- Open Access
Painful nerve injury increases plasma membrane Ca2+-ATPase activity in axotomized sensory neurons
Molecular Pain volume 8, Article number: 46 (2012)
The plasma membrane Ca2+-ATPase (PMCA) is the principal means by which sensory neurons expel Ca2+ and thereby regulate the concentration of cytoplasmic Ca2+ and the processes controlled by this critical second messenger. We have previously found that painful nerve injury decreases resting cytoplasmic Ca2+ levels and activity-induced cytoplasmic Ca2+ accumulation in axotomized sensory neurons. Here we examine the contribution of PMCA after nerve injury in a rat model of neuropathic pain.
PMCA function was isolated in dissociated sensory neurons by blocking intracellular Ca2+ sequestration with thapsigargin, and cytoplasmic Ca2+ concentration was recorded with Fura-2 fluorometry. Compared to control neurons, the rate at which depolarization-induced Ca2+ transients resolved was increased in axotomized neurons after spinal nerve ligation, indicating accelerated PMCA function. Electrophysiological recordings showed that blockade of PMCA by vanadate prolonged the action potential afterhyperpolarization, and also decreased the rate at which neurons could fire repetitively.
We found that PMCA function is elevated in axotomized sensory neurons, which contributes to neuronal hyperexcitability. Accelerated PMCA function in the primary sensory neuron may contribute to the generation of neuropathic pain, and thus its modulation could provide a new pathway for peripheral treatment of post-traumatic neuropathic pain.
Influx of Ca2+, the dominant second messenger in neurons, follows neuronal activation by membrane depolarization or ligand binding. Although much of this Ca2+ is initially buffered and sequestered in intracellular stores, including the endoplasmic reticulum and mitochondria, it must ultimately be discharged from the cell. The principal pathways for this process are the Na+/Ca2+ exchanger (NCX), which has high transport capacity but low affinity , and the plasma membrane Ca2+-ATPase (PMCA), which has high affinity for Ca2+ (200nM) but low capacity. For sensory neurons, PMCA is the dominant extrusion pathway for Ca2+ that enters during low amplitude transients (peak [Ca2+ c <400nM) [2, 3] such as those that accompany typical patterns of activation , PMCAs, predominantly the 2a and 4b isoforms in sensory neurons , generate Ca2+ efflux against an approximately 2 × 105-fold gradient through the consumption of ATP, with an obligatory influx of hydrogen ions from the extracellular space.
Calcium signals in neurons control critical functions including differentiation, growth, excitability, synaptic transmission, cytotoxicity and apoptosis. We have previously found that painful peripheral nerve injury is accompanied by disordered Ca2+ signaling in the traumatized sensory neurons of the dorsal root ganglion (DRG). Specifically, neurons axotomized by spinal nerve ligation (SNL) develop a decreased resting [Ca2+ c . This level is set by the balance of Ca2+ influx regulated by levels of stored Ca2+, so-called store-operated Ca2+ entry (SOCE) versus Ca2+ discharge by PMCA [7, 8]. Our observation that SOCE is amplified by axotomy in sensory neurons  therefore suggests that PMCA may be activated by injury. Furthermore, we have identified more rapid resolution of depolarization-induced transients after injury , which additionally implicates upregulation of PMCA. Finally, other research has shown that PMCA action reduces the size of the afterhyperpolarization (AHP) that follows action potentials (APs) [5, 10], possibly leading to elevated neuronal excitability. Reduced AHP and neuronal hyperexcitability are features of axotomized neurons after SNL , so these findings together suggest a possible role of PMCA in the pathogenesis of neuropathic pain.
To characterize the effect of nerve injury, we have measured PMCA function in rats subjected to SNL. This injury creates fully axotomized neurons in the 5th lumbar (L5) DRG. In contrast, the neurons of L4 remain intact but are exposed to inflammatory conditions in the distal sciatic nerve triggered by degenerating distal fragments of axotomized L5 neurons. Accordingly, we have evaluated these populations separately and compared them to control neurons from animals receiving only skin incision. The influence of PMCA upon excitability has not been directly examined, so we additionally evaluated this critical physiological role.
Rats were subjected to spinal nerve ligation or anesthesia and skin incision only (control group) and hyperalgesic behavior was assessed at three different time points by the response to mechanical stimulation with a 22 G spinal needle. Hyperalgesia-type responses developed in SNL rats (44 ± 10%, n = 14) but not in control rats (0 ± 0%, n = 20; P < 0.001). The accuracy of the SNL surgery was confirmed at the time of tissue harvest in all SNL animals.
