Aβ_1-42 (A1163-2) and scrambled Aβ_1-42 (A-1004-2) were purchased from rPeptide and monomerized as described previously (Broersen et al., 2011). First, 0.5 mL of hexafluoro-2-propanol (HFIP) was added to the vials containing the peptide. After vortexing for 30 s, HFIP was evaporated under a gentle stream of nitrogen gas. The DMSO-solubilized peptides were applied to a HiTrap desalting column (17-1,408-01, GE Healthcare) that was previously equilibrated with phosphate buffered saline (PBS) with 1 mM ethylenediaminetetraacetic acid (EDTA) and eluted with the same buffer to obtain a DMSO-free peptide solution. The Aβ_1-42 or scrambled Aβ_1-42 in PBS with 1 mM EDTA was stored in pre-cooled LoBind tubes (0030108442, Eppendorf) on ice and the peptide concentration was calculated via ultraviolet absorption at 280 nm with a Nanodrop (Nanodrop 2000, ThermoScientific). The peptide was then aliquoted in LoBind tubes, snap frozen in liquid nitrogen, and stored at −80°C until injection. The monomers were injected within 1 week after storage at −80°C.

The thioflavine T (ThT) assay was used to measure β-sheet formation over time with a fluorescence read-out. Different concentrations (0, 10, 20, 100 μM) of Aβ_1-42 and scrambled Aβ_1-42 were added to non-binding 96-well microplates (655,906, Greiner) with 12 μM of ThT in PBS with 1 mM EDTA. Fluorescence was measured at 5 min-intervals for 8 h on a plate reader (Victor 31,420 Multilabel Counter, Perkin Elmer) at 21°C using excitation and emission wavelengths of 440 nm and 480 nm, respectively, with an automated protocol.

Aβ_1-42 was allowed to aggregate for different time intervals. A 4 μL aliquot of 100 μM Aβ_1-42 in PBS 1 mM EDTA was absorbed on 150-mesh formvar coated cupper grids for 1 min before it was blotted. The grid was washed twice with milli-Q water, after which it was stained for 30 s with uranyl acetate 2% in veronal buffer. The grids were then washed four times with milli-Q water, after which they were allowed to dry and stored until imaging. Imaging was done with a Tecnai 10 Philips transmission electron microscope (TEM) at an operating voltage of 80 kV. Images were acquired with a mega viewG2 CCD camera (SIS-company) and visualized with iTEM software.

Six-week-old male NMRI mice were bought from Charles River (France). Mice were habituated for 1 week to our facility before starting the experiment and were group housed (4–5 mice per cage) for the entire experiment. The experimental procedures were approved by the ethical committee of the Vrije Universiteit Brussel (19-213-10 and 22-213-4). Both the ARRIVE guidelines and the Basel declaration were considered when designing the experiments.

General anesthesia was induced with 4% isoflurane in an induction chamber for 2 min. Mice were then fixed on a stereotaxic frame and the isoflurane concentration was reduced to 1–2% for the rest of the procedure. 5 mg/kg meloxicam (Metacam, Boehringer Ingelheim) and 1 mL NaCl 0.9% were administered subcutaneously. A 2 cm scalp incision was made, after which we verified skull flatness by ensuring that the dorsoventral (DV) deviation between bregma and +/− 1.00 mediolateral (ML) and lambda was less than 0.1 mm. We then drilled holes at −2.20 anteroposterior (AP) and +/− 1.40 ML for DG injection or at −0.34 AP and + 1.00 ML for the i.c.v. injection. The dura mater was punctured with a 29G needle and the microsyringe (700/1700 series 65,460–05, Hamilton) was slowly advanced through the drill holes to −2.10 DV for DG injection and − 2.70 DV for i.c.v. injection. The 100 μM Aβ_1-42 or 100 μM scrambled Aβ_1-42 in PBS with 1 mM EDTA was previously allowed to oligomerize for 1.5–2 h at room temperature. Mice in which we induced a seizure 1 week after Aβ_1-42 injection received a 1 μL (0.2 μL/min) injection in both DG. A 2 μL injection (0.4 μL/min) was performed in both DG of mice in which we evoked a seizure 1.5 h after Aβ_1-42 injection. Mice that were subjected to a seizure 3 weeks after the injection of Aβ_1-42 received a unilateral i.c.v. injection of 10 μL (2 μL/min). After injection, the microsyringe was left in place for 5 min, after which it was slowly taken out and the skin sutured. Finally, mice were allowed to recover in a heated recovery chamber until they regained full mobility. The duration of anesthesia ranged between 30 and 40 min.

