J Neurophysiol 103: 761–778, 2010. First published November 25, 2009; doi:10.1152/jn.00609.2009.

Global Expression Analysis of Rodent Motor 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 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 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 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 describes no “clonus” (a lar components of the spinal cord interact after injury to create rapid tremor of the end of the tail during spasms), no coiling, no the pathophysiological postinjury state including spasticity, response to light touch, and little or no hypertonus (no response to pinching); 2–3 describes strong flexor directed coiling spasms of the which will open up for the possibility of designing more whole tail in response to the stretch-rub maneuver (lasts Յ10 s), and specific treatments of spinal cord injury. some coiling, some clonus, hypertonus, and very sensitive to light Part of this study was previously published in abstract form touch (pinching gives some flicking, coiling, and clonus); 4–5 is (Wienecke et al. 2007). similar to 2–3 but with a greater amplitude response and longer

duration, showing both extensor and flexor coiling, resulting in an by 10.220.33.3 on March 28, 2017 S-shape curvature (lasting Ͼ tens of minutes), greater clonus, greater METHODS hypertonus, and greater flicking than that in 2–3 (pinching gives Spinal cord operation flicking and long-lasting coiling and clonus). All animals were as- sessed clinically by the same person. All handling of animals was approved by the Danish Animal After clinical testing the reflex was assessed with electromyo- Experiments Inspectorate. The handling and experimental procedures graphic (EMG) recordings, quantifying muscle activity as a response of the animals was conducted at University of Copenhagen (Denmark) to electrical stimulation of afferent fibers. The rats were briefly and the isolated spinal cord tissue was further processed at Karolinska anesthetized with isoflurane while four pairs of sterile steel wire Institutet in Stockholm (Sweden). (Ethicon, 4–0, Johnson & Johnson) electrodes were inserted in the Adult male Wistar rats (325–480 g) were used in this study. The tail, each pair passing subcutaneously on either side of the tail. Each animals used for microarray hybridization were separated into four wire electrode pair was joined, allowing bilateral stimulation or groups: spinalized for 21 days (Spi-21, n ϭ 8), spinalized for 60 days recording. The electrode configuration for stimulation and recording is (Spi-60, n ϭ 8), sham-operated controls for 21 days (ShamC-21, n ϭ illustrated in Fig. 1A. Electrophysiological recordings were performed 6), and sham-operated controls for 60 days (ShamC-60, n ϭ 5). This when the rat had fully recovered from the wire-mounting surgery study also includes data from clinical and electrophysiological quan- (ϳ20 min later). tifications of the reflex response performed on the day of termination. Stimulus–response curves were obtained by applying single stimula- To describe the full time course of the reflex response some rats (n ϭ tion pulses of 0.2 ms duration delivered every 2–5 s with increasing 15) were tested at several additional time points prior to termination. stimulus strength to cover a range from 0.2 to 10 mA. The maximal

Before operation the rats were initially anesthetized with isoflurane motor response (Mmax) was determined as maximal amplitude in the raw (Baxter) and thereafter a xylocaine–marcaine mix (xylocaine, 12.5 ␮g/ signal (measured peak-to-peak) following stimulation of all motor axons ml:marcaine, 2.5 mg/ml, 1:4, AstraZeneca) of 0.2–0.3 ml per animal was during the acquisition of the stimulus–response curve. The stimulus was injected intramuscularly. The second, and a few times also the third, normalized to the motor threshold (MT). EMG responses were quantified lumbar vertebrae (L2 and L3) were removed. For ShamC-21 and on the basis of a stimulus strength five times the motor threshold ShamC-60 the dura was left intact after laminectomy, where after the (5ϫMT). The EMG trace was band-pass filtered (100 Hz to 10 kHz), wound was closed suturing muscles, muscle fascia, and skin separately. rectified, integrated, and subsequently normalized with respect to Mmax For the spinalized animals the dura was opened and spinal cord tissue and Spi-2 rats (Fig. 1C). This normalization was performed to produce a corresponding to 1–2 mm was gently removed using a glass rod, forceps, standardized measure of the increase in activity/spasticity. We normal- and suction—creating a clear separation between the rostral and caudal ized to Spi-2 (“acute spinal”) rats instead of normal animals to avoid cord at the sacral S2 segment. Only rats in which the dorsal vein and the inflicting unnecessary pain to animals (which a 5ϫMT stimulation would ventral artery remained intact across the dissected gap were included in otherwise induce in normal rats with intact spinal cord). However, the the study. After spinalization, the wound was closed suturing muscles, reflex amplitude in normal and early acute spinal rats has been shown to muscle fascia, and skin separately. Care was taken to relieve pain be more or less identical (Bennett et al. 2004). The reflex response was postoperatively by administration of buprenorfin subcutaneously (0.1 quantified as the area under the curve in two separate intervals following

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stimulation: 1) 15–50 ms (i.e., initial reflex component) and 2) 50–500 Animals were anesthetized with pentobarbital (initially 20 and then ms (i.e., late tonic reflex component). 5 mg/kg every 30 min, Mebumal) before the sacrocaudal spinal cords were removed, after which the rats were killed with an overdose of Motor neuron labeling, spinal cord dissection, pentobarbital. The spinal cords were immediately frozen in liquid cryosectioning, and laser microdissection nitrogen and stored at Ϫ80°C until cryosectioning. The isolated and frozen spinal cords (the S3–S4 level, where most of Motor neurons were retrogradely labeled by injection of Fluoro- the tail motor neurons are located; Bennett et al. 2004) were cryosec- Gold dissolved in saline (Fluorochrome LLC) into both the tail tioned into 10 ␮m sections. The sections were mounted on nuclease and ␮ ␮ muscles (150 l) and intraperitoneally (30 l) (Leong and Ling 1990). human nucleic acid free 0.9 ␮m polyester (POL) membrane frame slides All animals (spinalized and sham operated) were injected with this (Leica Microsystems) and stored at Ϫ80°C until laser microdissection. dual-injection procedure under anesthesia (isoflurane) 5 to 7 days The Fluoro-Gold–labeled motor neurons were isolated from the spinal before the spinal cord was removed. We found that this dual-injection cord sections using the Leica AS laser microdissection system (Leica procedure enhanced the labeling of the sacral motor neurons. Microsystems) at room temperature (Fig. 2A before and Fig. 2B after laser microdissection). To reduce RNA degradation laser microdissection was performed within 1 h for each slide. Between 70 and 200 Fluoro- Gold–labeled neurons were laser microdissected per rat and the motor neurons were collected in the cap of a eppendorf tube by force of gravity. Downloaded from RNA degradation and contamination from external sources were mini- mized in all steps of the experimental procedure. Possible contamination of samples with material from surrounding nonmotor neurons during laser microdissection was also minimized as described by Ryge et al. (2008).

Total RNA isolation, mRNA amplification, and aRNA http://jn.physiology.org/ biotinylation for microarray hybridization Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus) and the messenger RNA (mRNA) fraction was amplified in a two-round T7 linear amplification process using the RiboAmp HS RNA Amplification Kit (Arcturus). The complementary DNA (cDNA) product from the second round of the amplification process was used to generate biotin-labeled antisense RNA (aRNA) (Gene- Chip Expression 3Ј-Amplification Reagents for IVT Labeling; Af- fymetrix). The integrity and concentration of the amplified and bio- by 10.220.33.3 on March 28, 2017 tinylated aRNA was assessed on an Agilent RNA chip with the Agilent 2100 bioanalyzer (Agilent Technologies) both before and after fragmentation. Only samples of good integrity were further used and 15 ␮g of the fragmented samples were hybridized to GeneChip Rat Genome 230 2.0 Arrays (Affymetrix, RAT230_2 chip) and subsequently scanned. Each array always originated from a single animal. The Agilent analysis and microarray hybridizations were conducted at the Affymetrix core facility at Novum (Bioinformatics and Expression Analysis Core Facility, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden).

Microarray preprocessing The microarray normalization and analysis for detection of signif- icantly differentially expressed (DE) genes were adopted from Ryge

FIG. 1. Spasticity development in the injury model. A: clinical evaluation of spasticity and subsequent quantification of tail reflexes were performed while the rat was pacified in a test tube. The electrodes placed in the tail for reflex quantification are indicated in the drawing: R, recording electrodes; G, ground electrodes; and S, stimulation electrodes. B: clinical evaluation score of the development of spasticity Յ8 wk following transection. The vertical lines indicate SDs. C: tonic reflex component (rectified electromyography [EMG]) evoked by 5 times the motor threshold (5ϫMT) 2 days (Spi-2, light gray trace), 2 wk (Spi-14, gray trace), and 8 wk (Spi-60, black trace) after transection. The inset shows a magnification of the first 50 ms of the Spi-60 trace illustrating the initial reflex component and the M-wave. This initial component is similar in Spi-60 and Spi-21 (dashed line in D). D: development of the initial (dashed line) and the late tonic (full line) reflex component at 5ϫMT with time. The size of the reflexes is standardized to the size in acute spinal rats 2 days after transection. Each point illustrates rectified and integrated averaged values of both reflex components. Vertical lines indicate SDs. The values for 2, 4, and 8 wk are significantly different (P Ͻ 0.05) from acute spinal values for both the initial and the late tonic reflex components.

