Research report 239

Validation of chronic mild stress in the Wistar-Kyoto rat as an

animal model of treatment-resistant depression
Paul Willnerb, Piotr Grucaa, Magdalena Lasona, Katarzyna Tota-Glowczyka,
Ewa Litwaa, Monika Niemczyka and Mariusz Pappa

A recent review proposed four criteria for an animal model responded to drug treatment with an increase in sucrose
of treatment-resistant depression (TRD): a phenotypic intake but WKY were nonresponsive to drug treatment on all
resemblance to a risk factor for depression; enhanced three behavioural tests. With one exception, DBS reversed
response to stress; nonresponse to antidepressant drugs the anhedonic, anxiogenic and dyscognitive effects of CMS
and response to treatments effective in TRD, such as deep in all groups of WKY rats. In a further experiment, subacute
brain stimulation (DBS) of the prefrontal cortex or ketamine. ketamine (10 mg/kg) also normalized behaviour on all three
Chronic mild stress (CMS) provides a valid model of tests. We conclude that WKY rats subjected to CMS meet all
depression; the Wistar-Kyoto (WKY) rat is considered to be four criteria for a valid model of TRD, and provide a basis for
nonresponsive to antidepressant drugs. Here, we applied studying the mechanism of action of DBS. Behavioural
CMS to WKY rats. WKY and Wistar rats were exposed to Pharmacology 30:239–250 Copyright © 2018
CMS, then treated with saline, imipramine, citalopram or Wolters Kluwer Health, Inc. All rights reserved.
venlafaxine. After 5 weeks of CMS and 3 weeks of drug Behavioural Pharmacology 2019, 30:239–250
treatment, all WKY groups were implanted unilaterally with
DBS electrodes in the prefrontal cortex, and examined in Keywords: antidepressant drugs, deep brain stimulation, ketamine,
prefrontal cortex, treatment-resistant depression, Wistar-Kyoto rat
sucrose intake, elevated plus maze (EPM; decreased
a
entries and time in the open arms) and novel object Department of Pharmacology, Polish Academy of Sciences, Institute of
Pharmacology, Krakow, Poland and bDepartment of Psychology, Swansea
recognition (decreased exploration) tests, following 2 × 2 h University, Swansea, UK
of DBS. CMS decreased sucrose intake, open arm entries
Correspondence to Mariusz Papp, PhD, Department of Pharmacology, Polish
on the EPM, and object recognition. Relative to Wistars, WKY Academy of Sciences, Institute of Pharmacology, 12 Smetna Street, 31-343
rats showed evidence of increased emotionality in the EPM Krakow, Poland
E-mail: nfpapp@cyfronet.pl
and novel object recognition tests, and a greater impact of
CMS on body weight gain and open arm entries. Wistars Received 23 May 2018 Accepted as revised 12 July 2018

Introduction two decades have seen remarkable progress in under-

It was identified almost 20 years ago that neuropsychia- standing the pathophysiology of depression, much of it
tric disorders carry by far the greatest global burden of based on the use of animal models of depression (Willner
disease, and that the largest single burden is carried by et al., 2013; Belzung et al., 2015), but this research has
people with major depression (Greden 2001; Ferrari et al., failed to discover novel drugs to extend the reach of
2013), which is projected to become the leading cause of antidepressant therapy into the populations of patients
global disease burden by 2030 (Lépine and Briley, 2011). who show little or no benefit. In relation to the problem
The most severely disabled group are those with of TRD specifically, current animal models of depression
treatment-resistant depression (TRD) (Greden, 2001). are not fit for purpose (Hendrie et al., 2013; Willner and
Prevalence studies typically report that in excess of 50% Belzung, 2015). Indeed, progress towards drug therapies
of depressed patients are resistant to antidepressant drug for TRD may require a paradigm shift towards models in
treatment (Nemeroff, 2007; Thase, 2011; Thomas et al., which conventional antidepressants are ineffective
2013). (Willner et al., 2013, 2014; Willner and Belzung, 2015).
But how to achieve such models when responsiveness to
Until very recently, there have also been no major antidepressant drug treatment is considered a sine qua
innovations in the identification of novel targets to non for a valid animal model of depression (Willner,
improve recovery rates, and where such innovations have 1984)?
been suggested by preclinical data, either they have not
progressed into the clinic, or clinical trials have been A potential way forward is provided by the recent dis-
unsuccessful. As a result, the pharmaceutical industry has covery of novel treatments that provide rapid and
abandoned or greatly scaled back its antidepressant drug sometimes prolonged relief to a high proportion of TRD
discovery programmes (Hendrie et al., 2013). This situa- sufferers. One such is high-frequency electrical stimula-
tion is particularly frustrating considering that the past tion of the anterior cingulate cortex and certain other
0955-8810 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/FBP.0000000000000431

