Interleukin-6 mediates the increase in NADPH-oxidase in the ketamine model of schizophrenia

Abstract

Adult exposure to NMDA receptor antagonists, such as ketamine, produces psychosis in humans, and exacerbates symptoms in schizophrenic patients. We recently showed that ketamine activates the innate immune enzyme NADPH-oxidase in brain, and that the superoxide produced leads to dysfunction of a subset of fast-spiking inhibitory interneurons expressing the calcium-binding protein parvalbumin (PV). Here we show that neuronal production of interleukin-6 (IL-6) is necessary and sufficient for ketamine-mediated activation of NADPH-oxidase in brain. Removal of IL-6 in neuronal cultures by anti-IL-6 blocking antibodies, or in vivo by use of IL-6-deficient mice, prevented the increase in superoxide by ketamine and rescued the interneurons. Accumulating evidence suggests that schizophrenia patients suffer from diminished antioxidant defenses, and a recent clinical trial showed that enhancing these defenses may ameliorate symptoms of the disease. Our results showing that ketamine-induced IL-6 is responsible for the activation of NADPH-oxidase in brain suggest that reducing brain levels of this cytokine may protect the GABAergic phenotype of fast-spiking PV-interneurons and thus attenuate the propsychotic effects of ketamine.

Figure 1.

Slow reversal of ketamine effects on PV-interneurons in vivo. C57BL/6 mice (3 month-old males) were treated with ketamine (30 mg/kg i.p.) on 1 or 2 consecutive days as described previously (Behrens et al., 2007). Animals were killed either 24 h after a single injection (1 injection), 24 h after the second injection (2 injections), or 3 and 10 d after withdrawal of the second ketamine injection. Coronal brain sections comprising the prelimbic region were analyzed by fluorescence immunohistochemistry for PV and GAD67, and expressed as percentage of the 1 d saline-treated controls. A slow increase in fluorescence intensity for both proteins is observed at 3 d after the second ketamine injection. *p < 0.05 by two-way ANOVA followed by Tukey's post hoc multiple comparisons. Each time point consisted of five animals treated with saline and five animals treated with ketamine. Data are means ± SD. Mean fluorescence intensity for 1 d of saline: PV = 160.6 ± 13.3; GAD67 = 110.4 ± 8.6.

Figure 2.

Absence of ketamine effects in the PFC of Nox2 knock-out mice. Three-month-old wild-type (C57BL/6) or gp91phox^−/− mice were treated with ketamine (30 mg/kg i.p. on 2 consecutive days) followed by DHE injections. Coronal sections comprising the prelimbic and infralimbic regions were analyzed for (A) oxidized DHE, and (B) PV immunofluorescence. Fluorescence intensity is expressed as percentage of saline-treated C57BL/6 animals. *p < 0.001 and ^#p < 0.05. Data are means ± SD; n = 4–5 for wild type and 5–7 animals for gp91phox. Mean baseline fluorescence intensity for saline C57BL/6 control: ox-DHE = 9.8 ± 1.5; PV = 147.2 ± 23.3.

Figure 3.

IL-6 exposure leads to the loss of phenotype of PV-interneurons in primary neuronal cultures. Neuronal cultures were treated with IL-6 (10 ng/ml) in the absence (control) or presence of the Nox2 inhibitor apocynin (0.5 mm) for 24 h. Fluorescence confocal images of representative fields depicting the expression of PV and GAD67 in PV-interneurons. Bar graph represents the quantification of fluorescence expressed as percentage of control. (*p < 0.01, ^#p < 0.001. n = 4 experiments per condition.) Data are means ± SEM. Baseline intensities: PV = 135 ± 32; GAD67 = 114 ± 26.

Figure 4.

IL-6 increases superoxide production and Nox2 expression in neurons. Neuronal cultures were treated with IL-6 (10 ng/ml) in the absence (control) or presence of the Nox2 inhibitor apocynin (0.5 mm) for 24 h. DHE (1 μg/ml) was added during the last hour of treatment. Images show the increase in Nox2 immunoreactivity and oxidized DHE on treatment with IL-6. Bar graphs show the results of quantification of oxidized DHE and Nox2 fluorescence expressed as percentage of control. (*p < 0.05. n = 5 experiments per condition.) Data are means ± SD. Baseline intensities: DHE = 21 ± 4.7; Nox2 = 18.7 ± 2.6.

Figure 5.

Ketamine increases IL-6 mRNA expression. Primary neuronal cultures were exposed to ketamine (0.5 μm) for the times indicated and the abundance of IL-6 mRNA was determined by PCR using specific primers after reverse-transcription of mRNA obtained from the cultures. Values for IL-6 mRNA abundance were obtained after normalization by the expression of GAPDH mRNA in the samples. (*p < 0.05 with respect to control. n = 3 experiments per time-point.)

Figure 6.

