In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.
摘要
在机械分离的、附着有天然突触前神经末梢的大鼠脊髓胶状质（SG）神经元中，在电压钳制条件下，使用制霉菌素穿孔膜片钳记录模式记录甘氨酸能微小抑制性突触后电流（mIPSCs）。在此条件下，测试了甘氨酸能突触前神经末梢上P2X受体亚型在出生后发育过程中是否发生变化。2. 在所有测试的发育阶段，ATP均以浓度依赖的方式促进甘氨酸能mIPSC频率，而αβ-亚甲基-ATP（αβ-me-ATP）仅在发育后期有效。3. 在无钙的外部溶液中，αβ-me-ATP引起的mIPSC频率促进作用完全被阻断，但添加10⁻⁴ M Cd²⁺对其无影响。4. 即使存在10⁻⁶ M毒胡萝卜素（一种钙泵阻滞剂），αβ-me-ATP仍能促进mIPSC频率。5. 在发育后期阶段，ATP引起的突触前或突触后反应可被10⁻⁵ M磷酸吡哆醛-5-磷酸-6-偶氮苯-2',4'-二磺酸（PPADS）可逆性阻断，但仅被10⁻⁷ M 2',3'-O-(2,4,6-三硝基苯基)-ATP（TNP-ATP）部分阻断。然而，αβ-me-ATP引起的突触前或突触后反应可被10⁻⁵ M PPADS或10⁻⁷ M TNP-ATP完全且可逆地阻断。6. αβ-me-ATP显著降低出生后28 - 30天神经元中诱发的甘氨酸能抑制性突触后电流（IPSC）幅度，而对10 - 12天神经元无影响。7. 得出的结论是，αβ-me-ATP敏感的P2X受体在发育后期阶段投射到SG神经元上的甘氨酸能突触前神经末梢上功能性表达。突触前P2X受体亚型的这种发育变化可能有助于突触可塑性，如调节神经元兴奋性和精细控制脊髓背角神经元的疼痛信号。
