The honeycomb maze provides a novel test to study hippocampal-dependent spatial navigation

Here we describe the honeycomb maze, a behavioural paradigm for the study of spatial navigation in rats. The maze consists of 37 platforms that can be raised or lowered independently. Place navigation requires an animal to go to a goal platform from any of several start platforms via a series of sequential choices. For each, the animal is confined to a raised platform and allowed to choose between two of the six adjacent platforms, the correct one being the platform with the smallest angle to the goal-heading direction. Rats learn rapidly and their choices are influenced by three factors: the angle between the two choice platforms, the distance from the goal, and the angle between the correct platform and the direction of the goal. Rats with hippocampal damage are impaired in learning and their performance is affected by all three factors. The honeycomb maze represents a marked improvement over current spatial navigation tests, such as the Morris water maze1,2,3, because it controls the choices of the animal at each point in the maze, provides the ability to assess knowledge of the goal direction from any location, enables the identification of factors influencing task performance and provides the possibility for concomitant single-cell recording.This work was supported by grants from the Wellcome Trust and the Gatsby Charitable Foundation to J.O. R.A.W. is an MRC Clinical Research Training Fellow, J.K. is a Wellcome Trust/Royal Society Sir Henry Dale Fellow and is supported by the Kavli Foundation Dream Team project and the Isaac Newton Trust. D.C. is funded by the Cambridge NIHR Biomedical Research Centre and by the Wellcome Trust

Inhibition of Stat3‐mediated astrogliosis ameliorates pathology in an Alzheimer's disease model

Abstract Reactive astrogliosis is a hallmark of Alzheimer's disease (AD), but its role for disease initiation and progression has remained incompletely understood. We here show that the transcription factor Stat3 (signal transducer and activator of transcription 3), a canonical inducer of astrogliosis, is activated in an AD mouse model and human AD. Therefore, using a conditional knockout approach, we deleted Stat3 specifically in astrocytes in the APP/PS1 model of AD. We found that Stat3‐deficient APP/PS1 mice show decreased β‐amyloid levels and plaque burden. Plaque‐close microglia displayed a more complex morphology, internalized more β‐amyloid, and upregulated amyloid clearance pathways in Stat3‐deficient mice. Moreover, astrocyte‐specific Stat3‐deficient APP/PS1 mice showed decreased pro‐inflammatory cytokine activation and lower dystrophic neurite burden, and were largely protected from cerebral network imbalance. Finally, Stat3 deletion in astrocytes also strongly ameliorated spatial learning and memory decline in APP/PS1 mice. Importantly, these protective effects on network dysfunction and cognition were recapitulated in APP/PS1 mice systemically treated with a preclinical Stat3 inhibitor drug. In summary, our data implicate Stat3‐mediated astrogliosis as an important therapeutic target in AD

Additional file 3: Figure S3. of Astroglial NF-kB contributes to white matter damage and cognitive impairment in a mouse model of vascular dementia

Histological changes in the internal and external capsule. Reactive astrogliosis (A, E), microgliosis (B, F), axonal degeneration (C, G) and demyelination (D, H) in sham and BCAS animals in the internal capsule (upper row) and external capsule (lower row). (PDF 118 kb

Primula kisoana Miquel

原著和名: カッコサウ キソコザクラ科名: サクラソウ科 = Primulaceae採集地: 群馬県 鳴神山 (上野 鳴神山)採集日: 1963/5/5採集者: 萩庭丈壽整理番号: JH024269国立科学博物館整理番号: TNS-VS-97426

The sphingolipid receptor S1PR2 is a receptor for Nogo-a repressing synaptic plasticity

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity

APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP

The in vivo function of the amyloid precursor protein (APP) and its related homologue APLP2 in the adult nervous system is not resolved. Here, the findings show that APLP2 and APP are required to mediate neuromuscular transmission, spatial learning, and synaptic plasticity

Localization of S1PR2 by immunohistochemistry in the adult mouse CNS.

<p>(A) S1PR2 expression in the hippocampus. CA, <i>cornu ammonis</i>; DG, dentate gyrus. (B) Magnification of the boxed region of CA1 depicted in (A). (C) S1PR2 expression in the cerebellum. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. (D) Magnification of the boxed region depicted in (C). (E) S1PR2 expression in the motor cortex. (F) Magnification of the boxed region depicted in (E). (G,H) S1PR2 expression in motoneuron cell bodies (arrows) and βIII-Tubulin-positive fibers (arrowheads) in the spinal cord. (I,J,K) S1PR2 expression in βIII-Tubulin-positive axons bundles (arrowheads) and cell bodies (arrows) of retinal ganglion cells. Scale bars: (A) 300 µm; (B) 30 µm; (C) 200 µm; (D) 15 µm; (E) 90 µm; (F) 30 µm; (G,H) 20 µm; (I–K) 15 µm.</p

S1PR2 is internalized upon Nogo-A-Δ20 binding.

