Involvement of dopaminergic system in the dentate gyrus of the hippocampus in modulating the orofacial pain-related behaviors in the rats.

Chemical stimulation of the lateral hypothalamus (LH) induces analgesia by forming neural circuitries with multiple brain regions. The involvement of hippocampal dopaminergic receptors in the LH stimulation-induced antinociception in specific pain models in animals has been documented. However, because the neural circuitries involved in the mediation of orofacial pain are not the same as those that mediate the other types of pain, the present study aims to detect the role of dopamine receptors within the dentate gyrus (DG) in the antinociceptive responses induced by LH stimulation in an animal model of orofacial pain. Male Wistar rats (220-250 g) were implanted with two separate cannulae into the LH and DG on the same side. D1- or D2-like dopamine receptor antagonist, SCH23390, or sulpiride (0.25, 1, and 4 µg) were microinjected into the DG, five minutes before intra-LH injection of carbachol (250 nM). The animals were then injected with formalin 1% (50 µL; sc) into the upper lip lateral to the nose and subjected to the orofacial formalin test. Intra-DG administration of SCH23390 or sulpiride attenuated the antinociceptive responses induced by intra-LH microinjection of carbachol during the orofacial formalin test. The findings of the current study suggest that chemical stimulation of the LH modulates orofacial pain, possibly through activation of the DG dopaminergic neurons. Due to the high incidence and prevalence of orofacial pain in the general population, understanding how such neuronal circuitry modulates nociceptive processing will advance the search for novel therapeutics.

Distinct roles for orexin-1 and orexin-2 receptors in the dentate gyrus of the hippocampus in the methamphetamine-seeking behavior in the rats.

Because of the relapsing properties of psychostimulants such as methamphetamine (Meth), there is no established pharmacotherapy for Meth addiction. The orexinergic system is a promising target for treating psychostimulant use disorders and relapse. However, to the best of our knowledge, no investigation regarding the role of orexin receptors in the dentate gyrus (DG) region of the hippocampus has been conducted in the extinction and reinstatement of Meth-seeking behavior. Two stainless-steel guide cannulae were bilaterally implanted into the DG of the rats' brains. The unbiased conditioned place preference (CPP) procedure was conducted to induce Meth conditioning. Following the five days Meth injections (1 mg/kg; sc), animals received intra-DG microinjection of SB334867 or TCS OX2 29, as orexin 1 (OX1) or orexin 2 (OX2) receptor antagonists, respectively (without Meth administration) during extinction phase to elucidate the role of orexin receptors in the latency of the extinction period in the Meth-conditioned rats. To evaluate the role of orexin receptors in the DG region in the reinstatement of Meth-seeking behavior, the extinguished rats received SB334867 or TCS OX2 29 before injecting a priming dose of Meth (0.25 mg/kg; sc). The results indicated two distinct roles for the OX1 and OX2 receptors in the DG region. TCS OX2 29 attenuated the extinction latency, and SB334867 considerably reduced the reinstatement of Meth-seeking behavior in this region. Therefore, the DG region's orexinergic system might be a potential therapeutic target for psychostimulant use disorders.

The modulatory role of dopamine receptors within the hippocampal cornu ammonis area 1 in stress-induced analgesia in an animal model of persistent inflammatory pain.

The intrinsic pain inhibitory mechanisms can be activated by fear, anxiety, and stress. Stressful experiences produce analgesia, referred to as stress-induced analgesia (SIA). Major components of the limbic system, including the ventral tegmental area, nucleus accumbens, amygdala, and hippocampus, are involved in the SIA. In this study, we tried to understand the role of dopamine receptors in the cornu ammonis area 1 (CA1) of the hippocampus in the forced swim stress (FSS)-induced analgesia. Stereotaxic surgery was unilaterally performed on 129 adult male Wistar rats weighing 220-280 g. SCH23390 (0.25, 1, and 4 µg/0.5 µl saline) or sulpiride (0.25, 1, and 4 µg/0.5 µl DMSO), as D1- and D2-like dopamine receptor antagonists, respectively, were microinjected into the CA1 area, 5 min before exposure to FSS for a 6-min period. The vehicle groups received saline or DMSO instead of SCH23390 or sulpiride, respectively. The formalin test was done using formalin injection (50 µl; 2.5%) into the plantar surface of the rat's hind paw immediately after exposure to FSS. The results demonstrated that FSS produces analgesia during the early and late phases of the formalin test. However, intra-CA1 microinjection of SCH23390 or sulpiride attenuated the FSS-induced analgesia in both phases of the formalin test. This study provides new insight into the role of D1- and D2-like dopamine receptors in the CA1 area in the FSS-induced analgesia during persistent inflammatory pain.

