Pathway-specific Intervention in Prelimbic Cortical Circuitry Decreases Cocaine-seeking

具体目标 对于患有物质使用障碍（SUD）的个人来说，一个主要的挑战是缺乏有效的药物治疗。 减少复发脆弱性的治疗。环境中的药物预测线索是 复发和理解这些持久的联系是如何形成和维持的是一个关键的焦点， 临床前SUD研究。在这一建议中，我们的基础是我们的发现，即在结束后不久， 可卡因自身给药（SA），将脑源性神经营养因子（BDNF）注入前边缘系统（PL） 前额叶皮质阻止可卡因SA诱导的关键神经元可塑性的去磷酸化， 相关蛋白（PRP），包括GluN 2A、GluN 2B、ERK MAP激酶和CREB 32，96。这种早期干预 与BDNF的结合也可以防止可卡因诱导的PL-NA核心神经递质传递的长期缺陷， 促进随后的可卡因寻求9.相反，在禁欲的第一周结束时，蛋白激酶A （PKA）依赖性GluA 1和CREB磷酸化增强出现。此时，PL内BDNF 对复发没有影响8，但PL内输注PKA抑制剂Rp-cAMPs， 过度磷酸化和减少复发70，92.最近，我们已经表明，双相变化， 戒断第一周内的GluA 1和pCREB与头部类似的双相变化相关 PL-NA核心神经元的树突棘的直径（dH）89.这些数据激发了一种整体假设 减少药物寻求的干预措施必须适应神经适应的动态变化， 在成瘾周期的不同阶段出现。 在化学遗传学研究中，研究源自PL皮质的特定途径对药物代谢的贡献。 通过对PL-NA核心通路和PL-丘脑室旁后核（pPVT）通路的研究，发现PL-NA核心通路和PL-丘脑室旁后核（pPVT 在提前退出时相互抵消影响。PL-NA的选择性cre依赖性DREADD抑制 在可卡因SA后立即用逆行转运的cre-AAV感染核心神经元， 其本身，但逆转PL内BDNF对随后药物寻找的抑制作用31。与此相反的是， 在可卡因SA后立即选择性抑制PL-pPVT途径减少了随后的可卡因- 寻找，这是由PL内BDNF 31阻止的效果。有趣的是，我们还发现，选择性抑制 PL-pPVT通路减少了戒断可卡因大鼠的焦虑相关行为， pPVT降低了对可卡因的条件性厌恶，这表明可卡因对焦虑的参与- 导致药物寻求的厌恶相关回路包括pPVT。 综上所述，我们的新发现提出了一个新的假设，即可卡因SA产生了差异化的 PL-NA核心和PL-pPVT途径的调节，以及这两种不同的回路共同支持未来 药物寻求在这个建议中，我们将使用特定的途径，组合化学遗传学方法，切片 PL-NA核心和PL-pPVT电路中的电生理学和最先进的树突棘和PRP成像 探索在这个网络中发生的影响可卡因寻求和厌恶的时间依赖性可塑性。 我们将使用这些方法在雄性和雌性大鼠中进行以下研究。 目标1. BDNF对PL-NA核心与PL-pPVT中PRPs的调节以及结构和突触可塑性 神经元在早期撤退。使用病毒载体的方法，我们将研究内PL的影响， BDNF输注对（A）树突棘可塑性和PRP变化以及（B）PL-NA核心与 早期戒断时的PL-PVT神经元。 目标二。PL-NA核心与PL-pPVT中PRPs的PKA调节以及结构和突触可塑性 神经元后7和30天的禁欲。(A)我们将研究抑制cAMP/PKA的作用， 信号传导对（A）树突棘可塑性和PRP变化和（B）PL-NA核心相对于PL-NA核心的突触可塑性的影响。 在戒断7或30天后，进行或不进行复发测试。 目标3：PL-NA核心和PL-pPVT神经元在可卡因诱导的焦虑和厌恶中的作用 与可卡因的关系寻求禁欲。我们将使用一个复古的Gi DREADD方法来测试 抑制PL-NA核心或PL-pPVT神经元（A）对戒断诱导的焦虑的作用 结束可卡因SA和（B）对可卡因的条件性回避在操作性跑道任务之前或之后 可卡因SA，然后进行线索诱导复发测试。 科学影响：这些研究将扩大我们对如何逆转关键神经适应的理解 在成瘾周期的不同阶段，潜在的前额叶功能障碍会引发药物复发， 寻找通过了解PL网络调节中的时间动态、差异神经适应， 奖励和厌恶，我们也许能够发现新的治疗靶点，以减少药物寻求。

Specific Aims A major challenge for individuals suffering from substance use disorders (SUDs) is the lack of effective treatments that reduce relapse vulnerability. Drug predictive cues in the environment are powerful triggers for relapse and understanding how these persistent associations are formed and maintained is a critical focus of preclinical SUD research. In this proposal, we have built on our discovery that immediately after the end of cocaine self administration (SA), an infusion of brain-derived neurotrophic factor (BDNF) into the prelimbic (PL) prefrontal cortex prevents the cocaine SA-induced dephosphorylation of key glutamatergic-related plasticity- related proteins (PRPs), including GluN2A, GluN2B, ERK MAP kinase, and CREB32,96. This early intervention with BDNF also prevents prolonged cocaine-induced deficits in PL-NA core glutamatergic transmission that promote subsequent cocaine seeking9. In contrast, by the end of the first week of abstinence, protein kinase A (PKA)-dependent augmentation of GluA1 and CREB phosphorylation emerges. At that time, intra-PL BDNF has no effect on relapse8 but intra-PL infusion of a PKA inhibitor, Rp-cAMPs, reverses the hyperphosphorylation and decreases relapse70,92. More recently, we have shown that the biphasic changes in GluA1 and pCREB within the first week of abstinence are associated with similar biphasic changes in the head diameters (dH) of dendritic spines of PL–NA core neurons89. These data have spurred the overall hypothesis that interventions to decrease drug seeking must be tailored to the dynamic changes in neuroadaptations