Journal of Psychiatric Research 151 (2022) 619–625

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Journal of Psychiatric Research

journal homepage: www.elsevier.com/locate/jpsychires

Sex differences in long-term fear and anxiety-like responses in a preclinical

model of PTSD
Delara Emtyazi a, Thallita Kelly Rabelo a, Hailey Katzman a, Ana Carolina Campos a,
Mustansir Diwan a, Darryl Gidyk a, Patricia Rabelo dos Santos b, Peter Giacobbe a, c, e,
Nir Lipsman a, c, d, Isabelle Aubert c, f, Clement Hamani a, c, d, *
a
Harquail Centre for Neuromodulation, Sunnybrook Health Sciences Centre, Sunnybrook Research Institute, Toronto, ON, M4N 3M5, Canada
b
Department of Medicine, Universidade Federal de Rondônia, Porto Velho, RO, 76801-059, Brazil
c
Hurvitz Brain Sciences Centre, Sunnybrook Research Institute, Toronto, ON, M4N 3M5, Canada
d
Division of Neurosurgery, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, M4N 3M5, Canada
e
Department of Psychiatry, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada
f
Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, ON, M5S 1A8, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: With a high prevalence of posttraumatic stress disorder (PTSD) in females, studying sex differences in preclinical
Sex models is of substantial importance. We have previously employed behavioural criteria to identify and charac­
Fear terize a subpopulation of rats that presented impaired fear extinction and long-term fear and anxiety responses
Anxiety
following fear conditioning. We now exposed male and female rats to fear conditioning and extinction and
Estrous cycle
segregated the animals into weak- (WE) and strong-extinction (SE) groups based on behavioural scores during
Post-traumatic stress disorder
Extinction extinction. Animals were subsequently tested for tone and context recall, as well as anxiety-like responses in the
Conditioning marble burying and novelty suppression of feeding (NSF) tests. Vaginal lavages were collected to characterize the
phase of the estrous cycle during fear extinction. We found that females had reduced freezing during tone recall
and a lower latency to feed in the NSF test. No differences were found in females undergoing extinction during
high and low estrogen phases of the cycle in any of the performed tests. Overall, the percentage of animals that
presented WE and SE phenotypes was similar in males and females. Both, WE males and females had increased
freezing during tone and context recall. Along with our previous reports, WE males presented anxiety-like re­
sponses, particularly in the NSF compared to SE animals. In contrast, WE females buried less marbles than their
SE mates. Future investigation including a larger number of behavioural tests are certainly required to corrob­
orate our findings and ascertain potential mechanisms to explain the differences observed in our study.

1. Introduction of rodents exposed to a traumatic event (e.g., fear conditioning) exhibit

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric
condition that emerges following exposure to a traumatic event. Females
are not only more likely to develop PTSD, but also tend to present more
severe and protracted symptoms (Breslau, 2002; Breslau et al., 1998;
Kilpatrick et al., 2013). While preclinical studies have been instrumental
to help us understand the mechanisms and circuitry of fear memory and
anxiety-type behaviours, they have mainly been conducted in males
(Cohen et al., 2004, 2006; Kessler et al., 1995; Miller and McEwen,
2006; Siegmund and Wotjak, 2006).
We have recently found that, similar to patients with PTSD, 20–30%
long-term fear and anxiety-type responses, have high baseline serum
corticosterone levels and a disrupted connectivity between the pre­
frontal cortical and the basolateral amygdala (Reznikov et al., 2015,
2018; Vimalanathan et al., 2020).
We compare long-term fear and anxiety-like responses between fe­
males and male rats, further subdividing the animals according to the
estrous cycle phase during extinction.

* Corresponding author. Sunnybrook Research Institute, 2075 Bayview Av, S126, Toronto, ON, M4N3M5, Canada.,
E-mail addresses: clement.hamani@sunnybrook.ca, c.hamani@sympatico.ca (C. Hamani).

https://doi.org/10.1016/j.jpsychires.2022.05.015
Received 5 March 2022; Received in revised form 14 May 2022; Accepted 19 May 2022
Available online 23 May 2022
0022-3956/© 2022 Elsevier Ltd. All rights reserved.
D. Emtyazi et al. Journal of Psychiatric Research 151 (2022) 619–625

2. Methods increase the statistical power while reducing the number of animals, rats
showing moderate levels of freezing (i.e., 33–67%) were not included in
2.1. Animals the analyses (Reznikov et al., 2015; Vimalanathan et al., 2020).

