Revisiting the antidepressant-like effects of desipramine in male and female adult rats: sex disparities in neurochemical correlates

Abstract

Background
The preclinical antidepressant-like characterization of desipramine relied almost exclusively in male rodents, with only a few contradictory reports done in females. Given that most experiments assessed a single dose and/or timepoint of analysis after-treatment, this study evaluated potential sex-differences in the length of the antidepressant-like response induced by different doses of desipramine as well as the molecular underpinnings driving the different responses by sex.

Methods
Male and female Sprague–Dawley rats were treated (i.p.) with 3 pulses of desipramine (5, 10 or 20 mg/kg) or vehicle (0.9% NaCl) within 24 h. The antidepressant-like effects were evaluated in the forced-swim test 1-h, 1- and 3-day post-treatment. The rate of cell proliferation and the regulation of key neuroplasticity markers (FADD, Cdk5, p35, p25) involved in antidepressant-like responses in the hippocampus were evaluated 1-h, 1-day and 5-day post-treatment.

Results
Desipramine induced similar antidepressant-like effects in male and female rats (effective doses of 10 and 20 mg/kg, with effects that lasted up to 1-day post-treatment), without altering the rate of cell proliferation. However, some sex-differences emerged when evaluating neuroplasticity markers in the hippocampus, while no changes were observed for female rats, desipramine regulated FADD, Cdk-5 and p25 in males in a way that suggested neuroprotective actions.

Conclusions
Our findings imply that while desipramine induced similar antidepressant-like responses for male and female rats, some differences emerged in the regulation of certain neuroplasticity markers, suggesting that distinctive molecular mechanisms might be participating in the therapeutic response of desipramine for both sexes.

Full text

Vol:.(1234567890)
Pharmacological Reports (2022) 74:626–636
https://doi.org/10.1007/s43440-022-00372-1
1 3
ARTICLE
Revisiting theantidepressant‑like effects ofdesipramine inmale
andfemale adult rats: sex disparities inneurochemical correlates
SandraLedesma‑Corvi1,2· M.JuliaGarcía‑Fuster1,2
Received: 4 April 2022 / Revised: 12 May 2022 / Accepted: 13 May 2022 / Published online: 2 June 2022
© The Author(s) 2022
Abstract
Background The preclinical antidepressant-like characterization of desipramine relied almost exclusively in male rodents,
with only a few contradictory reports done in females. Given that most experiments assessed a single dose and/or timepoint
of analysis after-treatment, this study evaluated potential sex-differences in the length of the antidepressant-like response
induced by different doses of desipramine as well as the molecular underpinnings driving the different responses by sex.
Methods Male and female Sprague–Dawley rats were treated (i.p.) with 3 pulses of desipramine (5, 10 or 20mg/kg) or
vehicle (0.9% NaCl) within 24h. The antidepressant-like effects were evaluated in the forced-swim test 1-h, 1- and 3-day
post-treatment. The rate of cell proliferation and the regulation of key neuroplasticity markers (FADD, Cdk5, p35, p25)
involved in antidepressant-like responses in the hippocampus were evaluated 1-h, 1-day and 5-day post-treatment.
Results Desipramine induced similar antidepressant-like effects in male and female rats (effective doses of 10 and 20mg/
kg, with effects that lasted up to 1-day post-treatment), without altering the rate of cell proliferation. However, some sex-
differences emerged when evaluating neuroplasticity markers in the hippocampus, while no changes were observed for female
rats, desipramine regulated FADD, Cdk-5 and p25 in males in a way that suggested neuroprotective actions.
Conclusions Our findings imply that while desipramine induced similar antidepressant-like responses for male and female
rats, some differences emerged in the regulation of certain neuroplasticity markers, suggesting that distinctive molecular
mechanisms might be participating in the therapeutic response of desipramine for both sexes.
Keywords Antidepressant· Rat· Hippocampus· Neuroplasticity· Sex
Introduction
There are well-known sex-differences in depression rates,
with women being almost twice as likely to suffer depres-
sion as men (e.g., [1–3]). Moreover, an extensive body of
research demonstrated sex differences in antidepressant
efficacy (reviewed by [4, 5]), although the direction of the
change is not completely clear; while generally speaking
women seem less responsive and/or suffering more adverse
effects than men, this appears especially true for tricyclic
antidepressants; however, other reports suggested a better
efficacy for females might also exist for drugs selectively
inhibiting the serotonin reuptake (reviewed by [6]). These
sex differences in efficacy might be explained by disparities
in the pharmacokinetics and, to a lesser extent, pharmacody-
namics effects of the drug (reviewed by [4–7]), which com-
bined with the shortage of preclinical studies performed in
female rats [8], justify the poor translation to the clinic, spe-
cially for female patients. Adjusting doses and/or timings,
and/or even changing medications should be considered for
each sex for an optimal clinical response.
In an effort to improve the scarce number of studies
including sex as a biological variable [8–10], our research
group (among several others) is centered in characteriz-
ing sex differences in antidepressant-like responses (e.g.,
[11–13]), including revisiting the antidepressant-like effects
of desipramine in male and female rats which was the goal
of the present study. Desipramine, a typical tricyclic anti-
depressant, is an effective and safe treatment prescribed for
major depressive and other psychiatric disorders [14, 15].
Although, desipramine showed no significant sex differences
* M. Julia García-Fuster
j.garcia@uib.es
1 IUNICS, University oftheBalearic Islands, Cra. de
Valldemossa km 7.5, 07122Palma, Spain
2 Health Research Institute oftheBalearic Islands (IdISBa),
Palma, Spain

