Gabapentinoids 藥物對於壓力誘導尼古丁渴求之小鼠動物模式的影響

Abstract

菸草是全世界中最常被濫用的其中一種成癮性物質，菸草中的主要成分尼古丁被認為是造成煙癮成癮主要的成分。許多臨床前試驗和臨床數據都顯示暴露於壓力情況之下和藥物成癮的敏感性增加呈現正相關。急性壓力可能導致尼古丁的渴求行為增加，回饋系統的迴路興奮，甚至對於戒煙後復發風險增加。然而其中的機制大部分是未知的。最近的研究也顯示濫用性藥物的暴露過程中，L型電壓鈣離子通道上的α2δ1次單元有增加的趨勢。因此，我們相信α2δ1次單元的表現或許參與了急性壓力誘導尼古丁的尋求行為的過程。實驗室近來研究也顯示急性束縛壓力會導致小鼠血漿中皮質酮量的增加，另外也會活化LH核區中的orexin 神經元，導致orexin 下游路徑的一連串活化。然而，目前並未有證據或是研究探討尼古丁渴求，α2δ1次單元和皮質酮之間的關係。另外，我們也想了解orexin pathway是否參與了急性壓力誘導尼古丁的尋求行為。因此，我們將在本文進一步探討這些因子者之間的關係。 Gabapentin和pregabalin 都是GABA (γ-aminobutyric acid) 的類似物，被統稱為gabapentinoid，同為市面上的處方藥物。它們的作用包括緩解神經痛，治療癲癇和焦慮等疾病。它們和α2δ1次單元有高度的親和力。許多研究指出當它們和α2δ1次單元結合，會造成鈣離子流量減少，調控神經元功能甚至參與藥物成癮的行為。 為了研究orexin pathway是否參與了急性壓力誘導尼古丁的尋求行為以及gabapentinoids是否可以在急性壓力誘導尼古丁成癮的病患當中成為新處方，我們使用動物模式來進行以下實驗。首先，在C57BL/6和ICR兩種不同品系的雄性小鼠中，我們利用場域偏好性試驗來建立一個有效率的壓力誘導尼古丁渴求模式，小鼠在喜愛箱和非喜愛箱待的時間差被定義為CPP 分數，它可以用為判斷小鼠是否對於尼古丁條件配對的箱子具有偏好性。我們嘗試利用急性束縛壓力模式來誘導小鼠對尼古丁有更明顯的渴求行為，其中我們試了不同時間點給予小鼠急性束縛壓力，包含條件配對下的壓力模式，條件配對前的壓力模式和條件配對後的壓力模式。接著，進而測試腹腔注射gabapentinoids (30 or 100 mg/kg)和SB334867 (orexin receptor antagonist, 15 mg/kg) 在場域性偏好試驗中對小鼠的影響。自主活動力試驗會被用來確認我們所給予的藥物劑量不會對小鼠活動力產生不良的影響。做完場域性偏好試驗後gabapentinoids組別的小鼠將會被犧牲，並取下大腦皮質組織和血漿樣本。使用西方墨點法分析壓力誘導尼古丁成癮的模式中，α2δ1次單元量的變化;並使用皮質酮酵素連結免疫吸附法來監測血漿中皮質酮的變化量。 實驗結果顯示，第一，小鼠可以用條件配對後壓力誘導模式，成功誘發尼古丁場域性偏好，我們發現給予小鼠兩天的尼古丁條件配對，無法使之產生尼古丁場域性偏好。但是在處於壓力的情形下，兩天的尼古丁條件配對卻可以成功使小鼠產生尼古丁場域性偏好。其他的模式中結果均是不成功的，在條件配對下壓力模式，ICR小鼠可能把尼古丁條件配對箱也就是非喜愛箱，和壓力做連結，產生懼怕的記憶;在條件配對前壓力模式下，ICR小鼠看起來對於環境比較敏感，具有較大的個體差異性，因此這兩種模式均無法有效的表現出壓力誘導尼古丁的場域偏好性。第二，我們發現，在給予C57BL/6小鼠急性束縛壓力前30分鐘，給予腹腔注射gabapentin (100 mg/kg) 可避免小鼠產生壓力誘發的尼古丁場域性偏好並且與對照組相比，此劑量的藥物並不造成小鼠的活動力下降;但是較低劑量的gabapentin (30 mg/kg) 和另一種gabapentinoids 藥物pregabalin (30 or 100 mg/kg) 以及orexin receptor antagonist SB334867這三種藥物處理的組別中均無此作用，推測orexin pathway應該不參與急性壓力誘導尼古丁的尋求行為。第三，大腦皮質中α2δ1次單元的量在給予小鼠急性束縛壓力後明顯增加，在經過gabapentin (100 mg/kg) 處理的組別中，因壓力而導致的α2δ1次單元增加量有明顯的下降;但較低劑量的gabapentin (30 mg/kg) 和pregabalin藥物的給予與否並不明顯影響壓力誘導增加的α2δ1次單元的量。最後，血漿中的皮質酮的量在壓力給予後也有明顯提升，但是無論是在給予尼古丁後或是給予藥物之後皮質酮的量並無顯著的被降低或是提高。這說明了急性尼古丁的給予並不會造成皮質酮量的改變，並且gabapentinoid藥物並不會減少因壓力增加的皮質酮的量。 總結，我們成功的建立了壓力誘發煙癮的小鼠動物模式。我們認為壓力給予前給予gabapentin (100 mg/kg) 腹腔注射可以避免壓力誘發的尼古丁渴求行為，機制或許是透過降低被壓力誘導增加的α2δ1次單元的量。這對於壓力誘發煙癮的病人可能具有臨床治療的潛力。另外α2δ1次單元在壓力誘發的尼古丁成癮中扮演著重要的角色，這是值得繼續進一步研究的。

