Perinatal Beta-Amyloid Treatment Decreases Hippocampal AcetylcholinesteraseTaylor J Ford and Ethan HuffingtonWinona State University, Winona, MN

Introduction Discussion

Acknowledgements

Subjects

Seventeen male and female C57BL/6j mice served as subjects. During all phases of the study, subjects were divided by gender and housed in groups of 3-4 animals each. Seven of the subjects received vehicle (controls) and the other 10 received 1 µg/µL β-Amyloid injected bilaterally into the dorsal hippocampus on day five post-partum. All subjects were maintained on a standard laboratory diet and housed in a colony room that was kept at 22 degrees C.

Behavioral Testing

a) Step-down Passive Avoidance Test:

All testing and scoring was completed with the researcher unaware of the subject’s treatment condition. The step-down passive avoidance test was used to evaluate memory loss. Subjects were placed on a raised platform (9.6 x 8.2 x 3.0 cm high) in a sound resistant test chamber (28.2 x 21.7 x 21.0 cm). A video camera was fixed in front of the test chamber to record behavior. Training for the step-down test was performed on day 120 post-partum. During this initial training phase each subject was placed on the platform. Mice have a natural tendency to step down within a few seconds. In order to inhibit this step-down response, a 0.18 mV shock was administered for 2 s when the subject had all four paws floor of the test chamber. The time until the animal stepped off the platform was recorded as the step-down latency. Twenty-four hours later, the animal was placed on the raised platform again and the step down latency was recorded in order to test for retention (inhibition of the step-down response or longer response times indicates stronger memory). This test phase was repeated seven days after training. No electrical impulse was given in either test phase. If the subject did not step down within five minutes the test was ended.

b) Light-Dark Tests of Anxiety:

Anxiety was evaluated in the light-dark test 24 h after the seven day retention test. Mice have a natural fear of entering bright illuminated areas. Subjects were individually placed in a dark chamber (19.5 x 14.5 x 20 cm) maintained at 0 lux for 30 s. A door allowing access to a chamber (50 x 40 x 20 cm) illuminated at 300 lux was then raised. A video camera was fixed in front of the test chamber to record behavior. When a subject placed all four paws in the light chamber emergence latency was recorded. If the subject did not step out within 5 minutes the test was ended.

Anatomical Testing

Brains were removed immediately after the last behavioral test on Day 128. Thin-sections (40 µm) were prepared from fresh unfixed tissue frozen at -18oC and stained overnight for acetylcholinesterase levels using the methods of Paxinos and Franklin (2001).

Methods

References

Thank you to Dr. R. Deyo for assistance with this project. Thank you to the Winona State University Psychology Department for use of resources. Funding was provided by the NAI Neuroscience Program.

Bäckman, L., Small, B. J., & Fratiglioni, L. (2001). Stability of the preclinical episodic memory deficit in Alzheimer's disease. Brain: A Journal Of Neurology, 124(1), 96-102. doi:10.1093/brain/124.1.96.Barage, S., & Sonawane, K. (2015). Amyloid cascade hypothesis: Pathogenesis and therapeutic strategies in Alzheimer's disease. Neuropeptides, 52, 1-18.Paxinos, G. and Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates (2nd ed., p. x ). New York: Academic Press.

Alzheimer’s disease (AD) is characterized behaviorally by anterograde amnesia and anxiety (Backman et al., 2001). The anatomical symptomology of AD is marked by ß-amyloid plaques and neurofibrillary tangles (Barage et al., 2015). One of the most well-known hypotheses in explanation of the pathogenesis of AD is based on cholinergic dysfunction. AD patient’s show a reduction in acetylcholinesterase in all areas of the brain compared with healthy patients (Barage et al., 2015). The present study investigated whether ß-Amyloid plays a role in development of these anatomical changes relative to adult behavioral deficits in an animal model of Alzheimer’s disease.

ß-amyloid1-42 was injected into the hippocampus of seventeen newborn mice. Behavioral testing began once the subjects had reached adulthood (120 days postpartum) using the step-down passive avoidance test (memory) and the light-dark test (anxiety). In order to access anatomical deficits, subjects’ brains were stained to identify acetylcholinesterase density. Cell loss, development of neurofibrillary tangles, and plaque formation would also be predicted but were not evaluated at this time as we elected to focus on the earliest stages of the disorder with the goal of developing early markers and prevention strategies. If the injection of ß-amyloid does play a role in the development of the anatomical and behavioral deficits involved in AD, we should see that the ß-amyloid treated subjects will display memory deficits, anxiety, and less acetylcholinesterase density in the hippocampus relative to controls.

As predicted, anatomical analyses revealed that the hippocampus of ß-Amyloid treated subjects showed significantly less density in acetylcholinesterase staining compared to controls (see Figure 1 and Table 1). However, our predictions were not supported by the results of the behavioral analysis (see Table 1). In fact, the results were opposite to our predictions. Control subjects had significantly higher levels of anxiety than ß-Amyloid treated subjects as illustrated by longer emergence latencies (see Table 1). Furthermore, in the 24 h step-down latency test controls exhibited significantly faster times to step down from the platform appearing as if they did not remember receiving the electrical impulse from the training session. This contradiction could be explained by the controls higher levels of anxiety (see Table 1). We noticed that controls bolted off the platforms at a rate that was 60% faster than training. Therefore, if controls were indeed more anxious during the learning experiment (supported by the anxiety data), memory for the aversive stimulus may have increased their anxiety even further leading to an escape response rather than the typical freezing response expected in this learning paradigm.

The decrease in acetylcholinesterase staining suggests that the subjects were in the early stages of an Alzheimer’s-like disease process. Further research will be required to determine the causes of the contradictory behavioral effects reported here.

Results

Figure 1. Acetylcholinesterase staining in the hippocampus at 10x magnification (a) control mouse exhibiting the highest level of density (b) control mouse exhibiting level of density closest to the mean (c) control mouse exhibiting the lowest level of density (d) β-Amyloid mouse exhibiting the highest level of density (e) β-Amyloid mouse exhibiting level of density closest to the mean (f) β-Amyloid mouse exhibiting the lowest level of density.

Table 1. Summary of Treatment Conditions and Results

Test Treatment Mean tEmergence latency Control

ß-Amyloid11.62 s 6.12 s

t(9)=2.404, p<.05

24 h step-down latency Control ß-Amyloid

4.63 s116.10 s

t(9)=2.147, p<.05

7 day step-down latency Control ß-Amyloid

48.26 s115.72 s

t(9)=1.122, p>.05

Acetylcholinesterase binding density

Control ß-Amyloid

175.33 units147.08 units

t(9)=1.899, p<.05

A summary of the descriptive and inferential statistical analyses are presented in Table 1. Analysis of the light-dark test of anxiety indicated a significant increase in anxiety in the control subjects compared to β-Amyloid injected subjects (t(9)=2.404, p<.05). Tests of memory at 24 h post-training indicated that control subjects stepped-down from the platform significantly faster than β-Amyloid injected subjects (t(9)=2.147, p<.05). Likewise, tests of memory at 7 days post-training showed control subjects stepping down faster but not significantly (t(9)=1.122, p>.05). Anatomical analysis showed higher density in acetylcholinesterase staining in β-Amyloid injected subjects compared with controls t(9)=1.899, p<.05.