Calcium Imaging of Central Amygdala Activity after Fentanyl Escalation

有证据表明，给予长时间接触(LGA)会话的大鼠自我给药(SA)类阿片类药物会使其 摄入，同时也显示出比大鼠更严重的戒断严重程度和药物诱导的恢复 在短时间访问(SAA)每日SA会话中维护。人们对此过程中发生的神经变化知之甚少 可能影响戒断和复发的偶发阿片类药物升级。过去使用SHA检查阿片类药物SA的工作 啮齿动物的SESSION已将中央杏仁核(CEA)确定为变得过度活跃的感兴趣区 在急性戒断中，可能参与了长期戒断后的渴求潜伏期。 然而，这些研究还没有研究使用LGA疗程增加阿片类药物摄入量对CEA的影响 在急性戒断期间的活动，当个体经历强烈的渴望和负面情绪时，或者 在长期禁欲期间长期戒断后，当个人经历更多 易复发。此外，还没有研究确定CEA活动的变化是否取决于 意外或仅因暴露于芬太尼本身而自行使用的芬太尼摄入量增加， 即使它是非偶然的给药。这项研究的具体目的是确定或有 芬太尼在急性戒断(目标1)或延长戒断后改变基础体外CEA活性 退出(目标2)。体外应用芬太尼对大鼠CEA脑片活性的影响 急性或长期戒断也将被确定。雄性和雌性Spraogue Dawley大鼠将被分为 分为三组：(1)联合芬太尼，(2)联合芬太尼，或(3)联合生理盐水。芬太尼麻醉中的大鼠 两组均接受7d芬太尼(2.5ug/kg/inf)静脉注射和21d 6h SA。带轭的老鼠将会收到 非偶然性输注芬太尼或生理盐水取决于偶发性芬太尼大鼠的反应。 在钙(Ca~(2+))成像的准备过程中，大鼠将在戒断后17小时或在戒断后30天被安乐死。 强迫禁欲。CA2成像将在基线和芬太尼(0.1um或1um)存在的情况下进行 以确定CEA钙荧光的差异，这是一种衡量神经活性的指标。我们假设，在两种情况下 在戒断阶段，芬太尼大鼠的钙瞬变时间将比束缚的生理盐水大鼠更大； 芬太尼大鼠也可能表现出比偏曲的芬太尼大鼠更大的效果。我们还假设，在这两个阶段 在禁欲后，所有的大鼠都会表现出芬太尼剂量依赖性的钙瞬变减少，这一效应将 生理盐水大鼠的作用大于芬太尼大鼠。最后，我们预测将观察到更大的基础CEA活动。 长期戒断后与急性戒断相比，特别是临时芬太尼大鼠。正在研究的 应用钙成像技术研究阿片类药物升级后戒断和复发的不同机制 并将阐明CEA在OUD中驱动戒断和复发的作用。这样做的长期目标是 工作是为OUD的新治疗方法的开发提供信息，特别是那些可能针对CEA到 减少在急性戒断和整个禁欲过程中出现的负面情绪和渴望。

Evidence suggests that rats given long access (LgA) sessions to self-administer (SA) opioids escalate their intake, while also showing greater withdrawal severity and drug-induced reinstatement compared to rats maintained on short access (ShA) daily SA sessions. Little is known about the neural changes that occur during contingent opioid escalation that may impact withdrawal and relapse. Past work examining opioid SA using ShA sessions in rodents has identified the central amygdala (CeA) as an area of interest that becomes hyperactive in acute withdrawal and may be involved in the incubation of craving that occurs after protracted withdrawal. However, these studies have not examined the impact of escalated opioid intake using LgA sessions on CeA activity during acute withdrawal, when individuals experience heightened craving and negative emotionality, or following protracted withdrawal during extended abstinence, when individuals experience an increased susceptibility to relapse. Furthermore, no study has determined if changes in CeA activity depend on the escalation of fentanyl intake that is self-administered contingently or merely from exposure to fentanyl per se, even if it were administered non-contingently. The specific aims of this research are to determine if contingent fentanyl escalation alters basal ex vivo CeA activity following either acute withdrawal (Aim 1) or protracted withdrawal (Aim 2). The effect of ex vivo application of fentanyl on the activity of CeA slices obtained following acute or protracted withdrawal will also be determined. Male and female Sprague Dawley rats will be divided into three groups: (1) contingent fentanyl, (2) yoked fentanyl, or (3) yoked saline. Rats in the contingent fentanyl group will undergo 7 days of fentanyl (2.5 ug/kg/inf) acquisition and 21 days of 6 h SA. Yoked rats will receive non-contingent infusions of fentanyl or saline dependent upon responding emitted by the contingent fentanyl rat. In preparation for calcium (Ca2+ ) imaging, rats will be euthanized either 17 h into withdrawal or after 30 days of forced abstinence. Ca2+ imaging will be conducted at baseline and in the presence of fentanyl (0.1 uM or 1 uM) to determine differences in CeA Ca2+ fluorescence, a measure of neural activity. We hypothesize that, at both stages of abstinence, fentanyl rats will demonstrate greater Ca2+ transients than yoked saline rats; contingent fentanyl rats may also display a greater effect than yoked fentanyl rats. We also hypothesize that, at both stages of abstinence, all rats will demonstrate a fentanyl, dose-dependent decrease in Ca2+ transients, an effect that will be greater in saline rats than fentanyl rats. Finally, we predict that greater basal CeA activity will be observed after protracted withdrawal compared to acute withdrawal, particularly for contingent fentanyl rats. Studying the mechanisms underlying differences in withdrawal and relapse after opioid escalation using Ca2+ imaging is highly novel and will elucidate the role of the CeA in driving withdrawal and relapse in OUD. The long-term goal of this work is to inform the development of novel treatments for OUD, particularly those that may target the CeA to reduce the negative emotionality and craving that occurs during acute withdrawal and throughout abstinence.