Brain Advance Access originally published online on August 18, 2005
Brain 2005 128(10):2359-2371; doi:10.1093/brain/awh623
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Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury
1 Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, 2 Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT and 3 Department of Surgery, Rhode Island Hospital, Brown University School of Medicine, Providence, RI, USA
Correspondence to: Stephen G. Waxman, MD, PhD, Department of Neurology, LCI-707, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA E-mail: stephen.waxman{at}yale.edu
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Spinal cord contusion injury (SCI) is known to induce pain-related behaviour, as well as hyperresponsiveness in lumbar dorsal horn nociceptive neurons associated with the aberrant expression of Nav1.3, a rapidly repriming voltage-gated sodium channel. Many of these second-order dorsal horn neurons project to third-order neurons in the ventrobasal complex of the thalamus. In this study we hypothesized that, following SCI, neurons in the thalamus undergo electrophysiological changes linked to aberrant expression of Nav1.3. Adult male Sprague-Dawley rats underwent contusion SCI at the T9 thoracic level. Four weeks post-SCI, Nav1.3 protein was upregulated within thalamic neurons in ventroposterior lateral (VPL) and ventroposterior medial nuclei, where extracellular unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, and expansion of peripheral receptive fields. Altered electrophysiological properties of VPL neurons persisted after interruption of ascending spinal barrage by spinal cord transection above the level of the injury. Lumbar intrathecal administration of specific antisense oligodeoxynucleotides generated against Nav1.3 caused a significant reduction in Nav1.3 expression in thalamic neurons and reversed electrophysiological alterations. These results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after SCI.
Key Words: pain; spinal cord injury; VPL; channel; sodium
Abbreviations: AS = antisense; BK = background; i.t. = intrathecal; MM = mismatch; ODNs = oligodeoxynucleotides; SCI = spinal cord injury; VPL = ventroposterior lateral; VPM = ventroposterior medial
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Received May 19, 2005. Revised July 11, 2005. Accepted July 26, 2005.
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Introduction |
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Human spinal cord injury (SCI) can result in severe chronic neuropathic pain (Widerstrom-Noga, 2003
) that can successfully be modelled in animal preparations (Hulsebosch et al., 2000; Lindsey et al., 2000; Hains et al., 2001, 2003a). Experimental SCI induces electrophysiological changes in dorsal horn discharge properties (Yezierski and Park, 1993; Drew et al., 2001; Hains et al., 2003b; Hao et al., 2004;), which contribute to pain-like behaviours in animals, but the origin of these changes is not understood.
Action potential generation and propagation rely on multiple isoforms of voltage-gated sodium channels, which contribute in different ways to electrogenic properties of neurons based on their biophysical characteristics (Waxman, 2000). The Nav1.3 sodium channel produces a rapidly repriming tetrodotoxin-sensitive sodium current that permits neuronal firing at higher-than-normal frequencies (Cummins and Waxman, 1997; Cummins et al., 2001). Nav1.3 is expressed at relatively high levels in the embryonic nervous system of the rat, but is barely detectable in adult rat dorsal respiratory group (DRG), spinal cord or brain (Waxman et al., 1994; Felts et al., 1997; Hains et al., 2002). However, expression of Nav1.3 is upregulated within DRG neurons following nerve injury (Black et al., 1999; Dib-Hajj et al., 1999; Hains et al., 2004), and is correlated with ectopic discharge generation (Kim et al., 2001). We have recently shown that after thoracic contusive SCI, expression of Nav1.3 is upregulated in NK1-positive (and thus presumably nociceptive) dorsal horn neurons, and that intrathecal (i.t.) administration of antisense (AS) oligodeoxynucleotides (ODNs) targeting Nav1.3 decreases expression of Nav1.3 mRNA and protein within these lumbar dorsal horn neurons, reduces their hyperresponsiveness and reverses mechanical allodynia and thermal hyperalgesia (Hains et al., 2003b). Questions remain, however, with respect to the role of Nav1.3 and changes in excitability at higher levels along the neuraxis after SCI, especially in the thalamus where most somatosensory information converges for processing and relay to the cerebral cortex.
Many second-order dorsal horn nociceptive neurons project rostrally within the spinothalamic tract, and synapse on third-order neurons of the ventroposterior lateral (VPL) nucleus of the thalamus (Jones, 1998; Willis and Coggeshall, 2004), which is involved in sensory discriminative aspects of pain processing. Although injured spinal somatosensory circuitry can be thought of as generating aberrant nociceptive impulses, thalamic integrative circuits may also act as a generator, as well as an amplifier of these signals (McCormick, 1999), which are interpreted by the brain as inappropriate and excessive pain (Yezierski, 2001). Thalamic changes have been associated with pain following SCI in humans (Lenz et al., 1989; Pattany et al., 2002), primates (Weng et al., 2000) and rats (Koyama et al., 1993; Gerke et al., 2003), but the molecular mechanisms contributing to these changes are still unclear.
