Activation of orexin neurons in dorsomedial/perifornical hypothalamus and antidepressant reversal in a rodent model of depression

Abstract

Chronic stressful life events are risk factors for depression often accompanied by homeostatic disturbances. Hypothalamic neuropeptides, such as orexins (OXs) and melanin-concentrating hormone (MCH), are involved in regulation of several autonomic functions that are altered in depression. However, little is known about the link between orexinergic or MCH-ergic systems and depression. Using double immunohistochemical labeling for OX- or MCH-containing neurons and Fos protein, we studied the effects of a chronic selective serotonin reuptake inhibitor antidepressant treatment (fluoxetine) on the OX and MCH neuronal activation in mice exposed to unpredictable chronic mild stress (UCMS), a rodent model of depression. Western blot was also performed to assess OX and MCH receptor expression in various brain areas. Finally, almorexant, a dual OX receptor antagonist, was assessed in the tail suspension test. UCMS induced physical and behavioral disturbances in mice reversed by 6-week fluoxetine treatment. Orexinergic neurons were more activated in the dorsomedial and perifornical hypothalamic area (DMH-PFA) of UCMS-subjected mice compared to the lateral hypothalamus (LH), and this increase was reversed by 6-week fluoxetine treatment. UCMS also reduced expression of OX-receptor 2 in the thalamus and hypothalamus, but not in animals chronically treated with fluoxetine. MCH neurons were neither affected by UCMS nor by antidepressant treatment, while UCMS modulated MCH receptor 1 expression in thalamus and hippocampus. Finally, chronic but not acute administration of almorexant, induced antidepressant-like effect in the tail suspension test. These data suggest that OX neurons in the DMH-PFA and MCH-ergic system may contribute to the pathophysiology of depressive disorders.

Introduction

Major depressive disorder (MDD) is characterized by different behavioral and neurobiological features, including mood disturbances, anhedonia, sleep abnormalities, significant weight changes, dysregulation of hypothalamic–pituitary–adrenal (HPA) axis and alteration of serotonin (5-HT) neurotransmission (Drevets et al., 2008). Interestingly, several studies have demonstrated that hypothalamic neuropeptides, such as orexins (OXs) (also known as hypocretins) and melanin-concentrating hormone (MCH), are involved in the regulation of homeostatic and autonomic functions such as energy balance, sleep–wake cycle, food/drug reward and emotions (Pissios et al., 2006, Mieda and Sakurai, 2009).

Neurons expressing orexin A (OX-A) and orexin B (OX-B) (de Lecea et al., 1998, Sakurai et al., 1998) are located in the posterior hypothalamus and send projections broadly all over the central nervous system (Peyron et al., 1998). OXs act through two receptors (OXR1 and OXR2) differentially distributed throughout the brain, especially in cortical regions, hippocampus, thalamic, hypothalamic and brain stem nuclei (Trivedi et al., 1998, Marcus et al., 2001). OXR1 selectively binds OX-A, whereas OXR2 is nonselective for both OXs (Sakurai et al., 1998). The OX-ergic system is well-known to promote behavioral arousal, and extracellular measurement of OX-A levels in the rat hypothalamus indicates a circadian fluctuation with an increase and a decrease of OX-A levels during active and rest phase respectively (Yoshida et al., 2001). In rodents, central administration of OX increases food intake (Sakurai et al., 1998), locomotor activity (Nakamura et al., 2000), and induces wakefulness (Hagan et al., 1999). Activation of OX neurons is also associated with consummatory rewards such as food, morphine and cocaine (Harris et al., 2005). In addition, OXs seem to regulate stress response since intracerebroventricular (i.c.v.) injection of OX-A increases HPA axis activity (Kuru et al., 2000, Al-Barazanji et al., 2001).

Recent studies underline the differential role of two putative sub-populations of OX neurons (Harris and Aston-Jones, 2006). OX-expressing neurons in the lateral hypothalamus (LH) seem to be involved in reward-related behaviors (Fadel et al., 2002, Harris et al., 2005, Harris et al., 2007), whereas those in the dorsomedial and perifornical hypothalamic area (DMH-PFA) seem to be involved in sleep/wake regulation and stress (Estabrooke et al., 2001, Sakamoto et al., 2004, Winsky-Sommerer et al., 2004).

MCH-containing neurons, intermingled with OX-expressing cells, have large projections throughout the brain (Adamantidis and de Lecea, 2008), and regulate a number of autonomic functions. Central administration of MCH also increases food intake (Qu et al., 1996) and enhances cocaine-induced hyperactivity (Chung et al., 2009). Furthermore, the administration of MCH into the rat paraventricular nucleus of the hypothalamus (PVN) increases plasmatic adrenocorticotropic hormone (ACTH) and corticosterone levels (Kennedy et al., 2003). Finally, it has been demonstrated that MCH neurons also play a role in arousal in a reciprocal manner to the OX-ergic system, with an increase of cell firing during REM sleep (Hassani et al., 2009).

Although alterations in MDD concern homeostatic and autonomic functions that are modulated by OXs and MCH, little is known about the link between these hypothalamic peptides and mood disorders. The involvement of OXs and MCH in the pathophysiology of depression was recently highlighted by several studies. Acute and chronic administration of MCH receptor 1 (MCHR1) antagonist (SNAP 94847) has an antidepressant-like effect in mice (David et al., 2007), and chronic mild stress induces an increase of hippocampal gene expression of MCHR1 in mice, reversed by chronic fluoxetine treatment (Roy et al., 2007). Moreover, some preclinical and clinical data suggests a reduction of number and size of OX neurons in a genetic animal model of depression and a low cerebrospinal fluid (CSF) level of OX-A in MDD patients (Allard et al., 2004, Brundin et al., 2007). However, the opposite was found in other studies, with increase of OX-A and OX-B expression in the hypothalamus in an animal model of depression, and a trend to a higher CSF level of OX-A in depressed patients (Salomon et al., 2003, Feng et al., 2008). Considering all these results, the putative involvement of OX-ergic and MCH-ergic system in the depressive-like state is still unclear.

We therefore undertook further studies to establish a role of OX and MCH neurons in an appropriate animal model of depression. The unpredictable chronic mild stress (UCMS) is particularly useful to investigate the neural mechanisms of MDD. This animal model of depression, consisting of chronic exposure to various social and environmental stressors of low intensity, presents a high predictive, face and construct validity (Surget and Belzung, 2008).

The objective of this study is to explore the neuronal activation, using Fos protein expression, in LH and DMH-PFA OX-ergic neurons during the depressive-like state of mice, with or without chronic selective serotonin reuptake inhibitor (SSRI, fluoxetine) antidepressant treatment. MCH-ergic neuronal activation as well as OX-receptors 1 and 2 and MCH-receptor 1 expression in various brain areas were also assessed. Finally, the effect of acute or chronic administration of dual OX receptor antagonist almorexant (ACT-078573, Brisbare-Roch et al., 2007) was investigated in the tail suspension test, a widely used paradigm for assessing antidepressant-like effect in mice.