure) without affecting locomotion (Figure). The acute 2-Br-LSD treatment (1 3 3 mg/kg) partially restored the effect of CVS because this group spent time in the center of the open field intermediate between the CVS-saline and naive-saline groups (Figure). CVS also reduced the time spent self-grooming in the splash test (Figure; 1-way ANOVA: F 3,40 = 3.016, p = 0.0410), an ecologically relevant measure of self-care that is sensitive to stress in mice.Female mice in the CVS-13-2-Br-LSD and CVS-43-2-Br-LSD groups had grooming levels intermediate between the naive-saline and CVS-saline groups, indicating a partial reversal of CVS effects (Figure). The same cohort of mice was tested 28 days after the last treatment. At this time, only the effects of CVS in the OFT were evident (Figure), while effects in the splash test appeared to have washed out (Figure). Indeed, just as during the day of treatment (Figuresand), the CVS-saline group had a persistent decrease in time exploring the center of the open field (Figure; 1-way ANOVA: F 3,42 = 4.337, p = 0.00095) that remained reversed by the 2-Br-LSD 4 3 1 mg/kg treatment regimen (Figure). Overall, these data support a therapeutic effect of 2-Br-LSD against the maladaptive effects of chronic stress. The effects of 2-Br-LSD on dendritogenesis and active coping behavior are mediated by 5-HT 2A activation Given the activity of 2-Br-LSD at the 5-HT 2A receptor, we treated primary cortical neurons with the selective 5-HT 2A antagonist volinanserin (M100907 [Vol]) at 0.1-1 mM prior to the administration of 2-Br-LSD (1 mM). Calcium flux assays conducted with 5-HTstimulated 5-HT 2 subtypes confirmed that Vol acts as a selective 5-HT 2A antagonist, with greater than 240-and 5,000-fold selectivity over 5-HT 2C and 5-HT 2B , respectively (Figure). Administered alone, Vol did not change any parameter linked to dendritic arbor complexity at any concentration assayed (Figures). Pre-treatment with Vol at every concentration tested blocked the effect of 2-Br-LSD on dendritic arbor complexity, as observed using Sholl intersection analysis, to levels seen in control neurons (Figuresand; 1-way In vitro, Vol was able to completely block the partial Gq and b-arrestin2 agonism of 2-Br-LSD at 5-HT 2A when tested at similar concentrations (Figuresand). In vivo, Vol pre-treatment (0.125 mg/kg) blocked the decrease in immobility induced by 2-Br-LSD (1 mg/kg) in the FST in females (Figure; pre-treatment 3 treatment interaction: F 1,43 = 5.32, p = 0.026) and males (Figure; pre-treatment 3 treatment interaction: F 1,44 = 5.441, p = 0.0243). Neither Vol nor a combination of Vol and 2-Br-LSD affected locomotion in the OFT (Figuresand).
DISCUSSION
Psychedelic drugs such as LSD and psilocybin induce intense hallucinogenic effects via 5-HT 2A receptor activation and show promise as potential treatments for depression and anxiety. Although LSD and psilocybin appear to have considerable therapeutic efficacy and are currently being evaluated as potential medications, developing psychedelic analogs that are therapeutic but have less hallucinogenic potential will be useful. The present investigation focused on the LSD analog 2-Br-LSD, which reportedly does not possess LSDlike activity in humans. We found that, like LSD, 2-Br-LSD acts as an agonist at a wide range of aminergic GPCRs. LSD is nearly a full agonist at the 5-HT 2A and 5-HT 2B subtypes, whereas 2-Br-LSD acts as a partial agonist at 5-HT 2A and a potent antagonist at 5-HT 2B . In addition to activating the 5-HT 2A receptor, LSD interacts with many other aminergic GPCRs, potentially resulting in side effects, but we found that 2-Br-LSD has less offtarget activity compared with LSD and possesses weak micromolar activity at other ion channels, including hERG channels. Taken together, 2-Br-LSD possesses a favorable profile as a drug candidate, with less potential for side effects compared with other serotonergic drugs, such as fenfluramine and methysergide. Importantly, 2-Br-LSD did not induce head twitches in mice despite acting as a potent 5-HT 2A partial agonist. The mouse HTR assay shows a high level of sensitivity to 5-HT 2A agonists,and hundreds of compounds have been tested in the assay. Lisuride, an LSD analog that acts as a partial 