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twitch response potentiation [F (3, 48)¼10.39, Po0.0001]. In this
study, FLX (10 mg/kg) significantly increased the 5-HTP induced
head twitch scores in mice (Po0.01 vs. control group), indicating
the predictive validity of the model. Further, post hoc analysis
revealed that, 4i (0.5 and 1 mg/kg) significantly elevated the head
twitch response induced by 5-HTP as shown by markedly increase
in head twitch scores in 4i treated group in mice (Po0.01 vs.
control group for 0.5–1 mg/kg), Fig. 5.
Mice subjected to light–dark box avoid the aversive light
illumination and prefer the dark chamber, which reflect the
anxious response of the animal, while increased exploration in
light chamber and latency to enter dark chamber are associated
with anti-anxiety effects (Hascoet et al., 2001). Acute treatment
with 4i increased latency to first passage in dark chamber and time
spent in light chamber, which corroborates the previous reports,
that 5-HT3 receptor antagonists increase animal exploration in
light chamber when exposed to light–dark box, evidently demon-
strating the anti-anxiety like effect of 4i (Costall et al., 1989).
Although acute behavioral studies substantially demonstrate the
pharmacological activity of the compounds, the tested doses do
not correspond to the clinical time course of their action. There-
fore, in the present study the effects of 4i in chronic behavioral
model namely, OBX was evaluated. Bilateral ablation of olfactory
bulb results in several neurobehavioral changes that correspond to
both depression and anxiety like symptoms (Glinka et al., 2012;
Kelly et al., 1997). In example, hyperactivity is one of the putative
indices of agitation like behavior in anxious patients whereas,
decreased reward related behavior reflects anhedonia or inability
to experience pleasure, which is one of the cardinal signs of
depression in humans (Kelly et al., 1997; Song and Leonard,
2005; Wang et al., 2007). In the current study, after 4-weeks of
bulbectomy, OBX rats exhibited abnormal behavioral pattern in
sucrose preference test and OFT used as the testing paradigms of
depression and anxiety respectively (Kelly et al., 1997; Wang et al.,
2007). Furthermore, FLX as a reference drug reversed the OBX
induced behavioral defects demonstrating high predictive validity
of the model (Zueger et al., 2005). In the sucrose preference test,
OBX rats showed pronounced anhedonic behavior (as indicated by
increased preference for sucrose consumption over water), which
was curtailed by chronic 4i treatment, the effect similar to that
shown by several antidepressants (Song and Leonard, 2005).
It demonstrated the potential influence of 4i in reward related
activity, which is specific to the diseased condition (since 4i had no
influence on hedonic behavior of sham rats) and revealed anti-
depressant like behavior following chronic treatment of 4i, in
agreement with its acute action (Romeas et al., 2009). In OFT, OBX
rats elicited the increased number of ambulation and rearing (as a
measure of horizontal and vertical locomotor activity, respectively)
which showed hyperactive behavior following bulbectomy and
revealed anxiety condition in association with depression like
behavior in rats (Song and Leonard, 2005; Wang et al., 2007).
Chronic 4i treatment on the other hand, reversed the hyper-
locomotor performance in OBX rats without any influence on the
motor behavior of sham rats, which revealed specificity of 4i
treatment in diseased condition. This is in agreement with the
activity of several antidepressants, including reference drug used
in the present study and several 5-HT3 antagonists (Devadoss
et al., 2010; Gautam et al., 2013; Song and Leonard, 2005;
Ramamoorthy et al., 2008), indicating the anti-anxiety like effect
of 4i on chronic treatment. Taking all the behavioral results into
account, 4i, when administered either acutely or chronically,
displayed robust antidepressant and anti-anxiety like effects in
multiple animal models in mice and rats.
4. Discussion
The complex pathophysiology of mood disorders (depression
and anxiety) and inconsistent therapeutic efficacy of present
antidepressants necessitate the research of new targets and novel
compounds with better effectiveness and promising treatment. In
the present study, 4i, a novel ligand with potential 5-HT3 receptor
antagonistic activity, demonstrated evident antidepressant and
anti-anxiety like effects in a series of acute and chronic rodent
behavioral assays.
In the preliminary study, the 5-HT3 receptor antagonistic
activity of 4i (in the form of pA2) was evaluated using guinea
pig-ileum and was found to be higher than the standard drug,
ondansetron, indicating potential 5-HT3 receptor affinity and
antagonistic action of 4i (MacKay, 1978; Mahesh et al., 2004).
Consequently, the current study utilized standard rodent beha-
vioral models to assess the effects of 4i, in vivo. With high
predictive validity, behavioral despair tests such as FST and TST
are the most widely used models across the laboratories to
evaluate the antidepressant activity of numerous compounds
(Cryan and Slattery, 2007; Millan et al., 2001; Petit-Demouliere
et al., 2005). The increased duration of immobility reflects a state
of hopelessness, which simulates the depressive symptoms seen in
humans (Castagne et al., 2011). 4i significantly reduced the
duration of immobility in mice after acute treatment in FST and
TST, a behavioral profile shown by several antidepressants in
clinical use (Millan et al., 2001) and several 5-HT3 receptor
antagonists (Bravo and Maswood, 2006; Devadoss et al., 2010;
Kos et al., 2006; Ramamoorthy et al., 2008). Since drugs with
central nervous system stimulant effects can also reduce the
duration of immobility in FST and TST, and may possibly give
false positive results (Boissier and Simon, 1965; Petit-Demouliere
et al., 2005), the effect of 4i on the SLA in mice was determined
and it was found that the antidepressant like effect of 4i (0.5 and
1 mg/kg) in these behavioral despair tests were independent of a
generalized increase in motor activity.
Anxiety is one of the most common mood disorders associated
with depression (Ressler and Nemeroff, 2000). Several antidepres-
sants such as fluoxetine and escitalopram, with serotonergic
modulatory activity have shown to be effective in both depression
and associated anxiety disorders (David et al., 2009; Davidson et al.,
2004; Dulawa et al., 2004; Pedersen, 2005). Therefore, the anti-
anxiety like effect of 4i in anxiety testing paradigms was estimated.
Hole board and light–dark box test are exploratory behavior
based, extensively used models for estimation of anxiety/anti-
anxiety effects (Bourin and Hascoet, 2003; Crawley, 1985; Takeda
et al., 1998; Van Gaalen and Steckler, 2000). In hole board test,
the number and duration of nose-poking (head dipping) are
putative indices of anxiety like behavior, which are shown to be
increased by various anti-anxiety agents (Takeda et al., 1998). In
the present study, similar to the positive control, 4i significantly
increased number of nose-poking as well as cumulative time
spent in nose-poking (duration of nose-poking) in mice. This is in
agreement with the previous findings that 5-HT3 receptor
antagonists have anti-anxiety like effects in rodents (Costall
and Naylor, 1992).
While considering the behavioral effects in rodents as well in
humans, several antidepressants act by enhancing synaptic con-
centration of monoamines particularly serotonin. Therefore an
estimate of serotonergic modulatory activity of 4i was evaluated
through interaction studies with an antidepressant (FLX, a SSRI) in
clinical use. Combination of sub-active doses of 4i (0.25 mg/kg)
and FLX (5 mg/kg) significantly reduced the duration of immobi-
lity in FST, which showed a synergistic action of 4i with FLX and
possibly demonstrated that, similar to FLX, 4i increased seroto-
nergic neurotransmission in mice. The results are in line with the
previous report that 5-HT3 receptor antagonists potentiate the
Gupta, Deepali
