Central amygdala input circuits control stress-induced anxiety after chronic ETOH

描述(申请人提供)：在戒酒期间，酗酒者的压力会导致负面情绪和渴望(1)，这种反应伴随着大脑功能磁共振成像(FMRI)的变化--压力的影响在社交饮酒者中看不到(2)。为了模拟戒酒期间应激引起的负面影响，长期接触慢性间歇性酒精(CIA)可以引起持久的适应，从而敏化戒酒期间应激诱导的负面影响。这一系列的临床和基本发现与酒精中毒的点燃/应激假说一致。虽然已知中央杏仁核(CEA)在中情局暴露后应激诱导的负面情绪中发挥重要作用，但尚不清楚CEA的输入回路在促进这种应激诱导的焦虑中所起的作用。除CRF外，CEA神经元的突触还有来自杏仁基底外侧核(BLA)的谷氨酸(GLU)终末、来自下丘脑的催产素(Oxy)终末和加压素(VP)终末。然而，在中情局暴露后，这些其他输入对应激诱导的焦虑的影响还没有确定。同样未知的是，向中脑导水管周围灰质(PAG)和其他部位输出终末的CEA内侧区(CEM)神经元是否是支持与CIA相关的应激性焦虑的神经回路的关键组成部分。为了指导解决这些未知因素，检验的假设是，压力通过影响由调节CEM输出的CEA外侧(CEL)和内侧(CEM)分区的终端输入组成的神经电路，在CIA暴露禁欲期间促进焦虑。首先，为了研究BLA终末在CIA暴露后促进应激性焦虑中的潜在作用，将含有视紫红质衍生物卤视紫质(NpHR3.0)或通道视紫红质(ChR2)的带有CaMKII启动子的AAV5-EYFP载体置于BLA中，以确认GLU终末从BLA到CEA外侧(CEL)和内侧(CEM)的定位。随后的光遗传学测试将是ChR2的激活或NpHR3.0抑制CEL神经元上GLU的BLA末端释放是否会影响CIA暴露后应激性焦虑的易化。后一种光发生策略将通过确定用Oxy受体激动剂激活CEL神经元是否会阻止CIA暴露后应激诱导的焦虑的易化来补充。在完成对CEL神经输入的研究后，对CEM神经元上含GLU的BLA终末突触的光发生抑制或兴奋将评估CIA暴露后应激诱导的焦虑效应是否受到影响。由于含有Vp的终末突触在CEM神经元上诱导焦虑样行为，推测Vp激活CEM神经元可能是CIA暴露后应激诱发焦虑的原因之一。为了证实CEM神经元直接参与VP的作用，在CEM神经元中表达NpHR3的AAV-EYFP载体将测试CEM神经元的光遗传抑制是否会改变CIA暴露后应激和VP诱导的焦虑。为了证实中情局暴露后应激性焦虑的发生与VP的作用有关，在应激前的中脑皮质对VP受体亚型进行药理拮抗，以确定中情局暴露后的应激性焦虑是否被预防。一旦证实CEM终末存在于PAG中，将通过光遗传抑制或兴奋这些CEM终末来评估这些CEM终末在CIA暴露后观察到的应激和VP诱导的焦虑中的参与-这是一种涉及CEM和PAG在CIA暴露后促进应激诱导焦虑的方法。总而言之，所提出的药理学和光遗传策略可以确定，CEL和CEM的特定神经输入，以及PAG和其他部位的CEM输出，是否形成影响CIA暴露后应激诱导的负面情绪易化的回路。这一涉及CEA的拟议回路被认为与神经病理有关，该神经病理学负责观察到的对压力的易化负面影响，促进了酗酒者在戒酒期间的渴望。关于CIA暴露后这一回路适应的新知识可能会提供线索，以最大限度地减少开发新的和改进的治疗方法的障碍，以治疗戒酒者观察到的应激反应的负面症状。

DESCRIPTION (provided by applicant): During abstinence from alcohol, stress in alcoholics can result in negative affect and craving (1), a response which is accompanied by a change in brain functional magnetic resonance imaging (fMRI)-effects of stress not seen in social drinkers (2). To model the negative affect induced by stress during abstinence in alcoholics, an extended period of chronic intermittent alcohol (CIA) exposure was found to cause an enduring adaptation that sensitizes stress-induced negative affect during abstinence. These series of clinical and basic findings are consistent with the kindling/stress hypothesis of alcoholism. Even though the central amygdala (CeA) is known to play an important role in stress-induced negative affect after CIA exposure, unknown is the role input circuits to the CeA have in facilitating this stress-induced anxiety. In addition to CRF, other inputs that synapse on CeA neurons are glutamate (GLU) terminals from the basolateral amygdala (BLA), oxytocin (OXY) terminals from the hypothalamus, and vasopressin (VP ) terminals. Nonetheless, the influence these other inputs have on stress-induced anxiety after CIA exposure has not been identified. Likewise unidentified is whether CeA medial division (CEM) neurons that output terminals to the periaqueductal gray (PAG) and other sites are a critical component of the neural circuit that supports stress-induced anxiety associated with CIA. To guide addressing these unknowns, the hypothesis tested is that stress facilitates anxiety during abstinence from CIA exposure by influencing a neural