Reports of Ca(2+) current I(Ca) loss after injury to peripheral sensory neurons do not discriminate between axotomized and spared neurons. The spinal nerve ligation model separates axotomized from spared neurons innervating the same site. The authors hypothesized that I(Ca) loss is a result of neuronal injury, so they compared axotomized L5 dorsal root ganglion neurons to spared L4 neurons, as well as neurons from rats undergoing skin incision alone.
After behavioral testing, dissociated neurons from L4 and L5 dorsal root ganglia were studied in both current and voltage patch clamp modes. The biophysical consequence of I(Ca) loss on the action potential was confirmed using selective I(Ca) antagonists. Data were grouped into small, medium, and large cells for comparison.
Reduced I(Ca) was predominantly a consequence of axotomy (L5 after spinal nerve ligation) and was most evident in small and medium neurons. ICa losses were associated with action potential prolongation in small and medium cells, whereas the amplitude and duration of after hyperpolarization was reduced in medium and large neurons. Blockade with Ca(2+) channel antagonists showed that action potential prolongation and after hyperpolarization diminution were alike, attributable to the loss of I(Ca).
Axotomy is required for I(Ca) loss. I(Ca) loss correlated with changes in the biophysical properties of sensory neuron membranes during action potential generation, which were due to I(Ca) loss leading to decreased outward Ca(2+)-sensitive K currents. Taken together, these results suggest that neuropathic pain may be mediated, in part, by loss of I(Ca) and the cellular processes dependent on Ca(2+).
FEW conditions resist effective treatment like pain resulting from nerve injury. For patients with limb trauma or chronic diseases such as diabetes or tumor formation, the cost of untreated pain is loss of function or total incapacity. Animal models in which the peripheral nerve is incompletely injured result in heightened reaction to mechanical stimulation indicative of neuropathic pain.1Explaining the molecular and chemical bases of these changes will suggest means of better treatment.2
We have reported that chronic constriction injury3of the sciatic nerve reduces calcium currents (ICa) in somata of primary afferent neurons in the dorsal root ganglia (DRG) proximal to the injury. These findings imply that decreased ICamay contribute to aberrant sensory processing, but it is not known whether ICaloss is solely a direct effect of axotomy or also an indirect effect of injury on adjacent, intact neurons. The spinal nerve ligation (SNL) model of neuropathic pain4segregates the somata of axotomized L5 neurons from the adjacent L4 neurons that share the sciatic nerve, where intact L4 axons are exposed to inflammatory mediators induced by the degeneration of distal segments of L5 neurons.5–7This model thus provides the opportunity to separately test the roles of neurons affected by these different pathogenic processes. Because the relative roles of axotomized and neighboring intact neurons are unresolved,8we compared neurons from the ligated L5 DRG and the adjacent L4 DRG.9,10We hypothesized that ICaloss is a generalized effect of painful sensory neuron injury and would be found after SNL as well as after chronic constriction injury. We further expected that the loss of current would be predominantly expressed in directly injured L5 neurons as compared with L4 neurons.
Voltage-activated Ca2+channels contribute to shaping the action potential (AP) profile and neuronal excitability.11,12To determine the biophysical role of decreased ICa, we measured the effects of injury on both the AP parameters and the underlying ICain the same neurons. The normal AP of sensory somata is only a few milliseconds long, so the response to conventional patch clamp test pulses lasting hundreds of milliseconds may not accurately depict ICaduring normal neuronal activity. By exposing each cell to a voltage command in the shape of an AP waveform, we measured total charge transfer as a representation of net Ca2+entry into the cell under natural physiologic conditions. Finally, complex interactions of various currents make prediction of net effects of the change in any one current difficult to predict. Specifically, inward Ca2+current depolarizes the membrane, but the arrival of Ca2+in the cytoplasm triggers the opening of Ca2+-activated K+channels that conduct an outward repolarizing current IK(Ca). The balance of these two actions dictates the outcome of decrease ICaon neuronal function. We therefore determined the direct consequences of ICaloss on AP characteristics and overall membrane currents using selective Ca2+channel antagonists.
