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

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


34
Psilocybin is a classical serotonergic psychedelic; a unique class of drugs capable of inducing 35 profound alterations of perception, cognition, and behaviour, commonly characterised as the 36 psychedelic state. A growing body of evidence suggests that, under appropriately controlled 37 conditions, acute psilocybin exposure can promote long-lasting positive effects on mood and 38 psychological well-being, offering a promising new treatment method for affective disorders 39  The activation of 5-HT2A receptors and induction of a psychedelic state is widely suggested to 58 promote neuroplasticity, which is theorised to be important for psilocybin's therapeutic efficacy 59 9 Technologies Inc., Florida, USA). Mice were habituated to their cables for three days before the 166 first baseline recording began. The recording room was kept on a 12 -12 hour light-dark cycle 167 (lights on at 9 am), at 22 ± 1 o C and 50 ± 20 % humidity. Food and water were provided ad libitum 168 throughout. 169

Experimental Design 170
Once fully recovered from surgery, each batch of animals underwent four injection experiments, 171 comprising all combinations of two sleep-wake conditions and two drug treatments. In one sleep-172 wake condition, mice were immediately returned to their home cage after injection and left 173 undisturbed. In the second condition, a sleep deprivation protocol was enforced for four hours 174 after injection. A within-subjects design was employed such that each mouse experienced all 175 combinations of psilocin vs. vehicle and undisturbed vs. sleep deprivation conditions exactly 176 once. The order of drug treatments and sleep-wake conditions was counterbalanced across all 177 eight animals. Each injection experiment was separated by three days, following a pattern of 178 baseline day, injection day, and recovery day. 179

Preparation of Psilocin 180
Psilocin (4-hydroxy-N,N-dimethyltryptamine, LGC Standards) was administered by 181 intraperitoneal injection at a dose of 2 mg/kg. Crystalline psilocin was dissolved in 50 mM tartaric 182 acid and subsequently diluted in saline (5% glucose) up to a concentration of 0.25 mg/ml. 183

Sleep Deprivation 184
Sleep deprivation was performed using the well-established gentle handling procedure (Fisher 185 et al., 2016). During this period, experimenters constantly monitored both the behaviour and 186 ongoing neurophysiological recordings of the mice. As soon as any animal showed signs of 187 sleepiness (such as immobility, or slow waves in the EEG), novel objects were introduced to the 188 cage (such as cardboard, colourful plastic, sponge, tin foil, wooden blocks and plastic wrap) in 189 order to encourage wakefulness. In some cases, towards the end of the sleep deprivation period, 190 Histology was only fully successful in four animals (two single shank and two array implanted). 241 Based on the analysed subset, the estimated distribution of targeted cortical regions was 242 approximately prelimbic (39%), cingulate (31%), secondary motor (12 %), medial orbital (10%) 243 and infralimbic (8%). Histological analysis of the remaining animals suggested that some 244 electrodes may have reached deeper and more posterior structures including the dorsal striatum 245 and lateral septal nucleus, however this could not be definitively confirmed. For analysis, all 246 quality LFP signals were grouped and treated as one population. 247

Data Inclusion/Exclusion 248
Out of the 8 animals in the study, frontal EEG recordings were successfully obtained from 7, 249 occipital EEG from 5, LFP from 6 and multi-unit spiking activity from 7. Lost signals were due to 250 damage to the electrode or connecting wires. All signals were obtained simultaneously from 5 251 animals. All individual LFP signals were manually examined and a total of 15 (out of 112) were 252 identified for exclusion based on the presence of frequent high amplitude artifacts or 253 unsystematic drift in signal amplitude during key analysis time windows. 254

Field Potential Spectral Analysis 255
The spectral properties of EEG and LFP signals were analysed with a discrete fast Fourier 256 transform (FFT) on segments of 4-second duration, applying a Hann window. Spectral power 257 values were averaged over epochs scored as the same vigilance state within the time window of 258 interest separately for each animal. An average power for each discrete frequency value in wake, 259 NREM and REM sleep was calculated for each animal from the whole 24-hour baseline day before 260 psilocin injection. For plotting spectra, values between 45 -55 Hz were interpolated for ease of 261 visualisation, since power in this frequency range was removed by a notch filter targeting 50 Hz 262 line noise. When specifically analysing slow wave activity, this was obtained for each epoch by 263 summing power over frequencies from 0.5 -4 Hz obtained from the FFT. 264 Statistics 265 13 Statistical tests were all performed using Matlab. ANOVA was performed using the Matlab 266 function anova2 and paired samples t-tests were conducted using the Matlab function ttest, after 267 confirming that data pass a Lillie's test for normality (function lilliestest) and in some cases, 268 where indicated, tests were run after applying a log transform to improve data normality. A 269 Wilcoxon test was performed (function ranksum) for highly skewed data. When testing for 270 differences between power spectra t-tests were run at all individual discrete frequency values, 271 applying a p < 0.05 significance threshold without correction for multiple comparisons. 272

