Fast-acting antidepressant-like effects of ketamine in aged male rats

Abstract

Background
The aging process causes anatomical and physiological changes that predispose to the development of late-life depression while reduces the efficacy of classical antidepressants. Novel fast-acting antidepressants such as ketamine might be good candidates to be explored in the context of aging, especially given the lack of previous research on its efficacy for this age period. Thus, the aim of the present study was to characterize ketamine’s effects in older rats.

Methods
The fast-acting (30 min) and repeated (7 days) antidepressant-like effects of ketamine (5 mg/kg, ip ) were evaluated in 14-month-old single-housed rats through the forced-swim and novelty-suppressed feeding tests. In parallel, the modulation of neurotrophic-related proteins (i.e., mBDNF, mTOR, GSK3) was assessed in brain regions affected by the aging process, prefrontal cortex and hippocampus, as well as possible changes in hippocampal cell proliferation.

Results
Acute ketamine induced a fast-acting antidepressant-like response in male aged rats, as observed by a reduced immobility in the forced-swim test, in parallel with a region-specific increase in mBDNF protein content in prefrontal cortex. However, repeated ketamine failed to induce antidepressant-like efficacy, but decreased mBDNF protein content in prefrontal cortex. The rate of hippocampal cell proliferation and/or other markers evaluated was not modulated by either paradigm of ketamine.

Conclusions
These results complement prior data supporting a fast-acting antidepressant-like effect of ketamine in rats, to further extend its efficacy to older ages. Future studies are needed to further clarify the lack of response after the repeated treatment as well as its potential adverse effects in aging.

Full text

RESEARCH
Pharmacological Reports
https://doi.org/10.1007/s43440-024-00636-y
also increasing between the ages of 60 and 90 [2]. Changes
during aging predispose to the development of the so-called
late-life depression [3], for which pharmacotherapy, psy-
chotherapy, and electroconvulsive therapy are the treat-
ments of choice [4]. However, classical antidepressants are
known to change in ecacy with age [4–6], with reduced
response rates ranging from 54% at age 54 to 42% at age
73 [6]. Despite these age-related dierences, and in the con-
text of our increasingly aging society, research focused on
characterizing classical or novel therapeutic options for this
age-group is really scarce (see some prior studies attempt-
ing to amend this [7–9]).
In this regard, the discovery of the rapid antidepressant
eects of ketamine, an N-methyl-D-aspartate (NMDA)
receptor antagonist, is considered one of the major break-
throughs in the treatment of depression in the past decades,
opening the door to a new class of fast-acting antidepres-
sant options [10]. While a single administration of ket-
amine has demonstrated rapid and potent reductions in
Introduction
Major depressive disorder is one of the most common and
debilitating mental illnesses and a major public health prob-
lem worldwide. After anxiety disorders, depression is the
most common mental health disorder among older adults,
aecting 5-7% of the elder population [1], with suicide rates
M. Julia García-Fuster
j.garcia@uib.es
1 IUNICS, University of the Balearic Islands, Cra. de
Valldemossa, Km 7.5, Palma E-07122, Spain
2 Health Research Institute of the Balearic Islands (IdISBa),
Palma, Spain
3 Department of Medicine, University of the Balearic Islands,
Palma, Spain
4 Present address: Department of Pharmacology, University of
the Basque Country (EHU/UPV), Leioa, Spain
Abstract
Background The aging process causes anatomical and physiological changes that predispose to the development of late-life
depression while reduces the ecacy of classical antidepressants. Novel fast-acting antidepressants such as ketamine might
be good candidates to be explored in the context of aging, especially given the lack of previous research on its ecacy for
this age period. Thus, the aim of the present study was to characterize ketamine’s eects in older rats.
Methods The fast-acting (30 min) and repeated (7 days) antidepressant-like eects of ketamine (5 mg/kg, ip) were evaluated
in 14-month-old single-housed rats through the forced-swim and novelty-suppressed feeding tests. In parallel, the modula-
tion of neurotrophic-related proteins (i.e., mBDNF, mTOR, GSK3) was assessed in brain regions aected by the aging pro-
cess, prefrontal cortex and hippocampus, as well as possible changes in hippocampal cell proliferation.
