Adenosine A2A Receptor Up-Regulation Pre-Dates Deficits of Synaptic Plasticity and of Memory in Mice Exposed to Aβ1–42 to Model Early Alzheimer’s Disease

The intracerebroventricular (icv) injection of amyloid peptides (Aβ) models Alzheimer’s disease (AD) in mice, as typified by the onset within 15 days of deficits of memory and of hippocampal long-term potentiation (LTP) that are prevented by the blockade of adenosine A2A receptors (A2AR). Since A2AR overfunction is sufficient to trigger memory deficits, we tested if A2AR were upregulated in hippocampal synapses before the onset of memory deficits to support the hypothesis that A2AR overfunction could be a trigger of AD. Six to eight days after Aβ-icv injection, mice displayed no alterations of hippocampal dependent memory; however, they presented an increased excitability of hippocampal synapses, a slight increase in LTP magnitude in Schaffer fiber-CA1 pyramid synapses and an increased density of A2AR in hippocampal synapses. A2AR blockade with SCH58261 (50 nM) normalized excitability and LTP in hippocampal slices from mice sacrificed 7–8 days after Aβ-icv injection. Fifteen days after Aβ-icv injection, mice displayed evident deficits of hippocampal-dependent memory deterioration, with reduced hippocampal CA1 LTP but no hyperexcitability and a sustained increase in synaptic A2AR, which blockade restored LTP magnitude. This shows that the upregulation of synaptic A2AR precedes the onset of deterioration of memory and of hippocampal synaptic plasticity, supporting the hypothesis that the overfunction of synaptic A2AR could be a trigger of memory deterioration in AD.


Introduction
Alzheimer's disease (AD) is the most common memory pathology.It is neurochemically characterized by an abnormal production of amyloid peptides (Aβ) and an hyperphosphorylation of microtubule-associated tau protein [1].One of the earliest alterations in AD evolution is a dysfunction and loss of synapses [2][3][4]-in particular, of glutamatergic synapses [5,6]-with aberrant patterns of synaptic plasticity in prominently afflicted regions, namely, in the hippocampus (e.g., [7,8]).However, the mechanisms triggering this synaptotoxicity, already present in mild cognitive impairment [9][10][11], considered a prodrome of AD, are still unknown.
The adenosine modulation system has emerged in recent years as a candidate target to alleviate the burden of AD.Adenosine is released in an activity-dependent manner [12,13] and is responsible for assisting in the encoding of information salience in neuronal networks through a combined action of inhibitory A 1 receptors (A 1 R) and facilitatory A 2A receptors (A 2A R) (reviewed in [14]).Both A 1 R and A 2A R are mainly located in excitatory synapses in the cerebral and limbic cortices [15][16][17].In particular, A 2A R increase glutamate release [18,19] and bolster NMDA receptor function in the hippocampus [20][21][22] to selectively control synaptic plasticity (e.g., [23,24]) as well as synaptotoxicity (e.g., [24][25][26]).Notably, the pharmacological, genetic or optogenetic overfunction of A 2A R is actually sufficient to deteriorate behavioral performance in tests of spatial reference memory [22,[27][28][29], and, conversely, the pharmacological or genetic blockade of A 2A R afford a general neuroprotection against brain damage (reviewed in [14,30,31]) and prevent memory dysfunction in different animal models of AD [8,22,25,[32][33][34].This is in notable agreement with human epidemiological findings showing that the regular intake of caffeinated coffee (caffeine is a selective antagonist of central adenosine receptors [35]) attenuates memory deficits and the onset of AD (e.g., [36][37][38]) and that A 2A R polymorphisms are associated with the incidence of AD [39,40].Importantly, in different models of neurodegeneration, A 2A R undergo a gain of function, typified by a parallel upregulation of cortical A 2A R (e.g., [8,22,[24][25][26]29,33]) and an increased formation of ATP-derived adenosine within cortical synapses [41][42][43][44][45], which is selectively directed at the activation of A 2A R [20,[41][42][43][44][45].Furthermore, in animal models of temporal lobe epilepsy [42] and of Parkinson's disease [45], A 2A R overfunction predates alterations of synaptic plasticity and of behavior that are characteristic of these diseases, prompting the hypothesis that A 2A R upregulation might actually be a trigger of brain dysfunction resulting from aberrant synaptic plasticity [46].
We took advantage of a model of early AD based on the intracerebroventricular (icv) administration of a suspension of soluble β-amyloid peptides monomers and oligomers (Aβ 1-42 ) to test if hippocampal A 2A R upregulation was a precocious event, i.e., pre-dating hippocampal-dependent memory deficits.Previous studies have shown that the selective A 2A R antagonist SCH58261 prevents Aβ-induced dysfunction of synaptic plasticity and of memory [25,41], and that there is time gap of at least 14 days between the icv administration of Aβ 1-42 and the establishment of memory dysfunction [25].This was exploited to test if A 2A R density was increased in hippocampal synapses before the onset of memory dysfunction and if this was coupled to adaptive compensatory alterations of synaptic transmission and plasticity in the AD presymptomatic period.

