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The Journal of Neuroscience, March 15, 2003, 23(6):2170

Synchronized Formation and Remodeling of Postsynaptic Densities: Long-Term Visualization of Hippocampal Neurons Expressing Postsynaptic Density Proteins Tagged with Green Fluorescent Protein

Tatsuhiko Ebihara¹, Izumi Kawabata², Shinichi Usui³, Kenji Sobue³, and Shigeo Okabe^{1, 2, 4}

¹ Molecular Neurophysiology Group, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8566, Japan, ² Department of Anatomy and Cell Biology, School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan, ³ Department of Neuroscience, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan, and ⁴ Core Research for Evolution Science and Technology, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

To explore mechanisms governing the formation and remodeling of postsynaptic density (PSD), we used dissociated cultures of hippocampal neurons isolated from transgenic embryos expressing green fluorescent protein (GFP)-tagged PSD proteins PSD-Zip45 (Homer 1c) and PSD-95. Expression of GFP-tagged PSD molecules was stable, and the remodeling process of PSDs could be followed for >1 week. A higher expression level of GFP-PSD-Zip45 enabled us to quantitatively analyze the amount of PSD-Zip45 clusters during development. Repetitive imaging of the same cell populations between 11 and 17 d in culture revealed an increase of the average PSD-Zip45 cluster density from 0.32 to 0.73/µm. Newly generated dendrites rapidly acquired GFP-PSD-Zip45 clusters, and their density reached the level of parental dendrites within a few days. Temporal profiles of GFP-PSD-Zip45 cluster density showed a variety of patterns. Some dendrites showed a monotonous increase of clusters, whereas others showed complex patterns, including short decremental stages. Analysis of long-term remodeling of PSD-95-GFP clusters confirmed that the decremental stages were not specific to the PSD-Zip45 clusters. Comparison of the temporal profiles of the cluster density among neurons indicated synchronization of both GFP-PSD-Zip45 and PSD-95 clustering within individual cells. Furthermore, activation of cAMP-dependent protein kinase suppressed the decremental stages of cluster remodeling. These observations suggest the presence of signaling mechanisms that can induce synchronized addition or elimination of PSD proteins throughout dendritic arborization of a single neuron.

Key words: postsynaptic density; green fluorescent protein; fluorescence microscopy; hippocampus; homer; transgenic mouse

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Copyright © 2003 Society for Neuroscience  0270-6474/03/2362170-12$05.00/0

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