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Spinal cord injury (SCI) results not only in motor deficits, but produces, in many patients, excruciating chronic pain (SCI-Pain). We have previously shown, in a rodent model, that SCI causes suppression of activity in the GABAergic nucleus, zona incerta (ZI), and concomitant increased activity in one of its main targets, the posterior nucleus of the thalamus (PO); the increased PO activity is correlated with the maintenance and expression of hyperalgesia after SCI. Here, we test the hypothesis that SCI causes a similar pathological increase in other thalamic nuclei regulated by ZI, specifically the mediodorsal thalamus (MD), involved in the emotional-affective aspects of pain. We recorded single and multi-unit activity from MD of either anesthetized or awake rats, and compared data from rats with SCI with data from sham-operated controls (anesthetized experiments) or with data from the same animals pre-lesion (awake experiments). Consistent with our hypothesis, MD neurons from rats with SCI show significant increases in spontaneous firing rates, and in the magnitude and duration of responses to noxious stimuli. In a subset of anesthetized animals, similar changes in activity of MD neurons were produced by pharmacologically inactivating ZI in naïve rats, suggesting that the changes in MD after SCI are related to suppressed inhibition from ZI. These data support our hypothesis that SCI-Pain results, at least in part, from a loss of inhibition to thalamic nuclei associated with both the sensory-discriminative and emotional-affective components of pain.
Every year there are some 12,000 new incidents of spinal cord injury (SCI) in the United States, and up to 80% of patients with SCI develop chronic pain (Siddall et al., 2003). Chronic pain after spinal cord injury (SCI-Pain) often presents as wide-spread allodynia, hyperalgesia, and spontaneous pain (Bowsher, 1996; Garcia-Larrea et al., 2002; Finnerup et al., 2003) that develop weeks or even months after the initial injury (Tasker et al., 1992; Falci et al., 2002). SCI-Pain has no cure, and, in most patients, is resistant to conventional pharmacological treatment (Baastrup and Finnerup, 2008). The delayed expression of SCI-Pain, the diffuse localization of painful symptoms, and the presence of pain below the denervated spinal segment strongly suggest the occurrence of maladaptive plasticity in not only the spinal cord, but also in supraspinal structures (Masri and Keller, 2011). Though the cause of SCI-Pain remains unknown, it has long been hypothesized that chronic pain results from abnormally suppressed inhibition in the thalamus (Head and Holmes, 1911; Boivie, 2005).
To this end, we recently demonstrated in an animal model of SCI that pain results in a significant decrease of neuronal activity in the zona incerta (ZI) (Masri et al., 2009), which provides potent inhibition to select thalamic nuclei and that the loss of ZI activity correlates with a pathological increase of neuronal activity in the posterior thalamus (PO), a somatosensory thalamic nucleus critical for processing nociceptive information (Masri et al., 2009). Further, we demonstrated that these maladaptive changes are causally related to the development of chronic pain after SCI (Masri et al., 2009; Davoody et al., 2011; Lucas et al., 2011).
Pain is not only a sensory-discriminative experience, but also an unpleasant emotional experience (Merskey and Bogduk, 1994). We therefore predicted that after SCI, maladaptive changes in ZI would correlate with pathological activity in both somatosensory and associative thalamic nuclei that are important for nociceptive processing, including the mediodorsal thalamus (MD). MD, like PO, receives dense GABAergic inputs from ZI (Bartho et al., 2002; Erickson et al., 2004) and is heavily connected to cortical areas involved in processing the affective aspects of pain (Price and Slotnick, 1983; Cornwall and Phillipson, 1988; Groenewegen, 1988). In humans, chronic pain is associated with abnormal activity in MD (Rinaldi et al., 1991). Similarly, lesions or inactivation of MD reduces both thermal and mechanical hyperalgesia in a rat model of peripheral neuropathic pain (Saade et al., 2007). These findings led to our hypothesis that pain after SCI is associated with abnormal increases in spontaneous and evoked activity of MD neurons.
All procedures were conducted according to Animal Welfare Act regulations and PHS guidelines. Strict aseptic surgical procedures were used, according to the guidelines of the International Association for the Study of Pain, and approved by the University of Maryland School of Medicine Animal Care and Use Committee. A total of 31 adult male Sprague-Dawley rats weighing 250-300 g rats were used in this study.
All animals were tested on three consecutive days before the spinal lesion surgery, at day three after surgery, at day seven after surgery, and at weekly intervals thereafter. To minimize the animals’ anxiety, they were habituated for two weeks before behavioral testing and were trained to stand upright with their forepaws on the experimenter's hand as described by Ren (1999). Calibrated von Frey filaments (Stoelting Co, Wood Dale, IL, USA) were applied in ascending order to the hindpaw. We applied the filaments to the dorsal surface of the paws based on studies demonstrating that the dorsal approach more reliably and consistently detects threshold changes (Ren, 1999). Mechanical withdrawal threshold was defined as the force at which the animal withdrew the paw to three of five stimuli delivered.
Sterile surgery was preformed as previously described (Masri et al., 2009; Lucas et al., 2011). Briefly, a laminectomy to expose the spinal cord immediately rostral to C7 was preformed and a quartz-insulated platinum electrode (5 μm tip) was targeted to the spinothalamic tract (STT). Electrolytic lesions were made by passing DC current (10 μA for 10 seconds, repeated four times) in two locations, 0.4 mm apart. Sham surgery was preformed without laminectomy. After completion of surgery the incision sites were approximated and sutured in layers.
