Imaging the motility of dendritic protrusions and axon terminals: roles in axon sampling and synaptic competition

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Dendritic spines and filopodia display actin-based morphological plasticity. The function of this rapid motility is unknown. Its ubiquitous expression during development has led to the hypothesis that motility plays a role in synaptogenesis. We investigated this by simultaneously imaging presynaptic boutons and dendritic protrusions in acute hippocampal slices from GFP-M transgenic mice loaded with FM 1–43 followed by immunostaining. Postsynaptic motility was inversely correlated with the presence of stable synaptic contacts. Filopodia were highly motile and made transient interactions, whereas spines were less motile and had stable contacts, although they could still move together with a synaptic terminal. “Head morphing” of spines was associated with interactions with more than one presynaptic terminal. Our data indicate that filopodia motility could serve to transiently sample the surrounding neuropil, while the motility of established spines could mediate interactions with two axonal terminals. Spine “morphing” could therefore be the morphological signature for synaptic input competition in central synapses.

Introduction

Dendrites of the majority of neurons in the adult mammalian central nervous system are decorated with spines (Ramón y Cajal, 1888, Ramón y Cajal, 1899), which receive most excitatory inputs (Harris and Kater, 1994). However, during development, dendrites are covered with immature forms of dendritic protrusions, such as filopodia (Miller and Peters, 1981). Recently, live imaging techniques have revealed that dendritic protrusions are motile and that this motility is downregulated as the brain matures (Dailey and Smith, 1996, Dunaevsky et al., 1999, Fischer et al., 1998, Lendvai et al., 2000, Lohmann et al., 2002, Portera-Cailliau et al., 2003, Wong et al., 2000, Ziv and Smith, 1996). Nevertheless, the function of this motility has not yet been elucidated. Some studies have shown that neuronal activity can regulate the motility of dendritic protrusions (Kirov and Harris, 1999, Wong et al., 2000) (Portera-Cailliau et al., 2003). Indeed, extension of filopodia and spines can be elicited by synaptic stimulation (Engert and Bonhoeffer, 1999, Maletic-Savatic et al., 1999). Also, filopodia and spines are also motile during development in vivo (Grutzendler et al., 2002, Lendvai et al., 2000, Trachtenberg et al., 2002), and that this motility can be reduced by sensory input deprivation (Lendvai et al., 2000). Together, these data have led to the hypothesis that the motility of dendritic protrusions has a function in synaptogenesis, whereby axonal terminals could stimulate the production and stabilization of spines, perhaps through a two-step process (Tashiro et al., 2003, Yuste and Bonhoeffer, 2004).

To address the relation between synaptogenesis and motility, we previously correlated spine motility with the presence or absence of a synapse using electron microscopy of slice cultures from cerebellum (Dunaevsky et al., 2001). Although spine motility was correlated with the absence of a synapse and the presence of free extracellular space, spines were surprisingly motile even in the presence of synaptic contact. In another study, using dissociated cultures (Korkotian and Segal, 2001), dendritic protrusion motility was inversely correlated with the presence of an active presynaptic bouton, as if boutons stabilized motility. These seemingly contradicting conclusions have left the question of the relation between motility and the presence of synapses unanswered.

To address this, we have imaged the motility of pre- and postsynaptic structures in acute slices, classifying their interactions, protrusion types, and quantifying their morphological changes. We find that, indeed, relatively mature protrusion types like spines and “hand-like” spines are still motile while preserving their stable contacts (Dunaevsky et al., 2001). On the other hand, highly motile filopodia make only transient interactions with presynaptic terminals. Also, a small percentage of presynaptic boutons are motile and interact with postsynaptic protrusions. Our results support the idea that filopodial motility has a role of contacting and possibly stabilizing a presynaptic region (Lohmann et al., 2002, Ziv and Smith, 1996). However, we also find that hand-like spines still retained some motility after contact, indicating a separate role for this type of head morphing than initial synapse recognition. Immunocytochemistry of presynaptic markers after imaging supports our hypothesis that head morphing may mediate interactions with more than one bouton at the same time and thus could implement synaptic competition.

Section snippets

Imaging pre- and postsynaptic motility

We chose postnatal ages P7–P10 in the CA1 region of the hippocampus. At these ages, protrusion morphology is diverse, synapse formation is extensive (Fiala et al., 1998), and dendritic protrusion motility is still very prominent. Within this age period, we found no age dependency of the pre- and postsynaptic motilities (not shown). First we measured the motility of presynaptic boutons and dendritic protrusions independently and next investigated the correlation between the potential pre- and

Discussion

Dendritic protrusion motility is a developmental phenomenon that begins with high filopodial extension–retraction activity at the first postnatal week, continues with more diverse motility expression including head morphing in the second postnatal week, and decreases sharply between second and third postnatal week, reaching a baseline level (Bonhoeffer and Yuste, 2002, Portera-Cailliau et al., 2003). The developmental decrease of spine motility, its high expression during peak periods of

Slice preparation

All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23; revised 1987) and with the Society for Neuroscience 1995 Statement. Acute (300-μm-thick) hippocampal slices were cut on a vibratome (Leica, Bannockburn, IL) from C-57 GFP-M strain mice (Feng et al., 2000) aged P7–P10. Slices were kept in artificial cerebrospinal fluid (ACSF) that contained (in mM) 126 NaCl, 3 KCl, 1 CaCl2, 3 MgSO4, 1.1

Acknowledgments

We thank Carol Mason for advice, Stanislav Zakharenko for help with FM loading, Volodymyr Nikolenko and Brendon Watson for image processing programs, and Carlos Portera-Cailliau and Peter Scheiffele for discussions and suggestions. We also thank Josh Sanes and Ania Majewska for kindly providing GFP-M mice. Funded by the National Eye Institute (EY13237), the New York STAR Center for High Resolution Imaging of Functional Neural Circuits, and the John Merck Fund.

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