Evoked bursting in injured Ab dorsal root ganglion neurons: A mechanism underlying tactile allodynia

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1 PAIN Ò 153 (2012) Evoked bursting in injured Ab dorsal root ganglion neurons: A mechanism underlying tactile allodynia Ying Song a,b,1, Hui-Ming Li a,1, Rou-Gang Xie a,1, Zhi-Feng Yue a,1, Xue-Jun Song c, San-Jue Hu a,, Jun-Ling Xing a, a Institute of Neuroscience, Fourth Military Medical University, Xi an, China b Jiangsu Provincial Key Lab of Anesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu, China c Department of Neurobiology, Parker University Research Institute, Dallas, TX, USA Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. article info abstract Article history: Received 1 July 2011 Received in revised form 28 November 2011 Accepted 29 November 2011 Keywords: Dorsal root ganglion Tactile allodynia Wide dynamic range neuron Evoked bursting Chronic compression of rat dorsal root ganglion (CCD) produced tactile allodynia accompanied with hyperexcitability of the myelinated Ab dorsal root ganglion (DRG) neurons. The Ab DRG neuron hyperexcitability exhibits as bursting discharges in response to peripherally evoked action potentials (evoked bursting [EB]). The incidence of EB was significantly increased after chronic compression of DRG (CCD) (43.3%) vs control (13.3%). EB was maintained by oscillation of the membrane potential, and its duration was increased when the membrane potential was depolarized. EB was found to coexist in some neurons with spontaneous bursting (SB), but EB always occurred at a more negative membrane potential than SB. Afterdischarges of the wide dynamic range neurons of the dorsal horn in the spinal cord in response to electrical stimulation of Ab afferent nerve fibers were suppressed by blocking EB of the DRG neurons. CCD neurons with EB exhibited increased current density of persistent sodium current (I Nap ) and hyperpolarization-activated cation current (I h ) and decreased a-dendrotoxin (a-dtx) sensitive current (I DTX ). The increased I h activated by afterhyperpolarization of peripheral afferent action potential was necessary for EB generation and a balance between I DTX and I Nap might be necessary for EB maintenance. This study may suggest a role of EB of myelinated DRG neurons in development of allodynia after nerve injury and a potential pharmaceutical therapy in treating neuropathic allodynia. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Tactile allodynia is one of the common features of neuropathy after peripheral nerve injury. Clinically, allodynia is intractable to the available approaches of pain treatment [5,31]. Although the underlying mechanisms of allodynia remain unclear, there is clear evidence that myelinated Ab fibers may contribute to tactile allodynia in animals and human beings with peripheral neuropathy [8,12,31]. Phenotype switch of the pain mediators such as substance P in the superficial dorsal horn of the spinal cord [27] and probably the substantial sprouting of the Ab fibers, although it has been argued [4,17,32], are promising targets that may be Corresponding authors. Address: Institute of Neuroscience, Fourth Military Medical University, 17# Changle West Road, Xi an , China. Tel.: ; fax: addresses: sjhu@fmmu.edu.cn (S.-J. Hu), xingjunl@fmmu.edu.cn (J.-L. Xing). 1 These authors contributed equally to this work. responsible for the mechanisms of tactile allodynia [22,26,38]. More recently, it has been suggested that phenotypic switch may not be the only process whereby the Ab input could render noxious sensation, and thus the direct action of Ab ectopic afferent barrage becomes one more important focus. After nerve injury, there is a sharp increase in the ectopic spontaneous discharges carried by dorsal root afferents into the segmental spinal cord [6,24]. These activities may act as a raw pain signal as well as an inducer of central sensitization [1,8,26]. These ectopic impulses could originate from the injured sites of the targeted nerve and the forward segments such as the somata, the peripheral and central branches of the axons of dorsal root ganglion (DRG) [13,33,35]. Peripheral injury-evoked ectopic firings exhibiting as various patterns originate from the DRG somata, predominantly in Ab neurons. These ectopic Ab firings may interact with afferent signals from the peripheral receptive field [3,24,33] rather than its function of propagating input signal with great fidelity in physiological condition. The remaining question is how this interaction occurs and whether some of the specific firing patterns could act as pain signals that would contribute to tactile allodynia /$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi: /j.pain

2 658 Y. Song et al. / PAIN Ò 153 (2012) Chronic compression of DRG (CCD model) has been used to mimic DRG compression in animals. CCD treatment induces allodynia in animals that is similar in some ways to that observed in patients with sciatica and/or other similar neuropathic disorders [16,34]. In CCD neurons, massive ectopic barrage develops in Ab-hyperexcitable neurons [16,19]. Bursting, considered to represent a distinct mode of neuronal signaling in sensory systems [21], is one of the multifarious firing patterns reported in injured DRG. In our recent experiments in CCD neurons, it was observed that Ab DRG neurons with a bursting firing pattern responded to afferent input in a seizure-like manner in which innocuous touch could evoke augmenting signals, which suggests that this new bursting series might contribute to behaviorally expressed allodynia. The above hypothesis was examined in the present study. First, the way that the stimuli evoke the new bursting patterns was examined through in vivo intracellular recording and some of its basic characters were revealed. Here, evoked bursting (EB) was defined as the bursting pattern evoked by stimulation of afferent Ab nerve fibers or touching on their sensory field. Thereafter, we investigated the relationship between EB and allodynia and identified the underlying ionic mechanisms of EB. This study may provide a strategy of treating allodynia in clinic. 