Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by several cognitive and behavioral troubles attacking particularly memory functions. Most of AD patients develop detectable symptoms after 65 year-old (sporadic cases). However, in familial forms of the disease representing less than 1% of AD cases, severe cognitive and memory deficits develop earlier, due to the presence of mutated forms of the Amyloid Precursor Protein (APP) or Presenilin (PS-1 or PS-2) genes [1,2,3,4]. Transgenic mouse models of AD, that overexpress at least one of these human mutated genes, reproduce the amyloidopathy and some anatomical and behavioral abnormalities found in AD patients [5], and thereby help to provide insights into the mechanisms underlying memory deficits.

Recently, new hypothesis have emerged about the contribution of network hypersynchrony in memory dysfunction in AD. Multiple lines of evidence point out an increased incidence of spontaneous seizures in patients with AD. In sporadic AD, an estimated 10 to 22% of patients exhibit unprovoked seizures [6,7,8]. In familial forms of AD or diseases in which APP gene is duplicated, 30 to 60% of patients develop seizures [9,10,11]. In patients diagnosed for both epilepsy and AD (or amnestic Mild Cognitively Impairment, aMCI), seizure onset preceded or coincided with the diagnosis of the disease in 83% of patients, suggesting an early onset of epilepsy in AD [12]. Using fMRI during a task involving the hippocampus, Bakker and colleagues showed that aMCI subjects displayed hippocampal hyperactivity [13]. Interestingly, in the same study, the antiepileptic drug levetiracetam reduced hippocampal hyperactivity and improved memory performances, suggesting a causal link between network hyperactivity and memory deficits [13].

Similar to human pathology, several mouse models of AD present a combination of features revealing network hypersynchrony and epilepsy. Initially, sporadic spontaneous seizures or behavioral stereotypies were fortuitously observed during behavioral phenotyping of AD transgenic lines [14,15]. Pro-convulsant agents that exacerbate neuronal excitation (kainate) or suppress inhibitory control (GABA_A receptor antagonist pentylenetetrazole, PTZ) trigger more severe seizures in several AD transgenic mice (TgCRND8, hAPPJ20, hAPPJ9) than in their respective non-transgenic (NTg) littermates [16,17,18,19]. Although very infrequent, spontaneous seizures in AD mice have also been objectively evidenced by using electroencephalographic (EEG) recordings [17,20,21,22]. Typically, chronic EEG recordings over very long period of time are required for observing a significant occurrence of seizures. For instance, 25% of APP_swexPS1_dE9 mice present seizures when recorded for 2 weeks [20], and about 10% of hAPPJ20 mice display seizures when recorded for 24 hours [23]. However, the vast majority—if not all—of the recorded hAPPJ20 and APP_swexPS1_dE9 mice show interictal spikes consisting of frequent high-voltage sharp events [17,20].

Aside from electroencephalographic markers of neuronal network hypersynchrony, neuropeptide Y (NPY) expression pattern has been proposed as a marker for chronic seizures. NPY is an inhibitory neuromodulator normally expressed by a subset of hippocampal interneurons, which can be expressed ectopically in mossy fibers after chronic seizures in animal models of epilepsy [24]. Such ectopic expression of NPY has been reported in Tg2576 [25,26,27], hAPPJ20 [17,28], and APP_swexPS1_dE9 mice [20], arguing for the existence of epilepsy in these AD models. Interestingly, chronic administration of the antiepileptic drug levetiracetam suppresses epileptiform activity and improves memory performances in hAPPJ20 mice, but also normalizes the expression of NPY in the mossy fibers [28].