Results Acute ketamine induced a fast-acting antidepressant-like response in male aged rats, as observed by a reduced
immobility in the forced-swim test, in parallel with a region-specic increase in mBDNF protein content in prefrontal cortex.
However, repeated ketamine failed to induce antidepressant-like ecacy, but decreased mBDNF protein content in prefron-
tal cortex. The rate of hippocampal cell proliferation and/or other markers evaluated was not modulated by either paradigm
of ketamine.
Conclusions These results complement prior data supporting a fast-acting antidepressant-like eect of ketamine in rats, to
further extend its ecacy to older ages. Future studies are needed to further clarify the lack of response after the repeated
treatment as well as its potential adverse eects in aging.
Keywords Ketamine · Fast-acting antidepressant · Aging · BDNF
Received: 29 May 2024 / Revised: 25 July 2024 / Accepted: 6 August 2024
© The Author(s) 2024
Fast-acting antidepressant-like eects of ketamine in aged male rats
ElenaHernández-Hernández1,2,4 · SandraLedesma-Corvi1,2 · JordiJornet-Plaza1,2 · M. JuliaGarcía-Fuster1,2,3
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E. Hernández-Hernández et al.
depressive symptomatology both in humans [11–14] and
in animal models of depression [15–20], repeated doses
seemed to sustain the observed short-term responses [21–
23]. Indeed, esketamine, the S-enantiomer of ketamine,
was rst approved by the FDA in US in 2019 and then by
EMA in Europe for the treatment of patients with resistant
depression [24–25]. Although ecacy in adult patients
seemed quite satisfactory, the potential eects of ketamine
to treat late-life depression has been poorly investigated. In
particular, and as reviewed by [26–27], only two random-
ized controlled trials have been conducted in an aging pop-
ulation (60 years and older) [28–29], with mixed results.
While one trial found a positive eect of ketamine admin-
istration both on response and remission rates [28], the
other one found no signicant dierences [29]; although
some benecial eects were observed (i.e., a trend towards
an improvement as measured by the Montgomery-Asberg
Depression Rating Scale). Additionally, a recent analy-
sis of an open-label clinical trial showed indications of
a lower antidepressant response (37.1% vs. 57.8%) and
remission rates (15.8% vs. 47.4%) in older vs. younger
depressed patients [30]. These promising but clearly insuf-
cient analyses highlight the need for increased research
on the potential antidepressant-like eects of ketamine in
the aging population, including its characterization at the
preclinical level.
In this context and to the best of our knowledge, the
evaluation of ketamine as an antidepressant in aged animals
is limited to a single study conducted in mice evaluating
the eects of a single administration [9], and another one
evaluating its prophylactic eects against stress-induced
behaviors [31]. However, neither of these studies [9, 31]
induced ecacy in aged animals using a dosing regimen
proven eective in adolescent and adult rodents (e.g., [19–
20]). Against this background the present study will fur-
ther characterize the potential antidepressant-like response
of ketamine in aging single-housed rats (e.g., [32]), since
this stress paradigm in rodents (i.e., social isolation due
to size requirements for the number of animals per cage)
reproduced several behavioral features mimicking late-
life depression (see our own studies phenotyping rats from
middle-age and onward [33–34, 8]). Moreover, the present
study will evaluate biomarkers of the antidepressant-like
response such as the activation of brain neurotrophic factor
(BDNF) (e.g., [10, 35]) and associated signaling partners
(e.g., mTOR and GSK-3; [36–38]) in several brain regions
(e.g., prefrontal cortex and hippocampus) impacted by aging
(e.g., [39]) and/or an increase in hippocampal neurogenesis
[40–42]; see a more recent study by [43]). A preliminary
report of a portion of this work was presented at the 34th
European College of Neuropsychopharmacology (ECNP)
Congress Hybrid [44].
Materials and methods
Animals
For the present study, a total of 48 male Sprague-Dawley
rats were used when they reached 14-month-old. Rats were
bred in the animal facility at the University of the Balearic
Islands and were housed under standard vivarium condi-
tions (22 ºC, 70% humidity, 12-h light/dark cycle, lights on
at 8:00 AM) with access to a standard diet and unlimited
tap water (except when otherwise specied, see Fig. 1a for
food deprivation prior to novelty-suppressed feeding test).