that emerge during different phases of the addiction cycle. In chemogenetic studies to investigate the contribution of specific pathways originating in PL cortex to drug seeking, we discovered that PL-NA core and PL-posterior paraventricular thalamic nucleus (pPVT) pathways oppose each others’ effects during early withdrawal. Selective cre-dependent DREADD inhibition of PL-NA core neurons infected with a retrogradely transported cre-AAV immediately after cocaine SA has no effect by itself, but reverses the suppressive effect of intra-PL BDNF on subsequent drug seeking31. In contrast, selective inhibition of the PL-pPVT pathway immediately after cocaine SA decreases subsequent cocaine- seeking, an effect that is prevented by intra-PL BDNF31. Interestingly, we also found that selective inhibition of the PL-pPVT pathway reduces anxiety-related behavior in rats withdrawing from cocaine and inactivation of pPVT decreases conditioned aversion to cocaine, suggesting that cocaine’s engagement of anxiety- and aversion-related circuitry that contributes to drug seeking includes pPVT. Taken together, our new findings suggest the novel hypothesis that cocaine SA produces differential regulation of PL-NA core and PL-pPVT pathways and that these two distinct circuits conspire to support future drug seeking. In this proposal, we will use pathway-specific, combinatorial chemogenetic approaches, slice electrophysiology, and state-of-the-art dendritic spine and PRP imaging in PL-NA core and PL-pPVT circuitry to explore the time-dependent plasticity occurring in this network that influences cocaine seeking and aversion. We will use these approaches in the following aims that will be performed in male and female rats. Aim 1. BDNF regulation of PRPs and structural and synaptic plasticity in PL-NA core vs. PL-pPVT neurons during early withdrawal. Using viral vector approaches, we will investigate the effects of intra-PL BDNF infusion on (A) dendritic spine plasticity and PRP changes and (B) synaptic plasticity in PL-NA core vs. PL-PVT neurons during early withdrawal. Aim 2. PKA regulation of PRPs and structural and synaptic plasticity in PL-NA core vs. PL-pPVT neurons after 7 and 30 days of abstinence. (A) We will investigate the effects of inhibiting cAMP/PKA signaling on (A) dendritic spine plasticity and PRP changes and (B) synaptic plasticity in PL-NA core vs. PL- PVT neurons after 7 or 30 days of abstinence with or without relapse testing. Aim 3. The role of PL-NA core and PL-pPVT neurons in cocaine-induced anxiety and aversion in relationship to cocaine seeking after abstinence. We will use a retro-Gi DREADD approach to test the effects of inhibiting PL-NA core or PL-pPVT neurons (A) on withdrawal-induced anxiety immediately after the end of cocaine SA and (B) on conditioned avoidance of cocaine in an operant runway task before or after cocaine SA followed by cue-induced relapse testing. Scientific Impact: These studies will expand our understanding of how to reverse critical neuroadaptations underlying prefrontal dysfunctions during different phases of the addiction cycle that trigger relapse to drug- seeking. By understanding temporally dynamic, differential neuroadaptations in the PL networks regulating reward and aversion, we may be able to discover novel therapeutic targets to decrease drug-seeking.