Investigating How Alcohol Withdrawal Dysregulates 'Reward' and 'Aversion' Neurons in the Basolateral Amygdala

杏仁基底外侧核(BLA)在酒精使用障碍(AUD)的发生发展中起关键作用。它的独特之处 在奖赏和厌恶相关回路中的位置使其能够调节酒精摄入量和戒断诱导 焦虑。谷氨酸能锥体神经元驱动这些行为反应，使得更高的锥体神经元 兴奋性使焦虑和寻求回报变得容易。锥体神经元接受来自中线的谷氨酸能传入 通过终纹(ST)的内侧前额叶皮质和更外侧的皮质区域等结构发出 谷氨酸能通过外囊(EC)输入。GABA能外侧囊旁细胞(LPCS)和 “局部”中间神经元调节锥体神经元。我的项目的目标是研究慢性间歇性 乙醇和戒断(CIE/WD)对投射到奖赏和厌恶相关区域的BLA神经元的调节失调 了解酒精摄入量增加的机制。伏隔核(NAC)参与了 终纹床核(BNST)调节焦虑。因此，我的项目 将把BLA-NAC神经元用作“奖赏”神经元，将BLA-BNST神经元用作“厌恶”神经元。我们的实验室 已证明WD通过ST输入的突触前和突触后机制增加谷氨酸能功能 和EC-BLA突触。我的初步数据显示，WD增加了ST的谷氨酸释放 传入BLA-BNST神经元不分性别，而WD介导的突触后功能增加 在EC-BLA，突触既是投射的，也是性别依赖的。我的初步数据还表明，WD会改变 GABA能传递受阻时BLA-NAC和BLA-BNST神经元的兴奋性在BLA-NAC中 神经元、女性具有较高的基础兴奋性，只有男性在CIE/WD后兴奋性增加。在BLA中- BNST神经元，WD增加兴奋性，不分性别。阻断谷氨酸和氨基丁酸能 传递消除了兴奋性的增加，强调了谷氨酸能传递在WD- 介导性的过度兴奋。这项提案将继续调查WD如何影响“奖励”和“厌恶” 大脑皮层神经元。具体目标1将使用电生理学和逆行标记技术检测GABA能 与BLA-NAC和BLA-BNST神经元的突触功能。实验室数据显示WD抑制LPC 局部神经元间突触的GABA释放和突触后GABA能功能。此外，降低了LPC GABA的释放是男性独有的。特殊目标1还将检查GABA能突触的结构可塑性 用免疫组织化学、共聚焦显微镜和逆行方法观察BLA-NAC和BLA-BNST神经元 贴标签。特定目标2将结合行为方法和化学遗传学来抑制特定的BLA回路 并评估他们在CIE后酒精摄入中的作用。这些实验将确定“奖励”和“厌恶” BLA神经元经历了明显的神经生理和结构变化，导致酒精摄入量增加 在CIE/WD之后。识别导致酒精摄入量增加的机制可能会改善对AUD的治疗。

The basolateral amygdala (BLA) is critical to the development of alcohol use disorder (AUD). Its unique position within reward- and aversion-related circuitry allows it to regulate alcohol intake and withdrawal-induced anxiety. Glutamatergic pyramidal neurons drive these behavioral responses such that higher pyramidal neuron excitability facilitates anxiety and reward-seeking. Pyramidal neurons receive glutamatergic inputs from midline structures like the medial prefrontal cortex through the stria terminalis (ST) and more lateral cortical areas send glutamatergic inputs through the external capsule (EC). GABAergic lateral paracapsular cells (LPCs) and ‘local’ interneurons regulate pyramidal neurons. The goal of my project is to examine how chronic intermittent ethanol and withdrawal (CIE/WD) dysregulates BLA neurons projecting to reward and aversion-related regions to understand the mechanisms driving increased alcohol intake. The nucleus accumbens (NAC) is involved in reward-seeking while the bed nucleus of the stria terminalis (BNST) regulates anxiety. Therefore, my project will use BLA-NAC neurons as ‘reward’ neurons and BLA-BNST neurons as ‘aversion’ neurons. Our laboratory has shown that WD increases glutamatergic function through pre- and postsynaptic mechanisms at ST inputs and EC-BLA synapses, respectively. My preliminary data reveal that WD increases glutamate release from ST inputs onto BLA-BNST neurons regardless of sex, whereas the WD-mediated increase in postsynaptic function at EC-BLA synapses is both projection- and sex-dependent. My preliminary data also indicate that WD alters the excitability of BLA-NAC and BLA-BNST neurons when GABAergic transmission is blocked. In BLA-NAC neurons, females have higher basal excitability and only males increase excitability after CIE/WD. In BLA- BNST neurons, WD increases excitability, regardless of sex. Blocking glutamatergic and GABAergic transmission abolished the increase in excitability, emphasizing the role of glutamatergic transmission in WD- mediated hyperexcitability. This proposal will continue to investigate how WD impacts ‘reward’ and ‘aversion’ BLA neurons. Specific Aim 1 will employ electrophysiology and retrograde labeling to examine GABAergic function in synapses with BLA-NAC and BLA-BNST neurons. Laboratory data reveal that WD suppresses LPC GABA release and postsynaptic GABAergic function in ‘local’ interneuron synapses. Moreover, reduced LPC GABA release is unique to males. Specific Aim 1 will also examine structural plasticity in GABAergic synapses with BLA-NAC and BLA-BNST neurons using immunohistochemistry, confocal microscopy, and retrograde labeling. Specific Aim 2 will combine behavioral approaches and chemogenetics to inhibit specific BLA circuits and evaluate their role in post-CIE alcohol intake. These experiments will establish that ‘reward’ and ‘aversion’ BLA neurons undergo distinct neurophysiological and structural changes, leading to increased alcohol intake after CIE/WD. Identifying the mechanisms driving increased alcohol intake could improve treatments for AUD.