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

描述（由申请方提供）：本研究将检查家族性ALS小鼠模型（SOD1G93A小鼠）运动神经元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（Ca 2+）的缓冲能力低，并且通过过量Ca 2+进入的兴奋性毒性是ALS中神经变性的可能机制。本研究将研究在成熟过程中的变化，在两个主要途径过量的Ca2+进入可能会发生：增加突触兴奋和/或增加运动神经元的内在兴奋性。内源性兴奋性由持续性Na+通道（NaP）和产生持续性内向电流（PIC）的Cav1.3 Ca2+通道介导。阿舒唑是FDA批准的唯一一种治疗ALS的药物，它以两种方式起作用，阻断NaP和减少突触前谷氨酸的释放。 不幸的是，随着时间的推移，利鲁唑失去了有效性，因此需要探索其他潜在的ALS治疗方法。将应用伊拉地平（一种有效阻断Cav1.3 Ca2+通道的二氢吡拉定拮抗剂），并在新生、幼龄和成年小鼠中评估运动神经元兴奋性。虽然在年轻大鼠中PIC的主要部分由NaP组成，但在成熟过程中，Ca 2+的贡献增加到约50%。成人ALS的发病可能与这种从Na+到Ca2+介导的PIC的转变有关。在帕金森氏病中，幼年黑质神经元中Na+依赖性起搏活性成熟为成年人中Cav1.3介导的活性导致变性，并且如果Na+依赖性活性在长期暴露于伊拉地平的成年人中恢复，则这些神经元对疾病不太敏感。SOD1G93A小鼠衰老运动神经元中也存在类似的Ca ++依赖性易感性，伊拉地平可能是一种有效的治疗方法。因此，我建议研究内在的和突触的兴奋性，包括体细胞和树突状细胞的Ca2+水平在正常和SOD1G93A小鼠和短期和长期的伊拉地平暴露对运动神经元过度兴奋的影响SOD1G93A小鼠从新生儿到成年年龄。采用全细胞膜片钳技术和锐电极细胞内记录技术记录腰骶部运动神经元的钙内流，并采用多光子成像技术和钙敏感染料检测各时间点树突状细胞内钙内流。 这项研究将评估ALS的新治疗方法伊拉地平的潜力，伊拉地平是目前FDA批准用于治疗高血压的药物。此外，这项工作可能会揭示更准确的方法，早期ALS诊断，通过评估反射反应从腿部肌肉。

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.