Intranasal cotinine improves memory, and reduces depressive-like behavior, and GFAP + cells loss induced by restraint stress in mice

Highlights

• Intranasal cotinine restored mood equilibrium after restraint stress in mice.

• Intranasal cotinine recovered recognition memory after restraint stress in mice.

• Intranasal cotinine recovered astrocytes morphology after restraint stress in mice.

Abstract

Posttraumatic stress disorder (PTSD), chronic psychological stress, and major depressive disorder have been found to be associated with a significant decrease in glial fibrillary acidic protein (GFAP) immunoreactivity in the hippocampus of rodents. Cotinine is an alkaloid that prevents memory impairment, depressive-like behavior and synaptic loss when co-administered during restraint stress, a model of PTSD and stress-induced depression, in mice. Here, we investigated the effects of post-treatment with intranasal cotinine on depressive- and anxiety-like behaviors, visual recognition memory as well as the number and morphology of GFAP + immunoreactive cells, in the hippocampus and frontal cortex of mice subjected to prolonged restraint stress. The results revealed that in addition to the mood and cognitive impairments, restraint stress induced a significant decrease in the number and arborization of GFAP + cells in the brain of mice. Intranasal cotinine prevented these stress-derived symptoms and the morphological abnormalities GFAP + cells in both of these brain regions which are critical to resilience to stress. The significance of these findings for the therapy of PTSD and depression is discussed.

Introduction

Although the effects of acute stress are usually brief and can be overcome quickly, exposure to chronic or extreme forms of unescapable stress can lead to neurochemical, morphological and functional changes in the brain that have been associated with posttraumatic stress disorder (PTSD) (North et al., 2016) and major depressive disorder (MDD) (Nestler et al., 2002). These stress-induced psychiatric diseases are characterized by symptoms such as intrusive distressing thoughts, anhedonia, irritability (McHugh et al., 2012), feelings of guilt, sleep disorders (Williams et al., 2015), cognitive impairment (Jak et al., 2016), anxiety (Wilder Schaaf et al., 2013) and sometimes treatment-resistant depression (Stander et al., 2014). Furthermore, these conditions have been associated with functional and structural changes in several regions of the brain including the amygdala (Laugharne et al., 2016), entorhinal cortex, prefrontal cortex and hippocampus (Meng et al., 2016, Sheynin and Liberzon, 2016, Yoon et al., 2017, Zhu et al., 2016). At cellular level, PTSD and depression are also associated with a decrease of glial fibrillary acidic protein (GFAP) immunoreactive cells (GFAP⁺) in the brain (Saur et al., 2016; Nestler et al., 2002; Fuller et al., 2010). GFAP is a family of proteins that includes eight isoforms expressed by different subpopulations of astrocytes as well as immature brain cells. These isoforms include GFAP^+ 1, GFAP delta and GFAP kappa. GFAP delta appears to be linked with neural stem cells (NSCs) and may be involved in migration.

Furthermore, the expression of GFAP has been reported to decrease in response to microgravity (Day et al., 1998). Another predominantly-astroglial enzyme, glutamine synthase, has been reduced in the frontal cortex following intraventricular injection of aluminum (Guo-Ross et al., 1999), which paralleled alterations in GFAP expression. These results suggest an impairment of astrocytic responsivity in frontal cortex following toxic insults. Stress-induced depression is associated with a reduction of neurogenesis, neuroinflammation and a decrease of astrocytes in the brain (Sanacora and Banasr, 2013), and reduced GFAP expression has been found associated with schizophrenia, bipolar disorder and depression (Cobb et al., 2016, Webster et al., 2005). In addition, dysfunction of GFAP expression has been reported in encephalopathy (Kretzschmar et al., 1985). Astrocytes have been implicated in brain and neuronal functions supporting learning and memory and emotional responses (Dienel, 2017, Gibbs et al., 2006, Lee et al., 2014). Astrocytes have been identified as playing a significant role in maintaining brain homeostasis and supporting neuronal function (Weinstein et al., 1991). Coherent with these roles, it has been found a dysfunction of astroglia in the hippocampus of subjects with depression (Cobb et al., 2016) and other psychiatric and neurological conditions (Saur et al., 2016).