Following animal housing regulations regarding the number
of animals allowed per cage in terms of size and weight,
14-month-old rats were single-housed in standard cages for
several months before testing started, which is a great model
of chronic stress. All procedures were performed during the
light period (between 8:00 AM and 3:00 PM), complied with
the ARRIVE guidelines [45], the EU Directive 2010/63/
EU for animal experiments, and the Spanish Royal Decree
53/2013, and thus were approved by the Local Bioethical
Committee (CEEA 58/04/16) and the Regional Government
(2016/08/AEXP). All eorts were made to minimize the
number of rats used and their suering. Unfortunately, since
no aged female rats were available at the time when this
experiment was performed, only male rats were included in
the present study, and therefore the conclusions are limited
to only one sex.
Behavioral procedures
All rats were handled several days prior to any behavioral
or administration procedure. For this experiment, a total
of 24 rats were used for the behavioral characterization of
ketamine (Fig. 1a). Rats were randomly allocated into two
groups and were treated daily for 7 consecutive days with
ketamine (7 × 5 mg/kg, ip, n = 12; Anesketin: 100 mg/ml
of ketamine from Dechra Pharmaceuticals, Northwich,
United Kingdom) or saline (7 × 1 ml/kg of 0.9% NaCl, ip,
n = 12). The dose of ketamine was chosen based on previ-
ous antidepressant results reported in the literature (e.g.,
[46–47]) and on our own results [19–20]. Antidepressant-
like responses were ascertained by diverse tests previ-
ously validated in the eld. We rst measured behavioral
despair under the stress of the forced-swim test (e.g., [48]),
30 min after the rst treatment-dose (D1, acute eects)
and 24 h after the last treatment dose (D8, repeated eects;
see Fig. 1a) as earlier described by our group [33–34, 19].
Briey, on pre-test day (D-2, see Fig. 1a), rats were placed
for 15 min in single tanks (32 cm diameter x 41 cm high)
lled with water (25 cm of depth, 25 ± 1 ºC). Then, on test
days (D1 and D8) rats were exposed for 5 min to the same
1 3

Fast-acting antidepressant-like eects of ketamine in aged male rats
conditions, and their performances were videotaped. Vid-
eos were analyzed by an experimenter blind to the specic
treatment groups with Behavioral Tracker software (CA,
USA) to ascertain the time each rat spent (s) immobile
(i.e., an indicative of despair) vs. active (i.e., escaping-
like behaviors such as swimming or climbing). Moreover,
the potential repeated antidepressant-like responses of
ketamine were also evaluated in the novelty-suppressed
feeding test 2 days after the last dose (D10). Following
standard procedures [19, 49], rats were food-deprived for
48 h (D8-D10) since motivation for food is required for
this particular test. During test day, each rat was placed for
5 min in a square open arena (60 cm x 60 cm, and 40 cm
in high) under housing lighting conditions with three food
pellets in the center [19]. Sessions were recorded and the
parameters latency to center (s), time in center (s), latency
to food (s), feeding time (s), and distance traveled (cm)
were blindly scored using the ANY-maze software (ver-
sion 7.37, Stoelting Co. Dublin, Ireland). Body weight
was monitored trough the treatment process and showed
no changes between experimental groups and/or across
time (data not shown). Notably, brains from the behav-
ioral procedures were not collected for neurochemical
studies.
Neurochemical procedures
For evaluating the neurochemical eects of ketamine, we
utilized a separate group of 24 aged rats who were randomly
assigned to the acute (1 dose; n = 12) or repeated (7 doses;
n = 12) treatment groups (Fig. 1b). Similarly, to the previous
experiment, each rat received the corresponding daily dose/
es of saline (1 or 7 × 1 ml/kg of 0.9% NaCl, ip, n = 6 per
group) or ketamine (1 or 7 × 5 mg/kg ip, n = 6 per group).
Rats were then sacriced without anesthesia by rapid decap-
itation with a large rodent guillotine following standard pro-
cedures in our group [8, 33] 30 min after the acute treatment
or 24 h after last dose for the repeated treatment paradigm
(D8). These time-points matched the particular times when
forced-swim tests were performed so direct comparisons
could be done between the potential antidepressant-like vs.
neurochemical eects of ketamine in aged rats.
