BRAIN-2021-02452.R1 Proof Hi

Brain
Chronic pain recruits hypothalamic dynorphin/kappa opioid

receptor signaling to promote wakefulness and vigilance
Journal: Brain
Manuscript ID BRAIN-2021-02452.R1
Manuscript Type: Original Article

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Date Submitted by the
n/a
Author:
Complete List of Authors: Ito, Hisakatsu; The University of Arizona, Pharmacology; University of
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Toyama, Anesthesiology
Navratilova, Edita; The University of Arizona, Pharmacology
Vagnerova, Barbora; The University of Arizona, Pharmacology
Watanabe, Moe; The University of Arizona, Pharmacology
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Kopruszinski, Caroline; The University of Arizona, Pharmacology

Moreira de Souza, Luiz; The University of Arizona, Pharmacology
Yue, Xu; The University of Arizona, Pharmacology
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Ikegami, Daigo; The University of Arizona, Pharmacology

Moutal, Aubin; The University of Arizona, Pharmacology
Patwardhan, Amol; The University of Arizona, Pharmacology
Khanna , Rajesh; University of Arizona Health Sciences Center,
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Pharmacology
Yamazaki, Mitsuaki ; University of Toyama, Anesthesiology
Guerrero, Miguel ; The Scripps Research Institute, Department of
Molecular Medicine
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Rosen, Hugh ; Scripps Research Institute

Roberts, Ed; Scripps Research Institute
Neugebauer, Volker; Texas Tech University,
Dodick, D; mayo clinic, neurology;
Porreca, Frank; The University of Arizona, Department of Pharmacology
Methodology: NEUROBIOLOGY OF DISEASE
Subject area: PAIN, SLEEP
ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 Support (434) 964 4100
Page 1 of 45 Brain
Chronic pain recruits hypothalamic dynorphin/kappa opioid

receptor signaling to promote wakefulness and vigilance
Hisakatsu Ito1,2†, Edita Navratilova1,5†, Barbora Vagnerova1, Moe Watanabe1, Carol

Kopruszinski1, Luiz H. Moreira de Souza1, Xu Yue1, Daigo Ikegami1, Aubin Moutal1, Amol
Patwardhan1, Rajesh Khanna1, Mitsuaki Yamazaki2, Miguel Guerrero3, Hugh Rosen3, Ed
Roberts3, Volker Neugebauer4, David W. Dodick5 and Frank Porreca1,5*
†These authors contributed equally to this work.
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Abstract
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Increased vigilance in settings of potential threats or in states of vulnerability related to pain is

important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the
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underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases
secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore
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hypothesized that hypothalamic dynorphin/KOR signaling may be a previously unknown

mechanism that is recruited in pathological conditions requiring increased vigilance.
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We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and
rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain
induced by partial sciatic nerve ligation (PSNL) using EEG/EMG recordings. Systemic continuous
administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased
the amount of NREM and REM sleep and increased sleep fragmentation in naive mice throughout
the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer
receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic
paraventricular nucleus (PVN), a key node of the hypothalamic-pituitary-adrenal (HPA) stress
response. Sustained activation of Gi-DREADD with clozapine-N-oxide (CNO) delivered in
drinking water over 4 days disrupted sleep in these mice in a similar way as systemic U69,593.
Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was
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normalized by systemic administration of two structurally different KOR antagonists,

norbinaltorphimine (nor-BNI) and NMRA-140, currently in phase II clinical development, or by
CRISPR/Cas9 editing of PVN KOR, consistent with endogenous KOR activation disrupting sleep
in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or
by CRISPR/Cas9 editing of PVN KOR in sham-operated mice.
Our findings reveal previously unknown physiological and pathophysiological roles of

dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signaling affects transitions
between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not
promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain,
revealing a pathophysiological role of KOR signaling that is selectively recruited to promote
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vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the
short-term, disruption of the homeostatic need for sleep over longer periods may become
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maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain
may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.
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Author affiliations:
1Department of Pharmacology, University of Arizona; Tucson, USA.
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2Department of Anesthesiology, University of Toyama; Toyama, Japan.

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3Department of Molecular Medicine, The Scripps Research Institute; La Jolla, USA

4Department of Pharmacology and Neuroscience and Garrison Institute on Aging, Texas Tech
University Health Sciences Center; Lubbock, USA.
5Departments of Neurology and Collaborative Research, Mayo Clinic; Phoenix, USA
Correspondence to:
Frank Porreca, Ph.D.
Department of Pharmacology
University of Arizona
Tucson, AZ 85724
frankp@arizona.edu
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Running title: Hypothalamic KOR promotes wakefulness
Keywords: sleep disruption; pain; vigilance; kappa opioid receptor; dynorphin; chronic pain;
hypothalamus
Abbreviations: AAV = Adeno-associated virus; CNO = clozapine-N-oxide: CPP = conditioned

place preference; CRISPR/Cas9 = clustered regularly interspaced short palindromic repeats -
associated endonuclease; DCN = descending control of nociception; DNIC = diffuse noxious
inhibitory controls; DREADD = designer receptor activated by designer drugs; EEG =
electroencephalogram; EMG = electromyography; KOR = kappa opioid receptor; nor-BNI =
norbinaltorphimine; NREM = non-rapid eye movement; PSNL = partial sciatic nerve ligation;
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PVN = paraventricular nucleus; REM = rapid eye movement
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Introduction
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The regulation of sleep is complex and involves many interacting neural systems that affect the
sleep-wake cycle.1 Neurons in the preoptic area of the anterior hypothalamus promote sleep while
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orexin and histamine containing neurons in the lateral and posterior hypothalamus, respectively,
promote wakefulness and are main components of the arousal system. These systems follow
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circadian rhythms and additionally respond to physiological demands such as those associated with
energy and fluid homeostasis. Additionally, however, disturbance of the sleep-wake cycle may
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occur in settings of threat to the organism that require increased vigilance to promote survival.2
The neural circuits that are specifically engaged to promote wakefulness in conditions of
vulnerability including injuries, stress or threats remain unknown.
A bidirectional relationship between sleep and pain is well established clinically. Approximately
50-80% of patients with chronic pain complain of poor sleep.3-6 The relative hazard ratio of sleep
disturbances in patients with chronic pain is two to five times higher than in healthy individuals7-
11 and the prevalence of severe sleep disturbances increases with pain severity.12,13 On the other
hand, people fulfilling the criteria for insomnia disorders have high comorbidity with chronic pain
and show higher sensitivity to nociceptive stimulation than those without insomnia.14 Chronic
exposure of healthy volunteers to restricted sleep was shown to increase sensitivity, decrease
habituation, and increase temporal summation to noxious stimuli.15 Even acute sleep-deprivation
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of only one day was reported to amplify pain reactivity16,17 and this finding has also been
demonstrated preclinically.18 Sleep disturbances are also a potential cause of mental and physical
health problems beyond pain. Sleep disturbances increase the risk for mental disorders such as
depression, anxiety, and substance misuse,19-21 and could be a significant factor in risk of obesity,
diabetes, hypertension and critical vascular diseases including stroke and myocardial infarction.22-
25 Quality of life is reduced by many factors that promote sleep disturbances including pain.26
Dynorphin, an endogenous ligand at the kappa opioid receptor (KOR), has been repeatedly
demonstrated to play a key role in stress responses.27,28 Antagonism of KOR signaling blocks
stress-related aversive behaviors23,28,29 and both preclinical and clinical studies have suggested that
KOR antagonists may be useful in blocking negative affective states associated with depression,
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anxiety and drug seeking behavior. Recent preclinical studies have suggested that KOR activation
promotes pain aversiveness and that KOR antagonists may be useful in targeting affective qualities
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of chronic pain.30-33 Additionally, KOR signaling promotes the loss of descending control of
nociception/diffuse noxious inhibitory controls (DCN/DNIC)34 observed in rodent models of
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chronic pain likely through the activation of stress-related circuits in the limbic system.35,36
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The disruption of sleep by chronic pain may reflect the need for increased vigilance in states of
vulnerability. We therefore hypothesized that dynorphin/KOR signaling in stress-related circuits
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may be a fundamental mechanism that promotes adaptive behaviors to increase survival. Our data
reveal previously unknown roles of hypothalamic dynorphin/KOR signaling in increasing arousal
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and wakefulness in both physiological and pathological conditions.
MATERIALS AND METHODS
Study Design
We recorded sleep/wake data continuously for one or more 24-hour light/dark cycles (starting at
07:00 am) in freely moving male mice that had been implanted with head mounts for EEG and
EMG recording electrodes and evaluated the amount of total, NREM and REM sleep and the
average duration of wake, NREM and REM episodes. We investigated the effects of chronic pain
on sleep in a model of chronic neuropathic pain induced by partial sciatic nerve ligation. The role
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of KOR signaling on sleep and pain was studied using: 1) sustained systemic administration of
KOR agonists or KOR antagonists by subcutaneously implanted osmotic minipumps; 2) cell
specific manipulation of KOR expressing neurons in the paraventricular nucleus (PVN) of KORCre
mice37 to mimic effects of KOR agonists in a specific brain region; or 3) CRISPR/Cas9 editing of
KOR expression in the PVN. We measured pain behaviors using evoked sensory thresholds
elicited by probing the hindpaws with von Frey filaments or with learning behaviors that capture
pain-related motivation to seek relief, i.e., conditioned place preference (CPP).38 Expression of
DREADDs or CRISPR/Cas9-mediated deletions were verified post-hoc.
Animals
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The study was conducted in accordance with the NIH guidelines for use of laboratory animals and
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approval from the Institutional Animal Care and Use Committee at the University of Arizona.
Male C57BL/6J mice (8 weeks) or KORCre mice (8-10 weeks) were used for all experiments.
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KORCre mice were generated as previously described37 and were backcrossed to C57BL/6
background for at least 8 generations. Mice were maintained under conditions with 12-hour
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light/dark cycle. Light onset and offset times were 07:00 and 19:00, respectively. Food and water
were available ad libitum. Every effort was made to minimize numbers and suffering of animals
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used in the experiments. Mice were randomly assigned to the treatment groups and the experiments
were conducted in a blinded fashion.
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Partial sciatic nerve ligation surgery

We produced a partial sciatic nerve ligation (PSNL) model as described previously.39 Mice were
anesthetized with 2-5% isoflurane. The right sciatic nerve was ligated by a tight ligature with 8–0
silk suture around approximately one-half of the diameter. In sham-operated mice, the nerve was
only exposed without ligation. The muscle was sutured with a 5-0 braided suture and the skin was
closed with an Auto-clip.
Pain measurements
Thermal pain responses were assessed using tail flick test (see supplementary methods for details).
Mechanical allodynia was assessed at different time points before and after PSNL/sham surgery
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using von Frey filaments (supplementary methods).40 Conditioned place preference (CPP) with a
five-day conditioning protocol involving: acclimation, baseline chamber preference, two
conditioning days using saline in one chamber and gabapentin (30 mg/kg, i.p.) in the opposite
chamber, and a CPP test day, was used to assess ongoing pain (supplementary methods).
EEG/EMG recording and sleep analysis