5-HT 2A agonist, fails to induce hallucinogenic effects in humans and is inactive in the HTR paradigm.One unanswered question is why lisuride and 2-Br-LSD lack hallucinogenic potential and fail to induce the HTR. One possible explanation is that the level of 5-HT 2A activation produced by 2-Br-LSD and lisuride may not be sufficient to induce head twitches. Our study determined that 2-Br-LSD is a weaker 5-HT 2A partial agonist compared with LSD and other psychedelic drugsand can partially antagonize 5-HT 2A . In previous studies, the peak HTR rate correlated with 5-HT 2A agonist efficacy,which suggests that weaker 5-HT 2A partial agonism may explain why 2-Br-LSD does not induce the HTR. These HTR results support 2-Br-LSD's lack of hallucinogenic potential in humans and provide evidence that 2-Br-LSD can block subjective responses to LSD in some human trialsvia occupation of 5-HT 2A . The HTR data with 2-Br-LSD are consistent with its reported effects in humans and support its classification as a non-hallucinogenic 5-HT 2A agonist. Five cluster headache patients who received 30 mg/kg 2-Br-LSD orally on three occasions experienced only minor side effects, such as feeling ''slightly tipsy.''Higher oral doses, ranging from 64-256 mg/kg, induced mild subjective responses, including restlessness, anxiety, drowsiness, impaired concentration, and euphoria.Intravenous infusion of 18-22 mg 2-Br-LSD produced more intense effects, such as depersonalization, derealization, and mild confusion.However, none of the subjects who received 2-Br-LSD orally or intravenously experienced visual hallucinations or profound cognitive alterations similar to those induced by LSD. Although 2-Br-LSD may produce some psychoactive effects in humans after administration of very high dosages, it clearly does not act as a psychedelic like LSD. Repeated treatment with psychedelic drugs downregulates 5-HT 2A receptor signaling and induces a rapid behavioral tolerance in rodentsand humans.The tachyphylaxis induced by LSD and psilocybin limits how frequently these drugs can be administered to patients. Notably, in our study, mice treated with 2-Br-LSD for 7 consecutive days did not show evidence of tachyphylaxis. A lack of tolerance to 2-Br-LSD may be a consequence of its weak recruitment of 5-HT 2A b-arrestin2, which have been shown to bind 5-HT 2A in vitro and are co-localized in pyramidal neurons.It was reported recently that b-ar-restin2 knockout mice show tolerance to the HTR-inducing effects of LSD after repeated treatment,indicating that b-ar-restin2 recruitment may not play a role in tolerance to psychedelics. However, the relevance of these findings to wild-type mice is unclear because there would likely be considerable re-organization or compensation of GPCR signaling after global deletion of the b-arrestin2 gene. The same line of knockout mice has been used to investigate the role of b-arrestin2 recruitment in respiratory depression induced by m-opioid receptor agonists,but the results have been called into question by subsequent studies.In summary, compared with psychedelic drugs, 2-Br-LSD produces less recruitment of b-arrestin2 via 5-HT 2A and fails to produce tolerance in vivo. Our results indicate that 2-Br-LSD has effects comparable with those of classical psychedelic drugs, which have been shown to produce lasting antidepressant-like effects. For example, psilocybin treatment increased dendritogenesis and spinogenesis in rodentsand produced rapid and lasting antidepressant effects in human clinical trials.LSD has also been shown to produce antidepressant effects in humans,enhance neuroplasticity in rat neuronal cultures, and promote hippocampal neuronal proliferation and spinogenesis.Finally, studies with the psychedelic drug 5-methoxy-DMT showed an increase in dendritic arbor complexity and spine density in cultured rat cortical neurons.These classic psychedelics share 5-HT 2A receptor agonism, considered to be a fundamental component in their antidepressant activity. We confirmed the likely 5-HT 2A dependency of their antidepressant-like effects 6 by testing whether the 5-HT 2A antagonist Vol can block 2-Br-LSD's activities. These results suggest that 2-Br-LSD has the potential to be an effective treatment for MDD, possibly