Cotinine reduces depressive-like behavior and hippocampal vascular endothelial growth factor downregulation after forced swim stress in mice

Behavioral Neuroscience

Cotinine reduces depressive-like behavior and hippocampal VEGF downregulationafter forced swim stress in mice

--Manuscript Draft--

Manuscript Number: BNE-2014-0037R1

Full Title: Cotinine reduces depressive-like behavior and hippocampal VEGF downregulationafter forced swim stress in mice

Abstract: Cotinine, the predominant metabolite of nicotine, appears to act as an antidepressant.We have previously shown that cotinine reduced immobile postures in the Porsolt'sforced swim and tail suspension tests while preserving the synaptic density in thehippocampus as well as prefrontal and entorhinal cortices of mice subjected to chronicrestraint stress. In this study, we investigated the effect of daily oral cotinine (5 mg/kg)on depressive-like behavior induced by repeated, forced swim (FS) stress for 6consecutive days in adult, male C57BL/6J mice. The results support our previousreport that cotinine administration reduces depressive-like behaviors in mice subjectedor not to high salience stress. In addition, cotinine enhanced the expression of thevascular endothelial growth factor (VEGF) in the hippocampus of mice subjected torepetitive FS stress. Altogether, the results suggest that cotinine may be an effectiveantidepressant positively influencing mood through a mechanism involving thepreservation of brain homeostasis and the expression of critical growth factors such asVEGF.

Article Type: Unmasked Article

Corresponding Author: Valentina Echeverria MoranBay Pines VAHCSUNITED STATES

Corresponding Author E-Mail: [email protected]

Corresponding Author SecondaryInformation:

Corresponding Author's Institution: Bay Pines VAHCS

Other Authors: J Alex Grizzell, MA

Michelle Mullins

Alexandre Iarkov, PhD

Sagar Patel

Ross Zeitlin

Adeeb Rohani

Laura Charry

Corresponding Author's SecondaryInstitution:

First Author: J Alex Grizzell, MA

Order of Authors Secondary Information:

Manuscript Region of Origin: USA

Order of Authors: J Alex Grizzell, MA

Michelle Mullins

Alexandre Iarkov, PhD

Sagar Patel

Ross Zeitlin

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Adeeb Rohani

Laura Charry

Valentina Echeverria Moran

Manuscript Classifications: 10.110: Neuropsychiatric disorders; 10.110.030: depression and anxiety disorders;20.70.040: motivated laboratory behavior (appetitive/aversive); 20.70.060: Otherbehavioral analysis

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Cotinine: stress-induced depression and VEGF

Cotinine reduces depressive-like behavior and hippocampal VEGF

downregulation after forced swim stress in mice

J. Alex Grizzellab

, Michelle Mullinsa†

, Alexandre Iarkovae

, Adeeb Rohania, Laura C. Charry

a and

Valentina Echeverria acde

*

a Research & Development Service, Department of Veterans Affairs, Bay Pines VA Healthcare System,

Bay Pines, Florida 33744, USA.

b Department of Psychiatry and Behavioral Neurosciences, Morsani College of Medicine, University of

South Florida, Tampa, FL, 33611, USA

c Research Service, Department of Veterans Affairs, Tampa VA Healthcare System, Florida 33612,USA

d Department of Molecular Medicine, University of South Florida, Tampa, Florida 33647, USA

e Universidad Autónoma de Chile, Carlos Antúnez 1920, Providencia, Santiago, Chile.

* Corresponding Author:

Valentina Echeverria Moran, Ph.D

10,000 Bay Pines Blvd. Bldg22, Rm123

Bay Pines, FL, USA 33744

Telephone: +1-727-398-6661 ext. 4425

Fax: +1-727-319-1161

[email protected]

Manuscript

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Cotinine: stress-induced depression and VEGF

Abstract

Cotinine, the predominant metabolite of nicotine, appears to act as an antidepressant. We have previously

shown that cotinine reduced immobile postures in the Porsolt’s forced swim and tail suspension tests

while preserving the synaptic density in the hippocampus as well as prefrontal and entorhinal cortices of

mice subjected to chronic restraint stress. In this study, we investigated the effect of daily oral cotinine (5

mg/kg) on depressive-like behavior induced by repeated, forced swim (FS) stress for 6 consecutive days

in adult, male C57BL/6J mice. The results support our previous report that cotinine administration

reduces depressive-like behavior in mice subjected or not to high salience stress. In addition, cotinine

enhanced the expression of the vascular endothelial growth factor (VEGF) in the hippocampus of mice

subjected to repetitive FS stress. Altogether, the results suggest that cotinine may be an effective

antidepressant positively influencing mood through a mechanism involving the preservation of brain

homeostasis and the expression of critical growth factors such as VEGF.

