Inhibition of NKCC1 Attenuated Hippocampal LTP Formation and Inhibitory Avoidance in Rat

Abstract

The loop diuretic bumetanide (Bumex) is thought to have antiepileptic properties via modulate GABAA mediated signaling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signaling, we sought to investigate whether they also affect hippocampal function. The current study was performed to evaluate the possible role of NKCC1 on the hippocampal function. Brain slice extracellular recording, inhibitory avoidance, and western blot were applied in this study. Results showed that hippocampal Long-term potentiation was attenuated by suprafusion of NKCC1 inhibitor bumetanide, in a dose dependent manner. Sequent experiment result showed that Intravenous injection of bumetanide (15.2 mg/kg) 30 min prior to the training session blocked inhibitory avoidance learning significantly. Subsequent control experiment's results excluded the possible non-specific effect of bumetanide on avoidance learning. We also found the phosphorylation of hippocampal MAPK was attenuated after bumetanide administration. These results suggested that hippocampal NKCC1 may via MAPK signaling cascade to possess its function.

Bumetanide blocks the acquisition of conditioned fear in adult rats

“…was injected 30 min prior to fear conditioning (Figure A). The dose chosen was based on our previous studies (Lu et al ., , ; Ko et al ., ). The results showed that bumetanide treatment blocked the acquisition of FPS [ t (1, 14) = 2.419] (Figure B).…”

“…The blots were incubated with anti‐phospho‐ERK (New England Biolabs, Hitchin, UK) and anti‐ERK antibodies (BD Biosciences, San Jose, CA, USA). Band detection was performed with Western blotting analysis system (RPN 2108; Amersham International, Amersham, UK) (Yang and Lu, ; Ko et al ., ).…”

“…30 min prior to the training of conditioned fear. The dose chosen (15.2 mg kg ‐1 ) was based on our previous experiments (Lu et al ., , ; Ko et al ., ; 15.2 mg·is equivalent to 42μmol). For extracellular recordings, bumetanide was first dissolved in 100% DMSO to make a 10 mM stock solution and then diluted to 5 and 10 μM with artificial CSF (ACSF); composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM NaHCO 3 , 2 mM NaH 2 PO 4 and 11 mM glucose; the pH value was maintained at 7.3–7.5 .…”

“…Cation–chloride co‐transporters (CCCs) are transmembrane proteins that mediate transport of chloride ions across the cell membrane (Blaesse et al ., ; Ko et al ., ). There are two main types of CCCs, in which chloride is co‐transported with sodium ions and/or potassium ions.…”

“…In adult neurons, [Cl − ] i is lower than extracellular chloride level, and the reversal potential for chloride currents is near the resting membrane potential of the neurons. Even slight changes in [Cl − ] i can significantly alter the magnitude, and even polarity, of the GABA‐mediated chloride current (Fiumelli and Woodin, ; Kahle and Staley, , ; Ko et al ., ).…”