This rat study (2016) found that LSD decreased the firing rate of dopamine neurons in the ventral tegmental area (VTA, in the midbrain) in rats at very high doses.
D-lysergic diethylamide (LSD) is a hallucinogenic drug that interacts with the serotonin (5-HT) system binding to 5-HT1 and 5-HT2 receptors. Little is known about its potential interactions with the dopamine (DA) neurons of the ventral tegmental area (VTA). Using in-vivo electrophysiology in male adult rats, we evaluated the effects of cumulative doses of LSD on VTA DA neuronal activity, compared these effects to those produced on 5-HT neurons in the dorsal raphe nucleus (DRN), and attempted to identify the mechanism of action mediating the effects of LSD on VTA DA neurons. LSD, at low doses (5-20 μg/kg, i.v.) induced a significant decrease of DRN 5-HT firing activity through 5-HT2A and D2 receptors. At these low doses, LSD did not alter VTA DA neuronal activity. On the contrary, at higher doses (30-120 μg/kg, i.v.), LSD dose-dependently decreased VTA DA firing activity. The depletion of 5-HT with p-chlorophenylalanine did not modulate the effects of LSD on DA firing activity. The inhibitory effects of LSD on VTA DA firing activity were prevented by the D2 receptor antagonist haloperidol (50 μg/kg, i.v.) and by the 5-HT1A receptor antagonist WAY-100,635 (500 μg/kg, i.v.). Notably, pretreatment with the trace amine-associate receptor 1 (TAAR1) antagonist EPPTB (5 mg/kg, i.v.) blocked the inhibitory effect of LSD on VTA DA neurons. These results suggest that LSD at high doses strongly affects DA mesolimbic neuronal activity in a 5-HT independent manner and with a pleiotropic mechanism of action involving 5-HT1A, D2 and TAAR1 receptors.
Papers cited by this study that are also in Blossom
Passie, T., Halpern, J. H., Stichtenoth, D. O. et al. · CNS Neuroscience and Therapeutics (2008)
Wallach, J. V. · Medical Hypotheses (2009)
Papers in Blossom that reference this study
O’Mahony, B., Harrington, C., Harkin, A. et al. · Journal of Psychopharmacology (2026)
Rieser, N. M. · Biological Psychiatry (2024)
LSD is a prototypical hallucinogen synthesised in 1938 that produces profound alterations of consciousness and psychotic-like phenomena. Previous work has primarily linked its psychotropic properties to serotonergic actions, notably partial agonism at 5-HT2A and agonism/partial agonism at 5-HT1A receptors, and in-vivo studies have shown LSD suppresses firing of dorsal raphe nucleus (DRN) 5-HT neurons while increasing cortical neuronal activity. In-vitro evidence also indicates LSD binds to dopamine (DA) receptors, especially D2, and to the trace-amine associated receptor 1 (TAAR1), but in-vivo interactions with mesolimbic DA neurons of the ventral tegmental area (VTA) and the role of TAAR1 had not been explored. De Gregorio and colleagues set out to characterise LSD's in-vivo effects on VTA DA neurons, to compare those effects with LSD's actions on DRN 5-HT neurons, and to test whether 5-HT1A, D2 and TAAR1 receptors mediate LSD's influence on VTA DA firing. The investigators hypothesised a pleiotropic mechanism involving both serotonergic and dopaminergic systems, and designed electrophysiological experiments in rats to probe dose-dependent effects and receptor involvement.
Adult male Sprague Dawley rats (300–330 g) were used. Animals were anaesthetised with chloral hydrate (400 mg/kg, i.p.; supplemental 100 mg/kg doses as needed) and placed in a stereotaxic apparatus for extracellular single-unit recordings. Single-barrelled glass micropipettes were used to record spontaneous activity of individual neurons in the DRN (putative 5-HT neurons) and VTA (putative DA neurons); neuronal identity was established by established electrophysiological criteria (firing rate, waveform and regularity for 5-HT; wide action potential, slow firing rate and waveform for DA). Burst activity for DA neurons was defined by interspike interval criteria (initial ISI ≤ 80 ms and maximum ISI 160 ms) and quantified as number of bursts per 200 s and percentage of spikes in bursts. Drugs were administered intravenously via a tail-vein catheter. The main test compound was cumulative doses of LSD; the extracted text reports dose ranges as "low" (5-20 g/kg, i.v.) and "high" (30-150 g/kg, i.v.), noting that the extraction uses the unit "g/kg". Receptor antagonists and probes included haloperidol (Halo, D2 antagonist), MDL 100,907 (5-HT2A antagonist), WAY-100,635 (5-HT1A antagonist), EPPTB (TAAR1 antagonist), apomorphine (Apo, tested as a D2 agonist probe), and PCPA for serotonin depletion (350 mg/kg, i.p., administered 48 h and 24 h before recordings). Only one neuron per rat was tested. Baseline firing was recorded for at least five minutes before drug administration; drugs were given sequentially every five minutes according to the experimental plan. The extracted methods include detailed equipment, electrode preparation and histological marking of recording sites to confirm placements. Data analysis treated neuronal responses as percentage change from vehicle (vehicle = 100%) and used Student t-tests, one-way ANOVA or two-way repeated-measures ANOVA with Bonferroni post hoc tests where appropriate; significance was set at P ≤ 0.05. ED50 values for LSD dose–response curves were estimated by non-linear regression after log-transforming doses.