through its effects on neuroplasticity. The loss of dendrite arbor complexity, retraction of neurites and dendritic spines, and reduced synaptic density represent negative structural changes observed in the PFC of patients suffering from depression and anxiety.Compounds modifying synaptic plasticity are considered promising therapies for these disorders. Indeed, a central hypothesis for the mechanism of action for the therapeutic antidepressant effects of psychedelics involves rapid induction of structural and functional neural plasticity and reversal of neuronal atrophy.While a direct link between neuroplasticity and the behavioral effects of psychedelic drugs has yet to be shown, the hypothesis of neuronal atrophy reversal may explain why psychedelics produce effects persisting after treatment has ceased. The NMDA receptor antagonist ketamine, a dissociative anesthetic drug with hallucinogenic effects, produces well-described anti-depressant effects after a few treatments, coupled with changes in arbor complexity and spine density, effects thought to be linked.While the primary receptor targets of dissociative anesthetics and psychedelic drugs are different, they show similar downstream effects in vivo and in vitro, suggesting that their effects on neuroplasticity and neuronal atrophy may serve as a common pathway for the treatment of MDD and anxiety disorders. We show that 2-Br-LSD induces spinogenesis in vivo and in vitro in two rodent species and produces effects on chronic stress, which together suggest that 2-Br-LSD may have therapeutic potential for the treatment of depression, anxiety, and potentially other psychiatric disorders. Finally, the lack of 2-Br-LSD tolerance and 5-HT 2B agonist activity may permit frequent dosing for mood disorders and other indications.
LIMITATIONS OF THE STUDY
Several limitations are noted for this study. First, in vitro GPCR assays may not reflect efficacy in vivo because of the measurement of specific G protein and b-arrestin subtypes, which may be cell type specific. Moreover, not all GPCR effectors and signaling pathways were studied. Assessment of hallucinogenic potential using HTR testing has limitations because it may not model the human psychedelic state. Although HTR data support classification of 2-Br-LSD as a non-hallucinogenic 5-HT 2A agonist, its activity ultimately must be defined based on human data. Although it cannot be excluded that the lack of hallucinogenic effects is a consequence of the dose range tested, pretreatment with 2-Br-LSD attenuated the response to LSD in some studies,further indicating that 2-Br-LSD is capable of engaging 5-HT 2A receptors in the brain. However, additional clinical studies are required to fully characterize the effects of 2-Br-LSD in humans and understand its subjective phenomenology. Finally, FST has been widely used for testing novel antidepressant drugs, but its utility has been questioned because of a lack of face and predictive validity.Therefore, we used additional tests to determine whether 2-Br-LSD produces antidepressant-like effects, including the chronic stress model, which has greater construct validity for the pathological alterations leading to depression. 107 Despite obtaining concordant results in both paradigms, the potential antidepressant effects of 2-Br-LSD must be confirmed in additional preclinical behavmodels of depression (e.g., chronic social defeat) and using additional physiological outcomes relevant to depression (e.g., inflammatory markers, hypothalamic-pituitary-adrenal axis activation).
STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: were obtained from Charles River Laboratories (as indicated below). Mice were housed in Carleton University's vivarium for at least 2 weeks of acclimatization before being experimental procedures started. Mice were housed in groups of four and kept in a 12:12 light/dark cycle with water and food pellets ab libitum, in temperature-and humidity-controlled rooms (21 C, $55% humidity), except for chronic stress experiments where housing conditions varied as described below. All experimental procedures involving animals were approved by Carleton University's Animal Care Committee, pursuant of the Canadian Council of Animal Care guidelines.
