Alteration of primary afferent activity following inferior alveolar nerve transection in rats
© Nakagawa et al; licensee BioMed Central Ltd. 2010
Received: 20 November 2009
Accepted: 3 February 2010
Published: 3 February 2010
In order to evaluate the neural mechanisms underlying the abnormal facial pain that may develop following regeneration of the injured inferior alveolar nerve (IAN), the properties of the IAN innervated in the mental region were analyzed.
Fluorogold (FG) injection into the mental region 14 days after IAN transection showed massive labeling of trigeminal ganglion (TG). The escape threshold to mechanical stimulation of the mental skin was significantly lower (i.e. mechanical allodynia) at 11-14 days after IAN transection than before surgery. The background activity, mechanically evoked responses and afterdischarges of IAN Aδ-fibers were significantly higher in IAN-transected rats than naive. The small/medium diameter TG neurons showed an increase in both tetrodotoxin (TTX)-resistant (TTX-R) and -sensitive (TTX-S) sodium currents (I Na) and decrease in total potassium current, transient current (I A) and sustained current (I K) in IAN-transected rats. The amplitude, overshoot amplitude and number of action potentials evoked by the depolarizing pulses after 1 μM TTX administration in TG neurons were significantly higher, whereas the threshold current to elicit spikes was smaller in IAN-transected rats than naive. Resting membrane potential was significantly smaller in IAN-transected rats than that of naive.
These data suggest that the increase in both TTX-S I Na and TTX-R I Na and the decrease in I A and I k in small/medium TG neurons in IAN-transected rats are involved in the activation of spike generation, resulting in hyperexcitability of Aδ-IAN fibers innervating the mental region after IAN transection.
Numerous papers have described how peripheral nerve injury causes a variety of functional deficits in sensory processing [1–7]. Neuropathic pain also may occur after nerve injury [8–11], and whereas the injured tissue does usually repair, the neuropathic pain frequently persists [12–14]. One mechanism that is considered to underlie the abnormal pain after nerve damage involves regenerating nerve fibers. Injured nerves regenerate several weeks after nerve damage [15–17]. Some clinical reports have noted that areas innervated by the regenerated nerves show an altered sensitivity to a variety of stimuli compared to areas innervated by intact nerve fibers [18–21]. The regenerated fibers are morphologically similar to normal nerve fibers and terminals [22, 23]. For example, periodontal sensory receptors are absent soon after inferior alveolar nerve (IAN) transection, but reappear more than 7 days after the transection, with morphological features similar to those in normal periodontal receptors . However, varieties of functional changes are induced in injured nerves.
The background activity of injured primary afferent fibers  and their mechanical and heat-evoked responses are enhanced after nerve injury [25–27]. It is also reported that regenerated cutaneous afferent nerve fibers exhibit ectopic discharges in the sural nerve [28, 29]. A variety of neuropeptides, such as neuropeptide Y or substance P, is also up- or down-regulated following peripheral nerve injury [30–32]. In the case of IAN transection for example, IAN fibers show significant increases in background activity and also trigeminal ganglion (TG) neurons show a change in the expression of several types of Na+ channels [33–37]. These changes in peripheral nerves may account for changes not only in the excitability of the primary afferent neurons but also may contribute to excitability changes in the central nervous system (CNS) [36, 38]. Hyperexcitability of peripheral nerves may be associated with sensitization of peripheral receptors , and CNS networks may become sensitized after long-lasting hyperexcitability of primary afferent neurons. Both peripheral and central sensitizations are thought to be involved in neuropathic pain following nerve injury [25, 38–43].