After acclimatization, mice were implanted with EEG and EMG electrodes for polysomnographic
recordings (Pinnacle Technology, Oregon, USA)41 as detailed in supplementary methods.
EEG/EMG recording was performed in individual recording chambers after at least 5 days
recovery. The collected EEG/EMG data were analyzed by Sleepsign software (Kissei Comtec,
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Japan) with every 5-second epoch automatically classified into either wakefulness, REM or NREM
sleep, according to standard criteria41 (see supplementary methods and supplementary Figure S1).
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Drug administrations
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U69,593 (Tocris, Bristol, United Kingdom) was dissolved in hydrochloric acid and adjusted to pH
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7.4 with sodium hydroxide. Mice received continuous infusion of U69,593 (30 mg/kg/day) or
vehicle by an implanted micro-osmotic pump (Model 2991, Alzet Cupertino, California, USA)
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under the skin (see supplementary methods for details). This dose of sustained U69,593 delivery
was determined by evaluating antinociceptive effects in the spinally mediated tail-flick test as in
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our previous report with a single bolus administration.42 Continuous delivery of U69,593 (30
mg/kg/day) produced persistent antinociception from days 1 to 6 after osmotic-pump implantation
(Fig. S2). Norbinaltorphimine (nor-BNI) was purchased from Tocris, Bristol, United Kingdom and
dissolved in 0.9% saline to 10 mg/kg just before injection. Mice received a single intraperitoneal
injection of nor-BNI or vehicle (saline) 1 day before EEG/EMG recording at 10 days after
PSNL/sham surgery. NMRA-140 (formerly BTRX-335140, or CYM-53093) was synthesized as
described previously42 and was dissolved in DMSO, tween and saline at a ratio of 1:1:8. Mice were
implanted subcutaneously with osmotic minipumps at 8 days after PSNL/sham surgery and
received vehicle or NMRA-140 at 10 mg/kg/day. EEG/EMG recording started after a 1-day
recovery. Gabapentin (Spectrum Chemical MFG; Gardena, CA) was dissolved in water and was
administered i.p. at 30 mg/kg.
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Design and in vivo transfection of CRISPR/Cas9 targeting KOR

To delete the KOR we targeted the second exon of the oprk1 common to all KOR splice variants
(ENSMUSG00000025905, gRNA: gTCCCCCATTCAGATCTTCCG, on-score 65.7, off-score
78.8). The indicated gRNA sequence was inserted into the Esp3I restriction site of the pL-
CRISPR.EFS.tRFP lentiplasmid (Cat# 57819, Addgene, Cambridge, MA) as described before.43-
45 Plasmids were verified by Sanger sequencing (Eurofins, Louisville, KY). Five hundred nL of
KOR CRISPR plasmid was injected into the PVN (bregma: 0.8 mm posterior, 1.2 mm lateral, 5.3
mm or 4.9 mm ventral, at an angle of 10). Decreased KOR expression was verified using western
blotting with the anti-KOR primary antibody (Cat# sc-9112, Thermo Fisher Scientific,
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Massachusetts, USA). See supplementary methods for details.
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Adeno-Associated Virus Vector (AAV) microinjection

AAV8-hSyn-DIO-hM4D(Gi)-mCherry (150 nL; Addgene, viral prep # 44362-AAV8) encoding
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hM4Di-mCherry was injected into the same PVN location as described for KOR CRISPR plasmid.
Expression of hM4D(Gi)-mCherry in the targeted brain region was verified using fluorescent
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microscopy on an Olympus BX51 microscope (Olympus, Massachusetts, USA). See

supplementary methods for details.
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CNO administration
After baseline EEG/EMG recordings, the water bottle was filled with water or 0.1 mg/ml CNO.
KORCre and KORWT mice weighed 20–25 g and consumed approximately 5 ml/day for an
approximate self-administered dose of 0.8-1.0 mg/kg/h CNO for 6 days. EEG/EMG was recorded
at test days 4 and 5 and the tail flick test was performed at test days 2 and 6.
Statistical Analysis
Data are expressed as the mean ± SEM. The statistical significance of differences between the
groups was evaluated with one-way or two-way repeated measures ANOVA followed by the
Bonferroni multiple comparisons test as appropriate. P < 0.05 was considered significant. All
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statistical analyses were performed with Prism 9 (GraphPad Software, California, USA) and are
reported in Table S1.
RESULTS
Sustained systemic administration of KOR agonist disrupts sleep in

naive mice
EEG/EMG was recorded in male C57Bl6 mice continuously to monitor wakefulness, NREM and
REM sleep across multiple days. Mice are nocturnal and typically sleep during the light phase and
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are active during the dark phase. In addition, mice show repeated short episodes of wakefulness
during the light phase and sleep during the dark phase. We recorded sleep for 5 days beginning 1
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day after s.c. implantation of an osmotic minipump delivering the KOR agonist U69,593 or saline
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(Fig. 1A). NREM and REM sleep were analyzed during the light and dark phases in 2, 6 or 12 h
bins as (a) percent of sleep time (for total sleep) and (b) amount of sleep time. Sleep fragmentation
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was assessed by calculating the average duration of individual NREM or REM sleep episodes for
each 6-hour period defining early (07:00-13:00) and late (13:00-19:00) light phase and early
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(19:00-01:00) and late (01:00-07:00) dark phase. Sleepiness (i.e., “daytime napping” in the dark
phase) was assessed by calculating the average duration of individual wake episodes for each 6-
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hour period. Fig. 1B shows an example of 24-h sleep cycles during baseline and at day 4 after
saline or U69,593 infusion. The percent of sleep in the 12-hour light and dark phases are
respectively plotted for all 5 days of infusion in Figs. 1C and 1D. The average duration of wake
episodes for the 6-hour early light and early dark period is shown in Figs 1E, F. The amount of
sleep and average duration of individual NREM and REM sleep episodes are shown in Figs. 1G-
K and Figs. 1L-P, respectively.
Under pretreatment baseline conditions, mice showed a typical nocturnal cycle with 70.6  2.3%
and 37.3  4.4% sleep during the 12-h light (white graph area) and dark (shaded graph area) phases,
respectively (Fig. 1C, D; see supplementary Table S1 for additional data and statistical analyses).
Mice showed short wake episodes during the light phase (mean duration of 2.1  0.2 min) and long
continuous wake episodes especially in the first half of the dark phase (mean duration of 22.5 
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2.0 min). They spent on average 7.8  0.3 h in NREM and 0.71  0.17 h in REM sleep during the
12-h light phase and 4.2  0.6 h in NREM and 0.30  0.09 h in REM sleep during the dark phase.
The duration of individual NREM episodes was similar between the light and dark phases (4.4 
0.3 min and 5.6  0.6 min, respectively). Likewise, the duration of REM episodes was similar in
the light and dark phases (1.0  0.1 min and 0.8  0.2 min, respectively). REM sleep episodes were
always preceded by NREM sleep.
Compared to baseline measures, U69,593, but not vehicle, reduced the percent of sleep (Fig. 1B-
D) and amount of NREM sleep (Fig. 1G-I) during both the light and dark phases (Table S1).
U69,593 reduced percent of sleep consistently throughout the 5-day recording period. U69,593
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did not significantly reduce amount of REM sleep during the light phase (Fig. 1L, M). However,
U69,593 decreased amount of REM sleep during the dark phase likely reflecting the little, if any,
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preceding NREM sleep in this active phase (Fig. 1L, N). The average duration of wake episodes
in the vehicle group was similar to baseline (Fig. 1E). In contrast, U69,593 significantly prolonged
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the duration of wake episodes in the dark phase when compared to the vehicle group (50.6 ± 20.2
min vs 22.3 ± 2.4 min at test day 2 and 110.8 ± 31.1 min vs 21.1 ± 2.4 min at test day 3, Fig. 1F)
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demonstrating less daytime sleep. Mice treated with U69,593 were unable to stay awake at test
day 5 with short duration of wake episodes (12.2 ± 4.2 min) likely reflecting homeostatic rebound
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sleep due to accumulated sleep debt (Fig. 1F). During all periods through the 5-day time course,
U69,593 produced shorter episodes of NREM sleep compared to baseline or vehicle-treatment
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revealing sleep fragmentation (Fig. 1J, K). Additionally, U69,593 produced fragmentation of REM
sleep in the dark phase also likely reflecting loss of NREM sleep (Fig. 1O, P). U69,593 decreased
the power density of slow δ waves suggesting a decrease in depth of NREM sleep (Fig. S2B, C).
Chemogenetic activation of hypothalamic KOR signaling disrupts

sleep in naive mice
Both the KOR and the paraventricular nucleus of the hypothalamus (PVN) are strongly associated
with stress responses. As U69,593 disrupted sleep, we evaluated whether this effect is mediated
by KOR signaling (i.e., inhibitory Gi-coupling) in the PVN. Prior to EEG/EMG head mount
implantation, we injected AAV8-hSyn-DIO-hM4D(Gi)-mCherry bilaterally into the PVN of
KORCre mice (Fig. 2A, B). Sleep recordings began after 4-weeks to allow expression of hM4D(Gi)-
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mCherry, a Gi coupled designer receptor activated by designer drugs (Gi-DREADD) which is

activated by clozapine-N-oxide (CNO). Expression in the PVN was verified post hoc by mCherry
fluorescence (Figs. 2C and S3A). We first collected baseline EEG/EMG recordings while mice
had water available for drinking (day 0). Mice then received CNO (approximately 20 mg/kg/day,
p.o.) in their drinking water for the 5-day experimental period. There were no significant
differences in CNO/water consumption between KORCre and KORWT mice (Fig. S3B, C) and this
treatment had no antinociceptive effect in the tail flick test (Fig. S3D). EEG/EMG was recorded
on days 4 and 5 after starting CNO and compared to sleep measures in the same animals during
water consumption (Fig. 2A). Reduced sleep time was observed on both days in KORCre but not
KORWT mice (Figs. 2D, E and S3E and Table S1). The average duration of wake episodes during
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either the light or dark phases was not significantly influenced (Fig. 2F). Gi-DREADD activation
of PVN KORCre neurons reduced the amount of NREM sleep (Figs. 2G, H and S3F) and the mean
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duration of NREM sleep episodes in both the light and dark phases (Figs. 2I and S3G), reflecting
sleep fragmentation. Notably, the effect of Gi-DREADD activation on NREM sleep in the dark
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phase was not as strong as that observed with systemic U69,593 (see Fig. 1I, K). Consequently,
CNO did not affect the amount of REM sleep nor the mean duration of REM episodes on day 4
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(Fig. 2J-L) although a small decrease in REM sleep was observed on day 5 (Fig. S3H). Also, unlike
U69,593 infusion, CNO did not change sleep depth (δ wave power) in KORCre and KORWT when
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compared to water (Fig. S3I-L).