METHOD DETAILS
Compounds (6aR,9R)-2-bromolysergic acid diethylamide (2:1) (+)-tartrate (2-Br-LSD; BETR-001) was obtained from BetterLife Pharma (Vancouver, BC, Canada). For in vivo studies, 2-Br-LSD was dissolved in 0.9% saline. For in vitro pharmacological studies, a 10 mM stock solution of 2-Br-LSD was prepared in DMSO and stored at - For in vivo assays, volinanserin was dissolved in 1 M HCl, the pH was adjusted to 7.2 using 1 M NaOH, and then 0.9 % saline was added to bring the solution up to full volume; the vehicle control consisted of 1 M HCl adjusted to pH 7.2 and brought up to full volume with 0.9% saline. All in vivo drug treatments were administered intraperitoneally (IP) with an injection volume of 5 mL/kg or 10 mL/kg body weight. GPCR G protein-dissociation and b-arrestin2 recruitment BRET assays All BRET assays were conducted using BRET 2 in HEK293T cells (ATCC CRL-11268; mycoplasma-free), which were subcultured in high-glucose DMEM (VWR) supplemented with 10% FBS (Life Technologies). Constructs in G protein-dissociation BRET assays were derived from the codon-optimized Tango pcDNA3.1 library 108 (Addgene) with V2tail/TEV/tTA encoding regions deleted to yield ''de-Tango'' constructs. All Ga-Rluc8 and GFP 2 -g constructs were derived from TRUPATH library(Addgene), and pcDNA3.1-Gb and human-b-Arrestin2 constructs were purchased from cDNA Resource Center; www.cDNA.org. N-terminal GFP 2 -fused human b-Arrestin2 constructs were created using templates from addgene and cdna.org, and subcloned into pcDNA3.1. 5-HT receptor constructs used in b-Arrestin2 recruitment BRET assays were also derived from the Tango library with V2tail/TEV/tTA encoding regions replaced with Renilla luciferase (Rluc8) using Gibson Assembly. Constructs from human GRK2 were synthesized from IDT and subcloned into pcDNA3.1. Approximately 48 hours before assays, cells were transfected using a reverse transfection method and plated in 1% dialyzed FBS (dFBS) at an approximate density of 15,000 cells per well into poly-L-lysine-coated 384-well white assay plates (Grenier Bio-One). For G protein-dissociation assays, cells were transfected in an indicated ratio of receptor: Ga-Rluc8: Beta: GFP 2 -g constructs (see supplemental information for ratios). For b-Arrestin2 recruitment assays, cells were transfected at a ratio of 5-HTR-Rluc8: GFP 2 -fused human b-Arrestin2 (see Table). All transfections were prepared in Opti-MEM (Invitrogen) and used a 3:1 ratio of TransIT-2020 (Mirus) uL:ug total DNA. On the day of the assay, plates were decanted and 20 uL of drug buffer per well (13 HBSS, 20 mM HEPES, pH 7.4) was added using a Multidrop (ThermoFisher Scientific), and plates were allowed to equilibrate at 37 C in a humidified incubator before receiving drug stimulation. Drug dilutions of all compounds were performed in McCorvy buffer (13 HBSS, 20 mM HEPES, pH 7.4, supplemented with 0.3% BSA fatty acid free (GoldBio), and 0.03% ascorbic acid). Drugs were dispensed using a FLIPR TETRA (Molecular Devices). Next, plates were incubated at 37 C in a humidified incubator for 60 minutes or specified time point (see supplemental information). Before reading, addition of coelenterazine 400a (5 uM final concentration; Nanolight Technology) was performed by the FLIPR TETRA .
CALCIUM FLUX ASSAYS
Stable-expressing 5-HT 2A/2B/2C receptor Flp-In 293 T-Rex Tetracycline inducible cell lines (Invitrogen, mycoplasma-free) were used for Gq-mediated calcium flux assays. Constructs used for these assays were derived from the codon-optimized Tango pcDNA3.1 library 108 (Addgene) with V2tail/TEV/tTA encoding regions deleted to yield ''de-Tango'' constructs, and then subcloned into pcDNA5/FRT/TO using Gibson Assembly. Cell lines were maintained in high-glucose DMEM (VWR) containing 10% FBS (Life Technologies), 10 mg/mL Blasticidin (GoldBio), and 100 mg/mL Hygromycin B (GoldBio). Approximately 24 hours before the assay, receptor expression was induced with tetracycline (2 ug/mL) and seeded into 384-well poly-L-lysine-coated black plates at a density of approximately 7,500 cells/well in DMEM containing 1% dialyzed FBS. On the day of the assay, plates were decanted and cells were incubated with Fluo-4 Direct dye (Invitrogen, 20 ml/well) for 1 h at 37 C, which was reconstituted in drug buffer (13 HBSS, Head-twitch response studies Head twitches were recorded using a head-mounted neodymium magnet and a magnetometer detection coil, as described previously.The mice were allowed to recover from the magnet implantation surgeries for at least 1 week prior to behavioral testing. HTR experiments were conducted in a well-lit room and the mice were allowed to habituate to the room for at least 1 h prior to testing. Immediately after drug treatment, mice were placed in a 12-cm diameter glass cylinder surrounded by a magnetometer coil and behavior was recorded continuously. Coil voltage was low-pass filtered (1 kHz), amplified, and digitized (20-kHz