This rat study with DMT found that it reduced anxiety by extinguishing of cued fear memory and reduces immobility in the forced swim test (a proxy for depression).
Depression and anxiety disorders are debilitating diseases resulting in substantial economic costs to society. Traditional antidepressants often take weeks to months to positively affect mood and are ineffective for about 30% of the population. Alternatives, such as ketamine, a dissociative anesthetic capable of producing hallucinations, and the psychoactive tisane ayahuasca, have shown great promise due to their fast-acting nature and effectiveness in treatment-resistant populations. Here, we investigate the effects of N,N-dimethyltryptamine (DMT), the principle hallucinogenic component of ayahuasca, in rodent behavioral assays relevant to anxiety and depression using adult, male, Sprague-Dawley rats. We find that while DMT elicits initial anxiogenic responses in several of these paradigms, its long-lasting effects tend to reduce anxiety by facilitating the extinction of cued fear memory. Furthermore, DMT reduces immobility in the forced swim test, which is a characteristic behavioral response induced by many antidepressants. Our results demonstrate that DMT produces antidepressant and anxiolytic behavioral effects in rodents, warranting further investigation of ayahuasca and classical psychedelics as treatments for depression and post-traumatic stress disorder.
Mood and anxiety disorders cause large amounts of disability worldwide and current antidepressant medications often take weeks to become effective, with about 30% of patients failing to respond. This therapeutic gap has prompted interest in fast-acting alternatives such as ketamine and classical serotonergic psychedelics. Ayahuasca, a traditional Amazonian tisane that combines DMT-containing Psychotria viridis with MAO-inhibiting Banisteriopsis caapi, has shown antidepressant and anxiolytic effects in humans and in some animal paradigms, but the contribution of individual constituents—particularly N,N-dimethyltryptamine (DMT), the principal hallucinogenic component—remains unclear. Cameron and colleagues set out to characterise the behavioural effects of a hallucinogenic dose of DMT in adult male Sprague-Dawley rats using standard assays relevant to anxiety, post-traumatic stress disorder (PTSD), and depression. The study aimed to determine both acute and longer-lasting behavioural consequences of DMT administration in paradigms including novelty-induced locomotion (NIL), the elevated plus maze (EPM), fear conditioning and extinction (cued and contextual), and the forced swim test (FST). This work seeks to clarify whether DMT alone can account for the anxiogenic, anxiolytic, or antidepressant effects attributed to ayahuasca and whether its profile resembles that of other fast-acting agents such as ketamine.
Papers cited by this study that are also in Blossom
Berman, R. M., Cappiello, A., Anand, A. et al. · Biological Psychiatry (2000)
Zarate, C. A., Brutsche, N. E., Ibrahim, L. et al. · Biological Psychiatry (2012)
Murrough, J. W., Perez, A. M., Pillemer, S. et al. · Biological Psychiatry (2012)
Feder, A., Parides, M. K., Murrough, J. W. · JAMA Psychiatry (2014)
Adult male Sprague-Dawley rats (8–14 weeks old) were used. Animals were housed two per cage with ad libitum food and water, maintained on a 12:12 light cycle, and experiments were performed during the light phase. The extracted text does not clearly report group sample sizes for each experiment; the paper indicates further details are provided in a Table that was not included in the extraction. All procedures were approved by the UC Davis IACUC. DMT was synthesised as the fumarate salt (2:1 DMT:fumaric acid), characterised by NMR and LC-MS, stored at −20ºC, and freshly prepared in 0.9% sterile saline for each experiment. DMT•fumarate was administered intraperitoneally at 10 mg/kg with an injection volume of 1 mL/kg. Ketamine•HCl (10 mg/kg, i.p.) served as a positive control in the FST; vehicle controls received 0.9% saline. The authors chose 10 mg/kg as a hallucinogenic dose in rats based on allometric scaling and prior drug discrimination literature. DMT’s half-life in rats following i.p. injection is reported as 5–15 min and it is cleared from tissues within 1 h; behavioural testing was therefore generally initiated 1 h after dosing to avoid confounding acute somatic effects. Behavioural assays comprised: novelty-induced locomotion (NIL) in an open-field chamber for 45 min with automated tracking of horizontal motion, rotations, rearings and stereotypies; the elevated plus maze (EPM) for 5 min to measure open-arm time and entries; auditory-cued and contextual fear conditioning followed by tests of memory and extinction using standard shock/tone protocols and automated Video Freeze scoring; and the forced swim test (FST) with a pre-test (15 min) followed 24 h later by a 5-min test. For the FST, rats received a subchronic dosing schedule of three administrations (23.5, 6, and 1 h before the test), a regimen noted to be effective for a range of antidepressants. Freezing and FST behaviours (immobility, swimming, climbing) were scored as specified. Statistical analyses used GraphPad Prism. Time-dependent data were analysed via two-way repeated measures ANOVA where appropriate. Comparisons between DMT and vehicle used two-tailed Student’s t-tests or two-way ANOVAs with Sidak post-hoc tests; the contextual extinction data, which showed a bimodal distribution, were analysed with a Mann–Whitney test. FST data involving multiple comparisons were analysed by one-way ANOVA with Tukey’s post hoc test. Data are reported as means ± SEM, with significance thresholds noted in the text.