At least 14 days after spinal lesion surgery, rats were anesthetized with urethane and prepared for extracellular recordings as previously described (Trageser et al., 2006; Masri et al., 2009). We selected urethane because it has no, or negligible, effects on glutamatergic and GABAergic transmission and therefore produces only minimal disruption of signal transmission (Sceniak and Maciver, 2006). For anesthetized experiments in sham and spinal-lesioned animals (n = 15 male animals), rats were initially anesthetized with ketamine/xylazine (100/8 mg/kg, intraperitoneal). We used intravenous infusion of a 6% urethane solution, delivered through a jugular catheter, to control the level of anesthetic, which was kept at level III-2 described by Friedberg et al. (1999). Anesthetic levels were monitored by continually recording electrocorticographs (ECoGs), obtained from a pair of screws implanted in the skull.
Extracellular recordings were obtained from MD contralateral to the spinal lesion, through quartz-insulated tungsten electrodes (2-4 MΩ). We recorded from well-isolated units, digitized (40 kHz) the waveforms through a Plexon (Dallas, TX) data-acquisition system, and sorted units off-line with Plexon's off-line sorter, using dual thresholds and principle component analyses. We generated autocorrelograms with Neuroexplorer software (Plexon) to confirm that we obtained recordings from single units. We exported time stamps of well-isolated units and stimulus triggers to Matlab (MathWorks, Natick, MA) for analyses using custom-written algorithms. Because we found wind-up-like responses in MD neurons (see Figures 1C & 2A) we analyzed responses to each stimulus separately using modified peristimulus time histograms (PSTHs, 100 ms bins) and defined significant stimulus-evoked responses as PSTH bins which significantly exceeded (99% confidence interval) spontaneous activity levels.
We defined bursts of action potentials as clusters of at least three spikes with inter-spike intervals of ≤ 5 ms in which the first spike in the burst has a preceding inter-spike interval of ≥ 100 ms, based on previous reports of bursting in thalamic nuclei (Rinaldi et al., 1991; Guido et al., 1995; Sherman, 1996). Extracellularly recorded bursts that meet these criteria are thought to correspond to calcium-dependent spike bursts characterized in thalamic neurons through intracellular recordings (Jahnsen and Llinas, 1984).
We recorded responses to noxious pinches (1 sec application, 200-225g) using electronic calibrated forceps (IITC Life Sciences, Woodland Hills, CA). Stimuli were applied to both hindpaws (at least two stimuli on each paw) because MD neurons have large, bilateral receptive fields (Dostrovsky and Guilbaud, 1990; Hsu et al., 2000; Wang et al., 2003) and because hyperalgesia occurs in both hindpaws in this animal model of SCI-Pain (Masri et al., 2009; Lucas et al., 2011). To minimize peripheral sensitization and avoid tissue inflammation, stimuli were applied to left and right hindpaws in an alternating pattern, with at least 20 sec between applications on each paw.
In a different group of naïve rats (n = 9), a microdialysis probe (CMA Microdialysis, Solna, Sweden) was implanted into the ventral portion of zona incerta (ZIv; stereotaxic coordinates: A: -3.9 mm, L: 2.8 mm, D: 7.1 mm (Paxinos and Watson, 1998). The microdialysis probe was used to administer 0.1 mL muscimol or saline (2.5 μL / min) to ZIv while we recorded single unit activity in MD of urethane anesthetized animals, as described above. To determine the effect of drug administration on neurons in MD, we compared mean firing rate during 30 seconds of baseline activity to mean firing rate during 30 seconds of activity after muscimol application.
In an additional group of naïve rats (n=6) we implanted a microdialysis probe in either the right ZIv (n=4) or the right substantia nigra (n=2). Several days after the rats completely recovered from surgery, we tested the effects of reversible inactivation on their responses to mechanical stimuli. To this end, we applied either muscimol (200 μM) or saline through these probes. Agents (50 μl volume) were applied with a micro-pump at a rate of 2.5 μl/min for 20 minutes. We then tested the effects of drug infusion on thresholds of withdrawals to mechanical stimuli, as described earlier.
In seven animals, a longitudinal incision was made along the midline of the skull to expose bregma and lambda. Bone overlaying MD was removed and custom-made 5-channel moveable (1.4-4 MΩ, 40-80 μm, insulated except at tip) recording electrodes were slowly (50 μm / 5 min) lowered into MD. Electrode locations were based on stereotaxic coordinates (A: –2.8 mm, L: 0.9 mm, D: 4.5-5.5 mm (Paxinos and Watson, 1998), based on data from our anesthetized recordings (see Figure 1D), and based on previously published anatomical and electrophysiological studies (Dostrovsky and Guilbaud, 1990; Ray and Price, 1992) of areas in MD that respond to noxious peripheral stimulation and project to limbic cortical areas. Electrodes were secured in place using six bone screws and acrylic resin and were protected by permanent custom-made shields. Before electrophysiological recording experiments began, animals were treated with oral antibiotics for two weeks after implant to reduce risk of infection.
During that time, animals were handled daily to become comfortable with experimenters and were trained to rest quietly in experimenters’ hands to minimize stress and ease headstage connection. To minimize anxiety and stress at the start of recording experiments, animals were acclimated to the recording room for at least 30 minutes before behavioral testing and recording. Recordings were obtained during three or more days before spinal lesions, and weekly after the lesions. Rats were fitted with a wireless headstage (Triangle BioSystems International, Durham, NC) and allowed to move freely in their home cage for 2-3 minutes, during which spontaneous activity of MD neurons was recorded. Next, the animals were placed on an elevated platform [0.25 m (w) × 0.6 m (l) × 0.5 m (h)] with a mesh floor, where they freely moved for 2 minutes before testing began to determine pain thresholds. During this unrestricted behavior, spontaneous activity of MD neurons was recorded.