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240 Behavioural Pharmacology 2019, Vol 30 No 2&3

brain areas [deep brain stimulation (DBS)]: over 50% of While important as a proof-of-principle in demonstrating
patients treated with DBS show a greater than 50% that DBS has antidepressant effects in animals that have
improvement in depression scores a year later, and DBS failed to respond to antidepressant drugs, these experi-
causes a normalization of functional brain activity in ments (Dournes et al., 2013; Papp et al., 2018) are inef-
treatment responders (Mayberg, 2009; Hamani et al., ficient, insofar as it is necessary to start with a large cohort
2011; McGrath et al., 2013; Delahoye and Holtzheimer, of animals in order to identify the minority, typically,
2014). Rapid improvements in antidepressant-refractory around 25–30% (Bisgaard et al., 2007; Bergström et al.,
patients are also reported following single intravenous 2007; Nieto-Gonzalez et al., 2015), of nonresponders to
infusions of the N-methyl-D-aspartate-receptor antagonist drug treatment. In order to meet the ethical requirement
ketamine (Berman et al., 2000; Zarate et al., 2006; to minimize the number of animals used, these experi-
Diazgranados et al., 2010). These innovations suggest two ments should properly employ a procedure in which the
major criteria that could be used to assess the predictive proportion of responders is much higher, that is, an ani-
validity of an animal model of TRD: the absence, in a mal model of TRD.
well-validated animal model of depression, of anti-
Willner and Belzung (2015) reviewed and evaluated 18
depressant drug effects, combined with a positive effect
potential animal models of TRD against the four criteria
of DBS and/or ketamine.
outlined above. The evidence was generally weak, and
Two further validation criteria for an animal model of only three models scored on all four criteria. Of those
TRD have been suggested (Willner and Belzung 2015). three front-runners, only one, the Wistar-Kyoto (WKY)
An obvious criterion, addressing the face validity of the rat, is commercially available. Here we have imple-
model, is the presence of some physiological or beha- mented the CMS model in the WKY rat and tested the
vioural phenotypic similarity between the model and effectiveness of three antidepressant drugs with different
human TRD. Less obvious, and relevant to the construct mechanisms of action, as well as DBS of the vm-PFC, to
validity of the model, is the proposal that an animal restore behaviour in a standard battery of behavioural
model of TRD should show a heightened response to tests (Papp et al., 2016, 2017a, 2018). For comparison, and
stress. This suggestion arises from the observation that, to confirm the appropriateness of the drug doses used, we
with the notable exception of female sex, common factors also tested the same drugs, and DBS, in antidepressant-
are associated clinically with sensitivity to stress and responsive Wistar rats.
resistance to antidepressant treatment, across a range of
developmental, personality, physiological and genetic Methods
variables (Willner et al., 2013, 2014). Subjects
The chronic mild stress (CMS) model is the most exten- Male Wistar (N = 96) and WKY (N = 152) rats were
sively validated animal model of depression (Willner, 1997, obtained from Charles River (Sulzfeld, Germany) at the
2017). Rats or mice are subjected over several weeks to a age of 4 (Wistar) or 5 (WKY) weeks, and weighing around
constant exposure to varying mild stressors, resulting in a 100 g. Body weight was next measured at the time of the
loss of responsiveness to rewards (anhedonia), typically final baseline sucrose intake test (see below) when the
studied as a decrease in the consumption of a weak sucrose animals were 11 and 12 weeks old, respectively. Except
solution, along with a panoply of other behavioural for the first 10 days after arrival when the animals were
(Willner, 1997, 2017) and physiological (Hill et al., 2012; housed in groups of 10 and as described below, they were
Willner, 2017) changes characteristic of depression, which housed singly with free access to food and water, and
respond to chronic but not acute treatment with anti- maintained on a 12-h light/dark cycle (lights on at
depressant drugs (Willner, 1997, 2017). As in the clinic, 08.00 h) in conditions of constant temperature (22 ± 20°C)
studies using the CMS model have shown that DBS of the and humidity (45 ± 5%). All procedures used conformed
ventro-medial prefrontal cortex (vm-PFC) causes a rapid to the rules and principles of EEC Directive 86/609 and
reversal of CMS-induced anhedonia and other depression- were approved by the Bioethical Committee at the
related behaviours (Hamani et al., 2012; Dournes et al., Institute of Pharmacology, Polish Academy of Sciences,
2013; Veerakumar et al., 2014; Lim et al., 2015). However, Krakow, Poland.
the translational value of these data in relation to TRD is
uncertain because the CMS model also responds reliably to Experimental design
antidepressant drug treatment. However, a study by A first series of four experiments in Wistars was con-
Dournes et al. (2013) reported that DBS normalized ducted, for comparison purposes, to confirm the effects of
behaviour in a subset of CMS-exposed mice that had failed CMS on sucrose intake and their reversal by anti-
to respond to chronic treatment with fluoxetine. We have depressant drug treatment. Each experiment included
recently reported similar findings: DBS had antianhedonic, control (CON) and CMS subgroups (n = 8/group), and ran
anxiolytic and procognitive effects in groups of Wistar rats for a total of 6 weeks, with weekly sucrose intake tests
that had failed to respond to chronic treatment with anti- and concomitant daily administration, in different
depressant drugs (Gruca et al., 2017; Papp et al., 2018). experiments, of saline (SAL, 1 ml/kg, intraperitoneally),

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 CMS in WKY as animal model of TRD Willner et al. 241

Fig. 1

(a) Timelines for CMS Electrode implantation Behavioural tests

Weeks of stress

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6

Weeks of drug treatment

(b) Timelines for behavioural tests in Week 8 (DBS experiments)

DBS morning DBS morning DBS morning

Sucrose test EPM NOR adapt NORT

DBS evening DBS evening NOR adapt DBS evening

Monday Tuesday Wednesday Thursday Friday Saturday

(c) Timelines for behavioural tests in Week 4 (ketamine experiment)