Fig. 1 Experimental design. (a) Behavioral procedures in single-
housed rats (14 months old) after an acute (1 dose of 5 mg/kg, i.p)
or a repeated (7 doses of 5 mg/kg, ip, 1 dose per day) treatment with
ketamine. Changes were evaluated 30-min (day, D1) or 24 h post-treat-
ment (D8) in the forced-swim test (FST) and 3 days post-treatment
(D10) in the novelty-suppressed feeding test (NSFT). (b) Neuro-
chemical procedures aimed at collecting brains at the same time points
(acute, D1, or repeated, D8) to evaluate molecular markers by western
blot (WB) and immunohistochemistry (IHC) experiments
1 3

E. Hernández-Hernández et al.
in each gel were calculated for each rat as compared to
control-treated samples. For loading control, low quan-
tities of total homogenates (15 µg) were run to detect
β-actin (dilution 1:10000; AC-15; Sigma-Aldrich, MO,
USA), since its content was not altered by the treatment
followed.
Immunohistochemical analysis
Cell proliferation was labeled in the hippocampus with
Ki-67 antibody (1:20,000; from Prof. Huda Akil and Stan-
ley J. Watson, University of Michigan, MI, USA) as pre-
viously described [50–52]. Experiments were performed
on 3 cryostat-cut sections (30 µm), one from the front,
middle or posterior parts of the hippocampus, and con-
taining 8 tissue-sections per slide. Briey, slide mounted
sections were post-xed in 4% paraformaldehyde and
processed through several steps including a series of
sequential incubation, with biotinylated anti-rabbit anti-
body (1:1000, BA-100, Vector Laboratories, CA, USA),
Avidin/Biotin complex (PK-6100, Vectastain Elite ABC
kit; Vector Laboratories, CA, USA) and the chromogen
3,3’-diaminobenzidine (H54000, Thermo Fischer, MA,
USA) for signal detection. Finally, tissue was counter-
stained in cresyl-violet (405760100, Thermo Fischer,
MA, USA), dehydrated in graded alcohols, submersed
in xylene and cover-slipped with Permount® (SP15-500,
Fisher Chemical, NH, USA). The number of positive
cells was counted by an experimenter blind to the treat-
ment groups with a Leica DMR light microscope (63x
objective lens) in a total of 3 slides per rat (8 sections
per slide; total of 24 tissue sections per rat) and focusing
through the depth of the tissue. The total number of cells
was then multiplied by the sampling factor 8 to provide
an overall estimate of the total number of Ki-67 + cells
per rat in the left hippocampus.
Data and statistical analysis
All data were analyzed and plotted with GraphPad Prism,
Version 10 (GraphPad Software, Inc, San Diego, CA, USA).
Results are expressed as mean values ± standard error of the
mean (SEM); symbols represent individual values for each
rat, following recommendations for displaying data and sta-
tistical results in pharmacology [57–58]. Potential changes
induced by the acute or repeated treatments with ketamine
for each behavioral feature and neurochemical marker ana-
lyzed were performed through two tailed Student’s t-tests.
All sets of data reported followed a normal distribution
according to Shapiro-Wilk normality test. The level of sig-
nicance was set at p ≤ 0.05.
Once brains were rapidly extracted, the whole prefrontal
cortex and the right hippocampus were freshly dissected,
fast frozen in liquid nitrogen, and saved at -80 ºC until
further processing to evaluate target proteins by Western
blot analysis. The left hemisphere was frozen in -30 ºC iso-
pentane (#143501,1611, Biolinea SL, Palma, Spain), and
kept at -80 ºC until the entire hippocampal extent (-1.72
to -6.80 mm from Bregma) was cryostat-cut in 30 μm sec-
tions. Consecutive sections were slide mounted in 24 slides
per animal with 8 tissue-sections per slide, divided in 3
series (8 slides per series), covering the most anterior part
of the hippocampus, the middle part and the most poste-
rior part of it. The rate of cell proliferation (Ki-67 + cells)
was then evaluated by immunohistochemical analysis as
previously performed [50–52] in a representative sample
of the whole hippocampus following a stereological proce-
dure that counts every 8-th section taken (1 slide from each
series containing the anterior, middle and posterior part of
hippocampus).
