Upregulation of the Hyperpolarization-Activated Cation Current after Chronic Compression of the Dorsal Root Ganglion
Departments of 1 Anesthesiology and 2 Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510q/#s*, 百拇医药
ABSTRACTq/#s*, 百拇医药
A chronic compression of the DRG (CCD) produces cutaneous hyperalgesia and an enhanced excitability of neuronal somata in the compressed ganglion. The hyperpolarization-activated current (Ih), present in the somata and axons of DRG neurons, acts to induce a depolarization after a hyperpolarizing event and, if upregulated after CCD, may contribute to enhanced neuronal excitability. Whole-cell patch-clamp recordings were obtained from acutely dissociated, retrogradely labeled, cutaneous, adult rat DRG neurons of medium size. Neurons were dissociated from L4 and L5 control DRGs or DRGs that had each been compressed for 5-7 d by L-shaped rods inserted into the intervertebral foramina. Ih, consisting of a slowly activating inward current during a step hyperpolarization, was recorded from every labeled, medium-sized neuron and was blocked by 1 mM cesium or 15 µM ZD7288. Compared with control, CCD increased the current density and rate of activation significantly without changing its reversal potential, voltage dependence of activation, or rate of deactivation. Because Ih activation provides a depolarizing current to the neuron, thus enhancing neuronal excitability, our results are consistent with the hypothesis that Ih contributes to hyperalgesia after CCD-induced nerve injury.
Key words: hyperpolarization-activated current; Ih; ion channels; dorsal root ganglion compression; neuropathic pain; ratsl4!1h, 百拇医药
Introductionl4!1h, 百拇医药
A chronic compression of the L4 and L5 dorsal root ganglia (CCD) produces cutaneous hyperalgesia and tactile allodynia on the ipsilateral foot in the rat (Song et al., 1999). The cell bodies (somata) of the dorsal root ganglia (DRGs) exhibit signs of hyperexcitability, such as a lower rheobase, and an increased incidence of spontaneous activity (Hu and Xing, 1998; Song et al., 1999; Zhang et al., 1999). These alterations in the membrane properties of DRG somata might contribute to the cutaneous hyperalgesia and allodynia after CCD. However, the underlying ionic mechanisms of the increased somal excitability after CCD are not well understood.l4!1h, 百拇医药
One type of current that might contribute to the presence and frequency of spontaneous activity after CCD is Ih. Ih current, carried by hyperpolarization-activated, cyclic nucleotide-gated channels (HCN), is a nonselective cation current activated by a membrane hyperpolarization occurring, for example, at the end of an action potential. The activation of Ih produces an inward current that slowly depolarizes the membrane (Pape, 1996). As a pacemaker current, Ih contributes to the rate of spontaneous rhythmic oscillations in the heart and in the brain (DiFrancesco, 1993; Luthi and McCormick, 1998). Ih is also present in DRG neurons (Mayer and Westbrook, 1983; Scroggs et al., 1994; Villiere and McLachlan, 1996; Yagi and Sumino, 1998; Cardenas et al., 1999) and is implicated in increasing discharge rate to excitatory stimuli by limiting membrane hyperpolarization and facilitating depolarization (Yagi and Sumino, 1998). Stimuli that modulate Ih can alter neuronal excitability. For example, certain inflammatory mediators, such as serotonin, increase intracellular cAMP, which, in turn, increases Ih current by binding to the HCN channel and shifting the voltage dependence of activation (Raes et al., 1997; Cardenas et al., 1999).
In the present study, we examined the biophysical properties of Ih current in cutaneous, medium-sized somata acutely dissociated from DRGs of CCD and unoperated control rats. Our hypothesis was that CCD induces an increase in the magnitude and/or an alteration in the kinetics of activation-deactivation of Ih.gk\!, 百拇医药
Materials and Methodsgk\!, 百拇医药
Labeling of cutaneous neurons and CCD surgery. Twenty-two female Sprague Dawley rats (140-160 gm) were anesthetized with pentobarbital (40 mg/kg, i.p.). Cutaneous afferent neurons were retrogradely labeled by injecting Fluorogold solution in saline (1%, 0.05 ml; Molecular Probes, Eugene, OR) subcutaneously into the right lateral plantar region (Oyelese and Kocsis, 1996). To determine whether there was dye leakage from the subcutaneous space to the neighboring muscle, True Blue (1.5%, 0.05 ml; Molecular Probes) was injected into the exposed gastrocnemius and soleus muscles (Liu et al., 2002). Immediately after the injections, in CCD rats (n = 11), the ipsilateral, right transverse process and intervertebral foramina of L4 and L5 were exposed as described previously (Hu and Xing, 1998; Song et al., 1999). A stainless steel L-shaped rod (0.63 mm in diameter and 4 mm in length) was inserted into each foramen, one at L4 and the other at the L5 ganglion. The remaining 11 rats received no additional surgery and were used as controls.
