Psilocin acutely disrupts sleep and affects local but not global sleep homeostasis in laboratory mice

This within-subjects animal study (n=8) investigated the effects of psilocin (2 mg/kg; 56mg/28g mouse weight) on the normal sleep-wake cycle and in combination with sleep deprivation. Results indicated that psilocin acutely disrupts sleep, suppressing the maintenance of both NREM and REM sleep, resulting in a pattern of fragmented sleep attempts and frequent brief awakenings which lasted up to 3 hours. No enduring effects of psilocin were observed on sleep-wake quantities, sleep duration, or the recovery from sleep deprivation.

Abstract

“Introduction: Serotonergic psychedelic drugs, such as psilocin (4-hydroxy-N,N-dimethyltryptamine), profoundly alter the quality of consciousness through mechanisms which are incompletely understood. Growing evidence suggests that a single psychedelic experience can positively impact long-term psychological well-being, with relevance for the treatment of psychiatric disorders, including depression. A prominent factor associated with psychiatric disorders is disturbed sleep, and the sleep-wake cycle is implicated in the regulation of neuronal firing and activity homeostasis. It remains unknown to what extent psychedelic agents directly affect sleep, in terms of both acute arousal and homeostatic sleep regulation.

Methods: Here, chronic in vivo electrophysiological recordings were obtained in mice to track sleep-wake architecture and cortical activity after psilocin injection.

Results: Administration of psilocin led to delayed REM sleep onset and reduced NREM sleep maintenance for up to approximately 3 hours after dosing, and the acute EEG response was associated primarily with an enhanced oscillation around 4 Hz. No long-term changes in sleep-wake quantity were found. When combined with sleep deprivation, psilocin did not alter the dynamics of homeostatic sleep rebound during the subsequent recovery period, as reflected in both sleep amount and EEG slow wave activity. However, psilocin decreased the recovery rate of sleep slow wave activity following sleep deprivation in the local field potentials of electrodes targeting medial prefrontal and surrounding cortex.

Discussion: It is concluded that psilocin affects both global vigilance state control and local sleep homeostasis, an effect which may be relevant for its antidepressant efficacy.”

Authors: Christopher W. Thomas, Cristina Blanco-Duque, Benjamin Bréant, Guy M. Goodwin, Trevor Sharp, David M. Bannerman, Vladyslav V. Vyazovskiy

Summary

Christopher W. Thomas1, Cristina Blanco-Duque1, Benjamin Bréant1, Guy M. Goodwin2, Trevor 4 Sharp3, David M. Bannerman4, Vladyslav V. Vyazovskiy1* 5 studied the effects of nicotine on brain activity in laboratory mice.

Serotonergic psychedelic drugs, such as psilocin, profoundly alter the quality of consciousness through mechanisms which are incompletely understood. Psilocin causes delayed REM sleep onset and reduced NREM sleep maintenance in mice, but no long-term changes in sleep-wake quantity were found. Psilocin did not alter the dynamics of homeostatic sleep rebound following sleep deprivation, but did decrease the recovery rate of sleep slow wave activity in the medial prefrontal and surrounding cortex.

Psilocybin is a classical serotonergic psychedelic, which induces profound alterations of perception, cognition, and behaviour. It may offer a promising new treatment method for affective disorders. The psychedelic effect of psilocybin depends on the ability of its metabolite, psilocin, to act as a partial agonist of 5-HT2A receptors, which are highly expressed on the dendrites of cortical layer V pyramidal neurones. Human neuroimaging studies with psychedelics identify widespread disruptions to thalamocortical and cortico-cortical connectivity, and changes in neuronal dynamic properties. However, which observed neuronal effects are specific to, and characteristic of, the psychedelic state remains to be further dissected.

While evidence exists that psychedelics can induce structural and functional synaptic plasticity, facilitate learning and memory, and exert long-lasting behavioural effects, the specific underlying neurophysiology remains unclear. Neuronal plasticity processes, such as cellular maintenance, synaptic scaling, firing rate homeostasis and systems-level memory consolidation, are shaped by ongoing neuronal activity. The sleep-wake cycle is strongly implicated in the regulation of plasticity of cortical function. The brain’s level of homeostatic sleep need is widely recognised to be reflected in the average levels of non-rapid eye movement (NREM) sleep slow wave activity in neurophysiological field potentials. However, the relationship between local neuronal activity and sleep-wake homeostasis is bidirectional.