sampling rate) using a Powerlab 8/35 with LabChart ver. 8.1.19 (ADInstruments, Colorado Springs, CO, USA). In the first HTR experiment, 6 groups of mice (n = 5-6/group, 36 total) were treated with vehicle, 2-Br-LSD (0.1, 0.3, 1, 3, or 10 mg/ kg), or LSD (0.1 mg/kg), and HTR activity was assessed for 60 min. In the second experiment, 5 groups of mice (n = 6-7/group, 31 total) were treated with vehicle or 2-Br-LSD (0.1, 0.3, 1, or 3 mg/kg); 10 min later, all of the mice were injected with (1 mg/kg) and then HTR activity was assessed for 30 min. In the third experiment, 2 groups of mice (n = 8/group, 16 total) were treated with vehicle or 2-Br-LSD (1 mg/kg); 10 min later, all of the mice were injected with DOI (1 mg/kg) and then HTR activity was assessed for 240 minutes. In the fourth experiment, 4 groups of mice (n = 6/group, 24 total) were treated with vehicle or WAY-100,635 (1 mg/kg); 20 min later, the mice were treated with vehicle or 2-Br-LSD (3 mg/kg) and then HTR activity was assessed for 30 min. In the fifth experiment, 4 groups of mice (n = 5/group, 20 total) were treated with vehicle or S-(-)-raclopride (1 mg/kg); 20 min later, the mice were treated with vehicle or 2-Br-LSD (3 mg/kg) and then HTR activity was assessed for 30 min. In the sixth experiment, 3 groups of mice (n = 5-6/group, 16 total) were treated with vehicle or (-)-quinpirole (0.025 or 0.25 mg/kg); 30 min later, all of the mice were injected with DOI (1 mg/kg) and then HTR activity was assessed for 30 min. In the seventh experiment, 3 groups of mice (n = 7/group, 21 total) received 7 daily injections of vehicle, DOI (10 mg/kg/day), or 2-Br-LSD (3 mg/kg day); 24 h after the final injection, all of the mice were injected with DOI (1 mg/kg) and then HTR activity was assessed for 30 min.
CORTICAL NEURONAL CULTURE
For primary neuronal cultures, gestating Sprague-Dawley rats were obtained from Charles River Laboratories, at gestation day (GD) 18. Briefly, embryos were removed from the uterus of a euthanized dam and placed in dissecting solution (Hank's balanced salt solution, 1 M HEPES, 1 % penicillin-streptomycin and glucose; Fisher Scientific). Cortices were immediately dissected from each pup and placed in chilled neurobasal medium (see below). Cortical tissue was then dissociated using TrypLE Express (Gibco) and mechanical disaggregation. Single cells were then plated at a density of 50 000 cells per well on poly-D-lysine coated glass coverslips. The initial plating medium consisted of 1% penicillin-streptomycin, 0.5 mM Glutamax (Thermo Fisher Scientific), and B-27 Plus supplement (Thermo Fisher Scientific) in Neurobasal Plus (Thermo Fisher Scientific). Plating medium was exchanged at 24 h for maintenance media with the same components, except Glutamax. Following this initial change, 50% media changes occurred at 3-day intervals. An additional 10% of the maintenance media was added during each change to account for evaporation,. Cultures were maintained at 37 C under 5% CO 2 . Overall, each treatment condition was replicated in 6 independent wells, from 2 different cultures (3 wells per culture). To assess dendritogenesis Cortical neurons maintained until day in vitro 3 (DIV3) and then treated with 2-Br-LSD (1 nM, 10 nM, 100 nM, 1 mM, 10 mM), ketamine (10 mM), or vehicle. Each treatment lasted 3 h, followed by a full media change. Plates were then maintained for an additional 69 h before fixation. Spinogenesis was assessed in a follow-up experiment; cortical neurons were maintained until DIV18 and treated with 2-Br-LSD (1nM, 10nM, 100nM, 1mM, 10mM), 10uM ketamine or vehicle (3 wells/plate). Neurons were fixed by replacing 80% of the medium with 4% paraformaldehyde for 20 min, then 0.2% Triton-X was added for another 20 min. Dendritogenesis plates were then blocked with 3% BSA, and a primary antibody against the microtubule-associated protein 2 (MAP2) was added (chicken polyclonal anti-MAP2 antibody; 1:5000, EnCor Biotechnology Inc., cat. # CPCA-MAP2) overnight, followed by an incubation with a secondary antibody for 1 h (goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 594; 1:2000; Invitrogen). Spinogenesis plates were also blocked with 3% bovine serum albumin, then fixed and stained on DIV 19, following the dendritogenesis protocol, with the addition of F-actin staining with phalloidin (Alexa Fluor 488 Phalloidin, Thermo Fisher). Culture coverslips were mounted using Vectashield mounting media containing DAPI nuclear staining (Vector Laboratories). In a different set of cultures, to test the effects of the 5-HT 2A antagonist volinanserin on 2-Br-LSD action, cultured cells were treated as follows: vehicle, volinanserin alone (100 nM, 500 nM, or 1 mM), 2-Br-LSD alone (1 mM), or volinanserin (100 nM, 500 nM, or 1 mM) plus 2-Br-LSD (1 mM) (3 wells/plate). Volinanserin or vehicle was applied for 1 h before being washed out with a full media change, which was immediately followed by 2-Br-LSD treatment for 3 h.
