This mouse study investigates the response to 5-MeO-DMT on cortical activity via genetic knockout of the serotonin 5-HT2A receptor in their test mice and the selective inhibition of 5-HT1A receptors via antipsychotic drugs. 5-MeO-DMT evoked marked alterations in the function of primary sensory areas (Au1, S1, V1) as well as in the highest association cortex (PFC), with a differential contribution of the 5-HT1A and 5-HT2A receptors that were selectively inhibited by antipsychotic drugs.
Introduction
5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural hallucinogen, acting as a non-selective serotonin 5-HT1A/5-HT2A-R agonist. Psychotomimetic agents such as the non-competitive NMDA-R antagonist phencyclidine and serotonergic hallucinogens (DOI and 5-MeO-DMT) disrupt cortical synchrony in the low frequency range (<4 Hz) in rat prefrontal cortex (PFC), an effect reversed by antipsychotic drugs.
Methods
Here we extend these observations by examining the effect of 5-MeO-DMT on low frequency cortical oscillations (LFCO, <4 Hz) in PFC, visual (V1), somatosensory (S1) and auditory (Au1) cortices, as well as the dependence of these effects on 5-HT1A-R and 5-HT2A-R, using wild type (WT) and 5-HT2A-R knockout (KO2A) anesthetized mice.
Results
5-MeO-DMT reduced LFCO in the PFC of WT and KO2A mice. The effect in KO2A mice was fully prevented by the 5-HT1A-R antagonist WAY-100635. Systemic and local 5-MeO-DMT reduced 5-HT release in PFC mainly via 5-HT1A-R. Moreover, 5-MeO-DMT reduced LFCO in S1, Au1 and V1 of WT mice and only in V1 of KO2A mice, suggesting the involvement of 5-HT1A-R activation in the 5-MeO-DMT-induced disruption of V1 activity. In addition, antipsychotic drugs reversed 5-MeO-DMT effects in WT mice.
Discussion
The present results suggest that the hallucinogen action of 5-MeO-DMT is mediated by simultaneous alterations of the activity of sensory (S1, Au1, V1) and associative (PFC) cortical areas, also supporting a role of 5-HT1A-R stimulation in V1 and PFC, in addition to the well-known action on 5-HT2A-R. Moreover, the reversal by antipsychotic drugs of 5-MeO-DMT effects adds to previous literature supporting the usefulness of the present model in antipsychotic drug development.
Papers cited by this study that are also in Blossom
Carter, O. L., Hasler, F. ;., Pettigrew, J. D. et al. · Psychopharmacology (2007)
Gonza ´lez-Maeso, J., Weisstaub, N. V., Zhou, M. et al. · Neuron (2007)
Halberstadt, A. L., Geyer, M. A. · Neuropharmacology (2011)
Kometer, M., Cahn, B. R., Andel, D. et al. · Biological Psychiatry (2011)
Serotonergic hallucinogens produce profound alterations in perception, thought and mood. Riga and colleagues note two chemical classes: indoleamines (for example LSD, psilocin, DMT and 5-MeO-DMT) which bind with high affinity to several serotonin receptor subtypes including 5-HT1A and 5-HT2A, and phenylalkylamines (for example mescaline and DOI) which are more selective for 5-HT2A/2C receptors. Previous preclinical work has shown that psychotomimetic agents such as PCP, DOI and 5-MeO-DMT disrupt low frequency cortical oscillations (LFCO, <4 Hz) in rodent prefrontal cortex (PFC), and that antipsychotic drugs can reverse these disruptions. However, the relative contributions of 5-HT1A and 5-HT2A receptors across different cortical regions remained unclear. The present study set out to characterise how 5-MeO-DMT affects LFCO in medial PFC and primary sensory cortices (somatosensory S1, auditory Au1 and visual V1) in anaesthetised mice, and to dissect the roles of 5-HT1A and 5-HT2A receptors. To achieve this, the investigators used a combination of genetic loss-of-function (5-HT2A receptor knockout mice, KO2A) and pharmacological tools (a selective 5-HT1A antagonist and antipsychotic drugs) together with electrophysiology and in vivo microdialysis to measure local field potentials and extracellular serotonin levels.
