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ANALGESIA

Modulators of Calcium Influx Regulate Membrane Excitability in Rat Dorsal Root Ganglion Neurons

Philipp Lirk, MD^*, Mark Poroli, BS^*, Marcel Rigaud, MD^*, Andreas Fuchs, MD^*, Patrick Fillip, MD^*, Chun-Yuan Huang, PhD^*, Marko Ljubkovic, MD^*, Damir Sapunar, MD, PhD^*, and Quinn Hogan, MD^*||

From the *Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin; Department of Anesthesiology and Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria; Department of Intensive Care and Anesthesiology, Medical University of Graz, Graz, Austria; University of Split Medical School, Split, Croatia; and ||Department of Anesthesiology, Milwaukee Veterans Administration Medical Center, Milwaukee, Wisconsin.

Abstract

BACKGROUND: Chronic neuropathic pain resulting from neuronal damage remains difficult to treat, in part, because of incomplete understanding of underlying cellular mechanisms. We have previously shown that inward Ca²⁺ flux (I_Ca) across the sensory neuron plasmalemma is decreased in a rodent model of chronic neuropathic pain, but the direct consequence of this loss of I_Ca on function of the sensory neuron has not been defined. We therefore examined the extent to which altered membrane properties after nerve injury, especially increased excitability that may contribute to chronic pain, are attributable to diminished Ca²⁺ entry.

METHODS: Intracellular microelectrode measurements were obtained from A-type neurons of dorsal root ganglia excised from uninjured rats. Recording conditions were varied to suppress or promote I_Ca while biophysical variables and excitability were determined.

RESULTS: Both lowered external bath Ca²⁺ concentration and blockade of I_Ca with bath cadmium diminished the duration and area of the after-hyperpolarization (AHP), accompanied by decreased current threshold for action potential (AP) initiation and increased repetitive firing during sustained depolarization. Reciprocally, elevated bath Ca²⁺ increased the AHP and suppressed repetitive firing. Voltage sag during neuronal hyperpolarization, indicative of the cation-nonselective H-current, diminished with decreased bath Ca²⁺, cadmium application, or chelation of intracellular Ca²⁺. Additional recordings with selective blockers of I_Ca subtypes showed that N-, P/Q, L-, and R-type currents each contribute to generation of the AHP and that blockade of any of these, and the T-type current, slows the AP upstroke, prolongs the AP duration, and (except for L-type current) decreases the current threshold for AP initiation.

CONCLUSIONS: Taken together, our findings show that suppression of I_Ca decreases the AHP, reduces the hyperpolarization-induced voltage sag, and increases excitability in sensory neurons, replicating changes that follow peripheral nerve trauma. This suggests that the loss of I_Ca previously demonstrated in injured sensory neurons contributes to their dysfunction and hyperexcitability, and may lead to neuropathic pain.

Activity of primary sensory neurons induced by natural stimulation or by processes such as trauma, inflammation, and nerve injury is the origin of all but a small fraction of painful sensations. Sensory neurons are additionally the afferent source of intraoperative nociceptive reflexes that trigger cardiovascular, ventilatory, and neurohumoral efferent pathways. The central role of these neurons in painful conditions and anesthesia makes it critical to understand the regulation of their excitability. The sensory neuron plasmalemma is equipped with a variety of voltage-activated ion channels conducting Na⁺, K⁺, or Ca²⁺, which together determine the biophysical function of the membrane. Currents through voltage-activated Ca²⁺ channels play a critical double role. First, inward Ca²⁺ flux (I_Ca) depolarizes the cell, and thus contributes to action potential (AP) formation. Once reaching the intracellular compartment, however, Ca²⁺ is also a key second messenger, controlling a broad range of neuronal functions including kinase activity, neurotransmitter release, cell differentiation and growth, genetic expression, and cell death. Membrane events are regulated by intracellular Ca²⁺ through depression of I_Ca,1 stimulation of Ca²⁺-activated Cl^– channels (I_Cl(Ca)),2 and opening of Ca²⁺-activated K⁺ channels (I_K(Ca)), which are widely distributed among dorsal root ganglion (DRG) neurons of all sizes.3

Pain resulting from neuronal damage is a special case in which the primary pathology involves disrupted regulation of sensory neuron excitability at the site of injury and in the DRG proximal to the injury.4–6 We and others have reported loss of both high-voltage activated (HVA)3,7–9 and low voltage-activated (LVA) subtypes10 of I_Ca in DRG neurons after peripheral nerve injury. Although the effects of decreased I_Ca upon neuronal membrane properties have been detailed in other cell types, the only studies examining I_Ca regulation of DRG neuronal excitability have used the patch-clamp electrophysiological technique on dissociated neurons.11,12 This is problematic, since dialysis of the cytoplasm by the solution in the patch pipette disrupts natural biophysical events, as demonstrated by substantially prolonged AP durations, more frequent after-depolarizations following the AP, and diminished after-hyperpolarization (AHP) amplitude, compared with microelectrode recording.2,6,13,14 For instance, although AP durations in control neurons are consistently less than 4 ms when recorded extracellularly or by high resistance intracellular microelectrodes,6,15 durations may be as long as 60 ms using the patch-clamp method,11 which indicates clearly abnormal recording conditions. Furthermore, intracellular regulation of Ca²⁺ concentrations is disrupted by the patch clamp technique,16 and amplifiers typically used for patch clamp recording are not ideally suited for recording APs.17 Finally, dissociation itself produces an injury-like effect on neuronal excitability.18

Since injury also produces substantial changes in voltage-gated Na⁺ and K⁺ channels of sensory neurons,19,20 the overall purpose of this study is to clarify the extent to which altered membrane functions after injury, especially increased excitability, are specifically attributable to diminished Ca²⁺ entry. Therefore, the present study examines the influence of changes in I_Ca and intracellular Ca²⁺ upon AP dimensions, threshold for initiation of APs, and repetitive firing. We used a technique designed to minimize disruption of cellular Ca²⁺ signaling, by using intracellular microelectrode recording from intact DRGs.