Nucleus accumbens dopamine receptors mediate the stress-induced analgesia in an animal model of acute pain.

Exposure to aversive stimuli such as stress results in profound analgesia named stress-induced analgesia (SIA). We previously showed that D1- and D2-like dopamine receptors within the nucleus accumbens (NAc) mediated the SIA in chronic pain. Since the neurophysiological mechanisms responsible for the various pain conditions are different, the present study aimed to examine the role of dopamine receptors within the NAc in the forced swim stress (FSS)-induced analgesia in the tail-flick test as an animal model of acute pain. Ninety-six adult male Wistar rats weighing 200-230 g were unilaterally implanted with a cannula into the NAc. SCH23390 or Sulpiride (0.25, 1, and 4 µg/0.5 µl vehicle), as D1- and D2-like dopamine receptor antagonists, respectively, were microinjected into the NAc, 5 min before exposure to FSS. The vehicle groups received saline or DMSO instead of SCH23390 or Sulpiride, respectively. The tail-flick test was performed in time set intervals after animals were subjected to FSS. The results showed that FSS produces analgesia during the tail-flick test. However, intra-accumbal injection of SCH23390 or Sulpiride attenuated the FSS-induced analgesia. D1-and D2-like dopamine receptor antagonists contributed almost equally to attenuating the antinociceptive responses induced by FSS. It seems that the mesolimbic dopamine system might act as a potential endogenous pain control system in stress conditions. Besides, an improved understanding of this endogenous pain inhibitory system can develop pharmacological and psychological approaches to treat pain.

Role of the Orexinergic System Within the Ventral Tegmental Area in the Development of Sensitization to Morphine Induced by Lateral Hypothalamus Stimulation.

Introduction: The Lateral Hypothalamus (LH) has long been known to implicate the addictive behaviors of drug abuse. The Ventral Tegmental Area (VTA) is a major area of the mesolimbic system that is strongly involved in developing morphine sensitization. The current study aimed to examine the role of intra-VTA orexin receptors in the LH stimulation-induced sensitization to the antinociceptive response of morphine. Methods: A total of 114 adult male Wistar rats underwent unilateral implantation of two separate cannulae in the LH and VTA using the stereotaxic apparatus. Intra-VTA administration of the Orexin-1 (OX1) and Orexin-2 (OX2) receptor antagonists, SB334867 and TCS OX2 29 (1, 3, and 10 nM/0.3 µL DMSO), respectively, was performed 5 min before concurrent microinjection of carbachol (250 nM/0.5 µL saline) into the LH and an ineffective dose of morphine (0.5 mg/kg; SC) during a 3-day sensitization period. After a 5-day free drug period, on the ninth day, for assessing the morphine sensitization, the nociceptive response was measured before and after morphine injection (1 mg/kg; SC) using the tail-flick test. Results: The results revealed that the concurrent administration of carbachol (250 nM) and an ineffective dose of morphine significantly induced morphine sensitization. Besides, the blockade of OX1 and OX2 receptors within the VTA before intra-LH carbachol injection attenuated morphine sensitization. Conclusion: These findings suggest that LH stimulation potentiates the sensitization to morphine antinociceptive responses via affecting orexin receptors located in the VTA. However, OX1 receptors contribute more than OX2 receptors in the VTA to morphine sensitization in rats. Highlights: LH stimulation enhances sensitization to the ineffective dose of morphineIntra-VTA OX1 receptor involves in morphine sensitization-induced by LH stimulationIntra-VTA OX2 receptor involves in morphine sensitization-induced by LH stimulation. Plain Language Summary: Behavioral sensitization, such as sensitization to the antinociceptive response of drugs, which defines as an enhanced systemic reaction to the same dose of addictive drugs, occurs in response to continuous and intermittent administration of these drugs. The Lateral Hypothalamus (LH) sends the orexinergic projections to the various regions of the brain and stimulation of LH induces sensitization to the antinociceptive response of morphine. The Ventral tegmental area (VTA) is a region of the brain that is strongly involved in developing morphine sensitization and receives orexinergic projections of LH. The current study aimed to examine the role of orexin receptors within the VTA in the LH stimulation-induced sensitization to the antinociceptive response of morphine in rats. In this study orexin-1 (OX1) and orexin-2 (OX2) receptors within the VTA region were blocked using their antagonists. After five minutes chemical stimulation of LH was done using carbachol microinjection into this area and ineffective dose of morphine was injected subcutaneously. These interventions were done for three consecutive days as sensitization period. After a 5-day free drug period, on the ninth day, for assessing the morphine sensitization, the nociceptive response was measured. The results revealed that the concurrent administration of LH stimulation and an ineffective dose of morphine significantly induced morphine sensitization. Besides, the blockade of OX1 and OX2 receptors within the VTA before LH stimulation attenuated sensitization to the antinociceptive response of morphine. Therefore, the orexinergic system plays an important role in morphine sensitization and can be considered as one of the potential targets to increase the analgesic effect of morphine.

