Introduction

Glutamate is the major excitatory neurotransmitter in the brain, and it exerts its functions trough different classes of receptors classified as N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5- methyl-4-isoxasole propionic acid (AMPA), and kainate (KA) receptors according to their selective agonist activation, pharmacological properties, and sequence similarity (Traynelis et al. 2010). Among the ionotropic glutamate receptors, the kainate receptors (KARs) have distinct role at the synapse compare to other glutamate receptors (Contractor et al. 2011). The KAR family is composed of five different members, GluK1-5. The GluK1–GluK3 subunits have low glutamate affinity and are capable of forming functional homomeric channels. GluK4 and GluK5, instead, bind glutamate with high affinity but require co-assembly with one or more GluK1–GluK3 subunits to form functional channels. Hetero-multimeric assembly of KARs, like many other ion channels, leads to the formation of receptors with unique pharmacological and functional properties, also through their action at both pre- and postsynaptic sites (Lerma 2003; Contractor et al. 2011). Recent advances in understanding of KARs function have revealed roles for KARs in neuronal differentiation, epilepsy, neurodegeneration, and neuronal cell death (Lerma 2006; Vincent and Mulle 2009). KARs have also been shown to modulate synaptic transmission and neuronal excitability through their action at both pre- and postsynaptic sites (Contractor et al. 2011).

Interestingly, as for AMPA receptors, the physiologic properties of KARs are controlled by post-transcriptional mechanisms such as RNA editing (Seeburg et al. 1998; Barbon and Barlati 2011), which modifies one or more translation codons, thus leading to functionally distinct proteins from a single gene. The predominant editing change in mammals is adenosine-to-inosine (A–I) catalyzed by the adenosine deaminase acting on RNA (ADAR) one and two enzymes (Orlandi et al. 2012). In particular, GluK1 and GluK2 are subjected to RNA editing at the so-called Q/R site, where glutamine can be substituted with arginine located in the channel pore forming the so-called P-loop. The functional consequence of editing at this site is the formation of receptor channels with markedly reduced divalent cations permeability (Egebjerg and Heinemann 1993) and low single channel conductance (Swanson et al. 1996). Two additional positions subjected to RNA editing have been identified in the M1 segment of GluK2 subunit, the I/V site, where isoleucine can be substituted with valine, and the Y/C site, where tyrosine can be replaced by cysteine. Editing at these positions modulates the effect of the Q/R site on calcium flow, such that the fully edited subunit exhibits null passage of this cation (Kohler et al. 1993). Editing of these sites begins during late embryonic development throughout the nervous system (Bernard et al. 1999; Paschen et al. 1997).

We have recently reported that RNA editing of ionotropic glutamate receptors is involved in the pathological sequence of events taking place after spinal cord injury (SCI) (Barbon et al. 2010). SCI is an acute neurodegenerative condition with consequent long-term disabilities (Frigon and Rossignol 2006). After SCI, complex ischemic and inflammatory responses occur involving excitatory neurotransmitter systems and intracellular signaling (Kwon et al. 2004). In animal models' SCI, extracellular glutamate levels are increased after trauma (Liu et al. 1991), inducing cell death through excessive Ca2+ and Na2+ influx through ionotropic GluR (Choi 1994). The involvement of AMPA and NMDA glutamate receptor subtypes has been extensively studied in SCI (Brown et al. 2004; Grossman et al. 1999). Less is known about the role of KARs, although important roles of KARs in spinal neurons physiology have been shown (Bhangoo and Swanson 2013).

Spinal neurons express KAR mainly in the dorsal horn and across many laminae (Tolle et al. 1993; Cui et al. 2012). In particular, GluK1 and GluK2 subunit-containing KARs contribute to whole cell KAR currents in cultured dorsal horn neurons (Kerchner et al. 2002). KARs have a prominent role in the modulation of excitatory signaling between sensory neurons and spinal cord neurons and mediate both sensory and nociceptive neurotransmission (Bhangoo and Swanson 2013). In the dorsal horn, KARs are expressed by spinal interneurons that regulate local inhibitory tone (Kerchner et al. 2001a). Both GluK1- and GluK2-containing KARs appear to contribute to heterosynaptic regulation of neuronal transmission in the dorsal horn (Kerchner et al. 2002).

