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Painful nerve injury leads to disrupted Ca2+ signaling in primary sensory neurons, including decreased endoplasmic reticulum (ER) Ca2+ storage. The present study examines potential causes and functional consequences of Ca2+ store limitation after injury.
Neurons were dissociated from axotomized fifth lumbar (L5) and the adjacent L4 dorsal root ganglia following L5 spinal nerve ligation that produced hyperalgesia, and were compared to neurons from control animals. Intracellular Ca2+ levels were measured with Fura-2 microfluorometry, and ER was labeled with probes or antibodies. Ultrastructural morphology was analyzed by electron microscopy of nondissociated dorsal root ganglia, and intracellular electrophysiological recordings were obtained from intact ganglia.
Live neuron staining with BODIPY FL-X thapsigargin (Invitrogen, Carlsbad, CA) revealed a 40% decrease in sarco-endoplasmic reticulum Ca2+-ATPase binding in axotomized L5 neurons and a 34% decrease in L4 neurons. Immunocytochemical labeling for the ER Ca2+-binding protein calreticulin was unaffected by injury. Total length of ER profiles in electron micrographs was reduced by 53% in small axotomized L5 neurons, but increased in L4 neurons. Cisternal stacks of ER and aggregation of ribosomes occurred less frequently in axotomized neurons. Ca2+-induced Ca2+ release, examined by microfluorometry with dantrolene, was eliminated in axotomized neurons. Pharmacologic blockade of Ca2+-induced Ca2+ release with dantrolene produced hyperexcitability in control neurons, confirming its functional importance.
After axotomy, ER Ca2+ stores are reduced by anatomic loss and possibly diminished sarco-endoplasmic reticulum Ca2+-ATPase. The resulting disruption of Ca2+-induced Ca2+ release and protein synthesis may contribute to the generation of neuropathic pain.
We have previously shown that disordered Ca2+ signaling contributes to the development of neuronal hyperexcitability and pain behavior following peripheral nerve injury.1–3 In the accompanying paper,4 we identified a deficit in Ca2+ stores in sensory neurons of the dorsal root ganglion (DRG) following axotomy. While a variety of organelles maintain Ca2+ storage pools, including the nuclear membrane, Golgi apparatus, and secretory vesicles,5–7 the endoplasmic reticulum (ER) contains the dominant storage pool available for release into the cytoplasm,8 and we will hereafter refer to the storage pool globally as the ER. Luminal Ca2+ in the ER regulates cellular protein synthesis through modulation of peptide assembly and protein folding, such that depletion of stored Ca2+ halts protein synthesis and leads to accumulation of unfolded proteins. This dependency of neuronal function upon ER Ca2+ stores provides a strong impetus for more complete understanding of injury-related processes leading to the loss of stores and the functional consequences of store depletion.
By direct microfluorimetric measurement with mag-Fura-2, we identified a diminished luminal Ca2+ concentration ([Ca2+]L) in the ER, probably on the basis of deficient function of the sarcoplasmic-ER Ca2+ ATPase (SERCA) that loads Ca2+ into the ER. While this alone may explain the decrease in releasable Ca2+ that we also observed, the storage capacity of the ER Ca2+ may also be limited by a diminished anatomical extent of the ER compartment. The ER is a dynamic structure9 and is reduced after neuronal trauma.10,11 Accordingly, we examined the hypothesis that peripheral injury of sensory neurons is associated with a loss of ER.
The activity-related Ca2+ signal in sensory neurons is initiated by Ca2+ entry through voltage-gated Ca2+ channels positioned in the plasma membrane (plasmalemma). Injury reduces this influx,12–14 and thereby depresses the sustained rise in cytoplasmic Ca2+ concentration ([Ca2+]c) that follows neuronal activity,3 often referred to as the Ca2+ transient. However, the signal initiated by Ca2+ entry is thereafter modulated by multiple intracellular processes that have not been examined as possible components of the pathogenesis of neuropathic pain. On the one hand, the transient is buffered and ultimately terminated by extrusion of Ca2+ from the cell and sequestration of Ca2+ into subcellular organelles, including the ER and mitochondria.15–17 Alternately, the ER Ca2+ stores may serve as a source that magnifies the transient by release of Ca2+ through the action of cytoplasmic Ca2+ upon the ryanodine receptors (RyRs) in the ER membrane, a process known as Ca2+-induced Ca2+ release (CICR). This discharge of calcium from the ER stores critically regulates neurotransmission, gene expression, and neuronal excitability,18 so we have additionally examined the influence of nerve injury on CICR in the present investigation. We have previously identified elevated excitability following axotomy of sensory neurons by spinal nerve ligation (SNL),19 which is associated with increased pain behavior.20 Since Ca2+ released through CICR in part generates the membrane afterhyperpolarization in other neuronal types,18 and since firing rate and pattern in DRG neurons is regulated by the afterhyperpolarization,21 we investigated the potential link between CICR and electrophysiological excitability in DRG neurons.