The sodium (Na+) currents (I Na) have an important role in generating action potentials and are also involved in the modulation of primary afferent activity [44, 45]. Therefore, I Na is thought to be important for modulating the excitability of primary afferent neurons after nerve injury [46–48]. The I Na is classified as either tetrodotoxin (TTX) -sensitive (TTX-S) or TTX-resistant (TTX-R) according to their sensitivity to TTX . Many researchers have reported that TTX-R I Na as well as TTX-S I Na modulates primary afferent activity following nerve injury [46, 48]. The potassium (K+) currents also are involved in modulation of the primary afferent neuronal excitability following nerve injury [24, 50]. Following IAN injury, both the fast inactivating transient K+ current (I A) and dominant sustained K+ currents (I K) in TG neurons are decreased . It is highly likely that I Na, I A and I K are up- and down-regulated in primary afferent neurons following IAN injury, resulting in hyperexcitability of the IAN fibers and induction of pain or other sensory abnormalities. However, the mechanisms underlying the functional changes in the transected IAN are not known.
Therefore, in the present study, the properties of the IAN innervating the mental region after IAN transection were analyzed using fluorogold (FG) tracing, nocifensive behavior monitoring, single fiber recording and patch-clamp recording from TG neurons in order to evaluate the changes in the excitability of TG neurons after IAN transection.
FG labeling in TG neurons
Mechanical nocifensive behavior
After successful completion of the training, in which rats allowed noxious mechanical stimulation (> 15 g) to be applied to the mental skin region, the IAN was transected. Figure 1E (ipsilateral side to IAN transection) and F (contralateral side to IAN transection) illustrate the mechanical threshold intensity for evoking escape behavior by mechanical stimulation of the mental skin region before and 7-14 days after IAN transection. The threshold value was significantly lower at 11-14 days after IAN transection compared to pre-operative values (11 days: p < 0.05, 12-14 days: p < 0.01) (median values, pre-operative, ipsilateral: 15 g, contralateral: 10 g; 7 days after transection, ipsilateral: 15 g, contralateral: 10 g; 14 days after transection, ipsilateral: 4 g, contralateral: 10 g, n = 5 in each group).
Primary afferent activity of the IAN
We analyzed the effect of mechanical stimulation of the RF on Aδ-fiber units only, because of the small population of C-fibers in IAN-transected rats. Aδ-fibers showed graded firing following increases in mechanical stimulus intensity from the non-noxious to the noxious range in naive and IAN-transected rats, as illustrated in Figure 3C. These fibers showed significantly larger responses to both non-noxious and noxious mechanical stimulation as compared to those of naive rats (Figure 3D).
Patch-clamp recording from TG neurons
Mean size of FG-labeled TG neurons for patch-clamp recording was 27.4 ± 0.8 μm in naive rats and 27.8 ± 0.7 μm in IAN-transected rats (n = 17 each). Since there is a positive correlation between neuronal cell size and conduction velocity of A- and C- afferents in DRG neurons , small-diameter TG neurons recorded in the present study were considered to be classified as small to medium Aδ-TG neurons (diameter 21-36 μm) for the patch-clamp recording experiment. Following perforation of the cell membrane with amphotericin B, the series resistance dropped to < 20 MΩ (naive: 17.2 ± 0.8 MΩ; IAN transection: 16.8 ± 0.8 MΩ, n = 17 each) within 5-12 min and remained stable for more than 15 min. In addition, the value for the cell capacitance was 23.1 ± 1.0 pF in naive rats and 23.1 ± 1.3 pF in IAN-transected rats (n = 17 in each group).
Change in INa, IA and Ik, and ability to generate action potentials in TG neurons
The present results can be summarized as follows: 1) Injection of FG into the mental region 2 weeks after IAN transection produced a notable increase in retrograde labeling of TG neurons. 2) The escape threshold to mechanical stimulation of the mental skin was significantly lower at 11-14 days after IAN transection. 3) The background activity and afterdischarge of IAN Aδ-fibers were significantly higher in IAN-transected rats than naive rats. 4) Mechanical evoked responses of these fibers were also significantly larger in IAN-transected rats compared to those of naive rats. 5) The spike amplitudes in small/medium diameter TG neurons were significantly larger in IAN-transected rats compared to those of naive rats. 6) Voltage clamp recordings from TG neurons after IAN transection demonstrated an increase in both TTX-R and TTX-S Na+ currents and a decrease in total K+ current, I K and I A. 7) The threshold current to elicit spikes in TG neurons was significantly smaller in IAN-transected rats than that of naive rats. 8) Current injection into TG neurons induced high frequency spike discharges in rats with IAN transection. 9) Resting membrane potential was significantly smaller in IAN-transected rats than that of naive rats.