Key words: Depressive disorders; Forced swim; Neurogenesis; Vascular endothelial growth factor

Highlights

Cotinine prevents depressive-like behavior after repetitive forced swim stress

Cotinine increases the expression of VEGF in the hippocampus of stressed mice

Cotinine may mediate the antidepressant effects of nicotine

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Cotinine: stress-induced depression and VEGF

Introduction

Depression is a mental disorder characterized by changes at the physiological, psychological, and

behavioral levels. Highly prevalent, this disorder affects about 25% of women and 12% of men and is the

leading cause of disability worldwide (Gelenberg, 2011; McIntyre et al., 2011). Depression often

manifests through multiple symptoms, including despair, guilt, anhedonia, psychomotor dysfunction,

reduced positive affect and memory impairment (Nutt et al., 2007). High co-morbidity rates between

depression and anxiety disorders as well as dementia have also been well described (Momartin et al.,

2004; Berger et al., 2005). The efficacy of treating depressive conditions is diminished by high rates of

treatment resistance and many fail to reach full remission (Philip et al., 2010b). Thus, a determined effort

to identify and develop new therapies on the basis of validated disease mechanisms to treat depression is

required.

Experiencing physical or psychological stress is one of the more frequent external causes of

depression (Bosch et al., 2012). The stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by

chronic stress leads to increased inflammation and oxidative stress (Zunszain et al., 2013) which

subsequently inhibits the expression of factors that contribute to the promotion of synaptic plasticity,

neurogenesis, and neuronal survival (Dwivedi, 2009). Increasing evidence suggests that this cascade of

events participates in both the development and maintenance of depression (Kawahara et al., 1997; Kim

et al., 2007; Lee & Kim, 2009; Shi et al., 2010). In fact, pro-inflammatory mediators produced by

activated immune cells induce many behavioral changes including depression, anxiety, impaired

cognitive function, diminished activity and reduced appetite (Hart, 1988). Cholinergic compounds related

to nicotine, such as anatabine, regulate cytokine production and display anti-inflammatory properties

(Paris et al., 2013). Since neuro-inflammation is a well-known phenomenon during depression, it is

possible that the modulation of inflammation by these factors is key in mediating its observed

antidepressant effects.

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Cotinine: stress-induced depression and VEGF

Cotinine, a component of tobacco leaves and the predominant metabolite of nicotine, is anti-

inflammatory (Rehani et al., 2008) and facilitates serotonin (5-hydroxytryptamine; 5-HT) release in the

brains of rats (Fuxe et al., 1979). Cotinine-treatment reduces anxiety in acute stress conditions (Zeitlin et

al., 2012) and depressive-like behavior in mice subjected or not to prolonged restraint stress (Grizzell et

al., 2014). These effects are accompanied by a cotinine-induced stimulation of the Akt/GSK3 pathway

(Echeverria et al., 2011a; Grizzell et al., 2014). The activation of this pathway stimulates neuronal

genesis and survival and decreases depressive-like behavior (Wada, 2009; Riadh et al., 2012). In fact,

most currently prescribed antidepressants, such as serotonin reuptake inhibitors (SSRIs), monoamine

oxidase (MAO) inhibitors and tricyclic antidepressants, also activate this pathway (Beaulieu et al., 2009;

Echeverria et al., 2011a) which, in turn, stimulates the expression of neurotrophic factors such as the

brain-derived neurotrophic factor (BDNF) (Beaulieu et al., 2009) and the vascular endothelial growth

factor (VEGF). Activation of this pathway also stimulates an upregulation of synaptic proteins such as

synaptophysin (King et al., 2013) and PSD95 (Xie et al., 2011). Indeed, cotinine treatment is also

associated with an increased expression of synaptophysin (Grizzell et al., 2014) and PSD95 (Patel et al.,

2014) in chronic stress and Alzheimer’s disease models, respectively. Here, we report the effect of

cotinine on depressive-like behavior induced by repetitive, daily forced swim stress and provide some

new insight about its potential mechanism(s) of action.