All methods and use of animals was approved by the Medical College of Wisconsin (Milwaukee, Wisconsin) Institutional Animal Care and Use Committee.
Male Sprague-Dawley rats weighing 160 to 180g (Taconic Farms Inc., Hudson, NY) were subjected to spinal nerve ligation in a manner derived from the original technique.22 Rats were anesthetized with 2% isoflurane in oxygen and the right paravertebral region was exposed. After removal of the 6th lumbar (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 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 lib.
Rats underwent sensory testing for hyperalgesic behavior on three different days between 10 d and 17 d after surgery, as previously described.2,20 Briefly, right plantar skin was mechanically stimulated with a 22G 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 incorporates sustained licking, chewing, grooming and elevation of the paw. The frequency of hyperalgesia responses was tabulated for each rat.
The L4 and L5 dorsal DRGs were excised after isoflurane anesthesia and decapitation 21 to 27 days after SNL or skin sham surgery, at which time hyperalgesia is fully developed.20 DRGs 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’s modified Eagle’s medium/F12 with glutaMAX (Invitrogen) for 1.5 hours, centrifuged and triturated with fire-polished pipettes in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5mM glutamine, 100ng/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, 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.
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. Agents were obtained as follows: dantrolene and dimethylsulfoxide from Sigma Aldrich, BODIPY FL-X Thapsigargin (BODIPY-TG), ER-Tracker Blue-White DPX, Fura-2-AM, ionomycin, and Pluronic F-127 from Invitrogen. Stock solutions of ionomycin, dantrolene and 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 recording chamber was superfused by a Tyrode’s solution (3ml/min) and agents were delivered by directed microperfusion through a 500 µm diameter hollow quartz fiber 300 µm upstream from the neurons.
Similar to our previously described technique,2,3 coverslips carrying the plated neurons were dye-loaded in Tyrode’s solution containing Fura-2-AM (5 µM) and Pluronic F-127 (0.04%) for 30 minutes, and left in regular Tyrode’s for deesterification for an additional 30 min, after which coverslips were mounted onto the recording chamber. Images were collected at 510nm using a cooled 12-bit digital camera (Coolsnap fx, Photometrics, Tucson, AZ), and excitation wavelengths were 340 and 380 nm. Recordings from each neuron were obtained as separate regions of interest (MetaFluor, Molecular Devices, Downingtown, PA). The [Ca2+]c was estimated by the formula [Ca2+]c = Kd•β• (R–Rmin)/(Rmax–R) where β = (I380max)/(I380min) and R = (I340)/(I380). Values of Rmin, Rmax and β were determined by in situ calibrations and were 0.38, 8.49 and 9.54, whereas 224 nM was used as Kd.23
Live, dissociated neurons were exposed to 1µM ER-Tracker Blue-White DPX in Tyrode’s for 30 min. After washing, images were acquired using an inverted microscope (Diaphot 200, Nikon Instruments, Melville, NY) with a 20x objective and a 12-bit digital camera (Coolsnap fx, Photometrics). Excitation was achieved with a 150W Xenon lamp (Lambda DG-4, Sutter, Novato, CA) at 380 nm and images were collected at 510 nm.
After 2 hours in culture, dissociated neurons were fixed in buffer containing 4% formaldehyde, 4% sucrose and 0.2% in phosphate buffered saline for 5 min and were permeabilized in methanol at −20°C for 10 min. After three washes in phosphate buffered saline with 0.2% glycine, neurons were incubated in gelatin diphosphate buffer, which contained 2% gelatin, 20% Triton X-100, 0.2 M phosphate buffer and 5 M NaCl in dH2O, with 8% normal goat serum for 1 hour. The coverslips were then incubated with a polyclonal rabbit calreticulin antibody (Abcam, Cambridge, MA) at 1:100 and 4% normal goat serum in gelatin diphosphate buffer overnight at 4°C. After washing with phosphate buffered saline they were incubated with a Texas-red goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight at 4°C. Following three rinses with phosphate buffered saline and mounting on slides, they were imaged at 600x on an Eclipse confocal microscope (Nikon Instruments, Melville, NY). For intensity analysis, a representative region of the cytoplasm, excluding the nucleus and the plasmalemma, was selected in each neuron for intensity measurement (MetaMorph, Molecular Devices). Neurons that showed sign of lysis or crenulation or were overlapped by glia were excluded by brightfield examination.
