Introduction

Despite advances in the management and care of stroke, ischemic-reperfusion injury is still a major cause of mortality and morbidity. Male stroke incidence rate and prevalence are significantly higher than females worldwide1, indicating that this gender difference may be the result of sex hormone, i.e., estrogen. Estrogen regulates oxytocin synthesis in many organs, including the brain. Oxytocin is a typical stress hormone that responds to several acute and chronic stressors, and, together with its receptors, modulates an important array of physiological and biological activities2 in central and peripheral nervous systems, such as facilitating birth3. Estrogen, interleukin (IL)-1β, IL-6, interferon τ, and oxytocin regulate the expression levels of oxytocin receptors4.

Plasma oxytocin increases the expression of the peroxisome proliferator-activated receptor gamma gene, a regulator of adipocyte differentiation, and regulates the activity of eukaryotic elongation factor 25, a translation-related protein. Oxytocin controls the differentiation of bone marrow-derived mesenchymal stem cells, regulates the cells’ proliferation and carbohydrate metabolism6, and promotes lipid metabolism as an energy substrate. Mice deficient in oxytocin receptors have been found to develop obesity7.

GABA is the principal inhibitory neurotransmitter in the brain and binds three receptors, GABAAR, GABABR, and GABACR. GABAAR plays a major role in fast synaptic inhibition in the central nervous system (CNS), and is activated by allosteric modulation of interfacial five compositions (2α/2β/1γ, δ, ε, θ, π) assembled from 16 subunits (α1–6, β1–3, γ1–3, δ, ε, θ, and π) consisting of 20 subtypes8. The GABAAR-mediated hyperpolarization of membrane potential is attributed to the direct activation of an integral anion channel, and the resultant influx of chloride ions along its electrochemical gradient9. The equilibrium shift of GABAAR subtype expression pattern is a key control point for the determination of receptor diversity of the neuronal plasma membrane. Appropriate equilibrium of inhibitory and excitatory neurotransmission regulates the neuronal network in normal brain function. Conversely, an imbalance between inhibitory and excitatory neurotransmission after an ischemic insult creates an excessive secretion of excitatory molecules and suppresses the GABAergic inhibition system, by selectively limiting the trafficking of GABAARs on the plasma membrane10.

Excitotoxicity has been well-documented as a causative factor in ischemia-induced neuronal cell death11. GABAARs on neuronal cell membrane are decreased when exposed to oxygen-glucose deprivation (OGD), suggesting that the number of membrane-bound GABAARs could be a pivotal process in the progression of ischemic-induced neuronal cell death12. Oxytocin regulates GABAAR-mediated synaptic signaling in the fetal brain during delivery, and reduces brain vulnerability to hypoxic damage13. Although oxytocin-induced neuroprotection has been demonstrated in ischemic-reperfusion injury models, the molecular mechanisms underlying such therapeutic benefit, especially how oxytocin interacts with individual GABAAR subtypes14, are still unknown.

In this study, we demonstrated that administration of oxytocin in primary rat neural cells (PRNCs) before OGD resulted in robust neuroprotective effects, but not when oxytocin was initiated during OGD/R. We also showed that oxytocin shifted the expression patterns of GABAAR subunit on the cells, accompanied by increased chloride ion influx. These observations provide evidence that oxytocin modulated GABAAR in exerting its neuroprotective effects against ischemia-induced neuronal cell death.

Results

Oxytocin exerts neuroprotection against OGD/R

As shown in Fig. 1, PRNCs were exposed to the OGD/R in vitro model of stroke15,16. Pretreatment with oxytocin increased cell viability (F(2,34) = 19.48; P < 0.0001; Fig. 2A), decreased mitochondrial damage (F(2,25) = 31.81, P < 0.0001; Fig. 2B), reduced oxidative stress (F(2,30) = 406, P < 0.0001; Fig. 2C), and prevented cell secretion of high mobility group box 1 (HMGB1), a mediator of ischemic progression11,17. Atosiban, the selective oxytocin receptor antagonist13, abolished these oxytocin receptor-mediated beneficial effects. Oxytocin administered during OGD/R had no neuroprotective effect (indicated as “With OXT” in Fig. 2).

