Sex differences in the antidepressant-like response and molecular events induced by the imidazoline-2 receptor agonist CR4056 in rats

Abstract

In searching for novel targets to design antidepressants, among the characterized imidazoline receptors (IR), I2 receptors are an innovative therapeutical approach since they are dysregulated in major depressive disorder and by classical antidepressant treatments. In fact, several I2 agonists have been characterized for their antidepressant-like potential, but the results in terms of efficacy are mixed, and were exclusively reported in male rodents. Since there are well-known sex differences in antidepressant-like efficacy, this study characterized the potential effects induced by two I2 drugs, CR4056 (i.e., most promising drug already in phase II clinical trial for its analgesic properties) and B06 (a compound from a new family of bicyclic α-iminophosphonates) under the stress of the forced-swim test in male and female rats exposed to early-life stress. Moreover, some hippocampal neuroplasticity markers related to the potential effects observed were also evaluated (i.e., FADD, p-ERK/ERK, mBDNF, cell proliferation: Ki-67 + cells). The main results replicated the only prior study reporting the efficacy of CR4056 in male rats, while providing new data on its efficacy in females, which was clearly dependent on prior early-life stress exposure. Moreover, B06 showed no antidepressant-like effects in male or female rats. Finally, CR4056 increased FADD content and decreased cell proliferation in hippocampus, without affecting p-ERK/t-ERK ratio and/or mBDNF content. Interestingly, these effects were exclusively observed in female rats, and independently of early-life conditions, suggesting some distinctive molecular underpinnings participating in the therapeutic response of CR4056 for both sexes. In conjunction, these results present CR4056 with an antidepressant-like potential, especially in female rats exposed to stress early in life, together with some neuronal correlates described in the context of these behavioral changes in females.

Full text

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
Available online 11 February 2023
0091-3057/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Sex differences in the antidepressant-like response and molecular events
induced by the imidazoline-2 receptor agonist CR4056 in rats
Elena Hern´
andez-Hern´
andez
a
,
b
,
1
,
2
, Sandra Ledesma-Corvi
a
,
b
,
1
, Fernando Y´
a˜
nez-G´
omez
a
,
b
,
Celia Garau
a
,
b
, Laura G´
alvez-Melero
a
,
b
, Andrea Bag´
an
c
, Carmen Escolano
c
,
M. Julia García-Fuster
a
,
b
,
*
a
IUNICS, University of the Balearic Islands, Palma, Spain
b
Health Research Institute of the Balearic Islands (IdISBa), Palma, Spain
c
Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food
Sciences, and Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain
ARTICLE INFO
Keywords:
I2 ligands
Sex differences
Depression
Hippocampus
Neurogenesis
Rat
ABSTRACT
In searching for novel targets to design antidepressants, among the characterized imidazoline receptors (IR), I2
receptors are an innovative therapeutical approach since they are dysregulated in major depressive disorder and
by classical antidepressant treatments. In fact, several I2 agonists have been characterized for their
antidepressant-like potential, but the results in terms of efcacy were mixed and exclusively reported in male
rodents. Since there are well-known sex differences in antidepressant-like efcacy, this study characterized the
potential effects induced by two I2 drugs, CR4056 (i.e., most promising drug already in phase II clinical trial for
its analgesic properties) and B06 (a compound from a new family of bicyclic
α
-iminophosphonates) under the
stress of the forced-swim test in male and female rats exposed to early-life stress. Moreover, some hippocampal
neuroplasticity markers related to the potential effects observed were also evaluated (i.e., FADD, p-ERK/ERK,
mBDNF, cell proliferation: Ki-67 +cells). The main results replicated the only prior study reporting the efcacy
of CR4056 in male rats, while providing new data on its efcacy in females, which was clearly dependent on
prior early-life stress exposure. Moreover, B06 showed no antidepressant-like effects in male or female rats.
Finally, CR4056 increased FADD content and decreased cell proliferation in hippocampus, without affecting p-
ERK/t-ERK ratio and/or mBDNF content. Interestingly, these effects were exclusively observed in female rats,
and independently of early-life conditions, suggesting some distinctive molecular underpinnings participating in
the therapeutic response of CR4056 for both sexes. In conjunction, these results present CR4056 with an
antidepressant-like potential, especially in female rats exposed to stress early in life, together with some neuronal
correlates described in the context of these behavioral changes in females.
1. Introduction
The past, present, and future of imidazoline receptors (IRs) was
recently reviewed by Bousquet et al. (2020), in the context of their
possible involvement in different brain functions, especially the ones
mediated through I2 receptors. In particular, the alteration of I2 re-
ceptors is not only linked with psychiatric disorders (i.e., major
depressive disorders, García-Sevilla et al., 1996a, 1996b), but also I2
receptors are regulated by classical antidepressant treatments (García-
Sevilla et al., 1999). The use of several I2 selective compounds has been
key in describing the roles that this receptor plays in mediating some
pharmacological effects. One recurrent discussion in the eld has always
been whether particular pharmacological effects induced by a specic I2
drug were truly mediated by I2 receptors (as discussed by Li, 2017). This
was considered the case when the drug selectively bound to I2 receptors
and the effects were blocked by idazoxan, the only known functional I2
receptor antagonist. In this context, several I2 selective agonists, which
are capable of inducing analgesia in animal models of chronic pain
* Corresponding author at: IUNICS, University of the Balearic Islands, Cra. de Valldemossa, Km 7.5, E-07122 Palma, Spain.
E-mail address: j.garcia@uib.es (M.J. García-Fuster).
1
These authors contributed equally to the present work.
2
Present address of EH-H: Department of Pharmacology, University of the Basque Country (EHU/UPV), Leioa, Spain.