Seizures were evoked by a continuous i.v. infusion (150 μL/min) of a chemoconvulsant in the lateral tail vein, as we previously published (Schallier et al., 2009; Portelli et al., 2012). We diluted 7.5 mg/mL KA, 7.5 mg/mL PTZ, or 4 mg/mL 4AP in 0.9% NaCl with 10 IU/mL heparin. Mice were put in a restrainer and the tail was briefly warmed in water at 37°C, after which the lateral tail vein was punctured with a 30G needle. The infusion pump was started to ensure correct i.v. delivery of the chemoconvulsant and the needle was fixed with tape to the tail. PTZ infusion resulted in (1) myoclonic twitch, (2) Straub tail, (3) forelimb clonus, (4) falling, (5) THE, and (6) death. KA-induced seizures resulted in the following phenotype: (1) behavioral arrest, (2) falling, (3) tonic hindlimb extension (THE), and (4) death. 4AP infusion resulted in (1) eye blinking, (2) jumping, (3) THE, and (4) death. Mice were then taken out of the restrainer into a transparent cage to assess the different seizure stages. Unsuccessful i.v. delivery of the infusion resulted in whitening of the tail and absence of seizure stages. These mice were discarded from the analysis. Mice were videotaped and the occurrence of seizures stages was evaluated by experimenters blinded to the test groups. The chemoconvulsant dose was calculated as follows: i n f u s i o n t i m e s × i n f u s i o n s p e e d m l s d o s e m g k g = × c h e m o c o n v u l s a n t m g m l b o d y w e i g h t k g

The statistical analysis was performed with Rstudio (2022.02.0). The results of i.v. seizure models were assessed with linear mixed-effects models in which the seizure stages and the injected peptide were fixed effects and the sequential stages within a mouse a random effect: dose ∼ seizure stage * injected peptide + (1|id). Normality of data was assessed with a qqplot, variance with a fitted plot. The evolution of the seizure stages was not linear over time, so we analyzed the different seizure stages as an ordinal categoric value, not as a numeric one. If a significant effect between groups or an interaction between seizure stage and group was present, multiple comparisons were assessed with Tukey’s test. When there was no interaction between seizure stage and the injected peptide, we simplified the model to: dose ∼ seizure stage + injected peptide + (1|id). Data were reported as mean ± standard deviation. α was set at 0.05.

We verified the successful solubilization and monomerization of Aβ_1-42 by characterizing the aggregation kinetics at 21°C with a ThT assay and TEM. The presence of a lag phase in the lower peptide concentrations (10 and 20 μM Aβ) in the ThT curve confirmed the monomeric state of our starting material, while the increase in ThT fluorescence indicates the formation of β-sheets, the main secondary structure of Aβ_1-42 oligomers and fibrils. ThT fluorescence increased gradually in a concentration-dependent manner in Aβ_1-42 over the 8 h of measurement (Figure 1A) (n = 3 technical replicates). No increase in fluorescence was noted in any of the concentrations of scrambled Aβ_1-42 (n = 3 technical replicates; Figure 1A). In addition to this biophysical measurement, we also visualized Aβ_1-42 aggregation with TEM. We confirmed that the 100 μM Aβ_1-42 solution oligomerized over time (Figures 1B,C). A TEM with PBS served as negative control (Figure 1D). These biophysical measurements confirm that we can replicate the standardized solubilization and aggregation of Aβ_1-42 that was previously described (Kuperstein et al., 2010; Broersen et al., 2011). To assess the biologic effect of Aβ_1-42 oligomers, we examined the intrinsic excitability of granular cells of the dentate gyrus in acute hippocampal slices from 6-week-old mice. After whole-cell patch-clamping these neurons, we looked at neuronal excitability before and after incubation of the slices in 500 nM Aβ_1-42 oligomers for 20 min (Supplementary Figure S1). As expected, increasing the injected current augmented the number of fired action potentials (F_(1,207) = 325, p < 0.001). Compared to baseline, the number of action potentials elicited by a 70 pA current injection was higher after incubation of the slices in Aβ_1-42 oligomers (F_(1,207) = 17, p < 0.001). There was a significant interaction between the injected current and the baseline or Aβ_1-42 oligomer condition (F_(1,207) = 6, p = 0.1). In addition to the in vitro characterization, this demonstrates a biologic effect of our Aβ_1-42 oligomers on neuronal excitability in acute hippocampal slices. Lastly, we validated successful Aβ_1-42 injection and correct location of injection in the DG with an anti-Aβ_1-42 DAB staining (Supplementary Figure S2).

