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Meeting Abstracts  |   June 2005
Painful Nerve Injury Decreases Resting Cytosolic Calcium Concentrations in Sensory Neurons of Rats
Author Affiliations & Notes
  • Andreas Fuchs, M.D.
    *
  • Philipp Lirk, M.D.
  • Cheryl Stucky, Ph.D.
  • Stephen E. Abram, M.D.
    §
  • Quinn H. Hogan, M.D.
    §
  • * Research Fellow, Department of Anesthesiology, Medical College of Wisconsin. Staff Anesthesiologist, Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Graz, Austria. † Research Fellow, Department of Anesthesiology, Medical College of Wisconsin. Resident, Department of Anesthesiology and Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria. § Professor, Department of Anesthesiology, ‡ Assistant Professor, Department of Cell Biology, Medical College of Wisconsin.
Article Information
Meeting Abstracts   |   June 2005
Painful Nerve Injury Decreases Resting Cytosolic Calcium Concentrations in Sensory Neurons of Rats
Anesthesiology 6 2005, Vol.102, 1217-1225. doi:
Anesthesiology 6 2005, Vol.102, 1217-1225. doi:
PAIN due to nerve injury may accompany diverse diseases, such as diabetes, herpes zoster, herniated nucleus pulposus, or direct nerve trauma.1–3 Patients may report either spontaneous pain, discomfort on innocuous stimulation (allodynia), or exaggerated pain on nociceptive stimulation (hyperalgesia).3,4 Neuropathic pain may continue for years after the original injury has healed, and currently available treatment is successful in only a minority of patients.5 
Widely distributed pathogenic processes contribute to neuropathic pain, including dysfunction at the site of injury and in the spinal cord and brain. In addition, important alterations contributing to increased excitability have been noted in the somata of sensory neurons proximal to the site of nerve trauma.6–8 Substantial membrane modifications have been identified in dorsal root ganglion (DRG) neurons in various models of neuropathic pain.9 Current through potassium channels is decreased,10 expression of various sodium channel isoforms is altered,11 and influx through high-voltage-activated12 and low-voltage-activated calcium channels13 is diminished. Calcium is particularly important because of its double role in neurons. Not only does the inward calcium current support action potential depolarization, but also, once in the cell, calcium serves as a key second messenger directing neuronal function and is closely regulated by a complex system of buffers, pumps, and release mechanisms.14 Although cytoplasmic calcium ([Ca2+]c) critically regulates neurotransmitter release, intracellular signaling cascades, membrane channel function, cell differentiation, and programmed cell death, there has been no investigation of effects of peripheral nerve injury on sensory neuronal [Ca2+]c.
The goal of this investigation was to identify effects of injury on resting [Ca2+]cas a starting point in understanding the role of disturbed intracellular calcium signaling in neuropathic pain. We used digital microfluorometry to measure [Ca2+]cin neurons dissociated from DRGs of control animals and animals with hyperalgesia from peripheral nerve trauma. Because there is uncertainty about the relative contributions of injured and adjacent neurons after nerve injury,9 we determined resting [Ca2+]cin neurons from the fourth (L4) and fifth lumbar (L5) DRGs after ligation and section of the fifth lumbar spinal nerve (SNL model). The L5 ganglion contains axotomized neurons, whereas the L4 DRG contains those that are intact but project axons that mingle in the sciatic nerve with degenerating fiber segments of L5 neurons (fig. 1), where they are exposed to an inflammatory milieu. DRG neurons are diverse, with cells responsive to low-threshold stimulation and others that are selectively responsive to high-intensity stimulation. We therefore categorized neurons by diameter because large somata size is an approximate indicator of nonnociceptive modality.15,16 In separate experiments, we categorized neurons by sensitivity to capsaicin,17 which is a hallmark of nociceptive modality.
Fig. 1. Spinal nerve ligation model  18 of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
Fig. 1. Spinal nerve ligation model  18of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
Fig. 1. Spinal nerve ligation model  18 of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
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Materials and Methods
All procedures were approved by the Animal Care and Use Committee of the Medical College of Wisconsin, Milwaukee, Wisconsin.
Injury Model
Male adult Sprague-Dawley rats (n = 42; Charles River Laboratories Inc., Wilmington, MA) weighing 160–180 g were randomly assigned to an SNL group or control group. SNL (n = 24 rats) was performed similar to the originally reported technique.18 During anesthesia with halothane (2–3%) in oxygen, the right lumbar paravertebral region was exposed. After subperiosteal removal of the sixth lumbar transverse process, both the right fifth and the sixth lumbar spinal nerves were tightly ligated with 6-0 silk suture and transected distal to the ligature (fig. 1). No muscle was removed, the intertransverse fascia were incised only at the site of the two ligations, and articular processes were not removed. The lumbar fascia was closed by 4-0 resorbable polyglactin suture, and the skin was closed with three staples. In control rats (n = 18), only lumbar skin incision and closure was performed. After surgery, the rats were returned to the colony, where they were kept in individual cages under normal housing conditions.
Sensory Testing
Identification of hyperalgesia was conducted as previously described.19 At least 1 day after arrival at the animal care facility, rats were brought to the testing area for 4 h of familiarization with handling and the environment. Hind paws were stimulated in random order with a 22-gauge spinal needle applied with pressure adequate to indent but not penetrate the plantar skin 2 days before surgery and on the 10th, 12th, and 14th postoperative days. Control rats showed only a brief withdrawal. SNL animals that displayed a hyperalgesia-type response with sustained lifting, licking, chewing, or shaking of the paw were considered to express a phenotype of neuropathic pain, whereas others without hyperalgesic responses were considered to lack neuropathic pain.