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compared with their respective sham-operated counterparts, ShamC-21 and ShamC-60. To determine the significantly DE genes within each time point, the false discovery rates (FDRs) of three statistical tests [Cyber- T, limma, and SAM described in Baldi and Long (2001), Smyth (2004), Tusher et al. (2001), and Wettenhall and Smyth (2004)] were used to create a conglomerate ranking of each gene, reflecting their degree of significance across all three tests. In short, for each test all genes were ranked on the basis of their test statistics, producing three ordered lists of genes from most to least significantly DE gene. The three rankings obtained from these lists were then used to produce an average rank for each gene, according to which all genes were FIG. 2. Motor neuron laser microdissection. A: picture of Fluoro-Gold–labeled reordered (Ryge et al. 2008). This procedure was carried out for both ␮ motor neurons from 10- m-thick sections on polyester membrane frame slides the Spi-21 and Spi-60 groups and the top-ranking genes that fall ready for laser microdissection. The 3 arrows indicate 3 motor neurons in the within a 1% FDR were extracted to produce two lists of significantly ventral horn. B: picture of the same section as in A after laser microdissection. DE genes, one for Spi-21 and one Spi-60 (these two lists of DE are and colleagues (2008). The RAT230_2 chip contains 31,099 probe provided in Supplemental Tables S1 and S2). For each test the number of DE genes that fall within a 1% FDR was calculated and the mean sets, whose annotations are continuously updated within the Ensembl Downloaded from genome database (Hubbard et al. 2007). We used the Affymetrix of these was used as the number of genes that on average pass the 1% probe sets verbatim, but discarded those not included in the Ensembl FDR to be extracted from the ordered list. For Spi-21 a number of database for the RAT230_2 chip prior to the statistical analysis. 1,502 genes pass a 1% FDR (Cyber-T ϭ 1,305, limma ϭ 1,336, Ensembl includes Affymetrix probe sets in their database only if more SAM ϭ 1,866), whereas for Spi-60 a number of 1,784 genes pass than half of its probes match the updated target gene sequence (Cyber-T ϭ 1,520, limma ϭ 1,516, SAM ϭ 2,317). MA and Volcano (http://mar2009.archive.ensembl.org/info/docs/microarray_probe_ plots of the injured groups versus their sham-operated counterparts set_mapping.html). This procedure reduced the set of probes from show that the ratios of expression between the two conditions are 31,099 to 12,919. The microarrays were then background compen- centered around 1.0 and well balanced throughout the intensity range http://jn.physiology.org/ sated, normalized, and RMA (robust multiarray average) expression (Supplemental Fig. S1, C–F), supporting the preprocessing of the summaries were calculated (Irizarry et al. 2003; Ryge et al. 2008). To data. Furthermore, the DE genes separate nicely from the non-DE validate the background correction and normalization procedure of all genes, supporting the robustness of the regulation of gene expression microarrays, the RMA distributions were examined together with the in the injured state compared with sham. To compensate for multiple average distribution of all microarrays. Additional background com- testing the P values of Cyber-T and limma were converted to FDRs pensation was carried out on the expression summaries since we using the approach of Allison and colleagues (2002), whereas the observed a discrepancy between the RMA distributions at low expres- FDRs of SAM are based on a methodology of permutation and sion levels that could not be eliminated with the probe level normal- resampling of the data (i.e., the FDRs are output from the SAM ization and background compensation. This discrepancy of RMA analysis directly). by 10.220.33.3 on March 28, 2017 distributions was manifested as a deviation from the diagonal of the quantile–quantile plots for low expression values between different Allen Brain Atlas samples. To compensate for this effect the expression summaries were transformed to normal scale (2RMA), the average of the first 10% of The expression of genes in the motor neurons of the normal spinal the RMA values (1,292 probe sets) was calculated for each microar- cord have not previously been described in the literature for all of the ray, and used as a microarray-specific background measure subtracted significantly DE genes contained in Tables 1, 2, and 3. To examine the from their corresponding microarray RMA values. To ensure that no presence of their transcripts in motor neurons located in the ventral negative expression summaries occurred after this procedure the uni- horn of normal rodents we therefore used the Allen Brain Atlas versal minima of all microarrays (negative) was subtracted from all (http://www.brain-map.org/). At the time of writing the Allen Brain RMA values. After this procedure the expression summaries were Atlas contained in situ hybridizations of close to 17,000 genes mapped across all anatomical segments of the cord for juveniles (P4) and transformed back to log2 scale and inspection of their distributions showed that all microarray RMA profiles followed the average dis- adults (P56) of the C57BL/6J mouse strain. Of the 68 genes contained tribution throughout the intensity range, validating the microarray in Tables 1–3, in situ hybridization data were available for 63 genes preprocessing steps (Supplemental Fig. S1A).1 To further examine the in the Allen Brain Atlas. These summaries are included in Tables 1, 2, separation of experimental groups principal components analysis was and 3, where the level of expression in the ventral horn is scaled from low performed on the RMA profiles, which showed a nice separation into to high as: low: *, medium: **, high: ***, very high: ****. Absent: minus two groups of injured and uninjured animals (Supplemental Fig. S1B). (Ϫ) and Not Available: NA. We assume that the expression of genes in RMA expression summaries based on alternative probe mappings motor neurons of the uninjured spinal cord of the mouse also provide to Ensembl exons were also used to examine the possible regulation strong evidence for their expression in the rat, an evolutionary closely of splice variants (Dai et al. 2005) (Brainarray project, Molecular and related species with very similar locomotor behavior. Behavioral Neuroscience Institute [MBNI], version 11). The RMA expression summaries together with the raw CEL files Real-time reverse-transcription PCR for all microarrays were submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) hosted by the National Real-time reverse transcription PCR (real-time RT-PCR) was used Center for Biotechnology Information and can be accessed under to validate the regulation of expression for 10 genes detected as accession number GSE16710. significantly DE in the array analysis (from Tables 1–3): Scn1b, Scn9a, Slc18a3, Cacng2, Grina, Grin3b, Kcnc3, Clcn3, Nrg1, and ␤ Differentially expressed genes Calm1. Sodium/potassium-transporting ATPase subunit -1 (Atp1b1) and 40S ribosomal S18 (Rps18) were chosen as normalization Differentially expressed (DE) genes were determined separately for genes (i.e., endogenous controls) based on the microarray data, where the two time points 21 and 60 days postinjury—i.e., Spi-21 and Spi-60 they showed a consistent and conserved level of expression across all samples. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and ␤-ac- 1 The online version of this article contains supplemental data. tin (Actb) are both frequently used as endogenous reference housekeeping

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TABLE 1. Significantly differentially expressed genes relating to ion channels

Allen Brain Atlas FDR Ratio Gene Protein Juvenile Adult Affy ID Symbols Symbols Description Inj-21 Inj-60 Inj-21 Inj-60 (P4) (P56)

Calcium Channels 1371175_a_at Cacna1b Cav2.2 Voltage-dependent N-type , subunit alpha-1B ϩϩϩ 1.38 ** ** 1369086_a_at Cacna1d Cav1.3 Voltage-dependent L-type calcium channel subunit alpha-1D ϩϩϩ Ϫ1.45 ** ** 1374717_at Cacnb2 CAB2 Voltage-dependent L-type calcium channel subunit beta-2 ϩϩ Ϫ1.45 * *** 1371039_at Cacnb4 CAB4 Voltage-dependent calcium channel, subunit beta 4 ϩϩϩ Ϫ1.67 ** *** Sodium Channels 1369662_at Scn2a1 Nav1.2 Voltage-gated Naϩ channel, type 2, subunit alpha ϩϩϩ Ϫ1.56 NA NA 1368539_at Scn9a Nav1.7 Voltage-gated Naϩ channel, type 9, subunit alpha ϩϩϩ ϩϩϩ Ϫ4.97 Ϫ3.29 * 1387010_s_at Scn1b SCN1B Voltage-gated Naϩ channel subunit beta-1 ϩϩϩ 1.66 *** **** 1383435_at Scn3b SCN3B Voltage-gated Naϩ channel subunit beta-3 ϩϩϩ Ϫ3.82 — **

Chloride Channels Downloaded from 1378658_at Clca6 CLCA6 calcium activated 6 ϩϩϩ 1.36 —* 1367893_a_at Clcc1 CLCC1 Chloride channel CLIC-like protein 1 ϩϩϩ Ϫ1.7 — * 1392453_at Clcn3 CLCN3 Chloride channel protein 3 ϩϩϩ ϩϩϩ 4.6 5.33 *** *** 1380547_at Clcn3 CLCN3 Chloride channel protein 3 ϩϩϩ ϩϩϩ 2.08 2.99 *** *** Potassium Channels 1370545_at Kcna1 Kv1.1 Voltage-gated Kϩ channel subfamily A () member 1 ϩϩϩ 1.96 *** *** 1369043_at Kcna4 Kv1.4 Voltage-gated Kϩ channel subfamily A (shaker) member 4 ϩϩϩ Ϫ2.24 — *