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Chronic pain disrupts sleep

We next investigated the impact of chronic pain on sleep using the partial sciatic nerve ligation
model (PSNL). Sleep was recorded at baseline and at 11 days after sham or PSNL surgery (Fig.
3A) when sham-operated mice fully recovered from surgery-related allodynia while mice with
PSNL demonstrate tactile allodynia (Fig. 3B) that lasts for many weeks.39 Ongoing pain was
demonstrated by conditioned place preference (CPP) to systemic administration of gabapentin (30
mg/kg, i.p.), an analgesic used clinically for treatment of neuropathic pain, in PSNL but not sham-
operated animals (Fig. 3C). While sleep did not change significantly in sham operated mice
compared to baseline, in mice with PSNL we observed significantly reduced percent of sleep
including reduction in the amount of both NREM and REM sleep during the light phase (Fig. 3D-I,
Table S1). More frequent wake episodes were observed during the light phase along with shorter
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average duration of NREM episodes, suggesting fragmented sleep in PSNL mice (Fig. 3J, K).
Additionally, more frequent sleep episodes and significantly reduced average duration of wake
episodes was observed during the dark (i.e., active) phase in animals with PSNL (Fig. 3 L, M),
indicative of increased “daytime” sleepiness (i.e., “napping”). We did not observe a change in
sleep depth (i.e., power density of delta wave) following PSNL surgery (Fig. S4A, B). Therefore,
chronic pain and KOR activation exert overlapping but not identical effects on sleep.
Nor-BNI normalizes pain-induced sleep disruption without

producing somnolence in sham-operated mice
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To determine the role of endogenous KOR signaling in chronic pain-induced sleep disruption, we
evaluated the sleep-wake cycle of sham and PSNL mice after systemic administration of a long
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lasting KOR antagonist, nor-BNI (10 mg/kg, i.p), or vehicle. We confirmed that PSNL reduced
the paw withdrawal threshold, and this was not affected by systemic administration of nor-BNI
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(Fig. S4C). The percent of sleep in the PSNL/vehicle group (46.1 ± 2.6 %) was significantly lower
than at baseline (69.2 ± 1.1 %; P < 0.0001) or in the sham/vehicle group (69.9 ± 3.1 %; P < 0.0001)
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during the light phase (Fig. 4A-C, Table S1). Nor-BNI normalized the percent of sleep (71.4 ± 1.3
%) of PSNL group to the same level as baseline and sham (Fig. 4A-C). This effect was due to
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normalization of the amount of NREM sleep (Fig. 4D-F) since nor-BNI had no effect on the
amount of REM sleep in PSNL animals (Fig. 4G-I).
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Importantly, systemic nor-BNI did not alter percent of sleep time in sham-operated animals (Fig.
4B, C) suggesting that dynorphin/KOR does not promote wakefulness in the absence of ongoing
pain. Additionally, in sham animals, there was no significant difference in the amount of NREM
sleep between nor-BNI and vehicle groups (Fig. 4E, F). Unexpectedly, however, nor-BNI
significantly reduced the amount of REM sleep in sham-operated mice (Fig. 4H, I) suggesting a
role of dynorphin/KOR in REM sleep. We compared the average duration of awake, NREM, and
REM sleep episodes across treatment groups. Although nor-BNI tended to reverse the decreased
wake duration observed in PSNL/vehicle mice in the dark phase (i.e., “daytime” sleepiness), this
was not statistically significant (Fig. 4J). However, the duration of NREM episodes was
normalized in PSNL/nor-BNI mice compared to PSNL/vehicle mice demonstrating improvement
of sleep fragmentation (Fig. 4K). Nor-BNI had no significant effect on the duration of REM
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episodes (Fig. 4L) and the depth of sleep (i.e., delta power) was unchanged by nor-BNI
administration in either PSNL or sham-operated animals (Fig. S4D-G).
Continuous systemic administration of NMRA-140 normalizes sleep

disruption in chronic pain
We also evaluated the effect of a selective KOR antagonist NMRA-140 currently in phase 2
clinical trials, on PSNL-induced sleep disruption. NMRA-140 (10 mg/kg/day) was delivered
continuously by osmotic minipump implanted 9 days after sham or PSNL surgery and sleep was
recorded at days 11 and 14 (Fig. 5A). The effects of NMRA-140 were similar to those found with
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nor-BNI with normalization of pain disrupted sleep but no somnolence in sham-operated animals.
We observed normalization of PSNL-induced reduction of percent of sleep on both days (Figs.
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5B, C, S5A, B, Table S1) and improvement of the decreased duration of wake episodes in the first
half of dark phase, i.e, “daytime” sleepiness (Figs. 5D and S5C). NMRA-140 also normalized the
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amount of NREM sleep (Figs. 5E, F and S5D, E) and PSNL-induced fragmented NREM sleep in
both the light and dark phases (Figs. 5G and S5F). Also like nor-BNI, NMRA-140 did not
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normalize PSNL-induced disruption of REM sleep and additionally decreased the amount of REM
sleep and the duration of REM episodes in sham-operated animals (Figs. 5H-J and S5G-I). The
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depth of sleep was not altered by NMRA-140 (Fig. S5J-M).

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CRISPR/Cas9 editing of PVN KOR normalizes chronic pain-induced

sleep disruption
To determine if the sleep disruption associated with chronic pain was due to hypothalamic KOR
signaling, we microinjected a CRISPR/Cas9 plasmid with a guide RNA (gRNA) targeting KOR
(i.e., KOR CRISPR) bilaterally into the PVN (Fig. 6A, B) and using western blot analysis
demonstrated decreased PVN KOR expression (Fig. 6C). PVN KOR CRISPR had no effect on
tactile withdrawal responses in either sham or PSNL mice (Fig. S6A). While PSNL mice injected
with a control CRISPR/Cas9 plasmid (i.e., control CRISPR) showed decreased percent of sleep,
this was not observed in PSNL mice injected with KOR CRISPR (Fig. 6D, E, Table S1), suggesting
prevention of chronic pain-induced sleep disruption. PVN KOR CRISPR but not control CRISPR
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also prevented PSNL-induced reduction in the duration of wake episodes during the dark phase
(i.e., “daytime” sleepiness) (Fig. 6F). Compared to control CRISPR treated PSNL mice, KOR
CRISPR treated PSNL mice showed normalization of NREM sleep (Fig. 6G, H) and increased
duration of NREM sleep episodes revealing decreased sleep fragmentation (Fig. 6I). Interestingly,
similar to treatment with KOR antagonists, PVN KOR CRISPR did not normalize REM sleep in
PSNL animals, and this treatment also disrupted REM sleep in sham-operated animals (Fig. 6J-L).
PVN KOR CRISPR had no effect on sleep depth (i.e., delta power) (Fig. S6B, C).
Discussion
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Our study reveals that dynorphin/KOR signaling is a previously unknown mechanism that
promotes arousal and wakefulness. We found that in naïve mice activation of brain KOR signaling
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with exogenous systemic agonists or by engagement of KORCre cells in the PVN promotes
wakefulness. Additionally, inhibition of KOR signaling or deletion of hypothalamic KOR (a)
ee
disrupts REM sleep without promoting somnolence in physiological conditions, and (b)
normalizes sleep in animals with chronic pain. Collectively, these observations suggest that KOR
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in brain hypothalamic circuits is a critical mediator of arousal that likely serves both physiological
functions and additionally represents a mechanism that can be recruited in situations of
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vulnerability that require increased vigilance. Finally, given the known homeostatic need for sleep
and the increase in pain that follows sleep disruption, continued dynorphin/KOR signaling may
iew
become maladaptive in chronic states resulting in a cycle that reinforces and sustains chronic pain.
Patients with insomnia complain of difficulty initiating sleep (sleep-onset insomnia), difficulty
maintaining sleep (sleep fragmentation), poor quality of sleep, circadian rhythm disorder
(abnormal sleep timing) and/or daytime impairment related to sleep difficulties such as sleepiness,
fatigue, reduced motivation and headache.46,47 Dynorphin, the endogenous opioid ligand at the
KOR, was found at higher levels in the rat hypothalamus when measured at night than in the day
suggesting possible circadian expression.48 Additionally, the secretion of dynorphin is facilitated
by stress.49 These observations suggested that dynorphin/KOR signaling might play a role in
promoting arousal. Consistent with this, we found that sustained administration of a selective KOR
agonist, U69,593, increased alertness during both light and dark phases, largely mimicking signs
of clinically observed insomnia (see Table 1 for summary). Uninjured animals treated with
Brain Page 14 of 45
U69,593 showed a reduction of NREM sleep with minimal direct effects on REM sleep. Unlike
humans, mice normally have short wake episodes lasting only for a few seconds during their sleep.
Treatment with U69,593 increased arousal as well as the frequency of these brief wake episodes
and decreased the duration of NREM episodes revealing increased sleep fragmentation. U69,593
treatment did not produce “daytime” sleepiness during the dark phase (i.e., no rebound sleep)
suggesting that continued exogenous KOR activation sustains wakefulness. The duration and
number of REM episodes in the dark phase were also decreased following U69,593. As the
occurrence of REM sleep requires preceding NREM sleep, the observed decreased amount of
REM sleep in the dark phase likely reflects the extreme decrease in preceding NREM sleep.
Furthermore, U69,593 decreased the power of slow (i.e., low frequency) δ waves characteristic of
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deep NREM sleep, suggesting a role of KOR signaling in reducing sleep depth and consistent with
increased overall arousal. While KOR agonists are uncommon in clinical practice, nalfurafine is
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marketed in Japan for the treatment of pruritis but is not used for pain control. Consistent with our
finding that KOR activation disrupts sleep in mice, one of the most frequently reported clinical
ee
side-effects of nalfurafine is insomnia.50,51

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Stress engages the HPA (hypothalamus-pituitary-adrenal) axis in part through the release of
dynorphin.27-29 Both dynorphin and KOR are localized in the PVN suggesting that chronic
ev
dynorphin/KOR activity may result in dysfunction of the HPA axis.27,52,53 We therefore

hypothesized that increased endogenous KOR signaling in this hypothalamic nucleus may promote
iew
sleep disturbances in conditions requiring increased vigilance such as chronic pain. The profile of
sleep disturbance following Gi-DREADD activation of PVN KORCre neurons was similar, but not
identical, to that seen with systemic administration of a KOR agonist (Table 1). Chemogenetic
PVN KOR activation reduced NREM sleep and increased sleep fragmentation without
significantly affecting REM sleep. Notably, while the effects of Gi-DREADD activation of PVN
KORCre neurons were similar to systemic U69,593, the magnitude of changes was smaller
suggesting that KOR mechanisms beyond the PVN likely play a role in arousal. The precise
mechanisms by which PVN KOR activation may disrupt sleep remain to be identified. However,
it is possible that hypothalamic KOR signaling could disrupt sleep through disinhibition of arousal-
related neurons including those that express orexin and/or histamine. Neurons expressing orexin,
a neuropeptide that promotes arousal,54 arise in the lateral hypothalamus and project to many brain
regions including the PVN.55 Dynorphin is often co-expressed in orexinergic neurons56 and has
Page 15 of 45 Brain
been reported to disinhibit these neurons.57 Furthermore, dynorphin disinhibits arousal-promoting