2. Methods 2.1. Animals and CCD models Experiments were performed in adult male and female Sprague Dawley rats ( g). Our experiments adhered to the guidelines of the Committee for Research and Ethical Issues of the IASP, and all the experimental protocols were approved by the University Health Network Research Ethics Board. The CCD model was prepared as described previously [16]. Briefly, under anesthesia with sodium pentobarbital (40 mg/kg, i.p.), the transverse process and intervertebral foramen at L4 and L5 on left side were exposed, and an L-shaped stainless steel rod was inserted into each foramen to produce a steady compression on the ganglia. Sham surgery was done in another group of rats and the surgical procedure was identical to that in CCD but without the rod insertion Behavioral tests The behavioral experiments were done in a blinded fashion (i.e., with the experimenter unaware of whether the animal was injured or was a control). Tactile allodynia was determined by quantifying the withdrawal threshold of the hind paw in response to von Frey filaments. The rats were placed in suspended individual chambers on a mesh floor and allowed to acclimate for 30 minutes. A series of calibrated von Frey filaments delivered in the order of increasing bending forces from 0.6 to 26 g (Stoelting, Wood Dale, IL) were applied perpendicularly to the central region on the glabrous surface of the hind paw for 5 seconds and at intervals of 15 seconds. Brisk withdrawal or paw flinching was considered as a positive response. The threshold was defined as the force eliciting a 50% withdrawal [34]. Sham-operated rats were used as controls In vivo intracellular recordings from DRG neurons Recordings were made from DRG neurons from 14 rats that previously received CCD for 2 to 8 days and from 11 control rats that received sham surgery. After rats were re-anesthetized with a mixture of anesthetics (a-chloralose 1.0 g/100 ml, urethane 17 g/ 100 ml i.p.; an additional injection at half dose was given in some animals as necessary), the laminectomy was performed to the segments of L4 and L5, and the rods were then carefully removed and L4 and L5 DRGs fully exposed. The capsules covering DRG were slit open with fine forceps. The animal was then transferred to a platform with its vertebral body clamped at the L3 and L6/S1 regions. A pool was prepared by suturing the skin to a metal ring and was filled with artificial cerebrospinal fluid (ACSF; content in mmol/l: NaCl 125, KCl 3.8, NaH 2 PO 4 1.2, NaHCO 3 26, glucose 10, MgCl 2 1.0, CaCl 2 2.0, ph 7.2, osmolarity 300 mosm) to cover the exposed tissue. The left sciatic nerve was exposed at mid-thigh and the exposed sciatic nerve was placed on a hook stimulating electrode, which was used to provide electronic stimulation as needed. The recording electrodes were fabricated from borosilicate glass (World Precision Instruments, Sarasota, FL) and pulled on a micropipette puller (P-97, Sutter Instrument, Novato, CA). Microelectrodes filled with 3 mol/l potassium acetate had a final resistance of 40 to 60 MX. Neurons were visualized with a 40 water-immersion objective under a microscope (BX51W, Olympus, Tokyo, Japan) equipped with infrared differential interference contrast optics and were impaled by advancing the microelectrode in steps, applying a small capacitance buzz when necessary. Data were collected with continuous current-clamp mode using an Axo- Clamp-700A (Axon Instruments, CA), stored digitally via a Digidata 1322A interface, and analyzed off-line. Electrode resistance was balanced by a bridge circuit in the amplifier. Intracellular hyperpolarizing or depolarizing currents were applied through the microelectrode amplifier. DRG neurons were classified, based on their conduction velocity measured at the sciatic nerve, as C (<2 m/s), Ad (2 12 m/s) or Ab/a (>12 m/s) [14,25]. Moreover, innocuous touch was applied to receptive field to identify Ab neuron. Cells that had stable (>3 min) membrane potentials ( 45 mv or more negative) after initial penetration were further tested. The heart rate and expiratory CO 2 concentration (3% 5%) were monitored throughout the experiment. Tetrodotoxin (TTX) (50 nmol/l) and lidocaine (2%) were obtained from Sigma Chemicals (St. Louis, MO) and were diluted from stock solutions before application In vivo extracellular recordings from the spinal wide dynamic range neurons As described previously, the rats were anesthetized by mixture anesthetics and spinal cord was transected at T9 to avoid supraspinal effects [28]. A laminectomy of vertebral segments T13 to L1 was then performed to expose the lumbar enlargement. The L4 and L5 DRGs were also exposed, and a surrounding pool was prepared for drug application. After fixing on a stereotactic frame, a pool on the exposed lumbar enlargement was made and filled with 2% warm agar (Sigma, St. Louis, MO) to a depth of 2 to 3 mm to prevent spinal cord movement and to protect the exposed spinal cord from drying. A small hole was made to expose the enlargement after the agar coagulated, and dura was excised, after which the exposed tissue was covered with ACSF. Extracellularly recordings were performed with glass microelectrodes with resistance of 8 to 10 MX at the depth of 300 to 900 lm below the surface of the dorsal horn. Neuronal activities were amplified and stored using pclamp 8.0 software. Only units with receptive field areas corresponding to the L4 and L5 spinal cord were chosen based on prior dermatomal mapping studies [36]. Wide dynamic range (WDR) neurons, which responded in a graded fashion both to innocuous and noxious stimuli, were considered for further study. Cells were further tested with electric current stimulation with steel hook electrode administered under sciatic nerve. The stimulus intensity used (0.1 ma, 0.05 ms, 1 Hz, 10 s) was for evoking the activity from Ab but not Ad or C fibers [37]. When testing on indentation of the skin with a set of von Frey filaments (4, 15, 60 g), each mechanical stimulus was applied for 10 seconds.

