Cotinine counteracts the CS-induced morphological changes in

astrocytes and stimulates the activity of the JAK/STAT after chronic

stress in mice.
Perez-Urrutia Nelsonᵃ, Gonzalez-Rivera Mairaᵃ, Oliveros-Matus Patriciaᵃ, Alvarez-Ricartes Nathalieᵃ, Iarkov
Alexandreᵃ, Echeverria Valentinaᵃb
ᵃ Universidad San Sebastián, Facultad de Ciencias de la Salud, Lientur 1457, Concepción, Chile.
b Bay Pines VA Healthcare System, Research and Development, Bay Pines, FL 33744, USA

Abstract Results
Chronic stress (CS) is a public health problem worldwide, it affects all social and age
groups. Intense, repeated or uncontrolled stress has been implicated in the appearance Porsolt’s test Sucrose preference test
of multiple neuropsychiatric conditions. In the hippocampus and prefrontal cortex, CS
decreases the density of astrocytes and alters their morphology and function. This type CTRL + PBS
ns
of disorder, which also causes depression, is treated with selective serotonin reuptake CS + PBS

inhibitors. CS + Cot

In our previous study, we demonstrated that oral and intranasal Cotinine (Cot) CS

administration alleviated posttraumatic stress disorder (PTSD) symptoms via an Cot - - +

astrocyte-related and anti-inflammatory mechanism. Cotinine, a tobacco-derived p-STAT3

alkaloid, facilitates the extinction of fear, decreases depressive and anxiety behaviors, STAT3

improves memory and restores astrocytic density in the hippocampus and frontal cortex
in mice subjected to fear conditioning or with chronic stress. Here, we investigate the
Fig 1: Effect of cotinine on depressive like behavior after chronic restraint stress. p<0.05. Fig. 2 The effects of Cotinine on the regulation of JAK/STAT3
effect of cotinine on behavior, morphology of astrocytes and the activity of the JAK/STAT activity p < 0.05.
pathway in the hippocampus of C57BL/6 male mouse subjected to chronic stress.
A B
CTRL CS + PBS CS + COTININE GFAP+ cell density / field

Methods CTRL + PBS

CS + PBS

CS + Cot
ns
CA1

ns

Animals: Mice C57BL/6 were obtained from CREAV (Universidad de Concepción, Chile) *
CA3

and maintained on a 12-h light-dark cycle with ad libitum access to food and water.
Experimental Design: This study investigated the effect of oral cotinine on depressive-
like behavior, JAK/STAT pathway, and morphological changes.
Drug Treatments: Mice received daily treatments with (1, 2) oral PBS (phosphate-
buffered saline, pH 7.4); (3) oral cotinine (10 mg/ml) dissolved in PBS. ns
Behavioral Analysis: Open field, Sucrose test preference and Porsolt’s Test. All tests
carried out with ANY-maze video tracking system.
Western Blot Analysis: Brain extracts were separated by PAGE-SDS 4-20% and
GD

**
transfered to PVDF membranes and probed with a rabbit STAT3 and p-STAT3 antibody
according our standard protocols.
Immunohistochemistry: Standard protocol were performed with rabbit GFAP antibody
on sagittal brain cryosections of 30um.
Skeleton Analyze: was performed on binary images using ImageJ. Fig 3. Effect of cotinine on GFAP expression after chronic stress in hippocampus of mice. (A) The images from the left represent GFAP+ in control (CTRL), restraint
stress (RS) and treated with Cotinine (10 mg/ml) (RS + COT); (B) Graph depicting the changes in the density of GFAP+ cells.

A B
Working Hypothesis CTRL CS + PBS CS + COTININE

nAchR
Skeleton Analyze process

Astrocyte
JAK
Activity of the JAK/STAT pathway C
CA1 CA3 GD
STAT3

Density of GFAP+ Astrocytes CS-induced morphological

STAT3
changes CTRL + PBS
STAT3 ns
ns ns CS + PBS

CS + Cot
Depressive-like behaviour ns
*
**

CBP/p300

STAT3 Smad1
STAT3 Smad4

Fig 4. Representation of the image analyze on GFAP+ cells (a); Diagrams represent the skeletonized GFAP + cells (B); . (B) Graph depicting the changes in the
Endpoints of GFAP+ skeletonized cells.

Acknowledges Conclusions
The authors were supported by the Grant Fondecyt 1150194, and the University San • Co-treatment with oral cotinine reduced depressive-like behavior.
Sebastián. • STAT3 activity in the hippocampus of CS mice is stimulated by co-treatment with oral cotinine.
• Co-treatment with oral cotinine counteracts CS-induced morphological changes
• Co-treatment with oral cotinine protect the decreasing density of CS-induced GFAP+ cell in the hippocampus