We recorded wideband activity from MD with the wireless headstage (Triangle BioSystems Intl.), and digitized the waveforms as described above. The wideband waveforms were filtered with a low-cut Butterworth filter (4-pole, 250 Hz), to obtain multiunit activity (MUA). The MUAs were classified off-line with Plexon's off-line sorter, using thresholds set at least 6 standard deviations higher than mean background activity.
To test for mechanical withdrawal thresholds, confirm development of hyperalgesia after spinal lesion, and produce stimulus evoked neuronal responses, von Frey filaments were applied to the plantar surface of the rats’ hindpaw through holes in the mesh floor of the elevated platform. Each filament (range: 26 to 180 g; intensities that in normal rats span from innocuous to noxious forces) was applied 5 times when the animal was calm and still. Manual triggers were used to store a digital timestamp of the stimuli. We recorded frequency of hindpaw withdrawal to ascending forces and threshold was defined as the force at which an animal withdrew to at least 3 to 5 of the stimuli. After a complete series of von Frey filaments was applied to the hindpaw, a noxious thermal stimulus (42-45 °C, 2 mm2 surface area, <5 sec application) was presented 5 times (10 sec intervals) to evoke a robust behavioral response from the animals and to confirm that the neurons respond to noxious stimuli.
Because we, and others, have reported that neurons in MD have large, bilateral receptive fields, (Fig. 5C) (Dostrovsky and Guilbaud, 1990; Wang et al., 2003), because our model of SCI-Pain presents with significant bilateral hyperalgesia below the site of the spinal lesion (Masri et al., 2009), and to help prevent peripheral sensitization, we applied stimuli to alternating hindpaws. We computed mean firing rate during spontaneous activity in both the home cage and on the elevated platform and in response to each level of stimulation delivered.
All animals were deeply anesthetized and transcardially perfused with buffered saline followed by 4% buffered paraformaldehyde. We obtained coronal brain and spinal cord sections (80 μm thick) and Nissl-stained them. The sections were examined with a transmission microscope to identify stimulation sites, lesions sites, microdialysis probe and recording implant location. For electrophysiological experiments, all recordings sites were reconstructed and only neurons with confirmed placement within the desired thalamic nucleus were included in analysis.
In all experiments preformed, we determined the appropriate sample size by performing a power analysis using α = 0.05 and power = 0.85. In all experiments, P < 0.05 was considered significant. We performed statistical analyses in STATA (StataCorp LP), and assessed, in recordings of individual units and MUAs, changes occurring in sensory-evoked activity using the Mann-Whitney U (MWU) test. Differences in neuronal activity between groups were analyzed using the MWU test. Comparisons of proportions of bursting neurons were made using the Pearson's χ2 test. P < 0.05 was considered significant.
For each group, extreme outliers were identified using the Fourth-Spread method and removed (Hoaglin et al., 1986).
MD neurons were particularly sensitive to level of anesthesia, such that when animals were deeper than level III-2 described by Friedberg et al. (1999), we recorded very few spontaneously active neurons in MD and virtually no neurons with peripherally evoked responses. To address this issue and to be sure we were making appropriate comparisons between sham and spinal-lesioned animals, we carefully monitored level of anesthesia in all experiments using several physiological metrics, including corneal reflex, breathing rate, pinch withdrawal as in (Friedberg et al., 1999), and electrocorticogram (ECoG) activity (described in detail in Methods).
We identified neurons in MD using two main criteria: their location, based on stereotaxic coordinates (Paxinos and Watson, 1998) and confirmed histologically post hoc, and their responses to noxious peripheral stimulation. We focused our recordings on neurons near the border between the central and lateral subdivisions of MD (Fig.1D) because these regions have a high density of thalamocortical neurons projecting to the anterior cingulate and insular cortices (Krettek and Price, 1977; Groenewegen, 1988) (see also Jones, 2007), regions which are important for nociceptive processing and pain perception (Treede et al., 1999; Price, 2000; Treede et al., 2000; Johansen et al., 2001; Wang et al., 2003; Jasmin et al., 2004; Wang et al., 2004; Shyu and Vogt, 2009; Zhang et al., 2011). In primates, this region of MD also receives dense inputs from several other regions in the frontal cortex (Erickson and Lewis, 2004). In addition to its relationship with the frontal cortex, primate lateral MD receives inputs from periaquedactal gray and from ZI (Erickson et al., 2004). Thus, lateral MD in both primates and rodents has extensive connections with pain related brain regions.
Most MD neurons had distinctive action potential waveforms. Figure 1A shows an extracellular recording from a representative MD neuron (asterisks) that has a large biphasic action potential with two positive peaks, and a greater than 2 msec duration. These waveform indices were characteristic of most (>85%) of the neurons recorded in MD. This likely reflects the rather homogeneous neuronal population of this nucleus, which contains stellate and fusiform excitatory neurons and, like some other thalamic nuclei in rodents, contains virtually no GABAergic interneurons (Ottersen and Storm-Mathisen, 1984; Kuroda et al., 1992). However, we occasionally recorded neurons with action potentials that did not have two positive peaks and that had a less than 1.5 msec duration (Fig. 1A, carets). Because these were rare (<15%) and because these cells were generally not responsive to peripheral stimulation, we excluded them from further analyses.