Immediately after a 10 mg/kg intraperitoneal dose, DMT produced acute somatic signs in the home cage—flat body posture and hind limb abduction—consistent with serotonin syndrome; head-twitches and wet-dog shakes were not observed. Somatic signs subsided within 30 min and by 1 h animals were qualitatively similar to vehicle-treated controls, so subsequent behavioural testing was conducted at 1 h when the compound is reported to be cleared from brain and plasma. In the novelty-induced locomotion test, DMT-treated rats showed reduced exploratory behaviour: total distance travelled was significantly shorter than in vehicle-treated animals, and both the number of rearings and time spent rearing were reduced. DMT decreased the time spent in stereotypies but did not alter the total number of stereotypic events, and it did not affect thigmotaxis. These results indicate an anxiogenic/neophobic effect in a novel open-field environment. Elevated plus maze results corroborated anxiogenic effects: DMT-treated animals spent a smaller percentage of time in open arms and made fewer open-arm entries compared with controls. Total distance travelled in the maze and average velocity were not statistically different between groups, and the number of closed-arm entries did not differ (Mean ± SEM; VEH = 9.375 ± 1.179; DMT = 8.556 ± 1.642; P = 0.6977), indicating no gross locomotor impairment at 1 h. In fear conditioning experiments, acute DMT given 1 h prior to conditioning did not alter baseline freezing before shocks but produced increased immediate freezing following training shocks, consistent with an anxiogenic effect during conditioning. When contextual and cued fear memory were assessed in the days after conditioning, no significant differences were found between DMT- and vehicle-treated groups, suggesting DMT did not alter consolidation of the original fear memories under the acute dosing regimen used. When the investigators administered DMT 1 h prior to cued-extinction training (animals conditioned drug-free), DMT-treated rats exhibited enhanced extinction: on a drug-free test the following day tone presentations produced significantly less freezing in the DMT group compared with controls, indicating stronger cued extinction memory. For contextual extinction using a stronger shock protocol to raise baseline freezing to approximately 60%, DMT was given 1 h prior to repeated extinction sessions on days 3–5; contextual extinction measured on day 6 (no drug) did not differ significantly between groups. The authors note the contextual extinction data had a bimodal distribution, suggesting presence of responders and non-responders. In the forced swim test, DMT significantly reduced immobility time and increased swimming counts relative to vehicle-treated animals, with no significant effect on climbing behaviour. The magnitude of the effects of DMT in the FST was indistinguishable from that of ketamine at the same dose, with no statistical differences between DMT- and ketamine-treated animals for immobility, swimming, or climbing. Across assays, the authors report that DMT produces an initial anxiogenic/neophobic profile in novel environments but later facilitates fear extinction (cued) and produces antidepressant-like effects in the FST. Quantitative details such as group sample sizes and exact p-values for many comparisons are not provided in the extracted text and appear to be listed in a Table not included here.