METHODS

All procedures used in the study were approved by the Animal Resource Center of the Medical College of Wisconsin. Experiments were performed on male Sprague-Dawley rats (200–300 g) obtained from a single vendor (Taconic Inc., Germanville, NY). Our general strategy was to record from intact DRGs, which avoids disruptive effects of cell dissociation, and to use multiple pharmacologic measures that together will delineate the role of I_Ca in sensory neuron activation.

Tissue Preparation
Rats were anesthetized with halothane in oxygen, and a laminectomy was performed up to the second thoracic level whereas the surgical field was perfused with artificial cerebrospinal fluid (aCSF: NaCl 128 mM, KCl 3.5 mM, MgCl₂ 1.2 mM, CaCl₂ 2.3 mM, NaH₂PO₄ 1.2 mM, NaHCO₃ 24.0 mM, and glucose 11.0 mM) aerated with 5% CO₂ and 95% O₂ to maintain a pH of 7.35. The fourth and fifth lumbar DRGs and attached dorsal roots were removed and the connective tissue capsule dissected away from the ganglia under 20x magnification. Ganglia were transferred to a glass-bottomed recording chamber and perfused with 35°C aCSF. The proximal cut end of dorsal roots was placed on a pair of platinum wire stimulating electrodes. DRG neurons were viewed using an upright microscope equipped with differential interference contrast optics and infrared illumination. Neuronal soma diameter was determined with the focal plane adjusted to reveal the maximum somatic area using a calibrated video image.

Electrophysiological Recording
Intracellular recordings were performed with microelectrodes fashioned from borosilicate glass (1 mm OD, 0.5 mm ID, with Omega fiber—FHC Bowdoinham) using a programmable micropipette puller (P-97, Sutter Instrument Company, Novato, CA). Microelectrode resistances were 80 to 120 M when filled with 2 M potassium acetate. Neurons were predominantly selected from the two outermost cell layers of the dorsal medial aspect of the DRG, and were impaled under direct vision by minimally indenting the membrane and then applying an oscillating current to the microelectrode. Data were acquired after stable recordings were achieved, typically within 5 min of puncture. Membrane potential was recorded using an active bridge amplifier (Axoclamp 2B, Axon Instruments, Foster City, CA), except in protocols requiring simultaneous current injection through the electrode, for which we used discontinuous current clamp recording mode. This allows accurate recording of voltage, although the noise level is increased compared with bridge mode. During discontinuous mode recording, the switching frequency from current injection to recording was 2 kHz, and full settling of the electrode charge was confirmed. Currents were filtered at 1 kHz (discontinuous mode) and 10 kHz (bridge mode), and then digitized at 10 kHz (discontinuous mode) or 40 kHz (bridge mode; Digidata 1322A and Axograph 4.9, Axon Instruments) for data acquisition and analysis. Somatic APs were produced by axonal conduction of APs from a dorsal root site 18–22 mm distant, where the root was stimulated with square-wave pulses of up to 90 mA lasting 0.06 ms via bipolar electrodes, except in specified protocols in which current was injected through the recording electrode.

Inclusion criteria were a resting membrane potential (RMP) negative to –50 mV, and an AP amplitude more than 40 mV. Together, these excluded approximately 6% of recordings. APs measures (Fig. 1) were obtained from single traces after consistency of dimensions was confirmed by comparison to 10 sequential APs. The presence of a hump or inflection on the descending limb of the AP was determined by examination of the differentiated trace (Figs. 1B and C). Determinations of AP and AHP durations and the area under the curve for the AHP (AHP area) were performed digitally. Input resistance was calculated from the minimum potential achieved during 100 ms hyperpolarizing current (0.5 nA) injection through the recording electrode.15 Sensory neurons show time-dependent rectification ("sag") in response to hyperpolarization, predominantly attributable to the hyperpolarization-induced H-current.21 If a neuron showed sag with 0.5 nA injection, additional traces during injection of lower depolarizing currents were examined to assure that the input resistance calculation was not affected. Sag was quantified as the fractional return from the peak hyperpolarized membrane potential back towards RMP during injection of a 1.2 nA hyperpolarization current for 100 ms. Rheobase was determined as the minimum current able to elicit an AP during incremental depolarizing current injection of 0.5 to 10 nA for 100 ms. The pattern of impulse generation was determined during current injection steps beyond rheobase, at which neurons either produced single APs or fired repetitively. The influence of bath Ca²⁺ level or drug upon AP firing pattern was measured at a depolarizing voltage that first produced a bath Ca²⁺- or drug-induced difference in the number of APs generated.