Male homozygous 5-HT2A knockout mice (KO2A) and wild-type (WT) littermates on a C57/BL6 background aged 9–16 weeks were used. Animal procedures followed EU regulations and stereotaxic coordinates were referenced to the mouse brain atlas. Drugs included 5-MeO-DMT, haloperidol (HAL), risperidone (RIS) and the selective 5-HT1A antagonist WAY-100635; doses are reported as free bases and most administrations were subcutaneous. For systemic electrophysiology experiments 5-MeO-DMT was given at 1 mg/kg s.c.; WAY-100635 was used at 0.5 mg/kg s.c.; HAL and RIS were used at 0.6 mg/kg and 1 mg/kg respectively. For local effects in mPFC, 5-MeO-DMT was applied via reverse microdialysis in artificial cerebrospinal fluid (aCSF). Electrophysiological recordings were performed under chloral hydrate anaesthesia (initial 400 mg/kg i.p., maintenance infusion 50–70 mg/kg/h). Local field potentials (LFPs) were recorded in medial PFC (mPFC) and electrocorticograms (ECoGs) from primary sensory cortices S1, Au1 and V1 using epidural electrodes. Signals were amplified, filtered (0.1–100 Hz) and after a 5-minute stable baseline a slow injection of 5-MeO-DMT was given; in some experiments antipsychotics or WAY-100635 were administered with defined inter-injection intervals. LFCO was defined as power between 0.15 and 4 Hz. Off-line analysis used Fast Fourier Transform on 18 consecutive ten-second epochs per condition, with LFCO power expressed as percentage of baseline. In vivo microdialysis measured extracellular serotonin (5-HT) in mPFC of freely moving mice 20–24 h after probe implantation. Probes were perfused with aCSF containing 1 μM citalopram to block reuptake, samples were collected every 20 minutes and analysed by HPLC-amperometry. Both systemic (1 mg/kg s.c.) and local (30–300 μM via reverse dialysis) applications of 5-MeO-DMT were tested. Behavioural head twitch response (HTR) was scored during microdialysis sessions in consecutive 5-minute blocks. Statistical analyses comprised Student’s t-test and two-way repeated-measures ANOVA with treatment and genotype (or area) as factors, followed by Newman–Keuls post-hoc tests; significance was set at p<0.05.
Baseline LFCO power did not differ significantly between WT and KO2A mice across mPFC, S1, Au1 and V1. In mPFC systemic 5-MeO-DMT (1 mg/kg) significantly reduced LFCO in WT mice from 0.054±0.004 to 0.030±0.002 μV2, equivalent to 51.1±2.5% of baseline (n=40). KO2A mice also showed a reduction but of smaller magnitude, from 0.064±0.004 to 0.041±0.004 μV2, or 61.4±3.3% of baseline (n=13). Two-way ANOVA indicated significant effects of 5-MeO-DMT treatment and genotype-related interactions. To probe 5-HT1A involvement, KO2A mice were pretreated with the selective antagonist WAY-100635 (0.5 mg/kg). WAY-100635 alone increased LFCO power and fully prevented the 5-MeO-DMT-induced LFCO reduction in KO2A mice. Two-way ANOVA showed significant main effects of 5-MeO-DMT and WAY-100635 and a significant treatment x pre-treatment interaction (for example F(2,34)=14.29, p<0.005 for 5-MeO-DMT; F(2,17)=32.75, p<0.0001 for WAY-100635). Microdialysis revealed comparable basal extracellular 5-HT in mPFC (WT 14.5±1.8 fmol/30 μl, n=15; KO2A 16.2±2.3 fmol/30 μl, n=11). Systemic 5-MeO-DMT decreased extracellular 5-HT similarly in both genotypes, with maximal reductions to 57.0±7.0% (WT) and 43.6±4.9% (KO2A) of baseline; two-way ANOVA showed a significant effect of 5-MeO-DMT (F(9,99)=8.35; p<0.00001) but no genotype interaction. Behaviourally, 5-MeO-DMT increased HTR in WT mice from 0.98±0.29 to 4.09±0.66 occurrences, whereas KO2A mice did not show this increase (0.92±0.34 to 0.39±0.15). Local reverse-dialysis application of 5-MeO-DMT to mPFC produced dose-dependent and genotype-dependent changes in extracellular 5-HT. At 30 μM both genotypes showed similar reductions. Higher concentrations diverged: 300 μM increased extracellular 5-HT to 149.5±22.1% of baseline in WT mice, whereas 100 μM decreased 5-HT to 38.8±8.1% of baseline in KO2A mice. ANOVA on normalized areas under the curve showed significant effects of concentration, genotype and their interaction. Antipsychotic drugs reversed the 5-MeO-DMT-induced LFCO reduction in WT mPFC. Both haloperidol and risperidone restored LFCO towards baseline; two-way ANOVA revealed significant main effects and interactions (for example F(2,26)=109.76, p<0.00001 for 5-MeO-DMT treatment; F(2,13)=4.62, p<0.05 for antipsychotic treatment). In primary sensory cortices, systemic 5-MeO-DMT reduced LFCO in WT mice in S1 to 67.1±4.3% of baseline, in Au1 to 59.3±4.1% and in V1 to 67.1±6.8%. In KO2A mice reductions were seen only in V1 (50.2±5.1% of baseline) but not in S1 or Au1. Statistical analyses indicated significant effects of treatment across areas in WT mice and genotype x treatment and area effects in KO2A mice, consistent with a regionally selective contribution of 5-HT1A and 5-HT2A receptors.
