J Neurophysiol 103: 761–778, 2010. First published November 25, 2009; doi:10.1152/jn.00609.2009. Global Gene Expression Analysis of Rodent Motor Neurons Following Spinal Cord Injury Associates Molecular Mechanisms With Development of Postinjury Spasticity J. Wienecke,2,* A-C. Westerdahl,1 H. Hultborn,2 O. Kiehn,1 and J. Ryge1,* 1Mammalian Locomotor Laboratory, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; and 2Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark Submitted 15 July 2009; accepted in final form 21 November 2009 Wienecke J, Westerdahl A-C, Hultborn H, Kiehn O, Ryge J. 2004). Significant effort has been devoted to understand the Global gene expression analysis of rodent motor neurons following pathological changes that occur as a response to spinal cord Downloaded from spinal cord injury associates molecular mechanisms with development injury as well as the mechanisms behind the subsequent de- of postinjury spasticity. J Neurophysiol 103: 761–778, 2010. First published November 25, 2009; doi:10.1152/jn.00609.2009. Spinal velopment of spasticity (Hultborn 2003; Little et al. 1999; cord injury leads to severe problems involving impaired motor, Nielsen et al. 2007). Research conducted on humans has sensory, and autonomic functions. After spinal injury there is an initial pointed toward changes in reflex transmission as a mechanism phase of hyporeflexia followed by hyperreflexia, often referred to as for the hyperreflexia (Nielsen et al. 2007; Pierrot-Deseilligny spasticity. Previous studies have suggested a relationship between the and Burke 2005). Animal studies suggest that many factors http://jn.physiology.org/ reappearance of endogenous plateau potentials in motor neurons and contribute to spasticity, including morphological changes such the development of spasticity after spinalization. To unravel the as axonal sprouting (Bareyre et al. 2004; Raisman 1969), molecular mechanisms underlying the increased excitability of motor neurons and the return of plateau potentials below a spinal cord injury denervation supersensitivity (Stavraky 1961), altered expres- we investigated changes in gene expression in this cell population. We sion of transmitter/receptor systems (Giroux et al. 1999; adopted a rat tail-spasticity model with a caudal spinal transection that Tillakaratne et al. 2002), as well as the expression of plateau causes a progressive development of spasticity from its onset after 2 potentials in motor neurons (Bennett et al. 1999; Eken et al. to 3 wk until 2 mo postinjury. Gene expression changes of fluores- 1989). Plateau potentials are seen in most vertebrate motor cently identified tail motor neurons were studied 21 and 60 days neurons, including man, and their activation leads to enhanced by 10.220.33.3 on March 28, 2017 postinjury. The motor neurons undergo substantial transcriptional and prolonged muscle contraction (Crone et al. 1988; Heckman regulation in response to injury. The patterns of differential expression show similarities at both time points, although there are 20% more et al. 2005; Hultborn 1999; Kiehn and Eken 1998; Schwindt differentially expressed genes 60 days compared with 21 days postin- and Crill 1977, 1980). The expression of plateaux in motor jury. The study identifies targets of regulation relating to both ion neurons is conditional and depends on metabotropic receptor channels and receptors implicated in the endogenous expression of activation, including activation of noradrenergic or serotoner- plateaux. The regulation of excitatory and inhibitory signal transduc- gic receptors (Alaburda and Hounsgaard 2003; Conway et al. tion indicates a shift in the balance toward increased excitability, 1988; Delgado-Lezama et al. 1997; Hounsgaard and Kiehn where the glutamatergic N-methyl-D-aspartate receptor complex to- 1989; Hounsgaard et al. 1988; Hultborn and Kiehn 1992; Lee gether with cholinergic system is up-regulated and the ␥-aminobutyric and Heckman 1999). The ability to generate plateaux disap- acid type A receptor system is down-regulated. The genes of the pears in motor neurons located caudal to an acute transection pore-forming proteins Cav1.3 and Nav1.6 were not up-regulated, (Crone et al. 1988; Hounsgaard et al. 1988), but reappears after whereas genes of proteins such as nonpore-forming subunits and intracellular pathways known to modulate receptor and channel traf- 2 to 3 wk. This reappearance coincides with the development ficking, kinetics, and conductivity showed marked regulation. On the of spasticity, which led to the proposal that the increased basis of the identified changes in global gene expression in motor extensor tonus and stretch reflexes may be related to the neurons, the present investigation opens up for new potential