We first investigated seizure susceptibility 1 week after the injection of Aβ_1-42 oligomers. As chemoconvulsant we used PTZ, a GABA_A-antagonist similar to bicuculline used by Bellugacci and colleagues. The injection of Aβ_1-42 was performed in the DG, the gateway to the large hippocampal neuronal pathways. In addition to scrambled Aβ_1-42 as our preferred control, we also included a control group that was injected with vehicle. This allows us to discard the possibility that any injected peptide would have impacted seizure susceptibility and not Aβ_1-42 or scrambled Aβ_1-42 specifically. The continuous i.v. PTZ infusion resulted in progressively worsening seizures (F_(5,141) = 62, p < 0.001) (Figure 2). Injecting Aβ_1-42, scrambled Aβ_1-42, or vehicle did not impact the required dose to reach any of the seizure stages (F_(2,29) = 1, p = 0.4). To confirm that the i.v. tail infusion of PTZ can detect the effect of an intracerebral injection of a proconvulsant, we set up a very similar experiment in a small group of mice (n = 6 per group). In these mice, we replaced the injection of Aβ_1-42 oligomers by a single unilateral injection of KA, a well-known chemoconvulsant, or vehicle in the DG. One week later, we performed an i.v. infusion of PTZ (Supplementary Figure S3). The infusion of PTZ resulted in progressively worsening seizures (F_(5,46) = 53, p < 0.001). There was no difference between groups in the dose required to reach seizure stage 1 (F_(1,10) = 4, p = 0.09), but there was a significant interaction between seizure stage and group (F_(5,46) = 4, p = 0.005). Post hoc pairwise comparison demonstrated that the group that received a KA injection reached stage 5 (THE) and 6 (death) more rapidly compared to the injection of vehicle (p = 0.01 and p = 0.006 respectively).

We then assessed the effect of a bilateral Aβ_1-42 oligomer injection in the DG on seizure susceptibility 1.5 h after injection. We chose this timeframe to keep the interval after injection as short as possible, still allowing mice to fully recover motor activity after anesthesia. As the seizure was evoked shortly after the Aβ_1-42 injection, we chose KA as chemoconvulsant since KA elicits seizures originating in the hippocampus where Aβ_1-42 was injected. The continuous infusion of KA resulted in gradually worsening seizures with focal temporal onset, progressing to bilateral motor seizures eventually resulting in death (F_(60,173) = 1, p < 0.001) (Figure 3). There was no difference between Aβ_1-42 or scrambled Aβ_1-42 in the dose required to attain the different seizure stages (F_(1,19) = 1, p = 0.4).

Finally, we attempted to replicate the experiments of Alcantara-Gonzalez and colleagues. Three weeks after the injection of Aβ_1-42 or scrambled Aβ_1-42, we submitted the mice to an infusion of 4AP, a K⁺ channel blocker, as did Alcantara-Gonzalez and colleagues. The infusion resulted in progressively worsening seizures over time (F_(3,53) = 391, p < 0.001) (Figure 4). However, the dose of 4AP required to reach the seizure stages was not different between the mice injected with Aβ_1-42 or scrambled Aβ_1-42 (F_(1,18) = 3, p = 0.1).

The research interest in Aβ originally stems from the fact that it is the main constituent of Aβ plaques in AD patients. The focus on Aβ oligomers exploded after they were found to be synapto- and neurotoxic and that they correlated better with cognitive decline in mouse models than other factors in the Aβ cascade (Ferreira et al., 2015). Much of the scientific attention was drawn to Aβ_1-42 as it is increased in people with autosomal dominantly inherited AD, aggregates rapidly due to its hydrophobicity, and reduces long term potentiation and synaptic density (Walsh et al., 2002; Shankar et al., 2008).

