Increased excitability in hippocampal neurons of synaptopodin-knockout mouse

Synaptopodin (SP) is localized within the spine apparatus, an enigmatic structure located in the neck of spines of central excitatory neurons. It serves as a link between the spine head, where the synapse is located, and the endoplasmic reticulum (ER) in the parent dendrite (Vlachos et al. 2009, Korkotian and Segal, 2011, Zhang et al. 2013). SP is also located in the axon initial segment, in association with the cisternal organelle, another structure related to endoplasmic reticulum. Extensive research using SP knockout (SPKO) mice suggests that SP has a pivotal role in structural and functional plasticity (Deller et al. 2003, Deller et al. 2007). Consequently, SPKO mice were shown to be deficient in cognitive functions, and in ability to undergo long term potentiation of reactivity to afferent stimulation (Deller et al. 2003). In contrast, neurons of SPKO mice appear to be more excitable than their wild type (wt) counterparts(Bas Orth et al, 2007). To address this discrepancy, we have now recorded activity of CA1 neurons in the mouse hippocampus slice, with both extracellular and patch recording methods. Electrophysiologically, SPKO cells in CA1 region of the dorsal hippocampus were more excitable than wt ones. In addition, exposure of mice to a complex environment caused a higher proportion of arc-expressing cells in SPKO than in wt mice hippocampus. These experiments indicate that higher excitability and higher expression of arc staining may reflect SP deficiency in the hippocampus of adult SPKO mice.

Innovative approach for the application of MTT, LDH and bis-ANS

The acute hippocampus slice-based models can serve as a feasible tool to gain deeper understanding on how pathological events in AD, in particular Aβ oligomerization, influence hippocampal functionality and therefore paving the way to develop and test related hypothesis but also to screen potential protective agents or validate results seen in vivo or predicted in silico. To overcome the shortcomings of the in vitro and in vivo methods, we developed a cost effective and simple apparatus for maintaining the viability of acute tissue slices, named: ExViS (Ex Vivo System; Universal Mini-Chamber Tube System for Acute Tissue Slices). The system allowed us to further develop methods that use acute (ex vivo) hippocampal slices to model AD-related patomechanisms. Over the course of my PhD studies we have developed the following techniques and ex vivo models based on the features of acute hippocampal slices: 1. Rapid, reliable and quantitative determination of Aβ1-42 toxicity in ageing (OGD) acute hippocampal slice model using MTT and LDH assays. 2. Quantitative determination of zinc-induced Aβ1-42 oligomerization toxicity in acute hippocampal slice model using MTT assay. 3. Fluorescent imaging of neurite cross-sections and detailed imaging of neurite structures in acute hippocampal slices via a novel application of bis-ANS and its co-staining variations. 4. Modelling the effect of Zn2+ released in glutamergic synaptic cleft in the hippocampus on Aβ1-42 aggregation and its consequences on cell viability and synaptic functionality, learning and memory (LTP).

Functional Dysregulations in CA1 Hippocampal Networks of a 3-Hit Mouse Model of Schizophrenia

For a better translation from treatment designs of schizophrenia to clinical efficiency, there is a crucial need to refine preclinical animal models. In order to consider the multifactorial nature of the disorder, a new mouse model associating three factors (genetic susceptibility—partial deletion of the MAP6 gene, early-life stress—maternal separation, and pharmacological treatment—chronic Δ-9-tetrahydrocannabinol during adolescence) has recently been described. While this model depicts a schizophrenia-like phenotype, the neurobiological correlates remain unknown. Synaptic transmission and functional plasticity of the CA1 hippocampal region of male and female 3-hit mice were therefore investigated using electrophysiological recordings on the hippocampus slice. While basal excitatory transmission remained unaffected, NMDA receptor (NMDAr)-mediated long-term potentiation (LTP) triggered by theta-burst (TBS) but not by high-frequency (HFS) stimulation was impaired in 3-hit mice. Isolated NMDAr activation was not affected or even increased in female 3-hit mice, revealing a sexual dimorphism. Considering that the regulation of LTP is more prone to inhibitory tone if triggered by TBS than by HFS, the weaker potentiation in 3-hit mice suggests a deficiency of intrinsic GABA regulatory mechanisms. Indeed, NMDAr activation was increased by GABAA receptor blockade in wild-type but not in 3-hit mice. This electrophysiological study highlights dysregulations of functional properties and plasticity in hippocampal networks of 3-hit mice, one of the mechanisms suspected to contribute to the pathophysiology of schizophrenia. It also shows differences between males and females, supporting the sexual dimorphism observed in the disorder. Combined with the previously reported study, the present data reinforce the face validity of the 3-hit model that will help to consider new therapeutic strategies for psychosis.