Cell preparation. Five to 7 d after surgery, the rats were deeply anesthetized with pentobarbital (40 mg/kg, i.p.), and the L4 and L5 lumbar DRGs were exposed. In CCD rats, the correct placement of each implanted rod was confirmed. Only one DRG was discarded because of incorrect placement of the rod. DRGs were removed from control or CCD rats and placed in complete saline solution (CSS) for cleaning and mincing. The CSS contained the following (in mM): 137 NaCl, 5.3 KCl, 1 MgCl2, 3 CaCl2, 25 sorbitol, and 10 HEPES. The DRGs were then digested for 15 min with collagenase A (1 mg/ml; Boehringer Mannheim, Mannheim, Germany) and for another 15 min with collagenase D (1 mg/ml; Boehringer Mannheim) and papain (30 U/ml; Worthington, Lakewood, NJ) in CSS containing 0.5 mM EDTA and 2 µg of cysteine at 37°C as described previously (Rizzo et al., 1995). The cells were dissociated by trituration in culture medium containing 1 mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor (Boehringer Mannheim) and plated on glass coverslips coated with polyornithine and laminin. The culture medium contained equal amounts of DMEM (Invitrogen, San Diego, CA) and F-12 (Invitrogen) with 10% FCS (HyClone, Logan, UT) and 1% penicillin and streptomycin (Invitrogen). The cells were incubated at 37°C (5% CO2 balance air) for 1 hr, after which culture medium without the inhibitor was added.
Electrophysiological recording and drug application. Coverslips were transferred to a recording chamber that was mounted on the stage of an upright microscope (BX50-WI; Optical, Tokyo, Japan). The chamber was filled with bath solution containing the following (in mM): 125 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 10 TEA-Cl, 3 4-AP, 2 MnCl2, 1 BaCl2, and 0.001-0.005 TTX. The pH was adjusted to 7.4 with NaOH, and osmolarity was adjusted with sucrose to 290 mOsm. Neurons selected for recording were 35-45 µm in diameter and were positive for Fluorogold fluorescence and negative for True Blue fluorescence. Electrodes were fabricated from borosilicate glass Sarasota, FL) and pulled on a Flaming/Brown micropipette puller (Sutter P-97; Sutter Instrument, Novato, CA). The pipette solution contained the following (in mM): 120 K-aspartate, 18 KCl, 1 CaCl2, 2 MgCl2, 5 EGTA, 10 HEPES, 5 Na2-ATP, and 0.4 Na-GTP. The pH was adjusted to 7.2 with KOH, and osmolarity was 295 mOsm (pipette resistance, 1-2 M).
Neurons were recorded at room temperature in the whole-cell mode (Multiclamp 700A; , Union City, CA) using pClamp 8.01 software . Data were filtered at 3 kHz and digitized at 20 kHz. The voltage drop across the access resistance was compensated at 40-80%, resulting in a voltage error of <8 mV. Cesium and ZD7288 were applied locally through a perfusion pipette (100 µm in diameter) using a drug application system (ALA Scientific Instruments, Long Island, NY). All of the chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated.0mg3%, 百拇医药
Data analysis. Data were fit using pClampfit 8.1 and Origin 6.0 (Microcal Software, Northampton, MA). The goodness of fit was determined either by the "model comparison" method in pClampfit 8.1 or the logarithm of the error ratio criterion (Horn, 1987). Data are expressed as mean ± SD unless otherwise indicated. Tests for statistical differences between means for CCD and control neurons were determined with either Student's t tests or repeated-measures ANOVA (RMANOVA), followed by post hoc pairwise comparisons [Tukey's honestly significant difference (HSD) test]. The criterion for statistical significance was a value of p <0.05.
