Hypoxia/reoxygenation impairs memory formation via adenosine-dependent activation of caspase 1

Abstract

After hypoxia, a critical adverse outcome is the inability to create new memories. How anterograde amnesia develops or resolves remains elusive, but a link to brain-based IL-1 is suggested due to the vital role of IL-1 in both learning and brain injury. We examined memory formation in mice exposed to acute hypoxia. After reoxygenation, memory recall recovered faster than memory formation, impacting novel object recognition and cued fear conditioning but not spatially cued Y-maze performance. The ability of mice to form new memories after hypoxia/reoxygenation was accelerated in IL-1 receptor 1 knockout (IL-1R1 KO) mice, in mice receiving IL-1 receptor antagonist (IL-1RA), and in mice given the caspase 1 inhibitor Ac-YVAD-CMK. Mechanistically, hypoxia/reoxygenation more than doubled caspase 1 activity in the brain, which was localized to the amygdala compared to the hippocampus. This reoxygenation-dependent activation of caspase 1 was prevented by broad-spectrum adenosine receptor (AR) antagonism with caffeine and by targeted A1/A2A AR antagonism with 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine. Additionally, perfusion of adenosine activated caspase 1 in the brain, while caffeine blocked this action by adenosine. Finally, resolution of anterograde amnesia was improved by both caffeine and by targeted A1/A2A AR antagonism. These findings indicate that amygdala-based anterograde amnesia after hypoxia/reoxygenation is sustained by IL-1β generated through adenosine-dependent activation of caspase 1 after reoxygenation.

Figure 1.

Restoration of memory recall after acute hypoxia. A, Wild-type mice were trained in memory formation using novel object recognition 1 h before hypoxia. Mice were then exposed to normoxia or hypoxia for 2 h. Memory recall (percent investigation) was measured at the reoxygenation time points indicated. Results are expressed as means ± SEM; n = 8. Bars without a common superscript are different (p < 0.05). B, Mice were treated as in A, and spontaneous locomotor activity (total distance traveled) was measured at the reoxygenation time points indicated. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05).

Figure 2.

Memory formation recovers more slowly than memory recall after acute hypoxia. A, Wild-type mice were exposed to normoxia (Norm) or hypoxia for 2 h. After hypoxia, mice were trained in memory formation using novel object recognition 1 h before the time points indicated. Memory recall (percent investigation) was measured at the reoxygenation time points indicated. Results are expressed as means ± SEM; n = 6–8. Bars without a common superscript are different (p < 0.05). B, Mice were treated as in A. Mice were trained in memory formation using cued fear conditioning after 4 h of reoxygenation. Memory recall (immobility) was measured after 5 and 52 h of reoxygenation. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). C, Mice were treated as in A. Perfect alternations were measured after 4, 52, and 76 h of reoxygenation. Results are expressed as means ± SEM; n = 4.

Figure 3.

Knockout of IL-1R1 improves memory formation and locomotion while blunting activation of ERK1/2 and p38 MAPK. A, WT or IL-1R1 KO mice were exposed to normoxia or hypoxia for 2 h. Mice were trained in memory formation using novel object recognition after 4 h of reoxygenation. Memory recall (percent investigation) was measured after 5 h of reoxygenation. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). B, WT mice treated with/without intraperitoneal IL-1RA were exposed to normoxia or hypoxia as in A, and memory was tested as in A. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). C, WT mice treated with/without intracerebroventricular Ac-YVAD-CMK were exposed to normoxia or hypoxia as in A, and memory was tested as in A. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). D, WT or IL-1R1 KO mice were treated as in A, and spontaneous locomotor activity (total distance traveled) was measured at the reoxygenation time points indicated. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). E, WT and IL-1R1 KO mice were exposed to normoxia or hypoxia as in A. Brain p-pERK1/2, p-p38 MAPK, and p-JNK were measured 1 h after hypoxia. Results are expressed as means ± SEM; n = 6–9. Bars without a common superscript are different (p < 0.05).

Figure 4.

AR blockade prevents hypoxia-dependent activation of caspase 1 in the brain. A, Wild-type mice were exposed to normoxia or hypoxia for 2 h. Caspase 1 activity was measured at the reoxygenation time points indicated. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). B, Wild-type mice treated with/without caffeine were exposed to normoxia or hypoxia as in A, and caspase 1 activity measured 1 h after reoxygenation. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). C, Wild-type mice treated with/without 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine (A1/A2A antag) were exposed to normoxia or hypoxia as in A, and caspase 1 activity was measured 1 h after reoxygenation. Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). D, Wild-type mice treated with/without NAC were exposed to normoxia or hypoxia as in A, and caspase 1 activity was measured 1 h after reoxygenation. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). E, Wild-type mice were perfused with the adenosine concentrations indicated. Caspase 1 activity was measured 1 h after perfusion. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). F, Wild-type mice were perfused with/without 50 μm adenosine plus 500 μm caffeine. Caspase 1 activity was measured 1 h after perfusion. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). G, Wild-type mice were perfused with/without 50 μm adenosine plus 500 μm NAC. Caspase 1 activity was measured 1 h after perfusion. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05).

Figure 5.

Hypoxia induces brain region-specific activation of caspase 1. A, Wild-type mice were exposed to normoxia or hypoxia for 2 h. Caspase 1 activity was measured in the amygdala and hippocampus 1 h after reoxygenation Results are expressed as means ± SEM; n = 4. Bars without a common superscript are different (p < 0.05). B, WT mice and IL-1R1 KO mice were treated as in A. Immunohistochemistry was performed for GFAP at 1 and 6 h after reoxygenation. Representative images of the amygdala (n = 3).

Figure 6.

AR blockade speeds recovery of memory formation after hypoxia. A, Wild-type mice treated with/without caffeine were exposed to normoxia or hypoxia for 2 h. Mice were trained in memory formation using novel object recognition after 4 h of reoxygenation. Memory recall (percent investigation) was measured after 5 h of reoxygenation. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05). B, Wild-type mice treated with/without 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine (A1/A2A antag) were exposed to normoxia or hypoxia as in A, and memory was tested as in A. Results are expressed as means ± SEM; n = 6. Bars without a common superscript are different (p < 0.05).