In line with the relevance of KARs in the physiology of the spinal cord and given that we have previously shown (Barbon et al. 2010) indicating a prominent role of RNA editing in SCI pathology in this study, we examined the molecular changes in GluK1 and GluK2 RNA editing set in motion by SCI.

Methods

Animal Treatments

All experimental protocols were approved by the Animal Review Committee of the University of Milan and met the Italian guidelines for laboratory animals which conform to the European Communities (EEC Council Directive 86/609 1987) and the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the United States National Institutes of Health. Adult Sprague–Dawley rats (Charles River Laboratories) weighing 240–260 g were kept under standard housing conditions (22 ± 2 °C, 65 % humidity, lights on from 06:00 to 20:00), with standard lab chow and water freely available. Traumatic spinal cord injury was performed as previously described (Gorio et al. 2005, 2007) using the UTS-Impactor at the core of which is a 2.3 mm diameter stainless steel rod that is precisely driven into the spinal cord with specified force and displacement. The movement and impact is monitored by means of a miniaturized piezoelectric dynamometer present within a section of the impacting rod and linked to a computer that drives the device, records, and manages the data. The impounding piston was positioned 1 mm above the exposed cord at T9 and set for an excursion of 3 mm. A force of 1 N was applied for 1 s followed by the automatic return of the impaction rod. Animals were maintained under halothane anesthesia and positioned over a mat kept at 38 °C and before awakening were treated with buprenorphine (0.03 mg/kg) for pain management and penicillin G (10.000 U/kg) to prevent infection. After spinal cord injury, the rats were housed two per cage and underwent manual bladder evacuation three times daily. Comparisons were made among animals that had no surgery (CTR), laminectomized animals that underwent surgery without spinal cord impaction (LAM), and lesioned animals (LES). At least five animals were used for each group.

Functional Assessment

Basal motor activity was evaluated the day before the lesion (day 1) to ensure that the animals enrolled in the study had no initial motor deficits. Motor function was examined at time points ranging from 1 to 28 days post-injury by four blinded observers, and values for each animal were averaged across observers using the methodology described in Basso et al. (1995).

Morphology

Three days after injury, animals were anesthetized by inhalation of halothane and transcardially perfused with 1 % paraformaldehyde in 0.1 M phosphate buffer pH 7.2 followed by 4 % paraformaldehyde for immunocytochemistry. Spinal cords were dissected and postfixed for 12 h in the same fixative used for perfusion, cryoprotected with 30 % sucrose, and quickly frozen and stored at –80 °C. At 0.5 mm caudal to the lesion epicenter, the spinal cord was serially cut by means of a cryostat (Carl Zeiss GmbH, Jena, Germany). Serial transverse sections of 40 μm from animals in each experimental group were stained with thionin and were used to estimate extent of tissue damage.

RNA Extraction and RT-PCR Reaction

Molecular analyses were performed at 3, 7, and 30 days after injury (n = 5 for each time point). Homogenates were prepared using a 1.5 mm length sample from the spinal cord taken at the epicenter (T9) of the lesion as well as 4 mm caudal and 4 mm rostral to the lesion; corresponding loci were also sampled from the control and laminectomized rats. The spinal cord regions of interest were dissected out rapidly, frozen on dry ice, and stored at −70 °C for further analyses. Total RNA was extracted using TRIZOL reagent (Life Technologies). RNA was recovered by precipitation with isopropyl alcohol, washed by 75 % ethanol solution, and dissolved in RNase-free water. RNA quantitation and quality controls were done using spectrophotometric analysis and the Agilent Bioanalyzer 2100. Reverse transcription (RT) was done using the Moloney murine leukemia virus-reverse transcriptase (MMLV-RT; Invitrogen). Total RNA of 2.5 μg from each spinal cord sample was mixed with 2.2 μl of 0.2 ng/μl random hexamer (Invitrogen), 10 μl of 5× buffer (Invitrogen), 10 μl of 2 mM dNTPs, 1 μl of 1 mM DTT (Invitrogen), 0.4 μl of 33 U/μl RNasin (Promega), and 2 μl MMLV-RT (200 U/μl) in a final volume of 50 μl. The reaction mix was incubated at 37 °C for 2 h, and then the enzyme was heat inactivated at 95° for 10 min. To perform the PCR reactions, 20 ng of retro-transcribed RNA were mixed with 2.5 μl 10× buffer (Polymed), 0.7 μl of 1.5 mM MgCl2, 2.5 μl of 2 mM dNTP, 0.7 μl of each forward and reverse primer, and 1.25 U of Taq polymerase in a final volume of 25 μl. Standard PCR cycle conditions were one denaturation step for 2 min at 95 °C followed by 25–35 cycles with 30 s of denaturation at 95 °C, 20 s of primer annealing at 60 °C, 30 s–1 min of elongation at 72 °C followed by a final extension of 1 min. Primer sequence are rGluK1-F: GTC AGT TGT GTA CTG TTT GTG ATT GC, GluK1-R: AAG GCA GCC AGG TTG GCC GTG, rGluK2-F: ACT TGG AAT AAG TAT TTT GTA CCG C, and rGluK2-R: CAA ATG CCT CCC ACT ATC CTG.