3 Y. Song et al. / PAIN Ò 153 (2012) TTX was applied in the solution of the pool made surrounding the L4 and L5 DRGs. Spontaneous and evoked WDR neuronal activities were measured at 1, 5, 15, and 25 min after. If the firing rate was stable, 2% lidocaine was applied on DRGs to determine whether the recorded WDR neurons received input from L4 or L5 DRG. The WDR neurons with unstable firing rate were not included in the database In vitro patch clamp recordings from DRG neurons At 2 to 8 days after CCD, the L4 and L5 DRGs with attached sciatic nerve were carefully removed. After the connective tissue was cleaned, the preparation was digested with a mixture of 0.4 mg/ml trypsin (Sigma) and 1.0 mg/ml type-a collagenase (Sigma) for 45 min at 37 C. Then it was transferred into ACSF to incubate at 28 C for at least 1 hour, and was agitated by gentle bubbling with 95% O 2 and 5% CO 2. Next, the preparation was moved into recording chamber. The whole ganglion was stabilized by a slice anchor, and the nerve was connected with a suction stimulate electrode. Whole-cell current and voltage clamp recordings were carried out by using Multiclamp 700B amplifier (Molecular Devices Corporation, Sunnyvale, CA) after a giga-ohm seal had been established. The electrode had a final resistance of 4 to 7 MX and was filled with normal intrapipette solution containing (in mmol/l) 120 potassium gluconate, 18 KCl, 2 MgCl 2, 5 EGTA, 10 HEPES, 5 Na 2 - ATP, 0.4 Na-GTP and 1 CaCl 2 (ph 7.2 adjusted with KOH, osmolarity 300 mosm). The series-resistance was 10 to 20 MX. Pipette offset current was zeroed immediately before contacting the cell membrane, and electrode capacitance was canceled after seal formation. After establishing the whole-cell recording mode, series resistance was compensated to 70% to 75%. The possible ionic mechanisms of EB were explored in patch clamp recording experiments. To identify an EB cell, the recordings were made with normal intracellular solution under current clamp mode, and voltage clamp mode was then used to test currents underlying EB. In some experiments (see I Nap measurement), a second patch was made on the cell to change to a different intracellular solution. To isolate I NaP, K-current was blocked using an intrapipette solution containing (in mmol/l): 130 CsF, 9 NaCl, 10 HEPES, 10 EGTA, 1 MgCl 2,3K 2 -ATP, and 1 Na-GTP. External solutions contained (in mmol/l): 131 NaCl, 10 HEPES, 3 KCl, 10 glucose, 1 CaCl 2, 2 MgCl 2, 10 tetraethylammonium (TEA)-Cl, 10 CsCl, 3 4-aminopyridine (4-AP), and 0.3 CdCl 2. Depolarizing voltage ramp from 90 to 10 mv was applied within 3 seconds in voltage clamp mode. The rising rate of voltage ramp was slow enough to inactivate I NaT [39].To isolate the a-dendrotoxin (DTX)-sensitive potassium current, normal intrapipette solution was used and Na current was blocked using external solution (in mmol/l): 150 choline chloride, 5 KCl, 2 CaCl 2, 3 KCl, 1 MgCl 2,10 HEPES, 1CdCl 2, and 10 D-glucose (ph 7.4 adjusted with KOH, osmolarity 320 mosm). The cell, in 0.5 lmol/l TTX and 100 lmol/l CdCl 2, was stepped for 300-millisecond intervals from 75 to 35 mv in increments of 10 mv, and was applied from a holding potential of 90 mv to elicit outward currents. These sets of commands were repeated in 100 nmol/l a-dtx and I DTX were obtained by subtraction. For recording of I h, ACSF and normal intrapipette solution mentioned above were used. To activate I h, hyperpolarizing potentials of 110 to 60 mv were delivered in increments of 10 mv from a holding potential of 50 mv for duration of 6 s. The current was obtained by the steady state current minus current at the beginning of the step. Membrane potential oscillation was sampled at 20 khz for 8 to 10 seconds (pclamp 8.0) and processed (Origin 8.0) for Fast-Fourier Transform (FFT) Statistical analysis Differences between CCD and control neurons were analyzed by Student s t test and v 2 tests. In the case of multiple comparisons, data were initially analyzed using a repeated-measures analysis of variance (ANOVA). If this revealed a significant difference, Bonferroni tests were applied for post hoc testing. Data were expressed as means ± SE. Statistical results were considered significant if p < Results 3.1. CCD-induced tactile allodynia We first tested the well recognized tactile allodynia in CCD rats. As shown in Fig. 1, in the sham-operated control rats, the paw withdrawal threshold (PWT) was ± 2.92 g on day 0 and ranged around 15 g throughout the period of 20 days tested. In the CCD rats, tactile allodynia was present on the operated side in 10 of 10 rats at the first test made 24 h after surgery, the PWT decreased from ± 1.88 g before surgery to 2.97 ± 1.68 g. Tactile allodynia reached a peak on postoperative day 2, remained at a plateau for at least 1 week (Fig. 1), and lasted for at least 20 days after CCD treatment (p < 0.05). The electrophysiological recordings were made on days 2 to 8 after CCD in the following experiments. Four of 10 CCD rats on the first postoperative day exhibited lifting and licking of the hind paw for several seconds when a cotton wisp was applied to the foot ipsilateral to the compressed DRGs. Similar results have been reported by other groups [34] EB and its characters recorded from the DRG neurons As reported previously, injured DRG neurons exhibited spontaneous firing in various patterns, in which most of the Ab neurons displayed burst pattern especially in the first week after injury, while majority of the Ad or C neurons displayed an irregular firing pattern. The cellular responses to peripheral stimuli were tested in vivo on 254 Ab DRG neurons, of which 120 neurons were from 11 control normal rats and 134 from 14 CCD rats. The results showed that Ab neurons in CCD rats displayed EB a periphery input spike followed by somatic bursting firing (Fig. 2A and B). EB could be identified by the first spike that had a peripheral input property with rapid-onset kinetics (Fig. 3Bb). EB was evoked in vivo by innocuous touch on the receptive field as shown in Fig. 2A, or by electrical stimulation (0.1 ma, 0.05 ms) that excited only Ab fibers of the sciatic nerve (Fig. 2B). In the following experiment, electrical stimuli were used for inducing EB. The occurrence of EB relied on the propagation of the periphery afferent spike; in other words, EB was evoked only by peripheral stimulation, either electrical or mechanical. In 6 cases, EB was prevented by topical application of 2% lidocaine to the sciatic nerve Fig. 1. Time course of tactile allodynia produced by chronic compression of dorsal root ganglion (CCD). Mechanical allodynia was quantitatively measured by von Frey filaments test. Each data point represents the mean paw withdrawal threshold (PWT) value on the selected day. Eight and 10 rats were included in CCD and shamoperation groups, respectively. P <.01 vs sham group.