DSTG: Deconvoluting Spatial Transcriptomics Data through Graph-based Artificial Intelligence

Recent development of spatial transcriptomics (ST) is capable of associating spatial information at different spots in the tissue section with RNA abundance of cells within each spot, which is particularly important to understand tissue cytoarchitectures and functions. However, for such ST data, since a spot is usually larger than an individual cell, gene expressions measured at each spot are from a mixture of cells with heterogenous cell types. Therefore, ST data at each spot needs to be disentangled so as to reveal the cell compositions at that spatial spot. In this study, we propose a novel method, named DSTG, to accurately deconvolute the observed gene expressions at each spot and recover its cell constitutions, thus achieve high-level segmentation and reveal spatial architecture of cellular heterogeneity within tissues. DSTG not only demonstrates superior performance on synthetic spatial data generated from different protocols, but also effectively identifies spatial compositions of cells in mouse cortex layer, hippocampus slice, and pancreatic tumor tissues. In conclusion, DSTG accurately uncovers the cell states and subpopulations based on spatial localization.

NLRC3 delays the progression of AD in APP/PS1 mice via inhibiting PI3K activation

Background/Aims: NLRC3 inhibits inflammatory responses. Epidemiological studies indicate that neuroinflammation induces and accelerates the onset of Alzheimer's disease (AD). This study was designed to determine whether NLRC3 plays a role in neuroinflammation, Aβ accumulation and neuroprotection in AD mice. Methods: Thirty 12-month-old APP/PS1 transgenic mice were randomized into three groups as model group, APP/PS1 +LVCON307 and APP/PS1 +LV-NLRC3 group. Ten 12-month-old wild-type C57 mice were chosen as control group. Mice in APP/PS1 +LVCON307 and APP/PS1 +LV-NLRC3 group were injected with LVCON307 or LV-NLRC3 through intracerebroventricular injection. Six months after LVCON307 or LV-NLRC3 injection, We carried out Morris water maze test on mice and harvested their brain tissues after the behavioral experiment. The deposition of amyloid protein and the changes of Nissle bodies were observed by ThS and Nissle staining. The expressions of NLRC3, 6E10, GFAP, Iba1, NeuN and PI3K were detected by immunohistochemistry or immunofluorescence. Western blot was used to analyze the expression of NLRC3, PI3K, GFAP and Iba1. Results: The expression of NLRC3 is down-regulated in brain tissues of APP/PS1 mice. Mice in APP/PS1 group had a significant attenuation of learning and memory ability compared to the control group, the ability of learning and memory was improved in APP/PS1 +LV-NLRC3 mice. The expression of 6E10, GFAP, Iba1 and PI3K in brain and hippocampus slice of APP/PS1 and APP/PS1 + LVCON307 mice were significantly higher than those of the control group, while the expression of NLRC3 and NeuN was significantly lower than that of the control group. After overexpression of NLRC3, the expression of 6e10, GFAP, Iba1 and PI3K in APP/PS1 + LV-NLRC3 group was significantly lower than that in APP/PS1 and APP/PS1 + LVCON307 group, while the expression of NLRC3 and NeuN was significantly higher than that in APP/PS1 and APP/PS1 + LVCON307 group. NLRC3 co-localized with NeuN. PI3K activation with 740YP increased the expression of GFAP and Iba-1 in hippocampus with exogenous NLRC3 protein. Conclusion: NLRC3 may play an important role in the development and progression of AD. Down-regulation of NLRC3 can lead to the activation of PI3K, resulting in abnormal plaque deposition, glial cell activation and neuron loss during AD. NLRC3 delays the progression of AD in APP/PS1 mice via inhibiting PI3K activation. Keywords: NLRC3 • inflammation • Aβ • neuron •PI3K •Alzheimer's disease

Design and Fabrication of a Three-Dimensional Multi-Electrode Array for Neuron Electrophysiology

Neural recording and stimulation with high spatial and temporal resolution are highly desirable in the study of neurocommunication and diseases. Planar multiple microelectrode arrays (MEA) or quasi-three-dimensional (3D) MEA with fixed height have been proposed by many researchers and become commercially available. In this paper, we present the design, fabrication, and test of a novel true 3D multiple electrode array for brain slice stimulation and recording. This MEA is composed of 105 microelectrodes with 50 μm diameter and 125 μm center-to-center spacing integrated in a 1.2 × 1.2 mm2 area. This “true” 3D MEA allows us to precisely position the individual electrodes by piezoelectric-based actuators to penetrate the inactive tissue layer and to approach the active neurons so as to optimize the recording and stimulation of electrical field potential. The capability to stimulate nerve fibers and record postsynaptic field potentials was demonstrated in an experiment using mouse brain hippocampus slice.