In this study we asked whether, in addition to producing pain-associated changes in sodium channel expression within neurons at spinal levels, SCI can also trigger supraspinal changes in sodium channel expression within thalamic neurons. We demonstrate that following SCI, thalamic neurons develop altered electrophysiological properties which persist after afferent barrage from the injured spinal cord is eliminated, and show that these changes are accompanied by upregulated expression of Nav1.3. Moreover, we show that these abnormal changes are reversed following administration of Nav1.3 AS. These results suggest a link between altered nociceptive processing and misexpression of Nav1.3 within the thalamus after SCI.
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Animal care
Experiments were carried out in accordance with National Institutes of Health guidelines for the care and use of laboratory animals; all animal protocols were approved by the Yale University Institutional Animal Use Committee. Adult male Sprague-Dawley rats (200–225 g) were used for this study. Animals were housed under a 12 h light– dark cycle in a pathogen-free area with free access to water and food. Three groups of animals were studied: SCI, SCI + MM (mismatch Nav1.3 AS ODN sequence) and SCI + AS (Nav1.3 AS sequence).
Spinal cord contusion injury
Rats were deeply anaesthetized with ketamine/xylazine (80/5 mg/kg i.p.). Spinal contusion injury was produced (n = 51 rats) at spinal segment T9 using the MASCIS/NYU impact injury device (Gruner, 1992). A 10 g, 2.0 mm diameter rod was released from a 25 mm height onto the exposed spinal cord. For sham surgery, animals (n = 20) underwent laminectomy and placement into the vertebral clips of the impactor without impact injury (intact). After SCI or sham surgery, the overlying muscles and skin were closed in layers with 4–0 silk sutures and staples, respectively, and the animals were allowed to recover on a 30°C heating pad. Post-operative treatments included saline (2.0 ml s.c.) for rehydration, and Baytril (0.3 ml, 22.7 mg/ml s.c., twice daily) to prevent urinary tract infection. Bladders were manually expressed twice daily until reflex bladder emptying returned, typically by 10 days post-injury. Following surgery, animals were maintained under the same preoperative conditions and fed ad libitum. Lesion histology was confirmed in perfused animals with cresyl violet on day 28.
ODN synthesis and delivery
AS ODN sequences corresponding to the translation initiation site of Nav1.3 (5'-CAGTGCCTGGGCCATCTTTTC-3') (SCI + AS, n = 12 rats), or its mismatch (MM, 5'-CGATCGCGTGCGCTATCTTCT-3') (SCI + MM, n = 12) were produced as previously described (Hains et al., 2003b, 2004). By BLAST search, the AS sequence did not show similarity, over the entire 21 nt, to the sequences for any other sodium channel or to other genes; BLAST search specifically demonstrated that there was no homology to Nav1.1, Nav1.2, Nav1.6, or other CNS sodium channels.
On day 28 after SCI, under ketamine/xylazine (80/5 mg/kg i.p.) anaesthesia, a sterile premeasured 32 G i.t. catheter (ReCathCo, Allison Park, PA, USA) was introduced through a slit in the atlantooccipital membrane, threaded down to the lumbar enlargement, secured to the neck musculature with suture and heat sealed to prevent infection and leakage of cerebrospinal fluid. Tip location was verified after killing the animal. Three days after catheter placement (day 31 after SCI), under brief (<1 min) halothane sedation (3% by facial mask), i.t. administration of AS or MM was initiated. For 4 days, 45 µg/5 µl of either AS or MM in artificial CSF (aCSF; 1.3 mM CaCl2·2H2O, 2.6 mM KCl, 0.9 mM MgCl, 21.0 mM NaHCO3, 2.5 mM Na2HPO4·7H2O, 125.0 mM NaCl, prepared in sterile H2O), was injected twice daily, followed by 10 µl aCSF flush. Animals were used for analysis (behavioural, histological, electrophysiological) at this timepoint (day 34).
Behavioural testing
Behavioural analysis (n = 8 animals/group) was performed 28 days after SCI to confirm that animals had developed behavioural signs of chronic neuropathic pain (for all experiments we used only animals that demonstrated the development of chronic pain) prior to MM or AS administration, and 4 days after to ensure that Nav1.3 AS was effective in reducing neuropathic pain as previously reported. On the day of behavioural testing 28 days after SCI, motor performance of rats with SCI recovered well enough to yield reliable withdrawal reflex measures, as shown in earlier studies (Hains et al., 2003b). After acclimation to the testing area (30 min), mechanical sensory thresholds were determined by paw withdrawal to application of a series of von Frey filaments (Stoelting, Wood Dale, IL, USA) to the glabrous surface of the paw. Following application of calibrated von Frey filaments (0.4–26 g) with enough force to cause buckling of the filament, a modification of the ‘up–down’ method of Dixon (1980) was used to determine the value at which paw withdrawal occurred 50% of the time (Chaplan et al., 1994), interpreted to be the mechanical nociceptive threshold.