4 660 Y. Song et al. / PAIN Ò 153 (2012) Fig. 2. Evoked bursting (EB) and its properties. (A and B) Examples of EB induced in Ab DRG neurons by touching the peripheral receptive field (A) or stimulating the sciatic nerve (B). In (A), touch stimulation evoked totally 3 EBs, which outlasted the stimulus duration (upper trace), In the bottom trace, EB could be clearly distinguished by an extension observation of the framed area in the upper trace. In (B), the discharge was in response to a single electrical stimulus. (C) Stimulating sciatic nerve-induced EB (upper trace as control) was blocked by 2% lidocaine (lower trace). (D) Duration of EB increased with depolarization of the membrane potential. Right: EB s voltagedependence plot (n = 9). (E) Application of tetrodotoxin (TTX; 50 nmol/l) abolished the repetitive discharges in an EB neuron. Arrows indicate the stimuli applied on the sciatic nerve. Fig. 3. Similarities and differences between spontaneous bursting (SB) and evoked bursting (EB). (A) Both the occurrence and duration of EB and SB were membrane potential dependent. The depolarization afterpotential could be observed after peripheral input-evoked spike (5th trace) or at the end of the burst as marked by arrowheads (4th trace). (B) Framed areas (a) and (b) in (A) are shown on an expanded scale. The first spike of EB in (b) exhibited steeper slope than that of SB (a) as indicated by the arrow. (C) EB can be produced at a more negative membrane potential level compared with SB. P <.05. between the stimulating electrode and DRG (Fig. 2C). A second property of EB was that the ability of a cell to develop EB in response to peripheral stimulation was dependent on the presence of subthreshold membrane potential oscillation (SMPO). Approximately 80% of neurons with EB showed SMPO at resting potential, the remaining 20% showed SMPO on depolarized membrane potential. Moreover, it was observed that the duration of EB increased with the level of membrane potential depolarization in each EB cell (Fig. 2D). The duration of EB lasted for 60 milliseconds to 1.8 seconds accompanied by membrane potential alteration from 60 mv to 45 mv (Fig. 2D, right). In addition, as shown in Fig. 2E, administration of TTX (50 nmol/l) on the DRG abolished the EB and SMPO without eliminating the peripherally evoked action potential. Such inhibition was reversible after washout.

5 Y. Song et al. / PAIN Ò 153 (2012) The incidence of EB in CCD neurons was significantly greater than that in the control DRG neurons (v 2 = 25.71, P <.01). Only 3.33% of Ab neurons (4 of 120) from intact group exhibited EB at resting membrane potential, and an additional 10% displayed EB (12 of 120) on depolarization (up to 30 mv). However, 13.4% (18 of 134) of injured Ab neurons exhibited EB at resting membrane potential in CCD group (Table 1), and an additional 29.9% (40/134) neurons exhibited EB on depolarization Similarities and differences between spontaneous bursting and EB In injured DRG neurons, the appearance of spontaneous bursting (SB) has previously been reported [3,42]. The voltage sensitivity of EB duration was also observed in SB neurons [3]. Both EB and SB were generated on the basis of SMPO. EB and SB would disappear once SMPO was abolished by either application of chemicals or alteration of membrane potential. Despite these similarities, EB could be distinguished from SB in at least two ways: the first is the shape of the first spike of EB or SB. In cells with SB, the first spike in each burst was triggered when an oscillatory sinusoid reached threshold (Fig. 3Aa), and thus there was a pre-potential phase at the foot of the first spike, with a rising speed of 2.34 ± 0.92 mv/ms (Fig. 3Ba). In contrast, EB was evoked by peripheral input spike at membrane potential, with a very steep rising slope of ± 6.46 mv/ms (Fig. 3Bb). The second is the membrane potential level at which the EB or SB started. In Fig. 3A, for instance, there was no SB and no EB at resting potential ( 60 mv) of the Ab cell; however, along with the membrane depolarization, EB first appeared at 51 mv, whereas SB started at 45 mv. This indicates that EB can be generated at more negative potential than SB (Fig. 3C). The voltage difference for EB and SB was 4.8 ± 0.63 mv (range, 2 8 mv; n = 11). However, SB was not induced in another 15 neurons with EB when membrane potential