Riga and colleagues interpret their data as confirming that 5-MeO-DMT disrupts low frequency cortical synchrony in PFC and primary sensory areas through actions at both 5-HT1A and 5-HT2A receptors. They highlight that systemic 5-MeO-DMT reduced LFCO in WT mice and produced a smaller but significant reduction in KO2A mice, indicating that receptors other than 5-HT2A contribute to the effect. Prevention of the LFCO reduction in KO2A mice by the 5-HT1A antagonist WAY-100635 supports a compensatory or unmasked role for 5-HT1A receptors when 5-HT2A receptors are absent. The investigators discuss mechanistic distinctions between presynaptic and postsynaptic 5-HT1A activation. Systemic 5-MeO-DMT decreased extracellular 5-HT similarly in WT and KO2A mice, suggesting a predominant presynaptic 5-HT1A autoreceptor-mediated inhibition of dorsal raphe neurons. By contrast, local application in mPFC revealed genotype-dependent concentration–response differences: low concentrations reduced 5-HT in both genotypes, while higher concentrations increased 5-HT in WT but not KO2A mice, implicating cortical 5-HT2A receptor-mediated effects on local serotonergic output. The concomitant increase in HTR in WT but not KO2A mice following systemic 5-MeO-DMT is interpreted as evidence of postsynaptic 5-HT2A activation at the behavioural dose used. Regional differences in receptor contribution are emphasised. The differential sensitivity of sensory cortices to 5-MeO-DMT in KO2A mice—marked in S1 and Au1 but preserved in V1—led the authors to propose a relatively greater role for 5-HT2A receptors in S1/Au1 and for 5-HT1A receptors in V1. They note dense expression and co-localisation of both receptor subtypes in visual cortex and cite prior evidence linking these receptors to modulation of thalamic visual inputs and visual plasticity. The apparent paradox that activation of both excitatory (5-HT2A) and inhibitory (5-HT1A) receptors can reduce LFCO is discussed in terms of complex cellular distribution and interactions, including expression in pyramidal cells and GABAergic interneurons and possible synergistic network effects. Limitations acknowledged by the authors include technical constraints in comparing systemic and local drug application: the dialysis membrane and extracellular clearance reduce effective concentrations reaching tissue, and the microdialysis probe samples only a small neuronal population. Finally, the reversal of 5-MeO-DMT effects by risperidone is attributed to direct competition at 5-HT2A receptors, whereas haloperidol reversal is interpreted at the network level via dopamine D2 receptor blockade affecting excitatory/inhibitory balance. The authors conclude that these findings illuminate receptor- and region-specific mechanisms by which indoleamine hallucinogens perturb cortical circuits related to hallucinations and support the LFCO disruption model as useful for antipsychotic drug development.
The authors conclude that 5-MeO-DMT produces marked disruptions of cortical function in both primary sensory cortices (Au1, S1, V1) and associative cortex (PFC), mediated by 5-HT1A and 5-HT2A receptors with differential regional contributions. They further state that 5-HT1A receptors play a major role in the drug’s effects on visual and prefrontal cortices, and that the ability of antipsychotic drugs to reverse the LFCO reduction supports the model’s relevance for antipsychotic drug development.
The serotoninergic hallucinogens evoke profound changes in perception, thought, mood and cognition. Chemically, these agents are divided in two main classes: a) indoleamines such as lysergic acid diethylamide (LSD), psilocin, psilocybin, N,N-dimethyltryptamine (DMT) and 5-Methoxy-N,Ndimethyltryptamine (5-MeO-DMT) which bind with high affinity to several 5-HT receptors (5-HT-R), namely 5-HT 1A -R 5-HT 2A -R and 5-HT 2C -R and, b) phenylalkylamines such as mescaline and 2,5-dimethoxy-4iodoamphetamine (DOI) which are highly selective for 5-HT 2A -R and 5-HT 2C -R. The interest in serotoninergic hallucinogens lies in their capacity to model some schizophrenia symptoms by inducing mental states that resemble psychoses, also helping to study brain areas/circuits altered in psychiatric disorders. Moreover, some of these agents were marketed in the past (e.g. LSD) as a therapeutic aid in psychoanalysis, and there is a growing interest in their therapeutic use for the treatment of mood and anxiety disorders. 5-MeO-DMT is a natural hallucinogen found in a variety of plant preparations (e.g., Virola snuffs) used for religious and recreational purposes. 5-MeO-DMT is a potent fast-acting hallucinogen with short duration of action in humans, and induces various physiological and behavioral changes in animal models. 