EPM

Sucrose test NOR adapt NOR adapt NORT Sucrose test

Ketamine Ketamine Ketamine Ketamine Ketamine Ketamine Ketamine

Monday Tuesday Wednesday Thursday Friday Saturday Monday Tuesday

Experimental timelines. The week number refers to the week before the number: that is, week 1 = 0–1, 2 = 1–2, etc. (a) Timelines for chronic mild
stress (CMS). Before the onset of CMS (week 0), animals were adapted for 3 weeks to laboratory and housing conditions followed by 6 weekly
baseline sucrose consumption tests (not shown). CMS (or no stress for the control group) was administered for 8 weeks, with drug treatment
(saline, imipramine, citalopram or venlafaxine, 10 mg/kg, each intraperitoneally) administered daily for 6 weeks, beginning after 2 weeks of CMS.
(b, c) Timelines for behavioural tests conducted in the final week 8 and 4, respectively. See text for further details. DBS, deep brain stimulation;
EPM, elevated plus maze; NOR, novel object recognition; NORT, novel object recognition test.

imipramine (IMI, 10 mg/kg, intraperitoneally), citalopram experiments (details below), followed by a final sucrose
(CIT, 10 mg/kg, intraperitoneally), or venlafaxine (VEN, intake test (Fig. 1c).
10 mg/kg, intraperitoneally), in weeks 3–6 (Fig. 1a).
At the end of the study, brain and blood samples were
A second series of four experiments in WKY began iden- harvested for biochemical analyses, which will be repor-
tically, but continued with two additional weeks of CMS ted separately.
and drug treatment (n = 16/group). In week 5 of CMS all
animals were implanted with an electrode in the vm-PFC,
and the sucrose intake test in week 6 was preceded by Behavioural procedures
DBS or sham stimulation (n = 8/group). Subsequently, Chronic mild stress
animals were tested during week 8 in the elevated plus The CMS procedure was conducted as previously
maze (EPM) and novel object recognition test (NORT), described (Papp, 2012). Briefly, after 3 weeks of habi-
also preceded by DBS or sham stimulation (Fig. 1a and b). tuation to laboratory and housing conditions, the animals
were trained to consume a 1% sucrose solution in six
A fifth DBS experiment replicated the DBS experiment, baseline tests conducted once weekly in the home cage.
exactly as described, in saline-treated Wistars. After 14 h food and water deprivation, the animals were
presented with a freshly prepared 1% sucrose solution for
A final experiment tested the effect of ketamine in WKY
1 h. Sucrose intake was calculated by weighing bottles
rats (n = 6/group). CMS was administered for 4 weeks; up
before and after the test. Subsequently, sucrose con-
to the end of week 2 of CMS, the procedure was identical
sumption was monitored once weekly, under similar
to that used in the earlier experiments. Ketamine (10 mg/
conditions, until the end of the study.
kg, intraperitoneally) was administered for a total of
7 days, commencing 1 day before the week 3 sucrose On the basis of their intakes in the final baseline test, the
intake test. During week 4, animals were tested in the animals were divided into two matched groups: CON and
EPM and NORT, on the same schedule as the DBS CMS. Each week of the stress regime consisted of two

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242 Behavioural Pharmacology 2019, Vol 30 No 2&3

periods of food or water deprivation, two periods of 45° Stimulation procedures

cage tilt, two periods of intermittent illumination (light Surgery
on and off every 2 h), two periods of soiled cage (250 ml Animals were anesthetized with ketamine (80 mg/kg) and
water in sawdust bedding), one period of paired housing, xylazine (5 mg/kg) and placed in a stereotaxic apparatus
two periods of low intensity stroboscopic illumination (Stoelting Co., Wood Dale, Illinois, USA). Monopolar
(150 flashes/min) and three periods of no stress. The stainless steel stimulating electrodes (Plastics One Inc.,
duration of all stressors was 10–14 h and they were Roanoke, Virginia, USA) consisted of two wires; one wire
applied individually and continuously, day and night. (250 µm in diameter, 0.75 mm of exposed surface) was
The control animals were housed in separate rooms and unilaterally implanted into the left vm-PFC (ante-
were deprived of food and water for 14 h before each roposterior: + 3.0 mm, lateral: ± 0.7 mm, dorsoventral:
sucrose test, but otherwise food and water were available − 4.2 mm from Bregma) according to the atlas of Paxinos
freely available. After 2 weeks, each of the CON and and Watson (1998) and served as the cathode. The sec-
CMS groups was split into subgroups, matched for ond wire had an epidural screw attached and was used as
sucrose consumption, which were administered different the anode. Control, sham rats were anesthetized and
drug treatments under continuing CMS exposure, as were implanted with inactive electrodes. The electrodes
described above. The drugs were administered at were connected to a plastic pedestal and were fixed to
~ 10.00 h and the weekly sucrose tests were carried out the skull with a dental cement (Adhesor Carboline;
24 h following the previous drug injections. SpofaDental, Jicin, Czech Republic).