Low-dose effects on DRN and VTA: Cumulative low doses of LSD (reported as 5–20 g/kg, i.v.) produced a dose-dependent decrease of DRN 5-HT firing. In four DRN neurons, 10 g/kg significantly reduced activity (P = 0.007) and 20 g/kg abolished firing (P < 0.001); the ED50 for DRN inhibition was 13.13 g/kg. The same low-dose range did not alter spontaneous or burst firing of VTA DA neurons (no significant change reported). Receptor blockade of low-dose effects: Pretreatment with haloperidol (50 g/kg, i.v.) prevented the inhibitory effects of low-dose LSD on DRN 5-HT firing (interaction and main-effect statistics reported, P < 0.001) while haloperidol alone did not affect 5-HT firing. Similarly, the 5-HT2A antagonist MDL 100,907 (200 g/kg, i.v.) blocked the suppressive effect of low doses of LSD on DRN 5-HT neurons. The 5-HT1A agonist 8-OH-DPAT (5–10 g/kg, i.v.) was able to silence DRN 5-HT neurons when administered after LSD, supporting a multi-receptorial mechanism for LSD's action on 5-HT cells. High-dose effects on VTA DA neurons: Increasing LSD doses (reported as 30–120 g/kg cumulative) produced a dose-dependent decrease of VTA DA firing in six recorded neurons. Significant suppression occurred at 60 and 90 g/kg (P < 0.001), and 120 g/kg completely silenced DA firing (P < 0.001). The ED50 for VTA inhibition was 71.80 g/kg. Burst-firing parameters (number of bursts per 200 s and percentage of spikes in bursts) were also significantly reduced by high doses. Role of serotonin depletion: Pre-treatment with PCPA (serotonin depletion > 89% at the VTA by the protocol used) did not alter the overall inhibitory effect of high-dose LSD on VTA DA firing: LSD reduced DA firing in both PCPA-treated and control animals (no interaction; P = 0.38), and ED50 values were not significantly different (59.24 g/kg in PCPA-treated v. 71.80 g/kg in controls). However, in 5-HT depleted rats a low dose of LSD (30 g/kg) decreased the number of DA bursts, an effect not seen in controls, indicating an influence of 5-HT tone on LSD's effects on burst firing. Effects of receptor antagonists on high-dose inhibition: Haloperidol pretreatment prevented the inhibitory effects of high-dose LSD on VTA DA firing and could reinstate firing when administered after LSD; statistical tests showed significant interactions and main effects (P < 0.001). Notably, in PCPA-pretreated animals haloperidol did not restore DA firing after LSD, suggesting haloperidol's action may depend on intact 5-HT signalling. WAY-100,635 (500 g/kg, i.v.; 5-HT1A antagonist) similarly blocked LSD-induced suppression of VTA DA firing; in WAY-100,635-pretreated neurons apomorphine still silenced DA cells, indicating the 5-HT1A blockade prevented LSD effects independently of D2 receptor capacity. TAAR1 involvement: Pretreatment with the TAAR1 antagonist EPPTB (5 mg/kg, i.v.) increased baseline VTA DA firing (single-injection effect P = 0.002, n = 7) and completely blocked the inhibitory effects of cumulative high-dose LSD on DA activity. EPPTB pretreatment also prevented apomorphine-induced changes in DA neurons in these experiments, suggesting a functional interaction between TAAR1 and D2-mediated mechanisms. Across these manipulations, burst-firing measures were variably affected; EPPTB showed a trend to increase bursts but cumulative LSD after EPPTB left burst parameters overall unchanged.
De Gregorio and colleagues interpret their findings as evidence that LSD exerts dose-dependent, biphasic electrophysiological effects: low doses primarily suppress DRN 5-HT neurons via a mechanism involving 5-HT2A and D2 receptors, whereas higher doses inhibit VTA DA neurons through a multi-receptorial mechanism engaging D2, 5-HT1A and TAAR1 receptors. They highlight that this study provides the first in-vivo electrophysiological indication of TAAR1 involvement in LSD's action on DA neurons. The investigators note that the higher potency of LSD at 5-HT versus DA neurons is consistent with prior literature and with behavioural data describing two temporal phases of LSD effects, an early serotonergic-mediated ‘‘psychedelic’’ phase and a later dopaminergic-mediated ‘‘paranoid’’ or psychotic-like phase. The result that serotonin depletion (PCPA) did not abolish LSD's suppression of DA firing supports a 5-HT-independent component for the high-dose DA effects, although depletion did sensitize burst-related responses and rendered haloperidol ineffective at reversing LSD in depleted animals; the authors suggest this may relate to mechanisms of neuroleptic-resistant psychosis and call for further research. Regarding TAAR1, the authors discuss evidence that TAAR1 is a modulator of aminergic systems and that TAAR1 antagonism (EPPTB) both increased basal VTA DA firing and blocked LSD-induced inhibition, implying a role for TAAR1 in mediating LSD's dopaminergic effects. They also point to cross-talk between TAAR1 and D2 receptors, citing that EPPTB prevented apomorphine effects in their preparation, and suggest further work is required to determine the precise TAAR1–D2 interaction. The authors acknowledge that further behavioural pharmacology and mechanistic studies are needed to clarify D2 involvement and the therapeutic implications, and they note that distinctions between TAAR1 antagonists and partial agonists require additional investigation. Overall, they position their findings as supporting a pleiotropic mechanism for LSD and as motivating exploration of combined receptor antagonism for drug-induced psychosis.
The study concludes that LSD operates via a pleiotropic, dose-dependent mechanism: at low doses it primarily affects the serotonergic system interacting with 5-HT2A and D2 receptors, whereas at higher doses it influences the dopaminergic system via 5-HT1A, D2 and TAAR1 receptors. The authors remark that this biphasic electrophysiological profile parallels biphasic psychotropic effects reported in humans, and they propose that combined antagonism at 5-HT1A, D2 and TAAR1 could represent a novel approach to manage drug-induced psychosis.
