Post-traumatic stress disorder (PTSD) is a neuropsychological disorder caused by exposure to traumatic events and is characterized by re-experiencing, avoidance, hyperarousal, and cognitive disturbances (Gou et al., 2023; Zhao et al., 2022) The pathophysiology of PTSD includes abnormalities in the hypothalamic-pituitary-adrenal (HPA)axis and monoaminergic system (Prajapati and Krishnamurthy, 2020). Typically, psychotherapy and selective serotonin reuptake inhibitors, such as sertraline and paroxetine, are prescribed for treatment (Frommberger et al., 2004). However, these drugs are only useful in approximately 30 % of patients with PTSD, whereas others do not respond to therapy due to the complexity of the pathological mechanism (Biltz et al., 2022; Garakani et al., 2020). Therefore, there is a need to identify novel pathological mechanisms to develop better treatment strategies for PTSD.

HPA axis dysfunction and monoaminergic deregulation are the primary causes of PTSD. However, several growing preclinical evidence suggested that mitochondrial dysfunction is crucial for PTSD (Garabadu et al., 2015). Since, the intracellular cholesterol reserve of steroidogenic cells controls the production of cortisol hormones, it is plausible that alterations in cortisol concentration linked to PTSD are connected to steroidogenesis inside mitochondria (Dmytriv et al., 2023). It is suggested that mitochondria regulate HPA axis functioning during chronic stress inferring the cross-talk between mitochondria and the HPA axis (Dmytriv et al., 2023). Earlier, it was demonstrated that mitochondrial dysfunction in the amygdala aggravates PTSD-like symptoms. Garabadu et al. demonstrated that improving mitochondrial function mitigates HPA-axis dysfunction and PTSD-like symptoms PTSD (Garabadu et al., 2015). During traumatic stress, there is a dysregulation of energy homeostasis which is regulated by mitochondrial dynamics through fission and fusion. Altered mitochondrial dynamics in the brain may cause mitochondrial dysfunction either through disturbance of energy regulation or by increasing oxidative stress (Perez-Ternero et al., 2017). However, there is limited information on the alterations in mitochondrial dynamics in experimental models of PTSD. Therefore, evaluating mitochondrial dynamics in an animal model of PTSD would provide in-depth knowledge of the pathophysiology of PTSD.

Mitochondrial dynamics are essential for cellular energy production and survival, and their disparity is a key mechanism in the development of neurological disorders (de la Monte and Wands, 2006). Mitochondrial dynamics involve fission and fusion processes, which are regulated by different proteins, such as dynamin-related protein-1 (Drp-1), mitochondrial fission protein-1 (Fis-1), optic atrophy-1 (Opa-1), and mitofusin-1 and -2 (Mfn-1 andMfn-2, respectively) (Flippo and Strack, 2017). Drp-1 and Fis-1 mediate mitochondrial fission, whereas Mfn-2 and Opa-1 control fusion (Chen and Chan, 2009; Zemirli et al., 2018). Dysfunction of mitochondrial dynamics directly impairs mitochondrial morphology, which plays a crucial role in maintaining healthy mitochondria (Bartolomé et al., 2017). Hence, evaluating mitochondrial dynamics could be a valuable strategy for regulating mitochondrial homeostasis in an experimental model of PTSD. Mitochondrial dynamics are directly regulated by the mammalian target of the rapamycin (mTOR) pathway (Morita et al., 2017). This mechanism underlies the phosphorylation of mitochondrial fission process-1 (MTFP-1), a downstream substrate that activates the mitochondrial fission protein Drp-1 (Morita et al., 2017). Recent studies have focused on the involvement of mTOR in the regulation of PTSD-related behavior (Gou et al., 2023; Zhao et al., 2022). During stressful situations, the mTOR pathway is mainly regulated by the orexinergic system (Wang et al., 2014). Orexin-A also regulates mitochondrial function and dynamics (Lassiter et al., 2015). We have previously reported the anti-PTSD-like effect of suvorexant, a non-selective orexin antagonist. Suvorexant attenuated the dysregulation of the HPA-axis and serotonergic abnormalities in a stress-re-stress (SRS) model of PTSD (Prajapati and Krishnamurthy, 2020).

However, the effect of suvorexant on mitochondrial dynamics and function in PTSD has not yet been investigated. Therefore, it would be interesting to determine whether the pharmacological antagonism of orexin affects mitochondrial dynamics using the SRS model of PTSD. It is crucial to use animal models that exhibit at least the key symptoms associated with human illness to develop novel treatment approaches. Generally, a set of criteria, including face, construct, and predictive validities, are used to evaluate the validity of animal models (Belzung and Lemoine, 2011). The rationality of using the SRS model of PTSD is because previously we have shown the validities of the SRS model in which symptoms last for more than a month following re-stress (Prajapati et al., 2020). According to clinical research, long-term therapy is necessary to recover from PTSD (Davis et al. 2006). As a result, the SRS paradigm has been modified to accommodate for the prolonged duration (>30 days) of PTSD-like symptoms. Further, the SRS model shows hypocortisolism as a clinical condition. The animals in this paradigm undergo three different types of varied stressors in quick succession: 2 hours of restraint, brief mild foot shock, and anaesthesia caused by halothane. A brief stress reminder caused rats to exhibit stable anxiety states and hormone anomalies resembling those seen in PTSD patients (Yehuda and Antelman, 1993).

In this study, the effects of suvorexant and rapamycin on PTSD-like phenotypes such as anxiety-like behavior, cognitive performance and fear response were measured using the elevated-plus-maze (EPM), Y-maze, and contextual-fear response tests respectively. Further, their effect on the HPA axis was measured by evaluating the plasma corticosterone levels. This study further investigates the role of mitochondrial dynamics in a SRS model of PTSD by assessing the mRNA and protein levels of fission (Drp-1 and Fis-1) and fusion (Mfn-2 and Opa-1) markers in the amygdala. Immunofluorescence assays were performed to confirm the mitochondrial fission and fusion marker localization in terms of the fluorescence intensity ratio in the amygdala. Transmission electron microscopy (TEM) analysis was performed to assess mitochondrial morphology. mTOR and MTFP-1 expression, along with plasma and cerebrospinal fluid (CSF) orexin-A levels, were measured to determine the relationship between the orexinergic system and mitochondrial dynamics in PTSD.