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Behavioural Brain Research xxx (xxxx) 114473
Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Ketamine supresses REM sleep and markedly increases EEG gamma
oscillations in the Wistar Kyoto rat model of treatment-resistant depression
Sandor Kantor a, b, ⁎, Michael Lanigan a, c, Lauren Giggins a, Lisa Lione c, Lilia Magomedova b,
Inés de Lannoy b, Neil Upton a, Mark Duxon a, b
a Transpharmation Ltd, 2 Royal College Street, London NW1 0NH, United Kingdom
b Transpharmation Canada, Fergus, ON N1M 2W8, Canada
c University of Hertfordshire, College Lane, Hatfield, Herts AL10 9AD, United Kingdom
A R T I C L E  I N F O
Keywords:
Wistar–Kyoto rats
Treatment-resistant depression
NMDA receptor antagonist
Ketamine
REM sleep
EEG gamma power
A B S T R A C T
Wistar–Kyoto (WKY) rats exhibit depression-like characteristics and decreased sensitivity to monoamine-based
antidepressants, making them a suitable model of treatment-resistant depression (TRD). Ketamine has emerged
recently as a rapidly acting antidepressant with high efficacy in TRD. Our aim was to determine whether sub-
anaesthetic doses of ketamine can correct sleep and electroencephalogram (EEG) alterations in WKY rats and
whether any ketamine-induced changes differentially affect WKY rats compared to Sprague-Dawley (SD) rats.
Thus, we surgically implanted 8 SD and 8 WKY adult male rats with telemetry transmitters and recorded their
EEG, electromyogram, and locomotor activity after vehicle or ketamine (3, 5 or 10 mg/kg, s.c.) treatment. We
also monitored the plasma concentration of ketamine and its metabolites, norketamine and hydroxynorketamine
in satellite animals. We found that WKY rats, have an increased amount of rapid eye movement (REM) sleep,
fragmented sleep-wake pattern, and increased EEG delta power during non-REM sleep compared to SD rats. Keta-
mine suppressed REM sleep and increased EEG gamma power during wakefulness in both strains, but the gamma
increase was almost twice as large in WKY rats than in SD rats. Ketamine also increased beta oscillations, but only
in WKY rats. These differences in sleep and EEG are unlikely to be caused by dissimilarities in ketamine metabo-
lism as the plasma concentrations of ketamine and its metabolites were similar in both strains. Our data suggest
an enhanced antidepressant-like response to ketamine in WKY rats, and further support the predictive validity of
acute REM sleep suppression as a measure of antidepressant responsiveness.
1. Introduction
Major depressive disorder (MDD) is a complex mental disorder char-
acterised by changes in mood and cognition that affects approximately
21 million people in the United States alone [1]. Disrupted sleep is one
of the most common early features of depression that often precedes the
core symptoms of the disease, and it is often an early sign of relapse pre-
ceding mood episode recurrences [2–4]. Still, sleep disturbances are of-
ten viewed as associated symptoms of the diseases, assuming that they
would resolve with the treatment of core symptoms. The available clini-
cal evidence suggests, however, that sleep disturbances contribute to
the disease process and their treatment improves the outcomes of de-
pression [5,6].
About 50% of people diagnosed with MDD receive conventional
monoamine-based antidepressant treatment, but around 30% of them
do not show improvement in their symptoms despite trying multiple,
structurally distinct antidepressants [7]. These patients constitute the
so-called ‘Treatment Resistant Depression (TRD)’ population. The N-
methyl-D-aspartate (NMDA)-receptor antagonist ketamine has emerged
recently as a rapidly acting antidepressant with high efficacy in TRD
[8–10]. Although the precise mechanism of action of ketamine remains
elusive [11], a growing body of clinical evidence suggests glutamater-
gic dysfunction in depression [12–14].
Endogenous animal models of TRD could provide a valuable tool for
investigating novel therapeutics against TRD. One such promising
model is the Wistar–Kyoto (WKY) rat, which shows depression-like neu-
Abbreviations: EEG, electroencephalogram; EMG, electromyogram; MDD, major depressive disorder; NMDA, N-methyl-D-aspartate; TRD, treatment-resistant
depression; LMA, locomotor activity; WKY, Wistar-Kyoto; SD, Sprague-Dawley; PRF, pontine reticular formation; LDT, laterodorsal tegmental; PPT,
pedunculopontine tegmental; REM, rapid eye movement; NREM, non-REM; GABA, gamma aminobutyric acid; s.c, subcutaneous
⁎ Correspondence to: Transpharmation Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, United Kingdom.