Dissociated neurons were stained with BODIPY-TG while still in culture. Coverslips were incubated with 1 µM of the dye in Tyrode’s for 5 min. The medium was changed to dye-free Tyrode’s and slips were immediately imaged on an inverted microscope (Diaphot 200, Nikon). Excitation was achieved with a 150W Xenon lamp (Lambda DG-4, Sutter) at 495nm using a fluorescein isothiocyanate filter cube (Nikon), images were acquired at 510 nm with a 12-bit digital camera (Coolsnap fx, Photometrics) and a 20x objective. Intensities were measured using a procedure similar to the one described above.
DRGs were harvested from control (L5 DRG from both sides, 19 d after skin incision surgery) and SNL rats (L4 and L5 DRGs from two rats, 19 d and 26 d after SNL), fixed for 1 hr in 2.5% glutaraldehyde in 0.1 M cacodylate buffer and post-fixed with 1% OsO4 in buffer for 1hr. Specimens were then washed with distilled water (2×5 min); dehydrated in methanol (50%, 70%, 85%, 95% and 3× 100%) followed by 2× 10-min acetonitrile; infiltrated with Epon 812 resin (Shell Chemical, Houston, TX) in acetonitrile 1:1 for 1hr followed by 100% Epon for 3 hrs; moved to fresh 100% Epon and polymerized at 70°C overnight. Sections 70 nm thick were cut and stained with saturated uranyl acetate in 50% ethanol and Reynold’s lead citrate. Images were generated from 6 cells in each DRG (3 larger and 3 smaller than 1000 µm2), using a transmission electron microscope (JEM2100, Japanese Electron Optics Limited, Tokyo, Japan) with a high resolution charge-coupled device digital camera (Ultrascan 1000, Gatan Inc., Pleasanton, CA). Each cell had 2 perinuclear and 2 peri-plasmalemmal fields (5.4 × 5.4 µm, and area of 29.2 µm2) imaged at 4,000x magnification, chosen to be distributed in four separate quadrants of the neuron in order to provide an unbiased sample of cytoplasmic contents. In each of the 144 images, the length of each ER profile was measured in a blinded fashion using an image analysis program (ImageJ, NIH, Bethesda, MD) to trace its running length. Only those profiles that had a typical tubular pattern with clear walls and a terminus indicating a closed compartment were included. The Golgi apparatus was easily distinguished by its characteristic curved series of flattened saccules that are expanded at their ends, and were not included in measurements. Thereafter, the perinuclear data for each neuron were pooled, as were the peri-plasmalemmal data.
DRGs were harvested from control rats weighing 200–250 g during anesthesia with isoflurane (2–3% in oxygen). A laminectomy was performed up to the sixth thoracic level while the surgical field was perfused with artificial cerebrospinal fluid (in mM: NaCl 128, KCl 3.5, MgCl2 1.2, CaCl2 2.3, NaH2PO4 1.2, NaHCO3 24.0, glucose 11.0) bubbled with 5% CO2 and 95% O2 to maintain a pH of 7.35. The L4 and L5 DRGs and attached dorsal roots were removed and the connective tissue capsule dissected away from the ganglia under 20x magnification. Ganglia were transferred to a glass-bottomed recording chamber and perfused with 35°C artificial cerebrospinal fluid. The proximal cut end of dorsal roots was placed on a pair of platinum wire stimulating electrodes. DRG neurons were viewed using an upright microscope equipped with differential interference contrast optics and infrared illumination. Neuronal soma diameter was determined with the focal plane adjusted to reveal the maximum somatic area, which was measured using a calibrated video image.
Intracellular recordings were performed with microelectrodes fashioned from borosilicate glass (1 mm OD, 0.5 mm ID, with Omega fiber – FHC Inc., Bowdoin, ME) using a programmable micropipette puller (P-97, Sutter Instrument Co., Novato, CA). Microelectrode resistances were 80–120 MΩ when filled with 2M potassium acetate. DRG neurons were impaled under direct vision and data were acquired after stable recordings were achieved. Membrane potential was recorded using an active bridge amplifier (Axoclamp 2B, Axon Instruments, Foster City, CA), except in protocols in which depolarizing current was passed through the recording electrode, for which we used discontinuous current clamp recording mode, during which the switching frequency was 2 kHz, and full settling of the electrode charge was confirmed. Currents were filtered at 1 kHz (discontinuous mode) and 10 kHz (bridge mode), and then digitized at 10 kHz (discontinuous mode) or 40 kHz (bridge mode; Digidata 1322A and Axograph 4.9, Axon Instruments) for data acquisition and analysis. Somatic action potentials (APs) were elicited by natural conduction from the site of dorsal root stimulation with square-wave pulses of up to 90 mA lasting 0.06 mS. To determine the rheobase current threshold for initiating an AP, and to identify the firing pattern during sustained membrane depolarization, current was injected directly into the neuronal soma through the recording electrode.