Figure 1
figure 1

Experimental design.

PRNCs; primary rat neural cells. OGD; oxygen and glycose deprivation. OXT; oxytocin. ATS; atosiban.

Figure 2
figure 2

Oxytocin confers neuroprotective effects and attenuates the oxidative stress against OGD.

(A) Cell viability tested by Calcine-AM/EthD-1 florescence and trypan blue dyes. (B) Mitochondrial activity by MTT assay. (C) GSSG activity. (D) Extracellular HMGB1 levels. *P < 0.05, **P < 0.01, and ***P < 0.001. Experiments were conducted in triplicate, with n = 6 per treatment condition in each run.

Biological activity readouts across treatments

Oxytocin acts as an anabolic hormone, and exhibits cell growth5,6 and anti-oxidative properties, suggesting its potential therapeutic application in stroke18. However, oxytocin administration did not alter cell growth of PRNCs compared with control treatment (Fig. 3A). Next, because peripheral oxytocin participates in glucose metabolism in modulating reactive oxygen species (ROS) production via NADPH (nicotinamide adenine dinucleotide phosphate) pathway, we examined the effects of oxytocin on glutathione (a major antioxidant), and glucose 6-phosphate dehydrogenase (G6PD) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (key enzymes that regulate NADPH production)19. Results revealed that G6PD, GAPDH, and glutathione disulfide (GSSG) activity levels did not significantly differ across treatment conditions (Fig. 3B–D). Pretreating the cells with oxytocin did not change the protein expression levels of G6PD and GAPDH in comparison with control values (Fig. 4).

Figure 3
figure 3

Effects of oxytocin treatment on biological activity before OGD.

(A) PRNCs’ cell growth. Oxytocin had no significant effects on the cell growth, although oxytocin has been shown to contribute to the differentiation of bone marrow-derived mesenchymal stem cells6 (B) G6PD activity. G6PD catalyzes the rate-determining step in the pentose phosphate pathway and produces NADPH to regulate GSH/GSSG levels. G6PD activities across treatments were not significantly different. (C) GAPDH activity. GAPDH regulates the ATP generation phase of glycolysis-derived NAD and functions as a reversible metabolic switch under oxidative stress. GAPDH activities across treatments did not significantly differ. (D) GSSG activity. GSSG is a biomarker of oxidative stress, and is generated by oxidized GSH with reduced NADPH. There were no statistical differences across conditions. Experiments were conducted in triplicate, with n = 6 per treatment condition in each run.

Figure 4
figure 4

Western blot analysis.

Following incubation of PRNCs in the absence of reagents (control; CONT), or with 1 μM oxytocin + 10 μM atosiban (OXT + ATS), or 1 μM oxytocin (OXT) for 3 days 37 °C. (A) Expression levels of GABAAR subunits, oxytocin receptor (OXR), bestrophin-1 (BST1), G6PD, and GAPDH. (B) Relative quantification of protein expression levels. Blue bars represent significantly increased protein expression levels, red bar showed significantly decreased levels, and white bars indicate no statistical differences between oxytocin-treated cells and control. *P < 0.05 and **P < 0.01. The dotted red line represents combined data from control and OXT + ATS, since these two groups did not significantly differ. Experiments were independently conducted in 3~6 times.

Oxytocin pretreatment shifts the GABAAR subunit expression patterns

Oxytocin has been found to alter the subtype expression patterns20 and function of GABAARs13,21. We therefore assessed whether oxytocin modulated the expression patterns of GABAARs subunit on PRNCs (Fig. 4). Treatment of PRNCs with oxytocin significantly increased α4, β3, δ, and ε GABAAR subunit expression levels, but decreased γ2 GABAAR subunit (Fig. 4). Although oxytocin receptor expression has been reported to be increased following oxytocin treatment4, we could not detect any differences in oxytocin receptor upregulation between control and oxytocin treatment in this experiment (Fig. 4). Moreover, oxytocin treatment did not affect the expression levels of Bestrophin-1 (BST1), a calcium-activated chloride ion channel normally distributed on synapses adjacent to soma22 and shown to mediate GABA release from astrocytes23.

