Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons' membrane potential is dependent on sodium ions. Depolarizing effect of 5·10-3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect - activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10-3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10-3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

中文翻译：

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2,4二氨基丁酸在水le Retzius神经元中引起广泛的去极化作用，这是两种不同的电生理机制。

最近的研究表明，几乎在所有环境中都存在的神经毒性兴奋性氨基酸2,4-DABA，但主要存在于水生生态系统中，可能是动物和人类神经退行性疾病发展的危险因素。尽管它具有神经毒性和潜在的环境重要性，但仍未探索2，4-DABA在神经元中的兴奋性和假定的兴奋性毒性作用的机制。我们之前曾报道过由2,4-DABA引起的水ech Retzius神经元发生广泛的两阶段膜去极化和功能紊乱。当前的研究对导致这种去极化的电生理过程进行了首次详细研究。使用玻璃微电极对水ech Haemopis sanguisuga的Retzius神经元进行细胞内记录，并通过向这些电极注入超极化电流脉冲来测量输入膜电阻（IMR）。结果表明，2,4-DABA对神经元膜电位的兴奋作用取决于钠离子。无钠溶液中5·10-3 mol / L 2,4-DABA的去极化作用显着降低了91％，将其降至3.26±0.62 mV，并取消了其两阶段性质。除了依赖钠离子外，该氨基酸诱导的膜电位去极化还伴随着膜渗透性的增加，因为2,4-DABA使IMR降低了8.27±1.47MΩ（67.60％）。由于目前的结果突出了钠离子的作用，我们研究了在2，4-DABA诱导的兴奋作用中两个推测的钠依赖性机制的作用-离子型谷氨酸受体的激活和中性氨基酸的电转运蛋白。5·10-3 mol / L 2,4-DABA的兴奋作用被非NMDA受体拮抗剂10-5 mol / L 6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）部分阻断。膜去极化的第一阶段显着降低了2.59±0.98 mV（40％），而第二阶段保持不变。此外，通过等摩尔共施用5·10-3 mol / L 2,4-DABA和该转运蛋白的竞争性抑制剂L-丙氨酸，研究了中性氨基酸的钠依赖性转运系统的参与。尽管L-丙氨酸对2,4-DABA引起的膜去极化的第一阶段没有影响，它将第二阶段（整个膜的去极化）从39.63±2.22 mV大幅降低到16.28±2.58 mV，降低了58.92％。因此，我们建议2,4-DABA对Retzius神经元的电生理作用是由两种不同的机制介导的，即通过离子型谷氨酸受体的激活来启动膜去极化的第一阶段，然后刺激电致钠依赖性中性氨基酸性转运蛋白，导致正电荷额外流入细胞，并进入去极化的第二阶段。