Interleukin-6 facilitates lipopolysaccharide-induced disruption in working memory and expression of other proinflammatory cytokines in hippocampal neuronal cell layers

Abstract

Proinflammatory cytokines inhibit learning and memory but the significance of interleukin-6 (IL-6) in acute cognitive deficits induced by the peripheral innate immune system is not known. To examine the functional role of IL-6 in hippocampus-mediated cognitive impairments associated with peripheral infections, C57BL6/J (IL-6(+/+)) and IL-6 knock-out (IL-6(-/-)) mice were trained in a matching-to-place version of the water maze. After an acquisition phase, IL-6(+/+) mice injected intraperitoneally with lipopolysaccharide (LPS) exhibited deficits in working memory. However, IL-6(-/-) mice were refractory to the LPS-induced impairment in working memory. To determine the mechanism by which IL-6 deficiency conferred protection from disruption in working memory, plasma IL-1beta and tumor necrosis factor alpha (TNFalpha), c-Fos immunoreactivity in the nucleus of the solitary tract (NTS), and steady-state levels of IL-1beta and TNFalpha mRNA in neuronal layers of the hippocampus were determined in IL-6(+/+) and IL-6(-/-) mice after injection of LPS. Plasma IL-1beta and TNFalpha and c-Fos immunoreactivity in the NTS were increased similarly in IL-6(+/+) and IL-6(-/-) mice after LPS, indicating high circulating levels of IL-1beta and TNFalpha and activation of vagal afferent pathways were not sufficient to disrupt working memory in the absence of IL-6. However, the LPS-induced upregulation of IL-1beta and TNFalpha mRNA that was evident in hippocampal tissue of IL-6(+/+) mice was greatly attenuated or entirely absent in IL-6(-/-) mice. Collectively, these data suggest that humoral and neural immune-to-brain communication pathways are intact in IL-6-deficient mice but that, in the absence of IL-6, the central cytokine compartment is hyporesponsive.

Figure 1.

Matching-to-place performance for IL-6^+/+ mice that received LPS (black line) or saline (gray line) 4 h before test session 3. A, Distance swam to platform across testing days. B, Latency to find the platform across testing days. C, Swim speed. Data are presented as mean±SEM. The arrow designates day of treatment. *Significant difference between groups as determined by Fisher's protected least-significant difference (p < 0.05).

Figure 2.

Matching-to-place performance for IL-6^+/+ mice that received a single intraperitoneal injection of LPS (100 μg; black line) or saline (gray line) 4 h before test session 9. A, Distance swam to platform across testing days. B, Latency to find the platform across testing days. C, Swim speed. Data are presented as mean±SEM. *Significant difference between groups as determined by Fisher's protected least-significant difference (p < 0.05).

Figure 3.

Matching-to-place performance for IL-6^+/+ (square symbols) and IL-6^−/− (diamond symbols) mice that received a single intraperitoneal injection of LPS (black lines) or saline (gray lines) 4 h before test session 9. A, Distance swam to platform across testing days. B, Latency to find the platform across testing days. C, Swim speed. Data are presented as mean±SEM. The arrow designates day of treatment. *Significant difference between groups as determined by Fisher's protected least-significant difference (p < 0.05).

Figure 4.

Plasma cytokine levels for IL-6^+/+ (solid filled bars) and IL-6^−/− (striped bars) mice that received a single intraperitoneal injection of LPS (black-filled bars) or saline (gray-filled bars) 4 h before kill. A, IL-1β. B, TNF-α. C, IL-6. D, IL-10. Data are presented as mean±SEM. nd, Cytokine levels were nondetectable. *Significant difference between groups as determined by Fisher's protected least-significant difference (p < 0.05).

Figure 5.

A–D, Laser capture microdissection of the hippocampal neuronal layer. A–D, Stained section (A), captured neuronal layer (B), section with removed hippocampus (C), and isolated hippocampal neuronal layer (D). E, F, Percent fold change for mRNA cytokine levels for IL-6^+/+ (solid filled bars) and IL-6^−/− (striped bars) mice that received a single intraperitoneal injection of LPS (black-filled bars) or saline (gray-filled bars) 4 h before kill. IL-1β mRNA (E) and TNF-α (F). Data are presented as mean±SEM. nd, Cytokine levels were nondetectable. *Significant difference between groups as determined by Fisher's protected least-significant difference (p < 0.05).

Figure 6.

A, Representative photomicrographs of c-Fos immunoreactivity in the NTS region for IL-6^+/+ (top panels) and IL-6^−/− (bottom panels) mice that received a single intraperitoneal injection of LPS (left panels) or saline (right panels) 4 h before sacrifice. B, Number of c-Fos-positive cells observed in the NTS. LPS induced Fos immunoreactivity similarly in both genotypes. Data are presented as mean±SEM. AP, Area postrema; CC, central canal. Scale bar, 50 mm.