As reported by others, we found that neurons in MD are responsive to peripheral stimulation, and that these responses occur at relatively long latencies (Sham: n = 52, interquartile range (IQR) = 0.2 - 1.7 sec; SCI: n = 58, IQR = 0.2 - 1.4 sec; calculated using 100 msec long bins) , possibly reflecting their role in the affective—as opposed to sensory-discriminatory—component of pain perception (Dostrovsky and Guilbaud, 1990; Wang et al., 2003; Zhang et al., 2011). Note also that the latencies reported here (and in previous studies) are affected by the relatively large temporal bins used in the analyses (100 ms in the present study). The receptive fields of these neurons were always bilateral and often included limbs and parts of the torso (Fig. 1B). In some cases, stimulation to the entire trunk and all four limbs evoked responses from these cells. Though we did not systematically study areas above the neck, some MD neurons responded to parts of the head, both forepaws, and the upper torso. We focused our recordings on neurons that responded to stimulation of the hindpaws because in this model of SCI-Pain bilateral thermal and mechanical hyperalgesia develops in dermatomes below the spinal lesion (Masri et al., 2009) and because in both anesthetized and awake experiments it was more feasible to control placement, duration, and intensity of stimulation to the hindpaws than to areas of the torso or the forepaws.
Consistent with previous reports (Dostrovsky and Guilbaud, 1990; Wang et al., 2003; Zhang et al., 2011), we found that MD neurons responded almost exclusively to noxious peripheral stimulation: In preliminary experiments in naive rats, fewer than 13% (4 of 31 cells) responded weakly to innocuous stimulation. We therefore did not quantify responses to innocuous touch in subsequent experiments. Figure 1C shows a rate histogram of typical spontaneous and evoked firing of MD neurons in control (sham) animals. This neuron had no spontaneous activity and was not activated by touch with a wooden probe (innocuous peripheral stimulation) on the hindpaws or lower trunk. Noxious pinch to the hindpaws elicited responses, and repeated pinches caused an increase in its firing rate (compare firing rate after arrowheads, Figure 1C). As a group, MD neurons in sham animals had similar response properties to those described above, and only frank noxious stimulation evoked increased firing rates. Individual neurons showed both increased firing rate and increased duration of response to later pinches compared to initial pinches, though the peripheral stimulation was always identical (1 second pinch, 200-225 g; compare firing rate and duration after arrowheads, Figure 1C; quantified below).
To test whether SCI affected neurons in MD, we compared spontaneous and evoked activity of MD neurons from sham and SCI rats. Figure 2A shows a rate histogram computed from activity of an MD neuron in a spinal-lesioned animal with confirmed hyperalgesia. Note the presence of spontaneous firing rate in this neuron, compared to that in sham-operated animas (compare Figure 1C “spontaneous” to Figure 2A “spontaneous”). MD neurons from animals with SCI (58 neurons from 6 animals) had, on average, a 2.5 fold increase in spontaneous firing rates, compared to those from sham animals (52 neurons from 3 animals) (sham: mean = 1.9 ± 0.26 Hz, median = 1.3; SCI: mean = 4.8 ± 0.75 Hz, median = 2.3; P = 0.011, MWU; Figure 2B).
Similar to sham animals, MD neurons in spinal-lesioned rats did not respond to innocuous peripheral stimulation, but they did respond robustly to noxious pinch of the hindpaws. As described above for neurons from sham animals, neurons in animals with SCI responded to repeated noxious stimulation with increasing firing rate and increasing duration of response (compare firing rate and duration after arrowheads, Figure 2B; quantified below).
To test whether animals with SCI had increased responses to peripheral stimulation, we compared magnitude and duration of neuronal activation in response to 1-sec pinches between sham-operated and spinal-lesioned animals. Total magnitude of response (combined responses to pinches 1 to 4) was nearly 7 fold higher in SCI animals than in sham-operated animals (sham: mean = 4.3 ± 1.2 spikes/stimulus, median = 1.7; SCI: mean = 29.0 ± 8.0 spikes/stimulus, median = 3.5; P < 0.0001, MWU; Figure 2C). Not only did MD neurons from SCI animals have significantly higher firing rates in response to noxious stimulation, but they also had elevated firing rates for significantly longer periods of time. Figure 2D shows total duration of elevated firing rates after noxious stimulation (pinches 1 to 4 grouped, sham: mean = 0.72 ± 0.15 sec, median = 0.30; SCI: mean = 2.6 ± 0.76 sec, median = 0.50; P = 0.0018, MWU).
Because we found augmenting responses in both sham and spinal-lesioned animals, such that later pinches produced significantly elevated firing rates for prolonged periods, we thought it insufficient to simply compare magnitude and duration of responses for all pinches grouped together. To address whether these wind-up like responses were enhanced in animals with SCI, we analyzed separately responses evoked by individual pinches. Response magnitude evoked by the first pinch was larger in spinal-lesioned animals when compared to that of sham animals, and this difference approached significance (sham: mean = 2.4 ± 0.35 spikes/stimulus; median = 1.5; SCI: mean = 33.3 ± 21.3 spikes/stimulus, median = 2.29; P = 0.068, MWU; Figure 2E). However, spinal-lesioned animals had significantly larger evoked responses to each of pinches 2 to 4 (Pinch 2: sham: mean = 2.52 ± 0.36 spikes/stimulus, median = 1.84; SCI: mean = 18.3 ± 5.88 spikes/stimulus, median = 3.62; P = 0.018, MWU. Pinch 3: sham: mean = 3.01 ± 0.51 spikes/stimulus, median = 1.64; SCI: mean = 48.6 ± 22.1 spikes/stimulus, median = 4.14; P = 0.009, MWU. Pinch 4: sham: mean = 9.48 ± 4.65 spikes/stimulus, median = 2.07; SCI: mean = 14.4 ± 4.00 spikes/stimulus, median = 4.17; P = 0.031, MWU. Figure 2D). A similar augmenting pattern was seen with response duration (data not shown).