Localization of GABAAR subunits

Electric current and localization patterns of GABAAR vary depending on the region of interest within the neuron, because glycogen (the main storage form of glucose in the body) is predominantly preserved in the soma where the main production of ATP occurs. In the ischemic brain, the rate of glycogen metabolism is significantly increased24. We observed the localization of α4, β3, γ2, δ, and ε GABAAR subunit expression within subcellular compartments of the neuron. Immunocytochemichal analysis showed that (i) δ GABAAR subunit was mainly located at the axon (Fig. 5A, indicated with box), (ii) ε GABAAR subunit predominantly resided in the soma (Fig. 5B, indicated with box), (iii) γ2, α4, and β3 GABAAR subunits were broadly expressed in the whole neuron (Fig. 5A,B,E,F), (iv) α4/δ- and β3/δ-GABAAR subunits showed co-localization (Fig. 5C,D, indicated with arrow), and (v) α4/ε- and β3/ε-GABAAR subunits were also co-localized in PRNCs (Fig. 5E,F, indicated with arrow).

Figure 5
figure 5

Localization of δ, γ2, α4, β3, and ε GABAAR subunits expression on neurons.

Following incubation of PRNCs in the absence of reagent (control; CONT) or with 1 μM oxytocin (OXT) for 3 days 37 °C. (A) Expression of γ2 and δ-subunits, (B) γ2 and ε-subunits, (C) δ and α4-subunits, (D) δ and β3-subunits, (E) ε and α4-subunits, and (F) ε and β3-subunits. Scale bars = 10 μm.

Intracellular chloride ion influx kinetics

After binding with GABA, GABAAR engages a chloride ion selective pore, resulting in chloride ion influx that inhibits the firing of neuron action potentials. The kinetic property of GABAAR depends on receptor subunit compositions, thereby providing a mechanism for neurons to regulate individual biological activities. We performed a time course study to reveal any differences in GABA-elicited chloride ion influxes between control- and oxytocin-treated PRNCs. Figure 6A revealed that chloride ion influx reached equilibrium at 10 min, but treatment with oxytocin significantly increased the influx at the 20 min period. To assess the differences of both GABAAR antagonistic conditions, we compared the inhibition dynamics of GABA-induced chloride ion influx in the presence of flumazenil (GABAAR antagonist, GABA + FLU) or picrotoxin (GABAAR channel blocker, GABA + PIC). Both reagents inhibited the GABA-induced chloride ion influx (control; Fig. 6B and oxytocin treatment; Fig. 6C). Interestingly, oxytocin-treated cells were more sensitive to picrotoxin inhibition, as evidenced by the Δ value of control cells = 14.0 ± 2.20 AU (Fig. 6B), and that of oxytocin-treated cells = 24.1 ± 1.70 AU (Fig. 6C), P < 0.001. Because δ GABAARs display increased sensitivity to THIP (δ-GABAAR specific agonist)25, we tested whether oxytocin-treated cells additively increased THIP-induced chloride ion influx. Results revealed that THIP-evoked chloride ion influx of oxytocin-treated cells was significantly higher than that of control (Fig. 6D).

Figure 6
figure 6

Characterization of chloride ion influx via GABAAR.

Following incubation of PRNCs in the absence of reagents (control; CONT) or with 1 μM oxytocin (OXT) for 3 days 37 °C. (A) Time course of chloride ion influx stimulated by 50 μM GABA. (B,C) The cells were pretreated with 1 μM flumazenil (GABA + FLU), or 1 μM picrotoxin (GABA + PIC), or PBS (GABA) for 45 min at 37 °C, and were stimulated by 50 μM GABA for 10 min at RT, and then fluorescence intensity was measured. (B,C) represent control- and oxytocin treated-cells, respectively. The Δ value was calculated from following equation: fluorescence intensity of (GABA – [GABA + PIC]). (D) Time course of chloride ion influx stimulated by 10 nM THIP. *P < 0.05, **P < 0.01, and ***P < 0.001. Experiments were conducted in triplicate, with n = 6 per treatment condition in each run.