Methods

Animals

Two-month-old male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA), weighing 25-30 g,

were maintained on 12-hours (h) of a light/dark cycle (light on at 07:00 h) with ad libitum access to food

and water at a regulated temperature (25 ± 1° C). Upon arrival mice were group-housed and acclimated

for 7 days before any intervention. Following behavioral testing, euthanasia was performed via cervical

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Cotinine: stress-induced depression and VEGF

dislocation under anesthesia with isofluorane (4% induction, 2% for maintenance). Experiments were

performed during the light period of the circadian cycle. All treatments and activities were conducted in

accordance with The Guidelines for Animal Experiments issued by the Ethics Committee of the Bay

Pines Veterans Affairs Healthcare System. These experiments also followed the National Institutes of

Health standards and were approved by the Institutional Animal Care and Use Committee of Bay Pines

Veterans Affairs Healthcare System.

Drugs and Route of Administration

Cotinine ((5S)-1-methyl-5-(3-pyridyl) pyrrolidin-2-one; Sigma-Aldrich Corporation, St. Louis, MO,

USA) solutions were prepared by dissolving the powdered compound in sterile phosphate buffered saline

(PBS). All mice were distributed in treatment groups per random assignment and then treated with vehicle

or cotinine (5 mg/kg) via gavage. The gavage technique was performed by well trained personnel and did

not induce significant stress in the mice. All investigators were blind to treatment groups and doses were

chosen based on previously conducted studies (Zeitlin et al., 2012; Grizzell et al., 2014). Treatments

were administered for 7 days before the induction of stress. For all animals, treatment continued from the

onset of treatment until euthanasia.

Behavioral Procedures

To induce depressive-like behavior, we employed a broadly used model of stress in rodents, the repetitive

FS stress paradigm (Furukawa-Hibi et al., 2011). FS mice were subjected to daily 6-min FS sessions for

6 days. After this time, all mice were tested for depressive-like behavior using the Porsolt’s forced swim

test (FST) (Porsolt et al., 1977a; Porsolt et al., 1977b; Bhatnagar et al., 2004) and the tail suspension test

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Cotinine: stress-induced depression and VEGF

(TST) (Cryan et al., 2005). Mice were monitored via closed-circuit video feed from an adjacent room and

all behavior recorded for later quantification.

Forced Swim Stress (FS)

The FS was performed as described (Furukawa-Hibi et al., 2011). Each mouse was placed in an

inescapable transparent plastic cylinder (40 cm high x 20 cm in diameter) filled with water to a depth of

30 cm. Water was changed between each trial and its temperature maintained at 23-24 °C. In all cases,

following exposure, animals were retrieved, dried with a hand towel and returned to their home cages.

Mice not exposed to stress (NES) serving as the control groups were removed from the animal housing

facility and taken to the behavioral testing room during the same period of time than FS mice but

remained in their home cages during the FS exposure period.

Porsolt’s Forced Swim Test (FST)

The FST was performed as previously described (Porsolt et al., 1977a; Porsolt et al., 1977b; Bhatnagar et

al., 2004; Grizzell et al., 2014). The FST was developed based on the fact that rodents, when exposed to

an inescapable stress, will engage in periods of fast movement followed by increasing periods of

immobile posture, which is considered to be a measure of depressive-like behavior. Each mouse was

placed in a transparent plastic cylinder filled with water at 23-24 °C as described above and behavior was

recorded for 5 min. Immobility times were scored by investigators blind to treatment levels. Immobility

was defined as the time spent floating making only the movements necessary to maintain the head above

the water.

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Cotinine: stress-induced depression and VEGF

Tail suspension test (TST)

The TST is a widely used instrument for quantifying depressive-like behavior in rodents (Cryan et al.,

2005; Grizzell et al., 2014). This test is predicated upon the fact that animals subjected to the short-term

unavoidable stress of being suspended by their tail will develop an immobile posture and/or cessation of

struggling behavior, which is considered analogous to depressive behavior. A strip of masking tape (20

cm x 1.9 cm) was positioned to encapsulate the tail of the mouse to prevent fall or injury. The end of the

tape was adhered to a blunt hook suspended upside-down such that the tip of nose was approximately 6

inches from the floor of the TST chamber. Immobility, defined as the summation of time the animal does

not struggle to escape, was measured during one 5 min trial and quantified separately by two investigators

blind to treatment groups.

