新生大鼠脊髓背角胶状质神经元的电生理学和形态学特性

Preterm and term neonates are exposed to repeated invasive procedures in the neonatal intensive care unit (NICU) which are frequently accompanied by local inflammation and tissue damage lasting for several hours to days. Multiple evidence indicates that nociceptive circuitry is both established and functional during late gestation, and that premature infants are indeed capable of mounting developmentally specific and distinct responses to noxious stimuli. Recent advances in neurobiology and clinical medicine have suggested that the consequences of repetitive or prolonged pain in the neonatal period include long-term changes in pain sensitivity and pain processing and may be associated with a variety of neurodevelopment, behavioral, and cognitive deficits that manifest in later childhood. Although newborns are more sensitive to pain than older infants, children, and adults, little is known about the neural circuitry of the spinal superficial dorsal horn (SDH) (lamina Ⅱ) and relationships between the electrophysiological and morphological properties of newborn individual substantia gelatinosa (SG) neuron. To identify the correlations between morphology and electrophysiological characteristics of the SG neurons, we perform tight-seal, whole-cell recordings with pipette microelectrodes filled with neurobiotin in horizontal slices from neonatal rat spinal cord to establish electrophysiological characteristics and to label the studied neurons. Voltage-clamp recordings are used to characterize some passive membrane properties such as resting membrane potential, membrane resistance, membrane capacitance, membrane time constant and spontaneous postsynaptic currents and current-voltage relationships. Current-clamp recordings are used to characterize active membrane properties such as action potential (AP) threshold, AP height, patterns of discharge to depolarizing pulses. We further compare the relationship among the transmitter phenotype, morphology and electrophysiological properties to categorize the SG neurons according to neurotransmitter content, location and size of the somata, the dendritic and axonal pattern of arborization, spontaneous synaptic potentials, pattern of discharge and current-voltage relationships. These information is important for understanding how injury affects nociceptive processing during early development and helping us to determine the short- and long-term goals of analgesia and sedation to newborn babies.

新生儿重症监护病房的新生儿生后反复经历动静脉穿刺、气管插管等致痛性操作，伴持续数小时至数日的局部炎症及组织损伤。近年国内外研究表明，新生儿的痛觉传导系统在解剖学和功能方面在孕晚期就均已完备，即使是未成熟儿对伤害性刺激亦具有痛感知的能力。生后早期的反复疼痛刺激可造成如心率加快、颅内压波动等近期和焦虑、注意力不集中等远期不良影响。尽管新生儿对疼痛较年长儿敏感，但机体处理伤害性信息的初级中枢- - 脊髓背角胶状质神经元（Ⅱ层）的电生理学和形态学及功能之间的关联目前尚不清楚。本研究应用全细胞膜片钳记录新生大鼠脊髓切片胶状质神经元膜内在特性，同时在电极内液加入生物素以标记所记录的神经元形态，并根据动作电位的类型、胞体及主要树突支的走向、尺寸及轴突终扣内所含神经递质进行分类，揭示胶状质神经元内在结构与功能之间的关系，为研究安全有效的减轻新生儿疼痛的干预方法，降低神经系统近期和远期的不良影响提供理论依据。

不同年龄阶段的人群对疼痛具有不同程度的反应，可能与疼痛调控的初级中枢，即脊髓背角II层（SG）的神经发育有关。为揭示不同发育阶段的SG神经元的形态学和电生理学特征，我们结合了全细胞膜片钳和免疫荧光技术，将染料通过玻璃微电极注入单个SG神经元，全细胞膜片钳记录神经元的电生理学特征后，常规固定，共聚焦显微镜下观察其形态，并进一步分析SG神经元形态特征与电生理学特征之间的关系。我们发现：1、新生和幼年大鼠SG区中vertical类神经元所占比例最高，其胞体多位于背角lamina II的浅层和中间层；相反，islet类神经元所占比例最少，其胞体在浅层、深层和中间层均有分布；2、islet神经元的比例随着年龄的增长逐渐增加，而vertical和radial神经元的比例随着年龄的增长却出现下降的趋势；3、不同放电模式的SG神经元的主动和被动膜特性各异，其中Tonic放电的神经元在新生和幼年大鼠所占比例均最高，而Gap-firing和Phasic-bursting放电的神经元所占比例最低。在新生大鼠中，Delay放电的神经元膜电容（Cm）最大；而在幼年大鼠中，Tonic放电的神经元Cm最大；4、Tonic放电神经元的比例随着年龄的增长逐渐降低，而Reluctant放电模式为成年大鼠特有。随着年龄增长，阈下电流IA、Ih、IT的比例均不同程度增加。由于以往的研究中认为islet神经元主要为抑制性神经元，而vertical和radial神经元主要为兴奋性神经元，因此我们的研究结果提示，随着年龄发育，大鼠脊髓背角浅层神经元的γ-氨基丁酸和/或甘氨酸能抑制性中间神经元的比例逐渐升高，而谷氨酸能兴奋性中间神经元的比例逐渐下降。这种在发育过程中由兴奋性神经元占主导到抑制性神经元占主导的现象对进一步深入研究其机制提供了科学基础，对临床上寻找疼痛治疗的新途径具有重要的科学意义。

内容获取失败，请点击重试