Chronic administration of sub‑hallucinogenic (microdose) LSD in mice produced no ventricular thickening or other ECG-detected cardiotoxic changes, whereas serotonin (positive control) caused significant ventricular thickening. In vitro assays showed similar 5‑HT2B affinity across mouse and human receptors and that low‑dose LSD yields only transient 5‑HT2B activation compared with the cardiotoxin d‑fenfluramine, together providing no evidence of cardiovascular risk from prolonged low‑dose LSD in this mouse model.
Summary Microdosing, the prolonged ingestion of psychedelics at sub-hallucinogenic doses, has gained popularity for its perceived cognitive and emotional benefits. Psychedelics have high affinity for 5-HT 2B receptors, which cause heart disease with strong chronic activation. We investigated the effects of microdosed psychedelics on cardiovascular health in mice using electrocardiography after chronically administering either serotonin as a positive control or lysergic acid diethylamide (LSD) at two sub-hallucinogenic doses. Serotonin produced significant ventricular thickening at 4- and 8-weeks. No significant changes were observed in vehicle or LSD groups. We determined the affinity and potency of LSD, psilocybin, and norfenfluramine at mouse and human 5-HT 2B Rs and observed no significant differences. We calculated that levels of 5-HT 2B activation by low-dose LSD were substantial, but short-lived, compared to the cardiotoxin d -fenfluramine. Together, these data provide no evidence of cardiovascular risk associated with prolonged administration of low-dose LSD in mice.
Psychedelic compounds such as psilocybin and LSD are increasingly used and, in some jurisdictions, decriminalised, prompting rising public interest in microdosing—the repeated ingestion of subhallucinogenic amounts of these drugs. Because many classical psychedelics are potent agonists at serotonin receptors including 5-HT2A and 5-HT2B, there is a safety concern: chronic activation of 5-HT2B receptors has previously been implicated in drug-induced valvular heart disease and pulmonary hypertension (for example, with d-fenfluramine). Although a single high dose of a psychedelic is unlikely to produce cardiac damage, the cardiovascular consequences of prolonged low-dose (microdose) use remain poorly characterised. This preclinical study set out to assess whether chronic microdosing with LSD produces structural or functional cardiac changes in mice. The investigators compared two low LSD doses against vehicle and positive controls (serotonin and d-fenfluramine) using serial echocardiography, measured behavioural evidence of psychedelic activity to define subhallucinogenic doses, characterised agonist activity at mouse and human 5-HT2B receptors in vitro, and modelled 5-HT2B receptor occupancy over time using published human pharmacokinetic data. The work aims to elucidate whether the pattern and magnitude of 5-HT2B activation with microdosing are sufficient to produce cardiotoxic effects in this mouse model.
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Vollenweider, F. X., Vollenweider-Scherpenhuyzen, M. F. I., Bäbler, A. et al. · NeuroReport (1998)
Animals and overall design: Male and female C57BL/6J mice (ordered at 8 weeks old; habituated 1 week) were used. Treatments for the chronic microdosing paradigm were given intraperitoneally (i.p.) on a 5 days on/2 days off schedule for 8 weeks, intended to model commonly used human microdosing regimens. Echocardiography was performed at baseline, 4 weeks, and 8 weeks. Body weight and heart rate were monitored during the treatment period. Doses and controls: For LSD the study used three doses to define behavioural and chronic exposure ranges: 0.01, 0.03, and 0.1 mg/kg (i.p.). Chronic cardiovascular experiments used the 0.01 and 0.03 mg/kg doses. Positive controls included serotonin hydrochloride at 40 mg/kg (i.p.) as a cardiac pathology control, and (+)-fenfluramine at 10 mg/kg (i.p.) as a known valvulopathic comparator. For in vitro pharmacology, compounds included 5-HT, (+)-norfenfluramine, LSD and psilocin. Behavioural assay and echocardiography: The head twitch response (HTR) was recorded with high-speed video and scored by an observer blinded to treatment to establish doses that are subhallucinogenic in mice (sample sizes for HTR assays were reported per dose group). Echocardiography used a Fujifilm Visualsonics F2 system with mice anaesthetised by isoflurane (initial bolus 3%, maintenance 1.5%) and temperature maintained at 37 °C. Left ventricular inner diameter, posterior wall diameter, volumes, ejection fraction and fractional shortening were measured using standard m-mode and software-based calculations. Aortic regurgitation was assessed at 4 weeks with colour Doppler, and