According to previous studies, orexins and their receptor antagonists were found to affect signal transmission and action potential of retinal neurons, specifically in M1 ipRGCs, which are closely associated with the SCN (Zheng et al., 2015, Zhou et al., 2021). In current study, we simulated the depletion of retinal orexins and their receptors, as observed in Alzheimer's patients (Savaskan et al., 2004). We then investigated the effect of this depletion on the expression of retinal Bmal1, hypothalamic Bmal1, PACAP, and Vip, as well as the clock-controlled gene C-fos, in both the retina and hypothalamus.
Our findings demonstrated that the IVI injection of specific antagonists of OX1R and OX2R resulted in significant changes in the expression of the studied genes, with the exception of retinal Bmal1. However, the effects were often canceled out when the antagonists were injected together. The present study demonstrated the overall impact of orexin receptor antagonists on rhythmic retinal and hypothalamic genes, disregarding cytotype or nucleus. This is due to the limitations of available cell separation techniques, and the verification of this hypothesis is still in its preliminary stages. The experiments in this study was conducted between 8 am (ZT-0) and 24 hours (ZT-24). This time frame was chosen based on prior research that demonstrated fluctuations in retinal clock genes, namely Bmal1, Clock, Per1, and Per3, within light-dark (LD) conditions (Schneider et al., 2010). Most likely, the expression of retinal orexins decreases in this time period. It was speculated that retinal orexins are likely elevated during the dark phase of the light-dark cycle (Zheng et al., 2015). This hypothesis of retinal OXA rising at night and decreasing during the day was proven recently via LC-MS/MS analysis of the vitreous body in rats (Chrobok et al., 2021). Savaskan et al. interpreted the absence of OX2R from human retinal tissue (Savaskan et al., 2004). However, this absence may be attributed to the retinal samples taken from elderly individuals, as age-related reductions in OX2R mRNA levels were observed in mouse brains (Terao et al., 2002). Furthermore, it was reported that TCS OX2 29 has a significant physiological impact on RBCs and the transmission of signals in the retina of rats (Zhang et al., 2018).
The fos and jun proto-oncogenes are members of the Fos and Jun families of DNA-binding proteins. These genes, called cellular immediate-early genes (IEGs), are quickly and temporarily activated in response to various cellular stimuli, especially in specific neuronal populations (Hoffman et al., 1993). For example, among the stimuli that increases the expression of retinal C-fos is light. Therefore, it is widely used as a cellular activation marker (Yoshida et al., 1993). The high expression of C-fos in the retina, as observed in this study, can be attributed to the findings of a previous study. IVI injection of the dual orexin receptor antagonist, TCS1102, increased the size pupil in mice by attenuating the pupillary constriction and reducing the spiking rates of M2 ipRGCs. These cells project to the olivary pretectal nucleus, which is responsible for pupillary light response (Zhou et al., 2021).
The Vip-expressing neurons in the ventral core region of the SCN are rhythmic oscillatory neurons that play a vital role in coordinating circadian rhythms throughout the body. However, VIP neurons receive inputs from the retina, (Ono et al., 2021). In response to changes in illumination conditions, the retinal ipRGCs neurons release glutamate and sometimes PACAP from their terminals in the ventral core of the SCN. This leads to an increase in the firing rate of SCN neurons (Lindberg et al., 2019). Although the mechanism is not yet clear, VIP is thought to be involved in the response of SCN to light information and photoentrainment. Additionally, in lighting conditions, the activity of SCNVIP neurons, which are stimulated by light in vivo, and the release of VIP were found to increase depending on the time of day and excitatory input (Jones et al., 2018). Interestingly, the findings of the current study indicate that blocking retinal orexin receptors led to increased expression of Vip in the hypothalamus at ZT-6 in the SB334867 group, as well as at ZT-12 and ZT-24 time points in the JNJ-10397049 groups. In contrast, the SB334867 + JNJ-10397049 groups did not show any noticeable changes. Although we do not currently have a satisfactory explanation for the delay in the response of the JNJ-10397049 groups, it was found that OXB, in experiments conducted on retinal slice preparations, exhibited a suppressive effect on inhibitory postsynaptic currents mediated by GABA receptors in RBCs located in the inner plexiform layer. However, the administration JNJ-10397049 eliminated this excitatory effect (Zhang et al., 2018). The disappearance of the response in the SB334867 + JNJ-10397049 group could be attributed to the possibility that the inhibitory effect of JNJ-10397049 impacted certain neurons, while the stimulatory effect of SB334867 affected other retinal neurons, resulting in a cancellation of each other's effects (Qiao et al., 2017). However, in previous study, the sleep-promoting effects of the OX2R antagonist were significantly attenuated when administered together with the OX1R (Dugovic et al., 2009).