Editing Level Quantitation

The editing level quantitation for AMPA GluK1, GluK2 transcripts was done by sequence analysis of gel purified PCR products (Barbon et al. 2003). Briefly, in the electropherogram obtained after RT-PCR and sequencing analysis of a pool of transcripts, the nucleotide that undergoes the editing reaction appears as two overlapping peaks: “A” from unedited transcripts and “G” from the edited ones. We previously determined that the editing level can be reliably calculated as a function of the ratio between the G peak area and A plus G peaks areas. The nucleotide areas were quantified by the Discovery Studio Gene 1.5 program (Accelrys Inc., San Diego, CA, USA). The means and standard errors from each group of animals were used for statistical analysis. In order to validate the system, several calibration curves obtained from the five 5-HTR2C editing sites and GluR sites have been already published (Barbon et al. 2003, 2010, 2011).

Statistical Analysis

Data were collected in individual animals (independent determinations) and are presented as means and standard errors. Data from editing experiments were analyzed using a two-way analysis of variance (ANOVA) with the site of the lesion (CTR, LAM, and LES) and the time of sacrifice (3, 7, and 30 days) as independent variables. When dictated by relevant interaction terms, the single contrast post hoc test Bonferroni was used. The statistical analysis of the data was carried out by means of GraphPad Prism 4 (GraphPad Software Inc., USA).

Results

Motor Function Recovery and Lesion Morphology

In accordance with our previous results (Gorio et al. 2005, 2007; Barbon et al. 2010), laminectomized animals showed only a minor initial effect on motor function with complete recovery within 4 days, whereas in the lesioned group, locomotion was compromised up to and including 28 days after injury (Table 1). SCI caused extensive damage to gray and white matter at site of injury with sparing of the ventral and lateral white matter. At 3 mm from the lesion epicenter, there is slight damage of the gray matter around the ependimal canal in the caudal portion, while there is no sign of injury rostrally (Fig. 1).

Table 1 Motor function recovery
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Fig. 1
figure 1

Thionin-staining of coronal sections at the lesion center (EPI) and 3 mm rostral (ROS) and caudal (CAU) to it from sham-operated (LAM) and injured rat at 3, 7, and 30 days post-injury

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RNA Editing

Since laminectomy did not cause any significant change in the editing of GluK1 and GluK2 at any of the sites of the spinal cord or time points examined, we will focus our description of results on those related to the lesion itself.

Figure 2a shows that SCI has profound effects on GluK1 receptor editing at Q/R site, primarily at the epicenter. Two-way ANOVA reported a significant effect of treatment (p < 0.0001), time (p < 0.01), and treatment × time interaction (p < 0.01). In particular, the lesioned group showed a strong downregulation, ranging from −33 to −38 % at 3 and 30 days post-injury, respectively, with the highest effect of −58 % observed 7 days after SCI (Bonferroni post test p < 0.001). A similar pattern of changes was observed caudally to the lesion, where two-way ANOVA reported a significant effect of treatment (p < 0.0001). The lesioned group showed a strong downregulation, being −22 % after 3 days, −23 % at day 7 (p < 0.001), and −22 % after 30 days (p < 0.01). Rostrally to the lesion, a downregulation of GluK1 Q/R levels was also evidenced (effect of treatment p < 0.001) persisting until the end of the time course: −35 % after 3 days (p < 0.001), −21 % after 7 days (p < 0.05), and −23 % after 30 days (p < 0.01).