targets expression of plateau potentials in motor neurons located for treatment of motor dysfunction following spinal cord injury. caudal to the chronic spinal injury (Bennett et al. 1999; Eken et al. 1989). The aim of the present study was to investigate possible molecular and cellular mechanisms underlying the INTRODUCTION increased motor neuron excitability and return of plateau po- tentials in the chronic spinal phase. Spinal cord injury leads to an immediate impairment of Plateau potentials in normal motor neurons are generated by motor and sensory functions that changes its manifestation persistent inward currents mediated by sodium and/or calcium over time. This involves an initial period of spinal shock with channels and the neuromodulators that enable the expression of reduced reflexes followed by the development of a disturbing plateaux act either through activation of the persistent inward hyperreflexia, often referred to as spasticity (Ditunno et al. currents or by reducing opposing outward currents (Carlin et al. 2000; Hounsgaard and Kiehn 1989; Lee and Heckman * These authors contributed equally to this work. 2001; Li and Bennett 2003; Li et al. 2004a; Simon et al. 2003; Address for reprint requests and other correspondence: J. Ryge and O. Kiehn, Mammalian Locomotor Laboratory, Department of Neuroscience, Svirskis and Hounsgaard 1998). Plateaux in chronic spinal Karolinska Institutet, Retzius va¨g 8, 171 77 Stockholm, Sweden (E-mail: animals are also mediated by calcium and sodium persistent [email protected] or [email protected]). inward currents (Harvey et al. 2006c; Li and Bennett 2003; Li www.jn.org 0022-3077/10 $8.00 Copyright © 2010 The American Physiological Society 761 762 WIENECKE, WESTERDAHL, HULTBORN, KIEHN, AND RYGE et al. 2004a). However, the molecular mechanisms underlying mg/kg, Temgesic, Schering-Plough) three times a day for the first 48 h. the increased motor neuron excitability remain elusive. To Until termination of the experiment the welfare of the rats was routinely shed new light on this unresolved conundrum we took advan- checked (e.g., for signs of infections, motor loss, or bladder dysfunction). tage of recently established methodologies to examine the Rats that showed signs of distress were immediately killed. Since the global transcriptional response of identified motor neurons spinal cord injury was inflicted at S2, only the motor and sensory (Cui et al. 2006; Ryge et al. 2008) in the rat tail-spasticity functions of the tail were affected, leaving the bladder, bowel, and hind model developed by Bennett and colleagues (1999). We com- limb functions intact. pare the global gene expression in identified tail motor neurons of transected animals with their sham-operated counterparts in Spasticity and polysynaptic reflex measures the late chronic phase 21 and 60 days postinjury, where the transected animals show clear signs of spasticity. We find that The development of tail spasticity was evaluated clinically and elec- the motor neurons undergo a very broad transcriptional re- trophysiologically acutely after operation (i.e., after 2–3 days) and at 1, 2, sponse to the injury, where 1,502 and 1,784 genes are signif- 4, and 8 wk (i.e., the intervals of 5–9, 14–15, 27–28, and 58–60 days) icantly differentially expressed 21 and 60 days postinjury, postinjury. These assessments were performed on awake rats after they respectively. Among these genes we identified several new were immobilized in a Plexiglas tube, leaving only the tail hanging out channel and receptor targets that are related to the increased and free to move (see Fig. 1A). A stretch-rub maneuver (as described in Downloaded from Bennett et al. 1999, 2004) was used, together with simple pinching of excitability and reappearance of plateaux. the tip of the tail, to clinically rate the degree of spasticity on a scale In summary, the present study focuses on the global tran- from 0 to 5. Briefly, the stretch-rub maneuver was performed by scriptional changes of a specific neuronal cell population in holding the base of the tail with thumb and index finger while the response to spinal cord injury and associates these changes other thumb and index finger were sliding a 37°C wet piece of gauze with mechanisms previously related to a distinct pathological down the tail, starting at the base of tail and finishing by sliding off the state of spinal cord injury—i.e., spasticity and the increased tip (three times in a row). After the stimulus the tail was released and excitability observed in motor neurons in the chronic phase. the tail movements were observed and the tail was subsequently http://jn.physiology.org/ This is a first step toward understanding how individual cellu- touched/pinched for a rating (Fig. 1B): 0–1