Population imaging of synaptically released glutamate in mouse hippocampal slices

Glutamatergic neurotransmission is a widespread form of synaptic excitation in the mammalian brain. The development of genetically encoded fluorescent glutamate sensors allows monitoring synaptic signaling in living brain tissue in real time. Here, we describe single-and two-photon imaging of synaptically evoked glutamatergic population signals in acute hippocampal slices express-ing the fluorescent glutamate sensor SF-iGluSnFR.A184S in CA1 or CA3 pyra-midal neurons. The protocol can be readily used to study defective synaptic glutamate signaling in mouse models of neuropsychiatric disorders, such as Alzheimer disease. For complete details on the use and execution of this protocol, please refer to Zott et al. (2019)

A vicious cycle of β amyloid-dependent neuronal hyperactivation

beta-amyloid (A beta)-dependent neuronal hyperactivity is believed to contribute to the circuit dysfunction that characterizes the early stages of Alzheimer's disease (AD). Although experimental evidence in support of this hypothesis continues to accrue, the underlying pathological mechanisms are not well understood. In this experiment, we used mouse models of A beta-amyloidosis to show that hyperactivation is initiated by the suppression of glutamate reuptake. Hyperactivity occurred in neurons with preexisting baseline activity, whereas inactive neurons were generally resistant to A beta-mediated hyperactivation. A beta-containing AD brain extracts and purified A beta dimers were able to sustain this vicious cycle. Our findings suggest a cellular mechanism of A beta-dependent neuronal dysfunction that can be active before plaque formation

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition.

We undertook longitudinal β-amyloid positron emission tomography (Aβ-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone in Aβ model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aβ-PET signal while promoting spatial learning and preservation of synaptic density. Here, we investigated longitudinally for 5 months PS2APP mice (N = 23; baseline age: 8 months) and App NL-G-F mice (N = 37; baseline age: 5 months) using Aβ-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. Surprisingly, Aβ-PET and immunohistochemistry revealed a shift toward higher fibrillary composition of Aβ-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. These translational data suggest that a shift toward higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the Aβ-PET signal upon immunomodulatory treatments targeting Aβ aggregation can thus be protective

Microglial Activation in the Right Amygdala-Entorhinal-Hippocampal Complex is Associated with Preserved Spatial Learning in AppNL-G-F mice.

BACKGROUND In Alzheimer`s disease (AD), regional heterogeneity of β-amyloid burden and microglial activation of individual patients is a well-known phenomenon. Recently, we described a high incidence of inter-individual regional heterogeneity in terms of asymmetry of plaque burden and microglial activation in β-amyloid mouse models of AD as assessed by positron-emission-tomography (PET). We now investigate the regional associations between amyloid plaque burden, microglial activation, and impaired spatial learning performance in transgenic mice in vivo. METHODS In 30 AppNL-G-F mice (15 female, 15 male) we acquired cross-sectional 18 kDa translocator protein (TSPO-PET, 18F-GE-180) and β-amyloid-PET (18F-florbetaben) scans at ten months of age. Control data were obtained from age- and sex-matched C57BI/6 wild-type mice. We assessed spatial learning (i.e. Morris water maze) within two weeks of PET scanning and correlated the principal component of spatial learning performance scores with voxel-wise β-amyloid and TSPO tracer uptake maps in AppNL-G-F mice, controlled for age and sex. In order to assess the effects of hemispheric asymmetry, we also analyzed correlations of spatial learning performance with tracer uptake in bilateral regions of interest for frontal cortex, entorhinal/piriform cortex, amygdala, and hippocampus, using a regression model. We tested the correlation between regional asymmetry of PET biomarkers with individual spatial learning performance. RESULTS Voxel-wise analyses in AppNL-G-F mice revealed that higher TSPO-PET signal in the amygdala, entorhinal and piriform cortices, the hippocampus and the hypothalamus correlated with spatial learning performance. Region-based analysis showed significant correlations between TSPO expression in the right entorhinal/piriform cortex and the right amygdala and spatial learning performance, whereas there were no such correlations in the left hemisphere. Right lateralized TSPO expression in the amygdala predicted better performance in the Morris water maze (β = -0.470, p = 0.013), irrespective of the global microglial activation and amyloid level. Region-based results for amyloid-PET showed no significant associations with spatial learning. CONCLUSION Elevated microglial activation in the right amygdala-entorhinal-hippocampal complex of AppNL-G-F mice is associated with better spatial learning. Our findings support a protective role of microglia on cognitive function when they highly express TSPO in specific brain regions involved in spatial memory

Beta secretase BACE1 promotes surface expression and function of Kv3.4 potassium channels in hippocampal mossy fiber synapses

