Male mice (3-week-old, ICR, Oriental Bio Service Inc., Nanjing, China) were used in this study. All animal procedures used in this study were performed in accordance with the Guidelines for Laboratory Animal Research of Nanjing Medical University and were approved by the Animal Care and Use Committee at Nanjing Medical University. All efforts were made to minimize the animals' suffering and to reduce the number of animals used.

The mice were anesthetized with ethyl ether and decapitated, and the brains were rapidly removed. Coronal brain slices (400 μm) were cut using a vibrating microtome (Microslicer DTK 1500, Dousaka EM Co., Kyoto, Japan) in ice-cold modified artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 126, CaCl₂1, KCl 2.5, MgCl₂ 1, NaHCO₃ 26, KH₂PO₄ 1.25, and D-glucose 20. The ACSF was oxygenated with a gas mixture of 95% O₂/5% CO₂. The hippocampal slices were incubated in ACSF for 1 h at 32°C to allow them to recover and were then transferred to a recording chamber.

All electrophysiological recordings were performed at room temperature (22–23°C). Hippocampal CA1 pyramidal neurons were viewed with an upright microscope equipped with an infrared-sensitive camera (DAGE-MTI, IR-1000) and in general, the second and the third layer of neurons in the slices were chosen for the patch clamp recording. I_GABA was recorded using an EPC-10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) sampled at 10 kHz and filtered (Bessel) at 2.9 kHz. The capacitance and series resistance were compensated (>90%) before recording. Data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors >5 mV were not used for subsequent analysis. The liquid junction potentials were compensated before patching.

To record I_GABA, the holding potential was set at −60 mV. The slices were continually perfused with the oxygenated ACSF containing 0.3 μM TTX. GABA was dissolved in the bath solution and was focally applied using a rapid drug delivery system directed toward the soma of the recorded neurons. Glass pipettes (No. 64-0817 [G85150T-3], Warner Instruments Inc., Hamden, CT, USA) were used with a resistance of 1–3 MΩ when they were filled with the pipette solution composed of (in mM) KCl 140, CaCl₂ 1, MgCl₂ 2, EGTA 10, HEPES 10, and Tris-ATP 5 at pH 7.3. The expression of TRPV4 receptors was functionally verified by recording the TRPV4 agonist (GSK1016790A)-activated current as previously reported (Hong et al., 2016).

Western blot analysis was performed at different time points (0, 15, 30 min, 1 and 2 h) after the slices were perfused with GSK1016790A. After the perfusion, the hippocampi were rapidly collected and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). Protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Rochford, IL, USA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature and were then incubated with an anti-phospho-AMPK antibody (1:1000, Cell Signaling Technology), an anti-AMPK antibody (1:1000, Cell Signaling Technology), an anti-phospho-Akt antibody (1:1000, Cell Signaling Technology), an anti-Akt antibody (1:1000, Cell Signaling Technology), or an anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) antibody (1:5000; Abcam) overnight at 4°C. After three washes with TBST, the membranes were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody and then developed using an ECL detection Kit (Amersham Biosciences, Piscataway, NJ). The Western blot bands were scanned and analyzed with image analysis software (NIH). The hippocampal samples obtained from three mice were considered as a set for the Western blot analysis, and the summarized data represent the average of three experimental sets.

The data are presented as means ± S.E.M. and were analyzed using PulseFit (HEKA Elektronik) and Stata 7.0 software (STATA Corporation, USA). All data came from neurons in which both I_GABA and GSK1016790A-evoked current could be recorded (Supplementary Figure 1). Paired or unpaired Student's t-tests or analysis of variance (ANOVA) followed by Bonferroni's post hoc test were used for the statistical analyses, and the significance level was set at either P < 0.05 or P < 0.01. In the dose-response curve, the I_GABA induced by different doses of GABA was normalized to the current induced by 300 μM GABA in the same neuron. The data were fitted to a Hill equation in which I = I_max/[1+(EC₅₀/C)ⁿ], with n being the Hill coefficient and EC₅₀ value being the concentration that produced a 50% maximal response. In the current-voltage relationship curve (I-V curve), I_GABA induced at different holding potentials was normalized to the current induced with a holding potential of −60 mV in the same neuron.

