A novel role for brain interleukin-6: facilitation of cognitive flexibility in rat orbitofrontal cortex

Abstract

Cytokines, small proteins released by the immune system to combat infection, are typically studied under inflammatory conditions. However, these molecules are also expressed in the brain in basal, nonpathological states, where they can regulate neuronal processes, such as learning and memory. However, little is known about how cytokine signaling in the brain may influence higher-order cognitive functions. Cognitive flexibility is one such executive process, mediated by the prefrontal cortex, which requires an adaptive modification of learned behaviors in response to environmental change. We explored the role of basal IL-6 signaling in the orbitofrontal cortex (OFC) in reversal learning, a form of cognitive flexibility that can be measured in the rat using the attentional set-shifting test. We found that inhibiting IL-6 or its downstream JAK/STAT signaling pathway in the OFC impaired reversal learning, suggesting that basal IL-6 and JAK/STAT signaling facilitate cognitive flexibility. Further, we demonstrated that elevating IL-6 in the OFC by adeno-associated virus-mediated gene delivery reversed a cognitive deficit induced by chronic stress, thus identifying IL-6 and the downstream JAK/STAT signaling pathway as potentially novel therapeutic targets for the treatment of stress-related psychiatric diseases associated with cognitive dysfunction.

Keywords: JAK/STAT; cognitive flexibility; depression; interleukin-6; orbitofrontal cortex; reversal learning.

Figure 1.

Effect of central IL-6 immunoneutralization on reversal learning and downstream signaling pathways in the OFC. a, Both CIC stress and treatment with an anti-IL-6 antibody (200 ng/d, i.c.v.) increased trials to meet criterion on the reversal learning stage of the attentional set-shifting test. *p < 0.05, main effect compared with IgG control groups. ⁺p < 0.05, main effect compared with nonstress control groups. b–e, Treatment with the IL-6 antibody decreased STAT3 and ERK1/2 phosphorylation in the OFC without affecting AKT phosphorylation. Fold change relative to No Stress, IgG Antibody control group. *p < 0.05, main effect compared with IgG control groups. f, Representative Western blot images. n = 9–11 per group.

Figure 2.

Effect of local IL-6 immunoneutralization on reversal learning performance. a, c, Representative images of cannula placement and injection sites in the OFC and dorsal striatum, respectively. *Injection site. b, Injection of the anti-IL-6 antibody into the OFC increased trials to meet criterion on the reversal learning stage of the attentional set-shifting test compared with the IgG control group. *p < 0.05, compared with IgG control group. d, Injection of the anti-IL-6 antibody into the dorsal striatum had no effect on reversal learning performance. n.s., Not significantly different from IgG control; Cg, cingulate cortex; Fr, frontal cortex; Pir, piriform cortex; CC, corpus collosum; CPu, caudate–putamen; Acb, nucleus accumbens. n = 6 or 7 per group.

Figure 3.

Effect of chronic IL-6 overexpression on reversal learning and downstream signaling pathways in the OFC. a, CIC stress increased trials to meet criterion on the reversal learning task, and AAV-IL-6 injection into the OFC restored reversal learning performance to nonstress levels. ⁺p < 0.05, main effect compared with no stress control groups. *p < 0.05, compared with the AAV-IL-6-injected animals subjected to CIC stress. b, AAV-GFP exclusively infected neurons, determined by GFP expression seen in neurons but not astrocytes or microglia. Scale bar, 10 μm. c, Representative micrograph showing the extent of infection in the OFC, with corresponding diagrams at AP levels from bregma 3.72 to 3.00 (Paxinos and Watson, 2006). Green represents GFP; red represents NeuN. d, e, AAV-IL-6 increased the expression of recombinant human IL-6 in the OFC, without affecting endogenous rat IL-6 levels. *p < 0.05, main effect compared with AAV-GFP-infected animals. f–i, AAV-IL-6 injection increased STAT3 phosphorylation in the OFC without affecting ERK1/2 or AKT phosphorylation. Fold change relative to No Stress, AAV-GFP control group. *p < 0.05, main effect compared with AAV-GFP-injected animals. j, Representative Western blot images. fmi, Forceps minor of the corpus callosum; rf, rhinal fissure; aca, anterior commissure; Pir, piriform cortex. n = 7 or 8 per group.

Figure 4.

Effect of acute IL-6 injection on reversal learning. CIC stress increased trials to criterion on the reversal learning stage of the attentional set-shifting test, and an acute microinjection of IL-6 (1.0 ng) into the OFC returned performance to nonstress control levels. ⁺p < 0.05, main effect of CIC stress compared with nonstress controls. *p < 0.05 vehicle compared with IL-6 injections in animals subjected to CIC stress. n = 8 or 9 per group.

Figure 5.

Effect of JAK/STAT pathway inhibition on reversal learning performance. Microinjection of the JAK/STAT inhibitor, JSI-124, into the OFC dose-dependently increased trials to criterion on the reversal learning task. *p < 0.05, compared with vehicle-injected animals. n = 6 per group.

Figure 6.

Basal IL-6 expression and IL-6 induced STAT3 phosphorylation in the OFC. a, The majority of IL-6-positive cells in the OFC also expressed the neuronal marker, NeuN. b, A smaller percentage of IL-6-positive cells also expressed the astrocytic marker, GFAP. c, IL-6 expression was not observed in CD11b-positive microglia. d, The majority of phospho-STAT3-positive cells in the OFC also expressed the neuronal marker, NeuN. e, In addition, astrocytes labeled with GFAP expressed phospho-STAT3. f, A very small number of Iba1-positive microglia were observed to express phospho-STAT3 in the OFC. Scale bar, 10 μm. n = 3 per group.