Results'@u#ds, 百拇医药
CCD increases the density of Ih current'@u#ds, 百拇医药
A total of 254 DRG neurons were recorded, 120 from 11 control and 134 from 11 CCD rats. There was no difference in cell size for the two groups. The whole-cell capacitance was 78.7 ± 3.0 pF (n = 51) for control and 74.8 ± 3.6 pF (n = 34) for CCD neurons (Student's t test; p = 0.41). To activate Ih, hyperpolarizing potentials of 60 to 120 mV were delivered in increments of 10 mV from a holding potential of 50 mV ( inset). Slowly activating, inward currents (top row) were blocked by the addition of cesium (1 mM) to the bath solution middle row), consistent with an Ih current. Cesium blocked 95.5 ± 2.4% of the inward current in control neurons (n = 12) and 95.9 ± 3.1% in CCD neurons (n = 12). In other experiments, ZD7288 (15 µM), a specific Ih channel blocker (BoSmith et al., 1993; Gasparini and DiFrancesco, 1997; Satoh and Yamada, 2000), showed a similar inhibitory effect, blocking 91.0 ± 5.2% of the inward current in CCD neurons (n = 12) and 96.5 ± 3.3% in control neurons (n = 12). The inhibition by cesium but not by ZD7288 was reversed during 30 min of washout (bottom row). The sensitivity of Ih to these inhibitors is consistent with that described previously for Ih (Yagi and Sumino, 1998; Cardenas et al., 1999). The magnitude of Ih current increased with increasing hyperpolarization (top row). At 60 mV, the mean magnitude of Ih was 22.9 ± 5.7 pA for control neurons and 53.6 ± 10.9 pA for CCD neurons. At 120 mV, Ih was 698.6 ± 85.2 pA (n = 34) for control and 1156.7 ± 115.1 pA (n = 51) for CCD neurons. When normalized to cell capacitance, CCD neurons had a significant increase in Ih current density by 66.6-61.5% compared with control neurons over the voltage range of 90 to 120 mV (control, n = 34; CCD, n = 51; two-way RMANOVA, p < 0.05) . For example, at 100 mV, the current density was 7.4 ± 0.9 pA/pF for control and 12.4 ± 1.3 pA/pF for CCD neurons, a 67% increase in the latter group
fig.ommtted?*dul2, 百拇医药
The effects of CCD on the density of Ih current. A, Current responses evoked by hyperpolarizing voltage steps from a holding potential of 50 mV in a control (left) and a CCD (right) DRG neuron. The voltage protocol is shown at the bottom. Ih current amplitude was calculated from the difference between the steady-state and the instantaneous currents (i.e., the difference between the two arrows shown as an example at the last trace of the left top traces). In both panels, families of current responses on the top were recorded in the control bath solution. Middle traces, Cs+ at 1 mM. Bottom traces, After 2 min of washout. Voltage protocol is shown as an inset at bottom. B, Ih current density versus membrane potential, Vm. Squares and circles represent the data summarized from 34 control and 51 CCD neurons, respectively. Error bars indicate SEM. * Indicates that means at the same membrane voltage are significantly different.?*dul2, 百拇医药
CCD increases the conductance but not the reversal potential of Ih current
The reversal potential and the maximal Ih conductance were measured by first applying a prepulse to 100 mV to fully activate Ih and then examining the tail currents after repolarization to test potentials from 90 to 50 mV ( inset). The time point for measurement of the peak tail current is shown in(arrows). For each test voltage, the peak tail current, after subtraction of the estimated leak current, was averaged for 31 control and 31 CCD neurons, and each was fitted with a linear regression equation, as follows: Itail = Gh.max(Vm Vrev), where Itail is the peak tail current, Vm is the membrane potential of the test pulse, Gh.max is the maximal conductance, and Vrev is the reversal potential of Ih current . This fitting yielded estimates of Gh.max, Vrev of 0.15 nS/pF, 21 mV for the control neuron and 0.23 nS/pF, 23 mV for the CCD neuron. The mean Vrev values of 21.3 ± 1.4 mV (n = 31) for control neurons and 22.3 ± 1.0 mV (n = 31) for CCD neurons were not significantly different (Student's t test; p = 0.55). As expected from the data in, CCD neurons exhibited a significant increase (76%) in maximal Ih conductance, from 0.13 ± 0.01 nS/pF (n = 31) in control neurons to 0.23 ± 0.02 nS/pF (n = 31) in CCD neurons (Student's t test; p < 0.01).