histaminergic neurons in the tuberomammillary nucleus.58 Notably, following Gi-DREADD
activation of PVN KORCre neurons we did not observe a change in power density of δ waves as
was seen with systemic U69,593 administration suggesting that KOR signaling in PVN is not
sufficient to impact the depth of sleep. It should be noted that other factors beyond stress and
dynorphin/KOR signaling undoubtedly contribute to sleep disturbances associated with chronic
pain. Previous reports suggest that the activity of ascending serotonin or noradrenaline neurons
from the brainstem reticular activating system may be altered in experimental neuropathic pain
resulting in facilitation of arousal.41,59
Patients with chronic pain show a high degree of co-morbidity with stress-related disorders
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including anxiety and depression, conditions that are also associated with insomnia. In our
preclinical model of chronic neuropathic pain induced by PSNL, mice showed long-lasting
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allodynia and ongoing pain. Consistent with clinical observation, mice with PSNL exhibited sleep
disturbances including reduced sleep and increased sleep fragmentation that largely mimicked
ee
those observed in uninjured mice treated with systemic U69,593 or by activation of Gi-DREADD
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signaling in PVN KORCre neurons (Table 1).
Supporting the conclusion that chronic pain-induced sleep disruption is mediated, in part, by
ev
increased KOR signaling, treatment with systemic nor-BNI, a long-acting KOR antagonist, or with
sustained infusion of NMRA-140, a relatively short-acting and selective KOR antagonist currently
iew
in phase 2 clinical trials, normalized disrupted sleep in PSNL mice (Table 1) to the same levels as
observed at pre-injury baselines and in sham-operated mice. In PSNL animals, both KOR
antagonists increased the duration of NREM sleep episodes in the light phase resulting in
decreased sleep fragmentation. KOR antagonists also increased the duration of wake episodes in
the dark phase suggesting improvement of daytime sleepiness. Critically, unlike blockade of
orexin or histaminergic signaling, KOR antagonists did not produce somnolence in sham-operated
mice suggesting that endogenous dynorphin/KOR signaling does not play a role in maintaining
wakefulness under physiological conditions.
Surprisingly, however, we found that KOR antagonists (a) decreased REM sleep in sham-operated
animals and (b) did not improve REM sleep in the PSNL model. Decreased REM sleep following
KOR antagonist administration suggests that endogenous dynorphin/KOR signaling promotes
Brain Page 16 of 45
REM sleep in normal physiological conditions. The failure of KOR antagonists to improve REM
sleep in conditions of chronic pain may indicate that dynorphin/KOR signaling likely does not
contribute to disruption of REM sleep by pain. The relationship between REM sleep and pain,
however, is not well understood and requires further study.60,61
To determine if the sleep disruption associated with chronic pain was due to hypothalamic KOR
signaling, we microinjected a CRISPR/Cas9 plasmid targeting KOR to decrease KOR expression
in the PVN. Unlike PSNL mice injected with control CRISPR, PSNL mice with PVN KOR
CRISPR did not show decreased amount of sleep and had a normal amount of NREM sleep.
Editing of PVN KOR in PSNL mice also increased the duration of NREM episodes, revealing an
improvement in PSNL-induced sleep fragmentation, and increased duration of wake episodes
Fo
during the dark phase suggesting normalization of chronic pain-induced day-time sleepiness. In
agreement with outcomes from KOR antagonist treatment, PVN KOR CRISPR did not normalize
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REM sleep in PSNL animals and additionally disrupted REM sleep in sham-operated animals.
ee
Translational implications
Pain is a warning alarm that promotes protective behaviors to avoid tissue damage. For this reason,
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pain interrupts deep sleep and increases vigilance. In conditions requiring alertness, sleep is
shallow allowing responses to external stimuli and behaviors associated with increased survival to
ev
be prioritized over sleep. In our studies, we observed shallower sleep in KOR agonist treated mice,
iew
but importantly, KOR antagonism improved sleep without altering the power density of δ (slow-
wave, NREM) and θ (REM) waves in animals with chronic pain. KOR antagonists can therefore
improve pain-disrupted sleep without impairment of protective behavioral responses.
Human studies suggest that fragmented sleep has a stronger influence on pain thresholds than
decreased amount of sleep.16 While increased wakefulness and arousal may be an adaptive
response to situations of organismal threat, there is a homeostatic need for sleep. Continued KOR
activation as a consequence of chronic pain can then lead to a cycle of sleep disruption that may
ultimately become maladaptive and reinforce and sustain the chronic pain state. We have
previously reported that nor-BNI reduces aversiveness of ongoing pain.30 In the present study, we
showed that nor-BNI, NMRA-140 and knock-down of KOR in PVN could improve pain-related
sleep disruption. As pain and sleep have a bidirectional relationship, the improvement in sleep that
we observed with blockade of KOR signaling could be due to relief of pain or, alternatively,
Page 17 of 45 Brain
improvement of pain could be the result of improved sleep. Future studies will be required to
determine whether the improvement in sleep is a direct effect of prevention of KOR signaling or
an indirect effect via the improvement of pain. Nevertheless, these findings support the conclusion
that targeting the KOR may break the detrimental vicious spiral of pain and sleep.
We also note that there are many overlapping clinical conditions including anxiety and depression
that are associated with sleep disruption. Such conditions are often co-morbid with chronic pain
and could also be amenable to treatment with KOR antagonists. KOR antagonists including
NMRA-140 and aticaprant (i.e., CERC-501) have not shown addictive properties and are currently
in clinical development for stress-related indications including anxiety and depression providing
an opportunity to rapidly explore the potential of this mechanism in sleep disruption associated
Fo
with chronic pain. We note that KOR antagonists, or genetic deletion of PVN KOR disrupted REM
sleep suggesting a potential side effect of KOR antagonist therapy. While the clinical impact of
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disruption of REM sleep with KOR antagonists remains unknown, such risks appear tolerable.
Many drugs in clinical use disrupt REM sleep including duloxetine, an SNRI used both for the
ee
treatment of depression as well as for chronic pain.62 In spite of the link between REM sleep and
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memory consolidation, antidepressants including SNRIs have been reported to have either no
effects or to even modestly improve memory.63 These observations suggest that the benefits of
ev
KOR antagonism in the dual modulation of chronic pain aversiveness and pain-related sleep
disruption may outweigh potential negative effects arising from disruption of REM sleep.
iew
It should also be emphasized that KOR antagonists did not produce direct hypnotic effects in
animals without pain suggesting that upregulation of dynorphin is a key factor in the maladaptive
consequences of chronic pain to induce insomnia. This observation provides an important
distinction between KOR antagonists and orexin antagonists that produce sleep in the absence of
chronic pain potentially limiting their daytime use. Finally, we note that the blockade of KOR
signaling may be useful in normalization of sleep disruption associated with stress-related
disorders beyond chronic pain including generalized anxiety, depression, and others.
Acknowledgements
We thank Dr. Sarah Ross for providing the KORCre mouse line.
Brain Page 18 of 45
Funding
This work was supported by grants from the National Institutes of Health: P01DA041307 (FP,
EN), R01 NS106902 (FP, EN, VN), R01 NS109255 (EN, VN, FP).
Competing interests
Frank Porreca has served as a consultant or received research funding from Voyager, Nektar,
Amgen, Acadia, Blackthorn, Teva, Eli Lilly, Hoba, Allergan, Abbvie, Ipsen, PeptideLogic and
Proximagen and is a founder of Catalina Pharma. Rajesh Khanna is a stakeholder in Regulonix
Fo
Holding Inc. and Eleutheria LLC. Amol Patwardhan is a founder of Catalina Pharma. David W.
Dodick reports the following conflicts within the past 12 months: Consulting: AEON, Amgen,
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Clexio, Cerecin, Cooltech, Ctrl M, Allergan, Alder, Biohaven, GSK, Linpharma, Lundbeck,
Promius, Eli Lilly, eNeura, Novartis, Impel, Satsuma, Theranica, WL Gore, Nocira, XoC, Zosano,
ee
Upjohn (Division of Pfizer), Pieris, Praxis, Revance, Equinox. Honoraria: Clinical Care Solutions,
CME Outfitters, Curry Rockefeller Group, DeepBench, Global Access Meetings, KLJ Associates,
rR
Academy for Continued Healthcare Learning, Majallin LLC, Medlogix Communications, MJH
Lifesciences, Miller Medical Communications, Southern Headache Society (MAHEC), WebMD
ev
Health/Medscape, Wolters Kluwer, Oxford University Press, Cambridge University Press.

Research Support: Department of Defense, National Institutes of Health, Henry Jackson
iew
Foundation, Sperling Foundation, American Migraine Foundation, Patient Centered Outcomes

Research Institute (PCORI). Stock Options/Shareholder/Patents/Board of Directors: Ctrl M
(options), Aural analytics (options), ExSano (options), Palion (options), Healint (Options),
Theranica (Options), Second Opinion/Mobile Health (Options), Epien (Options/Board), Nocira
(options), Matterhorn (Shares/Board), Ontologics (Shares/Board), King-Devick Technologies
(Options/Board), Precon Health (Options/Board). Patent 17189376.1-1466:vTitle: Botulinum
Toxin Dosage Regimen for Chronic Migraine Prophylaxis. Miguel Guerrero reports financial
interests in NMRA-140. Ed Roberts and Hugh Rosen are paid consultants and hold shares in
Blackthorn. Other authors declare no competing interests.
Page 19 of 45 Brain
Supplementary material
Supplementary material is available at Brain online.
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Figure legends
Figure 1. Sustained U69,593 administration disrupts sleep. (A) Experimental timeline:
Baselines were collected after EEG/EMG head mount implantation and habituation in recording
chambers. Mice then received osmotic minipumps delivering U69,593 or vehicle. Continuous
sleep recording during the light (white rectangle) and dark (grey rectangle) started at 7:00 am 1
day after the minipump implant and continued nonstop for 5 days. (B) Sleep time at baseline and
at day 4 is expressed in percent of total time in 2-h bins over a 24 h cycle. Dark phase from 7:00
pm to 7:00 am is shaded in grey. (C, D) Percent of sleep time in the 12-h light (C) and dark (D)
phases for all recorded days (BL, D1-5). (E, F) Average duration of wake episodes during the light
Fo
(E) and dark (F) phases for all recorded days. (G) Amount of NREM sleep time (in min) at baseline
and at day 4 in 2-h bins. (H, I) Total amount of NREM sleep time (in hours) during the light (H)
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and dark (I) phases for baseline and days 1-5. (J, K) Average duration of NREM episodes during
the light (J) and dark (K) phases. Fragmented sleep (i.e., reduced duration of NREM episodes) is
ee
indicated by down arrows. (L) Amount of REM sleep time at baseline and at day 4 in 2-h bins.
(M, N) Total amount of REM sleep time (in hours) during the light (M) and dark (N) phases. (O,
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P) Average duration of REM episodes during the light (O) and dark (P) phases. Fragmented sleep
is indicated by down arrows. Data represent mean  SEM; values for individual mice are shown
ev
as small symbols; N=6 for vehicle; N=5 for U69,593; baseline data are combined from both vehicle
iew
and U69,593 groups. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA

with Bonferroni post hoc test. Detailed statistics in Table S1.
Figure 2. Chemogenetic activation of KOR in the PVN disrupts sleep. (A) Experimental
timeline: KORCre mice received bilateral AAV8-hSyn-DIO-hM4D(Gi)-mCherry into the PVN and
(B) EEG/EMG head mount implantation. Sleep was recorded 4 weeks later (day 0) when all mice
had drinking water available. Water was replaced with a CNO and water solution and sleep was
recorded again on days 4 and 5 after starting CNO self-administration. (C) Verification of
hM4D(Gi)-mCherry expression by fluorescent microscopy. Scale bar, 100 μm. (D) Percent of
sleep time in 2-h bins is plotted in WT and KORCre mice at day 0 (water) and day 4 (CNO). (E)
Percent of sleep time in the light and dark phases. (F) Average duration of wake episodes during
Brain Page 24 of 45
the 6-h early and late light and early and late dark phases. (G) Amount of NREM sleep time in 2-
h bins over the 24 h day cycle. (H) Amount of NREM sleep time during the light and dark phases.
(I) Average duration of NREM episodes during the 6-h phases; fragmented sleep is indicated by
down arrows. (J) Amount of REM sleep time in 2-h bins. (K) Amount of REM sleep time during
the light and dark phases. (L) Average duration of REM episodes during the early/late light and
dark phases. Data represent mean  SEM; values for individual mice are shown as small symbols;
N=6 for WT; N=7 for KORCre. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM
ANOVA with Bonferroni post hoc test. Detailed statistics in Table S1.
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Figure 3. Chronic neuropathic pain disrupts sleep. (A) Experimental timeline: Top shows the
timeline for sleep measurements that were performed before PSNL/sham surgery and again on day
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11 after the surgery. All mice received saline (s.c.) on day 10 as part of the study presented in Fig.
4. Bottom timeline shows the times when pain behavior was measured in a different cohort of
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mice: von Frey (VF) testing was done before PSNL/sham surgery and on days 7, 11, and 15. A 5-
day CPP protocol started on day 11 after the surgery with final testing on day 15. (B) Tactile
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allodynia was measured as a reduction in withdrawal thresholds of the injured hindpaw. (C)
Ongoing pain was assessed by CPP to gabapentin. (D-F) Percent of sleep (D), and amount of
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NREM (E) and REM sleep (F) in 2-h bins is shown at baseline and on day 11 after PSNL or sham
injury. (G-I) The same data are presented for the light and dark phases. (J) Examples of EMG
iew
integral and EEG δ-power traces during the light phase at baseline and at day 11 after PSNL
demonstrating more frequent wake episodes (arrows) and shorter duration of NREM and REM
episodes after PSNL. (K) Average duration of NREM episodes during the 6-h early/late light and
dark phases; fragmented sleep is indicated by down arrows. (L) Examples of EMG integral during
the dark phase at baseline and after PSNL show more frequent and shorter wake episodes (i.e.,
“napping”) in mice after PSNL. (M) Average duration of wake episodes during the 6-h early/late
light and early/late dark phases. Data represent mean  SEM; values for individual mice are shown
as small symbols; N=11 for both groups in the pain behavioral study and N=8 for both groups in
the sleep study. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA with
Bonferroni post hoc test. Detailed statistics in Table S1.
Page 25 of 45 Brain
Figure 4. KOR antagonist nor-BNI restores sleep in mice with PSNL. The sleep study was
performed according to the timeline shown in Fig. 3A. Nor-BNI or vehicle was administered on
day 10 after PSNL/sham surgery, i.e., 1 day before EEG/EMG recording. (A, B) Percent of sleep
time in 2-h bins is shown at baseline and 11 days after PSNL (A) or sham (B) injury in mice treated
with nor-BNI or vehicle. (C) Summary of percent of total sleep data in light and dark phases for
all groups. (D, E) NREM sleep time in 2-h bins for mice with PSNL (D) or sham (E) injury. (F)
NREM sleep in the light and dark phases for all groups. (G, H) REM sleep time in 2-h bins for
mice with PSNL (G) or sham (H) injury. (I) REM sleep in the light and dark phases for all groups.
(J) Average duration of wake episodes during the 6-h early/late light and early/late dark phases.
“Daytime sleepiness” is indicated by a down arrow. (K) Average duration of NREM episodes
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during the 6-h early/late light and dark phases; fragmented sleep is indicated by down arrows. (L)
Average duration of REM sleep episodes during the 6-h early/late light and dark phases. Data
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represent mean  SEM; values for individual mice are shown as small symbols; N=6 for sham/nor-
BNI; N=8 for sham/vehicle and both PSNL groups. *P<0.05; **P<0.01; ***P<0.0001;
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****P<0.00001; 2-way RM ANOVA with Bonferroni post hoc test. Detailed statistics in Table
S1.
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Figure 5. KOR antagonist NMRA-140 restores sleep in mice with PSNL. (A) Experimental
timeline: mice were given PSNL or sham surgery and were instrumented with EEG/EMG head
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mounts. After habituation, on day 9, mice were implanted with osmotic minipumps delivering
NMRA-140 or vehicle and sleep was assessed on days 11 and 14 after PSNL/sham surgery. Day
11 recordings are shown in this figure, for day 14 data see supplementary Figure S5. (B) Percent
of sleep time in 2-h bins is shown for NMRA-140 or vehicle treated mice at 11 days after PSNL
or sham surgery. (C) Summary of total sleep data in light and dark phases. (D) Average duration
of wake episodes during the 6-h early/late light and early/late dark phases. “Daytime sleepiness”
is indicated by down arrows. (E, F) NREM sleep time in 2-h bins (E) and during light and dark
phases (F). (G) Average duration of NREM episodes during the 6-h early/late light and dark
phases; fragmented sleep is indicated by down arrows. (H, I) REM sleep time in 2-h bins (H) and
in the light and dark phases (I). (J) Average duration of REM sleep episodes during the 6-h
early/late light and dark phases. Fragmented sleep is indicated by down arrows Data represent
Brain Page 26 of 45
mean  SEM; values for individual mice are shown as small symbols; N=6 for sham/vehicle; N=8
for sham/NMRA; N=7 for PSNL/vehicle and N=9 for PSNL/NMRA groups. *P<0.05; **P<0.01;
***P<0.0001; ****P<0.00001; 2-way RM ANOVA with Bonferroni post hoc test. Detailed
statistics in Table S1.
Figure 6. Deletion of KOR in the PVN with CRISPR normalizes sleep in mice with PSNL.
(A) Experimental timeline: mice received KOR CRISPR or control plasmid bilaterally in the PVN
as well as an EEG/EMG implant. Seven days later, baseline sleep recordings were collected, mice
then underwent PSNL, or sham surgery and sleep was assessed on day 11 after the surgery. (B)
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Design of gRNA to target Oprk1 gene. (C) KOR levels in the PVN were assessed at the end of the
experiment by western blotting. KOR CRISPR reduced KOR expression by 47  15 % compared
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to control plasmid. (D) Percent of sleep time in 2-h bins is shown for at 11 days after PSNL or
sham surgery. (E) Total sleep time in light and dark phases. (F) Average duration of wake episodes
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during the 6-h early/late light and early/late dark phases. “Daytime sleepiness” is indicated by
down arrows. (G, H) NREM sleep time in 2-h bins (G) and during light and dark phases (H). (I)
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Average duration of NREM episodes during the 6-h early/late light and dark phases; fragmented
sleep is indicated by down arrows. (J, K) REM sleep time in 2-h bins (J) and in the light and dark
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phases (K). (L) Average duration of REM sleep episodes during the 6-h early/late light and dark
phases. Fragmented sleep is indicated by down arrows. Data represent mean  SEM; values for
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individual mice are shown as small symbols; N=8 for PSNL/CRISPR and both sham groups; N=6
for PSNL/control. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA with
Page 27 of 45 Brain
Treatment Total sleep1 NREM2 REM3 Fragmented Daytime Sleep

(percent) (amount) (amount) sleep4 sleepiness5 depth6
KOR agonist Reduced Reduced Reduced Yes No effect Reduced
(U69,593) (dark)
Naive
PVN KORCre Reduced Reduced No effect Yes No effect No effect

Gi-DREADD
PSNL Reduced Reduced Reduced Yes Yes No effect
(light) (light) (light) (dark)
PSNL
KOR antagonists Normalized Normalized No effect Normalized Normalized No effect
PVN KOR CRISPR Normalized Normalized No effect Normalized Normalized No effect

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KOR antagonists No effect No effect Reduced No effect No effect No effect

(light)
Sham
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PVN KOR CRISPR No effect No effect Reduced No effect No effect No effect