regurgitant jets were classified by jet length relative to left ventricular length (mild <25%, moderate 25–50%, severe >50%). In vitro pharmacology and receptor occupancy modelling: Bioluminescence resonance energy transfer (BRET) assays were used in HEK T cells to measure Gq-mediated 5-HT2B receptor dissociation (Gq and β3/γ9), evaluating potency (EC50) and efficacy (Emax) for compounds at both human and mouse 5-HT2B receptors. Concentration–response curves were analysed by nonlinear regression and normalised to 5-HT stimulation. Pharmacokinetic modelling used published human plasma concentration–time courses for LSD, the active fenfluramine metabolite d-norfenfluramine, and psilocin. The Hill equation was applied, together with the in vitro EC50/Emax values, to estimate fractional 5-HT2B receptor activation over time for single human doses (modelled doses: LSD 20 μg, psilocybin 3 mg, d-fenfluramine 30 mg). Other procedural details: Echocardiogram analyses were carried out using VevoLab software. Head twitch scoring was blinded. The extracted text does not clearly report the specific statistical tests used for group comparisons or the exact sample sizes for every treatment group in the chronic echocardiography experiments when pooled across sex; some group sizes are given (for example, several groups are described as n = 3 males/3 females).
Dose selection and head twitch response: Using the head twitch response as an index of 5-HT2A activation, LSD at 0.01 and 0.03 mg/kg produced only small increases in head twitch frequency relative to 0.1 mg/kg. The investigators therefore treated 0.01 mg/kg as a subhallucinogenic microdose and 0.03 mg/kg as a weakly hallucinogenic dose in mice. Structural and functional cardiac outcomes by echocardiography: Across the 8-week treatment period there were no treatment effects on heart rate or body weight, indicating no gross adverse health effects. Mice receiving serotonin (40 mg/kg) showed significant cardiac structural changes: reductions in left ventricular (LV) inner diameters (end diastolic and end systolic) at 4 weeks relative to baseline, significant increases in LV posterior wall diameter at 4 and 8 weeks, and decreases in LV end diastolic and systolic volumes at 4 weeks. These changes indicate ventricular thickening in the serotonin-treated group. By contrast, neither the vehicle-treated mice nor mice treated with chronic LSD at either 0.01 or 0.03 mg/kg showed significant changes in LV inner diameter, posterior wall thickness, or LV volume at 4 or 8 weeks. Measures of systolic function (ejection fraction and fractional shortening) did not show consistent treatment-related changes. No differences in ejection fraction were detected in vehicle or serotonin groups. The 0.01 mg/kg LSD group showed a statistical effect across time for both ejection fraction and fractional shortening, but post hoc comparisons found no differences from baseline at the follow-up time points. The 0.03 mg/kg LSD group showed no changes in these functional measures. Valvular function and fenfluramine control: Colour Doppler imaging after 4 weeks of dosing revealed a significant increase in aortic valve regurgitation in the d-fenfluramine (10 mg/kg) group compared with vehicle (groups reported as n = 3 males/3 females). No difference in aortic valve regurgitation was observed between the 0.03 mg/kg LSD group and vehicle. In vitro 5-HT2B pharmacology: BRET Gq dissociation assays at human and mouse 5-HT2B receptors produced potency and efficacy estimates. Reported EC50 values include norfenfluramine 6.78 nM, LSD 0.88 nM, and psilocin 3.1 nM. Relative efficacies (as a percentage of 5-HT stimulation) were higher for norfenfluramine (about 96%) compared with LSD (about 62%) and psilocin (about 52%). The extracted text does not present full statistical details for these assays, but these values were used for downstream modelling. Pharmacokinetic-based receptor occupancy modelling: Using published human plasma time–course data and the in vitro EC50/Emax parameters, the investigators estimated fractional 5-HT2B receptor activation over time for a single human microdose of LSD (20 μg), a single human dose of d-fenfluramine (30 mg), and a psilocybin microdose (3 mg). The model indicated that 5-HT2B receptor occupancy for microdoses of LSD and psilocybin peaked at roughly half the level produced by norfenfluramine and declined to negligible levels by about 10 hours after dosing. By contrast, d-norfenfluramine produced larger and more sustained plasma exposure, with estimated receptor activation remaining above 50% for 24 hours. The investigators note that daily fenfluramine dosing would therefore be expected to produce persistent and cumulative 5-HT2B activation, consistent with its known cardiotoxicity.