After acclimation to the test chamber, thermal hyperalgesia was assessed by measuring the latency of paw withdrawal in response to a radiant heat source (Dirig et al., 1997). Animals were placed in Plexiglas boxes on an elevated glass plate under which a radiant heat source (4.7 A) was applied to the glabrous surface of the paw through the glass plate. The heat source was turned off automatically by a photocell upon limb-lift, allowing the measurement of paw withdrawal latency. If no response was detected, the heat source was automatically shut off at 20 s. Three minutes were allowed between each trial and four trials were averaged for each limb.
Immunocytochemistry
Coronal sections were collected from the brain, corresponding to the ventrobasal complex of the thalamus (bregma –3.14 mm) of animals from the following groups: intact (n = 5), SCI (n = 5), SCI + MM (n = 6) and SCI + AS (n = 6). Thin (12 µm) cryosections (n = 4–7 sections/animal/group) were processed simultaneously. For detection of Nav1.3 protein, slides were incubated at room temperature in the following order: (i) blocking solution [phosphate-buffered saline (PBS) containing 5% NGS, 2% bovine serum albumin, 0.1% Triton X-100 and 0.02% sodium azide] for 30 min; (ii) subtype-specific primary antibody raised in rabbit against Nav1.3 (Hains et al., 2002, 2003b, 2004) overnight in blocking solution; (iii) PBS, 6 times for 5 min each; (iv) goat anti-rabbit IgG-Cy3 (1 : 2000; Amersham Biosciences, Piscataway, NJ, USA) in blocking solution, 2 h; and (v) PBS, 6 times for 5 min each. Anti-rabbit Cy3 (1 : 2000, Amersham) was used as a secondary antibody. Mouse anti-NeuN was used to counterstain for neurons (Chemicon 1 : 500), with anti-mouse Cy2 secondary antibody (1 : 2000; Amersham Biosciences). Control experiments were performed without primary or secondary antibodies, and blocking compounds, which yielded only background levels of signal.
Quantitative image analysis
Images were captured with a Nikon Eclipse E800 microscope equipped with epifluorescence and Nomarski optics, using a SPOT-RT camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Quantitative microdensitometry was performed using IPLab Spectrum software (Scanalytics, Fairfax, VA, USA). The number of positively labelled neurons was counted for each section. Signal intensity was obtained by outlining individual neurons (n = 15 cells/section) and using IPLab integrated functions to determine levels of signal. Neurons were sampled only if the nucleus was visible within the plane of section and if its profile exhibited distinctly delineated borders. Background levels of signal were subtracted, and control and experimental conditions evaluated in identical manners. Neurons were classified as Nav1.3-positive if their signal intensity was >25% above background tissue fluorescence levels.
Electrophysiological procedures
Animals from intact (n = 7), SCI (n = 5), SCI + MM (n = 7) and SCI + AS (n = 6) groups underwent extracellular single unit recording according to established methods (Hains et al., 2003b, 2004). The activity of 3–7 units/animal was recorded, yielding 12–36 cells/group. Rats were initially anaesthetized with halothane (4% in induction chamber), and maintained by tracheal intubation (1.1%, 2–2.5 ml tidal volume, 60–70 strokes/min). Halothane anaesthesia lasted 
2 h, until the end of each experiment. Rectal temperature was maintained at 37°C. The head was fixed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA) and skin incision and a limited craniotomy exposed the brain surface vertical to the recording sites within the thalamus.
Neuronal units were isolated from the VPL and ventroposterior medial (VPM) nuclei of the thalamus [respective stereotaxic coordinates in mm: bregma (–3.30, –2.12); lateral (2.6, 3.5); vertical (4.8, 6.8)]. Extracellular single-unit recordings were made with a low-impedance 5 M
tungsten insulated microelectrode (A-M Systems, Carlsborg, WA, USA). Electrical signals were amplified and filtered at 300–3000 Hz (DAM80, World Precision Instruments, Sarasota, FL, USA), processed by a data collection system (CED 1401+; Cambridge Instruments, Cambridge, UK), and stored on a computer (Pentium 4 PC, Dell, Austin, TX, USA) to construct peristimulus time histograms or Wavemark. The stored digital record of individual unit activity was retrieved and analysed off-line with Spike2 software (v3.13, Cambridge Electronic Design, Cambridge, UK).
