Synaptic and intrinsic exitability in motoneurons in a mouse model of ALS.

描述(申请人提供)：这项研究将从家族性肌萎缩侧索硬化症小鼠模型SOD1G93A小鼠中检测运动神经元内在和突触兴奋性的出生后发展。肌萎缩侧索硬化症是一种进展缓慢的疾病，其特征是运动神经元丢失，导致瘫痪，最终死亡。易受肌萎缩侧索硬化症影响的运动神经元对细胞内钙离子的缓冲能力较低，过度的钙离子内流引起的兴奋性毒性可能是肌萎缩侧索硬化症神经退变的机制之一。这项研究将探讨两条主要途径在成熟过程中的变化：突触兴奋增加和/或运动神经元的内在兴奋性增加。内源性兴奋性是由产生持久内向电流(PIC)的持续性Na+通道(NAP)和Cav1.3钙通道(Cav1.3)介导的。利鲁唑是FDA批准的唯一治疗肌萎缩侧索硬化症的药物，它有两种作用方式，阻止NAP和减少突触前谷氨酸的释放。不幸的是，随着时间的推移，利鲁唑失去了疗效，因此需要探索其他潜在的治疗ALS的方法。伊拉地平是一种有效阻断Cav1.3钙通道的二氢吡拉定拮抗剂，将用于评估新生、幼年和成年小鼠的运动神经元兴奋性。虽然幼年大鼠PIC的主要成分是NAP，但在成熟过程中，Ca~(2+)的贡献增加到约50%。成人肌萎缩侧索硬化症的发病可能与这种从钠离子到钙离子介导的PIC的转变有关。在帕金森病中，幼年黑质神经元中钠离子依赖的起搏器活动成熟到Cav1.3介导的活动会导致变性，如果长期暴露于伊拉地平的成年人恢复了钠离子依赖的活动，这些神经元就不太容易受到疾病的影响。SOD1G93A小鼠老化的运动神经元也可产生类似的钙依赖敏感性，伊拉地平可能是一种有效的治疗方法。因此，我建议研究正常和SOD1G93A小鼠的内源性和突触兴奋性，包括胞体和树突钙离子水平，以及短期和长期暴露伊拉地平对SOD1G93A小鼠从新生到成年的运动神经元过度兴奋性的影响。我将使用全细胞膜片钳和尖电极细胞内记录的方法记录腰椎和骶骨运动神经元，并使用多光子成像和钙敏感染料，在各个时间点检测树突状钙内流。这项研究将评估治疗ALS的新药物伊拉地平的潜力，伊拉地平是FDA目前批准用于治疗高血压的药物。此外，通过评估腿部肌肉的反射反应，这项工作可能揭示出更准确的ALS早期诊断方法。

DESCRIPTION (provided by applicant): This study will examine postnatal development of intrinsic and synaptic excitability in motoneurons from the mouse model for familial ALS, the SOD1G93A mouse. ALS is a slowly progressing disease marked by loss of motoneurons resulting in paralysis and finally death. Motoneurons that are vulnerable to ALS have low buffering capacity for intracellular calcium (Ca2+), and excitotoxicity through excessive Ca2+ entry is a possible mechanism of neurodegeneration in ALS. This study will examine changes during maturation in the two main routes excessive Ca2+ entry could occur: increased synaptic excitation and/or an increase in the intrinsic excitability of the motoneuron. Intrinsic excitability is mediated by both persistent Na+ channels (NaP) and Cav1.3 Ca2+ channels which produce the persistent inward current (PIC). Riluzole, the only FDA approved drug for ALS, works in two ways, blocking NaP, and decreasing presynaptic glutamate release. Unfortunately riluzole loses effectiveness over time, so exploration of other potential treatments for ALS is needed. Isradipine, a dihydropiradine antagonist effective in blocking Cav1.3 Ca2+ channels will be applied and motoneuron excitability will be assessed in neonatal, juvenile and adult mice. Although a major portion of the PIC consists of NaP in young rats, during maturation the contribution of Ca2+ increases to about 50%. The adult onset of ALS could be linked to this shift from a Na+ to Ca2+-mediated PIC. In Parkinson's disease, maturation of Na+-dependant pacemaker activity in juvenile substantia nigra neurons to Cav1.3- mediated activity in adults causes degeneration, and if Na+-dependent activity is brought back in adults with prolonged exposure to isradipine, these neurons were less susceptible to the disease. A similar Ca++ dependent susceptibility could develop in aging motoneurons of SOD1G93A mice, and isradipine could be an effective treatment. Therefore I propose to study intrinsic and synaptic excitability, including somal and dendritic Ca2+ levels in normal and SOD1G93A mice and the effects of short- and long-term isradipine exposure on motoneuron hyperexcitability in SOD1G93A mice from neonatal through adult ages. I will record from lumbar and sacral motoneurons using whole cell patch clamp and sharp electrode intracellular recording, and using multiphoton imaging and Ca2+ sensitive dye, examine dendritic Ca2+ influx at all timepoints. This study will assess the potential of a new treatment for ALS, isradipine, a drug currently FDA approved for management of hypertension. In addition, this work may reveal more accurate methods of early ALS diagnosis, through evaluation of the reflex responses from the leg muscles.