Effinger and colleagues interpret their findings to indicate that chronic administration of low doses of LSD, given on a 5‑days-on/2‑days-off schedule for up to 8 weeks in mice, did not produce echocardiographic evidence of ventricular remodelling or valvular dysfunction, whereas positive controls produced expected pathology: serotonin induced ventricular thickening and d-fenfluramine produced aortic valvular regurgitation. The lack of detectable ventricular or valvular changes in the LSD groups is attributed by the investigators to limited 5-HT2B receptor activation under the microdosing regimen rather than to an absence of intrinsic agonist activity at the receptor. The authors position these results in the context of earlier work linking sustained 5-HT2B activation to cardiac valvulopathy. Their in vitro assays showed that although LSD and psilocin bind 5-HT2B receptors with nanomolar affinity, their efficacy to activate Gq-dependent signalling was lower than that of norfenfluramine. Combined with pharmacokinetic modelling using published human plasma data, the researchers argue that LSD and psilocin achieve lower peak concentrations and much shorter durations of 5-HT2B receptor occupancy than d-norfenfluramine. This pharmacokinetic and pharmacodynamic profile is offered as an explanation for why chronic low-dose LSD did not reproduce the cardiotoxic effects seen with fenfluramine in this mouse model. Key limitations acknowledged by the investigators include the translational gap between mice and humans: species differences in drug absorption and metabolism may alter receptor occupancy, and although no species differences in 5-HT2B affinity or efficacy were detected in their assays, other species-specific pharmacokinetic or physiological factors may matter. The experiments were performed in presumably healthy mice, so the findings may not generalise to people with preexisting cardiovascular risk factors. The authors also note the possibility that longer treatment durations or different dosing regimens could produce different outcomes. They therefore caution that absence of observed pathology in mice should not be taken as proof of safety in humans and recommend prospective human studies to evaluate cardiac effects of microdosing in clinical populations. The discussion also briefly notes that other serotonergic drugs, for example MDMA, may elevate cardiac risk through related mechanisms.
P sychedelic drugs, such as psilocybin and LSD, have produced therapeutic effects in a variety of affective psychiatric disorders.Colorado and Oregon recently passed legislation decriminalizing possession and use of psilocybin. There has subsequently been a 43% increase in past-year initiation of psychedelic use, versus a 15% increase in other US states. Many other states are considering legislation regarding medical use of psychedelic compounds.The public health consequences of this 'experiment' are likely to be many and multifaceted. Because so much remains unknown about longterm psychedelic use, it is a challenge to advise the public and potential users about many key issues surrounding safety. Serotonergic psychedelics, such as psilocybin and LSD, are potent agonists at many serotonin receptors (5-HTRs), including the 5-HT 2A and 5-HT 2B receptors.Strong activation of 5-HT 2A Rs causes characteristic alterations in perception and consciousness.This psychedelic response is a potential barrier to widespread and cost-effective utilization of psychedelic compounds because it necessitates 6-8 h of psychological support in an inpatient setting during administration.For this reason, microdosing, or the chronic consumption of a subhallucinogenic dose of a psychedelic, typically LSD or psilocybin, has gained popularity due to anecdotal accounts of emotional and cognitive benefits. Between 2015 and 2023, internet searches related to microdosing increased by a factor of 13.4, with the greatest increases seen in Oregon and Colorado.A full understanding of the risks associated with chronic use of psychedelics is lacking, including cardiovascular risk. Serotonergic appetite suppressants, such as d-fenfluramine, were associated with a significant incidence of valvulopathies and pulmonary hypertension, causing their withdrawal from the market.This pathology is characterized by echocardiographic evidence of heart wall thickening,aortic and mitral valve regurgitation, and histological evidence of valve defects caused by cellular proliferation.5-HT 2B Rs are expressed at high levels in the embryonic and adult heart, where they are necessary for normal cardiac development,and their activation was subsequently identified as the cause of this cardiac valvulopathy and the pulmonary hypertension.Although the partially overlapping 5-HT 2B R pharmacological profile of these medications with serotonergic psychedelics is known, the cardiovascular risks associated with use of psychedelics remain uncertain. While transient 5-HT 2B R activation following a single acute administration of a high dose of psychedelics is unlikely to cause cardiac damage, it is not known whether longer-term consumption during microdosing causes damage. Given the increasing legalization, availability, and use of psychedelics, investigation into potential cardiovascular risks associated with prolonged psychedelic use is imperative.■ RESULTS