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior
journal homepage: www.elsevier.com/locate/pharmbiochembeh
https://doi.org/10.1016/j.pbb.2023.173527
Received 4 January 2023; Received in revised form 8 February 2023; Accepted 8 February 2023

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
2
(Ferrari et al., 2011; reviewed by Bousquet et al., 2020), inducing hy-
pothermia and neuroprotection, while improving cognition in rodents
(e.g., Gri˜
n´
an-Ferr´
e et al., 2019; Ab´
as et al., 2017, 2020; Vasilopoulou
et al., 2020, 2021; Rodríguez-Ar´
evalo et al., 2021), have shown mixed
results in terms of their antidepressant-like potential in preclinical
studies done in rodents under the stress of the forced-swim test. Some
prior reports suggested the induction of antidepressant-like responses
for several I2 agonists: 2-BFI in rats (Nutt et al., 1995; Hern´
andez-
Hern´
andez and García-Fuster, 2021) and mice (Tonello et al., 2012),
BU224 in rats (Finn et al., 2003), CR4056 in rats (Siemian et al., 2019),
and MCR5 in a senescence-accelerated mouse model (Vasilopoulou
et al., 2020). Contrarily, other studies reported negative data for several
other I2 drugs: e.g., BU224, BU239, BDF 8082 in mice (O'Neill et al.,
2001), 2-BFI or phenyzoline in rats (Siemian et al., 2019), LSL60101
(also known as Garsevil) in rats (Hern´
andez-Hern´
andez et al., 2021),
tracizoline (known as LSL61122 or Valldemossine) in aged rats
(Hern´
andez-Hern´
andez and García-Fuster, 2021). However, most of
these prior studies, if not all, used only male rodents and did not
consider sex as a biological variable (Miller et al., 2017; Beltz et al.,
2019; Docherty et al., 2019) , which is extremely important, especially
given the described sex differences in the efcacy of many antidepres-
sants (e.g., LeGates et al., 2019; García-Cabrerizo et al., 2020; Ledesma-
Corvi et al., 2022).
Therefore, in an attempt to gain knowledge on the potential sex
differences in antidepressant-like responses induced by some I2 drugs,
we further characterized the response of CR4056 (a compound that
features an imidazoline ring), whose efcacy was already described for
male rodents (see Siemian et al., 2019), since it is the most promising I2
agonist (i.e., phase II clinical trial for its analgesic properties, see for
example Vellani et al., 2020). Moreover, this study also aimed at char-
acterizing the potential antidepressant-like response induced by B06, a
compound from a new family of bicyclic
α
-iminophosphonates recently
depicted in terms of its structure (as compared to other I2 drugs
including CR4056), its afnity for I2 receptors, its modulation over brain
Fas-associated protein with death domain (FADD) and/or its agonistic
benecial effects in behavior and cognition in several murine models of
Alzheimer's disease (see Ab´
as et al., 2020; Vasilopoulou et al., 2021).
These drugs were evaluated in the forced-swim test (Slattery and Cryan,
2012), both in naïve and early-life stressed rats as a mild model with the
potential to mimic a negative impact at a preclinical level (see similar
designs but for other antidepressants: García-Cabrerizo et al., 2020; Bis-
Humbert et al., 2021a; Ledesma-Corvi et al., 2022).
In terms of the potential molecular markers behind the
antidepressant-like effects of I2 drugs, and similarly to what was pre-
viously evaluated in the context of other I2 agonists (Garau et al., 2013;
Gri˜
n´
an-Ferr´
e et al., 2019; Ab´
as et al., 2017, 2020; Hern´
andez-Hern´
andez
et al., 2021; Rodríguez-Ar´
evalo et al., 2021), the present study ascer-
tained the regulation of FADD, a cell fate marker within the apoptotic
pathway modulated by several classical antidepressant drugs (García-
Fuster and García-Sevilla, 2016). Moreover, since members of the MAPK
signaling (i.e., ERK1/2) showed a direct link in the regulation of FADD
by certain pharmacological agents (e.g., García-Fuster et al., 2007), we
Fig. 1. Experimental design. A Study I: Characterization of the potential antidepressant-like effect of CR4056. B Study II: Characterization of the potential
antidepressant-like effect of B06. For both studies, a total of 156 male and female rats were exposed in early-life to maternal deprivation (MD) or control condition.
During adulthood, rats were drug- or vehicle-treated (3 pulses in 24 h, i.p.) and scored in the forced-swim test. Groups of treatment including the number of rats per
group are shown for each drug and experimental condition under study. Abbreviations: CR, CR4056; D, day; DMI, desipramine; FST, forced-swim test; IHC,
immunohistochemistry; MD, maternal deprivation; PND, post-natal day; Veh, vehicle; WB, Western blot.
E. Hern´
andez-Hern´
andez et al.

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
3
also evaluated the regulation of p-ERK/t-ERK. Finally, mBDNF was
evaluated as a key molecular marker mediating antidepressant-like ac-
tions (e.g., Diniz et al., 2018; Casarotto et al., 2021), together with the
regulation of hippocampal cell proliferation (e.g., Numakawa et al.,
2018), since hippocampus is a key region modulating affect (reviewed
by Flores et al., 2022).
2. Material and methods
2.1. Animals
For this study, we utilized a total of 156 adult Sprague-Dawley rats
(85 males and 71 females; Fig. 1) that were bred in the animal facility at
the University of the Balearic Islands. Rats resided under standard
housing conditions (12 h light/dark schedule, lights on at 8:00 AM;
22 ◦C, 70 % humidity) and unlimited access to a standard diet and water.