CELL VIABILITY ASSAYS
To test the effect of 2-Br-LSD on cell viability, primary cortical neurons were cultured as for the dendritogenesis experiment above. On DIV6 (69 h after treatment), viability was assessed using the Neurite Outgrowth Staining Kit (Thermo Fisher). This assay consists of two stains, one for cell membranes in living and dead cells and one that only fluoresces when metabolized by a live cell.
IMAGING DENDRITOGENESIS AND SPINOGENESIS
Neurons from the dendritogenesis and spinogenesis experiments were imaged with a Zeiss confocal laser scanning microscope (LSM 700), using an oil immersion 633 objective at 23 zoom and 1024 3 1024 pixel resolution. The spectral detectors were adjusted to capture emission from a helium/neon laser at wavelengths of 488-594 nm for Alexa Flour staining of MAP2 and F-Actin, and the pinhole diameter was maintained at 1 Airy unit. The image acquisition was set at a range of 8 bits.
IN VIVO MODELS OF DEPRESSION OPEN FIELD TEST (OFT)
The open field test (OFT) consisted of mice being placed in 1:1.4 ratio rectangular transparent arenas (45 cm length, 30 cm width) in a well-lit room, with black cardboard barriers preventing mice from seeing conspecifics during testing. Mice were allowed to explore the arena for 10 minutes. Forced-swim test (FST) For FST, mice were placed in a four-liter cylindrical glass container filled with 3 L of water at 25±1 C and filmed using a digital camera for 6 minutes. To assess the in vivo effects of 2-Br-LSD, adult male and female C57BL/6J mice (Jackson laboratories) were treated with a single of 2-Br-LSD (0.3, 1 and 3 mg/kg) or vehicle. Mice were tested in the open field 24 h after drug treatment, followed by assessment in the FST 1 h later (n=12/group/sex). To investigate the role of the 5-HT 2A receptor in the effects of 2-Br-LSD, a separate cohort of male and female mice were treated with vehicle or the 5-HT 2A selective antagonist VOL (0.125 mg/kg) 15 min before treatment with vehicle or 2-Br-LSD (1 mg/kg). Twenty-four hours later, the mice were tested in the OFT, followed 1 h later by the FST. Immediately following behavioural testing, mice were sacrificed, and the brains were removed and bisected; the right hemisphere was flash frozen, while the left hemisphere was processed using the FD Rapid Golgi Stain Kit protocol (FD NeuroTechnologies, Inc).