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Revisiting theantidepressant‑like effects ofdesipramine inmale andfemale adult rats: sex…
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for the treatment of multiple illnesses in children and ado-
lescents [16], other prior pharmacological studies revealed
an impact of sex in hormonal responses to desipramine
in healthy young sibling pairs in a randomized cross-over
experiment [17], as well as sex differences in pharmacoki-
netics (reviewed by [6]). The preclinical characterization of
desipramine as an antidepressant relied almost exclusively in
male naïve rodents (e.g., [18–20]) or male rodents exposed
to stress [21], and generally utilizing the forced-swim test
as a well-accepted experimental tool to screen drugs for
its activity [22]. However, only a few preclinical studies
included females, and showed contradictory results, with
either signs of a significant antidepressant-like response [23]
or a lack of efficacy [24]. In the context of these inconsistent
effects, and given that most studies only assessed a single
dose and/or timepoint of analysis after treatment, the present
study evaluated potential sex differences in the length of
the antidepressant-like response (1h, 1day and up to 5-day
post-treatment) induced by different doses of desipramine (5,
10 and 20mg/kg) in rats exposed to the forced-swim test, a
model with strong predictive and discriminative validity, in
which to further characterize the molecular underpinnings
driving the potential different responses of desipramine by
sex.
In particular, neural adaptations taking place in the hip-
pocampus (reviewed by [25], and more recently by [26]),
such as neurogenesis, are modulated in depressive-like states
(neurogenic theory of depression; [27–29]), by most antide-
pressants [28, 30]), desipramine in particular [21, 31–33],
and its ablation reduced the effects of certain antidepressants
[34, 35], suggesting an important role for this particular pro-
cess in the antidepressant-like response of certain drugs.
Moreover, besides neurogenesis, other hippocampal plas-
ticity events [25, 26] induced by desipramine also deserve
further characterization, in terms of their role in controlling
cell fate (balance between cell death vs. survival/plasticity),
and since these processes might alter the basal rates of cell
genesis, and participate in mediating some of the behavio-
ral responses observed. For example, since depression has
been linked to certain neurotoxic events (aberrant apopto-
sis, [36]) in humans and animals (reviewed by [25]), the
role of antidepressants as neuroprotective agents (mediating
anti-apoptotic actions) generated continued interest. Particu-
larly, different antidepressant drugs, including desipramine,
decreased pro-apoptotic signals (e.g., [37]) and promoted
anti-apoptotic functions through the regulation of Fas-
associated protein with death domain (FADD), and inter-
estingly FADD levels correlated with the antidepressant-
like response exerted by desipramine [19]. Finally, cyclin
dependent kinase-5 (Cdk5), which is limited to post-mitotic
neurons [39] and is linked to hippocampal neurogenesis by
playing an important role in neuronal migration and hip-
pocampal cell survival [42], also regulates depression-like
behavior [38], and depends of co-factors p35 and p25 [40,
41]. While hippocampal Cdk-5 activity was increased in
animal models of stress, it was reduced by antidepressants
through redistribution of p35 from the cell membrane to the
cytoplasm [43], suggesting that the Cdk-5/p35–p25 complex
might play an important role in regulating antidepressant
actions. Against this background, and since most molecular
studies previously reported were performed almost exclu-
sively in male rodents, this study deepened in the molecular
sex differences induced by desipramine in male and female
rats and developing throughout time.
Experimental procedures
Animals
Adult Sprague–Dawley rats (n = 142; 90 males and 52
females) were bred at the University of the Balearic Islands
and housed in groups of 2–4 in standard cages in controlled
environmental conditions (22ºC, 70% humidity, and 12-h
light/dark cycle, lights on at 8:00 a.m.) with ad libitum
access to a standard diet and tap water. Prior to the experi-
mental procedures, rats were habituated to being handled
by the experimenter (for at least 2days). All animal pro-
cedures were performed during the light period, followed
ARRIVE guidelines [44, 45] and Directive 2010/63/EU of
the European Parliament and of the Council, and were pre-
viously approved by the Local Bioethical Committee (Uni-
versity of the Balearic Islands; CEEA 58/04/16 and CEEA
155/12/20) and the Regional Government (Conselleria Medi
Ambient, Agricultura i Pesca, Direcció General Agricultura
i Ramaderia, Govern de les Illes Balears; Exp.: 2016/08/
AEXP and 2021/05/AEXP) as stated in the Spanish Royal
Decree 53/2013. All efforts were made to minimize the num-
ber of rats used and their suffering. In this context, to avoid
inducing extra-stress in female rats during the experimental
procedure, and since only a minor influence on the baseline
response for naturally cycling females was reported in the
forced-swim test (reviewed by [46]), and the cyclicity of
females was not part of our research question (see [10]), the
specific stages of the estrous cycle were not examined.
Pharmacological treatments
In a series of independent experiments (i.e., female and
male rats were performed at different points in time), ran-
domly allocated rats from each sex received (i.p.), 3 pulses
(23, 5 and 1h prior to behavioral or neurochemical test-
ing; see Fig.1) of desipramine (5, 10 or 20mg/kg; doses
selected from [19]) or saline (0.9% NaCl, 1ml/kg, i.p.)
within a period of 24h, at the indicated times (see Fig.1).
The rats exposed to behavioral testing were sacrificed 5-day