Cell Isolation and Plating
The L4 and L5 DRG were removed from control rats as well as both hyperalgesic and nonhyperalgesic SNL rats (studied separately) after halothane anesthesia and decapitation. The operative field was perfused with cold, oxygenated, calcium and magnesium chloride free Hanks Balanced Salt Solution. Minced ganglia were enzymatically dissociated in a solution containing 0.018% liberase blendzyme 2 (Roche Diagnostics Corp., Indianapolis, IN), 0.05% trypsin (Sigma, St. Louis, MO), and 0.01% deoxyribonuclease 1 (150,000 U; Sigma, St. Louis, MO) in 4.5 ml Dulbecco's modified Eagle's medium F12 (Gibco, Carlsbad, CA) for 90 min in a shaker bath at 32°C. Cells were harvested by centrifugation and resuspended in a culture medium consisting of 0.5 mm glutamine, 0.02 mg/ml gentamicin, 100 ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel), 2% (vol/vol) B-27 supplement (Life Technologies, Rockville, MD), and 98% (vol/vol) neurobasal medium A 1X (Life Technologies) for plating onto poly-l-lysine-coated 12-mm glass coverslips (Deutsche Spiegelglas; Carolina Biologic Supply, Burlington, NC), plating from two to four slips per ganglion. Cells were incubated for 2–3 h in humidified incubator at 37°C with 95% air and 5% CO2before dye loading and were studied within 5 h of dissociation.
Calcium Microfluorometry
Cells were loaded with the ratiometric calcium indicator Fura-2 AM (2.5 μm in 0.1% Pluronic F-127; Molecular Probes, Eugene, OR) for 45 min at room temperature and then washed three times with a Tyrode's solution consisting of 140 mm NaCl, 4 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm glucose, and 10 mm HEPES. Cells were left in a dark environment for 30 min for dye deesterification. Coverslips were mounted in a 500-μl recording chamber superfused with room-temperature (22°C) Tyrode's solution at a gravity-driven flow rate of 2 ml/min and imaged at 400× magnification using an inverted microscope (Nikon Diaphot 200; Tokyo, Japan) and cooled charge-coupled device camera (Cool Snap fx-Photometrics; Tucson, AZ). Cell diameter was determined by calibrated video image. On bright-field examination, neurons were excluded from measurement if they showed evidence of lysis or crenulation of their surface, because these cells showed unstable recordings, and also if they had overlying glial satellite cells (fig. 2). Each neuron was specified as a region of interest in the digital image (MetaFluor; Universal Imaging Corporation, Downington, PA) for separate R measurement, and an additional background area was recorded in each field for on-line subtraction of background fluorescence. Only one field was studied per slip. Autofluorescence of unloaded cells had a signal strength of less than 5% of the fluorescence of loaded cells. Emitted Fura-2 fluorescence was recorded at 510 ± 20 nm wavelength during alternating 340- and 380-nm excitation (DG-4; Sutter, Novato, CA). The frame capture period was 200 ms at intervals of 10 s. The ratio (R) of fluorescence excited by 340 nm divided by fluorescence excited by 380 nm was determined on a pixel-by-pixel basis, and [Ca2+]cwas calculated as
Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
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in which Kdis the dissociation constant for Fura-2 (specified as 224 nm),20 Rminis the 340/380 fluorescence ratio in the presence of no calcium, Rmaxis the ratio in the presence of saturating concentrations of calcium, and β is the ratio of fluorescence during 380-nm excitation at zero and saturating calcium concentrations.
Resting R (Rrest) was measured by averaging 10 stable sequential determinations. In this study, Rmin, Rmax, and β were determined by in situ  calibration for each cell, rather than applying averaged values as is typically done. Bath application of a calcium-selective ionophore (fig. 3), either ionomycin (5 μm) or 4-bromo A-23187 (5 μm; Molecular Probes), permitted equilibration of [Ca2+]cwith bath calcium concentration during superfusion first with Tyrode's solution with 10 mm EGTA and no added calcium to determine Rminand then with normal Tyrode's external solution to determine Rmax.21,22 
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
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Drug Delivery
Drugs were delivered by bath change, except application of capsaicin, which was directed to the field through a microperfusion system 125 μm upstream from the imaged field. Using dye delivered through this system, onset time was measured as less than 200 ms, and there was no dilution of microperfusion solution despite bath flow.
Analysis and Statistics
Neurons were divided into two groups with diameters larger or smaller than 34 μm. This division is well established in similar studies of [Ca2+]cin dissociated DRG neurons.23,24 Although the size distribution of sensory neurons is a continuum (fig. 4),25 grouping large and small neurons partially segregates the overlapping nociceptive and nonnociceptive neuron categories.26–28 The chosen diameter for separating the large and small populations is derived from histologic studies.29,30 
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
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In separate experiments, neurons were categorized as sensitive to capsaicin if R increased by 50% over Rrest. Results are presented as mean ± SEM, and n  refers to the number of cells tested unless otherwise stated. For [Ca2+]cmeasurements, main effects were determined by two-way analysis of variance, and post hoc  assessment of within-group comparisons was performed conservatively using the Bonferroni test (Statistica 6.0; StatSoft, Tulsa, OK). Differences between two groups were considered to be significant when P  was less than 0.05. Significance of injury effects on frequency of capsaicin response was tested by nonparametric cross-tabulation. Because there is no established test for post hoc  comparisons within contingency tables, values for Fisher exact test are reported. Concentration-response analysis was performed using Prism 4.0 (GraphPad Software, Inc., San Diego, CA).
Results
Behavioral Responses
Of 40 rats subjected to SNL, 24 in which hyperalgesia developed were considered successful neuropathic models, and DRG were removed 19.1 ± 0.5 days after injury. In these rats, hyperalgesia-type responses occurred in 41.4 ± 4.8% of needle applications to the right foot. Post mortem examination confirmed accurate placement of ligatures and section for all SNL animals. Hyperalgesic behavior did not develop in control rats (n = 17; 0 ± 0% hyperalgesic response rate; P  < 0.001 vs.  SNL), and DRG were removed 18.9 ± 1.6 days after skin incision surgery. Four rats in which hyperalgesia did not develop (1.7 ± 1.7% hyperalgesic response rate; P  < 0.01 vs.  SNL) were also studied at 19.5 ± 0.9 days after injury.