E-mail address: sandor.kantor@transpharmation.co.uk (S. Kantor).
https://doi.org/10.1016/j.bbr.2023.114473
Received 9 November 2022; Received in revised form 10 March 2023; Accepted 20 March 2023
0166-4328/© 20XX
Note: Low-resolution images were used to create this PDF. The original images will be used in the final composition.
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S. Kantor et al. Behavioural Brain Research xxx (xxxx) 114473
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Fig. 1. Ketamine increased the amount of wakefulness and reduced REM sleep, but it had no effect on locomotor activity (LMA) in SD and WKY rats. The hourly
amounts of wake (A, B), NREM sleep (C, D), REM sleep (E, F) and LMA (G, H) are shown in SD (A, C, E, G) and WKY (B, D, F, H) rats after treatment (arrow) with ket-
amine (3, 5 and 10 mg/kg; s.c.) or its vehicle (0 mg/kg, s.c.). Data are presented as mean ± SEM. *P < 0.05 vs vehicle treatment (Dunnett post-test).
◀
rochemical and behavioural characteristics as well as decreased sensi-
tivity to monoamine-based antidepressants [15–19]. Compared to other
strains such as Wistar, Fisher 344, Lewis and Sprague-Dawley (SD), the
WKY rats display increased stress responses and anhedonia, learned
helplessness, and increased depressive-like behaviours, and they are
less responsive to monoamine-based antidepressants in the forced swim
test of depression [16,18–23]. WKY rats also display depression-like
sleep abnormalities that are less affected by serotonergic or noradrener-
gic antidepressants [24,25]. Alteration in glutamatergic signalling may
also contribute to the depressive phenotype of WKY rats. Decreased
NMDA receptor binding has been reported in WKY rats in brain regions
also implicated in depression such as the prefrontal cortex, caudate
putamen, nucleus accumbens and hippocampus [23,26,27].
The aim of the current study was to determine whether subanaes-
thetic doses of the glutamatergic NMDA receptor antagonist ketamine
can correct the sleep and electroencephalogram (EEG) alterations seen
in a rodent model of TRD (WKY) and whether the ketamine-induced
changes in sleep and EEG differentially affect WKY rats in comparison
to SD rats.
2. Methods
All experiments were conducted under the authority of United King-
dom Animals (Scientific Procedures) Act 1986 and with the approval of
Royal Veterinary Colleges’ Animal Welfare and Ethical Review Body
and are in compliance with the ARRIVE guidelines. Adult male Sprague
Dawley (SD; 200–250 g, Charles River, UK), and Wistar Kyoto (WKY;
200–250 g; Envigo, UK) rats were pair housed within their own strain
with environmental enrichment and maintained on a standard 12 h
light/dark cycle with food/water available ad libitum.
2.1. Surgery and EEG/EMG recordings
After a minimum of 2 weeks of acclimatisation to environmental
conditions, we implanted 8 SD and 8 WKY rats with telemetry transmit-
ters, and with EEG and electromyogram (EMG) electrodes under isoflu-
rane anaesthesia (2–5%). Specifically, we placed a telemetry transmit-
ter (HD-S02; Data Sciences International, St. Paul, MN, USA) into the
peritoneal cavity and the tip of EEG leads epidurally over the frontal
(2 mm anterior / 1 mm lateral to Bregma) and parietal (0 mm anterior
/ 1.5 mm lateral to Lambda) cortices of the left hemisphere for fron-
toparietal EEG recordings. EMG signals were acquired by a second pair
of stainless-steel spring wires inserted into the neck extensor muscles.
After a recovery period of 7–10 days, the rats were placed individu-
ally in recording chambers in a sound-attenuated room with a 12:12 h
light-dark cycle, constant temperature (21 ± 1 °C), with food and wa-
ter available ad libitum, and EEG, EMG and locomotor activity (LMA)
were recorded for 0.5 h before and 24 h after treatments. LMA was de-
tected by an antenna (RPC-1, Data Sciences International, St. Paul, MN,
USA) placed below the recording cages. EEG/EMG signals were ampli-
fied, analogue filtered (implants’ frequency response: 0.5–100 Hz), dig-
itized (500 Hz), and recorded alongside LMA on a computer for offline
analysis (Spike2; CED, Cambridge, UK).