fig.ommtted$., 百拇医药
Effects of CCD on the conductance and reversal potential of Ih current. A, In a control (top) and a CCD (bottom) neuron, Ih was fully activated by a hyperpolarizing prepulse to 100 mV for 1 sec, followed by a depolarization to test potentials of 90 to 50 mV (inset). Tail currents were masured at the start of the test potentials (arrows). B, Leak-subtracted tail current versus test potential. Squares and circles represent control and CCD neurons, respectively. The solid lines are the linear regressions fitted to the data points for each group. The slopes of the linear regressions were 0.15 and 0.23 nS/pF for control and CCD, respectively. The reversal potentials were 21 and 23 mV for control and CCD, respectively.$., 百拇医药
CCD does not change the voltage dependence of Ih activation$., 百拇医药
The activation curve of Ih current was estimated by measuring the tail current at 120 mV after application of prepulse potentials between 40 and 120 mV . Tail currents were normalized to the maximal current (obtained at a prepulse of - 120 mV). The activation curve for each neuron was fitted with a Boltzmann equation of the following form: Itail/Itail(max) = (1 + e((Vm V0.5)/k))1, where Vm is the membrane potential of the prepulse, V0.5 is the membrane potential at which Ih conductance is half-activated, k is a slope factor, Itail is the peak amplitude of the tail current recorded immediately after the prepulse, and Itail(max) is the tail current recorded after the maximal prepulse of 120 mV. V0.5 was measured from the activation curve , and values were not significantly different for control (74.2 ± 4.6 mV; n = 34) vs CCD (73.5 ± 4.6 mV; n = 51; Student's t test; p = 0.52) neurons. The slope factors for each group were not significantly different (6.2 ± 1.8 mV for control and 6.6 ± 1.4 mV for CCD neurons; Student's t test; p = 0.19).
fig.ommttedlk;v\y$, http://www.100md.com
Effects of CCD on the voltage dependence of Ih activation. A, Ih was activated from a holding potential of 50 mV to prepulse potentials ranging from 40 to 120 mV, followed by a step to a test potential of 120 mV (voltage protocol as shown at bottom). The magnitude of the tail current at the start of the test potential was used as an index of Ih activation (arrows in insets a and b). The tail currents of a control (top) and a CCD (middle) neuron are shown. B, Activation curves obtained by fitting the data of a control (squares) and a CCD (circles) neuron (as shown in A, top and middle, respectively) with single Boltzmann functions. The fitting yielded midpoint potentials, V0.5, and slope factors, k, of 76.2 and 5.3 mV for control and 77.4 and 6.5 mV for CCD neurons, respectively.lk;v\y$, http://www.100md.com
CCD increases the rate of activation of Ih currentlk;v\y$, http://www.100md.com
Ih was activated by a series of hyperpolarizing test potentials of 60 to 120 mV from a holding potential of 50 mV ( inset). Test potentials of shorter duration were used at more hyperpolarized potentials to avoid damaging the cell membrane. The time course of activation of Ih was fitted to first- and second-order exponential functions. On the basis of the criterion of Horn (1987), the current traces were not well described by a single exponential function but rather by the sum of two exponential functions. The equation was of the following form: Ih(t) = Af e(t/f) + Ase(t/s), where Ih(t) is the amplitude of the current at time t, and Af and As are the amplitude coefficients of the fast (f) and slow (s) time constants, respectively. The relative proportion of the total current in the slow and fast components did not differ significantly across activation potential or between control and CCD neurons (two-way RMANOVA, p > 0.05). In general, Ih current activated faster at more hyperpolarized potentials . For example, from 80 to 120 mV, the mean f decreased from 302 to 68 msec in control neurons and from 175 to 52 msec in CCD neurons . f was significantly faster in CCD than in control neurons for voltages of 70 to 90 mV (n = 17 for control; n = 21 for CCD; two-way RMANOVA; Tukey's HSD; p < 0.05) . From 80 to 120 mV, the mean s decreased from 1134 to 636 msec in control neurons and from 925 to 445 msec in CCD neurons. s was significantly faster in CCD than in control neurons for voltages of 90 to 110 mV (n = 17 for control; n = 21 for CCD; two-way RMANOVA; Tukey's HSD; p < 0.05) .
fig.ommtted)62t.ne, http://www.100md.com
Effect of CCD on the activation kinetics of Ih current. A, Current responses of a control (top) and a CCD (bottom) neuron each evoked by a series of hyperpolarizing voltage steps of 60 to 120 mV from a holding potential of 50 mV. Step duration was varied from 2150 to 1150 msec (inset at bottom right). Each solid line is a fitting of the sum of two exponentials to the data points (open circle) obtained at each hyperpolarizing voltage. Note the faster activation of Ih currents at each voltage in the CCD neuron. B, The mean ratio of Af to Af + As versus membrane potential for control (squares) and CCD (circles) neurons. C, D, The mean time constants f (C) and s (D) versus membrane potential for control (squares) and CCD (circles). * Indicates that means are significantly different.)62t.ne, http://www.100md.com
The deactivation kinetics were estimated using an envelope technique described by Mayer and Westbrook (1983). Neurons were subjected to a prepulse potential of 110 mV to fully activate Ih, followed by a repolarization (deactivation) to a test potential of varying duration, followed, in turn, by a return to 90 mV. The magnitude of deactivation was calculated by measuring the amount of Ih remaining after the test potential. The measurements were obtained at test potentials of 60, 50, 40, and 30 mV .