Criteria for inclusion of data were a resting membrane potential (RMP) negative to –5 0mV, and an AP amplitude greater than 40 mV. Using techniques described previously,19 AP measures including RMP, conduction velocity (CV), AP amplitude and duration, and afterhyperpolarization amplitude, duration and area were obtained from single traces after consistency of dimensions was confirmed by comparison to 10 sequential APs. Input resistance was calculated from the shift of RMP generated by a 100 ms injection of a hyperpolarizing current through the recording electrode, using a current amplitude (0.2–0.5 nA) that failed to show any time-dependent rectification.24 Rheobase was determined as the minimum current able to elicit an AP during incremental depolarizing current injection of 0.5–10 nA for 100 mS. The pattern of impulse generation during sustained depolarization was determined during incremental depolarizing current steps to twice rheobase, at which neurons either continued to produce single APs or fired repetitively. The influence of dantrolene upon AP firing pattern was determined at the depolarizing voltage that first produced a drug-induced difference in the number of APs generated, which is a sensitive indicator of drug effect upon excitability.
The following frequency is the fastest rate at which the neuronal soma may be excited by APs conducted along the axon, and represents the degree of afferent signal filtering by the impedance mismatch where the distal and proximal axons meet the T-branch that leads to the soma.25 Trains of 20 axonal stimuli were presented with 3 s intervals between trains, each train having progressively greater stimulation frequency. The maximal at which each stimulus in the train produced a full somatic AP was considered the somatic following frequency.
Classification of neurons24 used CV, measured by dividing the distance between stimulation and recording sites by the conduction latency. Neurons with dorsal root CV<1.5 m/s were considered C-type, neurons with CV>15 m/s were considered Aα/β-type, which are putative low-threshold receptor modality.26 Neurons with CV>1.5 m/s but CV<10 m/s were considered Aδ-type, which are putative nociceptor modality. For neurons with CV between 10 and 15 m/S, long AP duration was used to categorize the neurons as Aδ-types.24
Dantrolene was dissolved in bath solution and was delivered by microperfusion from a pipette with a 4 µm diameter tip positioned 100 µm from the impaled neuron. Preliminary experiments indicated an effective 4-fold dilution of pipette solution into the bath at the neuronal membrane, so the dantrolene concentration was approximately 25 µM at the DRG surface. Preliminary experiments showed a full onset of action within 3 min.
Statistical analyses were performed with Statistica (StatSoft, Tulsa, OK). Two-tailed Student´s t-tests were used to compare means. One-way ANOVA with Bonferroni’s post-hoc testing was employed to detect the influence of injury group on measured parameters. Quantifications of features in the analysis of EM images were treated as nonparametric data and therefore the Kruskal-Wallis test followed by multiple comparisons of mean ranks was used. Correlation between two observers was determined by linear regression. Paired, one-tailed t-tests were used in the analyses of the intracellular recordings, since we only compared between injury and control conditions and a hypothesis for the direction of the changes already existed. Results are reported as mean ± SD. A P<0.05 was considered significant.
Upon needle stimulation, all SNL animals (n=14) displayed a hyperalgesia response rate that was greater than 25% and averaged 42 ± 19%, whereas control animals (n=27) showed a hyperalgesic response 0 ± 0% (P<0.001).
We first characterized the distribution of ER with the selective dye ER-Tracker Blue-White DPX, which has the property of localizing to the membrane of ER through an unexplained mechanism. Dissociated, live neurons stained with ER-Tracker (1µM, 30 min, fig. 1A) showed distribution of the dye throughout the cytoplasm without nuclear staining without any differences between injured and control neurons.
In order to gain more consistent quantification of the ER, an immunocytochemical approach was used targeting the Ca2+ binding protein calreticulin, which functions as the dominant binding protein regulating the mass of stored Ca2+. It is largely excluded from the cytoplasm but does appear in the cell membrane in addition to the ER,27 so we avoided including this binding in our measure by quantifying signal only in cytoplasmic areas of confocal images. Staining with the calreticulin antibody (fig. 1B) was distributed comparably to ER-Tracker, and demonstrated no quantitative differences between groups (fig. 1C).
The specificity of the SERCA blocker TG may be used for fluorescent quantification of binding sites by conjugation to the fluorescein-like dye BODIPY FL-X. Staining of live neurons (fig. 1D, E) was distributed comparably to ER-Tracker, and showed fluorescence intensities that were decreased by 34% in L4 neurons and by 40% in L5 neurons compared to control neurons (fig. 1F). This finding is compatible with either a lower density of SERCA in the ER membrane of injured neurons, or alternatively less total ER.