1378738_at Kcnab1 KCAB1 Voltage-gated Kϩ channel subunit beta-1 ϩϩϩ Ϫ1.75 *** *** http://jn.physiology.org/ 1370439_a_at Kcnc2 Kv3.2 Voltage-gated Kϩ channel subfamily C (Shaw) member 2 ϩϩϩ Ϫ2.41 NA NA 1389120_at Kcnc3 Kv3.3 Voltage-gated Kϩ channel subfamily C (Shaw) member 3 ϩϩϩ ϩϩϩ 2 1.97 *** *** 1370958_at Kcnc3 Kv3.3 Voltage-gated Kϩ channel subfamily C (Shaw) member 3 ϩϩ ϩϩϩ 1.37 1.39 *** *** 1388162_a_at Kcnc3 Kv3.3 Voltage-gated Kϩ channel subfamily C (Shaw) member 3 ϩϩ 1.25 *** *** 1369133_a_at Kcnc3 Kv3.3 Voltage-gated Kϩ channel subfamily C (Shaw) member 3 ϩϩϩ 1.75 *** *** 1369134_x_at Kcnc3 Kv3.3 Voltage-gated Kϩ channel subfamily C (Shaw) member 3 ϩϩ 1.36 *** *** 1386770_x_at Kcne2 KCNE2 Voltage-gated Kϩ channel subfamily E (Isk) member 2 ϩϩ Ϫ1.41 ** ** 1368343_at Kcnh2 Kv11.2 Voltage-gated Kϩ channel subfamily H member 2 ϩϩϩ ϩϩϩ 1.34 1.38 — ** 1368793_at Kcnj2 Kir2.1 Inwardly rectifying Kϩ channel subfamily J member 2 ϩϩϩ 1.31 * ** 1369035_a_at Kcnj6 Kir3.2 G protein-activated inward rectifier Kϩ channel 2 (GIRK2) ϩϩϩ Ϫ1.59 —* by 10.220.33.3 on March 28, 2017 1387477_at Kcnk12 THIK-2 Kϩ channel subfamily K (Tandem pore domain) member 12 ϩϩϩ 1.52 NA NA 1370342_at Kcnk2 TREK-1 Kϩ channel, subfamily K (Tandem pore domain), member 2 ϩϩ ϩϩ Ϫ1.62 Ϫ1.51 * ** 1387441_at Kcnk3 TASK-1 Kϩ channel subfamily K (Tandem pore domain) member 3 ϩϩϩ 1.63 NA NA (Acid-sensitive) (KT3.1) 1370111_at Kcnn2 Kca2.2 Small conductance calcium-activated ϩϩϩ ϩϩϩ Ϫ1.67 Ϫ1.72 * * protein 2 1368751_at Kcns3 Kv9.3 Voltage-gated Kϩ channel subfamily S (Delayed-rectifier) ϩϩ 1.4 * * member 3 1385226_at Kctd11 KCD11 Potassium channel tetramerisation domain containing 11 ϩϩ Ϫ1.35 ** genes in real-time RT-PCR experiments, but both appear on the list of Real-time RT-PCR with TaqMan MGB probes was performed ac- significantly DE genes (Supplemental Tables S1 and S2). This is not cording to manufacturer’s instructions. Ϫ⌬⌬ unreasonable because injury not only may induce changes in cytoskeletal The 2( CT) method (Livak and Schmittgen 2001) was used to structures but also may regulate cellular metabolism. quantify the difference in mRNA levels between samples and endog- Real-time PCR was performed on cDNA reverse transcribed from enous controls. The analysis was done in R on data exported from the two linear round amplified and biotinylated aRNA (i.e., excess aRNA ABI Prism 7000 SDS software (Applied Biosystems). from microarray experiments not used for hybridization). TaqMan Reverse Transcription Reagents (Applied Biosystems) were used for Software the RT reaction and the cDNA was primed with random hexamers. TaqMan Gene Expression Assays and TaqMan Universal PCR Master The microarray and real-time RT-PCR analyses were done using R Mix were used in the real-time PCR reactions (Applied Biosystems). (http://www.r-project.org/) and Bioconductor (http://bioconductor.org). The TaqMan primers chosen for the TaqMan gene expression assays Cyber-T source code was obtained from the website http://cybert. all targeted the last exon boundary of their transcript to ensure microarray.ics.uci.edu/. Separate R scripts for filtering, analyses, ranking, detection after two rounds of linear amplification and their identifiers and plotting microarray data as well as for the real-time RT-PCR analysis are listed in Supplemental Table S3. Three individual samples from were developed and can be obtained from the authors on request. each experimental group (Spi-21, Spi-60, ShamC-21, and ShamC-60) were assayed in three replicates and the input amount of the respective RESULTS cDNA template was consistently 0.1 ␮g per 50 ␮l PCR reaction. The real-time PCR reactions were performed in a ABI Prism 7000 Se- Development of spasticity after spinalization in the rat quence Detection System (Applied Biosystems) under the following tail model thermal cycling conditions: 1) incubation at 50°C for 2 min, 2) incubation at 95°C for 10 min, and 3) amplification for 55 cycles of Clinical evaluation of spasticity as well as reflex quantification 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). were performed in all spinalized rats before removal of the spinal

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TABLE 2. Significantly differentially expressed genes relating to neuro-transmitter receptors

Allen Brain Atlas FDR Ratio Gene Protein Juveline Adults Affy ID Symbols Symbols Description Inj-21 Inj-60 Inj-21 Inj-60 (P4) (P56)

Glutamate Receptors and related genes 1396696_at Gria4 GluR4 Glutamate receptor, ionotropic, AMPA 4 ϩϩϩ ϩϩϩ Ϫ1.96 Ϫ1.48 **** **** 1369128_at Grik5 GRIK5 Glutamate receptor, ionotropic, kainate 5 ϩϩϩ ϩϩϩ 1.38 1.69 *** * 1368572_a_at Grin1 NR1 Glutamate receptor, ionotropic, NMDA ϩϩϩ 1.61 **** *** receptor subunit zeta-1 1387559_at Grin3b NMDAR3B Glutamate receptor, ionotropic, NMDA3B ϩϩϩ ϩϩϩ 2.2 2.37 ** * 1372724_at Grina NMDARA1 Glutamate receptor, ionotropic, NMDA- ϩϩϩ ϩϩϩ 1.63 2.42 *** *** associated protein 1 1398889_at Grinl1a GL1AD Glutamate receptor, ionotropic, ϩϩ Ϫ1.4 **** ** NMDA-like 1A 1387286_at Grm1 mGluR1 Metabotropic glutamate receptor 1 ϩϩϩ Ϫ2.64 * ** Downloaded from 1368690_a_at Grm4 mGluR4 Metabotropic glutamate receptor 4b ϩϩ 1.68 ** ** 1368759_at Cacng2 CCG2 Transmembrane AMPA receptor regulator ϩϩϩ ϩϩϩ 4.86 6.85 ** ** protein (TARP), stargazine Acetylcholine Receptors and Related Genes 1369252_a_at Chma4 ACHA4 Acetylcholine receptor subunit alpha-4 ϩϩϩ ϩϩ 1.37 1.27 **** *** 1369845_at Chma6 ACHA6 Acetylcholine receptor subunit alpha-6 ϩϩϩ 1.31 — * 1398928_at Cuta CUTA Acetylcholinesterase putative membrane ϩϩϩ 1.41 * *** anchor http://jn.physiology.org/ 1370607_a_at Nrg1 SMDF Pro-neuregulin-1, Acetylcholine ϩϩϩ ϩϩϩ 2.49 2.7 *** *** receptor-inducing activity 1371211_a_at Nrg1 SMDF Pro-neuregulin-1, Acetylcholine ϩϩϩ Ϫ1.53 *** *** receptor-inducing activity 1368615_a_at Slc18a3 VAChT Vesicular acetylcholine transporter ϩϩϩ 2.79 **** **** GABA Receptors and Related Genes 1369371_a_at Gabbr1 GABAR1 GABA-B receptor 1 ϩϩϩ 1.37 **** **** 1380828_at Gabra1 GBRA1 GABA(A) receptor subunit alpha-1 ϩϩϩ Ϫ1.69 —*

1371057_at Gabra5 GBRA5 GABA(A) receptor subunit alpha-5 ϩϩϩ Ϫ1.6 *** * by 10.220.33.3 on March 28, 2017 1391653_at Gabrg2 GBRG2 GABA(A) receptor subunit gamma-2 ϩϩϩ Ϫ1.49 ** *** 1378842_at Gabarapl1 GBRL1 GABA(A) receptor-associated protein-like 1 ϩϩϩ 1.63 **** **** 1380170_at Gabarapl2 GBRL2 GABA(A) receptor-associated protein-like 2 ϩϩϩ Ϫ1.98 —* 1370176_at Trak2 TRAK2 GABA(A) receptor-interacting factor 1 ϩϩϩ 1.59 ** ** (GRIF-1) Glycinergic Receptors 1371156_a_at Glra1 GLRA1 Glycine receptor subunit alpha-1 ϩ 1.26 *** *** Serotonergic Receptors 1369456_at Htr2b 5HT2BR Serotonin receptor 2B ϩϩ Ϫ1.49 —— Adrenergic Receptors 1368534_at Adra1d ADA1D Alpha 1D-adrenoreceptor ϩϩϩ 1.62 ** *** 1388757_at Adrbk1 ARBK1 Beta-adrenergic receptor kinase 1 ϩϩϩ 1.73 * * Dopaminergic Receptors and related genes 1387520_at Drd4 DRD4 Dopamine D4 receptor ϩ 1.28 — * 1376345_at Caly CALY D1 dopamine receptor-interacting protein ϩϩϩ 1.42 **** ** calcyon (Drd1ip) cord while the animals were immobilized in a test tube (Fig. 1A). tion of the reflexes shown in Fig. 1D (data from 15 rats). The two The clinical evaluation of spasticity was performed using the reflex components progress slightly differently. The initial reflex stretch-rub-maneuver and pinching (see METHODS). In the acute component increases gradually until 14–21 days postinjury, after phase the tail is completely flaccid and does not respond to the which it levels off and remains at a constant level. In contrast, the stretch-rub maneuver or pinching. After 1 wk the first signs of late reflex component continues to develop throughout the record- spasticity are observed, which then gradually develop Յ8wk ing period. These data show the presence of hyperreflexia at day following the lesion (Fig. 1B). Spi-21 rats all showed moderate 21 with a further increase in severity until 60 days postinjury in spasticity, characterized by hypertonus and occasionally clonus of accordance with previous studies (Bennett et al. 1999, 2004). the tail, and were rated 2–3. Spi-60 rats showed severe spasticity and were rated 4–5, characterized by severe hypertonus, clonus, Transcriptional response of motor neurons to the injury and coiling of the tail. After clinical evaluation the animals were tested electrophysiologically. All animals showed two reflex com- To determine the transcriptional response in motor neurons of ponents, an initial reflex with a delay of around 25–30 ms, and a spinalized rats we compared samples of Spi-21 or Spi-60 with late tonic reflex at 50–500 ms (Fig. 1C). The size of the reflexes their sham-operated counterparts, ShamC-21 and ShamC-60, re- was measured at stimulus strengths 5ϫMT, with the quantifica- spectively. To extract the significantly DE genes we used a