Animals
We used adult C57bl/6j mice, 3 months of age (24.2 ± 1.7 g) and of both sexes (20 females and 12 males for the data presented in Figures 1 and 2; 14 females and 18 males in the groups treated with the A 2A R antagonist in Figure 3, with similar number of mice from each sex in each of the two groups), obtained from Charles River (Barcelona, Spain).We did not carry out a de novo power analysis, since we relied on our previous experience of the variability associated with the model used and drugs tested to pre-define the sample size as 8 mice per group in the behavioral and electrophysiology experiments and a minimum of 6 mice per group in the binding experiments in order to be able to detect 20% changes with 95% confidence.Mice were housed under controlled temperature (23 ± 1 • C) and a 12 h light/dark cycle, with free access to food and water.Mice were handled following European Community guidelines (EU Directive 2010/63/EU) and the Portuguese law on animal care (1005/92), and all procedures were approved by the Ethical Committee of the Center for Neuroscience and Cell Biology of Coimbra (ORBEA-128/2015).

Behavioral Analysis
All behavior tests were carried out as previously described [8,24,33,41] from 9 AM until 5 PM by experimenters who were unaware of drug treatments in a sound-attenuated room maintained at 21-23 • C and 50-60% humidity with red lightening (8 lux light intensity) and visual cues on the walls, to which the animals were previously habituated for at least 1 h before beginning behavioral tests.The tests were video-recorded and analyzed with the ANY-maze Video Tracking Software (version 6.18; Stoelting Europe, Dublin, Ireland).The apparatuses were cleaned with 10% ethyl alcohol to remove odors after testing each mouse.Locomotion and exploratory behavior were monitored in the morning of either day 6 or day 14 after Aβ-icv injections, using an open-field arena, where each mouse was placed in the center of the open field to record during 10 min the following variables: number of peripheral squares (adjacent to the walls) crossed (peripheral locomotion); number of central squares (away from the walls) crossed (central locomotion); and total locomotion (peripheral locomotion plus central locomotion).
Hippocampal-dependent memory was evaluated using the object displacement test, carried out in the afternoon of either day 6 or day 14 after icv injections.Mice were exposed to two identical objects in the same open field apparatus in which they were habituated and were allowed to explore for 5 min the objects fixed in opposite corners 5 cm away from walls and 25 cm apart from each other.In a test trial carried out 2-h later, mice were again placed for 5 min in the open field arena, except that one of the objects was moved to a novel position.Memory performance was quantified with an object displacement index defined as the ratio between the time exploring the object in the novel location over the total time exploring both objects.Exploration of an object is defined as directing the nose to the object at a distance equal to or less than 1 cm from the object and/or touching it with the nose; rearing on to object was not considered exploratory behavior.
Spatial memory was further evaluated using a 2-trials Y-maze paradigm on the morning of either day 7 or day 15 after icv injections.The test consisted of two sessions of 8 min duration separated by a 90-min inter-trial interval.During the first session, the mouse was placed at the end of one arm and allowed to explore the two available arms since the third arm (the novel arm) was blocked by a guillotine door.During the second session, the 'novel' arm was open, and the mouse was placed in the start arm and allowed to explore the three arms.Memory performance was evaluated by measuring the time spent exploring the 'novel' arm compared to the exploration of the other two arms.An entry into an arm was defined as placement of all four paws into the arm.

