Intranasal Cotinine Plus Krill Oil Facilitates Fear Extinction, Decreases Depressive-Like

N° 35
Behavior, and Increases Hippocampal Calcineurin A Levels in Mice.
Patricia Oliveros-Matusᵃ , Nathalie Alvarez-Ricartes , Cristhian Mendozaᵃ , Nelson Perez-Urrutiaᵃ
Florencia Echeverriaᵃ , Alexandre Iarkovᵃ , George E. Barretoᵇᶜ , Valentina Echeverriaᵃᵈ

ᵃ Universidad San Sebastián, Facultad de Ciencias de la Salud, Lientur 1457, Concepción, Chile.
ᵇ.Pontifice Universidad Javeriana, Facultad de Ciencias, Departamento de Nutrición y Bioquímica, Bogotá D.C., Colombia.
ᶜ Universidad Autónoma de Chile, Instituto de Ciencias Biomédicas, Santiago, Chile.
ᵈ Bay Pines VA Healthcare System, Research and Development, Bay Pines, FL 33744, USA

Abstract Results
.
Failure in fear extinction is one of the more troublesome characteristics of
posttraumatic stress disorder (PTSD). Cotinine facilitates contextual fear
memory extinction and reduces depressive-like behavior when administered 24
h after fear conditioning in male C57BL/6 mice. In this study, the effects of
cotinine, and other antidepressant infused via intranasal (IN) on fear extinction
was investigated. Since calcineurin A (CaA) has been involved in facilitating fear
extinction in rodents, we investigated changes of CaA in the hippocampus, a
region key on contextual fear extinction. Short-term treatment with cotinine
formulations was superior to krill oil (KO) and oral sertraline in reducing
depressive-like behavior and fear consolidation and enhancing contextual fear
memory extinction in mice. Furthermore, KO slowed the extinction of fear. At
the molecular level, cotinine increased the protein expression of CaA in the
hippocampus of conditioned mice. The results, suggest that IN cotinine
formulations can be useful to enhance the extinction of contextual fear memory
and treatment of treatment-resistant depression in PTSD and other psychiatric
conditions showing deficits in fear extinction.

Methods Fig. 2 Effect of early posttreatment with cotinine, sertraline, and krill oil on the retention and extinction of fear
memory. Two hours after fear conditioning (FC), mice (n = 5–8 mice/group) received intranasal (IN) PBS, krill oil
(KO) IN, cotinine (Cot) IN, or Cot + KO IN, oral sertraline (2 mg/day). Next day after, mice were tested for contextual
fear memory (retention test) and subjected to daily trials of fear extinction until a minimun and stable freezing
behavior was reached. The graphs depict the freezing behavior during the retention test (a) and during the
extinction trials in mice treated with PBS IN, Cot IN, KO IN (b); PBS IN, Cot IN, Cot + KO (c); and PBS IN, Cot IN, oral
sertraline (d). Data was analyzed using one-way ANOVA and Tukey post hoc test. ns nonsignificant change; *p <
0.05; **p < 0.01.

Fig. 3 Effect of cotinine and krill oil on depressive-

like behavior in the forced swim tests. Two hours
after fear conditioning (FC), mice (n = 5–8
mice/group) received oral sertraline (2 mg/day),
intranasal (IN) krill oil (KO), IN cotinine (Cot) (24 μl, 10
Fig. 1 Diagram representing the experimental design. Male mice (n = 5–8/condition) were housed and habituated to
mg/ml), or IN Cot plus KO and subjected to fear
their cages before FC. After this period, mice were fear conditioned, treated, behaviorally tested for fear retention and
extinction. The graphs depict the effect of treatments
extinction, and depressive-like behavior, and euthanized. Calcineurin analysis was then performed in hippocampal
on freezing behavior as measure of depressive-like
extracts of mice by Western blot.
behavior. Data was analyzed using one-way ANOVA.
Drugs: Cotinine (Sigma-Aldrich), Sertraline hydrochloride (Sigma-Aldrich), Krill oil (KO) (Walgreens, Ns, non-significant change; *p < 0.05; **p < 0.01;
Superba USA). ***p < 0.001
Animals: Mice C57BL/6 were obtained from the University of Chile (Santiago, Chile) and maintained on a
12-h light-dark cycle with ad libitum access to food and water. Protocols were performed with the
approval of the institutional animal care and use committees of the University of San Sebastián, Chile.
Experimental Design: This study investigated the effect of intranasal cotinine alone or plus KO, krill oil,
and oral sertraline on depressive-like behavior, fear consolidation, and extinction as well as the expression
of CaA in the hippocampus of mice.
Drug Treatments: Mice received daily treatments with (1) PBS (phosphate-buffered saline, pH 7.4) via
intranasal; (2) cotinine (10 mg/ml) dissolved in PBS via intranasal (IN Cot, 24 μl); (3) krill oil dissolved in
PBS, via intranasal (48 mg/ml, 24 μl); (4) cotinine (10 mg/ml) plus KO dissolved in PBS, (48 mg/ml, 24 μl)
via intranasal; and (5) sertraline, via oral in PBS (3 mg/kg, 50 μl). The dose of sertraline was chosen to be
equivalent to a 200 mg/day in humans.
Intranasal Delivery: Mice were hand-restrained, placed in a supine position, and given two 12 μl drops of
cotinine solutions, or PBS, into both nares consecutively. Mice were given an extra 12 μl treatment drop if
the subject forcibly ejected or sneezed out the solution. Mice were held supine for 5–10 s after delivery to
ensure that all fluid was inhaled. Fig. 4 Cotinine increased calcineurin A in the conditioned mice after fear extinction. The hippocampal expression
Behavioral Analysis: Fear Conditioning, Fear Retention and Extinction Test. All tests carried out with ANY- of calcineurin A (CaA) was analyzed by Western blot in the mice after fear conditioning (FC) and extinction (FE).
maze video tracking system. The graphs represent the expression of CaA in the hippocampus of control non-exposed to stress (NS) and
Western Blot Analysis: Brain extracts were separated by PAGE-SDS 4-20% and transfered to nitrocellulose conditioned (FC) mice treated with PBS (vehicle) (a) and NS conditioned mice treated with PBS, plus cotinine (Cot)
membranes and probed with a rabbit CaA antibody according our standard protocols. (b). ns non-significant change; *p < 0.01; **p < 0.05.

Working Hypothesis Conclusions

• IN cotinine and cotinine plus KO, and sertraline but not KO reduced depressive-like behavior.
• IN KO delayed fear memory extinction.
• IN cotinine alone or plus krill oil inhibited fear consolidation and facilitated fear extinction.
• IN cotinine increased hippocampal calcineurin A levels in the fear conditioned mice.

Acknowledges
The authors were supported by the Grant Fondecyt 1150194, and the University San Sebastián.

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