5(6)-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-EET) and PKI were obtained from Cayman Chemical (Ann Arbor, MI, USA), and TTX was obtained from Enzo Life Science (Ann Arbor, MI, USA). Unless stated otherwise, all other chemicals were obtained from Sigma Chemical Company. GSK1016790A, 4α-PDD, HC-067047, RN1734, D-sphingosine, bisindolylmaleimide II (BIM), phorbol 12-myristate 13-acetate (PMA), H-89, PKI, 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP), LY294002, Compound C, AICAR, STO-609, 740 Y-P, KN62, and KN93 were prepared as stock solutions in DMSO. The final concentration of DMSO in the bath solution or pipette solution was < 0.1%. GSK1016790A, 4α-PDD, 5,6-EET, HC-067047, RN1734, BIM, PMA, 8-Br-cAMP, Compound C, AICAR, STO-609, 740 Y-P, and bicuculline were extracellulary applied by being added to the bath solution and the rapid drug delivery system. When exploring the effect of these chemicals on I_GABA, the slices were pre-incubated by these chemicals. D-Sphingosine, H-89, PKI, LY294002, KN62, and KN93 were present in the pipette solution and pre-applied by dialyzing into the neurons through the pipette. The concentrations of these drugs were chosen according to previous reports (Ben-Ari et al., 1997; Williams and Doherty, 1999; Chen et al., 2000, 2008b; Liu et al., 2007; Langelueddecke et al., 2012; Sisignano et al., 2012; Li et al., 2013a; Shen et al., 2014; Hong et al., 2016; Rahman et al., 2016).

In the present study, the synthetic TRPV4 agonists GSK1016790A and 4α-PDD and the endogenous TRPV4 agonist 5,6-EET were used to determine whether activation of TRPV4 could modulate I_GABA. We found that I_GABA (activated by 10 μM GABA) was markedly decreased by 41.1 ± 4.7% from −24.4±2.1 to −15.8±3.2 pA/pF after the application of the TRPV4 agonist GSK1016790A (0.3 μM; n = 25, paired t-test, P < 0.01; Figure 1A). The decrease in I_GABA was partially reversed after GSK1016790A was washed out. The GSK1016790A-induced inhibition of I_GABA was dose-dependent at concentrations ranging from 0.1 to 5 μM, with an IC₅₀ value of 0.1 ± 0.05 μM (Figure 1B). As 0.3 μM GSK1016790A significantly inhibited I_GABA, this dose was used in the subsequent experiments.

We then studied the effect of GSK1016790A on the dose-response of I_GABA. As shown in Figure 1C, in the absence of GSK1016790A, the EC₅₀ and n values of the dose-response curve were 12.0 ± 2.3 μM and 1.9 ± 0.4, respectively. In the presence of GSK1016790A, the maximal response to 300 μM GABA was markedly decreased (n = 7, paired t-test, P < 0.01), with EC₅₀ and n-values being 12.0 ± 2.8 μM and 2.4 ± 0.3, respectively (unpaired t-test, P > 0.05 in each case). According to the dose-response curve, 10 μM GABA was used to activate I_GABA in the following experiments. We then assessed the effect of GSK1016790A on the I-V curve of I_GABA. I_GABA was markedly inhibited by the application of GSK1016790A at voltages ranging from −80 mV to +60 mV. In the control group, the reversal potential of the I-V curve was 7.8 ± 0.7 mV, and the ratio of the current at +60/−80 mV (I_+60mV/I_−80mV) was −0.3. After the neurons were treated with GSK1016790A, the reversal potential of the I-V curve was 8.5 ± 0.5 mV and I_+60mV/I_−80mV ratio was −0.3 (n = 9, paired t-test, P > 0.05 in each case; Figure 1D).