Fig. 2
figure 2

Evaluation of RNA editing levels for the KA GluK1 Q/R (a) site and GluK2 Q/R (b), I/V (c), and Y/C (d) editing sites for control (CTR), laminectomized (LAM), and lesioned (LES) rats. Different portions of the spinal cord were analyzed: ROS rostral to the lesion; EPI at the epicenter; CAU caudal to the lesion. Data represent means and standard errors obtained from at least five animals in each group at each time point. Bonferroni correction was used after two-way ANOVA and asterisks denote the individual time points at which the lesioned group differs from the control groups

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As for Q/R editing site of GLUK, we also observed the most dramatic changes in GluK2/Q/R editing at the lesion epicenter (Fig. 2b). Two-way ANOVA analysis reported a significant effect of treatment (p < 0.0001). The lesioned group showed reduced editing level throughout the observational period (−57 %, p < 0.001 at day 3; −67 %, p < 0.001 at day 7, and −38 %, p < 0.001 at day 30 post-injury). Caudally to the lesion, we observed an effect of treatment (p < 0.01) and time (p < 0.01). SCI affected editing levels after 3 days (−22 % p < 0.01) and 7 days (−19 %, p < 0.01), whereas at day 30, the reduced editing was completely abolished. Rostrally to the lesion site, we observed an effect of treatment (p < 0.0001) and time (p < 0.05). In particular, at day 3 a downregulation of −50 % was present (p < 0.001) that persisted until day 7 (−21 %, p < 0.05). Also, in this case, the effect was terminated at 30 day post-injury.

The main effects were present at the epicenter (treatment p < 0.0001 and time p < 0.05) when the I/V site was examined (Fig. 2c). The lesioned group showed a strong downregulation that persisted through the observational period (−45 % at 3 days; −63 % at 7 days, and −42 % at 30 days, p < 0.001). Caudally to the lesion, the effect of treatment is significant (p < 0.0001), with a reduced editing level observed after 3 (−22 %, p < 0.01) and 7 days (−19 %, p < 0.01) while it vanished after 30 days. In the rostral area, two-way ANOVA reported effects of treatment (p < 0.0001), time (p < 0.05), and treatment × time interaction (p < 0.01). The main effect was found at day 3 (−37 %, p < 0.001), still present at day 7 (−19 %, p < 0.01), but recovered at day 30.

Concerning the Y/C site (Fig. 2d) at the epicenter, we found a treatment × time interaction (p < 0.01) and an extremely significant effect of time (p < 0.0001). The downregulation of editing level was −42 % at day 3 (p < 0.01), of −58 % at day 7 (p < 0.01), and of −35 % at day 30 (p < 0.001). Reduction in the editing level was also observed caudally to the lesion. ANOVA analysis showed a significant treatment × time interaction (p < 0.05) and an effect of treatment (p < 0.0001). The lesioned group showed a reduced Y/C editing level of about 24 % (p < 0.001) after 3 days post-injury and 20 % at day 7 (p < 0.001). At day 30, this effect had ceased. A similar pattern of editing changes was observed rostrally to the lesion. ANOVA analysis showed a significant treatment × time interaction (p < 0.0001) and a significant effect of treatment (p < 0.0001) and of time (p < 0.01). In particular, a downregulation of about −31 % (p < 0.001) was present at day 3, of about 15 % (p < 0.001) at day 7, while at day 30, the reduction of editing was terminated. The abovementioned editing downregulations are specific for glutamate receptors editing as confirmed by our previously reported data (Barbon et al. 2010) indicating the editing level of the 5-HT2c receptor five editing sites were not affected by SCI.

Discussion

This study reports that KAR RNA editing is involved in the molecular outcomes triggered by acute SCI. RNA editing levels of both GluK1 and GluK2 were decreased at the epicenter of the lesion but also caudal and rostral to it, although with distinct magnitude and kinetics. These data add complexity, as well as specificity, to our previous observations related to AMPA receptor RNA editing after SCI and further strengthen the role of post-transcriptional regulations of glutamate receptors after acute SCI.

