Nootropic effects of LSD: Behavioral, molecular and computational evidence

This double-blind placebo-controlled study (n=25) assessed the effects of LSD on metabolic pathways associated with neural plasticity, to gain insight into the relationship between neural plasticity, ageing and LSD-induced cognitive gains in both humans and rodents. LSD treatment in humans (50μg) enhanced performance in a visuospatial memory task, and in a novel object recognition task in rodents indicating that LSD has nootropic effects.

Abstract

“The therapeutic use of classical psychedelic substances such as d-lysergic acid diethylamide (LSD) surged in recent years. Studies in rodents suggest that these effects are produced by increased neural plasticity, including stimulation of the mTOR pathway, a key regulator of metabolism, plasticity, and ageing. Could psychedelic-induced neural plasticity be harnessed to enhance cognition? Here we show that LSD treatment enhanced performance in a novel object recognition task in rats, and in a visuospatial memory task in humans. Proteomic analysis of human brain organoids showed that LSD affected metabolic pathways associated with neural plasticity, including mTOR. To gain insight into the relationship between neural plasticity, ageing and LSD-induced cognitive gains, we emulated the experiments in rats and humans with a neural network model of a cortico-hippocampal circuit. Using the baseline strength of plasticity as a proxy for age and assuming an increase in plasticity strength related to LSD dose, the simulations provided a good fit for the experimental data. Altogether, the results suggest that LSD has nootropic effects.”

Authors: Isis M. Ornelas, Felipe A. Cini, Isabel Weißner, Encarni Marcos, Draulio de Araújo, Livia Goto-Silva, Juliana Nascimento, Sergio R. Silva, Marcelo Falchi, Rodolofo Olivieri, Fernanda Palhano-Fontes, Eduardo Sequerra, Daniel Martins-de-Souza, Amanda Feilding, Cesar Renno-Costa, Luis F. Tófoli, Stevens K. Rehen & Sidarta Ribeiro

Author Highlights

• LSD enhances neural plasticity signalling pathways in human brain organoids.
• LSD enhances novel object preference in rats.
• LSD enhances visuospatial memory in humans.
• LSD-induced neural plasticity explains cognitive gains in rats and humans.

Summary

‡ Current address:

Studies in rodents suggest that psychedelic substances such as LSD induce increased neural plasticity, which may be a key regulator of metabolism, plasticity, and aging. A computational model of the cortico-hippocampal circuit suggests that LSD has nootropic effects.

We used human induced pluripotent stem cells to generate brain organoids that recapitulate aspects of patterning, organization, and connectivity observed in the human embryonic brain. Nine thousand cells were plated per well in an ultra-low attachment 96 well plate in hESC medium containing 50 m ROCKi and 4 ng/ml b-FGF, and the medium was changed on days 3 and 5. Embryoid bodies were transferred to ultra-low attachment 24 well plates on day 7, and the media was changed on days 9 and 11.

We used three independent batches of 45-day old human brain organoids and randomly assigned 4-5 organoids per experimental group. Organoids were treated with 10 nM LSD in water for 24 hours, and then digested with trypsin in-gel overnight. Peptides were injected into a reverse-phase liquid chromatographer coupled to a Synapt G2-Si mass spectrometer. Peptides were loaded onto an M-Class BEH C18 Column, fractionated with 13%, 18%, and 50% acetonitrile, and then eluted using a 7 to 40% (v/v) acetonitrile gradient for 54 min at a flow rate of 0.4 L/min. MS/MS analyses were performed using a NanoLock Spray source.

We searched the Uniprot human proteome database for HDMSE proteins and peptides using Progenesis® QI for proteomics version 4.0, including software packages with Apex3D, peptide 3D, and ion accounting informatics. We used the Hi-N (3) method for comparing peptides.

The abundance of each individual protein was calculated and the fold change in abundance was normalized using the VSN package in RStudio. Significant hits were subjected to gene enrichment using the Metascape online bioinformatic tool.

Pathway enrichment analysis was carried out using KEGG Pathway and Reactome Gene Sets. Terms with a p-value 0.05, a minimum count of 3, and an enrichment factor >1.5 were collected and grouped into clusters based on their membership similarities.

The University Research Ethics Committee approved the study, and 76 Wistar male rats were used. They received 1 or 3 daily intraperitoneal injections of saline or LSD, and were then kept alone while being video recorded for ten hours following dosing.

