Contribution of the intra-hippocampal orexin system in the regulation of restraint stress response to pain-related behaviors in the formalin test

Stress-induced antinociception (SIA) is due to the activation of several neural pathways and neurotransmitters that often suppress pain perception. Studies have shown that the orexin neuropeptide system is essential in pain modulation. Therefore, this study aimed to investigate the role of orexinergic receptors in the hippocampal CA1 region in modulating SIA response during the formalin test as an animal model of inflammatory pain. The orexin-1 receptor (OX1r) antagonist, SB334867, at 1, 3, 10, and 30 nmol or TCS OX2 29 as an orexin-2 receptor (OX2r) antagonist at the same doses were microinjected into the CA1 region in rats. Five minutes later, rats were exposed to restraint stress (RS) for 3 h, and pain-related behaviors were monitored in 5-min blocks for the 60-min test period in the formalin test. Results showed that applying RS for 3 h reduced pain responses in the early and late phases of the formalin test. The main findings showed that intra-CA1 injection of orexin receptor antagonists reduced the antinociception caused by stress in both phases of the formalin test. In addition, the contribution of OX2r in mediating the antinociceptive effect of stress was more prominent than that of OX1r in the early phase of the formalin test. However, in the late phase, both receptors worked similarly. Accordingly, the orexin system and its two receptors in the CA1 region of the hippocampus regulate SIA response to this animal model of pain in formalin test.


Introduction
Stress-induced antinociception (SIA) refers to a natural pain-suppression response observed in mammals, which occurs either during or following exposure to a stressful stimulus SIA can be caused by electric shock, forced swimming, centrifugal rotation and restraint stress (RS; Lewis et al., 1980;Lester and Fanselow, 1985;Quintero et al., 2000;Gameiro et al., 2005).In the past decades, studies have been conducted on the mechanisms of SIA (Fields and Heinricher, 1985;Yamada and Nabeshima, 1995).At various levels of the nervous system, pain perception is influenced by the descending regulatory system, which can be categorized into two segments.One segment employs endogenous opioid peptides as neurotransmitters, while the other employs non-opioid neurotransmitters such as GABA (gamma-aminobutyric acid) monoamines, dopamine and the orexin system (Quintero et al., 2003;Chapman et al., 2008).
Orexin (hypocretin) is a 33 amino acid peptide derived from the precursor of prepro-orexin (De Lecea et al., 1998;Sakurai et al., 1998).Two orexin receptors exist: the orexin-1 receptor (OX1r) and the orexin-2 receptor (OX2r).These two receptors are expressed in various brain areas, such as the ventral tegmental area, nucleus accumbens, and hippocampus (Sargin, 2019).Orexin plays essential roles in regulating brain functions, such as energy homeostasis (Sweet et al., 1999;Sakurai, 2003), neuroendocrine processes, cardiovascular system, sleep (Hagan et al., 1999;Siegel, 1999), reward, and addiction (Zhou et al., 2006).Besides, several studies showed that the orexin system reduces mechanical, inflammatory, and thermal pains at supraspinal and spinal levels (Mobarakeh et al., 2005;Heidari-Oranjaghi et al., 2012;Azhdari-Zarmehri et al., 2014;Cady et al., 2014).Evidence suggests that the inhibition of orexin neurons in the hypothalamus results in inhibition of SIA responses (Bingham et al., 2001).Furthermore, intraventricular injection of an OX1r antagonist reduced the antinociceptive effect of RS in the formalin test (Sofiabad et al., 2011;Razavi and Hosseinzadeh, 2017).Studies have shown that activating the opioid or non-opioid systems of the brain plays a vital role in reducing pain caused by stress, with this effect mediated by brain areas such as the PAG, lateral hypothalamus, and hippocampus (Fields and Heinricher, 1985).
The hippocampus plays a vital role in brain learning, memory, and pain modulation (Lu et al., 2000;Cluderay et al., 2002).The hippocampus regulates the hypothalamic-pituitary-adrenal axis and the stress response (Groeneweg et al., 2011).Work by others has shown that the hippocampus receives orexinergic projections from the hypothalamus, and orexin receptors are excited in different hippocampus areas, such as the CA1 (Boss and Roch, 2015).There is a substantial body of evidence suggesting that the hippocampal formation plays a role in nociception (Sargin, 2019;Ghalebandi et al., 2022).Additionally, certain neural populations within the dorsal hippocampal Cornu Ammonis 1 (CA1) region have been observed to respond to persistent noxious stimuli.Moreover, the hippocampus has been firmly linked to the regulation of cognitive and emotional aspects of pain (Pourreza et al., 2018).It's worth noting that orexinergic projections are also present in the hippocampal formation, including regions such as CA1 and the Dentate gyrus (Pourreza et al., 2018).
Our recent studies showed that orexin receptors in the CA1 modulate formalin-induced orofacial pain and reduce acute stress-induced nociceptive responses in the tail-flick test (Haghparast et al., 2018;Ghalebandi et al., 2022).In 2017, Chen showed that orexin-A exerts excitatory effects on CA1 region neurons through OX1 receptors (Chen et al., 2017).Accordingly, this study aimed to measure the role of intra-hippocampal CA1 orexin receptors on stress-related immobility-induced antinociception using the formalin-induced pain.