273
In this study 8 mice were injected with either 2 mg/kg psilocin (4-hydroxy-N,N-274 dimethyltryptamine) or vehicle while EEG, cortical LFPs and neuronal activity were continuously 275 monitored over a period of 11 days, comprising 4 injection experiments. The within-subject 276 design incorporated two sleep-wake conditions (undisturbed vs. sleep deprivation) and two 277 treatments (drug vs. vehicle). 278

Sleep is acutely destabilised and fragmented after psilocin 279
In the first condition, the mice were left undisturbed in their home cage after injection, in order 280 to study arousal and spontaneous sleep-wake behaviour. The injection was administered shortly Psilocin injection did not change the essential features of electrophysiological signals in wake, 290 NREM or REM sleep in a way that was immediately visually identifiable (Figure 1), and it was 291 therefore possible to score sleep-wake episodes in both vehicle and psilocin-treated animals. 292 Analysis of this period using electrophysiological criteria for sleep-wake definition suggested that 293 the psilocin-injected animals were rapidly alternating between short wake and shallow NREM 294 sleep episodes (Figure 2A, 2B). The average latency from the time of injection to the first 4-second 295 epoch scored as NREM sleep, based on electrophysiological criteria, was 18.6 ± 6.1 minutes in the 296 vehicle condition and 26.3 ± 4.5 minutes in the psilocin animals, which was not significantly 297 different between conditions (p = 0.30, n = 8, paired t-test, Figure 2C). However, the mean latency 298 to the first NREM sleep episode, defined as continuous NREM sleep of at least 1-minute duration, 299 was 25.7 ± 5.4 minutes in vehicle animals compared to 43.4 ± 3.7 minutes in animals injected with 300 psilocin, which was a significant difference (p = 0.015, n = 8, paired t-test, Figure 2D). Similarly, 301 the latency to the initiation of any REM sleep was also increased by psilocin, from an average of 302 44.5 ± 5.1 minutes in vehicle condition to 74.6 ± 6.1 minutes in the psilocin condition (p = 0.013, 303 n = 8, paired t-test, Figure 2E). 304 Alterations to sleep-wake activity in the psilocin-treated animals were observed to be greatest 305 during the first hour after injection but could last up to approximately 3 hours, and so this time 306 window was analysed further. Over the 3 hours following injection, psilocin increased the average 307 proportion of time spent awake (Vehicle: 30.8 ± 2.0%, Psilocin: 44.3 ± 3.4%, p = 6.8 x 10 -4 , n = 8, 308 paired t-test), and correspondingly significantly decreased the time spent in NREM (Vehicle: 59.7 309 ± 1.7%, Psilocin: 50.6 ± 2.9%, p = 0.0011, n = 8, paired t-test), and REM sleep (Vehicle: 9.4 ± 0.5%, 310 Psilocin: 5.1 ± 0.8%, p = 0.0012, n = 8, paired t-test, Figure 2F). However, during this 3-hour period 311 after injection, the mean duration of continuous wake episodes was unchanged (Vehicle: 28.8 ± 312 4.5 secs, Psilocin: 29.8 ± 3.1 secs, p = 0.89, paired t-test), whereas episode duration was 313 significantly reduced for both NREM (Vehicle: 93.6 ± 12.9 seconds, Psilocin: 50.0 ± 2.6 seconds, p 314 = 0.0070 paired t-test) and REM sleep (Vehicle: 67.7 ± 7.0 secs, Psilocin: 47.6 ± 5.1 seconds, p = 315 0.019, Figure 2G). This form of sleep disruption resembles an increased propensity for brief 316 awakenings, usually defined in mice as periods of wakefulness lasting ≤ 20 seconds occurring 317 during NREM sleep and typically accompanied by small body movements. During the first hour 318 following injection, the frequency of brief awakenings per minute of NREM sleep was increased 319 by psilocin (Vehicle: 0.60 ± 0.32; Psilocin: 1.4 ± 0.48; p = 2.0 x 10 -5 , n = 8, paired t-test, Figure 2H). 320 These results suggest that the increased wakefulness produced by psilocin is due to an increased 321 drive to awaken from sleep, corresponding to an impairment of sleep maintenance rather than 322 an enhanced stability of wakefulness. 323 This period of rapidly alternating wake and NREM sleep is further illustrated in Figures 2I and 2J,  324 showing a time-frequency plot for the frontal EEG spectral power (relative to the baseline day), 325 from one representative example animal after both vehicle and psilocin administration. In the 326 vehicle condition, clear vigilance state boundaries are visible in the spectrogram ( Figure 2I), 327 including wake periods with heterogenous spectral composition, NREM sleep with increased low 328 frequency (< 30 Hz) and decreased high frequency power (> 30Hz) and REM sleep characterised 329 by reduced low frequency (< 6 Hz) and elevated upper theta (7 -10 Hz) power. In contrast, in the 330 psilocin condition, the wake to sleep transition was less distinct ( Figure 2J). In this example, 331

