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. 2011 Oct 26;31(43):15511-21.
doi: 10.1523/JNEUROSCI.3688-11.2011.

Microglia and memory: modulation by early-life infection

Lauren L Williamson  1 , Paige W Sholar, Rishi S Mistry, Susan H Smith, Staci D Bilbo
Affiliations

Microglia and memory: modulation by early-life infection

Lauren L Williamson et al. J Neurosci. .

Abstract

The proinflammatory cytokine interleukin-1β (IL-1β) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1β during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the "second hit," LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1β before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1β during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b(+) enriched cells are the source of IL-1β during normal HP-dependent learning. CD11b(+) cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1β ex vivo compared with controls. However, an exaggerated IL-1β response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.

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Figures

Figure 1.
Figure 1.
Learning increases IL-1β protein specifically within the hippocampus, and this increase is modulated by neonatal infection. a, Rats treated with PBS or E. coli on P4 were injected as adults with saline (SAL) or LPS, and fear conditioned 24 h later (n = 8/group). Memory was assessed 1 or 72 h after conditioning. A separate group of rats from each neonatal treatment were injected as adults with SAL or LPS, and 24 h later received Fear Conditioning, Shock only, or Context exposure only (n = 8/grp). Brains were collected 2 h later to assess IL-1β protein. b, Freezing to the context did not differ between groups at the 1 h test, although freezing was greater overall in LPS-injected rats (**p < 0.05). At the 72 h test, freezing to the context was significantly lowest in NI rats treated with LPS 24 h prior (*p < 0.001). Freezing to the tone cue (CS) in an altered environment was significantly greater than the pre-CS period, but there was no neonatal group difference (#p < 0.001). c, IL-1β was exaggerated within the HP of NI rats following LPS (*p < 0.02). Moreover, IL-1β was detectable only in HP following SAL injection (**p < 0.001), and concentrations were highest overall in HP (#p < 0.001). d, Neither Shock nor Context alone produced the same exaggerated response within the HP of NI plus LPS rats, and concentrations were undetectable 24 h after SAL. Error bars indicate SEM.
Figure 2.
Figure 2.
Microglial activation within the HP is exaggerated in NI rats, but only in response to learning. Rats treated with PBS or E. coli on P4 were injected as adults with SAL or LPS, and 24 h later received Fear Conditioning or Shock only (n = 8/group). Brains were collected 2 h later, and CD11b and GFAP were assessed. a, CD11b relative expression to GAPDH was increased by LPS (**p < 0.02) and exaggerated within the HP of NI rats (overall effect of E. coli, *p < 0.02). Shock alone did not produce the same exaggerated response within the HP of NI rats, although there was a significant effect of LPS (**p < 0.05). b, GFAP expression did not significantly differ among groups, either following Fear Conditioning or Shock alone (p > 0.05 for both). Error bars indicate SEM.
Figure 3.
Figure 3.
NI rats exhibit decreased neuronal inhibition within the HP compared with controls. Rats treated with PBS or E. coli on P4 were injected as adults with SAL or LPS, and 24 h later received Fear Conditioning or Shock only (n = 8/group). Brains were collected 2 h later, and target genes were assessed. a, CD200 and its receptor (CD200R) were reduced overall in NI rats compared with controls in the fear conditioned group (*p < 0.01 for both), whereas only CD200R was significantly decreased by LPS (**p < 0.02). b, There were no neonatal group differences in CD200 or CD200R in the Shock only group; however, both genes were decreased by LPS (**p < 0.02). c, d, Fractalkine receptor (CX3CR1) but not its ligand was reduced overall in NI rats in both Fear Conditioned and Shock only groups. (*p < 0.05 for all, compared with PBS). Error bars indicate SEM.
Figure 4.
Figure 4.