Ketamine effects on PV-interneurons do not require the presence of astrocytes. Primary neuronal cultures were grown on glass coverslips with “feet” as described (Kinney et al., 2006). After 21 d of development in vitro, the cultures were treated with ketamine (0.5 μm for 24 h) in the presence or absence of the astrocytic layer. For this, the coverslips containing neurons were separated from the astrocytic layer by transfer of the coverslip together with its media into an empty well. DHE was added for the last hour of treatment as described (Behrens et al., 2007). After treatment, neurons were fixed and processed for immunofluorescence for detection of either PV or GAD67 or for oxidized DHE. *^,#Statistical significant difference compared with control conditions at p < 0.001. n = 4–5 experiments per condition. Data are means ± SEM. Baseline intensities: PV = 210 ± 32; GAD67 = 195 ± 26.

Figure 7.

Blocking IL-6 with antibodies prevents ketamine effects on PV-interneurons. Primary neuronal cultures were exposed to ketamine in the absence of the astrocytic monolayer and in the presence of an anti-mouse IL-6 blocking antibody produced in goat (α-mIL-6). A, Increasing concentrations of α-mIL-6 prevented the decrease in PV and GAD67 after 24 h of ketamine exposure. Bar graph show results for fluorescence quantification of both antigens in PV-interneurons expressed as % of control. *^,**p < 0.05; ^#,## p < 0.001. n = 4 experiments per condition. Baseline intensities: PV = 165 ± 30; GAD67 = 127 ± 28. B, Neuronal cultures were treated as in A, and DHE was added for the last hour of treatment. After fixation, the coverslips were processed for immunocytochemistry for parvalbumin (PV, green). Bar graphs show results for oxidized DHE fluorescence (red) intensity analysis in all neurons including PV-interneurons. (*p < 0.001 compared with control and **p < 0.001 compared with ketamine. n = 3 experiments per condition.) Baseline intensities: DHE = 25.4 ± 5.4.

Figure 8.

CNS production of IL-6 mediates ketamine effects on Nox and PV-interneurons in vivo. A, Animals were treated with saline or ketamine (30 mg/kg) on two consecutive days and the brains extracted for mRNA preparation 24 h after the last ketamine injection. The abundance of IL-6, IL-1β, and TNFα mRNA was determined by PCR using specific primers after reverse-transcription of mRNA obtained from forebrains. Values for mRNA abundance were obtained after normalization by the expression of GAPDH mRNA in the samples. (*Statistically significant differences compared with control conditions. n = 4 animals per condition.) B, C, Three-month-old C57BL/6 (wt) or IL-6-deficient (IL-6^−/−) male mice were treated with ketamine (30 mg/kg) on 2 consecutive days, followed by DHE, as described previously (Behrens et al., 2007). Coronal sections comprising the prelimbic and infralimbic regions were analyzed by immunohistochemistry for PV and oxidized DHE fluorescence. Ketamine produced a substantial increase in oxidized DHE in wild-type mice but not in IL-6^−/− animals (*p < 0.001 wt-sal vs wt-ket; **p = 0.001 wt-ket vs IL-6^−/− sal or ket). The loss of parvalbumin expression induced by ketamine was prevented in the IL-6^−/− animals. (^#p < 0.001 wt-sal vs wt-ketamine; ^##p < 0.001, wt-ketamine vs IL-6. n = 4 animals per condition). Data are means ± SD. Mean fluorescence intensity for saline: wild type, PV = 111.6 ± 9.3; ox-DHE = 10.1 ± 2.5; IL-6^−/−, PV = 101.7 ± 10.2; ox-DHE = 11.7 ± 3.2.

Figure 9.

Ketamine-induced IL-6 release directly activates Nox. A, EPR assessment of superoxide production in live cultures on treatment with ketamine (0.5 μm). Primary cultures were exposed to ketamine for the times indicated in the absence or presence of an anti-mouse IL-6 blocking antibody produced in rat (αIL-6, 0.1 μg/ml). At the indicated times, the coverslips were transferred to a quartz chamber and superoxide production was followed by EPR spectroscopy using the spin-trap DIPPMPO. Ketamine induced a rapid increase in superoxide signals that were significantly reduced by the blocking antibody (*Statistically significant difference compared with control, p < 0.05 and p < 0.001 for 1 and 3 h, respectively. ^#Statistically significant difference compared with ketamine, p < 0.05. n = 3–6 experiments per condition.) B, IL-6 (100 ng/ml) increased basal NADPH oxidase activity in synaptosomes isolated from 3-month-old C57BL/6 male forebrains. IL-6 was preincubated with synaptosomal preparations for 5 min before triggering oxidase activity by addition of substrate, NADPH. Apocynin (0.4 mm) was applied 5 min before IL-6. Accumulation of superoxide during the first 6 min was analyzed using the spin trap DEPMPO. Data are means ± SEM (*p < 0.01. n = 6–7 experiments per condition.) Data are means ± SEM.