The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca signaling in primary sensory neurons after injury. The authors examined the effect of injury on intracellular Ca stores of the endoplasmic reticulum, which critically regulate the Ca signal and neuronal function.
Intracellular Ca levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats after L5 spinal nerve ligation, compared to neurons from control animals.
Endoplasmic reticulum Ca stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca-free bath solution, which removes the contribution of store-operated membrane Ca channels, and after blockade of the mitochondrial, sarco-endoplasmic Ca-ATPase and the plasma membrane Ca ATPase pathways. Ca released by the sarco-endoplasmic Ca-ATPase blocker thapsigargin and by the Ca-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca stores in axotomized neurons were not expanded by neuronal activation by K depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca-ATPase was normal. Luminal Ca concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation.
Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca stores and a reduced luminal Ca concentration. Depletion of Ca stores may contribute to the pathogenesis of neuropathic pain.
THE primary afferent neuron is the source of all somatic sensory experience, a target of regional anesthetics, the afferent pathway for pain during surgery, and an origin of abnormal activity in chronic pain. Cytoplasmic Ca2+is the dominant second messenger in sensory neurons, and it regulates diverse processes, including neuronal differentiation, neurotransmitter release, excitability, gene expression, enzymatic activation, and programmed cell death (apoptosis). We have previously shown that painful nerve injury leads to prolonged disruption of the Ca2+signal in primary afferent neurons. Specifically, after transection of their peripheral axons, neurons with their cell bodies in the dorsal root ganglia (DRGs) demonstrate a depressed resting cytoplasmic Ca2+concentration ([Ca2+]c)1as well as a diminished temporary increase of [Ca2+]cthat follows neuronal activation (the [Ca2+]ctransient).2These findings may be partially attributable to the decrease in activity-related Ca2+entry through the plasmalemma caused by axotomy of DRG neurons.3–5However, the cellular machinery that modulates the sensory neuron Ca2+signal is complex,6and its function in the context of injury has not been examined.
Sensory neurons possess channels and pumps that extrude Ca2+from the cell, sequester Ca2+into organelles within the cell, and release Ca2+from these stores. Specifically, the plasma membrane Ca2+-ATPase expels cytoplasmic Ca2+from the neuron, as does the Na+-Ca2+exchange pathway. Cytoplasmic Ca2+is pumped through the action of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) into high-concentration subcellular reservoirs, predominantly the endoplasmic reticulum (ER),6,7from which it may be released back to the cytoplasm through channels sensitive to ryanodine (the ryanodine receptor [RyR]) or inositol triphosphate (IP3). The amount of Ca2+accumulated in this store is functionally linked to plasma membrane channels (store-operated Ca2+channels [SOCCs]) that admit Ca2+into the cytoplasm at a rate inversely related to the store level.8Mitochondrial Ca2+uptake driven by the potential across the mitochondrial inner membrane buffers large Ca2+loads by uptake when the [Ca2+]crises above a critical threshold and subsequently releases this Ca2+as the [Ca2+]crecovers towards baseline levels. All of these processes are tuned by regulatory systems and may be sites at which neuronal injury perturbs the final Ca2+signal.
The quantity of stored Ca2+critically regulates Ca2+signaling in neurons. This parameter dictates the ability of the sensory neuron to receive Ca2+from the cytoplasm,9,10by which the cell limits excessive [Ca2+]clevels during neuronal activity. Stored Ca2+may also be released into the cytoplasm through the process of Ca2+-induced Ca2+release, by which an initial increase in [Ca2+]cactivates the RyRs to discharge stored Ca2+into the cytoplasm, resulting in amplification of [Ca2+]ctransients. Thus, the ER is at once both a Ca2+sink and source,11critically shaping the Ca2+signal.
Disordered homeostasis of the Ca2+store participates in the pathogenesis of various neurologic conditions, such as stroke and Alzheimer disease, as well as painful conditions such as diabetes, human immunodeficiency virus infection, and tissue inflammation.12–14The ER stores may likewise play a central role in the pathogenesis of neuropathic pain. Accordingly, the experiments reported here explored the influence of injury on the size of the intracellular Ca2+store. We employed the spinal nerve ligation (SNL) injury model; this allows separate evaluation of the axotomized neurons of the fifth lumbar (L5) DRG for comparison with the L4 neurons that are not directly injured but have axons that transit the sciatic nerve in the company of degenerating distal segments of the L5 neurons, and are thus exposed to mediators of inflammation.15,16The relative importance of the contribution to pain genesis of these two populations remains unresolved.17
Materials and Methods
All methods and use of animals were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee (Milwaukee, Wisconsin).