<p>(A) Representative confocal micrographs of 3T3 cells stained alive (Non-perm) or fixed (Perm) for S1PR2 before (control) and 30 min after Δ20 treatment at 37°C. (B) Mean fluorescence intensity quantification of the cell surface staining shown in (A). (C) Addition of Δ20 downregulates cell surface S1PR2 in 3T3 plasma membranes (PM): immunoblot and relative quantification thereof. Loading control: β-Actin. (D) Representative confocal micrographs of 3T3 cells incubated with 1 µM HA-tagged Δ20 for 1 h at 4°C (pulse), which were then subsequently chased for 15 and 30 min at 37°C. Cells were stained with an anti-HA (Δ20), S1PR2, or EEA1 antibody (early endosomes). Arrows indicate cell surface-bound Δ20 (top panel) or colocalization of Δ20 and S1PR2 in early endosomes (middle and bottom panel). The inset panel shows an enlarged view of the boxed region. (E) Western blot analysis of ubiquitinated and non-ubiquitinated protein fractions of 3T3 cells 30 min after Δ20 or S1P treatment. Data shown are means ± SEM (<i>n</i> = 3–6 experiments; **<i>p<</i>0.01, ***<i>p<</i>0.001). Scale bars: (A,D) 50 µm.</p

Nogo-A-Δ20 inhibition is mediated via the G₁₃-LARG-RhoA signaling axis and can be modulated by exogenous S1P.

<p>(A) 3T3 cells transfected with siRNAs against <i>G₁₂</i>, <i>G₁₃</i>, <i>G_q</i>, or <i>Larg</i>, or control (ctrl) siRNA were replated on a Nogo-A-Δ20 substrate and assessed for cell spreading. G_i/o was blocked with Pertussis Toxin (PTX) for which saline was used as control. JTE-013 was co-applied to <i>G₁₃</i>-siRNA-treated cells to investigate a cumulative effect. (B) Transfection of DIV4 E19 cortical neurons with siRNA against <i>G₁₃</i> but not <i>G₁₂</i> similarly rescued Nogo-A-Δ20-induced neurite outgrowth inhibition. (C,D) Nogo-A-Δ20-induced RhoA activation was assessed in JTE-013- versus DMSO-treated cells (C) or in cells carrying a stable knockdown of S1PR2 (sh-<i>S1pr2</i>) versus control vector (sh-Vec) (D). (E,F) Relative quantification of (C) and (D), respectively. (G,H) Competitive ELISA quantifications of extra- (EC) and intracellular (IC) S1P levels in 3T3 cells (G) and cerebellar granule neurons (H) before and after 30 and 60 min incubation with Nogo-A-Δ20. (I) Quantification of Nogo-A-Δ20-mediated cell spreading inhibition in the presence of the SphK-specific blocker D,L-<i>threo</i>-dihydrosphingosine (DHS) or in SphK1^−/− or SphK2^−/− MEFs. (J,K) 3T3 cells were plated on a Nogo-A-Δ20 substrate in the presence of the function blocking anti-S1P antibody Sphingomab (J) or of exogenous S1P (K) and assessed for cell spreading. Co-application of JTE-013 significantly reversed the modulatory effects obtained by S1P (K) but not anti-S1P (J). Anti-BrdU antibody or methanol was used as control in (J) and (K). Data shown are means ± SEM (<i>n</i> = 3–6 experiments; *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p<</i>0.001).</p

S1PR2 mediates Nogo-A-Δ20- and myelin-induced inhibition of cell spreading and neurite outgrowth.

<p>(A,C) Representative pictures of 3T3 fibroblasts treated with JTE-013 or vehicle (DMSO) (A), or stably carrying a <i>S1pr2</i> shRNA (sh-<i>S1pr2</i>) or empty vector (sh-Vec) construct (C) and plated on control, Nogo-A-Δ20 or myelin substrates. (B,D) Cell spreading quantification of (A) and (C). (E) Representative pictures of MEFs isolated from WT or S1PR2^−/− mice and plated on control, Nogo-A- Δ20, or myelin substrates. (F) Cell spreading quantification of (E). Cells were stained with Alexa488-conjugated Phalloidin in (A, C, and E). (G,I) Representative pictures of P5–8 cerebellar granule neurons treated with JTE-013 or DMSO (G), or isolated from S1PR2^−/− or WT mice (I) and plated on PLL (ctrl), Nogo-A-Δ20 or myelin substrates. (H,J) Normalized mean neurite length per cell quantification of (G) and (I). Neurons were stained with βIII-Tubulin in (G) and (I). Data shown are means ± SEM (<i>n</i> = 3–6 experiments; *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p<</i>0.001). Scale bars: 50 µM.</p