NI rats exhibit increased surface antigen expression of CD11b on isolated HP microglia. a, Adult rat HP were microdissected following cold saline perfusion and brought to single-cell suspensions using Miltenyi's Neural Dissociation Kit followed by myelin depletion. Myelin-depleted cells were stained with APC-conjugated CD11b and analyzed using flow cytometry. Representative presorted populations are shown as distinct peaks. An unstained control is overlaid for reference (shaded histogram). b, Myelin-depleted cells from rats in each neonatal treatment group were stained with APC-conjugated CD11b to assess expression on microglia independent of selection using magnetic beads, which could influence antigen expression. The bar graph shows greater average (±SEM) MFI for CD11b reactivity from NI (n = 10) compared with PBS (n = 9) rats (*p < 0.05). c, Myelin-depleted cells from rats in each neonatal treatment group were sorted into CD11b+ and CD11b− fractions using magnetic beads. Representative postsorted (MACS) populations are shown. Purity was consistently >93% CD11b+ and ∼99% CD11b− for all samples. A rightward shift can be seen in the population of CD11b+ cells from NI rats, indicating increased expression (vertical line is provided for comparison). d, Sorted CD11b+ cells were stained with APC-conjugated CD11b/c and CD45 (a marker highly expressed on infiltrating macrophages), which revealed a large, dense cluster of CD11bhigh/CD45low cells. In contrast, CD11bhigh cells that also stained brightly for CD45 accounted for only 4.2% of the population, indicating the perivascular macrophage population was minimal.
Figure 5.
Figure 5.
Isolated HP microglia from NI rats express exaggerated IL-1β mRNA ex vivo. a, Microglia were rapidly isolated from the adult HP of rats using myelin depletion and CD11b+ selection as described above in Figure 4. HP CD11b+ and CD11b− fractions were cultured with and without LPS (10 ng/ml) for 4 h at 37°C. Immediately following culture, cells were lysed and total RNA isolated for RT-PCR. Relative IL-1β gene expression to GAPDH is shown, which is increased in response to LPS and undetectable in the CD11b− population (n = 2/group). b, Microglia were isolated from the adult HP of rats from each neonatal treatment. CD11b+ cells from NI rats express greater relative expression of IL-1β with or without LPS stimulation ex vivo, compared with controls (*p = 0.02). The bars represent the average (±SEM) relative expression values for two rat HPs (pooled) per treatment group per day run as three independent experiments (n = 6/group).
Figure 6.
Figure 6.
HP microglia isolated 1 h after behavioral experience express exaggerated IL-1β mRNA in NI rats compared with controls, but only following learning. Rats treated with PBS or E. coli on P4 were injected as adults with SAL or LPS, and received Fear Conditioning or Shock only 24 h later. Microglia were rapidly isolated 1 h later using myelin depletion and CD11b+ selection as described above in Figure 4. Isolated microglia were immediately lysed and total RNA isolated for RT-PCR. Relative IL-1β gene expression to GAPDH is shown. a, IL-1β mRNA expression was higher overall in NI rats (*p = 0.04) and higher in response to LPS in both groups (#p = 0.02) (n = 6–7/group). b, There were no group differences 1 h following Shock alone (p > 0.05; n = 4–5/group). c, IL-1β mRNA was undetectable in CD11b− cells collected from a separate group of rats in each condition that also received fear conditioning (n = 2/group). d, GFAP and fractalkine/CX3CL1 were robustly expressed in CD11b− cells, indicating this population contained astrocytes and neurons, and was viable and responsive to LPS. Error bars indicate SEM.
Figure 7.
Figure 7.
Inhibiting microglia before LPS or before learning prevents the memory impairment and exaggerated IL-1β response in NI rats. a, Adult rats treated on P4 with PBS or E. coli were injected with LPS 24 h before fear conditioning. A subset of animals were killed 2 h later to assess HP IL-1β protein, and the remaining animals were tested for memory at 72 h. b, Rats in each neonatal treatment group (n = 10/group) received water or MINO 12 h and again 1 h before LPS in the experimental paradigm illustrated in a. Freezing was significantly lowest in NI rats that received water before LPS (*p < 0.03), but this decrease was prevented by MINO. c, A separate group of rats in each neonatal treatment group (n = 10/group) received MINO 12 h and again 1 h before Fear Conditioning (FC), but after LPS the previous day. A SAL-injected group was also included to ensure that MINO alone did not impair learning. There were no differences between groups. d, IL-1β protein was significantly highest in NI rats that received water before LPS (*p = 0.03) (left-most bars), but this increase was prevented by MINO, administered either before LPS (center bars), or in a separate group, after LPS but before FC (right-most bars). Error bars indicate SEM.
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