Novel, in Vivo Measures of Rat Spinal Cord Glutamate

描述（由申请人提供）：记录正常大鼠脊髓或创伤性损伤后体内谷氨酸释放和摄取的快速动态的能力受到可用方法的时间分辨率的限制。然而，由于谷氨酸在正常生理过程中作为一种必需的神经递质而在病理和创伤中作为一种神经毒素的不同作用，这一研究领域仍然是神经科学的主要兴趣。这项资助是一项试点研究，旨在建立一种新型的陶瓷基多位点微电极系统，作为一种体内电化学方法来测量正常大鼠脊髓中的快速谷氨酸神经传递。这种新颖的、高度专业化的传感器的应用提供了独特的快速、逐秒测量谷氨酸释放和吸收的能力，这些谷氨酸释放和吸收是在涂有谷氨酸氧化酶的铂电极上进行的。虽然这项技术已经成功地测量了大鼠脑组织中的谷氨酸，但在脊髓中的有效性尚未确定。第一个具体目标将验证记录脊髓灰质和白质中谷氨酸释放的基线和诱发水平的能力。这些实验将(i)表征细胞外信号并确认谷氨酸的测量，（ii）表征响应KCI诱发释放的信号动力学，（iii）比较脊髓灰质和白质中的这些参数。第二个目标将测量谷氨酸摄取动力学(i)响应于局部应用谷氨酸和（ii）响应于兴奋性氨基酸转运蛋白摄取抑制剂。药剂将通过连接在传感器上的微移液管输送。随着时间的推移，快速记录脊髓组织中“实时”体内谷氨酸事件的能力代表了常用微透析的一个有价值的替代方案。这一重要的方法学进展将极大地增强阐明正常大鼠脊髓中正常谷氨酸能神经传递的调节以及病理或创伤反应的能力。在将该方法应用于脊髓损伤之前，有必要在大鼠脊髓中进行验证。

DESCRIPTION (provided by applicant): The ability to record the rapid dynamics of in vivo glutamate release and uptake in normal rat spinal cord or after traumatic injury has been limited by the temporal resolution of available methodology. However, this area of research remains of major interest in neuroscience because of the contrasting roles of glutamate as an essential neurotransmitter in normal physiological processes yet as a neurotoxin in pathologies and trauma. This grant is a pilot study to establish a novel, ceramic based, multi-site microelectrode system as an in vivo electrochemical method to measure fast glutamate neurotransmission in normal rat spinal cord. The application of the novel, highly specialized sensors offers the unique capacity for rapid, second by second measures of glutamate release and uptake at platinum electrode sites coated with glutamate oxidase. While this technique has successfully measured glutamate in rat brain tissue, the efficacy in spinal cord has not been established. The first specific aim will validate the ability to record both baseline and evoked levels of glutamate release in spinal cord gray and white matter. These experiments will (i) characterize the extracellular signal and confirm the measure of glutamate, (ii) characterize the signal kinetics in response to KCI evoked release and (iii) compare these parameters in both spinal cord gray and white matter. The second aim will measure the kinetics of glutamate uptake (i) in response to locally applied glutamate and (ii) in response to excitatory amino acid transporter uptake inhibitors. Agents will be delivered through micropipettes attached to the sensors. The ability to rapidly record "real-time", in vivo glutamate events over time in spinal cord tissue represents a valuable alternative to commonly used microdialysis. This significant methodological advance will greatly enhance the ability to elucidate the regulation of normal glutamatergic neurotransmission in normal rat spinal cord and in response to pathology or trauma. It will be essential to validate the technique in rat spinal cord prior to application of the method to spinal cord injury.