Quiet wakefulness: the influence of intraperitoneal and intranasal oxytocin on sleep–wake behavior and neurophysiology in rats

Joel S Raymond; Nicholas A Everett; Anand Gururajan; Michael T Bowen

doi:10.1093/sleep/zsad112

. 2023 Apr 11;46(7):zsad112. doi: 10.1093/sleep/zsad112

Quiet wakefulness: the influence of intraperitoneal and intranasal oxytocin on sleep–wake behavior and neurophysiology in rats

Joel S Raymond ^1,², Nicholas A Everett ^3,⁴, Anand Gururajan ^5,⁶, Michael T Bowen ^7,^8,^✉

PMCID: PMC10334738 PMID: 37039749

Abstract

Study Objectives

Exogenous administration of the neuropeptide oxytocin exerts diverse effects on various neurobehavioral processes, including sleep and wakefulness. Since oxytocin can enhance attention to social and fear-related environmental cues, it should promote arousal and wakefulness. However, as oxytocin can attenuate stress, reduce activity, and elicit anxiolysis, oxytocin might also prime the brain for rest, and promote sleep. At present, little research has comprehensively characterized the neuropsychopharmacology of oxytocin-induced effects on sleep–wake behavior and no reconciliation of these two competing hypotheses has been proposed.

Methods

This study explored the effects of oxytocin on sleep–wake outcomes using radiotelemetry-based polysomnography in adult male and female Wistar rats. Oxytocin was administered via intraperitoneal (i.p.; 0.1, 0.3 and 1 mg·kg⁻¹) and intranasal (i.n.; 0.06, 1, 3 mg·kg⁻¹) routes. Caffeine (i.p. and i.n.; 10 mg·kg⁻¹) was administered as a wake-promoting positive control. To ascertain mechanism of action, pretreatment experiments with the oxytocin receptor (OXTR) antagonist L-368,899 (i.p.; 5 mg·kg⁻¹) followed by oxytocin (i.p.; 1 mg·kg⁻¹) were also conducted.

Results

In both male and female rats, i.p. oxytocin promoted quiet wakefulness at the cost of suppressing active wakefulness, non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Several i.p. oxytocin-induced sleep–wake effects were mediated by OXTR binding. In contrast, i.n. oxytocin did not alter most sleep–wake outcomes at any dose tested. Both i.p. and i.n. caffeine demonstrated wake-promoting effects.

Conclusions

These findings help reconcile competing hypotheses of oxytocin-induced effects on sleep–wake behavior: i.p. oxytocin promotes quiet wakefulness—a state of restful environmental awareness compatible with both oxytocin’s anxiolytic effects and its enhancement of processing complex stimuli.

Keywords: sleep, wake, oxytocin, caffeine, quiet wakefulness, intraperitoneal, intranasal, rat, rodent, oxytocin receptor

Graphical abstract

Statement of Significance.

The findings of the present study contribute important nuance to oxytocin’s wake-promoting effects in the preclinical literature; intraperitoneal oxytocin dose-dependently promotes quiet wakefulness over active wakefulness, NREM sleep, and REM sleep. Additionally, this research confirms that dose and route of administration, but not biological sex, impact oxytocin’s sleep–wake effects. Our finding that intranasal oxytocin demonstrated no major impacts on sleep–wake outcomes across a wide dose range is important as intranasal oxytocin is currently under investigation in clinical trials for several disorders with comorbid sleep problems. The promotion of quiet wakefulness holds potential clinical relevance for health conditions involving irritability, aggression, and excessive daytime sleepiness, which highlights the oxytocin system as a potential target for transdiagnostic therapeutics involving these symptoms.

Introduction

The neuropeptide oxytocin exerts diverse effects on numerous domains of mammalian neurobiology and behavior [1], including sleep and wakefulness [2]. Endogenous oxytocin is synthesized in the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, and is released into circulation via the neurohypophysis, and centrally via axonal projections to various forebrain, midbrain, and hindbrain regions [3, 4]. As both oxytocinergic projections and oxytocin receptor (OXTR) expression have been found in various hypothalamic and pontine sleep–wake regulatory brain regions [1, 4], the fundamental neural circuitry exists for oxytocin to impact sleep and wakefulness. However, current understanding of oxytocin’s influence on sleep–wake behavior and neurophysiology is limited [2].

Theoretically, it is difficult to hypothesize what impact oxytocin will exert on sleep–wake outcomes as two seemingly contradictory hypotheses emerge. From an environmental awareness-arousal perspective, oxytocin can orient attention towards environmental cues critical for survival and adaptation [5], both social [6, 7] and fear-related [8, 9]. Enhanced attention likely relies on sufficiently enhanced physiological arousal [10], so hypothetically oxytocin should promote arousal and consequently, promote wakefulness. In contrast, from a stress attenuation-quiescence perspective, oxytocin can reduce stress and anxiety [11–13], locomotor activity [14, 15], pain sensitivity [16, 17], heart rate [18], and aggression [19]. Taken together, this suggests that oxytocin physiologically and psychologically primes the brain and body for rest, which in turn should—hypothetically—promote sleep.

Empirically, based on our recent systematic review [2], exogenous oxytocin appears to exert mixed sleep–wake effects ranging from promoting wakefulness [20, 21] to promoting sleep [22] to exerting no sleep–wake effects [23, 24] across preclinical studies. However, when factors of dose and route of administration were considered, a relatively clear wake-promoting effect of oxytocin emerged. Additionally, a lack of studies investigating potential sex differences in oxytocin-induced sleep–wake effects was highlighted. Hence, the current study aimed to address the following research questions: (1) what effects does peripherally administered oxytocin exert on sleep–wake behavior and neurophysiology, (2) do any effects depend on dose, route of administration, or biological sex, and (3) are any observed effects mediated by the OXTR? Based on preclinical evidence from our systematic review [2], we hypothesized that oxytocin would exert dose-dependent wake-promoting effects.

Methods

An abridged methods section is presented below; for complete methodological details, see “Methods” section in Supplementary Material.

Animals and housing

Eight-week-old male and female Wistar rats (ARC, WA, Australia) were pair-housed in filter-top cages (58 × 38 × 20 cm; Able Scientific) containing corn cob bedding material (Bed-o’ Cobs, The Andersons), environmental enrichment, and ad libitum access to standard rodent chow and water. All experiments were conducted within a specific pathogen-free facility and rats were housed in a temperature- and humidity-controlled room (22 ± 0.5°C; 50%–60%) under a reverse light cycle (12L:12D; lights on at 1400, defined as ZT0). At transitions between light phases, illumination was slowly transitioned between 0 and 500 lux over a 16-minute period. All experiments were conducted in line with the Australian code for the care and use of animals for scientific purposes (8th edition, 2013) and were approved by the Animal Ethics Committee at The University of Sydney (AEC number: 2019/1615).

Drug preparation

Oxytocin (China Peptide, China) and caffeine (anhydrous; AK Scientific Inc., USA) were dissolved in physiological saline (0.9% w/v). The non-peptidergic OXTR antagonist L-368,899 hydrochloride (Santa Cruz Biotechnology, Texas) was dissolved in dimethylsulfoxide (5% v/v), Tween 80 (5% v/v), and saline (90% v/v).

Intraperitoneal drug administration

All drugs administered via intraperitoneal (i.p.) injection were administered at an injection volume of 1 mL·kg⁻¹ body weight. Oxytocin was administered at light onset (ZT0) at a range of doses (0.1, 0.3, and 1 mg·kg⁻¹ b.w.). These doses were selected to cover doses of oxytocin that do (1 mg·kg⁻¹) and do not (0.3 and 0.1 mg·kg⁻¹) suppress gross locomotor activity in rats [14]. Caffeine was administered at ZT0 at a dose of 10 mg·kg⁻¹ as a positive control, based on previous research demonstrating potent wake-promoting effects at this dose in rats [25, 26]. OXTR antagonist (L-368,899, 5 mg·kg⁻¹) pretreatment was administered 15 minutes prior to light onset (ZT23.75). L-368,899 was chosen as it is centrally penetrant and preferentially binds to OXTRs [27, 28]. The dose was chosen based on its ability to block the stress-attenuating effects of 1 mg·kg⁻¹ oxytocin in rats [29].

Intranasal drug administration

Oxytocin (0.06, 1, and 3 mg·kg⁻¹) and caffeine (10 mg·kg⁻¹) were administered intranasally (i.n.) according to methods adapted from Lukas and Neumann [30]. The 0.06 mg·kg⁻¹ dose represents an approximately four-to-five-fold higher dose than the highest i.n. dose administered in human clinical research 80 IU [31]; based on allometric scaling using the FDA guidance for conversion between animal and human doses Food and Drug [32], the human equivalent dose of 0.06 mg·kg⁻¹ in a rat is ~348 IU for a 60 kg human. This dose was selected to investigate whether doses closer to those administered in clinical research would impact sleep–wake behavior. The 1 and 3 mg·kg⁻¹ doses were chosen to facilitate comparisons between i.p. and i.n. routes of administration; the higher dose was based on a previous study demonstrating that i.p. oxytocin elicited approximately two-fold greater increases in plasma and central oxytocin levels compared to the same dose administered i.n [33].

Radiotelemetry probe implantation surgery

Briefly, after induction and administration of pre-operative analgesics, rats were surgically implanted with wireless radiotelemetry probes capable of polysomnographic (PSG)—electrocorticographic (ECoG) and electromyographic (EMG)—recording (HD-X02, Data Sciences International Inc.). The telemetry probe was implanted subcutaneously under the right flank, ECoG wires were secured around two screws contacting the dura at frontoparietal locations (measured from bregma: (1) anterior/posterior: +2 mm, lateral: +1.5 mm, (2) anterior/posterior: –7 mm, lateral: –1.5 mm), and EMG wires were threaded through and secured to trapezius muscle. Rats received post-operative analgesia and were allowed at least 14 days of recovery prior to experimentation.