A possible explanation for both diminished BODIPY-TG staining and loss of releasable Ca2+ stores after neuronal injury4 is loss of the structure that contains the stored Ca2+, which is predominantly ER.8,17 Accordingly, we examined dimensions of the ER in electron microscopy (EM) images of injured and control neurons categorized into large (area 1438 ± 295 µm2, n=16) and small (area 495 ± 185 µm2, n=16) types. Since injury in other neurons has been reported to lead to preferential ER displacement or loss in the perinuclear region,28 we examined perinuclear and peri-plasmalemmal areas separately. EM images displayed profiles that were readily identified as ER (fig. 2). There was a preponderance of smooth variety,28,29 with only relatively small segments endowed with ribosomes, as has been described in normal DRG neurons before.10,30 Quantification of linear dimensions (table 1) showed that the summated total length of ER segments is decreased in axotomized small neurons, with changes evident in both perinuclear and peri-plasmalemmal regions. In contrast, total ER increased in L4 neurons after SNL in both small and large neurons. This was attributable to increases in both cellular subregions in the large neurons, but selectively to an increase in the peri-plasmalemmal area in small neurons.
Initial examination of the EM images suggested that ER profiles in axotomized SNL L5 neurons were typically shorter, as has been reported.11,31 Therefore, we additionally evaluated the effect of injury on the lengths of each segment of ER represented by a discontinuous profile in the different groups in a blinded fashion (table 1). While median segment lengths were unaffected in large neurons, small neurons developed shorter ER profiles (fig. 2) after axotomy in both subcellular regions, and SNL L4 neurons showed longer profiles in the peri-plasmalemmal region.
The neuronal ER system has been shown to be organized into functionally and anatomically distinct elements, and cisternae that stack as plates may play a special role in Ca2+ management.32,33 We therefore quantified these specialized structures by grading each micrographic field as having no such cisternal stacks (score 0), minimal stack formation (one group with least 4 parallel ER profiles, score 1), clear stack formation with multiple groups (score 2), or extensive formation of stacks that fill at least one quarter of the field (score 3). Scores assigned separately by two blinded observers correlated at R=0.75 (P<0.001). Although others have observed preferential development of these structures in the peri-plasmalemmal region,34 we did not identify any preferential subcellular localization in control neurons, so data was combined for perinuclear and peri-plasmalemmal regions. Analysis of large and small neurons separately by Kruskall-Wallis ANOVA by ranks showed no effect of injury in large neurons (fig. 3A), but near total elimination of cisternal stacks in axotomized small neurons in the L5 DRG after SNL (fig. 2, ,3A),3A), which was significantly different from both control and L4 neurons (multiple comparisons of mean ranks).
The assembly of ribosomes into rosette-like polyribosomes (polysomes) takes place upon the initiation of peptide assembly, and loss of polysomes is a marker of interrupted protein synthesis. As a comparative index of protein synthesis,35 we characterized the relative abundance of polysomes in each EM field as having minimal or no polysomes (score 0), an approximately even presentation of ribosomes as free or assembled into rosettes (score 1), or predominantly polysomal rosettes (score 2). Scores assigned separately by two blinded observers correlated at R=0.55 (P<0.001). There was no influence of neuronal size or location within the cell. However, axotomy reduced the level of aggregation in both large (fig. 3B) and small neurons (fig. 2, ,3B)3B) in the L5 group compared to both the control and L4 neurons (Kruskall-Wallis ANOVA by ranks, multiple comparisons of mean ranks).
A substantial portion of the transient rise in [Ca2+]c triggered by neuronal activation is due to influx through voltage-gated Ca2+ channels. However, additional Ca2+ is released during neuronal depolarization through the activation of CICR. We quantified this component by blocking CICR with dantrolene,36 which acts directly and selectively on the RyR to decrease its sensitivity of Ca2+ and thereby inhibit channel activation.37 We first confirmed blockade of RyRs in sensory neurons by showing that prior application of dantrolene (10 µM, 10 min) reduced caffeine-induced transient area to 6% (1016 ± 819 nM•s, n=40) of control transients. Application of dantrolene (10 µM) for 10 min (fig. 4A) reduced K+-evoked transient area by 39% compared to a previous baseline transient (fig. 4B). In SNL L4 neurons, the area decreased by 26% only, whereas in SNL L5 neurons the effect of dantrolene was eliminated (−1%). Additional determination of transient amplitude confirmed a diminished inhibition by dantrolene in L5 neurons after SNL (−19 ± 15%, n=21; P<0.001 vs. C and L4) compared to SNL L4 (−36 ± 11%, n=12) and control neurons (−41 ± 9%, n=16). Transient slope also showed diminished inhibition by dantrolene in L5 neurons after SNL (−17 ± 24%, n=21; P<0.05 vs. C) compared control neurons (−37 ± 18%, n=16), with no effect on SNL L4 (−34 ± 15%, n=12). Together, these findings point to a complete elimination of CICR in axotomized neurons.