5-MeO-DMT is currently controlled in the United States as a Schedule I hallucinogen by the Drug Enforcement Administration. Like other indoleamine hallucinogens, 5-MeO-DMT shows high affinity for 5-HT 1A -R and 5-HT 2A -R) and both receptors participate in its behavioral effects actions. Preclinical and clinical evidence supports that the psychotomimetic action of classical hallucinogens is mainly mediated by their agonistic actions at cortical 5-HT 2A -R. Although this is the prevailing view, other findings indicate that 5-HT 1A -R also play an important role in the behavioral effects of indoleamine hallucinogensas well as in the mechanism of action of antipsychotic drugs. However, the exact role of 5-HT 1A-R activation in the psychotomimetic actions of indoleamine hallucinogens remains unclear. Cortical oscillations have a key role in brain function due to their involvement in input selection, synaptic plasticity, memory consolidation and information processing. Alterations in oscillatory activity have been associated with psychiatric disorders such as schizophreniaand have been found in healthy volunteers after the consumption of psychotomimetic agents. Moreover, alterations in ACCEPTED MANUSCRIPT 4 cortical oscillatory activity have been reported in neurodevelopmental and pharmacological models of schizophrenia. Hence, previous studies showed that psychotropic agents with different mechanism of action, such as the non-competitive NMDA receptor antagonist phencyclidine (PCP), the preferential 5-HT 2A -R agonist DOIand the non-selective 5-HT 1A/2A -R agonist 5-MeO-DMT, markedly disrupted the activity of rodent prefrontal cortex (PFC), increasing pyramidal neuron discharge and reducing low frequency cortical oscillations (LFCO, <4Hz). Classical and atypical antipsychotic drugs reversed these alterations in all cases. Given the limited knowledge of the brain areas/networks involved in hallucinogen action, the aim of the present study was to assess the effects of 5-MeO-DMT on cortical activity in anaesthetized mice. We used a combination of genetic (5-HT 2A -R knockout mice) and pharmacological approaches to 1) examine the effect of 5-MeO-DMT on LFCO in PFC and primary sensory areas, and 2) examine the role of 5-HT 1A -R and 5-HT 2A -R in the reduction of LFCO evoked by 5-MeO-DMT in the various cortical areas examined.
We used 9-16 week-old male homozygous 5-HT 2A -R knockout mice (referred as KO2A) and wild-type (WT) mice of the same genetic background (C57/BL6). Generation of KO2A strain has been reported elsewhere. Animal care followed the European Union regulations (directive 2010/63 of 22/09/2010) and was approved by the Institutional Animal Care and Use Committee. Stereotaxic coordinates were taken from bregma and duramater according to the mouse brain atlas.
5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT), risperidone (RIS) and WAY-100635 maleate were from Sigma/RBI (Natick, MA). Haloperidol (HAL) was from Laboratorios Esteve (Barcelona, Spain). Citalopram hydrobromide was from Tocris (Bristol, UK). Doses are expressed as free bases. All drugs were dissolved in saline and injected subcutaneously (s.c.). For the assessment of local effects in mPFC, 5-MeO-DMT was dissolved in the artificial cerebrospinal fluid (aCSF) used to perfuse the microdialysis probes (see below).
Electrophysiological procedures were performed as described elsewhere. Mice were anesthetized with chloral hydrate (400 mg/kg i.p.). Chloral hydrate was subsequently administered M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 using a perfusion pump (50-70 mg/kg/h) to maintain a constant level of anesthesia. Recordings of oscillatory activity (local field potential, LFPs) were carried out in the medial PFC (mPFC; AP+2.2 to +2.4, ML-0.2 to -0.4, DV-1.0 to -2.5 below brain surface; coordinates in mm). In most experiments simultaneous recordings of oscillatory activity in the primary somatosensory (S1, AP+0.5, ML+3.0), primary auditory (Au1, AP-2.8, ML+4.2) or primary visual (V1, AP-3.6, ML+2.5) cortices were also performed using epidural electrodes (electrocorticograms, ECoGs). LFP and ECoGs signal were amplified (X 1000 and X2000 respectively) and filtered between 0.1-100 Hz. After recording stable baseline activity for 5 min, drugs were administered. 5-MeO-DMT (1 mg/kg) was slowly injected followed by saline (in all genotypes) or the antipsychotics HAL (0.6 mg/kg) or RIS (1 mg/kg) in WT mice. Time between injections was 12 minutes. To further evaluate the role of the 5-HT 1A -R on 5-MeO-DMT induced disruption of prefrontal activity, KO2A mice were pretreated (5 minutes after recording stable baseline activity) with saline or the selective 5-HT 1A -R WAY-100635 (0.5 mg/Kg) before 5-MeO-DMT administration. WAY-100635 dose was chosen from the literature owing to its ability to antagonize behavioural effects of 5-MeO-DMT.