Synthesized in 1938 by A. Hoffmann, d-lysergic diethylamide (LSD) is a hallucinogen drug with potent psychotropic effects, described as "mystical experiences"including alterations of the state of consciousness, euphoria, enhanced capacity for introspection, and altered psychological functioning. In particular, LSD may produce psychotic-like symptoms such as visual, tactile, acoustic hallucinations, change in body perception, synaesthesia, thought disorders, time distortions, etc. For these reasons, LSDinduced psychosis is considered a pharmacological model to better understand the pathogenesis of psychosis and schizophrenia. Common wisdom has associated the psychotropic properties of LSD to its effects at the level of the serotonergic (5-HT) system acting as a 5-HT 2A receptor partial agonistand a 5-HT 1A receptor agonist/partial agonist, binding the 5-HT 2A receptor with an EC 50 value of 7.2 nM. In-vivo electrophysiological studies in rats have shown that LSD decreases the activity of 5-HT neurons in the dorsal raphe nucleus (DRN)and increases the firing rate of cortical neurons in the somatosensory cortex. However, several in-vitro studies have reported affinity not only for the 5-HT but also for dopamine (DA) receptors, in particular affinity for D 2 receptors in pig brainand human cloned D 2 receptor, and like others psychostimulants, it induces the incorporation of [ 35 S]GTP-␥-S into G i protein-coupled to D 2 receptors in homogenates of rat brain striatum. Little is known about the in-vivo effects of LSD in the mesolimbic DA neurons of the ventral tegmental area (VTA), which is the main source of DA neurons in the brain and is also implicated in the pathogenesis of psychosis, as well as the mechanism of action of antipsychotics. Intriguingly, in-vitro studies have shown that LSD has high affinity for the G protein-coupled trace-amine associated receptor 1 (TAAR 1 ), but its in-vivo interaction with TAAR 1 receptors has not yet been explored. Since TAAR 1 is expressed in the VTA, and mRNA and protein levels of D 2 receptors are over-expressed in the striatum of TAAR 1 knock-out mice, a close interaction between the DA system and TAAR 1 can be speculated. Based on this previous evidence, we have hypothesized that LSD may act with a pleiotropic mechanism of action, involving not only 5-HT but also DA systems. The main goals of this study were thus 1) to better characterize the in-vivo contribution of the DA system to the effects of LSD, 2) to compare the effects of LSD on VTA DA neurons vs. those on DRN 5-HT neurons, and 3) to test the involvement of 5-HT 1A , D 2 and TAAR 1 receptors in mediating the effects of LSD on VTA DA neurons.
Adult male Sprague Dawley rats (Charles River, Saint-Constant, Quebec, Canada) weighing 300-330 g were housed under standard laboratory conditions with a 12 h light-dark cycle (lights on at 07:00 h) with ad libitum access to food and water. All experimental procedures were conducted from 9:00 a.m. to 3:00 p.m. and were in accordance with the guidelines set by the Canadian Institute of Health Research for animal care and scientific use and the Animal Care Committee of McGill University.
Rats were anaesthetized with chloral hydrate (400 mg/kg, i.p.) in their housing room and then transported in light-free boxes to the procedural room. Rats were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) and a hole was drilled through the skull. Body temperature of the animals was measured using a rectal thermometer (Yellow Springs Instrument Co., Yellow Springs, OH, USA) and was maintained at 35-36.5 • C using an IR heating lamp (Philips, Infrared Heat). To maintain a full anesthetic state during the experiments, supplemental doses of chloral hydrate (100 mg/kg, i.p.) were periodically administered. Anesthesia was confirmed by the absence of nociceptive reflex reaction to a tail or a paw pinch and of an eye blink response to pressure. Extracellular single-unit recordings were performed using singlebarreled glass micropipettes pulled from 2 mm Stoelting (Wood Dale, IL) capillary glass on a Narashige (Tokyo, Japan) PE-21 pipette puller and preloaded with fiberglass strands to promote capillary filling with 2% Pontamine Sky Blue dye in 2 M NaCl for DRN 5-HT recordings and sodium acetate 0.5 M for VTA DA recordings. The micropipette tips were broken down to diameters of 1-3 m to reach an electrode impedance of 2-6 M . Single-unit activity was recorded as large-amplitude action potentials captured by a software window discriminator, amplified by an AC Differential MDA-3 amplifier (BAK electronics, INC.), post-amplified and band-pass filtered by a Realistic 10 band frequency equalizer, digitized by a CED 1401 interface system (Cambridge Electronic Design, Cambridge, UK), processed online, and analyzed off-line using Spike2 software version 5.20 for Windows PC. The spontaneous singlespike activity of neurons was recorded for at least 2 min; the first 30 s immediately after detecting the neuron was not considered to eliminate mechanical artifacts due to electrode displacement. Once the recordings were terminated, pontamine sky blue dye was injected iontophoretically by passing a constant positive current of 20 A for 5 min through the recording pipette to mark the recording site. Then rats were decapitated and their brains extracted and placed in a freezer at -20 Celsius ( • C). Subsequent localization of the labeled site was made by cutting 20 m-thick brain sections using a microtome (Leica CM 3050 S) and the electrode placement was identified with a microscope (Olympus U-TVO.5 × C-3).
In-vivo single-unit extracellular recordings of DRN 5-HT neurons were performed as previously described. The electrode was advanced slowly into the DRN, guided by coordinates from the rat brain atlas of Paxinos and Watson (2007): 1.2 mm anterior to interaural zero on the midline, 5.0-6.5 mm from the dura mater. Under physiological conditions, spontaneously active 5-HT neurons exhibit characteristic electrophysiological properties distinguishable from non-5-HT neurons. These 5-HT neurons exhibit a slow (0.1-4 Hz) and a prominently regular firing rate (coefficient of variation, C.O.V., ranges from 0.12 to 0.87) and a broad biphasic (positive-negative) or triphasic waveform (0.8-3.5 ms; 1.4 ms first positive and negative deflections). Although, these criteria may vary in response to pharmacological or environmental manipulations, some spike features (i.e., waveform, shape and spike duration) have been shown to be stable across conditions, and are therefore reliable indicators for 5-HT neurons.
