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

Abstract

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.

Keywords: Acute pain; Lateral hypothalamus; Morphine; Orexin; Rat; Ventral tegmental area.

Figure 1.

A Schematic timeline of the experimental protocol Carbachol was dissolved in 0.5 μL saline and was microinjected into the lateral hypothalamus region in the rat’s brain. SB334867 and TCS OX2 29 were dissolved in 0.3 μL DMSO (12%) and were microinjected into the ventral tegmental area region in the rat’s brain. TF: Tail-Flick; SC: Subcutaneous.

Figure 2.

Effect of morphine administration during the sensitization period on the induction of morphine sensitization measured by Tail-Flick Test Three consecutive days (sensitization period) of morphine administration (5 mg/kg; SC) followed by five days of no drug administration enhanced sensitization to the antinociceptive response of morphine (1 mg/kg; SC). The percentage mean of maximal possible effect (%MPE) was considered an antinociceptive index. Injection of saline instead of morphine did not induce morphine sensitization at any doses of morphine. Each point shows the mean ± SEM for seven rats in each group. ^***P<0.001 compared to the saline-control group.

Figure 3.

Effect of chemical stimulation of the Lateral Hypothalamus (LH) by carbachol before morphine injection during sensitization period on the induction of morphine sensitization measured by Tail-Flick Test Intra-LH microinjection of carbachol just before injection of an ineffective dose of morphine (0.5 mg/kg; SC) during the sensitization period (right panel) enhanced sensitization to the antinociceptive response of morphine. In contrast, concurrent administration of saline (1 mL/kg; SC) and different doses of intra-LH carbachol during the sensitization period (left panel) could not induce morphine sensitization. Each point shows the Mean±SEM for 6–7 rats in each group. P<0.01 and ^***P<0.001 compared to the saline-control group. P<0.01 and †††P<0.001 compared to the respective vehicle groups.

Figure 4.

The effect of intra-VTA injection of SB334867 on the morphine sensitization-induced by co-administration of carbachol and morphine Intra-VTA administration of OX1 receptor antagonist, SB334867 before co-administration of intra-LH carbachol (250 nM/0.5 μL saline) and ineffective dose of morphine (0.5 mg/kg; SC) during the sensitization period could decrease the morphine sensitization-induced by concurrent administration of carbachol and morphine. Morphine sensitization was measured by the tail-flick test. Each point shows the Mean±SEM for 7 rats in each group. ^**P<0.01 and ^***P<0.001 compared to the saline-control group. †P<0.05 and †††P<0.001 compared to the DMSO group.

Figure 5.

The effect of intra-VTA injection of TCS OX2 29 on the morphine sensitization-induced by co-administration of carbachol and morphine Intra-VTA administration of OX2 receptor antagonist, TCS OX2 29 (10 nM/0.3 μL DMSO) before co-administration of intra-LH carbachol (250 nM/0.5 μL saline) and ineffective dose of morphine (0.5 mg/kg; SC) during the sensitization period could decrease the morphine sensitization by concurrent administration of carbachol and morphine. Morphine sensitization was measured by the tail-flick test. Each point shows the Mean±SEM for 6–8 rats in each group. ^*P<0.05 and ^***P<0.001 compared to the saline-control group. †P<0.05 compared to the DMSO group.