Heart Rate, and Body Weight. For the purpose of this study, we defined a microdose of LSD as being devoid of known behavioral effects associated with hallucinogenic drugs and equivalent to ∼1/10th of what would be considered a hallucinogenic dose.In order to determine the mouse equivalent of a subhallucinogenic microdose, we used the head twitch response assay (HTR), as an indicator of 5-HT 2A R activation.Male and female C57BL/6J mice were given intraperitoneal (i.p.) injections of LSD at 0.01 mg/kg (n = 4m/ 3f), 0.03 mg/kg (n = 3m/2f), or 0.1 mg/kg (n = 3m/3f). Consistent with previous results,only small increases in head twitch frequency were seen at 0.01 mg/kg and 0.03 mg/kg, compared to a dose of 0.1 mg/kg (Figure). Use of 0.01 and 0.03 mg/kg are thus equivalent to a subhallucinogenic and weakly hallucinogenic dose, as might be used in human microdosing.
We next investigated the cardiovascular consequences of administering these two different doses of LSD (0.01/0.03 mg/kg, i.p.) for 8-weeks in mice, using a 5 days on/2 days off protocol, which is a widely used human microdosing paradigm,as compared to a positive control group receiving injections of serotonin (40 mg/kg, i.p.), which has been shown to induce cardiac pathology in rodents.Echocardiography was performed at baseline, 4-weeks, and 8-weeks. There was no effect of treatment on heart rate or body weight during the treatment period, indicating no overall adverse effects of the treatment on animal health. To assess potential hypertrophic effects within the heart, left ventricle (LV) inner diameter, posterior wall diameter, and volume were measured using echocardiography. No significant change in LV inner diameter was seen in vehicle control mice for end diastolic or end systolic (Figure). In the 5-HT treated animals (n = 3m/3f), there was a significant effect across time for end diastolic and systolic, with significant decreases in diameter compared to baseline seen at 4-weeks (Figure). No effects were seen in either the 0.01 or 0.03 mg/kg LSD groups (n = 3m/3f, Figure). For LV posterior wall diameter, no effect was seen in the vehicle control group (Figure). In the 5-HT group, there was a significant effect across time, with significant increases in wall diameter at 4-and 8-weeks compared to baseline (Figure). No effect was seen in either the 0.01 or 0.03 mg/kg LSD groups (Figure). For LV volume, no effect was seen in the vehicle control group (Figure). In the 5-HT group, there was a significant effect across time for both end diastolic and systolic, with significant decreases in volume seen at 4-weeks compared to baseline (Figure). No effect was seen in either the 0.01 or 0.03 mg/kg LSD groups (Figure). To assess functional consequences, an analysis of ejection fraction was conducted. No changes were detected in the vehicle or 5-HT control groups (Figure). In the 0.01 mg/ kg LSD group, there was an effect across time, however, post hoc analysis revealed no differences from baseline at follow up scans (Figure). In the LSD 0.03 mg/kg group, no changes were detected (Figure). Next, fractional shortening was assessed. No changes were detected in the vehicle and 5-HT control groups (Figure). In the 0.01 mg/kg LSD group, there was an effect of time, however, post hoc analysis found no differences between baseline or follow up time points (Figure). In the 0.03 LSD mg/kg group, there were no changes in fractional shortening or ejection fraction (Figure). Fenfluramine, but Not Vehicle or 0.03 mg/kg LSD, Produced Valvular Dysfunction after 4-Weeks of Chronic Administration. To assess potential valvular dysfunction following chronic administration, we utilized Doppler echocardiography to detect differences in aortic valvular regurgitation following 4-weeks of chronic dosing. Because d-fenfluramine has been shown to produce valvulopathy in both humansand rodents,10 mg/kg dose of dfenfluramine was used as a positive control in these studies. At 4-weeks, echocardiogram color Doppler analysis revealed a significant increase in valve regurgitation in the fenfluramine group compared to vehicle (Figure; n = 3m/3f/group), with no difference seen between 0.03 mg/kg LSD and vehicle.