Cameron and colleagues interpret their findings as indicating that a single, hallucinogenic dose of DMT produces an early anxiogenic response in novel or threatening contexts but also yields longer-lasting behavioural effects consistent with antidepressant and anxiolytic actions. Specifically, DMT reduced exploratory behaviour and promoted anxiety-like responses in the open field and elevated plus maze, yet it facilitated cued fear extinction and reduced immobility in the forced swim test—effects the authors equate with potential therapeutic benefits relevant to PTSD and depression. The authors position their results relative to prior work on ayahuasca and its constituents. They note that DMT reproduces several behavioural features previously ascribed to ayahuasca (reduced exploration and increased anxiety in some paradigms, reduced immobility in the FST), suggesting DMT may be the ayahuasca component responsible for decreased exploration and acute anxiety in rats. At the same time, other ayahuasca components (β-carbolines such as harmine) have distinct effects and may contribute to or modulate antidepressant outcomes; the authors highlight that harmine alone has antidepressant-like effects in the FST and that combined or interactive effects between DMT and β-carbolines warrant investigation. Mechanistically, the investigators caution that simple serotonin transporter inhibition is an unlikely sole explanation for the antidepressant-like effects because DMT’s reported Ki at SERT is much weaker than that of typical SSRIs. Instead, they raise the possibility that DMT and ketamine—although acting primarily at different receptor systems (5-HT receptors versus NMDA receptors)—might converge on downstream mechanisms that promote neural plasticity. Supportive observations include the rapid metabolism and clearance of DMT despite persistent behavioural effects, and external reports (cited by the authors) that 5-MeO-DMT can induce proteins associated with dendritic spine formation in human organoids. They therefore suggest future studies should test whether DMT promotes structural and functional plasticity in prefrontal cortical neurons, similar to proposed mechanisms for ketamine. The authors acknowledge several uncertainties and limitations within the extracted text. Differences between acute and chronic dosing paradigms may account for discrepancies with prior studies of ayahuasca; acute DMT did not alter fear memory consolidation in their hands but did enhance extinction when given before extinction training. The contextual extinction data displayed a bimodal distribution indicating heterogeneous responsiveness, and the extracted text does not provide sample sizes or full statistical detail for every comparison (these appear to be in tables not included here). Safety considerations are noted: transient serotonin syndrome-like signs were observed immediately after dosing. Finally, the authors call for additional work to determine persistence of antidepressant effects, potential synergistic actions with β-carbolines, and whether endogenous DMT has a physiological role in mood regulation. In sum, the paper concludes that DMT alone can produce a behavioural profile in rats that includes acute anxiogenesis in novel contexts together with facilitation of fear extinction and antidepressant-like effects in the FST, and the authors argue these findings strengthen the rationale for further mechanistic and translational research into classical serotonergic psychedelics as fast-acting therapeutics.
Mood and anxiety disorders are among the leading causes of disability worldwide,,2 yet we still lack effective treatments. Current medications typically require 2-4 weeks before displaying efficacy, with approximately 1/3 of patients remaining unresponsive even after experimenting with several different medications.This distinct lack of adequate treatments has led investigators to seek alternatives that are both fast-acting and effective in treatmentresistant populations. The archetype for this next-generation of neurotherapeutics has been the dissociative anesthetic ketamine, as it produces rapid clinical antidepressant effectsincluding in treatment-resistant populations.Additionally, it has shown promise for treating post-traumatic stress disorder (PTSD).There is a clear need to identify compounds that produce therapeutic effects comparable to ketamine in order to better understand the basic mechanisms of fast-acting antidepressants. Perhaps some of the most promising compounds capable of eliciting beneficial clinical effects similar to ketamine are the classical serotonergic psychedelics such as psilocybin and LSD. In the past 30 years, there have been numerous clinical trials examining the therapeutic effects of these drugs,with most reporting robust antidepressant and anxiolytic effectseven in treatment-resistant populations.Though their notorious effects on perception have limited their therapeutic potential in mainstream medicine, these compounds have been used for centuries by people from a variety of cultures who consume psychedelic-rich plants and fungi during religious or healing ceremonies.One such traditional botanical mixture, known as ayahuasca, has attracted considerable attention owing to its potent antidepressant and anxiolytic effects in humans.Ayahuasca is an Amazonian tisane, which can be prepared by boiling the Banisteriopsis caapi vine and the leaves of the shrub Psychotria viridis.The latter plant contains substantial quantities of the classical hallucinogen N,N-dimethyltryptamine (DMT) while the former contains β-carboline alkaloids capable of inhibiting monoamine oxidase (MAO). As DMT is readily oxidized by MAO in the gut, the β-carbolines present in ayahuasca make DMT orally bioavailable.Studies conducted on populations that regularly use ayahuasca for religious purposes have demonstrated that it is relatively safe and could possibly promote mental well-being by reducing various measures of psychopathology and improving cognitive performance on tasks such as the Stroop and Wisconsin Card Sorting Tests.In addition to having robust antidepressant and anxiolytic effects in humans,ayahuasca has also been tested in several behavioral paradigms relevant to depression and anxiety. While the tisane reduces immobility time in the forced swim test, it also leads to anxiogenic responses in the open field and elevated plus maze.As ayahuasca is a complex mixture of psychoactive chemicals including tryptamine and βcarboline alkaloids (Figure),delineating the exact roles of each of these compounds will be critical to understanding the behavioral effects of the concoction. Of the major alkaloids in ayahuasca, harmine and harmaline have received the most attention thus far. Both mice and rats treated with harmine display decreased immobility in the FST,and harmine produces anxiolytic effects in rats as measured by behavior in the EPM.Harmaline has been shown to have more complex effects on EPM behavior, as it produces anxiogenic or anxiolytic responses in mice depending on the dose.In terms of exploratory behavior, neither acute nor chronic administration of harmine to rats had any effect on spontaneous locomotor activity in the open field.In Table, we have summarized current literature assessing the effects of ayahuasca and its chemical constituents on rodent behaviors relevant to anxiety and depression. While there have been a number of studies characterizing the effects of individual βcarboline alkaloids (e.g., harmine, harmaline, and tetrahydroharmine) in animal models of anxiety and depression, relatively little is known about the effects of N,Ndimethyltryptamine, the principle hallucinogenic component of ayahuasca. Here, we investigate the effects of a hallucinogenic dose of DMT in rodent behavioral tests relevant to anxiety, PTSD, and depression.