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TABLE 3. Significantly differentially expressed genes relating to intracellular pathways affecting motor neuron excitability

Allen Brain Atlas FDR Ratio Gene Protein Juvenile Adult Affy ID Symbols Symbols Description Inj-21 Inj-60 Inj-21 Inj-60 (P4) (P56)

Calmodulin and CaM kinase 1369937_at Calm1 CALM Calmodulin (CaM) ϩϩϩ ϩϩϩ 1.78 1.45 NA NA 1369993_at Camk2g KCC2G CaM kinase II gamma ϩϩϩ 1.93 **** **** Calcium Binding Proteins 1369886_a_at Cabp1 CaBP1 Calcium-binding protein 1, Caldendrin ϩϩϩ 1.6 — * 1387685_at Freq NCS-1 Neuronal calcium sensor 1, Frequenin homolog ϩϩ 1.29 ** ** Protein Kinase A and Related Genes 1386950_at Ppp1cb PP-1B Serine/threonine-protein phosphatase PP1-beta catalytic ϩϩϩ ϩϩϩ Ϫ1.68 Ϫ2* ** subunit Downloaded from 1390990_at Ppp1cb PP-1B Serine/threonine-protein phosphatase PP1-beta catalytic ϩϩϩ ϩϩϩ Ϫ1.75 Ϫ2.18 * ** subunit 1367637_a_at Ppp1cc PP-1G Serine/threonine-protein phosphatase PP1-gamma catalytic ϩϩϩ ϩϩ Ϫ1.54 Ϫ1.5 ** *** subunit 1388805_at Ppp2ca PP2A-alpha Serine/threonine-protein phosphatase 2A catalytic subunit ϩϩϩ Ϫ1.72 *** *** alpha 1371578_at Prkaca PKA C-alpha cAMP-dependent protein kinase, alpha-catalytic subunit ϩϩϩ ϩϩϩ 1.58 2.17 **** **** 1384772_at Prkacb PKA C-beta cAMP-dependent protein kinase, beta-catalytic subunit ϩϩϩ ϩϩϩ Ϫ1.73 Ϫ1.4 **** *** Protein Kinase C http://jn.physiology.org/ 1370585_a_at Prkcb PKC-B Protein kinase C beta type ϩϩϩ ϩϩϩ Ϫ2.59 Ϫ2.92 ** ** Protein Tyrosine Phosphatase 1368516_at Ptpra R-PTP-alpha Receptor-type tyrosine-protein phosphatase alpha ϩϩ ϩϩϩ Ϫ1.32 Ϫ1.62 — * 1368412_a_at Ptpro R-PTP-O Protein tyrosine phosphatase, receptor type, O ϩϩϩ ϩϩϩ Ϫ1.71 Ϫ2.19 *** ** 1370362_at Ptpm R-PTP-N Receptor-type tyrosine-protein phosphatase-like N ϩϩϩ 1.68 *** *** 1368358_a_at Ptprr R-PTP-R Receptor-type tyrosine-protein phosphatase R ϩϩϩ ϩϩϩ Ϫ1.61 Ϫ1.6 — * Anion Transporters 1368772_at Slc4a3 AE3 Anion exchange protein 3, ClϪ transporter ϩϩϩ ϩϩϩ 1.41 2.01 ** ** by 10.220.33.3 on March 28, 2017 conglomerate classifier based on three statistical methods: minus prefix. These ratios are furthermore plotted in Fig. 3A limma, Cyber-T, and SAM (see METHODS). Each constitutes a for Spi-21 versus ShamC-21 and in Fig. 3B for Spi-60 versus modified t-test with alternative ways of calculating the expres- ShamC-60, including error bars to give a visual impression of sion variance and, taken together, they constitute a good the significance of regulation. Note that a gene needs only to be estimate of likely differential expression (Ryge et al. 2008). A classified as significantly DE at one time point to be included list of significantly DE genes was thus created for the two time in Table 1 (data included for genes only at time points of points, each containing the average number of genes that pass significant DE). Figure 3, A and B on the other hand includes a 1% FDR. For Spi-21 and Spi-60 this produces two lists of the ratios for all genes contained in Table 1 irrespective of 1,452 and 1,841 DE genes, respectively (Supplemental Tables significance at a give time point. This illustrates that although S1 and S2). the magnitude of gene regulation (i.e., ratios of Spi/ShamC) changes over time, most genes relating to ion channels are Differentially expressed genes affecting ionic conductances changing their expression in the same direction 21 and 60 days postinjury, even for genes not reaching a significance level of From the two lists of DE genes 21 and 60 days postinjury 1% FDR at one of the two time points. (Supplemental Tables S1 and S2), the candidates that may have Inward currents. Calcium and sodium ion channels conduct a direct effect on the intrinsic membrane excitability were inward currents and up-regulation of these channel complexes can extracted and are displayed in Table 1. These encompass the thus lead to increased membrane excitability. Calcium voltage- genes coding for the protein subunits that constitute the cal- gated channels are classified into five groups according to their cium channels, sodium channels, chloride channels, and potas- distinctive conductance properties, determined by their core pore- sium channels. Table 1 contains 31 probe sets representing 26 forming protein subunits. The five channel families contain the unique genes, where two genes are represented by two or more following protein subunits: L-type (Cav1.1–Cav1.4), P/Q-type probe sets (Clcn3 and Kcnc3). The level of significance within (Cav2.1), N-type (Cav2.2), R-type (Cav2.3), and T-type (Cav3.1– a 1% FDR of either Spi-21 or Spi-60 is indicated by: ϩ, one of the Cav3.3). Two genes coding for different ␣ subunits undergo three statistical methods classify the gene as significant; ϩϩ, two of regulation as a response to injury—i.e., Cacna1b (Cav2.2) and the statistical methods classify the gene as significant; or Cacna1d (Cav1.3). Both genes are significantly DE in Spi-60, ϩϩϩ, all three methods classify the gene as significant. The where Cacna1b is up-regulated and Cacna1d is down-regulated. ϩ ␤ ␤ absence of signifies no significant expression of that gene Two genes coding for calcium channel subunits are DE— 2 ␤ within a 1% FDR at that time point. The table also contains the (Cacnb2) and 4 (Cacnb4)—both of which are down-regulated. ratios of regulation, where down-regulation is indicated with a Cacnb4 is down-regulated in both Spi-21 and Spi-60, whereas

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Spi-21 A

Spi-60 B Downloaded from http://jn.physiology.org/ Spi-21 C by 10.220.33.3 on March 28, 2017