Cannabidiol microinjection into the nucleus accumbens attenuated nociceptive behaviors in an animal model of tonic pain.

Cannabidiol, the major non-psychoactive constituent of Cannabis, has attracted much attention as a therapeutic agent for intractable chronic pain in many conditions. Nucleus accumbens (NAc) as a major site of action of cannabinoids is one of the main mediators of several analgesic agents especially in the persistent pain condition. The present study aimed to investigate the effect of cannabidiol microinjection into the NAc on the modulation of nociception induced by formalin injection into the rat's paw. Adult male Wistar rats weighing 220-250 g were underwent stereotaxic surgery for unilateral (right or left side) cannula placement into the NAc. After one week recovery period, intra-NAc administration of the cannabidiol or its vehicle, DMSO was performed in a volume of 0.5 µl, five minutes before the formalin test. The formalin test was performed using 50 µl injection of formalin (2.5%) into the plantar surface of the rat's hind paw. Intra-accumbal administration of cannabidiol attenuated the nociceptive responses during the early and late phases of the formalin test in a dose-dependent manner. However, the antinociceptive effect of cannabidiol was significantly higher in the late phase of the formalin test than that in the early phase. Therefore, a non-psychoactive cannabinoid, cannabidiol may be developed as therapeutic agents in conditions, such as persistent inflammatory pain for which primary treatments are insufficient or not possible.

The role of dentate gyrus dopaminergic receptors in the lateral hypothalamic-induced antinociception during persistent inflammatory pain in male rats.

The lateral hypothalamus (LH) is one of the key brain areas involved in pain modulation. Also, the dentate gyrus (DG) of the hippocampus expresses various receptors, including dopaminergic receptors. Dopaminergic receptors play a key role in pain transmission and modulation within the brain. The present study aimed to investigate the involvement of DG dopaminergic receptors in the LH-induced antinociception during the presence of inflammatory pain. Male Wistar rats were used in this study. Cannulae were unilaterally implanted in their skull for microinjections into the LH and DG. The LH was chemically stimulated by carbachol injection (250 nM/0.5 µl saline). In separate groups, different doses (0.25, 1, and 4 µg/0.5 µl vehicle) of the D1- and D2-like dopamine receptor antagonists (SCH23390 and Sulpiride, respectively) were microinjected into the DG, 5 min prior to intra-LH injection of carbachol. Five min after the second injection, formalin test as a persistent inflammatory pain model in animals was done in all rats. The results revealed that carbachol could induce antinociception following formalin injection into rat's hind paw. The 4 µg dose of both antagonists significantly reduced the LH stimulation-induced antinociception in both phases of formalin pain responses. Although the 1 µg dose of sulpiride significantly reduced antinociception during both phases, 1 µg SCH23390 could only reduce this antinociception during the late phase. These findings demonstrate the involvement of DG dopaminergic receptors in the LH-induced antinociception. The results also suggest that the effectiveness of DG dopaminergic receptors is more pronounced during the late phase of formalin-induced pain responses.