Outbred male (n = 20; 250–275g) and female (n = 30; 200–250 g) 2.3. Anxiety-type behaviour
Sprague-Dawley rats (Charles River, Quebec) were housed in pairs on a
12 h light–dark cycle with water and standard rat chow available ad Marble burying test (MBT). On day 12, rats were placed in a stan­
libitum. All protocols were approved by the Animal Care committee of dard cage containing 5 cm of bedding and 15 identical marbles evenly
the Sunnybrook Research Institute. The timeline for the experiments is arranged in three columns. The number of marbles buried (<50% of
shown in Suppl Fig. 1. visible surface) in a 30 min session was quantified.
Novelty Suppressed Feeding (NSF). Animals were trained to
consume food treats (Honey Nut Cheerios®) in their home cages for 2
2.2. Fear conditioning, extinction and recall
days prior to testing. They were then food deprived for 16h prior to
testing. On the testing day, animals were placed in a novel Plexiglas cage
Fear conditioning and extinction were carried out in an operant
lined, which contained the previously habituated treat. Latency to begin
conditioning chamber (Coulbourn Instruments, Holliston, MA). On day
consuming the food was measured, as previously described (Reznikov
1, rats were presented with six conditioned stimuli (CS; 30sec, 85 dB, 4
et al., 2015).
kHz auditory tones), each co-terminating with a footshock (uncondi­
tioned stimulus, US; 0.8 mA, 0.5s), as previously described (Gidyk et al.,
2.4. Determining the phase of the estrous cycle
2021; Reznikov et al., 2015, 2018; Vimalanathan et al., 2020). On day 2,
rats underwent extinction training, which consisted of 12 presentations
Stages of the estrous cycle were identified based on cytology from
of the CS in the absence of shocks. On days 10 and 11, rats were exposed
vaginal lavages. Briefly, smears were obtained approximately 30 min
to 2 CS-only presentations in a novel context (tone recall) or in the
after fear extinction. Samples were collected using 300 μL of saline via a
original context in the absence of CS or shocks (context recall). %
1000 μL pipette and mounted onto positively charged slides. After
freezing was calculated as (duration of freezing/30sec)*100.
collection, samples were allowed to air-dry overnight prior to staining
with Giemsa. Stages of the estrous cycle were classified according to
2.2.1. Behavioural classification criteria
specific cell structures in the collected material (Ajayi and Akhigbe,
Similar to our previous work, animals were subdivided in two
2020; Marcondes et al., 2002).
groups: Weak Extinction (WE): average percentage of freezing >67%
during the last 2 blocks of extinction (4 trials); Strong Extinction (SE):
average percentage of freezing <33% freezing average during the last 2
extinction blocks (Reznikov et al., 2015; Vimalanathan et al., 2020). To

Fig. 1. Behavioural results according to sex. A) Fear Conditioning and Extinction. No differences were found in the percentage of freezing between females (n = 24)
and males (n = 16) across conditioning (C1–C3) and extinction sessions (E1-E6). B, C) Females had a significant decrease in the percentage of freezing compared to
males during tone recall, while no significant sex differences were found during context recall. D) No differences were observed in the marble burying test between
females and males. E) In the novelty supressed feeding test, females had a significantly lower latency to feed compared to males. Significant trial effects were not
indicated for clarity. Values represent means and SEM. **p ≤ 0.01, *p ≤ 0.05.

