Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease

Abstract

Neural network dysfunction may play an important role in Alzheimer's disease (AD). Neuronal circuits vulnerable to AD are also affected in human amyloid precursor protein (hAPP) transgenic mice. hAPP mice with high levels of amyloid-beta peptides in the brain develop AD-like abnormalities, including cognitive deficits and depletions of calcium-related proteins in the dentate gyrus, a region critically involved in learning and memory. Here, we report that hAPP mice have spontaneous nonconvulsive seizure activity in cortical and hippocampal networks, which is associated with GABAergic sprouting, enhanced synaptic inhibition, and synaptic plasticity deficits in the dentate gyrus. Many Abeta-induced neuronal alterations could be simulated in nontransgenic mice by excitotoxin challenge and prevented in hAPP mice by blocking overexcitation. Aberrant increases in network excitability and compensatory inhibitory mechanisms in the hippocampus may contribute to Abeta-induced neurological deficits in hAPP mice and, possibly, also in humans with AD.

Figure 1.. Remodeling of inhibitory circuits and alterations in the expression of NPY and its receptors in the dentate gyrus of hAPP-J20 mice.

(A) Brain sections were immunoperoxidase-stained for NPY. Compared with NTG controls, hAPP-J20 mice had an increase in NPY expression in the molecular layer of the dentate gyrus (arrow) and in the mossy fibers (arrowhead). (B) Confocal microscopic imaging of sections double-labeled for calbindin (red) and NPY (green) demonstrated sprouting of NPY axons in the molecular layer (arrow), ectopic expression of NPY in mossy fibers (arrowheads), and severe depletion of calbindin (CB) in granule cells of an hAPP-J20 mouse. Images represent the regions indicated by squares in panel A. (C–D) Calbindin depletions correlated with NPY increases in the dentate gyrus (C) and mossy fibers (D) in hAPP-J20 mice. (E) In situ hybridization revealed ectopic expression of NPY mRNA in granule cells (arrow) of an hAPP-J20 mouse (bottom panel). (F) Compared with NTG mice, hAPP-J20 mice had sprouting of somatostatin (SOM)-positive axons in the molecular layer (top panels), which was quantitated by densitometry (bottom panel). (G) Axonal sprouts in the molecular layer of hAPP-J20 mice were double-labeled for NPY (green) and SOM (red), indicating that they originated from hilar GABAergic cells. (H) NPY receptors in the dentate gyrus were also altered in hAPP-J20 mice, as determined by quantitative fluorogenic RT-PCR: Y1 receptors were decreased, whereas Y2 receptors were increased compared with NTG controls. Numbers in bars indicate number of mice per group. *P<0.05, **P<0.01, ***P<0.001 vs. NTG by Student’s t test.

Figure 2.. Aberrant innervation of inhibitory basket cells by glutamatergic mossy fiber collaterals.

Brain sections from hAPP-J20 and NTG mice were stained with the Timm method to detect glutamatergic synaptic vesicles in collateral mossy fibers, and/or immunostained for the GABAergic basket cell markers parvalbumin (PV) or NPY. (A) Sprouting of mossy fiber collaterals in the dentate gyrus was evident in hAPP-J20 mice but not in the NTG controls. Note the increase of Timm-positive axons in the granular layer (arrow) and the Timm-positive clusters in the inner molecular layer (arrowhead) of the hAPP-J20 mouse. (B) Quantitation of Timm-positive collateral mossy fibers in the granular layer of hAPP-J20 and NTG mice. (C) Collateral mossy fibers outlined specific cells in the granular layer of hAPP-J20 mice (left). Double-labeling for Timm/PV (center) or Timm/NPY (right) identified these cells as basket cells. (D) Somatostatin (SOM)-positive GABAergic interneurons (arrow) in the hilus of the dentate gyrus in an hAPP-J20 mouse and a NTG control. (E) Quantitation of SOM-positive interneurons in the hilus. Data represent the mean (± SEM) number of cells at different rostrocaudal levels. **P<0.01 vs. NTG by Student’s t test.

Figure 3.. Increase in mIPSC frequency and large-amplitude mIPSCs in dentate gyrus slices from hAPP-J20 mice.

(A) Representative traces of mIPSCs recorded from NTG (top) and hAPP-J20 (bottom) mice. (B) Average cumulative probability plot of mIPSC interevent intervals for NTG (thin line, n = 7 cells, 3 mice) and hAPP-J20 (thick line, n = 10 cells, 3 mice) mice shows decreased interevent intervals in hAPP-J20 mice. (C) mIPSC frequency was significantly increased in hAPP-J20 mice (NTG, n=7 cells, 3 mice; hAPP-J20, n=10 cells, 3 mice). (D) Average cumulative probability plot of mIPSC amplitudes recorded from NTG (thin line, n = 7 cells, 3 mice) and hAPP-J20 (thick line, n = 10 cells, 3 mice) mice shows enhanced number of large-amplitude mIPSCs. (E) The largest 25% of mIPSCs were increased in amplitude in the hAPP-J20 mice. (n=10 cells, 3 mice) relative to NTG controls (n=7 cells, 3 mice). *P<0.05 by Student’s t test.