View larger version (10K): [in this window] [in a new window]   Figure 1. A, Measurements determined from action potential (AP) trace. RMP, resting membrane potential; APamp., amplitude of AP; AP 95%, duration of AP at 95% of amplitude; t, latency following axonal stimulation; AHP amp., amplitude of after-hyperpolarization; AHP 80%, duration of after-hyperpolarization until 80% recovery to baseline. B, AP trace (above) and differentiated wave (below) from an A/β-type neuron that lacks an inflection on the descending limb of the AP. C, AP trace (above) and differentiated wave (below) from an A-type neuron, showing an inflection on the descending limb of the AP, as confirmed in the differentiated trace with an interval of decreased negative slope (arrows). Note the different voltage and time scales compared with B.

Neurons were classified by the method of Villiere and McLachlan.15 Conduction velocity (CV) was measured by dividing the distance between stimulation and recording sites by the conduction latency. Neurons with dorsal root CV <1.5 m/s were considered C-type, neurons with CV >15 m/s were considered A/β-type, and neurons with CV >1.5 m/s but CV <10 m/s were considered A-type. For neurons with CV between 10 and 15 m/s, long AP duration was used to categorize the cells as A-types.15 Too few C-type neurons were successfully recorded to provide statistical analysis, and are not reported here.

Ca²⁺ Current Modulation
The influence of I_Ca on membrane biophysical events was revealed by various techniques that alter I_Ca or intracellular Ca²⁺ concentration. Baseline electrophysiologic variables were measured in neurons in aCSF, after which they were exposed by bath change to an external solution identical except for a low Ca²⁺ concentration, in which CaCl₂ was substituted with MgCl₂ (n = 25 A/β, 6 A). This resulted in a measured Ca²⁺ concentration of 0.35 ± 0.02 mM (n = 3 measurements, by Ca²⁺-sensitive electrode, ABL 515, Radiometer, Copenhagen). Magnesium was added (final concentration 3.5 mM) to exclude the possible influence of changed surface charge, which is otherwise a potent source of neuronal stimulation,22 and to maintain a constant divalent cation effect on potassium channels.23 Stable recordings were achieved after a wash-in interval of 3 min. Using conventional patch-clamp recording of whole-cell I_Ca7 in dissociated DRG neurons, we confirmed that only 6 ± 4% of baseline I_Ca remains during these low bath Ca²⁺ conditions (n = 4; Fig. 2). In supplementary experiments, I_Ca was decreased in other neurons (n = 13 A/β, 7 A) by bath exchange to aCSF containing cadmium 200 µm, a widely used blocker of neuronal voltage-activated Ca²⁺ channels.13,24

View larger version (21K): [in this window] [in a new window]   Figure 2. Inward Ca2+ current under different bath Ca2+ concentrations. Recordings were made in a dissociated dorsal root ganglion neuron by conventional patch-clamp recording in voltage-clamp mode, using a voltage step command from –70 mV to 0 mV for 100ms. Compared with baseline conditions (Ca2+ concentration 2.3 mM, black trace), bath solution with no added Ca2+ (0.35 mM, red trace) decreased current, whereas high bath Ca2+ (7 mM, blue trace) raised current.

In other recordings (n = 6 A/β and 1 A neurons), ethylenediamine-tetraacetic acid (EDTA) 500 µM was added to the potassium acetate micropipette solution to chelate intracellular Ca²⁺ upon entry, using a previously established iontophoretic method in which injection is achieved using a 1 nA hyperpolarizing current for 60 s.25 Preliminary studies showed that current injection of this magnitude alone had no effect on measured variables. Further studies (n = 10 A/β, 1 A neurons) used the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as the cell-permeant acetoxymethyl ester (BAPTA-AM) to decrease intracellular concentration.26 This was dissolved in bath solution (200 µM) and delivered by a microperfusion technique from a pipette with a 10-µm diameter tip that was positioned 200 µm from the impaled neuron, and ejected continuously by pressure applied to the back end of the pipette (Picospritzer II, General Valve Corp., Fairfield, NJ). Preliminary experiments in which cell depolarization was measured during microperfusion application of solutions of various K⁺ concentrations indicated an effective fivefold dilution of pipette solution into the bath at the cell membrane, so that the BAPTA-AM concentration was approximately 40 µM at the cell surface.

To further test the role of intracellular Ca²⁺ on sensory neuron function, increased I_Ca was provoked in other neurons by bath change to a solution with Ca²⁺ concentration increased to 5 or 7 mM. Since the results of these 2 concentrations were comparable, the results were pooled for analysis (n = 8 A/β and 9 A neurons). Patch-clamp measurement of I_Ca showed a 35% ± 16% increase of I_Ca under these conditions (n = 4; Fig. 2).

Selective Modulation of Ca²⁺ Channel Subtypes
Sensory neurons express a variety of voltage-gated Ca²⁺ currents, including subtypes with both LVA and HVA activation characteristics.27 Since the methods described above affect all voltage-activated Ca²⁺ currents indiscriminately, we examined the contributions of specific Ca²⁺ channel subtypes to membrane activation before and after microperfusion application of selective blockers. Specifically, the agents and concentrations in the application pipette were as follows: nickel (2 mM) and mibefradil (5 µM) to block the T-type LVA current, nitrendipine (50 µM) to block the L-type HVA current, BayK 8644 (50 µM) to amplify L-type current, SNX-111 (2.5 µM) to block the N-type HVA current, -Aga-IVA (2.5 µM) to block the P/Q-type HVA current, and SNX-482 (2.5 µM) to block the R-type HVA current. Since the peptide toxins are irreversible, no more than two recordings were made from widely separated sites on each ganglion. In other experiments, all the selective blockers (nitrendipine, SNX-111, -Aga-IVA, SNX-482) were administered together to examine the effect of complete HVA I_Ca blockade. The vital dye fast-green, which is compatible with neuronal electrophysiological recording,28,29 was included at a final concentration of 0.5% to permit visual confirmation of drug delivery to the recorded cell during application, and to ensure that new recordings were made from parts of the ganglion that had received no previous drug exposure. Cytochrome C 0.1% was included with the peptide toxins to inhibit nonspecific binding to glass surfaces. The nitrendipine solution included dimethyl sulfoxide at a final concentration of 0.25% in the delivery pipette. Control experiments were performed to determine if pressure ejection of vehicle alone altered recorded variables. In this portion of the study, dimensions were measured from APs averaged from 10 sequential traces before and 5 min after initiation of drug administration, at which time stable changes had evolved.