Rats were given at least 1 week to acclimatize to the housing conditions
and the handling prior to the actual experiments, which were performed
during the light period. All procedures complied with the ARRIVE
Guidelines (Percie du Sert et al., 2020), were approved by the competent
authority responsible for ensuring compliance with the regulations
governing the use of animals in scientic experiments as updated in
2010 (Directive 2010/63/EU) and the Spanish Royal Decree 53/2013
for animal experiments, and were awarded ethical approval by the Local
Bioethical Committee and the Regional Government in the Balearic
Islands (Spain). In an effort to avoid unnecessary stress in female rats
during the experimental procedures, the specic stages of the estrous
cycle were not examined. This was also justied by the fact that female
rats are not more variable than their male counterparts in neuroscience
research due to hormonal periodicity (e.g., Becker et al., 2016; Kaluve
et al., 2022), but also because their cyclicity was not part of our research
question (see Beltz et al., 2019).
2.2. Early-life conditions
As a way of inducing early-life stress conditions we relied on a well-
dened paradigm of maternal separation that induces certain negative
psychophysiological effects on rodents (Ellenbroek et al., 1998, 2005;
Marco et al., 2009; also reviewed in Marco et al., 2015), but not
necessarily a depressive-like phenotype (reviewed by Schmidt et al.,
2011). Particularly, in our research group, we have reliably used this
specic paradigm of maternal separation (24 h, post-natal day, PND,
9–10) with reproducibility among different waves of experiments and
with hundreds of rats (García-Cabrerizo et al., 2020; Bis-Humbert et al.,
2021a, 2021b; Bis-Humbert and García-Fuster, 2021; Ledesma-Corvi
et al., 2022). In general, early-life stress modeled with maternal sepa-
ration failed to reproduce baseline changes in affective-like behavior
and locomotor activity, but proved differences in the effectiveness of
selected antidepressant drug treatments (see Ledesma-Corvi et al.,
2022), presenting itself as a suitable platform in which to characterize
novel antidepressant options. Therefore, our experimental design
exposed whole litters to a single episode of maternal deprivation (24 h,
PND, 9–10), during which time pups were left in their home cage with
no nutritional supplements, and the mother was placed in an adjacent
cage. The weights of the pups were monitored right before and after
maternal separation. Litters from the control groups received the same
amount of handling, since they were weighed on PND 9 and 10, but were
kept with the mother during the procedure. Rats were weaned at PND 22
and grouped by sex and early-life condition in cages of 2–4 rats.
2.3. Synthesis of the compounds CR4056 and B06
Compound CR4056 (6-(1H-imidazol-1-yl)-2-phenylquinazoline) was
prepared following the synthetic sequence described in the patent by
Giordani (2008). On the other hand, compound B06 (diethyl
(1RS,3aSR,6aSR)-5-(3-chloro-4-uorophenyl)-4,6-dioxo-1-phenyl-
1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole-1-phosphonate) was
synthetized as described in Ab´
as et al. (2020).
2.4. Pharmacological drug treatments
Study I aimed at characterizing the antidepressant-like response of
CR4056 in control and maternally deprived male and female rats as
described in Fig. 1A. The dose of CR4056 (30 mg/kg, i.p.) was selected
based on a preliminary study from our group, in which this dose was
capable of inducing hypothermia in male rats as measured 90 min post-
treatment (data not shown), and from a prior published study (dose of
32 mg/kg: Thorn et al., 2012).
Study II, which aimed at characterizing the antidepressant-like
response of another I2 drug, B06 (doses of 5, 10 or 20 mg/kg, i.p.;
Ab´
as et al., 2020) was performed in four independent experiments in
which control or maternally deprived, male or female rats were evalu-
ated separately (see groups of study in Fig. 1B). This was caused by
animal availability and/or procedural logistics. Desipramine hydro-
chloride (DMI, 20 mg/kg, i.p.), was used as a positive control of the
antidepressant-like response in either male (see García-Fuster and Gar-
cía-Sevilla, 2016) or both male and female rats (Ledesma-Corvi and
García-Fuster, 2022). In an attempt to reduce the number of animal and/
or experimental groups used, desipramine was not tested in maternally
deprived rats, since it was not part of our research question. The goal
was to use desipramine exclusively in male control rats as a positive
control during the experiment, to ensure that a lack of response by the
drugs under study was not due to something particular and/or
methodological.
2.5. Forced-swim test
This test was rst developed to assess acute antidepressant-like ac-
tivity in rodents (Slattery and Cryan, 2012), and has been extensively
used in our group to evaluate the antidepressant-like potential of several
drugs (e.g., García-Fuster and García-Sevilla, 2016; Bis-Humbert et al.,
2020, 2021a; Ledesma-Corvi et al., 2022; Ledesma-Corvi and García-
Fuster, 2022), including I2 agonists (Hern´
andez-Hern´
andez et al., 2021;
Hern´
andez-Hern´
andez and García-Fuster, 2021), as well as non-
pharmacological treatment options (e.g., electroconvulsive treatment,
see García-Cabrerizo et al., 2020), with reliable and reproducible results
across time. Prior to any drug treatment, rats were individually forced to
swim for 15 min (pre-test session, see Fig. 1) in cylinders (41 cm high ×
32 cm diameter) lled with water (25 ±1 ◦C) up to 25 cm in depth.
Then, rats were treated with 3 pulses of the indicated drug and dose of
study (CR4056, B06 or DMI; see Fig. 1) 24, 5 and 1 h before a 5 min test
session that was videotaped (see Fig. 1). Rats were dried off with a paper
towel at the end of each swimming session, before returning them to
their cage. Later on, each video was analyzed with the software
Behavioral Tracker (CA, USA) by an experimenter blind to the treatment
conditions that scored the time spent (s) immobile vs. active (climbing
or swimming) for each rat. It is important to remember that the forced-
swim test is a reliable test to measure antidepressant-like responses, but
not necessarily to phenotype a depressive-like response (reviewed by
Armario, 2021), and thus it might not be reliable to detect basal dif-
ferences in affective-like behavior when comparing maternally deprived
vs. control rats.
