High Behavioral Reactivity to Novelty as a Susceptibility Factor for Memory and Anxiety Disorders in Streptozotocin-Induced Neuroinflammation as a Rat Model of Alzheimer's Disease

Abstract

Individual differences in responsiveness to environmental factors, including stress reactivity and anxiety levels, which differ between high (HR) and low (LR) responders to novelty, might be risk factors for development of memory and anxiety disorders in sporadic Alzheimer's disease (sAD). In the present study, we investigated whether behavioral characteristics of the HR and LR rats, influence the progression of sAD (neuroinflammation, β-amyloid peptide, behavioral activity related to memory (Morris water maze) and anxiety (elevated plus maze, white and illuminated open field test) in streptozotocin (STZ)-induced neuroinflammation as a model of early pathophysiological alterations in sAD. Early (45 days) in disease progression, there was a more severe impairment of reference memory and higher levels of anxiety in HRs compared with LRs. Behavioral depression in HRs was associated with higher expression of β-amyloid deposits, particularly in the NAcS, and activation of microglia (CD68⁺ cells) in the hypothalamus, as opposed to less inflammation in the hippocampus, particularly in CA1, compared with LRs in late (90 days) sAD progression. Our findings suggest that rats with higher behavioral activity and increased responsivity to stressors show more rapid progression of disease and anxiety disorders compared with low responders to novelty in the STZ-induced sAD model.

Keywords: Morris water maze; cytokines; elevated plus maze; high and low responders to novelty; neuroinflammation; rat Alzheimer’s disease model; spatial memory and anxiety disorders; streptozotocin; white and illuminated open field; β-amyloid.

Figure 1

Behavioral activity associated with anxiety in the elevated plus maze (EPM) test measured as time spent in the open arms (a), time spent in the center (b), and time spent in the closed arms (c) of the maze in rats with high (HR) or low (LR) reactivity to novelty at baseline (before injections) and 45 and 90 days after intracerebroventricular injections of streptozotocin (STZ, n = 16) or citrate buffer (VEH, n = 14). Explanations: Data are presented as mean ± SD and were analyzed using a Mann–Whitney-U test; # in a painted circle—p ≤ 0.05, ## in a painted circle indicate significance of differences between HR and LR rats; ^—p ≤ 0.05, ^^—p ≤ 0.01 indicate significance of differences between test and re-test; &—p ≤ 0.05 indicates significance of differences between 45 day and 90 day after injection.

Figure 2

Behavioral activity associated with anxiety in the white and illuminated open field (OF) test measured as exploration (number of crossed lines) and time of freezing in rats non-divided (a,b) and divided into rats with high (HR) or low (LR) reactivity to novelty (c,d) at baseline (before injections) and 45 and 90 days after intracerebroventricular injections of streptozotocin (STZ, n = 16) or citrate buffer (VEH, n = 14). Explanations: Data are presented as mean ± SD and were analyzed using a Mann–Whitney-U test; # in a painted circle—p ≤ 0.05, ## in a painted circle indicate significance of differences between HR and LR rats; *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001 indicate significance of differences between STZ and VEH at 45 or 90 days after injection; &—p ≤ 0.05, &&—p ≤ 0.01, &&&—p ≤ 0.001 indicate significance of differences between 45 days and 90 days after injection; @ in brackets above the bars—p ≤ 0.05, @@ in brackets above the bars—p ≤ 0.01, @@@ in brackets above the bars—p ≤ 0.001 indicate significance of differences vs. baseline.