Western blot analysis
Total homogenates of brain regions (prefrontal cortex or
hippocampus) were prepared with minor modications
as previously described [53–54]. Each sample (40 µg of
total protein) was loaded in 7.5–14% SDS-PAGE mini-
gels (Bio-Rad Laboratories, Hercules, CA, USA) that
were resolved by electrophoresis and then processed fol-
lowing standard immunoblotting procedures [33, 55].
Membranes (0.2 μm: #10600001 or 0.45 μm: #10600002,
Merck SL, Barcelona, Spain) were incubated overnight
at 4 ºC in blocking solution containing the specic pri-
mary antibodies: anti-BDNF (N-20) (dilution 1:10000;
AB108319; Abcam, Cambridge, United Kingdom) for
identifying the mature form of BDNF (mBDNF) [56];
anti-pS2448mTOR (dilution 1:1000; #2971) and anti-
mTOR (dilution 1:1000; #2972) (Cell Signaling, MA,
USA); anti-pS21/9-GSK3 (dilution 1:1000; # 9331; Cell
Signaling) for detecting inhibitory phosphorylation and
anti-GSK3 (dilution 1:1000; 4G-1E; 05-412; Millipore)
for total protein. Membranes were then incubated with an
anti-rabbit (#7074S) or anti-mouse (#7076S) horserad-
ish peroxidase-conjugated secondary antibody (1:5000;
Cell Signaling, MA, USA) and the immunoreactivity of
target proteins was displayed on autoradiographic lms
(#28906837, Amersham ECL Hyperlm) using an ECL
detection system (#17652005, Amersham, Buckingham-
shire, United Kingdom). Autoradiograms were quantied
by densitometric scanning (GS-800 Imaging Calibrated
Densitometer, Bio-Rad). All samples were loaded at
least 2–3 times in dierent gels, and percent changes
1 3

Fast-acting antidepressant-like eects of ketamine in aged male rats
*p = 0.011 vs. control rats), no changes were observed in
hippocampus (t10 = 1.07, p = 0.310). When analyzing the
regulation of its downstream signaling partners (i.e., active
ratio of p-mTOR/mTOR and inhibitory ratio of p-GSK3/
GSK3), no changes were observed in prefrontal cortex or
hippocampus (Fig. 3a). Moreover, acute ketamine did not
change the rate of hippocampal cell proliferation (t9 = 1.17,
p = 0.273).
The eects of a repeated paradigm of ketamine on the neu-
rochemical markers under study are shown in Fig. 3b. The
data showed that repeated ketamine in aged rats decreased
the protein content of mBDNF in prefrontal cortex (-21 ±
9%, t10 = 2.35, *p = 0.041 vs. control rats), but not in hippo-
campus (t9 = 1.26, p = 0.239). Similar to what was observed
following an acute injection, no changes were observed fol-
lowing a repeated paradigm in p-mTOR/mTOR or p-GSK3/
GSK3 in both regions analyzed (Fig. 3b). Finally, repeated
ketamine did not change the rate of hippocampal cell pro-
liferation (t10 = 2.06, p = 0.067). Representative images of
selected western blot and immunohistochemistry experi-
ments are shown in Fig. 3c.
Discussion
The present study investigated the acute and repeated
antidepressant-like potential of ketamine in aged male rats
following the same administration paradigms previously
proven eective in adolescent and adult rats [19–20]. The
main results showed a rapid fast-acting antidepressant-
like eect of ketamine (observed 30 min post-treatment),
as reported by a decrease in immobility and an increase in
swimming behaviors in the forced-swim test, paired with
an increase in mBDNF protein content in prefrontal cortex.
Results
Ketamine induced a rapid antidepressant-like
response after an acute dose in aged rats: lack of
ecacy following a repeated treatment
When evaluating the acute antidepressant-like eects of ket-
amine under the stress of the forced-swim test, a two-tailed
Student’s t-test revealed a signicant reduction in immobil-
ity as observed 30 min post-ketamine administration (∼13%
reduction: -40 ± 15 s, t = 2.62, df = 21, *p = 0.016 vs. con-
trol rats; Fig. 2a), which paralleled an increase in swimming
behavior (∼8% increase: +25 ± 6 s, t21 = 4.29, ***p < 0.001
vs. control rats; Fig. 2a). Acute ketamine did not induce
changes in climbing behavior (t21 = 0.87, p = 0.394; Fig. 2a).
