Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy

Abstract

Alzheimer's disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid beta (Abeta) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Abeta1-42 or Abeta25-35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Abeta (proto-)fibrils. Overall, these data identify fibrillar Abeta as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

Figure 1.

Epileptiform activity in APdE9 mice. A, Example of interictal spiking in an APdE9 mouse (18). Time bar, 0.5 s; voltage bar, 0.5 mV. B, Examples of two spontaneous seizures in APdE9 mice. The first one (mouse 021) lasted for 35 s and was scored as 2 in the Racine scale based on behavioral observations, whereas the second one (mouse 251) lasted for 30 s and was scored as 3. The two EEG skull electrodes were located on the right (CxR) and left (CxL) frontal cortex. Both seizures began with a large-amplitude spike (arrowheads). The seizure in mouse 021 took place during waking immobility, whereas that in mouse 251 occurred during REM sleep. Both seizures were followed by regular low-amplitude oscillation of ∼7 Hz. Time bar, 1 s; voltage bar, 1 mV.

Figure 2.

Prevalence and characteristics of seizures in APdE9 mice. A, The cumulative proportion (percentage) of APdE9 mice (n = 20) remaining seizure free during the video-EEG monitoring as a function of their true biological age. The gap between 15 and 18 weeks of age indicates the interval between the first (2 weeks) and the second (1 week) recording periods. WT, Wild type. B, Prevalence and characteristics of seizures in APdE9 mice (n = 20). In this table, the video-EEG recording has been divided into three periods (Per) of 1 week each. Mean dur., Mean seizure duration; Mean beh. score, mean behavioral score in Racine's scale; Gen. sz. (%), percentage of generalized seizures of all seizures. The two rightmost columns represent alterations in hippocampal calbindin (CB) and NPY staining. For CB staining, + means presence of normal immunoreactivity; for NPY staining, + means presence of ectopic immunoreactivity in mossy fibers. N/A, Data not available [we chose 7 APdE9 mice with at least one unprovoked seizure and 7 APdE9 mice with no detectable seizures for immunostaining together with 7 wild-type mice (not included in the table)].

Figure 3.

Calbindin-D28k and NPY in the dentate gyrus of APdE9 mice. A, D, Wild-type mouse. B, E, APdE9 mouse with no seizures. C, F, APdE9 mouse (021) with multiple seizures. D–F are zoomed in from the rectangle shown in A–C. Note a dramatic decrease in calbindin (CB) staining in the dentate GCs of the APdE9 mouse with seizures. This corresponded with the decrease in calbindin immunoreactivity in the molecular layer. NPY immunostaining of the septal hippocampus revealed overexpression in mossy fibers in some APdE9 but in none of the wild-type animals (I; open arrow). G, Wild-type mouse. H, APdE9 mouse with no seizures (no sz). I, APdE9 mouse (251) with multiple seizures (sz). wt, Wild type; g, GC layer; m, molecular layer. Scale bars: A–C, G–I, 250 μm; D–F, 50 μm.

Figure 4.

Plaque distribution in telencephalic areas of APdE9 mice. Anti-human Aβ staining (W0-2) in APdE9 mice at the end of the study (age, 5 months) revealed a substantial number of amyloid plaques in the cortex, hippocampus, and amygdala, but hardly any plaques in the thalamus. A–D, The coronal sections of an APdE9 mouse are at the levels of motor cortex (A), amygdala and perirhinal cortex (B), septal hippocampus (C), and dorsal thalamus just below the septal hippocampus (D). E, A corresponding hippocampal section of a wild-type mouse showing even no intracellular staining for human Aβ. Scale bars, 500 μm. F, Amyloid burden expressed as immunoreactive surface area in the percentage of the total area of analyzed sections. Student's t test did not reveal significant differences in plaque load between APdE9 mice with or without seizures for motor, perirhinal, and entorhinal cortices (p = 0.34), hippocampus (p = 0.34), and amygdala (p = 0.08). *In the thalamus, more APdE9 mice without seizures had plaques than mice with seizures (p = 0.01, Mann–Whitney U test). Cx, Perirhinal and entorhinal cortices; Hc, hippocampus; Am, amygdale; Th, thalamus.

Figure 5.

Increased neuronal excitability in APdE9 mice. A, Resting membrane potential (V_R) of L2/3 pyramidal cells in APdE9 and wild-type mice at the age of 6 months. B, C, Incrementing current injection in the soma of L2/3 pyramids leads to premature action potential (AP) firing in APdE9 mice at subthreshold current steps compared with neurons from wild-type mice as early as 3.5 months of age. D, Representative recording traces. E, Schematic illustration of the L1 afferent stimulation paradigm. The inset shows a representative experimental setting. The bar graph indicates the relative distance of stimulating pipettes along the apical tuft of pyramidal cells from their somata. F, Representative recordings with incrementing extracellular stimulus strengths at 3.5 months of age. G, Cumulative analysis of neuronal excitability in response to afferent stimulation in APdE9 (n = 8) and wild-type (n = 6) slices. *p < 0.05, **p < 0.01 versus wild type.

Figure 6.

Fibrillar Aβ affects neuronal excitability. A, Transmission electron microscopy (EM) images of Aβ1–42 and Aβ25–35 fibrils dissolved in water (fibrillar) or in DMSO, a chaotic solvent. Note that DMSO pretreatment eliminated preformed Aβ (proto-)fibrils in both cases and generated small oligomers for Aβ1–42 (arrowheads) instead. DMSO-pretreated Aβ25–35 could not be observed by transmission electron microscopy because of the relatively low resolution of the applied protein staining technique, which made the Aβ25–35 monomers/low-molecular-weight oligomers indetectable (Shemer et al., 2006). B, C, Fibrillar but not oligomeric Aβ1–42 depolarizes both L2/3 pyramidal cells (B) and GCs in the dentate gyrus (DG; C). D, D′, Similarly, (proto-)fibrillar but not monomers/low-molecular-weight oligomers of Aβ25–35 lower the membrane potential threshold for action potential firing in L2/3 pyramidal cells. The arrow points to the steeper slope of the passive membrane charge in the presence of (proto-)fibrillar Aβ25–35 in V_ramp recordings. E, Membrane depolarization of 10 mV results in a significant change in pyramidal cell excitability in APdE9 cells. A L2/3 pyramidal cell was held at −80 mV [corresponding to membrane potential in wild-type mice (B)] or −70 mV [representative of Aβ1–42 effects (B) in APdE9 mice] while stimulating afferent fibers in L1. Note that a 10 mV depolarization converted polysynaptic EPSPs into reliable action potential firing. F, F′, Fibrillar Aβ1–42 increases network excitability in vitro. The field potential (neuronal population activity) recorded in L2/3 in response of L1 stimulation (the arrow indicates remnants of stimulation artifact after subtraction) is larger after acute bath application of fibrillar Aβ1–42 than in control. Both traces are the average of 10 sweeps. *p < 0.05, **p < 0.01 versus control.