Experimental conditions

Effect of cotinine on depressive-like behavior in mice

After one week of treatments, mice treated with vehicle or cotinine (5 mg/kg; n = 10/group) via gavage,

were exposed under continue treatment to 6-min FS stress for six consecutive days. Two additional

control groups that were not exposed to stress (NES) were treated with vehicle or cotinine via gavage (5

mg/kg; n=10/group). 24 and 48 h after the last FS session, mice in all groups were tested for depressive-

like behavior using the TST and FST, respectively (Figure 1a).

Western blot analysis of brain extracts

The Western blot analysis investigated the expression of VEGF (FS mice: n=8/group; NES mice: n=4-

6/group). Following euthanasia, mice were perfused with saline, and brain tissues were rapidly dissected

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Cotinine: stress-induced depression and VEGF

and stored at -80o

C. Brain tissues were then disrupted by sonication in cold lyses buffer (Cell Signaling

Technology, Denver, MA, USA) containing a complete protease inhibitor cocktail (Roche Molecular

Biochemicals). After sonication, brain extracts were incubated on ice for 30 min and centrifuged at

20,000 x g for 30 min at 4°C. Protein concentrations of supernatants were measured using the Bio-Rad

protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by gradient (4-

20%), SDS-PAGE, then transferred to nitrocellulose membranes (BA83 0.2 µm; Bio-Rad). The

membranes were blocked in TBS with 0.1% Tween 20 (TBST) containing 5% dry skim milk for 1h.

Membranes were incubated with primary antibodies in TBST overnight at 4°C and with secondary

antibodies for 1h at room temperature (RT) in a blocking buffer. Rabbit polyclonal antibodies directed

against VEGF was obtained from Abcam (Cambridge, MA, USA). A monoclonal antibody directed

against β-tubulin (Promega Corporation, Madison, WI, USA) was used to control protein sample loading

and transfer efficiency. Membranes were washed with TBST and incubated with LI-COR’s goat anti-

mouse IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE) for 1h at RT, washed with TBST

and TBS. Images were acquired using an Odyssey Infrared Imaging System (LI-COR Biosciences) and

analyzed using the NIH Image J software.

Statistical analysis

To analyze the group and treatment effects, differences between group means in the behavioral analyses

were assessed using a multifactorial, 2 (Treatment) x 2 (Stress) analysis of variance (ANOVA) and were

followed by Tukey’s post hoc multiple comparisons tests. A one-way ANOVA was used to identify

differences between groups in the analysis of VEGF protein expression between vehicle- and cotinine-

treated FS mice and vehicle-treated NES mice. Student’s t-test was use to compare protein expression

data between NES mice treated with vehicle or cotinine, as these were run on a separate gel. Statistical

analyses were conducted using statistical software packages (SPSS, Chicago, IL, USA and GraphPad

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Cotinine: stress-induced depression and VEGF

Prism, San Diego, CA, USA). For all comparisons, statistical significance was considered with α = 0.05.

All error bars shown in figures represent the standard error of the mean (SEM).

Results

Effect of cotinine over depressive-like behavior after repetitive FS stress

To investigate whether continuous daily treatments with cotinine beginning one week prior to the

induction of stress could prevent the increase in depressive-like behavior induced by 6 minutes/day for 6

days of FS stress, mice were subjected to the TST and FST 24 and 48 hours after the cessation of FS

stress (Fig. 1a). Results of a multifactorial, 2 (Treatment) x 2 (Stress) ANOVA of TST (Fig. 1b) revealed

significant main effects of treatment (F(1,36) = 19.39, p < 0.0001) and stress (F(1,36) = 48.13, p < 0.0001).

Results of a similar analysis on FST (Fig.1c) revealed significant main effects of treatment (F(1,36) = 29.54,

p < 0.0001) and stress (F(1,36) = 204.30, p < 0.0001) as well. Results of a Tukey’s post-hoc test revealed

that in the TST (Fig.1b), vehicle-treated, FS mice displayed higher levels of immobility than NES mice (p

< 0.0001) and cotinine-treated, FS mice (p < 0.01). A Tukey’s post-hoc test also revealed that in

Porsolt’s FST (Fig.1c), vehicle-treated, stressed mice displayed significantly more immobility than NES

(p < 0.0001) and cotinine-treated, FS cohorts (p < 0.0001).