KARs have a prominent role in the modulation of excitatory signaling between sensory and spinal cord neurons (Bhangoo and Swanson 2013). They are located both at the pre- and postsynaptic sites in spinal cord neurons, across many laminae of the dorsal horns (Cui et al. 2012). Presynaptic KARs regulate transmission at both excitatory and inhibitory synapses (Huettner 2003). At excitatory primary afferent sensory synapses, presynaptic KARs expressed by a subset of dorsal root ganglia (Hwang et al. 2001) and regulate glutamate release (Kerchner et al. 2001b). At inhibitory synapses within the dorsal horn, presynaptic KA receptors, which respond to glutamate released from dorsal root sensory fibers, regulate GABA and glycine release by depolarization of interneuron terminals (Kerchner et al. 2001a, 2002; Lerma 2003). In addition to their presynaptic actions at excitatory and inhibitory terminals, KA receptors also are found at the postsynaptic membrane of neurons that respond to high-threshold dorsal root fiber stimulation to integrate nociceptive inputs (Li et al. 1999). Our data show that SCI may strongly alter the functional properties of KARs via a direct modification of RNA editing reaction, thus resulting in a markedly altered functionality of SC neurons after the lesion. In particular, we found that both GluK1 and GluK2 Q/R editing levels were strongly reduced after the lesion, most notably at the epicenter, an effect that persisted at least for 1 week. Different effects were identified caudally and rostrally to the lesion; we observed a variable reduction of GluK1 Q/R at the different time points, whereas GluK2 showed complete recovery to normal state at 30 days after injury. These data differ from those described for AMPA GluA2 Q/R site that was unaffected (Barbon et al. 2010). However, whereas GluA2 is always fully edited, editing of the Q/R site in GluK1 and GluK2 mRNAs occurs at very low levels in the embryonic brain and increases within the first few days after birth in most regions of the brain (Belcher and Howe 1997; Paschen et al. 1997; Bernard et al. 1999; Barbon et al. 2003). This developmentally regulated Q/R editing modulates GluK Ca2+ permeability (Egebjerg and Heinemann 1993; Kohler et al. 1993), channel conductance (Swanson et al. 1996) (Vissel et al. 2001), and alter current voltage relationship (Bowie and Mayer 1995; Kamboj et al. 1995) during cell maturation. As indicated by a study of dorsal root ganglia neurons, the unedited GluK1 subunit could have a developmental role in synapse formation in the spinal cord dorsal horn (Kamboj et al. 1995). However, in later development, GluK1 with Q/R site-edited may acquire other functions in which Ca2+ influx is not required.

SCI heavily modulates the editing level also of GluK2 I/V and Y/C. The main changes were observed at the epicenter of the lesion where both editing sites were downregulated by more than 20 % at the different time points analyzed. In rostral and caudal to the lesion, a robust downregulation was observed in the first post-lesion period, while I/V and Y/C editing level returned to physiological condition. The I/V and Y/C sites, which are located in the first transmembrane domain, seem to be involved in a finer regulation of ion permeability along with the Q/R site (Kohler et al. 1993; Burnashev et al. 1995) although the electrophysiologic data reported so far are not exhaustive.

Although we cannot rule out the hypothesis that the reduced RNA editing at the epicenter may be due, at least in part, to the greater loss of neuronal cells than of white matter (Paschen et al. 1994; Kawahara et al. 2003); however, the evidence that it is also reduced caudally and rostrally to the epicenter, where the spinal cord morphology is preserved, suggests that the decreased RNA editing at the epicenter is not due to cell loss, but perhaps is more related to an adaptive response of the system. This possibility is strengthened by the evidence that RNA editing is partially recovered 30 days post-injury. Instead, as we have previously shown, the downregulation of KARs editing level might be due to a partial deactivation of ADAR2 enzymes (Barbon et al. 2010). Further work is, however, needed to better understand the molecular mechanisms modulating ADAR activity after SCI.

Our study cannot show whether the reduced editing levels is capable of affecting postsynaptic KARs in response to nociceptive stimuli or presynaptic KARs expressed by spinal interneurons and involved in the regulation of local inhibitory tone; however, independently from the receptor synaptic location, the downregulation of the editing level might facilitate the activation of ascending neurons and propagate sensory/nociceptive information from the spinal cord to higher brain centers. These data are in agreement with the evidence that blockers of post- and presynaptic KA receptors located on dorsal horn interneurons would be expected to have analgesic properties (Kerchner et al. 2001b).

Taken together, our data show that we might have unraveled a previously unappreciated modulation of KA receptors following acute SCI. While it is still premature to speculate on the potential functional relevance of these findings, our results, together with our previous findings related to the editing of AMPA receptor subunits, highlight new potential targets for pharmacological interventions in spinal cord injury.