Polysomnographic recordings

Rats were placed individually into telemetry cages (43 × 26 × 12 cm, Techniplast, Italy) located above telemetry receiver plates (RPC-1, Data Sciences International Inc.). Habituation to the telemetry recording cages and procedures were conducted over three 6-hour baselines PSG recordings prior to testing to ensure proper probe functioning and reception by telemetry receiver plates. For all experiments except experiment 2, rats were placed into recording cages at least 1 hour prior to light onset and recordings were started by 1300 (ZT23). Due to pretreatment with antagonist L-368,899 for experiment 2, rats were placed into recording cages at least 1.25 hours prior to light onset and recordings were started by 1245 (ZT22.75). At light onset (ZT0), rats were removed from the telemetry cage, administered an i.p. injection or i.n. application, placed back in the cage, left to behave freely for 6 hours (until ZT6), and then returned to their home cages (Figure 1A). For female rats, estrus phase determination was conducted according to methods adapted from Marcondes, Bianchi, and Tanno [34], immediately following the termination of PSG recording sessions (ZT6-6.5).

Figure 1. — Overview of polysomnographic session timeline, sleep–wake scoring procedure, and representative hypnograms from experiment 1. (A) Experimental schedule outlining the timing of procedures undertaken during recording sessions. The gradient and color of the bar represent the circadian phase and intensity of light conditions during the shift from the dark phase to the light phase. i.n.: intranasal; i.p.: intraperitoneal; OXT: oxytocin; OXTR: oxytocin receptor; PSG: polysomnography; ZT: zeitgeber, timing relative to light onset (ZT0). (B) Representative polysomnography traces (5-second duration) of each sleep–wake state category used for scoring. ECoG: electrocorticography; EMG: electromyography. (C) Representative hypnograms depicting sleep–wake state across 0–120 minutes of recording (starting at ZT0) for each dose of i.p. oxytocin administered (0, 0.1, 0.3, and 1 mg·kg⁻¹). (D) Representative hypnograms depicting sleep–wake state across 0–120 minutes of recording (starting at ZT0) for i.p. caffeine (0, 10 mg·kg⁻¹). Hypnograms for (C) and (D) were constructed using data from the subject with the median effect size of i.p. oxytocin 1 mg·kg⁻¹ on %QW 0–90 minutes.

Experimental design

All experiments were conducted across consecutive recording sessions using repeated-measures, counterbalanced designs to minimize inter-subject variability and increase statistical power. In all designs, dose sequences were generated using William’s Latin Square designs that control for first-order carryover effects [35], and rats were randomized to dose sequences using a random number generator, with the caveat that pair-housed rats could not be assigned to the same dose sequence. There was at least 90 hours of washout before the next recording session following oxytocin or L-368,899 administration, and 7 days following caffeine administration, which demonstrates more marked and prolonged effects on sleep [20, 26]. Hence, the caffeine condition was not incorporated into oxytocin dose–response William’s Latin square designs in Experiment 1 to preserve a consistent washout period between recording sessions.

Oxytocin i.p. dose–response (experiments 1A and 1B)

This experiment characterized the effects of i.p. oxytocin (vehicle [VEH], 0.1, 0.3, 1 mg·kg⁻¹; experiment 1A) and caffeine (VEH and 10 mg·kg⁻¹; experiment 1B) on sleep–wake outcomes. Male and female rats (N = 16; n = 8 per sex) were run as separate cohorts to avoid potential interference of opposite-sex pheromones in sleep–wake behavior and physiology during PSG recording sessions reviewed by [36].

Oxytocin i.p. and OXTR antagonism (experiment 2)

This experiment explored whether i.p. oxytocin-induced effects on sleep–wake outcomes are mediated by the OXTR. Since no significant sex-by-dose interactions were apparent during experiment 1 (see Supplementary Table S1), only female rats (n = 4) were used for experiment 2. The following conditions examined whether an OXTR antagonist inhibited oxytocin effects on sleep–wake outcomes: (1) VEH + VEH; (2) VEH + oxytocin (1 mg·kg⁻¹); (3) L-368,899 (5 mg·kg⁻¹) + VEH, and (4) L-368,899 (5 mg·kg⁻¹) + oxytocin (1 mg·kg⁻¹).

Oxytocin i.n. dose–response (experiments 3A and 3B)

Experiment 3A characterized the dose-dependent effects of i.n. oxytocin (0, 0.06, 1 mg·kg⁻¹ in 20 μL), and experiment 3B a higher dose of i.n. oxytocin (3 mg·kg⁻¹ in 20 μL) and caffeine (10 mg·kg⁻¹ in 20 μL), on sleep–wake outcomes. The goal was to examine whether i.n. oxytocin would recapitulate the effects of i.p. oxytocin observed in experiment 1. As with experiment 2, only female rats were used: n = 6 for experiment 3A and n = 5 for experiment 3B.

Data acquisition, processing, and analysis

Polysomnographic recording data (ECoG, EMG, body temperature, and activity) were acquired using Ponemah software (Version 6.41, Data Sciences International). NeuroScore (Version 3.2.1, Data Sciences International) was used to score sleep–wake states for each 10-second epoch of time: active wake (AW), quiet wake (QW), non-rapid eye movement sleep (NREMS), and rapid eye movement sleep (REMS). Pre-REM sleep, the transition state between NREMS and REMS characterized by high amplitude EEG activity within the theta and alpha power bands [37], was scored as NREMS.

ECoG and EMG signals were filtered prior to scoring: a band-pass filter (0.1–80 Hz) and high-pass filter (>0.1 Hz) were applied to the ECoG and EMG signal Data Sciences [38], respectively. Sleep scoring was conducted manually by a blinded experimenter via visual examination of various raw and derived signals: power spectral density (PSD) “periodogram” of ECoG signal (Fast Fourier Transform; 0–25 Hz), theta:delta ratio, ECoG trace, EMG trace, activity counts, and sleep–wake state of previous epoch. Briefly, scoring criteria adapted from [39] for each sleep–wake state were as follows (see Figure 1B for representative traces):

Active wake: (ECoG) low amplitude, low synchrony, high frequency; (EMG) high amplitude, irregular tone; (Activity) locomotion and movement present
Quiet wake: (ECoG) low amplitude, low synchrony, moderate-high frequency relative to AW; (EMG) moderate amplitude relative to AW, regular tone; (Activity) no locomotion and minimal movement
NREM sleep: (ECoG) moderate-high amplitude, high synchrony, very low-low frequency; (Periodogram) high power density within the delta frequency band (0–4 Hz); (EMG) low amplitude, regular tone; (Activity) no locomotion or movement
REM sleep: (ECoG) low amplitude, high frequency, “saw-tooth” profile; (Periodogram) high power density within the theta frequency band (4–8 Hz) and high theta:delta ratio; (EMG) low amplitude, regular tone; (Activity) no locomotion or movement
Artifact: epochs were scored as artifacts and excluded from analysis if no signals (ECoG, EMG, activity, etc.) were available during the epoch to indicate sleep–wake state.

After sleep scoring was completed, the following sleep architectural outcomes of interest were extracted: sleep onset latency (minutes), REM sleep onset latency (minutes), proportion of total time spent in each sleep state (%), bout frequency of each sleep state, mean duration of bout of each sleep state (minutes), and mean body temperature (ºC). These 7 hours of PSG data (1 hour prior to oxytocin administration during last hour of dark phase and 6 hours post-administration during light phase) were then compiled into 30-minute bins for statistical analysis. Sample sizes for each experiment and experimental condition are detailed in figure legends; some subjects were excluded due to surgical complications following probe implantation, while some data are absent due to issues with dropout during telemetry recordings. For complete details on subject attrition and data exclusion, see Supplementary Materials.

Absolute ECoG PSD values during AW, QW, NREMS, and REMS were extracted for each 1-Hz frequency band from 0 to 25 Hz using Discrete Fourier Transform within specified time windows. Artifact detection, both automated and manual, was conducted and those epochs containing artifacts were excluded from Data Sciences [38]. As large inter-individual differences existed in ECoG spectral outcomes, all subjects’ data were normalized by expressing values as a proportion of an outcome during the corresponding VEH condition of each respective experiment [20]. For further details on PSD data analysis, see Supplemental materials.

As sleep outcomes appear to be predominantly influenced by the estrus cycle during proestrus and estrus phases [40, 41], estrus stage was delineated into two categories: either proestrus/estrus or metestrus/dioestrus.

Statistical analysis

Linear mixed effects models (LMMs) were constructed for all sleep architectural outcomes with Dose, Time, and Dose × Time as within-subjects fixed effects factors. LMMs were chosen to avoid having to exclude subjects with missing data. A compound symmetry covariance matrix structure was employed and fitted using restricted maximum likelihood estimation. Greenhouse-Geisser corrections were applied to adjust for violations of sphericity. If random effects were zero, they were removed from the model and a simpler model was fitted.

For sleep architectural outcomes, the first hour of data (dark phase; pre-administration) were analyzed separately from the following 6 hours (light phase; post-administration) to test for baseline differences in outcomes. As part of the LMM analysis, trend analysis was conducted on Dose to determine if a linear dose-dependent effect was present. Based on the pharmacokinetics of acute peripherally administered oxytocin (t_1/2 = <5 minutes [42]), duration of action of i.p. oxytocin on body temperature and heart rate (i.e. 180 minutes duration [18]) and post hoc visual inspection of the sleep architectural outcomes, average values were calculated for periods of the peak effect for each outcome—AW: 0–30 minutes and 30–180 minutes, QW: 0–90 minutes, NREMS: 0–90 minutes, REMS: 30–180 minutes, and body temperature: 0–120 minutes. While this inconsistent application of time windows across each sleep–wake state complicates comparisons between sleep–wake states, this analytic approach prioritizes characterizing the temporal and directional nature of oxytocin’s effects within each sleep–wake state. LMM and trend analyses were conducted on these composite outcomes with Dose as a fixed effect.