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D. Emtyazi et al.
2.5. Statistical analyses
Mixed design ANOVA was used to analyze freezing scores during
conditioning and extinction sessions with group as between-subjects and
time as within-subjects factors (Sidak post hoc). In our initial experi­
ment, groups (between-subjects) were males vs females. In our second
experiment, groups were WE vs SE, with separate analyses conducted for
males and females. In our third experiment, groups were proestrus vs
metestrus/diestrus. Independent t-tests were used for recall trials, MBT,
and NSF. The Fisher’s exact test was used to compare the proportion of
WE/SE animals. Unless otherwise specified, data are reported as mean
± standard error of the mean (SEM). Statistical significance was set at p
≤ 0.05.
3. Results
3.1. Behavioural characteristics of female and male rats
During conditioning, trial x group interaction [F(2,76) = 6.56, p =
0.002] and trial block effect were found to be significant [F(2,76) =
279.6, p < 0.001], while no group effect was noted (Female vs. Male) [F
(1,38) = 0.001, p = 0.97] (Fig. 1A). During extinction, a significant trial-
block effect [F(5, 190) = 23.30, p < 0.0001] but no group effect [F
(1,38) = 1.05, p = 0.32] or trial x group interaction [F(5,190) = 1.86, p
= 0.10] were observed (Fig. 1A).
While females froze significantly less during the tone recall (females
21.2 ± 4.3% vs. males 44.1 ± 4.6%; p = 0.001; Fig. 1B) no sex differ­
ences were recorded during context recall (females 30.6 ± 5.7% vs.
males 38.8 ± 5.3%; p = 0.30; Fig. 1C). Both females (6.8 ± 0.7) and
males (6.4 ± 0.6) buried a similar number of marbles (p = 0.19;
Fig. 1D). The latency to feed in the NSF was significantly lower in fe­
males (females 242.0 ± 34.7 s) compared to males (347.9 ± 36.79 s; p
= 0.05; Fig. 1E).
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Journal of Psychiatric Research 151 (2022) 619–625
3.2. Weak vs strong extinction
Once freezing time during fear conditioning and extinction was
scored, animals were subdivided in strong-extinction, weak-extinction
or interthreshold groups (Reznikov et al., 2015; Vimalanathan et al.,
2020). In females, 7 and 17 out of 30 animals were respectively classi­
fied as WE (23%) and SE (57%). Six animals were characterized as
intrathreshold (20%) and excluded from further analyses. This propor­
tion was similar to that of WE, SE and intrathreshold males, which
comprised 25% (5/20), 55% (11/20) and 20% (4/20) of the population
(p = 0.99).
In females, significant trial-block [F(2,44) = 167.6, p < 0.001] and
group effects (strong vs. weak-extinction) [F(1,22) = 7.79, p = 0.01] but
no trial x group interaction [F(2,44) = 2.74, p = 0.08] were found
during conditioning (Fig. 2A). Males had a significant trial-block effect
[F(2,28) = 87.16, p < 0.001], but no group effect [F(1,14) = 0.67, p =
0.42] or trial x group interaction [F(2,28) = 0.21, p = 0.82; ] (Fig. 2B).
Post-hoc tests revealed that WE females froze significantly more than SE
animals in block C2 of the conditioning phase (p < 0.05).
During extinction, the effects of trial-block [F(5, 110) = 6.28, p <
0.0001], group [F(1,22) = 43.52, p < 0.0001] and interaction trial x
group [F(5, 110) = 2.60, p = 0.03] were all found to be significant in
females. In males, significant trial-block [F(5, 70) = 9.78, p < 0.0001]
and group effects [F(1,14) = 24.59, p = 0.0002], but no trial x group
interaction [F(5, 70) = 0.42, p = 0.83] were found. Post-hoc tests
revealed that WE females froze significantly more than SE animals in E2-
E6. In males, significant differences were found in E2, E3, E5 and E6
(Fig. 2A and B).
Freezing in SE females was significantly lower than in WE animals
during tone recall (13.7 ± 3.5% vs 39.4 ± 9.4%; p = 0.03; Fig. 3A) and
context recall (22.7 ± 5.9% vs 49.8 ± 10.9%; p = 0.004; Fig. 3B). In
males, freezing was also significantly lower in SE compared to WE ani­
mals during tone recall (34.9 ± 5.7% vs 64.4 ± 5.7%; p = 0.0006;
Fig. 3E) and context recall (32.1 ± 5.4% vs 58.0 ± 7.1%,; p = 0.008;
Fig. 3F).
Fig. 2. Fear Conditioning and Extinction in Weak
(WE) and Strong Extinction animals (SE). A) During
conditioning, WE females (n = 7) had a reduced
percentage of freezing compared to SE (n = 17) ani­
mals in C2. During extinction, WE females froze
significantly more than SE females in all sessions. B)
In males, no difference was found between the WE (n
= 5) and SE groups (n = 11) during conditioning. In
contrast, WE males froze significantly more than SE
animals during E2, E3, E5 and E6. Significant trial
effects were not indicated for clarity. Values represent
means and SEM. **p ≤ 0.01, *p ≤ 0.05.
D. Emtyazi et al. Journal of Psychiatric Research 151 (2022) 619–625