2.6. Brain sample collection and neurochemical evaluations
Brains were collected for the experimental groups in which an
antidepressant-like response was observed (i.e., CR4056) by rapid
decapitation 24 h post-treatment (see Fig. 1A). The regulation of the
selected neuroplasticity markers was evaluated by Western blot analysis
in the right hippocampus, which was freshly dissected. To do so, total
homogenates were prepared as previously described (e.g., García-Cab-
rerizo et al., 2015), and brain proteins (40
μ
g) were separated by
E. Hern´
andez-Hern´
andez et al.

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
4
electrophoresis on 10–12 % SDS-PAGE mini-gels (Bio-Rad Laboratories,
CA, USA). Membranes were incubated with the appropriate primary
antibody: anti-FADD (H-181) (1:2500) and anti-mBDNF (1:2500) were
both from Santa Cruz Biotechnology (CA, USA); anti-p-ERK1/2 (p44/
p42) (1:1000) and anti-t-ERK1/2 (1:1000), from Cell Signaling (MA,
USA); and anti-β-actin (clone AC-15) (1:10000; Sigma–Aldrich, MO,
USA). The next day, membranes were incubated with the secondary
antibody (anti-rabbit or -mouse IgG linked to horseradish peroxidase;
1:5000; Cell Signaling), and immunoreactivity was detected with ECL
reagents (Amersham, Buckinghamshire, UK) in autoradiographic lms
(Amersham ECL Hyperlm), which were then quantied by densito-
metric scanning (GS-800 Imaging Calibrated Densitometer, Bio-Rad).
For each marker and rat, we calculated percent changes in immunore-
activity with respect to male-control-vehicle samples (100 %) in various
gels, and the mean value was used as a nal estimate. β-actin was used as
a loading control as it did not change with the treatment conditions.
The left half-brain was snap-frozen with isopentane (−30 ◦C) and
stored at −80 ◦C until hippocampal cell proliferation was quantied
with Ki-67 antibody (1:20,000; provided by Drs. Huda Akil and Stanley
J. Watson, University of Michigan, MI, USA) by immunohistochemistry
(e.g., García-Fuster et al., 2010; Garcia-Fuster et al., 2017; García-Cab-
rerizo et al., 2020). Briey, experiments were performed in 1 slide per
rat containing 8 tissue-sections from the middle portion of hippocampus
that were cryostat-cut (30
μ
m sections), slide-mounted and post-xed in
4 % paraformaldehyde. The next steps included a series of sequential
incubations, including that with a biotinylated anti-rabbit antibody
(1:1000 respectively, Vector Laboratories, CA, USA), an Avidin/Biotin
complex (Vectastain Elite ABC kit; Vector Laboratories), and the chro-
mogen 3,3′-diaminobenzidine (DAB) for signal detection. Finally, tissue
was counterstained in cresyl violet, dehydrated in graded alcohols and
immersed in xylene before cover-slipping it with Permount®. Positive
cells were quantied in 8 sections/rat by an experimenter blind to the
treatment groups with a Leica DMR light microscope (63×objective
lens) and focusing through the thickness of the tissue. The quantied
area (mm
2
) in each section was measured with a densitometer (GS-800
Imaging Calibrated Densitometer, BioRad) to correct the total number of
proliferating cells (Ki-67+) in the dentate gyrus by the total area
analyzed (mm
2
) as previously described (e.g., García-Cabrerizo et al.,
2015; Garcia-Fuster et al., 2017).
2.7. Data analysis
GraphPad Prism, Version 9.5 (GraphPad Software, USA) was used for
data analysis and graph plotting was performed following the guidelines
in experimental pharmacology for displaying data and statistical
methods (e.g., Curtis et al., 2018; Michel et al., 2020). Results are re-
ported in bar graphs as mean values ±standard error of the mean
(SEM), with symbols shown for each individual rat. For Study I, and
depending on the presence of sex differences, data was either analyzed
with three-way ANOVAs (independent variables: Sex, Early-life Condi-
tion, Treatment) or two-way ANOVAs (independent variables: Early-life
Condition, Treatment) for each sex separately followed by multiple
comparisons tests to perform post-hoc pair-wise statistical evaluations
when appropriate. For Study II, and given that each sex and early-life
experimental condition was tested in separate experiments, statistical
analysis were performed through one-way ANOVAs (followed by Dun-
nett's multiple comparisons test when appropriate) or unpaired two-
tailed t-tests depending on the number of groups to compare. Signi-
cance was set at p ≤0.05.
3. Results
3.1. Characterizing the potential antidepressant-like effect of CR4056 in
rats (Study I)
When analyzing the results obtained in the forced-swim test through
three-way ANOVAs (Fig. 2A–C), specic sex differences emerged that
conditioned and/or justied the subsequent analysis for each sex sepa-
rately. In particular, there were sex differences in immobility (F
1,52
=
27.11, ###p <0.001; Fig. 2A) and climbing (F
1,52
=27.66, ###p <
0.001) behavior, as measured 1 h after the last treatment injection.
Female rats showed higher immobility (mean of 199 s vs. 133 s for
males; Fig. 2A), lower climbing (mean of 79 s vs. 144 s for males;
Fig. 2B), but no change in the time spent swimming (mean of 21 for both
sexes; Fig. 2C).