628 S.Ledesma-Corvi, M.J.García-Fuster
1 3
post-treatment (n = 74, see Fig. 1A), while other parallel
experiments were performed in rats from each sex (with no
behavioral testing) to collect brains at different timepoints
after treatment (1-h and 1-day post-treatment; see Fig.1B).
Forced‑swim test
A cohort of rats was used to evaluate the antidepressant-
like potential of desipramine (control, n = 17 males and 8
females; desipramine 5mg/kg, n = 9 males and 7 females;
desipramine 10mg/kg, n = 9 males and 8 females; desip-
ramine 20mg/kg; n = 8 males and 8 females; see Fig.1A).
To do so, we relied on the forced-swim test, with predic-
tive validity for assessing antidepressant-like efficacy [22],
and with prior reliable results from our research group (e.g.,
[19, 47, 48]), including the evaluation of the time-course
length of the response (e.g., [11–13]). To perform this test,
we used water tanks of 41cm high × 32cm diameter, filled
with clean water for each rat to a depth of 25cm and at a
temperature of 25 ± 1°C. Prior to any drug treatment, rats
were exposed to a pre-test forced-swim session (15min;
D1, see Fig.1A), followed by a sequence of 5-min tests
performed 1-h, 1-day and 3-day post-treatment to evaluate
duration of the antidepressant-like response of desipramine.
Sessions were videotaped and analyzed by an experimenter
blind to the treatment groups with Behavioral Tracker soft-
ware (CA, USA) that provided a measure of the time rats
spent immobile vs. active (i.e., climbing). Since experi-
ments were done in different waves throughout the year,
and given the described circannual changes in the duration
of the immobility response of rats in the forced-swim test
[49], among other factors also affecting baseline levels in
the forced-swim test (reviewed by [50]), direct comparisons
across studies (in terms of the individual seconds spent in
each behavior) were not applicable and thus, results were
calculated for each study as the percent change vs. the cor-
responding wave-control group for each sex.
Tissue collection
Brain tissue from different cohort of rats was collected at the
different timepoints of analysis (1h, 1 and 5days, see Fig.1)
in which to evaluate neuroplastic-like changes that might
emerge during the course of treatment and that could explain
the behavioral-like responses observed (control, 1h: n = 12
males and 6 females; 1day: n = 11 males and 4 females;
desipramine 10mg/kg, 1h: n = 6 males; 1day: n = 6 males;
desipramine 20mg/kg; 1h: n = 6 males and 6 females; 1day:
Fig. 1 Experimental design. A Time-course of the antidepressant-like
response induced by different doses of desipramine was evaluated in
the forced-swim test (FST) 1-h and 1- and 3-day post-treatment in
male and female rats (control-vehicle, n = 17 males and 8 females;
desipramine 5mg/kg, n = 9 males and 7 females; desipramine 10mg/
kg, n = 9 males and 8 females; desipramine 20mg/kg; n = 8 males and
8 females). Prior to any drug treatment, rats were handled for some
days and then exposed to a 15-min pre-forced-swim test (D1), fol-
lowed by 3 pulses within a 24-h period of the corresponding treat-
ment, and a sequence of 5-min forced-swim tests performed 1-h (D2),
1-day (D3) and 3-day (D5) post-treatment. Brains were collected
5-day post-treatment (D7) to evaluate potential neurochemical mark-
ers regulated by desipramine through immunohistochemistry (IHC)
or western blot (WB) analyses. B Potential neurochemical markers
regulated by desipramine were also evaluated by immunohistochem-
istry (IHC) or western blot (WB) analyses 1-h and 1-day post-treat-
ment in male and female rats treated with the same drug regimens
(control, 1h: n = 12 males and 6 females; 1 day: n = 11 males and 4
females; desipramine 10mg/kg, 1h: n = 6 males; 1 day: n = 6 males;
desipramine 20mg/kg; 1h: n = 6 males and 6 females; 1 day: n = 6
males and 5 females)

