Activity-dependent coordinated mobility of hippocampal inhibitory synapses visualized with presynaptic and postsynaptic tagged-molecular markers

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Abstract

Axonal varicosities and dendritic spines at excitatory synapses are dynamic structures essential for synaptic plasticity, whereas the behavior of inhibitory synapses during development and plasticity remains largely unknown. To investigate the morphology and dynamics of inhibitory synapses, we used two distinct pre- and postsynaptic fluorescent probes: one is a yellow fluorescent protein, Venus, incorporated into vesicular GABA transporter (VGAT) gene as a specific marker of presynaptic inhibitory neurons and the other red fluorescent protein (mCherry)-tagged gephyrin, a postsynaptic scaffolding protein, as a postsynaptic marker. Using primary culture of mouse hippocampal neurons and confocal laser-scanning microscopy, we established a system by which close contacts of Venus-positive axonal varicosities with mCherry-labeled gephyrin clusters in the dendritic shafts of dissociated hippocampal pyramidal neurons could be clearly visualized. Time-lapse imaging revealed that: (1) the presynaptic varicosities actively moved with marked changes in their shapes, and the postsynaptic scaffolding protein gephyrin clusters underwent coordinated movements in a tight association with the presynaptic varicosities, (2) the extents of morphological changes and movements depended on the developmental stages, reaching a stable level as the inhibitory synaptic connections matured, and (3) the motility indexes of the varicosity and its counterpart gephyrin cluster were well correlated. Furthermore, action potential blockade with tetrodotoxin treatment reduced the varicosity size, gephyrin cluster mobility as well as the amplitude of GABAergic synaptic currents in pyramidal neurons. Such a neural activity-dependent dynamic change in GABAergic synaptic morphology is likely to play a critical role in the regulatory mechanism underlying the formation and plasticity of inhibitory synapses.

Introduction

Excitatory synapses on certain central neurons are formed at contact sites between presynaptic varicosities and postsynaptic spines (Harris and Kater, 1994, Megias et al., 2001). Both dendritic spines and presynaptic varicosities undergo morphological changes in development- and neural activity-dependent manners (Colicos et al., 2001, Fischer et al., 1998, Konur and Yuste, 2004a, Korkotian and Segal, 2001, Nikonenko et al., 2003). Such structural changes and postsynaptic receptor trafficking likely provide the mechanism for plasticity of excitatory synapses (Engert and Bonhoeffer, 1999, Kasai et al., 2010, Konur and Yuste, 2004b, Maletic-Savatic et al., 1999, Matsuzaki et al., 2004, Umeda et al., 2005). As compared to excitatory synapses, relatively little is known about how inhibitory synapses develop and undergo structural and functional modifications during development and synaptic plasticity.

To investigate the mechanisms underlying the regulation of formation and plasticity of inhibitory synapses, it is essential to devise an experimental system by which morphology and dynamics of both presynaptic and postsynaptic structures could be reliably monitored for a considerable time period. For this purpose, we adopted two strategies: one is to use a genetically manipulated mouse strain where inhibitory neurons are labeled with a yellow fluorescent protein, Venus (Nagai et al., 2002), and the other labeling of the scaffolding protein gephyrin with a red fluorescent protein, mCherry (Shaner et al., 2004), using transfection of dissociated neurons under culture with recombinant adenovirus. At central inhibitory synapses, gephyrin has been identified to play a critical role in clustering of postsynaptic GABAA receptors (GABAARs) and glycine receptors (GlyRs) (Fuhrmann et al., 2002, Moss and Smart, 2001, Prior et al., 1992, Yu et al., 2007). Gephyrin has been shown to display a cytoskeleton-controlled lateral movement along the dendritic shaft of postsynaptic neurons in a synaptic activity-dependent manner (Hanus et al., 2006), and it forms clusters highly colocalized with GABAAR clusters, thereby regulating the mobility of GABAARs as well as GlyRs at inhibitory synapses (Bannai et al., 2009, Ehrensperger et al., 2007, Jacob et al., 2005, Maas et al., 2006, Meier et al., 2001, Yu et al., 2007).

By combined use of the double labeling technique, we found that dynamics of inhibitory synaptic connections could be clearly visualized in our hippocampal dissociated neurons in culture. Venus-positive presynaptic inhibitory varicosities exhibited dynamic changes in their morphology together with marked movements of postsynaptic mCherry-tagged gephyrin clusters in close association with presynaptic varicosities. More importantly, the mobility of contact sites between presynaptic varicosities and gephyrin clusters was dependent on development reflected by the culture period as well as the neural activity. Such dynamic changes of presynaptic and postsynaptic contact sites thus appear to underlie maturation and plasticity of inhibitory GABAergic synapses.

Section snippets

Visualization of inhibitory presynaptic and postsynaptic sites in hippocampal neurons

To visualize axons derived from inhibitory GABAergic interneurons, we exploited a transgenic mouse strain called VGAT-Venus mouse which is genetically manipulated to express Venus, a brighter YFP variant, in inhibitory neurons under the control of vesicular GABA transporter (VGAT) promoter (Wang et al., 2009). VGAT takes up inhibitory amino acid transmitters into synaptic vesicles at GABAergic and glycinergic nerve terminals (McIntire et al., 1997, Sagne et al., 1997). Wang et al. has recently

Discussion

By combined use of double labeling for the presynaptic axon varicosity and postsynaptic scaffolding protein, this study allowed us to clearly visualize the putative contact site of inhibitory GABAergic synapses on the dendritic shaft of hippocampal pyramidal neurons under the dissociated neuron culture conditions. Using this technique the results from this study demonstrate that GABAergic inhibitory synapses on hippocampal pyramidal neurons dynamically change their morphology with close

mCherry-gephyrin construct and adenovirus infection

Rat gephyrin cDNA has been described elsewhere (Fuhrmann et al., 2002, Prior et al., 1992). Gephyrin-P1 N-terminally labeled with mCherry was constructed by inserting full-length cDNA into the pmCherry-C2 derived from pmCherry-C1. The CAG promoter region was ligated with the mCherry-gephyrin coding region to generate the entry vector, pENTR-mCherry-gephyrin. To recombine mCherry-gephyrin into pAd/PL-DEST™, the LR recombination reaction was performed between pENTR-mCherry-gephyrin containing att

Acknowledgments

We would like to thank Drs. H. Betz and M. Kneussel for kindly providing Gephyrin-P1-peGFP-C2, Dr. R. Y. Tsien for pmCherry-C1, and Dr. A. Miyawaki for pCS2-Venus. This study was supported by Grant-in-Aid for Scientific Research (18022039, 18200026, 20021027 to S.K.; 20500296 to T.K.; 20019010, 22300105 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, Takeda Science Foundation Grant (to T.K., Y.Y.) and AstraZeneca Research Grant (to S.K.).

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