Fig. 3. Long-term fear and anxiety-like behaviour in Weak (WE) and Strong Extinction (SE) animals. Both WE females (n = 7) and males (n = 5) had significantly
higher freezing scores during tone recall (A, E) and context recall compared to SE animals (n = 17 females, n = 11 males; B, F). In the marble burying test, WE females
buried less marbles than their SE mates (C), whereas a trend was noticed for WE males to bury more marbles compared to SE animals (G). In the novelty supressed
feeding test, no differences were found between WE and SE females (D), whereas WE males showed a significantly higher latency to feed (H). Values represent means
and SEM. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

In the marble burying tests, SE females had a 77% significant in­
crease in the number of buried marbles compared to WE animals (7.8. ±
0.7 vs 4.4. ± 1.3; p = 0.03; Fig. 3C). In contrast, SE males buried 29%
less marbles than their WE mates (5.7 ± 0.7 vs 8.0 ± 1.1; p = 0.08;
Fig. 3G).
In the NSF test, while no differences were found between WE and SE
females (255.4 ± 88.0 s vs 236.4 ± 35.2 s; p = 0.81; Fig. 3D), SE males
had a 36% significantly lower latency to feed compared with WE ani­
mals (295.5 ± 40.3 s vs 463.0 ± 49.7 s; p = 0.03; Fig. 3H).
3.3. Estrous cycle
In the proestrus group (5 in total), 1 female was classified as WE
(20%) and 4 as SE (80%). Similarly (p > 0.99), 5 out of 15 metestrus/
diestrus females (33%) were classified as WE and 10 as SE (67%).
During conditioning, a significant effect of trial block [F(2,36) =
92.93; p < 0.0001], but no group effect (proestrus vs. metestrus/dies­
trus) [F(1,18) = 0.80; p = 0.38] or trial x group interaction [F(2,36) =
1.38; p = 0.27] were observed (Fig. 4A). During extinction, a significant
effect of trial-block [F(5, 90) = 7.09; p < 0.0001] but no group effect [F
(1,18) = 0.01; p = 0.92] or trial x group interaction [F(5,90) = 0.82; p =
0.54] were found (Fig. 4A).
Freezing during tone recall (proestrus 21.3 ± 10.6%; metestrus/
diestrus 22.6 ± 5.9%; p = 0.92; Fig. 4B) and context recall (proestrus
32.1 ± 12.2%; metestrus/diestrus 27.6 ± 6.9 &; p = 0.75; Fig. 4C) was
similar in females extinguished during proestrus or low estrogen phases
of the cycle. Similarly, no differences were found in the number of
buried marbles (proestrus 6.0 ± 1.6; metestrus/diestrus 7.4 ± 0.8; p =
0.42; Fig. 4D) or the latency to feed (proestrus 295.2 ± 71.8 s;
metestrus/diestrus 248.1 ± 44.50 s; p = 0.60; Fig. 4E) between groups.
4. Discussion
Experiments in the current study were conducted to test whether sex
differences could be observed in our proposed model of PTSD. Since
estrogen during fear extinction was shown to reduce freezing during
recall (Chang et al., 2009; Milad et al., 2009), we hypothesized that
females extinguished in high hormonal phases of the cycle would show
reduced fear and anxiety-type responses compared to animals extin­
guished during low hormonal phases. Along this line, we expected this
latter group to have a behavioural pattern similar to that observed in
males. These hypotheses, however, were not confirmed by our results, as
no behavioural differences were found in animals undergoing extinction
during different phases of the estrous cycle. In addition, females froze
less during tone recall and had a lower latency to feed in the NSF
compared to males. A novel aspect of our study was that males pre­
senting the extinction deficient phenotype developed long-term fear and
anxiety-like behaviour, whereas only the former was observed in fe­
males. In contrast to males, WE females buried less marbles. As defen­
sive burying is an innate behavioural response, this may suggest a
shifted coping strategy, rather than an increase in anxiety.
Sex differences have been a major subject of study in rodents and
humans undergoing fear conditioning and extinction paradigms (Dalla
and Shors, 2009; Stark et al., 2006). In the literature, a series of rodent
studies have shown enhanced conditioned fear in males, while others
demonstrated no sex differences (Maren et al., 1994; Pryce et al., 1999).
Similar to the former, human reports suggest that men exhibit a trend
towards an increased conditioned fear relative to women (Milad et al.,

