Systematic characterization of a non-transgenic Aβ1-42 amyloidosis model: synaptic plasticity and memory deficits in female and male mice

Abstract

Background: The amyloid-β (Aβ) cascade is one of the most studied theories linked to AD. In multiple models, Aβ accumulation and dyshomeostasis have shown a key role in AD onset, leading to excitatory/inhibitory imbalance, the impairments of synaptic plasticity and oscillatory activity, and memory deficits. Despite the higher prevalence of Alzheimer's disease (AD) in women compared to men, the possible sex difference is scarcely explored and the information from amyloidosis transgenic mice models is contradictory. Thus, given the lack of data regarding the early stages of amyloidosis in female mice, the aim of this study was to systematically characterize the effect of an intracerebroventricular (icv.) injection of Aβ_1-42 on hippocampal-dependent memory, and on associated activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse, in both male and female mice.

Methods: To do so, we evaluated long term potentiation (LTP) with ex vivo electrophysiological recordings as well as encoding and retrieval of spatial (working, short- and long-term) and exploratory habituation memories using Barnes maze and object location, or open field habituation tasks, respectively.

Results: Aβ_1-42 administration impaired all forms of memory evaluated in this work, regardless of sex. This effect was displayed in a long-lasting manner (up to 17 days post-injection). LTP was inhibited at a postsynaptic level, both in males and females, and a long-term depression (LTD) was induced for the same prolonged period, which could underlie memory deficits.

Conclusions: In conclusion, our results provide further evidence on the shifting of LTP/LTD threshold due to a single icv. Aβ_1-42 injection, which underly cognitive deficits in the early stages of AD. These long-lasting cognitive and functional alterations in males and females validate this model for the study of early amyloidosis in both sexes, thus offering a solid alternative to the inconsistence of amyloidosis transgenic mice models.

Keywords: Alzheimer’s disease; Amyloid-β; Aβ 1–42; Hippocampus; LTP; Sex differences; Spatial memory.

Fig. 1

Experimental design showing timeline and corresponding figure numbers. A A guide cannula was implanted for icv. drug administration on the left ventricle (histologic verification image on the right). A minimum of 8 days after surgery, a single injection of either Aβ_1–42 or controls (reverse Aβ_42–1 peptide or vehicle), were administered. Scale bars: 500 μm. B Habituation phase of Barnes maze task was conducted the day before icv. administration (day-1). 1 h after treatment injection (day 0, pre-training icv. injection), training in the Barnes maze began, which consisted of 3 trails per day for 4 consecutive days (days 0–3). Two memory tests were conducted, on days 7 and 11 post-injection. C The Open field habituation test was conducted on days 15 and 16 post-injection. D Finally, mice went through a test battery that included testing of stereotyped and locomotor behaviors by an automated LABORAS^® System to assess their overall spontaneous behavior and health, as well as rotarod, elevated plus maze and tail suspension tests (days 15–17). E Another cohort of animals was used to evaluate the effect of Aβ_1–42 on ex vivo hippocampal LTP by multi-electrode arrays (MEAs) electrophysiology (days 1–17 post-injection). Representative location of stimulation (St., green) and recording (Rec., red) electrodes in an hippocampal coronal slice. F A different cohort of animals underwent the Object location memory test (OLM) to study the effect of Aβ_1–42 on memory retrieval. After habituation (day 0), both training and retrieval (OLM1) sessions were conducted on day 1. On day 2, animals were icv. injected with the corresponding treatment (post-training injection) and, 1 h later, another retrieval session (OLM2) was performed. G The same animals also underwent the Open field habituation test, with post-training icv. treatment. D, dorsal; DG, dentate gyrus; icv., intracerebroventricular; L, lateral; V, ventricle; M, medial

Fig. 2

Aβ_1–42 impairs spatial learning and memory encoding in both female and male mice. A Representative traces of the path traveled during the first and last trial of the last training day (day 3) for Aβ_1–42 and controls: vehicle and reverse Aβ_42–1 peptide. B–D Escape latency (B; in s), number of errors (C) and distance traveled (D; in cm) during the four training days. Data is expressed as the mean ± SEM of the 3 trials per day. E Overview image of the test phase on the Barnes maze, with all holes closed. F Latency (in s) to reach the target hole of the latest training day for the first time, during the two test sessions. G Distance traveled (in cm) during the two test sessions. H Representative traces of the three possible search strategies: random, serial, and spatial. I Ratio of the use of each search strategy for all the experimental groups during training (Days 0 and 3) and tests (Days 7 and 11) sessions. Stacked bars are normalized so that the sum of the three strategies each day is 100%. N vehicles: males = 14 and females = 15; N Aβ_1–42: males = 14 and females = 16; N reverse Aβ_42–1 males = 7 and females = 6. Aβ, Amyloid-β; cm, centimeters; s, seconds. *p < 0.05, **p < 0.01, *** p < 0.001 vs. vehicle of the corresponding sex; # p < 0.05, ## p < 0.01 vs. Aβ_42–1 of the corresponding sex