Male Sprague-Dawley (Taconic Farms Inc., Hudson, NY) rats weighing 160–180 g were subjected to SNL in a manner derived from the original technique.18Rats were anesthetized with 2% isoflurane in oxygen, and the right paravertebral region was exposed. After removal of the L6 transverse process, the L5 and L6 spinal nerves were ligated with 6-0 silk suture and transected distal to the ligature. The fascia was closed with 4-0 resorbable polyglactin suture, and the skin was closed with staples. Control animals received anesthesia, skin incision, and stapling only. After surgery, the rats were returned to their cages and kept under normal housing conditions with access to pellet food and water ad libitum .
Rats underwent sensory testing for hyperalgesic behavior on three different days between 10 and 17 days after surgery, as previously described.1,19Briefly, right plantar skin was mechanically stimulated with a 22-gauge spinal needle with adequate pressure to indent but not penetrate the skin. Whereas control animals respond with only a brief reflexive withdrawal, rats after SNL may display a complex hyperalgesia response that incorporates sustained licking, chewing, grooming, and elevation of the paw. The frequency of hyperalgesia responses was tabulated for each rat. After SNL, only rats that displayed a hyperalgesia-type response after at least 20% of stimuli were used further in this study.
Neuron Isolation and Plating
Neurons were dissociated from L4 and L5 DRGs after isoflurane anesthesia and decapitation 21 to 27 days after SNL or skin sham surgery. This interval was chosen because hyperalgesia is fully developed by this time.19DRGs were incubated in 0.0625% trypsin (Sigma Aldrich, St. Louis, MO), 0.0125% DNAse (Invitrogen, Carlsbad, CA), and 0.01% blendzyme 2 (Roche Diagnostics, Indianapolis, IN) in Dulbecco modified Eagle´s medium (DMEM)/F12 with glutaMAX (Invitrogen) for 1.5 h, centrifuged, and triturated with fire-polished pipettes in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5 mm glutamine, 100 ng/ml nerve growth factor 7S (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 Biologic Supply, Burlington, NC) and maintained at 37°C in humidified 95% air and 5% CO2for 2 h, and were studied no later than 6 h after harvest.
Solutions and Agents
Unless otherwise specified, the bath contained Tyrode´s solution (in mm): NaCl 140, KCl 4, CaCl22, Glucose 10, MgCl22, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, with an osmolarity of 297–300 mOsm and pH 7.40. In some experiments, a Ca2+-free Tyrode’s was used that contained (in mm): NaCl 140, KCl 4, Glucose 10, MgCl22, HEPES 10, and ethylene glycol tetraacetic acid (EGTA) 0.2. In experiments with permeabilized neurons, a bath solution modeled upon intracellular solution was used that contained (in mm): KCl 120, EGTA 5, CaCl22.25, MgCl22, and HEPES 20. Osmolarity was adjusted to 295 with sucrose, and pH was adjusted to 7.40. This clamped Mg2+and Ca2+at physiologic concentrations20of 1.6 mm and 39 nm, respectively, calculated using a web-based EGTA calculator.**Adenosine-triphosphate was omitted to not trigger release of stored Ca2+. Agents were obtained as follows: caffeine, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), dimethylsulfoxide, lanthanum chloride, thapsigargin from Sigma Aldrich, saponin from Calbiochem EMD (Gibbstown, NJ), Fura-2-AM, mag-Fura-2-AM, ionomycin, and Pluronic F-127 from Invitrogen. Stock solutions of ionomycin, thapsigargin, FCCP, Fura-2-AM, and mag-Fura-2-AM were dissolved in dimethylsulfoxide and subsequently diluted in the relevant bath solution such that final bath concentration of dimethylsulfoxide was 0.1% or less, which has no effect on [Ca2+]c(n = 20, data not shown). The 500-μl recording chamber was constantly superfused by a gravity-driven bath flow at a rate of 3 ml/min. Agents were delivered by directed microperfusion 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 by delivery of bath solution, when specific agents were not being administered, through a computerized valve system. Solution changes were achieved within 200 ms.