was depolarized to 30 mv. high frequency discharge during electric stimulation followed by persistent firing after termination of the stimulation. A representative response is shown in Fig. 4A. These patterns have been reported as an after-discharge in a previous study [28]. Furthermore, topical administration of TTX at 50 nmol/l onto the DRG, which did not affect conduction of the evoked action potential as shown in DRG recording in vivo, produced a significant, reversible inhibition of the innocuous electric stimulus-evoked afterdischarges of WDR neurons in CCD rats (Fig. 4). The number of the afterdischarges was reduced 56.36% ± 5.29%, decreasing from 260 ± 5.7 to 119 ± 3.1 spikes/response (Fig. 4B) Ionic mechanisms of EB We first tested role of I Nap in EB formation. Following application of TTX (5 nm), the EB duration (Fig. 5A) and SMPO amplitude (Fig. 5B and C) were significantly reduced. In voltage-clamp experiments, the results showed that the TTX-sensitive I NaP current density was much higher in EB cells, compared to that in non-eb cells (Fig. 5D and E). We also tested the possible role of low threshold, a-dtx-sensitive K-current in EB. a-dtx was used to determine whether the a-dtx-sensitive component of the outward current participated in the EB formation. Application of a-dtx (1 nmol/l) increased duration of the EB and the membrane oscillation amplitude (Fig. 6A C). When a-dtx was used at higher concentration (50 nmol/l), the firing pattern was transferred from EB to tonic firing (data not shown). Voltage-clamp experiments were performed to test the a-dtx sensitive current (I DTX ) in EB and non-eb neurons (Fig. 6D and E). At certain levels of the membrane potential, the mean current 3.4. Blocking EB at DRG level suppressed responses of spinal WDR neurons A WDR neuron in the spinal dorsal horn can be identified by its response to both innocuous and noxious stimulation applied to the peripheral receptive field and the responses increase with the increment of stimuli intensity. Studies have suggested that the afterdischarge of WDR neurons to peripheral innocuous mechanical stimulation is a distinguishing feature of spinal neural correlate of tactile allodynia in a polyethylene cuff model [28]. In CCD rats with tactile allodynia, innocuous stimulation also could induce nociceptive-like afterdischarge that persisted 2 to 3 minutes [28]. This afterdischarge was not observed in control rats. We used this afterdischarge as a feature of the spinal neural correlate of tactile allodynia in CCD rats and investigated and compared responses of spinal WDR neurons to peripheral stimulation between the CCD and normal control groups. In normal rats, peripheral innocuous electrical stimulation (0.1 ma, 0.05 ms, 1 Hz, 10 s) evoked only initial high-frequency activities within the duration of the stimuli. However, in 37 WDR neurons from 13 CCD rats, there was an initial Table 1 Incidence of EB in Normal and CCD groups. Group With EB At Vt Depolarized to 6 30 mv Total Normal 4/120 (3.33) 12/120 (10) 16/120 (13.33) CCD 18/134 (13.4) 40/134 (29.9) 58/134 (43.3) ** CCD = chronic depression of dorsal route ganglion; EB = evoked bursting. Numbers in parentheses are percentages (%). ** P < 0.01 vs normal. Fig. 4. Ab-stimuli-induced afterdischarge of wide dynamic range (WDR) neuron was suppressed by blocking dorsal root ganglion (DRG) neuronal evoked bursting (EB). (A) Upper trace: high frequency discharges of a dorsal horn neuron induced by electrical sciatic nerve stimulation (SNS). Lower trace: topical application of tetrodotoxin (TTX; 50 nmol/l) onto the DRG suppressed the afterdischarges and spontaneous activity of the WDR neuron. Discharges shown in both traces were recorded from the same WDR neuron located in a depth of 750 lm from the dorsal surface of the spinal cord ipsilateral to injury. Vertical axis shows the firing frequency (each bin represents the rate of discharge in spikes/s) and the horizontal axis represents time (s). Sciatic nerve stimulation (SNS): 10 seconds. Inset singleunit discharges were chosen at the time indicated by the dotted lines. Dotted line under each trace indicates the time window (1 min) over which the afterdischarge were computed. (B) Histogram summarizing the inhibiting effect of TTX, topically administered at DRG level, on the afterdischarge of WDR neurons (n = 8, P <.05).

