Hippocampal neuronal nitric oxide synthase (nNOS) is regulated by nicotine and stress in female but not in male rats

Abstract

NO (nitric oxide) produced in limbic brain regions has important roles in the regulation of autonomic nervous system and HPA axis activity, anxiety, fear learning, long-term memory formation, and depression. NO is synthesized from l-arginine in a reaction catalyzed by nitric oxide synthase (NOS). Neuronal nitric oxide synthase (nNOS), one of the three isoforms of NOS, is synthesized constitutively in nerve cells. Increasing evidence indicates that nNOS expression in the nervous system may be regulated by stress and nicotinic receptors. Furthermore, data obtained from several studies suggest that signaling pathways induced by stress and nicotinic receptors may converge on various signal transduction molecules to regulate nNOS expression in brain. In the present study, we used Western Blot analysis to test the effect of forced swim stress, chronic nicotine administration, and the combined effect of both procedures on nNOS expression in the hippocampus, amygdala and frontal cortex of the male and female rat brain. Basal nNOS levels of the three brain regions examined did not show sex differences. However, forced swim stress and chronic nicotine administration increased nNOS expression in the hippocampus of female rats. When stress and nicotine were applied together, no additional increment was observed. Stress and nicotine did not regulate nNOS expression in the amygdala and the frontal cortex of either sex. Data obtained from the present study indicate that the regulation of stress and nicotine induced-nNOS expression in rat hippocampus shows sexual dimorphism and nNOS expression in the female rat hippocampus increases by nicotine and stress.

Introduction

Nitric oxide (NO) is a highly diffusible gas that functions as a signaling molecule in the central and peripheral nervous system. It has been implicated in the regulation of cognitive (Kanit et al., 2003, Koylu et al., 2005), emotional and behavioral processes such as long-term memory formation (Arancio et al., 1996), fear learning (Overeem et al., 2010), and anxiety (Spolidorio et al., 2007). NO also appears to mediate stress responses by regulating autonomic functions (Krukoff, 1999) and hypothalamo-pituitary–adrenal (HPA) axis activity (Rivier, 2001). In fact, recent studies suggest a role for NO in the pathophysiology of depression through the suppression of neurogenesis in the hippocampus (Packer et al., 2003, Zhou et al., 2007).

NO is synthesized from l-arginine in a reaction catalyzed by nitric oxide synthase (NOS). Three isoforms of NOS are expressed in the brain: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NOS. Nerve cells constitutively synthesize nNOS, which is activated by the binding of Ca²⁺/calmodulin complex (Roman and Masters, 2006). nNOS expressing neurons are widely distributed within the central nervous system including the limbic structures amygdala, hippocampus, and prefrontal cortex (Blackshaw et al., 2003, Kubota et al., 1994, McDonald et al., 1993). NO produced in these limbic regions has important roles in the regulation of autonomic nervous system (Resstel and Correa, 2006, Yao et al., 2007) and HPA axis activity (Seo and Rivier, 2001), anxiety (Forestiero et al., 2006, Spolidorio et al., 2007), fear learning (Overeem et al., 2010), and long-term memory formation (Arancio al., 1996).

Various stress procedures (novelty, restraint, osmotic stress) increase nNOS mRNA, nNOS immunoreactivity, and enzyme activity in the amygdala and hippocampus (de Oliveira et al., 2000, Echeverry et al., 2004, Krukoff and Khalili, 1997, Leza et al., 1998, Yao et al., 2007). Additionally, mild intermittent footshock stress and cytokine administration increase nitric oxide metabolites in the prefrontal cortex (Ishizuka et al., 2000, Ishizuka et al., 2008). Enhancement of nNOS synthesis and activity during the stress response will increase the production of NO, which regulates various neuroendocrine, cognitive, emotional, and behavioral processes in these limbic regions.

Studies report that nicotine administration regulates nNOS expression (Jang et al., 2002, Nakamura et al., 1998, Toborek et al., 2000, Yoo et al., 2005) and activity (Tonnessen et al., 2000) and NO release in the nervous system (Pogun et al., 2000, Smith et al., 1998). Nicotinic receptors are expressed at high levels in the hippocampus, amygdala, and frontal cortex (Tribollet et al., 2004). Additionally, nNOS and α7-nicotinic acetylcholine receptor subunits are co-expressed in the rat hippocampal interneurons (Adams and Freedman, 1997) and primate prefrontal cortex (Csillik et al., 1998). Regulation of NO production in these limbic regions through nicotinic receptors may affect learning and memory (Yilmaz et al., 2000) and HPA axis activity (Raber et al., 1995, Zhu et al., 2001).

Signaling pathways induced by the stress response and nicotinic receptors may converge on various signal transduction molecules to regulate nNOS expression in the brain. Studies report increased glutamatergic signaling in the frontal cortex, amygdala, and hippocampus during the stress response (Musazzi et al., 2010, Reznikov et al., 2007, Sunanda et al., 2000). Similarly, cholinergic signaling through nicotinic receptors enhances glutamate release and glutamatergic neurotransmission in these brain regions (Dani, 2001, Girod et al., 2000, Lambe et al., 2003). Glutamatergic signaling through NMDA, AMPA, and metabotropic receptors activate cAMP response element binding protein (CREB), a transcription factor that is reported to regulate nNOS gene transcription (Mao et al., 2004, Sasaki et al., 2000, Boulware et al., 2005). Additionally, stress also increases acetylcholine release and the expression of α-7 nicotinic receptor mRNA in hippocampus (Tajima et al., 1996, Hunter et al., 2010).

In view of the reported findings above, the aim of this study is to assess the effect of forced swim stress, chronic nicotine administration, and the combined effect of both procedures on nNOS expression in the hippocampus, amygdala and frontal cortex of the rat brain. Considering the sex differences observed in the regulation of the stress response (Solomon and Herman, 2009), central effects of nicotine (Pogun and Yararbas, 2009) and NO (Panzica et al., 2006, Pogun, 2001), sex was included as a factor.