Discussion

The present study revealed a novel molecular mechanism underlying oxytocin-mediated neuroprotection against ischemic stroke in a cell culture paradigm. We found that oxytocin-induced GABAAR subunit modification is a predominant factor in conferring neuroprotection against OGD. GABA is the principal inhibitory transmitter in the brain, and its functions are mediated by ubiquitously expressed ligand-opened chloride ion channel GABAARs26. Aberrant GABAergic inhibition is a key pathological feature displayed by ischemic neurons in the peri-infarct area (secondary damaged region) after stroke26. Our present results demonstrated that oxytocin reduced ischemic stroke deficits likely by modulating specific GABAAR subtype signal transduction14, which parallels studies showing that oxytocin improves stroke outcomes via social interaction pathways18.

We showed that oxytocin protected PRNCs against OGD (Fig. 2). Ischemic injury is mediated by ROS, generated primarily by damaged mitochondria27, which leads to apoptosis and necrosis. During OGD, cell viability and mitochondrial activity were decreased, and the GSSG activity and extracellular HMGB1 levels were increased. HMGB1, a non-histone DNA-binding protein, is released from necrotic neurons after 2 h OGD17, and its concentrations in serum are significantly increased in stroke patients due to blood brain barrier (BBB) disruption associated with the disease progression11,17. That oxytocin exerted neuroprotection in OGD, but not in the OGD/R model is consistent with in vivo evidence, demonstrating that the subsequent reperfusion after ischemia exacerbates neuronal functions and causes massive brain injuries when oxygen-saturated and nutrient-rich blood suddenly returns to the lesion after a period of ischemia11, suggesting that OGD/R is worse than OGD. Under the OGD condition, pretreatment with oxytocin increased cell viability and mitochondrial activity, decreased the GSSG activity, and prevented HMGB1 secretion from the cells. In the presence of atosiban, this neuroprotection was abolished, indicating that the therapeutic effect was likely mediated by oxytocin receptor signal transduction. HMGB1 is phosphorylated by protein kinase C28 and calcium/calmodulin-dependent protein kinase29. Although oxytocin is capable of activating both kinases3, we could not detect extracellular HMGB1 despite incubating the cells with oxytocin prior OGD. Altogether, these observations suggest that oxytocin could serve as a neuroprotective agent in the acute phase of stroke by acting as an ischemic preconditioning factor in modulating therapeutic protein synthesis.

Oxytocin regulates glucose uptake that is critical for stem cell growth6 and antioxidant activity30. However, cell growth of PRNCs was not affected by oxytocin (Fig. 3A). We thus tested whether oxytocin utilized its receptor signal transduction in regulating glutathione-related proteins (G6PD, GAPDH, GSSG). G6PD regulates the antioxidant activity of NADPH31, facilitating NADPH to maintain glutathione/GSSG recycling32. GAPDH is not only a key enzyme in glycolysis, but also phosphorylates the α1 GABAAR subunit for sustaining the GABAAR structure and stability, thereby establishing the link of GABAergic inhibition with glucose metabolism. Under ischemic condition, glycolytic flux increases, while GAPDH activity is reversibly decreased, inhibiting neuronal cells from producing NADPH31 as a result of increased G6PD activity. We speculated that oxytocin would normalize G6PD, GAPDH, GSSG activity, but no significant difference were detected across treatment conditions (Fig. 3B–D), suggesting that oxytocin’s neuroprotection is largely independent of G6PD, GAPDH, and GSSG signal transduction pathways.

Maternal oxytocin exerts neuroprotective action on fetal neurons during parturition (a perturbed physiological environment similar to hypoxic-ischemic brain) mediated by GABAAR signaling pathway13. In the present study, oxytocin likely engaged the GABAAR subunit expression patterns by enhancing α4, β3, δ, and ε GABAAR subunit expression levels while reducing γ2 GABAAR subunit on PRNCs (Fig. 4). Although most GABAAR subunit expressions were not significantly influenced in the presence of atosiban (OXT + ATS), α2 GABAAR subunit expression decreased and γ1 and π GABAAR subunits increased (Fig. 4A), implicating that redundant signal impedance of oxytocin receptor could activate alternative signal transduction of these subunit expressions. Conversely, an on/off signaling switch of oxytocin may be tightly regulated by engagement with GABAAR subunits.