Deep brain stimulation

Elevated plus maze
After a 1-week postoperative recovery period, animals
The animals were tested in a darkened room, using an
received stimulation at the following parameters: amplitude
apparatus comprising two open (50 × 11 cm) and two
250 μA, frequency 130 Hz; pulse width 90 µs. Stimulation was
nontransparent closed (50 × 11 × 40 cm) arms, elevated
administered using a custom-made multichannel stimulator
50 cm above the floor and illuminated by two 25 W bulbs
and digital-to-analogue converters, connected to the animals
located beneath the open arms. The animals were placed
through bipolar extension cables and commutators (Plastics
individually in the centre of the apparatus and the
One Inc.). Two 2 h DBS sessions were conducted, one on the
number of entries of all four feet into open and closed
previous evening and one on the morning before each
arms, and the time spent in each arm were recorded
behavioural test (i.e. sucrose intake, EPM and NORT). The
manually in a 5-min test by an observer who was blind to
stimulation parameters and the schedule of DBS administra-
treatments. A % open arm entries (%OAE) score was
tion were established in our unpublished preliminary studies.
calculated according to the following formula: number of
OAE divided by total number of entries multiplied
Drugs
by 100.
Imipramine HCl (Sigma-Aldrich, St Louis, Missouri, USA),
citalopram HBr and venlafaxine HCl (Carbosynth Ltd,
Novel object recognition test Compton, UK) and ketamine (Biowet Pulawy, Pulawy,
The animals were tested in an opaque circular open field Poland) were dissolved in 0.9% sterile saline, which was
(100 cm in diameter, 35 cm high, floor divided into used for vehicle administration. All injections were made
painted 16-cm squares). After a period of 2 days’ adap- intraperitoneally in a volume of 1 ml/kg of body weight.
tation (10 min daily), on day 3 of week 8, the animals Doses were as used in previous studies (Papp et al., 2016,
were allowed to explore two identical cylinder-shaped 2017a, 2017b).
white objects (7 cm in diameter, 11 cm high) for the time
required to complete 15 s of exploration of both objects Statistical analysis
(T1 session). In the retention trial (T2 session) con- For the period before the start of drug treatment, strain dif-
ducted 1 h later, one of the objects presented previously ferences between Wistar and WKY rats were evaluated by t-
was replaced by a novel prism-shaped black object (5 cm test (body weight) or repeated-measures analysis of variance
wide, 14 cm high). Rats were returned to the open field (ANOVA) (sucrose intakes in weeks 0–2 of stress), combin-
for 5 min and the duration of exploration of each object ing data across the four groups that later received different
(i.e. sitting in close proximity to the objects, sniffing or drug treatments. Sucrose intake in weeks 2–6 was analysed
touching them) was recorded by a trained observer who for each drug separately, using repeated-measures ANOVA
was blind to treatments. A NORT index was calculated with the factors strain, stress and weeks. t-Tests were used to
according to the following formula: time of novel object analyse strain differences in behaviour in the EPM and
exploration minus time of familiar object exploration, NORT. Effects of DBS were analysed for each experiment
divided by total exploration time (novel plus familiar separately using two-way ANOVA (DBS × CMS). For
objects). During the test session the number of line the ketamine experiment, the sucrose intake data were
crossings was also recorded as a measure of locomotor analysed by three-way ANOVA (ketamine × CMS × we
activity. eks) and the EPM and NORT data by two-way ANOVA

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 CMS in WKY as animal model of TRD Willner et al. 243

(ketamine × CMS). Because there was no prediction [t(14) = 3.21, P < 0.01] and were significantly less active
regarding the effect of DBS or ketamine in control animals, [t(14) = 3.47, P < 0.01].
the main statistics of interest were subsequent t-tests of the
a priori predictions that CMS would decrease behaviours in
Wistar-Kyoto rats show evidence of increased
the sucrose intake, EPM and NORT tests in sham-
responsiveness to stress
stimulated animals, and that DBS or ketamine would rein-
At baseline, WKY rats were 16% lighter in weight than
state those behaviours.
Wistars (despite being a week older): 275.3 ± 1.2 versus
328.6 ± 3.3 g [t(189) = 18.1, P < 0.001]. To take account of
Results this difference, the effects of CMS were computed as %
Wistar-Kyoto rats are more ‘emotional’ change from baseline. The increase in body weight over the
Figure 2 compares saline-treated and sham-stimulated next 2 weeks was very similar in control animals of the two
Wistar and WKY rats in the EPM (Fig. 2a) and NORT strains (mean: 7.2%). Body weight increases were smaller
(Fig. 2b). WKY rats made significantly fewer OAE in the in both groups of stressed animals [F(1,186) = 101.2,
EPM [t(14) = 3.8, P < 0.002] and spent correspondingly P < 0.001]. However, stressed WKY gained significantly less
less time in the open arms [t(14) = 4.8, P < 0.001]. Wistars weight than stressed Wistars [Fig. 3a: stress × strain inter-
and WKY did not differ in %OAE [t(14) = 0.5, NS, but action, F(1,186) = 5.84, P < 0.02; strain difference in CMS
the % time spent in the open arms was significantly lower groups: t(93) = 3.51, P < 0.001].
in WKY [t(14) = 4.8, P < 0.001] (results not shown) . The
WKY rats were also more responsive to CMS in the EPM.
two strains did not differ significantly in closed-arm
To take account of the fact that the two strains differed in
entries, a measure of locomotor activity [t(14) = 1.6,
their behaviour on the EPM under control conditions, as
NS]. In the NORT, the two strains showed similar levels
described above, scores were expressed as a proportion of
of recognition memory [t(14) = 0.81, NS], but WKY rats
the control mean. Stress decreased OAE and the % of
spent significantly less time exploring the objects
OAE to total arm entries by 40% in Wistars and by 80% in
WKY [t(15) = 2.80, P < 0.02 and t(15) = 3.72, P < 0.005,
Fig. 2 respectively; Fig. 3b].
Wistar and WKY rats did not, however, differ significantly
in their sensitivity to the effects of CMS on sucrose intake
(Fig. 4a–d; Fig. 4a also includes data from the Wistar
replication experiment, which are not included in the sta-
tistical analyses). Across the four experiments, baseline
sucrose intakes (week 0) were very similar [F(1,189) = 0.51,
NS, overall], and while there was a certain amount of