Identification of PMCA function
To characterize the role of PMCA in normal and injured neurons, we first evaluated whether PMCA is active in control neurons at rest. Ca2+ extrusion by PMCA requires the influx of hydrogen ions, and elevated extracellular pH can block the function of PMCA , with a peak effect at pH 8.8 , In acutely dissociated DRG neurons, we noted a progressive rise of [Ca2+ c (30 ± 41nM over 3 min, n = 50; Figure 1A) upon changing the bath solution to pH 8.8, whereas resting [Ca2+ c changed minimally in time controls (5 ± 4nM, n = 18, P < 0.01 vs. pH 8.8; Figure 1B). This indicates constitutive operation of the PMCA that contributes to the regulation of Ca2+ levels in resting sensory neurons. When the neurons were activated by depolarization by a 0.3 s K+ stimulus, blockade of PMCA by pH8.8 resulted in prolongation of the Ca2+ transient (increased τ and T95) compared to a prior depolarization in pH7.4 bath (Figure 1A, C). Time controls in pH7.4 also showed increased τ at the second depolarization (Figure 1B, D), but this increase was much smaller (23 ± 19%) than that produced by switching to pH8.8 (83 ± 99%, P < 0.05).
These observations reveal the participation of PMCA in regulating Ca2+ signaling after neuronal activation. However, the simultaneous operation of other clearance mechanisms precludes the use of this approach for the measurement of the specific activity of PMCA per se under different injury conditions. We therefore employed the strategy of measuring PMCA selectively after eliminating the function of other Ca2+ sequestration pathways. First, we blocked sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA), which pumps cytoplasmic Ca2+ into the ER, by exposure to thapsigargin (TG, 1 μM, 5 min). This resulted in transients that resolved more slowly than in the absence of TG (Figure 2), which indicates that SERCA assists PMCA in clearing activity-induced Ca2+ loads. Additionally, by using brief depolarizations, we limited transient amplitude to levels (400nM) that are insufficient to initiate mitochondrial buffering of cytoplasmic Ca2+ . Finally, traces that showed a shoulder or plateau of sustained Ca2+ elevation during the descending limb of the activity-induced transient were not included in the evaluation of PMCA, as this pattern represents the participation of mitochondrial buffering . These two criteria required exclusion of 25% of neurons.
To test if any residual mitochondrial Ca2+ uptake persisted under these conditions, we examined depolarization-induced transients in TG-treated neurons for sensitivity to mitochondrial blockers. Although the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) prolonged transients, this uncoupling of oxidative phosphorylation also leads to robust hydrolysis of cytoplasmic ATP , and thereby inhibits PMCA function. As an alternative approach, we therefore employed the combined application of inhibitors of mitochondrial ATP synthase (oligomycin, 10 μM) and mitochondrial electron transport (antimycin, 1 μM), thereby eliminating mitochondrial Ca2+ uptake without affecting ATP levels . These agents produced no effect on the rate of Ca2+ transient recovery (Figure 2A), which provides assurance that activity-induced transients under our baseline conditions do not engage the Ca2+ buffering function of mitochondria.
NCX contributes minimally to Ca2+ extrusion after neuronal activation in adult  and neonatal  sensory neurons during patch recording. We examined NCX in intact adult sensory neurons by replacement of bath Na+ with N-methyl-D-glucamine (NMDG), which similarly did not alter the Ca2+ recovery kinetics of transients in TG-treated neurons (Figure 2B). An effect of NCX on resting [Ca2+ c has been noted in DRG neurons during patch recording , but such changes were not noted in our recordings (data not shown).
On the basis of these initial observations, we adopted the approach of measuring PMCA function by determining the rate of recovery of small, depolarization-induced transients in TG-treated sensory neurons. To confirm that this successfully isolates PMCA as the only functioning mechanism for clearing Ca2+ from the cytoplasm, we compared the recovery from small activation-induced Ca2+ loads in TG-treated neurons before and after switching the bath pH to 8.8 (Figure 3A). Application of pH 8.8 solution substantially increased resting [Ca2+ c (Figure 3B), reflecting unopposed Ca2+ entry through store-operated Ca2+ channels [7, 8]. Transients induced by K+ depolarization did not recover in any neurons (n = 7; Figure 3A), which demonstrates that PMCA is the only available clearance pathway following test depolarizations under our study conditions.
Sensory neurons are a diverse population. For instance, neurons with small somatic area generally convey nociceptive traffic, while low threshold stimuli activate larger neurons . We examined whether PMCA function differed in resting sensory neurons of different sizes (Figure 2C), but found no significant difference in the rate of recovery from Ca2+ loads.
Effect of injury on PMCA
Following TG treatment, resting [Ca2+ c was depressed in axotomized neurons isolated from the L5 DRG after SNL compared to control neurons (Figure 4A). We have previously observed a similar injury-induced decrease in [Ca2+ c in the absence of TG treatment . These findings jointly indicate that injury reduces resting [Ca2+ c by affecting PMCA function. The current data do not confirm our previous finding that injury reduces transient amplitude (Figure 4B) . However, depolarizations in the present study were more brief, and we specifically excluded transients over 400nM, which may have reduced the apparent effect of injury on transient amplitude.
