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

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

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

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

<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