Unravelling the role of a pain facilitatory area of the brain during chronic opioid exposur

Opiates represent the most commonly used drugs for the treatment of moderate-to-severe postoperative and chronic pain. Chronic use of opioids can induce paradoxical hyperalgesia (opioid-induced hyperalgesia; OIH). OIH is characterized by hypersensitivity to innocuous or noxious stimuli during sustained opiate administration, and is reported both in clinical and pre-clinical settings but its molecular mechanisms are not fully understood. One of the mechanisms that contribute to OIH is the activation of brain areas involved in pain facilitation. Here we studied the involvement of an area located in the medulla oblongata, the dorsal reticular nucleus (DRt), which plays a unique and exclusive pain facilitatory role. The studies included in the present thesis aimed at i) determining whether chronic administration of morphine induces OIH in acute pain and the spared nerve injury (SNI) model of chronic neuropathic pain; ii) study the effects of chronic morphine on morphine reward and iii) evaluate the involvement of the DRt in the mediation of OIH and morphine reward. To determine the effects of chronic morphine administration the animals were implanted with osmotic mini-pumps filled with morphine (45 μg.μl.h) or saline, which released their content continuously for 7 days. The effects of chronic morphine administration on pain behavior were tested before and at 2, 4 and 7 days after the mini-pump implantation. In naïve animals pain behavior was tested by the von-Frey and hotplate test which evaluate mechanical allodynia and thermal hyperalgesia respectively. Pain assessment in SNI animals was performed by the von-Frey test, the pin-prick test which evaluate changes in mechanical hyperalgesia and the acetone test to assess cold allodynia. The continuous infusion of morphine induced OIH in naïve animals and, for the first time, we show that chronic morphine administration induced OIH in an animal model of neuropathic pain. To study the effects of chronic morphine on the reward behavior we used the conditioned place preference test (CPP). In animals chronically treated with morphine, the acute administration of morphine failed to induce CPP, unlike in control animals, which indicates a loss of the reward effect of morphine. To study the involvement of the DRt in the mediation of OIH, we first inactivated the DRt with lidocaine (0.5 μl; 4% w/v) in naïve animals. The injection of lidocaine was performed on day 7 after implantation of the osmotic pumps containing saline or morphine (45 μg.μl.h) and its effects were tested by behavioral tests mentioned above. The administration of lidocaine at the DRt fully reversed mechanical allodynia and thermal hyperalgesia in morphine-infused animals. Then we studied the ii FCUP Unravelling the role of a pain facilitatory area of the brain during chronic opioid exposure expression of the phosphorylated cAMP response element-binding protein (pCREB) and μ-opioid receptor (MOR), at the DRt, by immunohistochemistry. We show that chronic morphine treatment induces an increase of pCREB and MOR expression and that MOR immunoreactive cells co-localized with pCREB. Finally, we performed a lentiviral-mediated knock-down of the expression of MOR at the DRt. For that, the animals were stereotaxically injected with lentiviral vectors at the DRt and implanted with osmotic mini-pumps containing saline or morphine (45 μg.μl.h). The animals were tested before and 7 days after the lentiviral injections and mini-pump implantation by the behavioural tests mentioned above. The knock-down of MOR in control animals showed an increase of pain behaviours. In animals chronically treated with morphine, the knock-down of MOR prevented the development of OIH. We also evaluated the effects of MOR knock-down to assess the involvement of the DRt in morphine reward during chronic morphine exposure. Our preliminary results show that MOR knock-down results in a reversion of the loss of morphine reward. Our results indicate that chronic morphine exposure induces OIH in naïve and neuropathic animals and, that the DRt is involved in the mediation of OIH, likely through MOR activation whose effects appear switch from inhibitors to facilitation upon chronic morphine. Our results also indicate that chronic morphine treatment induces a loss of morphine reward and that the DRt might also be involved in the mediation of such effects through MOR activation. Given the increase in the expression of pCREB at the DRt, it would be interesting to explore the involvement of this transcription factor in pain transmission from the DRt, during opioid-induced hyperalgesia. It would also be interesting to explore the interactions between the DRt and brain areas involved both in pain and reward on morphine reward.