Once a cell was identified by a gentle probing of the body surface, its receptive field was mapped and stimulated by an experimenter blinded to the treatment of the animal. Background (BK) activity was measured followed by cutaneous receptive field mapping with von Frey filaments and/or brief pinches. Receptive fields for VPM units were exclusively mapped to the head, whereas those for VPL units were mapped to the rest of the body (upper body for receptive fields corresponding to dermatomes above lesion level, and lower body to those below lesion level). Three mechanical stimuli were routinely applied: (i) brush (BR) stimulation of the skin with a cotton brush; (ii) increasing intensity von Frey filaments (0.39 g; 1.01 g; 20.8 g forces); (iii) pressure (PR), by attaching a large arterial clip with a weak grip to a fold of the skin (144 g/mm2); and (iv) pinch (PI), by applying a small arterial clip with a strong grip to a fold of skin (583 g/mm2). Multireceptive units were identified by their responsiveness to brush, press and pinch, and with increasing responsiveness to incrementing strength von Frey stimuli. Low threshold or high threshold units were classified as such based on their high (>30%) rate of response to brush, von Frey stimulation or pinch. BK activity was recorded for 20 s and stimuli applied serially for 20 s, separated by 20 s of baseline activity. Care was taken to ensure that the responses were maximal, that each stimulus was applied to the unit's primary receptive field (except for expanded receptive fields after SCI where stimulation of a large body surface could induce discharges), and that isolated units remained intact and held for the duration of each experiment using Spike2 template matching routines. Neurons responding mainly to joint movement or to probing subcutaneous tissue were excluded from analysis. Evoked responses were calculated by subtracting the prestimulus baseline activity to yield net number of spikes per response. Afterdischarges were characterized by sustained increase in firing for several seconds beyond the stimulation period, in which case extended time was allowed for the activity to return back to baseline before application of the following stimulus.
Blockade of afferent barrage to the thalamus
Intact animals (n = 8) and animals 28 days after SCI (n = 16) were prepared for standard electrophysiological recording within the VPL. In these experiments, however, the spinal cord was also exposed by laminectomy rostral to the SCI site, and topical application of 2% lidocaine to the dorsal and lateral surfaces of the spinal cord followed by complete cord transection, was performed at a location between T1 and T6, 2–8 segments rostral to the SCI. For each animal one unit was isolated in the VPL that had an identifiable hind-limb receptive field. Unit activity was continuously recorded from the same VPL neuron for the duration of each experiment. Spontaneous activity and evoked responses were recorded for 200–250 s, which was followed by topical application of a pledget-soaked solution of 2% lidocaine (20 mg/ml, pH 6.5; Abbott Labs, North Chicago, IL, USA). At 650–700 s, the spinal cord was transected with iridectomy scissors at the same location while recording from the VPL neurons continued. In a second group of SCI animals, the activity of VPL units was recorded immediately before (n = 7), and for 2–4 h after lidocaine application and spinal cord transection (n = 6).
Statistical analysis
All statistical tests were performed at the alpha level of significance of 0.05 by two-tailed analyses using parametric tests. Data were tested for significance using one-way ANOVA to determine degree of variability within a sample and whether there was a difference between groups among the obtained means, followed by Bonferroni post hoc analysis. Tests of factors including pairwise comparisons were carried out where appropriate, with either the paired Student's t-test for before–after comparisons or the two sample Student's t-test to compare two groups. Data management and statistical analyses were performed using SAS (1992) statistical procedures with Jandel SigmaStat (v1.0), and graphed using Jandel SigmaPlot (v7.0) as mean ± standard deviation.
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Spinal cord injury and recording site histology
Histological sections showing the extent of SCI and the location of VPL recordings are shown in Fig. 1. Spinal contusion resulted in typical transection of descending dorsal corticospinal tract, with injury to the raphespinal tract and a large degree of grey matter at the lesion epicentre. The spinothalamic tract was spared bilaterally (Fig. 1A). A representative coronal brain section showing the recording site within the VPL nucleus is shown in Fig. 1B.
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Nav1.3 immunocytochemistry
Nav1.3 immunolabelling revealed very low levels of signal within the thalamus in intact animals (Fig. 2A). In contrast, 28 days after T9 SCI, Nav1.3 signal was increased within both the VPM nucleus and the VPL nucleus of the thalamus (Fig. 2B). The level of Nav1.3 signal was higher in the VPL nucleus than in the VPM nucleus in all cases, but some neurons were positive within the atlas boundaries of the VPM nucleus. Except for this, signal was restricted tightly to the VPL nucleus. Nav1.3 signal (Fig. 2C) tended to be colocalized with NeuN (Fig. 2D), indicating that the expression of Nav1.3 occurred primarily in neurons (Fig. 2E). The occasional expression of Nav1.3 in NeuN-negative profiles could be owing to differential labelling of cellular compartments in different planes of section or glial localization. The Nav1.3-positive neuronal cell bodies were
20–25 µm in diameter and displayed dendritic branching patterns characteristic of neurons. Of the NeuN-positive cells 47% were Nav1.3-positive. Within the VPL, there was no recognizable somatotopic distribution of Nav1.3-positive neurons.