It has been reported that the injured primary afferent nerve fibers regenerate 2-3 weeks after injury [15–17]. We observed that more than half of TG neurons were labeled with FG at 14 days after IAN transection, the number of labeled neurons was also significantly increased compared with that at earlier days after injury, suggesting that many transected IAN fibers were reinnervating the mental region at this time period. It is also possible that the transected IAN may reinnervate other intraoral structures as well as the mental skin. It has been reported for example that the jaw-opening reflex elicited by tooth pulp stimulation can be recorded at 6 weeks after IAN transection in cats and that more than half of the IAN-transected cats show lingual nerve innervations into the tooth pulp . However, in the present study we only analyzed TG neurons receiving input from the mental skin at 14 days after IAN transection, although we cannot rule out that some regenerated TG neurons become associated with inputs from intraoral or other facial structures as well as from mental skin. Furthermore, some previous studies have also reported that reinnervated nerve fibers change their response properties to a variety of peripheral stimuli such as mechanical and thermal stimuli [53–55]. It may be possible that the reinnervated IAN causes functional changes resulting in altered sensitivity to mechanical stimulation of the skin.
In the present study, there was a decrease in the escape threshold to mechanical stimulation of the mental skin following the IAN injury. Some previous clinical studies have reported that patients feel abnormal pain sensation after wound healing [18–20]. When considering the previous data and our findings, it is possible that an abnormal pain sensation occurs in the cutaneous tissues reinnervated by the injured nerves, but how the reinnervated nerves are involved in generation of an abnormal pain in the areas innervated by the injured IAN is not well understood. Therefore, we focused on the peripheral mechanisms underlying abnormal pain sensations in the mental region innervated following the IAN injury.
Our previous studies showed that A-fibers are predominantly involved in the hypersensitivity of the cutaneous RF innervated by the injured nerve as well as surrounding skin areas innervated by the uninjured nerves located close to the injured nerve [24, 50]. In the present study, we observed that Aδ-fibers showed significantly increased background activity, afterdischarges following noxious stimulation and mechanical-evoked responses (see in Figure 3). Furthermore, TG neurons lacking RFs showed very high background activity in IAN-transected rats (Figure 3A). The precise mechanisms underlying these observations are unclear but one possible process could involve increases in interleukin-6 (IL-6) and nerve growth factor (NGF). It has been shown that such increases may be related to the development of mechanical allodynia after trigeminal nerve injury  and that Schwann cells at the nerve injury site release chemical signals including IL-6 or NGF which are retrogradely transported to the primary sensory neurons . The possibility that the release of both IL-6 and NGF may be involved in the observed generation of ectopic discharges from injured IAN fibers lacking a RF is also supported by our findings that approximately a half of the TG neurons were unlabeled with FG at 14 days after IAN transection (Figure 1C). We have also reported recently that trigeminal spinal subnucleus caudalis (Vc) neurons display enhanced RF and response properties following reinnervation of their RFs by the transected IAN . Thus, it is very likely that the increase in excitability of TG neurons after IAN transection as documented in the present study makes an important contribution to the increase in the Vc neuronal excitability. Furthermore, we observed that the number of C-fiber responses was decreased in IAN-transected rats compared to naive rats, as illustrated in Figure 2C and 2D. Saito et al. have also reported that heat responsive units in the Vc are significantly decreased in the IAN-regenerated rats, suggesting that the IAN C-fibers may have less ability to regenerate after transection reflecting the decrease in the number of C-fiber responses after IAN regeneration .
Alterations in ion channels may also be associated with nerve injury. It has been reported that many different types of ion channels are expressed in dorsal root ganglion (DRG) neurons after peripheral nerve injury [59, 60] and TG neurons . Na+ and/or K+ channels are importantly involved in spike generation and also in the modulation of neuronal excitability following nerve damage [59, 60]. Chronic pain conditions are associated with altered Na+ channel activity and the change in the Na+ channel properties appears to be a critical feature of persistent pain following peripheral nerve injury [46–48].