In AD mouse models, neuropathological processes and memory deficits progress with age. So far, the presence of network hypersynchrony has been assessed when mice were old enough to exhibit memory deficits. We thus do not know if this abnormal brain activity occurs before, concomitantly or after the onset of memory dysfunction in AD mouse models. Learning and memory deficits have been extensively described at different ages in Tg2576 mice, which express a human APP (hAPP) gene carrying the double Swedish mutation (HuAPP696swe) [29]. No memory disorder has been reported at 1.5–2 months [30,31,32], while a few studies have evidenced impairments at 3 months [30,33,34,35], and most of authors agree on clear memory deficits after 6 months [32,36,37,38,39]. Interestingly, network hypersynchrony has not been evaluated before 6 months of age in Tg2576 mice. We thus gauged network hypersynchrony of Tg2576 mice at different ages (1.5, 3 and 6 months old) by assessing their seizure susceptibility to PTZ, and evaluating their spontaneous epileptiform activity through EEG recordings and hippocampal expression pattern of NPY. We show that Tg2576 mice display aberrant network activity as early as 1.5 month-old, that is to say before the onset of memory deficits.

Methods

Results

Tg2576 mice show higher susceptibility to pharmacologically-induced seizures from early age on

First, we determined seizure susceptibility of Tg2576 mice and their NTg littermates at ages of 1.5, 3 and 6 months. Injection of the GABA_A receptor antagonist PTZ, at the dose of 40 mg/kg, induced more severe seizures in Tg2576 mice than in NTg littermates at 1.5 and 6 months of age (Fig. 1A, Kruskal-Wallis: p<0.0001, Dunn’s post hoc tests: p<0.05 for NTg vs Tg2576 at 1.5 and 6 month-old). At 3 months of age, the difference in seizure severity between NTg and Tg2576 mice did not reach statistical significance as NTg mice showed higher seizure susceptibility than at 1.5 months of age (Dunn’s post hoc test: p<0.01; Fig. 1A). Some Tg2576 animals showed lethal seizures, whilst this was never observed among NTg animals (Fig. 1B). In summary, as early as 1.5 months of age, Tg2576 mice exhibit a higher susceptibility to pharmacological seizures than NTg littermates.

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Fig 1. Tg2576 mice show high susceptibility to pharmacologically-induced seizures from early age on.

(A) Seizure severity score of 1.5, 3 and 6 month-old Tg2576 male mice and non-transgenic (NTg) age-matched littermates. Whiskers boxes represent the interquartile distribution. Number of mice in each group is indicated below the boxes. Tg2576 mice exhibit more severe seizures than NTg at 1.5 and 6 months of age (Dunn’s tests: p<0.05 for Tg2576 vs NTg at 1.5 and 6 month-old). (B) Proportion of animals that died during PTZ-induced seizures in 1.5, 3 and 6 month-old Tg2576 and NTg mice. Note that only transgenic animals exhibit lethal seizures. Numbers over the horizontal axis indicate the number of mice used in each experimental group.

https://doi.org/10.1371/journal.pone.0119910.g001

Tg2576 mice exhibit spontaneous epileptiform activity as young as 1.5 months of age

Then, we examined to which extent such susceptibility might be associated to spontaneous electroencephalographic (EEG) abnormalities in Tg2576 mice. We recorded cortical EEG in Tg2576 and NTg mice at 1.5, 3 and 6 months of age. During the recording session, we did not observe any electroencephalographic seizures in NTg nor in Tg2576 mice. Since spontaneous seizures are relatively rare events in other mouse models of Alzheimer’s disease [20, 23] and given our recording time window, this was highly expected. Nevertheless, interictal spikes were observed in most of Tg2576 mice (63% of mice, regardless of their age). These events lasted 21.2 ± 2.6 ms and displayed the characteristic shape of interictal spikes [20] (Fig. 2A). In NTg mice, only 1 mouse out of 26 displayed spikes at a very low frequency (0.01 spike/minute) (Fig. 2B). Spike frequency was significantly influenced by the genotype but not by the age of the animals (Fig. 2B, two-way ANOVA; transgene effect: p = 0.013; age effect: p = 0.4091; interaction: p = 0.3865). These results clearly show that spontaneous epileptiform activity is already present in Tg2576 mice as early as 1.5 months of age.