Measurement of Cytoplasmic Ca2+Concentration
Cover slips holding plated neurons were transferred to a room-temperature, 5 μm solution of Fura-2-AM that contained 0.04% Pluronic F-127 to aid dispersion of the fluorophore. After 30 min, they were washed three times with regular Tyrode’s solution and left in a dark environment for de-esterification for 30 min and then mounted onto the recording chamber. The fluorophore was excited alternately with 340-nm and 380-nm wavelength illumination (150W Xenon, Lambda DG-4; Sutter, Novato, CA), and images were acquired at 510 nm by using a cooled 12-bit digital camera (Coolsnap fx; Photometrics, Tucson, AZ) and inverted microscope (Diaphot 200; Nikon Instruments, Melville, NY) through a 20× or 40× fluor oil-immersion objective. Neurons were visually examined in the brightfield mode, and those showing signs of lysis, crenulation, or superimposed glial cells were excluded. Recordings from each neuron were obtained as separate regions of interest by appropriate software (MetaFluor; Molecular Devices, Downingtown, PA). 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 calcium concentration was then estimated by the formula [Ca2+]c= Kd·β· (R − Rmin)/(Rmax− R) where β= (I380max)/(I380min). Values of Rmin, Rmax, and β were determined by in situ calibrations of 33 neurons from three different preparations as described before1and were 0.38, 8.49, and 9.54, respectively, and 224 nm was used as Kd.21
Determination of Ca2+Release
Release of Ca2+from stores was quantified by measuring the size of the resulting [Ca2+]ctransient. Using amplitude to quantify large transients, such as those induced by the action of caffeine, poorly reflects the magnitude of Ca2+release from stores because of the influence of mitochondrial buffering during intervals of high [Ca2+]c. Initial mitochondrial Ca2+uptake depresses the peak [Ca2+]cduring a transient and produces a plateau phase thereafter, during which Ca2+is released from mitochondria into the cytoplasm.22Thus, mitochondrial buffering has reciprocal effects on transient amplitude and duration.23Further, we have previously shown that transient area is proportionate to cytoplasmic Ca2+load.2We therefore chose to quantify caffeine-induced transients by calculating the area of the transient.
In other experiments, application of thapsigargin was used to produce transients, for which the amplitude of the rise from baseline to peak [Ca2+]cwas measured rather than area, because these transients had a more gradual onset without a clearly discernible beginning and endpoint, unlike those triggered by caffeine. Also, such transients do not reach [Ca2+]clevels that engage mitochondrial buffering, which we estimated from the level of the plateau in large K+– (316 ± 124 nm; n = 115) and caffeine-induced transients (310 ± 128 nm, n = 541).22,24Amplitude measurement was also used for transients induced by ionomycin; this ionophore to equilibrate [Ca2+] gradients between subcellular compartments, including cytosol, ER, and mitochondria.25Calcium was absent from the bath solution during thapsigargin application to remove the contribution of Ca2+entry through SOCCs26and similarly during ionomycin application to avoid entry of Ca2+through the ionophore.
Measurement of ER Luminal Ca2+Concentration
The low-affinity indicator mag-Fura-2-AM was used to measure the Ca2+concentration contained within the lumina of intracellular compartments ([Ca2+]L).27,28Dye loading and recording procedures were similar to those used with Fura-2-AM, although loading took place at 37°C to maximize compartmentalization of the fluorophore in intracellular organelles.29Calibration of the mag-Fura-2 signal was performed in situ on 26 neurons from 3 different dissociated DRGs and showed values of Rmin, Rmax, and β of 0.32, 3.63, and 13.09. A Kdof 53 μm was used for calculation of [Ca2+]L.30Attempts using patch-pipette dialysis in protocols requiring removal of cytoplasmic mag-Fura-2 proved ineffective, but successful permeabilization of neurons was achieved by 75- to 105-s bath application of 0.01% saponin.31Cytoplasmic washout of the fluorophore was confirmed by following the decline in both the fluorescence intensities at 340 nm and 380 nm and a concurrent increase in the ratio R. Only traces in which the decline in intensity during 380 nm illumination exceeded 40% were included for analysis.
Preliminary studies showed no differences between neurons dissociated from L4 and L5 ganglia, removed from the same control animals, in Ca2+release by caffeine with normal Tyrode’s bath solution (transient area 14.7 ± 11.3 nm · s × 103in L4 [n = 19]vs. 15.7 ± 11.4 nm · s × 103in L5 [n = 37]; P = 0.38). We therefore pooled findings from L4 and L5 neurons as the control group. Neurons characterized by size (large, greater than 34 μm; small, less than 34 μm) were considered separately. Statistical analyses were performed with Statistica (StatSoft Inc, Tulsa, OK). Two-tailed Student t test was used for comparing two means. One-way ANOVA was used to detect the influence of injury group on measured parameters. Consideration of additional factors such as time or pharmacologic interventions was achieved using a two-way ANOVA design. An ANCOVA model was used to account for variance due to a covariate after assuring linearity of the influence of the covariate upon the outcome measure. Bonferroni post hoc test was used to compare relevant means, and P < 0.05 was considered significant. Averages are reported ± SD.
Upon needle stimulation, SNL animals (n = 69) displayed a hyperalgesia response rate of 35 ± 21%, whereas control animals (n = 56) showed a hyperalgesic response 0 ± 1% (P < 0.001). Consistent with our previous findings,1sensory neuron axotomy depressed resting [Ca2+]c. Specifically, whereas [Ca2+]cwas 66 ± 27 nm in control neurons (n = 274), SNL of L5 neurons resulted in a [Ca2+]cof 46 ± 25 nm (n = 157; P < 0.001 vs. control). SNL had no effect on resting [Ca2+]cof adjacent L4 neurons, in which [Ca2+]cwas 63 ± 26 nm (n = 110).