Comparison of Calibration Techniques
Stable Rrestrecordings were achieved after 5–10 min of imaging for neurons dissociated from 65 DRGs (24 L4 and 24 L5 after SNL; 17 L4 or L5 control), including control cells (n = 343), SNL L4 cells (n = 252), and SNL L5 cells (n = 225). To determine whether extreme values of Rmin, Rmax, or β should be used as exclusion criteria in addition to visual features, we plotted [Ca2+]cacross the range of these parameters (data not shown). Because there were no outliers of [Ca2+]cwith the highest or lowest values of Rmin, Rmax, or β, we did not exclude any cells on this basis.
We noted that R continued to decrease during application of ionophore/low bath calcium for as long as 25 min (fig. 3). The descending trace of R during this interval fit an exponential decay curve (r  2= 0.98 ± 0.06), so that an extrapolated Rmincould be determined (Origin 7.0; Origin Lab Corp., Northampton, MA), which was lower than recorded Rminby an average of 0.10 ± 0.01 and resulted in an average increase of [Ca2+]cby 26.4 ± 1.6 nm. We compared calibration using the ionophores 4-bromo A-23187 (n = 165) or ionomycin (n = 264). Because [Ca2+]cdecreased more slowly during Rmindetermination and cells more frequently detached from the coverslip during Rmaxdetermination using 4-bromo A-23187, further studies used only ionomycin as an ionophore during calibration. This choice is also supported by findings indicating that ionomycin has a higher selectivity for calcium and is more effective than 4-bromo A-23187 in binding and transporting calcium.21 [Ca2+]cvalues determined using 4-bromo A-23187 (n = 70) were higher in control animals than those (n = 122) determined using ionomycin, but the effects of injury on [Ca2+]cwere comparable (n = 164; data not shown). For ionomycin, average values of extrapolated Rmin, measured Rmax, and β were 0.53 ± 0.01, 5.19 ± 0.10, and 5.81 ± 0.13, respectively. Comparison of [Ca2+]ccalculated with either cell-by-cell calibration or mean values for Rmin, Rmax, and β (r  2= 0.22; fig. 5) demonstrates the importance of determination of parameters for each cell. The absolute value of the difference between these two determinations is 58.7 ± 2.4 nm (overall for all cells), or 92 ± 6% of the fully calibrated [Ca2+]c.
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
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Resting [Ca2+]cDecreases after Injury in Large and Small DRG Neurons
Two-way analysis of variance showed significant main effects for both injury and neuronal size on [Ca2+]c, as well as a significant interaction of injury and size. Both large- and small-neuron groups showed significantly lower resting [Ca2+]cin axotomized L5 neurons after SNL compared with control (fig. 6), but large neurons showed a proportionately greater decrease to 54% of control. Large neurons also showed a decrease of [Ca2+]cin SNL L4 neurons to 29% of control.
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
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Resting [Ca2+]cDoes Not Decrease after Injury in Rats in Which Hyperalgesia Does Not Develop
To probe whether there is a relation between the decreased [Ca2+]cand altered sensory behavior after injury, we examined a small number of animals without behavior change. Unlike neurons from hyperalgesic rats, large neurons removed from these four rats did not show a decrease in resting [Ca2+]c(182 ± 25 nm, n = 13 for L4; 146 ± 35 nm, n = 5 for L5) compared with neurons from control rats (133 ± 11 nm, n = 25). This was also true for small neurons from such rats, which showed significantly higher resting [Ca2+]cin injured neurons (160 ± 15 nm, n = 22, P  < 0.001 for L4; 129 ± 10 nm, n = 31, P  < 0.001 for L5) compared with control (78 ± 4 nm, n = 96).
[Ca2+]cResponse to Capsaicin Increases in L4 Neurons but Decreases in L5 Neurons after SNL
Neurons with nociceptive and nonnociceptive modalities have broadly overlapping size distributions.15,16 Because previous studies have established capsaicin sensitivity as a hallmark of nociceptors,17 we categorized additional neurons according to their response to capsaicin, using a 50% increase of R to indicate sensitivity. A concentration-dependent response to capsaicin was evident between1 nm and 10 mm in uninjured neurons for both frequency of response at each dose and the response amplitude (fig. 7). A concentration of 100 nm resulted in an intermediate response rate comparable to the expected frequency of nociceptors among dissociated neurons and was used for further testing. For all control neurons, 63 of 130 (48%) responded to 100 nm capsaicin, with a smaller proportion in large neurons (5 of 20, 25%) than in small neurons (58 of 110, 53%; P  < 0.05). SNL decreased the overall frequency of response to capsaicin in L5 neurons (7 of 57, 12%) but increased the incidence in L4 neurons (51 of 77, 66%) compared with both control and SNL L5 (fig. 8). This influence of injury on capsaicin sensitivity was evident among both large and small neurons (data not shown).
Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
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Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
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Injury Decreases Resting [Ca2+]cin Capsaicin-insensitive DRG Neurons
Two-way analysis of variance showed significant main effects for both injury and capsaicin sensitivity on resting [Ca2+]c. Separate analysis of insensitive neurons (fig. 9) showed that resting [Ca2+]cfor SNL L5 neurons was 60% lower than in control neurons and was also lower than in SNL L4 neurons. For capsaicin-sensitive neurons (fig. 9), the influence of injury did not reach significance (P  = 0.077), due in part to the small number of SNL L5 neurons that were sensitive to capsaicin.
Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
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Discussion
Although calcium is the principal signaling pathway controlling cellular processes of sensory neurons, there has been only minimal examination of effects of peripheral nerve injury on [Ca2+]c. In this study, we demonstrate that axonal injury depresses resting calcium concentrations. This decrease is greater in directly axotomized neurons than in adjacent neurons exposed to degenerating axonal segments. The influence of injury is not uniform across neuronal types but is dominant in units presumed to have a nonnociceptive modality, as indicated by large somatic diameter and insensitivity to capsaicin.
We are not aware of other reports of resting [Ca2+]cin painful neuropathic conditions, except for experimental diabetes mellitus. Although some studies of streptozotocin-induced diabetes have found increased resting [Ca2+]c,23,24 the relevance to traumatic or inflammatory neuropathy is uncertain. For example, diabetes is associated with increased voltage-activated calcium currents in the DRG neuronal membrane,31 whereas we have determined that traumatic neuropathy decreases these currents.12,13 Also, there are contrasting reports that show unchanged sensory neuronal resting [Ca2+]cin streptozotocin-induced diabetic rats32–34 and in a spontaneously diabetic rat strain.35 
Response of [Ca2+]cto Capsaicin
Our intent in examining capsaicin response was to categorize cells according to sensory modality.17 We also confirmed an EC50for [Ca2+]cresponse to capsaicin in the 55–72 nm range,36,37 which is much less than the 350–728 nm necessary to produce a membrane current.38–41 Sensory neurons express the capsaicin receptor TRPV1 on endoplasmic reticulum,42 so release of calcium stored in endoplasmic reticulum43 may explain the responses to low capsaicin concentrations we observed. Supporting our belief that the [Ca2+]cresponse to capsaicin is a valid indicator of nociceptive modality, we noted a higher response rate in small cells, which tend to be nociceptors. Further, the response rate was substantially decreased in the axotomized neurons of the L5 DRG after SNL but increased in L4, consistent with immunohistochemical observations of TRPV1 expression.44,45 A generally higher resting [Ca2+]cin control cells tested with capsaicin compared with those categorized only by size may be due to an unexplained effect of capsaicin on ionophore calibration. Nonetheless, injury effects between groups persisted in cells treated this way.
Technique of Measuring [Ca2+]c
Determination of resting [Ca2+]cis highly sensitive to technical details. The use of a ratiometric indicator such as Fura-2 largely corrects for variations in dye loading, bleaching, or differences in fluorescent signal due to cell thickness. We have noted substantial variability between neurons in Rmin, Rmax, and β. This requires the determination of calibration parameters for each neuron if [Ca2+]cis to be compared between cells, although averaged calibration parameters may be adequate if repeated measures for a given cell are compared (e.g.  , response to an agonist).
Technical differences may in part explain the wide range of resting [Ca2+]creported for sensory neurons that spans from 60 to 207 nm.24,36,46–48 Further factors may influence measured [Ca2+]c, however, including choice of Kd, age of the animal,47 and variability in neuronal size and sensory modality of the sample population, as we have shown in the current study. Our finding of higher resting [Ca2+]cin large-diameter sensory neurons than in small neurons contrasts with previous findings in mice32,49 but is in accord with findings in rats.24 
Potential Mechanisms of Depressed [Ca2+]cafter Injury
Cytosolic calcium is regulated by the balanced actions of various membrane processes that acquire, store, and expel calcium. Although calcium entry through voltage-gated calcium channels does not influence [Ca2+]cof the inactive neuron,50 influx of calcium through voltage-independent channels regulated by calcium stores (capacitative calcium entry) is active at rest and sensitive to resting membrane potential.50,51 Uptake into endoplasmic reticulum via  the sarcoplasmic or endoplasmic reticulum calcium adenosine triphosphatase pump and expulsion of calcium from the cell by the plasma membrane calcium adenosine triphosphatase pump both regulate resting [Ca2+]c,52 and mitochondria also sequester calcium in the inactive neuron.53 In most sensory neurons, the plasmalemmal Na+–Ca2+exchanger is constitutively active such that removal of extracellular Na+increases resting [Ca2+]c.54 The decrease in resting [Ca2+]cafter peripheral nerve injury may result from a disturbance in any of these regulatory processes, but these have not been examined.
Withdrawal of target-derived neurotrophins may be an upstream trigger for depression of [Ca2+]c, because nerve growth factor,55 brain-derived neurotrophic factor, and neurotrophin-356 support resting [Ca2+]c. However, nerve growth factor in the ligated L5 DRG recovers after 2 days,57 and brain-derived neurotrophic factor expression is increased after nerve injury.58,59 An alternate cause of depressed resting [Ca2+]cmay be decreased neuronal firing, because activation of neurons increases resting [Ca2+]c.60,61 Decreased afferent traffic due to axotomy or disuse of the limb combined with injury-induced depression of voltage-activated calcium currents12,13 may decrease the cytoplasmic calcium load. Although spontaneous activity develops in injured DRG neurons,8,62 this is typically at very low rates.63,64 The normal or higher levels of resting [Ca2+]cin neurons from SNL rats that lacked hyperalgesia might reflect particularly high levels of spontaneous activity in these subjects. Differences we noted between putative nociceptive and low-threshold neurons may likewise be due to differences in neuronal activity between these modality categories.
Possible Consequences of Decreased [Ca2+]c
A role for decreased resting [Ca2+]cin generation of neuropathic pain is supported by our observation of a normal or increased resting [Ca2+]cin a small number of rats lacking hyperalgesia after SNL. Although the functional consequence of dynamic [Ca2+]ctransients is well established, the role of resting [Ca2+]chas been less studied. In various neuronal tissues, decreased [Ca2+]cprecipitates cell loss, including programed cell death by apoptosis.60,61,65 DRG neuronal loss has been noted as a feature of neuropathy after SNL,66,67 and induced cell activity may prevent cell loss after axotomy in the central nervous system, presumably by increasing resting [Ca2+]c.68 Therefore, the decrease in resting [Ca2+]cwe have observed may directly lead to loss of sensory neurons after injury.