SNX-111 was the kind gift of Dr. Scott Bowersox from Neurex Corporation (Menlo Park, CA), while -Aga-IVA was the gift of Dr. Nicholas Saccomano from Pfizer (Groton, CT), and SNX-482 was purchased from Peptides International (Louisville, KY). All other agents were purchased from Sigma-Aldrich Co. (St. Louis, MO).

Data Analysis and Statistics
The control group included cells from both L4 and L5 ganglia in animals having undergone skin incision only. It was not the purpose of the study to determine differences in biophysical variables between cell types or injury categories, as we did before,6 and so these comparisons were not tested statistically. Data were analyzed using Excel (Microsoft CO, Seattle) and Statistica 6.0 (StatSoft, Tulsa). Averaged results are expressed as means ± sd. The effect of drug application was evaluated using paired Student's t-test, which allows identification of significant drug effects upon measured variables in the context of natural variability between neurons that results in large sd and differences between group means under baseline conditions. Nonparametric data were evaluated by cross-tabulation with significance tested by Pearson ² for a main effect and planned post hoc paired comparisons by Fisher's exact test. The threshold for statistical significance was accepted at P < 0.05.

RESULTS

Electrophysiological data were obtained from a full range of DRG neuronal sizes (average diameter 39.7 ± 0.6 µm for A/β, 34.2 ± 0.8 µm for A). As shown in Figure 3, the distributions of neuronal diameters for A/β and A categories extensively overlap.

View larger version (22K): [in this window] [in a new window]   Figure 3. Cell size histogram indicating distribution of diameters of A/β-type neurons and A-type neurons included in the study. Although the average size is larger for A/β-type neurons (see text), there is substantial overlap between the groups.

The main results for altered bath Ca²⁺ are summarized in Table 1. The effects of decreased and increased external bath Ca²⁺ and cadmium administration were reversible on washout (data not shown).

Influence of I_Ca Modulators on AP variables
Low bath Ca²⁺ levels depolarized the RMP in A neurons and decreased AP amplitude in A/β and A neurons (Fig. 4). Bath Ca²⁺ withdrawal also led to the disappearance of the hump on the descending limb of the AP in 2 of 4 inflected A/β neurons and in 3 of 5 inflected A neurons. Similarly, cadmium application abolished the inflection in both of 2 inflected neurons, so that decreasing I_Ca eliminated the inflection in 7 of 11 inflected neurons overall (P < 0.01). However, high bath Ca²⁺ did not elicit the appearance of de novo inflections. Thus, while I_Ca is necessary to produce an inflection, not all cells can do so even if intracellular Ca²⁺ levels are increased. Bath Ca²⁺ withdrawal had no significant effect on AP duration in either A/β or A neurons, and cadmium similarly had no effect on AP duration in A/β neurons (from 0.85 ± 0.19 to 0.82 ± 0.18 ms; P = 0.25, n = 13) or in A neurons (from 0.96 ± 0.13 to 0.90 ± 0.12 ms; P = 0.08, n = 7). Contrasting effects on inflection and duration are attributable to our measurement of the duration at the base of the AP. Buffering of intracellular Ca²⁺ with BAPTA-AM, did prolong the AP duration (from 0.88 ± 0.08 to 0.96 ± 0.11 ms; P < 0.05, n = 9). BAPTA-AM, also decreased the AP upstroke velocity (from 425 ± 54 to 366 ± 58 mV/ms; P < 0.05, n = 9), as did cadmium blockade of I_Ca in A neurons (from 256 ± 51 to 246 ± 52 mV/ms; P < 0.05, n = 7).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of manipulations that decrease inward Ca2+ flux. A, Lowering bath Ca2+ concentration (*, red trace) from 2.3 mM to 0.35 mM in an A/β-type neuron results in decreased action potential amplitude (arrow) and more rapid resolution of the after-hyperpolarization. B, Action potential traces (above) and differentiated traces (below) showing the effects of lowering bath Ca2+ (red traces) in an A-type neuron. The action potential duration is shortened and the inflection on the descending limb of the action potential (arrowheads) is lost in low bath Ca2+. C, Bath application of cadmium (200 µM) to an A-type neuron decreased the amplitude and duration of the after-hyperpolarization (*, red trace). D, Iontophoretic injection of the Ca2+ chelator ethylenediamine-tetraacetic acid (EDTA; *, red trace) in an A-type neuron decreased amplitude and duration of the after-hyperpolarization.