Electrophysiological Recordings
Following sacrifice, the mouse brain was quickly removed and placed in ice-cold, oxygenated (95% O 2 , 5% CO 2 ), artificial cerebrospinal fluid (ACSF; in mM: 124.0 NaCl, 4.4 KCl, 1.0 Na 2 HPO 4 , 25.0 NaHCO 3 , 2.0 CaCl 2 , 1.0 MgCl 2 , 10.0 glucose).One hemisphere was used for electrophysiological experiments and the other hemisphere was used for receptor binding studies (in 6 out of the 8 mice per group).Slices (400 µm-thick) from the dorsal hippocampus were cut transverse to the long axis of the hippocampus with a McIlwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK) and maintained for at least 1 h prior to recording in a holding chamber with oxygenated ACSF at room temperature.Slices were then transferred to a submerged recording chamber and superfused at 3 mL/min with oxygenated ACSF kept at 30.5 • C. Electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) were carried out as previously described (e.g., [24,33,35,41]) with the recording electrode, filled with 4 M NaCl (2-5 MΩ resistance), placed in the CA1 stratum radiatum targeting the distal dendrites of pyramidal neurons and the stimulating bipolar concentric electrode placed in the proximal CA1 stratum radiatum.Rectangular pulses of 0.1 ms were delivered every 20 s through a Grass S44 or a Grass S48 square pulse stimulator (Grass Technologies, Singapore).After amplification (ISO-80, World Precision Instruments, Singapore), the recordings were digitized (Pico ADC-42, Pico Technologies Ltd., St. Neots, UK), averaged in groups of 3 and analyzed using the WinLTP version 2.10 software [50].
Input/output (I/O) curves, in which the percentage of the maximum fEPSP slope was plotted versus stimulus intensity, were first performed in all slices to evaluate changes in basal synaptic transmission and to determine the adequate level of electrical stimulation required to trigger between 50-60% of maximal fEPSP response, which was used for the remainder of the experimental protocol.Long-term potentiation (LTP) was induced by a high-frequency stimulation train (100 Hz for 1 s).LTP was quantified as the percentage change between the average slope of the 10 fEPSPs between 50 and 60 min after LTP induction in relation to the average slope of the fEPSPs measured during the 10 min that preceded LTP induction.The effect of SCH58261 (50 nM) on LTP was assessed by comparing LTP magnitude in the absence and presence of SCH58261 in experiments carried out in different slices from the same animal.

