Amygdalar orexinergic–GABAergic interactions regulate anxiety behaviors of the Syrian golden hamster

Abstract

At present neurobiological interests are directing more attention towards the major role of the amygdalar GABA_A receptor on orexin-dependent behaviors. This telencephalic region has been widely studied especially in view of its control on various psychiatric disorders such as anxiety and depression. Recently, cross-talking relationships between these two specific neuroreceptor systems of the central-cortical amygdalar complex has been considered an important element for anxiety type of behaviors. In the present study, we investigated the effects of central amygdalar infusions with orexin-A, orexin-B ± GABA_A receptor α₂ subunit agonist (flunitrazepam) on elevated plus-maze and light–dark explorative behaviors of the facultative hibernating Syrian hamster. In a first case, it seemed that doses of orexin administered directly into the central nucleus were responsible for greater anxiogenic type of effects as shown by more time being spent both in the dark compartment and the closed arm of the elevated plus-maze, whereas, these effects were suppressed in the presence of flunitrazepam. At the cellular level, the effects of orexin accounted for evident argyrophilic reactions (neurodegeneration phenomena) including altered cell membrane and loss of cytoplasmic architecture in most amygdalar and hippocampal neuronal fields, while in the presence of flunitrazepam these reactions resulted to either be unappreciable or absent. Overall the actions of α₂-dependent inhibitory signals tend to corroborate, for the first time, a neuroprotective role against the over-excitatory orexinergic neurodegeneration reactions and thus its abnormal anxiety-like indications may prove to be therapeutically useful for orexin-dependent sleeping disorders.

Introduction

The family of orexin (ORX) neuropeptides also known as hypocretins consists mainly of two subtypes recognized as Orexin-A and -B (ORX-A and ORX-B), which derive from a common precursor prepro-orexin following post-translational proteolytic cleavage [51]. Studies have largely shown that ORX producing neurons are localized in the lateral hypothalamic area (LHA) and in posterior portions of the hypothalamus [40]. Both neuropeptides were first identified as novel peptide ligands of two orphan G protein-coupled receptors and namely ORX1 receptor (ORX1R) and ORX2 receptor (ORX2R). In the case of the former subtype it displays a greater affinity for ORX-A (almost 50 times greater than B) while ORX2R shows a comparable affinity for both neuropeptides [51]. At the brain level such ORX producing neurons widely project to various limbic regions among which the cerebral cortex, olfactory bulb, hypothamamic areas, hippocampus (HIP) and amygdala (AMY) [40]. The activities of these two orphan G protein-coupled receptors appear to require the participation of several other neuronal receptor systems such as serotonin [34], noradrenaline [23], histamine [15], corticotrophin-releasing hormone (CRH) [55] and above all GABA_A receptor (GABA_AR)/glutamate subtypes [1], [38]. As far as the major neuroinhibitory GABA_AR complex is concerned, the benzodiazepines, which are a highly noted family of anxiolytics are capable of mediating their sedative, amnesic and anxiolytic actions through the binding of the α subunits of this receptor complex [28], [29]. In particular, α₂/α₃ appear to be the major subunits involved with the regulation of anxiety-like behaviors [46]. In addition, it appears that the inhibitory GABAergic effects are mostly responsible for the blocking of AMY ORXergic fibers and consequently a decrease of anxiety state [26]. Consistent with this, reduced central GABAergic activity has been reported in subjects with panic disorder, and drugs like α₂/α₃ GABAergic subunit agonist that are involved with the restoration of inhibition responses have proven to be clinically effective for similar disorders. As a matter of fact, acute disruption of GABAergic signals in panic-generating brain sites such as the dorsomedial–perifornical hypothalamus, the AMY or the dorsal periaqueductal gray in rats, leads to panic-like behavior and increased cardiovascular responses [45].

Recent behavioral studies have displayed that stressful conditions tend to alter ORXergic-dependent awakening processes [2], [26]. Indeed, this specific neuropeptide is known to control some of the major circadian rhythms including feeding and sleep-wake cycle underlining that ORXergic fibers are widely associated with the maintenance of homeostasis [4], [33], [48]. Interestingly, ORXergic neurons have shown to be very sensitive as exhibited by their ability to activate arousal promoting transmitters, including the corticotrophin releasing factor (CRF), a peptide involved in stress responses [59]. The increased activity of ORX seems to cause a greater frequency of awakening state that could contribute to several neuropsychological functions such as vulnerability drug seeking events [12]. Consequently, the blocking of ORX1 attenuates stress-induced reinstatement of extinguished cocaine [7] and alcohol [47] seeking stimuli. ORX-synthesizing neurons have also been shown to be involved in not only mobilizing sympathetic responses plus desensitizing parasympathetic mediated baroreflex stimuli, but also a simultaneous increases of blood pressure and heart rate, which are typical of panic attacks [26].

At the brain level, it seems that most of environmental-derived stimuli are responsible for the activation of distinct encephalic ORXergic fibers [52] very likely through the major limbic olfactory target and precisely AMY. This telencephalic region is composed of different anatomically interconnected amygdaloid nuclei that include basolateral (BLA), cortico-medial and centromedial nuclear groups, [44], [48] which are capable of influencing emotional and mnemonic functions [17], vary likely through extensive visceral (hypothalamus and olfactory lobes) and autonomic-somatomotor connections [50]. Even the central AMY nucleus (Ce) that is involved with aversive learning, escaping responses and mnemonic capabilities of stressful experiences [3], [32] has been identified as a critical site for the mediation of responses to anxiogenic stimuli or stress-related behaviors [41], [52] deriving from elevated-plus maze (EPM), shock probe burying, and social anxiety tests [53]. Curiously, numerous evidences have demonstrated that Ce is mostly innervated by ORX neurons as displayed by dense levels of ORX1R and ORX2R fibers throughout this AMY nucleus [60]. As a consequence of this feature, plus Ce not only being largely responsible for stressful type of behaviors via its interaction with BLA [49] but also of it being directly involved with learning, feeding and fear behaviors [31] constitutes such an AMY nucleus as an important limbic site for emotional events.

On the basis of these features it was our intention to evaluate the role of intracerebroventricular (i.c.v.) ORX-A infusions alone or together with α₂ subunit agonist flunitrazepam, in specific AMY sites on anxiety-like behaviors of a facultative hibernating rodent by using light–dark exploration (LDT) and EPM tests. These effects were compared to animals treated with ORX-B especially since this neuropeptide has recently shown to play a key role on synaptic plasticity [5], [54]. The results of predominating and distinct effects exerted by both neuropeptides may supply further insights regarding the role of olfactory communicating channels operating via AMY stations on plasticity events that are known to be gradually but constantly changing.