Fig. 3

Responses of Wistar (n = 64) and Wistar-Kyoto (WKY) (n = 128) rats

when exposed to chronic mild stress (CMS). (a) Body weight gain from
week 0 to week 2 of CMS. Because the groups differed in body weight
at week 0 (WKY rats 16% lighter), the change in body weight is
Behaviour of Wistar and WKY rats in the elevated plus maze (a) and the expressed as a % of baseline. (b) Behaviour of sham-stimulated rats in
novel object recognition test (b). The data are from animals that were the elevated plus maze (EPM) after 8 weeks of CMS (n = 8/group).
nonstressed (i.e. the controls for CMS animals) and sham-stimulated Because nonstressed Wistar and WKY rats differed in their behaviour
(i.e. the controls for animals receiving DBS). Values are mean ± SEM; on the EPM (Fig. 2), the results for stressed animals are expressed as a
n = 8/group; **P < 0.01; ***P < 0.001. CMS, chronic mild stress; DBS, % of the control mean. Values are mean ± SEM; *P < 0.05; **P < 0.01;
deep brain stimulation; WKY, Wistar-Kyoto. ***P < 0.001. CON, control.

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244 Behavioural Pharmacology 2019, Vol 30 No 2&3

Fig. 4

Sucrose intake (ml) in control (white circles) and CMS (black circles) Wistar (dashed lines) and WKY (solid lines) rats during exposure to CMS (from
week 0) and treatment (from week 2 onwards) with saline (a), imipramine (B), citalopram (c) or venlafaxine (d). The dotted lines in (a) show a
replication of the Wistar saline experiment. Note that drug treatment (b–d) causes a gradual increase in sucrose intake in Wistars (black circles,
dashed lines) but not in WKY (black circles, solid lines). Values are mean ± SEM; n = 16/group, except for the original Wistar experiment for which
n = 8/group.

variability in the control values, particularly in WKY ani- three drug-treated groups [F(1,140) = 14.11, P < 0.001], but
mals, week 1 and 2 sucrose intakes in stressed animals not in saline-treated animals [F(1,47) = 0.79, NS].
were very similar between the two strains [weeks 0–2:
As described below, WKY rats also failed to respond to
week × strain interaction, F(2,186) = 1.73, NS].
drug treatments in the EPM and NORT.
Wistar-Kyoto rats do not respond to antidepressant drug
treatment Wistar-Kyoto rats do respond to deep brain stimulation
Weeks 3–6 in Fig. 4 represent the period of drug treat- Sucrose intake
ment before DBS. In saline-treated animals (Fig. 4a) Figure 5a shows results of the five DBS experiments,
sucrose intakes remained low throughout, in both strains. conducted at week 7 of CMS. Confirming the data
The sucrose intakes of drug-treated animals (Fig. 4b–d) reported above, sucrose intake was significant lower in
were somewhat variable, but in each case the sucrose CMS-sham groups compared with CON-sham groups in
intake of stressed animals recovered in Wistars but did all experiments (i.e. drug treatment continued to be
not recover in WKY. ineffective). DBS significantly increased sucrose intake
in CMS animals, relative to sham stimulation, in both
In all four experiments there was a significant stress × Wistar and WKY SAL-treated groups, and also in CIT-
strain × weeks interaction [SAL: F(4,172) = 3.98, P < 0.005; treated and VEN-treated WKY. However, DBS did not
IMI: F(4,176) = 5.57, P < 0.001; CIT: F(4,176) = 3.19, increase sucrose intake in IMI-treated WKY.
P < 0.02; VEN: F(4,176) = 9.84, P < 0.001]. For all three
antidepressant drugs, the linear component of the three-way Statistical analysis confirmed a significant main effect of
interaction is highly significant, indicating that the diver- DBS for SAL-treated Wistars [F(1,28) = 7.28, P < 0.02],
gence between drug-treated Wistar and WKY increases over SAL-treated WKY [F(1,27) = 9.97, P < 0.005] and VEN-
time [IMI: F(1,44) = 12.14, P < 0.001; CIT: F(1,44) = 14.10, treated WKY [F(1,28) = 9.42, P < 0.005], and a signi-
P < 0.001; VEN: F(1,44) = 19.59, P < 0.001]. However, for ficant stress × DBS interaction for CIT-treated WKY
SAL-treated animals there is no such gradual divergence [F(1,28) = 6.79, P < 0.02].
[F(1,43) = 0.10, NS]: the significant interaction arises largely
from an irregular value in week 5 (Fig. 4a), with both groups Elevated plus maze
of stressed animals maintaining a similar downward trajec- In all five experiments, CMS significantly decreased %
tory. (Indeed, the WKY-CMS data are almost identical to OAE on the EPM (Fig. 5b), extending the ineffective-
those seen in the Wistar-CMS replicates). Consequently, at ness of antidepressant drug treatment in WKY animals to
week 6 there is a significant stress × strain interaction in the the anxiolytic effect in the EPM. And in all five