Because we were interested in assessing the effects of a hallucinogenic dose of DMT on rodent behaviors, we chose to use a 10 mg/kg dose of DMT for all of our studies. This dose, when converted to a human equivalent dose using allometric scaling,is expected to be hallucinogenic based on the pioneering human studies conducted by Strassman and coworkers.Furthermore, a preponderance of evidence from rodent drug discrimination studies using DMT and related compounds suggested that a 10 mg/kg dose would reliably produce effects characteristic of hallucinogens.Immediately following administration of an intraperitoneal dose of DMT (10 mg/kg), rats displayed flat body posture and hind limb abduction in their home cages (Figure), which is indicative of acute serotonin syndrome.We did not observe any head-twitches or wet-dog shakes. After 30 minutes, DMT-treated rats began to engage in normal behaviors, and by one hour post-administration, they were qualitatively indistinguishable from vehicletreated control animals. In order to specifically avoid any confounding effects from initial DMT-induced serotonin syndrome, all subsequent behavioral tests were conducted 1 h following DMT administration. DMT has a half-life of 5-15 min in rats following intraperitoneal injectionand is rapidly metabolized and cleared from brain, liver, and plasma within 1 h.This ensured that any behavioral effects observed would be due to persistent changes and not simply from the acute effects of the drug. Animals were tested in both novelty-induced locomotion (NIL) and elevated plus maze (EPM) paradigms to assess the effects of compound on anxiety (Figure). When exposed to a novel open space, DMT-treated animals displayed reduced exploratory behavior and traveled a significantly shorter total distance (Figureand) than vehicle-treated controls, but the drug did not affect thigmotaxis (Figure). Both the number of rearings and time spent rearing were significantly reduced by DMT (Figureand). Furthermore, the drug reduced the time spent engaging in stereotypies but not the total number, suggesting that each event occurred for a shorter period of time (Figureand). Taken together, an acute dose of DMT can have anxiogenic effects even after the drug has been cleared from the body.To corroborate these findings, we next assessed the effects of DMT on EPM behavior. DMTtreated animals spent a decreased percentage of time in the open arms of the maze (Figure) and had a fewer number of open arm entries (Figure). The total distance traveled in the maze (Figure) and the average velocity of the rodents (Figure) were not statistically different between DMT-treated and vehicle-treated groups. The number of closed arm entries was also not statistically different between the treatment groups (Mean ± SEM; VEH = 9.375 ± 1.179; DMT = 8.556 ± 1.642; P value = 0.6977, Student's t test). These data indicate that DMT administration does not grossly impair locomotion 1 h after dosing, but does have anxiogenic affects. We find that the behavioral effects in the NIL and EPM paradigms of a single hallucinogenic dose of DMT administered to rats are consistent with those of ayahuasca reported by Pic-Taylor and coworkers.Specifically, both DMT and ayahuasca reduce exploratory behavior (e.g., total distance traveled and number of rearings) in the open field, but do not influence thigmotaxis, a more traditional measure of anxiety in the open field.Furthermore, both DMT and ayahuasca tend to decrease the amount of time rats spend in the exposed areas of an elevated plus maze. These results contrast with the effects of harmine in these paradigmsand suggest that DMT is the component of ayahuasca that decreases exploratory behavior and promotes acute anxiety in rats. This conclusion is further supported by the seminal work of Geyer and coworkers, as they found that DMT decreased exploratory behaviors, decreased rearing, and promoted avoidance of the center of the arena in the behavioral pattern monitor.Seeing as these behaviors did not occur in familiar environments, they concluded that DMT and related compounds potentiate neophobia. The majority of studies regarding ayahuasca have focused on its ability to regulate mood and anxiety, and thus, very little is known about its effects on memory. Recently, Oliveira and co-workers discovered that chronic administration of ayahuasca did not impair spatial memory as measured by the Morris water maze, nor did it impact cued fear memory.However, chronically administered ayahuasca did enhance both foreground and background contextual fear memory.Therefore, we tested the effects of DMT on fear memory. Rodents were subjected to a fear conditioning paradigm (Figureand Figure) and both contextual and cued fear memory was assessed. Administration of DMT had no effect on initial freezing behavior prior to receiving foot shocks, however, it