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Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons.
Aquat Toxicol. 2020 Mar; 220:105398.AT

Abstract

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.

Authors+Show Affiliations

Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia. Electronic address: svetolik.spasic@med.bg.ac.rs.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.Institute for Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine University of Belgrade, Dr Subotica 1/III, 11000, Belgrade, Serbia.Institute for Pathological Physiology "Ljubodrag Buba Mihailovic", Faculty of Medicine University of Belgrade, Dr Subotica 1/II, 11000, Belgrade, Serbia.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31891816

Citation

Spasic, Svetolik, et al. "Two Distinct Electrophysiological Mechanisms Underlie Extensive Depolarization Elicited By 2,4 Diaminobutyric Acid in Leech Retzius Neurons." Aquatic Toxicology (Amsterdam, Netherlands), vol. 220, 2020, p. 105398.
Spasic S, Stanojevic M, Nesovic Ostojic J, et al. Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons. Aquat Toxicol. 2020;220:105398.
Spasic, S., Stanojevic, M., Nesovic Ostojic, J., Kovacevic, S., Todorovic, J., Dincic, M., Nedeljkov, V., Prostran, M., & Lopicic, S. (2020). Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons. Aquatic Toxicology (Amsterdam, Netherlands), 220, 105398. https://doi.org/10.1016/j.aquatox.2019.105398
Spasic S, et al. Two Distinct Electrophysiological Mechanisms Underlie Extensive Depolarization Elicited By 2,4 Diaminobutyric Acid in Leech Retzius Neurons. Aquat Toxicol. 2020;220:105398. PubMed PMID: 31891816.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons. AU - Spasic,Svetolik, AU - Stanojevic,Marija, AU - Nesovic Ostojic,Jelena, AU - Kovacevic,Sanjin, AU - Todorovic,Jasna, AU - Dincic,Marko, AU - Nedeljkov,Vladimir, AU - Prostran,Milica, AU - Lopicic,Srdjan, Y1 - 2019/12/26/ PY - 2019/09/26/received PY - 2019/12/11/revised PY - 2019/12/25/accepted PY - 2020/1/1/pubmed PY - 2020/1/1/medline PY - 2020/1/1/entrez KW - 2,4-diaminobutyric acid KW - CNQX KW - Ionotropic glutamate receptors KW - L-alanine KW - Membrane potential KW - Transport system A SP - 105398 EP - 105398 JF - Aquatic toxicology (Amsterdam, Netherlands) JO - Aquat. Toxicol. VL - 220 N2 - 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. SN - 1879-1514 UR - https://www.unboundmedicine.com/medline/citation/31891816/Two_distinct_electrophysiological_mechanisms_underlie_extensive_depolarization_elicited_by_2,4_diaminobutyric_acid_in_leech_Retzius_neurons L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-445X(19)30797-0 DB - PRIME DP - Unbound Medicine ER -