Influence of I_Ca Modulators upon AHP
Low bath Ca²⁺ levels caused a decrease in AHP duration and area for both A/β and A neurons (Fig. 4, Table 1). This observation was supported by decreased AHP area when I_Ca was blocked by cadmium in A/β neurons (from 238 ± 269 to 147 ± 152 mV · ms; P = 0.01, n = 13), although a change was not confirmed in A neurons (from 227 ± 234 to 147 ± 161 mV · ms; P = 0.11, n = 7). Combined application of all HVA I_Ca blockers to neurons (n = 7 A/β and 5 A neurons) similarly reduced AHP duration (from 21.1 ± 22.0 to 13.0 ± 15.2 ms; P < 0.01) and AHP area (from 397 ± 570 to 204 ± 344 mV · ms; P < 0.05). The importance of Ca²⁺ influx for generating the AHP was further substantiated by chelating intracellular Ca²⁺ with EDTA iontophoresis, which similarly decreased AHP area (from 103 ± 76 to 79 ± 67mV · ms; P = 0.05, n = 7; Fig. 4), as did BAPTA-AM application (from 187 ± 163 to 134 ± 111mV · ms; P < 0.05, n = 9). Reciprocally, bath change to high Ca²⁺ concentration increased the AHP duration and area in A/β neurons (Fig. 5), providing confirmation of the dependence of the AHP on I_Ca. Variability in baseline AHP dimensions between groups reflects natural heterogeneity of I_K(Ca) ion sensory neurons.30

View larger version (14K): [in this window] [in a new window]   Figure 5. Effects of increasing inward Ca2+ flux. Elevation of bath Ca2+ concentration from 2.3 to 5 mM increased the after-hyperpolarization amplitude (*, blue trace) of an A-type neuron.

Influence of I_Ca Modulators upon Excitability
Low bath Ca²⁺ solution decreased rheobase in A/β neurons (Fig. 6, Table 1), which also followed blocking I_Ca using cadmium (from 1.77 ± 1.6 nA to 1.45 ± 1.27 nA; P = 0.02, n = 13), and by application of BAPTA-AM (from 3.10 ± 1.7 nA to 2.39 ± 1.72 nA; P < 0.01, n = 7). Three of 38 A/β and 1 of 10 A neurons fired repetitively during sustained depolarization under baseline conditions, but 9 additional A/β neurons fired repetitively in low Ca²⁺ solution (P < 0.05). The average number of APs in A/β neurons during these sustained (100 ms) depolarizations followed a similar pattern, increasing from 1.3 ± 0.9 to 10.9 ± 13.4 (P = 0.01) with decreased bath Ca²⁺. Cadmium similarly provoked novel repetitive firing in A/β neurons (from 3 to 6 of 13 cells), and A neurons (from 2 to 4 of 6 cells, Fig. 7), as did BAPTA-AM (from 2 to 5 of 7 cells; average rate increased from 1.1 ± 0.8 to 13.1 ± 5.6, P < 0.05, n = 7). During combined application of HVA blockers, 4 of 4 nonaccommodating neurons fired more rapidly.

View larger version (15K): [in this window] [in a new window]   Figure 6. Influences on repetitive firing behavior of ICa modulation in three sensory neurons. In each case, the top panel shows traces recorded during baseline conditions (2.3 mm bath Ca2+), whereas the middle panel shows the response to altered bath conditions. The current commands are the same for each pair and are shown in the bottom panel. Increased noise in these current injection protocols is due to discontinuous current clamp recording mode that is employed for accurate voltage measurement. A, Effect of lowering bath Ca2+ concentration to 0.35 mM on firing behavior of an A/β-type neuron. The rheobase current at which an action potential first appears was lowered, and the number of action potentials during depolarization was increased in low bath Ca2+. B, Effect of bath cadmium (200 µM) application on firing behavior of an A-type neuron. Cadmium caused burst firing during a current injection that previously only elicited a single action potential. C, Effect of elevating bath Ca2+ concentration to 5 mM on firing behavior of a repetitively firing (nonaccommodating) A/β-type. The neuron fired repetitively under baseline conditions but fired only a single action potential with increased bath Ca2+.

View larger version (20K): [in this window] [in a new window]   Figure 7. Dependence of hyperpolarization-induced voltage sag on intracellular Ca2+ accumulation. A, Switching from baseline bath Ca2+ (2.3 mM) to lowered bath Ca2+ (0.35 mM, red trace) caused decreased voltage sag in an A-type neuron. (B). Iontophoretic injection of the Ca2+ chelator ethylenediamine-tetraacetic acid (EDTA; red trace) in an A/β-type neuron likewise decreased the sag towards resting membrane potential during a hyperpolarizing current injection. C, Current command for both neurons. Recording by discontinuous current clamp.

Changing the bath to high Ca²⁺ solution had the opposite effect on the pattern of neuronal firing, causing 2 of 7 repetitively firing A/β neurons (Fig. 6) and 1 of 2 repetitively firing A neurons to become adapting and fire only once during sustained depolarization.

Hyperpolarization-Induced Voltage Rectification
The depolarizing sag in membrane potential during hyperpolarization was diminished by low Ca²⁺ bath solution in both A/β and A neurons (Fig. 7A, Table 1), as did combined application of HVA I_Ca blockers (from 21.8 ± 18.0% to 12.4 ± 18.0%; P < 0.05, n = 7 A/β and 5 A neurons), cadmium application (from 37.6 ± 17.6% to 23.2 ± 11.4%; P < 0.05, n = 5 A neurons), and chelation of intracellular Ca²⁺ with either EDTA (from 19.23 ± 17.2% to 14.9 ± 16.8%; P < 0.05, n = 5; Fig. 7B) or BAPTA-AM (from 28.0.6 ± 19.5% to 13.9 ± 17.6%; P < 0.05, n = 8). Together, these observations indicate a Ca²⁺dependency for the hyperpolarization-induced inward current.

