Presynaptic NMDA receptors and GABA release in cortical dysplasia

皮质发育不良中突触前 NMDA 受体和 GABA 释放

• 批准号: 7516663
• 负责人: John J. Hablitz
• 金额: $ 19.03万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7516663
• 关键词: Age; Animal Model; Animals; Anticonvulsants; Brain; Cells; Condition; Cortical Dysplasia; Cortical Malformation; Development; Disruption; Dyes; Epilepsy; Freezing; Frequencies; Generations; Human; Image; Imagery; Imaging technology; In Vitro; Interneurons; Lead; Lesion; Location; Methods; Microgyria; Microscopy; Modeling; Monitor; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Neocortex; Neonatal; Nerve; Neuronal Migration Disorder; Neurons; Numbers; Operative Surgical Procedures; Patients; Pharmacotherapy; Presynaptic Terminals; Public Health; Rattus; Receptor Activation; Regulation; Resected; Role; Sampling; Seizures; Slice; Sorting - Cell Movement; Staining method; Stains; Techniques; Testing; Therapeutic; Thinking; Tissue Sample; Tissues; gamma-Aminobutyric Acid; human tissue; ifenprodil; insight; neuromechanism; novel strategies; postnatal; postsynaptic; presynaptic; receptor; receptor expression; research study; two-photon; voltage clamp

DESCRIPTION (provided by applicant): Intractable seizure disorders in humans are often associated with cortical dysplasia, microgyria, and heterotopias resulting from neuronal migration disorders. We have used the rat freeze lesion model to examine neural mechanisms underlying hyperexcitability in focal cortical dysplasia. N-methyl-D-aspartate (NMDA) receptors are involved in generation of epileptiform discharges in this model and in human cortical dysplasia. The location of the NMDA receptors has not been determined and functional studies in human tissue are limited. Proposed studies will use electrophysiological and two-photon microscopy techniques to test specific hypotheses about the role of NMDA receptors in the freeze lesion model of cortical dysplasia. The applicability of these finding to human epilepsy will be directly tested in tissue slices from patients with focal cortical dysplasia using imaging methods. It is hypothesized that NMDA receptors are located on presynaptic nerve terminals of GABAergic interneurons in the early postnatal period and these receptors are normally developmentally downregulated but persist in cortical dysplasia. Experiments will investigate (1) if presynaptic NMDA receptors are present on inhibitory nerve terminals in neocortex. Whole-cell voltage-clamp recordings of pharmacologically isolated IPSCs will be obtained under conditions where postsynaptic NMDA receptors are blocked. It is hypothesized that NMDA receptor antagonists will decrease the frequency of miniature IPSC in young (PN 12-16) but not older (PN 26-30) sham operated animals. We will also use styryl dye (FM1-43) staining and multiphoton excitation microscopy to visualize vesicular release from inhibitory GABAergic terminals and study NMDA receptor modulation of GABA release. It will be determined if NMDA receptor activaton selectively modulates distinct vesicular pools, the readily releasable pool and the reluctant pool. We will also examine (2) if functional presynaptic NMDA receptors are present in human cortical dysplasia. Previous studies of human cortical dysplasia tissue has shown an increase in NMDA receptor expression, particularly NR2B receptors. It is hypothesized that NMDA receptors are located presynaptically on inhibitory nerve terminals and modulate vesicular release of GABA. These studies will provide important new information about presynaptic NMDA receptors and regulation of GABA release in cortical dysplasia. New insights into the role of presynaptic NMDA receptors in development and regulation of epileptiform activity will be forthcoming. These studies will also increase our understanding of basic mechanisms of transmitter release and its' developmental regulation in human and animal models of focal cortical dysplasia. PUBLIC HEALTH RELEVANCE: Cortical dysplasia is associated with intractable seizure disorders in humans. Up to 43% of patients receiving surgical treatment for intractable seizures have some sort of cortical malformation. Anticonvulsant drug therapy is often ineffective in these patients. Studies in vitro of brain slices prepared from human dysplastic neocortex have demonstrated that this tissue displays intrinsic hyperexcitability. The mechanisms responsible for the inherent epileptogenicity of dysplastic cortex have been incompletely defined. It is proposed to directly examine a previously unexplored mechanism, presynaptic NMDA receptors, using an animal model of cortical dysplasia and tissue samples from human cortical dysplasia. New insights into mechanisms regulating presynaptic release could lead to novel strategies for therapy.

描述（由申请人提供）：人类中的顽固性癫痫发作疾病通常与神经元迁移障碍引起的皮质发育不良，小胶质细胞和异内植物有关。我们已经使用大鼠冻害病变模型来检查局灶性皮质发育不良中过度兴奋性的神经机制。 N-甲基-D-天冬氨酸（NMDA）受体参与该模型和人皮质发育不良中的癫痫样放电。 NMDA受体的位置尚未确定，人体组织中的功能研究受到限制。拟议的研究将使用电生理学和两光子显微镜技术来测试有关NMDA受体在皮质发育不良病变模型中的作用的特定假设。这些发现对人癫痫的适用性将直接在使用成像方法的局灶性皮质发育异常患者的组织切片中进行测试。假设NMDA受体位于产后早期的GABA能中间神经元的突触前神经末端，并且这些受体通常发育下调，但在皮质不良症中持续存在。实验将研究（1）如果新皮层中的抑制神经末端存在突触前NMDA受体。在突触后NMDA受体被阻断的条件下，将获得药理学分离的IPSC的全细胞电压钳记录。假设NMDA受体拮抗剂将降低Young（PN 12-16）中微型IPSC的频率，但不会降低（PN 26-30）假手术动物。我们还将使用Styryl染料（FM1-43）染色和多光子激发显微镜来可视化抑制性GABA能末端的水泡释放，并研究GABA释放的NMDA受体调制。将确定NMDA受体Activaton是否选择性调节不同的囊泡池，易于释放的池和不情愿的池。我们还将检查（2）是否在人皮质发育不良中存在功能性突触前NMDA受体。先前对人皮质发育不良组织的研究表明，NMDA受体表达，尤其是NR2B受体的增加。假设NMDA受体位于抑制性神经末端，调节GABA的囊泡释放。这些研究将提供有关突触前NMDA受体的重要新信息，并提供皮质发育不良的GABA释放的调节。对突触前NMDA受体在发育和调节癫痫样活动的调节中的作用的新见解将即将到来。这些研究还将提高我们对发射机释放的基本机制及其在局灶性皮质发育不良模型中的发育调控。公共卫生相关性：皮质发育不良与人类棘手的癫痫发作疾病有关。接受手术治疗的患者中，多达43％的患者具有某种皮质畸形。抗惊厥药物治疗在这些患者中常常无效。在人类发育不良新皮层制备的脑切片的体外研究表明，这种组织表现出内在的过度刺激性。未完全定义了负责发育不良皮质固有的癫痫病的机制。提议使用人类皮质发育不良的皮质发育不良和组织样品的动物模型直接检查先前未开发的机制，突触前NMDA受体。对调节突触前释放的机制的新见解可能导致新的治疗策略。