Impairment of Dendrodendritic Inhibition in the Olfactory Bulb of APP/PS1 Mice

Abstract

Olfactory dysfunction is an early event in Alzheimer's disease (AD). However, the mechanism underlying the AD-related changes in the olfactory bulb (OB) remains unknown. Granule cells (GCs) in the OB regulate the activity of mitral cells (MCs) through reciprocal dendrodendritic synapses, which is crucial for olfactory signal processing and odor discrimination. Nevertheless, the relationships between the morphological and functional changes of dendrodendritic synapses, particularly the local field potentials (LFPs) as a consequence of olfactory disorders in patients with AD have not been investigated. Here, we studied the morphological and functional changes induced by dendrodendritic inhibition in GCs onto MCs in the OB of amyloid precursor protein (APP)/PS1 mice and age-matched control mice during aging, particular, we focused on the effects of olfactory disorder in the dendrodendritic synaptic structures and the LFPs. We found that olfactory disorder was associated with increased amyloid-β (Aβ) deposits in the OB of APP/PS1 mice, and those mice also exhibited abnormal changes in the morphology of GCs and MCs, a decreased density of GC dendritic spines and impairments in the synaptic interface of dendrodendritic synapses between GCs and MCs. In addition, the aberrant enhancements in the γ oscillations and firing rates of MCs in the OB of APP/PS1 mice were recorded by multi-electrode arrays (MEAs). The local application of a GABA_AR agonist nearly abolished the aberrant increase in γ oscillations in the external plexiform layer (EPL) at advanced stages of AD, whereas a GABA_AR antagonist aggravated the γ oscillations. Based on our findings, we concluded that the altered morphologies of the synaptic structures of GCs, the dysfunction of reciprocal dendrodendritic synapses between MCs and GCs, and the abnormal γ oscillations in the EPL might contribute to olfactory dysfunction in AD.

Keywords: Alzheimer’s disease; dendrodendritic inhibition; olfactory bulb; olfactory dysfunction; γ oscillations.

Figure 1

Atypical olfactory and cognitive behaviors of amyloid precursor protein (APP)/PS1 mice. (A) Design of the buried food test. (B) Quantitative statistical analysis of latency of 3–4-, 6–7 and 9–10-month-old (mo) APP/PS1 and C57 mice to find the hidden food particle. (C) Design of the fine odor discrimination test. Mice were deprived of water for 24 h and trained to identify distilled water. In one dish, the distilled water was coupled with an odor mixture of mango and almond, in which mango was the main component. The other dish contained bitter water with an odor mixture in which almond was the main component. Different odor proportions of mango and almond were used in the performance test. (D–F) Performance of APP/PS1 and C57 mice at 3–4 (D), 6–7 (E), and 9–10 months of age (F). (G) Design of the Morris water maze (MWM) test. (H) Quantitative statistics of the crossing platform times of APP/PS1 and C57 mice at 3–4, 6–7 and 9–10 months of age. (I–K) Escape latencies of APP/PS1 and C57 mice at 3–4 (I), 6–7 (J) and 9–10 months of age (K). The data are presented as the means ± standard errors of the means (SEMs). *p < 0.05; **p < 0.01.

Figure 2

Spatiotemporal pattern of amyloid-β (Aβ) depositions in the olfactory bulb (OB) of APP/PS1 mice. (A) Image of the OB coronal plate indicating the bulbar cell layers [glomerular layer (GL), external plexiform layer (EPL), MCL, inner plexiform layer (IPL) and granule cell layer (GCL)]. (B) No Aβ was observed in the OB of C57 mice at 9–10 months of age. (C–E) Representative images of Aβ staining (white arrows) in the OB of APP/PS1 mice at 3–4, 6–7 and 9–10 months of age. Scale bars = 200 μm in (E; applies to B–E). (F) The mean percentage density of deposited Aβ was elevated in the GCL within the OB cell layers of APP/PS1 mice. The error bars indicate the SEMs.

Figure 3

Unchanged laminar organization of the OB in APP/PS1 mice. (A–F) No significant change in the laminar organization of the OB was observed between APP/PS1 and age-matched C57 mice at different ages. (A1–F1) High-magnification images showing the structure of the GCL. Scale bars = 500 μm in (F; applies to A–F) and 100 μm in (F1; applies to A1–F1).