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Lumbar i.t. administration of either Nav1.3 MM or AS was performed 31 days after SCI. Four days after start of ODN administration, levels of Nav1.3 expression in the VPL were unaffected in the SCI + MM group (Fig. 2F). In contrast, in the SCI + AS group, the level of Nav1.3 expression within VPL was reduced (Fig. 2G).
Quantification of the number of VPL neurons that expressed Nav1.3 signal showed that intact animals demonstrated very limited expression of Nav1.3-positive neurons (4.1 ± 2.3 per section), whereas after SCI, VPL neurons demonstrated significant (P < 0.05) upregulation of Nav1.3-positive neurons (95.7 ± 12.6 per section) (Fig. 2H). Nav1.3 signal intensity was significantly increased (P < 0.05) within Nav1.3-expressing VPL neurons after SCI compared with intact animals (138.1 ± 24.9 versus 49.9 ± 9.6) (Fig. 2I). Administration of MM resulted in no change in the number of Nav1.3 immunopositive profiles (101.5 ± 15.8) compared with SCI (95.7 ± 12.6). However, AS administration resulted in a significant (P < 0.05) reduction in the number of Nav1.3 immunopositive profiles within the VPL (66.4 ± 6.3) (Fig. 2H). In contrast to the SCI + MM group (152.6 ± 18.9), the SCI + AS group (139.2 ± 16.4) showed decreased signal intensity of Nav1.3-positive neurons when compared with SCI, although the result was not statistically significant (Fig. 2I).
Spontaneous thalamic activity independent of spinal afferent barrage
Recordings from VPL neurons with hind-limb receptive fields demonstrated a high rate of background firing (Fig. 3) 28 days after SCI. To ascertain whether this high rate of firing was the result of increased afferent barrage from neurons close to the SCI lesion, we recorded from VPL neurons before and after application of 2% lidocaine and subsequent cord transection at high thoracic levels (T1–T6) (Fig. 3A). In a representative record from an animal with SCI (Fig. 3B), spontaneous BK activity was present in VPL and occurred at 5–11 Hz, and a response was evoked upon brush stimulation of the hindpaw. Topical lidocaine application (at t = 230 s) at T1, eight spinal segments rostral to the SCI, had no effect on spontaneous firing rates of thalamic units which remained high (5–12 Hz), although it abolished the evoked response to brush and pinch stimulation of the hindpaw. Similarly, complete cord transection at T1 (at t = 690 s) had no effect on spontaneous firing rates, although brush or pinch stimulation could not evoke a response (Fig. 3C; pinch responses not shown). In this series of experiments, increased spontaneous activity was still present for as long as 10 min after blockade of evoked activity from the hindpaw by lidocaine and 2.5 min following spinal cord transection.
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Quantification of results from 24 units confirmed that brush activity could only be evoked prior to lidocaine or cord transection in intact and SCI groups, and that BK activity continued at a higher rate (9.2 ± 2.1 spikes/s prior to lidocaine, 8.7 ± 1.6 spikes/s following lidocaine, 8.5 ± 1.2 spikes/s following spinal cord transection) than in intact animals (3.0 ± 1.1 spikes/s, P < 0.05) throughout the experiment, for as long as 10 min following lidocaine and 2.5 min following transection (Fig. 3D).
Following up on these experiments, we recorded from VPL neurons from SCI rats at longer intervals following T1 spinal cord transection, to determine whether the high rates of spontaneous activity persisted over the course of hours. Figure 3E shows a continuous recording for a VPL neuron which was followed for >4 h after spinal cord transection. As in the short-term experiments, cord transection abolished the thalamic response to brush, however; a high rate of BK activity (7–11 Hz) persisted and was continuously present for as long as the unit was held. In another VPL unit, which was similarly followed for >2.5 h after thoracic cord transection, a high rate of spontaneous activity (9–13 Hz) could be seen to continue throughout the recording period. We also recorded from 7 VPL units from spinally interrupted rats at intervals of 30 min after the continuous recording period (i.e. at 3–4.5 h after cord transection) and observed high frequency spontaneous firing (7–14 Hz) in each of these units (see Fig. 3H1 and H2 for examples), at a much higher rate than in VPL neurons from intact animals (Fig. 3G).