Interestingly, following the repeated treatment with ket-
amine (7 days of a daily 5 mg/kg injection), no signicant
changes were observed in the forced-swim test (immobility:
t22 = 1.14, p = 0.266; swimming: t22 = 1.20, p = 0.244; climb-
ing: t22 = 0.50, p = 0.620) and/or the novelty-suppressed
feeding test (latency to center: t22 = 0.17, p = 0.870; time
in center: t22 = 0.77, p = 0. 452; latency to food: t22 = 0.33,
p = 0.743), denotating the loss of antidepressant-like poten-
tial (Fig. 2b). Moreover, it is worth mentioning that none
of the rats evaluated spend any feeding time (despite prior
food deprivation) and no changes were observed in distance
travelled among treatment groups (data not shown).
Region-specic neurochemical eects after acute or
repeated ketamine administration in aged rats
Acute ketamine induced a region-specic modulation
of mBDNF protein content (Fig. 3a); while it increased
mBDNF in prefrontal cortex (+ 42 ± 13%, t9 = 3.23,
Fig. 2 Exploring the antidepressant-like eects of ketamine in aged
rats. (a) Fast-acting acute eects of ketamine (1 single dose of 5 mg/
kg, ip, D1) as measured in the forced-swim test (FST) 30 min post-
treatment. (b) Lack of ecacy following a repeated treatment with
ketamine (5 mg/kg, ip, 7 days, D1-D7) as evaluated in the FST 24 h
post-treatment (D8), and in the novelty-suppressed feeding test
(NSFT) 3 days post-treatment (D10). Columns represent mean ± SEM
of time spent in each behavior. Individual values are shown in symbols
for each rat. ***p < 0.001, *p < 0.05 when comparing ketamine-treated
rats (Ket) vs. control-treated rats (C) through two-tailed Student’s
t-tests
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E. Hernández-Hernández et al.
1 3

Fast-acting antidepressant-like eects of ketamine in aged male rats
model in which to validate the fast-acting acute eects of
ketamine administration.
Contrarily to prior reports demonstrating that under
stressful situations repeated ketamine induced antidepres-
sant-like eects in adolescent [19] and/or adult rats [20, 23,
60–64], the present results showed a lack of response in
aged male rats. Disparities in the type of stressor and/or dif-
ferences in ketamine pharmacokinetics due to age might be
behind these discrepancies, as studies with anesthetic doses
of ketamine showed a signicant increase in half-life, drug
availability, and duration of anesthesia in aged Sprague-
Dawley rats compared to young rats [62]. Although test rep-
etition might have been behind the lack of eects observed
following the repeated paradigm, this seems unlikely, since
we followed a standard procedure previously used in other
studies from our group that had proven eects across time in
the forced-swim test (e.g., [19–20, 52]). Moreover, similar
to the low dose we tested (5 mg/kg), the same dose also ren-
dered inecacious in another study [61], suggesting dose-
dependent eects as mentioned in some of the other reports,
and the potential need for a much higher dose and/or an
increasing-dosage regimen to observe a benecial response
after a repeated treatment, and overcome potential adaptive
mechanisms. For example, pharmacodynamic responses
caused by the daily repeated administration might be play-
ing a role in this lack of ecacy (i.e., tachyphylaxis). In this
context, we aimed at exploring the dierences in the molec-
ular responses elicited after an acute or repeated dosing
paradigm in an attempt to further understand the behavioral
results. Particularly, we explored markers of antidepressant-
like responses (i.e., mBDNF and associated partners, as
well as the rst stage of hippocampal neurogenesis) in key
brain areas mediating aective-like responses and impacted
by the aging process (i.e., prefrontal cortex and/or hippo-
campus) (see [63–64] and references therein).
Concurrent with the observed fast-acting antidepressant-
like response, a single administration of ketamine increased
mBDNF expression in the prefrontal cortex of aged male
rats. This eect was not observed in hippocampus, sug-
gesting a region-specic role for prefrontal cortex in the
molecular actions behind ketamine’s response, and in line
with prior results (reviewed by [35] and recently by [10]).