Figure 4

Reduced dendritic spine density of GCs in APP/PS1 mice. (A) Representative images of Golgi-stained apical dendritic spines of GCs in APP/PS1 and C57 mice at 3–4, 6–7 and 9–10 months of age. Scale bar = 10 μm. (B–D) Quantitative analysis of the dendritic spine density (B), mature spine density (C) and immature spine density (D) from randomly selected dendritic segments of GCs in APP/PS1 and age-matched C57 mice. (E–H) Western blotting assays of postsynaptic density95 (PSD95; F), synapsin I (G) and synaptophysin (H) in the OB; the levels of these proteins were reduced in APP/PS1 mice compared with age-matched C57 mice. The data are expressed as the means ± SEMs. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Ultrastructural characterization of dendrodendritic synapses in the EPL. (A) The morphology of asymmetric synapses between GC dendrites and mitral cell (MC) dendrites in the EPL of APP/PS1 and C57 mice. (B,C) The synaptic clefts and diameter of the vesicles in asymmetric synapses were not significantly different between APP/PS1 and age-matched C57 mice. (D) The APP/PS1 mice showed a reduced thickness of the PSD in asymmetric synapses compared with the age-matched C57 mice. (E) Morphological structures of symmetric synapses between GC dendrites and MC dendrites in the EPL of APP/PS1 and C57 mice. (F) The diameters of the vesicles in symmetric synapses were not significantly different between APP/PS1 and age-matched C57 mice. (G) The synaptic clefts were wider in APP/PS1 mice than in age-matched C57 mice. The data are presented the means ± SEMs. The scale bar represents 200 nm. *p < 0.05; **p < 0.01.

Figure 6

Spontaneous γ oscillations in the EPL of OB slices. (A) Schematic showing the locations of MCs and GCs and the dendrodendritic synapses between MC and GC dendrites. Dendrites of MCs receive signals from olfactory sensory neuron termini in the GCL and form synaptic connections with GCs in the EPL. (A1) Action potential (AP) propagation in the MC lateral dendrites release glutamate (black vesicles) to activate postsynaptic AMPA and NMDA receptors in the GC dendrites. In turn, NMDA receptors trigger GABA release (red vesicles) and postsynaptic activation of GABA_AR in MCs. In addition, glutamate released from MCs also activates extrasynaptic glutamate autoreceptors on MC lateral dendrites, resulting in spontaneous excitatory transmission. (B) Example of a 60-channel multi-electrode array (MEA). (C) Photograph of an OB slice placed on an MEA chip; the subregions of the OB are indicated. (D) Representative traces of local field potential (LFP) signals (1–100-Hz bandwidth) and filter traces (γ, low-γ and high-γ) of the EPL in the C57 group, APP/PS1 group, APP/PS1 + muscimol group and APP/PS1 + bicuculline group at 3–4 months of age. (E–G) Muscimol weakened the aberrantly increased γ oscillations in 3–4-mo APP/PS1 and C57 mice. Bicuculline enhanced the aberrantly elevated γ oscillations in 3–4-mo APP/PS1 and C57 mice. (H) Representative traces of LFP signals (1–100-Hz bandwidth) and filter traces of the EPL in 9–10-mo C57 and APP/PS1 mice. (I–K) Muscimol weakened the aberrantly increased γ oscillations in 9–10-mo APP/PS1 and C57 mice, and bicuculline enhanced the aberrantly elevated γ oscillations in 9–10-mo APP/PS1 and C57 mice. The data are presented as the means ± SEMs. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

Increased MC spontaneous firings in APP/PS1 mice. (A) Spontaneous firing rates of MCs in APP/PS1 and C57 mice at 3–4 months of age. (B) Quantitative analysis of spontaneous firing rates of MCs in APP/PS1 and C57 mice at 3–4 months of age. (C) Spontaneous firing rates of MCs in APP/PS1 and C57 mice at 9–10 months of age. (D) Quantitative analysis of spontaneous firing rates of MCs in APP/PS1 and C57 mice at 9–10 months of age. The data are presented as the means ± SEMs. **p < 0.01; ***p < 0.001.