Spi-60 D

Spi-21 E

Spi-60 F

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Cacnb2 is significant only in Spi-21. The gene Cacng2 also shows tors were extracted from the two lists of DE genes and are a response to the injury. This gene was initially classified as a shown in a separate table (Table 2). This extraction includes ␥ ␥ calcium channel subunit ( 2), but has recently been shown to receptors of glutamate, acetylcholine, glycine, and ␥-aminobu- belong to the TARP family and will therefore be described in the tyric acid (GABA) as well as monoamine receptors. Table 2 subsequent section Regulation of excitatory synaptic transmission. contains 27 distinct genes belonging to this class, where only For the voltage-gated sodium channels 2 of the 11 known ␣ one gene is represented with more than one probe set (Nrg1). subunit genes (Scn1a through Scn11a) are DE: Scn2a1 Levels of significance and expression ratios are indicated as in (Nav1.2) and Scn9a (Nav1.7). Both are down-regulated. Two Table 1 for Spi-21 and Spi-60. The ratios of expression are ␤ genes of the subunits are also DE: Scn1b and Scn3b. Scnb1 plotted for all genes in Table 2 and for Spi-21 and Spi-60 in is up-regulated in both Spi-21 and Spi-60, whereas Scnb3 is Fig. 3, C and D, respectively. Note again the similar pattern of down-regulated in Spi-60. gene regulation at the two time points, even for genes not Outward currents. Chloride and potassium ion channels significantly DE in both because absolute ratios may differ. conduct outward, hyperpolarizing currents on activation, op- Excitatory neurotransmitter receptors. Glutamate and acetyl- posing the effect of sodium and calcium channels. The potas- choline receptors belong to the excitatory class of receptor chan- sium channel family constitutes the largest group in this set nels. For the DE genes relating to ionotropic glutamate receptors, (Table 1, 15 of 31 genes). the gene coding for the ␣-amino-3-hydroxy-5-methyl-4-isox- Downloaded from Among the genes coding for potassium channel subunits that azolepropionic acid (AMPA) glutamate receptor 4 (Gria4)is undergo regulation as a response to injury, we find transcripts down-regulated, whereas the gene coding for the kainate gluta- of calcium-activated, inward-rectifying, voltage-gated, and mate receptor 5 (Grik5) is up-regulated. Up-regulation is also seen tandem pore domain (leak) channels. Genes relating to the for one gene coding for a protein belonging to the TARP family voltage-gated type family seem to constitute the largest group (Cacng2), known to regulate the localization of AMPA receptors of DE potassium channels (8 of 15 genes). Genes belonging to ␥ as well as modulate their channel conductivity: 2 (stargazin). http://jn.physiology.org/ the voltage-gated family include two genes of the Shaker Four genes related to the N-methyl-D-aspartate (NMDA) subfam- subfamily (Kcna1 and Kcna4), two genes of the Shaw subfam- ily are DE (Grin1, Grin3b, Grina, and Grinl1a), suggesting that ily (Kcnc2 and Kcnc3), one gene of the Isk-related subfamily the NMDA complex undergoes strong regulations as a (Kcne2), one gene of the subfamily H (Kcnh2), and one gene response to the injury. Grinl1a is down-regulated, whereas of the delayed-rectifier family (Kcns3). One gene that codes for the other three are up-regulated. Two genes belonging to the ␤ the modulatory subunits belonging to the Shaker subfamily metabotropic glutamate receptor subfamily are DE: Grm1 is also found to be DE (Kcnab1). Of the other types, one gene (mGluR1) is down-regulated, whereas Grm4 (mGluR4) is belongs to the calcium-activated potassium channels of the SK 2ϩ up-regulated. (small conductance Ca -activated) subfamily (Kcnn2), two For acetylcholine receptors, primarily genes coding for the by 10.220.33.3 on March 28, 2017 genes belong to the inward-rectifying family (Kcnj2 and nicotinic receptors undergo regulation in response to the injury. Kcnj6), and three genes belong to the tandem domain family Genes of two ␣ subunits are up-regulated (Chrna4 and (Kcnk2, Kcnk3, and Kcnk12). Finally, one potassium channel Chrna6). One gene possibly involved in the anchoring of tetramerization-domain-containing gene is DE (Kctd11). Note ϩ acetylcholinesterase in the membrane (Cuta) together with the here that 8 of the 15 genes related to K channels are clearly gene coding for the vesicular acetylcholine transporter down-regulated in either Spi-21 (Kcna1b, Kcnc2, Kcnj6, and (VAChT, Slc18a3) are up-regulated as a response to the injury. Kctd11), Spi-60 (Kcna4 and Kcne2), or both (Kcnk2 and One transcript related to maturation and maintenance of ace- Kcnn2). The rest are up-regulated in either Spi-60 (Kcna1, tylcholine receptor system has two probe sets that show op- Kcnj2, Kcnk3, Kcnk12, and Kcns3) or both (Kcnc3 and Kcnh2). posing direction of regulation (Nrg1), indicating regulation of The chloride channels are not as well characterized as the different splice variants (see REGULATION OF SPLICE VARIANT other classes of ion channels. One gene (Clca6) coding for a TRANSCRIPTS). Taken together these DE genes indicate that the calcium-sensitive chloride channel is up-regulated in Spi-21 nicotinic receptor complex undergoes regulation as well as the animals. Two other genes coding for voltage-gated chloride machinery for cholinergic synaptic transmission. channels were DE as a response to the injury, where one is Inhibitory neurotransmitter receptors. GABA and glycine up-regulated (Clcn3) and one is down-regulated (Clcc1). receptors belong to the inhibitory class of receptor channels. Most of the regulated genes coding for GABA-receptor sub- Differentially expressed genes related to units relate to the ionotropic GABAA subfamily, i.e., two genes neurotransmitter receptors of the ␣ subunits (Gabra1 and Gabra5) and one gene of the ␥ subunit (Gabrg2) are down-regulated. Three genes putatively In a similar fashion to Table 1, the genes relating to recep- involved in GABAA-receptor trafficking and clustering in the tors of the most common neurotransmitters and neuromodula- membrane are DE, with two being up-regulated (Gabarapl1

FIG. 3. Average expression ratios of genes affecting motor neuron excitability. The average ratios of expression for genes contained in Tables 1, 2, and 3 for Spi-21 and Spi-60 with respect to their sham-operated counterpart. Error bars signify SE of expression ratios and horizontal dotted lines are drawn at Ϯ1.3 ratio of expression. A: expression ratios of genes relating to ion channels (Table 1) for Spi-21 vs. ShamC-21. Color code: red, calcium channels; green, sodium channels; blue, chloride channels; dark blue, potassium channels. B: the same as A but for Spi-60 vs. ShamC-60. C: expression ratios of genes relating to neurotransmitter receptors (Table 2) for Spi-21 vs. ShamC-21. Color code: red, glutamate receptors; yellow, acetylcholine receptors; green, GABA receptors; turquoise, glycine receptor; light blue, serotonin receptors; dark blue. adrenergic receptors; purple: dopamine receptors. D: the same as C but for Spi-60 vs. ShamC-60. E: expression ratios of genes relating to intracellular pathways affecting motor neuron excitability (Table 3) for Spi-21 vs. ShamC-21. Color code: red, calmodulin and CaM kinase; yellow, Ca2ϩ binding proteins; green, protein kinase A and related genes; light blue, protein kinase C; dark blue, protein tyrosine phosphatases; purple, ClϪ cotransporters. F: same as E but for Spi-60 vs. ShamC-60.

J Neurophysiol • VOL 103 • FEBRUARY 2010 • www.jn.org 770 WIENECKE, WESTERDAHL, HULTBORN, KIEHN, AND RYGE and Trak2) and one being down-regulated (Gabarapl2). Only The expression of the gene coding for the protein tyrosine one gene belonging to metabotropic GABAB subfamily is kinase Src, known to modulate the function of both NMDA- up-regulated (Gabbr1). One of the four ␣ subunit genes be- and GABAA-receptor complexes, was not changed after spinal longing to glycine receptors is up-regulated (Glra1). cord injury. Several genes coding for protein tyrosine phos- Modulatory neurotransmitter receptors. Monoaminergic phatase (PTP) receptor subunits were down-regulated both in receptors are important for modulating motor neuron excit- Spi-21 and Spi-60. These include PTP receptor type ␣ (Ptpra), ability and were therefore included in Table 2. Four of the PTP receptor type O (Ptpro), PTP receptor type N (Ptprn), and five DE genes related to this class of receptors are up- PTP receptor type R (Ptprr). One gene coding for the anion exchange protein 3 (Slc4a3) regulated as a response to injury. One gene of the serotonin Ϫ receptor family (Htr2b) is down-regulated in Spi-21. Two of that may accumulate Cl in the motor neurons was up-regu- lated in Spi-21 and Spi-60. the DE genes belong to the adrenergic receptor family, which comprise two classes of G-protein–coupled recep- Regulation of splice variant transcripts tors, ␣ and ␤. One gene belonging to the ␣ receptor ␣ subfamily is up-regulated, 1 (Adra1d). A transcript of the In many cases the microarray contains several probe sets ␤ that target the same gene at various locations. For two genes receptor kinase that phosphorylates the -2 adrenergic re- Downloaded from ceptor is also up-regulated (Adrbk1). The D2-like dopamine relating to ion channels (Table 1: Clcn3 and Kcnc3), one receptor gene (Drd4) is up-regulated in Spi-60 together with gene relating to receptors (Table 2: Nrg1), and one gene a gene of the D1 receptor interacting protein (Caly) up- relating to intracellular pathways (Table 3: Ppp1cb), multi- regulated in Spi-21. ple probe sets show significant DE. One of these genes has probe sets that exhibit opposing direction of regulation (Nrg1). This apparent discrepancy could be due to a differ-

Differentially expressed genes related to intracellular ential regulation of different splice variants. To investigate http://jn.physiology.org/ pathways affecting motor neuron excitability this possibility we had to use alternative probe set mappings where the probes have been mapped specifically to Ensembl Channel and receptor functions are subject to modulation exons (Dai et al. 2005), since the probe sets of the by various intracellular pathways. In particular, we focus on RAT230_2.0 chip are neither transcript nor exon specific. DE genes coding for proteins involved in intracellular path- RMA summaries were calculated for these new probe sets. ways that have been shown to modulate persistent inward Overall, this analysis confirmed our findings based on the currents as well as NMDA- and GABA -mediated synaptic A original probe sets showing similar pattern of regulation in transmission, identified as the major receptor targets of the Ensembl exons and DE of the genes contained in Tables 1, transcriptional injury response (previous section). These by 10.220.33.3 on March 28, 2017 2, and 3 (data not shown). This analysis therefore supports genes are included in Table 3. The extracted categories the initial filtering conducted on Affymetrix probe sets include genes affecting 1) persistent inward calcium cur- ϩ based on the Ensembl annotations. Two of the exons of rents: calmodulin and Ca2 /calmodulin (CaM)-dependent ϩ Nrg1 were significantly DE within a 1% FDR exhibiting protein kinase as well as additional Ca2 -binding proteins; opposing ratio of expression, suggesting that Nrg1 most 2) persistent inward currents as well as GABAergic trans- likely undergo splice variant regulation 21 and 60 days mission: protein kinase A (PKA), protein kinase C (PKC), postinjury. and their counterbalancing phosphatases; and 3) NMDA and GABA transmission: protein tyrosine kinase and phospha- Validation with real-time RT-PCR tases. The table also contains DE genes coding for anion transporters, possibly affecting the reversal potential of To validate the results of the microarray analysis ten genes inhibitory ionotropic receptors, such as GABAA. In total were chosen for real-time RT-PCR (see also Ryge et al. 2008) Table 3 contains 16 probe sets representing 15 distinct among the significantly DE genes of Tables 1 and 2 (Scn1b, genes, where expression ratios and levels of significance are Scn9a, Slc18a3, Cacng2, Grina, Grin3b, Kcnc3, Clcn3, Nrg1, indicated as in Tables 1 and 2 for Spi-21 and Spi-60. The and Calm1). For the Nrg1 target transcript as well as Scn9a the ratios of expression are plotted for these probe sets in Fig. microarray analysis showed down-regulation, whereas all the 3, E and F for Spi-21 and Spi-60, respectively. other tested genes showed an up-regulation (see Tables 1 and The gene coding for Calmodulin 1 (Calm1) and the gene 2). The real-time RT-PCR validation of the microarray ratios coding for the CaM kinase II ␥ subunit (Camk2g) were up- 21 days postinjury is shown in Fig. 4A. The error bars of Fig. regulated. Two other genes coding for Ca2ϩ-binding proteins 4A indicate SEs of expression ratios. The same genes are were also up-regulated: Caldendrin (Cabp1) and Neuronal shown in similar fashion for Spi-60 compared with ShamC-60 calcium sensor 1 (Freq). The gene coding for the ␤-catalytic (Fig. 4B). Of the tested genes four exhibited amplification subunit for PKA (Prkacb) was down-regulated in both Spi-21 curves that leveled off below the signal threshold set for CT and Spi-60, whereas the gene for the ␣-catalytic subunit for determination, indicating the presence of a low number of PKA (Prkaca) was up-regulated at both time points. Three transcript copies for these genes. Of the remaining six genes all genes coding for phosphatases that may counteract PKA ac- show the same direction of regulation in the real-time RT-PCR tivity were all down-regulated: PP1-␤ (Ppp1cb), PP1-␥ (Ppp1 as in the microarray analysis in Spi-60. In Spi-21 the genes ml), and PP2-␣ (Ppp2ca). The gene for PKC beta (Prkcb1) was Scn1b, Grina, and Nrg1 are not DE because they show rather strongly down-regulated both in Spi-21 and Spi-60. Genes large variation of expression, whereas the remaining genes relating to phosphatidylinositol 3-kinase (PI3K) were not dif- Cacng2, Clcn3, and Calm1 confirm the microarray results at ferentially expressed. this time point.