The β3 and γ2 GABAAR subunits on the neuronal membrane are vulnerable to ischemic stroke10,33. In the late stage of rat pregnancy the maternal brain displays increased expression of the ε GABAAR subunit, which is responsible for the respiratory function34. The α4 GABAAR subunit, on the other hand, is associated with dendritic development35, and is well co-expressed with δ-subunit in the brain8,36. The α4, δ, and ε GABAAR subtypes are extrasynaptic GABAARs8,36, which mediate tonic inhibition upon activation by GABA spillover from synaptic sites, as well as by ambient GABA in the extracellular space. The majority of GABAARs are α1β2γ2, α2β3γ2, and α3β2/3γ2, approximately occupying 80% of total GABAAR expressions in the brain8, in contrast, α4, δ, and ε GABAARs represent less than 5%8. Oxytocin-treated cells significantly increased α4, δ, and ε GABAAR subunit expression levels and decreased γ2 GABAAR subunit on PRNCs (Fig. 4). It is conceivable that oxytocin pretreatment led to an upregulation of specific GABAAR subtype expression levels, which in turn might have modified the neuronal networks towards neuroprotection.

Electrical properties of a neuron vary along the segments of subcellular organization (soma, dendrites, and axon), which is essential for orchestrating cellular function and structure preservation37. GABAAR-mediated chloride ion fluctuation substantially changes the intracellular chloride ion concentration in the soma and spreads into the dendrites38. Hypoxic ischemia causes retrograde neurodegeneration, which shortens the axonal and dendritic lengths and swells the soma, and produces a rapid and significant loss of axon in the acute phase of injury39. Therefore, it is an important to elucidate the GABAAR subunit localizations at the subcellular neuron. Interestingly, δ GABAAR subunit is mainly expressed at axon, and ε GABAAR subunit is primarily distributed on soma which concurred with previous report40. In contrast, α4, β3 and γ2 GABAAR subunits were broadly expressed throughout the whole neuron8. Here, we found that α4/δ- and β3/δ-GABAAR subunits, and α4/ε- and β3/ε-GABAAR subunits were well co-localized on neurons (Fig. 5). That appropriate distribution and specific expression of GABAARs subtypes exist in neurons suggest that oxytocin could elicit neuroprotection by subcellularly targeting specific GABAAR subunits within ischemic neurons.

Under hypoxic ischemia, the extracellular GABA concentration on/around the synaptic cleft increases and elevates neuronal intracellular chloride ion, which functions as a counter-reaction of depolarization41. We demonstrated here that oxytocin modulated discrete GABAAR subunits tasked to monitor chloride ion influx. The kinetic of GABA-stimulated chloride ion influx on oxytocin-treated cells was altered (Fig. 6A), in that while the response to GABA by the control cells was saturated at 10 min, oxytocin-treated cells continuously evoked the ion influx for 20 minutes, suggesting that boosting GABAARs-mediated neuronal inhibition can afford substantial protection while minimizing the extent of neuronal cell loss during OGD. Following incubation of cells with 50 μM GABA for 10 min (Fig. 6A), the values of chloride ion influx were similar. We next assessed GABAAR antagonism and found that Flumazenil inhibited the γ contained GABAARs in response to GABA, but was not able to antagonize the δ and ε GABAARs42. Picrotoxin directly binds the ion pore of GABAAR, thereby regulating the influx of chloride ion, and inhibiting the whole GABAAR channel activity42. The inhibition ability of flumazenil (GABA + FLU) did not significantly differ between conditions, implicating that oxytocin administration had no effect on the expression levels of γ GABAAR subtypes (Fig. 6B,C). In contrast, oxytocin-treated cells exhibited significant inhibition of chloride ion influx following picrotoxin treatment (GABA + PIC) compared to control, suggesting that oxytocin significantly increased total GABAAR expression, especially δ and ε GABAAR subtypes. Of note, to date, there is no specific antagonist for δ and ε GABAAR subunits. The present observation of specialized GABAARs antagonism is also supported by δ-GABAAR specific agonist THIP significantly elevating the chloride ion influx of oxytocin-treated cells compared to control (Fig. 6D). In summary, oxytocin induced the shift of GABAAR subunit expression in cultured PRNCs, which likely changed the kinetics of chloride ion influx in response to GABA.