Response to Selective Blockers of Ca²⁺ Channel Subtypes
The regulation of AP dimensions and neuronal excitability by individual I_Ca subtypes was further examined using selective blockers on 59 A/β neurons (Table 2, Fig. 8). Mibefradil, in contrast to low Ca²⁺ bath solution, hyperpolarized the RMP, which may be attributable to nonspecific effects of this agent.31,32 AP amplitude was increased by nickel and SNX-482 but decreased by nitrendipine. All agents prolonged AP duration, and all agents except nickel and nitrendipine slowed the CV. AHP area was decreased by SNX-111, nitrendipine, -Aga-IVA, and less so by SNX-482. All blockers except nitrendipine decreased rheobase, indicating an excitatory influence, whereas repetitive firing during sustained depolarization was not affected by individual application of the selective blockers. Voltage sag after membrane hyperpolarization was decreased only by mibefradil, -Aga-IVA, and SNX-482. None of the agents produced a consistent change in the presence or absence of inflection. The only effect of pressure ejection of vehicle alone (n = 9 A/β neurons and 1 A neuron) was to decrease the slope of the ascending limb of the AP (dV/dt), which was decreased to a greater extent by all agents.

View larger version (15K): [in this window] [in a new window]   Figure 8. Representative AP traces showing effects of subtype-specific blockers of ICa. Traces before and 5 min after (*, red traces) blocker are superimposed at the same resting potential (dotted line). Peak amplitude is indicated by an arrow in each case. AP duration was substantially prolonged by T-type current block with mibefradil, N-type block with SNX-111, P/Q-type block with Aga-IVA, and R-type block with SNX-482. AHP area was diminished by L-type block with nitrendipine, SNX-111, Aga-IVA, and SNX-482. Approximate doses at the neuron (and resting membrane potential for the demonstration traces before/after drug application) were nickel 0.5 mM (–73.2/–72.0 mV), mibefradil 1 µM (–70.2/–74.6 mV), nitrendipine 10 µM (–70.3/–70.1 mV), SNX-111 0.5 µM (–66.3/–62.7 mV), Aga-IVA 0.5 µM (–55.0/–52.4 mV), and SNX-482 0.5 µM (–75.5/–73.8 mV).

We extended our examination of the role of Ca²⁺ entry by amplifying I_Ca with the L-current agonist BayK 8644 (n = 10 A/β neurons and 1A neuron; Table 2), which slowed the CV, increased the AP amplitude, and substantially prolonged the AP. The effect on the AHP showed divergent patterns. Maximum AHP amplitude was decreased in 10 neurons (Figs. 9A and B), but increased in another (Fig. 9C). Expansion of the AHP area was also seen in two other neurons that developed new AHPs following the after-depolarization, which were more delayed than the AHP present at baseline (Fig. 9). Although there were no after-depolarizations under baseline conditions, these appeared in 7 of 11 neurons during application of BayK 8644, either with loss (Fig. 9A) or expansion (Fig. 9B) of the AHP. In contrast, only 2 small after-depolarizations developed during application of the selective blockers to 59 neurons (one cell each for SNX-111 and -Aga-IVA, P < 0.001). BayK 8644 decreased rheobase in all neurons, and 9 of 10 neurons either initiated repetitive firing during application of BayK 8644 or increased firing rate.

View larger version (8K): [in this window] [in a new window]   Figure 9. Representative AP traces showing divergent effects of ICa amplification with the L-type current agonist BayK 8644 (approximate concentration 10 µM) in three different neurons. Traces before and after (#1min, * 5 min, both represented as blue traces) BayK 8644 are superimposed at the same resting potential (dotted line). Display of the AP spike is truncated in B and C. In (A) the AHP was replaced by an after-depolarization (arrowhead) during Bay K 8644. Other neurons developed a new, late AHPs, either with an initial after-depolarization (B), or without one (C). Peak AP amplitude is indicated by arrows in A.

DISCUSSION

This study was designed to identify changes in membrane electrical properties of sensory neurons attributable to the loss of inward I_Ca, and thereby indicate the consequences of injury-related I_Ca loss, which we have previously identified. To this end, we manipulated ionic conditions and applied a variety of pharmacologic agents to DRG neurons in their nondissociated state, while recording membrane events in a manner least likely to disrupt intracellular Ca²⁺ handling. This approach lacks the ability to identify effects on isolated specific currents that patch-clamp membrane control provides, but it allows characterization of the natural interactions of the cell's full complement of membrane voltage-gated and Ca²⁺-activated channels. Moreover, processes that sequester and extrude intracellular Ca²⁺ remain largely intact. Therefore, the role of I_Ca can be put into a physiologically relevant context in the sensory neuron soma. Our observations collectively indicate that loss of I_Ca and depression of the intracellular Ca²⁺ level are accompanied by increased excitability of DRG neurons. Although similar events are likely in C-type neurons, this cannot be proved as those were not examined here.