fig.ommtted]o9l|, 百拇医药
Effects of CCD on the deactivation of Ih current. A, Current response (top) of a control neuron evoked by a triple-pulse voltage protocol (bottom). A hyperpolarizing potential of 110 mV was followed by different durations of a depolarization to 30 mV to deactivate Ih current and a hyperpolarization to 90 mV to reactivate Ih (the number of traces was reduced for clarity). The amplitude of Ih current (shown as an example in the right trace) activated by the third pulse was used to calculate the deactivation time constant. B, Current responses of a control cell (same cell as in A) to the triple protocol but with deactivating potentials of 30, 40, 50, and 60 mV (a-d, respectively). Current traces were truncated at the end of b-d to keep the same time scale as that in a. The number of data points in each current trace in both A and B were reduced to . C, The effect of potential on the time course of Ih current deactivation. Data points are fit by a single exponential. D, Mean time constant of deactivation of Ih current versus membrane potential for control and CCD neurons (squares and circles, respectively). The single exponential functions fitted to the data points for each group are not significantly different for CCD and control neurons.
Because the data were not equally spaced in this experiment, the Levenberg-Marquardt method (Clampfit; ) was used for curve fitting. A single exponential equation fitted the data best as determined using a model comparison procedure (pClampfit) (. The deactivation of Ih became faster when membrane potentials were more depolarized. For example, in a control neuron, the deactivation time constants were 512.3, 342.6, 228.2, and 141.4 msec at potentials of 60, 50, 40, and 30 mV, respectively . The deactivation time constants of Ih were plotted as a function of test potential for control and CCD neurons. The functions were well described by (msec) = 15.8 + 9.9 exp(Vm/17) for control and (msec) = 15.6 + 12.1 exp(Vm/18) for CCD neurons. The deactivation time constants of Ih from both groups showed a clear voltage dependence, with an e-fold decrease of 17 mV (control) or 18 mV (CCD) depolarization . However, at each depolarizing voltage, the deactivation time constants were not significantly different between the two groups (n = 19 for control and n = 23 for CCD neurons; two-way RMANOVA; p > 0.05) . For example, mean time constants were 73.4 ± 7.0 (control) and 77.0 ± 6.2 (CCD) msec at 30 mV.
Ih does not contribute to the resting membrane potentials of control or CCD neurons998e%|{, 百拇医药
To determine whether Ih contributes to resting membrane potential, neurons were recorded in the current-clamp mode with no holding current. A periodic hyperpolarizing pulse (0.3 to 0.7 nA, 200 msec) was applied every 3 sec for the measurement of membrane resistance and to demonstrate the presence of "depolarization sag," the latter an index of Ih current. Under control conditions, there was no significant difference between the mean resting potentials of control and CCD neurons (58 ± 4 mV (n = 24) and 57 ± 6 mV (n = 17), respectively; Student's t test; p = 0.42). Application of Ih channel blockers Cs+ (1 mM) and ZD7288 (15 µM) eliminated the depolarization sag ( bottom) but did not change resting membrane potential (top).998e%|{, 百拇医药
fig.ommtted998e%|{, 百拇医药
The effects of Ih on the resting membrane potential. Current-clamp recordings of the resting membrane potential (Vrest) of a control (A) and a CCD (B) neuron. The respective initial values of Vrest for the control and CCD neurons were 53 and 56 mV. Current pulses of 0.6 nA, 200 msec, were injected into each cell every 3 sec to elicit a hyperpolarization and sag in voltage response. The horizontal bars indicate the durations of application of the Ih blockers Cs+ (1 mM) and ZD7288 (15 µM). A, B, Top trace, Voltage responses to the current injection; bottom trace, enlarged voltage traces to show the reduction in sag produced by the blocker. Note that, in both control and CCD neurons, both 1 mM Cs+ and 15 µM ZD7288 did not alter resting potential but had similar effects in abolishing the sag.