We used the novel object preference task to measure the subacute effects of LSD pretreatment on hippocampus-dependent memory processes. Animals were habituated to the arena for three days before the test, and were then presented with two different objects. Rats’ novelty preference was calculated using the discrimination index (DI), and the animals were randomly assigned to each treatment (drug/saline).

The behavioral data from rats was tested for normality, and the effects of different doses of LSD were analyzed using ANOVA. The Tukey HSD post-hoc test was used to assess the potential interactions of Age, Treatment, Dose, and Washout.

Twenty-five healthy volunteers were randomized to receive 50 g of LSD in one session and 50 g of an inactive placebo in the other session. They provided written informed consent before participation.

Participants had to be over 21 and have experienced LSD, and be abstinent from other psychedelics and alcohol for two weeks.

To assess LSD-induced overnight memory consolidation and sub-acute LSD effects on immediate and delayed memory recall, two visuospatial tasks were applied the morning after dosing.

In a paper version of the visuospatial two-dimensional object-location task, 15 pairs of cards with colored pictures of animals and everyday objects were placed covertly on squares on a whiteboard. At the learning stage, participants were shown a pair of cards for 1 second and the other card for 3 seconds. The procedure was repeated until 60% of the cards were correctly recalled, and at final recall, the percentage of recalled card locations was used to indicate memory consolidation.

After treatment, participants were presented with a white sheet of paper with several black lines forming a complex geometric figure, and were asked to copy, recall and draw the same figure from memory.

The immediate recall and delayed recall points were calculated to take into consideration prior performance, and evaluate spontaneous memory retention and visuospatial recall skills. We used IBM SPSS Statistics Version 22 to examine the effects of LSD and placebo on the 2D object-location task, points, and the Complex Figures Test. We also evaluated the effects of LSD and placebo on the effects of psychedelics in different age groups.

To explore how age-dependent plasticity could promote the effects attributed to LSD, we built a computational model of the brain that included a memory module and a decision-making module.

In the computational model, the activity of 200 neurons feeds the hippocampal module, and the weight of synapses projecting to 500 memory neurons is incremented by the presynaptic neuron’s level of activity multiplied by a parametrized learning rate.

The decision-making module consists of two groups of excitatory neurons with recurrent connections that compete through mutual inhibition. The activity of the excitatory groups is described by a mean-field approximation of a realistic spiking network.

In our simulations, the population’s activity saturates at fmax, and the decision is made when the difference in activity between the two excitatory groups is above 30 spikess-1.

We ran 20 experiments to emulate the rat experiment and to emulate the human experiment, we used new patterns that shared 60% of the activity of one equivalent familiar object for novel objects and a constant input that reflected the mean activity observed in the memory module for familiar objects.

The decision-making process was different for excitatory group 1 and 2, with group 2 receiving the memory module’s summed activity for patterns that shared 70% with the reference pattern. Human iPSC-generated brain organoids were treated with 10 nM LSD (3.23 ng/mL) for 24 h to study the effects of LSD treatment at the protein level. LSD treatment affects 234 proteins in human neural cells, and these proteins are involved in several biological processes. These processes include DNA replication, axon guidance, synaptic vesicle cycle, mTOR pathway, long-term depression and dopamine neurotransmitter release cycle.

mTOR is a protein kinase crucial for long-lasting synaptic plasticity, and is regulated by LSD. The mTOR pathway is overrepresented in the LSD-treated samples, suggesting that this signaling pathway is overrepresented in our dataset.

To assess whether LSD has nootropic effects dependent on age, rats were pre-treated with 0.13 mg/kg LSD or saline and then explored a novel object more than adult or old rats. However, there was no significant interaction between age and LSD dosing. LSD increased novel object preference in adult animals at a dose of 0.13 mg/kg, but not at a higher dose or with a longer interval.

We recruited 25 healthy adult volunteers of various ages to receive LSD and placebo, and then subjected them to two learning tasks. We hypothesized that different memory processes would be improved by LSD treatment, and examined the effects of 50 g LSD on the performances on the two tasks.

LSD treatment increased the percentage of recalled card locations in a 2D object-location task, indicating that LSD supported visuospatial memory consolidation overnight.

LSD significantly increased recall in immediate recall points and percentage compared to placebo, but there was no carryover effect. There was a period effect for immediate recall points and percentage, with better performance in the second than the first session.