It has been demonstrated that both TTX-S and TTX-R Na+ currents were increased in small- and medium-diameter DRG neurons 2-7 weeks after sciatic nerve transection . In agreement with this finding, the present study revealed that both TTX-R I Na and -S I Na densities of TG neurons were significantly larger in IAN-transected rats (14 days after transection) compared with those in naive rats (Figure 4). Our findings are consistent with the evidence that spike amplitudes of TG neurons were also significantly larger in IAN-transected rats following current injection. The threshold current for spike generation was significantly smaller in IAN-transected rats than that of naive rats and current injection into TG neurons induced high-frequency spike discharges in rats with IAN transection. Although the increase in the magnitude of TTX-S I Na was larger than that of TTX-R I Na in the TG neurons after IAN transection, it is possible that the hyperexcitability of TG neurons innervated by the regenerated IAN is augmented by an increase in TTX-R Na+ current densities, resulting in abnormal excitation of the CNS networks and nociceptive behavior. However, further studies are needed to address this possibility.
In order to evaluate the changes in neuronal excitability associated with the demonstrated changes in sodium and potassium currents following IAN transection, we analyzed action potentials properties under current-clamp conditions. In this study, action potentials were generated by supra-threshold current injection. Current injection into TG neurons induced action potentials with larger amplitude in IAN-transected rats compared with those of naive rats. The current injection into TG neurons also induced high-frequency spike discharges and the threshold intensity to generate action potentials was significantly lower in IAN-transected rats. These changes in the physiological properties of TG neurons indicate that the excitability of TG neurons is increased in IAN-transected rats. There are several possible mechanisms that could explain the increase in action potential amplitude and frequency. One is an increase in the density of Na+ channels and the other is a change in channel kinetics and permeability.
Voltage-gated K+ channels are important physiological regulators of membrane potential in excitable tissue, including sensory ganglia [61, 62]. TG neurons express two distinct classes of K+ currents at varying levels, involving the I K and I A currents [63, 64]. In this study, we found that the IAN-transection significantly decrease in the density of both I K and I A in the FG-labeled TG neurons. In agreement with this finding, we found that the resting membrane potential was significantly decreased following IAN-transection. In addition to increase in I Na, we can raise the possibility that the reduction of both I K and I A contributes to the hyperexcitability of IAN-transected TG neurons. The change in the excitability of TG neurons associated with the change in the I Na and K+ current (I K and I A) may be involved in central sensitization of the Vc neurons that results in pain abnormalities following IAN transection.
The present findings suggest that TTX-R I Na and -S I Na, and I K and I A in the reinnervated Aδ-IAN-TG neurons are involved in an increase in spike generation, resulting in the hyperexcitability of the reinnervated Aδ-IAN fibers. The findings further suggest that this hyperexcitability of the reinnervated Aδ-IAN fibers is involved in a development of mechano-allodynia in the area IAN are reinnervated following IAN transection.
Inferior alveolar nerve transection
A total of 94 male Sprague-Dawley rats weighing 100-250 g was used for the present study. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed on a warm mat. A small incision was made on the surface of facial skin over the masseter muscle and the alveolar bone was reached through the masseter muscle. The surface of the alveolar bone was exposed and the bone covering the IAN was removed and the IAN was exposed. The IAN was transected at 7 mm proximal from the angle of alveolar bone and then immediately replaced into the inferior alveolar canal . For patch clamp recording, FG dye (2%, 10 μl; Fluorochrome, Englewood, CO, USA) was subcutaneously injected into the mental region 2 days before the recording experiment. After surgery, Penicillin G potassium (20,000 units, i.m.; Eli Lilly, Indianapolis, IN) was injected to prevent infection.