Membrane Binding Assays
Following sacrifice, one hippocampus of each mouse was dissected and homogenized in ice-cold sucrose solution (0.32 M D-sucrose; 1 mM EDTA-Na; 10 mM HEPES; 0.015 mM bovine serum albumin; pH 7.4 at 4 • C) for subsequent preparation of synaptosomes and total membranes, as previously described (e.g., [16,17]).The synaptosomal fraction and the total membrane fraction were each resuspended in 300 µL of a pre-incubation solution (50 mM Tris, 1 mM EDTA, 2 mM EGTA, pH 7.4) to determine their protein content with the Bio-Rad protein assay (Bio-Rad, Amadora, Portugal) and stored at −80 • C until used for membrane binding assays.
Binding assays to estimate A 2A R density in synaptosomal membranes were carried out as previously described (e.g., [8,24,26,33]).Briefly, the synaptosomes were lysed in a Tris/Mg solution (50 mM Tris, 10 mM MgCl 2 , pH 7.4) and pelleted.Both synaptosomal membranes and total membranes were incubated for 30 min at 37 • C with adenosine deaminase (2 U/mL; Roche Molecular Biochemicals, Indianapolis, IN, USA) to remove endogenous adenosine.After centrifugation at 24,000× g for 15 min at 4 • C, the pellets were resuspended in 600 µL of Tris/Mg solution with 4 U/mL of adenosine deaminase.A 2A R binding density was determined with 217-345 µg of synaptosomal membrane protein exposed during 1 h at room temperature to a single supra-maximal concentration (2 nM) of the selective antagonist 3 H-SCH58261 (specific activity of 77 Ci/mmol, prepared by GE Healthcare Life Sciences and generously offered by E. Ongini, Schering-Plough, Italy) in a final volume of 200 µL.The binding reactions were stopped by addition of 5 mL of ice-cold Tris/Mg and vacuum filtration through glass fiber filters (Whatman GF/C filters, GE Healthcare Life Sciences, Carnaxide, Portugal), followed by a washing step with 5 mL of ice-cold Tris/Mg.Filters were then placed in vials with 2 mL of scintillation liquid (AquaSafe 500Plus; Zinsser Analytic GMBH, Eschborn, Germany) to measure radioactivity in a 2900TR Tricarb β-counter (PerkinElmer, Lisbon, Portugal) after at least 12 h.Specific binding was expressed as fmol/mg of protein and determined via subtraction of the non-specific binding, measured in the presence of the mixed A 1 /A 2A receptor antagonist XAC (Sigma-Aldrich, Lisbon, Portugal) at a concentration (12 µM) over 1000 times higher than that of the radioligand.Total binding measurements were conducted in triplicate, and nonspecific binding in duplicate.Negative controls in hippocampal membranes of A 2A R knockout mice have previously ensured the selectivity of the tested concentration of 3 H-SCH58261 [51].

Statistical Analyses
The values are presented as mean ± S.E.M. with the number of determinations (n, preparations from different mice).A Grubbs' test was first used to detect putative outliers.The comparison of two experimental conditions was performed using a two-tail Student's t test with Welsh correction.Otherwise, statistical analysis was performed by two-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison post hoc test.p < 0.05 was considered to represent statistical significance.Statistical analysis was performed using GraphPad Prism software (version 6.0; GraphPad Software, La Jolla, CA, USA).

Memory Deficits Are Present at 14 Days but Not at 7 Days after Aβ Administration
The Aβ-icv mouse model of AD is characterized by selective hippocampal-dependent memory deficits emerging 12 days after Aβ-icv administration without motor or emotional alterations [25,41].Accordingly, compared to vehicle-treated mice, we now observed that 6-7 days after Aβ-icv administration, mice were devoid of alteration of (i) spontaneous locomotion in the open field test (number of line crossing: 92.5 ± 4.3 for vehicle and 100.3 ± 4.5 for Aβ-icv, n = 8, p = 0.236 with Student's t test (Figure 1A)); (ii) emotional-like behavior assessed as the percentage of time spent in the more aversive central zone of the open field arena (22.0 ± 1.9% for vehicle and 19.3 ± 1.4% for Aβ-icv, n = 8, p = 0.280 with Student's t test (Figure 1B)); (iii) hippocampal-dependent memory performance in the object displacement test (discrimination ratio: 65.0 ± 2.0 for vehicle and 64.7 ± 2.1 for Aβ-icv, n = 8, p = 0.915 with Student's t test (Figure 1C)); and (iv) memory performance in the modified Y-maze test (percentage time in the new arm: 41.0 ± 1.4% for vehicle and 40.5 ± 1.2% for Aβ-icv, n = 8, p = 0.806 with Student's t test (Figure 1D)).In contrast, 14-15 days after Aβ-icv administration, mice displayed a selective deficit of hippocampaldependent memory performance both in the object displacement test (discrimination ratio: 64.1 ± 1.6 for vehicle and 52.7 ± 1.7 for Aβ-icv, n = 8, p < 0.001 with Student's t test (Figure 1C); with similar discrimination ratio during the training period: 50.7 ± 1.0 for vehicle and 50.3 ± 1.0 for Aβ-icv, n = 8, p = 0.778 with Student's t test), as well as in the modified Y-maze test (percentage time in the new arm: 39.6 ± 1.0% for vehicle and 33.8 ± 0.8% for Aβ-icv, n = 8, p < 0.001 with Student's t test (Figure 1D)), without evident alterations of locomotion (number of line crossings: 93.9 ± 2.7 for vehicle and 92.0 ± 3.3 for Aβ-icv, n = 8, p = 0.663 with Student's t test (Figure 1A)) or anxiety in the open field test (percentage time in the center: 20.9 ± 1.6% for vehicle and 21.9 ± 1.5% for Aβ-icv, n = 8, p = 0.638 with Student's t test (Figure 1B)).Thus, Aβ-icv administration only triggers a selective hippocampal-dependent memory deficit at 14-15 days, without evident behavioral modification 6-7 days after Aβ-icv administration.