Discussion48c2v|, 百拇医药
The major finding of this study is that CCD increased the density of Ih current in DRG somata because of an increase in Ih conductance and also increased the rate of activation of Ih current.48c2v|, 百拇医药
Characterization of Ih in DRG neurons48c2v|, 百拇医药
The kinetics of activation of Ih observed in this study are generally consistent with that described previously by other laboratories. We found that the activation kinetics of Ih are voltage dependent and are best described by a double exponential in rat DRG neurons. The f and s were similar to values reported for neonatal rat DRG neurons (Wang et al., 1997). The complicated kinetics of activation may reflect the presence of multiple isoforms of the Ih channel. Four members of a gene family encoding mammalian HCN channels (HCN1-HCN4) have been cloned in recent years. When expressed in HEK 293 cells, recombinant HCN1-HCN4 channels demonstrate unique activation kinetics, i.e., with the fastest time constant for HCN1 and gradually slower time constants for HCN2-HCN4 (Moosmang et al., 2001). The two distinct time constants obtained in our preparation may indicate that two kinds of homomeric channels exist in DRG neurons, as has been proposed for sinoatrial node cells (Moosmang et al., 2001). This is consistent with the observation of Chaplan et al. (2001) that both HCN1 and HCN3 isoforms are present in rat DRG.
The parameters of steady-state activation of Ih observed in our recordings are consistent with those reported previously for DRG neurons in rodents. We obtained a half-activation voltage of 74 mV, consistent with the value (73 mV) obtained previously for medium-sized rat DRG neurons (Cardenas et al., 1999). Our V0.5 value is more positive than those of neonatal rat DRG neurons (Wang et al., 1997), possibly because of the absence of ATP in their recording pipette. ATP has been shown to cause a depolarizing shift of V0.5 in mouse DRG neurons (Raes et al., 1997). The Ih conductance of 0.13 nS/pF at 100 mV is within range of values obtained in a variety of neuronal types (Pape, 1996), and our reversal potential value of 21 mV is virtually identical to those reported previously for DRG neurons in rodents (Raes et al., 1997; Wang et al., 1997).3, 百拇医药
Effect of CCD on Ih3, 百拇医药
A major finding of our study is that the magnitude of Ih increased after CCD. The immediate cause of the increase in Ih may be complicated, because this model is presumably associated with ischemia-hypoxia and inflammation. A local inflammation and acidosis could recruit mast cells and macrophages that release inflammatory mediators, such as 5-HT, prostaglandin E2, ATP, NGF, and cytokines (Millan, 1999. These mediators can activate cellular mechanisms that alter the expression of HCN channels, change the intrinsic properties of HCN channels, and/or increase the amount of cAMP binding to the Ih channel or enhancing the phosphorylation processes. Results using other models of neuropathic pain have had inconsistent effects on Ih. After transection of the sciatic nerve, Ih was found to decrease in one study (Abdulla and Smith, 2001), but, in another study, transection of the spinal nerve caused Ih to increase (Chaplan et al., 2001). A reconciliation among these models and observations is not immediately obvious.
There are several possible explanations for the increase in magnitude of Ih observed in this study. (1) There may be a change in HCN isoform, as occurs for sodium currents after peripheral axotomy (Dib-Hajj et al., 1999; Ishikawa et al., 1999; Kim et al., 2001). However, we believe this to be unlikely, because CCD did not alter the ratio of Af/(Af + As). Thus, if there are two homomers of the HCN channel, neither of them became dominant after CCD. (2) A "beta " -subunit for the HCN channel family may exist (Yu et al., 2001), and changes in its expression could modify the Ih current properties, as has been observed in Na+, Ca2+, and K+ channels (Isom et al., 1994). (3) Intracellular messengers, particularly cAMP, may have modified Ih. HCN channels have a cyclic binding domain located in the C terminal (Wainger et al., 2001) and PKA consensus phosphorylation sites (Santoro et al., 1998; Gauss and Seifert, 2000; Vargas and Lucero, 2002). Electrophysiological studies have shown that cAMP enhances Ih by altering the voltage dependence of activation (Ingram and Williams, 1996; Cardenas et al., 1999). However, we did not observe a change in activation properties after CCD in our experiments. These possibilities provide several possible paths for the pursuit of additional experiments.
Functional implications of increased Ih in CCD neurons&*, http://www.100md.com
CCD increases the excitability of DRG neurons, as indicated by an increased incidence of spontaneous activity and subthreshold oscillations in membrane potential and lowered current and action potential thresholds (Hu and Xing, 1998; Song et al., 1999; Zhang et al., 1999; Xing et al., 2001). The increased Ih current conductance we observed may account for the higher incidence of spontaneous activity and contribute to neuropathic pain. The latter is supported by the observation that ZD7288 blocked neuropathic pain caused by spinal nerve ligation (Chaplan et al., 2001).&*, http://www.100md.com
In CCD neurons, a repolarization of an action potential will produce a greater depolarization because of increased Ih and thus have a greater chance of evoking another action potential, possibly enhancing the occurrence of repetitive discharge either autonomously or in response to excitatory stimuli. For example, in rat DRG neurons, an increase of Ih by 5-HT increased the number of anode break discharges. A reduction of Ih by clonidine decreased the rate of evoked multispike intervals (Yagi and Sumino, 1998; Cardenas et al., 1999). In hippocampal neurons, inhibition of Ih by its blocker ZD7288 reduced the frequency of spontaneous activity and blocked the evoked multispike discharge (Maccaferri and McBain, 1996; Gasparini and DiFrancesco, 1997). An increase of Ih mediated through the -adrenergic receptor by the -agonist isoproterenol (ISP) increased the frequency of spontaneous spikes recorded from rat cerebellar basket cells, whereas ZD7288 decreased the spike frequency and abolished the ISP-induced increase in spike discharges (Saitow and Konishi, 2000).