BDNF-mediated activation of tropomyosin receptor kinase
B (TrkB) induces the activation of several signaling path-
ways, including the inhibitory phosphorylation of the glyco-
gen synthase kinase-3 (GSK-3) [36–37], which ultimately
activates the mechanistic target of rapamycin complex 1
(mTORC1) [38]. However, no changes were detected in
mBDNF-associated downstream partners in either brain
region under evaluation (i.e., p-mTOR/mTOR and p-GSK3/
GSK3 ratios). Interestingly, and in line with the present
results, increased BDNF expression in the prefrontal cortex
However, these acute eects were no longer observed after
a repeated treatment paradigm, as measured by a lack of
antidepressant-like response in two independent behavioral
tests, combined with a decrease in mBDNF in prefrontal
cortex. These results proved an acute fast-acting antidepres-
sant-like response for ketamine in aged male rats (observed
both behaviorally and at the neurochemical level), while
suggested that its repeated administration might lead to
molecular adaptive changes preventing its ecacy.
Ketamine induced a rapid antidepressant-like response,
observed 30 min after a single administration in aged rats,
consistent with previous studies [15–16]. Interestingly our
prior studies which utilized the same acute dose of ketamine
(5 mg/kg) showed dierences in antidepressant-like ecacy
depending on the age of animals, the biological sex and
prior stress exposure [19–20]. Particularly, acute ketamine
induced an antidepressant-like response in adolescent rats
in the forced-swim test, an eect that was observed for both
sexes, but that depended on prior stress exposure (see fur-
ther details in [19]). However, acute ketamine in adult rats
was inecacious for both sexes and only showed ecacy
when male rats were priory pretreated with letrozol (an aro-
matase inhibitor that blocks the biosynthesis of estrogens;
[20]). Overall, these results suggest that there may be some
similarities in the antidepressant-like response between ado-
lescent and aged rats, with a similar dose needed in aged [8]
and adolescent rats, but with the need for a higher dose to
induce ecacy in adult rats [59]. Remarkably, it is worth
mentioning that the parallelisms observed for adolescent
and aged rats were under dierent sources of stress (i.e.,
early maternal separation for the adolescent study [19] vs.
social isolation due to size and cage requirements in the
present study with aged rats), suggesting that ketamine
proved good ecacy for stress-related conditions. In partic-
ular, the present experimental paradigm of physiologically
aged rats, individually housed for several months, and with
an expected phenotype mimicking depressive-like manifes-
tations (see characterization at [33]) proves to be a great
Fig. 3 Exploring the neurochemical eects of ketamine in aged rats.
(a) Fast-acting acute (30 min post-treatment) or (b) repeated (24 h
post-treatment, D8) eects of ketamine in prefrontal cortex (PFC)
and hippocampus (HC) of the selected protein markers evaluated by
western blot (mBDNF, p-mTOR/mTOR and pGSK3/GSK3) or immu-
nohistochemistry (Ki-67 + cells). Columns represent mean ± SEM of
protein content (% Control) or Ki-67 + cells per group. Individual val-
ues are shown in symbols for each rat. *p < 0.05 when comparing ket-
amine-treated rats (Ket) vs. control-treated rats (c) through two-tailed
Student’s t-tests. (c) Representative images of selected western blot or
immunohistochemistry experiments. Left panels: immunoblots depict-
ing the labeling of each protein and loading control β-actin. For unpro-
cessed full western blot images check Supplementary Figures S1-S6.
Right panels: representative images of Ki-67 + cells (brown labeling
in the blue granular layer) taken with a light microscope using a 40x
objective lens. A magnied window is shown at 63x. Scale bar: 30 μm.
For other representative images check Supplementary Figures S7-S10
1 3

E. Hernández-Hernández et al.
age-related depression would include characterizing the
duration of the acute antidepressant-like response, evaluat-
ing other doses and/or administration regimens, as well as
including sex as a biological variable. In any case, ketamine
seems like a great novel fast-acting option to be further
explored for our aged population in which classical antide-
pressants showed reduced ecacy.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s43440-
024-00636-y.
Acknowledgements The authors would like to thank Júlia Valor-Blan-
quer for assisting EH-H with the ANY-maze analysis. Also, we would
like to recognize Drs. Huda Akil and Stanley J. Watson (University of
Michigan, Ann Arbor, MI, USA) for providing the primary antibody
for Ki-67 labeling and Dr. Alfredo Ramos-Miguel (University of the
Basque Country UPV/EHU, Leioa, Spain) for allowing us to use the
ANY-maze behavioral video tracker software.