circuit composed of terminal inputs to the lateral (CEL) and medial (CEM) divisions of the CeA that modulate CEM output. First, to permit optogenetic investigations of the potential role BLA terminals have in facilitating stress-induced anxiety after CIA exposure, an AAV5-eYFP vector with a CAMKII promoter containing the rhodopsin derivatives halorhodopsin (NpHR3.0) or channel- rhodopsin (ChR2) will be placed into the BLA to confirm localization of GLU terminals from the BLA to the lateral (CEL) and the medial (CEM) divisions of the CeA. Subsequently tested with optogenetics will be whether ChR2 activation or NpHR3.0 inhibition of BLA terminal release of GLU on CEL neurons will influence facilitation of stress-induced anxiety after CIA exposure. This latter optogenetic strategy will be complemented by determining if activation of CEL neurons with an OXY receptor agonist will block facilitation of stress-induced anxiety after CIA exposure. Upon completing investigations of CEL neural inputs, optogenetic inhibition or excitation of GLU-containing BLA terminals synapsing on CEM neurons will assess if the stress-induced anxiogenic action after CIA exposure is affected. Because VP-containing terminals synapse on CEM neurons to induce anxiety- like behavior, it is reasoned that activation of CEM neurons with VP may contribute to the anxiety induced by stress following CIA exposure. To confirm a direct involvement of CEM neurons in VP action, an AAV-eYFP vector with the NpHR3 expressed in CEM neurons will test if optogenetic inhibition of CEM neurons will alter both stress- as well as VP-induced anxiety after CIA exposure. To confirm that VP action contributes to stress-induced anxiety after CIA exposure, VP receptor subtypes will be pharmacologically antagonized in the CEM prior to stress to determine if the stress- induced anxiety after CIA exposure is prevented. Upon confirmation of CEM terminal presence in the PAG, involvement of these CEM terminals in the PAG in stress- and VP-induced anxiety observed after CIA exposure will be assessed by optogenetic inhibition or excitation of these CEM terminals- an approach to implicate both the CEM and the PAG in facilitation of the stress-induced anxiety after CIA exposure. Collectively, the proposed pharmacological and optogenetic strategies utilized can be expected to define whether involvement of specific neural inputs to the CEL and CEM, which are accompanied by CEM output to the PAG and other sites, form a circuit that influences facilitation of stress-induced negative affect after CIA exposure. This proposed circuit involving the CeA is felt to be associated with the neuropathology responsible for the facilitated negative affect observed to stress that facilitates craving in alcoholics durin abstinence. New knowledge concerning adaptations in this circuit after CIA exposure could possibly provide clues by which to minimize the barrier to developing new and improved therapeutics to treat the negative symptoms to stress observed in abstinent alcoholics.