Subjects
In this study, 118 adult male rats weighing approximately 230-270 g, which were Wistar albino (Pasteur Institute, Tehran, Iran), were used.In this study, in each cage (14 × 21 × 27 Cm), three rats were cohabited within a controlled environment maintained at 22 •C under a 12/12 h light/dark cycle.Prior to commencing the experimental procedure, the animals underwent a 1-week acclimatization period, during which they had unrestricted access to both food and tap water, and were accustomed to their unfamiliar surroundings.All experiments were carried out by the Guide for the Care and Use of Laboratory Animals (National Research Council. 2011.Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC: The National Academies Press), confirmed by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.PHNS.REC.1400.181),Tehran, Iran.

Stereotaxic surgery
For cannulation, after being anesthetized with ketamine 10% and xylazine 2%, the animals were placed in a stereotaxic apparatus (Stoelting, USA), and the skin of the head area was cut.After drying and cleaning of the skull around Bregma and Lambda according to the Paxinus Atlas (Charles Watson, 2007), the coordinates for the CA1 were AP = 3.50 ± 0.15 mm caudal to the bregma, Lat = ±2.2(±0.7) mm lateral to the midline and DV = 2.7 mm ventral to the skull surface.The guide cannulae (23gauge needle) were bilaterally secured 1 mm above the appropriate injection place.Then, a stainless steel screw and dental acrylic cement were used to fix the guide cannula to the skull.All experiments were performed after the recovery period of 5 to 7 days.

Drug administration
In the present study, the OX1r antagonist, SB334867, (1, 3, 10 and 30 nmol) and the OX2r antagonist, TCS OX2 29, (1, 3, 10 and 30 nmol) (Tocris Bioscience, Bristol, UK) were prepared in a volume of 0.5 μL DMSO (12%) and injected into the CA1 region (Zareie et al., 2022).All microinjections were performed using an injector connected to a 1-μl Hamilton syringe through polyethylene tubing.The dose of drugs was prepared fresh on the day of the experiment and was injected into the CA1 region within 60 s.Five minutes after the injection of drugs, a subcutaneous injection of 50 microliters of 1% formalin was performed in the animal's hind paw.

Stress induction
Plexiglas tubes of 6 × 25 cm adjusted with pistons were used to immobilize the animals.After drugs or vehicle injection, rats were subjected to RS for 3 h.During this time, animals were placed in plastic containment containers without access to food or water, and then 5 min later, formalin was injected into the plantar surface of the right hind paw (Fig. 1).

Formalin test
The formalin test is a suitable model for measuring and evaluating pain behavior in rodents.In this test, a transparent chamber with dimensions of 35 × 35 × 35 cm and made of Plexiglas with a mirror embedded under it was used to observe and check the animal's behavior.Following formalin injection, the animal exhibits a series of pain behaviors.The pain behaviors were given a score of 0 to 3 as follows: 0, the animal's foot is forward on the surface; 1, the weight is reduced on the formalin-injected paw; 2, the complete removal of the animal's foot from the surface; 3, biting, shaking and licking the foot.
These behaviors consist of two phases, early (1-5 min) and late phases (15-60 min), and these two phases are separated from each other by the interphase (reduction of pain behavior) (Tjølsen et al., 1992).During the assessment, the duration of time (in seconds) spent in each stage was meticulously recorded at 5-minute intervals, spanning a total duration of 60 min.Subsequently, the weighted pain score, which ranged from 0 to 3, was computed using the provided formula: This method's utilization resulted in the creation of an ordinal scale representing nociceptive scores, which ranged from 0 to 3 (Haghparast and Ahmad-Molaei, 2009).