Fifteen rats were used for the FG tracing study (naive: n = 5, 7 days after transection: n = 5, 14 days after transection: n = 5). Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and 10 μl of 2% FG was subcutaneously injected into the mental skin 2 days before perfusion in IAN-transected rats. Two days after the FG injection, rats were deeply anesthetized with the same anesthetic and perfused with 200 ml 0.9% saline followed by 500 ml of 4% paraformaldehyde. The TG was removed and post-fixed in the same fixative for 2 days and the tissue was then transferred to 20% sucrose (w/v) in phosphate-buffered saline (PBS) for several days for cryoprotection. Thirty-micron-thick sections were cut with a freezing microtome and sections were collected in PBS. FG immunohistochemical staining of TG neurons was carried out as previously described by Saito et al. . TG neurons were defined as FG positive if the cytoplasm was stained with a black deposit. The FG-labeled TG neurons with clear nuclei were counted and their areas were measured. The number of FG-labeled TG neurons at the root of the third branch of the trigeminal nerve was counted in 3 sections (one section with the largest number of FG labeled neurons and next two sections) from each rat.
In daily sessions, rats were trained to stay in a plastic cage and to keep their snout protruding through a hole on the wall during mechanical stimulation of the mental skin with von Frey filaments (Touch-Test, North Coast Medical, Inc., CA, USA). Rats under this condition can escape from von Frey stimulus freely. When rats escaped from the von Frey stimulus, the escape behavior was defined as nocifensive. The maximum intensity used before IAN transection was 15 g. The escape threshold was measured before the IAN was transected and then the IAN of those rats was transected. The escape threshold to mechanical stimulation of the mental skin was measured daily at 8 days before and 7-14 days after IAN transection. Only rats with IAN transection which showed a significant decrement in mechanical escape threshold at 14 days after IAN transection were used for single fiber and patch clamp recording experiments (see below). Quantitative mechanical stimuli were applied to the mental skin region in ascending and descending orders to evaluate the escape threshold. Each von Frey filament was applied 5 times. When rats showed an escape response to a filament, the bending force of that filament was defined as the escape threshold intensity . The median threshold intensity was calculated from the values following one ascending and one descending trial.
Single fiber recording after IAN transection
We used IAN-transected rats which showed hypersensitivity to mechanical stimulation of the mental skin and IAN-transected rats without behavior changes which did not show any behavioral changes after IAN transection for single fiber recording experiments. Rats were divided into 3 groups: ipsilateral to IAN transection (n = 16), ipsilateral to IAN without behavioral changes after IAN transection (n = 5) and naive groups (n = 22). For IAN fiber recording, each group of rats was anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the trachea and left femoral veins were cannulated to allow artificial respiration and intravenous administration of drugs, respectively. Anesthesia was maintained with halothane (2-3%) mixed with oxygen during surgery. Rats were mounted in a stereotaxic frame and a craniotomy was performed, 1-4 mm lateral to the midline and 2-5 mm anterior to the interaural line. The skull was rigidly secured to a head holder by stainless-steel screws and dental acrylic resin, and the nose holder was removed. This setup allowed convenient access to fibers which responded to stimulation of the orofacial RFs innervated by the IAN.
After surgery, anesthesia was maintained throughout the experiment by continuous inhalation of halothane (1-2%) mixed with oxygen. During recording sessions, rats were immobilized with pancuronium bromide (1 mg/kg/h, i.v.) and ventilated artificially. Expired CO2 concentration was monitored (Capstar-100, Cwe, Bioseb, USA) and maintained between 3.0-4.0%. Rectal temperature was maintained at 37-38°C by a thermostatically-controlled heating pad (ATB-1100, Nihon Kohden, Tokyo, Japan) and an electrocardiogram was monitored.