We then examined the effect of 4α-PDD, another TRPV4 agonist, on I_GABA. As shown in Figures 2A,B, after treatment with 10 μM 4α-PDD, I_GABA was decreased by 20.0 ± 2.2% from −25.2±3.2 to −19.9±1.9 pA/pF (n = 15, paired t-test, P < 0.01). I_GABA recovered to −22.6±1.1 pA/pF after 4α-PDD was washed out. By examining the dose-response curve, we found that the EC₅₀ and n-values were 12.3 ± 2.5 μM and 2.0 ± 0.2 during 4α-PDD treatment, respectively, which were not significantly different from the control values (unpaired t-test, P > 0.05; Figure 2C). By examining the I-V curve in the presence of 4α-PDD, we found that I_GABA was inhibited at the voltages ranging from −80 mV to +60 mV (unpaired t-test, P < 0.01 at potential being −80, −60, −40, −20, and +60 mV; unpaired t-test, P < 0.05 at potential being +40 mV), with the reversal potential being 8.3 ± 1.0 mV (n = 10) and I_+60mV/I_−80mVratio being −0.3 (n = 10). Neither the reversal potential nor I_+60mV/I_−80mV ratio was markedly different from the value in the absence of 4α-PDD (unpaired t-test, P > 0.05 in each case; Figure 2D). These results indicate that the activation of TRPV4 by the synthetic TRPV4 agonists GSK1016790A and 4α-PDD induces similar inhibitory effects on I_GABA.

5,6-EET is a metabolite of arachidonate and has been identified as an endogenous TRPV4 agonist (Vincent and Duncton, 2011). In the present study, we also examined the effect of 5,6-EET on I_GABA. Figures 3A,B show that after treatment with 300 nM 5,6-EET, I_GABA was decreased by 28.1 ± 4.9% from −24.9±4.2 to −16.3±3.3 pA/pF (n = 20, paired t-test, P < 0.01), and the inhibitory effect of 5,6-EET on I_GABA was partially reversed after washout. As shown in Figure 3C, similar to the effect of the synthetic agonists of TRPV4, 5,6-EET inhibited the maximal response to 300 μM GABA (n = 17, paired t-test, P < 0.01), without affecting the EC₅₀ (15.0 ± 3.1 μM) or n (2.1 ± 0.1) value of the dose-response curve (unpaired t-test, P > 0.05 in each case). In the I-V curve, the reversal potential (8.0 ± 0.8 mV, n = 8) and I_+60mV/I_−80mV ratio (−0.3, n = 8) were statistically the same as the values before the 5′6′-EET treatment (unpaired t-test, P > 0.05 in each case; Figure 3D). These results indicate that activation of TRPV4 by either synthetic or endogenous agonists could inhibit I_GABA.

Specific TRPV4 antagonists, HC-067047 (1 μM) and RN1734 (10 μM), were used to further demonstrate the role of TRPV4 in I_GABA inhibition. I_GABA was −22.7±2.4 and −20.5±2.7 pA/pF before and during HC-067047 treatment, respectively (n = 8, paired t-test, P > 0.05). I_GABA was −23.2±1.0 and −21.7±1.3 pA/pF in the absence and presence of RN1734, respectively (n = 7, paired t-test, P > 0.05). As shown in Figure 4, in the presence of HC-067047 or RN1734, the inhibition caused by GSKA1016790, 4α-PDD, or 5,6-EET was markedly ameliorated (unpaired t-test, P < 0.01 in each case; Figures 4A,C,E).

Application of bicuculline (10 μM), a specific GABA_A receptor antagonist, markedly reduced I_GABA by 93.6 ± 1.8% (n = 21, paired t-test, P < 0.01). In the presence of bicuculline, I_GABA was statistically the same before and during treatment with TRPV4 agonists (Figures 4B,D,F). Together, these results imply that GABA_A receptor is inhibited by activation of TRPV4.