We demonstrated that oxytocin exerts neuroprotection against ischemic stroke, but requires its treatment initiation prior to injury induction. Oxytocin may serve as a pharmacological ischemic preconditioning factor that can engage GABAAR towards neuroprotection. The present results provide evidence that oxytocin altered the expression patterns of GABAAR subunit and the kinetics of GABA-induced chloride ion influx. Our study highlights a close interaction between oxytocin and GABAAR that should aid in our understanding of stroke pathology and its treatment.

Methods

Cell culture and oxygen-glucose deprivation-reperfusion (OGD/R) progression

Primary rat neural cells (PRNCs; consisted of 40% neurons and 60% astrocytes) were obtained from BrainBit (E18 rat cortex; Springfield, IL, USA). As described elsewhere15, cells (4 × 104 cells/well) were suspended in 200 μl Neural Medium (NbActive 4, BrainBit) containing 2 mM l- glutamine and 2% B27 in the absence of antibiotics and grown in poly-l-lysine-coated 96-well plates at 37 °C in humidified atmosphere containing 95% O2 and 5% CO2. After 3 days in culture, PRNCs were exposed to 1 μM oxytocin (O4375, Sigma-Aldrich, St. Louis, MI, USA), 1 μM oxytocin + 10 μM atosiban (A3480, Sigma-Aldrich), 10 μM atosiban, and the absence of regents (control) for 3 days at 37 °C. After 6 days in culture (Fig. 1), RPNCs were exposed to OGD as described previously15. The cells were initially exposed to OGD medium (glucose-free Dulbecco’s Modified Eagle Medium), then placed in an anaerobic chamber containing 95% N2 and 5% CO2 for 15 min at 37 °C (preincubation), for 90 min at 37 °C (culture medium pH 6.7~6.8; mimicking the acidic environment of ischemic brain in vivo). OGD was terminated by adding 5 mM glucose to medium and cell cultures were re-introduced to the regular CO2 incubator at 37 °C for 2 h. Control cells were incubated in the same buffer containing 5 mM glucose at 37 °C in a regular 95% O2 and 5% CO2 incubator.

Measurement of cell viability

Measurement of cell viability was performed using fluorescent live/dead cell assay and trypan blue exclusion method15,43. Following treatment, the cells were incubated with 2 μM Calcein-AM and 4 μM EthD-1 (L3224 Invitrogen, Waltham, MA, USA) for 45 min at room temperature (RT) in dark. After washing once with phosphate buffer saline (PBS), the green fluorescence of the live cells was measured by the Gemini EX florescence plate reader (Ex/Em = 490/520; Molecular Devices, Sunnyvale, CA). In addition, trypan blue (15250, Gibco, Waltham, MS, USA) exclusion method was conducted and mean viable cell counts were calculated in 16 randomly selected areas (1 mm2, n = 10) to reveal the cell viability. Briefly, within 5 min after adding trypan blue, we digitally captured under microscope (200x) 10 pictures (approximately 100 cells/picture) for each condition, then randomly selected 5 pictures, and counted the number of cells for each individual treatment condition. Normalized cell viability was calculated from the following equation: viable cells (%) = [1.00 – (Number of blue cells /Number of total cells)] × 100. To precisely calibrate the cell viability, the values were standardized from fluorescence intensity and trypan blue data.

Measurement of mitochondrial activity

Following OGD/R, reduction of 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT; 11465007001, Roche, Basel, Switzerland) by mitochondrial dehydrogenases was used as a measure of mitochondrial activity as previously described43. The optical density of solubilized purple formazan was measured at 570 nm on a Synergy HT plate reader (Bio-Tex, Winooski, VT, USA).