The changes in PACAP expression were essentially harmonious to those in Vip expression. This harmony between PACAP and Vip expression can be explained by the fact that the paraventricular (PVN) and supraoptic (SON) nuclei are the primary locations of PACAP neurons in the hypothalamus (Piggins et al., 1996), and these PVN-containing PACAP neurons are innervated by SCNVIP neurons (Teclemariam-Mesbah et al., 1997). In addition, a partial co-localized immunoreactivity of PACAP and VIP in the SON and PVN nuclei has been reported (Vereczki et al., 2003). It is evident from the results that the hypothalamic Vip, PACAP, and Bmal1 respond to the inhibition of retinal orexin receptors in a coordinated manner. In the SB334867 groups, almost all of these genes were up-regulated at ZT03 and ZT06, and down-regulated at ZT12 and ZT24 time points, and vice versa in the JNJ-10397049 groups. Bmal1 is expressed in various hypothalamic nuclei (Frederick et al., 2017). Several studies indicated a relationship between Vip and Bmal1 expression in the hypothalamus, where the absence of one can impact the other. In mice, deletion of Bmal1 in GLAST-positive SCN astrocytes resulted in up-regulation of Vip at ZT0 and ZT12 (Barca-Mayo et al., 2017). Furthermore, VIP KO mice showed a down-regulation of Bmal1 in the SCN under conditions of constant darkness (DD) (Loh et al., 2011). Regarding the association between changes in the expression of PACAP and Bmal1 in the hypothalamus, it is important to note that PACAP is considered a “master regulator” neuropeptide for the stress response (Stroth et al., 2011). In neonatal rats, during stress conditions, the expression of Bmal1 in the SCN was found to be affected (Olejnikova et al., 2018).
The inhibition of retinal orexin receptors resulted in a down-regulation of hypothalamic C-fos, despite the fact that these antagonists produced retinal results similar to those observed under light conditions. This finding contradicts with the induced expression of C-fos in the hypothalamus under light conditions (Munch et al., 2002). In contrast, it was noted that GABA is expressed in most SCN neurons and is the most prevalent neurotransmitter in the SCN. GABA exerts excitatory and inhibitory activity in the shell and core, respectively, depending on the circadian phase (Ono et al., 2018). It was proposed that Vip and GABA are co-expressed to modify inhibitory synaptic transmission within the SCN, and the SCNVIP neurons trigger functional GABAergic responses through the GABAA receptor (Fan et al., 2015). However, since the Vip expression in the JNJ-10397049 groups at ZT-3 and ZT-6 time points is nearly identical to that of the control group, it raises the question of why C-fos is down-regulated in this group. Another factor that may be contributing to the decrease in hypothalamic C-fos expression is the gene expression level of hypothalamic Bmal1. There was a reported lack of neuronal activity in the bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA) in Bmal1 knockout mice. These brain regions are involved in generating a behavioral response to the scent of the female mice (Schoeller et al., 2016). On the other hand, it is possible that the significantly reduced expression of Bmal1 in the hypothalamus of the JNJ-10397049 groups led to oxidative stress, resulting in the impairment of neural signaling cascades (Musiek et al., 2013).
The current study is the first step in examining the potential role of retinal orexin receptors in regulating rhythmicity in the retina and hypothalamus. Future research is required to identify the specific retinal neurons and hypothalamic nuclei involved, and to further investigate the effects of these receptors on disorders associated with circadian rhythms, such as neurodegenerative diseases and sleep disorders.