The cellular energy-sensing kinase AMPK is known to be activated in neurons in response to either metabolic insults or increased [Ca²⁺]_i through CaMKKβ. Activation of AMPK is related to modulating PI3K/Akt signaling, and the latter is involved in the modulation of GABA_A receptors (Amato et al., 2011; Nakamura et al., 2015). As TRPV4 is permeable to Ca²⁺, we examined whether activation of TRPV4 could affect AMPK-PI3K/Akt signaling. The protein levels of phosphorylated AMPK (p-AMPK) and phosphorylated Akt (p-Akt) in the hippocampi were assessed after the slices were perfused with ACSF containing GSK1016790A for 15, 30 min, 1 and 2 h. The protein level of p-AMPK was increased 15 min to 2 h after GSK1016790A treatment; the level peaked 15 min after GSK1016790A treatment and then declined (Figure 5A). The protein level of p-Akt decreased from 30 min to 2 h after GSK1016790A treatment (Figure 5B). Based on the changes in the levels of p-AMPK and p-Akt and the acute effect of TRPV4 activation, the changes in protein levels were examined 30 min after GSK1016790A treatment in the subsequent experiments. Here, it was noted that both the GSK1016790A-mediated increase in the protein level of p-AMPK and the GSK1016790A-mediated decrease in the protein level of p-Akt were markedly attenuated when the slices were perfused with Ca²⁺-free ACSF. In the presence of 10 μM STO-609, a CaMKKβ antagonist, the GSK1016790A-induced changes in the protein levels of p-AMPK, and p-Akt were markedly inhibited (Figures 5C,D). The GSK1016790A-induced decrease in the protein level of p-Akt was significantly reversed by the application of 10 μM Compound C, an AMPK antagonist (Figure 5D). These results indicate that TRPV4-induced activation of AMPK is Ca²⁺- and CaMKKβ-dependent and this action then down-regulates Akt signaling.

We then evaluated whether the regulation of AMPK and Akt signaling was involved in TRPV4-induced inhibition of I_GABA. I_GABA was reduced by 20.55 ± 2.57% when 1 mM AICAR, an AMPK agonist, was applied to the bath solution (n = 18, paired t-test, P < 0.01). We also found that I_GABA was increased by 11.84 ± 3.15% when the slices were exposed to Compound C (n = 10, paired t-test, P < 0.05). These results indicate that activation of AMPK plays a role in the regulation of I_GABA. As shown in Figure 6A, after pre-application of Compound C, GSK1016790A reduced I_GABA by 10.0 ± 2.1%, which was markedly different from the inhibition caused by GSK1016790A alone (unpaired t-test, P < 0.01).

In this study, I_GABA was increased 19.1 ± 3.1% by the application of 20 μM 740 Y-P (a PI3K agonist; n = 11, paired t-test, P < 0.01) and was inhibited 22.3 ± 4.2% by the application of 50 μM LY294002 (a PI3K antagonist; n = 7, paired t-test, P < 0.01). In the presence of 740 Y-P, GSK1016790A-induced inhibition of I_GABA was reduced to 12.5 ± 3.6% (n = 11, paired t-test, P < 0.01); however, the GSK1016790A-induced inhibition of I_GABA was virtually unaltered (37.8 ± 2.1%; n = 9) by the application of LY294002 (unpaired t-test, P > 0.05; Figure 6A). Collectively, these results indicate that the activation of AMPK and the subsequent down-regulation of the PI3K/Akt signaling pathway are likely responsible for the inhibition of I_GABA caused by acute activation of TRPV4.

We also investigated whether PKC, PKA, or CaMKII signaling was involved in TRPV4-induced inhibition of I_GABA. After the application of PKC antagonist D-sphingosine (20 μM) or BIM (1 μM), I_GABA was inhibited by 21.5 ± 3.1% (n = 10) or 22.3 ± 3.4% (n = 10) by GSK1016790A, which was significantly different from the inhibition induced by GSK1016790A alone (unpaired t-test, P < 0.01; Figure 6B). As shown in Figure 6C, in the presence of the PKA antagonist PKI (10 μM) or H-89 (10 μM), GSK1016790A treatment inhibited I_GABA by 45.4 ± 2.0% (n = 10) or 40.5 ± 3.1% (n = 11), respectively. Both inhibition levels were similar to the inhibition caused by GSK1016790A alone (unpaired t-test, P > 0.05). Figure 6D shows that when CaMKII antagonist KN62 or KN93 was added in the pipette solution, GSK1016790A inhibited I_GABA by 39.1 ± 3.7% (n = 12) or 38.3 ± 3.0% (n = 11), respectively. These results indicate that in addition to AMPK-PI3K/Akt signaling, the PKC signaling pathway is also involved in GSK1016790A-induced inhibition of I_GABA.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.