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FIG. 4. Real-time RT-PCR validation. Ratios of expression for 6 genes (Scn1b, Cacng2, Grina, Clcn3, Nrg1, and Calm1) 21 and 60 days postinjury compared with their sham counterparts. A: gene expression ratios of Spi-21 compared with ShamC-21. B: the same as A but for Spi-60 compared with ShamC-60. Error bars illustrate the SE for each ratio. DISCUSSION chosen for real-time RT-PCR analysis validated the mi- Spasticity model and gene expression analysis croarray results. The four remaining gene transcripts did not by 10.220.33.3 on March 28, 2017 amplify above the signal threshold set for CT determination. For the purpose of the present project it was crucial to study The four transcripts did exhibit weak signal at late cycles the segmental effects of spinal cord injury in isolation without (cycles 35–45, not shown) and we speculate that in our very interference of compensatory or plastic effects resulting from small samples (100–200 cells) the amplification is limited any remaining descending fibers. We therefore used an injury for transcripts with relative low to medium copy numbers. model where the spinal cord is fully transected, effectively This included the genes Slc18a3 (VAChT) and Grin3b, causing a degeneration of all descending fibers, rather than which are both expressed ubiquitously in motor neurons using the other commonly used contusion or compression (Allen Brain Atlas), thus supporting the specificity and spinal cord injury model, in which descending fibers may be sensitivity of the microarray analysis. The low signal ob- spared (see Grill 2005 for a commentary of those models). To served with the TaqMan probes of the real-time RT-PCR for analyze the transcriptional regulation of motor neurons we these genes therefore seems to indicate that they are ex- adopted the method of Ryge and colleagues (2008), where it pressed in lower levels compared with the other real-time was shown that reliable differences in gene expression could be RT-PCR positive genes. This was also reflected in the detected between neuronal populations of the spinal cord based microarray intensities (not shown). We therefore conclude on microarrays originating from laser microdissected cells that the microarrays are sensitive to low expressed genes identified by retrograde labeling and with minimal contamina- including ion channels and receptors, enabling us to exam- tion from neighboring cells such as glia (Cui et al. 2006; Ryge ine the regulation of these as a response to injury. et al. 2008). Two strategies were used to support the microarray anal- Transcriptional response related to postinjury spasticity and ysis. To validate the presence of transcripts in motor neu- the return of plateau potentials rons of uninjured animals we examined in situ hybridization data obtained from Allen Brain Atlas. Of the 68 distinct The emergence of plateau potentials and increased motor genes shown in Tables 1, 2, and 3, 63 genes were available neuron excitability is likely to be influenced by many factors, in the database and 62 of these were present at some level in which can be divided into a few functional categories relating the ventral horn of the rodent spinal cord of either juvenile to 1) configuration that define the intrinsic mem- or adult animals, suggesting a role for these genes in motor brane properties, 2) receptors mediating fast synaptic transmis- neurons under normal physiological conditions. Since in situ sion or acting on “neuromodulatory” pathways, and 3) intra- hybridization cannot be used to quantify small changes in cellular pathways modulating membrane excitability and sig- gene expression within a given cell population, real-time nal transduction. RT-PCR was used on a small subset of genes to validate The patterns of regulation for the genes associated with these their regulation in the injured animals. Six of the 10 genes categories (Tables 1–3) are quite similar between the two time

J Neurophysiol • VOL 103 • FEBRUARY 2010 • www.jn.org 772 WIENECKE, WESTERDAHL, HULTBORN, KIEHN, AND RYGE points—i.e., whereas the magnitude of gene regulation might ing and gating of Cav1.2 in the absence of the ␤ subunits, differ, the transcriptional response largely changes in the same effectively resulting in slow inactivation of the channel (Ravind- direction 21 and 60 days postinjury (Fig. 3). The magnitudes of ran et al. 2008). CaM is known to be tethered constitutively to the regulation (ratios) seem to increase progressively for many L-type Ca2ϩ channels Cav1.2 and Cav1.3, where it works as a genes from Spi-21 to Spi-60, signified by a 20% increase of calcium detector responsible for both Ca2ϩ-dependent facilitation genes being classified as significantly DE in the motor neurons and inactivation (Halling et al. 2005; Perrier et al. 2000; Peterson 60 days (1,784 genes) compared with 21 days (1,502 genes) et al. 1999), balancing a positive and negative feedback regulation postinjury. This strongly suggests that the molecular mecha- of the persistent inward current, probably acting on different nism underlying the increased motor neuron excitability is timescales to prevent Ca2ϩ overload as well as refine channel shared 21 and 60 days postinjury, whereas the response is more signaling. The functional membrane targeting and gating of extensive 60 days postinjury, supporting the observed progres- Cav1.2 by CaM works through another mechanism independent sive increase in the late-phase hyperreflexia (Fig. 1). of Ca2ϩ binding, but relying on relatively high levels of CaM. The up-regulation of calmodulin (Calm1) together with the Ion channels down-regulation of the ␤ subunits could therefore indicate an altered membrane targeting of the Ca2ϩ channels with a po-

REGULATION OF INWARD CURRENTS. Our study shows that the tentially slower inactivation. The up-regulation of a gene Downloaded from expression of genes coding for both sodium and calcium coding for an alternative Ca2ϩ binding protein (Cabp1), which channels undergoes changes as a response to the injury. It is affects the inactivation of L-type channels, could also play a not known which type of L-type channels is involved in part in the observed Ca2ϩ-mediated persistent inward current generating the plateau in normal animals, although it has been seen in the chronic spinal phase. Cabp1 codes for a neuronal suggested that Cav1.3 may conduct the persistent Ca2ϩ current calmodulin-like Ca2ϩ binding protein that has been described in rat motor neurons (Li and Bennett 2003). Based on immu- to substitute for calmodulin as the Ca2ϩ sensor of both Cav1.2 nohistochemistry the Cav1.2 channel has been shown to be and Cav1.3, prolonging Ca2ϩ currents by preventing Ca2ϩ- http://jn.physiology.org/ up-regulated postinjury, compared with sham-operated animals dependent inactivation (Cui et al. 2007; Zhou et al. 2004). The with open dura (Anelli et al. 2007). Here we find that of the up-regulation of the gene coding for CaM kinase II gamma L-type channels the gene coding for Cav1.3 is down-regulated, (Camk2n1) further suggests that Cav1.2 and/or Cav1.3 con- but we observed no expressional change in the gene coding for ductivity may be enhanced. CaM kinase II has been shown to Cav1.2. It therefore seems unlikely that it is an increased cause a negative shift in L-type Ca2ϩ current activation (Calin- amount of Ca2ϩ channel ␣ subunits that are directly responsi- Jageman and Lee 2008; Gao et al. 2006). Persistent inward ble for the expression of the Ca2ϩ part of the plateau in the Ca2ϩ currents have also been shown to be promoted by injury state. Although the reports by the Bennett group focus stimulation of PI3K (Viard et al. 2004) and PKA pathways by 10.220.33.3 on March 28, 2017 on the supersensitivity to monoaminergic transmitters and their (Cav1.3; Dai et al. 2009; Qu et al. 2005), whereas PKC inhibits agonists following transection (Harvey et al. 2006a; Li et al. Cav1.3 (Baroudi et al. 2006). The concomitant up- and down- 2007), they also present evidence that suggests that the return- regulation of genes associated with PKA and PKC and down- ing persistent inward currents following spinal transection regulation of their counterbalancing phosphatases (Table 3) reach higher values than what can be achieved in the control therefore suggest a conglomerate modulatory effect by these on animals even after maximal monoaminergic facilitation. This is the persistent inward calcium current, effectively increasing the case both for the Naϩ PIC (Harvey et al. 2006c) and the conductance without affecting the channel composition. Ca2ϩ PIC (Li et al. 2007). However, the conductance kinetics One other gene coding for an ␣ Ca2ϩ-channel subunit not and activation curves of ␣ subunits can be modulated, both directly related to persistent Ca2ϩcurrent is up-regulated: the through physical modulations such as phosphorylation or N-type channel Cav2.2. This channel has been implicated with through altered composition of the channel complex with its synaptic release (Pardo et al. 2006; Schenning et al. 2006) and ancillary subunits. In light of this it therefore seems more could therefore be related to the apparent increase in the relevant to focus on the modulatory mechanisms affecting the machinery for acetylcholine release (see REGULATION OF EXCI- channels conducting the persistent inward current rather than TATORY SYNAPTIC TRANSMISSION). on the expression of the pore-forming ␣-subunits themselves. No separate sodium ion channel that solely conducts persis- Interestingly, we find the genes coding for the calcium channel tent inward currents has been described. It has therefore been ␤ subunits to be down-regulated, which could indicate both a hypothesized that the persistent inward currents conducted by regulation of L-type channel conductance dynamics as well as an sodium ion channels most likely are mediated through the same altered expression in the membrane. Calcium ␤ subunits can have channels that conduct transient currents, which are thought to facilitatory effect on the persistent inward current by shifting the have an additional slowly inactivating state (Ulbricht 2005). activation curve to more hyperpolarized potentials. The ␤ subunits This additional conductive state is also modulated by the ␤ also interact with other proteins involved in second-messenger subunits (Catterall et al. 2005a; Isom et al. signaling pathways as well as cytoskeletal structures, causing 1995; Zhou and Goldin 2004). Several genes pertaining to reorganization and incorporation of the target channels into the sodium channels were found to undergo regulation as a re- membrane and thus increasing the density of the channels con- sponse to injury. The genes coding for ␣ channel subunits ducting persistent Ca2ϩcurrent (Arikkath and Campbell 2003; Nav1.2 and Nav1.7 are both down-regulated. The sodium ␤ Catterall et al. 2005b; Dolphin 2003). Both Cabnb2 ( 2) and channel Nav1.6 is the major component of the sodium current ␤ Cacnb4 ( 4) are down-regulated, suggesting a modulation of the of spinal neurons of the rodent, but the expression of the gene Ca2ϩ channel gating and membrane targeting. Interestingly, it has coding for Nav1.6 was unaltered. PKA and PKC are known to been shown that Calmodulin (CaM) can induce membrane target- reduce the current in Nav1.2 but they have no reported regu-