For ECoG PSD outcomes, PSD values were averaged over all epochs of a specified sleep–wake state within a specified time window, normalized to the respective vehicle condition for that experiment (%) for each 1-Hz frequency band and then transformed using a logarithmic (log₁₀) transformation. Subsequently, mean values across each power band (delta: 0.1–4 Hz, theta: 4–8 Hz, alpha: 8–12 Hz, sigma: 12–16 Hz, beta: 16–25 Hz), and then compared to the baseline (VEH) value using a two-tailed one-sample t-test. For estrus phase data, differences in the proportion of rats in proestrus/estrus and metestrus/dioestrus phases during each experiment between each dose and VEH were analyzed using two-sided Fisher’s Exact tests. Fisher’s least significant difference test was used to control type I error rate. Statistical analyses were conducted using GraphPad Prism (Version 9.3.1). The level of significance for all tests was p < .05.

Results

For experiment 1, a small sex difference was observed; female rats demonstrated greater %QW and %REMS and lower %NREMS than male rats. However, data from male and female rats were combined for all analyses based on the lack of significant Sex × Dose interaction effects observed across all proportions of time spent in sleep–wake state outcomes for periods of peak effect for experiment 1a (for more details, see Supplementary Table S1).

Experiment 1—effects of i.p. oxytocin dose range and i.p. caffeine on sleep–wake outcomes

Figure 1 depicts the timeline of sleep recording sessions (1A), representative PSG traces for each sleep–wake state (1B), and representative hypnograms of results from experiment 1 illustrating the temporal nature of oxytocin- and caffeine-induced effects on sleep–wake state (1C and D). Summarized statistical results for experiments 1a and 1b are presented in Tables 1 and Supplementary Table S2.

Table 1.

Effects of i.p. Oxytocin Dose Range and i.p. Caffeine Dose on Sleep–Wake Outcomes

Wake outcome	Oxytocin dose–response (0, 0.1, 0.3, 1 mg·kg⁻¹)			Caffeine 10 mg·kg⁻¹ (positive control)
Wake outcome	Linear trend	Main effect of dose	Dose × Time interaction	Main effect of dose	Dose × Time interaction
Active wake
Proportion of time	F(1, 38) = 0.87 p = .3570	F(2.302, 29.93) = 0.55 p = .6070	F(8.546, 108.0) = 3.85 p = .0004	F(1, 11) = 68.56 p < .0001	F(5.006, 55.06) = 6.79 p < .0001
Bout frequency	F(1, 38) = 13.90 p = .0006	F(2.847, 37.01) = 4.13 p = .0139	F(7.608, 96.13) = 3.57 p = .0014	F(1, 11) = 67.86 p < .0001	F(5.079, 55.86) = 3.38 p = .0095
Bout duration	F(1, 38) = 3.00 p = .0911	F(1.891, 24.58) = 0.89 p = .4189	F(3.016, 38.11) = 2.76 p = .0549	F(1, 11) = 0.88 p = .3679	F(3.018, 33.20) = 2.59 p = .0692
AW 0–30 min
Proportion of time	F(1, 38) = 43.46 p < .0001	F(2.116, 26.80) = 15.37 p < .0001	—	t(11) = 5.20 p = .0003	—
Bout frequency	F(1, 38) = 15.64 p = .0003	F(2.437, 30.87) = 6.52 p = .0027	—	t(11) = 1.71 p = .1148	—
Bout duration	F(1, 38) = 15.49 p = .0003	F(1.259, 15.95) = 5.56 p = .0253	—	t(11) = 1.49 p = .1653	—
AW 30–180 min
Proportion of time	F(1, 38) = 44.09 p < .0001	F(2.313, 29.30) = 15.25 p < .0001	—	t(11) = 7.45 p < .0001	—
Bout frequency	F(1, 38) = 35.75 p < .0001	F(2.513, 31.83) = 12.86 p < .0001	—	t(11) = 9.96 p < .0001	—
Bout duration	F(1, 38) = 5.83 p = .0207	F(1.836, 23.26) = 2.41 p = .1154	—	t(11) = 1.40 p = .1892	—
Quiet wake
Proportion of time	F(1, 38) = 22.23 p < .0001	F(2.451, 31.87) = 7.55 p = .0011	F(7.515, 94.96) = 8.06 p < .0001	F(1, 11) = 14.31 p = .0030	F(4.655, 51.21) = 2.79 p = .0293
Bout frequency	F(1, 38) = 12.11 p = .0013	F(2.372, 30.84) = 1.98 p = .1491	F(8.618, 108.9) = 2.41 p = .0172	F(1, 11) = 33.24 p = .0001	F(5.113, 56.24) = 2.67 p = .0303
Bout duration	F(1, 38) = 9.02 p = .0047	F(2.254, 29.30) = 3.79 p = .0301	F(7.566, 95.61) = 3.88 p = .0007	F(1, 11) = 0.75 p = .4057	F(4.970, 54.67) = 1.85 p = .1192
QW 0–90 min
Proportion of time	F(1, 38) = 116.40 p < .0001	F(2.237, 28.34) = 39.22 p < .0001	—	t(11) = 2.66 p = .0221	—
Bout frequency	F(1, 38) = 41.00 p < .0001	F(2.210, 27.99) = 14.41 p < .0001	—	t(11) = 4.21 p = .0015	—
Bout duration	F(1, 38) = 49.79 p < .0001	F(2.484, 31.46) = 18.47 p < .0001	—	t(11) = 1.57 p = .1460	—
NREM sleep
Sleep onset latency	F(1, 38) < 0.01 p = .925	F(2.107, 26.69) = 0.06 p = .945	—	t(11) = 4.57 p = .0008	—
Proportion of time	F(1, 38) = 11.90 p = .0014	F(2.414, 31.38) = 1.82 p = .1721	F(8.038, 101.6) = 3.23 p = .0026	F(1, 11) = 56.21 p < .0001	F(5.327, 58.60) = 6.80 p < .0001
Bout frequency	F(1, 38) = 0.76 p = .3877	F(1.921, 24.97) = 0.50 p = .6073	F(8.652, 109.3) = 1.66 p = .1111	F(1, 11) = 3.30 p = .0967	F(4.948, 54.43) = 3.44 p = .0092
Bout duration	F(1, 38) = 0.40 p = .5305	F(2.286, 29.72) = 0.29 p = .7822	F(6.848, 86.54) = 1.46 p = .1955	F(1, 11) = 26.40 p = .0003	F(4.972, 54.69) = 4.51 p = .0017
NREM sleep 0–90 min
Proportion of time	F(1, 38) = 69.01 p < .0001	F(2.442, 30.93) = 23.61 p < .0001	—	t(11) = 8.50 p < .0001	—
Bout frequency	F(1, 38) = 13.74 p = .0007	F(1.758, 22.26) = 5.33 p = .0156	—	t(11) = 5.15 p = .0003	—
Bout duration	F(1, 38) = 9.72 p = .0035	F(2.057, 26.06) = 3.70 p = .0374	—	t(11) = 6.34 p < .0001	—
REM sleep
REM sleep onset latency	F(1, 51) = 105.70 p < .0001	F(1.898, 32.26) = 35.72 p < .0001	—	t(11) = 7.55 p < .0001	—
Proportion of time	F(1, 38) = 97.25 p < .0001	F(2.300, 29.90) = 13.83 p < .0001	F(8.829, 111.6) = 5.08 p < .0001	F(1, 11) = 63.97 p < .0001	F(4.618, 50.80) = 2.93 p = .0238
Bout frequency	F(1, 38) = 36.64 p < .0001	F(2.234, 29.04) = 9.53 p = .0005	F(9.211, 116.4) = 3.39 p = .0009	F(1, 11) = 20.45 p = .0009	F(4.223, 46.46) = 3.32 p = .0165
Bout duration	F(1, 38) = 29.56 p < .0001	F(2.053, 26.69) = 9.87 p = .0006	F(7.762, 98.09) = 5.01 p < .0001	F(1, 11) = 47.37 p < .0001	F(5.539, 60.93) = 2.959 p = .0155
REM sleep 30–180 min
Proportion of time	F(1, 38) = 247.90 p < .0001	F(2.371, 30.03) = 83.28 p < .0001	—	t(11) = 8.30 p < .0001	—
Bout frequency	F(1, 38) = 169.30 p < .0001	F(2.335, 29.58) = 57.23 p < .0001	—	t(11) = 5.46 p = .0002	—
Bout duration	F(1, 38) = 174.50 p < .0001	F(2.200, 27.86) = 58.70 p < .0001	—	t(11) = 7.90 p < .0001	—

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There were no significant differences in the proportion of female rats in different estrus phases between dose conditions (see Supplementary Tables S3 and S4). Additionally, there were no significant main effects of dose across %sleep–wake state outcomes at baseline (see Supplementary Tables S5 and S6).

Wake outcomes

The effects of i.p. oxytocin (0, 0.1, 0.3, and 1 mg·kg⁻¹) and caffeine (0, 10 mg·kg⁻¹) on wake outcomes are displayed in Figure 2 and corresponding statistical outcomes are reported in Tables 1 and Supplementary Table S2.

Active wake.

For %AW, over the 360 minutes post-dose period, there was an oxytocin Dose × Time interaction (Figure 2A_i). During 0–30 minutes, oxytocin dose-dependently reduced %AW (Figure 2A_ii); this effect was produced through a dose-dependent reduction in mean AW bout duration, which was, interestingly, accompanied by a dose-dependent increase in bouts of AW (Figure 2A_iii). In contrast, during 30–180 minutes, oxytocin dose-dependently increased %AW (Figure 2B_ii) due to a dose-dependent increase in AW bouts and mean AW bout duration (Figure 2B_iii).

Overall, caffeine (i.p.; 10 mg·kg⁻¹) increased %AW and there was a Dose × Time interaction (Figure 2B_i). During 0–30 minutes, caffeine increased %AW (Figure 2A_iv); however, it was unclear whether this was due to increased bouts of AW or increased mean AW bout duration (Figure 2A_v). During 30–180 minutes, caffeine also increased %AW (Figure 2B_iv), through increased AW bouts but not mean AW bout duration (Figure 2B_v).