Comparison of PMCA function in axotomized SNL L5 neurons and neighboring SNL L4 neurons to control neurons (treated with TG) revealed an activated PMCA state selectively in axotomized neurons. Specifically, measurement of both τ (Figure 4C) and T95 (Figure 4D) revealed an accelerated recovery of the depolarization-induced transient in the SNL L5 population. PMCA activity is regulated by the concentration of Ca2+ in the cytoplasm . The diameter of the axotomized SNL L5 neurons (24.4 ± 3.3 μm, n = 50) was smaller than both the control (27.5 ± 5.1 μm, n = 173, P < 0.001) and the SNL L4 neurons (29.4 ± 5.5 μm, n = 45, P < 0.001), consistent with prior observations on the effects of sensory neuron axotomy [11, 18]. To eliminate a possible influence of neuronal size per se, we examined PMCA function in neuronal populations with diameters between 23 μm and 29 μm (average for Control 26.2 ± 18 μm, n = 103; SNL L4 26.3 ± 1.6, n = 29; SNL L5 25.7 ± 1.1, n = 20). In these restricted populations, the τ for transient recovery was still significantly depressed in the SNL L5 neurons (10.2 ± 8.5 s) compared to the SNL L4 (16.3 ± 9.6 s) and control neurons (16.9 ± 9.1 s; P < 0.05). This indicates that injury, rather than a shift in neuronal size, activates PMCA after axotomy.
Since injury depresses Ca2+ entry through voltage-gated Ca2+ channels , we examined whether the amount of Ca2+ accumulation during activation may itself affect PMCA function. Regression analysis showed no influence of transient amplitude upon τ (Figure 5), which supports a direct effect of injury on PMCA independent of the level of Ca2+ influx. To further examine whether the reduced transient amplitude in injured neurons affects recovery, we compared recovery kinetics in the different injury groups using only transients with amplitudes between 50 and 200nM. Limited this way, groups showed comparable average amplitudes (Control 118 ± 40nM, n = 103; SNL L4 121 ± 48nM, n = 26; SNL L5 118 ± 46 nM, n = 36), but SNL L5 neurons still had more rapid PMCA function (τ of 10.2 ± 7.5 s) compared to Control (16.9 ± 10.2 s, P < 0.01) or SNL L4 (16.8 ± 9.9 s, P < 0.05). These findings strongly indicate that accelerated function of PMCA after injury is due to alteration of the pump and not a result of loading conditions.
Influence of neuronal activation on PMCA
Axotomized neurons may be quiescent due to being disconnected from their receptive fields, or alternatively may be hyperactive from ectopically generated activity due to membrane instability or mechanical and inflammatory influences . To identify whether PMCA function is sensitive to neuronal activity level, we exposed neurons to repeated Ca2+ loads similar to those we have previously recorded during AP trains conducted to the neuronal soma . Specifically, we generated repeated 200-400nM Ca2+ transients in TG-treated sensory neurons by 0.3 s K+ depolarizations at 2 min intervals, and examined the effect of this activity on the rate of transient recovery (Figure 6A). This revealed a progressive acceleration of PMCA function in TG-treated control neurons, such that τ was decreased to 68 ± 12% of baseline after 3 preceding periods of activation (P < 0.05, n = 6), suggesting that neuronal hyperactivity could account for the increased PMCA function found in sensory neurons after nerve injury. Although activity-induced facilitation of PMCA function was also observed in neighboring L4 neurons after SNL (Figure 6B), axotomized SNL L5 neurons showed no acceleration of PMCA function (Figure 6C), which may indicate saturation of this form of plasticity after neuronal injury.
Regulation of neuronal excitability by PMCA
Since cytoplasmic Ca2+ regulates sensory neuron excitability [21, 22], we speculated that PMCA function may modulate excitability through controlling the pace of Ca2+ extrusion from the cytoplasm after activity-induced influx. Electrophysiological studies were performed by patch technique in dissociated sensory neurons that did not differ between groups for diameter (30.5 ± 1.7 μm) or resting membrane potential (−60.7 ± 4.4 mV, n = 28). We first evaluated effects on the afterhyperpolarization (AHP), since this regulates repetitive firing behavior in sensory neurons [23, 24]. We used intracellular vanadate, delivered by dialysis from the patch electrodes, to block PMCA function [2, 5]. Vanadate also blocks SERCA function, so selective effects on PMCA were isolated by pretreatment with TG (1 μM). After allowing 10 min of dialysis for delivery of vanadate (Na+ orthovanadate, 200 μM) to the cytoplasm, trains of APs (50 Hz for 4 s)  were generated by stimulation at current amplitude of 2-fold above threshold, which assured AP generation for each stimulus (Figure 7A). Vanadate significantly prolonged the AHP, compared to separate control neurons in which vanadate was not included in the patch pipette (Figure 7B). This confirms prior findings in neonatal sensory neurons cultured for 2-3d .