The electrodes were descended into the VTA using a hydraulic micropositioner (David Kopf Instruments, Tujunga, CA, USA) according to the stereotaxic coordinates described in the rat brain atlas of Paxinos and Watson (2007): A-P: 3.4-4.1 mm from the interaural line; lateral: 0.6-1.1 mm from the midline; ventral: 7.5-8.8 mm from the brain surface. Putative DA neurons were identified based on well-established electrophysiological properties: a wide action potential (>2.5 ms), biphasic or triphasic waveform and slow firing rate (0.5-10 Hz). Neuronal activity was measured by calculating the mean firing rate frequency, expressed as the number of spikes per second or Hz. Additionally, the burst activity of DA neurons was analyzed using a script for the Spike 2 software (available on line at www.ced.co.uk). Based on criteria previously described, a burst was defined as a train of at least two spikes with an initial interspike interval (ISI) ≤ 80 ms and a maximum ISI of 160 ms, within a regular low-frequency firing pattern and decreased amplitude from the first to the last spike within the burst. The parameters analyzed with the Spike 2 script included the number of bursts found and the percentage of spikes fired in burst calculated for 200 s. All these parameters were expressed as percentage of vehicle injection. (EPPTB) (Tocris Bioscience, Missouri, USA) was dissolved in a vehicle of 60% polyethylene glycol 400 (Sigma-Aldrich, Oakville, Canada) and 40% NaCl solution (0.9%). Apo, LSD, WAY, Halo and EPPTB were freshly prepared the day of the experiment. Drugs were injected intravenously (i.v.) using a 24G x 3/4" catheter (Terumo Medical Corporation, Elkton, MD, USA) inserted into the lateral vein of the tail. The maximum volume used for a single i.v. injection was 0.1 mL. Cumulative doses of LSD, 120 and 150 g/kg, i.v.), Apo (25, 50, 100 g/kg, i.v.) Halo (50, 100, 150 and 200 g/kg, i.v.) and 8-OH-DPAT (5, 10 g/kg, i.v) were tested. Once a stable DRN 5-HT or VTA DA neuron was found, its basal firing activity (CTRL) was recorded for at least five minutes. PCPA (350 mg/kg, i.p.,) was administered 48-h and 24-h before VTA DA recordings. Rats were i.v. injected with VEH and then every five minutes with sequential doses of one of the four drugs (LSD, Apo, Halo or 8-OH-DPAT) or with a singular injection of MDL 100 907 (200 g/kg, i.v.), WAY (500 g/kg, i.v.) or EPPTB (5 mg/kg, i.v.) until all doses and drug were tested according to the experimental plan. The dose of EPPTB was chosen following its pharmacokinetic parameters (at the dose of 2.5 mg/kg i.v., clearence 87 mL/min/kg, t 1/2 = 1.9 h, brain/plasma = 0.5). Only one neuron per rat was tested.
Data were analyzed using SigmaPlot 13 (Systat Software, Inc.). Neuronal responses to cumulative administration of drugs were calculated as percentage of change from VEH injection (100%), were reported as mean (% of VEH) ± standard error of the mean (SEM), and were computed using Student t-test or one-way ANOVA or two-way ANOVA for repeated measures followed by Bonferroni post hoc comparisons where appropriate. Statistical values of P ≤ 0.05 were considered significant.
A log transformation of each LSD dose was computed. ED 50 values were then determined by non-linear regression analysis using GraphPad Prism version 5.04 (GraphPad Software) following the method by Ford et al..
Based on previous findings. and our experience testing LSD activity upon DRN 5-HT neurons, we first confirmed the effects of 5-20 g/kg LSD in four DRN 5-HT neurons. Second, we investigated whether these doses were active in four VTA DA neurons. Fig.andreport the example of an integrated histogram of spontaneous firing rate of a DRN 5-HT and a VTA DA neuron, respectively, following i.v. injection of cumulative doses of LSD. LSD (5-20 g/kg) produced a dose-dependent decrease of DRN 5-HT cell firing frequency (F(3, 8) = 28.090, P < 0.001; Fig.) as previously reported. In particular, compared to VEH injection, 10 g/kg LSD significantly decreased DRN 5-HT activity (P = 0.007), and 20 g/kg LSD completely shut down 5-HT firing activity (P < 0.001). The ED50 value was 13.13 g/kg (Fig.). No significant effects were observed with 5 g/kg LSD. Around 20% of DRN 5-HT neurons in rats usually display burst firing activity; accordingly, we found only 1 out of 4 DRN 5-HT neurons discharging in bursts. The changes in 5-HT burst-firing parameters occurring in this neuron are reported in Table. On the contrary, 5-20 g/kg LSD did not affect either spontaneous (F(3, 9) = 0.93, P = 0.463; Fig.) or burst VTA DA firing activities (data not shown). After the injection of 20 g/kg LSD, we tested the previously demonstrated inhibitory effects of cumulative doses of Apo (25-100 g/kg, i.v.) on VTA DA neurons, and we found that at the dose of 50 g/kg, the DA neural activity was completely shut down (Fig.). Fig.reports an example of the histological control of a recording site in the DRN. We have defined this range of LSD doses (5-20 g/kg) affecting 5-HT but not DA neurotransmission as "low doses". 