During the 0–30 minutes post-dose time window, relative to vehicle, 1 mg·kg⁻¹ oxytocin reduced ECoG PSD in the alpha frequency band, while caffeine reduced power in the delta, sigma, and beta bands and increased power in alpha band (Figure 2A_vi). From 30 to 180 minutes post-dose, no effect of oxytocin on ECoG PSD was observed at any dose at any frequency band, whereas caffeine reduced ECoG PSD within the delta, sigma, and beta frequency bands and increased power in alpha band, relative to vehicle (Figure 2B_vi).

Quiet wake.

Overall, oxytocin impacted %QW and this changed over time during the sleep recording (Figure 2C_i). During 0–90 minutes, oxytocin dose-dependently increased %QW (Figure 2C_ii) by increasing both QW bouts and mean QW bout duration (Figure 2C_iii). Overall, caffeine increased by %QW and this differed over the course of the sleep recording (Figure 2D_i). During 0–90 minutes, caffeine increased %QW (Figure 2C_iv), which was due to increased QW bouts but not mean QW bout duration (Figure 2C_v).

During the 0–90 minutes post-dose period, relative to vehicle, oxytocin reduced ECoG PSD in the theta (1 mg·kg⁻¹), alpha (0.3 and 1 mg·kg⁻¹), and sigma (1 mg·kg⁻¹) frequency bands, while caffeine reduced ECoG PSD across all frequency bands (Figure 2C_vi).

Sleep outcomes

NREM sleep.

Overall, for %NREMS, there was an oxytocin Dose × Time interaction (Figure 3A_i). During 0–90 min, oxytocin dose-dependently reduced %NREMS (Figure 3A_ii), due to both reduced bouts of NREMS and reduced mean NREMS bout duration (Figure 3A_iii). However, there was no effect of oxytocin on sleep onset latency (Figure 3A_iv). Oxytocin dose-dependently reduced the frequency of transitions from NREMS to REMS, but not to AW and QW states, and reduced the proportion of NREMS bouts terminating in REMS from 0 to 90 minutes (Supplementary Figure S2A–D). Likewise, from 90 to 180 minutes post-dose, oxytocin dose-dependently reduced the number of transitions from NREMS to REMS and reduced the proportion of NREMS bouts terminating in REMS, despite longer exerting effects on %NREMS, NREMS bout frequency, and mean duration of NREMS bouts (Supplementary Figure S2E-G).

Overall, caffeine (i.p.; 10 mg·kg⁻¹) reduced %NREMS, and this differed over time during the recording session (Figure 3B_i). During 0–90 minutes, caffeine greatly reduced %NREMS (Figure 3A_v), due to both reduced NREMS bouts and reduced mean NREMS bout duration (Figure 3A_vi). Additionally, caffeine increased sleep onset latency (Figure 3A_vii).

During the 0–90 minutes post-dose period, relative to vehicle, oxytocin increased ECoG PSD in the delta (0.3 mg·kg⁻¹) and theta (0.1 and 0.3 mg·kg⁻¹) frequency bands, while caffeine reduced ECoG PSD across the delta and alpha frequency bands (Figure 3B_ii). Note that while 1 mg·kg⁻¹ oxytocin did not appear to impact ECoG PSD outcomes during this period, analysis of an earlier period (i.e. 0–60 min) demonstrates that the highest dose of oxytocin reduced power across all frequency bands (Supplementary Figure S1). During the remaining recording period (90–360 minutes post-dose), oxytocin increased ECoG PSD in the delta (all doses), theta (all doses), alpha (0.3 and 1 mg·kg⁻¹), and beta (0.3 mg·kg⁻¹) frequency bands, whereas caffeine had no impact on ECoG PSD (Figure 3B_iii).

REM sleep.

Overall, oxytocin influenced %REMS, and this differed over time during the recording session (Figure 3C_i). During 30–180 minutes, oxytocin dose-dependently reduced %REMS (Figure 3C_ii), due to reductions in both REMS bouts and mean REMS bout duration (Figure 3C_iii). Additionally, oxytocin dose-dependently increased REMS onset latency (Figure 3C_iv).

Overall, caffeine reduced %REMS, and this effect varied over the course of the recording (Figure 3D_i). During 30–180 minutes, caffeine reduced %REMS (Figure 3C_v), due to both reduced REMS bouts and mean REMS bout duration of REM bouts (Figure 3C_vi). Furthermore, caffeine increased REMS onset latency (Figure 3C_vii).

During the 30–180 minutes post-dose period, relative to vehicle, oxytocin increased ECoG PSD across all frequency bands at all doses. In contrast, caffeine reduced ECoG PSD in the theta, alpha, sigma, and beta frequency bands (Figure 3D_ii).

Body temperature.

Administration of i.p. oxytocin dose-dependently reduced body temperature (Supplementary Figure S3A and B). In contrast, i.p. caffeine (10 mg·kg⁻¹) slightly elevated body temperature (Supplementary Figure S3C and D).

Experiment 2—influence of oxytocin receptor antagonism on oxytocin-induced effects on sleep–wake outcomes

No differences were observed in the proportion of rats in estrus phases between dose conditions and their respective controls (Supplementary Table S8). Summarized statistical results for experiment 2 are presented in Table 2 and Supplementary Table S7.

Table 2.

Influence of Oxytocin Receptor Antagonism on i.p. Oxytocin-Induced Effects on Sleep–Wake Outcomes

Sleep–wake outcome	Main effect of dose	Main effect of antagonism	Dose × antagonism interaction
Active wake (0–30 min)
Proportion of time	F(1, 3) = 2.12 p = .2414	F(1, 3) = 7.40 p = .0725	F(1, 3) = 1.02 p = .3870
Bout frequency	F(1, 3) = 9.56 p = .0536	F(1, 3) = 17.70 p = .0245	F(1, 3) = 13.65 p = .0344
Bout duration	F(1, 3) = 1.17 p = .3594	F(1, 3) = 0.37 p = .5884	F(1, 3) = 1.12 p = .3677
Active wake (30–180 min)
Proportion of time	F(1, 3) = 5.27 p = .1055	F(1, 3) = 0.01 p = .9207	F(1, 3) = 10.89 p = .0457
Bout frequency	F(1, 3) = 3.28 p = .1680	F(1, 3) = 0.45 p = .5504	F(1, 3) = 13.11 p = .0362
Bout duration	F(1, 12) = 0.14 p = .7148	F(1, 12) = 2.91 p = .1139	F(1, 12) = 2.20 p = .1641
Quiet wake (0–90 min)
Proportion of time	F(1, 3) = 39.46 p = .0081	F(1, 3) = 3.40 p = .1624	F(1, 3) = 18.30 p = .0235
Bout frequency	F(1, 3) = 31.99 p = .0109	F(1, 3) = 0.46 p = .5467	F(1, 3) = 14.68 p = .0313
Bout duration	F(1, 3) = 9.79 p = .0521	F(1, 3) = 2.19 p = .2358	F(1, 3) = 6.47 p = .0844
NREM sleep (0–60 min)
Sleep onset latency	F(1, 12) = 1.60 p = .2295	F(1, 12) = 1.88 p = .1951	F(1, 12) = 0.26 p = .6177
Proportion of time	F(1, 12) = 44.01 p = .0001	F(1, 12) = 14.56 p = .0025	F(1, 12) = 7.10 p = .0206
Bout frequency	F(1, 3) = 6.33 p = .0864	F(1, 3) = 0.67 p = .4726	F(1, 3) = 0.02 p = .903
Bout duration	F(1, 3) = 22.47 p = .0178	F(1, 3) = 4.12 p = .1355	F(1, 3) = 1.51 p = .3064
REM sleep (30–180 min)
REM sleep onset latency	F(1, 3) = 23.95 p = .0163	F(1, 3) = 19.44 p = .0216	F(1, 3) = 72.76 p = .0034
Proportion of time	F(1, 3) = 28.91 p = .0126	F(1, 3) = 0.16 p = .7168	F(1, 3) = 9.95 p = .0511
Bout frequency	F(1, 3) = 32.93 p = .0105	F(1, 3) = 1.63 p = .291	F(1, 3) = 13.05 p = .0364
Bout duration	F(1, 3) = 20.69 p = .0199	F(1, 3) = 0.67 p = .4745	F(1, 3) = 7.17 p = .0751

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Active wake.

From 0 to 30 minutes, pre-administration of the selective oxytocin receptor antagonist L-368,899 (i.p.; 5 mg·kg⁻¹) inhibited the oxytocin-induced (i.p.; 1 mg·kg⁻¹) increase in bouts of AW (Figure 4A_ii) but not in %AW (Figure 4A_i) or mean duration of AW bouts (Figure 4A_iii). Administration of L-368,899 alone significantly reduced AW bouts during this time window [t(3) = 3.628, p = .036]; however no effects were observed for any other AW outcomes (all p > .05). During the 0–30 minutes post-dose period, relative to vehicle, oxytocin increased ECoG PSD in the delta frequency band, and this effect was not observed when L-368,899 was administered before oxytocin or when L-368,899 was administered alone (Figure 4A_iv).

