Remodeling of synaptic structures in the motor cortex following spinal cord injury

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Abstract

After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI.

Introduction

Spinal cord injury (SCI) removes supraspinal input to the spinal motor networks and thus results in a severe and permanent loss of motor function below the injury site. Some degree of functional recovery, however, can be observed without intervention (Bregman and Goldberger, 1983, Burns et al., 1997). Since regeneration of injured axons is extremely limited in mature CNS, spontaneous recovery in motor function appears to be mediated by structural reorganization of spared motor system. This compensatory remodeling occurs at multiple levels of the neuraxis including spinal motor centers, descending supraspinal motor tracts, brainstem, and the motor cortex (Raineteau and Schwab, 2001).

The sensorimotor cortex in adults retains the capability to reorganize in response to alteration in peripheral sensory input or behavioral manipulation (Clark et al., 1988, Nudo et al., 1996). A much greater extent of structural and functional plasticity can be observed after large-scale injuries such as limb amputation or SCI (Jain et al., 1997, Pons et al., 1991). Several lines of evidence suggest that synaptic connectivity in the sensorimotor cortex can be modified after SCI. Spinal lesions in cats and primates reshape the sensory representational map in the cortex (Jain et al., 1997, McKinley et al., 1987). Functional imaging (Bruehlmeier et al., 1998) and transcranial magnetic stimulation (Topka et al., 1991) demonstrated significant alterations in connectivity between the motor cortex and spinal cord in human SCI as well.

The cellular and/or anatomical substrates subserving injury-induced cortical plasticity are not fully understood. Unmasking or potentiation of existing connections may contribute to the plasticity within a short time scale (Hess and Donoghue, 1994, Jacobs and Donoghue, 1991), whereas alteration in cortical connectivity over months to years may involve new growth of axonal and dendritic processes (Darian-Smith and Gilbert, 1994, Florence et al., 1998, Volkmar and Greenough, 1972). Structural remodeling at the level of individual synapses may be another mechanism (Stepanyants et al., 2002). The density of dendritic spines, postsynaptic specializations at the excitatory synapses, is sensitive to a variety of experience or environmental stimuli (Globus et al., 1973, Gould et al., 1990, Moser et al., 1994). Recent in vivo imaging studies have suggested that the appearance and disappearance of spines underlie experience-dependent plasticity during development as well as in adults (Lendvai et al., 2000, Trachtenberg et al., 2002). Not only spine density but also morphology of spine structures is sensitive to various stimuli that are associated with synaptic plasticity (Hayashi and Majewska, 2005, Yuste and Bonhoeffer, 2001). For example, changes in spine neck length were observed after social stimulation or electrical stimulation to evoke long-term potentiation (Coss and Globus, 1978, Fifkova and Anderson, 1981), and changes in morphology or size of spine head were also associated with synaptic plasticity (Desmond and Levy, 1983, Desmond and Levy, 1986, Matsuzaki et al., 2004). It is not known, however, whether remodeling of dendritic spines, particularly spine morphology, is involved in the injury-induced cortical reorganization, and if so, with what time scale this remodeling proceeds after injury.

In this study, we sought to determine whether SCI leads to remodeling of synaptic structures in the motor cortex. For this end, we visualized dendritic spines in the motor cortex using confocal microscopy in fixed slice preparations and examined the spine density and morphology in detail. Since spine morphology such as spine length and head diameter is closely related to functional characteristics of an individual synapse (Kasai et al., 2003, Yuste et al., 2000), the analysis of spine morphology from a large population of dendritic spines also allowed us to infer the potential changes in overall functional properties in the motor cortical network following SCI. Furthermore, we examined correlative changes in expression of various synapse-associated proteins in the motor cortex after SCI, attempting to define a temporal profile of the remodeling process.

Section snippets

Animals and spinal lesion

Adult female Sprague–Dawley rats (200–250 gm; Zivic Inc., Zelienople, PA) were used in this study. They were housed in the Georgetown University Division of Comparative Medicine Facility and all protocols were approved by the Georgetown University Animal Care and Use Committee. Animals received a right cervical overhemisection injury at the C4 level using a procedure modified from that described previously (Bregman et al., 1997). This injury removes the right hemicord plus the left dorsal

Remodeling of dendritic spines in the motor cortex following SCI

To determine whether axotomy at the spinal level alters synaptic structures in the motor cortex, we focused on postsynaptic spine structures and measured density (number per unit length), head diameter, and length of dendritic spines. To more accurately quantify those variables from a large number of spines, we opted to use confocal microscopic imaging on fluorescently labeled neuronal processes, instead of using traditional Golgi staining or electronmicroscopy (EM) analysis. Golgi staining

Discussion

We made several observations that collectively provide evidence that dynamic remodeling of synaptic structures occurs in the motor cortex following SCI. First, the density of postsynaptic spines decreases in the motor cortex at 7 days after SCI followed by a partial recovery by 28 days. Second, spine head diameter increases after SCI with a different time course depending on the layer. Third, SCI leads to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10%

Acknowledgments

We thank Dr. Bogdan Stoica for technical advice for confocal microscopy, Dr. Zhanyan Fu for assistance in DiI labeling, and Dr. Barry Wolfe and Dr. Robert Yasuda for generous supply of NMDA receptor subunit antibodies and technical comments on the immunoblot experiment. We also thank Dr. Thomas Finn and Dr. Daniel Pak for helpful comments on the manuscript, and Dr. Shibao Feng for statistical consultation. This study was supported by NIH grant NS27054.

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    Current address: Brain Disease Research Center, Ajou University School of Medicine, Suwon 443-721, Republic of Korea.

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