Receptor Occupancy after Microdoses. LSD is an ergoline psychedelic and affinity values between human and mouse 5-HT 2B Rs may differ by more than an order of magnitude for ergolines such as methysergide and mesulergine.We therefore used a previously described bioluminescence resonance energy transfer (BRET) assayto measure G q dissociation activity for 5-HT, LSD, d-norfenfluramine, and psilocin at both human and mouse 5-HT 2B Rs. Only small). To better understand why low-dose LSD did not induce 5-HT 2B R-mediated valvulopathy, we estimated the fraction of 5-HT 2B Rs activated by a single human microdose of LSD (20 μg) and compared it to the 5-HT 2B R activation expected from a single human dose d-fenfluramine (30 mg) or a human microdose of psilocybin (3 mg). The time course of the plasma levels of LSD, the active d-fenfluramine metabolite, dnorfenfluramine, and the active psilocybin metabolite, psilocin, were taken from published papers.Fractional receptor activation was then computed from the Hill equation, using EC 50 and E max values for 5-HT 2B activation determined above. Consistent with the larger dose, the pharmacokinetic profile of d-norfenfluramine was considerably larger and longer than for LSD (Figure). Comparing 5-HT 2B R activation across time, we found that 5-HT 2B R occupancy for microdoses of LSD and psilocybin peaked at about half the level achieved with norfenfluramine consumption and was absent by 10 h, whereas d-norfenfluramine-induced 5-HT 2B R activation remained >50% for 24 h (Figure). Given that fenfluramine was a daily prescribed treatment, it is likely that 5-HT 2B R activation would have been constant and increasing, which could therefore explain its greater cardiotoxic effects. Together, these data suggest that the risk of LSD-or psilocybin-induced cardiotoxicity is lower than for d-fenfluramine due to their lower concentration and half-life in plasma.