The function and prevalence of bursts in thalamic cells during conditions of pain have been debated. Some have suggested that chronic pain conditions are associated with increased incidence of bursting activity in somatosensory thalamic nuclei in both humans and rats (Lenz et al., 1989; Vierck et al., 1990; Weng et al., 2003; Hains et al., 2005; Lee et al., 2005; Hains et al., 2006; Iwata et al., 2011). However, this hypothesis is not universally accepted (Canavero and Bonicalzi, 2007; Dostrovsky, 2007). Bursting activity has been reported in MD of humans with chronic pain (Rinaldi et al., 1991), though these authors acknowledge that without similar recordings from healthy individuals it is impossible to determine the significance of this finding. To our knowledge, nothing has been reported about bursting activity in MD of healthy individuals, and there are no published reports about bursts in MD of normal rats.
We therefore compared the prevalence of bursting cells between sham and spinal-lesioned animals. Because the prevalence of bursts varies as animals transition through stages of wakefulness (McCarley et al., 1983; Ramcharan et al., 2000; Swadlow and Gusev, 2001; Massaux and Edeline, 2003), we carefully controlled and monitored the level of anesthetic for each animal, as described above.
We based our burst criteria on previously published analyses of bursts in somatosensory thalamic neurons (Guido et al., 1995; Lu et al., 1995; Sherman, 1996; Masri et al., 2009) and on burst properties reported from MD neurons in humans with chronic deafferentation pain (Rinaldi et al., 1991). We defined bursts as clusters of at least three action potentials with inter-spike intervals of ≤ 5 ms in which the first spike in the burst has a preceding inter-spike interval of ≥ 100 ms during spontaneous activity.
In sham animals, 28% (15/52) of neurons exhibited burst firing during spontaneous activity. This was not different from the proportion of cells that exhibited burst firing in spinal-lesioned animals (33%, 19/58; P = 0.576, Pearson's x2).
Figure 3A shows a burst of action potentials from an MD neuron. Burst firing was typically present during spontaneous activity, but the frequency of bursts was reduced when the cell was activated by pinch. In sham animals, frequency of bursts was reduced by half during pinch evoked responses (spontaneous: mean = 0.209 ± 0.075 Hz, median = 0.033; evoked: mean = 0.147 ± 0.07 Hz, median = 0.013; P = 0.02, Mann-Whitney U; Figure 3A) compared to spontaneous activity. Similarly, in spinal-lesioned animals frequency of bursts was significantly lower during pinch evoked responses than during spontaneous activity (spontaneous: mean = 0.26 ± 0.056, median = 0.15; evoked: mean = 0.14 ± 0.044, median = 0.073; P = 0.02, MWU; Figure 3B). There was no difference in frequency of bursts between sham and spinal-lesioned animals during either spontaneous or evoked activity (spontaneous: P = 0.12, MWU; evoked: P = 0.3, MWU). Taken together, these findings are not consistent with the hypothesis that SCI-Pain is associated with increased bursting in MD neurons.
We have previously shown that both spontaneous and evoked activity in ZI are significantly reduced after spinal lesion (Masri et al., 2009) and others have shown that ZI projects to thalamocortical neurons in MD. In monkey, Erickson et al. (2004) report that up to 10% of all neurons that project to MD originate from ZI; this likely represents a major inhibitory input to MD. In their original manuscript, Bartho et al. (2002) report that in the rat there are “scattered” ZI terminals in MD. In a personal communication the senior author of that manuscript suggests that they likely underestimated the density of these inputs due to their restricted injection sites in ZI. Their more recent, unpublished, data in mice demonstrate dense projections from more rostral ZI sectors to MD (László Acsády, personal communication).
Therefore, we hypothesized that the significant increase of neuronal activity in MD after spinal lesion was a result of decreased inhibition from ZI. However, to the best of our knowledge, functional inhibition of MD by ZI activity has never been demonstrated. To test whether ZI exerts significant tonic inhibition on neurons in MD, in naïve animals we transiently inactivated ZI neurons with the GABAA receptor agonist muscimol, while recording spontaneous activity from MD neurons (described in detail in Methods). Figure 4A shows a rate histogram (5 sec bins) of a representative MD neuron before and during ZI inactivation. The infusion of 0.1 mL muscimol (arrow) into ZI caused a significant increase in MD firing rate. Infusion of the same volume of saline (arrowhead) did not significantly alter firing rate. On average, inactivation of ZI caused a 2.5 fold increase of spontaneous neuronal activity in MD neurons when compared to baseline activity (baseline: mean = 1.61 ± 2.62 spikes/sec, median = 0.85; after muscimol: mean = 4.37 ± 5.04 spikes/sec, median = 1.95; n = 36; P = 0.0002, MWU; Figure 4B). Infusion of an equivalent volume of saline (before or after the muscimol injections) had no significant effect on the activity of MD neurons (baseline: mean = 1.03 ± 1.14 spikes/sec, median = 0.667; after saline: mean = 1.09 ± 0.758 spikes/sec, median = 0.9; n = 11; P = 0.532, MWU). The significant increase in MD spontaneous activity during ZI inactivation indicates that, under normal conditions, ZI tonically inhibits MD, and is consistent with our hypothesis that decreased activity of ZI is causally related to the increase of MD activity in conditions of chronic pain. We note, however, that we cannot exclude the possibility that muscimol inadvertently inactivated pathways other than the one from ZI to MD. We describe, below, the results of a control experiment that addresses this possibility.
To further test the causal relationship between ZI and MD activities, we asked whether reversible, pharmacological inactivation of ZI affects behavioral hyperalgesia. In rats implanted with microdialysis probes over the right, ventral ZI, we compared responses to mechanical stimuli before and after infusion of muscimol (as described above). Muscimol infusion in ZI resulted in a large and significant (p<0.01, t-test) reduction in withdrawal threshold of both the left and right hindlimbs (Fig. 6). That ipsilateral ZI inactivation had bilateral effects may be related to the bilateral projections from ZI to thalamic nuclei in both hemispheres (Power and Mitrofanis, 2001). This is consistent also with our findings that unilateral spinal lesions result in bilateral hyperalgesia (Masri et al., 2009), and unilateral stimulation of motor cortex results in bilateral pain relief (Lucas et al., 2011).