Locomotor activity measurement
To investigate the locomotor activity caused by the effect of surgery, drugs, and RS; the animals were subjected to the open field (60 × 60 cm).SB334867 or TCS OX2 29 were injected bilaterally into the CA1.A video tracking system and Ethovision software recorded the total distance covered over 60 min.

Experimental design
To evaluate the effect of formalin injection on pain scores and how the stress protocol affects them, the control groups were initially divided into three groups: the saline control group, the formalin control group, and the RS + formalin group.In addition, in the next part, rats were divided into four groups: No DMSO + No RS, No DMSO + RS (without any vehicle microinjection), DMSO + No RS, and DMSO + RS groups.Furthermore, the effects of surgery, microinjection volume, and stress on pain-related behaviors were investigated.To investigate the effect of OX1r and OX2r antagonists on antinociceptive-related behavior in the formalin test, intra-CA1 administration of SB334867 (1, 3, 10, and 30 nmol/0.5 μL DMSO; n = 7-8 per group) or TCS OX2 29 (1, 3, 10, and 30 nmol/0.5 μL DMSO; n = 7-8 per group) was performed in different doses 5 min before exposure to RS (3h/rat).Then, the formalin test was performed to evaluate the animal's pain scores in 5-minute blocks for 60 min (Fig. 1).In addition, to consider the effect of OX1r or OX2r antagonists alone on antinociceptive response, the highest dose of SB334867 or TCS OX2 29 (30 nmol/0.5 μL DMSO) was microinjected in CA1 (n = 8).

Histological verification
To confirm the correct position of the cannulae in the brain, after the end of the experiment, the animals were deeply anesthetized with a mixture of ketamine and xylazine and perfused with formaldehyde solution (10%) through the heart.After decapitation, the 50-μm transverse brain sections were prepared, and the location of the tip of the guide cannula was compared with the position of CA1 in the rat brain atlas (Fig. 2a and b).Only animals with correct cannulae were included in the data analysis.Eight animals were excluded from the data analysis.

Statistics
In this study, GraphPad Prism 6.0 (GraphPad Software, CA, USA) commercial software was used for statistical analysis.The results were expressed as mean ± SEM (SEM).The normality of distribution was evaluated with the Kolmogorov-Smirnov test.A two-way analysis of variance with repeated measures followed by the Bonferroni post-hoc test was employed to compare the effects of treatment and time on pain scores in the formalin test.Furthermore, each group's AUC (area under the curve) values were calculated as raw pain scores × time by linear trapezoidal method with a one-way analysis of variance followed by the Tukey's test.
One-way ANOVA followed by post-hoc Dunnett multiple comparison tests and the Newman-Keuls post-hoc test were used to calculate the percentage decrease in the AUC values, calculated for nociceptive scores of the experimental groups compared to the AUC values of DMSO + RS group during the early and late phases.
For the AUC values that did not pass Gaussian distribution according to Kolmogorov-Smirnov test, the non-parametric Kruskal-Wallis test, and Mann-Whitney test were used for analysis.Likewise, using the trend line equation option in Excel software (version 2013), the best line was drawn to display the data in the scatter diagram to evaluate the 50% effective dose of drugs.P-values less than 0.05 were statistically considered significant.

Effects of restraint stress on nociceptive behaviors in the formalin test
Figure 3a shows that formalin injection increases pain behavior in the early and the late phases and RS significantly reduces the pain scores in both the early (Fig. 3b) and late phases (Fig. 3c) compared to those in the control group (P < 0.0001).No significant difference in formalin pain scores was found between the no DMSO+ no RS and DMSO+ no RS groups (Fig. 3d).The results revealed that microinjection of 12%DMSO (0.5 μL) did not affect the antinociceptive response induced by RS in early (Fig. 3e) and the late phase (Fig. 3f).