The effect of Aβ_1-42 oligomers on neuronal activity has been extensively studied in vitro, with conclusions that are not completely congruent. In hippocampal slices, Aβ_1-42 oligomers increased paired pulse ratios of population spikes in the DG, and increased the amplitude of population spikes in the presence of bicuculline in the DG (Costa et al., 2016). Also we showed that incubation of acute hippocampal slices in a solution with synthetic Aβ_1-42 oligomers increased the neuronal excitability of granule cells of the DG. In pyramidal neurons of slices of the anterior cingulate cortex, Aβ_1-42 oligomers increased the frequency of action potentials induced by current injections and decreased the frequency and amplitude of miniature inhibitory post-synaptic potentials (Ren et al., 2018). Intracellular infusion of Aβ_1-42 oligomers, but not monomers, increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-regulated excitatory post-synaptic currents within minutes (Whitcomb et al., 2015). The intracellular injection of Aβ_1-42 oligomers increased miniature excitatory postsynaptic currents in primary neuronal cultures and increased the number of action potentials in cornu ammonis 1 neurons in vivo in anesthetized mice (Fernandez-Perez et al., 2021). However, another study showed that Aβ_1-42 oligomers reduced neuronal activity in vitro in cultures measured with multiple electrode array (Kuperstein et al., 2010). In that same study Aβ_1-40 on the other hand increased neuronal activity.

Besides Aβ_1-42 oligomers, Aβ_1-40 oligomers thus also received much attention. Aβ_1-40 oligomers increased neuronal activity assessed with electrophysiology and calcium imaging in hippocampal cultures (Cuevas et al., 2011). Applying 500 nM Aβ_1-40 dimers with a S26C mutation to the cornu ammonis 1, increased neuronal calcium transients within 15 min in vivo (Busche et al., 2012; Zott et al., 2019). The authors argued that the increase in neuronal activity required baseline activity, as in slices this effect was only present after increasing basal neuronal activity with bicuculline, glutamate, or increased K⁺ concentration. This again stresses that in vitro findings on neuronal activity cannot per se be translated in vivo. Moreover, Aβ_1-40 application also resulted in reduced neuronal activity in neuronal cultures in a study (Sepúlveda et al., 2009). Some studies have even questioned the effect of Aβ in general on epileptiform activity in vivo. Johnson and colleagues assessed the effect of early and prolonged reduction of Aβ on epileptiform activity on EEG in vivo (Johnson et al., 2020). Reduction of Aβ (including Aβ oligomers) with a BACE inhibitor did not change epileptiform activity on EEG. The impact of Aβ oligomers on neuronal network excitability is thus still unclear.

What are the proposed mechanisms by which Aβ_1-42 would lead to increased seizure susceptibility? The very rapid response in vitro would suggest a fast receptor-dependent mechanism. There is much evidence for a direct interaction of Aβ_1-42 with N-methyl-D-aspartate, AMPA and GABA receptors (Ferreira et al., 2015; Palop and Mucke, 2016; Fernandez-Perez et al., 2021). Another frequently postulated rapid mechanism is that its hydrophobic properties allow Aβ_1-42 to form cationic channels in neuronal membranes. Only Aβ_1-42 oligomers, not Aβ_1-42 monomers or Aβ_1-40 oligomers, did so in the membrane of HEK293 cells (Bode et al., 2017). As Aβ_1-40 also alters neuronal activity, it is improbable that the Aβ pores are the cause of the bulk of the biological mechanism. Aβ_1-42 was proposed to exert its effect by increasing the release of glutamate by astrocytes in culture and reducing the inhibitory tone in slices and in mice (Ulrich, 2015; Sanz-Blasco et al., 2016; Calvo-Flores Guzmán et al., 2020). Pinpointing the exact mechanism is very difficult, as alterations in excitatory tone will immediately result in compensatory changes in the inhibitory tone and vice versa. As these compensatory mechanisms occur very rapidly, the chicken or egg question is very complex to answer experimentally.