In the brain, expression of BACE1 is high during development and declines subsequently. One remarkable exception is the hippocampus, in parts of which BACE1 levels remain elevated into adulthood. The sites of elevated BACE1 in mature hippocampus show a striking overlap with those of the voltage-gated K+ channel 3.4 (Kv3.4), which has also been linked to AD. This presynaptically located K+ channel gives rise to fast activating and inactivating currents that serve to repolarize action potentials that invade the terminals, thereby shaping the kinetics of transmitter release. In view of the prominent parallel enrichment of BACE1 and Kv3.4 in the hippocampus, we investigated whether BACE1 plays a role in Kv3.4 expression and function. Our main findings were: 1) BACE1 and Kv3.4 strictly co-localize in the mossy fiber tract of the hippocampus. 2) Kv3.4-dependent synaptic transmission is altered in BACE1 knockout mice. 3) Hippocampal cell surface levels and synaptic levels (Figure 1) of Kv3.4 are reduced in BACE1 knockout mice. 4) In a cell line, co-expressed BACE1 drastically increases Kv3.4 channel density at the plasma membrane when proteolytically active and, most importantly, also when pharmacologically rendered inactive. 5) BACE1 was immunoprecipitated (IP) with Kv3.4 and vice versa in co-IP experiments in the expression system. The last two points argue in favor of a non-proteolytic interaction between BACE1 and Kv3.4 as already demonstrated for other K+ channels (Agsten et al., 2015; Hessler et al., 2015). We therefore hypothesized that pharmacological inhibition of the enzymatic activity of BACE1 should not affect its interaction with Kv3.4. This hypothesis, however, was based on over- expression experiments in cell lines. To substantiate the notion that BACE1 interacts with Kv3.4 in a non-enzymatic manner, we planned to investigate synaptic Kv3.4 protein level in C57BL/6 mice which were either fed with BACE1 inhibitor NB-360 containing food pellets or with control pellets for four week

Early and longitudinal microglial activation but not amyloid accumulation predict cognitive outcome in PS2APP mice

Neuroinflammation may have beneficial or detrimental net effects on the cognitive outcome of Alzheimer's disease patients (AD). 18kDa translocator protein (TSPO) imaging by positron-emission-tomography (PET) enables longitudinal monitoring of microglial activation in vivo. We compiled serial PET measures of TSPO and amyloid with terminal cognitive assessment (water maze) in an AD transgenic mouse model (PS2APP) from eight to 13 months of age, followed by immunohistochemical analyses of microglia, amyloid and synaptic density. Better cognitive outcome and higher synaptic density in PS2APP mice was predicted by higher TSPO expression at eight months. The progression of TSPO activation to 13 months also showed a moderate association with spared cognition, but amyloidosis did not correlate with the cognitive outcome, regardless of the timepoint. This first PET investigation with longitudinal TSPO- and amyloid-PET together with terminal cognitive testing in an AD mouse model indicates that continuing microglial response seems to impart preserved cognitive performance

Pre-therapeutic microglia activation and sex determine therapy effects of chronic immunomodulation.

Modulation of the innate immune system is emerging as a promising therapeutic strategy against Alzheimer's disease (AD). However, determinants of a beneficial therapeutic effect are ill-understood. Thus, we investigated the potential of 18 kDa translocator protein positron-emission-tomography (TSPO-PET) for assessment of microglial activation in mouse brain before and during chronic immunomodulation. Methods: Serial TSPO-PET was performed during five months of chronic microglia modulation by stimulation of the peroxisome proliferator-activated receptor (PPAR)-γ with pioglitazone in two different mouse models of AD (PS2APP, AppNL-G-F ). Using mixed statistical models on longitudinal TSPO-PET data, we tested for effects of therapy and sex on treatment response. We tested correlations of baseline with longitudinal measures of TSPO-PET, and correlations between PET results with spatial learning performance and β-amyloid accumulation of individual mice. Immunohistochemistry was used to determine the molecular source of the TSPO-PET signal. Results: Pioglitazone-treated female PS2APP and AppNL-G-F mice showed attenuation of the longitudinal increases in TSPO-PET signal when compared to vehicle controls, whereas treated male AppNL-G-F mice showed the opposite effect. Baseline TSPO-PET strongly predicted changes in microglial activation in treated mice (R = -0.874, p < 0.0001) but not in vehicle controls (R = -0.356, p = 0.081). Reduced TSPO-PET signal upon pharmacological treatment was associated with better spatial learning despite higher fibrillar β-amyloid accumulation. Immunohistochemistry confirmed activated microglia to be the source of the TSPO-PET signal (R = 0.952, p < 0.0001). Conclusion: TSPO-PET represents a sensitive biomarker for monitoring of immunomodulation and closely reflects activated microglia. Sex and pre-therapeutic assessment of baseline microglial activation predict individual immunomodulation effects and may serve for responder stratification