Injections of saline had no effect on withdrawal thresholds of either limb.
We observed also, in 3 of 4 rats, that immediately after muscimol injections they displayed typical nociceptive behaviors, including vigorous shaking of a hindlimb and repetitive facial grooming. These behaviors—which were not quantified—ended within 30 minutes after drug application. They were never observed in animals that received saline injections.
As a negative control, in 2 rats we infused muscimol into the substantia nigra (pars reticulata). We chose this nucleus because it neighbors ZI, allowing us to test the possible diffusion of drugs distal to the intended injection region (ZI). Further, the substantia nigra contains inhibitory neurons that may affect MD activity indirectly, through their projections to the superior colliculus (Edwards et al., 1979), which provides significant inputs to MD (Groenewegen, 1988). In contrast to the effects of injections into ZI, muscimol injection into substantia nigra had no discernible effects on paw withdrawal thresholds, nor on the rats’ behavior (Fig. 6).
These findings are consistent with the conclusion that decreased activity of ZI is causally related to the increase of MD activity in conditions of chronic pain.
Pain is a complex sensation, the individual components of which can be modified and modulated by behavioral states such as arousal and attention (Miron et al., 1989; Buffington et al., 2005). For this reason, we next tested whether SCI affected neuronal activity in MD of awake, behaving animals. We obtained extracellular multiunit recordings from MD of seven awake animals both before and after spinal lesion. All animals included in this study developed clear and statistically significant mechanical hyperalgesia after the spinal lesion. We identified multiunit activity (MUA) in MD using two main criteria: recording location, based on stereotaxic coordinates (Paxinos and Watson, 1998) and confirmed histologically post hoc, and responses to mechanical and thermal peripheral stimulation. After histological reconstruction of recording sites, MUA recordings outside of MD were excluded from further analysis. Figure 5A depicts a rate histogram (100 ms bins) from a representative MUA recording taken from an awake animal before spinal lesion. Both spontaneous activity and activity evoked in response to a series of von Frey filaments are shown. All traces were taken from the same continuous recording, and depict an increased firing rate in response to increased von Frey force. The experimental procedure (see Methods), did not allow us to record from the same site before and after SCI. However, to our knowledge, our is the first demonstration of spontaneous and evoked activity—to innocuous and noxious stimuli— in MD of a normal, awake animal.
We emphasize that MUA reflect the summed activity of multiple neurons. Therefore, changes in the firing rate of MUAs may reflect changes in the firing rates of individual neurons in the recorded cluster, as well as changes in the number of neurons recorded in that cluster.
To test whether neurons in MD of behaving animals had increased spontaneous activity after spinal lesions, we compared spontaneous firing rates of MUA in MD during two different behavioral conditions. First, we recorded two minutes of spontaneous activity while animals were in their home cages and while animals were freely moving on the testing platform (described in detail in Methods). After spinal-lesion there was a significant increase in MUA spontaneous firing rate in MD when the animals were in the home cage (before lesion: mean = 2.69 ± 0.44 spikes/sec, median = 1.82, n = 29 MUA; after lesion: mean = 5.30 ± 0.65 spikes/sec, median = 5.0, n = 31 MUA; P = 0.0033, MWU; Figure 5B). There was also a 3-fold increase in spontaneous firing rate recorded while animals were freely moving on the elevated testing platform (before lesion: mean = 3.45 ± 0.50 spikes/sec, median = 3.21, n = 29 MUA; after lesion: mean = 10.78 ± 1.71 spikes/sec, median = 7.80, n = 31 MUA; P = 0.0000, MWU, Figure 5B).
These data show that spontaneous neuronal activity is increased after SCI both in environments associated with noxious peripheral stimulation and in less anxiety-producing environments such as the animals’ home cage. To our knowledge this is the first report of enhanced spontaneous neuronal activity in MD of awake behaving animals with hyperalgesia after SCI.
We tested for changes in pain thresholds while the animals were on an elevated platform with a mesh floor (described in Methods). Briefly, we applied von Frey filaments in ascending order (range: 26-180 g) five times each to the plantar surface of the rats’ hindpaws to evoke neuronal activity in MD neurons and to obtain pain thresholds for each animal, both before and after spinal lesion.
For each stimulus intensity, we compared response magnitude of MUAs that had significant stimulus evoked increases in firing rate (exceeding 99% confidence interval relative to spontaneous firing rates) for animals before lesion to those after spinal lesion. In response to 26 g von Frey filament stimulation there was a large increase in mean firing rate after lesion and the difference between the two groups approached statistical significance (before lesion: mean = 10.2 ± 3.62 spikes/stimulus, median = 4.61; after lesion: mean = 26.7 ± 5.59 spikes/stimulus, median = 23.4; P = 0.069, MWU; Figure 5C).
When 60 g of force was applied to the hindpaw of animals with SCI, there was a 6-fold increase in MUA response magnitude compared to responses from the same animals before spinal-lesion (before lesion: mean = 6.60 ± 1.95 spikes/stimulus, median = 1.52, n = 24 MUA; after lesion: mean = 44.7 ± 13.5 spikes/stimulus, median = 10.0, n = 31 MUA; P = 0.0083, MUA; Figure 5C). Similarly, there was, on average, a 3.5-fold increase in response magnitude when 100 g of force was applied the hindpaws of animals with SCI, compared to before lesion responses (before lesion: mean = 7.62 ± 2.54 spikes/stimulus, median = 4.89, n = 24 MUA; after lesion: mean = 26.5 ± 5.22 spikes/stimulus, median = 28.1, n = 23 MUA; P = 0.0023, MUA, Figure 5C).