Extracellular serotonin (5-HT) concentrations were measured by in vivo microdialysis as previously described. Briefly, one concentric dialysis probe (membrane 2 mm long) was implanted in mPFC (AP +2.2; ML -0.2; DV -3.4 from skull). Microdialysis experiments were carried out in freely moving mice 20-24 h after surgery. Probes were continuously perfused with aCSF (in mM: NaCl, 125; KCl, 2.5; CaCl2, 1.26 and MgCl2, 1.18) pumped at 1.5 μl/min and containing 1 μM citalopram to prevent 5-HT reuptake. In these conditions, the extracellular 5-HT concentration is representative of the spontaneous 5-HT release by nerve terminals. Dialysate samples were collected every 20 min. After an initial 60 min stabilization period, four baseline samples were collected before systemic or local (intra-mPFC) pharmacological treatments. 5-HT concentrations was analysed by HPLC-amperometric detection (Hewlett Packard-1049, Palo Alto, CA, USA) at +0.60 V, with detection limit of 2 fmol/sample. Moreover, side-to-side head weaving (head twitch response, HTR) was scored for 4 consecutive 5min periods by direct observation of mice undergoing in vivo microdialysis, in basal conditions and after 5-MeO-DMT administration. At the end of experiments, mice were killed by anesthetic overdose. Brain sections were stained according to standard procedures, to verify recordings sites and proper probe placement.
Off-line analysis of electrophysiology results was performed using the Spike2 software. Drug effects on LFCO were analyzed, as follows. For each condition (baseline, 5-MeO-DMT, WAY-100635 or saline + 5-MeO- DMT and 5-MeO-DMT + antipsychotic or saline), the power spectrum of 3 minutes signal was analyzed offline using Spike2 software built-in and self-developed routines. Eighteen consecutive ten-second periods were subjected to a Fast Fourier Transformation, for frequencies from 0.15 to 80 Hz, with a resolution of 0.15 Hz. For statistical analyses, the mean values of the LFCO power (0.15-4 Hz) were quantified. Data were expressed as percentage of baseline and are given as mean ± SEM. Microdialysis data are expressed as fmol/30 μl for 5-HT and shown in the figures as percentages of basal values, averaged from four fractions collected before treatment. Normalized areas under curve values (AUCs) were also calculated to compare genotypes. Stereotypes were rated during the last 20 min before drug administration and the first 20 min postdrug administration and were divided in four 5-min blocks. HTR was quantified as the number of occurrences during the observation period. Total scores for each animal were calculated by averaging the individual values during each 5-min period. Results are given as (mean±SEM). All data were analyzed by Student's t-test or two-way repeatedmeasures analysis of variance (ANOVA), with treatment (or area) and genotype as factors, followed by Newman-Keuls post-hoc test, as appropriate. The level of significance was set at p<0.05.
As previously reported, the power spectra of LFCO in mouse mPFC did not differ between genotypes (WT: 0.054±0.004; KO2A: 0.064±0.004 μV 2 ; n.s Student's t-test; n=40 and 22, respectively). Similarly, there were no differences among genotypes in the power spectra of LFCO in S1 (WT: 0.053±0.007; KO2A: 0.087± 0.019 μV 2 ; n.s Student's t-test; n=11 and 10, respectively), Au1 (WT: 0.041±0.012; KO2A: 0.027±0.018 μV 2 ; n.s Student's t-test; n=11 and 10, respectively) and V1 (WT: 0.065± 0.011; KO2A: 0.073±0.023 μV 2 ; n.s Student's t-test; n=10 and 6, respectively).
As reported in rats, systemic 5-MeO-DMT administration significantly reduced LFCO in the mPFC of WT mice. Interestingly, 5-MeO-DMT differently reduced LFCO in WT and KO2A mice (WT: from 0.054±0.004 to 0.030±0.002 μV 2 (51.1±2.5% of baseline), n=40; KO2A: from 0.064±0.004 to 0.041±0.004 μV 2 (61.4±3.3% of baseline), n=13). Two-way ANOVA revealed significant effects of 5-MeO-DMT
Given the differential effect of 5-MeO-DMT in WT and KO2A mice, we examined the potential involvement of 5-HT 1A -R. Pretreatment with the 5-HT 1A receptor antagonist WAY-100635 (0.5 mg/kg s.c.) fully prevented the 5-MeO-DMT-evoked reduction of LFCO in the mPFC of KO2A mice (Figure). Interestingly, WAY-100635 increased the power of LFCO by itself. Two-way ANOVA revealed a significant effect of 5-MeO-DMT treatment (F(2,34)=14.29, p<0.005), WAY-100635 pre-treatment (F(2,17)=32.75, p<0.0001) and of treatment x pre-treatment interaction (F(4,34)=7.28, p<0.0002), with significant post-hoc differences between saline and WAY-100635 pre-treatments and between saline+5-MeO-DMT and WAY-100635+5-MeO-DMT treatments (Figure).