Because astrocytes support neuronal function and neurogenesis, it is thought that a decrease in astrocytic function is a critical factor underlying maladaptive responses in individuals with posttraumatic stress disorder (PTSD). Interestingly, treatment with antidepressants such as fluoxetine improved mood and induced a restoration of astroglia, suggesting that a restoration of astrocytes function is associated with changes in behavior.

An excellent model to investigate PTSD-induced, treatment-resistant depression (TRD) is the prolonged restraint stress paradigm (Perrine et al., 2016). Using this model, it has been shown that chronic stress induces cognitive deficits and depressive-like behavior, and morphological changes such as a decrease in the length and number of dendrites and synapses in neurons of the CA3 region of the hippocampus (Watanabe et al., 1992). Indeed, it has been shown that a two-hour immobilization stress combined with forced swim stress induced a decrease in the number of astrocytes in the hippocampus of rats (Imbe et al., 2012). Chronic restraint stress (6 h/day for 3 weeks), but not short-term restraint stress (6 h/day for 3 days), caused mechanical hypersensitivity and aggressive behavior. The chronic restraint stress induced a significant decrease of GFAP protein levels in the ventrolateral periaqueductal gray (Imbe et al., 2012). This decrease in astrocyte immunoreactivity (IR) was accompanied by a parallel decrease in glutamate transporter EAAT2 expression. The EAAT2 protein levels in the 3 week-stress group was significantly lower (− 20%) than in the control group. In contrast, there was no significant differences in the GFAP and EAAT2 protein levels between the three-day stress groups control in the periaqueductal gray matter (Imbe et al., 2012). It is thought that a deficit in glutamate transport after chronic stress may trigger neuronal dysfunction due to excitotoxicity in the brain.

Cotinine, a positive modulator of the α7 nicotinic acetylcholine receptors (nAChRs) (Echeverria et al., 2016b), has shown to decrease anxiety and to improve the extinction of fear in rodents subjected to fear conditioning a model of PTSD (de Aguiar et al., 2013, Zeitlin et al., 2012). Furthermore, continue treatment with oral cotinine prevented working memory loss and depressive-like behavior, as well as increased synaptic density in the hippocampus and frontal cortex of mice subjected to chronic immobilization stress (Grizzell et al., 2014a, Grizzell et al., 2014b). Cotinine has a long plasma half-life (19 to 24 h) and shows minor side effects in humans (Grizzell and Echeverria, 2015). Cotinine also has anti-inflammatory properties, overriding the production of cytokines that are under transcriptional control of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) system (transforming neurotrophic factor alpha (TNF-α), Interleukin (IL)-1β, IL-6, IL-12, IL-23) (Rehani et al., 2008).

The targets of cotinine are the most abundant nicotinic receptors in the brain, the low affinity (α7) and high affinity (α4β2) nAChRs (Colquhoun and Patrick, 1997). The activation of α7 receptors at the synapse triggers an increase in permeability to Na⁺ and Ca^2 + ions (Pichon et al., 2004). These currents depolarize the cell and activate different neuronal signaling cascades (Exley and Cragg, 2008). Furthermore, Na⁺ and Ca^2 + currents depolarize the pre-synaptic membrane, inducing the release of neurotransmitters such as dopamine, serotonin, glutamate, and gamma-amino butyric acid (GABA) (d'Incamps and Ascher, 2014, Exley and Cragg, 2008). In this way, the stimulation of the α7 and α4β2 receptors in the prefrontal cortex can promote the activation of glutamatergic neurons that normally inhibit the activity of the amygdala after chronic stress (Bencherif et al., 2014, Broide and Leslie, 1999). Thus, by acting on these receptors, cotinine may alleviate TRD in patients with PTSD.

Intranasal (IN) administration of CNS drug is acquiring increasing attention because of therapeutic advantages in reducing time of action of drugs, decreasing systemic effects and preventing hepatic first-pass elimination (Hanson and Frey, 2007, Hanson and Frey, 2008). In this study, in the search of new delivery methods for cotinine, we tested the behavioral effects of IN cotinine on depressive-like behavior, anxiety and working visual recognition memory when administered after prolonged restraint stress. In addition, to understand the mechanism of action of IN cotinine, we also examined its effect on the levels of GFAP⁺ cells in the hippocampus and frontal cortex of mice subjected or not to restraint stress. The significance of these results for the treatment of PTSD and TRD is discussed.