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 CMS in WKY as animal model of TRD Willner et al. 245

Fig. 5

Effects of deep brain stimulation (DBS) or sham stimulation of the vm-PFC on Wistar rats treated with saline (SAL) (left-most columns) and WKY rats
treated with saline (SAL), imipramine (IMI), citalopram (CIT) or venlafaxine (VEN). White bars: control (CON); grey bars, exposure for 7–8 weeks to
chronic mild stress (CMS). Daily saline or drug treatments commenced at week 3. (a) Sucrose intake at week 7; (b) % Open arm entries on the
elevated plus maze (week 8); (c) Novel object recognition (week 8). CMS impaired all behaviour in all three tests; these impairments were not
reversed by chronic antidepressant drug treatments, but with the single exception of sucrose intake in the IMI-treated group (a: middle columns),
behaviour in all tests in all groups was improved by DBS. Values are mean ± SEM.*P < 0.05; **P < 0.01; ***P < 0.001: small stars, sham, CON versus
CMS; large stars, CMS, sham versus DBS.

experiments, DBS significantly increased %OAE, that is, Statistical analysis confirmed a significant CMS × DBS
DBS was effective not only in SAL-treated Wistars, but interaction for SAL-treated Wistars [F(1,28) = 10.1,
also in WKY rats treated either with saline or with inef- P < 0.005], and for WKY treated with SAL [F(1,27) = 25.68,
fective antidepressant drugs. P < 0.001] or VEN [F(1,28) = 6.78, P < 0.02]. For IMI-

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246 Behavioural Pharmacology 2019, Vol 30 No 2&3

treated WKY, in which DBS failed to increase sucrose et al., 1988), including increased emotionality in a novel
intake, there was a marginally significant interaction environment (Berton et al., 1997; Baum et al., 2006), an
[F(1,28) = 4.05, P = 0.054] but a significant effect of DBS in anxiogenic profile on the elevated plus maze (Mileva and
stressed animals [t(14) = 2.69, P < 0.02]. In CIT-treated Bielajew, 2015; Zubcevic et al., 2017), and increased
WKY the interaction was nonsignificant [F(1,28) = 1.61], immobility in the forced swim test (FST) (Armario et al.,
but the effects of CMS in sham animals [t(14) = 5.25, 1995; Paré, 1989; Rittenhouse et al., 2002; Nam et al.,
P < 0.001] and of DBS in CMS animals [t(14) = 6.31, 2014; Kin et al., 2017). WKY rats also show enhanced
P < 0.005] were significant, as predicted. physiological responses to stress (Rittenhouse et al., 2002;
Pardon et al., 2002; Morilak et al., 2012) and an increased
Novel object recognition test propensity to develop learned helplessness (Paré, 1989;
Recognition memory in the NORT was effectively Belujon and Grace, 2014). This evidence is consistent
abolished by CMS (Fig. 5c). Novel object recognition in with the two minor criteria for a model of TRD.
CMS animals was untouched by antidepressant treat-
ment, but in all five experiments, recognition in CMS The present study was not designed to test system-
animals was significantly increased by DBS. atically the emotionality of the WKY rat, nor its respon-
siveness to stress, but some of the data obtained are
Statistical analysis confirmed a significant DBS × CMS
relevant to these two issues. Nonstressed WKY rats
interaction in SAL-treated Wistars [F(1,28) = 25.67,
showed an anxiogenic profile on the EPM (decreased
P < 0.001], IMI-treated WKY [F(1,28) = 11.61, P < 0.002],
activity in the open arms; Fig. 2a), with normal locomotor
and CIT-treated WKY [F(1,28) = 12.70, P < 0.05]. In
SAL-treated and VEN-treated WKY the interaction was activity in a safe environment (closed-arm entries;
nonsignificant [F(1,28) = 1.51 and 1.37], but DBS sig- Fig. 2a). In the NORT their recognition memory was
nificantly increased the recognition index in the CMS unimpaired, but, in this large arena, they also showed a
groups [t(14) = 2.68, P < 0.02 and t(14) = 2.32, P < 0.05]. decrease in locomotor activity, and they avoided the
objects placed in the open field, suggestive of increased
Wistar-Kyoto rats do respond to ketamine
fearfulness (Fig. 2b). WKY rats were also more responsive
CMS decreased sucrose intake from week 0 to week 2; to stress, relative to the Wistar control strain, showing
this decline was reversed by acute (week 3) and subacute lower body weight gain (Fig. 3a) and a greater anxiogenic
(week 4) ketamine (Fig. 6a), resulting in a significant response (Fig. 3b). These data are consistent with the
three-way interaction [F(4,80) = 3.71, P < 0.01]. In CMS earlier literature.
animals, a single dose of ketamine significantly increased
We did not, in the present study, detect abnormalities in
sucrose intake relative at week 3 relative to week 2 [t-
the sucrose consumption measure, either at baseline or
(5) = 3.61, P < 0.02] but the increase was nonsignificant
following exposure to CMS. The literature shows a
relative to saline-treated animals [t(10) = 1.78, P = 0.10];
mixed picture, with lower responsiveness of WKY to
however, a significant effect relative to saline-treated
animals was present after 8 days of ketamine treatment sucrose or saccharin reported in some studies (De La
[t(10) = 3.71, P < 0.005]. Garza, 2005; Malkesman et al., 2005; Burke et al., 2016;
D’Souza and Sadananda 2017) but no difference relative
CMS significantly decreased %OAE in the EPM, and to Wistars in others (Dommett and Rostron, 2013; Mileva
this effect was reversed by ketamine, tested after 2 days and Bielajew, 2015; Burke et al., 2016; Kin et al., 2017).
of treatment (Fig. 6b), resulting in a significant WKY were also somewhat less responsive than control
ketamine × CMS interaction [F(1,20) = 7.26, P < 0.02], strains to rewarding effects of nicotine (Rauhut et al.,
reflecting a significant effect of ketamine in CMS animals 2008) or morphine (Dennis et al., 2016) in place con-
[t(10) = 3.26, P < 0.01]. ditioning procedures. Overall, there is some evidence for
Recognition memory in the NORT was abolished by CMS, decreased reward sensitivity in WKY, but the effect is
but restored by three doses of ketamine (Fig. 6c). The two- somewhat variable, and was not apparent in the present
way interaction was nonsignificant [F(1,20) = 2.94, P = 0.10]; study. The discrepancies may to some extent reflect
however, the effect of ketamine to increase NOR was sig- methodological differences, such as the use of intake
nificant overall [F(1,20) = 6.17, P < 0.025], and in the CMS measures, as here, versus preference measures (Burke
group [t(10) = 2.95, P < 0.02], but not in nonstressed animals et al., 2016; D’Souza and Sadananda, 2017). Another
[t(10) = 0.55, NS]. relevant factor may be age: an anhedonic phenotype may
be more prominent in adolescent WKY (Malkesman
Discussion et al., 2005; D’Souza and Sadananda, 2017). Interestingly,
WKY rats were originally bred as a normotensive control adolescent WKY also show decreased levels of social
strain for the spontaneously hypertensive rat strain, but play, leading to the suggestion that they could be con-
were later found to display physiological and behavioural sidered as a model of childhood depression (Malkesman
features suggestive of heightened emotionality (Gentsch and Weller, 2009).