Intra-CA1 administration of OX1 receptor antagonist attenuated antinociceptive effects induced by restraint stress in the formalin test
Statistical analysis using two-way analysis of variance and followed by Bonferroni post-hoc test showed that the intra-CA1 injection of SB334867 (1, 3, 10 and 30 nmol) reduced the antinociceptive effects after exposure to stress [treatment effect:   On the other hand, no significant difference in AUC was found between SB334867 30 nmol+ RS and SB334867 30 nmol groups in both phases of the formalin test.As shown in Fig. 4d, the percentage of increase in AUC values in the SB334867 + RS groups (10 and 30 nmol) compared to the DMSO + RS group was significant in both phases of the formalin test.

Intra-CA1 administration of OX2 receptor antagonist attenuated antinociceptive effects induced by restraint stress in the formalin test
The two-way analysis of variance followed by the Bonferroni post-hoc test showed the effect of intra-CA1 injection of TCS OX2 29 (1, 3, 10, and 30 nmol) on reducing pain-related responses during the 60-min period of the formalin test after a 3-h exposure to RS [Effect of treatment: F (5, 440) = 18.69,P < 0.0001; Time effect: F (11, 440) = 14.14, P < 0.0001; Interaction of treatment and time: F (55, 440) = 2.45, P < 0.0001; Fig. 5a].. On the other hand, one-way ANOVA followed by Dunnett posthoc test in comparison of pain score AUC values showed that the antinociceptive response of RS was suppressed by injection of TCS OX2 29 in both phases of formalin test; [early phase: F (5, 46) = 13.16,P < 0.0001; Fig. 5b] and [late phase: F (5, 46) = 13.12,P < 0.0001; Fig. 5c].Correspondingly, the results demonstrated that the intra-CA1 microinjection of the TCS OX2 29 with doses of 10 and 30 nmol reduced the antinociceptive effects of RS in both phases of the formalin test, but the dose of 3 nmol inhibited the antinociceptive effects caused by RS only in the first phase (Fig. 5b and c).On the other hand, there was no significant difference in AUC between TCS OX2 29 (30 nmol) + RS and TCS OX2 29 (30 nmol) groups in both phases of the formalin test.The percentage of increase in AUC values in the TCS OX2 29+ RS groups (10 and 30 nmol) compared to the DMSO + RS group was shown in Fig. 5d.Results showed that the percentage of increase in AUC significantly increased in both phases of the formalin test ([early phase: F (4, 39) = 13.85,P < 0.0001; Fig. 5d, left panel] and [late phase: F (4, 39) = 22.87, P < 0.0001; Fig. 5d, right panel]).

A log dose-response curve of the effect of intra-CA1 administration of SB334867 and TCS OX2 29 on the antinociceptive responses induced by RS in the formalin test
The OX1r in the CA1 region seems equally involved in the early and late phases of the formalin test because the results show that the effective dose of SB334867 in the early phase (11.07) was almost equal to the late phase (12.27).Also, the effect size (Eta-squared) in the early phase (η 2 = 0.97) was almost equal to the late phase (η 2 = 0.96).
The results revealed that the effective dose of TCS OX2 29 in various doses was different between the early (7.21) and late phase (12.13) of the formalin test (Fig. 6a).Thus, OX2r in the CA1 seems to be not equally involved in the modulation of pain during both phase of formalin test.Also, the effect size (Eta-squared) in the early phase (η 2 = 0.61) was not equal to the late phase (η 2 = 0.06) (Fig. 6b).

Intra CA1 microinjection of SB334867 or TCS OX2 29 had no significant effect on locomotor activity
The results indicated that the movement activity of the animals in 60 min during the immobility stress and the administration of drugs in the CA1 had no significant effect on the control group F (4, 38) = 0.09, P > 0.05; Supplementary Fig. 1, Supplemental digital content 1, http://links.lww.com/BPHARM/A102].