Tobacco is one of the most abused substances in the world. Nicotine is considered the major component of tobacco that is responsible for addiction. Several preclinical also clinical data point to a positive correlation between exposure to stress and increased vulnerability to drug addiction. Acute stress can potentiate nicotine craving, increasing rewarding effects, even increasing relapse risk of smoking. The mechanisms that underlie this relationship are, however, unclear. Recent data suggest that L-type voltage-gated calcium channel alpha 2 delta 1 subunit (α2δ1) is increased after chronic abuse drug exposure. Hence, we believed that α2δ1 subunit expression might involve in acute stress induced nicotine seeking behavior. Previous studies in our lab indicated that acute restraint stress can increase corticosterone level in mice plasma, and activate orexin neuron in lateral hypothalamic (LH), lead to orexin downstream pathway activation. However, there is study focusing on the association of stress-induced nicotine seeking with α2δ1 subunit, corticosterone and orexin. Therefore, we try to investigate their associations in this study. Gabapentin and pregabalin, both are gamma-aminobutyric acid (GABA) analog, also called gabapentinoid, are a kind of prescriptive drugs. They are used to relieve neuropathic pain, treat epilepsy and anxiety. They have high affinity at the α2δ1 subunit of voltage-dependent calcium channels. Some studies indicated that when binding to the α2δ1 subunit, they reduce calcium current, modify neuronal function, and may be involved in drug addiction. To investigate if gabapentinoids can be as a new indication for stress-induced nicotine addiction patient, we use animal model to conduct below experiment. First, we use conditioned place preference (CPP) test to establish an effective model of stress-induced nicotine CPP in C57BL/6 and ICR mice. The time differences in preferred chamber and in non-preferred chamber is defined CPP score, it can judge that whether the mice seeking to the drug. We tried different time when should be given restraint stress, including conditioning-paired stress model, pre-conditioning stress model and post-conditioning stress induced nicotine CPP model. Then, we used acute restraint stress model to induce nicotine craving in mice and investigated whether gabapentinoids (30 or 100 mg/kg, i.p.) and SB334867 (orexin receptor antagonist, 15 mg/kg, i.p.) were effective in this model. Spontaneous locomotor activity test was also examined to make sure effects of tested drugs are not due to an impairment of motor activity. After conducting CPP experiment, mice in the gabapentinoids group were sacrificed and collected cortex tissue and plasma sample respectively. The western blot test was used for examining whether the level of α2δ1 subunit change in the cortex during the stress-induced nicotine CPP. Corticosterone levels in the plasma were also analyzed by a corticosterone ELISA kit. The results suggested that firstly, an acute restraint stress can induce nicotine CPP in mice when stress was applied after nicotine conditioning. That is, mice did not develop nicotine CPP after 2 days’ nicotine-pairing but expressed nicotine CPP under stressful condition. In the conditioning-paired stress model, mice may produce fearing memory to induce an association between the non-preferred chamber and stress environment. In the pre-conditioning stress model, ICR mice seemed to be more vulnerable to the environment. They were easily induced nicotine CPP with big variation individually. Thus, it is not easy to establish a stress-induced nicotine CPP model in ICR mice. Second, pretreatment of 100 mg/kg, but not 30 mg/kg (i.p.) of gabapentin, neither pregabalin nor orexin receptor antagonist (SB334867, 15 mg/kg, i.p.), can prevent acute stress-induced nicotine craving. All of these drugs had no effects on locomotor activity. Third, the level of α2δ1 subunit in the cerebral cortex was not changed after 2 days’ nicotine-pairing, consistent with a previous study where chronic, but not acute, nicotine administration increased the α2δ1 level in the cortex. However, after receiving acute restraint stress, the α2δ1 level in mouse cerebral cortex was significantly increased, and pretreatment with gabapentin (100 mg/kg) significantly decreased the elevated α2δ1 levels. Neverless, in gabapentin (30 mg/kg) and pregabalin groups, the α2δ1 level was not significantly changed in the stress group. Finally, the corticosterone level in the plasma of mice was increased after receiving stress. However, the elevated corticosterone level was not significantly changed during or after nicotine treatment in the stress-induced nicotine CPP experiment. It suggested that acute nicotine administration didn’t change the level of corticosterone, and the pretreatment of drug can’t decreased corticosterone level induced by acute restraint stress. Taken together, we have established an acute restraint stress-induced nicotine CPP model by giving C57BL/6 mice a 30 min-restraint stress after 2 day nicotine CPP training. Our results for the first time showed that gabapentin (100 mg/kg) can prevent stress-induced nicotine CPP, suggesting gabapentin may be a potential therapeutic agent for stress-induced nicotine craving. Its action mechanism may be mediated through normalizing the elevated α2δ1 level in the brain induced by acute restraint stress. Corticosterone and the orexin system may not be involved in acute stress-induced nicotine seeking behavior.