In a second step, we additionally conducted an explorative analysis to investigate possible treatment effects depending on different age groups. We found that LSD decreased recalled card location percentage, marginally increased immediate recall points, and significantly increased immediate recall percentage and delayed recall points.

To gain insight into the relation between neural plasticity and the behavioral changes induced by LSD, we emulated the experimental protocols with a neural network model of a cortico-hippocampal circuit. The model produced an aggregated memory trace from the projection of an input population vector into a memory space.

After training, the network with reference patterns in the simulated rat experiment elicits more robust memory output if compared to the response to partial memories (Novel object 60% of reference) or noise. Moreover, plasticity level strongly modulates the memory output activity for different input patterns. The model can replicate the differences across ages and LSD doses, and can be used to simulate human experiments including the memory output dependence on the plasticity level and the impact on the memory task performance.

The proteomic data from human brain organoids suggest that LSD regulates multiple processes involved in neural plasticity, including DNA replication and axon guidance, as well as synaptic plasticity.

We found that LSD increases the expression of the mTOR pathway, which is involved in multiple neural plasticity events, including the extinction of aversive memories. The 5-HT2A receptor is also involved in LSD-induced plasticity, but we did not use receptor antagonists to determine which receptors are involved.

Endosomal trafficking of neurotrophins regulates the neuronal function and synaptic plasticity. LSD regulates the mTOR pathway associated with endosomal/lysosomal vesicles, and BDNF/TrkB-induced endosomal activation of mTOR is recently implicated in synaptic plasticity by suppressing autophagy in mice.

LSD pre-treatment increases novelty preference in rats several days after dosing, with a significant single dose effect. The results suggest that LSD-induced plasticity enhanced novelty-seeking, or that LSD may increase the time that the rat takes to become familiarized with the new object.

The contrasting results likely reflect the fact that the animals were still under acute effects while training, which jeopardized learning. However, the long interval between administration and task allowed for neural plasticity to take place.

LSD has long-lasting anxiolytic effects in rodents, but the results of our experiment indicate that the anxiolytic effect of LSD did not drive the increase in time exploring the novel object in the LSD group.

The authors note that the rats were isolated for 10 h after the LSD pretreatment, and that the results may reflect the rescue of a stress-induced cognitive deficit caused by the isolation stressor.

This study shows that LSD improves subacute memory in humans, including consolidation, encoding, and recall. The effects were not very strong, likely due to the single, relatively low dose applied, and the study was merely measured once after treatment (+24 h).

LSD enhanced photographic memory but did not prevent forgetting. Alternatively, the results may be interpreted in terms of enhanced bottom-up visual attention, as psilocybin decreases the functional coupling within its nodes and LSD increases its entropy levels.

LSD reduced consolidation, enhanced spontaneous encoding, and improved enduring retention in different age groups, with reduced consolidation and enhanced spontaneous encoding in the older age group and enhanced spontaneous encoding and improved enduring retention in the intermediate age group. The acute effects of psychedelics on memory are mostly unknown, but include impairments in subjective and behavioral cognitive functioning, including memory retention, recall, recognition, associative learning, working memory, and other executive functions.

Microdosing has shown to increase subjective cognition, including memory and attention, and behavioral measures of cognitive functioning. Additionally, subacute effects and chronic use of psychedelics have been associated with increased subjective and behavioral cognitive performance.

The effects of psychedelics on cognition are dose-dependent and depend on the cognitive domain. A low dose of LSD improves sub-acute memory on several levels.

We produced in silico simulations of rat and human behavior using a computational model that explained how changes in neural plasticity affect behavior. The simulations showed that a system with reduced plasticity cannot learn to differentiate relevant patterns from noise, and suffers from poor recall.

Simulations indicate that plasticity in the decision network underlies learning and implementation of rules, a stage prior to the LSD intervention in our study. This provides a plausible mechanism by which LSD pre-treatment affects behavior in rats and humans.

The antidepressant and anxiolytic effects of serotonergic psychedelics may be explained by a boost in synaptogenesis, improving both local and long-range connectivity. Alternatively, the effects may be explained by an increase in salience, via a process more connected to motivation than learning.