(light)
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Table 1. Summary of main effects of KOR signaling on sleep in uninjured and PSNL mice.
Measures indicating disrupted sleep are highlighted in orange; green highlights denote
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normalization of sleep disruption observed in PSNL. 1Percent of sleep time; 2amount of NREM
sleep time; 3amount of REM sleep time; 4sleep fragmentation was assessed as significant decrease
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in the average duration of individual NREM or REM sleep episodes; 5sleepiness (i.e., “daytime
napping”) was assessed as significant decrease in the average duration of individual wake episodes
iew
in the dark phase; 6reduced depth of sleep signifies a shift in the power of delta waves to higher
wavelengths. Light/dark indicates whether the effect is limited to a specific sleep phase.
Brain Page 28 of 45
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Figure 1. Sustained U69,593 administration disrupts sleep. (A) Experimental timeline: Baselines were
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collected after EEG/EMG head mount implantation and habituation in recording chambers. Mice then
received osmotic minipumps delivering U69,593 or vehicle. Continuous sleep recording during the light
(white rectangle) and dark (grey rectangle) started at 7:00 am 1 day after the minipump implant and
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continued nonstop for 5 days. (B) Sleep time at baseline and at day 4 is expressed in percent of total time in
2-h bins over a 24 h cycle. Dark phase from 7:00 pm to 7:00 am is shaded in grey. (C, D) Percent of sleep
time in the 12-h light (C) and dark (D) phases for all recorded days (BL, D1-5). (E, F) Average duration of
wake episodes during the light (E) and dark (F) phases for all recorded days. (G) Amount of NREM sleep
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time (in min) at baseline and at day 4 in 2-h bins. (H, I) Total amount of NREM sleep time (in hours) during
the light (H) and dark (I) phases for baseline and days 1-5. (J, K) Average duration of NREM episodes during
the light (J) and dark (K) phases. Fragmented sleep (i.e., reduced duration of NREM episodes) is indicated
by down arrows. (L) Amount of REM sleep time at baseline and at day 4 in 2-h bins. (M, N) Total amount of
iew
REM sleep time (in hours) during the light (M) and dark (N) phases. (O, P) Average duration of REM
episodes during the light (O) and dark (P) phases. Fragmented sleep is indicated by down arrows. Data
represent mean  SEM; values for individual mice are shown as small symbols; N=6 for vehicle; N=5 for
U69,593; baseline data are combined from both vehicle and U69,593 groups. *P<0.05; **P<0.01;
***P<0.0001; ****P<0.00001; 2-way RM ANOVA with Bonferroni post hoc test. Detailed statistics in Table
S1.
185x112mm (300 x 300 DPI)
Page 29 of 45 Brain
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Figure 2. Chemogenetic activation of KOR in the PVN disrupts sleep. (A) Experimental timeline: KORCre mice
received bilateral AAV8-hSyn-DIO-hM4D(Gi)-mCherry into the PVN and (B) EEG/EMG head mount
implantation. Sleep was recorded 4 weeks later (day 0) when all mice had drinking water available. Water
was replaced with a CNO and water solution and sleep was recorded again on days 4 and 5 after starting
CNO self-administration. (C) Verification of hM4D(Gi)-mCherry expression by fluorescent microscopy. Scale
bar, 100 μm. (D) Percent of sleep time in 2-h bins is plotted in WT and KORCre mice at day 0 (water) and
day 4 (CNO). (E) Percent of sleep time in the light and dark phases. (F) Average duration of wake episodes
during the 6-h early and late light and early and late dark phases. (G) Amount of NREM sleep time in 2-h
bins over the 24 h day cycle. (H) Amount of NREM sleep time during the light and dark phases. (I) Average
duration of NREM episodes during the 6-h phases; fragmented sleep is indicated by down arrows. (J)
Amount of REM sleep time in 2-h bins. (K) Amount of REM sleep time during the light and dark phases. (L)
Average duration of REM episodes during the early/late light and dark phases. Data represent mean  SEM;
values for individual mice are shown as small symbols; N=6 for WT; N=7 for KORCre. *P<0.05; **P<0.01;
***P<0.0001; ****P<0.00001; 2-way RM ANOVA with Bonferroni post hoc test. Detailed statistics in Table
S1.
185x183mm (300 x 300 DPI)
Brain Page 30 of 45
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ev
iew
Figure 3. Chronic neuropathic pain disrupts sleep. (A) Experimental timeline: Top shows the timeline for
sleep measurements that were performed before PSNL/sham surgery and again on day 11 after the surgery.
All mice received saline (s.c.) on day 10 as part of the study presented in Fig. 4. Bottom timeline shows the
times when pain behavior was measured in a different cohort of mice: von Frey (VF) testing was done
before PSNL/sham surgery and on days 7, 11, and 15. A 5-day CPP protocol started on day 11 after the
surgery with final testing on day 15. (B) Tactile allodynia was measured as a reduction in withdrawal
thresholds of the injured hindpaw. (C) Ongoing pain was assessed by CPP to gabapentin. (D-F) Percent of
sleep (D), and amount of NREM (E) and REM sleep (F) in 2-h bins is shown at baseline and on day 11 after
PSNL or sham injury. (G-I) The same data are presented for the light and dark phases. (J) Examples of EMG
integral and EEG δ-power traces during the light phase at baseline and at day 11 after PSNL demonstrating
more frequent wake episodes (arrows) and shorter duration of NREM and REM episodes after PSNL. (K)
Average duration of NREM episodes during the 6-h early/late light and dark phases; fragmented sleep is
indicated by down arrows. (L) Examples of EMG integral during the dark phase at baseline and after PSNL
show more frequent and shorter wake episodes (i.e., “napping”) in mice after PSNL. (M) Average duration of
Page 31 of 45 Brain
wake episodes during the 6-h early/late light and early/late dark phases. Data represent mean  SEM;
values for individual mice are shown as small symbols; N=11 for both groups in the pain behavioral study
and N=8 for both groups in the sleep study. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way
RM ANOVA with Bonferroni post hoc test. Detailed statistics in Table S1.
185x237mm (300 x 300 DPI)
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ev
iew
Brain Page 32 of 45
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ev
iew
Figure 4. KOR antagonist nor-BNI restores sleep in mice with PSNL. The sleep study was performed
according to the timeline shown in Fig. 3A. Nor-BNI or vehicle was administered on day 10 after PSNL/sham
surgery, i.e., 1 day before EEG/EMG recording. (A, B) Percent of sleep time in 2-h bins is shown at baseline
and 11 days after PSNL (A) or sham (B) injury in mice treated with nor-BNI or vehicle. (C) Summary of
percent of total sleep data in light and dark phases for all groups. (D, E) NREM sleep time in 2-h bins for
mice with PSNL (D) or sham (E) injury. (F) NREM sleep in the light and dark phases for all groups. (G, H)
REM sleep time in 2-h bins for mice with PSNL (G) or sham (H) injury. (I) REM sleep in the light and dark
phases for all groups. (J) Average duration of wake episodes during the 6-h early/late light and early/late
dark phases. “Daytime sleepiness” is indicated by a down arrow. (K) Average duration of NREM episodes
during the 6-h early/late light and dark phases; fragmented sleep is indicated by down arrows. (L) Average
duration of REM sleep episodes during the 6-h early/late light and dark phases. Data represent mean 
SEM; values for individual mice are shown as small symbols; N=6 for sham/nor-BNI; N=8 for sham/vehicle
and both PSNL groups. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA with
185x197mm (300 x 300 DPI)
Page 33 of 45 Brain
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Figure 5. KOR antagonist NMRA-140 restores sleep in mice with PSNL. (A) Experimental timeline: mice were
iew
given PSNL or sham surgery and were instrumented with EEG/EMG head mounts. After habituation, on day
9, mice were implanted with osmotic minipumps delivering NMRA-140 or vehicle and sleep was assessed on
days 11 and 14 after PSNL/sham surgery. Day 11 recordings are shown in this figure, for day 14 data see
Supplementary Figure S4. (B) Percent of sleep time in 2-h bins is shown for NMRA-140 or vehicle treated
mice at 11 days after PSNL or sham surgery. (C) Summary of total sleep data in light and dark phases. (D)
Average duration of wake episodes during the 6-h early/late light and early/late dark phases. “Daytime
sleepiness” is indicated by down arrows. (E, F) NREM sleep time in 2-h bins (E) and during light and dark
phases (F). (G) Average duration of NREM episodes during the 6-h early/late light and dark phases;
fragmented sleep is indicated by down arrows. (H, I) REM sleep time in 2-h bins (H) and in the light and
dark phases (I). (J) Average duration of REM sleep episodes during the 6-h early/late light and dark phases.
Fragmented sleep is indicated by down arrows Data represent mean  SEM; values for individual mice are
shown as small symbols; N=6 for sham/vehicle; N=8 for sham/NMRA; N=7 for PSNL/vehicle and N=9 for
PSNL/NMRA groups. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA with Bonferroni
post hoc test. Detailed statistics in Table S1.
185x167mm (300 x 300 DPI)
Brain Page 34 of 45
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iew
Figure 6. Deletion of KOR in the PVN with CRISPR normalizes sleep in mice with PSNL. (A) Experimental
timeline: mice received KOR CRISPR or control plasmid bilaterally in the PVN as well as an EEG/EMG
implant. Seven days later, baseline sleep recordings were collected, mice then underwent PSNL, or sham
surgery and sleep was assessed on day 11 after the surgery. (B) Design of gRNA to target Oprk1 gene. (C)
KOR levels in the PVN were assessed at the end of the experiment by western blotting¬¬. KOR CRISPR
reduced KOR expression by 47  15 % compared to control plasmid. (D) Percent of sleep time in 2-h bins is
shown for at 11 days after PSNL or sham surgery. (E) Total slee¬p time in light and dark phases. (F)
Average duration of wake episodes during the 6-h early/late light and early/late dark phases. “Daytime
sleepiness” is indicated by down arrows. (G, H) NREM sleep time in 2-h bins (G) and during light and dark
phases (H). (I) Average duration of NREM episodes during the 6-h early/late light and dark phases;
fragmented sleep is indicated by down arrows. (J, K) REM sleep time in 2-h bins (J) and in the light and
dark phases (K). (L) Average duration of REM sleep episodes during the 6-h early/late light and dark
phases. Fragmented sleep is indicated by down arrows. Data represent mean  SEM; values for individual
mice are shown as small symbols; N=8 for PSNL/CRISPR and both sham groups; N=6 for PSNL/control.
*P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA with Bonferroni post hoc test.
Detailed statistics in Table S1.
185x175mm (300 x 300 DPI)
Page 35 of 45 Brain
Treatment Total sleep1 NREM2 REM3 Fragmented Daytime Sleep

(percent) (amount) (amount) sleep4 sleepiness5 depth6
KOR agonist Reduced Reduced Reduced Yes No effect Reduced
(U69,593) (dark)
Naive
PVN KORCre Reduced Reduced No effect Yes No effect No effect

Gi-DREADD
PSNL Reduced Reduced Reduced Yes Yes No effect
(light) (light) (light) (dark)
PSNL
KOR antagonists Normalized Normalized No effect Normalized Normalized No effect
PVN KOR CRISPR Normalized Normalized No effect Normalized Normalized No effect
KOR antagonists No effect No effect Reduced No effect No effect No effect

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(light)
Sham
PVN KOR CRISPR No effect No effect Reduced No effect No effect No effect

(light)
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Table 1. Summary of main effects of KOR signaling on sleep in uninjured and PSNL mice.
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Measures indicating disrupted sleep are highlighted in orange; green highlights denote
normalization of sleep disruption observed in PSNL. 1Percent of sleep time; 2amount of NREM
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sleep time; 3amount of REM sleep time; 4sleep fragmentation was assessed as significant decrease
in the average duration of individual NREM or REM sleep episodes; 5sleepiness (i.e., “daytime
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napping”) was assessed as significant decrease in the average duration of individual wake episodes
in the dark phase; 6reduced depth of sleep signifies a shift in the power of delta waves to higher
iew
wavelengths. Light/dark indicates whether the effect is limited to a specific sleep phase.
Brain Page 36 of 45
Supplementary material
Detailed methods
Tail flick test: Mice were restrained gently and 1/2 to 2/3 of the tail was submerged into 50°C hot
water bath. The latency to a rapid flick of the tail was recorded. The cutoff time was set at 15 s to
prevent tissue damage.
Von Frey testing: Mechanical allodynia was assessed using von Frey filaments at different time
points before and after PSNL/sham surgery. Mice were placed in suspended chambers with wire
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mesh floors and allowed to habituate for 30 min. Calibrated von Frey filaments were applied to
the ipsilateral hindpaw until the filament buckled. Withdrawal threshold was determined using the
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up-down method as previously described.40

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Conditioned place preference (CPP): A five-day conditioning protocol was used to assess
ongoing pain. On acclimation day (day 1), mice were placed into the CPP boxes (Panlab, Harvard
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Apparatus - LE896/76-0376 and LE989/76-0441) for 15 min with free access to all chambers. The
next day, mice were placed in the chambers again and baseline time spent in each chamber across
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15 min was analyzed using automated software (Panlab, Harvard Apparatus - PPCWIN). On
following two conditioning days (days 3 and 4), mice received a saline injection (i.p.) in the
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mornings and were confined in one conditioning chamber for 30 min. Four hours later, mice
received gabapentin (30 mg/kg, i.p.) and were placed into the opposite chamber for 30 min.
Chamber assignment was counterbalanced. On test day (day 5), mice were placed in the CPP box
and were allowed to explore all chambers freely for 15 min; the time in chambers was recorded to
determine chamber preference. Difference scores were calculated as test time minus
preconditioning time spent in the gabapentin-paired chamber.
Subcutaneous minipump implantation: Mice were lightly anesthetized with 2% isoflurane, a

horizontal incision was made at the shaved base of the neck and osmotic minipumps (Model 2991,
Alzet Cupertino, CA, USA) (1μl/h) delivering saline, U69,593 (at 10 or 30 mg/kg/day) or NMRA-
140 (at 10 mg/kg/day) for 7 days were inserted into the underlying subcutaneous space. The
minipump implantation lasted approximately 1 min and no analgesics were administered.
Page 37 of 45 Brain
EEG/EMG recording and sleep analysis: Mice were placed in the recording chambers (Pinnacle
Technology) and acclimatized for 2 days. Under 2-5% isoflurane anesthesia, mice were then
implanted with EEG and EMG electrodes for polysomnographic recordings (Pinnacle Technology,
Oregon, USA) as described previously.41 Briefly, mice were mounted in a stereotaxic head holder.
Approximately 2 cm incision was made down the midline from just behind the eyes to expose the
cranium. Four holes that were positioned 1 mm anterior to the bregma or lambda, both 1.5 mm
lateral to the midline, were drilled avoiding visible blood vessels to allow for insertion of electrode
screws. Dedicated 2 EEG/1 EMG mouse head-mounts (Pinnacle Technology) were implanted with
four EEG electrode screws. Two Teflon-coated stainless-steel wires were placed bilaterally into
both trapezius muscles to record EMG. The head-mount is secured to the skull with 2 anchor
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screws and covered by dental acrylic. Animals were placed in the recording chambers again for
additional 5 days to acclimate and recover from the surgery, resulting in one week of
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acclimatization prior to the recording in all of our experiments. All data were taken from the EEG
electrodes positioned on the frontal lobe.
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The collected EEG/EMG data were analyzed by Sleepsign software (Kissei Comtec, Japan). Every
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5-second epoch was automatically classified into three stages, i.e., wakefulness, REM and NREM
sleep, according to the standard criteria.41 As a final step, defined sleep–wake stages were
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examined visually for any recording artifacts and corrected, if necessary (less than 1% of all data).
The vigilance states were assessed as follows: 1) wakefulness was defined by a high EMG
iew
amplitude, low EEG amplitude; 2) REM sleep was defined by a low EMG amplitude, low EEG
amplitude, and high θ wave (5.0-10.0 Hz) activity; and 3) NREM sleep was defined by a low EMG
amplitude, high EEG amplitude and high δ wave (0.65-4.5Hz) activity. Normalized power
spectrum in each frequency was calculated by dividing the EEG power (μV2) of each frequency
band by the sum of EEG power (μV2) of all frequencies and expressed as percent. A single 5-sec
epoch of wake in between two long NREM episodes was counted as a wake episode.
Intracranial plasmid microinjection: KORcre or wild type littermates (KORWT) were

anesthetized with 2-5% isoflurane and placed in a stereotaxic head holder. A glass micropipette
(10 m diameter; World precision instruments, Florida, USA) was inserted at the stereotactic
coordinates of the PVN of hypothalamus relative to bregma: 0.8 mm posterior, 1.2 mm lateral, 5.3
or 4.9 mm ventral, at an angle of 10. The pipette was left in place for 5 min after injection to
Brain Page 38 of 45
minimize backflow. Sleep recording was performed at least 4 weeks after AAV injection or 1 week
after plasmid injection.
Verification of CRISPR/Cas9 editing of KOR: To verify that KOR CRISPR/Cas9 effectively

reduced KOR expression in the targeted brain region, we quantified KOR expression in the PVN
brain punches using western blotting. Animals were euthanized and fresh brains were quickly
dissected. Following removal of the brains, 2 mm coronal sections at approximately bregma -0.8
mm containing the PVN were cut on ice using a brain matrix and a 2 mm punch of the PVN was
collected using the 3rd ventricle as a guide indicator. The tissue was homogenized with lysis buffer
containing protease inhibitors, phosphatase inhibitors, and benzonase. Extracted proteins were
separated by electrophoresis and transferred to PVDF membranes 0.45 μm (Millipore, Darmstadt,
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Germany). The membranes were blocked with TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1%
Tween 20, pH 7.4), 5% non-fat dry milk, then incubated with the anti-KOR primary antibody (Cat#
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sc-9112, Thermo Fisher Scientific, Massachusetts, USA) and anti-βIII-tubulin antibody (Cat#
G7121, Promega) in TBST, 5% bovine serum albumin. Blots were incubated in HRP-conjugated
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secondary antibody, followed by enhanced luminescence (Millipore, Darmstadt, Germany) and