Analysis of the protein expression of VEGF in the hippocampus of mice after repetitive forced swim

stress

VEGF is a neurotrophin that modulates blood flow and angiogenesis and is involved in neurogenesis (Jin

et al., 2002; Fabel et al., 2003; Cao et al., 2004). In a pilot mRNA expression RT-PCR assay analysis, we

found that the mRNA expression of Vegfa was up-regulated in the hippocampus of cotinine-treated FS

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Cotinine: stress-induced depression and VEGF

mice when compared to vehicle-treated FS mice (p < 0.01; data not shown). Therefore, we investigated

their associated protein expression levels in the hippocampus of the same mice using Western blot

analysis. The results show that the groups differed significantly from one another (F(2,18) = 14.93, p =

0.0002; Fig. 2b) and Tukey-Kramer’s post hoc analyses revealed that vehicle-treated mice subjected to

repetitive FS stress had a significant decrease in the expression of VEGF in the hippocampus (p < 0.001).

On the other hand, FS mice treated with cotinine showed significantly higher levels of VEGF expression

in the hippocampus than vehicle-treated, FS mice (p < 0.05), to reach levels not significantly different

than those of NES mice (p > 0.05; Fig.2b). Finally, in the absence of stress, a Student’s t-test revealed

that cotinine induced no changes in VEGF expression between cotinine and vehicle-treated mice (t =

0.2910, p = 0.7795; Fig.2a).

Discussion

We have previously shown that cotinine prevented depressive-like behavior in rodent models of

mental health conditions (Grizzell et al., 2014; Patel et al., 2014). The present study investigated the

molecular mechanisms of cotinine’s antidepressant effects in a model of stress-induced impairment, the

repetitive forced swim stress. The behavioral tests showed that cotinine reduced depressive-like postures

in the FST and TST and neurochemical analysis revealed that cotinine prevented the stress-induced

decrease of VEGF in the hippocampus of stress-exposed mice.

The antidepressant properties of cotinine found in this study are in agreement with our recent reports

showing that cotinine reduced depressive-like behavior in mice subjected to chronic restraint-stress

(Grizzell et al., 2014) as well as mice developing Alzheimer’s disease (AD)-like pathology (Patel et al.,

2014). The antidepressant effect of cotinine in the restrained mice was accompanied by an increase in

synaptic density in the CA1, CA3, and dentate gyrus of the hippocampus as well as in the prefrontal and

entorhinal cortices (Grizzell et al., 2014). Consistent with an effect of cotinine on synaptic function,

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Cotinine: stress-induced depression and VEGF

cotinine’s positive effects on depressive-like behavior corresponded with an increase in the expression of

the postsynaptic density protein 95 (PSD95) in the brain of AD mice (Patel et al., 2014).

The TST and FST are highly regarded as valid and reliable tests of depressive-like behaviors,

particularly in screening the efficacy of antidepressant compounds (Bourin & Hascoet, 2003; Ramos,

2008). In this study, both tests were included to control for individual test limitations and to ensure a

reliable antidepressant-like effect, as some commonly prescribed antidepressants have only shown

efficacy in one but not both. Following one week of pre-treatment, we observed significant decreases in

depressive-like behavior in both the TST and FST in the mice treated with cotinine. Because the

interpretations of these tests are inherently reliant on sensorimotor abilities, it would be plausible to

attribute this effect to a cotinine-induced alteration of locomotor activity. However, based on previously

reported evidence, the possibility that the observed decrease in immobility in these tests was due to a

cotinine-induced decrease in locomotion is unlikely. A cotinine treatment regimen similar to that used in

this study did not influence ambulation or sensorimotor abilities in the open field test either with or

without exposure to an acute stressor (Zeitlin et al., 2012). In another study, cotinine-treatment did result

in a marginal, though statistically insignificant reduction of ambulation in the open field, however these

same animals also displayed more escape-oriented behavior in both the FST and TST (Grizzell et al.,

2014). If cotinine were to influence locomotor behavior in a manner that would confound the

interpretation of depressive-like behavioral tests, one would therefore expect to instead see an increase in

immobile postures in the cotinine treated groups, particularly in stressful conditions. We therefore

conclude that the observed differences between cotinine- and vehicle-treated mice in the TST and FST

reported here are not due to a cotinine-induced sensitization of locomotor activity.