Discussion
This study aimed to evaluate the involvement of the orexin receptors within the CA1 in antinociceptive responses induced by RS during the formalin test in rats.The main findings were: (a) RS significantly reduced the pain scores in both the early and late phases of the formalin test.(b) Microinjection of OX1r and OX2r antagonists into the CA1 attenuated the antinociceptive effect of RS in the early and late phases of the formalin test.(c) Intra-CA1 administration of SB334867 or TCS OX2 29 alone could not significantly change nociceptive behaviors.(d) The contribution of intra-CA1 OX2r in mediating the antinociceptive effect of stress was more prominent than that of OX1r in the early phase of the formalin test.(e) The role of OX1r and OX2r in CA1 was not equal in the early phase of formalin nociception.(f) Nevertheless, in the late phase of the formalin test, the role of OX1r and OX2r in CA1 was somewhat equal.
Many studies have provided convincing evidence that various stresses cause antinociceptive responses in animal models.For example, studies have shown that acute exposure to stressors induces antinociception in experiments, such as tail-flick and plantar tests in rats (Wideman et al., 1996;Wolf et al., 2007;Lafrance et al., 2010).Furthermore, previous studies have shown that immobility stress in rats can reduce pain in the formalin test (Gameiro et al., 2005;Heidari-Oranjaghi et al., 2012).However, some studies have also reported that acute and severe stress can cause pain in some conditions.For example, frequent exposure to a cold environment (4 °C for 30 min) causes hyperalgesia, or being in a high ambient temperature environment for 1 hour every day for 40 days causes thermal hyperalgesia (Satoh et al., 1992;Torres et al., 2001).This difference can be caused by the type, period, and stress intensity in different trials involving transitional systems.The present study showed that 3 hours a day of immobility stress could increase antinociception in the formalin test in rats.
Several morphological studies have shown that orexin neurons distributed in various brain regions are involved in pain and stress behaviors (Peyron et al., 1998;Nambu et al., 1999).It has been shown that during pain and stress, the activity of orexin neurons changes, and subsequently, the activity of some neural circuits relates to stress-evoked changes.Furthermore, orexin receptor expression increases after exposure to stress.Therefore, the change in orexin expression may cause a change in pain threshold during stress (Furlong et al., 2009;Berridge et al., 2010).Gerashchenko et al. showed that inhibition of orexin neurons in the lateral hypothalamus reduces the inhibitory effect of stress on pain behavior (Gerashchenko et al., 2011).Besides, the orexin gene knockout mice showed a higher degree of hyperalgesia caused by peripheral inflammation and a lower degree of SIA than normal mice (Watanabe et al., 2005).The present study revealed that the orexinergic system plays a role in SIA.Hence, the block of orexin receptors in the CA1 reduced the pain-related behaviors in the formalin test after exposure to immobility stress.These results are consistent with other studies that reported that foot electric shock stress activates orexinergic neurons and induces antinociception (Watanabe et al., 2005).On the other hand, our studies show that orexin receptors in different brain areas modulate antinociception caused by various stresses, such as the stress of forced swimming in acute and chronic pain (Heidari-Oranjaghi et al., 2012;Ghalebandi et al., 2022).Hypothalamic orexin neurons have been shown to activate and release orexins (Ho et al., 2011).Another study showed that orexin regulates pain in a mouse model of SIA through interaction with nociceptin/orphanin FQ (N/OFQ) systems (Xie et al., 2008).
On the other hand, evidence suggests that the hippocampus modulates pain through orexin (Pourreza et al., 2018).Furthermore, the hippocampus is involved in opioid and non-opioid SIA (Ghasemzadeh and Rezayof, 2015;Poznański et al., 2019).In the present study, SIA was blocked by the intra-CA1 injection of OX1r and OX2r antagonists in both phases of the formalin test.These results are consistent with previous findings showing that swimming stress affects both phases of formalin-induced pain, and SIA is blocked by the intra-CA1 injection of OX1r and OX2r antagonists in both phases of the formalin test (Ghalebandi et al., 2022).In the present study, the maximal dose of SB334867 and TCS OX2 29 alone were injected into the CA1 region and were shown to have no significant effect on the formalin test's early and late phases.
The percentage increase in AUC values in the early and late phases compared to the DMSO-RS control group is shown in Fig. 6a and b, respectively.The Log Dose-Response curve showed that TCS OX2 29 as an OX2r antagonist was more effective in the reducing the percentage increase in AUC values than SB334867 as an OX1r antagonist in the early phase of the formalin test, but in the late phase, there was no significant difference between SB334867 and TCS OX2 29.The different distribution of orexin receptors in the CA1 area or the different affinity of orexin receptors for orexin may be the reason for this phenomenon.Results revealed that intra-CA1 administration of OX1r and OX2r antagonists reduced the effects of SIA in the early phase more than in the late phase.Therefore, it is suggested that CA1 orexinergic receptors play a more critical role in the early phase of pain behavior.However, further research with immunohistological, electrophysiological, and molecular approaches is needed to elucidate the exact mechanisms of orexin receptors and their involvement in pain modulation and SIA.Data will be made available upon request.The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.