At any moment, the neuronal transmembrane potential is the result of ionic conductance across the membrane through multiple channel types. The function of these channels is linked, since membrane voltage and intracellular Ca²⁺ regulate opening of these channels, but levels of both voltage and Ca²⁺ concentration are simultaneously controlled by currents through the various channels. Thus, it is impossible a priori to predict the final effect on AP dynamics of a change in a single channel. Central to this study is the specific case of divergent electrical effects that may arise from a loss of I_Ca. On the one hand, I_Ca flux depolarizes the membrane such that decreasing I_Ca should inhibit generation of APs and diminish axonal conduction success. However, the entry of a diminished amount of Ca²⁺ results in less recruitment of I_K(Ca), which in turn will lead to the excitatory effects of membrane depolarization and increased membrane resistance.33 In cerebellar Purkinje neurons, for instance, block of I_Ca enhances firing since the diminished outward K⁺ flux outweighs the decreased I_Ca flux.24 Further complexity is added by participation of I_Cl(Ca),2 a depolarizing current that competes with inward I_K(Ca). Even membrane hyperpolarization may either directly stabilize the neuron or create conditions for repetitive firing by phasically reactivating Na⁺ currents and LVA Ca²⁺ currents. As a result of the intricate interplay of these various currents, experimental observation is necessary to resolve the balance of effects in a natural system.

To determine the end result of decreased I_Ca, we modulated inward I_Ca using techniques each of which has imperfections. Bath Ca²⁺ withdrawal dependably decreases the driving force for ionic flux through the Ca²⁺ channels but may also change the surface potential and thus disrupt voltage gradients in the immediate microdomain of the channel.22 We compensated for this by reciprocal Mg²⁺ addition, although divalent cations may differ in extent of effects on various channels.23 A factor that further complicates interpretation of Ca²⁺ withdrawal experiments is the presence in neurons of a nonselective cation channel that is suppressed by external Ca²⁺,34,35 particularly in nociceptive sensory neurons.36 Calcium withdrawal may thus initiate a separate excitatory inward current, which potentially accounts for differences between findings with bath Ca²⁺ withdrawal and I_Ca blockade. Also, a direct interaction of Ca²⁺ with the Na⁺ channel pore has been demonstrated in which Na⁺ channels that are not Ca²⁺-occupied fail to close.37 We elevated bath Ca²⁺ to predictably increase the concentration gradient driving Ca²⁺ entry. This, however, does not necessarily produce opposite electrophysiological effects compared with Ca²⁺ withdrawal, for instance on AP duration,38 as may be the case if Ca²⁺-dependent processes are saturated in the baseline condition. Cadmium inhibits I_Ca globally, but lacks perfect selectivity since it may also minimally modulate Na⁺ currents24 and K⁺ currents.23 Although peptide toxins for HVA I_Ca subtypes are highly specific, there is no comparably specific toxin for T-type currents. We therefore used both nickel and mibefradil,39 which provided largely congruent results. Nitrendipine was chosen to block L-type current because of its greater specificity in DRG neurons compared to other dihydropyridine blockers.40 Despite these precautions, overlapping effects are noted even with the most selective toxins.41 Finally, modulation of excitability may conceivably also result through I_Ca regulation of release of regulatory peptides from glia and neurons in the DRG.42 Because of these various limitations, we used multiple complementary techniques for decreasing and increasing Ca²⁺ entry to resolve the role of I_Ca.

AP Inflection is Regulated by I_Ca Modulators
Withdrawal of bath Ca²⁺ and application of cadmium both reduced the incidence of inflection of the descending limb of sensory neuron APs, presumably through the depression of I_Ca. Blockade of individual I_Ca subtypes had no effect on AP inflection, perhaps indicating the partial contribution of several subtypes to this feature. Our findings confirm an observation on cranial nerve sensory neurons that identified a contribution of I_Ca to the inflection.38,43 This contrasts with the effect of nerve injury, which is accompanied by an increase in the frequency of AP inflection,6 despite the development of a concurrent loss of HVA and LVA Ca²⁺ currents.7,10 Since I_K(Ca) participates in repolarization of the AP and competes with I_Ca in the formation of the inflection,30,44 a possible explanation is that injury induces a proportionately greater loss of I_K(Ca) than I_Ca, through a decrease of Ca²⁺-activated K⁺ channel number or function. Indeed, we have recently identified such a postinjury deficit.45 Loss of voltage-gated K⁺ current20 might also contribute to more frequent inflections after injury. Although slow tetrodotoxin-resistant Na⁺ current also contributes to the AP inflection,38,46 injury is associated with a decrease of this current.47

Regulation of AP Variables by I_Ca Modulators
The RMP is depolarized and the AP amplitude is diminished by low bath Ca²⁺ levels but not by cadmium administration. This may be due to activation of an inward current during low extracellular Ca²⁺ conditions as noted above, although combined administration of HVA blockers also diminishes AP amplitude. Individual selective blockers have a divergent effect on AP amplitude, perhaps indicative of their variable contributions of I_Ca versus I_K(Ca) during AP depolarization and repolarization. The decrease of AP amplitude after nerve injury6 may thus be attributed either to loss of L-type current or simultaneous changes in Na⁺ channels. Selective blockers of I_Ca consistently prolong AP duration, presumably by a dominant indirect inhibition of inward I_K(Ca),24 and thus duplicate the effect of nerve injury.6 The lack of effect of low bath Ca²⁺ and cadmium on AP duration may result from a less complete loss of I_Ca. Alternatively, the difference may be due to secondary effects of low extracellular Ca²⁺ noted above. Increased AP duration may contribute to neuropathic pain by enhancing synaptic neurotransmitter release.48

Decrease of Inward Ca²⁺ Flux Diminishes AHP and Increases Excitability
Although Ca²⁺-independent K⁺ currents may contribute to the formation of the AHP,49,50 previous studies have shown a critical dependence of the AHP upon I_Ca,51–53 which our observations now confirm in adult DRG neurons. AHP regulates a critical aspect of neuronal excitability by determining spike frequency adaptation and the ability of sensory neurons to maintain rapidly firing pulse trains.15,54,55 In this study, repetitive firing was amplified by decreasing extracellular Ca²⁺, whereas elevated Ca²⁺ had the opposite effect, indicating that the increased excitability found in neurons after axonal injury may be the result of diminished I_Ca.