Materials and Methods
Two hundred forty-five cells were derived from 98 adult male Sprague-Dawley rats weighing 125–150 g provided by a single vendor (Charles River Laboratories Inc., Wilmington, MA), after approval of the Medical College of Wisconsin Institutional Animal Care and Use Committee (Milwaukee, Wisconsin). An additional 67 cells obtained from 21 adult male Sprague-Dawley rats (Taconic Farms, Inc., Hudson, NY) were studied in a separate group. Rats received either ligation and section of the right L5 and L6 spinal nerves (SNL) approximately 5 mm distal to the DRG4or control surgery with skin incision only. After fully recumbent recovery from halothane anesthesia, rats were returned to individual cages under climate- and light-controlled conditions for 10 days before behavioral testing as previously described.9Rats were weighed and placed on a 0.25-in wire grid in clear plastic enclosures and allowed to rest for 30 min. Five applications of a 22-gauge spinal needle were made to the plantar skin of each paw and repeated 3 min later, using a force adequate to indent the skin but not to puncture it. These mechanical stimuli produced either a normal brief flinch, or a hyperalgesic-type response characterized by sustained (> 2 s) paw lifting, shaking, and licking. Only SNL rats with an ipsilateral response rate of at least 20% averaged over 3 test days and normal contralateral responses were used for study. The validity of this approach to behavioral testing is supported by the observation that control rats never exhibited characteristic hyperalgesic responses.
Cell Isolation and Solutions
After a postsurgical interval of 15.8 ± 1.4 days, the L4 and L5 ganglia were removed after decapitation during halothane anesthesia, placed into separate 35-mm Petri dishes containing Ca2+and Mg2+-free iced Hanks’ Balanced Salt Solution, and minced with iris scissors. Using separate sterile, silicone-coated (Sigmacote; Sigma, St. Louis, MO) Pasteur pipettes, each ganglion was placed into a 25-ml sterile tissue culture flask containing 0.0625% trypsin (Boehringer-Mannheim, Indianapolis, IN), 0.0125% DNase (Sigma), and 0.01% blendzyme 2 (Roche Molecular Biochemicals, Indianapolis, IN) in DMEM/F12 with glutaMAX (Invitrogen Corporation, Carlsbad, CA) for 1.5 h in a tissue shaking bath at 32°C and 70 rpm. Neurons were resuspended in adult neural basal media (1×; Invitrogen Corporation, Carlsbad, CA) containing 2% (vol:vol) B27 supplement (50×; Invitrogen Corporation), 0.5 mm glutamine, 0.02 mg/ml gentamicin, and 100 ng/ml nerve growth factor 7S (Alomone Labs, Ltd., Jerusalem, Israel). Dissociated L4 and L5 cells from each tube were plated onto separate poly-L-lysine (70–150 kd)–coated coverslips and placed in a 95:5 oxygen–carbon dioxide, water-jacketed incubator and allowed to plate for 2 h before study. All cells were studied within 8 h after plating.
Current clamp solutions duplicated natural cytosolic and extracellular conditions. A modified Tyrode solution was used externally in the bath during current clamp protocol, consisting of the following: 140 mm NaCl, 4 mm KCl, 2 mm CaCl, 2 mm MgCl, 10 mm D-glucose, and 10 mm HEPES at a pH of 7.4 with NaOH and an osmolarity of 300 mOsm. Internal pipette solution contained 120 mm KCl, 5 mm na-ATP, 0.4 mm na-GTP, 5 mm EGTA, 2.25 mm CaCl2, 5 mm MgCl2, and 20 mm HEPES at a pH of 7.4 with KOH and an osmolarity of 296 mOsm. Because internal pipette solutions cannot be changed after seal formation, ICawas isolated by external blockade of other voltage-sensitive currents. The following solution provided adequate separation from other currents: 2 mm BaCl2, 1 mm 4-aminophyridine, 10 mm HEPES, 160 mm tetraethylammonium chloride, and 0.1 mm tetrodotoxin at a pH of 7.4 with tetraethylammonium hydroxide and an osmolarity of 300 mOsm. Tetrodotoxin blocked tetrodotoxin-sensitive INa, and 4-aminophyridine, tetraethylammonium chloride, and Ba2+blocked potassium currents. Because the change in external solutions results in a +4 mV shift in junction potential, no equivalent offset was added after switching from current clamp to voltage clamp. Cadmium (200 μm) was added at the end of some studies to verify the efficacy of ICaisolation. In a separate protocol examining AP parameters and whole cell currents before and after ICablockade, Ca2+channel antagonists were applied directly to the cell through a pressure regulated microperfusion system (ALA-BP8 system; ALA Scientific Instruments, Westbury, NY). Complete ablation of high voltage–activated Ca+channels was achieved with a cocktail of antagonists that contained nisoldipine (200 nm), SNX-111 (200 nm), agatoxin IVA (200 nm), and SNX-482 (200 nm) to block L-, N-, P/Q-, and R-type calcium channel subtypes, respectively. Tetrodotoxin was obtained from Alomone Labs. SNX-111 was the kind gift of Dr. Scott Bowersox, Ph.D. (Department of Pharmacology, Neurex Corporation, Menlo Park, CA), agatoxin IVA was a gift of Dr. Nicholas Saccomano, Ph.D. (Assistant Director, Medical Chemistry Research at Pfizer, Groton, CT), and SNX-482 was purchased from Peptides International (Louisville, KY).