Contribution of Nav1.3 to thalamic hyperactivity
Recordings were obtained from a total of 126 units in intact, SCI, SCI + MM and SCI + AS animals. In addition, activities from four units with spontaneous bursts but no identified receptive fields were also recorded in SCI animals. Typical evoked firing activity is shown in Fig. 4. The response rate of all units to natural stimuli was increased after SCI (Fig. 4B) compared with intact animals (Fig. 4A). Quantification revealed that this increase was significant for all stimuli except for brush (Fig. 5). When these responses were separated based on receptive field properties of isolated units, similar trends were observed in units with receptive fields mapped to the upper and lower body. Increases in responses to all stimuli except press were significant in neurons with lower body receptive fields. Increases of the order of 200–300% (ranging up to 21.6 ± 3.1 spikes/s in SCI animals) were observed. No significant changes were found in neurons with a receptive field in the head, except for a significant increase in response to pinch in animals with SCI (4.8 ± 0.5 spikes/s) compared with intact animals (2.4 ± 0.9 spikes/s, P < 0.05 compared with SCI group).
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Evoked responsiveness after Nav1.3 MM administration was not significantly different from SCI animals (Fig. 4C). However, administration of Nav1.3 AS significantly (P < 0.05) reversed increases in evoked activity to natural stimuli for the population of all units studied, suggesting reversal of hyperresponsiveness after SCI (Fig. 4D). Evoked discharge rates were not significantly different from intact discharge rates for von Frey filaments, press or pinch, whereas in response to brush, evoked rates after AS administration (11.6 ± 2.3 spikes/s) were below those for intact rates (16.3 ± 1.9 spikes/s) (Fig. 5). Responses to natural stimuli were reduced in brush and multireceptive unit classifications, but not in neurons with receptive fields in the head (16.4 ± 4.1 spikes/s).
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Behavioural testing
Behavioural testing was performed to confirm that animals had developed pain-related behaviours following SCI, and that Nav1.3 AS was effective at reducing mechanical allodynia and thermal hyperalgesia as previously described. Following SCI, animals demonstrated significantly reduced hind-limb mechanical thresholds (3.4 ± 1.6 g) when compared with intact animals (22.1 ± 3.4 g) (Fig. 4E). Thermal paw withdrawal latencies were also significantly reduced after SCI (6.9 ± 1.1 s) relative to intact animals (10.6 ± 1.2 s) (Fig. 4F). After 4 days of ODN administration, Nav1.3 AS resulted in a significant increase in mechanical thresholds (16.2 ± 2.1 g) compared with the SCI + MM (4.0 ± 1.7 g) groups (Fig. 4G). Similarly, thermal latency was significantly longer in SCI + AS animals (9.2 ± 0.9 s) compared with SCI and SCI + MM animals (6.5 ± 1.1 s) (Fig. 4H).
BK activity
After SCI, although there was an overlap between the SCI and intact groups (Fig. 6A), mean spontaneous BK activity of the entire population of units studied was significantly increased (6.7 ± 1.2 spikes/s) compared with the activity in intact animals (3.1 ± 0.9 spikes/s) (Fig. 6B), suggesting an increased rate of ongoing neuronal activity. When BK activity was segregated according to the receptive field and neuronal phenotype (Fig. 6B), this increase manifested mostly in multireceptive units (6.9 ± 1.6 spikes/s in SCI animals compared with 2.7 ± 0.9 spikes/s in intact animals, P < 0.05) and those with lower body (corresponding to dermatomes below the lesion level) receptive fields (7.4 ± 1.2 spikes/s in SCI animals compared with 1.7 ± 0.6 spikes/s in intact animals, P < 0.01).
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Following Nav1.3 MM or AS administration, the distribution of individual background rates was wide (Fig. 6A). However, analysis of spontaneous firing activity (Fig. 6B) showed that administration of Nav1.3 AS significantly reduced the average increase in BK activity in all units (3.2 ± 0.2 spikes/s compared with 6.7 ± 1.2 spikes/s in SCI, P < 0.05, and 6.1 ± 0.6 spikes/s in SCI + MM animals), multireceptive units (2.9 ± 0.7 spikes/s compared with 6.9 ± 1.6 spikes/s in SCI, P < 0.05 and 6.4 ± 0.6 spikes/s in SCI + MM animals), and lower body receptive field units (4.5 ± 0.2 spikes/s compared with 7.4 ± 1.2 spikes/s in SCI and 7.0 ± 1.5 spikes/s in SCI + MM animals, P < 0.05), indicating reversal of hyperresponsiveness.