Therefore, the antidepressant-like effects induced by CR4056 in the
forced-swim test were evaluated 1 h after the administration of 3×
pulses given in a 24-h period for each sex separately. In male rats, a two-
way ANOVA showed a signicant effect of Early-Life Condition (F
1,24
=
4.90, p =0.037) and of Treatment (F
1,24
=9.11, p =0.006), but no
interaction (F
1,24
=0.01, p =0.944). In particular, rats exposed to
maternal deprivation showed lower rates of immobility than controls
(general drop of −42 ±19 s, MD vs. Control groups, Fig. 2A), and
CR4056 induced an antidepressant-like effect independently of early-life
condition (drop in immobility of −57 ±19 s vs. Veh-treated groups,
Fig. 2A). Interestingly, in females, besides the signicant effect of Early-
Life Condition (F
1,28
=5.70, p =0.024) and Treatment (F
1,28
=27.56, p
<0.001), there was a signicant interaction between Early-Life Condi-
tion x Treatment (F
1,28
=4.60, p =0.041). Post-hoc comparisons
(Tukey's multiple test) revealed that CR4056 induced an antidepressant-
like effect in maternally-deprived rats (−125 ±24 s immobile, ***p <
0.001 vs. MD-Veh-treated rats; Fig. 2A), but also when comparing
CR4056 effects depending on the prior Early-Life Condition, since it
showed a higher efcacy in rats previously exposed to maternal depri-
vation (−77 ±23 s immobile, $p =0.013 vs. Control-CR; Fig. 2A).
We later evaluated what active behavior was behind the changes
observed in immobility. In male rats, there was a signicant effect of
Treatment (F
1,24
=7.99, p =0.009), but no effect of Early-Life Condition
(F
1,24
=4.08, p =0.055) or interaction (F
1,24
=0.16, p =0.690).
Therefore, the antidepressant-like effect of CR4056 in male rats was
driven by an increase in climbing (+52 ±18 s, CR vs. Veh-treated
groups, Fig. 2B). In females, besides the signicant effect of Treatment
(F
1,28
=26.67, p <0.001), there was a signicant interaction between
Early-Life Condition x Treatment (F
1,28
=4.00, p =0.05), but no effect
of Early-Life Condition (F
1,28
=3.51, p =0.071). Post-hoc comparisons
(Tukey's multiple test) revealed that CR4056 induced an antidepressant-
like effect in maternally-deprived rats by increasing climbing (+121 ±
24 s, ***p <0.001 vs. MD-Veh-treated rats; Fig. 2B), but also when
comparing CR4056 effects depending on the prior Early-Life Condition,
since it showed a higher efcacy in rats previously exposed to maternal
deprivation (+66 ±23 s, $p =0.040 vs. Control-CR; Fig. 2B). Finally, no
relevant changes were observed for swimming behavior (Fig. 2C).
3.2. Characterizing the potential antidepressant-like effect of B06 in rats
(Study II)
The effects of B06 in the forced-swim test were evaluated, as
mentioned earlier, in separate experiments (control rats separately than
maternally-deprived, and male separately than female rats). Therefore,
the potential effects were evaluated for each experiment separately.
Particularly, B06 at the doses tested did not induce an antidepressant-
like effect as measured through immobility time in male control (one-
way ANOVA: F
3,32
=1.28, p =0.300) or maternally-deprived (t =1.05,
df =12, p =0.314; Fig. 2D) rats. Similarly, no effects were observed for
female control (one-way ANOVA: F
2,23
=2.00, p =0.158) or maternally
deprived (t =0.88, df =11, p =0.396; Fig. 2D) rats. Moreover, when
evaluating activity time, no signicant changes were observed for
climbing or swimming behaviors both for male or female rats (statistical
analyses not shown; see Fig. 2E and F). As a positive control, we used
desipramine at a dose known, in our experimental conditions, to induce
a signicant antidepressant-like effect for male (García-Fuster and
García-Sevilla, 2016) or for both male and female rats (Ledesma-Corvi
E. Hern´
andez-Hern´
andez et al.

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
5
and García-Fuster, 2022). As expected desipramine induced a positive
response by decreasing immobility (t =2.85, df =14, *p =0.013 vs.
Veh-treated rats; Fig. 2D) and increasing climbing (t =2.21, df =14, *p
=0.045 vs. Veh-treated rats; Fig. 2E), while no changes were induced on
swimming behavior (Fig. 2F).
3.3. Characterizing the molecular effects of CR4056 in rats (Study I)
Since CR4056 showed a clear effect on antidepressant-like behaviors,
we wanted to further study this effect on a molecular level. The analysis
of the molecular events induced by CR4056 through three-way ANOVAs
(Fig. 3A–E), disclosed basal sex differences in the hippocampal protein
content of FADD (F
1,51
=41.63, ###p <0.001; Fig. 3A) and mBDNF
(F
1,51
=9.36, ##p =0.004; Fig. 3C), but not of p-ERK/t-ERK ratio (F
1,39
=0.28, p =0.603; Fig. 3B) nor for the rate of cell proliferation (Ki-67 +
cells/mm
2
: F
1,52
=3.37, p =0.072; Fig. 3E). Based on some sex differ-
ences, the subsequent analyses were performed for each sex separately.
While in male rats no signicant effects of Early-Life Condition or
Treatment were observed for FADD modulation (statistical analyses not
shown; see Fig. 3A), there was a signicant effect of Treatment in female
rats (F
1,27
=7.37, p =0.011; Fig. 3A). In particular, CR4056 induced an
increase in hippocampal FADD content in females independently of
early-life condition (+21 ±8 % vs. Veh-treated groups, Fig. 3A). When
analyzing p-ERK/t-ERK ratio or mBDNF protein levels in hippocampus,
no signicant effects were observed by Early-Life Condition or Treat-
ment both for male or female rats (statistical analyses not shown; see
Fig. 3B and Fig. 3C). Similar to what was observed for FADD, when
analyzing the number of Ki-67 +cells/area, there were no effects of
Early-Life Condition or Treatment for male rats, but a signicant effect
of Treatment for female rats (F
1,28
=8.45, p =0.0071); CR4056 induced
a decrease in cell proliferation in females independently of early-life
condition (−6.8 ±2.3 +cells/mm
2
vs. Veh-treated groups, Fig. 3E).