Basal extracellular concentrations of 5-HT in dialysed samples of mPFC were WT: 14.5±1.8 (n=15); KO2A: 16.2±2.3 (n=11) fmol/30 μl. Non-significant differences between genotypes were found in basal 5-HT concentrations. The systemic administration of 5-MeO-DMT (1 mg/kg s.c) decreased extracellular 5-HT concentration comparably in the mPFC of WT and KO2A mice (Figure). The maximal decreases were to 57.0±7.0% and 43.6±4.9% of baseline for WT and KO2A mice, respectively. Two-way ANOVA revealed a significant effect of 5-MeO-DMT (F(9,99)=8.35; p<0.00001) with no significant effects of genotype and genotype x treatment interaction. In parallel, 5-MeO-DMT produced a significant increase in the spontaneous HTR rate in WT but not in KO2A mice (from 0.98±0.29 to 4.09±0.66 in WT and from 0.92±0.34 to 0.39±0.15 in KO2A mice after 5-MeO-DMT administration) (Figure). Two-way ANOVA showed a significant effect of 5-MeO-DMT
5-HT 1A autoreceptors play a major role in the control of the ascending serotonergic system. Likewise, there is an additional control of serotoninergic activity by postsynaptic 5-HT 1A -R via direct descending inputs from PFC to the raphe nuclei)". Therefore, in order to discriminate the involvement of presynaptic and postsynaptic 5-HT 1A -R in the reduction of 5-HT release induced by 5-MeO-DMT, we locally applied the compound in mPFC by reverse dialysis. The local perfusion of 5-MeO-DMT (30, 100, 300 μM) by reverse dialysis dose-dependently altered the 5-HT concentration differently in the mPFC of WT and KO2A mice (Figure). Two-way ANOVA revealed
12 significant effects of 5-MeO-DMT (F(18,198)=5.11; p<0.00001), genotype (F(1,11)=9.27; p<0.02) and genotype x treatment interaction (F(18,198)=3.73; p<0.00001). The lower concentration used (nominal 30 µM) evoked a similar reduction of extracellular 5-HT in WT and KO2A mice. However, higher concentrations clearly discriminated between WT and KO2A mice (Figure). Hence, 300 µM 5-MeO-DMT increased extracellular 5-HT to 149.5±22.1% of baseline in WT mice and 100 µM 5-MeO-DMT decreased 5-HT to 38.8±8.1% of baseline in KO2A mice. Two-way ANOVA of normalized AUCs (Figure) of the different experimental periods used revealed significant effects of 5-MeO-DMT (F(3,30)=6.17; p<0.03), genotype (F(1,10)=10.17; p<0.01) and genotype x treatment interaction (F(3,30)=5.94; p<0.03).
As previously showed in ratswe examined in WT mice whether the antipsychotic drugs haloperidol (HAL) and risperidone (RIS) could reverse the disruption alteration of mPFC activity induced by 5-MeO-DMT. Figureshows the reversal of 5-MeO-DMT effects on LFCO by HAL and RIS. Two-way ANOVA analysis revealed a significant effect of the 5-MeO-DMT treatment (F(2,26)=109.76, p<0.00001), antipsychotic treatment (F(2,13)=4.62, p<0.05) and of 5-MeO-DMT x antipsychotics treatments interaction (F(4,26)=3.47, p<0.03). Post-hoc analysis revealed significant differences between baseline and 5-MeO-DMT and between 5-MeO-DMT+saline and 5-MeO-DMT+antipsychotic treatments.
To examine whether sensory cortical are affected by 5-MeO-DMT, we recorded LFCO in S1, Au1 or V1 using ECoGs. 5-MeO-DMT reduced LFCO in S1, Au1 and V1 (S1: 67.1±4.3%; Au1: 59.3%±4.1%; V1: 67.1±6.8% of baseline) of WT mice, but not in S1 and Au1 of KO2A mice. Interestingly, 5-MeO-DMT reduced LFCO in V1 of KO2A mice (50.2±5.1% of baseline). Figureshows representative examples of the effect of 5-MeO-DMT on LFCO in S1, Au1 and V1 in the two genotypes. In WT mice, two-way ANOVA analysis revealed a significant effect of 5-MeO-DMT (F(1,30)=138.08; p<0.00001), with no effects of area and area x treatment interaction. In KO2A mice, two-way ANOVA analysis revealed a significant effect of 5-MeO-DMT
ACCEPTED MANUSCRIPT 14 (F(1,22)=17.63; p<0.0005), area (F(2,22)=7.01; p<0.005) and area x treatment interaction (F(2,22)=7.61; p<0.005). Thus, 5-MeO-DMT disrupts differently S1-LFCO and Au1-LFCO in WT and KO-2A mice. On S1-LFCO two-way ANOVA analysis revealed a significant effect of 5-MeO-DMT (F(1,19)=12.17; p<0.003), genotype (F(2,22)=7.01; p<0.005) and genotype x treatment interaction (F(1,19)=6.18; p<0.03); on Au1-LFCO two-way ANOVA analysis revealed a significant effect of 5-MeO-DMT (F(1,14)=28.95; p<0.0001) and genotype x treatment interaction (F(1,14)=7.88; p<0.02). In contrast, two-way ANOVA analysis of LFCO in V1 revealed a significant effect of 5-MeO-DMT on LFCO (F(1,19)=12.17; p<0.003) with no effects of genotype and genotype x treatment interaction. Post-hoc analysis showed significant differences between 5-MeO-DMT treatment in S1 and Au1 in the two genotypes, but not in V1 (Figure).