Hippocampal Excitability Increases at 7-8 Days and Decreases at 15-16 Days after Aβ Administration
In spite of the absence of behavioral alteration at days 6-7 after Aβ-icv, there were alterations of synaptic excitability in hippocampal slices of these mice.Thus, the recording of synaptic transmission in Schaffer fibers-CA1 pyramid synapses, assessed as field excitatory synaptic potentials (fEPSP), revealed an increase in the input/output (I/O) curves in slices of mice at days 7-8 after Aβ-icv compared to vehicle-treated controls (Figure 2A,B).This increased excitability is likely to be a transient compensatory feature since it disappeared in slices of mice at days 15-16 after Aβ-icv, which displayed I/O superimposable to these of vehicle-treated mice (Figure 2C,D).
We next investigated if A 2A R blockade would be able to prevent the hyperexcitability observed in the hippocampus in the presymptomatic period, i.e., 7-8 days after Aβ-icv injection.The administration of a supramaximal concentration of SCH58261 (50 nM) [20] prevented the increased synaptic efficiency in the hippocampus 7-8 days after Aβ-icv administration, bringing the I/O curve to a profile nearly superimposable to that observed in vehicle-treated mice ((Figure 3B); note that the vehicle-icv and Aβ-icv groups in Figure 3B are the same as in Figure 2A).Moreover, the tendency for an increased LTP magnitude observed in hippocampal slices collected 7-8 days after Aβ-icv administration was eliminated by 50 nM SCH58261 ((Figure 3D); note that the vehicle-icv and Aβ-icv groups in Figure 3D,E are the same as in Figure 2C-E); thus, in slices collected 7-8 days after Aβ-icv administration, LTP magnitude was 62.7 ± 2.4% over baseline (n = 8) in the absence and was significantly lower (p < 0.001, Student's t test) in the presence of SCH58261 (49.4 ± 2.1% over baseline, n = 8).A Tukey's multiple comparison test after a two-way ANOVA (effect of Aβ: F 1,28 = 18.82, p < 0.001; effect of SCH58261: F 1,28 = 48.61,p < 0.001) revealed no significant difference between LTP magnitude in vehicle-treated and Aβ-icv-treated mice in the presence of SCH58261 (p = 0.291), although SCH58261 also decreased LTP magnitude in saline-treated mice (p < 0.001, Student's t test), as previously reported (e.g., [26,35,41]).