622
D. Emtyazi et al.
Fig. 4. Behavioural results according to the estrous cycle. Females were subdivided into proestrus (n = 5) and metestrus/diestrus groups (n = 15). No differences
were found between groups during A) fear conditioning and extinction, B) tone recall, C) context recall, D) marble burying, and E) novelty supressed feeding.
Significant trial effects were not indicated for clarity. Values represent means and SEM.
2006, 2009, 2010). Our study corroborates these findings, inasmuch as
males presented slightly higher freezing scores, albeit not significantly
different, compared to females in C3.
In rodents, sex differences during fear extinction were largely re­
ported in studies that considered the estrous cycle and hormonal profiles
(Cossio et al., 2016; Maeng et al., 2015; Milad et al., 2009). Proestrus
extinguished females had less freezing during recall than
metestrus-extinguished animals (Milad et al., 2009). The administration
of estrogen to rodents either systemically or focally (e.g., into the hip­
pocampus) facilitates extinction and reduces freezing during recall
(Chang et al., 2009; Milad et al., 2009). In contrast, a few studies re­
ported no relationship between fear extinction and the estrous cycle
(Graham, 2018; Lesuis et al., 2018). Along with these findings, we show
no sex differences or an influence of the estrous cycle during extinction.
One important sex difference observed in our study was the lower
freezing scores recorded in females during tone recall. In the literature, a
few reports showed no apparent differences in recall between sexes or
even an increased freezing in females (Fenton et al., 2014, 2016; Leb­
ron-Milad et al., 2013). Differences between our study and others may
be related to the number of extinction sessions and the timeframe be­
tween extinction and recall. In addition, freezing during recall may also
vary according to reproductive behaviour, as only nulliparous females
have lower freezing during recall in the proestrus phase (Tang and
Graham, 2020).
In the current study, we show that a similar proportion of females
undergoing fear conditioning had extinction deficits and prolonged fear
responses (WE phenotype) compared to males. Despite these findings,
however, major sex differences were observed in defensive burying and
in the novelty supressed feeding test. While WE males had a significantly
higher latency to feed compared to SE animals, WE females had a
reduction in defensive burying. Performance in neither test was
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Journal of Psychiatric Research 151 (2022) 619–625
associated with the phase of the estrous cycle during extinction.
Controversial findings have been reported on the potential associa­
tion between the estrous cycle and defensive burying. While a few
studies have shown that females in estrus exhibit less impulsive burying
compared to animals in the diestrus (Llaneza and Frye, 2009), others
failed to show an influence of the estrous cycle on this behavioural trait
(Zhao et al., 2021). Corroborating the former, the administration of
progesterone or estrogen + progesterone to ovariectomized rats
decreased impulsive burying (Llaneza and Frye, 2009), suggesting a
hormonal influence. Overall, we found neither sex differences nor a
strong influence of the estrous cycle on marble burying, but an opposite
trend in WE females and males. While the former showed an
anxiolytic-like response, SE males buried 29% less marbles (non-sig­
nificant trend) than WE animals. Overall, the use of defensive burying as
a surrogate of anxiety-like behaviour has limitations (de Brouwer et al.,
2019; Gidyk et al., 2021; Nicolas et al., 2006; Njung’e and Handley,
1991). Though animals receiving anxiolytic and antidepressant treat­
ments tend to bury less marbles, burying objects considered to be
dangerous, threatening or associated with an unpleasant experience is
considered an innate rodent behaviour (de Brouwer et al., 2019; Gidyk
et al., 2021; Nicolas et al., 2006; Njung’e and Handley, 1991; Reznikov
et al., 2016). At present, we do not have a clear explanation for the sex
differences observed in our study. It is possible that, instead of an in­