The present study confirms and extends previous observations in rat brain, indicating that 5-MeO-DMT decreases LFCO in PFC by stimulating 5-HT 1A -R and 5-HT 2A -R. We also show that this effect is reversed by classical (haloperidol) and atypical antipsychotic drugs (risperidone). Moreover, in addition to PFC, 5-MeO-DMT reduced LFCO in primary sensory areas (S1, Au1 and V1) of WT -yet only in V1 of KO2A micesupporting the involvement of 5-HT 1A -R in the visual alterations induced by 5-MeO-DMT. Overall, these observations shed further light on the neurobiological mechanisms involved in the brain areas/circuits related to psychotic symptoms, such as hallucinations. Despite the interest of serotonergic hallucinogens as models of schizophrenia symptoms, few studies examined 5-MeO-DMT effects on brain activity (de Montigny and. In recent years, our group has characterized the reduction of LFCO in rodent PFC as a common trait of psychotomimetic agents, including PCP and serotonergic hallucinogens. These actions are countered by classical and atypical antipsychotic drugs. The action of serotonergic hallucinogens has been attributed to the activation of 5-HT 2A -R, for which they show high affinity. However, behavioral studies with WAY-100635 and KO1A mice support the additional involvement of 5-HT 1A -R on the action of indolamine hallucinogens -and in particular 5-MeO-DMT. In the pre-pulse inhibition (PPI) model, 5-HeO-DMT has opposite effects in rats (decrease;and mice (increase; Halberstadt and Geyer 2011), as observed for selective 5-HT 1A -R agonists. Irrespectively of this species difference, the effect of 5-MeO-DMT on PPI was blocked or attenuated by the 5-HT 1A -R antagonist WAY-100635, supporting the involvement of 5-HT 1A -R. 5-MeO-DMT reduced LFCO and the BOLD signal in PFC and V1 of the rat. The fall in LFCO was prevented or reversed by selective 5-HT 1A -R and 5-HT 2A -R antagonists. Here, we extended the observations to mice and examined the involvement of 5-HT 1A -R and 5-HT 2A -R. Interestingly, 5-MeO-DMT reduced LFCO in the PFC of WT mice, as previously observed in rats. It also reduced LFCO in KO2A mice, yet to a smaller extent, which suggests an additional role for other 5-HT-R. Subsequent but not in WT mice. The differential effect of WAY-100635 in WT and KO2A mice cannot be ascribed differences in 5-HT 1A -R density. More convincingly, given the high cellular co-expression and interactions between these receptors in PFC (see below), a functional compensatory change in the control of LFCO by 5-HT 1A -R may occur in KO2A mice. 5-MeO-DMT markedly reduced the discharge of 5-HT neurons (de Montigny and. Therefore, some of the observed changes might be due to the activation of presynaptic 5-HT 1A -R in the midbrain raphe and the subsequent reduction of 5-HT release in PFC. However, 5-HT 1A -R and 5-HT 2A -R in the mPFC also control serotonergic activity and the local 5-HT release via direct inputs to the raphe nuclei. These effects are due to the stimulation of 5-HT 1A -R and 5-HT 2A -R in pyramidal neuronsprojecting to the DR. Hence, we examined the relative contribution of pre-and postsynaptic 5-HT 1A -R, by comparing the effects of 5-MeO-DMT on 5-HT release in PFC after systemic and local application. The comparable reduction PFC 5-HT release in WT and KO2A mice after systemic 5-MeO-DMT administration (1 mg/kg s.c.) suggests a predominant role of presynaptic 5-HT 1A -R in this effect. Interestingly, the fall in 5-HT release was accompanied by an increase in the HTR in WT -not KO2A-mice indicating a parallel activation of postsynaptic 5-HT 2A -R at the dose used. On the contrary, local 5-MeO-DMT application in PFC evoked a differential concentration-response curve in WT and KO2A mice. At the lower concentration used (30 µM), 5-MeO-DMT evoked a similar reduction of the local 5-HT release in WT and KO2A mice, most likely due to the activation of 5-HT 1A -R in midbrain-projecting pyramidal neurons. The 5-HT reduction persisted in KO2A mice after the subsequent administration of higher 5-MeO-DMT concentrations (100 and 300 µM). However, local 5-MeO-DMT application evoked a concentration-dependent increase of 5-HT release in WT mice. The 5-HT increase in WT -not KO2A-mice is likely attributable to the activation of 5-HT 2A -R in PFC. These results suggest that 5-MeO-DMT acts preferentially on 5-HT 1A -R at low doses, occupying both receptors at higher doses. A limitation of these experiments is the difficulty to compare the activation of postsynaptic 5-HT-R produced by systemic and local 5-MeO-DMT administration. Despite of the nominal concentrations applied exceed the in vitro affinity of 5-MeO-DMT for the 5-HT-R several factors dramatically reduce the effective concentration once in the brain compartment. Thus, the passage of the dialysis membrane may reduce it by one order of magnitude and once in the extracellular compartment, 5-MeO-DMT is continuously cleared by the CSF. Finally, the reduced size of the dialysis membrane makes that only a small population of PFC neurons are affected. As previously observed for PCP, DOI and 5-MeO-DMT in rats, the effects of 5-MeO-DMT were countered by antipsychotic drugs. The reversal by risperidone can be easily explained by direct displacement of 5-MeO-DMT from 5-HT2A-R. However, the