Fig. 3

Aβ_1–42 administration similarly alters non-associative exploratory habituation memory encoding in both female and male mice. A An OF habituation test was carried out by submitting the animals to the same OF arena twice, on consecutive days 15 and 16 post-icv. injection. B Total distance traveled during the two OF sessions (training -OF1- and retrieval -OF2- sessions). Data is expressed as the percentage (%) of the distance traveled in the training session (OF1). C Examples of mice movement tracked during OF1 and OF2 for the different treatment groups. N vehicles: males = 7 and females = 12; N Aβ_1–42: males = 9 and females = 10; N reverse Aβ_42–1: males = 7 and females = 6. Aβ, Amyloid-β; icv., intracerebroventricular; OF, open field. * p < 0.05, ** p < 0.01 vs. vehicle of the corresponding sex; # p < 0.05, ## p < 0.01 vs. Aβ_42–1 of the corresponding sex; ‡ p < 0.05, ‡‡ p < 0.01, ‡‡‡ p < 0.001 vs. OF1

Fig. 4

Aβ_1–42 administration also alters spatial and non-associative memory retrieval in both female and male mice. A An OLM test was performed, changing the location of one object between the training and each memory test (OLM1 and OLM2). Treatment was administered icv. between OLM1 and OLM2 to evaluate memory retrieval. B Discrimination index during the training, OLM1, and OLM2 sessions. Data is expressed as the mean ± SEM. C An OF habituation test was carried out by submitting the animals to the same OF arena twice, administering the icv. injection between the training and the retrieval sessions. D Total distance traveled during the two OF sessions (training -OF1- and retrieval -OF2- sessions). Data is expressed as the percentage (%) of the distance traveled in the training session (OF1). E Examples of mice movement tracked during OF1 and OF2 for the different treatment groups. N vehicles: males = 8–9 and females = 7; N Aβ_1–42: males = 9–10 and females = 7–8; N reverse Aβ_42–1: males = 8–9 and females = 6. Aβ, Amyloid-β; icv., intracerebroventricular; OF, open field; OLM, object location memory. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle of the corresponding sex; #p < 0.05, ##p < 0.01 vs. reverse Aβ_42–1 of the corresponding sex; ‡‡p < 0.01, ‡‡‡p < 0.001 vs. OF1

Fig. 5

Aβ_1–42 administration does not induce alterations in locomotor activity, anxiety, and depression-like behavior. Behavioral tasks to evaluate general health state were carried out on days 15–17 post-icv. injection. A Stereotyped behaviors were assessed using a LABORAS^® system, measuring the time (in s) spent performing each type of activity (locomotion, climbing, rearing, and grooming). B Latency (in s) to fall off the Rotarod during the six trials (left) and the whole session (right). C Number of entries in closed and total arms were used as measure of locomotor activity (left), while anxiety levels were assessed by the percentage (%) of entries and time spent on open arms in an elevated plus maze (right). D Depression-like behavior was assessed by measuring the immobility time (in s) during a single session in the tail suspension test. N vehicles: males = 8–11 and females = 11–12; N Aβ_1–42: males = 8–11 and females = 9–12; N reverse Aβ_42–1: males = 7 and females = 6). Aβ, Amyloid-β; s, seconds

Fig. 6

Aβ_1–42 inhibits ex vivo hippocampal LTP and induces LTD in both female and male mice. A–C I/O curve with paired fEPSPs collected at increasing stimulus intensities (from 0.075 to 0.4 mA) from control vehicle (A), Aβ_1–42 (B) and Aβ_42–1 reverse control (C) slices, respectively. Data is expressed as a percentage (%) of the maximum amplitude obtained. D PPF curve with paired fEPSPs collected at interstimulus intervals of 10, 20, 40, 100, 200 and 500 ms. Data is expressed as mean ± SEM amplitude of the second fEPSP as a percentage of the first [(second/first) × 100] for each inter-pulse interval used. E Representative averaged (n = 5) traces of fEPSPs recorded in the CA1 area, collected during the baseline (1) and ≈50 min post-HFS (2) in hippocampal slices from the different groups. F Time course of LTP evoked in the CA1 area after HFS in hippocampal slices from the different groups. Recordings were obtained from day 1 to 17 post-icv. injection. G, H Bars illustrate mean ± SEM fEPSPs amplitude of the last 10 min of the recording, to show acute (G; 24–48 h post-icv. injection) vs. long-term (H; 3–17 days post-icv. injection) effects on LTP. N (slices) vehicles: males = 6–5 and females = 5–5; N Aβ_1–42: males = 7–7 and females = 6–7; N reverse Aβ_42–1: males = 5–7 and females = 6–7. Aβ, amyloid-β; HFS, High frequency stimulation; LTP, long-term potentiation; mA, milliamperes; ms, milliseconds; min, minutes. ***p < 0.001 vs. vehicle of the corresponding sex; ###p < 0.001 vs. Aβ_42–1 of the corresponding sex