Influenza infection induces neuroinflammation, alters hippocampal neuron morphology, and impairs cognition in adult mice

Abstract

Influenza is a common and highly contagious viral pathogen, yet its effects on the structure and function of the CNS remain largely unknown. Although there is evidence that influenza strains that infect the brain can lead to altered cognitive and emotional behaviors, it is unknown whether a viral strain that is not neurotropic (A/PR/8/34) can result in a central inflammatory response, neuronal damage, and neurobehavioral effects. We hypothesized that neuroinflammation and alterations in hippocampal neuron morphology may parallel cognitive dysfunction following peripheral infection with live influenza virus. Here, we show that influenza-infected mice exhibited cognitive deficits in a reversal learning version of the Morris water maze. At the same time point in which cognitive impairment was evident, proinflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-α) and microglial reactivity were increased, while neurotrophic (BDNF, NGF) and immunomodulatory (CD200, CX3CL1) factors were decreased in the hippocampus of infected mice. In addition, influenza induced architectural changes to hippocampal neurons in the CA1 and dentate gyrus, with the most profound effects on dentate granule cells in the innermost portion of the granule cell layer. Overall, these data provide the first evidence that neuroinflammation and changes in hippocampal structural plasticity may underlie cognitive dysfunction associated with influenza infection. In addition, the heightened inflammatory state concurrent with reduced neurotrophic support could leave the brain vulnerable to subsequent insult following influenza infection. A better understanding of how influenza impacts the brain and behavior may provide insight for preventing inflammation and neuronal damage during peripheral viral infection.

Figure 1.

A–D, Representative examples of Golgi-stained neurons along with corresponding 3D reconstruction and Sholl ring analysis in the CA1 (A, B) and dentate gyrus (C, D) subfields of the hippocampus. Arrows indicate neuron selected for tracing. Magnification = 20×; scale bars, 20 μm.

Figure 2.

Sickness behavior following influenza infection. A, B, Influenza-infected mice (FLU) lost a significant amount of body weight (A), and locomotor activity rapidly declined (B) over the course of influenza infection. Data are represented as means ± SEM [*p < 0.05 compared with controls (CON)].

Figure 3.

Morris water maze acquisition and probe trial. A, Influenza-infected (FLU) and control (CON) animals demonstrated similar performance during acquisition of the task as measured by distance to reach the hidden platform over 5 d of testing. B, A probe trial (24 h following the last day of acquisition testing) indicated no significant difference in the amount of time influenza mice spent in the target quadrant compared with controls.

Figure 4.

Morris water maze reversal testing. A, Influenza-infected mice (FLU) demonstrated an increase in distance (pathlength) to reach the hidden platform when it was moved to the opposite quadrant during reversal testing. B, Influenza-infected mice spent less time searching the correct target quadrant across trials during reversal testing when compared with control mice (CON). Data are represented as means ± SEM (*p < 0.05, **p < 0.01 compared with controls).

Figure 5.

Influenza infection altered hippocampal gene expression. A, Inflammatory cytokines (IL-1β, IL-6, TNF-α) and MHCII (marker of microglial activation) were increased in the hippocampus of influenza-infected mice. B, Influenza infection induced increased hippocampal expression of anti-viral cytokine IFN-α. C, Neurotrophic (BDNF, NGF) and immunomodulatory factors (CD200, CX3CL1) were decreased in the hippocampus of influenza-infected mice (FLU). All data are from day 7 after inoculation. Data are represented as means ± SEM [*p < 0.05, **p < 0.01 compared with controls (CON)].

Figure 6.

Influenza infection increased microglia reactivity. A, Influenza infection increased the proportional area of Iba-1 staining in the hippocampus at day 7 after infection. B, Influenza infection increased the number of Iba-1-positive cells in the hippocampus at day 7 after inoculation. C, D, Representative examples of Iba-1-positive cells in the dentate gyrus of a control (CON, C) and influenza-infected (FLU, D) animal. Inset includes enlarged image of Iba-1-positive cell indicated by arrow. Magnification = 40×; scale bar, 50 μm. Data are represented as means ± SEM (*p < 0.05, **p < 0.01, ^#p = 0.08 compared with controls).

Figure 7.

Influenza infection altered CA1 pyramidal neuron morphology. A, Influenza infection (FLU) did not affect the total number of apical or basal tree dendritic intersections of CA1 pyramidal neurons. B, Influenza infection induced retraction of dendrites in the distal portion of the CA1 apical tree (reduced intersections 200–240 μm from the soma). Data are represented as means ± SEM [*p < 0.05 compared with controls (CON)].

Figure 8.

Influenza infection altered dentate granule neuron morphology. A, Influenza infection (FLU) reduced the total number of dendritic intersections of dentate granule neurons in the outer portion of the GCL. B, Influenza infection induced retraction of distal dendrites (160–220 μm from the soma) while increasing dendritic extent proximal to the cell body (40–80 μm from the soma) of dentate granule neurons residing in the inner portion of the GCL. C, Influenza infection induced retraction of distal dendrites (140–200 μm from the soma) of dentate granule cells located in the outer portion of the GCL. Data are represented as means ± SEM [*p < 0.05 compared with controls (CON)].

Figure 9.

Effects of influenza infection on spine density of hippocampal dendrites. A, Representative image of Golgi-stained dendritic spines in the dentate gyrus. Magnification = 100×; scale bar, 10 μm. B, Influenza infection (FLU) did not alter spine density of apical or basal dendrites in the CA1 region. C, Influenza infection decreased dendritic spine density of dentate granule cells located in the inner portion of the GCL. Data are represented as means ± SEM [*p < 0.05 compared with controls (CON)].