Though recent efforts have greatly advanced the pharmacokinetic and pharmacodynamic properties of

cotinine, the mechanism(s) of action is still not completely understood. We have proposed that cotinine is

a positive allosteric modulator of the homomeric 7nAChRs (Moran, 2012; Grizzell & Echeverria, 2014).

Recent reports have shown evidence in vivo suggesting that cotinine’s effects are mediated by 7 and

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Cotinine: stress-induced depression and VEGF

42 nAChRs (Aguiar et al., 2013; Wildeboer-Andrud et al., 2014). Further pharmacological studies are

required to determine whether these are the targets mediating cotinine’s antidepressant effects. The role

of cotinine’s precursor, nicotine, in influencing depressive- and anxiety-like behavior in rodents appears

to be somewhat equivocal (Picciotto et al., 2002; Philip et al., 2010a; Fernandez et al., 2013). For

example, it has been reported that repetitive doses of nicotine reduced (Semba et al., 1998; Djuric et al.,

1999; Tizabi et al., 2009) and induced (Hayase, 2007; 2008; 2013) depressive-like behavior. However, it

has been suggested that nicotine’s effects in the central nervous system may be due to complex

relationships with its metabolites, most likely, cotinine (Crooks & Dwoskin, 1997; Grizzell & Echeverria,

2014).

There is consensus that the deleterious effects of stress result, at least in part, from a deregulation of

the central monoamine systems. A decrease in MAO activity has been shown in the brains tobacco

smokers (Fowler et al., 1996a, Fowler et al., 1996b and Fowler et al., 1998). Since MAO degrades

dopamine, noradrenalin, and serotonin, it has been suggested that certain tobacco constituents, nicotine

excluded, may acts as MAO inhibitors (Fowler et al., 1996a, Fowler et al., 1996b and Fowler et al., 1998).

Cotinine has been shown to increase the release and reduce the uptake of 5-HT in the brains of rats (Fuxe

et al., 1979). Thus, our results may be explained by an enhancement of the serotonergic system which

decreased the impact of stress thereby reducing the engagement in depressive-behavior in the FS-exposed

mice treated with cotinine.

At the neurochemical level, we found that cotinine upregulated VEGF expression in the hippocampus

of mice subjected to FS stress. VEGF is a cytokine that plays an important role in modulating

neurogenesis and angiogenesis (Schanzer et al., 2004; Galvan et al., 2006; Jin et al., 2006; Sun et al.,

2006; Antequera et al., 2012). Stress reduces hippocampal neurogenesis (Gould & Tanapat, 1999) and the

enhancement of hippocampal neurogenesis buffers the stress response as well as associated depressive-

like behaviors (Snyder et al., 2011). VEGF is also neuroprotective (Gora-Kupilas & Josko, 2005). For

example, increased VEGF levels prevent motor neuron degeneration induced by expression of a mutant

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Cotinine: stress-induced depression and VEGF

form of the superoxide dismutase 1 (Lunn et al., 2009). Furthermore, environmental enrichment,

considered of therapeutic value against depression (Hannan, 2014), enhances neurogenesis as well as

hippocampal VEGF levels (Cao et al., 2004). Moreover, increases in VEGF expression not only stimulate

adult hippocampal neurogenesis, but also confer antidepressant-like effects in rodents (Fournier &

Duman, 2012). Recent studies suggested that VEGF may be deregulated during depression (Kahl et al.,

2009; Fournier & Duman, 2012), and the induction of its expression in the hippocampus is required for

the effects of various antidepressants (Warner-Schmidt & Duman, 2007; Greene et al., 2009).

This report provides novel evidence that cotinine prevents the decrease in the expression of VEGF in

the hippocampus of mice exposed to high salience stress. A modulation of VEGF expression by cotinine

was previously suggested by an in vitro study using isolated carotid arteries that showed that both

cotinine and nicotine increased the mRNA and protein expression of VEGF in endothelial cells (Conklin

et al., 2002). However, because cotinine did not induce any changes in VEGF expression in the control

mice in this report, it is possible that cotinine is restoring VEGF expression by positively influencing

molecular mechanisms of homeostasis under conditions of stress only. Taken together, we postulate that

cotinine promotes restorative cerebral changes by stimulating signaling factors such as VEGF, which

may, in turn, promote plasticity processes such as neurogenesis, and therefore, enhanced stress resiliency

and mood. These studies support the hypothesis that cotinine acts as an antidepressant, probably through a

mechanism that involves the stimulation of synaptic plasticity processes such as neurogenesis and

neuroprotection, by preserving the expression of neurogenesis factors such as VEGF.