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Revisiting theantidepressant‑like effects ofdesipramine inmale andfemale adult rats: sex…
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n = 6 males and 5 females; see Fig.1). All rats were killed by
rapid decapitation and their brains removed at the indicated
times. The left half-brain was quickly frozen in isopentane
at – 30ºC and stored at – 80ºC, until it was cryostat-cut
in 30μm sections starting at around − 0.72mm and up to
-6.80mm from Bregma, to cover the whole hippocampal
extent (starting around − 1.72 from Bregma). Sections were
slide-mounted and kept at – 80ºC for the later analysis of
the early stages of hippocampal neurogenesis (i.e., Ki-67
for recent cell proliferation) by immunohistochemistry. The
right hippocampus was freshly dissected, frozen in liquid
nitrogen, and kept at – 80ºC until neuroplasticity markers
(i.e., FADD, Cdk-5, p35-p25) were examined by Western
blot experiments.
Immunohistochemistry
The rate of cell proliferation was quantified in tissuesections
(30μm) that were post-fixed in 4% paraformaldehyde and
exposed to several steps (i.e., antigen retrieval, blocking),
including the incubation with the primary antibody anti-
Ki-67 (1:40,000, kindly provided by Professors Huda Akil
and Stanley J. Watson, University of Michigan, MI, USA)
and as previously described (for further details see [51, 52]).
Ki-67 + cells were quantified in the dentate gyrus with a
Leica DMR light microscope (63 × objective lens) in every
8th section throughout the entire extent of the hippocampus
following a modified unbiased stereological procedure [51,
52], and then the overall number of cells was multiplied
by the sampling factor 8 to provide an estimate of the total
number labeled cells per animal (e.g., [11, 51, 52]).
Western blot
Following standardized protocols from our research group
(e.g., [19, 53]), total brain proteins (40μg) from rat hip-
pocampal homogenates were resolved by electrophoresis
on 10–12% SDS–PAGE minigels (Bio-Rad Laboratories,
Hercules, CA, USA) and then transferred to nitrocellulose
membranes that were incubated overnight at 4 ºC in block-
ing solution containing the appropriate primary antibodies
(previously characterized, see [19, 53]). The vendors and
conditions of the primary antibodies were the following: (1)
Santa Cruz Biotechnology (CA, USA): anti-FADD (H-181)
(dilution 1:5000) and anti-p35 (Ab-C19) (dilution 1:3000),
(2) Lab Vision Corporation (CA, USA): anti-Cdk-5 (DC17)
(dilution 1:1000), and (3) Sigma-Aldrich (MO, USA): anti-
β-actin (AC-15) (dilution 1:10,000). Following the incu-
bation (1h) at room temperature with the corresponding
secondary antibody (anti-rabbit or anti-mouse, 1:5000 dilu-
tion; Cell Signaling, MA, USA), target proteins were visual-
ized by exposing membranes incubated with ECL reagents
(Amersham, Buckinghamshire, UK) to an autoradiographic
film (Amersham ECL Hyperfilm) for 1–60min. The signal
was quantified by densitometric scanning (GS-800 Imaging
Calibrated Densitometer, Bio-Rad) and immunoreactivity
of target proteins (i.e., FADD, Cdk-5, p35–p25 and β-actin)
for each rat was calculated in each gel as the percent (%)
change vs. the corresponding control group (1 or 5days).
Each sample was loaded at least 3 times in different gels and
the mean value was used as a final estimate. β-actin served
as a loading control, since its content was not altered by any
treatment conditions (data not shown).
Data analysis anddata availability statement
Data were analyzed with GraphPad Prism, Version 9.3.1
(CA, USA). Bar graphs represent mean values ± standard
error of the mean (SEM), and contain individual symbols
for each rat, in line with the guidelines for displaying data
and statistical methods in experimental pharmacology [54,
55]. Normal distribution was evaluated with Shapiro–Wilk
normality test and thus parametric tests were used for data
evaluation. Behavioral changes in the forced-swim test were
evaluated by two-way repeated measures (RM) ANOVAs
followed by Dunnett’s multiple comparisons tests when
appropriate, in which Treatment (control vs. desipramine:
5, 10 and 20mg/kg) and Time (1-h, 1- and 3-day post-treat-
ment) were used as independent variables. The regulation
of hippocampal cell genesis (Ki-67 + cells) and neuroplas-
ticity markers were evaluated at each timepoint of analysis
for male rats by one-way ANOVAs followed by Dunnett’s
multiple comparison tests when appropriate, and for female
rats by Student's t tests (control vs. 20mg/kg groups). The
variable Time was not included in the neurochemical analy-
sis, since brains were collected at each timepoint of analy-
sis from independent experiments (see Fig.1), and given
the potential basal differences in the regulation of certain
molecular markers with the particular conditions at the time
of sample collecting. The level of significance was set at
p ≤ 0.05. The data that supports the findings of this study
will be available upon reasonable request to the correspond-
ing author.
Results
Desipramine induced similar antidepressant‑like
responses inmale andfemale rats
The antidepressant-like effect of desipramine was evaluated
across time (1-h, 1- and 3-day post-treatment) in the forced-
swim test in male rats by two-way RM ANOVAs. The results
showed significant Treatment x Day interactions for immo-
bility (F6,68 = 4.82, p < 0.001) and climbing (F6,68 = 10.78,
p < 0.001; Fig.2A). Dunnett’s post-hoc comparisons revealed