All cells were recorded in current clamp before changing to voltage clamp. Cells with resting membrane potentials more depolarized than −45 mV were excluded from study. Efficacy of potassium and sodium channel blockade was confirmed by observation of a reversal of negative membrane potential while still in current clamp and a lack of brief, inward currents during voltage clamp recording. Terminal application of Cd2+to block ICashowed no residual voltage-activated currents. Membrane capacitance was measured and access resistance compensated from 80 to 95%. Cells with greater than 10 MΩ access resistance were rejected. Most recordings were made on a List EP-7 amplifier (ALA Scientific Instruments) modified by the addition of a 150-kΩ resistor to the current injection circuit to increase maximum stimulus to 5 nA. An A/D converter (Axon 1200B; Axon Instruments, Union City, CA) attached to a personal computer running pClamp 8 (Axon Instruments) was used to record data. A second series of experiments on large and medium cells with ICaof up to 72.5 nA were performed on an Axon 200B amplifier (Axon Instruments) with a different A/D converter (Axon 1320; Axon Instruments).
Initial observation of resting membrane potential determined cell viability before evoked potentials. To determine AP characteristics, a 1-s step protocol was used with current sufficient (between 10 pA and 2 nA) to evoke an AP (fig. 1). AP voltage threshold (APthresh) was defined as the level of the inflection at the beginning of the rapid upstroke,13whereas AP duration was measured at 50% of peak (AP50). Inflection on the repolarization phase of the AP was observed, and the number of APs during 1 s current injection was determined to characterize the neuron as either adapting or repetitive firing. Continuous current injection and positive feedback from patch clamp head stage amplification prolongs AP duration.14Nevertheless, the relative effect of injury is still detectable. To avoid distortion from continuous current injection during measurement of the afterhyperpolarization (AHP), additional APs were evoked by a brief, 2-ms, 5-nA depolarizing pulse, and peak hyperpolarization (AHPampl) and duration at 50% amplitude (AHP50) were recorded, as well as the time constant for resolution of the AHP (AHPτ) by fitting the recovery phase to a single exponential (fig. 1).
All cells were exposed to both a sustained square wave protocol and a standardized AP waveform voltage command protocol. The square wave protocol consisted of 200-ms commands from a holding potential of −90 to +50 mV in 10-mV increments with 5-s intervals between steps. Measured inward current was normalized by membrane capacitance, which results in a current density corrected for cell size. Peak inward current was determined, and linear leak was subtracted post hoc by fitting the first three steps before significant ICaactivation to a linear function. Maximum conductance (Gmax) was determined by fitting peak inward current to a Boltzmann function, as previously described.15Some large cells produced currents in excess of the bandwidth of the List EP-7 amplifier. Therefore, maximal ICafor large cells was calculated by linear fit of current-voltage peaks from the three traces before and at the reversal potential. Gmaxdetermined by this procedure differed from that determined by Boltzmann fit by ±3% in 12 nonsaturating cells.
A standardized AP waveform command was used to reveal the effects of naturally rapid depolarization and repolarization on ICaacross all cells. The basic features of the standardized wave were an inflection on the repolarization phase and a prolonged AHP to mimic standard features of a nociceptor (fig. 1). A modified P8 wave was used to subtract capacitance; namely, the AP waveform was inverted and divided by 8 to acquire membrane capacitance with minimal channel activation. Each P8 wave was subsequently multiplied by −8 and subtracted from the current in response to an AP waveform voltage command. Peak inward current and total charge transfer (current integrated over time) were determined by cursor measurements after current traces crossed 0 mV at the beginning and end of a wave (fig. 1). Charge transfer (coulombs) was normalized to cell area by converting cell capacitance to the specific capacitance of plasma membranes (0.01 pF/μm2) to compare Ca2+flux between cells of different sizes. Because cells reliably produced a triangular current waveform (fig. 1), peak and area for large cells with saturating currents were calculated by linear extrapolation of the nonsaturating portion of the trace. The reliability of this method was tested on artificially truncated traces of 12 nonsaturating cells, with results varying ±3% from actual measured values.