Figure 4.. Pharmacological induction of neuronal overexcitation in NTG mice triggers molecular and anatomical alterations resembling those observed in untreated hAPP_FAD mice.

(A-E) NTG mice were injected IP with saline or kainate (KA) (5, 10, 20, or 25 mg/kg) and analyzed 3 days later. (A) Representative photomicrographs depicting KA-induced sprouting of NPY-positive terminals in the molecular layer of the dentate gyrus (arrow) and ectopic expression of NPY in the mossy fibers (arrowhead). (B) Quantitation of KA-induced increases in NPY expression demonstrating dose-dependence. (C) Typical KA-induced reduction of Arc expression in granule cells of the dentate gyrus. (D) This effect was also dose-dependent. (E) Representative western blots of dentate lysates and quantitations of signals, illustrating KA-induced changes in the levels of STEP (left), α7 nAchR (center), and calbindin (right). Each lane represents an individual mouse. (F-H) NTG mice were injected IP with pilocarpine (Pilo) (250 mg/kg) or kainate (25 mg/kg) and euthanized 2 h or 3 days later for analysis of NPY and Arc expression. (F) At these concentrations, both drugs elicited similar overall seizure severity within 20 min after their injections. (G) By 3 days after the injection, KA, but not pilocarpine, increased NPY expression in mossy fibers. (H) By 2 h after the injection, both drugs elicited comparable increases in Arc expression in the neocortex, but only KA elicited marked Arc expression in granule cells. ^#P<0.05, ^##P<0.01, ^##P<0.01 by ANOVA and contrasts test. *P<0.05, **P<0.01, ***P<0.001 vs. Sal by Student’s t test or Tukey-Kramer test.

Figure 5.. Tau reduction prevents KA- and hAPP/Aβ-induced changes in NPY.

(A-B) Tau^−/− and Tau^+/+ mice were injected IP with saline or 17 mg/kg of kainate (KA), and brains were analyzed 3 days later. (A) Compared with Tau^+/+ mice, Tau^−/− mice displayed less KA-induced freezing. (B) In Tau^+/+ mice, but not in Tau^−/− mice, KA induced robust increases in NPY in the dentate gyrus (left) and the mossy fibers (right). (C-G) Brain sections from hAPP-J20 mice with or without Tau expression were immunostained for calbindin or NPY; immunoreactivity was quantified by densitometry. (C) Increased NPY expression was prominent in hAPP-J20/Tau^+/+ mice, but absent in hAPP-J20/Tau^−/− mice. (D, E) Quantitation of NPY-IR in the molecular layer (D) (hAPP x Tau interaction, P < 0.02; ***P < 0.001, **P < 0.01 vs. groups without hAPP) and mossy fibers (E) (hAPP x Tau interaction, P < 0.02; **P < 0.01 vs. groups without hAPP). (F) Calbindin depletion in the dentate gyrus was observed in hAPP-J20/Tau^+/+ mice, but not in hAPP-J20/Tau^−/− mice. (G) Quantitation of calbindin immunoreactivity (CB-IR) in the molecular layer (hAPP x Tau interaction, P < 0.0001; ***P < 0.001 vs. groups without hAPP.)

Figure 6.. Aβ-dependent alterations in the dentate gyrus are associated with increased susceptibility of hAPP_FAD mice to seizures induced by a GABA_A antagonist.

Mice were injected IP with PTZ (40 or 80 mg/kg). Brain tissues were collected 20 min thereafter, or earlier if they developed fatal seizures. Behavior was video-recorded, and seizure severity was scored off-line. (A) Compared with NTG controls, hAPP-J20 mice had shorter latencies to reach a given seizure severity (left), greater overall seizure severity (center), and more seizure-associated deaths (right). (B) hAPP-ARC48 mice and hAPP-J9/FYN mice also had increased overall seizures severity compared with littermate controls. (C) hAPP-J9/FYN mice also had a greater incidence of seizure-associated death than the control groups. (D) All NTG mice treated with 80 mg/kg of PTZ developed fatal seizure activity. (E-G) Protein levels of NPY (E), calbindin (F), and Fos (G) in the dentate gyrus were not altered by fatal seizures. (H-K) Compared with hAPP-J20 mice that survived the PTZ injection, hAPP-J20 mice that developed fatal seizures had higher levels of NPY in the dentate gyrus and mossy fibers (H), lower dentate calbindin levels (I), and lower numbers of granule cells expressing Fos (J) or Arc (K). *P<0.05, **P<0.01, ***P<0.001 vs. NTG by Student’s t test or Tukey-Kramer test. ^#P<0.05, ^##P<0.01 by Fisher’s exact test.