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Fig 2. Tg2576 mice exhibit spontaneous epileptiform activity as young as 1.5 months of age.

(A) Representative electroencephalographic (EEG) traces from non-transgenic (NTg) (top) and Tg2576 (bottom) mice from left and right parietal cortices. Note that only transgenic animals displayed sharp, high-voltage spikes that characterize epileptiform activity (inset). (B) Quantitative analysis of the frequency of interictal spikes (mean ± SEM). Two-way ANOVA shows a significant genotype effect (p = 0.013) but no age effect (p = 0.4091) and no interaction (p = 0.3865). Numbers over the horizontal axis indicate the number of mice used in each experimental group.

https://doi.org/10.1371/journal.pone.0119910.g002

Ectopic expression of NPY in the mossy fibers of young Tg2576 mice

To assess the occurrence of chronic seizures in Tg2576 mice, we looked for NPY ectopic expression in mossy fibers of the dentate gyrus by using NPY immunohistochemistry (Fig. 3). While never observed in the mossy fibers of NTg animals, NPY ectopic expression was found in a significant proportion of Tg2576 animals (Table 1, Chi square test for genotypes, regardless of the age: p = 0.0002). NPY ectopic expression was observed as young as 1.5 month of age. The proportion of mice showing NPY ectopic expression was significantly higher in Tg2576 than in NTg littermates at 3 and 6 months of age but not at 1.5 months of age (Table 1). Although not statistically significant in this sample, the proportion of Tg2576 mice showing NPY ectopic expression seemed to increase with age (Table 1, Chi square test for trend: p = 0.16). These results suggest that chronic seizures occur at very early stages in the course of the disease, and that their incidence likely increases with age among the Tg2576 population.

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Fig 3. Ectopic expression of NPY in the mossy fibers of young Tg2576 mice.

Photographs of the dorsal hippocampus immunostained for NPY in 1.5, 3 and 6 month-old non-transgenic (NTg) and Tg2576 mice. Left: In NTg animals, NPY staining is visible in the soma of hilar interneurons. Their axons display a faint staining visible in the molecular layer (ml), where these axons form synapses onto the dendrites of granular cells. Right: Typical ectopic NPY expression in the mossy fibers (in hilus and stratum lucidum) of a Tg2576 mouse. CA1 and 3, Cornu Ammonis 1 and 3, GCL: Granular Cell Layer, mf: mossy fibers, ml: molecular layer. Scale bar: 200 μm.

https://doi.org/10.1371/journal.pone.0119910.g003

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Table 1. Proportion of mice with NPY ectopic expression among Tg2576 mice and NTg littermates.

https://doi.org/10.1371/journal.pone.0119910.t001

Discussion

This study reports that at 1.5, 3 and 6 months of age, Tg2576 mice exhibit high susceptibility to pharmacologically-induced seizures, EEG epileptiform activity and NPY ectopic expression in mossy fibers, this later marker of chronic seizures showing an increased incidence with age. Our work provides the first evidence that such network dysfunction precedes the onset of memory deficits.

A few studies have previously assessed network hypersynchrony in the Tg2576 mouse line. To date, there is no data concerning the sensitivity of Tg2576 mice to convulsive agents, except a study showing an increased sensitivity to PTZ in Tg2576 mice bred on a pure C57bl/6 background, which exhibit an unusually high mortality rate (40% died before they reached 2 months of age) and thus cannot be considered as a typical Tg2576 line [41]. In the original Tg2576 line, EEG abnormalities were reported at 5–7 months of age but were not clearly described. The authors related longer durations of “higher frequency brain activity” (6 to 10 Hz), which they interpret as an increased synchrony, but did not report any obvious spike or seizure [27]. Thus, our data constitute the first clear demonstration of higher sensitivity to convulsive agents and occurrence of spontaneous interictal spikes in this mouse line. Ectopic expression of NPY in the mossy fibers has often been associated with epileptic activity in the hippocampus or with the occurrence of a generalized seizure in the days before sacrifice and can thus be used as a marker for chronic seizures [24,42]. Ectopic expression of NPY in the Tg2576 mice was evidenced at 5–7 months of age [27] and previous data from our group showed that the proportion of mice presenting this marker of chronic seizures increases with age (11% at 3 months, 60% at 18 months) [25]. Our present data confirm this observation and further point out that this marker can already be observed as soon as 1.5 months of age. Altogether, our data strongly suggest that Tg2576 mice present a precocious epileptic phenotype. Interestingly, previous work showed that Tg2576 exhibit normal memory performances at 1.5–2 months of age [30,31], memory deficits appearing in this mouse line between 3 and 6 months of age [30,31,32]. Thus, network hypersynchrony and expression of markers of chronic seizures occur before the onset of memory deficits in Tg2576 mice.

