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.

Figure 1. 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.

Figure 2. Systemic administration of bumetanide blocked inhibitory avoidance learning.

(A) Rats were administered vehicle and bumetanide intravenously 1 hr prior to training session. The test was carried out 24 hrs later. Result showed that retention latency of the bumetanide-treated group was decreased significantly compared with vehicle-treated group (***p<0.001), n = 10 for each group. Data represent the medians ± interquartile range. (Mann-Whitney U-tests). (B) No significant difference had been found in locomotor activity among vehicle-treated and bumetanide-treated group. n = 8 for each group. Data represent the mean ± SEM.

Figure 3. Systemic administration of bumetanide blocked the long-term memory but not the short-term memory formation.

(A) Rats were administered vehicle and bumetanide intravenously 1 hour before training session. The 1^st and 2^nd test were carried out 1 hr and 24 hrs after training session respectively. Result showed that retention latency of the bumetanide-treated group was distinguishable with the vehicle-treated group. Significant difference had been found in the 2^nd test (**p<0.01) n = 8 for each group. Data represent the medians ± interquartile range (Mann-Whitney U-tests).

Figure 4. Systemic administration of bumetanide blocked avoidance learning induced MAPK activation in hippocampus.

(A) Representative Western blots among different groups. (B) Densitometric analysis of the activation of MAPK in the hippocampus under different treatments. MAPK phosphorylation was significantly elevated in hippocampus after inhibitory avoidance learning. Bumetanide treatment significantly reduced the phosphorylation level of MAPK compared with vehicle-treated group (Values are mean ± SEM, * P<0.05 versus vehicle-treated group).

Figure 5. Picrotoxin enhanced hippocampal fEPSPs were disrupted by suprafusion of bumetanide in a dose dependent manner.

(A) From Left to right, representative traces of fEPSPs recorded extracellularlly under control condition, after exposure to 10 µM picrotoxin (PTX), PTX + 5 µM bumetanide (BUT), and PTX + 10 µM BUT. Application of 10 µM PTX significant enhanced fEPSP amplitude in hippocampus CA1 Schaffer collateral fiber (178±8%, ***p<0.001 compared with control fEPSP). The picrotoxin enhanced fEPSPs were attenuated by bumetanide treatment in dose dependent manner (5 µM bumetanide: 149±10%, P = 0.066 compared with PTX group; 10 µM bumetanide: 117±9%, ***p<0.001 compared with PTX group, P = 0.453 compared with control group).