A second role of resting [Ca2+]cis control of responsiveness to ligand stimulation. Increased resting [Ca2+]cattenuates receptor-triggered calcium signaling in lymphocytes69 and central nervous system microglia.70 In DRG neurons, increased [Ca2+]cinactivates responses to capsaicin.37,38,71 In contrast, responsiveness to heat is potentiated when [Ca2+]cis increased by capsaicin or a calcium ionophore,72 but this is not found after [Ca2+]cincrease by membrane depolarization.73 Depressed resting [Ca2+]c, as we have measured in sensory neurons after injury, may thus modulate the transduction of membrane receptor activation into intracellular calcium signals for a broad range of ligands and stimuli, including catecholamines, purines, pH, and inflammatory mediators such as bradykinin, complement, and cytokines. Altered expression of receptors may have a competing influence, however, as in the injury-induced changes in capsaicin responsiveness measured in this study. Low resting [Ca2+]cmay itself produce complex genetic effects, increasing expression of inducible nitric oxide synthase in chondrocytes while depressing synthesis of cyclooxygenase II,74 and increasing the expression of sodium channels in the surface membrane of adrenal chromaffin cells.75 
Finally, enzymatic signaling cascades sensitive to calcium, such as calmodulin, phosphatases, and protein kinases A and C, modulate neuronal activity. Through regulation of calcium-activated K+channels76 and hyperpolarization-activated cation channels,77 resting [Ca2+]ccontrols membrane excitability and neuronal activity. Calcium/calmodulin-dependent protein kinase II has particularly diverse phosphorylation targets and may be an important pathway generating functional and genetic changes from shifts in resting [Ca2+]c.78 
Conclusion
Our findings indicate that nerve injury associated with hyperalgesia depresses resting [Ca2+]cin sensory neurons. This change is most evident in nonnociceptive axotomized neurons of L5 after SNL, which supports a central role of this neuronal group in the genesis of neuropathic pain.9 However, these observations do not eliminate the possibly critical contributions of functional changes in calcium signaling of neurons that remain in continuity to their receptive fields, since we also identified decreased resting [Ca2+]cin large L4 neurons after SNL.
References
Koltzenburg M, Scadding J: Neuropathic pain. Curr Opin Neurol 2001; 14:641–7Koltzenburg, M Scadding, J
Woolf CJ: Pain. Neurobiol Dis 2000; 7:504–10Woolf, CJ
Rasmussen PV, Sindrup SH, Jensen TS, Bach FW: Symptoms and signs in patients with suspected neuropathic pain. Pain 2004; 110:461–9Rasmussen, PV Sindrup, SH Jensen, TS Bach, FW
Woolf CJ, Mannion RJ: Neuropathic pain: Aetiology, symptoms, mechanisms, and management. Lancet 1999; 353:1959–64Woolf, CJ Mannion, RJ
Sindrup SH, Jensen TS: Efficacy of pharmacological treatments of neuropathic pain: an update and effect related to mechanism of drug action. Pain 1999; 83:389–400Sindrup, SH Jensen, TS
Devor M, Janig W, Michaelis M: Modulation of activity in dorsal root ganglion neurons by sympathetic activation in nerve-injured rats. J Neurophysiol 1994; 71:38–47Devor, M Janig, W Michaelis, M
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; 17:321–39Wall, PD Devor, M
Kajander KC, Wakisaka S, Bennett GJ: Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat. Neurosci Lett 1992; 138:225–8Kajander, KC Wakisaka, S Bennett, GJ
Gold MS: Spinal nerve ligation: What to blame for the pain and why. Pain 2000; 84:117–20Gold, MS
Everill B, Kocsis JD: Reduction in potassium currents in identified cutaneous afferent dorsal root ganglion neurons after axotomy. J Neurophysiol 1999; 82:700–8Everill, B Kocsis, JD
Wood JN, Akopian AN, Baker M, Ding Y, Geoghegan F, Nassar M, Malik-Hall M, Okuse K, Poon L, Ravenall S, Sukumaran M, Souslova V: Sodium channels in primary sensory neurons: relationship to pain states. Novartis Found Symp 2002; 241:159–68Wood, JN Akopian, AN Baker, M Ding, Y Geoghegan, F Nassar, M Malik-Hall, M Okuse, K Poon, L Ravenall, S Sukumaran, M Souslova, V
Hogan QH, McCallum JB, Sarantopoulos C, Aason M, Mynlieff M, Kwok WM, Bosnjak ZJ: Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons. Pain 2000; 86:43–53Hogan, QH McCallum, JB Sarantopoulos, C Aason, M Mynlieff, M Kwok, WM Bosnjak, ZJ
McCallum JB, Kwok WM, Mynlieff M, Bosnjak ZJ, Hogan QH: Loss of T-type calcium current in sensory neurons of rats with neuropathic pain. Anesthesiology 2003; 98:209–16McCallum, JB Kwok, WM Mynlieff, M Bosnjak, ZJ Hogan, QH
Berridge MJ, Lipp P, Bootman MD: The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1:11–21Berridge, MJ Lipp, P Bootman, MD
Harper AA, Lawson SN: Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones. J Physiol 1985; 359:31–46Harper, AA Lawson, SN
Koerber HR, Druzinsky RE, Mendell LM: Properties of somata of spinal dorsal root ganglion cells differ according to peripheral receptor innervated. J Neurophysiol 1988; 60:1584–96Koerber, HR Druzinsky, RE Mendell, LM
Holzer P: Capsaicin as a tool for studying sensory neuron functions. Adv Exp Med Biol 1991; 298:3–16Holzer, P
Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992; 50:355–63Kim, SH Chung, JM
Hogan Q, Sapunar D, Modric-Jednacak K, McCallum JB: Detection of neuropathic pain in a rat model of peripheral nerve injury. Anesthesiology 2004; 101:476–87Hogan, Q Sapunar, D Modric-Jednacak, K McCallum, JB