Figure 4. — Influence of OXTR antagonism on oxytocin-induced effects on sleep–wake outcomes. Influence of pre-administration of the OXTR antagonist L-368 899 (i.p.; 5 mg·kg⁻¹) on oxytocin-induced (i.p.; 1 mg·kg⁻¹) effects on: %AW (A_i), AW bouts (A_ii), mean duration of AW bouts (A_iii), and ECoG PSD during AW (A_iv) during first period of AW peak effect (0–30 minutes); %AW (A_v), AW bouts (A_vi), mean duration of AW bouts (A_vii), and ECoG PSD during AW (A_viii) during the second period of AW peak effect (30–180 minutes); %QW (B_i), QW bouts (B_ii), mean duration of QW bouts (B_iii), and ECoG PSD during QW (B_iv) during the period of QW peak effects (0–90 minutes); %NREMS (C_i), NREMS bouts (C_ii), mean duration of NREMS bouts (C_iii), sleep onset latency (C_iv), and ECoG PSD during NREMS (C_v) during the period of NREM peak effects (0–60 minutes); %REMS (D_i), REMS bouts (D_ii), mean duration of REMS bouts (D_iii), REMS onset latency (D_iv), and ECoG PSD (D_v) during period of peak REMS effect (30–180 minutes). Sample size for all dose conditions is n = 4 females. Data represent mean values ± S.E.M., individual data points represent individual subject data (closed circles—females). For A_iii, A_vii, B_iii, C_iii, and D_iii, data points represent individual subject data with lines connecting same subject data. OXTR antagonist was administered at ZT23.75 and oxytocin was administered at ZT0. For ECoG PSD figures, data represent mean percentage change in ECoG PSD (0.1–25 Hz) ± S.E.M. Statistical significance is indicated by the following symbols: * – dose × antagonist interaction effect; # – VEH-oxytocin versus VEH-VEH (baseline) comparison. For graphs with two y-axes, the alignment of the significance symbol represents which outcome the significance refers to: left—bout frequency; right—mean bout duration. Level of statistical significance is indicated by the number of symbols: one—p < .05, two—p < .01, three—p < .001, four—p < .0001.

From 30 to 180 minutes, pre-administration of L-368,899 attenuated the oxytocin-induced increase in %AW and bouts of AW (Figures 4A_v and A_vi) but not mean duration of AW bouts (Figure 4A_vii). During this period, no significant effect of any treatment condition on ECoG PSD was detected at any frequency band (Figure 4A_viii), and no effects of L-368,899 alone were detected for any AW outcomes (all p > .05).

Quiet wake.

Pre-administration of L-368,899 prevented the oxytocin-induced increase in %QW (Figure 4B_i) and QW bout frequency (Figure 4B_ii), but did not reach significance for mean QW bout duration (Figure 4B_iii). No significant effects of any treatment condition on ECoG PSD were detected at any frequency band (Figure 4B_iv), and no effect of L-368,899 alone was detected for any QW outcomes (all p > .05).

NREM sleep.

Pre-administration of L-368,899 antagonized the oxytocin-induced reduction in %NREMS (Figure 4C_i); effects of antagonism on NREMS bout frequency, mean duration of NREMS bouts, and sleep onset latency were not clear (Figures 4C_ii, C_iii, and C_iv). Relative to vehicle, oxytocin reduced ECoG PSD during NREMS across all frequency bands, and these changes were not observed when L-368,899 was administered before oxytocin or when L-368,899 was administered alone (Figure 4C_v). No effects of L-368,899 alone were detected for any NREMS outcomes (all p > .05).

REM sleep.

Pre-administration of L-368,899 antagonized the oxytocin-induced reduction in REMS bout frequency (Figure 4D_ii) and increase in REMS onset latency (Figure 4D_iv), but did not reach significance for %REM sleep (Figure 4D_i, p = .051) or mean REMS bout duration (Figure 4D_iii). Relative to vehicle, while oxytocin significantly increased ECoG PSD within the delta frequency band, this effect was not observed when L-368,899 was administered before oxytocin or when L-368,899 was administered alone (Figure 4D_v). No effect of L-368,899 alone was detected for any REMS outcomes (all p > .05).

Body temperature.

Administration of i.p. oxytocin (1 mg·kg⁻¹) reduced body temperature (Supplementary Figure S4). Notably, pre-administration of L-368,899 attenuated this oxytocin-induced reduction in body temperature (see Supplementary Material for details). No effect of L-368,899 alone on body temperature was detected (p > .05)

Experiment 3—effects of i.n. oxytocin and i.n. caffeine on sleep–wake outcomes

There were no differences in the proportion of rats in different estrus phases between oxytocin dose conditions and VEH. However, the proportion of rats in different estrus phases significantly differed between caffeine and VEH (see Supplementary Tables S11 and S12). No main effects of dose across %sleep–wake state outcomes at baseline were observed (see Supplementary Table S13 and S14). Summarized statistical results for experiments 3a and 3b are presented in Tables 3 and Supplementary Table S10, respectively.

Table 3.

Effects of i.n. Oxytocin Dose Range and i.n. Caffeine Dose on Sleep–Wake Outcomes

Wake outcome	Oxytocin dose range (0, 0.06, 1 mg·kg⁻¹)			Pairwise comparison
Wake outcome	Linear trend	Main effect of dose	Dose × Time interaction	VEH vs. 3 mg·kg⁻¹ OXT	VEH vs. 10 mg·kg⁻¹ caffeine
Active wake
Proportion of time	F(1, 10) = 0.13 p = .7248	F(1.971, 9.857) = 0.15 p = .8573	F(3.809, 19.04) = 0.80 p = .5376	t(3) = .083 p = .9393	t(4) = 6.36 p = .0031
Bout frequency	F(1, 10) < 0.01 p = .9486	F(1.931, 9.657) = 0.01 p = .9867	F(3.425, 17.13) = 0.75 p = .5551	t(3) = 1.60 p = .2086	t(4) = 5.16 p = .0067
Bout duration	F(1, 10) = 0.84 p = .3821	F(1.168, 5.842) = 0.71 p = .4537	F(1.511, 7.553) = 0.97 p = .3967	t(3) = 1.69 p = .1892	t(4) = 3.28 p = .030
AW 0–30 min
Proportion of time	F(1, 10) = 1.47 p = .2534	F(1.630, 8.151) = 0.74 p = .482	—	t(3) = .44 p = .6927	t(4) = 5.42 p = .0056
Bout frequency	F(1, 10) = 1.33 p = .2756	F(1.738, 8.689) = 0.76 p = .4774	—	t(3) = .78 p = .4904	t(4) = 2.28 p = .0845
Bout duration	F(1, 10) = 1.65 p = .2278	F(1.032, 5.162) = 1.02 p = .3611	—	t(4) = .36 p = .7415	t(4) = .91 p = .4146
AW 30–180 min
Proportion of time	F(1, 15) < 0.01 p = .9635	F(1.990, 14.93) = 0.01 p = .9945	—	t(3) = .01 p = .993	t(4) = 3.68 p = .0213
Bout frequency	F(1, 15) = 0.16 p = .6918	F(1.664, 12.48) = 0.15 p = .8248	—	t(3) = .35 p = .7479	t(4) = 3.49 p = .0252
Bout duration	F(1, 10) < 0.01 p = .9934	F(1.588, 7.938) = 0.07 p = .8966	—	t(3) = 1.48 p = .235	t(4) = 1.27 p = .2735
Quiet wake
Proportion of time	F(1, 10) = 0.22 p = .6489	F(1.891, 9.454) = 0.12 p = .8763	F(3.961, 19.81) = 1.35 p = .2885	t(3) = .45 p = .6846	t(4) = 1.876 p = .1339
Bout frequency	F(1, 10) = 1.91 p = .1972	F(1.807, 9.035) = 1.92 p = .203	F(3.433, 17.17) = 0.69 p = .5901	t(3) = .09773 p = .9283	t(4) = 4.519 p = .0107
Bout duration	F(1, 10) = 0.08 p = .7816	F(1.653, 8.263) = 0.23 p = .7619	F(3.870, 19.35) = 1.54 p = .2302	t(3) = .6057 p = .5875	t(4) = .1051 p = .9214
QW 0–90 min
Proportion of time	F(1, 10) = 9.33 p = .0121	F(1.645, 8.223) = 4.67 p = .0491	—	t(3) = .42 p = .7011	t(4) = 2.12 p = .1014
Bout frequency	F(1, 15) = 5.35 p = .0354	F(1.703, 12.77) = 2.82 p = .1026	—	t(3) = .25 p = .8219	t(4) = 2.78 p = .0497
Bout duration	F(1, 10) = 2.00 p = .1878	F(1.766, 8.832) = 1.64 p = .2465	—	t(3) = 1.48 p = .235	t(4) = 1.27 p = .2735
NREM sleep
Sleep onset latency	F(1, 10) = 0.03 p = .8758	F(1.793, 8.966) = 0.04 p = .9452	—	t(3) = 0.99 p = .396	t(4) = 1.92 p = .1267
Proportion of time	F(1, 10) = 1.48 p = .2515	F(1.517, 7.583) = 0.98 p = .3923	F(3.593, 17.97) = 0.52 p = .7043	t(3) = .43 p = .6955	t(4) = 7.03 p = .0022
Bout frequency	F(1, 10) = 0.15 p = .7072	F(1.184, 5.920) = 0.10 p = .8038	F(4.092, 20.46) = 0.57 p = .6905	t(3) = .63 p = .5728	t(4) = 2.54 p = .064
Bout duration	F(1, 10) = 0.46 p = .5145	F(1.434, 7.168) = 0.24 p = .7253	F(3.462, 17.31) = 0.68 p = .5966	t(3) = .21 p = .8473	t(4) = 6.46 p = .003
NREM sleep 0–90 min
Proportion of time	F(1, 10) = 1.34 p = .2735	F(1.857, 9.285) = 0.76 p = .4839	—	t(3) = .08 p = .9378	t(4) = 4.36 p = .012
Bout frequency	F(1, 15) = 0.31 p = .5875	F(1.979, 14.84) = 0.17 p = .8446	—	t(3) = .65 p = .5595	t(4) = 1.74 p = .1571
Bout duration	F(1, 10) = 0.28 p = .6059	F(1.810, 9.051) = 0.21 p = .7918	—	t(3) = .17 p = .8740	t(4) = 7.13 p = .0020
REM sleep
REM sleep onset latency	F(1, 15) = 0.03 p = .8747	F(1.445, 10.84) = 1.60 p = .2416	—	t(3) = .75 p = .5087	t(4) = 2.15 p = .0975
Proportion of time	F(1, 10) = 1.79 p = .2111	F(1.289, 6.446) = 1.05 p = .3657	F(4.114, 20.57) = 1.36 p = .2815	t(3) = .6323 p = .5721	t(4) = 5.57 p = .0051
Bout frequency	F(1, 10) = 0.48 p = .5054	F(1.292, 6.460) = 0.190 p = .7366	F(4.094, 20.47) = 0.43 p = .7925	t(3) = .89 p = .4409	t(4) = 2.49 p = .0674
Bout duration	F(1, 10) = 3.14 p = .1067	F(1.265, 6.325) = 2.21 p = .1876	F(3.901, 19.51) = 1.30 p = .3060	t(3) = 0.75 p = .5054	t(4) = 4.28 p = .0128
REM sleep 30–180 min
Proportion of time	F(1, 15) = 0.64 p = .4375	F(1.767, 13.25) = 0.58 p = .5534	—	t(3) = .23 p = .834	t(4) = 3.89 p = .0176
Bout frequency	F(1, 10) = 1.54 p = .2432	F(1.726, 8.631) = 0.77 p = .4745	—	t(3) = .93 p = .4208	t(4) = 2.91 p = .0436
Bout duration	F(1, 15) = 0.36 p = .5555	F(1.671, 12.53) = 0.33 p = .6863	—	t(3) = .63 p = .5723	t(4) = 3.25 p = .0315

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Wake outcomes

Active wake.