did significantly increase freezing immediately after the rodents received the training foot shocks (Figure). This anxiogenic effect is consistent with our NIL and EPM experiments, however, DMT did not have a lasting impact on fear memory, as both contextual and cued fear memory, assessed in the days following conditioning, were indistinguishable between DMT-and vehicle-treated animals (Figure). There are several possible explanations for the differing results between our study and that of Oliveira and co-workers.One possibility is that the effects of ayahuasca on fear conditioning are mediated by other compounds in the concoction, and not DMT. Alternatively, there could be differences between acute and chronic treatment paradigms. While DMT did not significantly impact fear conditioning, it did promote cued fear extinction. Animals were fear conditioned as described previously in the absence of drug, and DMT was administered 1 h prior to cued fear extinction training (Figure). The following day, cued extinction memory was assessed in the absence of drug. Tone presentations caused the DMT-treated group to freeze significantly less than the control group, indicating that DMT administered prior to extinction training resulted in a stronger extinction memory (Figureand). Patients with PTSD exhibit deficits in cued extinction recall,and thus, compounds capable of enhancing fear extinction learning/memory could prove to be effective therapeutics. Furthermore, our discovery that DMT promotes fear extinction in rats is consistent with previous reports demonstrating that psilocybin and MDMA promote cued fear extinction in mice,and adds to the growing body of literature suggesting that classical psychedelics and entactogens might be useful for treating PTSD in the clinic.In fact, MDMA was recently granted "breakthrough therapy" status by the Food and Drug Administration in order to expedite the approval process for this promising therapeutic. In a second experiment, animals were subjected to foreground contextual conditioning using additional and more intense foot shocks in order to raise their baseline contextual freezing levels (Figure). Contextual fear memory was extinguished on subsequent days 1 h following administration of DMT. Finally, contextual extinction memory was assessed on day 6 in the absence of drug. The DMT-treated animals were not statistically different from the vehicle control group (Figure). Interestingly, the data from the contextual extinction experiment had a bimodal distribution, suggesting that there were responders and nonresponders to contextual extinction training. Similar results in rat fear extinction experiments have been observed previously.Due to the known antidepressant properties of ayahuasca,we next assessed the effects of DMT in the forced swim test (FST) (Figure), a behavioral paradigm used to identify novel antidepressants, using ketamine as a positive control. We found that DMT significantly decreased the amount of time the rodents spent immobile and increased the amount of time they spent swimming (Figure). There was no significant difference in climbing behavior between DMT-and vehicle-treated animals. Notably, the effects of DMT in the FST were indistinguishable from those of the fast-acting antidepressant ketamine (Figure). These results suggest that DMT has antidepressant properties in rodents. While ayahuasca is known to produce anxiogenic effects in the open field and in the EPM, it also displays robust antidepressant properties in the FST.Similarly, we find that DMT also decreases immobility and increases swimming behavior in this behavioral paradigm (Figure). Because DMT and ayahuasca actually reduce locomotor activity in the open field, the increased swimming behavior observed in the FST can be interpreted as a true antidepressant effect, and not simply due to a confounding effect from changes in general activity levels. Finally, as harmine alone has produced similar results in the FST,it is likely that both DMT and the β-carboline alkaloids contribute to the antidepressant effects of ayahuasca. It will be interesting to see if DMT and harmine have any synergistic or additive effects that go beyond harmine's known capacity to increase DMT bioavailability through inhibition of monoamine oxidase.DMT is known to inhibit the serotonin transporter,which could possibly explain its antidepressant effects in the forced swim test. However, its K i value is quite low (4 μM) as compared to common selective serotonin reuptake inhibitors such as fluoxetine (K i value for fluoxetine = 7 nM).Furthermore, ayahuasca produces rapid antidepressant effects in treatment-resistant populations, suggesting that it works through a different mechanism than traditional antidepressants.Perhaps the most intriguing aspect of the results presented here is the remarkable similarity between