This experiment was validated with a second immunohistochemistry
study that reported identical results.
4. Discussion
The main results of the study reinforced, along with prior literature,
the importance of including both sexes when characterizing pharma-
cological agents at the preclinical level. Indeed, male and female rats
differed basally in the response induced in some behavioral tests, con-
ditioning and/or justifying the subsequent analysis for each sex sepa-
rately. For example, in the forced-swim test, female rats were more
immobility and less active (i.e., climbing) than their male counterparts.
Some basal differences were also observed in the expression of certain
neurochemical markers (i.e., females showed higher hippocampal FADD
and mBDNF protein content). As for the potential antidepressant-like
effects of the drugs tested in the forced-swim test, the results repli-
cated the only prior study reporting the efcacy of CR4056 in male rats,
while providing some new data on its efcacy in female rats, which was
clearly dependent on prior early-life stress exposure. However, B06
showed no antidepressant-like effects in male or female rats. Finally,
CR4056 increased levels of FADD protein content and decreased cell
proliferation in hippocampus as measured 24 h post-treatment, without
affecting p-ERK/t-ERK ratio and/or mBDNF content. Interestingly, these
effects were exclusively observed in female rats, and independently of
Fig. 2. A–C Study I: Characterization of the potential antidepressant-like effect of CR4056. D–F Study II: Characterization of the potential antidepressant-like effect
of B06. Antidepressant-like responses were measured in the forced-swim test after treatment in male and female rats with or without early-life stress (maternal
deprivation). Columns represent mean ±SEM of the time spent (s) A, D immobile, B, E climbing or C, F swimming (individual symbols are shown for each rat).
Three-way ANOVAs were used to detect sex differences: ###p <0.001. Antidepressant-like effects were evaluated for each sex separately by two-way ANOVAs
followed by Tukey's multiple comparisons test when appropriate: *p <0.05 when compared with Control-Veh-treated rats, ***p <0.001 when compared with MD-
Veh-treated rats, and $p <0.05 when compared with Control-CR-treated rats. (For interpretation of the references to colour in this gure legend, the reader is
referred to the web version of this article.)
E. Hern´
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Pharmacology, Biochemistry and Behavior 223 (2023) 173527
6
early-life conditions, suggesting some distinctive molecular un-
derpinnings taking place right after the therapeutic response of CR4056
for both sexes.
The antidepressant-like effects induced by CR4056 in the forced-
swim test were evaluated (1 h after the administration of 3×pulses
given in a 24-h period) for each sex separately, given the clear basal sex
differences described in behavior in the present study and as previously
reported (see for example Ledesma-Corvi et al., 2022 for our prior
results). In male rats, CR4056 induced an antidepressant-like effect
independently of early-life condition (i.e., decreased immobility and
increased climbing). An improvement in the test through climbing
behavior was previously reported for other I2 agonists (e.g., Hern´
andez-
Hern´
andez and García-Fuster, 2021), in line with the expected increase
in brain noradrenaline produced by these drugs known to mediate some
of their responses (e.g., Ferrari et al., 2011; Siemian et al., 2018), and
similarly to antidepressants that enhance noradrenaline
Fig. 3. Study I: Characterization of the molecular effects of CR4056. A–D Changes in the modulation of hippocampal proteins were measured by western blot 24 h
after treatment in male and female rats with or without early-life stress (maternal deprivation). A FADD, B p-ERK/t-ERK ratio, and C mBDNF proteins. Columns
represent mean ±SEM of n experiments per group and expressed as % Male-Control-Vehicle (individual values are shown for each rat in symbols). Three-way
ANOVAs were used to detect sex differences ##p <0.01 and ###p <0.001. A two-way ANOVA detected a signicant increase in FADD content for female rats
treated with CR4056. No other changes were observed. D Representative immunoblots depicting labeling of FADD, p-ERK, t-ERK, mBDNF and the corresponding for
β-actin as a loading control. E–F Changes in hippocampal cell proliferation were measured by immunohistochemistry 24 h after treatment in male and female rats
with or without early-life stress (maternal deprivation). Columns represent mean ±SEM of Ki-67+cells/area (mm
2
) (individual symbols are shown for each rat). No
statistical differences were detected. F Representative images showing individual Ki-67 +cells (brown labeling in the blue granular layer) taken with a light mi-
croscope (63×objective lens). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
E. Hern´
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Pharmacology, Biochemistry and Behavior 223 (2023) 173527
7
neurotransmission (see Detke et al., 1995, 1997; R´
en´
eric et al., 2002).
