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Inhibition of NKCC1 attenuated hippocampal LTP formation and inhibitory avoidance in rat.

Ko MC, Lee MC, Amstislavskaya TG, Tikhonova MA, Yang YL, Lu KT - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

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.

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Superfusion of bumetanide blocked the formation of hippocampal LTP in a dose dependent manner.(A) From Left to right, representative superimposed traces of fEPSPs recorded extracellularlly under control conditions and after exposure to 0, 5, 10, and 20 µM bumetanide. Application of 10 µM bumetanide blocked LTP formation in hippocampus CA1 Schaffer collateral fiber. Bumetanide was applied from −10 min∼10 min. n = 10 for each trial (B) fEPSP recorded in the stratum radiatum and evoked by stimulation of the Schaffer collateral—commissural pathway with different intensities in vehicle-treated and bumetanide-treated slices (10 µM). fEPSP slopes are comparable between vehicle-treated (filled circle, n = 8) and bumetanide-treated (open circle, n = 8) for a given range of stimulus intensities. (C) Fiber volley amplitudes are similar between vehicle-treated and bumetanide-treated (10 µM) slices for a given range of stimulus intensities.
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pone-0106692-g001: Superfusion of bumetanide blocked the formation of hippocampal LTP in a dose dependent manner.(A) From Left to right, representative superimposed traces of fEPSPs recorded extracellularlly under control conditions and after exposure to 0, 5, 10, and 20 µM bumetanide. Application of 10 µM bumetanide blocked LTP formation in hippocampus CA1 Schaffer collateral fiber. Bumetanide was applied from −10 min∼10 min. n = 10 for each trial (B) fEPSP recorded in the stratum radiatum and evoked by stimulation of the Schaffer collateral—commissural pathway with different intensities in vehicle-treated and bumetanide-treated slices (10 µM). fEPSP slopes are comparable between vehicle-treated (filled circle, n = 8) and bumetanide-treated (open circle, n = 8) for a given range of stimulus intensities. (C) Fiber volley amplitudes are similar between vehicle-treated and bumetanide-treated (10 µM) slices for a given range of stimulus intensities.

Mentions: Hippocampal long-term potentiation (LTP) has been widely accepted as a candidate cellular mechanism for associative learning and memory. In order to investigate the possible role of NKCC1 on hippocampus function, brain slices containing hippocampus were subjected to extracellular recording. High frequency stimulus (100 Hz) was applied in a 20 second interval for a total of three times. Vehicle or bumetanide was applied 10 min prior to stimulation and sustained for 10 min after stimulation. Resulted showed that suprafusion of bumetanide blocked hippocampal LTP formation comparing with the vehicle group (vehicle group: 187% ± 11% bumetanide group: 102% ± 8%, p<0.001) (5 µM: 154% ± 8%, 20 µM: 105% ± 2%). Our results also showed that basal synaptic transmission is preserved in bumetanide treated rats. We first analyzed basal synaptic transmission by applying isolated stimuli of increasing intensity to the Schaffer collaterals (Figure 1B). Input/output curves for extracellular fEPSP were indistinguishable between slices from vehicle-treated group and bumetanide-treated. For a range of stimulation intensities, the slopes of bumetanide-treated slice's fEPSP responses were not significantly different from the fEPSP responses of vehicle-treated slices. Likewise, measurements of the fiber volleys amplitude from vehicle and bumetanide slices were similar (P>0.05), and there was no difference in an input/output curve (Fig. 1C). This result indicates that the given dose of bumetanide treatment does not modify the basal synaptic transmission at the postsynaptic level.


Inhibition of NKCC1 attenuated hippocampal LTP formation and inhibitory avoidance in rat.

Ko MC, Lee MC, Amstislavskaya TG, Tikhonova MA, Yang YL, Lu KT - PLoS ONE (2014)

Superfusion of bumetanide blocked the formation of hippocampal LTP in a dose dependent manner.(A) From Left to right, representative superimposed traces of fEPSPs recorded extracellularlly under control conditions and after exposure to 0, 5, 10, and 20 µM bumetanide. Application of 10 µM bumetanide blocked LTP formation in hippocampus CA1 Schaffer collateral fiber. Bumetanide was applied from −10 min∼10 min. n = 10 for each trial (B) fEPSP recorded in the stratum radiatum and evoked by stimulation of the Schaffer collateral—commissural pathway with different intensities in vehicle-treated and bumetanide-treated slices (10 µM). fEPSP slopes are comparable between vehicle-treated (filled circle, n = 8) and bumetanide-treated (open circle, n = 8) for a given range of stimulus intensities. (C) Fiber volley amplitudes are similar between vehicle-treated and bumetanide-treated (10 µM) slices for a given range of stimulus intensities.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219661&req=5

pone-0106692-g001: Superfusion of bumetanide blocked the formation of hippocampal LTP in a dose dependent manner.(A) From Left to right, representative superimposed traces of fEPSPs recorded extracellularlly under control conditions and after exposure to 0, 5, 10, and 20 µM bumetanide. Application of 10 µM bumetanide blocked LTP formation in hippocampus CA1 Schaffer collateral fiber. Bumetanide was applied from −10 min∼10 min. n = 10 for each trial (B) fEPSP recorded in the stratum radiatum and evoked by stimulation of the Schaffer collateral—commissural pathway with different intensities in vehicle-treated and bumetanide-treated slices (10 µM). fEPSP slopes are comparable between vehicle-treated (filled circle, n = 8) and bumetanide-treated (open circle, n = 8) for a given range of stimulus intensities. (C) Fiber volley amplitudes are similar between vehicle-treated and bumetanide-treated (10 µM) slices for a given range of stimulus intensities.
Mentions: Hippocampal long-term potentiation (LTP) has been widely accepted as a candidate cellular mechanism for associative learning and memory. In order to investigate the possible role of NKCC1 on hippocampus function, brain slices containing hippocampus were subjected to extracellular recording. High frequency stimulus (100 Hz) was applied in a 20 second interval for a total of three times. Vehicle or bumetanide was applied 10 min prior to stimulation and sustained for 10 min after stimulation. Resulted showed that suprafusion of bumetanide blocked hippocampal LTP formation comparing with the vehicle group (vehicle group: 187% ± 11% bumetanide group: 102% ± 8%, p<0.001) (5 µM: 154% ± 8%, 20 µM: 105% ± 2%). Our results also showed that basal synaptic transmission is preserved in bumetanide treated rats. We first analyzed basal synaptic transmission by applying isolated stimuli of increasing intensity to the Schaffer collaterals (Figure 1B). Input/output curves for extracellular fEPSP were indistinguishable between slices from vehicle-treated group and bumetanide-treated. For a range of stimulation intensities, the slopes of bumetanide-treated slice's fEPSP responses were not significantly different from the fEPSP responses of vehicle-treated slices. Likewise, measurements of the fiber volleys amplitude from vehicle and bumetanide slices were similar (P>0.05), and there was no difference in an input/output curve (Fig. 1C). This result indicates that the given dose of bumetanide treatment does not modify the basal synaptic transmission at the postsynaptic level.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

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.

Show MeSH
Related in: MedlinePlus