the effects of DMT in rats and those of the fast-acting antidepressant ketamine. In addition to having acute anxiogenic effects in the EPM,ketamine facilitates fear extinction learning and reduces immobility in the FST.These known effects of ketamine are the same as what we report here for DMT. In fact, when compared directly, we found that there was no statistical difference between DMT-and ketamine-treated animals in terms of the amount of time they spent immobile, swimming, or climbing in the FST. As the antidepressant effects of ketamine are known to be long-lasting, future studies should investigate if DMT produces similar persistent effects. Ketamine is believed to exert its therapeutic effects by promoting structural and functional plasticity in the prefrontal cortex (PFC)-a key brain region involved in both extinction learningand the top-down control of mood.While the primary receptors targeted by ketamine and DMT are different (i.e., NMDA and 5-HT receptors, respectively), it is intriguing to speculate that they might produce similar downstream effects on neural plasticity ultimately underlying their behavioral effects. Two pieces of evidence suggest that this might be the case. First, DMT is rapidly metabolized,however, its anxiolytic and antidepressant effects are observed long after the compound has been cleared from the body suggesting that plasticity might result in long-lasting changes contributing to its behavioral effects. Second, the related molecule 5-MeO-DMT was recently shown to promote the expression of proteins involved in the formation and maturation of dendritic spines in human cerebral organoids.An important question that remains is whether or not DMT and/or ayahausca, like ketamine, is capable of promoting structural and functional plasticity in prefrontal cortical neurons. Finally, our studies on the effects of DMT in rats raise important questions about the possible roles of endogenous DMT in regulating mood and anxiety. A preponderance of evidence suggests that DMT is a hallucinogenic compound produced by a variety of animals including humans.Due to its rapid metabolism, exact in vivo quantification of DMT has proven challenging. Assuming that its endogenous production yields sufficient quantities, the results presented here suggest that it might play a natural role in the regulation of anxiety and mood. In conclusion, we report the effects of DMT-the principle hallucinogenic component of ayahuasca-on rodent behaviors related to anxiety and depression. To the best of our knowledge, we describe the first example in rats of a classical serotonergic psychedelic facilitating fear extinction learning and producing antidepressant effects in the FST. Importantly, we find that like ketamine, DMT produces behavioral responses in several paradigms relevant to anxiety and depression. Our work coupled with the fast-acting clinical effects of ayahuasca, strengthens the growing evidence that classical serotonergic psychedelics might serve as fast-acting antidepressants and anxiolytics.
Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA, USA), were housed two per cage, and were given ad libitum access to food and water. Lights in the vivarium were turned on at 07:00 hours and turned off at 19:00 hours. All experiments were performed on 8-to 14-week-old male rats. Studies were performed during the light-on phase, with experiments taking place between 08:00 and 18:00 hours. All experimental procedures involving animals were approved by the UC Davis Institutional Animal Care and Use Committee (IACUC) and adhered to principles described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The University of California, Davis is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
The DMT utilized in these studies was synthesized in our laboratory using the following procedure. To an ice-cold solution of tryptamine (0.50 g, 3.1 mmol), and glacial acetic acid (0.89 mL, 15 mmol, 5.0 equiv) in MeOH (49 mL) was added sodium cyanoborohydride (0.39 g, 6.2 mmol, 2.0 eq.) followed by 37% formaldehyde (aq) (0.66 mL, 8.1 mmol, 2.6 equiv). The reaction was stirred at room temperature for 5 h before being concentrated under reduced pressure. The unpurified material was diluted with CH 2 Cl 2 (50 mL) and 1M NaOH (aq) (100 mL). The phases were separated and the aqueous phase was extracted twice with CH 2 Cl 2 (50 mL). The organic phases were combined, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The unpurified material was dissolved in acetone (15 mL) and added to a boiling solution of fumaric acid (0.26 g, 2.2 mmol, 0.7 equiv) in acetone (50 mL). A precipitate formed immediately, and the solution was cooled to room temperature prior to being filtered. The resulting white solid was dried under reduced pressure to yield the pure compound as the fumarate salt (2:1 DMT:fumaric acid) (white solid, 