Although it was suggested that I2 receptor activation might stimulate
locomotion activity in rats (Barrot et al., 2000), prior studies reported, at
a dose-range that include the dose tested in the present study, either no
changes in the locomotor response induced by CR4056 in the open eld
in Wistar rats (Ferrari et al., 2011) or 5xFAD mice (Mota et al., 2022), or
even a decrease in locomotion in Sprague-Dawley rats (Thorn et al.,
2012). These prior data ensured that the increased activity observed in
the forced-swim test by CR4056 was not due to potential increases in
locomotor activity. Interestingly, CR4056 also induced an
antidepressant-like effect in female rats by decreasing immobility and
increasing climbing in the forced-swim test. However, this benecial
effect was mainly driven by how it interacted with early-life stress
exposure, since efcacy was higher in females with a prior exposure to
maternal deprivation; the signicant effect was observed both when
comparing the response vs. maternally deprived vehicle-treated rats, or
vs. control CR4056-treated rats. This interaction aligned with prior
studies demonstrating that certain benecial effects of I2 agonists
needed a prior insult to be effective (e.g., CR4056 improved cognition
only in 5xFAD but not in wild-type mice; Mota et al., 2022) and/or with
the effects described for other antidepressant drugs (i.e., low dose of
ketamine) only observed in female rats exposed to early-life stress
(Ledesma-Corvi et al., 2022). Therefore, together with the prior study
that characterized the effects induced by CR4056 in the forced-swim
test, but exclusively in male reserpinized rats (dose of 32 as opposed
to 10 mg/kg that was not efcacious; Siemian et al., 2019), our data
validated the antidepressant-like potential for this drug (dose of 30 mg/
kg), while including both sexes, and suggested a better efcacy for fe-
males, especially in the presence of a previous early-life stressor. The
fact that CR4056 worked so well for females is of relevance, since most
antidepressants previously tested failed to show efcacy in females
when scored in the forced-swim test at doses that would otherwise work
for males (e.g., García-Cabrerizo et al., 2020; Ledesma-Corvi et al.,
2022), and given the lack of knowledge regarding the effects of I2 ag-
onists on female rats.
Contrarily to the potential benecial effects of CR4056, another I2
drug from a new structural family of bicyclic
α
-iminophosphonates
(B06; see Ab´
as et al., 2020 for its structural characterization, and also
Fig. 4) did not induce signs of an antidepressant-like potential, at the
doses tested, and as measured through immobility time in the forced-
swim test in male or female rats (both for control or maternally-
deprived conditions). Since no prior studies have been done attempt-
ing to characterize this family of new compounds in terms of their
antidepressant-like potential, a positive control was evaluated in par-
allel. In particular, desipramine at a dose known to induce, in prior
studies from our group and with equivalent experimental conditions, a
signicant antidepressant-like response of similar magnitude both in
male (García-Fuster and García-Sevilla, 2016) and female rats (Ledesma-
Corvi and García-Fuster, 2022), also showed decreased immobility in
the forced-swim test paired with increased climbing. The present lack of
antidepressant-like potential of B06 aligned with prior data, in terms
that not all I2 drugs are capable of displaying antidepressant-like
responses (e.g., O'Neill et al., 2001; Siemian et al., 2019; Hern´
andez-
Hern´
andez et al., 2021). The differences in antidepressant-like responses
observed for I2 agonists might rely, as previously discussed (Hern´
andez-
Hern´
andez et al., 2021) in their chemical structures. For example, as
shown in Fig. 4, CR4056 features the atom of the nitrogen of the imid-
azole linked to the rest of the heteroaromatic ring, while LSL60101
embodies a 2-imidazolebenzofuranyl, and 2-BFI a 2-imidazolinebenzo-
furanyl (imidazoline is a nucleus with one of the double bonds of the
imidazole reduced) (Nutt et al., 1995; Tonello et al., 2012). However,
B06 does not contain the imidazole/imidazoline nucleus (Fig. 4). As
mentioned in a prior study (Ab´
as et al., 2020), the structure of B06
differed from the restricted substitution pattern of I2-IR drugs, with its
bicyclic-iminophosphonate structure being the rst described non-
imidazoline/non-imidazole- containing compound, showing
outstanding afnity to I2-IR. In fact, a 3D-QSAR study revealed key
structural parameters for the design of future promising structures,
proposing a pharmacophore for certain standard agonists (i.e.,
LSL60101, tracizoline, idazoxan and BU99008; see Ab´
as et al., 2020). A
prior study reported that there are differences in efcacy among existing
I2 receptor agonists (e.g., Qiu et al., 2015), suggesting that the limited
efcacy induced by B06 on I2 receptors might be insufcient to produce
an antidepressant-like response in this particular model. Interestingly,
this lack of efcacy might not be generalized, since B06 has proven to
cross the blood brain barrier in sufcient quantities to induce pharma-
cological effects, both behavioral and neurochemical (Ab´
as et al., 2020).
In fact, B06 levels correlated between plasma and the brain when the
drug was administered at a dose of 10 mg/kg (Bag´
an et al., 2022), a dose
within the range tested in the present study (5–20 mg/kg). Particularly,
SAMP8 mice treated with 5 mg/kg of B06 during 3 weeks showed
improved cognitive rates through a neuronal pathway (Vasilopoulou
et al., 2021), and B06 was capable of inducing behavioral improvements
at a dose of 5 mg/kg in a 5xFAD mouse model (Ab´
as et al., 2020).
The molecular events induced by CR4056 as measured 24 h post-
treatment also revealed sex differences in the basal content of some of
the markers evaluated in hippocampus (i.e., FADD and mBDNF), while
others showed similar contents for both sexes (i.e., p-ERK/t-ERK ratio,
Ki-67 +cells). Interestingly, the only changes induced by CR4056 were
observed exclusively in female rats, and independent of early-life con-
ditions. Particularly, FADD was increased and cell proliferation was
decreased in hippocampus 24 h after treatment. No changes were
observed in p-ERK/t-ERK ratio or mBDNF protein levels in hippocampus
by CR4056 in rats. Preliminary non-published data from our group
proved that a single dose of 30 mg/kg of CR4056 induced hypothermia
(90 min post-treatment) while reduced hippocampal FADD in male rats
as measured 2 h post-treatment, similarly to most I2 agonists previously
evaluated (e.g., Ab´
as et al., 2017, 2020; Rodríguez-Ar´
evalo et al., 2021).