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440–50Grynkiewicz, G Poenie, M Tsien, RY
Kao JP: Practical aspects of measuring [Ca2+] with fluorescent indicators. Methods Cell Biol 1994; 40:155–81Kao, JP
Malgaroli A, Milani D, Meldolesi J, Pozzan T: Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J Cell Biol 1987; 105:2145–55Malgaroli, A Milani, D Meldolesi, J Pozzan, T
Kruglikov I, Gryshchenko O, Shutov L, Kostyuk E, Kostyuk P, Voitenko N: Diabetes-induced abnormalities in ER calcium mobilization in primary and secondary nociceptive neurons. Pflugers Arch 2004; 448:395–401Kruglikov, I Gryshchenko, O Shutov, L Kostyuk, E Kostyuk, P Voitenko, N
Huang TJ, Sayers NM, Fernyhough P, Verkhratsky A: Diabetes-induced alterations in calcium homeostasis in sensory neurones of streptozotocin-diabetic rats are restricted to lumbar ganglia and are prevented by neurotrophin-3. Diabetologia 2002; 45:560–70Huang, TJ Sayers, NM Fernyhough, P Verkhratsky, A
Willis WD, Coggeshall RE: Sensory Mechanisms of the Spinal Cord. New York, Plenum Press, 1991, pp 47–78Willis, WD Coggeshall, RE New York Plenum Press
Lee KH, Chung K, Chung JM, Coggeshall RE: Correlation of cell body size, axon size, and signal conduction velocity for individually labeled dorsal root ganglion cells in the cat. J Comp Neurol 1986; 243:335–46Lee, KH Chung, K Chung, JM Coggeshall, RE
Mu X, Silos-Santiago I, Carroll SL, Snider WD: Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J Neurosci 1993; 13:4029–41Mu, X Silos-Santiago, I Carroll, SL Snider, WD
Sugiura Y, Hosoya Y, Ito R, Kohno K: Ultrastructural features of functionally identified primary afferent neurons with C (unmyelinated) fibers of the guinea pig: classification of dorsal root ganglion cell type with reference to sensory modality. J Comp Neurol 1988; 276:265–78Sugiura, Y Hosoya, Y Ito, R Kohno, K
McDougal DB Jr, McDougal SH, Johnson EM Jr: Effect of capsaicin upon fluoride sensitive acid phosphatases in selected ganglia and spinal cord and upon neuronal size and number in dorsal root ganglion. Brain Res 1985; 331:63–70McDougal, DB McDougal, SH Johnson, EM
Schmalbruch H: The number of neurons in dorsal root ganglia L4-L6 of the rat. Anat Rec 1987; 219:315–22Schmalbruch, H
Hall KE, Sima AA, Wiley JW: Voltage-dependent calcium currents are enhanced in dorsal root ganglion neurones from the Bio Bred/Worchester diabetic rat. J Physiol 1995; 486:313–22Hall, KE Sima, AA Wiley, JW
Kostyuk E, Pronchuk N, Shmigol A: Calcium signal prolongation in sensory neurones of mice with experimental diabetes. Neuroreport 1995; 6:1010–2Kostyuk, E Pronchuk, N Shmigol, A
Kostyuk E, Voitenko N, Kruglikov I, Shmigol A, Shishkin V, Efimov A, Kostyuk P: Diabetes-induced changes in calcium homeostasis and the effects of calcium channel blockers in rat and mice nociceptive neurons. Diabetologia 2001; 44:1302–9Kostyuk, E Voitenko, N Kruglikov, I Shmigol, A Shishkin, V Efimov, A Kostyuk, P
Li F, Obrosova IG, Abatan O, Tian D, Larkin D, Stuenkel EL, Stevens MJ: Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats. Am J Physiol Endocrinol Metab 2005; 288:E29–36Li, F Obrosova, IG Abatan, O Tian, D Larkin, D Stuenkel, EL Stevens, MJ
Hall KE, Sima AA, Wiley JW: Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat. J Clin Invest 1996; 97:1165–72Hall, KE Sima, AA Wiley, JW
Stucky CL, Abrahams LG, Seybold VS: Bradykinin increases the proportion of neonatal rat dorsal root ganglion neurons that respond to capsaicin and protons. Neuroscience 1998; 84:1257–65Stucky, CL Abrahams, LG Seybold, VS
Cholewinski A, Burgess GM, Bevan S: The role of calcium in capsaicin-induced desensitization in rat cultured dorsal root ganglion neurons. Neuroscience 1993; 55:1015–23Cholewinski, A Burgess, GM Bevan, S
Koplas PA, Rosenberg RL, Oxford GS: The role of calcium in the desensitization of capsaicin responses in rat dorsal root ganglion neurons. J Neurosci 1997; 17:3525–37Koplas, PA Rosenberg, RL Oxford, GS
Su X, Wachtel RE, Gebhart GF: Capsaicin sensitivity and voltage-gated sodium currents in colon sensory neurons from rat dorsal root ganglia. Am J Physiol 1999; 277:G1180–8Su, X Wachtel, RE Gebhart, GF
McLatchie LM, Bevan S: The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons. Br J Pharmacol 2001; 132:899–908McLatchie, LM Bevan, S
Marsh SJ, Stansfeld CE, Brown DA, Davey R, McCarthy D: The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro. Neuroscience 1987; 23:275–89Marsh, SJ Stansfeld, CE Brown, DA Davey, R McCarthy, D
Karai LJ, Russell JT, Iadarola MJ, Olah Z: Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons. J Biol Chem 2004; 279:16377–87Karai, LJ Russell, JT Iadarola, MJ Olah, Z
Liu M, Liu MC, Magoulas C, Priestley JV, Willmott NJ: Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons. J Biol Chem 2003; 278:5462–72Liu, M Liu, MC Magoulas, C Priestley, JV Willmott, NJ
Michael GJ, Priestley JV: Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 1999; 19:1844–54Michael, GJ Priestley, JV
Hudson LJ, Bevan S, Wotherspoon G, Gentry C, Fox A, Winter J: VR1 protein expression increases in undamaged DRG neurons after partial nerve injury. Eur J Neurosci 2001; 13:2105–14Hudson, LJ Bevan, S Wotherspoon, G Gentry, C Fox, A Winter, J