To determine the functional consequences of loss of CICR during neuronal activity, we investigated the effects of application of dantrolene during electrical activation of neurons in intact DRGs for neurons of different CV categories. Dantrolene, at an approximate concentration of 25µM at the DRG surface, decreased afterhyperpolarization duration in both Aα/β and Aδ neuronal populations and decreased the afterhyperpolarization area in the Aδ neurons (fig. 5A, table 2). Because the late afterhyperpolarization is particularly dependent upon Ca2+ released by CICR,38,39 we measured the afterhyperpolarization amplitude at 350 and 1,350 ms following the onset of the AP. In Aα/β neurons, the late afterhyperpolarization amplitude was not affected by dantrolene, whereas the late afterhyperpolarization was diminished in Aδ neurons. Dantrolene also increased the firing rate during membrane depolarization in both neuron types (fig. 5B, table 3), and accelerated the following frequency in Aδ neurons (fig. 5C). At baseline, 6/13 Aα/β and 5/13 Aδ neurons generated repetitive APs during sustained depolarization beyond rheobase. Separate consideration of these nonaccommodating neurons showed that administration of dantrolene shifted the relationship between firing rate and degree of depolarization in all of these neurons such that repetitive firing started at a lower depolarization voltage and/or the firing rate increased at each voltage. An additional Aα/β neuron that fired only singly at baseline also developed repetitive firing during dantrolene. At the depolarizing voltage that first produced a dantrolene-induced difference in the number of APs, the firing rate among nonaccommodating neurons was increased by dantrolene in both Aα/β neurons (from 2.7 ± 4.1 100 ms−1 at baseline to 4.9 ± 3.9 100 ms−1 during dantrolene, n=7; P<0.01) and Aδ neurons (from 1.4 ± 0.9 100 ms−1 at baseline to 3.2 ± 1.3 100 ms−1 during dantrolene, n=5; P<0.01).
Similar recordings were made from nonmyelinated C-type neurons that were recognized by their CV of less than 1.5 m/s. In this population, application of dantrolene at a concentration of approximately 25 µM significantly (P<0.001) depolarized the RMP by 12.6 ± 9.5 mV, in contrast to the lack of effect on RMP in Aα/β (P<0.001) and Aδ (P<0.01) neurons. Full AP generation was lost concurrent with this depolarization in 7 of 9 neurons, which was due to the effect of depolarization inactivating voltage-gated Na+ channels, as repolarization by injection of conditioning current through the recording pipette resulted in return of the AP (fig. 6). This return of the AP with repolarization alone is consistent with a selective action of dantrolene on ryanodine receptors rather than voltage-gated conductances.40 Because of the greater sensitivity of C-type neurons to dantrolene, we decreased the applied concentration to approximately 12 µM. In this group of experiments, depolarization was less (7.7 ± 8.1 mV). For further analysis, the influence of dantrolene on excitability was isolated from its effect upon RMP by using conditioning current to repolarize RMP in 4 C-type neurons.
Dantrolene prolonged the AP duration in C-type neurons (table 3), possibly due to the loss of Ca2+-activated K+ currents that contribute to repolarization.41 Dantrolene also decreased afterhyperpolarization duration and area (table 2). The late afterhyperpolarization amplitude at baseline was greater in C-type neurons than in both the Aα/β and Aδ neurons, and the inhibition of the late afterhyperpolarization by dantrolene was greater than in the other neuronal groups, indicating a greater contribution of CICR in the C-type neurons. At baseline, only 1 of 12 C-type neurons generated repetitive APs during sustained depolarization beyond rheobase, but during application of dantrolene, 6 additional neurons fired repetitively during depolarization. At the depolarizing voltage that first produced a dantrolene-induced difference in the number of APs, the firing rate of these nonaccommodating neurons was increased by dantrolene (from 1.3 ± 0.8 100 ms−1 at baseline to 2.7 ± 1.1 100 ms−1 during dantrolene, n=7; P<0.001). Following frequency was not tested in C-type neurons as this category of neuron is capable of following only very slow and highly variable frequencies.