Psilocin affects the sleep homeostatic process in a region-specific manner 361
The sleep-wake history of an individual is tracked by physiological processes in the brain in order 362 to homeostatically regulate global vigilance states, such that, for example, sleep deprivation is 363 compensated by increased subsequent sleep duration and intensity. This phenomenon is termed 364 "Process S", and with an underlying biological substrate that is not completely certain, measures 365 the magnitude of the homeostatic drive to sleep, and can predict with high accuracy EEG slow The effects of psilocin on EEG and LFP spectra in different states of vigilance was then explored. In NREM sleep, no well-defined band-specific differences were identified between vehicle and 430 psilocin conditions. A trend existed in all EEG and LFP signals for decreased low frequencies (< 4 431 Hz), perhaps reflecting that sleep was less intense ( Figure 5C, 6C, 7C). During REM sleep after 432 psilocin, high frequencies (> 30 Hz) tended to be reduced, whereas low frequencies (< 8 Hz and 433 10-20 Hz) were mostly increased ( Figure 5D, 6D, 7D). These differences might be interpreted as 434 a bleeding of NREM-like activities (delta waves, spindles, reduced gamma) into REM sleep.

Effects of serotonergic agents and antidepressants on sleep 448
The role of the serotonin system in sleep-wake control is complex and somewhat controversial 449 sleep-wake architecture in mice, at least at the 2 mg/kg dose given here, and that it is unlikely 472 that modulation of REM sleep quantity is a core mechanism of the psychological benefits of 473 psychedelics. It remains possible, however, that REM sleep is affected in a more subtle way in 474 terms of the underlying network activity. 475

Significance of the 3 -5 Hz oscillation 476
Analysis of frontal EEG and LFP spectra in these recordings identified a prominent 3 -5 Hz peak 477 amplified by psilocin. This peak was present in baseline wakefulness in both conditions, so likely 478 corresponds an enhancement of a particular form of existing activity. Oscillatory activity in this 479 frequency range has been previously associated with breathing during wakefulness in mice in the that these coincide with behavioural inactivity, although breathing was not measured (Contreras 488 et al., 2021). That this psilocin-associated oscillation is related to respiration remains to be 489 directly tested, and moreover its functional significance within the context of both endogenous 5-490 HT2A receptor activation and the psychedelic phenomenon is unclear. Dissecting the behavioural 491 and pharmacological influences on this oscillation would require more carefully controlled 492 experiments. 493

Does psilocin affect Process S? 494
One of the main aims of this study was to determine the effects of psilocin on Process S. 495 Considering the ability of psilocybin to disrupt normal neuronal dynamics and to promote 496 widespread functional plasticity, it can be predicted that psilocin injection would lead to a net 497 increase in cortical synaptic strengths. According to the synaptic homeostasis hypothesis, this is 498 identical to an increase in Process S (Tononi & Cirelli, 2003). However, it has been alternatively 499 argued that the neuroplastic influence of psychedelics might actually complement or even 500 substitute the effects of sleep, therefore reducing Process S, since both involve a temporary 501 relaxation of functional constraints followed by self-organised re-optimisation (Froese et al., 502