Stimulus afterdischarge activity
Afterdischarges were detected in units sampled after SCI, an example of which is shown in Fig. 7A. Afterdischarges were not modality-specific and were observed in response to all stimuli, but were observed in more animals in response to brush, possibly suggesting a contribution to mechanical allodynia. Only one unit from intact animals (out of a total of 31 neurons studied or 3%) exhibited afterdischarge, compared with nine units recorded from rats with SCI (out of a total of 36 neurons studied, or 25%) (Fig. 7B). SCI + MM animals exhibited a similar degree of after-discharge as SCI animals (22% of 32 total units). Afterdischarge was seen in neurons with receptive fields in the lower body, upper body and head after SCI and in SCI + MM animals. A majority of neurons displaying afterdischarge in the SCI and SCI + MM groups were of the multireceptive type. Afterdischarge activity was observed up to 15 s following termination of stimulation in SCI and SCI + MM animals. In contrast, afterdischarge was never seen in the AS group (0% of 35 total units).
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Receptive field expansion
Most receptive fields mapped to the body of intact animals were clearly identified and restricted in dimensions (typically 1–2 mm2 surface area). Receptive field areas were classified as expanded when the isolated neuron responded to stimuli applied over a wider area than those measured in intact animals (either whole joint, whole limb, or even separate body parts). Representative mapped receptive field areas are shown in Fig. 8A. Receptive fields in six neurons, out of a total population of 36 units mapped (17%) were dramatically expanded in SCI animals compared to intact (0 out of 31 units, P < 0.05) (Fig. 8B). Expansion of peripheral receptive fields was observed in only 3 neurons out of a population of 35 units mapped (9%) in SCI + AS animals, in contrast to results shown for untreated animals with SCI (17%). In SCI + MM animals, there was no effect on receptive field size, which was comparable to SCI (21%).
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Discussion |
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Spinal cord injury can alter nociceptive circuitry within the spinal dorsal horn, and this can contribute to chronic neuropathic pain in both humans and animal models. We have previously documented the development of hyperresponsiveness of lumbar dorsal horn nociceptive neurons associated with pain-related behaviour after T9 SCI in rats and have showed the contribution of upregulated expression of Nav1.3 within these nociceptive neurons to this hyperresponsiveness (Hains et al., 2003b
). Second-order dorsal horn nociceptive neurons receive input from the periphery via the dorsal root ganglia, and project rostrally to third-order neurons within a pain-signalling pathway, located within the ventrobasal complex of the thalamus. Most of these spinothalamic projections terminate in the VPL nucleus. In the present study, we hypothesized that in addition to producing pain-associated modifications of spinal nociceptive circuitry, SCI would also induce supraspinal changes within VPL neurons that contribute to the processing of nociceptive signals.
In this study, together with Nav1.3 upregulation, we observed an increase in the BK activity and responses to natural stimuli in thalamic neurons following SCI. Abnormally upregulated Nav1.3 within VPL neurons, following SCI, might carry significant functional implications because Nav1.3 reprimes (recovers from inactivation) rapidly and produces a depolarizing response to small stimuli close to resting potential, increasing the excitability of cells that express Nav1.3 (Cummins and Waxman, 1997; Cummins et al., 2001). Lumbar i.t. administration of Nav1.3 AS in SCI animals reduced the number of neurons displaying abnormal Nav1.3 upregulation within the VPL, and reversed thalamic electrophysiological abnormalities caused by SCI. It is possible that other factors also contribute to the reconfiguration of the firing properties of thalamic units following SCI since the magnitudes of change of Nav1.3 expression levels are not the same as the magnitude of the changes in hyperexcitability. Nonetheless, our results demonstrate for the first time, molecular changes within the thalamus that are associated with abnormal sensory processing and chronic neuropathic pain after SCI.
A few studies have shown that injury to the spinal cord can affect thalamic neurons. Morrow et al. (2000) using autoradiographic estimates of regional cerebral blood flow to identify alterations in the activation of forebrain structures reported increases in rCBF within the VPL, as well as the VPM, after excitotoxic SCI. Dysrhythmic thalamic firing has been demonstrated within VPL neurons after experimental SCI (Weng et al., 2000; Gerke et al., 2003), and in humans with SCI, a bursting pattern of thalamic firing emerges (Lenz et al., 1989, 1994). However, mechanisms underlying thalamic burst firing have been relatively uninvestigated. VPL neurons may become hyperactive after spinothalamic tract transection through recruitment of N-methyl-D-aspartate receptors (Koyama et al., 1993), which have been shown to modulate thalamic pain signalling after inflammation-induced hyperalgesia (Kolhekar et al., 1997). Moreover, magnetic resonance spectroscopy has shown alterations in metabolites such as N-acetyl and myo-inositol, interpreted as reflections of neuronal dysfunction in thalami of patients with post-SCI pain (Pattany et al., 2002).
