Regulation of hippocampal synapse remodeling by epileptiform activity
Introduction
Most excitatory synapses in the CNS are formed at tiny dendritic protrusions, termed spines, that extend from dendrite shafts. The formation and remodeling of spines occurs during neuronal maturation (Dailey and Smith, 1996, Yuste and Bonhoeffer, 2004) and in response to changes in neural activity (Hering and Sheng, 2001, Yuste and Bonhoeffer, 2001). Indeed, different patterns of neural activity appear either to promote spine formation and structural remodeling or to induce spine resorption (see Kasai et al., 2003, Nikonenko et al., 2002, Yuste and Bonhoeffer, 2001). For example, epileptiform activity (EA), which is characterized by repetitive synchronized firing in groups of neighboring neurons, is commonly associated with reduced spine density in hippocampal (Bothwell et al., 2001, Drakew et al., 1996, Isokawa and Levesque, 1991, Jiang et al., 1998, Muller, 1993, Muller et al., 1993) and cortical (Bothwell et al., 2001, Multani et al., 1994) neurons. Because most excitatory synapses in mature brain tissues are formed on dendritic spines, changes in spine structure and density are thought to indicate changes in synaptic structures. However, some spines and spine-like structures do not bear synapses, some bear multiple synapses, and some synapses are located on dendritic shafts (Fiala and Harris, 1999, Fiala et al., 1998). Therefore, effects of neural activity on synaptic remodeling cannot be directly inferred solely from observations of spines. Thus, although changes in dendritic spine density are well documented in a variety of epilepsy models (Fiala et al., 2002), there is little direct information on the fate of synapses.
We ask here what synaptic remodeling events occur coincident with changes in dendritic spines in response to EA in hippocampal tissue slices. Are synapses transferred from spines to shafts, or are spine synapses lost completely? Does the relative distribution of spine and shaft synapses change? What is the time course and scale of these changes? To address these questions, we visualized synapses in neurons using a marker of postsynaptic densities (PSDs), PSD95. PSD95 is a scaffolding protein involved in the assembly of the PSD of excitatory synapses (El-Husseini et al., 2000, Kennedy, 2000, McGee and Bredt, 2003, Sheng, 2001, Tomita et al., 2001). A fluorescent PSD95-GFP fusion protein is clustered at postsynaptic sites when expressed in hippocampal pyramidal neurons (Marrs et al., 2001, Okabe et al., 1999, Okabe et al., 2001). We used PSD95-GFP to directly observe synaptic sites on dendrites in rat organotypic hippocampal slice cultures in conjunction with a diffusible fluorescent protein, which allows us to resolve the effects of EA on synapses directly as well as on spines and filopodia.
To investigate the effect of EA on synapses, we induced EA in slices by blocking GABAA receptors. We verified that spine density decreased following EA and that this decrease depended on TTX-sensitive neural activity. Analysis of PSD95-GFP showed that EA induced a decrease in spine but not shaft synapses, whereas activity blockade caused a decrease in shaft but not spine synapses. Time-lapse imaging showed that EA-induced loss of synapses resulted from a shift in the balance between synapse gain and loss. It also showed formation of filopodia-like dendritic protrusions (lazypodia) that lacked synapses and that persisted for much longer than typical filopodia. These observations identify changes in synaptic distribution and dendritic remodeling that may occur during, and contribute to, epilepsy-associated pathological conditions.
Section snippets
Gabazine induces epileptiform activity in hippocampal slice cultures
Previous studies have shown that hippocampal slice cultures derived from neonatal rats develop an epileptiform pattern of activity following GABAA receptor inhibition (Badea et al., 2001, Dailey, 1996, Grinvald et al., 1982, Scanziani et al., 1994). Here we used a specific GABAA receptor blocker, SR-75531 (gabazine, Gbz), to induce EA. Because one consequence of neuronal activity is an increase in intracellular Ca2+ concentration ([Ca2+]i) that is temporally correlated with neuronal firing (
Discussion
We have systematically assessed changes in hippocampal neuronal spines, synapses, and filopodia as a consequence of epileptiform activity (EA). EA was generated in organotypic hippocampal slice cultures by adding Gbz. These observations confirm previous reports of an EA-induced decrease in spine density in hippocampal neurons. By directly labeling PSDs via expression of PSD95-GFP/YFP, we could further show that this decrease in spine density was accompanied by a selective loss of spine but not
Constructs and reagents
The PSD95-GFP construct was kindly provided by Dr. David Bredt (UCSF). PSD95-YFP constructs were kindly provided by Drs. Alaa El-Husseini (University of British Columbia) and Bonnie Firestein (Rutgers University). EGFP-C1, ECFP-C1, and DsRed2 expression vectors were purchased from Clontech. Culture media and reagents were purchased from Gibco. Other reagents used: SR-75531 (gabazine, Gbz) from Sigma or RBI, tetrodotoxin (TTX) from RBI, and Fluo-4 AM dye from Molecular Probes.
Hippocampal slice culture and transfection
Rat hippocampal
Acknowledgments
We thank Rui Qin at the University of Iowa Statistical Consulting Center for help on χ2 analysis. This research was supported equally by NIH grants #NS37159 (MED) and #DC02961 (SHG).
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Present address: Department of Internal Medicine, Howard Hughes Medical Institute, University of Iowa Carver College of Medicine, 500 EMRB, Iowa City, IA 52242, USA.