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detected luminescence by the Odyssey Fc Imager (LI-COR, Nebraska, USA), and quantified using
ImageJ.
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Verification of hM4D(Gi)-mCherry expression: Anesthetized animals were perfused with 4%

paraformaldehyde from the left ventricle of the exposed heart. The brain was removed and post-
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fixed for 4 hours with 4% paraformaldehyde and dehydrated with 15% and 30% sucrose for 24
hours in each solution. Brains were sectioned coronally at 20 m thickness. To evaluate the
expression of mCherry, sectioned samples were examined and photographed on an Olympus BX51
microscope (Olympus, Massachusetts, USA) equipped with a Hamamatsu C8484 camera.
Page 39 of 45 Brain
Supplementary figures
Waveform
EEG
EMG High amplitude Low amplitude

5 sec
Fast Fourier Transform
50 μV 250 μV 250 μV
δ θ α β δ θ α β δ θ α β
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0 20 Hz 0 20 Hz 0 20 Hz
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Wakefulness REM sleep NREM sleep
Figure S1. Classification of the sleep/wake vigilance states. The collected EEG/EMG data were
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analyzed and automatically classified into three stages, i.e., wakefulness, REM and NREM sleep
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by Sleepsign software. The vigilance states were assessed as follows: 1) wakefulness was defined
by a high EMG amplitude, low EEG amplitude; 2) REM sleep was defined by a low EMG
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amplitude, low EEG amplitude, and highθwave (5.0-10.0 Hz) activity; and 3) NREM sleep was
defined by a low EMG amplitude, high EEG amplitude and highδwave (0.65-4.5Hz) activity. Data
iew
shown are from baseline sleep recording.
Brain Page 40 of 45
A Gi-DREADD-mCherry expression
A U69,593 analgesic dose response B Power density of delta (light) C Power density of delta (dark)
15 Vehicle 8 10
Power spectrum (%)

10 mg/kg/day
Power spectrum (%)

# 30 mg/kg/day vehicle 8
6
**** ****
Latency (s)
10 U69,593
** **** ****
*** 6
* ** *** ** 4
** ** 4
5
2
2
0 0 0
BL 1 2 3 4 5 6 7 8 0 5 10 15 20 0 5 10 15 20
Time (days) Frequency (Hz) Frequency (Hz)
B CNO/water consumption C CNO/water consumption D Tail flick latency

12 50 15
WT WT
KORCre 40 KORCre
Dose (mg/kg/day)
Latency (s)
Volume (ml)
8 10
30
Fo
20
4 5
10 WT
KORCre CNO
0 0 0
water day1 day2 day3 day4 day5 day1 day2 day3 day4 day5 BL 0 2 6
rP
CNO Time (days)
REM sleep
sleep
E total sleep.
Percent day5
of sleep F NREM sleep G DurationofofNREM
Duration NREMepisodes
periods H REM
80 ** 10 10 1.5
WT/CNO ** Fragmented sleep
Duration of NREM (min)
ee
*
NREM sleep time (min)
REM sleep time (min)

KOR-cre/CNO 8 8
60
1.0
% of sleep
** 6 ** 6
40
4 4
*** 0.5
20 ****
rR
****
2 2
0 0 0 0.0
light phase dark phase light phase dark phase 7-13 13-19 19-1 1-7 light phase dark phase
ev
I Power density of delta (light) J Power density of delta (dark) K Power density of delta (light) L Power density of delta (dark)
8 8 8 8
KORCre/CNO
Power spectrum (%)
Power spectrum (%)
WT/CNO
Power spectrum (%)
KORCre/CNO
Power spectrum (%)
WT/CNO
6 WT/water 6 WT/water 6 KORCre/water 6 KORCre/water
iew
4 4 4 4
2 2 2 2
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz)
Figure S2 (supplementary data to Fig. 1). Effects of sustained infusion with U69,593 on
spinally mediated analgesia and sleep depth. (A) Tail flick latency was determined daily after
s.c. implantation of osmotic minipumps to naïve mice, delivering saline or U69,593 at 10 or 30
mg/kg/day. (B, C) Power density of δ wave was analyzed from EEG/EMG sleep recording shown
in Fig. 1. Normalized power spectra during the light (B) and dark (C) phases at day 4 after
minipump implant (delivering saline and 30 mg/kg/day U69,593) are shown.
Page 41 of 45 Brain
Figure S3 (supplementary data to Fig. 2). Chemogenetic activation of KOR in the PVN
disrupts sleep. (A) Verification of hM4D(Gi)-mCherry expression in the PVN for all KORCre
mice using fluorescent microscopy for mCherry. Scale bar, 100 μm. (B) Consumption of water
(day 0) and 0.1 mg/ml CNO/water (days 1-5) in the KORCre and KORWT mice. (C) The dose of
CNO consumed on days 1-5 was calculated from the weight of individual mice and their
CNO/water consumption. (D) Tail flick latency was assessed prior to EEG/EMG head mount
implantation (BL), on day 0 (before baseline sleep recordings with water available for drinking)
and at days 2 and 6 after the start of the CNO/water regiment. (E) Total percent of sleep time on
day 5 in the light and dark phases. (F) Total amount of NREM sleep time during the light and dark
phases on day 5. (G) Average duration of NREM episodes during the 6-h phases on day 5;
Fo
fragmented sleep is indicated by down arrows. (H) Total amount of REM sleep time during the
light and dark phases. (I-L) Normalized power spectra for KORWT (I, J) and KORCre (K, L) mice
rP
on day 4 during the light and dark phases. Data represent mean  SEM; values for individual mice
are shown as small symbols; N=6 for WT; N=7 for KORCre.
ee
rR
A B C Power density of theta (light) D Power density of theta (dark)

ev
Power density of delta (light) Power density of delta (dark)

8 8 8 10
Baseline
Sham 8
Power spectrum (%)
Power spectrum (%)
Power spectrum (%)
Power spectrum (%)
6 6 6
PSNL
6
4 4 4
4
iew
2 2 2
2
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz)
E Tactile allodynia F Power density of delta (light) G Power density of delta (dark) H Power density of delta (light) I Power density of delta (dark)
4 8 8 8 8
PSNL/Vehicle
Withdrawal threshold (g)
Sham/vehicle
Power spectrum (%)
Power spectrum (%)

Power spectrum (%)
Power spectrum (%)
Sham/nor-BNI PSNL/nor-BNI
3 6 6 6 6
2
* 4 4 4 4
1
* 2 2 2 2
0 0 0 0 0
vehicle norBNI vehicle norBNI 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Sham PSNL Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz)
Figure S4 (supplementary data to Figs. 3 and 4). Effects of PSNL and nor-BNI on power
density of δ wave. (A, B) Normalized power spectra during the light (A) and dark (B) phases at
baseline and on day 11 after PSNL or sham surgery in mice receiving vehicle day prior. (C)
Mechanical withdrawal thresholds were collected after sleep recording in vehicle or nor-BNI
treated sham or PSNL mice. (D, E) Comparison of normalized power spectra during the light (D)
Brain Page 42 of 45
and dark (E) phases in sham mice receiving vehicle or nor-BNI. (F, G) Comparison of normalized
power spectra during the light (F) and dark (G) phases in PSNL mice receiving vehicle or nor-
BNI.
Fo
rP
ee
rR
ev
iew
Page 43 of 45 Brain
A Percent
Percent of sleep
of sleep in 2 h B Percent of
Percent of sleep
sleep C Duration
Duration of
of wake
wakeepisodes
periods
100 100 ####
60
## Sham/Vehicle Sham/Vehicle Sleepiness
Duration of wake (min)

# Sham/NMRA Sham/NMRA
80 80 ####
## PSNL/Vehicle PSNL/Vehicle
40
% of sleep
% of sleep
PSNL/NMRA PSNL/NMRA
60 60 ***
40 40
20
* ****
20 20
0 0 0
7 9 11 13 15 17 19 21 23 1 3 5 Light Phase Dark Phase 7-13 13-19 19-1 1-7
Clock time Clock time
D NREM
NREM sleep
sleep in 2 h E NREM sleep
NREM F Duration
Durationof
ofNREM
NREMepisodes
periods
120 12 10
### #### Fragmented sleep
Duration of NREM (min)

NREM sleep time (min)
# ####
NREM sleep time (h)

#
100 8
## ### ### *
###
80 8
* 6
60
4
40 4 ** *
*
20 2
Fo
0 0 0
G REM sleep H REM sleep I Duration of REM episodes

rP
REM sleep in 2 h REM sleep Duration of REM periods
15 1.5 1.5
Fragmented sleep
Duration of REM (min)

REM sleep time (min)
REM sleep time (h)
10 1.0 **** 1.0

ee
**
**
**
**** **
** **
5 ** *** *** 0.5 **** 0.5

* ****
rR
0 0.0 0.0
J Power density of delta (light) K Power density of delta (light)

ev
8 8
Sham/Vehicle PSNL/Vehicle
Sham/NMRA PSNL/NMRA
Power spectrum (%)
Power spectrum (%)
6 6
iew
4 4
2 2
0 0
0 5 10 15 20 0 5 10 15 20
Frequency (Hz) Frequency (Hz)
L Power density of delta (dark) M Power density of delta (dark)