2.2. Drug treatment
We treated both SD and WKY rats with 3 different doses of keta-
mine [3, 5 and 10 mg/kg, subcutaneously (s.c.); Sigma-Aldrich,
Gillingham, UK] or vehicle (0.9% saline; 2 ml/kg, s.c.) 3 h after light
onset [Zeitgeber time 03:00 (ZT03)]. The different doses of ketamine
and its vehicle were given to SD and WKY rats in a crossover design
and in a randomized order, with 6–8 days between treatments. The
doses were chosen based on the literature [28,29] and our pilot ex-
periments (data not shown).
In addition, we treated a satellite group of SD and WKY rats (n = 9
each) with ketamine (3, 5 and 10 mg/kg, s.c.) and collected blood sam-
ples by venepuncture at 0.5, 1, 2 and 3 h post-treatment for pharmaco-
kinetics profiling of ketamine an its major metabolites. 4 h after keta-
mine treatment, the rats were euthanized by CO2 exsanguination and
terminal blood samples were collected by cardiac puncture into
K2EDTA tubes for plasma isolation.
2.3. Quantification of ketamine and its metabolites by LC-MS/MS
Ketamine is metabolised to norketamine (NK), and norketamine is
further metabolised to the hydroxynorketamine (HNK), and both
metabolites are pharmacologically active [30]. Thus, alongside keta-
mine we also measured the concertation of its metabolites, NK and
HNK, in the plasma samples. We analysed the plasma samples on an AB
Sciex API4000 QTRAP liquid chromatography/mass spectrometry (LC-
MS/MS) system equipped with an ESI source in positive ion mode and
coupled to an Agilent 1200 liquid chromatographic system. The chro-
matographic separation was performed on an Aquasil C18 column
(2.1 × 50 mm, 5 µm) at 25 °C using gradient elution with a 0.8 mL/
min flow rate. The mobile phases A and B were 10 mM ammonium ac-
etate in water and acetonitrile, respectively. The multiple reaction mon-
itoring (MRM) parameters for ketamine and its metabolites, NK and
HNK, with their respective internal standards (IS, shown in brackets)
were: ketamine m/z 238.1 →125.1 (ketamine-d4 IS m/z 242.1 →129.1),
NK m/z 224.1→125.1 (NK-d4 IS m/z 228.1 →129.0) and HNK m/z
240.1→125.0 (fenoterol IS m/z 304.1 →107.1). The calibration dy-
namic range was 0.2–5000 ng/mL for ketamine and 0.5–5000 ng/mL
for NK and HNK.
2.4. Data analysis and statistics
EEG and EMG signals were resampled at 256 Hz, digitally filtered
(EEG: 0.5–100 Hz; EMG: 5–100 Hz), and semi-automatically scored as
wake, rapid eye movement (REM) sleep or non-REM (NREM) sleep, in
10-s epochs using SleepSign (Kissei Comtec, Matsumoto, Japan). An ex-
perienced scorer, blinded to treatment condition, visually inspected
these preliminary scorings and made corrections when appropriate. We
then measured the duration and number of bouts, and calculated the
time spent in each behavioural state after treatment. To measure the
propensity for NREM and REM sleep, we also calculated the latency to
NREM and REM sleep onset for each rat. This was measured from the
time of drug administration to the first six continuous 10 s epochs
scored as NREM sleep or to the first three continuous 10 s epochs scored
as REM sleep, respectively.
To reveal the changes in the frequency content of the recorded sig-
nal, we also performed a power spectral analysis of the EEG. EEG power
spectra were computed for artifact-free 2-s epochs in the 0.5–100 Hz
frequency range by fast Fourier transformation with a frequency resolu-
tion of 0.5 Hz. Before fast Fourier transformation, a window weighting
function (Hanning) was applied. Epochs with movement-induced and
other artefacts were discarded on the basis of the polygraph records.