Ryanodine Receptor-mediated Ca2+Release by Caffeine
An estimate of stored Ca2+may be made by measuring the rise of [Ca2+]cduring Ca2+release from stores triggered by application of a high concentration of caffeine (20 mm), which sensitizes the RyR to Ca2+to such an extent that resting levels of Ca2+produce sustained channel opening.32For caffeine-induced Ca2+release, we chose to measure transient area rather than amplitude to limit the influence of mitochondrial buffering (see Materials and Methods), and we tested the assumption in control neurons by comparing transient parameters with and without blockade of mitochondrial Ca2+buffering with FCCP (1 μm).22Whereas amplitude of the caffeine-induced transient increased by 108% in the presence of FCCP (from 779 ± 807 nm [n = 273] to 1615 ± 2075 nm [n = 198]), area of the transient increased by only 20% (from 18.2 ± 11.1 nm · s × 103[n = 268] to 21.9 ± 16.6 nm · s × 103[n = 198]).
To measure stores, caffeine was administered continuously until the [Ca2+]creturned to baseline to assure that the stores were fully emptied, which was confirmed by the observation that reapplication of caffeine 30 s after washout produced no further Ca2+release (n = 10; data not shown). Sustained application also precluded SERCA function so that this process did not affect the measure of Ca2+release. Application of caffeine (20 mm) in this fashion produced a rapidly rising transient that included a prolonged plateau phase followed by resolution to a level close to resting [Ca2+]c(fig. 1A). All but 1 of 542 neurons responded with an increase of at least 50% above resting [Ca2+]c.
DRG neurons are a heterogeneous population representing different sensory modalities. Although small neurons are broadly associated with nociceptive sensory modality, neurons with large somata conduct low-threshold sensory information.33,34In small neurons, the transient areas evoked by caffeine were significantly decreased by axotomy in L5 neurons after SNL, as well as in adjacent L4 neurons (fig. 1B). Caffeine-induced transients had areas in control large neurons that were smaller than in control small neurons (P < 0.001). However, there was no effect of injury on large neuron Ca2+release measured this way (fig. 1C).
Caffeine release of stored Ca2+triggers influx of Ca2+across the plasmalemma through store-operated Ca2+channels (SOCCs),35which could be independently influenced by injury. We therefore performed additional experiments in small neurons, eliminating this factor by using Ca2+-free bath solution.36Under these conditions, transients triggered by caffeine application showed influences of injury that were congruent with the findings above (fig. 1D). The transient area in all groups was less in the absence of bath Ca2+compared to experiments conducted with bath Ca2+, which likely indicates a contribution by SOCC to the transient generated by caffeine release of Ca2+stores.35
To further compare Ca2+stores and injury effect in putative nociceptors versus low-threshold afferents, we examined additional small neurons that were further categorized by sensitivity to capsaicin. Sensitivity to capsaicin is typical of peptidergic polymodal nociceptors, and insensitivity is a characteristic of IB4-binding mechanosensitive nociceptors.37In a separate preliminary study (data not shown), the EC50for capsaicin necessary to achieve a 50% increase in [Ca2+]cwas 7.9 nm (total n = 175 neurons, used only for this determination). Using 10 nm, 45 of 85 (53%) of control neurons responded to capsaicin by this criterion (fig. 1E), comparable to the typical proportion of nociceptive neurons.38In control neurons, [Ca2+]ctransient areas were significantly larger in capsaicin-responsive cells than in nonresponsive ones (P < 0.001; fig. 1F), as reported previously. SNL decreased the proportion of neurons sensitive to capsaicin in the L4 group (10 of 37, 27%) and especially the L5 group (8 of 53, 15%; P < 0.001), as expected from previous findings that axonal injury causes loss of the capsaicin receptor, transient receptor potential vanilloid-1.1,39In both capsaicin-responsive and nonresponsive populations, axotomy reduced the area of the caffeine-induced transient (fig. 1F), indicating loss of Ca2+stores. Specifically, in capsaicin-responsive neurons, the average area under the transient curve was decreased by 59% in axotomized compared to control neurons, and axotomy of capsaicin-nonresponsive neurons decreased the area by 33%. This indicates that both subgroups of small nociceptive sensory neurons have decreased releasable Ca2+stores after injury.
