Classification of MD Neurons
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 () 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. 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 (, 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 (). 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. 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, ). 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, ; quantified below).
Enhanced neuronal activity in MD
To test whether SCI affected neurons in MD, we compared spontaneous and evoked activity of MD neurons from sham and SCI rats. 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 “spontaneous” to “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; ).
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, ; 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; ). 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. 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; ). 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. ). A similar augmenting pattern was seen with response duration (data not shown).
Burst properties of MD neurons
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 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).
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; ) 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; ). 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.
Burst activity in MD neurons is not altered in animals with SCI
Reversible ZI inactivation: electrophysiology
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). 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; ). 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.
Neuronal activity in MD is enhanced during transient ZI inactivation
Reversible ZI inactivation: behavior
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 (). 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
ZI inactivation in naïve rats produces hyperalgesia
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 ().
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.
Increased spontaneous neuronal activity in MD of awake animals
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. 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; ). 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, ).
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.
Increased responses to peripheral stimulation in MD of behaving animals
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; ).
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; ). 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, ).
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’.