Dopaminergic receptors in the ventral tegmental area modulated the lateral hypothalamic stimulation-induced antinociception in an animal model of tonic pain.

The role of the ventral tegmental area (VTA) and the lateral hypothalamus (LH) in the modulation of formalin-induced nociception is well documented individually. The present study aimed to investigate the role of dopamine receptors of the VTA in the modulation of the LH stimulation-induced antinociception during both phases of the formalin test as an animal model of tonic pain. In this study, male Wistar rats were unilaterally implanted with two guide cannulae in the VTA and LH. In two separate groups, animals received different doses (0.25, 1, and 4 µg/rat) of D1- or D2-like dopamine receptor antagonists (SCH-23,390 or Sulpiride, respectively) into the VTA before intra-LH injection of carbachol (22.83 ng/rat) following formalin injection (50 µL; s.c.) into their contralateral hind paws. The blockade of these two receptors reduced intra-LH carbachol-induced antinociception during both phases of the formalin test. This reduction during the late phase of the formalin test was more than that of the early phase. The results indicated that LH stimulation-induced antinociception was mediated by D1- and D2-like dopamine receptors in the VTA, and so, the neural pathway projecting from the LH to the VTA contributes to the modulation of formalin-induced nociception in the rats.

Restraint Stress Potentiated Morphine Sensitization: Involvement of Dopamine Receptors within the Nucleus Accumbens.

Sensitization to psychostimulant drugs, as well as morphine, subjected to cross-sensitization with stress. The development of morphine sensitization is associated with enhancements in dopamine overflow in the Nucleus accumbens (NAc). This study aimed to examine the role of accumbal D1/D2-like dopamine receptors in restraint stress (RS) induced sensitization to morphine antinociceptive effects. Adult male Wistar rats weighing 220-250 g underwent stereotaxic surgery. Two stainless steel guide cannulae were bilaterally implanted, 1 mm above the NAc injection site. Different solutions of SCH-23390, as a D1-like receptor antagonist or sulpiride, as a D2-like receptor antagonist, were microinjected into the NAc five min before exposure to RS. Restraint stress lasted for 3 h, 10 min after RS termination; animals received a subcutaneous injection of morphine (1 mg/kg) for 3 consecutive days. The procedure was followed by a 5-day drug and/or stress-free period. After that, on the 9th day, the nociceptive response was evaluated by the tail-flick test. The results revealed that intra-NAc administration of D1/D2-like dopamine receptor antagonists, SCH-23390 or sulpiride, respectively, blocked morphine sensitization-induced by RS and morphine co-administration in rats for three consecutive days. This work provides new insight into the determinant role of accumbal dopamine receptors in morphine sensitization produced by RS-morphine co-administration.

Role of D1- and D2-like dopamine receptors within the dentate gyrus in antinociception induced by chemical stimulation of the lateral hypothalamus in an animal model of acute pain.

The dentate gyrus (DG) as the main gateway of the hippocampal formation (HF) plays a crucial role in pain modulation. For this purpose, the HF receives dopaminergic inputs originated from the substantia nigra and the ventral tegmental area. It has previously been shown that the lateral hypothalamus (LH) stimulation produces antinociception via orexinergic projections of the LH to the DG region. So, given the presence of dopamine receptors in the DG and the undeniable role of the dopaminergic system in pain modulation, the current study was conducted to investigate the role of dopamine receptors located within the DG in the LH stimulation-induced pain modulation. Adult male Wistar rats weighing 220-250 g were unilaterally implanted with two separate cannulas into the LH and DG. Intra-DG administration of D1- or D2-like dopamine receptor antagonist (0.125, 0.25, 1, and 4 µg) was performed just 5 min before chemical stimulation of the LH by carbachol (250 nM). Nociceptive assay was done using the tail-flick apparatus immediately after the last microinjection. The results demonstrated that the administration of SCH23390 or Sulpiride into the DG decreased the intra-LH carbachol-induced antinociceptive responses and decreased the tail-flick latency times. The role of D2-like dopamine receptor of the DG was more prominent than that of D1-like dopamine receptor in antinociceptive response produced by the LH stimulation. It seems to be a complex neural circuitry in which the LH produces antinociceptive effects, in part, via dopamine receptors located in the DG region.