J Neurophysiol • VOL 103 • FEBRUARY 2010 • www.jn.org DEVELOPMENT OF POSTINJURY SPASTICITY 773 latory effect on Nav1.6 (Cantrell and Catterall 2001; Dai et al. erate plateaux or at least contribute to a higher input resistance, 2009). The only reported regulation of Nav1.6 is by MAP which has been reported in Harvey et al. (2006a). The remain- kinase p38-␣ (Wittmack et al. 2005), which was not affected ing significantly DE genes relating to potassium channels by spinalization. Thus any effect on this channel appears to suggest a rather complex regulation of potassium currents in reside in the regulation of its subunits. motor neurons postinjury and the appearance of plateaux does ␤ not seem to be a result of a clear reduction in overall outward The up-regulation of the gene coding for the sodium 1 ϩ subunit (Scnb1) together with the down-regulation of the gene K currents. ␤ coding for the sodium 3 subunit (Scnb3) indicate altered intracellular trafficking of sodium channels as well as modu- Neurotransmitter receptors lation of their conductance kinetics. Experiments have shown ␤ ␤ ␣ REGULATION OF EXCITATORY SYNAPTIC TRANSMISSION. Several that coexpression of either 1 or 3 with sodium channel subunits can shift the steady-state inactivation curve as well as genes relating to ionotropic glutamate receptors were regulated the voltage dependence of channel activation in the hyperpo- after injury. The gene coding for one kainate receptor (Grik5) ␤ was up-regulated, whereas the gene coding for the AMPA larizing direction (Isom et al. 1995; Meadows et al. 2002). 1 also causes an additional increase in the expression of the ␣ receptor (Gria4) was down-regulated. Another gene up-regu- subunits in the plasma membrane not observed for ␤ in lated as a response to injury could affect the expression of Downloaded from 3 ␥ mammalian cell lines. The ostensible larger expression of AMPA receptors in the membrane, Cacng2 ( 2). It codes for a sodium channels in the plasma membrane could therefore transmembrane AMPA receptor regulatory protein (TARP) increase overall sodium conductances and a concomitant redi- (Chen et al. 2007) and its up-regulation suggests an increase of rection of channel trafficking to soma and dendrites, rather than AMPA receptor trafficking as well as in its expression in the to axons and terminals, could also increase the role of sodium plasma membrane. Most interesting among the glutamate re- ceptor regulations is the apparent strong regulation of the persistent inward currents. http://jn.physiology.org/ In summary, from previously published results showing NMDA-receptor complex. Four genes relating to this complex nonconditional expression of persistent inward currents in are regulated as a response to injury: Grin1 (NR1), Grin3b motor neurons through both Ca2ϩ and Naϩ channels in chronic (NR3B), Grina (NRA1), and Grinl1a (GL1AD). The up- spinal animals (Li and Bennett 2003), we might have expected regulation of the gene for NR1 suggests an increase in the to find an up-regulation of the genes coding for the pore- expression of the channel itself and promotes increased sensi- forming ␣ subunits of Cav 1.3 and the Naϩ channels that have tivity to synaptic glutamate transmission. Grinl1a is part of a a persistent component. Our results certainly demonstrate that complex transcript unit that is known to modulate NR1 and the changes in these channel complexes are far more complex seems to facilitate glutamatergic signal transduction and also and not least targeting modulatory subunits and intracellular possibly to serve a protective role against glutamate-mediated by 10.220.33.3 on March 28, 2017 pathways. excitotoxicity (Roginski et al. 2008). NR3B is a motor-neuron– specific NMDA-receptor modulatory subunit that reduces the REGULATION OF OUTWARD CURRENTS. Only one of the DE NMDA conductance as well as calcium permeability (Matsuda genes belonging to the family of chloride channels are involved et al. 2003). NRA1 also interacts with the NMDA-receptor in membrane conductance, i.e., a calcium-sensitive chloride complex, but its role in modulating signal transmission is not channel Clca6 (Piirsoo et al. 2009); this gene is up-regulated. known. Src protein tyrosine kinase enhances and potentiates Little is known about the electrophysiological properties and NMDA current, whereas protein tyrosine phosphatases (PTPs) the functional significance for neuronal signaling of this class have an opposing action (Salter and Kalia 2004). Although we of channels (Hartzell et al. 2005), but Ca2ϩ-activated ClϪ found no change in Src there was a clear down-regulation of channels have been implicated with the switch of action po- PTPs pathways, which potentially could further enhance the tential afterhyperpolarization to afterdepolarization in axoto- NMDA-receptor–mediated activity. Thus the overall regula- mized sympathetic ganglion cells (Sanchez-Vives and Gallego tion of the NMDA complex suggests a rather intricate regula- 1994) and vagal ganglion neurons (Lancaster et al. 2002). This tion, with increased sensitivity to glutamate resulting in in- depends on the reversal potential of ClϪ, which could be creased signal transmission at the same time as the Ca2ϩ affected toward a more depolarizing potential in motor neurons permeability decreases, possibly serving a neuroprotective postinjury, possibly contributing to the observed increase in excit- role. ability (see REGULATION OF INHIBITORY SYNAPTIC TRANSMISSION). The For acetylcholine receptors the up-regulation of the genes ␣ ␣ two remaining chloride channel genes are not known to influ- coding for the nicotinic receptors Chrna4 ( 4) and Chrna6 ( 6) ence membrane conductance, but are expressed in subcellular suggests an increased sensitivity to acetylcholine transmission organelles (Clcc1 and Clcn3). in the motor neurons. Three other genes related to acetylcho- The potassium channels comprise the largest ion channel line transmission are DE: Nrg1, Cuta, and Slc18a3 (VAChT). family that was regulated. A little more than half of the genes Slc18a3 and Cuta are up-regulated, whereas the two DE probe coding for potassium channels are down-regulated (see Table sets of Nrg1 indicate a regulation of splice variants, where one 1). Among these we find the gene Kcnn2, which codes for a is down-regulated and another is up-regulated. Nrg1 is known small-conductance calcium-activated potassium channel that as a gene coding for a transynaptic growth factor that increases generates the afterhyperpolarization in motor neurons. This acetylcholine receptor activity, stimulating the expression of conductance has been shown to be blocked by serotonin in acetylcholine receptor genes, the insertion of the receptors into motor neurons, leading to increased expression of plateaux the plasma membrane, and their assembly into clusters (Falls (Grunnet et al. 2004; Hounsgaard and Kiehn 1989). Its down- 2003; Sandrock Jr et al. 1997). Thus the altered expression of regulation could therefore signify an increased ability to gen- an Ngr1 splice variant might play a role in the up-regulation of