The values of consecutive 2-s EEG epochs per vigilance states were av-
eraged over 2 h after the treatments. To reveal the changes in specific
frequency bands after the treatments, we compared the discrete
changes in the delta (0.5–4 Hz), theta (4–10 Hz), sigma (10–15 Hz),
beta (15–30 Hz) and gamma band (30–100 Hz) of the EEG during
wakefulness and NREM sleep. Due to the limited number of REM sleep
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Fig. 2. Ketamine suppressed the abnormally increased amount of REM sleep in
WKY rats in a dose-dependent manner. Changes in the percentage of wakeful-
ness (A), NREM sleep (B), REM sleep (C) and LMA (D) are shown in SD and
WKY rats during the first 4 h after treatment with vehicle (0 mg/kg, s.c.) or ket-
amine (3, 5 and 10 mg/kg; s.c.). Data are presented as mean ± SEM. *P < 0.05
vs vehicle treatment (Dunnett post-test); #P < 0.05 SD vs WKY of the same
treatment (Sidak post-test).
◀
episodes available, no power spectral analysis was performed on REM
sleep EEG during the 2 h post-treatment period. In addition to vigilance
state specific analysis, we also calculated the spectral values in the
gamma frequency band regardless of vigilance states 0.5 h before and
5 h after treatments. For graphical presentation, we normalized the
gamma power values of ketamine (3, 5 and 10 mg/kg, s.c.) treatment to
the values of vehicle treatment in the same animal (100%).
To compare statistically the vigilance state parameters and raw EEG
power spectral values, we used multivariate analysis of variance with
repeated measures or mixed effects model where values were missing,
and that was followed by Dunnett or Šidak tests for post hoc compar-
isons (Prism 9.4; GraphPad, San Diego, CA USA). The results were con-
sidered statistically significant at p < 0.05. All results were expressed
as means ± SEM.
3. Results
3.1. Ketamine suppresses REM sleep in WKY rats
At subanaesthetic doses, ketamine increased the time spent awake
at the expense of NREM and REM sleep in both SD and WKY rats mainly
within the first 4 h after the treatment [drug effects: F(2.21,15.48) = 8.69,
p < 0.01 (Fig. 1A); F(2.15,15.06) = 11.11, p < 0.01 (Fig. 1B); F(2.25,15.75)
= 5.99, p < 0.01 (Fig. 1C); F(2.35,16.48) = 6.17, p < 0.01 (Fig. 1D);
F(2.22,15.51) = 9.130, p < 0.01 (Fig. 1E); F (1.76,12.31) = 7.68, p < 0.01
(Fig. 1F)]. Although the amounts of wakefulness and NREM sleep were
similar in SD and WKY rats after vehicle treatment (Fig. 2A and B),
WKY rats had 41% more REM sleep than SD rats early in the light pe-
riod (ZT03–07), which is the passive phase in the rats [strain effect:
F(1,14) = 13.38, p < 0.01 (Fig. 2C)]. In addition, WKY rats had a frag-
mented sleep-wake pattern that was shown by the shorter and in-
creased number of NREM sleep bouts (−35% and +47%, respectively)
and by the increased number of wake bouts (+67%) compared to SD
rats [strain effect: F(1,14) = 39.42, p < 0.01; F(1,14) = 89.14, p < 0.01;
and F(1,14) = 85.60, p < 0.01, respectively (Table 1)].
Acute treatment with subanaesthetic doses of ketamine decreased
the amount of REM sleep in both SD and WKY rats in a dose-dependent
manner [drug effect: F(3,42) = 18.89, p < 0.01 (Fig. 2C)]. Ketamine re-
duced the amount of REM sleep by supressing its initiation, as shown by
the dose-dependent increase in the latency to REM sleep onset, and by
the decrease in REM sleep bout numbers in both SD and WKY rats [drug
effects: F(3,42) = 34.23, p < 0.01 (Fig. 3B) and F(3,42) = 23.21, p
< 0.01 (Table 1), respectively]. Besides supressing REM sleep, keta-
mine also increased the time spent awake and reduced the amount of
NREM sleep in a dose-dependent manner during the first 4 h after the
treatment in both SD and WKY rats [drug effects: F(3,45) = 18.33, p
< 0.01 and F(3,42) = 10.18, p < 0.01, respectively (Fig. 2A and B)]. Ke-
tamine supressed the initiation of NREM sleep, as shown by the dose-
dependent decrease in the number of NREM sleep bouts in both SD and
WKY rats [drug effect: F(3,42) = 20.44, p < 0.01 (Table 1)]. In WKY
rats, ketamine also increased the latency to NREM sleep onset [drug ef-
fects: F(3,42) = 5.89, p < 0.01 (Fig. 3A)]. Interestingly in SD rats, keta-
mine (10 mg/kg, s.c.) not only decreased the number of wake, NREM
and REM sleep bouts, but it also increased their mean duration produc-
ing a more consolidated sleep-wake pattern [drug effects: F(3,42)
= 11.35, p < 0.01; F(3,42) = 8.80, p < 0.01 and F(3,42) = 3.87, p
< 0.05, respectively (Table 1)]. In contrast, the fragmented sleep-wake
behaviour seen in WKY rats improved only partially after ketamine
treatment as ketamine decreased the number of NREM and REM sleep
bouts, but it had no effect on their durations [drug effects: F(3,42)
= 20.44, p < 0.01 and F(3,42) = 23.21, p < 0.01, respectively (Table
1)].