After obtaining mechanical thresholds with von Frey filaments, we applied a clearly noxious heat stimulus (42-45 °C, 10 sec intervals), using a heat probe (see Methods), 5 times to alternating hindpaws of the animals. In all animals, both before and after spinal surgery, the heat probe elicited a clear nocifensive response, including hindpaw withdrawal, licking, biting, and shaking. There was no significant difference in the magnitude of neuronal responses elicited by the heat probe between animals before and after lesion (before lesion: mean = 14.5 ± 4.49 spikes/stimulus, median = 0.82, n = 24 MUA; after lesion: mean = 9.55 ± 2.04 spikes/stimulus, median = 5.95, n = 31 MUA; P = 0.64, MWU; data not shown). Fifty-four percent (13/24) of MUAs in MD of before lesion animals and 70% (21/30) of MUAs in MD of after lesion animals had significant increases in firing rate in response to the heat probe; this difference was not significant (P = 0.231, Pearson's x2). A possible explanation for the lack of effect of SCI on MD responses to noxious thermal stimuli may be that in both groups of rats these stimuli saturated the responses of the peripheral, heat-sensitive receptors; that is, that these results are confounded by a ‘ceiling effect’.
We have previously shown that hyperalgesia after SCI is associated with a reduction in neuronal activity in the GABAergic zona incerta (ZI) and that this correlates with an increase in neuronal activity in somatosensory thalamic nuclei, such as the PO (Masri et al., 2009). Because pain is both a sensory-discriminative experience and an unpleasant emotional experience (Merskey and Bogduk, 1994), we tested the hypothesis that chronic pain produces an increase of activity also in associative thalamic nuclei involved in processing affective components of pain. We focus on the mediodorsal thalamus (MD) which has been proposed to be involved in integrating somatosensory and affective components of pain (Price, 2002). Consistent with this hypothesis, we found that neurons in MD are strongly driven by noxious peripheral stimulation in both anesthetized animals and awake behaving animals. Consistent with this hypothesis, we observed that SCI causes a robust increase in both spontaneous and evoked MD activity in both anesthetized and awake behaving animals.
The subdivisions and cortical projections of MD are largely conserved between species, though the anatomical nomenclature can vary. Considering thalamocortical projections and cellular architecture, Ray and Price (1993) identified the primate MD pars caudo-dorsalis as analogous to the rat medial and dorsolateral MD studied here. Although there is evidence that MD plays a critical role in processing affective components of pain, there have been few studies on the extracellular electrophysiological properties of MD neurons (Mogenson et al., 1987; Lavin and Grace, 1998; Vaculin et al., 2000), and even fewer focused on responses to noxious peripheral stimuli (Dostrovsky and Guilbaud, 1990; Wang et al., 2003; Zhang et al., 2011). To the best of our knowledge, no studies have been conducted on the activity of MD neurons in animal models of SCI-pain.
Besides its role in processing noxious stimuli, MD has been implicated in diverse functions, including learning and memory and other cognitive functions (Jones, 2007). We therefore cannot exclude the possibility that the electrophysiological changes we report in MD might be related also to other consequences of SCI.
In humans, SCI-Pain presents not only as exaggerated responses to peripheral stimuli, but also as chronic spontaneous pain (Bowsher, 1996; Garcia-Larrea et al., 2002; Finnerup et al., 2003). We have shown that in this model of SCI-Pain rats develop mechanical and thermal hyperalgesia as well as a tonic aversive state, analogous to the human condition of spontaneous pain (Davoody et al., 2011). Here, we report that spinal lesions result in significant increases in spontaneous firing of MD neurons. Because MD is densely interconnected with several areas of the limbic circuit that are critical for nociceptive processing (Treede et al., 1999; Price, 2000; Treede et al., 2000; Johansen et al., 2001; Wang et al., 2003; Jasmin et al., 2004; Wang et al., 2004; Shyu and Vogt, 2009; Zhang et al., 2011), it is possible that this contributes to the ongoing spontaneous pain in this condition.
We hypothesized that the increase in spontaneous activity is causally related to the dramatic loss of spontaneous activity we have recorded from ZI in animals with SCI (Masri et al., 2009). Consistent with this, here we show that, in naïve animals, ZI exerts significant tonic inhibition over MD. Even so, we considered the possibility that in SCI-Pain there is a reduction in other inhibitory inputs to MD. There are no GABAergic interneurons within MD (Kuroda et al., 1992) and, therefore, all GABAergic inhibition is mediated by extrinsic afferents. An important source of GABAergic afferents is the reticular nucleus of the thalamus (TRN). Unlike ZI, TRN does not receive spinothalamic inputs, and its major source of excitatory input is the somatosensory cortex (Liu and Jones, 1999). Moreover, we found that responses evoked by innocuous stimuli in the ventrobasal somatosensory nuclei that receive inhibition exclusively from TRN are unaffected by spinal lesions (Masri et al., 2009). These findings suggest that the enhanced activity in the thalamus after SCI is related primarily to thalamic nuclei that normally receive strong inhibition from ZI.
However, other thalamic nuclei may also have a causal role in SCI-Pain. We have previously reported that, after SCI, spontaneous activity increases modestly in ventrobasal thalamus (4 fold increase, compared with 30 fold increase in PO thalamus), and that SCI is associated with an increase in the incidence of bursting cells in ventrobasal thalamus (Masri et al., 2009). These findings are consistent with reports of similar changes in the ventrobasal thalamus from the Waxman group (Hains et al., 2005; Hains et al., 2006).