We observed that reversal of Nav1.3 expression with AS functionally influenced the thalamus, suggesting a contribution of Nav1.3 to an increase in the intrinsic excitability of thalamic neurons following SCI. Our AS experiments, however, could not definitively confirm a direct action on VPL neurons (as opposed to a primary effect on dorsal horn neurons, with ascending effect on their thalamic targets), because our previous studies (Hains et al., 2003b, 2004) have shown that i.t. administration of Nav1.3 AS has a direct effect on dorsal horn neurons. Thus, our AS experiments cannot, in themselves, demonstrate that persistent upregulation of Nav1.3 in thalamic neurons is responsible for their autonomous hyperexcitability. We cannot rule out the possibility that an increased barrage to thalamic neurons from dorsal horn neurons contributes to increased activity or other changes in these neurons after SCI, and note that it is possible that increased input from dorsal horn neurons triggers molecular changes in upstream neurons within the thalamus.
Importantly, however, we observed that increased spontaneous activity persists within VPL neurons, even after complete spinal cord transection (which presumably completely abolishes ascending afferent barrage that might otherwise have originated in spontaneously active and hyperresponsive dorsal horn neurons near or below the injury site). This suggests that the electrophysiological abnormalities within the thalamus are independent of tonic ascending spinal input, at the time of cord block (28 days after SCI). Consistent with this argument, spinal cord conduction block with local anaesthetic (Loubser and Donovan, 1991) above lesion level is often ineffective in alleviating pain following SCI in humans, arguing that, if indeed pain is coupled to thalamic neuronal hyperresponsiveness, the thalamus can act as an intrinsic ‘pain generator’ irrespective of spinal input.
Our observations of thalamic hyperresponsiveness cannot be explained by deafferentation of ascending inputs (Faggin et al., 1997; Jain et al., 1998). Whereas deafferentation of the thalamus has been suggested to contribute to changes in firing properties and chronic pain syndromes (Weng et al., 2000), in our preparation, some spinothalamic inputs to sampled units were intact because we were able to elicit evoked unit activity by stimulating peripheral receptive fields. However, it is possible that within the VPL, reorganization of inputs or disruption of the distributed GABAergic inhibitory system (Roberts et al., 1992; Lee et al., 1994; Ferreira-Gomes et al., 2004), which has been demonstrated after dorsal column transection in primates (Ralston et al., 1996), could potentially contribute to penetration of subthreshold inputs. It is also possible that perturbations in ascending serotoninergic raphe-thalamic inputs (Koyama and Yokota, 1993; Losier and Semba, 1993) may contribute to abnormal firing in the thalamus after SCI. Contusion SCI spinally transects the descending raphe-spinal circuitry (Jakeman et al., 1998) and consequently disinhibits nociceptive neurons within the dorsal horn (Willcockson et al., 1984; Hains et al., 2003a), which become hyperexcitable after injury (Hains et al., 2001, 2002). Perhaps, the damage and the loss of raphe neurons that occurs following SCI (Kim et al., 2002) can result in reduced inhibitory tone within the VPL as well.
Although to a lesser degree than in the VPL, we found an upregulation of Nav1.3 within some neurons of the VPM after SCI. We observed afterdischarges (which were not seen in neurons from intact animals) in neurons with receptive fields in the head, typically associated with the VPM, after SCI. There are also widespread changes throughout the thalamus in non-human primates (Jain et al., 1998), and in humans after SCI (Melzack and Loeser, 1978; Lenz et al., 1994). Our results are consistent with the clinical observation that above-level pain can sometimes involve the head in human subjects after SCI (Defrin et al., 2001; Finnerup et al., 2003).
Our findings demonstrate changes in excitability and expression of Nav1.3 within VPL neurons following SCI. We also report that hyperexcitability of thalamic neurons following SCI is to at least some degree autonomous, since it persists following spinal cord transection which abolishes ascending barrages from spinal cord neurons near or below the injury site. Together with our earlier results on dorsal root ganglion and dorsal horn neurons (Hains et al., 2003b), these results provide evidence suggesting that dysregulation of sodium channel Nav1.3 expression at both supraspinal and spinal levels after SCI contributes to altered processing of somatosensory information and chronic neuropathic pain.
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* These authors contributed equally to this work
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The authors thank Dr Joel Black for the valuable experimental advice and Mr Bart Toftness for technical assistance. This work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs and the National Multiple Sclerosis Society, and by generous gifts from J. Pelkey and K. and R. Kimball. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association. B.C.H. was funded by The Christopher Reeve Paralysis Foundation (HB1-0304-2), the NIH/NINDS (1 F32 NS046919-01), and Pfizer (Scholar's Grant in Pain Medicine).
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