630 S.Ledesma-Corvi, M.J.García-Fuster
1 3
that desipramine decreased immobility in a dose-dependent
manner as observed 1-h (5mg/kg: -8 ± 10%, p = 0.799, n.s.;
10mg/kg: − 39 ± 6%, ***p < 0.001; 20mg/kg: − 40 ± 11%,
*p = 0.013) and 1-day post-treatment (5mg/kg: + 10 ± 8%,
p = 0.554, n.s.; 10mg/kg: − 38 ± 11%, *p = 0.011; 20mg/
kg: − 35 ± 14%, p = 0.062, n.s.) and as compared with
control-treated rats. Similarly, Dunnett’s post-hoc com-
parisons for climbing showed significant increases 1-h
(5mg/kg: + 8 ± 21%, p = 0.971, n.s.; 10mg/kg: + 55 ± 16%,
*p = 0.010; 20mg/kg: − 314 ± 94%, *p = 0.030) and 1-day
post-treatment (5 mg/kg: − 25 ± 20%, p = 0.496, n.s.;
10mg/kg: + 88 ± 28%, *p = 0.017; 20 mg/kg: + 157 ± 52%,
*p = 0.039) and as compared with control-treated rats
(Fig.2A). For female rats, similar results were observed:
Treatment x Day interactions for immobility (F6,54 = 3.49,
p = 0.006) and climbing (F6,54 = 4.32, p = 0.001; Fig.2B) and
significant Dunnett’s post-hoc comparisons for immobility
(1h: 5mg/kg, − 4 ± 5%, p = 0.756, n.s.; 10mg/kg, -26 ± 9%,
*p = 0.037; 20mg/kg, − 50 ± 11%, **p = 0.005; 1day: 5mg/
kg, + 1 ± 4%, p = 0.997, n.s.; 10mg/kg, − 4 ± 6%, p = 0.830,
n.s.; 20mg/kg, − 27 ± 8%, *p = 0.015) and climbing (1h:
5mg/kg, + 33 ± 47%, p = 0.820, n.s.; 10mg/kg, + 244 ± 80%,
*p = 0.035; 20 mg/kg, + 470 ± 106%, **p = 0.005; 1day:
5mg/kg, 0 ± 53%, p > 0.999, n.s.; 10 mg/kg, + 64 ± 79%,
p = 0.767, n.s.; 20mg/kg, + 339 ± 97%, *p = 0.015). All the
changes induced bydesipramine on immobility and climbing
in male and female rats returned to normal 3-day post-treat-
ment (see Fig.2A and 2B).
Desipramine did notregulate therate ofcell
proliferation inthedentate gyrus ofmale
andfemale rats
The effect of desipramine on regulating an early marker of
hippocampal neurogenesis (i.e., cell proliferation by labeling
Ki-67 + cells) was evaluated 1-h and 1-day post-treatment
(timepoints with an observed antidepressant-like effect).
The results showed that desipramine did not significantly
alter the number of Ki-67 + cells observed in the hippocam-
pus of male (1h: F2,20 = 2.67, p = 0.094; 1day: F2,20 = 0.34,
p = 0.715) and female (1h: t = 2.21, df = 10, p = 0.052; 1day:
t = 0.77, df = 7, p = 0.467) rats (see Fig.3).
Desipramine modulated certain neuroplasticity
markers inthehippocampus ofmale butnotfemale
rats
When assessing FADD hippocampal regulation, one-way
ANOVAs detected significant effects 1 h (F2,20 = 4.81,
p = 0.020) and 1day (F2,20 = 18.11, p < 0.001), but not
5days (F2,29 = 0.25, p = 0.779) post-treatment (Fig. 4A).
Dunnett’s multiple comparisons tests revealed that the dose
of 20mg/kg of desipramine decreased FADD content 1-h
Fig. 2 Evaluating the time-course of the antidepressant-like response
of different doses of desipramine in male and female rats. Groups of
treatment (i.p., 3 pulses within a 24-h period): control (C: 0.9% NaCl,
1 ml/kg; n = 17 males and 8 females), desipramine (DMI) 5 mg/kg
(n = 9 males and 7 females), 10 mg/kg (n = 9 males and 8 females),
or 20 mg/kg (n = 8 males and 8 females). Columns represent
mean ± SEM of the % time spent immobile or climbing (individual
symbols are shown for each rat). Two-way RM ANOVAs followed
by Dunnett's multiple comparisons tests: ***p < 0.001, **p < 0.01 or
*p < 0.05 when compared to control rats at the indicated timepoint of
analysis