Ca2+Release during Inhibition of Sequestration and Extrusion
To examine Ca2+release in the absence of the buffering by uptake into mitochondria, we applied caffeine (20 mm) during the continued presence of FCCP (1 μm; fig. 2A). Peak of the transient in L4 neurons was not different from controls, while the axotomized L5 neurons decreased by 51% compared to controls (fig. 2B). This indicates that smaller caffeine-induced [Ca2+]ctransients in axotomized neurons are not due to greater mitochondrial buffering in this neuronal group.
We also eliminated the potential influence of variable Ca2+reuptake by the SERCA, Ca2+extrusion via the plasma membrane Ca2+-ATPase and Ca2+-Na+exchange mechanism, and Ca2+influx by inducing Ca2+release with caffeine (20 mm) in the context of multiple pharmacological blockers (FCCP, 1 μm; thapsigargin, 1 μm to block SERCA, administered only with and after caffeine to avoid an initial thapsigargin-induced store release; La3+, 1 mm to block plasma membrane Ca2+-ATPase, Ca2+-Na+exchange, and Ca2+entry pathways, discontinued during caffeine in order to avoid precipitation). This resulted in transients in which an initial increase of [Ca2+]cwas not followed by recovery (fig. 2C). Injury had a significant effect on the caffeine-induced transient amplitude (fig. 2D), in which the L4 group was unchanged but the L5 group showed 30% less Ca2+release compared to control.
Constitutive Ca2+Release Revealed by Thapsigargin
It is unclear whether ryanodine receptor-sensitive Ca2+stores fully overlap with Ca2+stores sensitive to inositol-3P.6For this reason and because receptor-mediated Ca2+release may be influenced by altered expression of the receptor after injury without actually affecting Ca2+stores per se , we also determined the effect of injury on Ca2+stores using techniques that do not rely on receptor activation. Thapsigargin provides such an alternate method by blocking SERCA, which exposes a constitutive Ca2+leak from stores. This effect is dose-related26; although a previously published study using 20 nm thapsigargin failed to produce transients in sensory neurons,40we observed slow [Ca2+]ctransients with application of 1 μm thapsigargin in all neurons (fig. 3A). Full emptying of RyR-sensitive stores by thapsigargin was demonstrated by reduction of the subsequent caffeine-induced Ca2+release transient area in control neurons to 165 ± 353 nm · s (n = 20), which is 1% of caffeine-induced Ca2+release reported above. However, thapsigargin-sensitive Ca2+stores encompass more than just the RyR-sensitive pool; a thapsigargin-induced transient (amplitude 23 ± 17 nm, n = 19) persists after caffeine-induced Ca2+release, which constitutes 22% of the thapsigargin-induced transient in the absence of preceding caffeine (107 ± 69, n = 85). An effect of injury is evident in the thapsigargin-sensitive pool; the thapsigargin-induced transient in SNL L5 neurons had a 25% lower transient amplitude than control or SNL L4 neurons (fig. 3B).
The rise of [Ca2+]cduring thapsigargin application was reliably a straight line (fig. 3A, inset) indicative of a zero-order process, consistent with the observation that the leak rate from Ca2+stores is independent of [Ca2+]Luntil the stores are nearly exhausted.10To evaluate the rate of constitutive release from thapsigargin-sensitive stores, we measured the slope of this rising phase of the thapsigargin-induced [Ca2+]ctransient. Leak rate measured this way showed a tight dependence on total releasable Ca2+content, as indicated by amplitude of the thapsigargin release transient (R2= 0.85 for all groups together, P < 0.001; n = 95 neurons, fig. 3C). For this reason, an analysis of covariance (ANCOVA) model was used to examine the average leak rate, which was depressed in SNL L5 neurons compared to control and SNL L4 neurons (fig. 3D). This was attributable to the effect of the depressed releasable Ca2+in the L5 neurons, as demonstrated by the comparable relationship between leak rate and releasable Ca2+in control and L5 neurons (fig. 3C). However, L4 neurons had a slightly depressed relationship of leak versus level of releasable Ca2+.
Ca2+Release by Ionomycin
Application of the Ca2+ionophore ionomycin (1 μm) is an additional technique that releases stored Ca2+without requiring receptor activation12and eliminates the influence of mitochondria.25A Ca2+-free bath solution was used for 3 min before ionomycin application and thereafter to avoid the influence of Ca2+entry through the plasmalemma. Transients showed an exponential resolution that lacked the plateau attributable to mitochondria (fig. 4A). After SNL, ionomycin released more Ca2+in the adjacent L4 neurons, but releasable Ca2+was reduced in axotomized L5 neurons compared to control (fig. 4B).