0.48 g, 62%): Melting Point = 140-142ºC; TLC R f (DMT free base) = 0.35 (9:1 CH 2 Cl 2 :MeOH with 1% NH 4 OH (aq) ); 1 H NMR (2:1 DMT:fumaric acid, 600 MHz, DMSOd 6 ) δ 10.8 (brs, 1H), 7.5 (d, 1H, J = 7.9 Hz), 7.3 (d, 1H, J = 7.9 Hz), 7.1 (s, 1H), 7.0 (t, 1H, J = 7.9 Hz), 6.9 (t, 1H, J = 7.9 Hz), 6.5 (s, 1H), 2.9 (t, 2H, J = 8.6 Hz), 2.8 (t, 2H, J = 8.6 Hz), 2.4 (s, 6H) ppm;C NMR (2:1 DMT:fumaric acid, 100 MHz, CD 3 OD) δ 173.8, 138.2, 136.9, 128.1, 124.2, 122.7, 120.1, 119.0, 112.5, 110.0, 59.2, 43.4, 21.9 ppm; IR (2:1 DMT:fumaric acid, diamond, ATR) ν 3483, 3146, 3107, 3045, 2927, 2881, 1561, 1226, 749cm -1 . The compound was stored at -20ºC in the dark prior to use. The prepared DMT was judged to be analytically pure by both LC-MS and NMR spectroscopy. For each experiment, a solution of DMT•fumarate (2:1) in 0.9% sterile saline was freshly prepared and passed through a 0.2 μm syringe filter. For all experiments, DMT•fumarate (2:1) was administered at 10 mg/kg via intraperitoneal injection using an injection volume of 1 mL/kg. Ketamine•HCl was purchased from Fagron (St. Paul, MN; batch# 13B28-U03-010128) and administered at 10 mg/kg (in 0.9% sterile saline) via intraperitoneal injection using an injection volume of 1 mL/kg. We used this subanesthetic dose of ketamine as it has been shown previously to elicit robust effects in the forced swim test(Browne and Lucki, 2013) without impairing motor function.For our vehicle controls, 0.9% sterile saline solution was utilized.
Drug-naïve rats were administered either DMT or vehicle 1 hr before behavioral testing. Animals were allowed to acclimate to the test room for 10 mins prior to being gently placed into the center of an AccuScan Instruments (Columbus, OH, USA) open field chamber (Digiscan Animal Activity Monitor Model #RXYZCM()CCD) and allowed to freely explore the chamber for 45 mins. At the conclusion of the test, animals were returned to their home cages and the test chambers cleaned with 10% Nolvasan. Horizontal motion, rotations, and stereotypies (repetitive beam breaks) were recorded in 1-min intervals for the duration of the test and analyzed using the program Integra. The margin of the arena was defined as being 10 cm from the wall. The open field chamber measured 41.9 cm L x 41.9 cm W x 28.6 cm H and was illuminated to between 25 and 30 lux.
The EPM apparatus consisted of a plus-shaped black plastic platform positioned 50 cm above the ground and was illuminated to between 20 and 25 lux. Two opposite arms of the maze were bordered by vertical walls measuring 31.75 cm high, with the other two arms possessing unprotected edges. The same animals were used in both the NIL and EPM tests. Two days following the NIL test, rats were administered DMT or vehicle (consistent with how they were dosed previously) 1 hr before being placed into the center of the maze facing an open arm and allowed to explore freely for 5 min. At the conclusion of the test, rats were returned to their home cages and the apparatus was cleaned with 10% Nolvasan. Animal movement was recorded and analyzed during the trial using EthoVision XT (version 9) software. The EPM is an established method for measuring anxiety in rodents.(and Frye, 2007).
On day 1, drug-naïve animals were administered either DMT or vehicle 1 hr prior to conditioning. They were placed in a fear conditioning apparatus (Med Associates model # MED-VFC2-SCT-R) for 3.5 min prior to three presentations of auditory cues (80 dB white noise, 30 s), each co-terminating with a foot shock (0.8 mA, 2 s.) and spaced 90 s apart. The fear conditioning apparatus consisted of a 30.5 cm x 24.1 cm x 21 cm internal soundproof chamber, with metal grated floors, an infrared camera, a sound generator, and a light source. After the last shock, the animals remained in the chambers for an additional 2 min before being returned to their home cages. During fear conditioning, the apparatus was illuminated to 100 lux and did not contain any additional odor cues. On day 2, contextual fear memory was assessed by exposing the animals to the conditioning context for 10 min before returning them to their home cages. On day 3, cued fear memory was assessed by exposing the animals to a novel context (lights off, A-frame insert, smooth plastic floor insert, additional vanilla odor) for 2 min prior to eight presentations of auditory cues (80 dB white noise, 30 s) spaced 30 s apart. Freezing responses for cue testing (day 3) are presented as the percentage of time spent freezing during the tone presentations. Fear conditioning experiments were performed between the hours of 08:00-11:00. Freezing behavior was scored using Med Associates Video Freeze software v2.25 (motion threshold = 18 au, detection method = linear, minimum freeze duration = 30 frames, which is equal to a 1 sec freeze). The apparatus was cleaned with 70% EtOH in between trials.