Although Ca2+ signaling regulates nearly all critical neuronal functions, there has been only minimal exploration of the influence of painful nerve injury on Ca2+ stores in sensory neurons. The investigations reported here extend our findings, reported in the accompanying paper,4 of depleted Ca2+ stores and depressed [Ca2+]L after axotomy. In the present study, we have found that injury diminishes the anatomical extent of ER and reduces levels of SERCA expression, both of which may contribute to diminished stores. Our data also demonstrate substantial functional impairment attributable to this loss of Ca2+ stores. Specifically, we have observed direct evidence of injury-induced loss of CICR that regulates neuronal excitability, and indirect evidence of disruption of protein synthesis.
Calreticulin is the dominant buffer within the ER27,42 and is distributed uniformly throughout neuronal ER.43 Its role in regulating the size of the Ca2+ pool is demonstrated by expansion of releasable Ca2+ stores in cells overexpressing calreticulin.43 Each calreticulin molecule possesses up to 50 binding sites for Ca2+, thus accommodating a large amount of Ca2+, while its low affinity permits the generation of high [Ca2+]L that are necessary for rapid Ca2+ release and facilitates longitudinal transport of Ca2+ within the ER lumen.17 We examined calreticulin levels by semiquantitative immunocytochemistry rather than Western blotting in order to avoid contamination by the numerous glial cells in the DRG. The lack of difference in injured neurons that we observed probably indicates that calreticulin is not the pathway through which injury influences Ca2+ stores.
The most direct approach for measuring the anatomic extent of the ER is morphometric analysis at the ultrastructural level by EM. By this means, we have identified a reduction of total ER length in small neurons after axotomy, which may in part account for reduced ability to store Ca2+. A potential link between stored Ca2+ mass and ER dimensions is supported by two observations. First, a deficit in both of these was found selectively only in small neurons. Second, the extent of these deficits is comparable, with 53% loss in total ER length per neuron and a loss of releasable Ca2+ ranging from 25% to 59% for the various techniques used in the accompanying paper.4 In addition to the total ER length per neuron, the length of individual profiles of ER segments is also diminished in axotomized small neurons. This may represent fragmentation of the ER tubules into disconnected segments. Alternatively, this appearance may result from increased tortuosity of the ER components, which then enter and leave the plane of the section, producing shorter ER profiles. Our data cannot resolve between these two possibilities, but fragmentation would restrict the coordinated function and signal distribution role of the ER.18 The finding of undiminished total cellular calreticulin is consistent with either an unchanged ER volume, or the calreticulin is concentrated in a smaller total ER volume.
Although the most prominent findings are in the axotomized neurons of the L5 DRG after SNL, certain distinct features appear in the adjacent L4 neurons. Total ER length is increased in both large and small L4 neurons, in contrast to the decrease seen in small L5 neurons. This may explain our finding in the accompanying paper of a greater capacity for expanding the Ca2+ stores by neuronal activation in L4 neurons than in control neurons, whereas the opposite occurs in L5 neurons.4
A striking contrast is evident between axotomized and control small neurons in the organization of the ER elements. After axotomy, there is almost complete elimination of the formation of ER into stacks of cisternae. Calcium mapping by electron energy loss spectroscopy has identified these cisternae as regions in the ER system with particularly high levels of Ca2+.44 Thus, selective elimination after injury could disproportionately depress levels of releasable Ca2+.
In addition to dissolution of ER elements, our findings show depression of BODIPY-TG binding after axotomy. This agent identifies SERCA and thus indicates a decrease in the cellular density of SERCA pumps that generate Ca2+ influx into the ER from the cytoplasm. This finding supports our conclusion in the accompanying paper that compromised SERCA function contributes to depressed [Ca2+]L after axotomy.4 However, the concordance of the extent of loss of BODIPY-TG binding sites (40% in axotomized neurons compared to control) and the loss of ER (50%) suggests that the loss of SERCA may be simply due to net loss of ER membrane. This proportionality would predict that the density of SERCA in the ER membrane is only minimally changed by injury, in which case depressed [Ca2+]L after axotomy cannot be attributed to loss of SERCA. Our data are not adequate to resolve this mechanistic difference, and we also note that BODIPY-TG staining decreases in L4 neurons whereas [Ca2+]L does not.4 While we cannot therefore explain the decrease in [Ca2+]L after axotomy, our data point to this feature plus the loss of anatomic ER as combined causes of decreased Ca2+ stores after axotomy.
Our study also examined key functional consequences that might follow from a loss of Ca2+ stores in neurons. While published evidence is not conclusive for all neuronal types, there is a consensus that activation of peripheral sensory neurons is accompanied by CICR.17 It is expected that injury-induced loss of stores should compromise the ability of CICR to amplify the Ca2+ signal initiated by neuronal activation. Our findings confirm that this is the case. The amount of CICR in dissociated neurons was gauged by sensitivity to blockade with the selective RyR blocker dantrolene, which revealed elimination of CICR after axotomy. This complete loss of functional CICR despite an only partial loss of Ca2+ stores and ER total length per neuron may indicate a disproportionate dependence of CICR on Ca2+ residing in the cisternal stack pool, which is also nearly eliminated by injury. The close stacking of adjacent ER components in cisternal stacks is thought to provide cooperative interactions that trigger regenerative Ca2+ release,42 which may be particularly important for CICR.