The AHP and underlying channels are known to regulate neuronal excitability, particularly repetitive firing behavior [23, 25]. To directly test if regulation of the AHP by PMCA affects excitability, we examined the ability of sensory neurons to generate APs during trains of depolarization stimuli with and without vanadate. Trains (50 Hz for 2 s) were generated in patched neurons (Figure 7C) by depolarizing stimuli at 1.5-fold above threshold (2.0 ± 0.2 nA, n = 6, for Control; 1.8 ± 0.4 nA, n = 7, for vanadate; P = 0.2). After 10 min of dialysis, AP generation was significantly less in neurons treated with vanadate (Figure 7D). Further evidence of an effect by vanadate is the observation in the vanadate group that the ability to generate trains of APs decreased 26 ± 27% after 10 min of dialysis compared to AP generation by an identical protocol immediately after breakthrough, whereas no change was evident during this time interval in the control group (−4 ± 4%, P < 0.05 vs. vanadate). These findings indicate that clearance of cytoplasmic Ca2+ by PMCA facilitates rapid firing in sensory neurons. Axotomized SNL L5 neurons stimulated by the same protocol without vanadate (50 Hz for 2 s, 1.5x threshold, 2.3 ± 0.4 nA, n = 8 Figure 7E) developed trains that on average contained more APs than control neurons, and included the maximum 100 APs in 7 of 8 cases. In other SNL L5 neurons (stimulus intensity 2.1 ± 0.4 nA, n = 9), vanadate reduced the number of APs (Figure 7F) to a level that was comparable to vanadate-treated control neurons. This suggests that a component of the elevated excitability of injured sensory neurons is attributable to their accelerated PMCA activity.
Prior studies by others have found that PMCA is a critical process by which sensory neurons clear Ca2+ from the cytoplasmic compartment after activity [2, 3, 5]. Since these studies used neurons from neonatal rats after prolonged culture (2-14d), their results may be specific to these conditions. For instance, animal age and duration of culture may affect the level of neuronal differentiation, which regulates PMCA expression . Our findings extend these prior observations to neurons examined within hours following dissociation from adult animals, and confirm a key role of PMCA in the reestablishment of [Ca2+ c homeostasis following activity. Of particular interest, we have shown that painful peripheral nerve injury stimulates PMCA function in sensory neurons.
PMCA is generally thought to be the principal mechanism of Ca2+ extrusion from sensory neurons, although there have been conflicting reports regarding the relative contribution of the NCX to Ca2+ clearance in sensory neurons. While a contribution of NCX to Ca2+ extrusion from sensory neurons has been proposed by some studies [15, 27], others have found no evidence of its participation [5, 28, 29], with which our findings agree. This supports the contention that after activation of sensory neurons, total cellular Ca2+ balance is reestablished predominantly by the action of PMCA.
The main finding of this investigation is that painful nerve injury results in increased PMCA function. In previous studies, we have demonstrated severe disruption of sensory neuron Ca2+ handling following axotomy, such as diminished Ca2+ influx through VGCCs during neuronal activation, decreased level of stored Ca2+ within the ER, and reduced [Ca2+ c in resting neurons. Since the dynamic balance between PMCA and SOCE establishes the resting [Ca2+ c, our prior observation of increased Ca2+ entry through store-operated Ca2+ channels after injury  suggested that PMCA function must be elevated to an even greater degree. This is borne out by our present findings. We have also previously noted that activity-induced transients resolve more quickly following axonal injury [4, 9], but the specific cause could not be ascertained without isolating selective Ca2+-handling components. The findings reported here indicate that accelerated PMCA function is at least one factor producing this effect. Although increased SERCA function could potentially contribute to more rapid resolution of transients following injury, our previous findings make this unlikely. Specifically, we have noted that injury produces a decreased intraluminal ER Ca2+ concentration while the constitutive leak of Ca2+ from the ER is unchanged , Together, these findings suggest that SERCA activity is diminished, but this has not been directly investigated.
There are several mechanisms by which PMCA function may be elevated following injury. Our data here indicate that a period of neuronal activity is followed by accelerated PMCA function. This resembles the conditioning effect previously noted in neonatal neurons after prolonged culture . Following peripheral nerve injury, excessive neuronal firing through ectopic spontaneous activity [32, 33] may be sufficient to condition PMCAs in sensory neuron somata. Neuronal activation may additionally lead elevated expression of PMCAs . Finally, PMCAs are a regulatory target of kinase signaling, such as protein kinase C [5, 35], protein kinase A , and protein tyrosine kinases . These kinase pathways may be activated in sensory neurons by injury [37–39].
The physiological consequences of accelerated PMCA function in injured sensory neurons may be diverse. At the sensory neuron central synapse in the spinal dorsal horn, accelerated PMCA clearance of Ca2+ from the presynaptic terminal may diminish excitatory neurotransmission, as has been shown in the hippocampus . This action would not be expected to contribute to hyperalgesia after nerve injury. However, in the peripheral portion of the sensory neuron, our present electrophysiological data show that suppression of PMCA with vanadate inhibits repetitive firing behavior, revealing a pro-excitatory regulatory role for PMCA function in the sensory neuron soma. Therefore, increased clearance of cytoplasmic Ca2+ by accelerated PMCA function after nerve injury will elevate neuronal excitability. Possible downstream mechanisms include diminished activation of Ca2+-activated K+ channels  and depressed CaMKII function . Ectopic activity originates in the soma after peripheral nerve injury , so these impulse trains would be accelerated by the concurrent increased PMCA activity. A similar effect at the axonal T-junction, a site of impulse failure during high frequency afferent activity , may lead to elevated conduction rates for AP trains originating from sources in the periphery, such as neuromata . These changes could all contribute to pain generation by increasing afferent neuronal traffic following peripheral injury. Finally, low resting [Ca2+ c may predispose neurons to the apoptosis  consistent with the progressive DRG neuron loss observed in the setting of chronic peripheral nerve injury .