Both Aβ_1-42 injections in the DG and i.c.v. have been frequently used as experimental models and were shown to be neurotoxic and result in cognitive impairments in rodents (Chambon et al., 2011). Aβ_1-42 oligomers (but not fibrils) penetrate from the cerebrospinal fluid into the brain parenchyma via the ventricular wall within 5 min (Kasza et al., 2017). Although we attempt to make our procedures minimally invasive, the ventricular wall will be damaged from the i.c.v. injection, further facilitating the entry of Aβ_1-42 oligomers into the brain parenchyma. It is unclear if the effect of an injection of Aβ_1-42 oligomers into the DG results from a local effect in the DG or if Aβ_1-42 oligomers need to diffuse. After a single injection of Aβ_1-42 oligomers in the DG, Aβ_1-42 is no longer detectable at the DG after a few days (Brouillette et al., 2012). Still, several studies found cognitive impairments in mice several weeks after a single injection of Aβ_1-42 (Chambon et al., 2011; Karthick et al., 2018). It is possible that Aβ_1-42 oligomers resulted in structural damage (although Brouillette and colleagues did not detect neuronal death) or that it initiated downstream events that exert their effect later on, independent of Aβ_1-42. Aβ_1-42 oligomers would be the match that ignites the fire that can then rage out of control afterwards. An interesting approach that could unravel the changes induced by Aβ_1-42 oligomers in these local injection models would be to perform “-omics” on punch biopsies of the DG region at different intervals post injection.

The next question is how much Aβ_1-42 one should inject to create these models. It is important to first consider the origin of the injected Aβ_1-42. Chemically synthesized Aβ_1-42 requires a much higher concentration to exert a similar biological effect compared to human-derived Aβ_1-42 from brain samples of patients with AD (Varshavskaya et al., 2022). These varying biological effects probably result from differences in aggregation dynamics. Indeed, subtle changes in medium may affect, not only the speed of aggregation, but even guide Aβ_1-42 to a very different aggregation end product (Brody et al., 2017; McAllister et al., 2020). Even within patient-derived Aβ preparations there are big differences. For example, Aβ_1-42 from brain homogenates are potent seeds for Aβ plaques, whereas Aβ_1-42 from human cerebrospinal fluid has no seeding capacity (Fritschi et al., 2014). We used a very similar methodology as Brouillette and colleagues to generate our Aβ_1-42 with the same Aβ_1-42 manufacturer, an identical monomerization process and aggregation time, and equivalent amount of moles of injected Aβ_1-42 (Brouillette et al., 2012). Their Aβ_1-42 preparation induced memory deficits and neurotoxicity. We validated the aggregation state of our Aβ_1-42 oligomers with two performant techniques of which the results were perfectly in line with previously published methodologic papers and ensured that we injected the Aβ_1-42 oligomers at the correct location (Broersen et al., 2011). Validating the biophysical properties of the used Aβ_1-42 preparations is crucial, because of the well-known complex behavior of this sticky and aggregating peptide that is sensitive to the smallest changes. In addition, we demonstrated that the Aβ_1-42 oligomers have a biologic effect on neuronal excitability of granular cells of the dentate gyrus in acute slice electrophysiology.

Knowing whether the amount of Aβ_1-42 oligomers we injected is similar to the concentration in AD patients is impossible. One would have to know the concentration of Aβ_1-42 intracellularly and in the interstitial fluid of patients, consider regional differences of Aβ concentration in the brain, have a precise knowledge of the fraction of each aggregation form, know the diffusion dynamics and half-life of Aβ_1-42 oligomers, and know which Aβ_1-42 oligomer pools are relevant to its (patho)biologic effect (Cirrito et al., 2003; Koffie et al., 2009; Keskin et al., 2017). The dose we used for i.c.v. injection was identical to that of Alcantara-Gonzalez and colleagues and so was the dose we injected in the DG for the mice subjected to a seizure 1.5 h later. For the mice that were subjected to a seizure 1 week after Aβ_1-42 injection in the DG, we used the dose of Brouillette and colleagues, which is only half the dose of Bellingacci and colleagues (see Supplementary Table S1 for an overview) (Brouillette et al., 2012; Bellingacci et al., 2022). It is unclear if this two-fold difference is relevant in a context where doses of Aβ_1-42 oligomers of different orders of magnitude all showed an effect (Berry et al., 2018). Compared to Bellingacci and colleagues, we also reduced the stress that the injection causes on the DG brain tissue by splitting our injection over both hemispheres.