crease in anxiety, WE females may present a shift in coping strategy,
rather than an increase in anxiety. This, however, remains to be
demonstrated in future studies.
Also interesting were the sex differences observed in novelty
supressed feeding. This test is proposed for the study of anxiety-like
behaviour because rodents have neohypophagia when exposed to a
novel stressful environment. As commonly conducted, however, it also
has a motivational component (Bambico et al., 2015; Bodnoff et al.,
D. Emtyazi et al.
1988; Dulawa and Hen, 2005; Santarelli et al., 2003; Stedenfeld et al.,
2011). Since animals are often food-deprived, they experience the
conflict of being driven to eat in a fearful novel open space. Several
antidepressant treatments reduce the latency to feed in this paradigm
(Bambico et al., 2015; Bodnoff et al., 1988; Dulawa and Hen, 2005;
Santarelli et al., 2003; Stedenfeld et al., 2011). In our study, females had
a lower latency to feed compared to males, with no effect of the estrous
cycle being recorded. In addition, only SE males had an anxiolytic-like
effect compared to WE animals. We note, however, that the latency to
feed in both WE and SE females was similar to that observed in SE males,
and approximately 45–50% lower than in WE males. In previous reports,
female rats were shown to consume a higher amount and derive a more
rewarding experience from palatable foods than males (Klump et al.,
2013; Sinclair et al., 2017). That said, when exposed to a novel context,
females seem to habituate as well as males, consuming less food in a
novel context (Greiner and Petrovich, 2020). The contrasting results
between these latter findings and ours may be related to the fact that we
have habituated the animals to palatable foods before testing in a novel
context. Similar to a previous report (Zhao et al., 2021), we found no
association between the estrous cycle and latency to feed.
One aspect that needs to be discussed regarding our estrous cycle
experiments is that group assignment was based on vaginal lavage
samples collected during extinction, and not during tests to measure
anxiety-type behaviour. We chose to collect vaginal samples at that time
point due to the strong influence of the estrous phase on extinction
(Milad et al., 2009). The estrous cycle in female rodents lasts 4–5 days,
but small individual variations may occur (Ajayi and Akhigbe, 2020;
Auta and Hassan, 2016). Under these circumstances, though it is likely
that differences in the estrous phase between groups extinguished in
high vs low estrous phases would be maintained during anxiety testing,
we cannot guarantee that females were tested in the same phases of the
cycle during marble burying and NSF.
In summary, we found that sex differences in tone recall, and the NSF
were unrelated to the estrous cycle. In addition, major sex differences
were observed between WE and SE, with males presenting long-term
anxiety-type behaviour in the NSF and females presenting an
anxiolytic-type response in the marble burying test. Future investigation
including a larger number of tests, oophorectomized animals and hor­
monal administration is certainly required to corroborate our findings
and ascertain potential mechanisms to explain the sex differences
observed in our study.
Role of funding
The funders had no role in study design, data collection, analysis, or
preparation of the manuscript.
Contributors
DE was responsible for executing the experiments, analyzing data
and preparing the manuscript. TR, ACC, PRS, HK, MD and DG helped
conducting some of the behavioural experiments. PG, NL and IA pro­
vided critical input. CH has planned the studies, analyzed data and
prepared the final version of the manuscript.
Declaration of competing interest
The authors declare no conflicts of interest related to this study.
Acknowledgments
This work was supported by funds from Veteran Affairs Canada and
the Harquail Centre for Neuromodulation. IA is supported by a Tier 1
Canada Research Chair in Brain Repair and Regeneration.
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Journal of Psychiatric Research 151 (2022) 619–625
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jpsychires.2022.05.015.
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