Outside of our laboratory, very little research has been conducted investigating the influence of

cotinine over symptoms associated with depression. To our knowledge, no peer-reviewed literature has

been reported documenting the investigation of cotinine on depressive-like behavior. Evidence of

cotinine’s influence over mood at the clinical level has been reported (Keenan et al., 1994b). In this study

the authors conducted a randomized, double-blind, placebo-controlled, counterbalanced-order design

experiment which investigated the effects of cotinine (30 mg base) given intravenously after 48 hours of

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Cotinine: stress-induced depression and VEGF

abstinence from cigarette smoking. Serum cotinine concentrations increased to levels commonly achieved

during daily cigarette smoking. Cotinine apparently produced subjective differences in self-reported

ratings of restlessness, anxiety and tension, insomnia, sedation, and pleasantness when compared to

placebo, though depression scores were unchanged from baseline. Not without controversy, the authors

concluded that cotinine was behaviorally active in the setting of cigarette abstinence. Almost two decades

later though, further clinical studies of the effect of cotinine on mood are almost non-existent and no

results are available on non-smokers, stable smokers, or previous smokers outside of the withdrawal

period.

Aside from mood, cotinine has consistently been shown to elicit general memory enhancing effects

(Buccafusco et al., 2009; Buccafusco & Terry, 2009; Echeverria et al., 2011b; Echeverria & Zeitlin,

2012; Grizzell et al., 2014) and these extend to conditions of prolonged stress (Grizzell et al., 2014). This

said, one pilot clinical study did report an impairment of verbal recall on a long but not short list

following cotinine treatment at doses up to 1.5 mg cotinine base/kg (Herzig et al., 1998). In the 16

individuals tested, there were no reported changes in the profile of mood state (POMS) scores following

cotinine treatment as well (Herzig et al., 1998). Due to the size of this study though, its results must be

taken with caution. In addition cotinine has been shown to reduce anxiety in abstaining cigarette smokers

(Keenan et al., 1994a) and anxiety-like behavior in rodents following acute-stress exposure (Zeitlin et al.,

2012).

The consistency of cotinine in reducing depressive-like behavior with and without exposure to chronic

stress conditions (Zeitlin et al., 2012; Grizzell et al., 2014; Patel et al., 2014) highlights the need to further

investigate its ability to treat depression. This is particularly true when considering that cotinine has good

pharmacokinetic properties (De Schepper et al., 1987) and a positive safety profile in humans, which

includes no habit-forming properties or withdrawal effects, among others (Hatsukami et al., 1997;

Hatsukami et al., 1998a; Hatsukami et al., 1998b; Echeverria Moran, 2012). Further investigation of

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Cotinine: stress-induced depression and VEGF

cotinine’s mechanistic role in modulating depressive-like states at the preclinical level as well as studies

aimed at determining the therapeutic efficacy at the clinical level are therefore warranted.

Conclusions

Currently used antidepressants only effectively alleviate the symptoms in a portion of depressed

patients and most have a delayed therapeutic onset. Neuroplasticity theories of depression have postulated

that a failure of multiple aspects of plasticity processes underlie the etiology and maintenance of

depression (Duman & Aghajanian, 2012). Accordingly, new antidepressant agents are in need of further

investigation. Here, we provide evidence that cotinine prevents FS-induced depressive-like behavior.

Previous studies using repetitive FS as a stressor have shown that depressive-like behaviors were

accompanied by decreased neurogenesis and synaptic plasticity in rodents (Wainwright & Galea, 2013).

This study shows that cotinine’s antidepressant effects were accompanied by a restoration in the

expression of hippocampal VEGF, a factor promoting adult neurogenesis. Cotinine benefits synaptic

plasticity, learning and memory, and enhanced the expression of the neurogenesis factor VEGF under

conditions of stress, which are effects similar to those of other antidepressants. Altogether this evidence

suggests that cotinine has the potential to be a new antidepressant agent and warrants clinical

investigation.