Measurement of extracellular high mobility group box1 (HMGB1) levels and glutathione disulfide (GSSH) activity

After OGD/R, culture medium was centrifuged at 3,000 g, 4 °C for 15 min, and the supernatant was processed for detection of HMGB1 using an ELISA kit (amin416082, Antibody, Atlanta, GA, USA) with absorbance measured at 450 nm on a Synergy HT plate reader (Bio-Tex). Cells were treated with oxidized glutathione lysis reagent (V6611, Promega, Fitchburg, WI, USA), and GSSG activity, a biomarker of reactive oxygen species (ROS) production, was measured by luciferase activity on Spectra Max Gemini EM plate reader (Molecular Devices, Sunnyvale, CA, USA).

Measurement of cell growth, glucose 6-phosphate dehydrogenase (G6PD)-, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-activity

Following cell culture, the cleavage of the tetrazolium salt, WST-1 (4-[3(4-lodophenyl)-2-(4-nirtophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; 05015944011, Roche) formazan was used as a measure cell growth. The optical density was measured at 450 nm on a Synergy HT plate reader (Bio-Tex). The levels of G6PDH- and GAPDH-activity were performed according to the manufacturer’s protocols for G6PD assay kit (ab102529, Abcam, Cambridge, MA, USA) and GAPDH ELISA kit (ab119627, Abcam), respectively.

Western blot analysis

PRNCs were treated with CelLytic MT mammalian lysis reagent (C3228, Sigma-Aldrich) with protease inhibitor cocktail (I3786, Sigma-Aldrich). The lysate was centrifuged at 3,000 g, 4 °C for 15 min, and the supernatant was stored at −80 °C until analysis. Protein samples (4~35 μg/lane) were processed on 4~14% Tris-Glycine SDS-PAGE gel and then transferred onto a nitrocellulose membrane (162–0112, Bio-Rad, Hercules, CA, USA) at 30 V, 4 °C for 14 h. The nitrocellulose membranes were treated with PBS containing 0.1% Tween-20 and 3% non-fat milk (170–6404, Bio-Rad) for 45 min at RT. Membranes were then incubated with the primary antibodies, anti-oxytocin receptor rabbit antibody (1/10,000, ab181077, Abcam), anti-GABAAR α1 subunit rabbit antibody (1/3,000, ab3299, Abcam), anti-GABAAR α2 subunit rabbit antibody (1/1,000, ab72445, Abcam), anti-GABAAR α3 subunit rabbit antibody (1/1,000, ab72446, Abcam), anti-GABAAR α4 subunit rabbit antibody (1/1,000, ab4120, Abcam), anti-GABAAR α5 subunit rabbit antibody (1/3,000, ab10098, Abcam), anti-GABAAR α6 subunit goat antibody (1/1,000, ab117100, Abcam), anti-GABAAR β1 subunit rabbit antibody (1/3,000, ab154822, Abcam), anti-GABAAR β2 subunit rabbit antibody (1/30,000, ab16213, Abcam), anti-GABAAR β3 subunit rabbit antibody (1/1,000, ab4046, Abcam), anti-GABAAR γ1 subunit rabbit antibody (1/5,000, AMIN485542, Antibodies), anti-GABAAR γ2 subunit rabbit antibody (1/1,000, ab16213, Abcam), anti-GABAAR γ3 subunit rabbit antibody (1/500, ab13861, Abcam), anti-GABAAR δ subunit rabbit antibody (1/1,000, ab11048, Abcam), anti-GABAAR ε subunit rabbit antibody (1/500, ab35971, Abcam), anti-GABAAR π subunit rabbit antibody (1/5,000, ab26055, Abcam), anti-GABAAR θ subunit rabbit antibody (1/5,000, ARP5283, Antibodies), anti-bestrophin-1 (BST1) mouse antibody (1/3,000, NB300-164, Antibodies), anti-G6PD rabbit antibody (1/10,000, ab993, Abcam), and anti-GAPDH mouse antibody (1/10,000, ab8245, Abcam) at 4 °C for 14 h. After washing with PBS containing 0.1% Tween-20 (PBST), the nitrocellulose membrane was incubated with donkey anti-mouse IRDye800®CW secondary antibody (1/5,000, 926-32212, LI-COR, Lincoln, NE, USA), or donkey anti-rabbit IRDye800®CW secondary antibody (1/5,000, 926-32213, LI-COR), or donkey anti-goat IRDye800®CW secondary antibody (1/5,000, 926-32214, LI-COR) for 90 min at RT in dark. Immunoreactive detection using near-infrared fluorescence was performed according to the protocol of Odyssey® Infrared Imaging System (LI-COR®).