Network hypersynchrony and hyperexcitability have been described in other mouse models of AD. In the TgCRND8 line, seizure susceptibility to PTZ was evidenced at 6–8 weeks of age [16] when these mice already present memory deficits [43]. In APP_SwexPS1_dE9 mice, electroencephalographic seizures and epileptiform activity were found at 3–4 months of age [20], corresponding to the earliest description of memory deficits in this mouse line [44]. In hAPPJ20 mice, susceptibility to pharmacological seizures, spontaneous seizures, interictal spikes and NPY ectopic expression were described at 4–7 months of age [17,18,23,28], when mice already exhibit memory deficits from 2–3 months of age [45]. Thus, although it has never been assessed, network hypersynchrony might also happen before the onset of memory deficits in these models. Interestingly, this is also supported by data from human cases suggesting that epileptic events can precede the onset of memory impairments in AD and aMCI [12].

Several hypotheses can be proposed to explain the precocity of epileptic activity in Tg2576 mice. The Tg2576 mouse model expresses a mutated form of hAPP inducing an excessive production of Aβ1–42 peptide and its accumulation into amyloid plaques [46]. In the APP_swexPS1_G384A mice model, neurons located in the vicinity of amyloid plaques were reported to be hyperactive [47]. However, Tg2576 mice are completely devoid of amyloid plaques at 1.5 months of age [46], ruling out the possibility that plaques could be responsible for the early onset of network hyperactivity nor hypersynchrony in these mice. Nevertheless, the brains of new born Tg2576 mice, but not of NTg mice, already contain soluble Aβ1–42 [48]. At the cellular level, soluble Aβ1–42 species were suggested to play a role in neuronal hyperexcitability of AD mice. For instance, bath application of soluble Aβ decreases depolarization threshold and thus increases excitability of pyramidal cortical neurons or granule cells of the dentate gyrus [20,21] and intra-hippocampal injection of Aβ oligomers was found to increase population spikes evoked by perforant path stimulation in rats [49]. However, the molecular and cellular mechanisms underlying the effects of Aβ on neuronal hyperexcitability are still unclear. Recently, Lee et al. reported that Aβ disturb mitochondrial function in 1.5 month-old Tg2576 mice, leading to a slower decay of Ca²⁺ transients in granule cells of the dentate gyrus [50]. Further work is needed to determine if this increase in intracellular Ca²⁺ signal in granule cells could participate to network hypersynchrony in 1.5 month-old Tg2576 mice.

Interestingly, the role of Aβ in network hypersynchrony has been recently challenged. The APP_Swe/LonxPS1_M146V mouse line, which produces high amounts of Aβ, does not exhibit any epileptiform activity even as late as 23 months of age [22]. Born and colleagues proposed that mutant APP itself could be involved in network hypersynchrony. Indeed, APP is a transmembrane protein cleaved by secretases as the β-site APP cleaving enzyme 1 (BACE1). In transgenic models of AD that overexpress mutant APP, excessive levels of this full-length APP may hijack a significant portion of BACE1, thus reducing its ability to process other substrates such as sodium channels subunits. Indeed, BACE1 cleaves the Navβ2 subunit of Nav1.1 channels, which regulates the expression of the functional α-subunits of these channels [51] that control excitability of parvalbumin-expressing interneurons [52]. Reduced levels of Nav1.1 channels were reported in association with impaired function of interneurons leading to network hypersynchrony and memory deficits in hAPPJ20 [23] and 5- to 7-month-old Tg2576 mice [27]. To which extent high levels of mutant APP reduce Nav1.1 levels in parvalbumin-expressing interneurons in young Tg2576 mice remains to be determined.