Figure 7.. Aberrant synchronous neuronal network activity and spontaneous non-convulsive seizures in hAPP-J20 mice.

Chronic cortical and hippocampal EEG recordings were performed in freely moving, untreated hAPP-J20 mice and NTG controls. L, left; R, right; F, frontal; T, temporal; P, parietal; O, posterior-parietal; and H, hippocampal, indicate the position of recording electrodes. (A) In contrast to NTG mice, which showed normal EEG activity (left), hAPP-J20 mice exhibited frequent (5–50/min) generalized cortical epileptiform (interictal) spike discharges (right). (B) Bilateral depth electrode recordings from the hippocampus detected occasional discharges of isolated hippocampal origin. In this example, a prominent left-sided discharge remains focal with minimal spread to the neocortex. (C) Initiation (upper panel) and termination (lower panel) of a cortical seizure in an hAPP-J20 mouse. Concurrent bilateral hippocampal depth electrode recordings show abnormal synchronization beginning as spike discharges (arrowheads) in the hippocampus and overlying posterior-parietal neocortex (LO-RO). Synchronous seizure activity then generalizes in the neocortex without behavioral signs of convulsive activity, followed by profound electroencephalographic postictal depression. Calibration: 1 sec and 400 mV. (D) Model of Aβ-induced network dysfunction. We propose that high levels of Aβ induce aberrant excitatory neuronal activity, which triggers compensatory inhibitory mechanisms to counteract overexcitation. Both aberrant excitatory neuronal activity and compensatory inhibitory mechanisms may contribute to AD-related network dysfunction.

Figure 8.. Alterations in Arc expression and in NMDA and AMPA receptors in the dentate gyrus of hAPP-J20.

Brain sections from 151 untreated hAPP-J20 mice and 142 NTG controls were immunostained for Arc, calbindin, and NPY. (A-C) Photomicrographs of the dentate gyrus showing three distinct representative patterns of Arc expression we identified. (A) Normal pattern of Arc expression found in virtually all NTG mice. (B) Typical reduction of Arc expression observed in the majority of hAPP-J20 mice. (C) Roughly 9% of hAPP-J20 mice showed abnormally increased Arc expression in the granule cells, suggestive of recent seizure activity (Lyford et al., 1995). (D-E) Calbindin depletions (D) and NPY increases (E) were even more severe in hAPP-J20 mice with excessive Arc expression than in hAPP-J20 mice with reduced Arc expression. (F-G) Representative western blots of dentate lysates and quantitations of signals illustrating reduced levels of tyrosine-phosphorylated NR2B (pY1472), but not total NR2B levels (F), and reduced levels of AMPA receptor subunits GluR1 and GluR2 (G) in hAPP-J20 mice compared to NTG controls. Each lane represents an individual mouse. **P<0.01, ***P<0.001 by Student’s t test.

Figure 9.. Region-specific electrophysiological alterations in hippocampal slices from hAPP-J20 mice.

(A) LTP at the medial perforant pathway synapse within the dentate gyrus (DG) was significantly depressed in hAPP-J20 mice (6 slices, 6 mice) compared with NTG mice (9 slices, 8 mice) (P<0.01 by repeated measures ANOVA on data collected from minutes 51-60). (B) At the medial or lateral (not shown) perforant pathway, paired-pulse ratio, a common measure of presynaptic function, was significantly different in hAPP-J20 mice (15 slices, 7 mice) compared with NTG mice (15 slices, 8 mice) (P<0.01 by Student’s t test at all interpulse intervals). (C) The slope of the input-output relationship, a measure of synaptic transmission, along the medial or lateral (not shown) perforant pathway was similar in hAPP-J20 (14 slices, 7 mice) and NTG (12 slices, 7 mice) mice. (D) LTP at the Schaffer collateral synapse within the CA1 region (CA1) was similar in hAPP-J20 (5 slices, 5 mice) and NTG (8 slices, 6 mice) mice. (E) Paired-pulse ratio at this synapse was similar in hAPP-J20 (14 slices, 6 mice) and NTG (16 slices, 7 mice) mice. (F) In contrast, synaptic transmission along the Schaffer collateral synapse was significantly less in hAPP-J20 mice (14 slices, 5 mice) than in NTG mice (11 slices, 5 mice) (P<0.01 by ANCOVA). TBS, theta-burst stimulation; HFS, high frequency stimulation.