3.2. The D 2 antagonist haloperidol and the 5-HT 2A antagonist MDL 100 907 prevent the inhibitory effects of low doses of LSD on DRN 5-HT firing activity Fig.reports the integrated histogram of spontaneous firing rate of a DRN 5-HT neuron following the injection of Halo (50 g/kg, i.v.) prior to cumulative low doses of LSD. Since no effects were observed until the injection of 20 g/kg LSD, 30 g/kg LSD was also injected. Since the maximal dose of 30 g/kg LSD was also blocked by Halo, cumulative doses of the 5-HT 1A agonist 8-OH-DPAT (5-10 g/kg, i.v.) were injected, inducing a total decrease of 5-HT firing activity. These findings suggest that the blockade of D 2 receptors prevents the inhibitory effects of low doses of LSD on DRN 5-HT firing independently from 5-HT 1A receptors. As shown in Fig., Halo pre-treatment prevented the inhibitory effects of cumulative low doses of LSD on 5-HT firing activity (interaction: F(5,30) = 11.06, P < 0.001; Halo pre-treatment: F(1,6) = 17.55, P = 0.006; LSD treatment: F(5,5) = 20.31, P < 0.001). Bonferroni post-hoc comparisons revealed a different effect of LSD on Halo pre-treated and non pre-treated neurons (P < 0.001). In particular, while we found a dose-response decrease of DRN 5-HT firing activity after 10, 20, 30 g/kg LSD compared to vehicle (P < 0.001), the pre-treatment with Halo blocked such effect (Fig.). Halo (50 g/kg) alone did not affect 5-HT firing activity (Fig.). Pretreatment with Halo also prevented the inhibitory effect of low dose LSD administration on DRN 5-HT burst-firing activity (Table; number of bursts per 200 s: F(5,12) = 0.77, P = 0.58; % of spikes in bursts: F(5,12) = 1.08, P = 0.41). Fig.reports the integrated histogram of spontaneous firing rate of a DRN 5-HT neuron following the injection of MDL 100 907 (200 g/kg, i.v.) prior to cumulative low doses of LSD. As shown in Fig., MDL 100 907 blocked the effect of cumulative low doses of LSD (interaction: F(5,30) = 31.20, P < 0.001; MDL 100 907 pre-treatment: F(1,5) = 66.63, P < 0.001; LSD treatment: F(5,30) = 30.95, P < 0.001). Bonferroni post-hoc comparisons revealed a different effect of LSD on MDL 100 907 pre-treated and non pre-treated neurons (P < 0.001), namely, a decrease of DRN 5-HT firing activity with 10, 20, 30 g/kg LSD (P = 0.007, P < 0.001, P < 0.001, respectively) in non-pretreated neurons, and no effects of LSD in MDL 100 907 pre-treated neurons. MDL 100 907 (200 g/kg) alone did not affect 5-HT firing activity (Fig.). The injection of cumulative doses of 8-OH-DPAT (5-10 g/kg, i.v.) after 30 g/kg LSD silenced DRN 5-HT neurons (Fig.) suggesting that the blockade of 5-HT 2A receptors prevents the inhibitory effects of low doses of LSD independently from 5-HT 1A receptors. One out of four DRN 5-HT neurons pre-treated with MDL 100 907 was discharging in bursts. No changes in DRN 5-HT burst-firing activity of this neuron were observed after either MDL 100 907 (200 g/kg) or low doses of LSD (Table).
Since 5-20 g/kg LSD did not alter VTA DA neural activity, we then examined whether increasing the dose of LSD (30-120 g/kg) any effect on VTA DA firing activity could be elicited. The acute effect of cumulative doses of LSD (30-120 g/kg) was therefore tested in 6 VTA DA neurons. Fig.reports the example of an inte-
Burst-firing activity of VTA DA neurons following cumulative high doses of LSD (30-150 g/kg, i.v.) in rats receiving vehicle (VEH), treated with PCPA, or pre-treated with haloperidol (Halo, 50 g/kg, i.v.), WAY (500 g/kg, i.v.) or EPPTB (5 mg/kg, i.v.). Data (mean ± SEM) are reported as% of change vs. VEH injection (100% grated histogram of spontaneous firing rate of a VTA DA neuron following injection of 30-120 g/kg LSD. This range of LSD doses (30-120 g/kg) active on VTA DA neurons has been defined as "high doses" in contrast to the "low doses" range (5-20 g/kg) that affects 5-HT but not DA neurotransmission. Fig.shows that cumulative high doses of LSD significantly decreased and silenced VTA DA firing activity, while subsequent cumulative injections of the selective D 2 antagonist Halo (50-150 g/kg, i.v.) were able to reinstate DA firing activity. As illustrated in Fig., cumulative high doses of LSD induced a dose-dependent decrease in VTA DA neural activity (F(4, 20) = 29.833, P < 0.001). Compared to VEH, the decrease in firing rate was significant after administration of 60 g/kg and 90 g/kg LSD (P < 0.001), and importantly, 120 g/kg LSD completely shut down VTA DA activity (P < 0.001). 30 g/kg LSD did not significantly modify DA firing rate. The decrease produced by 120 g/kg LSD was also significantly higher compared to that induced by 30 and 60 g/kg LSD (P < 0.001 and P = 0.003, respectively). The ED 50 value was 71.80 g/kg (Fig.). Linear regression analysis comparing the dose-response curve of the effect of LSD upon DRN 5-HT and VTA DA neurons revealed that the slopes of the two lines were significantly different (F(2,7) = 39.34, P < 0.0001; Fig.). Therefore, LSD acts with higher potency toward 5-HT than toward DA neurons. A main effect of high doses of LSD was also detected when analyzing the effects of LSD on VTA DA burst-firing activity ((Table; number of bursts per 200 s: F(5,18) = 35.0, P < 0.001; % of spikes in bursts: F(5,18) = 7.6, P < 0.001)). An example of the histological control of a recording site in the VTA is shown in Fig..