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post-treatment (− 93 ± 33%, *p = 0.022), while increased it
1-day post-treatment (+ 90 ± 16%, ***p < 0.001) as com-
pared to their respective control treated rats (Fig.4A).
No significant effects were observed for female rats (1h:
t = 1.86, df = 10, p = 0.093; 1day: t = 2.03, df = 7, p = 0.082;
5days: t = 0.06, df = 13, p = 0.956; Fig.4A).
As for Cdk5, one-way ANOVAs detected signifi-
cant effects 1-day (F2,20 = 12.72, p < 0.001), but not 1-h
(F2,20 = 1.82, p = 0.189) or 5-day (F2,29 = 0.17, p = 0.848)
post-treatment (Fig.4B). Dunnett’s multiple compari-
sons tests revealed that the dose of 20mg/kg of desipra-
mine increased Cdk5 protein content 1-day post-treatment
(+ 33 ± 7%, *p = 0.022) as compared to their respective
control treated rats (Fig.4B). No significant effects were
observed for female rats (1h: t = 0.07, df = 10, p = 0.948;
1day: t = 0.25, df = 7, p = 0.812; 5 days: t = 2.06, df = 14,
p = 0.059; Fig.4B).
The analysis of hippocampal p35 revealed a significant
effect 1h (F2,20 = 4.05, p = 0.033), but not 1-day (F2,20 = 2.92,
p = 0.077) or 5-day (F2,29 = 2.24, p = 0.125) post-treatment
(Fig.4C). However, no significant pairwise comparisons
were revealed by Dunnett's test 1-h post-treatment. Simi-
larly, to the regulation of the other proteins, no significant
effects were observed for female rats (1h: t = 1.66, df = 10,
p = 0.129; 1day: t = 0.94, df = 7, p = 0.380; 5days: t = 0.31,
df = 13, p = 0.759; Fig.4C).
Finally, when assessing p25 hippocampal regulation, one-
way ANOVAs detected significant effects 1h (F2,20 = 7.70,
p = 0.003) and 1day (F2,20 = 9.47, p = 0.001), but not 5-day
(F2,29 = 2.52, p = 0.098) post-treatment (Fig. 4D). Dun-
nett’s multiple comparisons tests revealed that the dose of
20mg/kg of desipramine decreased p25 content as meas-
ured 1-h (− 43 ± 12%, **p = 0.005) and 1-day (− 31 ± 9%,
**p = 0.003) post-treatment, and as compared to their
respective control groups (Fig.4C). Again, no significant
effects were observed for female rats (1h: t = 0.04, df = 10,
p = 0.968; 1day: t = 0.65, df = 7, p = 0.534; 5days: t = 0.24,
df = 12, p = 0.812; Fig.4D).
Fig. 3 Evaluating the effects
of desipramine on the rate of
hippocampal cell prolifera-
tion in male and female rats as
measured 1-h and 1-day
post-treatment by immunohis-
tochemistry. A Quantitative
analysis of Ki-67 + cells in the
left dentate gyrus, performed
in a light microscope with a
63 × lens. Groups of treatment
(i.p., 3 pulses within a 24-h
period): control (C) (0.9% NaCl,
1ml/kg: 1h, n = 12 males and 6
females; 1day, n = 11 males and
4 females), desipramine (DMI)
10mg/kg (1h: n = 6 males;
1day: n = 6 males) or 20mg/
kg (1h: n = 6 males and 6
females; 1day: n = 6 males and
5 females). Columns represent
mean ± SEM of Ki-67 + cells
(individual symbols are shown
for each rat). One-way ANO-
VAs did not detect any signifi-
cant changes. B Representative
images showing individual
Ki-67 + cells (brown labeling in
the blue granular layer) taken
with a light microscope using
a 40 × objective lens to identify
individual cells