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 CMS in WKY as animal model of TRD Willner et al. 247

Fig. 6

Effects of ketamine in WKY rats exposed to CMS. (a) Sucrose intake in controls (CON) and during 4 weeks of chronic mild stress (CMS). Ketamine
(KET) or saline (SAL) were administered daily for 8 days, starting 1 day before the week 3 sucrose test and ending 1 day before the week 4 sucrose
test (arrows). (b) % Open arm entries on the elevated plus maze (EPM) after two doses of ketamine. (c) Object recognition in the novel object
recognition test (NORT) after three doses of ketamine. Values are mean ± SE; n = 6; *P < 0.05; **P < 0.01; SAL versus KET in CMS animals.

An anhedonic phenotype is not a necessary feature of an WKY rats were insensitive to chronic treatment with the
animal model of predisposition to depression; rather, a tricyclic antidepressant imipramine, the selective ser-
tendency to develop anhedonia may only become otonin reuptake inhibitor citalopram, and the serotonin
apparent under exposure to stress (Willner and Mitchell, noradrenaline reuptake inhibitor venlafaxine. The posi-
2002; Willner et al., 2014). We are aware of only one tive effects of all three drugs in Wistar rats (Fig. 4b–d)
previous study comparing reward responsiveness in WKY confirm that their ineffectiveness in WKY rats did not
and Wistars following exposure to stress; Paré (2000) result from an inappropriate choice of doses. (In this
reported that acute stress exposure decreased investiga- context, the absence of a strain difference in sucrose
tion of females by male WKY, with no effect in Wistars. intake in the present study, either before or following
The authors interpreted these data as consistent with a CMS exposure, is a positive feature.) It has long been
greater propensity to anhedonia in WKY, but there are known that the ineffectiveness of antidepressants in
many other potential explanations. The lack of a strain WKY rats does not have a pharmacokinetic explanation,
difference in the suppression of sucrose intake by CMS because on some measures, neurochemical effects are
could reflect a ‘floor effect.’ But the literature linking similar in WKY or Wistar rats (Lahmame et al., 1997;
antidepressant treatment resistance to the presence of Getachew et al., 2017). Moreover, the three drugs appear
risk factors for depression (Willner et al., 2014) does not to be equally ineffective; there is no suggestion in the
refer to anhedonia specifically, so there is no strong data (Figs 4 and 5) that imipramine or venlafaxine, which
prediction that WKY rats should be more susceptible to are potent noradrenaline uptake inhibitors, were any
suppression of sucrose intake by CMS. more effective in WKY rats than the purely serotonergic
agent citalopram. Hence, the present data do not support
Although WKY rats are generally considered to be non- the suggestion that WKY rats have a specific insensitivity
responsive to antidepressant drugs, this understanding to serotonergic drugs (Lopez-Rubalcava and Lucki, 2000;
rests on a limited evidence base, relying largely on Tejani-Butt et al., 2003), at least, following chronic
responses to acute antidepressant treatment in the FST. treatment. The results also demonstrate the ineffective-
Some studies reported a specific subsensitivity to ser- ness of all three drugs in WKY rats in three behavioural
otonergic antidepressants (Lopez-Rubalcava and Lucki, tests that have greater ecological validity than the FST or
2000; Tejani-Butt et al., 2003), while others reported a rapid eye movement sleep suppression. Although we
more general effect (Lahmame et al., 1997). WKY rats tested only one behaviour in each of the three domains,
have also been found to be subsensitive to suppression of the fact that similar results were seen across all tests
rapid eye movement sleep by antidepressants (Ivarsson suggests that the effects are generalizable across beha-
et al., 2005). However, to our knowledge, the respon- viours. Taking together, the pharmacological and beha-
siveness of WKY rats to conventional antidepressants has vioural data provide strong support for the proposition
not previously been tested in a validated animal model of that the WKY rat exposed to CMS is indeed non-
depression. responsive to antidepressant drugs.