Guiard et al.previously demonstrated that the selective lesion of DRN 5-HT neurons produced by 5,7-dihydroxytryptamine (5,7-DHT) enhanced the firing activity of VTA DA neurons by 36%, thereby indicating an inhibitory influence of the 5-HT input upon DA neurons. On the other hand, the selective lesion of DA neurons elicited by 6-hydroxydopamine (6-OHDA) decreased the sponta-neous firing activity of DRN 5-HT neurons by 60%, thus revealing the excitatory effect of the DA input upon 5-HT neurons. Given this reciprocal interaction between DRN 5-HT and VTA DA neurons and the effects of LSD on both neurotransmissions (see above), we examined a possible involvement of 5-HT in the effects of high doses of LSD on VTA DA neurons. We thus performed a 5-HT depletion using PCPA (350 mg/kg, i.p.,) injected 48-h and 24-h before testing cumulative high doses of LSD (30-120 g/kg LSD). Using this protocol, 5-HT depletion induced by PCPA at the level of the VTA is more than 89%. Fig.reports an example of an integrated histogram of the spontaneous firing rate of a VTA DA neuron in a rat pretreated with PCPA and injected with cumulative high doses of LSD. Cumulative high doses of LSD equally decreased VTA DA neural activity in both PCPA pretreated and nonpretreated rats (no interaction (F(4,31) = 1.08, P = 0.38), no PCPA pre-treatment (F(1,8) = 1.051, P = 0.335), and an effect of LSD treatment (F(4,31) = 54.32, P < 0.001)). In particular, compared to VEH, we observed a decrease of VTA DA firing activity following 60, 90 and 120 g/kg LSD (Fig., P < 0.001). No effect of 30 g/kg LSD was found (P = 0.20). ED50 value for the PCPA-LSD dose-response curve was 59.24 g/kg, and was not different than the ED50 of the dose-response curve of LSD in non-PCPA pre-treated animals (71.80 g/kg). VTA DA burst-firing activity was decreased by cumulative high doses of LSD also in 5-HT depleted animals (Table; number bursts per 200 s: F(3,7) = 45.2, P < 0.001; % of spikes in bursts: F(3,7) = 19.6, P < 0.001). While LSD(30 g/kg) did not significantly affect VTA DA firing rate in both 5-HT depleted and control animals, it decreased the # of bursts (200 s) (P < 0.001) in 5-HT depleted rats but not in controls, suggesting that 5-HT is very likely involved in the effects of LSD upon VTA DA burst-firing activity. Of note, unlike in the normal condition (Fig.), the injection of Halo (50-150 g/kg) after cumulative high doses of LSD in PCPA pretreated animals did not restore VTA DA firing (Fig.), suggesting that Halo may need an intact 5-HT system for its action on DA firing activity. i.v.) before that of cumulative high doses of LSD. Since no effects were observed until the injection of 120 g/kg LSD, we increased the dose up to 150 g/kg LSD. After the injection of 150 g/kg LSD (which did not change firing activity), we tested the inhibitory effects of cumulative doses of Apo (25-100 g/kg, i.v.) on VTA DA neurons, and we found that VTA DA neurons did not respond to Apo, thus confirming that haloperidol blocked LSD effects via DA D 2 receptors (Fig.). As shown in Fig., Halo pretreatment prevented the inhibitory effects of cumulative high doses of LSD on DA cell firing frequency (interaction: F(6,46) = 13.93, P < 0.001; Halo pre-treatment: F(1,9) = 54.03, P < 0.001; LSD treatment: F(6,6) = 13.93, P < 0.001). Bonferroni post-hoc comparisons revealed a different effect of LSD on Halo pre-treated and non pretreated neurons (P < 0.001). Indeed, while we found a decrease of VTA DA firing activity with 60, 90, 120 and 150 g/kg LSD compared to vehicle in non pre-treated neurons (P < 0.001), no effects of high doses of LSD were present in Halo pre-treated neurons (Fig.). Interestingly, while 50 g/kg Halo, prior LSD, induced only a slight but not significant increase of VTA DA firing activity(Fig.), it did significantly increase the number of bursts per 200 s compared to vehicle (Table; P = 0.01). On the other hand, Halo pretreatment prevented the ability of high doses of LSD to modify VTA DA burstfiring activity ((Table; number of bursts per 200 s: F(6,24) = 3.2, P = 0.018); % of spikes in bursts: F(6,24) = 0.4, P = 0.830).
3.6. The 5-HT 1A receptor antagonist WAY-100,635 prevents the inhibitory effects of high doses of LSD on VTA DA firing activity Fig.reports the integrated histogram of the spontaneous firing rate of a VTA DA neuron following the injection of WAY-100,635 (500 g/kg, i.v.) prior to cumulative high doses of LSD (30-150 g/kg LSD). VTA DA firing activity was not altered by LSD in WAY-100,635-treated rats. Similar to the previous experiment with Halo pre-treatment, after the injection of 150 g/kg LSD, we tested the inhibitory effects of cumulative doses of Apo (25-100 g/kg, i.v.). Unlike Halo pre-treatment, we found that Apo silenced VTA DA neurons. This finding suggests that the blockade of 5-HT 1A receptors prevents the inhibitory effects of high doses of LSD on VTA DA neurons independently of D 2 receptors. WAY-100,635 (500 g/kg) alone did not affect DA neural activity (Fig.). As shown in Fig., cumulative high doses of LSD did not affect VTA DA neural activity in neurons pre-treated with WAY-100,635. On the contrary, compared to vehicle, it significantly reduced DA firing at the doses of 30 (P = 0.011) and 60-150 (P < 0.001) g/kg in non pre-treated neurons (interaction: F(5,40) = 15.98, P < 0.001; WAY-100,635 pre-treatment: F(1,8) = 58.23, P < 0.001; LSD treatment: F(5,40) = 25.05, P < 0.001). No variation after high doses of LSD was observed on VTA burst-firing activity after pre-treatment with WAY-100,635 (Table; number of bursts per 200 s: F(6,24) = 1.9, P = 0.116; % of spikes in bursts:F(6,24) = 1.7, P = 0.162).
