Regulation of voltage-gated calcium channels during chronic BZ treatment in rats

大鼠慢性 BZ 治疗过程中电压门控钙通道的调节

• 批准号: 8247019
• 负责人: Damien Eugene Earl
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247019
• 关键词: Accident and Emergency department; Address; Adolescent; Aminobutyric Acids; Anxiety; Anxiety Disorders; Benzodiazepines; Brain; Calcium; Calcium Channel; Calcium Channel Inhibition; Cells; Central Nervous System Depressants; Chronic; Clinical; Dependence; Diazepam; Dihydropyridines; Electrons; Embryo; Epilepsy; Ethanol; Excision; Fluorescent Dyes; Flurazepam; Fura-2; Glutamate Receptor; Half-Life; High voltage activated calcium channel; Hippocampus (Brain); Human; Image; Imaging Techniques; Immunoblotting; Kidney; Label; Lead; Light; Link; Measures; Mediating; Membrane; Membrane Potentials; Methods; Microscopic; Modeling; Monitor; Neuraxis; Neurons; Nimodipine; Patch-Clamp Techniques; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Physical Dependence; Physiological; Play; Protocols documentation; Rattus; Recombinants; Regulation; Reporting; Role; Sleeplessness; Slice; Societies; Substance Withdrawal Syndrome; Surface; T-Lymphocyte; Techniques; Testing; Time; Visit; Water; Withdrawal; density; dihydropyridine; drug of abuse; drug withdrawal; gamma-Aminobutyric Acid; hippocampal pyramidal neuron; in vivo; kidney cell; new therapeutic target; nonmedical use; novel; prevent; prototype; ratiometric; receptor; receptor function; trafficking; voltage

DESCRIPTION (provided by applicant): Benzodiazepines (BZs) enhance inhibitory gamma-aminobutyric acid type A receptors (GABARs) in the central nervous system and are clinically used to treat insomnia, anxiety, and seizure disorders. Misuse of CNS depressant drugs, such as the BZs, is of growing concern. There has been increased non-medical use of CNS depressants among adolescents since 2001 and BZ misuse and abuse resulted in over 150,000 emergency room visits in 2004. Of particular concern is the fact that chronic BZ use can lead to physical dependence manifested by a withdrawal syndrome, which limits the clinical usefulness of BZs. Our lab models BZ dependence using a well-established protocol of chronic treatment of rats with the BZ, fluraze- pam (FZP). We have linked withdrawal anxiety to enhanced excitatory receptor function in the rat hippocam- pal CA1 region. We also discovered that voltage-gated calcium channel (VGCC) current density doubles during chronic FZP treatment and remains elevated following withdrawal. Interestingly, selectively blocking calcium influx through L-type VGCCs with nimodipine prevented the excitatory receptor changes and associated withdrawal anxiety, suggesting that L-VGCCs play a critical role in BZ withdrawal phenomena. The overall objective of this project is to determine the mechanisms of L-VGCC modulation during FZP treatment and withdrawal, as this could suggest a previously unknown mechanism of developing BZ physiological dependence. The specific aims of this project are to: 1) Determine if GABAR-mediated depolarization, known to be induced by chronic BZ treatment, activates L- VGCCs during FZP withdrawal using electrophysiologic and fluorescent calcium-imaging techniques. 2) Investigate changes in L-VGCC subunit expression and phosphorylation after chronic FZP treatment, using immunoblot, immunofluorescent, and electron microscopic techniques. 3) Investigate the often unrecognized ability of BZs to directly modulate L-VGCC function. Direct inhibition of recombinant L-VGCCs by BZs will be measured using whole-cell patch clamp techniques. Chronic misuse of prescription benzodiazepines (BZs) can result in physical dependence and remains a major concern in today's society. The results of this project could reveal a novel mechanism of neuroadaptive changes mediating the BZ withdrawal phenomenon, which current evidence suggests may be similar to other frequently abused CNS depressants, such as ethanol. Elucidation of this mechanism could reveal new therapeutic targets for BZ withdrawal, and possibly withdrawal from other drugs of abuse.

描述(申请人提供)：苯二氮卓类药物(BZS)增强中枢神经系统中抑制性伽马-氨基丁酸A型受体(GABAR)，临床上用于治疗失眠、焦虑和癫痫障碍。滥用中枢神经系统抑制药，如BZS，越来越令人担忧。自2001年以来，青少年中非医疗用途的中枢神经系统镇静剂的使用有所增加，2004年滥用和滥用BZ导致150,000多人到急诊室就诊。尤其值得关注的是，长期使用BZ会导致身体依赖，表现为戒断综合症，这限制了BZS的临床应用。我们的实验室使用一种公认的BZ慢性治疗方案来建立BZ依赖的模型，FZP是用BZ慢性治疗大鼠的方案。我们将戒断焦虑与大鼠海马CA1区兴奋性受体功能增强联系起来。我们还发现，电压门控钙通道(VGCC)电流密度在慢性FZP治疗期间增加一倍，并在停药后保持升高。有趣的是，尼莫地平选择性阻断L类VGCC的钙内流可阻止兴奋性受体的变化和相关的戒断焦虑，提示L-VGCC在BZ戒断现象中起关键作用。本项目的总体目标是确定L-VGCC在FZP治疗和停药过程中的调节机制，因为这可能暗示了一种先前未知的形成BZ生理依赖的机制。该项目的具体目的是：1)利用电生理和荧光钙成像技术，确定慢性BZ治疗引起的GABA介导的去极化是否激活FZP戒断过程中的L-VGCC。2)应用免疫印迹、免疫荧光、电子显微镜等技术，研究慢性扶正冲剂治疗后L-血管内皮生长因子受体亚单位表达及磷酸化的变化。3)研究BZS对L-VGCC功能的直接调节作用，这种能力往往不为人所知。采用全细胞膜片钳技术检测补肾活血方对重组L-VGCCs的直接抑制作用。长期滥用处方苯二氮卓类药物(BZS)会导致身体依赖，在当今社会仍然是一个主要问题。该项目的结果可能揭示一种新的神经适应性变化调节BZ戒断现象的机制，目前的证据表明，这可能类似于其他经常滥用的中枢神经系统抑制剂，如乙醇。阐明这一机制可能会揭示BZ戒断的新的治疗靶点，甚至可能是其他滥用药物的戒断。