GOLGI STAINING
To test the effects of 2-Br-LSD on spine density in vivo, we prepared the left hemisphere of brains from the stress-naı ¨ve experimental animals and subjected them to Golgi-Cox staining (FD Rapid GolgiStain Kit, FD NeuroTechnologies, INC). Briefly, brains were placed into the impregnation solution A/B (5% potassium dichromate, 5% mercuric chloride and 5% solution of potassium chromate), in the dark at $25 o C for 14 days. Brains were then placed in solution C (5% potassium dichromate, 5% mercuric chloride), and stored for a further 7 days. Following this, brains were removed from solution and stored at -80 o C. Then 150 mm coronal cryosections were collected using a cryostat (Epredia Microm HM525 NX, Fisher Scientific), focusing on the prefrontal cortex. After cryosection, final treatment consisted of rinsing with distilled water twice for 10 min, 50 % ethanol dehydration, ammonia incubation for 15min, 5% sodium thiosulfate incubation in the dark, gradient ethanol dehydration (50-100%), xylene clearing and mounting. Imaging was done using an Olympus BX51 brightfield microscope with a 100x objective, running the Neurolucida imaging analysis software suite (Neurolucida 360/Explorer). Chronic variable stress (CVS) CVS consisted of mild stressors administered twice daily for 33 days to single-housed female mice. Stressors' order of application was pseudo-randomized and consisted of cage tilt (at a 45 angle for 4-8 h), restraint (10 min), wet bedding (overnight), forced swim (10 min), odour exposure (animals exposed to a cotton swab embedded with lemon or cinnamon extract for 4-8 h) and/or disrupted light cycle (lights left on overnight). Control mice were also singled-housed and left undisturbed, except for a weekly handling and weighing session. Control and stressed mice were housed in separate rooms. On day 28 of stress, mice were randomly grouped into 4 groups and subjected to the following treatments (n=12/group): naı ¨ve-vehicle, CVS-vehicle, CVS-1X 2-Br-LSD (3 mg/kg) or CVS-4X 2-Br-LSD (1 mg/kg). Mice were injected every 48 h until day 34 (24 h after the last stressor for the CVS groups), for a total of 4 injections. The CVS-1X 2-BrLSD group received three vehicle injections and one injection of 2-Br-LSD, while the group CVS-4X 2-BrLSD received 4 doses of 2-Br-LSD. Behavioral testing began 2 h following the final injection and consisted of the splash test (ST), followed by OFT (performed 2 h after the ST, as described above). A similar series of tests were performed 4 weeks later to determine whether the effect of 2-Br-LSD is long lasting. For the ST, mice were placed in a new cage similar to their home cage for 10 min, 500 mL of a 30% sucrose solution was applied to their back, and their behavior was video recorded for 10 min.
QUANTIFICATION AND STATISTICAL ANALYSIS GPCR G PROTEIN-DISSOCIATION AND B-ARRESTIN2 RECRUITMENT BRET ASSAYS
Plates were read at 400 nm Rluc8 and 510 nm GFP 2 emission filters for 0.8 seconds per well using a PheraStarFSX (BMB Lab Tech). The BRET ratios of 510/400 luminescence were calculated per well and were plotted as a function of drug concentration using Graphpad Prism 5 or 9 (Graphpad Software Inc., San Diego, CA). Data were analyzed using nonlinear regression ''log(agonist) vs. response'' to yield E MAX and EC 50 parameter estimates. Relative activities (log (E MAX /EC 50 ) were calculated based on normalized data. Antagonist affinities (K B ) were calculated according to the method by Cheng utilizing EC 50 and competing agonist concentration for each individual receptor subtype. 109 Data were normalized to % positive control stimulation and a positive control concentration-response curve was present on every plate.
CALCIUM FLUX ASSAYS
Fluorescence in each well was normalized to the average of the first 10 reads for baseline fluorescence, and then either maximumfold peak increase over basal or area under the curve (AUC) was calculated. Both peak and AUC was plotted as a function of drug concentration, and data were normalized to percent 5-HT stimulation. Data were plotted and non-linear regression was performed using ''log(agonist) vs. response'' in Graphpad Prism 9 to yield E MAX and EC 50 parameter estimates. Data were normalized to % 5-HT response, and a positive control concentration-response curve was present on every plate. For FST quantification, an experimenter blinded to treatment manually recorded immobility time during the last 4 minutes of the test. Immobility was defined as the animal lacking any lateral movement with not more than one limb moving in full swim rotation, for at least 1 second. For the ST quantification, self-grooming time was scored for the 10 min period following sucrose application by an experimenter blind to the treatment. Data were plotted using GraphPad Prism (Ver. 9.5.0, San Diego, CA, USA) and are presented as mean±SEM. Data were analyzed using one-way or two-way ANOVAs, followed by Bonferroni's post hoc tests for post hoc comparisons; significance was demonstrated by surpassing an a level of 0.05.
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Study Details
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- Populationrodentscells
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