Fig.reports the integrated histogram of the spontaneous firing rate of a VTA DA neuron following the injection of EPPTB (5 mg/kg, i.v.) prior to cumulative high doses administration of LSD. LSD did not affect VTA DA firing activity in the presence of EPPTB. The subsequent injection of cumulative doses of Apo did not alter DA neural activity suggesting that TAAR 1 antagonism also involves D 2 receptors. Interestingly, the single injection of 5 mg/kg EPPTB induced a significant increase of VTA DA firing activity (P = 0.002, n = 7, Fig.). The inhibitory effects of cumulative high doses of LSD were completely prevented by EPPTB pre-treatment (Fig.; interaction: F(6,53) = 8.39, P < 0.001; EPPTB pretreatment: F(1,10) = 15.26, P = 0.003;LSD treatment: F(6,53) = 7.31, P < 0.001). Bonferroni post-hoc analysis revealed that compared to vehicle, none of the doses of LSD significantly altered firing activity of VTA DA neurons pretreated with EPPTB, whereas at the doses of 60-150 g/kg it reduced VTA DA neural activity in non pre-treated neurons (P < 0.001). Although there was a trend towards an increase of the number of bursts per 200 s induced by EPPTB 5 mg/kg, overall VTA DA burst-firing activity resulted unchanged when cumulative high doses of LSD were injected after EPPTB pre-treatment (Table; number of spikes in bursts per 200 s: F(6,30) = 2.1, p = 0.086; % of spikes in bursts: F(6,30) = 0.9, P = 0.534).
In this study, we have demonstrated that the hallucinogenic drug LSD strongly influences dopaminergic neural activity of the VTA. In particular, at the doses inhibiting DRN 5-HT firing activity, LSD does not affect VTA DA neurons, but at higher doses, decreases VTA DA neural activity. Using selective antagonists, we have also demonstrated that the decrease in VTA DA neural activity induced by high doses of LSD occurs through a complex and multi-receptorial mechanism including activation of D 2 , 5-HT 1A , and TAAR 1 receptors. Noteworthy, this is the first in-vivo evidence highlighting the involvement of TAAR 1 receptors in the action of LSD. In addition, we highlight that low doses of LSD also act on DRN 5-HT neurons with a multi-receptorial mechanism including activation of 5-HT 2A and D 2 receptors. As previously reported, LSD acts on the serotonergic system by decreasing the firing rate of 5-HT neurons in the DRN. In agreement, we found that LSD at low doses (5-20 g/kg) ceased DRN 5-HT firing activity, but was ineffective at modulating DA neurons in VTA at this dose. Besides the well-known 5-HT 1A receptor-mediated effects of LSD, in keeping with previous studies describing an involvement of 5-HT 2A receptors on the effects produced by LSDand in the inhibition of 5-HT neurons, we found that the decrease of 5-HT firing activity following LSD administration was blocked by the 5-HT 2A antagonist MDL 100 907. Considering the ineffectiveness of low doses of LSD on DA neurons, we performed electrophysiological recordings of VTA DA neurons with higher doses of LSD (30-120 g/kg), revealing that cumulative injections of high doses of LSD significantly decreased VTA DA neural activity. Intriguingly, the experiments show that the D 2 antagonist haloperidol prevented the decrease in VTA DA firing rate following cumulative injections of high doses of LSD, and also reinstated the basal DA firing activity when injected after the drug. In addition, haloperidol also blocked the inhibitory effect of LSD on DRN 5-HT neurons, an effect likely due to the presence of D 2 receptors within the DRN. Collectively, in keeping with previous studies, these experiments may suggest that D 2 receptor is involved in the mechanism of action of LSD. In particular, in vitro experiments demonstrated that LSD has a k i of 2 nM for the human cloned dopamine D 2 receptorand that it inhibited prolactin secretion by the pituitary cells in a concentration-dependent manner, and this effect was antagonized by the D 2 selective antagonist spiperone, but not by the D 1 selective antagonist SKF83566. However, behavioral pharmacology studies are warranted to better understand the possible D 2 receptor-mediated mechanism of LSD. Our results are partially in keeping with a previous study by White and Wangwho found that cumulative doses of LSD (1-500 g/kg) decreased A10 VTA DA cell firing activity in 54% of the tested cells, and increased or had no effect on the remaining neurons. This discrepancy in percentage is likely due to the fact that today, compared to 30 years ago, we use computerized systems and software allowing us to recognize with high precision neuronal length, shape, duration, amplitude and bursts of DA neuron signals vs non-DA neurons. Given the established role of 5-HT 1A receptors on the mechanism of action of LSD on 5-HT firing activity, we tested whether pre-treatment with the5-HT 1A antagonist WAY 100,635 could affect the activity of high doses of LSD on VTA DA neurons. We found that blockade of 5-HT 1A receptors prevented the inhibitory effects of high doses of LSD. Of note, this is the first in-vivo electrophysiological demonstration of the influence of 5-HT 1A receptors on the effect of LSD on VTA DA neuronal activity. Given the presence of 5-HT 1A receptors in the VTA, one can hypothesize that LSD acts directly on 5-HT 1A receptors within VTA without any 5-HT mediated effect; indeed, 5-HT depletion with PCPA did not affect LSD responses on the firing activity of VTA DA neurons. On the other hand, we found that the depletion of 5-HT with PCPA sensitized the LSD-induced burst response, since a low dose of LSD (30 g/kg) significantly reduced the number of bursts in 5-HT depleted animals compared to controls, shifting the