There were no significant effects of i.n. oxytocin (at any dose) on any AW outcome (Figure 5A_i-vi and B_i-vi). Averaged across the 6-hour recording, caffeine (i.n., 10 mg·kg⁻¹) increased %AW (Figure 5B_i). During 0–30 minutes, caffeine increased %AW (Figure 5A_iv), but effects on AW bouts, mean AW bout duration, and ECoG PSD during AW were not statistically significant (Figure 5A_v-vi). From 30 to 180 minutes, caffeine increased %AW (Figure 5B_iv) and AW bouts (Figure 5B_v), but did not increase mean AW bout duration. During this time window, relative to vehicle, caffeine reduced ECoG PSD within the beta frequency band (Figure 5B_vi).

Quiet wake.

Averaged across the entire recording, there was no significant effect of oxytocin at any dose on %QW (Figure 5C_i and D_i). From 0 to 90 minutes post-dose, oxytocin significantly—albeit slightly—increased %QW in a dose-dependent manner across 0, 0.06, and 1 mg·kg⁻¹ doses (Figure 5C_ii), due to dose-dependent increases in QW bouts rather than QW bout duration (Figure 5C_iii). However, this increase in %QW and QW bouts was not found for the higher 3 mg·kg⁻¹ oxytocin dose (Figure 5C_iv and C_v). Additionally, no effects of oxytocin on ECoG PSD during QW were found at any dose within this 0–90 minutes time window (Figure 5D_vi).

Averaged across the entire session, no significant effect of caffeine on %QW was observed (Figure 5D_i). However, from 0 to 90 minutes, caffeine increased the number of QW bouts but not %QW or mean QW bout duration (Figures 5C_iv and C_v). During this time window, relative to vehicle, caffeine reduced ECoG PSD within the sigma and beta frequency bands (Figure 5B_vi).

Sleep outcomes

NREM sleep.

During the 0–90 minutes post-dose period, relative to vehicle, i.n. oxytocin (3 mg·kg⁻¹) reduced ECoG PSD within the beta frequency band (Figure 6B_iii); however, no other significant effects of i.n. oxytocin (at any dose) on any NREMS outcomes were found (Figure 6A_i-vii and B_i-ii). Averaged across the 6-hour session, i.n. caffeine (10 mg·kg⁻¹) significantly reduced %NREMS (Figure 6B_i); the increase in sleep onset latency did not reach statistical significance (Figure 6A_vii). During 0–90 minutes, i.n. caffeine reduced %NREMS (Figure 6A_v) and reduced duration of NREMS bouts (Figure 6A_vi), but did not significantly reduce bout frequency. During the 0–90 minutes time window, relative to vehicle, caffeine appeared to reduce ECoG PSD within the delta frequency band but this did not reach statistical significance (p = .06, Figure 6B_ii). Note that while caffeine did not appear to impact ECoG PSD outcomes during this period, analysis of earlier periods (i.e. 0–60 minutes) demonstrates that i.n. caffeine reduced power in the delta frequency band (Supplementary Figure S5). No effect of caffeine on ECoG PSD was detected over the remaining 90–360 minutes recording period (Figure 6B_iii).

REM sleep.

No significant effects of i.n. oxytocin (at any dose) on any REMS outcomes were found (Figure 6C_i-vii and 6D_i-ii). Overall, i.n. caffeine (10 mg·kg⁻¹) significantly reduced %REM sleep (Figure 6D_i); however, the increase in REMS onset latency did not reach statistical significance (Figure 6C_vii). During 30–180 minutes, i.n. caffeine reduced %REM sleep (Figure 6C_v), due to both reduced REMS bout frequency and mean REMS bout duration (Figure 6C_vi). During the 30–180 minutes post-dose period, relative to vehicle, caffeine reduced ECoG PSD within the theta and beta frequency bands (Figure 6D_ii).

Body temperature.

Administration of i.n. oxytocin did not significantly affect body temperature at any dose. Likewise, no significant effect of i.n. caffeine on body temperature was found (Supplementary Figure S6).

Discussion

The current study explored the acute effects of exogenous oxytocin and caffeine, administered via two routes, on sleep–wake outcomes, and whether observed effects were mediated by the OXTR. As hypothesized, i.p. oxytocin promoted wakefulness and suppressed sleep in a dose-dependent manner in both female and male rats. I.p. oxytocin predominantly promoted a state of quiet wakefulness, initially reduced AW, suppressed NREMS and REMS, and delayed REMS onset. Relative to oxytocin, caffeine effects on AW and NREMS appeared more pronounced, on QW less pronounced, and on REMS of similar magnitude. I.p. oxytocin-induced sleep–wake effects were at least partially mediated by the OXTR. I.n. oxytocin failed to recapitulate most i.p. oxytocin-induced effects on sleep–wake behavior across the dose range tested, whereas i.n. and i.p. caffeine had similar effects. Oxytocin-induced sleep–wake effects, therefore, appear dependent on dose and route of administration, but not biological sex.

Sleep architectural and power spectral outcomes

I.p. oxytocin exerted clear dose-dependent effects on almost all sleep–wake outcomes. Immediately post-administration, oxytocin facilitated a dose-dependent exchange of AW for QW, without impacting sleep; in contrast, caffeine increased AW primarily at the cost of NREMS. This oxytocin-induced promotion of QW at the cost of AW is driven by a combination of longer and more frequent bouts of QW, and AW fragmentation—more frequent but shorter AW bouts reflective of disrupted activity. Although the dose-dependent increase in AW during the 30–180 minutes period may appear to contradict oxytocin’s primary effect of elevating QW from 0 to 90 minutes, examination of the AW time course (Figure 1A_i) reveals that this reflects a dose-dependent delay in the natural progression of reduced AW across the time course (as observed in the VEH condition). When averaged across oxytocin dose, AW decreases from 30 to 180 minutes (Figure 1B_ii: 8%–22% of time spent in AW) relative to the 0–30 minutes period (Figure 1A_ii: 30%–60% of time spent in AW). Hence, the observed dose-dependent increase in AW from 30 to 180 minutes is neither a rebound effect—no increase beyond the prior 0–30 minutes timepoint—nor a delayed effect of oxytocin—since no change in effect direction from the initial reduction is evident within each dose condition.

In comparison to AW, QW increased from baseline and relative to VEH in an oxytocin-induced dose-dependent manner, acted only in one direction (i.e. increased) during the entire time course, and returned to baseline levels after the 90-minute post-dose period. From 30 to 90 minutes, oxytocin maintained substantial elevations in QW, primarily at the cost of NREMS and REMS sleep. During QW and NREMS within the 0–90 minutes wake-dominant period, both caffeine and oxytocin (1 mg·kg⁻¹) reduced ECoG PSD across frequency bands (Figures 2C_vi and Supplementary Figure S1) reflecting reduced homeostatic sleep pressure [26, 43, 44]. Notably, no rebound in NREMS was observed following this initial sleep loss (Figures 3A_i and Supplementary Figure S2E–G), despite sleep assessment being conducted from ZT0-6, a period when rats should experience relatively higher circadian and homeostatic drive for sleep under normal conditions [45]. However, whether this rebound-free NREM sleep disturbance would occur in cases of markedly elevated homeostatic sleep drive (e.g. following 6 hours of sleep deprivation [46]) is uncertain and future research should investigate the interplay between oxytocin-induced effects and homeostatic sleep pressure.

Although oxytocin-induced NREMS suppression abated by approximately 90 minutes post-administration with no observed rebound sleep, ECoG PSD in lower frequency bands (particularly delta and theta) remained elevated for the remaining 4.5 hours of sleep recording. This aligns with Lancel, Krömer, and Neumann [20] who found similar effects of i.c.v. oxytocin on EEG PSD. Notably, this elevation in lower frequency PSD occurred for all i.p. oxytocin doses tested and likely reflects increased sleep pressure [47], sleep intensity [39], and sleep propensity [43], and may also indicate improved sleep quality [48]. While a brief elevation following oxytocin-induced NREMS suppression might suggest a transient rebound in sleep pressure, the protracted nature of this effect is unlikely due to homeostatic sleep drive. Tobler and Borbély [46] demonstrated that 3-hour total sleep deprivation in rats—a relatively longer, more potent sleep deprivation than the current study—had no significant impact on EEG PSD within the delta frequency (0.75–4.5 Hz) band during NREMS. Hence, oxytocin may induce persisting increases in sleep intensity during NREMS, independent of initial NREMS suppression. Caffeine reduced sleep pressure during NREMS, as expected [26].

