DOPAMINE/GLUTAMATE INTERACTIONS IN NUCLEUS ACCUMBENS

描述：（来自申请人摘要） 了解和治疗药物成瘾的一个重要的新假设是 这可能是一种不恰当的学习方式。这个假设是基于 研究表明，谷氨酸，一种对学习和 记忆，是神经适应性发展所必需的， 是成瘾和复发的基础我们已经证明，重复管理 精神兴奋剂（安非他明或可卡因）的使用会导致 谷氨酸系统。这些包括AMPA受体表达的改变， 神经核（Nac）的反应，这是一个对大脑的反应至关重要的大脑区域。 精神兴奋剂的奖励和成瘾特性。中型多棘 含有GABA的神经元，占所有神经元的95%， 来自中脑多巴胺（DA）神经元和谷氨酸神经元的会聚输入 起源于大脑皮层和边缘区域我们的长期目标是了解 最初针对多巴胺转运蛋白的精神兴奋剂， 产生适应性的谷氨酸盐传输。这是一个重要 问题，因为谷氨酸系统很可能介导神经元 从药物实验到药物治疗的可塑性 依赖不幸的是，我们对多巴胺和多巴胺受体之间的慢性相互作用知之甚少。 和谷氨酸受体。逻辑起点是确定 急性DA受体刺激如何影响谷氨酸传递。这 本申请将使用出生后的Nac培养物来检验DA 受体调节AMPA受体亚基GluR 1的磷酸化。我们 之所以关注AMPA受体，是因为它们是 兴奋性传递中的中型多刺神经元和磷酸化作为一个 调节机制，因为GluR 1磷酸化导致显著增强 AMPA受体介导的电流。将检测GluR 1的磷酸化 使用GluR 1磷酸化位点特异性抗体， 理查德·胡加尼尔一种选择性识别Ser-845上磷酸化的GluR 1 [蛋白激酶A（PKA）位点]。另一个识别磷酸化的GluR 1， ser-831 [蛋白激酶C（PKC）/Ca 2 + -钙调蛋白依赖性蛋白激酶型 II（CaMKII）位点]。第一个目的是表征调节基础 GluR 1的磷酸化和参与其磷酸化的激酶。 首先，将对照培养物与在不存在培养基中孵育的培养物进行比较。 Ca 2+，突触活动的缺乏（TTX，荷包牡丹碱，MK-801，CNQX）， 无抑制活性（荷包牡丹碱），无兴奋性 活性（MK-801，CNQX）。其次，我们将使用PKA的激活剂和抑制剂， PKC和CaMKII来确定哪些激酶使我们培养物中的GluR 1磷酸化。 第二个目的是确定DA受体刺激是否调节GluR 1 磷酸化实验将被设计为检测刺激和 D1和D2 DA受体激活的抑制作用。的身份 残基磷酸化，结合涉及选择性蛋白质的实验 激酶抑制剂，将有助于确定激酶参与DA 受体介导的效应。

DESCRIPTION: (from applicant's abstract) An important new hypothesis for understanding and treating drug addiction is that it may be an inappropriate form of learning. This hypothesis is based on studies demonstrating that glutamate, a transmitter critical for learning and memory, is required for the development of neuroadaptations believed to underlie addiction and relapse. We have shown that the repeated administration of psychostimulants (amphetamine or cocaine) causes profound alterations in glutamate systems. These include alterations in AMPA receptor expression and responsiveness in the nucleus accumbens (Nac), a brain region critical for the rewarding and addictive properties of psychostimulants. Medium spiny GABA-containing neurons, which represent 95% of all neurons in the Nac, receive convergent inputs from midbrain dopamine (DA) neurons and glutamate neurons originating in cortex and limbic regions. Our long-term goal is to understand how psychostimulants, which initially target the DA transporter, ultimately produce adaptations in glutamate transmission in the Nac. This is an important question, because glutamate systems are likely to mediate the neuronal plasticity underlying the transition from drug experimentation to drug dependency. Unfortunately, we know little about chronic interactions between DA and glutamate receptors in the Nac. A logical starting point is to determine how acute DA receptor stimulation can influence glutamate transmission. This application will use postnatal Nac cultures to test the hypothesis that DA receptors modulate the phosphorylation of the AMPA receptor subunit GluR1. We are focusing on AMPA receptors because they are the primary mediators of excitatory transmission in medium spiny neurons and on phosphorylation as a regulatory mechanism because GluR1 phosphorylation leads to marked enhancement of AMPA receptor-mediated currents. Phosphorylation of GluR1 will be detected using phosphorylation site-specific antibodies to GluR1 developed by Dr. Richard Huganir. One selectively recognizes GluR1 phosphorylated on ser-845 [protein kinase A (PKA) site]. The other recognizes GluR1 phosphorylated on ser-831 [protein kinase C (PKC)/Ca2+ -calmodulin dependent protein kinase type II (CaMKII) site]. The first Aim is to characterize conditions regulating basal phosphorylation of GluR1 and the kinases involved in its phosphorylation. First, control cultures will be compared with cultures incubated in the absence of Ca2+, the absence of synaptic activity (TTX, bicuculline, MK-801, CNQX), the absence of inhibitory activity (bicuculline), and the absence of excitatory activity (MK-801, CNQX). Second, we will use activators and inhibitors of PKA, PKC and CaMKII to determine which kinases phosphorylate GluR1 in our cultures. The second Aim is to determine if DA receptor stimulation modulates GluR1 phosphorylation. Experiments will be designed to detect both stimulatory and inhibitory effects of D1 and D2 DA receptor activation. The identity of the residue phosphorylated, combined with experiments involving selective protein kinase inhibitors, will help identify the kinases involved in DA receptor-mediated effects.