Suto K, Gotoh H: Calcium signaling in cold cells studied in cultured dorsal root ganglion neurons. Neuroscience 1999; 92:1131–5Suto, K Gotoh, H
Kirischuk S, Pronchuk N, Verkhratsky A: Measurements of intracellular calcium in sensory neurons of adult and old rats. Neuroscience 1992; 50:947–51Kirischuk, S Pronchuk, N Verkhratsky, A
Thayer SA, Miller RJ: Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro. J Physiol 1990; 425:85–115Thayer, SA Miller, RJ
Shmigol A, Kostyuk P, Verkhratsky A: Role of caffeine-sensitive Ca2+ stores in Ca2+ signal termination in adult mouse DRG neurones. Neuroreport 1994; 5:2073–6Shmigol, A Kostyuk, P Verkhratsky, A
Usachev YM, Thayer SA: Ca2+ influx in resting rat sensory neurones that regulates and is regulated by ryanodine-sensitive Ca2+ stores. J Physiol 1999; 519(pt 1): 115–30Usachev, YM Thayer, SA
Nohmi M, Hua SY, Kuba K: Intracellular calcium dynamics in response to action potentials in bullfrog sympathetic ganglion cells. J Physiol 1992; 458:171–90Nohmi, M Hua, SY Kuba, K
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(pt 1):83–101Wanaverbecq, N Marsh, SJ Al-Qatari, M Brown, DA
Svichar N, Kostyuk P, Verkhratsky A: Mitochondria buffer Ca2+ entry but not intracellular Ca2+ release in mouse DRG neurones. Neuroreport 1997; 8:3929–32Svichar, N Kostyuk, P Verkhratsky, A
Verdru P, De Greef C, Mertens L, Carmeliet E, Callewaert G: Na(+)-Ca2+ exchange in rat dorsal root ganglion neurons. J Neurophysiol 1997; 77:484–90Verdru, P De Greef, C Mertens, L Carmeliet, E Callewaert, G
Pandiella-Alonso A, Malgaroli A, Vicentini LM, Meldolesi J: Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells. FEBS Lett 1986; 208:48–51Pandiella-Alonso, A Malgaroli, A Vicentini, LM Meldolesi, J
Berninger B, Garcia DE, Inagaki N, Hahnel C, Lindholm D: BDNF and NT-3 induce intracellular Ca2+ elevation in hippocampal neurones. Neuroreport 1993; 4:1303–6Berninger, B Garcia, DE Inagaki, N Hahnel, C Lindholm, D
Lee SE, Shen H, Taglialatela G, Chung JM, Chung K: Expression of nerve growth factor in the dorsal root ganglion after peripheral nerve injury. Brain Res 1998; 796:99–106Lee, SE Shen, H Taglialatela, G Chung, JM Chung, K
Michael GJ, Averill S, Shortland PJ, Yan Q, Priestley JV: Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei. Eur J Neurosci 1999; 11:3539–51Michael, GJ Averill, S Shortland, PJ Yan, Q Priestley, JV
Zhou XF, Chie ET, Deng YS, Zhong JH, Xue Q, Rush RA, Xian CJ: Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat. Neuroscience 1999; 92:841–53Zhou, XF Chie, ET Deng, YS Zhong, JH Xue, Q Rush, RA Xian, CJ
Galli C, Meucci O, Scorziello A, Werge TM, Calissano P, Schettini G: Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF-1 through distinct mechanisms of action: The involvement of intracellular calcium and RNA synthesis. J Neurosci 1995; 15:1172–9Galli, C Meucci, O Scorziello, A Werge, TM Calissano, P Schettini, G
Collins F, Schmidt MF, Guthrie PB, Kater SB: Sustained increase in intracellular calcium promotes neuronal survival. J Neurosci 1991; 11:2582–7Collins, F Schmidt, MF Guthrie, PB Kater, SB
Burchiel KJ: Spontaneous impulse generation in normal and denervated dorsal root ganglia: Sensitivity to alpha-adrenergic stimulation and hypoxia. Exp Neurol 1984; 85:257–72Burchiel, KJ
Tal M, Eliav E: Abnormal discharge originates at the site of nerve injury in experimental constriction neuropathy (CCI) in the rat. Pain 1996; 64:511–8Tal, M Eliav, E
Study RE, Kral MG: Spontaneous action potential activity in isolated dorsal root ganglion neurons from rats with a painful neuropathy. Pain 1996; 65:235–42Study, RE Kral, MG
Johnson EM Jr, Koike T, Franklin J: A “calcium set-point hypothesis” of neuronal dependence on neurotrophic factor. Exp Neurol 1992; 115:163–6Johnson, EM Koike, T Franklin, J
Schmalbruch H: Loss of sensory neurons after sciatic nerve section in the rat. Anat Rec 1987; 219:323–9Schmalbruch, H
Vestergaard S, Tandrup T, Jakobsen J: Effect of permanent axotomy on number and volume of dorsal root ganglion cell bodies. J Comp Neurol 1997; 388:307–12Vestergaard, S Tandrup, T Jakobsen, J
Shahar T, House SB, Gainer H: Neural activity protects hypothalamic magnocellular neurons against axotomy-induced programmed cell death. J Neurosci 2004; 24:6553–62Shahar, T House, SB Gainer, H
Healy JI, Dolmetsch RE, Timmerman LA, Cyster JG, Thomas ML, Crabtree GR, Lewis RS, Goodnow CC: Different nuclear signals are activated by theB cell receptor during positive versus negative signaling. Immunity 1997; 6:419–28Healy, JI Dolmetsch, RE Timmerman, LA Cyster, JG Thomas, ML Crabtree, GR Lewis, RS Goodnow, CC
Hoffmann A, Kann O, Ohlemeyer C, Hanisch UK, Kettenmann H: Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): Suppression of receptor-evoked calcium signaling and control of release function. J Neurosci 2003; 23:4410–9Hoffmann, A Kann, O Ohlemeyer, C Hanisch, UK Kettenmann, H
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 1997; 389:816–24Caterina, MJ Schumacher, MA Tominaga, M Rosen, TA Levine, JD Julius, D
Guenther S, Reeh PW, Kress M: Rises in [Ca2+]i mediate capsaicin- and proton-induced heat sensitization of rat primary nociceptive neurons. Eur J Neurosci 1999; 11:3143–50Guenther, S Reeh, PW Kress, M
Kress M, Distler C: Differences in calcium signalling in rat peripheral sensory neurons. Neurosci Lett 2004; 354:127–30Kress, M Distler, C