SERCA-dependent Recovery of Ca2+Stores at Rest
One possible reason that Ca2+stores cannot be filled in injured neurons may be dysfunction of the SERCA mechanism that pumps Ca2+into the ER. Repeat application of caffeine (20 mm) produces a diminished Ca2+release until adequate time has elapsed to allow refilling of the stores (fig. 5A). We therefore tested the pace at which neurons in the different groups recovered from caffeine-induced depletion of stored Ca2+. Complete release of stored Ca2+was achieved by sustained application of caffeine (20 mm), which was followed by a 1-, 3-, or 10-min recovery period, and then caffeine was applied again. Each neuron was used to test only one time interval. After 10-min recovery time, the area of the second transient in control neurons had recovered to 79 ± 23% of the first (fig. 5B), and there was no difference in the proportionate rate of recovery after injury, normalized to the initial baseline Ca2+release. We confirmed that replenishment during rest was SERCA-dependent; thapsigargin applied during the refilling interval reduced 10-min recovery to 0.3 ± 1.4% (n = 20). These findings indicate that SERCA function is adequate after neuronal injury to provide recovery to the original baseline level of stores.
Role of Activity in Regulating Ca2+Stores
Ca2+enters sensory neurons predominantly through voltage-gated Ca2+channels during neuronal activity. After SNL, axotomized L5 neurons receive no afferent activity generated from natural stimulation of the receptive field, and this loss may thereby lead to depleted Ca2+stores. We therefore examined whether membrane depolarization with K+, which in turn leads to Ca2+influx across the plasmalemma and store repletion, might increase Ca2+stores in axotomized neurons to the levels of control neurons. Priming transients were generated by 5-s application of 50 mm K+followed 3 min later by sustained application of 20 mm caffeine. Compared to caffeine-induced transients without prior in vitro activation, caffeine-induced transients that followed activation by K+(fig. 6) were larger by 94% in control neurons and by 182% in SNL L4 neurons, whereas activation produced no expansion (0%) of the caffeine-induced transient in axotomized L5 neurons. However, the K+-induced transient in SNL L5 neurons was also smaller (13.0 ± 12.6 × 103nm · s, n = 24) than in control neurons (31.4 ± 27.9 × 103nm · s, n = 40) or SNL L4 neurons (44.1 ± 23.5 × 103nm · s, n = 14), indicating that these groups may have received a greater Ca2+influx and boost in stores than the SNL L5 population. Application of K+lasting only 2 s resulted in K+-induced transients in control neurons (10.2 ± 7.2 × 103nm · s, n = 30) that were comparable in area to those produced by 5 s K+activation in SNL L5 neurons, but these similar Ca2+loads still showed a greater priming effect in control neurons (28.7 ± 20.1 × 103nm · s, n = 29; 58% greater than baseline caffeine-induced transient) than in axotomized L5 neurons (0%). These observations indicate that the deficit in Ca2+stores after injury cannot be repaired by activity, and therefore is not due to neuronal disuse.
Direct Measurement of [Ca2+]L
Pharmacological control of Ca2+release, sequestration, and extrusion is inevitably imperfect, due to incomplete drug specificity and efficacy. We therefore devised a technique for directly measuring Ca2+concentration in the subcellular stores using the low-affinity Ca2+fluorophore mag-Fura-2.31Although this approach defines the concentration of unbound Ca2+in the membrane-delimited Ca2+stores rather than their net mass, it can be assumed that the concentration of free luminal Ca2+is directly related to total stored Ca2+.
We initially determined [Ca2+]Lin nonpermeabilized neurons.41Although such neurons contain mag-Fura-2 in the cytoplasm, the conditions of loading (see Materials and Methods) were optimized to preferentially accumulate mag-Fura-2 in organelle compartments. Under these conditions, calculated [Ca2+]Lwas found to be significantly lower in L4 neurons and especially L5 neurons after SNL compared to control (fig. 7A). Caffeine (20 mm) was applied to the loaded neurons to validate the location of mag-Fura-2 in the stored Ca2+compartment. This produced a decrease of calculated [Ca2+]Lby at least 10% in 107 of 131 (82%) of the neurons, although this may be an underestimation resulting from the influences of caffeine.42If the principal source of fluorescence emission was in fact cytoplasmic rather than compartmentalized mag-Fura-2, release of Ca2+stores would have produced an increase in the calculated [Ca2+] value, so these findings confirm that the fluorophore is preferentially located in the compartment from which Ca2+is released by caffeine.