Immunocytochemistry analysis

PRNCs (8 × 104 cell/well) were cultured in 400 μl Neural medium containing 2 mM l- glutamine and 2% B27 in the absence of antibiotics in poly-l-lysine 8-chamber (354632, BD Bioscience, Franklin Lakes, NJ, USA) for 3 days, then the cells were exposed to 1 μM oxytocin in the absence of reagents (control) for 3 days and fixed in 4% paraformaldehyde15. The cells were washed 5 times for 10 min in PBST. Then they were blocked by 5% normal goat serum (50062Z, Invitrogen, Carisbad, CA, USA) in PBST for 1 h at RT. Primary antibodies included anti-GABAAR α4 subunit mouse antibody (1/100, SMC-489, StressMarq Bioscience Inc., BC, Canada), anti-GABAAR β3 subunit mouse antibody (1/500, ab98968, Abcam), anti-GABAAR γ2 subunit mouse antibody (1/250, MABN263, Millipore, Billerica, MA, USA), anti-GABAAR δ subunit rabbit antibody (1/200, ab111048, Abcam), anti-GABAAR ε subunit rabbit antibody (1/200, ab35971, Abcam), and anti-microtubule-associated protein 2 (MAP2) chicken antibody (1/10,000, ab5392, Abcam). The cells were incubated overnight at 4 °C in primary antibody with 5% normal goat serum. The cells were washed 5 times for 10 min in PBST and then soaked in 5% normal goat serum in PBST containing corresponding secondary antibodies goat anti-mouse IgG-Alexa 405 (blue; 1/1,000, A31553, Invitrogen), goat anti-rabbit IgG-Alexa 405 (blue; 1/1,000, A31556, Invitrogen), goat anti-mouse IgG-Alexa 488 (green; 1/1,000, A11029, Invitrogen), goat anti-rabbit IgG-Alexa 488 (green; 1/1,000, A11034, Invitrogen), and goat anti-chicken IgG-Alexa 594 (red; 1/1,000, A11042, Invitrogen) for 90 min in the dark. Immunofluorescent images were visualized using confocal microscope (FV1000, Olympus, Tokyo, Japan). Control experiments were performed with the omission of the primary antibodies yielding negative results.

Measurement of intracellular chloride ion influx

The quinolinium salt-based halide-sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE; ab145418, Abcam) was used as a measure of chloride ion influx activity13. Following cell culture, the PRNCs were incubated with 5 mM MQAE for 2 h at RT in the dark, and subsequently washed twice with NbActive 4 (BrainBit). Cells were then treated with 50 μM GABA (A2129, Sigma-Aldrich) or 10 nM GABA analog THIP, δ-GABAAR specific agonist8 (T101, Sigma-Aldrich), then fluorescence intensity was consequently measured at 0, 5, 10, and 20 min at RT. For inhibition assay, the cells were pretreated with 1 μM flumazenil (F6300, Sigma-Aldrich) or 1 μM picrotoxin (P1675, Sigma-Aldrich) or PBS (control) for 45 min at RT, and then stimulated with 50 μM GABA for 10 min at RT. Intercellular MQAE is quenched by 10 μM tributyltin chloride (T50202, Sigma-Aldrich) and 10 μM nigericin sodium salt (N7143, Sigma-Aldrich). The fluorescence intensity was measured by the Gemini EX florescence plate reader (Ex/Em = 360/460; Molecular Devices). The kinetic analysis was performed by using GraphPad Prism 6® software.

Data analysis

Data were evaluated using one-way analysis of variance (ANOVA) followed by post hoc compromised t-tests (GraphPad Prism 6® software). Statistical significance was preset at P < 0.05. Data are represented as means ± SD from quintuplicates of each treatment condition.

Additional Information

How to cite this article: Kaneko, Y. et al. Oxytocin modulates GABAAR subunits to confer neuroprotection in stroke in vitro. Sci. Rep. 6, 35659; doi: 10.1038/srep35659 (2016).