Role of orexin receptors within the dentate gyrus in antinociception induced by chemical stimulation of the lateral hypothalamus in an animal model of inflammatory pain.

Pain is a complex experience consisting of sensory, affective-motivational, and cognitive dimensions. Hence, identifying the multiple neural pathways subserving these functional aspects is a valuable task. The role of dentate gyrus (DG) as a relay station of neocortical afferents in the hippocampal formation (HF) in persistent pain is still controversial. The lateral hypothalamus (LH)-HF neural circuits are involved in numerous situations such as anxiety-like behavior, reward processing, feeding, orofacial as well as acute pain. Nonetheless, to our knowledge, the involvement of the LH-DG neural circuit in persistent pain has already remained unexplored. Adult male Wistar rats weighing 220-250 g were undergone stereotaxic surgery for unilateral implantation of two separate cannulae into the LH and DG. Intra-DG administration of the orexin-1 (OX1) and orexin-2 (OX2) receptor antagonists, SB334867 and TCS OX2 29, respectively, was performed 5 min before intra-LH microinjection of carbachol. Animals were then undergone the formalin test using 50 µl formalin injection (2.5%) into the plantar surface of the hind paw. Microinjection of SB334867 or TCS OX2 29 into the DG region attenuated the antinociceptive effect produced by carbachol microinjection into the LH. The preventive effect of SB334867 and TCS OX2 29 on intra-LH carbachol-induced antinociception was approximately equal in both early and late phases of formalin nociception. The results suggest a neural pathway from the LH to the DG, which contributes to the modulation of formalin-induced inflammatory pain through the recruitment of OX1 and OX2 receptors within the DG.

The Potential Role of Glycogen Synthase Kinase-3ß in Neuropathy-Induced Apoptosis in Spinal Cord.

INTRODUCTION: Glycogen Synthase Kinase-3ß (GSK-3ß) participates in several signaling pathways and plays a crucial role in neurodegenerative diseases, inflammation, and neuropathic pain. The ratio of phosphorylated GSK-3ß over total GSK-3ß (p-GSK-3ß/t-GSK-3ß) is reduced following nerve injury. Apoptosis is a hallmark of many neuronal dysfunctions in the context of neuropathic pain. Thus, this study aimed to evaluate the contribution of p-GSK-3ß/t-GSK-3ß ratio in spinal dorsal horn apoptosis following peripheral nerve injury. METHODS: In this study, adult male Wistar rats (220-250 g) underwent Spinal Nerve Ligation (SNL) surgery. Mechanical allodynia and thermal hyperalgesia were assessed before the surgery (day 0); then, every other day up to day 8. GSK-3ß selective inhibitor, AR-014418 [0.3 mg/kg, Intraperitoneal (IP)] was administrated 1 h prior to SNL on day 0, then daily up to the day 8. The GSK-3ß activity and apoptosis in the lumbar section (L4, L5, or L6) of the study rat's spinal cord were assessed by immunohistochemical and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining, respectively on day 8 post-SNL. RESULTS: Following the SNL, the mechanical allodynia and thermal hyperalgesia increased on day 2 up to day 8 post-SNL. The ratio of p-GSK-3ß/t-GSK-3ß decreased, and the number of apoptotic cells increased in the spinal dorsal horn on day 8. However, AR-A014418 administration could increase the p-GSK-3ß/t-GSK-3ß ratio and decreased apoptosis in the SNL rats. In addition, AR-A014418 decreased the mechanical allodynia from day 4 up to day 8; however, it did not affect thermal hyperalgesia. CONCLUSION: The study findings suggested that increasing the p-GSK-3ß/t-GSK-3ß ratio might be a helpful strategy for reducing the apoptotic cells and subsequent neuropathic pain during peripheral nerve injury.

The Effect of Rosmarinic Acid on Apoptosis and nNOS Immunoreactivity Following Intrahippocampal Kainic Acid Injections in Rats.