In conclusion, the increase in Ih current density that we observed after CCD could contribute to an increased incidence of spontaneous activity and subthreshold oscillations in membrane potential and thus to the genesis of neuropathic pain.ay9@[, 百拇医药
Referencesay9@[, 百拇医药
Abdulla FA, Smith PA (2001) Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons. J Neurophysiol 85:644-658 .ay9@[, 百拇医药
BoSmith RE, Briggs I, Sturgess NC (1993) Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br J Pharmacol 110:343-349 .ay9@[, 百拇医药
Cardenas CG, Mar LP, Vysokanov AV, Arnold PB, Cardenas LM, Surmeier DJ, Scroggs RS (1999) Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J Physiol (Lond) 518:507-523 .ay9@[, 百拇医药
Chaplan SR, Guo HQ, Lee DH, Butler MP, Velumian A, Dubin AE (2001) Markedly increased DRG Ih and HCN mRNA accompany neuropathic pain in the rat. Soc Neurosci Abstr 27:54.11 .
Dib-Hajj SD, Fjell J, Cummins TR, Zheng Z, Fried K, LaMotte R, Black JA, Waxman SG (1999) Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic pain. Pain 83:591-600 .4q, http://www.100md.com
DiFrancesco D (1993) Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55:455-472 .4q, http://www.100md.com
Gasparini S, DiFrancesco D (1997) Action of the hyperpolarization-activated current (Ih) blocker ZD 7288 in hippocampal CA1 neurons. Pflügers Arch 435:99-106 .4q, http://www.100md.com
Gauss R, Seifert R (2000) Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels. Chronobiol Int 17:453-469 .4q, http://www.100md.com
Horn R (1987) Statistical methods for model discrimination: applications to gating kinetics and permeation of the acetylcholine receptor channel. Biophys J 51:255-263 .4q, http://www.100md.com
Hu SJ, Xing JL (1998) An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain 77:15-23 .4q, http://www.100md.com
Ingram SL, Williams JT (1996) Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons. J Physiol (Lond) 492:97-106 .
Ishikawa K, Tanaka M, Black JA, Waxman SG (1999) Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy. Muscle Nerve 22:502-507 .qx, 百拇医药
Isom LL, De Jongh KS, Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12:1183-1194 .qx, 百拇医药
Kim DS, Yoon CH, Lee SJ, Park SY, Yoo HJ, Cho HJ (2001) Changes in voltage-gated calcium channel alpha(1) gene expression in rat dorsal root ganglia following peripheral nerve injury. Brain Res Mol Brain Res 96:151-156 .qx, 百拇医药
Liu CN, Devor M, Waxman SG, Kocsis JD (2002) Subthreshold oscillations induced by spinal nerve injury in dissociated muscle and cutaneous afferents of mouse DRG. J Neurophysiol 87:2009-2017 .qx, 百拇医药
Luthi A, McCormick DA (1998) H-current: properties of a neuronal and network pacemaker. Neuron 21:9-12 .qx, 百拇医药
Maccaferri G, McBain CJ (1996) The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J Physiol (Lond) 497:119-130 .
Mayer ML, Westbrook GL (1983) A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones. J Physiol (Lond) 340:19-45 .y*), 百拇医药
Millan MJ (1999) The induction of pain: an integrative review. Prog Neurobiol 57:1-164 .y*), 百拇医药
Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A (2001) Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem 268:1646-1652 .y*), 百拇医药
Oyelese AA, Kocsis JD (1996) GABAA-receptor-mediated conductance and action potential waveform in cutaneous and muscle afferent neurons of the adult rat: differential expression and response to nerve injury. J Neurophysiol 76:2383-2392 .y*), 百拇医药
Pape HC (1996) Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58:299-327 .y*), 百拇医药
Raes A, Wang Z, van den Berg RJ, Goethals M, Van de Vijver G, van Bogaert PP (1997) Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons. Pflügers Arch 434:543-550 .