Acute seizures elicited by chemoconvulsants (frequently PTZ and KA) successfully demonstrated increased susceptibility in genetic AD mouse models (Jolas et al., 2002; Del Vecchio et al., 2004; Palop et al., 2007). KA is an agonist of glutamatergic KA-receptors. We chose this chemoconvulsant to induce seizures rapidly after injection into the DG because KA results in seizures with hippocampal onset (Coppola and Moshé, 2012). As for both experiments with delayed seizures, in which the Aβ_1-42 already had the occasion to diffuse, we used PTZ and 4AP that model generalized seizures as did Alcantara-Gonzalez and colleagues and Bellingacci and colleagues. The experiment is based on the “two-hit” model of seizures in which a first epileptogenic substance is injected into the brain, followed by a different method to elicit the seizure. This was first described with an injection of KA into the hippocampus, followed by amygdala kindling. The group of rats that received a KA injection first, developed seizures much quicker (Feldblum and Ackermann, 1987). This two-hit phenomenon is not limited to classic chemoconvulsants. Increasing or decreasing the concentration in the hippocampus of molecules that affect general excitability can also affect seizure susceptibility in this two-hit paradigm (Vezzani et al., 1999; Kelley et al., 2018).

The two above mentioned studies that assessed the effect of intracerebral Aβ_1-42 injection on seizure susceptibility used vehicle as control (Alcantara-Gonzalez et al., 2019; Bellingacci et al., 2022). Since various peptides alter seizure susceptibility, we used both a vehicle and a scrambled Aβ_1-42 control group to rule out this possibility (Clynen et al., 2014; Manavi, 2022). Our data suggest that the effects they found would not have been induced by any peptide, as our scrambled Aβ_1-42 and vehicle groups did not differ in seizure susceptibility. However, even more crucially, their Aβ_1-42 solution contained 2% DMSO, which was not present in any of their vehicle groups. As DMSO impacts seizure susceptibility in vivo, this seems a potentially crucial difference (Bauwens et al., 2005; Kovács et al., 2011).

We used a different species than Alcantara-Gonzalez and colleagues, and a different mouse strain than Bellingacci and colleagues, who used outbred Wistar rats and inbred C57/Bl6 mice, respectively. We deliberately opted for outbred mice, as their genetic heterogeneity better represents a real-world situation. It is well-known that genetic background affects susceptibility to seizures, both in wildtype mice and AD mouse models (Jackson et al., 2015; Leclercq and Kaminski, 2015). The genetic background of AD mouse models also affects their Aβ dynamics (Jackson et al., 2015). However, it is difficult to attribute the contrasting conclusions between our studies only by the different species and strain we used since both a study on inbred mice and a study on outbred rats detected an increased seizure susceptibility after Aβ_1-42 oligomer injection.

The major limitation of our study is inherent to the fact that we were not able to demonstrate any difference between the Aβ_1-42 and scrambled Aβ_1-42 groups in vivo. However, we showed that the i.v. PTZ tail infusion seizure model is able to detect the effect of a single intracerebral injection of the established proconvulsant KA under similar experimental conditions. In addition, we showed that a 20 min incubation of acute hippocampal slices in 500 nM Aβ_1-42 oligomers increased the number of action potentials fired by patched granular cells of the DG. This way, we confirmed the sensitivity of the seizure model in vivo and a biological action of our Aβ_1-42 oligomer preparation ex vivo in slices. A post hoc power calculation of the Aβ_1-42 experiments only has limited value. When comparing our sample sizes to the two papers that demonstrated an effect of Aβ_1-42 oligomer injection on seizure susceptibility, we have more data points per group than Alcantara-Gonzalez and colleagues, but less than Bellingacci and colleagues.

As discussed throughout this paper, the effect of Aβ varies greatly in literature and probably depends on multiple factors that are not yet considered or cannot be controlled for. Publishing these results that contradict earlier work is key, especially in the field of Aβ_1-42 in which conclusions diverge much between studies. This again stresses the importance of thorough biophysical characterization of the used Aβ_1-42 preparation, to allow comparison between studies. Only the publication of experiments in which significant effects are found, and those in which they aren’t, can provide readers with the most complete representation of scientific findings. Here we can only deduce that earlier seizure threshold lowering effects of Aβ_1-42 cannot be generalized to all seizure models and experimental conditions.

In conclusion, we did not find an increased seizure susceptibility in mice injected with Aβ_1-42. This contrasts with two other published reports. Our biophysical validation of Aβ_1-42 oligomers was however state-of-the-art, we demonstrated its effects on ex vivo neuronal excitability and showed successful in vivo intracerebral Aβ_1-42 injection in mice.