Limits...
PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

Nikonenko I, Boda B, Steen S, Knott G, Welker E, Muller D - J. Cell Biol. (2008)

Bottom Line: Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs.NOS blockade also reduced spine and synapse density in developing hippocampal cultures.These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Neuroscience, Geneva Neuroscience Center, University of Geneva School of Medicine, CH-1211 Geneva, Switzerland.

ABSTRACT
Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Show MeSH

Related in: MedlinePlus

PSD-95–overexpressing pyramidal neurons in hippocampal slice cultures. (A) Low magnification confocal view of a CA1 pyramidal cell 2 d after transfection with a PSD-95–EGFP construct. (B) Higher magnification of a dendritic segment illustrating the characteristic punctate aspect of PSD staining obtained after PSD-95–EGFP overexpression. (C) Low magnification electron microscopic view of a section through a dendrite (dark) of a transfected neuron upon revelation of EGFP immunostaining. (D) Higher magnification serial sections of a dendritic spine from an EGFP-immunostained neuron (the arrow points to a dendrite). Note the presence of a clearly visible PSD (arrowheads). (E) Contour representation of the multiple sections obtained from a three-dimensionally reconstructed dendritic segment of a PSD-95–EGFP-transfected cell. Note the presence of many large dendritic spines. (F) Three-dimensionally reconstructed dendritic segments (dark contours) from nontransfected neurons located in the vicinity of the PSD-95–transfected cell (light gray contours). Bars: (A) 10 μm; (B, E, and F) 5 μm; (C) 1 μm; (D) 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2600742&req=5

fig1: PSD-95–overexpressing pyramidal neurons in hippocampal slice cultures. (A) Low magnification confocal view of a CA1 pyramidal cell 2 d after transfection with a PSD-95–EGFP construct. (B) Higher magnification of a dendritic segment illustrating the characteristic punctate aspect of PSD staining obtained after PSD-95–EGFP overexpression. (C) Low magnification electron microscopic view of a section through a dendrite (dark) of a transfected neuron upon revelation of EGFP immunostaining. (D) Higher magnification serial sections of a dendritic spine from an EGFP-immunostained neuron (the arrow points to a dendrite). Note the presence of a clearly visible PSD (arrowheads). (E) Contour representation of the multiple sections obtained from a three-dimensionally reconstructed dendritic segment of a PSD-95–EGFP-transfected cell. Note the presence of many large dendritic spines. (F) Three-dimensionally reconstructed dendritic segments (dark contours) from nontransfected neurons located in the vicinity of the PSD-95–transfected cell (light gray contours). Bars: (A) 10 μm; (B, E, and F) 5 μm; (C) 1 μm; (D) 0.5 μm.

Mentions: To examine the effect of PSD-95 overexpression on spine morphology, we transfected hippocampal organotypic slice cultures with a PSD-95 tagged with EGFP using a biolistic approach. 2 d after transfection, neurons showed the classical punctuate PSD-95 staining of synapses (Fig. 1, A and B). The tissue was then processed for EM analyses using anti-EGFP antibodies to reveal the transfected neurons and their dendritic spines (Fig. 1, C and D). Through serial sectioning and 3D reconstruction of labeled dendritic segments (Fig. 1 E), we examined the ultrastructural characteristics and distribution of synapses. As controls, we analyzed EGFP-transfected neurons and also nontransfected neurons located close to the PSD-95– or EGFP-transfected cells (Fig. 1 F). In total, 1,314 spine synapses from 72 different cells obtained under 12 different conditions were three-dimensionally reconstructed in this study (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1).


PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

Nikonenko I, Boda B, Steen S, Knott G, Welker E, Muller D - J. Cell Biol. (2008)

PSD-95–overexpressing pyramidal neurons in hippocampal slice cultures. (A) Low magnification confocal view of a CA1 pyramidal cell 2 d after transfection with a PSD-95–EGFP construct. (B) Higher magnification of a dendritic segment illustrating the characteristic punctate aspect of PSD staining obtained after PSD-95–EGFP overexpression. (C) Low magnification electron microscopic view of a section through a dendrite (dark) of a transfected neuron upon revelation of EGFP immunostaining. (D) Higher magnification serial sections of a dendritic spine from an EGFP-immunostained neuron (the arrow points to a dendrite). Note the presence of a clearly visible PSD (arrowheads). (E) Contour representation of the multiple sections obtained from a three-dimensionally reconstructed dendritic segment of a PSD-95–EGFP-transfected cell. Note the presence of many large dendritic spines. (F) Three-dimensionally reconstructed dendritic segments (dark contours) from nontransfected neurons located in the vicinity of the PSD-95–transfected cell (light gray contours). Bars: (A) 10 μm; (B, E, and F) 5 μm; (C) 1 μm; (D) 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600742&req=5

fig1: PSD-95–overexpressing pyramidal neurons in hippocampal slice cultures. (A) Low magnification confocal view of a CA1 pyramidal cell 2 d after transfection with a PSD-95–EGFP construct. (B) Higher magnification of a dendritic segment illustrating the characteristic punctate aspect of PSD staining obtained after PSD-95–EGFP overexpression. (C) Low magnification electron microscopic view of a section through a dendrite (dark) of a transfected neuron upon revelation of EGFP immunostaining. (D) Higher magnification serial sections of a dendritic spine from an EGFP-immunostained neuron (the arrow points to a dendrite). Note the presence of a clearly visible PSD (arrowheads). (E) Contour representation of the multiple sections obtained from a three-dimensionally reconstructed dendritic segment of a PSD-95–EGFP-transfected cell. Note the presence of many large dendritic spines. (F) Three-dimensionally reconstructed dendritic segments (dark contours) from nontransfected neurons located in the vicinity of the PSD-95–transfected cell (light gray contours). Bars: (A) 10 μm; (B, E, and F) 5 μm; (C) 1 μm; (D) 0.5 μm.
Mentions: To examine the effect of PSD-95 overexpression on spine morphology, we transfected hippocampal organotypic slice cultures with a PSD-95 tagged with EGFP using a biolistic approach. 2 d after transfection, neurons showed the classical punctuate PSD-95 staining of synapses (Fig. 1, A and B). The tissue was then processed for EM analyses using anti-EGFP antibodies to reveal the transfected neurons and their dendritic spines (Fig. 1, C and D). Through serial sectioning and 3D reconstruction of labeled dendritic segments (Fig. 1 E), we examined the ultrastructural characteristics and distribution of synapses. As controls, we analyzed EGFP-transfected neurons and also nontransfected neurons located close to the PSD-95– or EGFP-transfected cells (Fig. 1 F). In total, 1,314 spine synapses from 72 different cells obtained under 12 different conditions were three-dimensionally reconstructed in this study (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1).

Bottom Line: Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs.NOS blockade also reduced spine and synapse density in developing hippocampal cultures.These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Neuroscience, Geneva Neuroscience Center, University of Geneva School of Medicine, CH-1211 Geneva, Switzerland.

ABSTRACT
Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Show MeSH
Related in: MedlinePlus