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D. Gupta et al. / Steroids 96 (2015) 95–102
chronic CORT treated control mice. On the other hand inhibition of
synaptic reuptake of serotonin could be associated with the antide-
pressant effects the positive control FLX (a SSRI). It has been
already mentioned in our earlier reports that, 5-HT3 receptor
antagonists facilitate synaptic serotonergic neurotransmission
[19]; while other reports have shown that pre-treatment of 5-
HT3 receptor antagonists potentiate the antidepressant effects of
SSRIs [27]. It implies that one of the modes of antidepressant-like
action of 4i in CORT-treated mice could be in the direction of
improvement of serotonin functions in the brain.
considering that the activities of these enzymes might be mark-
edly decreased for scavenging ROS in the neuronal cells. This
contention is supported by the earlier findings that abnormally
elevated GCs levels stimulate oxidative stress in the brain [50].
Furthermore, previous reports have documented that GCs may
damage hippocampus and other brain areas by generating ROS,
which are potentially related to the regulation of mood and
behavioral activity [51]. Therefore, the inhibited hippocampal
function due to elevated oxidative damage plausibly provides
positive correlation between abnormal GCs activity and depres-
sion. Thus, increased oxidative stress due to chronic CORT could
be related to the depressive-like behavior observed in mice dur-
ing behavioral testing paradigms. Interestingly, 4i similar to FLX
ameliorated CORT induced oxidative load in the brain. This is in
line with the earlier reports that several antidepressants reverse
oxidative damage in the brain; one of the pathogenic causes of
depression, which indicates the possible mechanism of antide-
pressant like effects of 4i [49].
It is important to note that additional pathological pathways
might modulate the depressogenic behavior of CORT or antide-
pressant-like effects of 4i and FLX. It is evident that enhanced
glutamate neurotransmission is associated with the pathogenesis
of depression. Stimulated glutamate activity can cause excitotox-
icity and neuronal damage in discrete areas of the brain [52].
Moreover, a considerable body of work carried out by in vivo
microdialysis has shown that both stress and chronic CORT treat-
ment induce glutamate release and transmission in the brain,
which may result in direct damage of cellular proteins and lipids
that consequently lead to loss of membrane fluidity and receptor
functions [51,53,54]. On the other hand, antidepressant drugs
(like FLX, reboxetine, desipramine and venlafaxine) have shown
to inhibit stress evoked glutamate release in hippocampus and
prefrontal cortex [55–57]. In addition, reports have demonstrated
that stimulation of pre-synaptic 5-HT3 receptors facilitate the
brain glutamatergic synaptic inputs, while 5-HT3 antagonists
markedly suppress the same [58]. Therefore, considering that
abnormal GCs functions due to repeated CORT administration
may affect glutamate activity and that 4i and FLX may have mod-
ulating effects on glutaminergic system and may impose an
inhibiting effects on oxidative stress induced brain damage, one
could suppose the involvement of aforementioned events in the
inducing effects of CORT and attenuating ability of 4i and FLX
on depressive-like behavior. However, additional studies are war-
ranted to confirm such hypothesis.
It has been well established that depression is often associated
with anxiety and that the abnormal GCs activity can also induce
anxiety-like behavioral deficits [9], the present study investigated
the effects of CORT and 4i treatment on anxiety-like behavior in
mice using light–dark aversion test. The model is based on an
approach/avoidance conflict of mice between the drive to explore
novel areas and an aversion to brightly illuminated environment
[48]. Anxiolytic drugs abolish the apparent apprehension of light
area and increase the latency time for the first passage from light
to dark chamber as well as total time spent in light chamber
[38]; while compounds with anxiogenic behavior decrease latency
and total time spent in light chamber [48]. In consistent with the
previous reports, chronic CORT administration resulted in
increased anxiety-like effects in mice subjected to light–dark test
with significantly increased aversion and decreased exploratory
behavior in light chamber [9]. However, few reports have demon-
strated that repeated CORT dosing has no significant effect on anx-
iety-like behavior in open field and social interaction tests [10].
The inconsistency in the results could be attributed to many fac-
tors, among them, the dose of CORT, the duration of CORT dosing,
the difference in anxiety models employed and finally due to fac-
tors related to each animal species. It is noted that 4i (only at
higher dose of 1 mg/kg), similar to FLX, increased exploratory
behavior of CORT treated mice in brightly lit light chamber. How-
ever, the lower dose of the test compound was ineffective, while it
has shown anxiolytic-like effects in untreated mice during the
behavioral paradigm [33]. The reason for this could be attributed
to the fact that dose was not effective enough in attenuating the
CORT induced anxiety in mice. Besides, this suggests that 4i has
potential to ameliorate anxiety-like behavior in addition to
depression-like state associated with abnormal GCs activity and
particularly disinhibition of the HPA axis.
While assessing the antidepressant and anxiolytic like effects of
compounds based on the despair behavior and exploratory based
models, the psychomotor stimulant/sedative effects can lead to
false positive/negative results, thus the influence of the test drug
on the locomotor activity of the animal is a governing concern
[34,43]. In the present study, to eliminate the influence of 4i and
CORT on the non-specific motor activity of mice (that could affect
the behavior in FST and light–dark aversion test), the effects on the
locomotor activity of mice were evaluated. Neither CORT nor did
the 4i affected the basal locomotion significantly in actophotome-
ter test. Therefore, it is unlikely that the depressogenic behavior of
CORT and antidepressant-like effects of 4i and FLX is an artifact due
to changes in locomotor activity.
Taken together, the finding presented herein point to
a
hypothesis that dysregulated HPA axis as a result of the exces-
sive CORT, present severe depressive-like behavior that can be
attributed, at least in part, to a stimulated brain damage due
to enhanced oxidative stress. The present study revealed that
4i, a novel 5HT3 receptor antagonist can reverse CORT induced
depressive-like behavior in mice. Furthermore, this effect can
be related to the decrease in oxidative stress induced neuronal
damage as the results of the study demonstrate that it decreases
pro-oxidant markers and increases anti-oxidant defense system
in the brain. However, further studies are required to understand
the molecular mechanism such as signal transduction pathways
and post receptor action and to elucidate the correlation
between the behavioral activity and regulatory effects on the
oxidative and anti-oxidant defense system produced by the test
compound.
The fact that brain has comparatively greater vulnerability to
oxidative damage, increased oxidative stress is one of the key
pathogenic mechanisms of depressive disorders [49]. Previous
reports have shown that repeated CORT injections lead to
elevated oxidative load in the brain that culminates in the
development of depressive symptomology [18]. In the present
study, chronic CORT administration exhibited increased TBARS
and nitrite levels in the brain demonstrating increased oxidation
of cellular lipids and generation of ROS. Subsequently, CORT
elicited reduced antioxidant enzyme actions as indicated by
decreased catalase activity and reduced glutathione levels,
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.