Cells were categorized according to cell size and surgical preparation. As noted elsewhere,16,17cell size correlates roughly to neuronal types, so we classified cells as small (C type, < 30 μm), medium (Aδ type, 30–40 μm), or large (Aα/Aβ type, > 40 μm). Neurons were further grouped according to surgical preparation (control or SNL) and DRG level (L4 or L5). Because there were no differences in responses from L4 and L5 ganglia from control rats, these cells were combined, resulting in a control group, an SNL L4 group, and an SNL L5 group. Recordings were evaluated post hoc using Clampfit 8 (Axon Instruments), and data were summarized in Excel (Microsoft Co., Redmond, WA) and analyzed with Statistica 6 (StatSoft, Tulsa, OK) for analysis of variance and paired t tests or GraphPad Prism 4 for Mac (GraphPad Software, San Diego, CA) for correlations. Values are expressed as mean ± SEM. For each dependent variable, the main effect of a group was tested with standard univariate analysis of variance, followed by planned Bonferroni post hoc comparisons between groups. Normal distribution was not assumed for large cells in voltage clamp mode (confirmed by Kolmogorov-Smirnov test for normalcy) because of occasional outliers with very large currents. For this size group, a nonparametric Kruskal-Wallis analysis was used with the Dunn test for post hoc comparisons. Significance was estimated at P ≤ 0.05 versus control. Drug effects were evaluated with paired t tests for dependent samples before and after drug administration. Pearson tests were calculated to determine the coefficient of correlation between Gmaxin voltage clamp and AP or AHP parameters in current clamp. Two-tailed P values and confidence levels were computed to determine whether covariance (r 2) was different from zero.
Injury was associated with longer action potential duration (AP50) in small and medium neurons from L5 ganglia compared with control cells, whereas medium and large cells from the same ganglion were characterized by AHPs with lower amplitudes and shorter durations after injury (table 1and fig. 2). The APthreshwas significantly reduced only in small cells from the L5 DRG of SNL rats compared with control neurons.
Inflection of the descending limb of the AP identifies nociceptors.18Overall, inflected cells had a longer AP50(6.4 ± 0.6 vs. 3.3 ± 0.5 ms, P < 0.05) and depolarized threshold (−15.4 ± 0.8 vs. −23.2 ± 1.2 mV, P < 0.05). Comparing neurons with and without inflection did not show any difference in the response of AP parameters to injury in any surgical group.
Cells from adjacent, noninjured L4 ganglia after SNL were statistically indistinguishable from control neurons in most parameters, except for isolated changes after injury in AHP amplitude in medium neurons and AHP duration in large neurons. Therefore, cells from adjacent, nonligated ganglia did not show the consistent pattern of AP or AHP parameter changes demonstrated in cells from the L5 ganglia of injured rats.
Voltage clamp studies were conducted in the same cells after fluid change. Efficacy of potassium and sodium channel blockade was confirmed by observation of elimination of the negative membrane potential while still in current clamp and lack of brief, inward currents during voltage clamp recording. In addition, terminal application of Cd2+to block ICashowed no residual voltage-activated currents.
Spinal nerve ligation reduced Ca2+conductance in L5 neurons across all size groups compared with neurons from control rats. Specifically, Gmaxwas reduced across all three size groups from 36 to 37% in cells from L5 SNL ganglia compared with neurons from control rats. Reduced ICawas also reflected in responses to AP waveform voltage commands. Specifically, peak current amplitude and the integral of Ca2+flux over the course of the AP waveform (total charge transfer) fell between 37 and 60% in L5 ganglia from SNL rats compared with control. Less consistent decreases were found during voltage clamp examination of L4 neurons (table 1), which showed reduced Gmaxonly in large neurons and altered AP waveform responses only in medium and large neurons.
Only L5 neurons showed decreased ICaafter injury in the small neuron group, whereas there were changes in both L4 and L5 neurons in the medium and large groups. However, this part of the study was not specifically designed to compare L4 and L5 neurons. To directly assess the possibly distinct effects of injury on L4 and L5 neurons, we devised additional studies to optimally compare currents in these two groups. In these experiments, we examined currents in paired L4 and L5 medium and large neurons isolated from the same animal under identical conditions. This focused examination also eliminated inconsistencies that accrue due to data collection over a span of time (2 yr for the data set above), including genetic drift in rat breeding colonies and different batches of dissociation enzymes. Further, we used a different amplifier with expanded bandwidth to clamp whole cell recordings with up to 72.5 nA peak inward current. These experiments identified contrasting injury responses in medium and large L4 and L5 neurons. Specifically, ICain L4 neurons from medium cells was unchanged, whereas L5 neurons displayed reduced ICacompared with L4 and control neurons (fig. 3). ICain large L4 and L5 neurons did not differ from control.