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Table 1
Vigilance state parameters in SD and WKY rats after vehicle or ketamine treatment.
Strain SD WKY
Dose Vehicle 3 mg/kg 5 mg/kg 10 mg/kg Vehicle 3 mg/kg 5 mg/kg 10 mg/kg
Mean bout duration (min)
WAKE 13.4 ± 2.8 14.9 ± 1.1 21.9 ± 2.4 39.4 ± 8.0a 6.7 ± 0.6 7.7 ± 0.7 9.3 ± 0.9b 13.8 ± 1.3b
NREM sleep 20.2 ± 1.3 19.3 ± 1.2 23.7 ± 2.3 29.5 ± 2.8a 13.1 ± 1.0b 12.5 ± 0.8b 15.0 ± 1.1b 14.9 ± 0.9b
REM sleep 7.6 ± 0.3 7.9 ± 0.6 8.6 ± 0.7 10.4 ± 1.0a 9.7 ± 0.7 9.5 ± 0.4 10.9 ± 0.3 9.7 ± 0.8
Number of bouts
WAKE 33.1 ± 2.6 34.6 ± 2.4 27.1 ± 1.4 20.6 ± 3.0a 55.4 ± 3.6b 59.4 ± 5.7b 50.3 ± 3.3b 44.9 ± 2.6b
NREM sleep 45.3 ± 2.9 43.3 ± 2.6 32.8 ± 1.7a 25.4 ± 2.8a 66.4 ± 3.2b 68.4 ± 3.8b 56.3 ± 3.6a,b 50.3 ± 2.8a,b
REM sleep 19.0 ± 1.8 15.0 ± 1.3 11.3 ± 0.6a 7.5 ± 0.8a 21.1 ± 1.6 17.8 ± 1.8 14.1 ± 1.5a 11.4 ± 1.4a
Mean duration and number of bouts in each state during the first 4 h after vehicle or ketamine treatment. Results are shown as mean ± SEM. aP < 0.05 compared to
vehicle (Veh) treatment of the same strain. bP < 0.05 SD vs WKY of the same treatment (Dunnett post-test).
Fig. 3. Treatment with ketamine produced a dose-dependent increase in REM
sleep latency in both SD and WKY rats. Changes in latencies to NREM (A) and
REM (B) sleep onset are shown in SD and WKY rats after treatment with vehi-
cle (0 mg/kg, s.c.) or ketamine (3, 5 and 10 mg/kg; s.c.). Data are presented as
mean ± SEM. *P < 0.05 vs vehicle treatment (Dunnett post-test).
Although ketamine may induce robust behavioural changes in ro-
dents especially at higher doses [31], it did not alter LMA in either SD
or WKY rats in this study at the doses tested (Figs. 1G and H, and 2D).
3.2. Eminent increase in EEG gamma oscillations in WKY rats after
ketamine treatment
Since ketamine virtually abolished REM sleep in both SD and WKY
rats for about two hours post- treatment in our study (Fig. 1E and F), we
analysed the changes in EEG power spectra only during wakefulness
and NREM sleep. Apart from the increased delta oscillations in WKY
rats during NREM sleep [strain effect: F(1,14) = 13.12, p < 0.01 (Fig.