6 662 Y. Song et al. / PAIN Ò 153 (2012) Fig. 5. Correlation between persistent sodium current (I Nap ) and evoked bursting (EB) as well as subthreshold membrane potential oscillation (SMPO). (A) The EB protocol stimulation (the bottom trace)-induced EB and SMPO in a neuron, which was silent at its resting membrane potential. Bath application of TTX (5 nmol/l) abolished the oscillations and the activities followed the first spike in EB within 5 minutes. Washout of TTX restored both the oscillations and EB. (B) Enlarged displays of SMPO from the framed area of a to d in (A). (C) FFT profile illustrates the decrease in oscillation amplitude (power) after TTX application. (D) Example of I NaP recordings in an EB neuron before and after TTX (5 nmol/l) treatment. Protocol for I NaP recording is shown above the current traces. (E) Average peak current density of I NaP in EB and non-eb neurons (n = 8, P <.05). density of I DTX was much smaller in EB neurons (n = 21) than that in non-eb neurons (n = 21, p < 0.05) (Fig. 6E). Hyperpolarization-activated current (I h ) was thought to contribute to hyperalgesia after CCD-induced nerve injury [45]. We tested the possible role of I h in EB. The results showed that administration of ZD 7288, a blocker of I h, shortened the duration of EB without altering action potential conduction in the peripheral nerve. The current density was significantly increased in EB cells, compared with those in non-eb cells (Fig. 7). 4. Discussion 4.1. Characteristics of EB We report here that DRG Ab neurons in rats develop an EB firing pattern following a CCD neuropathic injury, which seldom occurs in normal conditions. There are 3 characteristics of these ectopic discharges. First, EB is peripheral input dependent, triggered by a periphery input spike, and followed by somatic bursting. This peripheral input-dependent EB can be canceled by blocking the nerve conduction with topical application of lidocaine at the peripheral afferent nerves (Fig. 2C). Second, generation of EB depends on SMPO. Whenever SMPO was abolished by either chemicals or change of membrane potential, EB disappeared accordingly (Fig. 2E). Third, EB duration is voltage dependent (Fig. 2D). Another discharge pattern, namely, SB, also has the second and third characteristics listed above, but EB is different from SB in 2 points: first, the first spike of SB with a pre-potential phase at the foot of the first spike in the wave form was triggered by an oscillatory sinusoid which reached threshold [3] (in contrast, EB was evoked by peripheral input spike leading to a steep upstroke of the first spike in the burst, as shown in Fig. 3B); and second,

7 Y. Song et al. / PAIN Ò 153 (2012) Fig. 6. Correlation between a-dendrotoxin (a-dtx) sensitive current (I DTX ) and evoked bursting (EB) as well as subthreshold membrane potential oscillation (SMPO). (A) EB protocol stimulation (bottom trace)-induced EB and SMPO in a neuron, which was silent at its resting membrane potential. Bath application of 1 nmol/l DTX enhanced the oscillations and prolonged the duration of EB. (B) Enlarged displays of SMPO are from the framed area of a to d in (A). (C) FFT profile illustrates the increase in oscillation amplitude (power) after DTX application. (D) Typical responses to a series of voltage command before (top trace, a) and after application of 1 nmol/l a-dtx (middle trace, b). Bottom traces: the command voltage steps from 75 to 35 mv, in increments of 10 mv, were applied from a holding potential of 90 mv to elicit outward currents. The a- DTX-sensitive component was obtained subtracting (b) from (a) [(a) (b)]. (E) The average current density of I DTX in EB neurons was significantly smaller than that in non-eb neurons at the same membrane potential checked (n = 21, p < 0.05). EB can be generated at a more negative level of membrane potential than SB (Fig. 3A). These features of EB and SB indicate that they belong to 2 distinct firing patterns. EB is a bursting firing in essence, and the information conveyed with the bursts is qualitatively different from that conveyed with single spike or spikes fired tonically [21]. EB has at least 2 distinct amplifying effects. One is that EB can augment the afferent input from receptive field and powerfully reinforce synaptic links at the central terminal of the DRG [23]. The other is that, through the possible neuron-to-neuron cross-excitation that was proposed by other investigators [2,9] and also was confirmed in our laboratory (unpublished observation), a localized, non-noxious stimulusevoked EB could induce a sensory response that spreads to cover a large contiguous area of ganglion, and thus the neighboring neurons could be depolarized and firing probably simultaneously [2,9]. Ignition in trigeminal neuralgia was first proposed in 1994 [29], in which self-sustaining discharge leads to temporal and spatial amplification and become a possible candidate for generation of allodynia. Here, we proposed that EB might have a similar role in the production of ignition. However, the SB from an individual cell may not have a power to induce such amplification through the neuron neuron excitation EB might be an underlying signal for allodynia EB can be supported as an allodynic signal by the responses of WDR neurons in the spinal dorsal horn. In a polyethylene cuff model, the Ab afferent-induced afterdischarges of WDR neurons are found to be matched with the time course of mechanical allodynia [28]. Moreover, the NK-1 receptor antagonist CP-99,994, depressed the innocuous pressure-evoked afterdischarge but not the brief initial discharge of wide dynamic range neurons, so it is hypothesized that afterdischarge in WDR neurons demonstrates a spinal cellular correlate of tactile allodynia [28]. In the present study, the innocuous stimuli-evoked afterdischarges of WDR neurons were observed only in CCD but not in normal animals. Moreover, blockade of EB at DRG level could suppress this afterdischarge of WDR neurons, suggesting that EB may contribute to tactile allodynia. In other words, EB may be a candidate for pain signal of tactile allodynia. It is quite possible that both temporal and spatial amplification effects of EB in DRG level may be involved in the process of innocuous stimuli induced afterdischarge of WDR neurons. However, the exact interaction within DRG is totally unknown, although is worth further examination.