632 S.Ledesma-Corvi, M.J.García-Fuster
1 3
Discussion
This study compared the course of the antidepressant-like
potential of different doses of desipramine administration
and deepened in the putative sex differences in neuroplasti-
city signaling partners modulated in the hippocampus, a key
region for affect regulation. The main results showed that
desipramine induced antidepressant-like effects of similar
magnitude and duration for both sexes (effective doses of
10 and 20mg/kg, with responses that lasted up to 1-day
post-treatment), without altering the rate of cell prolifera-
tion. However, desipramine regulated some neuroplasticity
markers exclusively in male rats (FADD, Cdk5 and p25), in
a way that suggested neuroprotective actions, proposing that
distinctive molecular mechanisms might be participating in
the therapeutic response of desipramine for both sexes.
Fig. 4 Evaluating the effects of
desipramine on hippocampal
neuroplasticity markers in male
and female rats as measured 1-h
and 1- and 5-day post-treatment
by western blot. A FADD, B
Cdk5, C p35 and D p25 protein
content in hippocampal sam-
ples. Groups of treatment (i.p.,
3 pulses within a 24-h period):
control (C) (0.9% NaCl, 1ml/
kg; 1h: n = 12 males and 6
females; 1day: n = 11 males and
4 females), desipramine (DMI)
10mg/kg (1h: n = 6 males;
1day: n = 6 males) or 20mg/
kg (1h: n = 6 males and 6
females; 1day: n = 6 males and
5 females). Columns represent
mean ± SEM of n experiments
per group and expressed as %
control (individual values are
shown for each rat in symbols).
One-way ANOVAs at each
timepoint of analysis followed
by Dunnett's multiple com-
parisons tests when appropri-
ate: *p < 0.05, **p < 0.01 and
*p < 0.05 vs. control rats at the
indicated timepoint of analysis.
E Representative immunoblots
depicting labeling of target
proteins at each timepoint of
analysis

633
Revisiting theantidepressant‑like effects ofdesipramine inmale andfemale adult rats: sex…
1 3
The antidepressant-like response of desipramine was
observed with a similar magnitude in male and female
rats, as decreased rates of immobility and increased climb-
ing in the forced-swim test, therefore, presenting preclini-
cal evidence of efficacy for both sexes. The effective doses
were 10 and 20mg/kg (3 pulses within 24h) and efficacy
was observed 1h and up to 1-day post-treatment. Our data
extended the published literature in the field by showing the
time-course of the antidepressant-like response, since most
previous studies only evaluated a single timepoint (mostly
1-h post-treatment) while also including efficacious doses
for female rats. In particular, desipramine induced a potent
antidepressant-like response as early as 1-h post-treatment,
which was still present with a similar magnitude 1day later,
but dissipated 3-day post-treatment. Some prior studies
suggested that lower doses of desipramine were ineffec-
tive when administered acutely (2 doses of 1, 2 or 5mg/kg
over 24-h, test performed 1-h post-injection), but induced
an antidepressant-like effect only when given chronically
(5mg/kg, 15 injections [18]) in male Sprague–Dawley rats.
However, other acute studies with a higher dose range (5,
10 and 20mg/kg × 3 doses in 24h), similar to the one evalu-
ated here, also demonstrated diminished immobility in the
forced-swim test in male rats [56] (see [19] for our own prior
results with 3 × 20mg/kg). As for female rats, only a few
preclinical studies included females; while one demonstrated
a significant antidepressant-like response with a 3 × 10mg/
kg dose in ovariectomized rats [23], the other one showed
no effects for female Sprague–Dawley rats, even though it
tested doses up to 3 × 10mg/kg, which were effective in
the present study [24]. The authors of that study discussed
that their experimental groups had a limited sample size that
might have prevented observing subtle differences (see [24]).
Interestingly, as expected for antidepressants that enhance
norepinephrine neurotransmission [18, 56, 57], desipramine
increased climbing in the forced-swim test for male rats, but
interestingly, the same pattern was also observed for female
rats. Therefore, contrary to other treatments that had clearly
shown sex differences in antidepressant-like responses, with
a drop in efficacy and/or even an inefficacious response for
females (i.e., electroconvulsive therapy, see [11]; or more
novel compounds, such as ketamine and cannabidiol, see
[12, 13]), desipramine has proven a similar behavioral
response in male and female rats.
In terms of the potential molecular mechanisms mediat-
ing the therapeutic response, the study evaluated an early
stage of hippocampal neurogenesis (cell proliferation,
Ki-67 + cells), but showed no changes for either sex. These
negative effects were consistent with prior findings sug-
gesting the need for a repeated drug treatment (14days) to
promote hippocampal cell proliferation [21, 32]. So far, the
results suggested that while acute desipramine is sufficient to
induce an antidepressant-like response of similar magnitude
for males and female rats, longer treatments might be needed
to increase cell proliferation, suggesting this mechanism
could not fully mediate and/or explain the behavioral out-
come. In this line of thought, previous studies with other
antidepressants, both from our group [47] and others [58],
showed a dissociation between emotional behavior and neu-
rogenesis regulation, suggesting that these responses might
occur independently or, at least, at different time-courses
and/or lengths of treatment. These results contradict prior
studies suggesting the need of a neurogenesis activation for
the antidepressant-like response to occur [34], or for the
observed parallel regulation of both effects [59–61], indi-
cating that other factors/molecular players might partici-
pate in the observed antidepressant-like response induced
by desipramine.
In this context, we evaluated the regulation of several key
hippocampal cell-fate markers. Overall, the present results
complemented prior data from our group showing that acute
desipramine induced neuroprotective actions by decreasing
FADD 1-h post-treatment in rat brain cortex [19], to include
another brain region (hippocampus) regulated in a similar
fashion, as well as the course of FADD regulation (up to
5-day post-treatment), and the lack of effects observed in
female rats. Following the initial decrease in FADD 1-h
post-treatment, desipramine increased FADD content 1-day
post-treatment (i.e., rebound effect after the acute initiation
of an anti-apoptotic response, see prior similar results by
[62]) and all levels were normalized 5-day post-treatment,
suggesting a role for this neuroplasticity marker in the effects
induced by desipramine. Moreover, this is the first study
evaluating FADD hippocampal regulation by desipramine
in female rats, proving sex disparities in its regulation that
deserve further characterization.
As for the rest of the hippocampal markers evaluated
(Cdk5, p35 and p25), some indications of neuroprotective
actions were also observed as measured 1-h post-treatment
exclusively in male rats, such as the decrease in neuro-
toxic p25 marker. Interestingly, these effects were also
present 1-day post-treatment, together with an increase
in hippocampal Cdk5. These effects normalized 5-day
post-treatment, when the behavioral response dissipated.
In this context, the activation of the apoptotic-pathway
through FasL, in which FADD is the intracellular adaptor
to Fas receptor, increased the transcription of p35 through
ERK1/2 pathway [63], thus suggesting an indirect connec-
tion for FADD and Cdk5, p35–p25 signaling molecules.
In addition, the regional inhibition of Cdk-5 in the den-
tate gyrus blocked depressive-like behavior (induced by
chronic mild stress) in rats, whereas the overexpression
of p35 blocked the antidepressant-like effect induced by
venlafaxine [43], suggesting a key role for the complex
Cdk5/p35–p25 in the regulation of antidepressant-like
responses. Moreover, Cdk-5 activity was not required for