Because reduced ICamay have diverging effects on the AP depending on whether the direct inward Ca2+current or the induced outward IK(Ca)dominates, we correlated Gmaxagainst AP parameters measured during current clamp in the same cells to determine the role of ICa. AP50correlated negatively with Gmaxin small cells from L5 of injured rats (fig. 4A) but positively in large control and SNL L5 neurons (fig. 4B), indicating opposite contributions of ICato AP duration in these two neuronal groups. While Gmaxdid correlate positively with AP50in large L5 neurons after SNL, this correlation was also seen in large cells from control ganglia without difference in means as determined by analysis of variance (table 1). Small control neurons showed minimal dependence of AP repolarization on Ca2+-dependent processes (fig. 4A). Gmaxcorrelated negatively with AHPamplin large cells from L5 ganglia of injured rats (fig. 4C). Gmaxdid not correlate with any parameters in L4 and medium-sized neurons.
To identify the biophysical consequences of reduced ICa, we duplicated the loss of ICaseen after injury by application of Ca2+channel blockers and measured the effect of blockade on AP parameters and whole cell currents in the same cell (table 2). Medium or small neurons (n = 9) from nonoperated rats were studied in normal Tyrode solution before and after complete I2+ablation with a cocktail of high voltage–activated Ca2+channel antagonists containing nisoldipine (200 nm), SNX-111 (200 nm), agatoxin IVA (200 nm), and SNX-482 (200 nm) to block L-, N-, P/Q-, and R-type calcium channel subtypes, respectively. An AP was elicited with a brief (0.5-ms) pulse ranging from 1 to 3 nA. This same AP was used as a voltage command to elicit whole cell currents in the same cell. After capacity transients, an inward current representing calcium and sodium was followed by an outward current representing potassium (fig. 5). Calcium channel blockade significantly prolonged average AP50and decreased AHPamplduring current clamp recordings (fig. 5A), whereas underlying inward and outward currents were significantly reduced. It is noteworthy to observe the time course of ionic changes (fig. 5B). Reduced inward ICacorrelated with the delayed pace of AP depolarization, whereas outward current, presumably IK(Ca), was reduced during the repolarization phase of the AP and the onset of AHP. This provides evidence of a critical role of ICain production of outward currents during AP repolarization in sensory neurons.
In this study, we have observed decreased ICain the somata of sensory neurons after peripheral nerve injury by SNL. In combination with our previous findings after more peripheral sciatic injury by chronic constriction,19this indicates that ICais a general feature of nerve injury. The chronic constriction injury model of neuropathic pain is limited by the inability to distinguish the contribution of axotomy versus inflammatory mechanisms, because somata of axotomized neurons cannot be readily distinguished from those with intact axons. Incomplete injury is a prerequisite for provoked pain behavior, so we chose a model of neuropathic pain in which neurons injured by complete axonal transection might be compared with anatomically segregated neighboring neurons. We have found that reduced ICais predominantly a consequence of axotomy (L5 after SNL) and is most evident in small and medium neurons.
Various voltage-activated currents, especially N and T types, inactivate during sustained depolarization,20,21so dynamic aspects of voltage commands may substantially affect patterns of recorded ICa. Studies of ionic currents underlying neuronal membrane function typically rely on prolonged command pulses to detect voltage sensitivity and kinetic properties of channel gating. However, natural neuronal activity leads to opening of voltage-gated channels in response to only brief membrane depolarization events, under which conditions the specific activation, inactivation, and channel closure after repolarization (deactivation) of various currents critically controls Ca2+flux. We therefore used a voltage command in the form of an AP wave to determine whether an injury would produce a loss of ICaas has been observed with prolonged step protocols. We find that injury reduces ICain response to an AP waveform to an even greater extent than ICameasured in response to 200-ms step pulses, which extends observations from traditional sustained square wave protocols and confirms that loss of ICarepresents a true biophysical change associated with neuropathic pain.