The depressed state is characterised by an impairment in sleep homeostasis, which may explain why acute sleep deprivation alleviates low mood in approximately 60% of depressed patients, but a relapse in depressive symptoms typically occurs after subsequent sleep. Currently, very little is known about the effects of psychedelic substances on the regulation of sleep. Here, we show that psilocin acutely disrupts sleep maintenance and promotes quiet wakefulness in mice, but is not associated with long-term changes to sleep-wake-related behaviour.

Eight young adult male C57BL/6J mice were surgically implanted with electrodes for the continuous recording of electroencephalography (EEG) and electromyography (EMG), as well as with a microwire array (n=4) or single-shank electrode (n=4) targeting the medial prefrontal cortex. Custom-made head stages were constructed in advance of each surgery, which comprised three EEG bone screws and two stainless steel EMG wires, soldered to an 8-pin surface mount connector. Four animals were implanted with a single-shank probe in left anterior medial cortex.

The four remaining animals were implanted with a custom-designed polyimide-insulated tungsten microwire array, spanning a larger area of left anterior medial cortex, with 16 wire channels of 33 mm diameter arranged in 2 rows of 8, with row separation 375 m, columnar separation 250 m, and tip angle 45 degrees. For the single-shank probe, an additional hole was drilled into the skull, and for the arrays, a craniotomy window was drilled. A silicone gel was applied to seal the craniotomy and protect the exposed brain. Mice were housed in separate ventilated cages following surgery, and their recovery was closely monitored. They were then placed inside ventilated sound-attenuated Faraday chambers, and video was recorded continuously during the light period.

Mice were habituated to their cables for three days before the first baseline recording began. Four injection experiments were conducted, with each animal experiencing all combinations of two sleep-wake conditions and two drug treatments exactly once. Sleep deprivation was performed using the well-established gentle handling procedure. Novel objects were introduced to the cage to encourage wakefulness.

Mice were awakened by gentle physical stimulation instead of novel objects, and sleep deprivation lasted 4 hours. Electrophysiological signals were acquired using a multichannel neurophysiology recording system and processed online using the software package Synapse. Signals were read from tanks into Matlab, filtered with a zero-phase 4th order Butterworth filter, then resampled at 256 Hz. Spiking activity was cleaned offline for artefacts using the Matlab spike sorting software Wave_clus, and then normalised by expression as a percentage of the mean spike rate within the same channel.

On the baseline day before psilocin injection, vigilance states were scored manually by visual inspection at a resolution of 4 seconds using the software SleepSign. NREM sleep was identified by high amplitude EEG and LFP slow waves coincident with synchronous spiking multi-unit off periods. After all experiments, animals were euthanised and their brains were prepared for histological analysis. Four channels were sequentially stimulated with 10 A of current for 20 seconds, and then the brains were suspended in PFA for 24 – 48 hours before being stored in PBS with sodium azide.

The study used 8 animals, 7 with frontal EEG recordings, 5 with occipital EEG recordings, 6 with LFP recordings and 7 with multi-unit spiking activity recordings. 15 out of 112 LFP signals were excluded based on the presence of frequent high amplitude artifacts or unsystematic drift in signal amplitude during key analysis time windows. The spectral properties of EEG and LFP signals were analysed with a discrete fast Fourier transform on segments of 4-second duration, applying a Hann window. The average power for each discrete frequency value in wake, NREM and REM sleep was calculated for each animal from the whole 24-hour baseline day before injection.

Statistical tests were performed using Matlab, including ANOVA, paired samples t-tests, Wilcoxon tests, and ranksum tests, after confirming that data pass a Lillie’s test for normality.

8 mice were injected with psilocin or vehicle over 11 days and their EEG, cortical LFPs and neuronal activity were continuously monitored. Psilocin acutely destabilizes and fragments sleep, and disrupts animals’ first attempts at initiating sleep. Animals affected by psilocin spend a significant amount of time in their nests, adopting a posture compatible with sleep, but still apparently awake according to electrophysiological criteria. Psilocin injection did not change the essential features of electrophysiological signals in wake, NREM or REM sleep in a way that was immediately visually identifiable, but instead caused animals to alternate rapidly between short wake and shallow NREM sleep episodes.

The latency to the first NREM sleep episode was increased by psilocin, as was the latency to the initiation of any REM sleep. Psilocin increased the proportion of time spent awake and decreased the proportion of time spent in NREM and REM sleep in rats over the first 3 hours after injection. Psilocin increased the frequency of brief awakenings during NREM sleep, but did not affect the duration of continuous wake episodes. This suggests that psilocin increased the drive to awaken from sleep rather than impairing sleep maintenance.

Approximately 10 minutes after psilocin injection, an active wake period is followed by a quiet wake period containing frequent NREM sleep attempts but dominated by wakefulness. Approximately 35 minutes post-injection, consolidated NREM sleep becomes more distinct, although frequent brief awakenings persist. Psilocin does not affect sleep-wake architecture over the long term, except for a brief increase in wake and suppression of sleep, particularly REM sleep, in the first few hours after injection.

Within one day of injection, the quantity of wake, NREM and REM sleep was not significantly different between psilocin and vehicle conditions, and the quantity of wake, NREM and REM sleep was not significantly different in the dark period after injection. Psilocin affects the sleep homeostatic process in a region-specific manner. Its acute effects on sleep-wake states were further explored by determining whether sleep quantity or slow wave activity levels would differ in subsequent recovery sleep between drug and vehicle conditions.

After 4 hours of sleep deprivation, the latency to NREM sleep was not significantly different between psilocin and vehicle groups. There was an effect of time, but not of drug condition or interaction, on hourly sleep quantities throughout the remainder of the light period. While psilocin administration did not affect Process S, changes may still be visible at the level of localised cortical activity. The time course of average slow wave activity in NREM sleep over the remainder of the light period after sleep deprivation shows no effect of psilocin, only of time. Psilocin significantly reduced the time course of mean LFP 398 slow wave activity in the prefrontal 401 cortex, and this effect was repeated in the prefrontal 402 cortex.

The Process S recovered more slowly after psilocin, but only on a local level in prefrontal and adjacent cortex. Psilocin induces sleep in both undisturbed and sleep deprivation conditions, and NREM and REM sleep are analysed in the undisturbed condition from the first episode of NREM sleep after injection of at least 1-minute duration. Psilocin increased power in the 3-5 Hz range in frontal EEG and LFP, and decreased power in the gamma range in frontal EEG and LFP. This increase was stronger in the undisturbed condition, and accompanied by an increase in high frequency power (> 60 Hz) particularly during sleep deprivation.

In NREM sleep, no well-defined band-specific differences were identified between vehicle and 431 psilocin conditions. However, in REM sleep, high frequencies were reduced, whereas low frequencies were mostly increased.

Psilocin, a psychedelic, disrupted sleep in mice, resulting in fragmented sleep attempts and frequent brief awakenings. The sleep homeostatic process was not disrupted, but there was evidence for a slower decline of slow wave activity in the LFP in recovery sleep following sleep deprivation combined with psilocin injection. The role of serotonin in sleep-wake control is complex and somewhat controversial. Optogenetic studies in mice suggest that serotonin may contribute to the build-up of sleep need, while psychedelic 5-HT2A receptor agonists may suppress sleep.

REM sleep suppression is a common effect of many different antidepressants, including selective serotonin reuptake inhibitors, serotonin-463 noradrenaline reuptake inhibitors, monoamine oxidase inhibitors and tricyclic antidepressants. Psilocin does not necessarily induce long-term changes in sleep-wake architecture in mice. A prominent 3 – 5 Hz oscillation was identified in baseline wakefulness in both conditions, and psilocin enhanced this activity. This oscillation has been linked to breathing during wakefulness in mice, as well as other areas, and serotonergic signalling is implicated in breathing regulation.

Mice treated with a 5-HT2A receptor agonist showed oscillatory activity at around 4 Hz, although breathing was not measured. This oscillation may be related to respiration, but its functional significance is unclear. Psilocin injection can increase cortical synaptic strengths, which is identical to an increase in Process S, but the neuroplastic influence of psychedelics might actually complement or even substitute the effects of sleep, therefore reducing Process S. Although no change in EEG slow wave activity was found after ingestion of psilocybin, ayahuasca, or ketamine, this does not mean that neuroplasticity does not widely occur or that psychedelics do not affect sleep regulation.

Psilocin’s acute sleep-inhibiting effects may have confounded inference into Process S, and a ceiling effect on slow wave activity may have reduced the ability to discriminate between psilocin and vehicle conditions in the frontal EEG. The recovery rate of Process S locally in the LFP targeting prefrontal cortex was slowed, which suggests that the recovery of neuronal homeostasis after exposure to psychedelics is in some way slower in prefrontal regions. Psychedelic drugs offer a novel approach to study the basic science underpinning sleep regulation, providing a means to manipulate the content of wakefulness and associated brain dynamics.

Sleep represents an overlooked aspect of physiology in the efforts to understand how psychedelic mediated mechanisms yield psychological benefits. It is likely that the effects of psychedelics on arousal and sleep regulation might depend on circadian time and preceding sleep-wake history. The greatest challenge in understanding psychedelic action will be to better understand how it translates between animals and humans. This will require careful use of comparator drugs and translational tools.

Borbély, Daan, Wirz-Justice, Cameron, Benson, Dunlap, and Olson (2016) reappraise the two-process model of sleep regulation and find that it is more accurate. Carhart-Harris, Robin L., Mark Bolstridge, James Rucker, Camilla M J Day, David Erritzoe, Mendel Kaelen, Michael Bloomfield, et al. 2017. ‘Psilocybin with Psychological Support for Treatment-Resistant Depression: An Open-Label Feasibility Study’.

‘A role for enhanced functions of sleep in psychedelic therapy?’ and ‘Cortical region specific sleep homeostasis in mice: effects of time of day and waking experience’ are discussed. In the article, Halberstadt, Adam L, Koedood, Susan B Powell, and Geyer (2011) discuss the neuropsychopharmacology of serotoninrgic hallucinogens, and Hibicke, Meghan, Alexus N. Landry, Hannah M. Kramer, Zoe K. Talman, and Charle s D. Nichols discuss the role of serotonin in respiratory function and dysfunction.

Kay, D. C., Martin, W. R., Kometer, Michael, Schmidt, Jäncke, Vollenweider, F. X., 2017. Sleep regulation of cortical firing rates. Psychedelic effects are mediated through the glutamatergic system and involve the hippocampal sharp-wave ripples, which are entrained by respiration. REM sleep homeostasis is also affected by psilocybin, which inhibits REM sleep.

A review of the current knowledge on the 5-HT2A Receptor and its role in sleep and wakefulness, as well as the potential therapeutic use of selective 5-HT2A Receptor Antagonists and Inverse Agonists for the Treatment of an Insomnia Disorder. The authors of the paper are Felix Müller, Friederike Holze, Patrick Dolder, Laura Ley, Patrick Vizeli, Alain Soltermann, Matthias E. Liechti, and Stefan Borgwardt. They have found that increased 737 Thalamic Resting-State Connectivity is a core driver of LSD-induced hallucinations.

The serotonergic raphe promotes sleep in zebrafish and mice, and downward firing rate homeostasis is promoted by sleep in zebrafish and mice. Sleep also promotes emotional functions, and changes in global and thalamic brain connectivity are attributable to the 5-HT2A receptor. Psilocybin induces time-dependent changes in global functional connectivity in the brain, and is involved in memory acquisition, retrieval, and consolidation in the rat.

Researchers have found that the neuropeptide Galan is required for homeostatic rebound sleep following increased neuronal activity. This study was conducted in free-moving mice and examined the effects of psilocybin and MDMA on between-network resting state functional connectivity. Steiger, Axel, Mayumi Kimura, Christopher W Thomas, Mathilde CC Guillaumin, Laura E McKillop, Peter Acher mann, and Vladyslav Vyazovskiy. 2010. ‘Wake and sleep EEG provide biomarkers in depression’.

Figure 1 shows an example segment of 5 seconds duration of frontal electroencephalogram after injection with vehicle solution or psilocin.

A representative example of slow wave activity derived from frontal electroencephalogram, occipital EEG, and mean local field potential, alongside total recorded spike firing rate, variance of the electromyogram, and scored vigilance states, all over a period of 2 hours after injection with vehicle or psilocin.

Brief awakenings per minute of NREM sleep were observed in animals injected with vehicle or psilocin during the first hour after injection.

Figure 3 shows the percentage of time scored as wake, NREM sleep and REM sleep in successive non-overlapping windows of one hour up to 24 hours after injection with vehicle (blue) and psilocin (yellow), and the mean length in seconds of wake, NREM and REM sleep episodes in this time.

Frontal electroencephalogram, occipital electroencephalogram, mean local field potential, electromyogram and scored vigilance states were recorded, and slow wave activity was measured from the end of sleep deprivation until the end of the light period.

Figure 5 shows the mean power spectra of frontal EEG in four different vigilance state conditions following injection with vehicle (blue) and psilocin (yellow). The difference in spectral power between vehicle and psilocin conditions was significant (p 0.05) according to paired t-tests.

Figure 6 shows the mean power spectra of occipital EEG in four different vigilance state conditions following injection with vehicle (blue) and psilocin (yellow). The difference in spectral power between vehicle and psilocin conditions was significant (p 0.05) according to paired t-tests.

Figure 7 shows the mean power spectra of mean LFP in four different vigilance state conditions following injection with vehicle (blue) and psilocin (yellow). The difference in spectral power between vehicle and psilocin conditions was significant (p 0.05) according to paired t-tests.