The most prolonged behavioral effect of i.p. oxytocin was the suppression of REMS. Since NREMS typically precedes REMS during normal sleep architecture [37], oxytocin-induced disruption to NREMS would be expected to impact REMS. However, while caffeine delayed both sleep- and REMS-onset latency, and effects on NREMS and REMS were mostly temporally co-occurring, oxytocin only impacted REMS-onset latency, and REMS effects endured considerably beyond NREMS disruptions. Since oxytocin dose-dependently reduced the proportion of NREMS bouts terminating in REMS during the period when REMS suppression was present while NREMS suppression had ceased (Supplementary Figure S2), this suggests that oxytocin can dose-dependently suppress transitions from NREMS to REMS, independent of disruptions to NREMS. Additionally, oxytocin markedly increased ECoG PSD across all frequency bands during this 30–180 minutes period involving considerable REMS disruption. This elevation of theta power during REMS could suggest improved facilitation of emotional memory consolidation [49, 50]. However, as temporally cooccurring elevations in PSD across higher frequency power bands—indicative of cortical hyperarousal [51–53]—were also observed (Figure 3D_ii), increased theta power is likely a consequence of REMS deprivation and compensatory REM sleep drive [54, 55].

A small impact of biological sex on sleep–wake outcomes were observed: on average, female rats demonstrated greater %QW and %REMS and lower %NREMS than male rats. This is in contrast to some previous studies which found that gonadally intact female rodents typically spend less time in sleep states than males [56], and female rats typically experience less %REMS than males [57, 58]. It has been suggested that these effects are primarily driven by comparisons between males and females during the proestrus phase [59]. In the present study, male and female rats were tested as separate cohorts due to limitations with space and equipment availability within the facility, so it remains possible this apparent sex difference reflects a cohort effect, and given the misalignment with previous findings, the effects of sex on %REMS and %NREMS should be interpreted with caution.

Body temperature

Oxytocin exerted dose-dependent hypothermic effects (nadir of ~1.5°C reduction at 1 mg·kg⁻¹, Supplementary Figure S2) within the 0–120 minutes period, replicating previous research demonstrating i.p. oxytocin-induced hypothermia in rats at the same dose [18]. However, in contrast to prior research, the current study indicates that oxytocin-induced hypothermia was mediated by the OXTR, rather than the V1aR [18]. As this time window coincides with most sleep–wake effects, several plausible explanations may account for the relationship between sleep–wake and temperature effects: (1) oxytocin impacts sleep–wake behavior and temperature independently, (2) oxytocin alters temperature and consequently impacts sleep–wake behavior, or (3) oxytocin alters sleep–wake behavior and consequently impacts temperature.

Under normal conditions, onset of NREMS coincides with a rapid reduction in body temperature, which is maintained during continued NREM sleep and reversed upon transition back to wakefulness [60–62]. This NREM sleep-evoked hypothermia is driven by reduced sympathetic activity that facilitates peripheral vasodilation and consequent heat loss, alongside diminished heat production from metabolic and activity-induced thermogenesis [60, 63, 64]. During the sleep initiation period, for both humans and rats, sleep onset is most likely to occur during the steepest drop in body temperature [62, 63], suggesting that body cooling and NREM sleep are linked. Since i.p. oxytocin reduced body temperature in the current study, which is typically associated with improved NREMS induction [63], it seems unlikely that the observed oxytocin-induced wake-promotion is due to hypothermia. Moreover, as oxytocin can cause vasodilation of small, peripheral arteries [65, 66] and consequent distal cooling, if sleep–wake effects were temperature-mediated, oxytocin should have promoted sleep, not wakefulness. However, as the magnitude of i.p. oxytocin-induced hypothermia was greater than normal sleep-related reductions [62], and since cold exposure can promote wakefulness and inhibit sleep [67], it is possible that oxytocin-induced hypothermia contributed to sleep suppression (particular REMS which is highly sensitive to fluctuations in temperature [68]).

The effect of oxytocin on body temperature (i.e. hypothermia) did not align with its effects on sleep—promoting quiet wakefulness—as this would be expected to be associated with increased temperature compared to the vehicle control condition that are primarily sleeping [62]. Hence, it is unlikely that oxytocin-induced hypothermia was due to differences in the time spent in different sleep–wake states. In contrast, the effects of caffeine on body temperature (i.e. mild hyperthermia) did align with its effects on sleep–wake behavior (i.e. promoting active wakefulness) and replicate previous research observing mild hyperthermia following administration of a similar dose of caffeine (i.p., 12.5 mg·kg⁻¹) to rats [69]. While the current study does not elucidate whether this caffeine-induced increase in temperature is pharmacological or an indirect consequence of increased activity, the magnitude of the increase (~0.5°C, Supplementary Figure S3) is possible to achieve with exercise-induced hyperthermia in rats [70]. Thus, it is likely that caffeine-induced hyperthermia was an indirect consequence of the increased time spent in active wakefulness.

Quiet wakefulness and mechanisms

Oxytocin-induced promotion of quiet wakefulness incorporates aspects of both competing hypotheses that motivated this study: enhanced environmental awareness (wake-promotion) and stress attenuation-quiescence (sleep-promotion). The observed quiet wakefulness state consisted of rats sitting or lying stretched out with their eyes open alongside minimal locomotion and physical activity (i.e. no eating, drinking, or grooming). This behavioral quiescence and adoption of sleep postures represent normal sleep preparatory behaviors and—under normal conditions—typically represent a brief transitional state preceding sleep onset [71]. This result aligns with Lancel, Krömer, and Neumann [20], who found central administration of oxytocin in rats promoted wakefulness at the cost of sleep. However, that study did not delineate between active and quiet wakefulness. The current findings may also align with research by Mahalati, Okanoya, Witt, and Carter [22], who found i.c.v. oxytocin promoted sleep-like postures in prairie voles. While this was interpreted as oxytocin-induced sleep-promotion, the lack of polysomnographic assessment and reliance on postural observation would have rendered quiet wakefulness difficult to distinguish from NREMS.

Within the preclinical literature concerning the QW state, various titles are used (i.e. resting wake, awake quiescence, and attentive wake) and defining criteria are typically vague, differing within and between species. However, most definitions converge on the fundamental criterion of “relaxed” wakefulness with no locomotion [72–75]. Recently, studies using PSG-based definitions demonstrated important neurobiological differences between active and quiet wakefulness: beta activity (15–35 Hz)—during QW but not AW—couples with slow-wave activity to reveal homeostatic sleep drive [76]. Furthermore, Moriya, Kanamaru, and Okuma et al. [75] showed that both QW and NREM sleep share similar patterns of acetylcholine and glutamate signaling in the hippocampus, and that experimental interventions can independently impact active wakefulness without influencing quiet wakefulness.

Previous studies have implicated the pontine parabrachial nucleus (PBn) in quiet wakefulness. Qiu, Chen, Fuller, and Lu [77] demonstrated that chemogenetic activation of parabrachial neurons elicited a “behaviorally quiet” wakefulness and “alert but minimally active arousal” at the cost of NREMS and REMS during the light phase. This wake-promoting effect was mediated via parabrachial projections to the lateral hypothalamus (LH) and basal forebrain (BF). Notably, although these authors do not use the term QW, when compared to spontaneous wake, representative traces of the “quiet” state indicate lower EMG amplitude and more regular EMG tone [77], consistent with our QW definition in the current study. More recently, Xu, Wang, and Dong et al. [78] found that chemogenetic and optogenetic activation of medial parabrachial neurons in rats induced an attentive wake state (i.e. wake with no locomotion, some head movement, and low EMG tone) via glutamatergic projections to the LH and BF. Since OXTRs are expressed by PBn neurons and as the PBn receives direct projections from PVN oxytocinergic neurons [79], it is possible that exogenous oxytocin induces quiet wakefulness through PBn-mediated mechanisms. While unspecific for quiet wakefulness, several other potential mechanisms for oxytocin-induced wakefulness exist. These include an oxytocin-orexin positive feedback interaction within the LH [21] and co-transmission of glutamate and corticotropin-releasing hormone from oxytocinergic neurons [2].

The OXTR antagonist L-368,899 blocked some, but not all sleep–wake effects. However, L-368,899 attenuated oxytocin-induced effects on at least one outcome from each sleep–wake state, which suggests that the OXTR mediates a broad-spectrum of oxytocin’s impact on sleep and wake behavior. Notably, this interpretation is limited by the small sample size (n = 4); it is possible that some analyses were statistically underpowered and that OXTR antagonism may have significantly blocked more (if not all) oxytocin-induced effects had the sample size been increased. Alternate explanations could be that a higher dose of the antagonist was necessary for complete blockade, (although the dose used in the current experiment has previously been sufficient to antagonize behavioral effects of i.p. oxytocin at the 1 mg·kg⁻¹ dose [29]) or that some effects are mediated by oxytocin’s actions at other targets.

Given that oxytocin demonstrates considerable affinity for the V1aR [80] and since some effects of exogenous oxytocin are V1aR-mediated [15, 19, 81, 82], it is also possible that oxytocin-induced activation of V1aRs may contribute to oxytocin’s effects on sleep–wake behavior. Additionally, chronic administration of vasopressin promotes wakefulness in rats [83], and activation (chemogenetic and optogenetic) of PVN vasopressinergic neurons can promote arousal and suppress both NREM and REM sleep via projections to lateral hypothalamic orexinergic neurons in mice [84]. The current study found that most i.p. oxytocin-induced sleep–wake effects were not V1aR-mediated through antagonism using the V1aR antagonist SR49059 (Supplementary Figure S7 and Tables S15 and S16). However, due to various experimental limitations (i.e. brain penetrance [85] and inconsistent findings with previous studies [18]), this conclusion should be interpreted with various caveats (see Supplementary Material for complete details). Thus, future studies should further explore the potential co-contribution of OXTR-V1aR activation in oxytocin’s sleep–wake effects.