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2ϩ Chrna4 and Chrna6. The neuronal Ca sensor NCS-1 (Freq) of GABAA receptors. The intracellular regulation of GABAA has been shown to have a facilitating effect on synaptic receptors is complex and depends on the subunit composition transmission (Hilfiker 2003) and the up-regulation of its gene of the receptor complex. The functional effect of PKA and together with the increase of the gene coding for the acetyl- PKC phosphorylation of Ser/Thr residues can either increase or choline vesicular transporter, VAChT, indicates increased decrease the receptor transmission depending on the identity of ␤ ␤ ␤ ␤ transmitter release both at central synapses and at the neuro- the subunit in the receptor complex, i.e., 1, 2,or 3 muscular junctions. Together, these regulations of genes relat- (Poisbeau et al. 1999). Phosphorylation of tyrosine residues by ing to both pre- and postsynaptic cholinergic transmission Src has been shown to potentiate the GABAA-receptor function suggest an increased acetylcholine signaling at central syn- (Moss et al. 1995). Since little is known about the exact subunit apses that may enhance the recurrent excitation among motor composition of GABAA-receptor complexes of motor neurons, neurons, both by motor axon collaterals terminating directly it seems difficult to predict the conglomerate modulatory onto motor neurons (Nishimaru et al. 2005) and by a polysyn- effect on the GABAA-receptor function based on the ob- aptic positive feedback loop, which was recently described served complex regulation of the genes associated with (Machacek and Hochman 2006). The latter pathway seems to these intracellular pathways. It has also been shown in the be unmasked by the presence of noradrenaline. Local release of rat tail model that the reflex response in the chronic phase noradrenaline has been described recently in the spinal cord lacks an initial inhibitory phase present in normal animals, Downloaded from following transection, where a group of cells seems to absorb indicating that part of the reason for the pathologically this transmitter from the bloodstream through the damaged long-lasting motor discharge may be due to a lack of blood brain barrier, subsequently releasing it in the spinal cord inhibition failing to terminate the activity (Bennett et al. (Cohen et al. 2009; Rank et al. 2007, 2008). This might provide 1999; Li et al. 2004b). In combination with our present a mechanism that could unmask the polysynaptic feedback findings this implies that regulation of the GABAergic

loop in the injured cord, creating a very powerful positive system plays a part in the chronic pathological state. Inter- http://jn.physiology.org/ feedback loop between motor neurons. We also note that this estingly, a recent modeling study including a small network feedback mechanism can work via the peripheral synapses of motor neurons and recurrent inhibition through Renshaw through the reflex loop, where an increased cholinergic release cells has shown that a selective decrease in the GABAA at the neuromuscular junction that results in enhanced muscle inhibition can unmask motor neuron plateau potentials tone and activation of Ia afferents projecting directly onto (Venugopal et al. 2009), suggesting a role for the observed motor neurons could provide a similar powerful source of regulation in GABAA receptors in the expression of pla- feedback excitation. teaux in the injury state. The effect of inhibitory synapses can be further altered if the REGULATION OF INHIBITORY SYNAPTIC TRANSMISSION. Genes re- reversal potential for chloride is changed indirectly, leading to by 10.220.33.3 on March 28, 2017 lating to GABA receptors underwent a striking regulation that increased motor neuron excitability. Recent experiments have ϩ Ϫ seems to target GABAA receptors and their expression in the shown that the K -Cl cotransporter 2 (KCC2) responsible for membrane. Only one gene not relating to this family is DE, a ClϪ extrusion is down-regulated as a response to spinal cord gene coding for a GABAB receptor (Gabbr1), which is up- injury, effectively decreasing the reversal potential for chloride regulated. All the DE genes coding for GABAA receptor and pushing the effect of chloride channel activation toward ␣ subunits are down-regulated: two subunits (Gabra1 and more depolarizing potentials (Jean-Xavier et al. 2006; Vinay ␥ Gabra5) and a subunit (Gabg2). There are six members of and Jean-Xavier 2008). The gene for KCC2 is not included in ␣ ␣ ␣ the GABAA receptor family ( 1– 6), which form functional the Ensembl annotations used to filter the probe sets and could ␤ ␥ receptor complexes in combination with and subunits. therefore not be investigated in the present study. Two other Their distribution have been described in spinal interneurons ClϪ transporters have been shown to accumulate ClϪ in the (Geiman et al. 2002), but there are presently limited data embryonic motor neurons, Na-K-Cl cotransporter 1 (NKCC1) ␥ available on their distribution in spinal motor neurons. The 2 and anion exchange protein 3 (EA3), and their up-regulation is ubiquitously expressed throughout the brain and presumably could therefore also shift the ClϪ reversal potential (Gonzalez- participates in most functional combinations of the GABAA- Islas et al. 2009). We find no change in transcript levels for the receptor complex (Sieghart and Sperk 2002). Its down-regula- gene coding for NKCC1 (Slc2a2), but the gene coding for EA3 ␣ ␣ tion together with the genes coding for the 1 and 5 GABAA (Slc4a3) seems to be subject to up-regulation as a response to subunits suggest a general down-regulation of GABAA-recep- the injury. The combined effect of KCC2 down-regulation with tor complexes in the motor neurons postinjury. The GABAA- AE3 up-regulation could very well shift the reversal potential ␥ Ϫ receptor subunit 2 also interacts with the GABARAP family for Cl increasing the excitability of motor neurons through (GABAA receptor interacting proteins) that are involved in GABAergic as well as glycinergic synaptic transmission from GABAA-receptor trafficking and clustering (Chen and Olsen spinal interneurons. 2007) and it has previously been shown to be down-regulated in postural motor neurons postinjury in the neonatal rat REGULATION OF NEUROMODULATORY RECEPTORS. Increased (Khristy et al. 2009). The DE of the genes Gabrg2, Gabarapl1, sensitivity to 5-HT (Barbeau and Bedard 1981; Harvey et al. and Gabarapl2 indicates a strong regulation of GABAA loca- 2006a; Li et al. 2007) and noradrenalin (Rank et al. 2007) have tion and membrane expression. Trak2 is also involved in been observed after spinalization. This hypersensitivity is GABAA-receptor trafficking, further stressing that trafficking thought to contribute to the expression of plateaux in the is subject to a substantial regulation in response to the injury chronic spinal phase through serotonergic and noradrenergic for this receptor subfamily. Together this response points to mechanisms since 10% of 5-HT and noradrenalin levels remain reduced GABAergic transmission and a substantial regulation in the spinal cord in the chronic spinalized state (Cohen et al.

J Neurophysiol • VOL 103 • FEBRUARY 2010 • www.jn.org DEVELOPMENT OF POSTINJURY SPASTICITY 775

2009; Hadjiconstantinou et al. 1984; Newton and Hamill 1988; change is primarily caused by posttranscriptional modulation, Rank et al. 2008). One gene coding for the adrenergic ␣ leaving this modulation undetected in the present study if the receptor 1D (Adra1d) is up-regulated together with the gene underlying regulation of transcript expression remains un- coding for an adrenergic receptor kinase (Adrbk1), indicating a changed. However, if the transcript splice variants are subject role for these in the noradrenaline hypersensitivity. It has to expressional regulation, the changes in their transcript levels ␣ recently been shown that the activation of the adrenergic 1 will be detected, irrespective of the splice form expressed, and receptor result in a strong activation of both Ca2ϩ and Naϩ we therefore remain assured that we do detect the majority of persistent inward currents in the rat tail model (Harvey et al. the interesting changes in transcript regulation as a response to 2006b; Rank et al. 2007), suggesting a strong role for the injury. up-regulation of the gene Adra1d in increasing the excitability of motor neurons postinjury. One gene coding for a serotonin Conclusion receptor, Htr2b, is down-regulated. The 5-HT2b receptor has In the present study the global transcriptional response to been described in frog and respiratory rat motor neurons, spinal cord injury has been examined for a specific cell where it contributes to increased activity (Gunther et al. 2006; population in the rat spinal cord, the motor neurons. The Holohean and Hackman 2004). The down-regulation of the focus of the study was to correlate the changes in gene gene coding for this receptor seems to suggest a decreased expression of this cell population with the pathological state Downloaded from sensitivity to 5-HT, although the posttranscriptional modula- of injury-induced spasticity—i.e., to elucidate the molecular tion of other genes relating to the serotonin receptor family, mechanisms behind the increased motor neuron excitability possibly remaining undetected in the present work, has been and the appearance of plateaux in motor neurons following shown to increase motor neuron excitability (Murray et al. injury. Our analysis shows a complex regulation of genes 2008) (see Regulation of splice variants). There has been little related to modulation of inward depolarizing currents and a focus on the role of dopamine transmission in the expression of massive regulation of outward currents. Receptors involved http://jn.physiology.org/ plateaux in the postinjury phase, but the expression of its in ionotropic synaptic transmission are subject to even more receptors in the motor neurons of the spinal cord and its substantial lesion-related regulation. For all of the channels influence on motor neuron excitability have been documented and receptors that are subject to regulation the nonpore- (Han et al. 2007; Madriaga et al. 2004; Zhu et al. 2007). The forming modulatory subunits as well as intracellular path- up-regulation of two genes coding for the dopamine receptor 4 ways that affect their function are intricately involved in the (Drd4)andaD1 dopamine receptor-interacting protein (Caly), injury response. The channel ancillary subunits affect con- could therefore contribute to an increased excitability of the ductivity of existing channels and channel clustering in the motor neurons. plasma membrane and, until now, have not been associated with the enhanced excitability of the motor neurons in the by 10.220.33.3 on March 28, 2017 Regulation of splice variants injury state. These subunits can therefore be very potent candidates for some of the observed changes relating to the One gene, Nrg1, has two probe sets with opposing direction of expression of plateaux and may serve as new targets for expression, indicating the differential expression of different future studies of spinal cord injury. splice variant transcripts. Since the probe sets on the RAT230_2.0 chip are not designed to target specific exons of their target REFERENCES transcript, we remapped the probe sets to Ensembl exons and Alaburda A, Hounsgaard J. 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