Psychedelic drugs can attenuate age-related changes in cognition and affect, including the novel object recognition memory, and are correlated with defects in the late phase of hippocampal long-term potentiation in vitro. LSD-25 induces psychosis in normals, and facilitation of avoidance acquisition by LSD-25 is known. THC restores cognitive function in old mice. Ayahuasca use is associated with differences in brain structure and personality in humans, including differences in executive function between experienced and occasional users, and differences in personality and psychopathology between long-term users of psychedelic drugs.

Signaling promotes synapse elimination in the developing cerebellum and autophagy is activated by targeting the vacuolar H -ATPase. Cox J, Mann M, Dakic V, Minardi Nascimento J, Maciel R de M, Ribeiro S, Martins-de-Souza D, Rehen SK. LSD reverses stress-induced anxiety-like behavior, cortical synaptogenesis deficits and serotonergic neurotransmission decline, and promotes social behavior through mTORC1 in the excitatory neurotransmission. A review of the literature on the effects of lysergic acid diethylamide (LSD) on the brain, body and memory of healthy young adults, as well as the effects of LSD on the brain, body and memory of older adults.

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Ayahuasca has been shown to have antidepressant effects in patients with recurrent depression, and its “afterglow” may also improve mindfulness and cognitive flexibility in ayahuasca drinkers. In 1944 Osterrieth PA developed a test to copy a complex figure, and in 2018 researchers found that ayahuasca can help treat treatment-resistant depression. Ayahuasca affects the salience and default mode networks in the brain, and lysergic acid diethylamide affects Rag GTPases. LSD acutely impairs working memory, executive functions, and cognitive flexibility, but not risk-based decision-making, in healthy adults. Prochazkova L, Lippelt DP, Colzato LS, Kuchar M, Sjoerds Z, Hommel B.

A computational modeling study found that feedforward inhibition regulates gamma-frequency oscillation in the hippocampus, and that place and grid cells in a loop are associated with enhanced mindfulness capacities in ayahuasca users. Ayahuasca has antidepressant effects in patients with recurrent depression, and mTORC1 signaling is palmitoylation-dependent in hippocampal neurons and non-neuronal cells. In 2018, Schmid Y, Liechti ME, Seeley WW, Menon V, Schatzberg AF, Keller J, Glover GH, Kenna H, Reiss AL, Greicius MD reported long-lasting subjective effects of LSD in normal subjects. Shavitt T, Johnson INS, Batistuzzo MC reported 2020.

A single inhalation of 5-MeO-DMT from dried toad secretion is related to sustained enhancement of satisfaction with life, mindfulness-related capacities, and a decrement of psychopathological symptoms. Ayahuasca influences affect, cognitive thinking style, and ego dissolution in humans. A systematic evaluation of normalization methods in quantitative label-free proteomics is presented. LSD increases novelty, symbolic thinking, and divergent thinking, and decreases utility and convergent thinking. It also increases discontinuity of mind, deep thoughts, and abstract flow, and can be prevented by a single dose of 5-MeO-DMT. BDNF signaling transmission from basolateral amygdala to infralimbic prefrontal cortex is involved in conditioned taste aversion extinction.

The intensity ratio of proteins identified from brain organoids treated with LSD and controls from each experimental batch is shown in Supplementary Table S1. The top terms from the 20 most relevant clusters are shown in Supplementary Table S2.

The group was defined as follows: animal treated with saline or LSD, dose applied, number of days the animal was treated with LSD or saline, age group (Young, Adult or Old), and for one group there is a greater interval between last day of application and the behavior training and test.

All treatment and time effects were significant with p 0.05, except for immediate recall points (p 0.06). Period effects were evaluated by the interaction treatment order * treatment.

LSD treatment increased novelty preference across the entire weight range and increased time exploring novelty across the entire age range in adult rats.

Supplementary Figure S3 shows the effect of varying levels of plasticity on memory output in a neural model of memory detailed by memory pattern.

Synapse-memory correlations in a neural model of memory with varying levels of plasticity are analyzed. The standard deviation from the mean for all synapse-pattern correlation values is computed separately for each pattern.

We hypothesized that the ability of the decision-making module to detect novelty is reduced by intense levels of plasticity. We observed that the memory output for different memory patterns is virtually the same level after learning.

One or a few patterns become more preeminent than the others, making it impossible to detect different levels of pattern matching to memory.

The dominance of a few patterns reflects the uneven synaptic weights that unevenly represent the different memory patterns. The growing inequality manifests as a reduction in the overall level of entropy of the correlation variability per input pattern.