4B)], there were no other differences in EEG power spectra between the
two strains. Ketamine, however, differentially affected the EEG oscilla-
tions in SD and WKY rats (Figs. 4 and 5). While ketamine had no effect
on EEG theta power in any of the strains during wakefulness (Fig. 4 A),
it approximately doubled the wake-EEG gamma power in SD rats
(+110%), and it more than tripled it in WKY rats (+244%) within the
first two hours post-treatment [drug x strain interaction: F(3,42) = 7.53,
p < 0.01 (Fig. 4E)]. In addition, ketamine also increased beta power in
the wake-EEG of WKY rats, but not of SD rats [drug effect: F(3,42)
= 5.822, p < 0.01 (Fig. 4 C)]. Ketamine’s effect on EEG power spectra
was not only strain, but also vigilance state specific. While ketamine in-
creased gamma and supressed sigma power in SD rats during NREM
sleep, it had no effect on EEG oscillations in WKY rats, leaving the in-
creased delta power in WKY rats largely unaffected during NREM sleep
[drug effects: F(3,42) = 3.34, p < 0.05 and F(3,42) = 9.93, p < 0.01, re-
spectively (Fig. 4B, D and F)].
To examine whether the strain specific differences in the ketamine-
induced gamma power increase are due to a delayed response in SD rats
compared to WKY rats, we also analysed the time course of changes in
this frequency band before and after the treatment and irrespective of
vigilance states. We found that the maximum increase in EEG gamma
power occurred within the 1st h, and extended into the 2nd h post-
treatment in both SD and WKY rats, suggesting a similar pharmacoki-
netics profile of ketamine in the both WKY and SD strains [drug x time
interactions: F(1.34,9.37) = 12.69, p < 0.01 and F(1.151,8.055) = 29.53, p
< 0.01, respectively (Fig. 5A and B)].
3.3. The plasma concentrations of ketamine and its metabolites were
generally similar in both SD and WKY rats after treatment
To determine whether there are strain-specific differences in keta-
mine metabolism, we measured the plasma concentrations of ketamine
and its major metabolites, NK and HNK, during the first 4 h post-
treatment (Fig. 6). The plasma concentrations of ketamine were similar
between SD and WKY rats after all three (3, 5 and 10 mg/kg, s.c.) doses
of the drug and at any measured time points (Fig. 6A-B). Compared to
SD rats, the plasma level of NK increased transiently in WKY rats 0.5 h
after treatment with 5 mg/kg (s.c.) ketamine [time x strain interaction:
F(4,14) = 19.58, p < 0.01 (Fig. 6E)]. WKY rats also had a higher plasma
level of HNK than SD rats 4 h after treatment with the highest (10 mg/
kg, s.c.) dose of ketamine [strain effect: F(1, 4) = 8.691, p < 0.05 (Fig.
6I)], but the plasma levels of both NK and HNK were similar in both
strains at any other measured time points.
4. Discussion
We found that the amount of REM sleep is increased in WKY rats
compared to SD rats. WKY rats also have a fragmented sleep-wake pat-
tern and increased delta oscillations during NREM sleep. Subanaes-
thetic doses of the glutamatergic NMDA receptor antagonist ketamine
decreased the amount of REM sleep in both WKY and SD rats, primarily
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Fig. 4. Ketamine increased high frequency (beta and gamma) EEG oscillations in WKY rats during wakefulness. Changes in wake-EEG theta (A), beta (C) and
gamma power (E) as well as in NREM sleep-EEG delta (B), sigma (D) and gamma power (F) are shown in SD and WKY rats during the first 2 h after treatment with
vehicle (0 mg/kg, s.c.) or ketamine (3, 5 and 10 mg/kg; s.c.). Data are presented as mean ± SEM. *P < 0.05 vs vehicle treatment (Dunnett post-test); #P < 0.05 SD
vs WKY of the same treatment (Šidák post-test).
by supressing its initiation. This was shown by an increase in the la-
tency to REM sleep onset, and a decrease in the number of REM sleep
bouts after ketamine treatment. Ketamine also increased the time spent
awake and EEG gamma power during wakefulness in both SD and WKY
rats, but it had no effect on LMA. More importantly, the ketamine-
induced increase in EEG gamma power was almost twice as high in
WKY rats as in SD rats despite of the generally similar plasma levels of
ketamine and its metabolites, NK and NHK, in both strains.