2018). 503
Since the relationship between plasticity and sleep regulation is neither straightforward nor fully 504 understood (Frank & Heller, 2019), the absence of a change in EEG slow wave activity found here 505 should not be interpreted as evidence that neuroplasticity does not widely occur, or that 506 psychedelics do not affected sleep regulation. A previous study in humans reported elevated EEG 507 slow wave activity during sleep 11 hours after ingestion of ayahuasca, a traditional psychedelic 508 drink containing dimethyltryptamine (Barbanoj et al., 2008). However, another human study 509 with psilocybin reported that EEG slow wave activity was suppressed in the first cycle of NREM 510 sleep (Dudysová et al., 2020). These differences may be simply due to species or drug dose, or 511 even the possibility that the compound remained in the system at sleep onset, exerting acute 512 effects on arousal and the expression of the sleep slow wave itself, confounding inference into 513 Process S per se. In this study a sleep deprivation duration was chosen which exceeded the 514 duration of observable acute effects of the drug wake and NREM sleep. It is possible that the 515 duration of sleep deprivation was too long, and as such a ceiling effect on slow wave activity 516 reduced the ability to discriminate the effects on Process S between psilocin and vehicle 517 conditions in the frontal EEG. Of course, reducing the sleep deprivation exacerbates the risk that 518 the acute sleep-inhibiting effects of psilocin remain present, so this would be difficult to 519 disentangle. 520 In this regard, the finding of a reduced recovery rate of Process S locally in the LFP targeting 521 prefrontal cortex is of potential importance. The global Process S which manifests in slow wave 522 activity at the EEG level has been suggested to result from the integration across the brain of many 523 local Processes S, which in turn each reflect the recent history of local neuronal activities (Thomas 524 et al., 2020). If the rate of Process S recovery is slowed locally in prefrontal regions, it is possible 525 that recovery may occur more quickly elsewhere in the brain, such as more posterior cortex. The 526 functional significance of this result is not certain and a more in-depth mapping of Process S 527 across the cortical surface would be valuable, however, it does imply that the recovery of neuronal 528 homeostasis after exposure to psychedelics is in some way slower in prefrontal regions. Increased 529 slow wave activity indicates elevated neuronal synchronisation, so this result may well be linked 530 to neuroplasticity of functional networks and is consistent with the widely formed hypothesis 531 that the prefrontal cortex is a key cortical region affected by psilocybin. 532

Future outlook 533
Psychedelic drugs such as psilocin provide a novel approach to study the basic science 534 underpinning sleep regulation, offering a means to manipulate the content of wakefulness and 535 associated brain dynamics. Psychedelic stimulation offers important advantages compared to 536 other manipulations of waking brain activity, such as optogenetic activation of cortical neurones, 537 owing to its relative simplicity, physiological validity, pharmacological specificity, applicability to 538 humans, and comprehensibility in terms of the associated conscious experience. Similarly, sleep 539 represents an overlooked aspect of physiology in the efforts to understand how psychedelic-540 mediated mechanisms yield psychological benefits. 541 It is likely that the effects of psychedelics on arousal and sleep regulation might depend on 542 circadian time and preceding sleep-wake history. For example, it is known in humans that the 5-543 HT2A receptor density increases after sleep deprivation (Elmenhorst et al., 2012). These factors 544 would be easy to control for and manipulate in future animal studies. Furthermore, the time scale 545 over which psilocybin-associated plasticity (and putative associated effects on Process S) occurs 546 is not known. While it is often assumed that plasticity must be induced during the acute 547 experience, this is not necessarily guaranteed to be the case. If plasticity unfolds gradually over 548 many days, or even selectively during sleep, this will not lead to changes visible in the EEG slow 549 wave activity. 550 Perhaps the greatest challenge here will be to first better understand the extent to which the 551 physiology of psychedelic action translates between animals and humans. This is particularly 552 important, since therapeutic benefits in humans are dependent on appropriate psychological 553 support, such as from a trained therapist. Identifying common mechanisms of action of 554 psychedelics in both humans and rodents would be a great advance, and will require careful use 555 of comparator drugs, with known pharmacological profiles and subjective effects, alongside 556 translational tools such as EEG. Although many essential questions remain, a consistent picture 557 of psychedelic biology is gradually forming, carrying great potential to inform neuroscience in 558 areas spanning basic neurobiology to clinical practice.