These acute effects reverted when measured 24 h post-treatment (Ab´
as
et al., 2020), in line to what we report here for CR4056 in male rats. The
higher content of hippocampal FADD in female rats, as compared to
males, was expected since it was previously reported (e.g., Ledesma-
Corvi and García-Fuster, 2022; ; as well as other unpublished data from
our group). However, this is the rst time that we measured FADD in
hippocampus of female rats following the administration of an I2 drug.
Interestingly, the results demonstrated increased FADD levels 24 h post-
treatment in adult female rats treated with CR4056; sex-specic effects
that emerged after the observed antidepressant-like response. This
pattern of FADD regulation was previously observed for other antide-
pressants (García-Fuster and García-Sevilla, 2016), including desipra-
mine (Ledesma-Corvi and García-Fuster, 2022). It was suggested, as it
could be speculated for CR4056, that following an acute antidepressant
administration, certain neuroprotective actions might be initiated that
cause a decrease in FADD (1 or 2-h post-treatment), to latter increase its
content (1-day post-treatment, i.e., rebound effect after the acute initi-
ation of an anti-apoptotic response, see prior similar results by García-
Fuster et al., 2009), followed by a normalization of all levels 5-days post-
treatment (e.g., Ledesma-Corvi and García-Fuster, 2022). Similarly,
Fig. 4. Chemical structure of CR4056, LSL60101, 2-BFI and B06 (2-imidazole
nucleus in green, 2-imidazoline in blue, and 3,4-dihydro-2H-pyrrole in red).
(For interpretation of the references to colour in this gure legend, the reader is
referred to the web version of this article.)
E. Hern´
andez-Hern´
andez et al.

Pharmacology, Biochemistry and Behavior 223 (2023) 173527
8
earlier studies showed that while a single drug injection with other I2
drugs decreased FADD in hippocampus (Ab´
as et al., 2017, 2020;
Rodríguez-Ar´
evalo et al., 2021), repeated (5 days) administration
showed no changes on FADD (Ab´
as et al., 2020), in line with the lack of
effects observed for male rats in the present study (3 pulses in a 24 h
period) and the ones observed in other brain regions (i.e., cerebral
cortex, see Garau et al., 2013).
Finally, CR4056 decreased cell proliferation in hippocampal samples
of female rats as measured 24 h post-treatment, in conjunction with
FADD increases, and in line with a prior study that reported increased
FADD (both at protein and mRNA levels) with impaired cell prolifera-
tion rates (Ki-67+mitotic progenitor cells; García-Fuster et al., 2011). In
that study, the decrease in cell proliferation was speculated to be asso-
ciated with a neurotoxic effect induced by cocaine, although, in line
with the prior discussion on FADD, it could also be justed by an
adaptive response following a putative speculated acute increase. In this
context, our initial hypothesis was to nd increased proliferation levels
since most antidepressants increase neurogenesis (see reviews over the
past 20 years by Duman et al., 2001 and Kot et al., 2022) but also
mBDNF (e.g., Diniz et al., 2018; Casarotto et al., 2021), which was also
unaltered. However, we did not collect samples when the
antidepressant-like effect was observed (1 h post-treatment), which
probably, in line with the previous literature, would show an acute in-
crease in cell proliferation and mBDNF. Also, most of these markers were
previously evaluated almost exclusively for male rodents, and thus the
lack of detailed molecular data on female brains together with the dis-
crepancies observed in the neurochemical events taking place for this
sex might still justify some level of neuroplasticity associated and/or
emerging after the positive behavioral response observed.
In conjunction, these results present CR4056, an I2 agonist, as a
potential candidate to be further studied as an antidepressant, especially
in female rats in which their condition might be stress-related. More-
over, some neuronal correlates were dysregulated right after these
behavioral changes in females, which will require a deeper analysis in
terms of their meaning and potential targeting for the development of
future drugs.
Role of funding source
This research was partly supported by several grants to MJG-F:
PID2020-118582RB-I00 (MCIN/AEI/10.13039/501100011033) and
PDR2020/14 (Comunitat Aut`
onoma de les Illes Balears through the
Direcci´
o General de Política Universit`
aria i Recerca with funds from the
Tourist Stay Tax Law ITS 2017–006). Moreover, this study was also
funded by PID2019-107991RB-I00 (MCIN/AEI) and PDC2022-133441-
I00 (MCIN/AEI/10.13039/501100011033 and by the “European Union
NextGenerationEU/PRTR”) to CE. These entities had no further role in
study design; in the collection, analysis and interpretation of data; in the
writing of the report; and in the decision to submit the paper for pub-
lication. The salaries of personnel were covered by several research
programs: predoctoral scholarships to EH-H (FPI/2102/2018) from
Consejería de Innovaci´
on, Investigaci´
on y Turismo del Gobierno de las
Islas Baleares y del Fondo Social Europeo, who is currently funded by the
Margarita Salas Program (Ministerio de Universidades; Plan de Recu-
peraci´
on, Transformaci´
on y Resilencia; NextGenerationEU) with the
participation of the University of the Balearic Islands; JUNIOR Program
to SL-C from “TALENT PLUS Construint Salut, Generant Valor” (IdISBa,
GOIB); CG is funded by the María Zambrano Program (Ministerio de
Universidades; Plan de Recuperaci´
on, Transformaci´
on y Resilencia;
NextGenerationEU) at the University of the Balearic Islands; AB received
nancial support from Institute of Biomedicine UB_2018.
Declaration of competing interest
The authors declare that they have no conict of interest.
Data availability
Data will be made available on request.
Acknowledgments
We would like to thank Professors Huda Akil and Stanley J. Watson
(Michigan Neuroscience Institute, University of Michigan, Ann Arbor,
USA) for providing Ki-67 primary antibody.
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