Moreover, TRN neurons can be inhibited by noxious stimuli (Yen and Shaw, 2003), and the inhibitory drive from TRN upon ventrobasal neurons regulates bursting in ventrobasal neurons (Steriade, 2000; Halassa et al., 2011). Therefore, SCI-associated alterations in inhibitory inputs from TRN may also affect the activity of ventrobasal (‘first order’) thalamic nuclei and contribute to chronic pain.
An early study by Dostrovsky and Guilbaud (1990) examined electrophysiological activity in several midline thalamic nuclei, including MD, in anesthetized normal and arthritic rats. In agreement with our findings, they found MD neurons that respond exclusively to nociceptive stimulation over large, bilateral receptive fields. Similar to the long duration, augmenting responses we describe (Fig. 1B, ,2B,2B, ,5C),5C), these authors also report that MD neurons have long after-discharges. Though the authors’ description of normal electrophysiological activity in MD agrees with what we have presented here, they found no change in spontaneous firing rate or magnitude of response in arthritic animals.
There are two critical differences between the Dostrovsky study and ours. First, they used an arthritic rat model in which inflammation related hyperalgesia, lasts several weeks (Colpaert, 1987). This hyperalgesia is largely driven by peripheral inflammation and can be relieved by administration of NSAIDs (Hirose and Jyoyama, 1971; Winter et al., 1979; Capetola et al., 1980), indicating that peripheral tissue damage is a driving force for maintenance of pain. In contrast, we use an animal model of SCI-Pain to produce long lasting pain characterized by a tonic aversive state, and that has significant central component (Masri et al., 2009). Second, the authors presented continuous noxious stimulation for 15 seconds, which may have maximized the responses, producing a ‘ceiling effect’. Because we found that MD neurons quickly changed their responses to repeated stimulation, we limited the duration of peripheral stimulation to less than 1 second of stimulation.
We found no difference in the proportion of MD neurons that had spontaneous burst activity between spinal-lesioned and sham-operated groups. Neither was there a difference in the number or frequency of bursts during spontaneous activity or of any burst property that we tested (number of action potentials per burst, inter-spike interval during burst, etc). This is consistent with our previous report on posterior thalamus, where we found no change in bursting activity in animals with SCI (Masri et al., 2009), though we and others have reported a moderate increase of bursting in first-order somatosensory nuclei (ventrobasal thalamus) in this model of SCI-Pain (Wang and Thompson, 2008; Masri et al., 2009). This selective change in burst activity following lesion of the STT may be a result of differential anatomical connections or physiological properties between first-order and higher-order thalamic nuclei (see Sherman and Guillery, 2005).
MD neurons from both sham and spinal-lesioned animals had significantly lower frequency of burst firing in response to noxious peripheral stimulation, compared to spontaneous bursts. Because thalamic burst firing requires de-inactivation of T-type calcium channels, neurons only fire in burst mode when they are relatively hyperpolarized (see Sherman and Guillery, 2005). It is possible that after noxious pinch, ascending excitation from the STT prevents thalamocortical neurons in MD from becoming sufficiently depolarized, thereby preventing neurons from firing in burst mode in response to peripheral simulation.
As discussed above, ZI potently regulates the activity of both PO and MD thalamus. We have previously shown that SCI is associated with significant decreases in the activity of ZI neurons (Masri et al., 2009). Here, we demonstrate that reversible inactivation of ZI results in immediate and profound hyperalgesia, as well as in increased activity of MD neurons. Further, we previously demonstrated that stimulation of ZI in rats with SCI results in immediate reversal of behavioral signs of hyperalgesia (Lucas et al., 2011). Thus, the present demonstration of abnormal neuronal activity in MD, taken together with our previous reports, support the conclusion that the pathophysiology of SCI-Pain involves abnormal reduction in GABAergic inhibition of thalamic nuclei that are regulated by ZI (Masri and Keller, 2011).
We are actively investigating the mechanisms through which SCI results in suppressed ZI activity. Preliminary findings (Keller, 2011) suggest that SCI is associated with a reduction of approximately 30% in the expression of GAD (the rate limiting enzyme in GABA synthesis) in ZI neurons, and a subsequent, gradual apoptosis of GABAergic neurons in ZI. Our preliminary findings also suggest that the changes result in a significant decrease in the frequency of miniature inhibitory postsynaptic potentials recorded from PO and MD neurons, supporting the conclusion that SCI results in suppression of inhibitory control of these thalamic nuclei by ZI.
We also cannot exclude the possibility that the enhanced activity of thalamic neurons reflects increased nociceptive inputs from upstream structures, such as sensitized dorsal horn neurons. Preliminary findings (Keller, 2011) suggest that this may not be the case, because acute suppression of spinal activity with injections of tetrodotoxin did not reverse the enhanced spontaneous activity of thalamic neurons in SCI rats.
It is also possible that the sensitization of thalamic neurons reflects not only disinhibitory mechanisms. For example, Waxman and collaborators reported that sensitization of VPL neurons after SCI is related to up-regulation of chemokines, microglia and unique sodium channels (Zhao et al., 2007; Dib-Hajj et al., 2010). Thus, chronic pain after SCI likely represents a multifaceted pathophysiology, involving converging processes in several thalamic nuclei.
This work was supported by NINDS Fellowship F31NS-070458 to J.L.W., Research Grants R01-051799 to A.K. and R01-NS069568 to R.M., Department of Defense grant SC090126 to R.M., and the Christopher and Dana Reeve Foundation.
Conflict of Interest: The authors declare no conflicts of interest.