It has been argued the determination of [Ca2+]Lmay be vitiated by even small amounts of residual mag-Fura-2 in the relatively low Ca2+concentration environment of the cytoplasm.28This view is supported by our observation of [Ca2+]Lin these intact neurons that are lower than the range reported by others.43,44We therefore permeabilized the neurons to provide a more accurate estimation of [Ca2+]L. Using this measurement technique (fig. 7B), [Ca2+]Lmeasurements were higher than in nonpermeabilized neurons. A luminal origin of the signal was confirmed by demonstration of decreased [Ca2+]Lupon ionomycin application (1 μm) in a subset of neurons (fig. 7B), during which [Ca2+]Ldecreased by 42 ± 16% (n = 6, P < 0.01). This incomplete equilibration of Ca2+between stores and cytoplasm may be the result of either an equilibration rate slower than our technique could observe or the existence of subcompartments that ionomycin failed to affect. Other studies using the same ionomycin concentration have shown comparable levels of store depletion.35,45Small neurons again showed a marked decrease after SNL in L5 neurons compared to control (fig. 7C). These [Ca2+]Lfindings, combined with observations on Ca2+release, reveal a deficit of stored Ca2+after injury of sensory neurons.
As has been done in other studies,43,44we included Mg2+in the bath to maintain a natural intracellular concentration20after permeabilization. However, entry of bath Mg2+acting on residual cytoplasmic mag-Fura-2 could contribute to our observed increase of [Ca2+]Lduring permeabilization and bias our determination of [Ca2+]L. This is an unlikely explanation, because this would also cause fluorescence intensities to increase during permeabilization, whereas we observed them to decrease. However, to also test this factor, permeabilization was performed in a set of control neurons using Mg2+-free bath solution, which produced a [Ca2+]L(132 ± 90 μm, n = 28) that was minimally different from experiments in which the bath solution contained Mg2+(105 ± 69 μm, n = 94; P = 0.16).
The somata of sensory neurons are known to be substantially affected by injury of their peripheral axons.46Our current findings extend this recognition by showing that a fundamental feature, the intracellular Ca2+store, is depleted after injury. Specifically, the amount of Ca2+that can be released from stores through a variety of pathways is diminished after axotomy, and the concentration of Ca2+in the luminal compartment is also reduced as estimated by direct imaging.
Intracellular Ca2+stores in neurons reside in several organelles. The best characterized is the ER store, where Ca2+also critically regulates the luminal processes of protein assembly and folding.47,48Additional sites of Ca2+storage include the nuclear envelope49,50and the Golgi apparatus.51The contribution of storage in secretory vesicles52and so-called calciosomes53remains poorly defined. Recent evidence indicates the dominant site for the Ca2+pool is in the ER lumen.7Although subcellular localization may play a role in directing released Ca2+to particular targets,54current techniques do not allow these stores to be functionally distinguished.
We used standard techniques to identify the magnitude of the Ca2+store.11Caffeine freely diffuses through the cell membrane and, by activating the RyR, completely discharges Ca2+from the stores available to this release pathway. Although caffeine interacts with Ca2+-sensitive fluorophores, these effects are minor when using ratiometric indicators such as in the current studies.42,55Stored Ca2+measured this way is affected selectively for specific subgroups of neurons. Axotomy by SNL has no effect on caffeine-releasable Ca2+in large neurons that mostly transduce and transmit low-threshold sensory information. However, axotomy substantially depresses caffeine-releasable stores in the small neuron population, especially in capsaicin-sensitive putative nociceptors. Capsaicin nonresponsive small neurons, which probably represent a population of nonpeptidergic, isolectin B4-binding mechanosensitive nociceptors,37also have depleted stores after injury, but are less affected than capsaicin sensitive neurons. Loss of Ca2+store in only certain neuron subtypes after axotomy may be due to ER damage selectively in these neurons, as is explored in the companion paper.
Sensory neurons possess additional intracellular Ca2+stores that may be released through activation of an IP3-sensitive channel. There is not full agreement about whether the Ca2+pools sensitive to IP3 and caffeine overlap in peripheral sensory neurons,6,56although experiments using sequential activation of these pathways mostly support the view that the pools are one and the same.57,58We did not measure Ca2+release by IP3 because injury of peripheral sensory neurons directly affects IP3 signaling,59,60and membrane permeant or caged IP3 compounds are not readily available. We therefore turned to an alternate strategy of discharging stores indiscriminately either by the Ca2+ionophore ionomycin or by constitutive leak from stores during SERCA inhibition by thapsigargin. Observations using both of these techniques support the findings from caffeine experiments and show an axotomy-induced deficit of Ca2+in stores that is independent of the release mechanism.
The Ca2+transient during release of stores is supplemented by Ca2+influx through SOCCs.26Therefore, a loss of this capacitative Ca2+entry process due to injury could create the appearance of a deficit in stores measured by release. However, we observed that the depressant effect of axotomy on store release persists in Ca2+-free bath solution, so this explanation is unlikely. Also, we considered whether neuronal inactivity might have led to store depletion after axotomy. However, cytoplasmic Ca2+loads provided by activation of voltage-gated Ca2+channels minimally expanded stores in axotomized neurons compared to control neurons. Together, our findings support the conclusion that the capacity of the intracellular Ca2+storage reservoir is compromised after axotomy in sensory neurons.
