Impact of early locus coeruleus lesions in the TgF344 Alzheimer's disease rat model

Figures

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  FIGURE 1. Repeated DSP‐4 administration depletes forebrain NE and damages LC cell bodies. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline, and the effects of DSP‐4 administration on the LC‐NE system were evaluated using HPLC and immunohistochemistry. (A) DSP‐4 administration reduced hippocampal NE levels. (B) Representative images (20×) of NET+ fibers in the DG with DAPI counterstain. Quantification of NET+ fibers in the (C) DG, (D) CA1, (E) CA3, and (F) PFC. (G) Representative images (20×) of TH staining in the LC. (H) Quantification of TH immunoreactivity in the LC expressed as mean gray value. Females are represented by open circles and males by closed circles. Scale bar = 50 µm. Data are mean ± SEM; n = 4–6 animals per group (A) or n = 5–6 animals per group (C–F, H). *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

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  FIGURE 2. TgF344‐AD animals display impaired social discrimination. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline, and the ratio of time spent interacting with (A) a novel object versus a novel animal and (B) a familiar versus novel animal was calculated. (A) All animals displayed a preference for novel animals versus novel objects, (B) but only WT animals displayed a preference for novel versus familiar animals. Females are represented by open circles and males by closed circles. Data are mean ± SEM; n = 9–12 animals per group. **p ≤ 0.01.

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  FIGURE 3. Circadian rhythms and arousal are affected by both genotype and DSP‐4 treatment. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline. (A) Circadian activity was evaluated during a 23‐hour locomotor behavioral paradigm. Total ambulations were binned over the (B) light phase, (C) dark phase, (D) entire 23‐hour task, and (E) first 30 minutes. (F) Arousal was evaluated during sleep latency, where the latency to fall asleep was quantified. Females are represented by open circles and males by closed circles. Data are mean ± SEM; n = 9–12 animals per group. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

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  FIGURE 4. Repeated DSP‐4 treatment ablates immediate stress‐induced repetitive behavior and causes apathy‐like behavior. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline, and stress‐induced repetitive and apathy‐like behavior was evaluated using nestlet shredding. Quantification of the percentage of nestlet shredded after (A) 90 minutes and (B) 6 days. Apathy‐like behavior was also evaluated using a stick‐chewing behavioral paradigm. Shown are the percentage of stick chewed after (C) 24 hours and (D) 1 week. Animals treated with DSP‐4 show a lack of nestlet shredding and stick chewing behavior. Females are represented by open circles and males by closed circles. Data are mean ± SEM; n = 9–12 animals per group. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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  FIGURE 5. Treatment and genotype cause increased anxiety‐like behaviors in a task‐dependent manner. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline. (A) Anxiety‐like behavior was evaluated using an open field behavioral paradigm, where the percentage of time spent in the center of the arena was quantified. Anxiety‐like behavior was also evaluated during a novelty‐suppressed feeding behavioral paradigm. Shown are the (B) latency to eat in the novel cage, (C) a comparison of the number of TgF344‐AD and WT animals that timed out of the task, (D) the latency to eat in the home cage, (E) and the total amount of pellet consumed after 1 hour. Animals treated with DSP‐4 showed increased anxiety‐like behaviors in the open field task, while TgF344‐AD animals displayed elevated anxiety‐like behavior during novelty‐suppressed feeding. Females are represented by open circles and males by closed circles. Data are mean ± SEM; n = 9–12 animals per group. *p ≤ 0.05, ****p ≤ 0.0001.

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  FIGURE 6. Treatment and genotype increase freezing during contextual and cued fear conditioning. WT and TgF344‐AD animals received monthly injections of DSP‐4 or saline, and learning and memory were evaluated using fear conditioning. The percentage of time spent freezing was quantified during (A) training, (B) contextual fear conditioning, and (C) cued fear conditioning. Despite similar rates of associative learning, TgF344‐AD rats and animals treated with DSP‐4 displayed higher freezing in both contextual and cued fear conditioning. BL ‐ baseline, Int ‐ interval. Data are mean ± SEM; n = 9–12 animals per group. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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  FIGURE 7. Neither treatment nor genotype impacted AD‐related pathology. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline, and amyloid‐β and hyperphosphorylated tau pathology were quantified. (A) Representative images (20×) of amyloid‐β pathology in the DG with a DAPI counterstain. Quantification of amyloid‐β pathology in the (B) PFC, (C) DG, (D) CA1, and (E) CA3. (F) Representative images (20×) of CP13 staining in the PFC with a DAPI counterstain. Quantification of hyperphosphorylated tau pathology in the (G) PFC, (H) DG, (I) CA1, and (J) CA3 regions expressed as the mean gray value within a standardized ROI. Neither treatment nor genotype exacerbated AD‐related neuropathology. Females are represented by open circles and males by closed circles. Scale bar = 50 µm. Data are mean ± SEM; n = 5–6 animals per group.

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  FIGURE 8. Levels of neuroinflammation depend on treatment and genotype. WT (black) and TgF344‐AD (pink) animals received monthly injections of DSP‐4 or saline. IBA1 and GFAP immunoreactivity was evaluated. (A) Shown are representative images (20×) of IBA1 staining in the PFC with a DAPI counterstain. Quantification of IBA1 in the (B) PFC, (C) DG, (D) CA1, and (E) CA3. (F) Representative images (20×) of GFAP staining in the PFC with a DAPI counterstain. Quantification of GFAP in the (G) PFC, (H) DG, (I) CA1, and (J) CA3. WT rats displayed more IBA1+ cells compared to TgF344‐AD rats in the PFC and CA1, while DSP‐4 treatment caused increased GFAP+ cells in the PFC. Females are represented by open circles and males by closed circles. Scale bar = 50 µm. Data are mean ± SEM; n = 5–6 animals per group. *p ≤ 0.05, **p ≤ 0.01.