Harnessing Calcium Influx to Visualize and Regulate Seizure Networks

利用钙流入来可视化和调节癫痫网络

基本信息

批准号：
10449225
负责人：
Matthew Alexander Stern
金额：
$ 3.14万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10449225
关键词：
Ablation Acute Affinity Animal Model Awareness Bioluminescence Calcium Calcium Binding Cells Cephalic Chronic Convulsions Dependovirus Development Disease Drug Targeting Electrodes Electroencephalography Electrophysiology (science)Enzymes Epilepsy Event Excision Exposure to Fire - disasters Freedom Future Generations Genetic Glutamates Head Image Implant In Vitro Individual Infection Informal Social Control Ion Channel Ion Pumps Knowledge Lead Light Luminescent Proteins Measures Mediating Medical Methods Microelectrodes Modeling Molecular Mus Neurons Operative Surgical Procedures Opsin Pathologic Patients Pattern Persons Pharmaceutical Preparations Population Positioning Attribute Prevalence Property Proteins Proxy Recurrence Regulation Reporter Resolution Risk Rodent Model Role Seizures Source Specificity Tetanus Toxin Tissues Transgenic Organisms United States Variant Viral Vector Work awake brain abnormalities calcium indicator cell type density drug discovery experience imaging modality in vivo in vivo two-photon imaging mouse model nano neocortical nervous system disorder neural network neuroregulation novel optical imaging preservation promoter red fluorescent protein relating to nervous system selective expression side effect spatiotemporal tool two photon microscopy two-photon

项目摘要

PROJECT SUMMARY. Epilepsy, with a prevalence in the United States of 7 per 1000 people, is the fourth most common neurological disorder. While many patients achieve relief from disabling seizures through medication, about one third of epilepsy cases are pharmacoresistant. For many of these medically intractable cases, surgical intervention is indicated and while seizure freedom is obtained in as many as three quarters of these patients, the surgical approaches have substantial limitations and drawbacks. Open resection and ablation are destructive and permanent, and neurostimulation, necessitating implanted hardware, presents a risk for hardware malfunction and infection. Furthermore, these methods lacking cell-type specificity impact all tissue in the region targeted, carrying a risk for off target effects. Hence there is a need for an epilepsy treatment that is specific to the neurons initiating or propagating the pathologic neuronal activity. Development of such a treatment would be greatly informed by a better understanding of seizure activity dynamics, especially at a cellular resolution. The majority of the current understanding of seizure dynamics come from electrophysiology, including EEG, which only offers population resolution of activity, and single unit recordings. While single units record activity from individual neurons the density of neurons recorded is sparse and it is exceedingly difficult to know what subtype of neuron is being recorded. Genetically encoded calcium indicators (GECI) circumvent many of these drawbacks and allow for observation of the activity of individual neurons through the use of two-photon microscopy in animal models. While this approach has been taken to visualize acute seizures in a rodent model, this work has yet to capture seizures in spontaneous seizure models, which better approximate epilepsy. Thus, we first aim to examine spatiotemporal firing of neurons within seizure networks in a chronic mouse model of neocortical seizures and parse the activity by neuronal subtype (Aim 1). The light emitted during this activity will then be harnessed to develop an activity responsive neuromodulatory agent to allow networks to self-regulate. Our lab developed opto-chemogenetic agents, luminopsins, which we have used for cell subtype specific, hardware independent in vitro and in vivo neuromodulation. Specifically, luminopsins are light responsive ion channels or pumps fused with their own light source, a bioluminescent enzyme. We aim to modify the luminopsin construct, exchanging the bioluminescent enzyme with a bioluminescent GECI, which will result in a luminopsin whose functionality is contingent upon sufficient intracellular calcium and thus activity. The calcium binding affinity for these responsive luminopsins will be selected such that they are only responsive to high intracellular levels of calcium, such as those experienced during seizures, which would allow for preservation of non- pathologic neuronal activity. Responsive luminopsins will then be introduced in vivo in a spontaneous seizure model to observe if they are able to enable these networks to self-regulate (Aim 2). Ultimately the development of such knowledge and tools could be used to inform development of future treatments for epilepsy.

项目摘要。癫痫症在美国的患病率为每 1000 人 7 例，是第四大疾病常见的神经系统疾病。虽然许多患者通过药物治疗使癫痫发作得到缓解，大约三分之一的癫痫病例具有耐药性。对于许多此类医学上棘手的病例，需要进行手术需要进行干预，虽然这些患者中多达四分之三的患者不再癫痫发作，手术方法有很大的局限性和缺点。开放切除和消融具有破坏性永久性的神经刺激需要植入硬件，这会给硬件带来风险故障和感染。此外，这些缺乏细胞类型特异性的方法会影响该区域的所有组织有针对性，但存在产生脱靶效应的风险。因此，需要一种针对癫痫症的治疗方法。启动或传播病理性神经元活动的神经元。开发这种治疗方法将是通过更好地了解癫痫活动动态，特别是在细胞分辨率下，获得了极大的信息。这目前对癫痫发作动力学的大部分理解来自电生理学，包括脑电图，仅提供活动的群体分辨率和单个单位记录。虽然单个单元记录的活动来自单个神经元记录的神经元密度稀疏，很难知道是什么亚型神经元的数量正在被记录。基因编码钙指示剂 (GECI) 规避了其中许多因素缺点并允许通过使用双光子观察单个神经元的活动动物模型中的显微镜检查。虽然这种方法已被用来可视化啮齿动物模型中的急性癫痫发作，这项工作尚未捕获自发性癫痫发作模型中的癫痫发作，该模型更接近癫痫。因此，我们首先的目的是在慢性小鼠模型中检查癫痫网络内神经元的时空放电新皮质癫痫发作并按神经元亚型解析活动（目标 1）。在此活动期间发出的光将然后用于开发一种活动响应神经调节剂，使网络能够自我调节。我们的实验室开发了光化学遗传学剂、发光蛋白，我们已将其用于细胞亚型特异性、硬件独立的体外和体内神经调节。具体来说，发光蛋白是光响应离子通道或泵与自己的光源（一种生物发光酶）融合。我们的目标是修饰发光蛋白构建体，将生物发光酶与生物发光 GECI 交换，这将产生发光蛋白其功能取决于足够的细胞内钙和活性。钙结合将选择对这些响应性发光蛋白的亲和力，使得它们仅响应于高细胞内钙水平，例如癫痫发作期间经历的钙水平，这将允许保存非病理性神经元活动。然后，在自发性癫痫发作时将响应性发光蛋白引入体内模型来观察它们是否能够使这些网络进行自我调节（目标 2）。最终发展这些知识和工具可用于为未来癫痫治疗的开发提供信息。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Bioluminescence-Optogenetics.

生物发光-光遗传学。

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Berglund, Ken;Stern, Matthew A;Gross, Robert E
通讯作者：
Gross, Robert E

One Ring to Bind Them: The Annulus of GABAergic Inhibitory Restraint Fades at Seizure Emergence.

一环将其束缚：GABA能抑制约束环在癫痫发作时消失。

DOI：
发表时间：
2024
期刊：
影响因子：
3.6
作者：
Stern, Matthew A;Dingledine, Raymond
通讯作者：
Dingledine, Raymond

Applications of Bioluminescence-Optogenetics in Rodent Models.

生物发光-光遗传学在啮齿动物模型中的应用。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Stern, Matthew A;Skelton, Henry;Fernandez, Alejandra M;Gutekunst, Claire;Gross, Robert E;Berglund, Ken
通讯作者：
Berglund, Ken

Seizure Event Detection Using Intravital Two-Photon Calcium Imaging Data.

使用活体双光子钙成像数据检测癫痫事件。

DOI：
发表时间：
2023-09-29
期刊：
影响因子：
0
作者：
Stern, Matthew A;Cole, Eric R;Gross, Robert E;Berglund, Ken
通讯作者：
Berglund, Ken

Bioluminescence-Optogenetics: A Practical Guide.

生物发光-光遗传学：实用指南。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Stern, Matthew A;Skelton, Henry;Fernandez, Alejandra M;Gutekunst, Claire;Berglund, Ken;Gross, Robert E
通讯作者：
Gross, Robert E