This study aimed to compare chronic and acute stress and investigate the effects of stress and noradrenergic activation on the orexin and glucocorticoid receptor systems in the rat hippocampus. The study examined plasma CORT levels, prepro-OX expression, OXr1 expression, GR expression, and hippocampal neuronal populations. The key outcomes of the study are as follows: 1. The study revealed that both chronic and acute stress elevated corticosterone levels and reduced hippocampal CA1 neuronal count. However, chronic stress had a more pronounced effect on the expression of stress-related genes, including prepro-orexin, orexin receptor 1, and glucocorticoid receptor. 2. Chronic stress and noradrenergic activation profoundly elevated plasma CORT level and also prepro-OX expression, each compared to its respective controls. However, chronic stress combined with noradrenergic activation reduced prepro-OX expression compared to chronic stress alone. Rats subjected to chronic stress or yohimbine administration or their combination exhibited considerably higher hippocampal OXr1 expression. Yet, the combination of chronic stress and noradrenergic activation had notably lower OXr1 expression relative to the chronic stress group. Chronic stress substantially increased hippocampal glucocorticoid receptor expression compared to controls. In contrast, the combined chronic stress and noradrenergic activation condition exhibited significantly lower expression compared to chronic stress alone. Chronic stress and their combination of chronic stress and noradrenergic activation meaningfully reduced hippocampal neuronal populations compared to controls. However, chronic stress combined with noradrenergic activation increased neuronal numbers compared to chronic stress alone.
The release of glucocorticoids, such as CORT in rodents, plays a crucial role in the HPA axis and serves as a biomarker of stress response (Tsigos et al., 2000; Bekhbat et al., 2018). The HPA axis is a vital component of the stress system, and glucocorticoids play a fundamental role in maintaining both resting and stress-related homeostasis (Nicolaides et al., 2015).
The study showed that both chronic and acute stress exposure activated the HPA axis, as indicated by the elevation of plasma CORT levels in response to chronic and acute stress (Sapolsky et al., 2000; Mokhtarpour et al., 2016), along with a decrease in neuronal population in the CA1 region of the hippocampus. However, chronic stress had a more profound impact on the expression of stress-related genes, such as prepro-OX, OXr1, and GR, compared to acute stress. Specifically, chronic stress exposure resulted in a significantly greater fold-change increase in the expression of these genes compared to both the control group and the acute stress group. Given that chronic stress had a more pronounced effect on the expression of stress-related genes involved in neuroendocrine and neuronal functions, it appears to be a more suitable model for further investigating the activation of the noradrenergic system by yohimbine. The chronic stress model seems to recapitulate better the molecular and cellular changes associated with stress-related disorders, making it a more relevant choice for studying the mechanisms underlying noradrenergic system dysregulation.
The study on stress and noradrenergic activation demonstrated that chronic stress and noradrenergic activation via yohimbine all increased plasma CORT compared to controls, implicating activation of the HPA axis stress response (Sapolsky et al., 2000). The stress- induced CORT build- up is a well-known effect which has been confirmed by many studies (Bhatnagar et al., 2006; Marin et al., 2007; Rabasa et al., 2011; Sheng et al., 2020), as well as ours (Mokhtarpour et al., 2016; Eghtesad et al., 2022). The study found that yohimbine mimics sympathetic activation by increasing norepinephrine, which is the first line of stress response. The data demonstrated an increase in CORT levels in the control animals injected with yohimbine, confirming this effect. This aligns with evidence that noradrenaline potentiates HPA axis signalling (Herman et al., 2003).