The amount of stored Ca2+is dictated by the concentration of Ca2+in the store, [Ca2+]L, the bound Ca2+in equilibrium with this free fraction, and the extent of the compartment. Therefore, although mag-Fura-2 microfluorimetry reflects [Ca2+]L, it cannot indicate the total mass of stored Ca2+per se . Although the concentration of the free fraction should be proportionate with the total Ca2+store, this method cannot account for changes in the amount or performance of various Ca2+-binding proteins in the compartment, which could alter also the mass of stored Ca2+without changing [Ca2+]L. Our findings with mag-Fura-2 reveal a decrease in resting [Ca2+]Lassociated with axotomy. Caffeine produces a decrease in [Ca2+]Lmeasured by mag-Fura-2; therefore, it is likely that the mag-Fura-2 technique quantifies the state of the same Ca2+pool as does caffeine-induced release. The combined results of these two approaches point to a model in which axotomy decreases the ability of the storage compartment, predominantly the ER, to concentrate Ca2+within its lumen. This level of [Ca2+]Lis set by the dynamic balance between SERCA and constitutive Ca2+leakage from the ER.10Although resting axotomized neurons show no delay in fractional recovery of Ca2+stores back to their baseline after release, this is nonetheless compatible with compromised SERCA function because baseline store levels are comparatively depleted. We showed that the relationship between constitutive Ca2+leak rate and Ca2+store level is normal in axotomized neurons, which points to a normal leak pathway. This leaves compromised SERCA function as the cause of decreased [Ca2+]L, which could be the result of altered regulation by kinases.41Our data do not reveal whether injury also diminishes the extent of the stores, which is addressed in the companion paper.
The effect of painful conditions on neuronal Ca2+stores has previously been examined only in models of diabetes mellitus. Those studies found that Ca2+transients induced by ionomycin12or caffeine61,62are decreased in diabetic animals, similar to our observations after peripheral nerve trauma. However, a substantially different pathogenic mechanism in diabetes is suggested by differences in other aspects of the Ca2+signaling pathway in sensory neurons, including an increased resting [Ca2+]c12,61,63,64and prolonged activity-induced transients,61,62,64which contrast with decreased resting [Ca2+]cand decreased activity-induced transients after axotomy.1,2,65Like diabetes, painful peripheral inflammation is associated with elevated resting [Ca2+]cand transients in DRG neurons,14but the state of the Ca2+stores in this condition has not been determined.
The SNL model provides the opportunity to separately evaluate neurons in the L5 DRG that are axotomized versus the adjacent L4 neurons. After SNL, features of Ca2+storage and release in these two populations are divergent. In contrast to axotomized L5 neurons, ionomycin release of stored Ca2+is elevated in L4 neurons, although this may represent increased release from the mitochondrial component of stored Ca2+released. The L4 neurons also show an expanded Ca2+storage capacity, as shown by a supranormal level of releasable Ca2+after cytoplasmic Ca2+loading. Finally, whereas axotomy diminishes Ca2+release by caffeine or thapsigargin and decreases [Ca2+]Lmeasured by mag-Fura-2 in permeabilized L5 neurons, these are unaffected in the L4 population. These divergent findings may result from the difference in predominant influences of SNL on the L5 versus L4 neurons. Whereas the SNL results in axotomy of the L5 population, the peripheral axons of the adjacent L4 neurons remain intact but are exposed to inflammatory mediators triggered by Wallerian degeneration of detached L5 axonal segments.15,16,66Other studies have shown divergent influences on Ca2+handling in inflammation compared to axotomy. Specifically, models of inflammatory pain reveal increased activity-induced Ca2+transients in DRG neurons,14whereas the opposite occurs after axotomy.2Our current findings also suggest that Ca2+stores are also differentially regulated by axotomy and inflammation. A degree of inconsistency in our L4 findings may be attributable to a variable admixture of axotomy among the L4 neuronal population following L5 SNL.67
Disruption of the level of Ca2+stores in neuronal subcellular compartments is a fundamental pathogenic factor in a variety of diseases, including ischemia, acquired immunodeficiency syndrome, and Alzheimer disease.6In addition to storing Ca2+, the ER is the site assembly and posttranslational modification of proteins by glycosylation and folding. Depletion of luminal Ca2+triggers highly conserved stress responses involving accumulation of unfolded protein, global suppression of protein synthesis, and activation of a variety of transcription factors, resulting in neuronal dysfunction and death.68For instance, Ca2+store depletion by caffeine or thapsigargin exposure suppresses protein synthesis and induces neuronal apoptosis independent of increased [Ca2+]c.69,70The current findings suggest that similar processes may take place in sensory neurons after peripheral nerve injury and that they may contribute to long-term changes associated with chronic pain.