Discussion
The present study shows that an increased density and functional impact of A 2A R in the hippocampus predates the Aβ-icv-induced deterioration of synaptic plasticity and of reference memory that are characteristic of this mouse model of AD.Thus, it was shown that the activity of these A 2A R-with increased density in the presymptomatic period of AD that is maintained throughout the phenotypic expression of synaptic and memory deficits characteristic of early AD-is required both for the adaptive hyperexcitability before memory deficits as well as for the expression of synaptic and memory deficits present at the onset of symptomatic AD.
We first characterized experimental conditions indicative of a presymptomatic period of AD, where an increase in hippocampal excitability was observed at 7-8 days after Aβicv, without any evident alteration of memory-related behavioral performance.This is in agreement with several studies reporting an initial hyperexcitability in the hippocampus in early AD and pre-dating the onset of memory deficits (reviewed in [54][55][56]), as well as with the presence of subclinical epileptiform activity in the AD prodrome [57,58] and its association with the onset of AD [59].This initial hippocampal hyperexcitability is linked to a deregulation of the inhibitory GABAergic network [60-62], involving a parallel alteration of the excitability of different interneurons [60, 61,63] as well as an altered astrocytic support [64,65].Importantly, this initial hippocampal hyperexcitability may contribute to the subsequent deterioration of information processing in hippocampal networks and the onset/aggravation of memory symptoms of AD [54][55][56], as heralded by the ability of drugs refraining from excessive excitation, such as levetiracetam, to improve performance on spatial memory tasks in early AD [66].
The exploitation of this tentative mouse model of presymptomatic AD then allowed us to conclude that A 2A R undergo an early upregulation and seem to be paramount to controlling the increased excitability during this prodrome-like period in this Aβ-icv model of AD.In fact, we observed an increased density of A 2A R in the synapses, where cortical A 2A R are most abundant [17], but also in total membranes that mostly include non-synaptic and astrocytic membranes.Moreover, we showed that a selective A 2A R antagonist, SCH58261 [51], abrogated the hippocampal hyperexcitability present before the onset of memory deficits in this Aβ-icv model of AD.Although the precise mechanism underlying hippocampal hyperexcitability in the pathogenesis of AD is still unclear, it has been proposed to involve a deregulation of GABAergic interneurons [60, 61,63] and an altered astrocytic support [64,65].Remarkably, hippocampal A 2A R can control the excitability of GABAergic neurons [67], the release of GABA [68,69] and the stability of GABAergic synapses [70].A 2A R are also located in astrocytes in the hippocampus (e.g., [71][72][73]), controlling GABA uptake [74], and astrocytic A 2A R control neuronal excitability and memory performance [73,75,76].These mechanistic links are in agreement with the observed upregulation of A 2A R in synapses as well as in total membranes of the hippocampus during the presymptomatic phase of AD; however, future work is required to unveil the relative contribution of these different mechanism(s) operated by A 2A R to control hippocampal hyperexcitability present during the presymptomatic phase of AD.
The pattern of alteration of hippocampal synaptic plasticity in our tentative presymptomatic Aβ-icv model of AD is also in agreement with the currently accepted evolution of hippocampal activity during the evolution of AD, starting with a hyperexcitability in the presymptomatic phases that gradually evolves into a hypofunction with the onset and aggravation of memory deficits [55,77,78].The density of synaptic A 2A R is increased before and remains increased after the onset on synaptic plasticity and memory deficits in this Aβicv model of AD.Furthermore, A 2A R blockade reverted the deficits of synaptic plasticity in a manner similar to that previously reported in the normalization of abnormal synaptic plasticity in the hippocampus in different animal models of AD [8,22,32,33,41] and those of other brain diseases [24,26,42,44,[79][80][81][82][83].The effects of A 2A R in the control of abnormal synaptic plasticity likely reflect the key role of A 2A R in the control of memory dysfunction, as heralded by the previous observations that A 2A R activation is sufficient to trigger memory dysfunction [27][28][29], whereas A 2A R blockade dampens memory dysfunction in AD models [8,22,25,[32][33][34]41], specifically in the currently used Aβ-icv model of AD [25,41,52].The mechanisms underlying this beneficial effect of A 2A R antagonists are probably related to the impact of A 2A R on the release of glutamate [18,19], on AMPA [84] and NMDA receptors [20][21][22]85,86], as well as the ability of A 2A R to control astrocytic function [71][72][73][74][75][76] and microglia [87][88][89][90], impacting neuroinflammation (e.g., [91][92][93]).Future work is needed to clarify the contribution of these different pools of A 2A R to controlling these different processes known to impact synaptic plasticity and memory performance in order to begin tackling the mechanisms associated with the ability of A 2A R blockade to normalize synaptic plasticity.These will probably involve the engagement of different heteromers containing A 2A R (reviewed in [94]) and the coupling of A 2A R to different transducing mechanisms, which may change with the evolution of brain diseases and the upregulation of A 2A R (reviewed in [14]).Thus, understanding the spatiotemporal dynamics of A 2A R signaling in different compartments-which often have opposite effects on synaptic and behavioral outputs (see [95][96][97][98])-will be critical to understand this apparently paradoxical ability of A 2A R blockade to prevent aberrant hyperexcitability and normalize synaptic plasticity both when it is increased as well as when it is decreased.