It is also important to consider the potential for peripheral mediation of oxytocin-induced wakefulness effects. Although peripheral oxytocin administration can increase central oxytocin levels [33, 86], peripheral oxytocin also exerts some effects through stimulation of the ascending vagal afferent pathway [87–89] and vagal stimulation can promote wakefulness [90–92]. However, given previous work [20] found i.c.v. oxytocin also promotes wakefulness, central mediation of at least some of the observed effects of i.p. oxytocin in the present study seems likely. Nevertheless, future research might investigate the relative contribution of central and peripheral targets in oxytocin sleep–wake effects.

Oxytocin and route of administration—intraperitoneal versus intranasal

Exogenous oxytocin-induced sleep–wake effects were dependent on route of administration. At equivalent or higher doses, i.n. oxytocin failed to replicate i.p. oxytocin effects on AW, NREMS, and REMS outcomes, and only exerted a relatively small increase in QW compared to the large effect induced by i.p. oxytocin. Since equivalent doses of i.p. and i.n. caffeine exerted comparable effects, this route of administration effect should not be interpreted as an issue with the i.n. administration procedure. Rather, this finding may be due to poorer bioavailability and/or differential distribution of oxytocin to sleep-relevant central or peripheral targets with i.n. oxytocin administration [93].

There is equivocal evidence regarding both the differential bioavailability and distribution of i.n. administered oxytocin, compared to other routes. Compared to oxytocin administered via i.p. and/or intravenous (i.v.) routes, i.n. oxytocin administered at the same dose has been found to produce lower (1.5-fold lower [33], 20-fold lower [94]), or comparable [86] plasma oxytocin levels in rodents. Higher oxytocin levels tend to be observed in olfactory regions following i.n. administration [94, 95] and one study reported higher oxytocin levels in the amygdala and hippocampus with i.p. compared to i.n. oxytocin [33], although this was not observed in another study [86].

Hence, it is possible that in the current study, i.p. oxytocin reached functionally relevant concentrations in sleep–wake regulatory brain regions or peripheral targets and i.n. oxytocin did not. The comparable effects of i.n. and i.p. caffeine suggests similar distribution via both routes of administration and possible fundamental differences in pharmacokinetics between caffeine and oxytocin [96]. It could simply be that substantially higher doses of i.n. oxytocin are required. Clearly, more research is needed examining the differential bioavailability and distribution of oxytocin with different routes of administration.

Conclusions

The current study possessed several strengths in design and methodology: (1) the inclusion of caffeine as a wake-promoting positive control facilitated crucial informative comparisons to oxytocin, (2) both male and female rats were tested [97], (3) co-administration of oxytocin with a centrally penetrant OXTR antagonist facilitated determination of OXTR involvement, (4) i.n. administration was conducted without anesthetic to avoid known interference with sleep and circadian rhythms [98], and (5) wireless telemetry was used for PSG recording to avoid potential interference of tethering and restriction of movement with sleep−wake behavior [99].

Understanding the impact of oxytocin—both endogenous and exogenous—on sleep–wake behavior holds valuable implications for basic and applied research. Our research confirms exogenous oxytocin can induce wakefulness and that mixed preclinical evidence on oxytocin effects on sleep–wake outcomes is likely due to inter-study differences in dose and route of administration [2]. Given growing interest in the development of oxytocin systems targeting pharmacotherapeutics [100] and use of i.n. oxytocin in clinical trials for psychiatric disorders [101], this study also offers novel insight into potential therapeutic applications. Inducing quiet wakefulness could prove therapeutically valuable for disorders involving agitation and aggression (e.g. dementia) [102]. Future research should expand upon extant evidence of oxytocin’s serenic effects [19] by investigating whether oxytocin is capable of inducing quiet wakefulness in rats under stressful conditions such as following a cage change [103] or acute restraint [104]. Similarly, inducing wakefulness, potentially without undesirable side-effects experienced with current wakefulness-promoting medications [105], holds clinical value for disorders involving excessive daytime sleepiness (e.g. idiopathic hypersomnolence, OSA, Prader-Willi Syndrome, and narcolepsy) [106].

More broadly, while the present study focused on the effects of exogenous oxytocin, it highlights the potential for the endogenous oxytocin system to regulate sleep–wake behaviors. Mammalian sleep mostly occurs within a social context (i.e. co-sleeping) [107], which often includes affiliative behaviors that involve endogenous oxytocin signaling [108, 109]. Thus, understanding the role of endogenous oxytocin in sleep–wake behavior may elucidate how co-sleeping impacts sleep quality, both objectively and subjectively [107], and how alterations to endogenous oxytocin may impact sleep.

The current study reconciles the apparent contradictory environmental awareness-arousal (wake-promotion) and stress attenuation-quiescence (sleep-promotion) hypotheses of oxytocin-induced influences on sleep–wake behavior. Exogenous oxytocin acutely promotes quiet wakefulness, a state of restfulness, and sustained arousal to support environmental awareness, at the cost of active wakefulness, NREMS, and REMS. Importantly, these effects are dependent on dose and route of administration, but not biological sex.

Supplementary Material

zsad112_suppl_Supplementary_Material

Click here for additional data file.^{(1.8MB, docx)}

Acknowledgments

We are grateful to Erin Lynch and Cassandra Hanbury-Brown for their insight and assistance with interpretation of ECoG data, and to technical staff Stephan Martin and Yann Abéguilé at Data Science International for their advice on conducting sleep scoring and analysis of ECoG data. We would like to thank Jennifer McKenna, Vincent Zappala and Laboratory Animal Services administrative staff at The University of Sydney for their assistance during the study and provision of animal welfare. The graphical abstract was created using BioRender.com (Academic License).

Contributor Information

Joel S Raymond, Faculty of Science, School of Psychology, University of Sydney, Sydney, NSW, Australia; Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia.

Nicholas A Everett, Faculty of Science, School of Psychology, University of Sydney, Sydney, NSW, Australia; Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia.

Anand Gururajan, Faculty of Science, School of Psychology, University of Sydney, Sydney, NSW, Australia; Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia.

Michael T Bowen, Faculty of Science, School of Psychology, University of Sydney, Sydney, NSW, Australia; Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funding from The University of Sydney and the National Health and Medical Research Council (NHMRC; APP1166044, APP1092046).

Author Contributions

Author contributions, as defined by the Contributor Roles Taxonomy (CRediT) matrix, are detailed as follows: conceptualization (JSR and MTB), ethics application (JSR, NAE, AG, and MTB), data curation (JSR and NAE), formal analysis (JSR, AG, and MTB), funding acquisition (MTB), investigation (JSR and NAE), methodology (JSR, NAE, AG, and MTB), project administration (JSR and MTB), resources (NAE, AG, MTB), software (JSR), supervision (NAE, AG, MTB), validation (JSR and MTB), visualization (JSR and MTB), writing—original draft (JSR and MTB), and writing—review and editing (JSR, NAE, AG, and MTB).

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure Statement

Financial disclosure statement: MTB is an inventor on patents and patent applications covering oxytocin-based therapeutics. He is co-founder and Chief Scientific Officer of Kinoxis Therapeutics Pty Ltd, a company commercializing some of this intellectual property. The other author(s) declared no financial arrangements or connections with respect to the research, authorship, and/or publication of this article. Non-financial disclosure statement: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

The experiments reported in this study were conducted in line with the guidelines laid out in the Australian code for the care and use of animals for scientific purposes (8th edition, 2013) and were approved by the Animal Ethics Committee at The University of Sydney (AEC number: 2019/1615). All experiments are reported in adherence with guidelines of ARRIVE 2.0 guidelines.

Prior Deposit of Preprint Manuscript

An earlier version of this manuscript was deposited as a preprint manuscript on bioRxiv (https://doi.org/10.1101/2022.11.23.514802) on November 24th, 2022: Raymond, J.S., Everett, N.A., Gururajan, A. and Bowen, M.T., 2022. Quiet wakefulness: The influence of intraperitoneal and intranasal oxytocin on sleep-wake behaviour and neurophysiology in rats. bioRxiv, pp.2022-11.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Quiet wakefulness: the influence of intraperitoneal and intranasal oxytocin on sleep–wake behavior and neurophysiology in rats

Joel S Raymond

Nicholas A Everett

Anand Gururajan

Michael T Bowen

Abstract

Study Objectives

Methods

Results

Conclusions

Graphical abstract

Graphical Abstract.

Statement of Significance.

Introduction

Methods

Animals and housing

Drug preparation

Intraperitoneal drug administration

Intranasal drug administration

Radiotelemetry probe implantation surgery

Polysomnographic recordings

Figure 1.

Experimental design

Oxytocin i.p. dose–response (experiments 1A and 1B)

Oxytocin i.p. and OXTR antagonism (experiment 2)

Oxytocin i.n. dose–response (experiments 3A and 3B)

Data acquisition, processing, and analysis

Statistical analysis

Results

Experiment 1—effects of i.p. oxytocin dose range and i.p. caffeine on sleep–wake outcomes

Table 1.

Wake outcomes

Figure 2.

Active wake.

Quiet wake.

Sleep outcomes

NREM sleep.

Figure 3.

REM sleep.

Body temperature.

Experiment 2—influence of oxytocin receptor antagonism on oxytocin-induced effects on sleep–wake outcomes

Table 2.

Active wake.

Figure 4.

Quiet wake.

NREM sleep.

REM sleep.

Body temperature.

Experiment 3—effects of i.n. oxytocin and i.n. caffeine on sleep–wake outcomes

Table 3.

Wake outcomes

Active wake.

Figure 5.

Quiet wake.

Sleep outcomes

NREM sleep.

Figure 6.

REM sleep.

Body temperature.

Discussion

Sleep architectural and power spectral outcomes

Body temperature

Quiet wakefulness and mechanisms

Oxytocin and route of administration—intraperitoneal versus intranasal

Conclusions

Supplementary Material

Acknowledgments

Contributor Information

Funding

Author Contributions

Data Availability

Disclosure Statement

Ethical Approval

Prior Deposit of Preprint Manuscript

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS