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

描述（由申请人提供）：本研究将研究来自家族性ALS小鼠模型SOD1G93A小鼠的运动神经元的内在和突触兴奋性的出生后发育。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的丧失，导致瘫痪并最终死亡。易受ALS影响的运动神经元对细胞内钙（Ca2+）的缓冲能力较低，过量Ca2+进入的兴奋性毒性是ALS神经退行性变的可能机制。本研究将检查成熟过程中过量Ca2+进入可能发生的两种主要途径的变化：突触兴奋增加和/或运动神经元固有兴奋性增加。内在兴奋性是由持续Na+通道（NaP）和Cav1.3 Ca2+通道介导的，它们产生持续向内电流（PIC）。利鲁唑是FDA唯一批准的治疗ALS的药物，它有两种作用方式，阻断NaP，减少突触前谷氨酸释放。不幸的是，随着时间的推移，利鲁唑会失去疗效，因此需要探索其他潜在的治疗ALS的方法。Isradipine是一种有效阻断Cav1.3 Ca2+通道的二氢吡拉定拮抗剂，研究人员将对新生、幼年和成年小鼠的运动神经元兴奋性进行评估。虽然在幼鼠中，PIC的主要部分由NaP组成，但在成熟过程中，Ca2+的贡献增加到约50%。成人发病的ALS可能与这种从Na+到Ca2+介导的PIC的转变有关。在帕金森病中，青少年黑质神经元中Na+依赖的起搏器活性成熟到成人中Cav1.3介导的活性会导致变性，如果长期暴露于isradipine的成人中Na+依赖的活性恢复，这些神经元对该疾病的易感性就会降低。SOD1G93A小鼠衰老运动神经元中也可能出现类似的ca2 +依赖性易感性，isradipine可能是一种有效的治疗方法。因此，我建议研究正常小鼠和SOD1G93A小鼠的内在兴奋性和突触兴奋性，包括染色体和树突Ca2+水平，以及短期和长期暴露于isradipine对SOD1G93A小鼠从新生儿到成年期运动神经元高兴奋性的影响。我将使用全细胞膜片钳和细胞内尖锐电极记录腰椎和骶部运动神经元，并使用多光子成像和Ca2+敏感染料，在所有时间点检查树突状Ca2+内流。这项研究将评估一种治疗ALS的新药物isradipine的潜力，isradipine是目前FDA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，这项工作可能揭示更准确的早期ALS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.