Bipolar electrodes (interpolar distance: 0.5 mm) were inserted into the spinal trigeminal subnucleus caudalis (Vc) 0.8 mm deep from the brainstem surface at the obex level to elicit antidromic spikes from Vc (Figure 7A). An enamel-coated tungsten microelectrode (impedance = 10 MΩ, 1000 Hz) was then advanced carefully through the cortex about 2.5-3.0 mm lateral to the midline and 3.0-4.0 mm anterior to the interaural line until an electrode tip reached the IAN trunk. Then, the electrode was advanced at 1 μm steps and single fiber activity was recorded. IAN unit activities were searched for by applying mechanical stimulation (pressure or brush) to the mental region. When single unit activity was isolated, responses to mechanical stimulation of the facial skin were carefully examined. Because of technical difficulties to approach intraoral structures, only cutaneous facial RFs were mapped. To identify antidromic responses, 1 ms electrical pulses (0.1-0.5 mA) were applied to the Vc. Each neuron was classified as Aβ- (> 7 m/s), Aδ- (7-2 m/s) or C- (< 2 m/s) fibers according to the conduction velocity of the action potentials calculated from the antidromic latency and the distance between recording and stimulating sites .
Graded mechanical stimuli were applied to the most sensitive areas of RFs. Mechanical stimuli consisted of quantitative pressure with von Frey filaments (1, 6, 15, 26 and 60 g), brushing with a camel hair brush and pinch produced by a small arterial clip. After identification of a neuron by brushing the face, graded mechanical stimuli were applied to RFs. To avoid sensitization by noxious stimulation, we did not use repeated noxious stimuli to search for high-threshold mechanosensitive neurons. Neuronal responses were saved on computer disk for subsequent off-line analysis of signals.
The waveforms of each neuron were amplified using a differential amplifier (AB-601G, Nihon Kohden, Tokyo, Japan, high cut: 10 KHz, low cut: 150 Hz) and identified using Spike 2 software (CED, Cambridge, UK). Peristimulus time histograms (bin width = 1 s) were generated in response to each stimulus. Background discharges were first recorded for 120 s before the application of the mechanical stimulation, and they were subtracted from the neuronal responses during the analysis. The mean firing frequency was calculated during mechanical stimulation. Afterdischarges were recorded for 10 s after pinching of the RF. The mechanical stimulation of the RFs was considered to have induced an effect when the mean firing frequency at 5 s after mechanical stimulation differed from mean background discharge rate by ± 2SD.
Acute dissociation of TG neurons
The IAN-transected rats were tested for mechanical stimulation of the mental region at 14 days after nerve injury. Rats that showed allodynia-like responses to non-noxious mechanical stimulation of the mental skin were used for patch clamp recording experiments. For mental skin stimulation, mechanical stimulation was applied to adjacent regions more than 1 mm distant from the FG injection site.
Rats with mechano-allodynia were sacrificed by decapitation and TG neurons were used for the electrophysiological studies. Acute dissociation of TG neurons was performed as described previously [63, 64]. Briefly, rats were anaesthetized with sodium pentobarbital (45 mg/kg, i.p.) and decapitated. The left TG was rapidly removed and incubated for 15-25 min at 37°C in modified Hank's balanced salt solution (130 mM NaCl, 5 mM KCl, 0.3 mM KH2PO4, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM glucose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3) containing collagenase type XI and type II (each 2 mg/ml; Sigma-Aldrich, MO, USA). The cells were dissociated by trituration with a fire-polished Pasteur pipette and then plated onto poly-L-lysine-coated coverslips in 35-mm dishes. The plating medium contained Leibovitz's L-15 solution (Invitrogen, Carlsbad, CA, USA) supplemented with 10% newborn calf serum, 26 mM NaHCO3, and 30 mM glucose. The cells were maintained in 5% CO2 at 37°C and used for recordings between 2 and 8 h after plating. After incubation, the coverslips were transferred to the recording chamber in a standard external solution containing 155 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 20 mM glucose, pH 7.3.
Recording solution and drugs
Composition of extracellular and intracellular solution.