Figure 3

Behavioral activity associated with reference memory performance in the Morris water maze (MWM) test measured as latency to reach the platform, the percentage of time in the critical quadrant, and the total distance swum in the critical quadrant in a probe test in rats non-divided (a) and divided into rats with high (HR) or low (LR) reactivity to novelty (b) at baseline (before injections) and 45 and 90 days after intracerebroventricular injections of streptozotocin (STZ, n = 16) or citrate buffer (VEH, n = 14). Explanations: latency to reach the platform: latency to reach the critical quadrant in which the platform was located in the previous sessions of spatial learning in the MWM (Days 1–3); probe test: single trial without the platform (Day 4); data are presented as mean ± SD and were analyzed using a Mann–Whitney-U test; # in a painted circle—p ≤ 0.05 indicates significance of differences between HR and LR rats; *—p ≤ 0.05 indicates significance of differences between STZ and VEH at 45 or 90 days after injection; &—p ≤ 0.05, &&—p ≤ 0.01 indicate significance of differences between 45 day and 90 day after injection; @ in brackets above the bars—p ≤ 0.05, @@ in brackets above the bars—p ≤ 0.01 indicate significance of differences vs. baseline, @@@—p ≤ 0.001 indicate significance of differences vs. baseline.

Figure 4

Number of activated microglia cells (CD68⁺ cells) (a) and β-amyloid counts (b) in the hippocampus (CA1, CA2, CA3, DG) and nucleus accumbens (NAcC, NAcS) in non-divided rats and divided into rats with high (HR) or low (LR) reactivity to novelty 90 days after intracerebroventricular injections of streptozotocin (STZ, n = 16). Explanations: Data are presented as mean ± SD and were analyzed using a Mann–Whitney-U test; # in a painted circle—p ≤ 0.05, ## in a painted circle p ≤ 0.01 indicate significance of differences between HR and LR rats; *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001 indicate significance of differences between STZ and VEH 90 days after injection.

Figure 5

(A) Representative photomicrographs showing activated microglia cells (CD68⁺ cells) in the CA1 area of the hippocampus of STZ HR (a), STZ LR (b), VEH HR (c), and VEH LR rats (d) 90 days after intracerebroventricular injections of streptozotocin (STZ) or citrate buffer (VEH). Scale bar = 100 μm. (B) Representative photomicrographs showing amyloid beta (indicated by arrows) in the CA1 area of the hippocampus of STZ HR (a), STZ LR (b), VEH HR (c), and VEH LR rats (d) 90 days after intracerebroventricular injections of streptozotocin (STZ) or citrate buffer (VEH). Scale bar = 100 μm.

Figure 6

The total number of T, B, NK, TCD4⁺, and TCD8⁺ lymphocytes in the peripheral blood mononuclear cells analyzed by the flow cytometric method 45 and 90 days after intracerebroventricular injections of streptozotocin (STZ, n = 16) or citrate buffer (VEH, n = 14) in non-divided rats (a,b) and divided into rats with high (HR, n = 8) or low (LR, n = 8) reactivity to novelty after intracerebroventricular injections of STZ (c,d). Explanations: Data are presented as mean ± SD and were analyzed using a Mann–Whitney-U test; # in a painted circle—p ≤ 0.05 indicate significance of differences between HR and LR rats; *—p ≤ 0.05, **—p ≤ 0.01 indicate significance of differences between STZ and VEH 45 and 90 days after injection; &—p ≤ 0.05, &&—p ≤ 0.01 indicate significance of differences between 45 day and 90 day after injection.

Figure 7

Scheme of experimental procedure and group assignments. Explanations: NOV—novelty test; EPM—elevated plus maze test: test and re-test after 1 h; OF—white and illuminated open-field test; MWM—Morris water maze test: phase of acquisition of reference memory (Day 1–Day 3), probe test (single trial without the platform (reference memory evaluation), working memory performance (Days 5–8); HR—high responders to novelty; LR—low responders to novelty; ICVSTZ—intracerebroventricular (ICV) injections of streptozotocin (STZ) (a total dose of 3 mg/kg b.w. divided into 2 injections to both lateral ventricles: 0.75 mg/kg STZ in 2 μL of citrate buffer per ventricle)—sAD model; ICVVEH—intracerebroventricular (ICV) injections of citrate buffer (VEH) (2 μL per ventricle); B—blood collection; S—1 h after the last MWM trial euthanasia and blood, brain, spleen, and thymus collection for further analysis (A).