Conclusions
It is concluded that A 2A R upregulation is an early event in the presymptomatic phase of AD that is maintained throughout the onset of memory deficits.This prompts consideration of the cortical A 2A R upregulation as a putative novel biomarker of the risk of developing AD [46], and eventually as a general biomarker of neuropsychiatric diseases [99] in view of the association of A 2A R overfunction with different neuropsychiatric diseases [99].Moreover, the present observation that A 2A R blockade prevents both the initial hyperexcitability during the presymptomatic phase of AD and the opposite decreased synaptic plasticity emerging with the onset of memory deficits confirms the prophylactic and therapeutic potential of A 2A R antagonists and stresses the need to understand the different signaling mechanisms operated by A 2A R in different cellular compartments.

17 Figure 1 .
Figure 1.Mice challenged with an intracerebroventricular (icv) injection of β-amyloid (1-42) (Aβ) displayed a selective alteration of hippocampal-dependent spatial memory after 14-15 days without behavioral alterations after 6-7 days.Compared to vehicle-icv-injected mice (veh, thin bars and open symbols), Aβ-icv-injected mice (Aβ, thick bars and filled symbols) analyzed at 6-7 days (green bars and symbols) or 14-15 days (red bars and symbols) after icv injections displayed a preserved spontaneous locomotion (A) and a preserved pattern of anxiety (B) in an open field arena Memory performance in the object displacement test (C) and in the modified Y-maze test (D) was preserved after 6-7 days and deteriorated at 14-15 days.Data are mean ± SEM; n = 8 mice per group *** p < 0.001, Student's t test with Welsh correction.

Figure 1 .
Figure 1.Mice challenged with an intracerebroventricular (icv) injection of β-amyloid (1-42) (Aβ) displayed a selective alteration of hippocampal-dependent spatial memory after 14-15 days, without behavioral alterations after 6-7 days.Compared to vehicle-icv-injected mice (veh, thin bars and open symbols), Aβ-icv-injected mice (Aβ, thick bars and filled symbols) analyzed at 6-7 days (green bars and symbols) or 14-15 days (red bars and symbols) after icv injections displayed a preserved spontaneous locomotion (A) and a preserved pattern of anxiety (B) in an open field arena.Memory performance in the object displacement test (C) and in the modified Y-maze test (D) was preserved after 6-7 days and deteriorated at 14-15 days.Data are mean ± SEM; n = 8 mice per group; *** p < 0.001, Student's t test with Welsh correction.

Figure 2 .
Figure 2. Hippocampal slices collected from mice during the presymptomatic period 7-8 days after an intracerebroventricular (icv) injection of β-amyloid 1-42 (Aβ-icv) displayed an hyperexcitability that was converted to a decreased pattern of synaptic plasticity upon onset of memory deficits at 15-16 days after Aβ-icv.At 7-8 days after Aβ-icv, and when compared to vehicle-icv injected mice (black open symbols/lines), Aβ-icv mice (filled green symbols/lines) displayed an hyperexcitability assessed by the shift to left of the input/output curve of the field excitatory post-synaptic potentials (fEPSP) slope (A,B) and a tendency towards an increased magnitude of long-term potentiation (LTP) triggered by a high-frequency stimulation train (HFS, 100 Hz for 1 s) in Aβ-icv mice (E,G).In contrast, in the symptomatic phase, there was no difference in the input/output curve between control and Aβ-icv mice (filled red symbols/lines) (C,D); however, LTP magnitude was lower in Aβicv mice (F,G).Data are mean ± SEM of 8 mice per group, the same as in Figure1; *** p < 0.001 between indicated groups using Student's t test with Welsh correction.