Geng Y, Lotz M: Increased intracellular Ca2+ selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability. J Cell Biol 1995; 129:1651–7Geng, Y Lotz, M
Shiraishi S, Shibuya I, Uezono Y, Yokoo H, Toyohira Y, Yamamoto R, Yanagita T, Kobayashi H, Wada A: Heterogeneous increases of cytoplasmic calcium: Distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels. Br J Pharmacol 2001; 132:1455–66Shiraishi, S Shibuya, I Uezono, Y Yokoo, H Toyohira, Y Yamamoto, R Yanagita, T Kobayashi, H Wada, A
Nelson AB, Krispel CM, Sekirnjak C, du Lac S: Long-lasting increases in intrinsic excitability triggered by inhibition. Neuron 2003; 40:609–20Nelson, AB Krispel, CM Sekirnjak, C du Lac, S
Schwindt PC, Spain WJ, Crill WE: Effects of intracellular calcium chelation on voltage-dependent and calcium-dependent currents in cat neocortical neurons. Neuroscience 1992; 47:571–8Schwindt, PC Spain, WJ Crill, WE
Molloy SS, Kennedy MB: Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices. Proc Natl Acad Sci U S A 1991; 88:4756–60Molloy, SS Kennedy, MB
Fig. 1. Spinal nerve ligation model  18 of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
Fig. 1. Spinal nerve ligation model  18of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
Fig. 1. Spinal nerve ligation model  18 of partial peripheral nerve injury, in which the spinal nerves (ventral primary ramus) at lumbar levels 5 and 6 are ligated and transected. The proximal site of this partial sciatic injury segregates axotomized L5 sensory neurons from intact neurons of the neighboring L4 ganglion, which share the sciatic nerve with the degenerating distal fragments of the L5 neurons (  dotted line  ). 
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Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
Fig. 2. Exclusion criteria by visual examination. Neurons were not used for data collection if there were overlying satellite cells (labeled  1  ) or if the cell surface appeared wrinkled and collapsed (labeled  2  ). All other neurons (labeled  3  ) were included. Most fields showed a predominance of usable neurons. The full width of the image is 180 μm. 
×
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
Fig. 3. Method of determination of calibration parameters for ratiometric microfluorometric cytosolic calcium concentration determination. The ratio (R) of emitted fluorescence intensity during 340-nm excitation divided by intensity during 380-nm excitation was measured at rest (Rrest), during ionophore and zero bath calcium, to determine the minimum fluorescence ratio (Rmin) and during ionophore with 2 mm bath calcium to saturate the Fura-2 fluorophore and produce the maximum fluorescence ratio (Rmzx). Because R continues to decrease during Rmindetermination, an extrapolated value (Extrapolated Rmin) was also determined by fitting an exponential decay curve, which in this case fit the data points with an  r  2value of 0.995. 
×
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
Fig. 4. Size histogram for recorded neurons. Total n = 1,057 cells. 
×
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
Fig. 5. Scatterplot comparing cytosolic calcium concentration ([Ca2+]c) calculated using either averaged calibration parameters Rmin, Rmax, and β (  ordinate  ) or calibration parameters determined for each cell (  abscissa  ). There is a poor correlation (  solid line  ) between values (  r  2= 0.219), which is nonetheless significant (  P  < 0.001). The line of identity is shown in  dashes  . 
×
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
Fig. 6. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in large and small sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . *  P  < 0.05, ***  P  < 0.001. 
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Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
Fig. 7. Dose-response curves for increase of cytosolic calcium ([Ca2+]i) during capsaicin administration. (  A  ) Frequency of at least 50% increase of fluorescence ratio (R340/380) at each capsaicin concentration. (  B  ) Magnitude of peak increase in R340/380at each capsaicin concentration. Line is fit by sigmoid dose-response curve (Prism 4.0; GraphPad Software Inc.). 
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Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
Fig. 8. Effect of injury on proportion of neurons that respond to capsaicin with an increase of cytosolic calcium. The threshold defining a sensitive neuron was a 50% increase of fluorescence ratio during 100 nm capsaicin administration. Control neurons were compared with neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. Number of neurons for each group is shown in the  bars  . **  P  < 0.01, ***  P  < 0.001. 
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Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
Fig. 9. Injury decreases resting cytosolic calcium concentration ([Ca2+]c) in capsaicin-insensitive sensory neurons. Control neurons were compared to neurons from the L4 dorsal root ganglion (SNL4) and L5 ganglion (SNL5) after L5 and L6 spinal nerve ligation. There was no significant main effect of injury (analysis of variance) in capsaicin-sensitive neurons. Number of neurons for each group is shown in the  bars  . ***  P  < 0.001. 
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