INTRODUCTION: Kainic Acid (KA) is an ionotropic glutamate receptor agonist. KA can induce neuronal overactivity and excitotoxicity. Rosmarinic Acid (RA) is a natural polyphenolic compound with antioxidant, anti-apoptotic, anti-neurodegenerative, and anti-inflammatory properties. This study aimed to assess the effect of RA on apoptosis, nNOS-positive neurons number, as well as Mitogen-Activated Protein Kinase (MAPK) and Cyclooxygenase-2 (COX-2) immunoreactivity, following intrahippocampal Kainic acid injection in rats. METHODS: The study rats were randomly assigned to three groups of sham, KA (KA was injected into the right side of the hippocampus) and KA+RA (a dose of 10 mg/kg/day through a gavage needle for one week before KA injection). Then, histopathological changes, including apoptosis [Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay], nNOS-positive neurons number, as well as COX-2 and MAPK immunoreactivity were evaluated in the hippocampus. RESULTS: In the RA pretreated group, nNOS-positive neurons and TUNEL-positive cells were significantly reduced compared to the KA group (P<0.05). COX-2and MAPK immunoreactivity demonstrated no significant changes compared to the KA group. They indicated a significant higher reactivity for COX-2 (P<0.01) and MAPK (P<0.005) versus the sham group. CONCLUSION: RA had neuroprotective effects, compared to KA, through reduced apoptosis and nNOS-positive neurons, but not MAPK and COX-2.

Role of orexin receptors within the dentate gyrus of the hippocampus in antinociception induced by chemical stimulation of the lateral hypothalamus in the tail-flick test as a model of acute pain in rats.

The dentate gyrus (DG) as a part of the hippocampal formation is considered one of the brain areas involved in pain perception. The orexin neurons in the lateral hypothalamus (LH) also show connections to regions engaged in the circuitry of pain modulation and many studies have shown their potential to alter pain transmission through the nervous system. However, the role of orexin receptors (OXRs) of the DG in mediation of antinociceptive responses induced by the LH stimulation in rats has not yet been investigated. Hence, the present study was an attempt to examine the role of OXRs located within the DG in the pain modulation induced by chemical stimulation of the LH. Adult male Wistar rats weighing 220-250 g were unilaterally implanted with two separate cannulae into the LH and DG. Intra-DG administration of the orexin-1 receptor (OX1R) antagonist, SB334867 or the orexin-2 receptor (OX2R) antagonist, TCS OX2 29 was performed just 5 min before intra-LH carbachol microinjection. Antinociceptive effects were measured using the tail-flick apparatus. The results showed that OX1R and OX2R antagonists dose-dependently decreased the antinociceptive effect of carbachol. In addition, the effective dose of SB334867 was lesser than that of TCS OX2 29, which indicates the more prominent role of OX1R of the DG in carbachol-induced antinociception. None of the antagonists had any significant effect when administrated without intra-LH carbachol microinjection. It seems to be a neural circuit includes the LH and DG, which plays a major role in the neural basis of pain modulation.

GABAA receptors are involved in the analgesic effects of morphine microinjected into the central nucleus of the amygdala.

The central nucleus of the amygdala (CeA) has an important role in pain perception and analgesia. Opioid and GABAA receptors, which are both involved in pain modulation, are found in high concentration in the CeA. The present study was designed to examine the interaction of opioidergic and GABAergic systems in the CeA during modulation of acute thermal pain. In the present study, different doses of morphine (25, 50 and 100 µg/rat), either alone or after 5 min pretreatment with the selective GABAA receptor agonist muscimol (60 ng/rat) or the selective GABAA receptor antagonist bicuculline (50 ng/rat), were injected bilaterally into the CeA of each rat. Tail-flick latencies (TFL) were measured every 5 min for 60 min. The results revealed that microinjection of morphine into the CeA significantly increased TFL in a dose-dependent manner. Microinjection of bicuculline or muscimol in combination with morphine into the CeA increased and decreased TFL, respectively. It seems that morphine in the CeA facilitates the function of descending inhibitory systems by interacting with the activity of local GABAA receptors.