Figure 2 .
Figure 2. Hippocampal slices collected from mice during the presymptomatic period 7-8 days after an intracerebroventricular (icv) injection of β-amyloid 1-42 (Aβ-icv) displayed an hyperexcitability that was converted to a decreased pattern of synaptic plasticity upon onset of memory deficits at 15-16 days after Aβ-icv.At 7-8 days after Aβ-icv, and when compared to vehicle-icv injected mice (black open symbols/lines), Aβ-icv mice (filled green symbols/lines) displayed an hyperexcitability assessed by the shift to left of the input/output curve of the field excitatory post-synaptic potentials (fEPSP) slope (A,B) and a tendency towards an increased magnitude of long-term potentiation (LTP) triggered by a high-frequency stimulation train (HFS, 100 Hz for 1 s) in Aβ-icv mice (E,G).In contrast, in the symptomatic phase, there was no difference in the input/output curve between control and Aβ-icv mice (filled red symbols/lines) (C,D); however, LTP magnitude was lower in Aβ-icv mice (F,G).Data are mean ± SEM of 8 mice per group, the same as in Figure1; *** p < 0.001 between indicated groups using Student's t test with Welsh correction.

Biomolecules 2023 , 17 Figure 3 .
Figure 3. A2AR are upregulated from the presymptomatic phase of AD onward and control both hippocampal hyperexcitability in the presymptomatic phase and LTP deficits upon onset of memory deficits.(A) Binding of the A2AR antagonist 3 H-SCH58261 (2 nM) in vehicle-icv mice (v, open symbols and thin bars) and in Aβ-icv mice (Aβ, filled symbols and thick bars) in hippocampal synaptosomal or total membranes at 7-8 days (green) or 15-16 days (red) after icv injections.Data are mean ± SEM of 6 mice per group; * p < 0.05 and ** p < 0.01 between indicated groups using Student's t test with Welsh correction.(B) The addition of SCH58261 (50 nM) to hippocampal slices prevented the hyperexcitability in Aβ-icv mice and had no effect in vehicle-icv mice (veh) at 7-8 days, whereas SCH58261 was devoid of effects on hippocampal excitability in Aβ-icv and vehicleicv mice at 15-16 days (C).SCH58261 decreased LTP magnitude triggered by a high-frequency stimulation train (HFS, 100 Hz for 1 s) in vehicle-icv-treated mice and normalized LTP magnitude in Aβ-icv both in the presymptomatic (D,E) and symptomatic phase (F,G).The vehicle-icv and Aβicv groups in Figure 3 are the same as in Figure 2. Data in (B-G) are mean ± SEM of 8 mice per group; * p < 0.05 between indicated groups using Tukey's test after a two-way ANOVA.

Figure 3 .
Figure 3.A 2A R are upregulated from the presymptomatic phase of AD onward and control both hippocampal hyperexcitability in the presymptomatic phase and LTP deficits upon onset of memory deficits.(A) Binding of the A 2A R antagonist 3 H-SCH58261 (2 nM) in vehicle-icv mice (v, open symbols and thin bars) and in Aβ-icv mice (Aβ, filled symbols and thick bars) in hippocampal synaptosomal or total membranes at 7-8 days (green) or 15-16 days (red) after icv injections.Data are mean ± SEM of 6 mice per group; * p < 0.05 and ** p < 0.01 between indicated groups using Student's t test with Welsh correction.(B) The addition of SCH58261 (50 nM) to hippocampal slices prevented the hyperexcitability in Aβ-icv mice and had no effect in vehicle-icv mice (veh) at 7-8 days, whereas SCH58261 was devoid of effects on hippocampal excitability in Aβ-icv and vehicle-icv mice at 15-16 days (C).SCH58261 decreased LTP magnitude triggered by a high-frequency stimulation train (HFS, 100 Hz for 1 s) in vehicle-icv-treated mice and normalized LTP magnitude in Aβ-icv both in the presymptomatic (D,E) and symptomatic phase (F,G).The vehicle-icv and Aβ-icv groups in Figure 3 are the same as in Figure 2. Data in (B-G) are mean ± SEM of 8 mice per group; * p < 0.05 between indicated groups using Tukey's test after a two-way ANOVA.