In various neuronal types, the source of Ca²⁺ that activates I_K(Ca) is not uniformly distributed among Ca²⁺ channel subtypes, due to colocalization of specific isoforms of Ca²⁺ channels and Ca²⁺-activated K⁺ channels.56,57 In contrast, we found in sensory neurons that N-type, P/Q-type, and L-type currents all contribute to the generation of the AHP, with a lesser role for R-type current, and that LVA current (T-type) contributes minimally to activation of channels producing the AHP. This may explain why selective blockade of I_Ca subtypes minimally contributed to repetitive firing (two cells of 43 became nonaccommodating), since burst firing is regulated by the combined contributions of more than a single channel subtype alone.

Enhancement of L-type current with the agonist BayK 8644 is associated with substantial prolongation of the AP and generation of after-depolarizations, which may result directly from sustained I_Ca flux or from activation of Cl^– channels. This latter option is supported by previous observations of large after-depolarizations through activation of I_Cl(Ca),2 and BayK 8644 has been reported to activate Ca²⁺-sensitive Cl^– current in rat DRG neurons.58 Repetitive firing was induced or enhanced by BayK 8644, and can be attributed to the influence of after-depolarizations.59 Heterogeneity is evident, however, since increased Ca²⁺ influx from BayK 8644 initiates a new, sustained AHP in a subset of neurons, which highlights the complexity of competing Ca²⁺ effects.

The rheobase current, which is the depolarizing current just adequate to initiate an AP, is decreased in A/β neurons during low bath Ca²⁺ and application of cadmium and most selective I_Ca blockers. We cannot explain the decrease in rheobase subsequent to lowering of I_Ca, but we note that it is not due in all cases to an increase in input resistance, such as might occur with decreased I_K(Ca) or I_h. Therefore, loss of I_Ca must lead to an increased intrinsic excitability of the neuronal membrane, making it easier for generator currents at natural and pathologic sensory transduction sites to initiate neuronal activity. The ubiquitous actions of Ca²⁺/calmodulin60 and Ca²⁺-sensitive kinases61–64 on a variety of membrane channels may contribute to membrane hyperexcitability during disruption of intracellular Ca²⁺ levels. Increased Ca²⁺ loading during Bay K 8644 application also destabilizes the sensory neuron, however, which indicates a system in which normal membrane excitability is maintained only within a certain range of intracellular Ca²⁺ concentrations.

Blockers of I_Ca Reduce Hyperpolarization-Induced Voltage Sag
Voltage sag as recorded in this study in sensory neurons is mostly due to the hyperpolarization-induced current I_h,15 which aids in setting the RMP and may generate excitability by contributing to the pacemaker potential of spontaneously active cells. However, I_h can also suppress excitation by decreasing input resistance in the resting neuron. In our study, depression of I_Ca decreased hyperpolarization-induced voltage sag. The loss of I_h in sensory neurons after bath Ca²⁺ withdrawal, application of I_Ca blockers, and intracellular Ca²⁺ buffering with EDTA and BAPTA-AM is similar in this respect to the effect of peripheral nerve injury.6 These findings indicate that cytoplasmic Ca²⁺ level regulates the hyperpolarization- induced inwardly rectifying current in sensory neurons, as has been reported for cortical and brainstem neurons,65,66 possibly by acting through the Ca²⁺-calmodulin signaling pathway and Ca²⁺/calmodulin-dependent protein kinase II activation.67 Nerve injury may additionally decrease expression of the channel underlying the I_h current.68

Implications for Neuropathic Pain
The findings of this study reveal that suppression of I_Ca increases DRG neuron excitability. Therefore, the loss of I_Ca associated with peripheral nerve injury may in part account for the hyperexcitability of the sensory pathway in neuropathic pain states. Furthermore, processes that decrease extracellular Ca²⁺ concentration may be expected to result in neuronal excitation. Both neural trauma69 and intense neuronal activity, such as happens at the moment of peripheral nerve injury, depress extracellular Ca²⁺ concentrations,70,71 and therefore may initiate processes that amplify sensory processing even in the absence of changes in Ca²⁺ channels.

These findings may appear to contradict reports of analgesia after intrathecal administration of Ca²⁺ channel blockers in experimental nerve injury72 or clinical neuropathic pain.73 However, neurophysiological processes, including Ca²⁺ signaling, are spatially distinct. For instance, relief of pain by intrathecal I_Ca blockade may result from interference with neurotransmitter release in the spinal cord, which is not applicable at peripheral sites. Block of I_Ca at the spinal nerve ligation injury site has no analgesic effect.72 Our findings point to the possibility that medications anatomically and pharmacologically targeted at increasing I_Ca flux of sensory neurons in the DRG may provide novel treatments for neuropathic pain.

Footnotes

Accepted for publication March 27, 2008.

Supported by National Institutes of Health grant NS-42150.

Address for correspondence and reprint requests to Quinn Hogan, MD, Department of Anesthesiology, MEB, Department of Anesthesiology, 8701 Watertown Plank Rd, Milwaukee, WI 53226. Address e-mail to qhogan{at}mcw.edu.

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