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Acknowledgements

This material is the result of work supported with resources and the use of facilities at the Bay Pines VA

Healthcare System and the James A. Haley Veterans’ Hospital. The contents do not represent the views of

the Department of Veterans Affairs or the United States Government. This work was also supported by

the Bay Pines Foundation, Inc. and a grant obtained from the James and Esther King Biomedical

Research Program 1KG03-33968 (to VE). We will be forever indebted to Ms. Rosalee Holmes, a member

of our team that passed away before submission of this manuscript.

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Cotinine: stress-induced depression and VEGF

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Cotinine: stress-induced depression and VEGF

Fig.1. Effect of cotinine on depressive-like behavior induced by repetitive forced swim stress in

mice. A timeline (a) depicts: mice were pretreated with 5 mg/kg of cotinine (Cot 5) or vehicle (Veh) daily

for one week before 6 min daily for 6 days repetitive forced swim (FS) and continuously treated until

euthanized. 24 and 48 hours following the cessation of FS exposure, depressive-like behaviors were

assessed using the tail suspension (TST) and Porsolt’s forced swim (FST) test, respectively. FS exposure

induced a significant increase in immobile postures and cotinine significantly reduced these postures in

both the TST (b) and FST (c). Tukey’s post hoc analyses revealed the reported differences between FS-

exposed vehicle and cotinine-treated mice: **, p < 0.01; ****, p < 0.0001.

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Cotinine: stress-induced depression and VEGF

Fig.2. Cotinine treatment prevents the stress-induced decrease in the expression of VEGF in the

hippocampus of mice subjected to repetitive forced swim stress. The levels of vascular endothelial

growth factor (VEGF) and -tubulin in the hippocampus of mice were analyzed by Western blot

(n=5/group). The plots represent VEGF immunoreactivity values (IRs) in the hippocampus of mice. a,

effect of cotinine on VEGF expression in mice not exposed to repetitive forced swim (FS) stress (NES); b,

comparison of the expression of VEGF in vehicle-treated NES, vehicle-treated FS mice (FS + Veh) and

cotinine-treated FS mice (FS +Cot 5), IRs were normalized to -tubulin and expressed as a percentage of

the average value found in vehicle-treated, NES mice. Western blot images are seen beneath each

comparison. c, diagram representing potential molecular mechanisms underlying cotinine’s

antidepressant effects. Cotinine, by positively modulating the a7nAChR or through other unidentified

Fig. 2

VEGF

CREBP

c

VEGFSynaptophysin

PSD95

Akt

Akt

GSK3

Promotes

synaptic

plasticity

and mood

stability Impaired synaptic

plasticity and

depression

7nAChR Cotinine

PI3K?

ACh

a bVEGF

NES NES +Cot 5 NES FS + Veh FS + Cot 5

CRE

CBP

CREB

P

P

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Cotinine: stress-induced depression and VEGF

receptors, activates Akt which stimulates CREB transcriptional activity and the expression of synaptic

proteins such as PSD95, Synaptophysin and VEGF. Akt, protein kinase B; GSK3, glycogen synthase 3;

CBP, CREB binding protein; CRE, cAMP-response element; CREB, CRE binding protein; 7nAChR,

nicotinic acetylcholine receptor; ns, no significant difference; PI3K, phosphoinositide 3- kinase;PSD95,

postsynaptic density protein 95. ***, p < 0.001.

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Cotinine: stress-induced depression and VEGF

Fig.3. Diagram describing the postulated effects of stress and cotinine on depressive-like behavior

and neuroplasticity. Stress decreases the expression of neurogenesis and neuroplasticity factors.

Cotinine restores and/or preserves these cellular neuroplasticity processes and decreases stress-induced

depressive-like behavior.

Fig.3

COTININESTRESS

Depression

Reduces synaptic density

Reduces neuroplasticity

and brain homeostasis

Mood stability

Increases synaptic density

Restores or preserves

neuroplasticity and brain

homeostasis

Increases or preserves

the expression

of synaptic and

neurogenesis proteins(e.g. VEGF, Synaptophysin, PSD95)

Decreases the expression

of synaptic and

neurogenesis proteins

Figure1

Figure2

Figure3