8 8
Sham/Vehicle PSNL/Vehicle
Sham/NMRA PSNL/NMRA
Power spectrum (%)
Power spectrum (%)
6 6
4 4
2 2
0 0
0 5 10 15 20 0 5 10 15 20
Figure S5 (supplementary data to Fig. 5). KOR antagonist NMRA-140 restores sleep in mice
with PSNL. (A-I) Sleep data recorded on day 14 after PSNL/sham surgery. (A) Percent of sleep
Brain Page 44 of 45
time in 2-h bins. (B) Percent of total sleep in light and dark phases. (C) Average duration of wake
episodes during the 6-h early/late light and early/late dark phases. “Daytime sleepiness” is
indicated by down arrows. (D) NREM sleep time in 2-h bins (E) NREM sleep time during light
and dark phases. (F) Average duration of NREM episodes during the 6-h early/late light and dark
phases; fragmented sleep is indicated by down arrows. (G) REM sleep time in 2-h bins. (H) REM
sleep time in the light and dark phases. (I) Average duration of REM sleep episodes during the 6-
h early/late light and dark phases. (J-M) The effect of NMRA-140 on normalized power spectra
recorded on day 11 after sham (J, L) or PSNL (K, M) surgery during the light and dark phases.
Data represent mean  SEM; values for individual mice are shown as small symbols; N=6 for
sham/vehicle; N=8 for sham/NMRA; N=7 for PSNL/vehicle and N=9 for PSNL/NMRA groups.
Fo
rP
A Tactile allodynia B Power density of delta (light) C Power density of delta (dark)
ee
4 8 8
Control/sham
Power spectrum (%)
Power spectrum (%)
Control/sham
CRISPR/sham
3 6 6 CRISPR/sham
PWT (g)
rR
2 4 4
PSNL/CRISPR
1 PSNL/Control 2 2
Sham/CRISPR ****
Sham/Control
0 0 0
ev
BL CRISPR PSNL 0 5 10 15 20 0 5 10 15 20
iew
Figure S6 (supplementary data to Fig. 6). Deletion of KOR in the PVN with CRISPR has no
effect on sleep depth. (A) Mechanical withdrawal thresholds were collected at baseline, after PVN
injection of control plasmid or KOR CRISPR and again after PSNL/sham surgery. (B, C)
Comparison of normalized power spectra in sham mice injected into the PVN with control or KOR
CRISPR plasmids. N=8/group. N=6 for sham/control and N=5 for sham/CRISPR. Data represent
mean  SEM.
Page 45 of 45 Brain
The ARRIVE Guidelines Checklist

Animal Research: Reporting In Vivo Experiments
1 2 3 4 5
Carol Kilkenny , William J Browne , Innes C Cuthill , Michael Emerson and Douglas G Altman
1 2
The National Centre for the Replacement, Refinement and Reduction of Animals in Research, London, UK, School of Veterinary
Science, University of Bristol, Bristol, UK, 3School of Biological Sciences, University of Bristol, Bristol, UK, 4National Heart and Lung
Institute, Imperial College London, UK, 5Centre for Statistics in Medicine, University of Oxford, Oxford, UK.
Section/
ITEM RECOMMENDATION Paragraph
Title 1 Provide as accurate and concise a description of the content of the article Title
as possible.
Abstract 2 Provide an accurate summary of the background, research objectives, Abstract
including details of the species or strain of animal used, key methods,
principal findings and conclusions of the study.
INTRODUCTION
Fo
Background 3 a. Include sufficient scientific background (including relevant references to

Introduction
previous work) to understand the motivation and context for the study,
and explain the experimental approach and rationale.
b. Explain how and why the animal species and model being used can
rP
address the scientific objectives and, where appropriate, the study’s

relevance to human biology.
Objectives 4 Clearly describe the primary and any secondary objectives of the study, or Introduction
ee
specific hypotheses being tested.

METHODS
Ethical statement
rR
5 Indicate the nature of the ethical review permissions, relevant licences (e.g. Methods/
Animal [Scientific Procedures] Act 1986), and national or institutional Animals
guidelines for the care and use of animals, that cover the research.
Study design 6 For each experiment, give brief details of the study design including: Methods/
ev
a. The number of experimental and control groups. Study Design

b. Any steps taken to minimise the effects of subjective bias when
allocating animals to treatment (e.g. randomisation procedure) and when
iew
assessing results (e.g. if done, describe who was blinded and when).
c. The experimental unit (e.g. a single animal, group or cage of animals).
A time-line diagram or flow chart can be useful to illustrate how complex
study designs were carried out.
Experimental 7 For each experiment and each experimental group, including controls, Drug
procedures provide precise details of all procedures carried out. For example: administration
a. How (e.g. drug formulation and dose, site and route of administration, s
anaesthesia and analgesia used [including monitoring], surgical
procedure, method of euthanasia). Provide details of any specialist
equipment used, including supplier(s).
b. When (e.g. time of day).
c. Where (e.g. home cage, laboratory, water maze).
d. Why (e.g. rationale for choice of specific anaesthetic, route of
administration, drug dose used).
Experimental 8 a. Provide details of the animals used, including species, strain, sex, Animals
animals developmental stage (e.g. mean or median age plus age range) and
weight (e.g. mean or median weight plus weight range).
b. Provide further relevant information such as the source of animals,
international strain nomenclature, genetic modification status (e.g.
knock-out or transgenic), genotype, health/immune status, drug or test
naïve, previous procedures, etc.
The ARRIVE guidelines. Originally published in PLoS Biology, June 20101
Brain Page 46 of 45
Housing and 9 Provide details of: Animals

husbandry a. Housing (type of facility e.g. specific pathogen free [SPF]; type of cage or
housing; bedding material; number of cage companions; tank shape and
material etc. for fish).
b. Husbandry conditions (e.g. breeding programme, light/dark cycle,
temperature, quality of water etc for fish, type of food, access to food
and water, environmental enrichment).
c. Welfare-related assessments and interventions that were carried out
prior to, during, or after the experiment.
Sample size 10 a. Specify the total number of animals used in each experiment, and the Results,
number of animals in each experimental group.
figure legends
b. Explain how the number of animals was arrived at. Provide details of any
sample size calculation used.
c. Indicate the number of independent replications of each experiment, if
relevant.
Allocating 11 a. Give full details of how animals were allocated to experimental groups, Animals
animals to including randomisation or matching if done.
experimental b. Describe the order in which the animals in the different experimental
groups
Fo
groups were treated and assessed.

Experimental 12 Clearly define the primary and secondary experimental outcomes assessed Results
outcomes (e.g. cell death, molecular markers, behavioural changes).
rP
Statistical 13 a. Provide details of the statistical methods used for each analysis.
Statistical
methods b. Specify the unit of analysis for each dataset (e.g. single animal, group of analysis
animals, single neuron).
ee
c. Describe any methods used to assess whether the data met the
assumptions of the statistical approach.
RESULTS
rR
Baseline data 14 For each experimental group, report relevant characteristics and health Animals
status of animals (e.g. weight, microbiological status, and drug or test naïve)
prior to treatment or testing. (This information can often be tabulated).
ev
Numbers 15 a. Report the number of animals in each group included in each analysis. Animals
2
analysed Report absolute numbers (e.g. 10/20, not 50% ).
b. If any animals or data were not included in the analysis, explain why.
iew
Outcomes and 16 Report the results for each analysis carried out, with a measure of precision Results
estimation (e.g. standard error or confidence interval).
Adverse events 17 a. Give details of all important adverse events in each experimental group. No adverse
b. Describe any modifications to the experimental protocols made to effects
reduce adverse events.
DISCUSSION
Interpretation/ 18 a. Interpret the results, taking into account the study objectives and Discussion
scientific hypotheses, current theory and other relevant studies in the literature.
implications b. Comment on the study limitations including any potential sources of bias,
any limitations of the animal model, and the imprecision associated with
2
the results .
c. Describe any implications of your experimental methods or findings for
the replacement, refinement or reduction (the 3Rs) of the use of animals
in research.
Generalisability/ 19 Comment on whether, and how, the findings of this study are likely to Discussion
translation translate to other species or systems, including any relevance to human
biology.
Funding 20 List all funding sources (including grant number) and the role of the Funding
funder(s) in the study.
References:
1. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving Bioscience Research Reporting: The ARRIVE Guidelines
for Reporting Animal Research. PLoS Biol 8(6): e1000412. doi:10.1371/journal.pbio.1000412
2. Schulz KF, Altman DG, Moher D, the CONSORT Group (2010) CONSORT 2010 Statement: updated guidelines for reporting parallel
group randomised trials.
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Brain

Chronic pain recruits hypothalamic dynorphin/kappa opioid

Manuscript Type: Original Article

Kopruszinski, Caroline; The University of Arizona, Pharmacology

Ikegami, Daigo; The University of Arizona, Pharmacology

Rosen, Hugh ; Scripps Research Institute

Methodology: NEUROBIOLOGY OF DISEASE

Subject area: PAIN, SLEEP

Chronic pain recruits hypothalamic dynorphin/kappa opioid

Hisakatsu Ito1,2†, Edita Navratilova1,5†, Barbora Vagnerova1, Moe Watanabe1, Carol

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is

hypothesized that hypothalamic dynorphin/KOR signaling may be a previously unknown

normalized by systemic administration of two structurally different KOR antagonists,

Our findings reveal previously unknown physiological and pathophysiological roles of

2Department of Anesthesiology, University of Toyama; Toyama, Japan.

3Department of Molecular Medicine, The Scripps Research Institute; La Jolla, USA

Running title: Hypothalamic KOR promotes wakefulness

Abbreviations: AAV = Adeno-associated virus; CNO = clozapine-N-oxide: CPP = conditioned

and wakefulness in both physiological and pathological conditions.

MATERIALS AND METHODS

Partial sciatic nerve ligation surgery

EEG/EMG recording and sleep analysis

Design and in vivo transfection of CRISPR/Cas9 targeting KOR

Adeno-Associated Virus Vector (AAV) microinjection

microscopy on an Olympus BX51 microscope (Olympus, Massachusetts, USA). See

Sustained systemic administration of KOR agonist disrupts sleep in

Chemogenetic activation of hypothalamic KOR signaling disrupts

mCherry, a Gi coupled designer receptor activated by designer drugs (Gi-DREADD) which is

compared to water (Fig. S3I-L).

Chronic pain disrupts sleep

Nor-BNI normalizes pain-induced sleep disruption without

Continuous systemic administration of NMRA-140 normalizes sleep

depth of sleep was not altered by NMRA-140 (Fig. S5J-M).

CRISPR/Cas9 editing of PVN KOR normalizes chronic pain-induced

side-effects of nalfurafine is insomnia.50,51

dynorphin/KOR activity may result in dysfunction of the HPA axis.27,52,53 We therefore

been reported to disinhibit these neurons.57 Furthermore, dynorphin disinhibits arousal-promoting

signaling in PVN KORCre neurons (Table 1).

Health/Medscape, Wolters Kluwer, Oxford University Press, Cambridge University Press.

Foundation, Sperling Foundation, American Migraine Foundation, Patient Centered Outcomes

7. Karaman S, Karaman T, Dogru S, et al. Prevalence of sleep disturbance in chronic pain.

Psychiatr Res. 2005;39(2):151-159.

diabetes incidence in a large U.S. sample. Sleep. 2007;30(12):1667-1673.

optogenetic tool: possible involvement of the activation of dorsal raphe nucleus-

Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140). J Med Chem.

Neurofibromatosis type 1 related pain. Channels (Austin). 2018;12(1):47-50.

Pain. Neuroscience. 2018;381:79-90.

Lacosamide. Pain. 2017;158(12):2301-2319.

noradrenergic neurons in pain-related sleep disorders. Neurosci Lett. 2015;589:200-206.

and U69,593 groups. *P<0.05; **P<0.01; ***P<0.0001; ****P<0.00001; 2-way RM ANOVA

Treatment Total sleep1 NREM2 REM3 Fragmented Daytime Sleep

PVN KORCre Reduced Reduced No effect Yes No effect No effect

KOR antagonists Normalized Normalized No effect Normalized Normalized No effect

PVN KOR CRISPR Normalized Normalized No effect Normalized Normalized No effect

KOR antagonists No effect No effect Reduced No effect No effect No effect

PVN KOR CRISPR No effect No effect Reduced No effect No effect No effect

185x112mm (300 x 300 DPI)

185x183mm (300 x 300 DPI)

185x237mm (300 x 300 DPI)

185x197mm (300 x 300 DPI)

185x167mm (300 x 300 DPI)

185x175mm (300 x 300 DPI)

and U69,593 groups. P<0.05; P<0.01; P<0.0001; ****P<0.00001; 2-way RM ANOVA

5 * * 0.5 ** 0.5