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. 2018 Jan:67:355-363.
doi: 10.1016/j.bbi.2017.09.016. Epub 2017 Sep 28.

Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1β

Meghan E Jones  1 , Christina L Lebonville  1 , Jacqueline E Paniccia  1 , Megan E Balentine  1 , Kathryn J Reissner  1 , Donald T Lysle  2
Affiliations

Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1β

Meghan E Jones et al. Brain Behav Immun. 2018 Jan.

Abstract

Post-traumatic stress disorder (PTSD) is associated with immune dysregulation. We have previously shown that severe stress exposure in a preclinical animal model of the disorder, stress-enhanced fear learning (SEFL), is associated with an increase in hippocampal interleukin-1β (IL-1β) and that blocking central IL-1 after the severe stress prevents the development of SEFL. Here, we tested whether blocking hippocampal IL-1 signaling is sufficient to prevent enhanced fear learning and identified the cellular source of stress-induced IL-1β in this region. Experiment 1 tested whether intra-dorsal hippocampal (DH) infusions of interleukin-1 receptor antagonist (IL-1RA, 1.25µg per hemisphere) 24 and 48h after stress exposure prevents the development of enhanced fear learning. Experiment 2 used triple fluorescence immunohistochemistry to examine hippocampal alterations in IL-1β, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker, and ionized calcium binding adaptor molecule -1 (Iba-1), a microglial-specific marker, 48h after exposure to the severe stressor of the SEFL paradigm. Intra-DH IL-1RA prevented SEFL and stress-induced IL-1β was primarily colocalized with astrocytes in the hippocampus. Further, hippocampal GFAP immunoreactivity was not altered, whereas hippocampal Iba-1 immunoreactivity was significantly attenuated following severe stress. These data suggest that hippocampal IL-1 signaling is critical to the development of SEFL and that astrocytes are a predominant source of stress-induced IL-1β.

Keywords: Astrocytes; Cytokines; Fear; Hippocampus; Microglia; PTSD; SEFL; Stress.

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Figures

Figure 1
Figure 1. Intra-dorsal hippocampal IL-1RA is sufficient to prevent SEFL
A. Schematic shows experiment 1 timeline of surgical procedures, intra-DH microinfusions, and severe stress exposure and contextual fear learning in the SEFL paradigm. B. Paxinos and Watson (2007) schematics of the rat brain show approximate cannulae placement. Coordinates −3.0 through −3.6 from Bregma are shown. Each circle represents where damage from the cannula tract was observed for all animals included in the analysis. C. DH-IL-1RA significantly attenuated SEFL. There were no differences between groups in freezing to Context B prior to the single shock. Stress-enhanced fear learning was observed within vehicle-treated groups in that rats that received foot shock in Context A followed by vehicle exhibited significantly more fear learning to Context B than rats that received no foot shock in Context A followed by vehicle. Critically, animals that received foot shock in Context A followed by IL-1RA exhibited significantly less freezing than animals that received foot shock in Context A followed by vehicle. Thus, IL-1RA prevented the expression of SEFL. * Foot Shock in Context A/Vehicle vs. Foot Shock in Context A/IL-1RA, p < 0.05.
Figure 2
Figure 2. Severe stress increases hippocampal IL-1β immunoreactivity
The stress-induced increase in hippocampal IL-1β that we previously reported is replicated here. Representative images of IL-1β immunoreactivity in the dentate gyrus of the DH acquired at 10X are shown from stressed (Foot Shock in context A) and non-stressed (No Foot Shock in Context A) rats. Top panel shows a tiled 10X image, while bottom panel shows a single 10X image. For the bottom panel, Bitplane Imaris was used for background subtraction to better visualize individual cells presented. Paxinos and Watson (2007) schematic shows the approximate region of the DH where images were acquired, AP −3.36 from bregma. Quantification of IL-1β immunoreactivity revealed that exposure to severe stress (15 foot shocks) significantly increased IL-1β immunoreactivity in the DH 48 hours post-stress. * p < 0.05.
Figure 3
Figure 3. Dorsal hippocampal Iba-1 immunoreactivity, but not GFAP immunoreactivity, is attenuated 48 hours after severe stress
A. Representative images of GFAP and Iba-1 immunoreactivity acquired at 10X (tiled image presented) and 20X are shown from stressed (Foot Shock in context A) and non-stressed (No Foot Shock in Context A) rats. Images were acquired in the DH, AP −3.36 from bregma. B. Both Imaris quantification and individual GFAP-positive cell counts indicated there was no effect of foot shock on GFAP immunoreactivity. C. In contrast, Imaris quantification and individual Iba-1 positive cell counts revealed that stress exposure significantly attenuated Iba-1 immunoreactivity 48 hours post-stress. * p < 0.05.
Figure 4
Figure 4. IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals
A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p < 0.05. C. Similarly, the Pearson’s correlation coefficient between the IL-1β signal intensity and the GFAP signal intensity was significantly higher than that for the IL-1β signal and Iba-1 signal or NeuN signal, respectively. * p < 0.05.
Figure 5
Figure 5. IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals
Representative images from the DH acquired at 63X (scale bar presented is 10μm) show colocalization of IL-1β with GFAP, Iba-1, and NeuN. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Colocalization panels (white, labeled ‘Coloc’) show Imaris-generated image of colocalized voxels in each Z stack image presented. Colocalization scatter plots show the signal intensity for each voxel in the Z stack. Specifically, color of each point represents the frequency, the Y axis represents IL-1β signal (Alexa-488) intensity, and the X axis represents GFAP (Alexa- 405), Iba-1 (Alexa- 568), or NeuN signal (Alexa- 568) intensity, respectively. In the top panel, the colocalization scatter plot between IL-1β and GFAP shows a high proportion of voxels that were high in both IL-1β and GFAP signal (selected region), and a high observed correlation, r = 0.3997, demonstrates that for any given voxel, as IL-1 signal increased, GFAP signal was also likely to increase. In contrast, scatter plots for both IL-1β with Iba-1 and IL-1β with NeuN show a high proportion of voxels that were high in only IL-1β or Iba-1 and NeuN signal, respectively (outside of selected region). In addition, there was a lower correlation for the IL-1β and Iba-1 signal, r = 0.1915, and IL-1β and NeuN signal, r = 0.0373, suggesting a much weaker relationship than that with GFAP.
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