634 S.Ledesma-Corvi, M.J.García-Fuster
1 3
the proliferation or differentiation of neuronal stem cells,
but was essential for the survival, migration and matura-
tion of newborn granule cells [64]. Overall, the molecular
results suggested the involvement of FADD, Cdk5 and
p35–p25 complex in the neuroplastic actions taking place
in the hippocampus [26] and mediated by desipramine but
exclusively in male rats, demonstrating that although the
behavioral effects were similar, clear sex differences were
observed in the molecular events driving those responses,
that deserve further study.
Taken together, these results showed that while acute
desipramine was capable of inducing antidepressant-like
effects of the same magnitude and duration in male and
female rats, the regulation of some neuroplasticity mark-
ers, that paralleled the behavioral response, was only
detected in male rats. Overall, the results suggested that
the regulation of FADD, Cdk-5 and p35–p25 proteins
could be common markers mediating some of the early
mechanisms taking place right after drug exposure and up
to 1-day post-treatment while matching the course of the
observed behavioral response. On the contrary, the regu-
lation of an early stage of adult hippocampal neurogen-
esis, such as cell proliferation, might require a prolonged
treatment exposure to desipramine, since no effects were
observed following this acute paradigm. Overall, these
molecular correlates were exclusively regulated in male
rats, suggesting other markers might be participating in
the behavioral response observed in females. Further stud-
ies are required to complement these results and deepen
into the neurochemical differences driven by sex in the
response mediated by desipramine, since these dispari-
ties might be responsible for the poor clinical translation
occasionally described.
Acknowledgements We would like to thank Drs. Huda Akil and Stan-
ley J. Watson (University of Michigan, Ann Arbor, MI, USA) who
kindly provided Ki-67 antibody.
Author contributions MJG-F was responsible for the study concept
and design with the contribution of SL-C. SL-C conducted the experi-
ments, analyzed the behavioral and molecular data and drafted the fig-
ures. MJG-F revised and interpreted all the data, as well as reformatted
the figures. MJG-F wrote the manuscript. Both authors have critically
contributed and have approved the final version of the manuscript for
publication.
Funding Open Access funding provided thanks to the CRUE-CSIC
agreement with Springer Nature. Funding for this study was provided
by Grants SAF2014-55903-R and PID2020-118582RB-I00 (MCIN/
AEI/10.13039/501100011033), as well aspartially by PDR2020/14
(Comunitat Autònoma de les Illes Balears through the Direcció General
de Política Universitària i Recerca with funds from the Tourist Stay Tax
Law ITS 2017–006), and Fundación Alicia Koplowitz to MJG-F; 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 deci-
sion to submit the paper for publication. The ‘García-Palmer’ Summer
Research Program (2019) and JUNIOR Program (IdISBa, GOIB) sup-
ported SL-C’s salary.
Declarations
Conflict of interest The authors have nothing to disclose.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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