In this investigation, we found an upregulation of PMCA in adult DRG neurons after painful nerve injury. PMCA may be considered a novel site for altering mechanisms leading to the generation of neuropathic pain. The continuing development of molecular therapies and the tolerance of the DRG as a site for intraneuronal injections [45, 46] make modulation of PMCA function in sensory neurons a potential future therapy for chronic pain.
All methods and use of animals were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.
Male Sprague–Dawley (Taconic Farms Inc., Hudson, NY) rats weighing 160 to 180 g were subjected to SNL modified from the original technique . Specifically, rats were anesthetized with 2% isoflurane in oxygen and the right paravertebral region was exposed. The L6 transverse process was removed, after which the L5 and L6 spinal nerves were ligated with 6–0 silk suture and transected distal to the ligature. The muscular fascia was closed with 4–0 resorbable polyglactin suture and the skin closed with staples. Control animals received skin incision and closure only. After surgery, rats were returned to their cages and kept under normal housing conditions with access to pellet food and water ad lib.
Rats underwent sensory testing for a pattern of hyperalgesic behavior that we have previously documented to be associated with conditioned place avoidance [48, 49]. Briefly, on 3 different days between 10d and 17d after surgery, right plantar skin was touched with a 22 G spinal needle with adequate pressure to indent but not penetrate the skin. Whereas control animals respond with only a brief reflexive withdrawal, rats following SNL may display a complex hyperalgesia response that includes licking, chewing, grooming and sustained elevation of the paw. The average frequency of hyperalgesia responses over the 3 testing days was tabulated for each rat.
Neuron isolation and plating
Between 21d and 28d after surgery, ganglia from rats were rapidly harvested following isoflurane anesthesia and decapitation and were incubated in 0.01% blendzyme 2 (Roche Diagnostics, Indianapolis, IN) for 30 min followed by incubation in 0.25% trypsin (Sigma Aldrich, St. Louis, MO) and 0.125% DNAse (Sigma) for 30 min, both dissolved in Dulbecco´s modified Eagle´s medium (DMEM)/F12 with glutaMAX (Invitrogen, Carlsbad, CA). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5 mM glutamine, 10 ng/ml nerve growth factor 7 S (Alomone Labs, Jerusalem, Israel) and 0.02 mg/ml gentamicin (Invitrogen). Dissociated neurons were plated onto poly-L-lysine coated glass cover slips (Deutsches Spiegelglas, Carolina Biological Supply, Burlington, NC) and maintained at 37°C in humidified 95% air and 5% CO2 for 2 hours, and were studied no later than 6 hours after harvest.
Measurement of cytoplasmic Ca2+ concentration
Unless otherwise specified, the bath contained Tyrode´s solution (in mM): NaCl 140, KCl 4, CaCl2 2, Glucose 10, MgCl2 2, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, with an osmolarity of 297-300 mOsm and pH 7.40. Agents were obtained as follows: Fura-2-AM from Invitrogen, and thapsigargin (TG), antimycin and oligomycin from Sigma Aldrich. Stock solutions of TG and Fura-2-AM were dissolved in DMSO, and subsequently diluted in the relevant bath solution such that final bath concentration of DMSO was 0.2% or less, which does not effect [Ca2+]c (n = 20, data not shown). The 500 μl recording chamber was superfused by gravity-driven flow at a rate of 3 ml/min. Agents were delivered by directed microperfusion controlled by a computerized valve system through a 500 μm diameter hollow quartz fiber 300 μm upstream from the neurons. This flow completely displaced the bath solution, and constant flow was maintained through this microperfusion pathway by delivery of bath solution when specific agents were not being administered. Dye imaging shows that solution changes were achieved within 200 ms.
Neurons plated on cover slips were exposed to Fura-2-AM (5 μM) at room temperature in a solution that contained 2% bovine albumin to aid dispersion of the fluorophore. After 30 min, they were washed 3 times with regular Tyrode’s solution, given 30 minutes for de-esterification, and then mounted in the recording chamber. Neurons were first examined under brightfield illumination, and those showing signs of lysis, crenulation or superimposed glial cells were excluded. For Ca2+ recording, the fluorophore was excited alternately with 340 nm and 380 nm wavelength illumination (150 W Xenon, Lambda DG-4, Sutter, Novato, CA), and images were acquired at 510 nm using a cooled 12-bit digital camera (Coolsnap fx, Photometrics, Tucson, AZ) and inverted microscope (Diaphot 200, Nikon Instruments, Melville, NY) through a 20x objective. Recordings from each neuron were obtained as separate regions (MetaFluor, Molecular Devices, Downingtown, PA) at a rate of 3 Hz. After background subtraction, the fluorescence ratio R for individual neurons was determined as the intensity of emission during 340 nm excitation (I340) divided by I380, on a pixel-by-pixel basis. The Ca2+ concentration was then estimated by the formula Kd·β· (R–Rmin)/(Rmax–R) where β = (I380max)/(I380min). Values of Rmin, Rmax and β were determined by in-situ calibration and were 0.38, 8.49 and 9.54, and Kd was 224 nm . Only neurons with stable baseline R traces were further evaluated. Traces were analyzed using Axograph X 1.1 (Axograph Scientific, Sydney, Australia). Sensory neurons somatic diameter is broadly associated with specific sensory modalities . We therefore stratified neurons as large (> 34 μm), which represent predominantly fast conducting non-nociceptive neurons, or small (< 34 μm), which represent predominantly nociceptive neurons. Unless otherwise stated, small neurons were examined. Transients were generated by depolarization produced by microperfusion application of K+ (50 mM) for 0.3 s. This duration was determined in preliminary studies to generate transient amplitudes less than 400nM in most cases, to limit involvement of mitochondrial Ca2+ buffering . Unless otherwise noted, transient measures were used only from the first depolarization following neuronal dissociation, in order to avoid amplification of PMCA function that might follow prior Ca2+ influx .