Adjusted to pH = 7.4 with TEAOH
Adjusted to pH = 7.3 with NaOH
Methane Sulfonic Acid
Adjusted to pH = 7.2 with CsOH
Adjusted to pH = 7.2 with KOH or HCl
FG-labeled TG neurons were identified by applying a short pulse of UV light (340-380 nm) and capturing the image of fluorescent cells with a microscope (Nikon, Tokyo, Japan). Locally developed software permitted the superposition of a tracing of the perimeter of the fluorescent cell onto the image of the same cell in the TG visualized with visible light. Whole cell recordings were conducted with the rapid perforated-patch technique [24, 50, 67–69]. Fire-polished patch pipettes (2-5 MΩ) were filled with an internal solution (Table 1) and amphotericin B. In the case of the potassium current recording under voltage clamp, we used the same internal solution as described in current clamp condition (Table 1). Both current- and voltage-clamp recordings were conducted with an Axopatch 200B amplifier (Axon Instr., Foster City, CA, USA). Signals were low-pass filtered at 1 or 5 kHz and digitized at 10 kHz.
Neurons were always bathed in a flowing stream of external solution, except during the application of drugs. After seal formation and membrane perforation, leakage and capacitive transients were reduced by analog circuitry. A series resistance compensation (> 80%) was employed . The recording chamber (volume, 0.5 ml) was mounted on an inverted microscope (Nikon, Tokyo, Japan) equipped with phase-contrast, a video camera and two micromanipulators. The chamber was perfused under gravity with an external solution at approximately 0.5 ml/min. Current density was determined by dividing the peak current evoked by the cell capacitance.
In the voltage-clamp mode, TTX-R I Na was recorded in naive and IAN-transected rats. TTX-S I Na was isolated by digitally subtracting TTX-R I Na from the total I Na. The current-voltage (I-V) relationship was first monitored by using step pulses (50 ms) from the holding potential of -80 mV to +80 mV in 5 mV increments at 5 s intervals.
In the current-clamp mode, we firstly determined the threshold (1T) for evoking action potential (overshoot of action potential > 30 mV). The threshold was defined as the current values for eliciting depolarizing single pulses (10-400 pA, 300 ms). The firing rate of action potentials was assessed by counting the number of action potentials evoked by the depolarizing pulses (1T, 2T and 3T). The spike amplitude and height of overshoot, and the threshold current, were also assessed in naive and IAN-transected rats as illustrated in Figure 5A.
We also analyzed the K+ current in this model. We used voltage protocols modified from a previous study [24, 63, 64]. Outward K+ currents were elicited by stepping to a conditioning voltage of either -40 mV or -120 mV from a holding potential of -60 mV; then the membrane was depolarized from -60 mV to +60 mV in increments of 10 mV; +60 mV produced the largest peak current in each recording. The transient A currents was determined by subtraction of -40 mV protocol from -120 mV protocol. Activation of the currents in standard solution was rapid and decayed only partially during 300 ms depolarization pulses. The amplitudes and rates of rise in the absolute current increased with increasing depolarization.
Results are presented as median for behavioral test, and mean ± SEM for single fibers analysis, patch clamp analysis and FG immunohistochemistry. One-way ANOVA followed by Dunnett's test was used for data from the FG labeling of TG neurons. ANOVA on rank with post-hoc Student-Newman-Keuls test was used for the behavioral data. The Mann-Whitney U test, two-way ANOVA followed by the Dunn's and Holm-Sidak tests were used for data from the single fiber recording experiments, and Duncan's new multiple range test was used for the data from the patch clamp recording experiments. Differences were considered significant at p < 0.05.
We thank Dr. Binshtok for commenting on the manuscript. This study was supported in part by Research Grants from Sato and Uemura Funds from Nihon University School of Dentistry, and a grant from the Dental Research Center, Nihon University School of Dentistry; Nihon University multidisciplinary research grant for KI and Individual Research Grant for KN; grants from the Ministry of Education, Culture, Sports, Science, and Technology to promote multidisciplinary research project "Translational Research Network on Orofacial Neurological Disorders" at Nihon University School of Dentistry, and Japan-Canada (CIHR) Joint Health Research Program. Dr. Sessle is the recipient of a Canada Research Chair. We thank Dr. D.A. Thomas for correcting English usage in this manuscript.
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