Given that serotonergic psychedelics, including psilocybin LSD, are potent agonists of the 5-HT 2B R, like the known cardiotoxic anorexogenic drug d-fenfluramine, there is concern about the potential for use of these compounds to have deleterious cardiotoxic effects. Although this seems unlikely following a single high-dose administration, the concerns are greater for chronic consumption of psychedelics, even with small microdoses.We first used echocardiography in mice to assess the potential cardiovascular risk associated with prolonged administration of two low doses (0.01 and 0.03 mg/ kg, i.p.) of the psychedelic LSD, compared to prolonged dosing of 5-HT (40 mg/kg, i.p.) as a positive control.The 5-HTtreated group had a significant thickening of the left ventricular wall and decreased ventricular volume. However, no changes in fractional shortening or ejection fraction were found. These results are consistent with studies showing that overexpression of 5-HT 2B Rs in mice leads to increases in ventricular wall thickness, accompanied by increased cellular proliferation at 12-weeks of age, without changes in ventricular volume, fractional shortening, blood pressure, or heart rate; presumably due to compensatory remodeling.No significant effects of treatment on ventricular inner diameter, posterior wall thickness, or ventricular volume were seen after injections of saline or in either low-dose LSD group at 4-or 8-weeks posttreatment. We also assessed risks of chronic LSD consumption on valve function, comparing vehicle and 0.03 mg/kg LSD with the known valvulopathic drug, d-fenfluramine (10 mg/kg, i.p.). As expected, we observed that fenfluramine produced significantly greater valvular regurgitation at 4-weeks compared to vehicle, with no difference seen between vehicle and LSD. These results are also comparable to pathological changes observed in humans following chronic use of d-fenfluramine and other serotonergic appetite suppressants.Finally, chronic administration of neither 5-HT nor LSD produced differences in body weight or heart rate across the 8-week time frame. Given the high affinity of LSD for 5-HT 2B Rs, why did 4-to 8-weeks of chronic administration fail to induce cardiovascular pathology? One potential explanation is that the treatment period was not long enough. Given the pathological changes observed with 4-to 8-weeks of 5-HT administration, chronic activation of 5-HT 2B Rs over this time-period would seem sufficiently long to detect pathological changes if they were triggered by microdosed LSD. An alternative explanation is that the pharmacokinetics of LSD at these doses are insufficient to produce significant 5-HT 2B R activation. The two doses of LSD used here are presumably comparable to the nonhallucinogenic doses used in human microdosing. The low doses used (0.01 and 0.03 mg/kg) produced a very modest head twitch response in mice, a behavioral readout associated with strong 5-HT 2A receptor activation by classical psychedelic drugs.5-HT 2B R-activation of G q signaling-initiated second messenger pathways was identified as the cause of the proproliferative, cardiotoxic effects of fenfluramine.We determined the potency and efficacy of LSD, psilocybin, and norfenfluramine to activate 5-HT 2B R-dependent signaling via a G q dissociation activity assay at both human and mouse receptors. Observed differences in affinity between norfenfluramine (EC 50 = 6.78 nM), LSD (0.88 nM), and psilocin (3.1 nM) were small, but there were larger differences in potency, with norfenfluramine capable of 96% of the activation produced by 5-HT, but LSD and psilocin only capable of 62% and 52% of the activation produced by 5-HT. These data were then combined with previously published plasma concentration data in humans to estimate the fraction of available 5-HT 2B Rs activated. A single dose of d-fenfluramine results in plasma concentrations of its active metabolite that are significantly higher and more prolonged than plasma levels of LSD or psilocin following a low dose of LSD or psilocybin. The fraction of 5-HT 2B R activation was estimated to be sizable for all three compounds, but was greater and more prolonged for d-fenfluramine than for LSD or psilocybin. We thus attribute the absence of cardiovascular pathology after microdoses of LSD in our experiments to insufficient levels of 5-HT 2B R receptor occupancy and concomitant of activation of G q signaling. It should be noted that MDMA may also elevate risk for cardiac disease through these same mechanisms.An essential caveat of this preclinical work is that, although we have no evidence that microdosing LSD results in cardiac pathology in mice, we cannot therefore conclude that the human microdosing of LSD is safe. While there is evidence that the 5-HT 2B R has species specific binding properties,we did not detect any species differences in affinity or efficacy for LSD or psilocin in 5-HT 2B receptor stimulated G q dissociation. There are significant differences in the rate at which LSD is absorbed or metabolized in mice versus humans,thus changing the degree of 5-HT 2B receptor occupancy. Furthermore, our studies were performed on presumably healthy mice and the risks for significant cardiotoxicity from microdosed psychedelics could be greater in people with preexisting cardiovascular risk factors. Prospective studies should be performed to determine whether microdosed psychedelics produce evidence of cardiac pathology in humans.