8 664 Y. Song et al. / PAIN Ò 153 (2012) to I DTX ) are considered to be necessary for bursting or burst-like activities in Mesencephalic V Neurons [15]. In the present study, the conductances of these 2 channels exhibit opposite effects on SMPO and EB, namely, I NaP increases SMPO and EB, whereas I DTX suppresses oscillations and spike production in EB series. I h, which is usually considered as a pacemaker current, may also be involved in EB formation. I h increases markedly in EB neurons (Fig. 7B and C), this is consistent with previous study [6] and agrees with their possible functions in the neuropathic pain [20,45]. It was also reported that elimination of the I h increased excitability of both the soma and the peripheral sensory terminals [10]. This seems to conflict to our observation that elimination of I h by ZD7288 shortened the duration of EB (a decreased excitability). A possible explanation is that the previous study was performed on a preparation of nodosal ganglion or dissociated cells, while our study was on CCD neurons with EB. These different experimental conditions and/or selection of different cells might account for the different outcomes of the effect of I h. The DRG neuron with EB has the oscillatory behavior, which was different from the normal nodosal ganglion cell. Actually, I h could act as an amplification factor for increasing the amplitude of resonance based on I Nap conductance that underlies oscillation behavior and is also a characteristic of EB neurons [18]. Therefore, conductances of these 3 ion channels would contribute to EB formation. Peripheral input evokes the first spike of EB and the afterhyperpolarization of first spike activates I h conductance. The I h inward current then depolarizes the cell and activates I Nap and I DTX. Thus the integration of these actions may result in initiation of soma spikes. A critical balance between I DTX and I Nap may be necessary for burst maintenance Potential therapy implications Fig. 7. Correlation of hyperpolarization-activated cation current (I h ) current and evoked bursting (EB). (A) Bath application of ZD 7288 (10 lmol/l) abolished oscillations and discharges after the first spike in EB. The bottom trace shows the EB-inducing protocol. (B) Typical I h current recordings from a non-eb and EB neuron. Bottom traces: voltage commands, in which hyperpolarizing potentials of 110 to 60 mv were delivered in increments of 10 mv from a holding potential of 50 mv with the duration of 6 seconds. (C) The average current density of I h was significantly greater in EB neurons compared with that in non-eb neurons (n = 12, P <.05) Ion channel mechanism underlying EB Our results suggest that I Nap, I DTX, and I h are involved in EB formation. The I NaP was observed in a variety of neuronal types and activated at around the level of resting membrane potential and associated with the control of membrane excitability [7,18]. We here have provided evidence that I NaP may play a key role in EB formation through its action on SMPO. This is consistent to the previous reports from our laboratory [11,43] and others [30,40]. I NaP and low-threshold DTX-sensitive K + conductance (corresponding As mentioned above, after injury, some DRG Ab neurons became hyperexcitable, in which single innocuous afferent spike could induce EB, a distinct firing pattern. Its temporal and spatial amplification effects would make injured Ab DRG neurons an important source for generating allodynia signals. Furthermore, the conductances of 3 channels, I Nap, I DTX, and I h, were identified to be involved in EB formation. In addition, in the present study, selective antagonists of these channels were administrated at very low concentration, especially in the current mode experiments. This makes it possible to observe the fine alternation of the ion channel currents in the whole process. For instance, DTX at 1 nmol/l exposed a subtle enhancing effect on the amplitude of SMPO, whereas at 100 nmol/l it caused an obvious firing pattern transformation [15]. This chemical and those indicated in our previous studies including gabapantin [44,43], riluzole [41] as well as lidocaine [11] targeting I Nap may suggest a potential therapeutic prospect in treating neuropathic allodynia. Conflict of interest statement There are no conflicts of interest associated with this study. Acknowledgements The authors are grateful to Dr. Victor Z. Han for the helpful discussion in the research work and Dr. Robert H. LaMotte for critical comments on the manuscript and English editing. This work was supported by the National Scientific Foundation of China (Grant Nos and ). References [1] Allen BJ, Li J, Menning PM, Rogers SD, Ghilardi J, Mantyh PW, Simone DA. Primary afferent fibers that contribute to increased substance P receptor

9 Y. Song et al. / PAIN Ò 153 (2012) internalization in the spinal cord after injury. J Neurophysiol 1999;81: [2] Amir R, Devor M. Chemically mediated cross-excitation in rat dorsal root ganglia. J Neurosci 1996;16: [3] Amir R, Michaelis M, Devor M. Burst discharge in primary sensory neurons: triggered by subthreshold oscillations, maintained by depolarizing after potentials. J Neurosci 2002;22: [4] Bao L, Wang HF, Cai HJ, Tong YG, Jin SX, Lu YJ, Grant G, Hokfelt T, Zhang X. Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord. Eur J Neurosci 2002;16: [5] Baron R. Mechanisms of disease: neuropathic pain a clinical perspective. Nat Clin Pract Neurol 2006;2: [6] Chaplan SR, Guo HQ, Lee DH, Luo L, Liu C, Kuei C, Velumian AA, Butler MP, Brown SM, Dubin AE. Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain. J Neurosci 2003;23: [7] Crill WE. Persistent sodium current in mammalian central neurons. Annu Rev Physiol 1996;58: [8] Devor M. Ectopic discharge in Abeta afferents as a source of neuropathic pain. Exp Brain Res 2009;196: [9] Devor M, Wall PD. Cross-excitation in dorsal root ganglia of nerve-injured and intact rats. J Neurophysiol 1990;64: [10] Doan TN, Stephans K, Ramirez AN, Glazebrook PA, Andresen MC, Kunze DL. Differential distribution and function of hyperpolarization-activated channels in sensory neurons and mechanosensitive fibers. J Neurosci 2004;24: [11] Dong H, Fan YH, Wang YY, Wang WT, Hu SJ. Lidocaine suppresses subthreshold oscillations by inhibiting persistent Na(+) current in injured dorsal root ganglion neurons. Physiol Res 2008;57: [12] Gracely RH, Lynch SA, Bennett GJ. Painful neuropathy: altered central processing maintained dynamically by peripheral input. Pain 1992;51: [13] Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model. Pain 2000;84: [14] Harper AA, Lawson SN. Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones. J Physiol 1985;359: [15] Hsiao CF, Kaur G, Vong A, Bawa H, Chandler SH. Participation of Kv1 channels in control of membrane excitability and burst generation in mesencephalic V neurons. J Neurophysiol 2009;101: [16] Hu SJ, Xing JL. An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain 1998;77: [17] Hughes DI, Scott DT, Todd AJ, Riddell JS. Lack of evidence for sprouting of Abeta afferents into the superficial laminas of the spinal cord dorsal horn after nerve section. J Neurosci 2003;23: [18] Hutcheon B, Yarom Y. Resonance, oscillation and the intrinsic frequency preferences of neurons. Trends Neurosci 2000;23: [19] Jian Z, Xing JL, Yang GS, Hu SJ. A novel bursting mechanism of type a neurons in injured dorsal root ganglia. Neurosignals 2004;13: [20] Jiang YQ, Sun Q, Tu HY, Wan Y. Characteristics of HCN channels and their participation in neuropathic pain. Neurochem Res 2008;33: [21] Krahe R, Gabbiani F. Burst firing in sensory systems. Nat Rev Neurosci 2004;5: [22] Lever IJ, Bradbury EJ, Cunningham JR, Adelson DW, Jones MG, McMahon SB, Marvizon JC, Malcangio M. Brain-derived neurotrophic factor is released in the dorsal horn by distinctive patterns of afferent fiber stimulation. J Neurosci 2001;21: [23] Lisman JE. Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci 1997;20: [24] Liu CN, Michaelis M, Amir R, Devor M. Spinal nerve injury enhances subthreshold membrane potential oscillations in DRG neurons: relation to neuropathic pain. J Neurophysiol 2000;84: [25] Ma C, LaMotte RH. Multiple sites for generation of ectopic spontaneous activity in neurons of the chronically compressed dorsal root ganglion. J Neurosci 2007;27: [26] Malcangio M, Ramer MS, Jones MG, McMahon SB. Abnormal substance P release from the spinal cord following injury to primary sensory neurons. Eur J Neurosci 2000;12: [27] Neumann S, Doubell TP, Leslie T, Woolf CJ. Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons. Nature 1996;384: [28] Pitcher GM, Henry JL. Nociceptive response to innocuous mechanical stimulation is mediated via myelinated afferents and NK-1 receptor activation in a rat model of neuropathic pain. Exp Neurol 2004;186: [29] Rappaport ZH, Devor M. Trigeminal neuralgia: the role of self-sustaining discharge in the trigeminal ganglion. Pain 1994;56: [30] Reboreda A, Sanchez E, Romero M, Lamas JA. Intrinsic spontaneous activity and subthreshold oscillations in neurones of the rat dorsal column nuclei in culture. J Physiol 2003;551: [31] Sandkuhler J. Models and mechanisms of hyperalgesia and allodynia. Physiol Rev 2009;89: [32] Santha P, Jancso G. Transganglionic transport of choleragenoid by capsaicinsensitive C-fibre afferents to the substantia gelatinosa of the spinal dorsal horn after peripheral nerve section. Neuroscience 2003;116: [33] Seburn KL, Catlin PA, Dixon JF, Lee MH, Matteson MS, Cope TC. Decline in spontaneous activity of group Aalphabeta sensory afferents after sciatic nerve axotomy in rat. Neurosci Lett 1999;274:41 4. [34] Song XJ, Hu SJ, Greenquist KW, Zhang JM, LaMotte RH. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia. J Neurophysiol 1999;82: [35] Song XJ, Vizcarra C, Xu DS, Rupert RL, Wong ZN. Hyperalgesia and neural excitability following injuries to central and peripheral branches of axons and somata of dorsal root ganglion neurons. J Neurophysiol 2003;89: [36] Takahashi Y, Nakajima Y, Sakamoto T. Dermatome mapping in the rat hindlimb by electrical stimulation of the spinal nerves. Neurosci Lett 1994;168:85 8. [37] Wall PD, Woolf CJ. Muscle but not cutaneous C-afferent input produces prolonged increases in the excitability of the flexion reflex in the rat. J Physiol 1984;356: [38] Weissner W, Winterson BJ, Stuart-Tilley A, Devor M, Bove GM. Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection. J Comp Neurol 2006;497: [39] Wu N, Enomoto A, Tanaka S, Hsiao CF, Nykamp DQ, Izhikevich E, Chandler SH. Persistent sodium currents in mesencephalic v neurons participate in burst generation and control of membrane excitability. J Neurophysiol 2005;93: [40] Wu N, Hsiao CF, Chandler SH. Membrane resonance and subthreshold membrane oscillations in mesencephalic V neurons: participants in burst generation. J Neurosci 2001;21: [41] Xie RG, Zheng DW, Xing JL, Zhang XJ, Song Y, Xie YB, Kuang F, Dong H, You SW, Xu H, Hu SJ. Blockade of persistent sodium currents contributes to the riluzoleinduced inhibition of spontaneous activity and oscillations in injured DRG neurons. PLoS One 2011;6:e [42] Xing JL, Hu SJ, Long KP. Subthreshold membrane potential oscillations of type A neurons in injured DRG. Brain Res 2001;901: [43] Yang RH, Wang WT, Chen JY, Xie RG, Hu SJ. Gabapentin selectively reduces persistent sodium current in injured type-a dorsal root ganglion neurons. Pain 2009;143: [44] Yang RH, Xing JL, Duan JH, Hu SJ. Effects of gabapentin on spontaneous discharges and subthreshold membrane potential oscillation of type A neurons in injured DRG. Pain 2005;116: [45] Yao H, Donnelly DF, Ma C, LaMotte RH. Upregulation of the hyperpolarizationactivated cation current after chronic compression of the dorsal root ganglion. J Neurosci 2003;23:

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