Activation of neurons by depolarization with brief bath application of K+ (50 mM, 0.3 s) resulted in transients that were fit well by a mono-exponential curve (Figure 1), from which the time constant (τ) was derived as a measure of the pace of recovery. PMCA function was additionally characterized by the duration of the transient from its peak to the point at which it is 95% resolved (T95).
Small to medium size neurons were studied following dissociation, using the whole cell configuration of the patch-clamp technique at room temperature. Tyrode’s solution (see above) was used for external bath solution. The internal pipette solution contained (in mM): potassium gluconate 135, KCl 5, MgCl2 2, EGTA 0.2, HEPES 10, Na2-phosphocreatine 10, Mg-ATP 4, Na2-GTP 0.3, at pH of 7.2 with KOH and osmolarity of 296 to 300 mOsm. Patch pipettes, ranging from 2-4MΩ resistance, were formed from borosilicate glass (King Precision Glass Co., Claremont, CA) and fire polished. Whole-cell recordings were made with an Axopatch 700B amplifier (Molecular Devices). Signals were filtered at 2 kHz and sampled at 10 kHz with a Digidata 1440 A digitizer and pClamp 10 software (Molecular Devices). Series resistance (7 – 14 MΩ) was monitored before and after the recordings, and data were discarded if the resistance changed by 20%. The threshold for generating APs was determined for each neuron by injecting a series of depolarizing currents with variable amplitudes lasting 1 ms. The duration of the AHP that followed a train of APs was characterized by the time constant τ determined by fitting a single exponential function of y = y 0 + k × exp (−x/τ) (Sigmaplot 11.0, Systat Software Inc., San Jose, CA).
Statistical analyses were performed with Statistica (StatSoft Inc, Tulsa, OK). Student’s T-test, Mann Whitney test, one-way ANOVA with Bonferroni’s post-hoc test, or repeated measures ANOVA with Dunnett’s post-hoc test was used to test significance of differences between groups. Where main effects were observed in ANOVA, was used to compare relevant means, and a P value less than 0.05 was considered significant. Data are reported as mean ± SD.
DiPolo R, Beauge L: Physiological role of ATP-driven calcium pump in squid axon. Nature 1979, 278: 271–273. 10.1038/278271a0
Benham CD, Evans ML, McBain CJ: Ca2+ efflux mechanisms following depolarization evoked calcium transients in cultured rat sensory neurones. J Physiol 1992, 455: 567–583.
Werth JL, Usachev YM, Thayer SA: Modulation of calcium efflux from cultured rat dorsal root ganglion neurons. J Neurosci 1996, 16: 1008–1015.
Gemes G, Rigaud M, Koopmeiners AS, Poroli MJ, Zoga V, Hogan QH: Calcium signaling in intact dorsal root ganglia: new observations and the effect of injury. Anesthesiology 2010, 113: 134–146. 10.1097/ALN.0b013e3181e0ef3f
Usachev YM, DeMarco SJ, Campbell C, Strehler EE, Thayer SA: Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4. Neuron 2002, 33: 113–122. 10.1016/S0896-6273(01)00557-8
Fuchs A, Lirk P, Stucky C, Abram SE, Hogan QH: Painful nerve injury decreases resting cytosolic calcium concentrations in sensory neurons of rats. Anesthesiology 2005, 102: 1217–1225. 10.1097/00000542-200506000-00023
Gemes G, Bangaru ML, Wu HE, Tang Q, Weihrauch D, Koopmeiners AS, Cruikshank JM, Kwok WM, Hogan QH: Store-operated Ca2+ entry in sensory neurons: functional role and the effect of painful nerve injury. J Neurosci 2011, 31: 3536–3549. 10.1523/JNEUROSCI.5053-10.2011
Wanaverbecq N, Marsh SJ, Al-Qatari M, Brown DA: The plasma membrane calcium-ATPase as a major mechanism for intracellular calcium regulation in neurones from the rat superior cervical ganglion. J Physiol 2003, 550: 83–101. 10.1113/jphysiol.2002.035782
Fuchs A, Rigaud M, Hogan QH: Painful nerve injury shortens the intracellular Ca2+ signal in axotomized sensory neurons of rats. Anesthesiology 2007, 107: 106–116. 10.1097/01.anes.0000267538.72900.68
Ghosh B, Li Y, Thayer SA: Inhibition of the plasma membrane Ca2+ pump by CD44 receptor activation of tyrosine kinases increases the action potential afterhyperpolarization in sensory neurons. J Neurosci 2011, 31: 2361–2370. 10.1523/JNEUROSCI.5764-10.2011
Sapunar D, Ljubkovic M, Lirk P, McCallum JB, Hogan QH: Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats. Anesthesiology 2005, 103: 360–376. 10.1097/00000542-200508000-00020
Carafoli E: Intracellular calcium homeostasis. Annu Rev Biochem 1987, 56: 395–433. 10.1146/annurev.bi.56.070187.002143
Friel DD, Tsien RW: An FCCP-sensitive Ca2+ store in bullfrog sympathetic neurons and its participation in stimulus-evoked changes in [Ca2+]i. J Neurosci 1994, 14: 4007–4024.