Alterations in REM sleep, including shortened REM sleep latency
and increased REM sleep amount, are among the most consistent find-
ings in depression [32,33]. Total sleep deprivation as well as selective
REM sleep deprivation rapidly alleviate the symptoms of depression
[34,35]. Furthermore, most antidepressants that are structurally and
pharmacologically different, suppress REM sleep in both healthy volun-
teers and depressed patients early in the treatment, which effect gradu-
ally diminishes after repeated administration of the drug [32]. There-
fore, it has been proposed that acute REM sleep suppression may pre-
dict the therapeutic efficacy of antidepressant drugs [36–40]. In this
study, WKY rats showed an increased amount of baseline REM sleep
compared to SD rats. A similarly increased amount of REM sleep has
been shown in WKY rats compared to SD and Wistar rats previously
[24,25]. Acute treatment with antidepressants (including ketamine) in-
hibits REM sleep in both SD and Wistar rats as we have shown here and
others demonstrated previously [25,41,42]. Similarly, ketamine re-
duces the amount of REM sleep in patients with severe burns but inter-
estingly, increases it in MDD patients [43,44]. In contrast to SD or Wis-
tar rats, WKY rats are less sensitive to the suppression of REM sleep by
serotonin reuptake inhibitors or tricyclic antidepressants [25,41]. Sub-
anaesthetic doses of ketamine, however, effectively supressed REM
sleep in WKY rats during the first 3–4 h post-treatment in this study that
may indicate antidepressant-like properties of the drug in a rodent
model of TRD. Indeed, ketamine’s antidepressant-like effect has been
demonstrated in the forced swim test, learned helplessness and sucrose
preference test in WKY rats previously [28,29,45]. Therefore, these
data together with our results further support the predictive validity of
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Fig. 5. The ketamine-induced increase in EEG gamma power is nearly twice as high in WKY rats than in SD rats. The time course of changes in EEG gamma power
are shown in SD (A) and WKY rats (B) during the first 5 h after treatment with vehicle (0 mg/kg, s.c.) or ketamine (3, 5 and 10 mg/kg; s.c.). Data are presented as
mean ± SEM. *P < 0.05 vs vehicle treatment (Dunnett post-test).
Fig. 6. The pharmacokinetics profile of ketamine and its metabolites is similar between SD and WKY rats. The time course of changes in the plasma concen-
tration of ketamine (A, B, C), norketamine (NK; D, E, F) and hydroxynorketamine (HNK; G, H, I) are shown in SD (solid line) and WKY (dotted line) rats af-
ter treatment with ketamine (3, 5 and 10 mg/kg; s.c.). Data are presented as mean ± SEM. *P < 0.05 SD vs WKY of the same treatment (Šidák post-test).
acute REM sleep suppression as an index of antidepressant-like efficacy
in TRD.
Although the precise mechanism through which subanaesthetic
doses of ketamine suppresses REM sleep is not known, most likely this is
achieved by acting on NMDA receptors expressed in neurons of the
brain stem. It is well established that REM sleep is generated in the
brain stem by the interaction between the glutamatergic neurons of
pontine reticular formation (PRF) and cholinergic neurons of laterodor-
sal/pedunculopontine tegmental (LDT/PPT) nuclei [46]. Injection of
muscarinic receptor agonists into PRF induces REM sleep [47]. Simi-
larly, low doses of glutamate injected into PPT increase REM sleep, but
high doses of it increase wakefulness presumably through different
mechanisms [48,49]. Furthermore, systemic administration of keta-
mine reduces acetylcholine release in the PRF [50]. Therefore, it seems
most likely that the subanaesthetic doses of ketamine supressed REM
sleep in this study by indirectly reducing the cholinergic tone in the
PRF.