The prepro-OX precursor protein is a 130 amino acid pre-pro-peptide encoded by the gene HRCT and located on chromosome 17 (17q21). Orexin-A and orexin-B are excitatory neuropeptides produced by cleavage of the prepro-OX protein (Sakurai, 2014). The study found that hippocampal prepro-OX expression was enhanced by chronic stress and yohimbine alone or in combination with chronic stress, similar to the effect of chronic stress (Eghtesad et al., 2022) and acute stress (Mokhtarpour et al., 2016) shown in previous studies focused on the lateral hypothalamus. This is consistent with the involvement of orexin signalling in stress pathways (Heydendael et al., 2012; Sokolowska et al., 2014). The enhanced expression of prepro-OX in the hippocampus may reflect activation of the HPA axis influencing orexinergic activity (Zhao et al., 2021) or even a direct impact of stress on the likely pathologic expression of prepro-OX in the hippocampus. Although this condition might happen as an adaptation compensation to pathologic changes in the LH orexinergic system, it may also aggravate the situation in the hippocampus. Yohimbine infusion could only partially mimic previous findings suggesting noradrenergic modulation of prepro-OX (Sakurai, 2014). The reversal of prepro-OX expression levels observed with the combination of chronic stress and yohimbine administration suggests the existence of complex regulatory mechanisms between neurotransmitters and neuropeptides. It is hypothesized that prepro-OX expression may be reduced following yohimbine-induced norepinephrine increase in stressed animals. However, yohimbine alone emulates the stress effect on the expression.
OXr1 is a G protein-coupled receptor that is widely expressed throughout the brain, including the hippocampus. The activation of OXr1 has been shown to induce long-term structural and functional changes in the hippocampus (Song et al., 2015; Elahdadi Salmani et al., 2022). OXr1 expression increased in all stress-exposed and yohimbine groups, fitting with orexin's role in stress (Heydendael et al., 2012). This likely enhances hippocampal sensitivity to heighten arousal and vigilance during stress (Sakurai, 2014). The study found that the chronic stress and yohimbine group showed lower OXr1 expression compared to chronic stress alone, suggesting a compensatory mechanism preventing excessive signalling and an interplay of the signal transduction pathways for norepinephrine and orexin. In accordance, a selective OXr1 antagonist has been shown to attenuate stress-induced hyperarousal without hypnotic effects, indicating the potential of OXr1 as a therapeutic target for stress-related disorders (Grafe & Bhatnagar, 2018). Therefore, blocking the OXr1 by an antagonist or its downregulation using an internal agent (adrenergic system) may help retaliate the increased receptor expression.
The GR is a nuclear receptor that functions as a ligand-activated transcription factor mediating the diverse physiological effects of glucocorticoids, such as CORT, in the hippocampus (Timmermans et al., 2019). Chronic stress, similar to acute stress, increased hippocampal GR expression, aligning with the established function of this receptor in negative feedback regulation of the HPA axis (de Kloet et al., 2005). In contrast, the combination of chronic stress and yohimbine administration significantly reduced GR expression. This implicates compensatory adaptation to mitigate potential GR overstimulation during prolonged stress exposure (Reul & de Kloet, 1985; George et al., 2013). The observed effect in the study may be attributed to the expressions of prepro-OX and OXr1 following yohimbine infusion, as well as the interaction between norepinephrine and orexin signalling pathways. However, the yohimbine-induced norepinephrine increase, which imitates mild stress, may have only employed mineralocorticoid receptors and not GRs in such a condition.
Stress has been shown to have a significant impact on the structure and function of the hippocampus (McEwen et al., 2016). Chronic stress exposure has been found to cause shrinkage of dendrites of hippocampal CA3 and dentate gyrus neurons, as well as loss of spines in CA1, which may contribute to the cognitive deficits observed in stress-related disorders (Kim et al., 2015; McEwen et al., 2016). As expected, chronic and acute stress reduced the hippocampal neuron population, consistent with stress-induced structural alterations in this region (McEwen & Seeman, 1999; Chenani et al., 2022). Intriguingly, the chronic stress and yohimbine group showed greater neuronal populations versus chronic stress alone, suggesting a neuroprotective effect of noradrenergic signalling (Arnsten & Li, 2005). The increased expression of orexin receptor type-2 (OXr2) observed in a previous study (unpublished data) following electric shock acute stress may be responsible for the effect observed in this study, indicating a neuroprotective mechanism. Future studies may need to use a different stress model for the acute condition to understand better the interplay of stress and the noradrenergic system in the hippocampus.
Overall, chronic stress, as the selected stress model, elicited an increase in HPA activity, orexin and glucocorticoid expression profiles, and a reduction of structural neuronal changes aligned with prior research. Unique findings emerged when examining interactions between chronic stress and noradrenergic stimulation. The combination revealed potential compensatory mechanisms not seen with either factor alone, including modulation of prepro-OX levels, orexin receptor expression, glucocorticoid receptor expression, and hippocampal neuronal viability.