Budd SL, Nicholls DG: A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis. J Neurochem 1996, 66: 403–411.
Verdru P, De Greef C, Mertens L, Carmeliet E, Callewaert G: Na(+)-Ca2+ exchange in rat dorsal root ganglion neurons. J Neurophysiol 1997, 77: 484–490.
Ma C, Shu Y, Zheng Z, Chen Y, Yao H, Greenquist KW, White FA, LaMotte RH: Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons. J Neurophysiol 2003, 89: 1588–1602.
Colegrove SL, Albrecht MA, Friel DD: Dissection of mitochondrial Ca2+ uptake and release fluxes in situ after depolarization-evoked [Ca2+](i) elevations in sympathetic neurons. J Gen Physiol 2000, 115: 351–370. 10.1085/jgp.115.3.351
Oyelese AA, Eng DL, Richerson GB, Kocsis JD: Enhancement of GABAA receptor-mediated conductances induced by nerve injury in a subclass of sensory neurons. J Neurophysiol 1995, 74: 673–683.
McCallum JB, Kwok WM, Sapunar D, Fuchs A, Hogan QH: Painful peripheral nerve injury decreases calcium current in axotomized sensory neurons. Anesthesiology 2006, 105: 160–168. 10.1097/00000542-200607000-00026
Devor M: Response of nerves to injury in relation to neuropathic pain. In Wall and Melzack´s Textbook of Pain. 4th edition. Edited by: McMahon S, Koltzenburg M. Churchill Livingston, London; 2006:905–927.
Lirk P, Poroli M, Rigaud M, Fuchs A, Fillip P, Huang CY, Ljubkovic M, Sapunar D, Hogan Q: Modulators of calcium influx regulate membrane excitability in rat dorsal root ganglion neurons. Anesth Analg 2008, 107: 673–685. 10.1213/ane.0b013e31817b7a73
Luscher C, Lipp P, Luscher HR, Niggli E: Control of action potential propagation by intracellular Ca2+ in cultured rat dorsal root ganglion cells. J Physiol 1996,490(Pt 2):319–324.
Scholz A, Gruss M, Vogel W: Properties and functions of calcium-activated K + channels in small neurones of rat dorsal root ganglion studied in a thin slice preparation. J Physiol 1998, 513: 55–69. 10.1111/j.1469-7793.1998.055by.x
Hogan Q, Lirk P, Poroli M, Rigaud M, Fuchs A, Fillip P, Ljubkovic M, Gemes G, Sapunar D: Restoration of calcium influx corrects membrane hyperexcitability in injured rat dorsal root ganglion neurons. Anesth Analg 2008, 107: 1045–1051. 10.1213/ane.0b013e31817bd1f0
Sah P: Ca(2+)-activated K + currents in neurones: types, physiological roles and modulation. Trends Neurosci 1996, 19: 150–154. 10.1016/S0166-2236(96)80026-9
Usachev YM, Toutenhoofd SL, Goellner GM, Strehler EE, Thayer SA: Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32. J Neurochem 2001, 76: 1756–1765. 10.1046/j.1471-4159.2001.00169.x
Lu SG, Zhang X, Gold MS: Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons. J Physiol 2006, 577: 169–190. 10.1113/jphysiol.2006.116418
Gover TD, Moreira TH, Kao JP, Weinreich D: Calcium regulation in individual peripheral sensory nerve terminals of the rat. J Physiol 2007, 578: 481–490.