We categorized neurons by size, whereas other studies have used AP characteristics, expression of membrane markers, or sensitivity to neuropeptides and algogenic agents. Clearly, sensory neurons show a high degree of heterogeneity, with some investigators describing seven subtypes in small cells alone.22However, nerve injury causes shifts in all these markers,23,24making their use problematic for categorizing neurons. A further limitation is that the exact electrophysiologic changes induced by injury in AP parameters are technique dependent. Specifically, intracellular recordings from nondissociated sensory neurons by us and others24,25contrast with the current patch recordings from dissociated neurons in showing prolonged AP duration after injury in large but not small neurons. To address these limitations, the current study examined Ca2+membrane currents together with AP characteristics in the same neuron, thus directly highlighting the role of ICa.
The functional implications of ICaloss are not fully understood. Previous reports have shown that Ca2+contributes a large inward current during the repolarization phase of the AP, leading to a prolongation or inflection of the descending limb, whereas removing Ca2+shortens AP duration.26–28Accordingly, we have observed a direct relation between AP duration and ICain large neurons (fig. 3B).
Among small and medium neurons, however, diminished ICaprolongs rather than shortens the AP (table 1and fig. 4). Blockade with specific Ca2+channel antagonists indicates that AP prolongation in these neurons is controlled by a shift in the balance of currents such that the loss of inward ICais overwhelmed by a much greater loss of outward IK(Ca)(fig. 5). This relation by which decreased ICaleads to prolonged AP duration is mirrored in the findings of others who have shown that a net outward current can result from Ca2+entry in neuronal cell types with a predominant expression of large conductance voltage- and Ca2+-gated K+channels.29,30Taken together, these results demonstrate the fine balance between ionic conductances and firing patterns which are altered in different ways by trauma. AP prolongation in small and medium neurons after injury may give rise to increased neurotransmitter release at synaptic junctions in the dorsal horn and thereby contribute to hyperalgesia.31
We have also shown that ICaloss is associated with AHP shortening in medium and large neurons after injury. Because currents through Ca2+-activated K+channels contribute substantially to the AHP,32our finding of shortened AHP provides further evidence of decreased outward K+current secondary to ICaloss in these cell groups, although injury and inflammation could also have direct effects on these K+channels. Blocking Ca2+currents with specific toxins similarly caused a reduction of AHP amplitude and duration through diminished activation of Ca2+-dependent outward current. AHP loss primes the neuron for increased repetitive firing,33as we have demonstrated in excised ganglia after injury.24Therefore, the deficiency of ICawe have observed in sensory neurons after injury may substantially contribute to increased excitability and production of neuropathic pain. Further demonstration of this mechanism may be the antinociception provided by intrathecal Ca2+injection in mice that is eliminated by blockers of calcium-activated potassium currents (IK(Ca))34and the burning pain in humans after delivery of a solution containing the Ca2+buffer EDTA to the DRG by epidural injection.35
Calcium signaling performs different functions in different neuronal types. Accordingly, our finding of decreased ICain sensory neurons after nerve injury in rats with hyperalgesic behavior contrasts with relief of neuropathic pain in animal models by intrathecal administration of Ca2+channel blockers.36,37However, neuraxial blockade will affect both primary and secondary sensory neurons and interferes with neurotransmitter release in the spinal cord. Moreover, subarachnoid injection of blockers would not block sensory ganglia because of incomplete penetration into the DRG.38Finally, loss of internal Ca2+in the DRG has been shown to accelerate afferent conduction, whereas increased intracellular Ca2+reduced AP propogation.39
We observed the greatest ICaloss in axotomized L5 neurons after SNL. Although these transected sensory neurons lack a peripheral receptive field, they nonetheless may be an important source of conditioning activity that produces dorsal horn hypersensitivity (fig. 6).8Even in the absence of natural stimulation, axotomized neurons are particularly subject to chemical activation by catecholamines, proinflammatory cytokines, bradykinin and neurotrophins, and mechanical stimulation.40,41Cross-excitation from adjacent intact neurons,42such as those in the dorsal primary ramus of the segmental spinal nerve that remains intact after SNL, also excites the axotomized somata. Our findings indicating membrane hyperexcitability may amplify this induced L5 traffic, leading to hyperalgesic sensory events when naturally triggered L4 activity arrives in the hyperexcitable dorsal horn.
The authors thank Ksenija Modric-Jednacak, M.D. (Resident), Mike Bode, B.A. (Technician), and Mark Poroli, B.S. (Research Technologist), for technical assistance (all from the Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin).