Asymmetric, selectively postsynaptic expression of N-cadherin impairs basal function of glutamatergic synapses.

<p>(<b>A</b>) Scheme of N-cadherin+EGFP expression in individual, transfected N-cadherin knockout neurons (Ncad; green) innervated by surrounding, not transfected N-cadherin knockout neurons (−/−; derived from mouse ES cells). (<b>B</b>) Overlay of a phase contrast image of cultured N-cadherin knockout neurons and the corresponding fluorescence image of a N-cadherin+EGFP transfected neuron (12 DIV, 2 days after transfection). Scale bar represents 30 µm. (<b>C</b>) <i>Left:</i> Fluorescence images (VAMP2 immunostaining) of a knockout (Ncad−/−) and a transfected (Ncad−/− + N-cadherin) neuron immunostained for VAMP2 and N-cadherin. The dendritic segment indicated in the whole cell image is shown enlarged and thresholded. <i>Center:</i> Corresponding N-cadherin immunostaining of the same dendritic segment. <i>Right:</i> Overlay of VAMP2 and N-cadherin immunostaining of the same dendritic segment. Scale bars represent 15 µm and 5 µm. (<b>D, E</b>) Spontaneous, AMPA receptor mediated miniature EPSCs recorded from an EGFP transfected, N-cadherin knockout neuron (GFP, control) and from a N-cadherin+EGFP transfected, N-cadherin knockout neuron (Ncad, mis-match). Patch-clamp recordings 2 days after transfection; holding potential −60 mV. (<b>E</b>) Quantification of the frequency and the amplitude (cells without mEPSCs not included) of AMPA mEPSCs (11–13 DIV). Cumulative distribution of mEPSC amplitudes is shown in the right panel. Note the strong reduction in mini frequency in neurons with asymmetric N-cadherin expression. (<b>F</b>) Control postsynaptic overexpression of N-cadherin in primary cultured cortical neurons. Patch-clamp recordings 2 days after transfection; holding potential −60 mV. Quantification of the frequency and the amplitude of AMPA mEPSCs with cumulative distribution of mEPSC amplitudes shown in the right panel. Means ± SEM. <i>n</i> (cells) is indicated on bars. **, <i>P</i><0.001 Students t-test.</p

Symmetric, pre- and postsynaptic expression of N-cadherin at autapses does not impair synaptic function.

<p>(<b>A</b>) Autaptic AMPA EPSCs recorded from N-cadherin knockout neurons transfected with either EGFP (GFP) or N-cadherin+EGFP (GFP+N-cadherin, Ncad). Autaptic AMPA EPSCs were elicited by action currents induced by depolarizing pulses in the same neuron. Holding potential −60 mV. 3 traces superimposed. Stimulation artefacts and Na⁺ currents are truncated. (<b>B</b>) Quantification of autaptic AMPA EPSCs. <i>Left:</i> Probability of occurrence of autaptic AMPA EPSCs (5mM Ca²⁺). 11–13 DIV, 2 days after transfection. <i>Center:</i> Peak amplitudes. <i>Right:</i> Failure rates. (<b>C, D, E</b>) Incidence of autapses is not significantly affected in N-cadherin knockout neurons expressing N-cadherin. (<b>C</b>) Autapses were identified by coexpression of DsRed2-VAMP2 and PSD-95-EGFP in N-cadherin knockout neurons (Ncad−/−, control) and in N-cadherin knockout neurons expressing N-cadherin (Ncad−/− + N-cadherin). Overlays of corresponding DsRed2-VAMP2 (red) and PSD-95-EGFP (green) fluorescence images (8 days after transfection; 22 DIV, same cells as in Fig. 2B) that were strongly thresholded to visualize the rare autapses (arrows). Scale bars: 2,5 µm. (<b>D</b>) Quantification of dendritic density of DsRed2-VAMP2 puncta (autapses) 2 days (2d) and 8 days (8d) after transfection at 14 DIV. −/−: N-cadherin knockout neurons. Ncad: N-cadherin knockout neurons expressing N-cadherin. Same cells as in Fig. 2. Note the strong (non-significant) trend to a general reduction in the number of autapses with time in culture. (<b>E</b>) No significant changes in the dendritic density of glutamatergic autapses (colocalized DsRed2-VAMP2 puncta and PSD-95-EGFP puncta) were induced by N-cadherin expression (Ncad). Normalized to values of N-cadherin knockout neurons (control) at matched times in culture. Means ± SEM. <i>n</i> (cells) is indicated on bars. Students t-test, ANOVA (D).</p

NPSR1-mediated IP₃ receptor activation triggers calcium-induced calcium release via ryanodine receptors.

<p>(A) Fluorescence recording from a single neuron treated with ryanodine [50 μM] (black) and an untreated control (grey). (B) Mean fluorescence intensities calculated from 136 individual neurons. (C) Mean amplitudes calculated from the dataset shown in (B). (D) Fluorescence as averaged from 155 control neurons for two consecutive NPS applications in the absence of ryanodine. (E) Mean amplitudes calculated from the dataset shown in (D). (F) Mean amplitudes for the second application of NPS normalized to the preceding first NPS-administration in control cells (grey) and in the presence of ryanodine (black). NPS was used at 500 nM in all experiments.</p

Asymmetric N-Cadherin Expression Results in Synapse Dysfunction, Synapse Elimination, and Axon Retraction in Cultured Mouse Neurons

<div><p>Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an <em>in vivo</em> asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.</p> </div

NPS-evoked calcium signals in hippocampus neurons expressing NPSR1.

<p>(A) Mean fluorescence images of Fluo4-stained hippocampus neurons transfected with AAV-NPSR1-TS-mCherry-ER during baseline-conditions, application of 500 nM NPS, washout and application of 300 μM glutamate. Arrows mark NPS-responsive neurons. Mean fluorescence images were calculated from seven raw images each. Scale bar indicates 25 μm. (B) Normalized mean fluorescence values for the first NPS-administration calculated from 229 individual neurons after peak alignment. (C) Fluorescence trace of a single NPSR1-expressing neuron stained with Fluo4-AM. NPS [500 nM] was applied for 90 seconds as indicated. (D) Comparison of normalized peak amplitudes for three consecutive applications of NPS. (E) Dose-response curve for NPSR1-mediated calcium mobilization in neurons, depicting an EC₅₀ of 19.8 ± 1.3 (SEM, n = 318 neurons).</p

<i>In vivo</i> expression of N-cadherin in the superior colliculus

<p>(<b>SC</b>) <b>during postnatal mouse development.</b> (<b>A</b>) In situ hybridization (ISH) to detect N-cadherin (Cdh2) mRNA expression. Note the strong expression of N-cadherin in neurons of the superficial collicular layers (prospective superficial gray layer [SGL] and optic layer [OL]) where most visual cortical axons terminate. (<b>B, C</b>) Immunohistochemistry (IHC) to detect N-cadherin protein (B) and calbindin D28k protein (C), a marker for SGL and OL. (<b>D</b>) Nissl stain of a section adjacent to that shown in A and B. Coronal sections through the postnatal day 6 superior colliculus are shown. Scale bar: 200 µm (for A–D).</p

<i>In vivo</i> expression of N-cadherin in the cerebral cortex during postnatal mouse development.

<p>(<b>A, B</b>) Immunohistochemistry (IHC) and in situ hybridization (ISH) to detect expression of N-cadherin (Cdh2) at the protein level and mRNA level, respectively. Coronal sections of the somatosensory cortex (A) and the visual cortex (B) of the mouse at postnatal day 6. Note the almost complete lack of N-cadherin mRNA expression in layer V somatosensory neurons (A, ISH) and the relatively strong expression in a subpopulation of layer V visual neurons (B, ISH). IHC did not show layer-specific expression, because pyramidal cell dendrites extend over several layers. For identification of cortical layers, a corresponding Nissl stain (Thio) is shown in the right panels. Other abbreviations: I–VI, cortical layers I–VI; wm, white matter. Scale bar: 200 µm (for A, B).</p

Vector design and expression of NPSR1.

<p>(A) Construct design of the AAV-NPSR1-TS-mCherry-ER vector. NPSR1 was expressed fused to mCherry. A plasma membrane targeting signal (TS) and an ER-export signal (ER) were used to enhance correct trafficking. (B,C) Immunocytochemistry of cultured hippocampus neurons, 9 days after plating and 7 days after viral transfection with AAV-NPSR1-TS-mCherry-ER. Green: MAP2-immunoreactivity (-IR), red: mCherry-IR. Punctual localization of NPSR1-mCherry is marked in (C). (D,E) Immunocytochemistry of cultured hippocampus neurons, 9 days after plating and 7 days after viral transfection with a control vector expressing mCherry but not NPSR1. Punctual localization of mCherry could not be detected. Scale bars indicate 10 μm. (F) Surface expression of NPSR1-mCherry. Immunoblot with anti-mCherry and anti-neuronal class III β-tubulin for the biotinylated plasma membrane fraction of proteins and the non-biotinylated intracellular fraction. Biotinylation of cultured hippocampus neurons was performed 6 days after transfection with AAV-NPSR1-TS-mCherry-ER and untransfected neurons were used as control. Exposure times were 4 s for the biotinylated fractions and 0.45 s for the unlabeled fractions.</p

Long-term asymmetric expression of N-cadherin induces elimination of glutamatergic synapses.

<p>(<b>A, B</b>) Immunocytochemical stainings for VAMP2 and fluorescence images of coexpressed PSD-95-EGFP in N-cadherin knockout neurons (Ncad−/−) and in N-cadherin knockout neurons expressing N-cadherin (Ncad−/− + N-cadherin) 2 days (A) and 8 days (B) after transfection at 14 DIV. Segments of proximal dendrites are shown. Scale bars: 2,5 µm. Upper panels are fluorescence images and lower panels are thresholded (black or white) images used to quantify puncta. Dendrites were identified using the PSD-95-EGFP images. Merge: Overlay of thresholded VAMP2 (red) and PSD-95-EGFP (green) puncta; >80% of PSD-95 puncta colocalized with VAMP2. (<b>C, E, F</b>) Quantification of dendritic density of VAMP2 puncta (including few autaptic puncta, C), VAMP2 puncta area (E), and VAMP2 puncta average intensity (F). −/−: N-cadherin knockout neurons; Ncad: N-cadherin knockout neurons expressing N-cadherin. (<b>G</b>) Quantification of dendritic density of PSD-95-EGFP puncta. (<b>H</b>) Relative changes of dendritic density of glutamatergic synapses (colocalized VAMP2 and PSD-95-EGFP puncta, excluding autaptic puncta) in N-cadherin knockout neurons expressing N-cadherin. Normalized to values of N-cadherin knockout neurons (control) at matched times in culture. Note that selectively at 8 days after transfection (8d, 22 DIV), but not at 2 days after transfection (2d, 16 DIV), the dendritic density of synapses is reduced. (<b>D</b>) Quantification of dendritic density of VAMP2 puncta 2 days (2d) and 8 days (8d) after transfection at an earlier stage in culture (9–11 DIV). GFP: control N-cadherin knockout neurons. Ncad: N-cadherin knockout neurons expressing N-cadherin. Note the reduced increase in synapse density in N-cadherin knockout neurons expressing N-cadherin at 8 days after transfection. Means ± SEM. <i>n</i> (cells) is indicated on bars. *, <i>P</i><0.05; **, <i>P</i><0.01 and <i>P</i><0.001 (in C, G) Students t-test.</p

Model for the intracellular mechanisms underlying NPSR1 activation in cultured mouse hippocampus neurons.

<p>Calcium is released from the endoplasmic reticulum via IP₃ and ryanodine receptors, which can be blocked by U73122, 2-APB and ryanodine, respectively. This decrease in the ER calcium content activates store-operated calcium entry (SOCE), which can be visualized by using Ca²⁺-free extracellular solution, the underlying signal cascade can be blocked by ML-9 and SKF96365.</p