All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Male and female C57BL/6J mice were obtained from Jackson Laboratories and ordered to arrive at 8-weeks old. Animals were allowed to habituate for 1-week prior to initiating echocardiograms. Drugs. For cardiovascular end points, serotonin hydrochloride (Tocris Bioscience) was dissolved in saline and injected at 40 mg/kg (i.p.). The serotonin dose was chosen based on the study of Gustafsson et al.,which demonstrated evidence of cardiac pathology. (+)-Lysergic acid diethylamide tartrate was provided by the National Institute on Drug Abuse Drug Supply Program (NDSP) and was dissolved into saline prior to being injected at 0.01, 0.03, or 0.1 mg/kg (i.p.). (+)-Fenfluramine hydrochloride (Millipore Sigma) was dissolved in saline and injected at 10 mg/kg (i.p.). For pharmacological end points, 5-HT and (+)-norfenfluramine were purchased from Sigma-Aldrich, and LSD and psilocin were purchased from Cayman Chemical Company. Head Twitch Response. Mice were recorded with a highspeed video camera and head twitches was scored manually by an observer blinded to treatment conditions. Rodent Echocardiograms. Echocardiographic recordings were conducted using FujiFilm Visualsonics F2 Imaging system and analyzed using VevoLab Imaging Analysis software. Animals were first weighed and then anesthetized with an initial bolus of 3% isoflurane and then maintained with 1.5% isoflurane during the echo procedure. The animals were laid supine on a warming pad to maintain body temperature at 37 °C; temperature was monitored and controlled with a rectal thermometer in series with a heated platform throughout the experiments. A thin layer of Nair was placed on the skin of the chest for 15 s. Wet gauze was used to wipe away the Nair and hair from the chest. The chest was then covered with warm ultrasound gel. Echocardiographic images were obtained while the echo probe was held gently against the chest. The echocardiography procedure lasted between 15-30 min, depending on the number of individual images obtained. Measurements of the left ventricular posterior wall (LVPW) were taken from the inner (endocardial) to the outer (epicardial) borders during both systole and diastole. The internal diameter of the left ventricle (LVID) was determined by measuring the distance between the endocardial surfaces of the anterior and posterior walls. Left ventricular volume, ejection fraction (EF), and fractional shortening (FS) were calculated using the system's built-in software based on these parameters. At the end of the procedure, the ultrasound gel was removed with wipes and water. Animals were allowed to recover on a table by the echo instrument in a cage placed over a warming pad before returning to the housing room. Aortic Regurgitation Protocol. Mice anesthetized with a maintenance level of 1.5% isoflurane are placed supine on a heated platform with paws secured to electrode pads. Parasternal long-axis views of the left ventricle and aortic root/valve were obtained using high-frequency echocardiography (Vevo F2, Visualsonics, Toronto, Canada). A transducer with a 57-25 MHz range is placed medially on the chest, then tilted 15-20°to the left for all imaging. Numerous m-mode images are acquired to measure systolic aortic valve cusp dimensions, as described in previous publications.Anatomical m-mode imaging is gathered by placing the measurement gate vertically across the aortic valve, scanning across the length of the valve cusps. Five second cineloops were analyzed using Visualsonics VevoLab software. Echocardiographic regurgitation was classified using color Doppler of the diastolic regurgitant (indicated in blue) produced at the aortic valve into the left ventricle. The length of the jet is then divided by measured end-diastolic long-axis length of the left ventricle. Mild regurgitation is considered <25% of left ventricle length, moderate 25-50%, and severe >50%.5-HT 2B R G q Dissociation BRET Assays. Bioluminescence resonance energy transfer G protein dissociation assays (BRET) measuring 5-HT 2B Gq and β3/γ9 dissociation were conducted in HEKT cells (ATCC CRL-11269; mycoplasmafree). Cells were transfected and compounds were incubated with cells for 60 min at 37 °C and read on a PheraStarFSX (BMG LabTech), as described previously.Human and mouse 5-HT 2B R constructs were subcloned into vectors, as described previously.Data were analyzed using nonlinear regression "log(agonist) vs. response" in GraphPad Prism 10 to yield Emax and EC50 parameter estimates. Data were normalized to percent 5-HT stimulation, with a concentration-response curve was present on every plate. Pharmacokinetic Modeling. The time course of human plasma drug levels was used to generate 5-HT 2B R activation profiles for LSD, psilocybin, and norfenfluramine using the Hill equation
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