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248 Behavioural Pharmacology 2019, Vol 30 No 2&3

Concerning their response to emerging novel treatments Pollier et al., 2000; De La Garza and Mahoney, 2004). Of
for TRD, there is one report that learned helplessness particular relevance to depression, WKY rats also have
was reversed by ketamine in WKY rats (Belujon and reduced hippocampal volume relative to Wistars (Tizabi
Grace, 2014). There was no comparison with control et al., 2010), and lower levels of hippocampal neurogenesis
strains in this study; however, WKY were more respon- and cell proliferation (Kin et al., 2017); decreased hippo-
sive than Wistar rats to ketamine reversal of immobility in campal volume is a well-established feature of depression
the FST (Tizabi et al., 2010, 2012; Akinfiresoye and (Videbech and Ravnkilde, 2004) and is associated with
Tizabi, 2013). A study in WKY rats found that DBS of the treatment resistance (MacQueen et al., 2008; Fu et al.,
nucleus accumbens, a site at which DBS is effective 2013). However, while there are many intriguing parallels
clinically in TRD (Schlaepfer et al., 2008), had locomotor between WKY rats and depressed people that may be
stimulant effects (Falowski et al., 2011). Another study relevant to antidepressant treatment resistance, reliable
reported that induction of long-term potentiation in the translation from the laboratory to the clinic requires a valid
hippocampus, a conceptually related procedure, induced animal model of TRD, which is not achieved by testing the
a rapid antidepressant-like effect in the FST (relative to a WKY rat in the FST or open field, as has typically been
more slowly developing effect of the noradrenergic the case.
antidepressant desipramine) (Kanzari et al., 2018).
Conclusion
Previous studies have demonstrated that DBS of the vm-
In the present study we have combined use of the WKY
PFC causes a rapid reversal of CMS-induced anhedonia
rat with the well-validated CMS model of depression. We
and other depression-related behaviours in antidepressant-
conclude from the data that WKY rats under CMS
responsive rat and mouse strains (Hamani et al., 2012;
meet all four of the criteria proposed for an animal model
Dournes et al., 2013; Veerakumar et al., 2014; Lim et al.,
of TRD (Willner and Belzung, 2015). The data support
2015). Here, we report that, with a single exception, DBS
the findings of earlier studies that WKY rats show a
of the vm-PFC has antianhedonic, anxiolytic, and procog-
relevant phenotype and evidence of greater sensitivity to
nitive effects in antidepressant-non-responsive WKY rats
stress, add a strong confirmation that WKY rats do not
displaying the anhedonic, anxiogenic and dyscognitive
respond to antidepressant drugs, and demonstrate that
effects of CMS (Fig. 5). The single failure of DBS to
WKY rats do respond to DBS and ketamine. Overall, the
reverse effects of CMS was on sucrose intake in IMI-
findings establish CMS in WKY rats as a valid model for
treated animals. Curiously, we have observed the identical
TRD. Two important questions that can now be
pattern of effects, a comprehensive reversal of CMS effects
addressed are as follows: ‘What critical differences
except for sucrose intake in IMI-treated animals, in a
between Wistar and WKY rats are responsible for the
subgroup of Wistar rats that had failed to respond to anti-
effectiveness of antidepressants in Wistars and their
depressant treatment (Papp et al., 2018). Perhaps the anti- ineffectiveness in WKY?’ and ‘What do DBS and keta-
muscarinic effect of IMI, which is not shared by CIT or mine do in the brains of WKY rats that antidepressant
VEN, might antagonize the effect of DBS. While this drugs do not?’ It is likely that the answers to these and
hypothesis merits investigation, its scope is limited, related questions would be readily translatable to the
because concurrent IMI treatment did not block the anti- clinic, with good prospects to improve the understanding,
anxiety or procognitive effects of DBS. and hence, the treatment, of TRD.
We have previously reported that, in Wistar rats, keta-
mine was partially effective to reverse the effect of CMS Acknowledgements
in sucrose intake after 3 days of treatment, with full The study was financially supported by the NCN grant
reversal after 7 days (Papp et al., 2017b). Here, we saw no. 2015/17/B/NZ7/02979.
essentially the same picture in WKY rats: partial reversal
after a single dose of ketamine, with full reversal after Conflicts of interest
8 days of treatment. Ketamine also reversed the effects of There are no conflicts of interest.
CMS in the EPM and NORT in Wistars, after 4 and
5 days of treatment, respectively (Papp et al., 2017b), as
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