Gover TD, Moreira TH, Kao JP, Weinreich D: Calcium homeostasis in trigeminal ganglion cell bodies. Cell Calcium 2007, 41: 389–396. 10.1016/j.ceca.2006.08.014
Rigaud M, Gemes G, Weyker PD, Cruikshank JM, Kawano T, Wu HE, Hogan QH: Axotomy depletes intracellular calcium stores in primary sensory neurons. Anesthesiology 2009, 111: 381–392. 10.1097/ALN.0b013e3181ae6212
Pottorf WJ, Thayer SA: Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons. J Neurochem 2002, 83: 1002–1008. 10.1046/j.1471-4159.2002.01221.x
Wall PD, Gutnick M: Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Exp Neurol 1974, 43: 580–593. 10.1016/0014-4886(74)90197-6
Wall PD, Devor M: Sensory afferent impulses originate from dorsal root ganglia as well as from the periphery in normal and nerve injured rats. Pain 1983, 17: 321–339. 10.1016/0304-3959(83)90164-1
Guerini D, Garcia-Martin E, Gerber A, Volbracht C, Leist M, Merino CG, Carafoli E: The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+. J Biol Chem 1999, 274: 1667–1676. 10.1074/jbc.274.3.1667
Verma AK, Paszty K, Filoteo AG, Penniston JT, Enyedi A: Protein kinase C phosphorylates plasma membrane Ca2+ pump isoform 4a at its calmodulin binding domain. J Biol Chem 1999, 274: 527–531. 10.1074/jbc.274.1.527
Baggaley E, McLarnon S, Demeter I, Varga G, Bruce JI: Differential regulation of the apical plasma membrane Ca(2+) -ATPase by protein kinase A in parotid acinar cells. J Biol Chem 2007, 282: 37678–37693. 10.1074/jbc.M703416200
Obata K, Yamanaka H, Kobayashi K, Dai Y, Mizushima T, Katsura H, Fukuoka T, Tokunaga A, Noguchi K: Role of mitogen-activated protein kinase activation in injured and intact primary afferent neurons for mechanical and heat hypersensitivity after spinal nerve ligation. J Neurosci 2004, 24: 10211–10222. 10.1523/JNEUROSCI.3388-04.2004
Mao J, Price DD, Mayer DJ, Hayes RL: Pain-related increases in spinal cord membrane-bound protein kinase C following peripheral nerve injury. Brain Res 1992, 588: 144–149. 10.1016/0006-8993(92)91354-H
Hua XY, Chen P, Yaksh TL: Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neurosci Lett 1999, 276: 99–102. 10.1016/S0304-3940(99)00818-6
Jensen TP, Filoteo AG, Knopfel T, Empson RM: Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3. J Physiol 2007, 579: 85–99. 10.1113/jphysiol.2006.123901
Nelson AB, Gittis AH, du Lac S: Decreases in CaMKII activity trigger persistent potentiation of intrinsic excitability in spontaneously firing vestibular nucleus neurons. Neuron 2005, 46: 623–631. 10.1016/j.neuron.2005.04.009
Luscher C, Streit J, Lipp P, Luscher HR: Action potential propagation through embryonic dorsal root ganglion cells in culture II. Decrease of conduction reliability during repetitive stimulation. J Neurophysiol 1994, 72: 634–643.
Collins F, Schmidt MF, Guthrie PB, Kater SB: Sustained increase in intracellular calcium promotes neuronal survival. J Neurosci 1991, 11: 2582–2587.
Vestergaard S, Tandrup T, Jakobsen J: Effect of permanent axotomy on number and volume of dorsal root ganglion cell bodies. J Comp Neurol 1997, 388: 307–312. 10.1002/(SICI)1096-9861(19971117)388:2<307::AID-CNE8>3.0.CO;2-5
Pfirrmann CW, Oberholzer PA, Zanetti M, Boos N, Trudell DJ, Resnick D, Hodler J: Selective nerve root blocks for the treatment of sciatica: evaluation of injection site and effectiveness–a study with patients and cadavers. Radiology 2001, 221: 704–711. 10.1148/radiol.2213001635
Fischer G, Kostic S, Nakai H, Park F, Sapunar D, Yu H, Hogan Q: Direct injection into the dorsal root ganglion: technical, behavioral, and histological observations. J Neurosci Methods 2011, 199: 43–55. 10.1016/j.jneumeth.2011.04.021
Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992, 50: 355–363. 10.1016/0304-3959(92)90041-9
Hogan Q, Sapunar D, Modric-Jednacak K, McCallum JB: Detection of neuropathic pain in a rat model of peripheral nerve injury. Anesthesiology 2004, 101: 476–487. 10.1097/00000542-200408000-00030
Wu HE, Gemes G, Zoga V, Kawano T, Hogan QH: Learned avoidance from noxious mechanical simulation but not threshold semmes weinstein filament stimulation after nerve injury in rats. J Pain 2010, 11: 280–286. 10.1016/j.jpain.2009.07.011
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985, 260: 3440–3450.
The study was supported by National Institutes of Health Grants NS-42150 (to Q.H.) and DA-K01 024751 (to H.-E.W.).
The authors declared that they have no competing interests.
Conception and design of the experiments were performed by GG, KO, HW, and QH. Collection, analysis and interpretation of data were performed by GG, KO, BP, HW, MB, QT, and QH. Drafting the article and revising it critically for important intellectual content was performed by GG, KO, HW, and QH. All authors approved the final version of the manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Gemes, G., Oyster, K.D., Pan, B. et al. Painful nerve injury increases plasma membrane Ca2+-ATPase activity in axotomized sensory neurons. Mol Pain 8, 46 (2012). https://doi.org/10.1186/1744-8069-8-46
- Dorsal root ganglion
- Nerve injury