Author contributions EH-H and MJG-F were responsible for the study
concept and design. EH-H, SL-C and JJ-P conducted the experiments
and analyzed the behavioral and molecular data. EH-H wrote the rst
draft of the manuscript, and MJG-F edited it to its nal version. All
authors approved the nal version of the manuscript.
Funding This research was funded by PID2020-118582RB-I00
(MCIN/AEI/https://doi.org/10.13039/501100011033; Madrid, Spain)
and partially sponsored and promoted by the Comunitat Autònoma de
les Illes Balears through the Servei de Recerca i Desenvolupament and
the Conselleria d’Educació i Universitats (PDR2020/14 - ITS2017-
006) to MJG-F. EH-H was rst funded by the Margarita Salas Program
(Ministerio de Universidades; Plan de Recuperación, Transformación y
Resilencia; NextGenerationEU) with the participation of the University
of the Balearic Islands, and currently holds grant FJC2022-048338-I,
funded by MCIN/AEI/https://doi.org/10.13039/501100011033 and by
the European Union NextGenerationEU/PRTR. The predoctoral pro-
gram JUNIOR (IdISBa, GOIB) covered SL-C’s salary and JJ‐P. was
funded by a predoctoral scholarship from Conselleria de Fons Europe-
us, Universitat i Cultura, Govern de les Illes Balears (FPI_022_2022).
Open Access funding provided thanks to the CRUE-CSIC agreement
with Springer Nature.
Data availability Data will be made available upon request.
Declarations
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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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
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org/licenses/by/4.0/.
has been shown to be necessary for the acute antidepressant-
like eects of ketamine [10, 65]). However, during aging,
changes in TrkB expression may be involved in the lack of
downstream signaling [66]. Also, since ketamine activates
mTOR signaling within 1 h in the prefrontal cortex [67],
another possible explanation for these results could be that
the chosen time point of study might be too early to observe
changes in the TrkB downstream pathway.
Curiously, repeated ketamine treatment reduced mBDNF
expression in the prefrontal cortex as evaluated 24 h after the
last daily dose, without altering its content in hippocampus
and/or its downstream partners and hippocampal cell prolifer-
ation. These eects could be related to the lack of antidepres-
sant-like ecacy after repeated ketamine administration and
might imply certain adaptive molecular changes to the acute
increase in mBDNF and caused by the repeated administra-
tion of the drug. Moreover, since some studies in the literature
described that the sustained antidepressant ketamine response
seemed to require hippocampal progenitor dierentiation
through a TrkB-dependent mechanism [68], the observed lack
of BDNF modulation in hippocampus after repeated ketamine
treatment may predict its lack of eect on cell proliferation.
Moreover, previous studies have shown that aging abolishes
the neurogenic eect of classical (e.g., uoxetine; [69]) and
novel antidepressants (e.g., cannabidiol [8]), possibly due to
changes in the neurogenic niche during aging [70].
A limitation of the present study is the fact that it was
conducted exclusively in male rats, especially since depres-
sion is twice as common in women as in men [71] and
given that ketamine has demonstrated sex-dependent anti-
depressant-like eects in rats of dierent ages [46, 19–20].
Unfortunately, the logistical burden of individually housing
animals as they age for several months severely hampered
the inclusion of sex as a biological variable in the present
study. Therefore, future studies should ascertain sex dier-
ences when characterizing the potential antidepressant-like
eects of ketamine in aging, as preclinical animal models
must be truly representative of the aging population. Of spe-
cial relevance would be to ascertain how potential sex dif-
ferences regarding the functioning of the glutamate system
(e.g., reviewed by [72]), and especially the NMDA receptor
[73] might be aecting the antidepressant-like response of
ketamine in aged male vs. female rats.
In conclusion, our study contributes by increasing the
existing body of knowledge on the role of ketamine as
an antidepressant through its eects in aged rats. Overall,
acute ketamine administration showed a fast-acting anti-
depressant-like response in aged rats (behavioral and bio-
marker responses). Future studies are needed to clarify the
lack of response after the repeated treatment as well as its
potential adverse eects. Moreover, other aspects to further
study when trying to nd a safe and eective treatment for
1 3

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