Since CICR amplification of the Ca2+ signal during firing of APs contributes to triggering currents that underlie the afterhyperpolarization, 45,46 we suspected that the loss of CICR in injured neurons might contribute to their hyperactivity, as has been shown in other tissues.39,47 We modeled this in intact DRGs by blocking CICR in control neurons with dantrolene, and observed decreased afterhyperpolarization and increased neuronal excitability. The greater sensitivity of C-type presumed nociceptive neurons is likely due to their greater expression of the low conductance SK isoform of Ca2+-activated K+ channels,48 which are particularly dependent upon Ca2+ derived from CICR for activation.38,49 The dominant component of cytoplasmic Ca2+ transient that accompanies an AP is derived from entry through voltage-gated Ca2+ channels, as is demonstrated by the persistence of the majority of the transient after RyR blockade with dantrolene. However, the loss of the CICR component clearly increases neuronal excitability during dantrolene application, so it is likely that a similar loss of CICR in axotomized L5 neurons, particularly in nociceptors, may lead to excess neuronal excitability after injury as well. By this means, loss of Ca2+ stores in sensory neurons may contribute to the generation of pain after peripheral nerve injury. This speculation is supported by the ability of dantrolene to replicate injury effects19, specifically reduction of afterhyperpolarization dimensions and increased repetitive firing.
Apart from providing a source for Ca2+ release, it is now recognized that maintenance of normal Ca2+ levels in the ER lumen is required for normal synthesis and maturation of proteins, including the processes of translational initiation, glycosylation, and protein folding.50 It is particularly the RyR-sensative Ca2+ pool that regulates these functions in neurons.51 Initiation of translation and peptide assembly is associated with aggregation of ribosomes into clusters (rosettes), whereas disaggregation is a hallmark of impaired translational initiation and arrest of protein synthesis.52 Others have also observed ribosomal disaggregation as an early response to axotomy in other neuronal tissues,10,11,53 and we now report the phenomenon after spinal nerve axotomy. Various proteins show decreased expression after peripheral sensory neuron axotomy.54,55 Since substantial selective modulation of gene expression after injury occurs at the transcription level, 56–58 it is likely that additional translational and posttranslational influences result from depletion of Ca2+ in the ER.
Our data do not offer direct insights into mechanisms that may produce the ultrastructural findings we have reported. However, these changes show morphological similarities with the process of apoptosis, which occurs in adult DRG neurons within one week of axotomy, peaks in 2 months, and continues as late as 6 months.59,60 Comparable to our findings in DRG neurons, apoptosis in cultured sympathetic neurons diminishes the extent of ER.61 In forebrain neurons, various apoptotic models, as well as natural apoptosis during brain development, produce fragmentation of the ER membrane and disaggregation of polysomes.62 The cause for apoptosis following axotomy is not known, but several possibilities are suggested by prior findings. Although dependence of DRG neurons upon neurotrophic factors for survival diminishes with postnatal age, axotomy-induced apoptosis is inhibited by glial-derived neurotrophic factor, 63 and glial-derived neurotrophic factor reverses sensory changes.64 Alternatively, activation of glia and production of cytokines following nerve injury may initiate DRG neuron apoptosis,65 and depletion of Ca2+ stores may lead directly to apoptosis in neurons.66 The contribution of apoptosis to generation of chronic pain after peripheral nerve injury is shown by prevention of delayed pain through suppression of DRG neuron apoptosis with the hematopoietic cytokine erythropoietin n.67
Prevention or correction of ER Ca2+ depletion may be a novel target for treatment for chronic pain following nerve injury. For instance, various proteins such as Bcl-2, grp78, and grp94 prevent ER Ca2+ depletion and subsequent cytotoxicity,68–70 and upregulate SERCA function.71 Molecular techniques may become available to overexpress these proteins in DRG neurons as a new therapy for chronic neuropathic pain.
The authors express their gratitude for the expert assistance of Clive Wells, M.I.Biol., C.Biol., Electron Microscopy Program Manager, Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin.
Funding: a) grant NS-42150 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland to QH; b) Erwin Schroedinger Fellowship from the Austrian Science Fund, Vienna, Austria (project J2695) to MR.
Summary Statement: Anatomic loss of endoplasmic reticulum contributes to diminished Ca2+ stores in injured primary sensory neurons, causing diminished Ca2+-induced Ca2+ release and neuronal hyperexcitability.