On day 1, drug-naïve animals were fear conditioned as described above, but in the absence of drug, and allowed to rest on day 2. On day 3, the animals were administered either DMT or vehicle 1 hr prior to extinction training. Extinction training consisted of exposure to a novel context (lights off, A-frame insert, smooth plastic floor insert, additional vanilla odor) for 2 mins prior to 8 presentations of auditory cues (80 dB white noise, 30 s) spaced 30 s apart. After the extinction training, animals were returned to their home cages. The procedure was repeated on day 4 in the absence of drug. Fear extinction experiments were performed between the hours of 08:00-11:00. Freezing responses for cue testing (day 4) are presented as the percentage of time spent freezing during the tone presentations. Freezing behavior was scored using Med Associates Video Freeze software v2.25 (motion threshold = 18 au, detection method = linear, minimum freeze duration = 30 frames, which is equal to a 1 sec freeze). The apparatus was cleaned with 70% EtOH in between trials.
First, drug-naïve animals were subjected to an optimal foreground contextual fear conditioning protocol. Rats were placed into the fear conditioning apparatus for three mins before being subjected to six foot shocks (1.0 mA, 2 s) spaced 58 s apart. The use of this strong foot shock protocol was necessary to sufficiently increase baseline contextual freezing levels to ~60% so that extinction (i.e. reduction in freezing) could be effectively measured. After initiation of the last shock, the animals remained in the chambers for an additional 2 min before being returned to their home cages. The animals were allowed to rest on day 2. On day 3, they were administered either DMT or vehicle 1 hr prior being placed in the fear conditioning context for 10 min. This procedure was repeated on days 4 and 5. On day 6, the animals were placed in the fear conditioning context without receiving any injections. Contextual fear conditioning experiments were performed between the hours of 08:00-11:00. Freezing behavior was scored using Med Associates Video Freeze software v2.25 (motion threshold = 18 au, detection method = linear, minimum freeze duration = 30 frames, which is equal to a 1 sec freeze) and reported as the percentage of time spent freezing over the entire 10 min session. The apparatus was cleaned with 70% EtOH in between trials.
The FST apparatus consisted of a clear Plexiglas cylinder measuring 80 cm tall, 20 cm in diameter and filled with 30 cm of 24 ± 1ºC water. Fresh water was used for every rat. Drugnaïve animals were subjected to a pre-test phase in which they were placed in the cylinder for 15 mins before being dried and returned to their home cage. Twenty-four hours later, rats were again placed in the FST apparatus for 5 mins and their activity was video recorded. Each rat received three administrations of DMT, ketamine, or vehicle at 23.5, 6, and 1 hr before the test phase. This subchronic dosing paradigm has proven effective for a wide range of antidepressant compounds.Each video was scored for immobility, swimming, and climbing behavior by a trained observer. The dominant behavior of the animal (i.e., immobility, swimming, or climbing) was determined every 5 sec and quantified as a "count." As the experiment lasted for 5 mins, the sum of the counts for all four behaviors equals 60.
Statistical analyses were performed using GraphPad Prism (version 7.0a). Data exhibiting a time dependency (e.g., 2b and S3) were analyzed using a two-way repeated measures analysis of variance (ANOVA). Comparisons of DMT-and vehicle-treated groups were accomplished using two-tailed Student's t-tests or two-way ANOVAs with a Sidak Post-Hoc test. As data from the contextual extinction experiment (Figure) displayed a bimodal distribution, a Mann-Whitney Test was used. To analyze data from the FST (Figure), which involved multiple comparisons, a one-way ANOVA was utilized with Tukey's post hoc test. Details of all statistical tests performed in this study are provided in Table. All data are represented as means ± SEM, NS = not significant, *p < 0.05, **p < 0.01
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