reversal by haloperidol needs to be interpreted at network level, since it shows low occupancy of 5-HT2A-R at the dose used. Given the presence of dopamine D2-R in pyramidal and GABAergic neurons of mPFC, and their control of excitatory neurotransmission in PFC, D2-R blockade by HAL may normalize the excitatory/inhibitory balance altered by 5-MeO-DMT. Thus, antipsychotic drugs with different pharmacological profiles can equally restore the physiological state of LFCO, acting via different signalling pathways and/or cortical networks. Various brain areas involved in the processing of sensory information show an altered activity in schizophrenia patientsas well as in healthy individuals and rodents treated with serotonergic hallucinogens. In addition to PFC, 5-MeO-DMT reduced LFCO in S1, Au1 and V1 of WT mice and only in V1 of KO2A mice. Interestingly, the contribution of 5-HT 1A -R to the LFCO reduction differed among the cortical areas examined. Hence, the differential effect of 5-MeO-DMT in WT and KO2A mice was maximal in S1 and Au1, and minimal in V1, suggesting the preferential involvement of 5-HT 2A -R in Au1/S1 and of 5-HT 1A -R in V1. 5-HT 1A -R and 5-HT 2A -R are densely expressed in V1suggesting a central role of these receptors in visual processing. Interestingly, [3H]-5-HT labeled a dense population of 5-HT1 receptors (5-HT1A+5-HT1B+5-HT1D) in layer IVß of the human primary visual cortex. Similarly 5-HT2 receptors are also expressed in layer IVc in the same area. These observations suggest that both receptors are involved in the modulation of thalamic visual inputs from the lateral geniculate nucleus. To our knowledge, there are no similar detailed studies in the visual cortex of the rodent brain. Both receptors inhibit NMDA-induced LTP in visual cortex via different mechanisms. Interestingly, 5-HT 1A -R activation evokes plasticity phenomena in adult rats. Thus, 5-HT 2A -Rs have been implicated in the pathogenesis of visual hallucinationsand both receptors participate in the sensory alterations evoked by psilocybin. Likewise, the marked effect of 5-MeO-DMT in V1 found in the present study is consistent with the changes evoked by this drug on visual processing (de Araujo et al, 2012). However, the exact reason for the preferential action of 5-MeO-DMT on 5-HT1A-R in V1 is not fully understood. It may appear contradictory that the activation of excitatory (5-HT 2A -R) and inhibitory (5-HT 1A -R) receptors contribute to reduce LFCO. However, there is a complex interplay between both receptors in PFC, which are expressed in pyramidal and GABAergic interneuronsand show a high cellular co-expression and functional interaction (Amargós-. Hence, despite endogenous 5-HT, released at PFC sites by the electrical stimulation of the DR, inhibit pyramidal neuron activity through activation of 5-HT 1A -R (Amargós-, the systemic administration of 5-HT 1A -R agonists increases pyramidal neuron discharge, an effect likely due to the preferential activation of 5-HT 1A -R in GABAergic interneurons (Lladó-. This effect could add to the excitatory effects of 5-HT 2A -R activation, resulting in a synergistic interaction between both receptors. Thus, the above regional differences may depend on the proportion of 5-HT 2A -R and 5-H 1A -R in pyramidal and GABAergic neurons in the different cortical areas examined.
The present data indicate that the indoleamine hallucinogen 5-MeO-DMT evokes marked alterations in the function of primary sensory areas (Au1, S1, V1) as well as in the highest association cortex (PFC). These alterations are mediated by 5-HT1A-Rs and 5-HT2A-Rs, with a differential contribution of each receptor in the various areas examined. Thus, 5-HT1A-Rs play a major role on 5-MeO-DMT effect on visual and prefrontal cortices. These observations help to elucidate the neurobiological basis of hallucinations. Moreover, as previously observed with other pychotomimetic agents (PCP, DOI), the fall in LFCO induced by 5-MeO-DMT was countered by antipsychotic drugs, supporting the usefulness of the reversal of psychotomimetic effects on LFCO in antipsychotic drug development.
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