curve to the left. Intriguingly, Halo did not reverse the effects of LSD in 5-HT depleted animals. Altogether, this data suggested that the lack of 5-HT enhances vulnerability to the hallucinogenic effects of LSD and make these animals resistant to the neuroleptic Halo, thus suggesting that 5-HT depletion can be a factor that influences neuroleptic-resistant psychosis. This is in agreement with a previous work in which it has been demonstrated that the cataleptic effect of haloperidol is reduced after lesions of midbrain raphe serotonergic neurons or depletion of serotonin stores by PCPA. On the other hand, Balsara et al.have demonstrated that Quipazine, a 5-HT agonist, and clomipramine, a selective 5 HT neuronal uptake blocker, potentiated the cataleptic effect of Halo. However, more studies are needed to understand the neurobiological basis of neurolepticresistant psychosis and the role-played by 5-HT dysfunction. The dose-dependent electrophysiological effects of LSD on 5-HT and DA neurotransmission are in keeping with previous workshowing that the discriminative stimulus effects of LSD in rats occur in two temporal phases, with initial activation of 5-HT 2A receptors and a mediation of D 2 receptors in a second phase. Importantly the biphasic effects of LSD have also been reported in humans; while a "psychedelic experience" with "meaningfulness and portentousness" is experienced in the early phase, a later phase is described as "a clearly paranoiac state, with ideas of references and paranoia". Consequently, in light of these results one can hypothesize that LSD produces a mood elevation and creativity mediated by its initial serotonergic effect at low doses followed by a psychotic-like status at more elevated doses. Importantly, these psychotic-like symptoms are treated in emergency settings with the D 2 receptor antagonist haloperidol or chlorpromazine, further supporting the involvement of D 2 receptors in the effects of high doses of LSD. For the first time, the activity of a TAAR 1 receptor antagonist was explored in-vivo, and we found that the TAAR 1 receptor is involved in the effect of LSD on DA neurons. Discovered in 2001, TAAR1 is an aminergic G protein-coupled receptorthat is an important modulator of the dopaminergic and serotoninergic system and potentially of the glutamatergic system. It binds the so-called trace-amines, a subgroup of biogenic amines, such as beta-phenylethylamine, p-tyramine or tryptamineand also endogenous hallucinogensand exogenous hallucinogens such as LSD. Recent studies found that it could be linked to psychosis. TAAR 1 knockout mice, don't have a specific phenotype, but compared to wildtype controls, they have an increased firing activity of VTA DA neurons, and are more sensitive to a challenge of amphetamine, releasing more DA and 5-HT. In addition, mRNA and protein levels of DA D 2 receptors are over-expressed in the striatum of TAAR 1 knockout mice. Importantly, the TAAR 1 agonist RO 5256390 and more potently the TAAR 1 receptor partial agonist RO 5263397 can mediate cocaineinduced hyperlocomotion and improve performance on an object retrieval task, but only the partial agonist is effective at decreasing immobility in the forced swim test and increasing wakefulness. Furthermore, using a visual whole-cell current clamp technique in brain slices, the TAAR 1 partial agonist RO5263397, but not the agonist RO5256390, augmented the firing frequency of DA VTA neurons, similar to other classes of antipsychotics. The full agonist RO5166017 inhibits the firing frequency of DA neurons in vitro. In our experiment, we pre-treated rats with the TAAR 1 antagonist EPPTB prior to the injection of LSD. EPPTB significantly increased the firing rate of VTA DA neurons, and blocked the inhibitory effects of LSD, suggesting that the effects of LSD over the DA system are also mediated by TAAR 1 receptor and further confirm the ability of TAAR 1 antagonism to counteract the inhibitory effects of hallucinogenic drugs on DA firing activity. As a consequence, a potential role for TAAR 1 antagonists in the management of psychotic-like effects of LSD deserves to be investigated.However, more studies are needed to understand the differential pharmacological role of TAAR 1 antagonists and partial agonists in the treatment of pharmacological and non-pharmacologicalinduced psychosis. The complex mechanism underlying the modulation of VTA DA activity by LSD was also confirmed by Apo. Pre-treatment with WAY 100,635 prevented the responsiveness of VTA DA neurons to the decreasing effect of LSD but not to those of Apo (Fig.), which occur through D 2 receptors. On the contrary, pre-treatment with EPPTB completely blocked not only the inhibiting effects of LSD but also those of Apo (Fig.), suggesting a close relationship between TAAR 1 and D 2 receptors. In agreement with this finding, Sukhanov et al.recently reported an involvement of TAAR 1 receptors in Apo-induced climbing in the forced swim test in mice. Further studies are needed to determine the exact mechanism underlying the interaction between TAAR 1 and D 2 receptors.
In conclusion, our results show that LSD acts with a pleiotropic mechanism of action and at low doses affects the 5-HT system interacting also with 5-HT 2A and D 2 receptors, while at higher doses it affects the DA system via 5-HT 1A, D 2 and TAAR 1 receptors. Interestingly, this biphasic dose-dependent mechanism of action is paralleled by a similar biphasic psychotropic effect in humans. A combination of 5-HT 1A, D 2 and TAAR 1 receptor antagonism could thus represent a novel avenue for drug-induced psychosis.
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