Recent studies clearly suggest a role of network hypersynchrony in memory deficits. In aMCI subjects, the antiepileptic drug levetiracetam improves memory performances [13]. In hAPPJ20 mice, one month of chronic levetiracetam treatment suppresses epileptiform activity, normalizes hippocampal NPY expression and improves memory performances [28]. The beneficial effect of levetiracetam on memory performances in hAPPJ20 mice may result from the suppression of epileptiform activity or the normalization of NPY expression in the hippocampus or both. In a rat model of epilepsy and epileptic patients, hippocampal interictal spikes occurring during memory retrieval impair memory performances [53,54]. Here we report epileptiform activity in 1.5 month- old Tg2576 mice, an age when these mice present normal memory performances [30,31]. Thus, memory deficits may not result from hypersynchrony and epileptiform activity themselves, but rather from the consequences of their chronicity. Chronic hypersynchrony triggers seizures which incidence increases with age as reported in the APP_swexPS1_dE9 model of AD (15% of mice had seizures at 3 months vs 50% at 4 months of age) [20]. Consistent with these results, the present study and previous data from our laboratory [25] report an age-related increase in the incidence of NPY ectopic expression which is significant from NTg mice at 3 months and clearly rises around 6 months of age. This parallels the progression of memory deficits, which begin at 3 months, and progressively worsen with age [30,31,32]. Expression of NPY in granule cells decreases glutamatergic synaptic transmission [55,56]. If it prevents a spread of neuronal overexcitation in a context of chronic seizures, it could also impair hippocampal function required for learning and memory processes. Thus, early network hypersynchrony induces seizures which in turn trigger neuroadaptations in the hippocampus, including NPY ectopic expression, which might cause a progressive degradation of hippocampal function explaining the age-dependent decline in memory performances in AD mice [57].

Network hypersynchrony could also cause memory impairment by altering neurogenesis. Adult hippocampal neurogenesis, a process by which new granule cells of the dentate gyrus are generated throughout life, contributes to learning and memory [58,59]. In AD mice, altered neurogenesis seems to be an early event in the course of the disease [25,60]. We recently described an impairment of adult hippocampal neurogenesis at 3 months of age in Tg2576 mice [25]. At this specific age, adult-generated neurons of Tg2576 mice exhibit impaired neuronal maturation and reduced dendritic spine density and dendritic length. Similar observations were made in hAPPJ20 mice [61]. In Tg2576 mice and hAPPJ20, altered neurogenesis has been described at the onset of memory deficits at 3 months of age, but it cannot be excluded that such alterations may begin earlier. In hAPPJ20 mice, Sun and colleagues showed that the excitation/inhibition imbalance contributes to adult neurogenesis impairments [61]. Whether alteration of hippocampal neurogenesis is present before memory deficits and whether it relates to hypersynchrony in Tg2576 mice remains to be established.

Conclusion

Here we evidence network hypersynchrony before the onset of memory deficits in Tg2576 mice and an age-related increase of the incidence of ectopic NPY in the mossy fibers of Tg2576 mice revealing an increasing frequency of seizures with age. This early network dysfunction could initiate progressive modifications of hippocampal network leading in fine to overt memory dysfunction. In human, network hypersynchrony would thus potentially represent an early diagnosis marker to predict memory decline. However, extrapolations of these findings to sporadic forms of the disease still remain to be investigated.

Acknowledgments

We thank A. Krezymon and E. DiDonato for their help and technical support. We also thank the ABC facility and ANEXPLO for housing mice.