Both clinical and pre-clinical studies have demonstrated that acute
treatment with subanaesthetic doses of ketamine and with its metabo-
lite, HNK, induces robust increase in EEG gamma power and rapid anti-
depressant effects in TRD, but the potential causal relationship between
the two has not been fully explored yet [51–56]. Although the acute in-
crease in gamma oscillations is primarily linked to the transient disso-
ciative and psychotomimetic effects of ketamine [57,58], the gamma
increase is also viewed as a potential marker of synaptic potentiation
and neural network adaptation, which are necessary to develop the sus-
tained antidepressant effects of ketamine [59,60]. Gamma oscillations
are primarily generated by networks of glutamatergic and gamma
7
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S. Kantor et al. Behavioural Brain Research xxx (xxxx) 114473
aminobutyric acid (GABA)-ergic interneurons of the cortex and con-
trolled by cortically projecting GABA-ergic neurons of the basal fore-
brain [61]. Numerous studies found alterations in gamma oscillations
in both MDD and bipolar disorder [62–69]. Although the functional sig-
nificance of gamma power increase in the antidepressant effect of keta-
mine is unknown, depressed patients with lower baseline gamma power
levels have better antidepressant responses if they also have a large in-
crease in gamma power after ketamine treatment [62]. WKY rats show
reduced EEG gamma power at REM sleep transitions and decreased
gamma coherence between brain regions implicated in depression that
is normalized by ketamine [63,64]. In our study, the baseline EEG
gamma power was similar in SD and WKY rats during wakefulness and
NREM sleep. However, the ketamine-induced increase in EEG gamma
power was almost twice as high in WKY rats as in SD rats. Since the
plasma concentrations of ketamine and its metabolites were similar be-
tween both strains, it is unlikely that the eminent increase in EEG
gamma oscillations is due to altered ketamine metabolism in WKY rats.
Thus, we hypothesize that the larger increases in gamma power could
indicate an enhanced antidepressant-like response to ketamine in the
WKY strain.
Subanaesthetic doses of ketamine reduce the symptoms of depres-
sion within hours after the treatment, and the antidepressant effects
may last three to seven days in TRD patients [8–10]. This acute anti-
depressant effect of ketamine, however, can be extended for up to 6
weeks with repeat doses [73–75]. Given its short half-life (~45 min),
ketamine’s sustained antidepressant efficacy, however, cannot simply
be attributed to the acute changes induced by the drug. A growing
body of evidence suggests that the sustained antidepressant effects of
ketamine are achieved by reversing the glutamatergic and GABAergic
dysfunction found in patients with depression [10,12–14]. It has been
suggested that ketamine increases glutamate neurotransmission in
the prefrontal cortex by blocking the NMDA receptors on GABAergic
neurons, which leads to increased synaptic plasticity through down-
stream molecular cascades and long-term changes in neural circuits
[76,77].
Apart from REM sleep alterations, depressed patients also have
frequent nocturnal awakenings resulting in fragmentated sleep and
poor sleep efficiency [78]. In our study, WKY rats had fragmented
NREM sleep as shown by the shorter and increased number of NREM
sleep bouts compared to SD rats. WKY rats also had increased EEG
delta power during NREM sleep compared to SD rats that could re-
flect an increased propensity for sleep resulting from frequent awak-
enings [79]. However, apart from decreasing the number of NREM
sleep bouts, ketamine had no effect on either EEG delta power or
NREM sleep bout duration in WKY rats, suggesting that ketamine and
its analogues may not address all aspects of disordered sleep in de-
pression, and that additional treatment may be required. This is par-
ticularly important as untreated sleep disturbances are seen as risk
factors for developing depression or relapse [80].
5. Conclusions
We show for the first time that the increased amount of REM sleep in
WKY rats is supressed by subanaesthetic doses of ketamine, and this
could be an indicative of antidepressant-like efficacy of the drug in
TRD. Although ketamine efficiently supresses REM sleep, it can only
partially correct the fragmented NREM sleep seen in WKY rats suggest-
ing that an adjuvant therapy may be required to address all aspects of
the disrupted sleep seen in depression. Furthermore, we show that sub-
anaesthetic doses of ketamine induce a more robust increase in EEG
gamma power in WKY rats than in SD rats that is unlikely to be caused
by strain-specific differences in ketamine metabolism. This eminent in-
crease in EEG gamma oscillations may suggest an enhanced antidepres-
sant response to ketamine in WKY rats. Thus, the ketamine-induced
changes in sleep and EEG in WKY rats shown here may serve as key
translational tools in an effort to discover novel therapeutics against
TRD.
Funding
ML is a PhD student of the Hertfordshire Knowledge Exchange
Programme supported by the Hertfordshire Local Enterprise Partner-
ship, European Regional Development Fund and Transpharmation
Ltd.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
We would like to extend our sincere thanks to Dr Guy Higgins for his
valuable advice, guidance and practical suggestions. We also thank Ju-
lia Izhakova for her technical assistance.
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