Rizzo MA, Kocsis JD, Waxman SG (1995) Selective loss of slow and enhancement of fast Na+ currents in cutaneous afferent dorsal root ganglion neurones following axotomy. Neurobiol Dis 2:87-96 .:?, 百拇医药
Saitow F, Konishi S (2000) Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells. J Neurophysiol 84:2026-2034 .:?, 百拇医药
Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR (1998) Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717-729 .:?, 百拇医药
Satoh TO, Yamada M (2000) A bradycardiac agent ZD7288 blocks the hyperpolarization-activated current (I(h)) in retinal rod photoreceptors. Neuropharmacology 39:1284-1291 .:?, 百拇医药
Scroggs RS, Todorovic SM, Anderson EG, Fox AP (1994) Variation in IH, IIR, and ILEAK between acutely isolated adult rat dorsal root ganglion neurons of different size. J Neurophysiol 71:271-279 .:?, 百拇医药
Song XJ, Hu SJ, Greenquist KW, Zhang JM, LaMotte RH (1999) Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia. J Neurophysiol 82:3347-3358 .
Vargas G, Lucero MT (2002) Modulation by PKA of the hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons. J Membr Biol 188:115-125 .9, 百拇医药
Villiere V, McLachlan EM (1996) Electrophysiological properties of neurons in intact rat dorsal root ganglia classified by conduction velocity and action potential duration. J Neurophysiol 76:1924-1941 .9, 百拇医药
Wainger BJ, DeGennaro M, Santoro B, Siegelbaum SA, Tibbs GR (2001) Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature 411:805-810 .9, 百拇医药
Wang Z, Van Den Berg RJ, Ypey DL (1997) Hyperpolarization-activated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture. J Neurophysiol 78:177-186 .9, 百拇医药
Xing JL, Hu SJ, Long KP (2001) Subthreshold membrane potential oscillations of type A neurons in injured DRG. Brain Res 901:128-136 .9, 百拇医药
Yagi J, Sumino R (1998) Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons. J Neurophysiol 80:1094-1104 .9, 百拇医药
Yu H, Wu J, Potapova I, Wymore RT, Holmes B, Zuckerman J, Pan Z, Wang H, Shi W, Robinson RB, El-Maghrabi MR, Benjamin W, Dixon J, McKinnon D, Cohen IS, Wymore R (2001) MinK-related peptide 1: a beta subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ Res 88:E84-E87 .9, 百拇医药
Zhang JM, Song XJ, LaMotte RH (1999) Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion. J Neurophysiol 82:3359-3366 .(Hang Yao David F. Donnelly Chao Ma and Robert H. LaMotte)
ABSTRACTq/#s*, 百拇医药
A chronic compression of the DRG (CCD) produces cutaneous hyperalgesia and an enhanced excitability of neuronal somata in the compressed ganglion. The hyperpolarization-activated current (Ih), present in the somata and axons of DRG neurons, acts to induce a depolarization after a hyperpolarizing event and, if upregulated after CCD, may contribute to enhanced neuronal excitability. Whole-cell patch-clamp recordings were obtained from acutely dissociated, retrogradely labeled, cutaneous, adult rat DRG neurons of medium size. Neurons were dissociated from L4 and L5 control DRGs or DRGs that had each been compressed for 5-7 d by L-shaped rods inserted into the intervertebral foramina. Ih, consisting of a slowly activating inward current during a step hyperpolarization, was recorded from every labeled, medium-sized neuron and was blocked by 1 mM cesium or 15 µM ZD7288. Compared with control, CCD increased the current density and rate of activation significantly without changing its reversal potential, voltage dependence of activation, or rate of deactivation. Because Ih activation provides a depolarizing current to the neuron, thus enhancing neuronal excitability, our results are consistent with the hypothesis that Ih contributes to hyperalgesia after CCD-induced nerve injury.
Key words: hyperpolarization-activated current; Ih; ion channels; dorsal root ganglion compression; neuropathic pain; ratsl4!1h, 百拇医药
Introductionl4!1h, 百拇医药
A chronic compression of the L4 and L5 dorsal root ganglia (CCD) produces cutaneous hyperalgesia and tactile allodynia on the ipsilateral foot in the rat (Song et al., 1999). The cell bodies (somata) of the dorsal root ganglia (DRGs) exhibit signs of hyperexcitability, such as a lower rheobase, and an increased incidence of spontaneous activity (Hu and Xing, 1998; Song et al., 1999; Zhang et al., 1999). These alterations in the membrane properties of DRG somata might contribute to the cutaneous hyperalgesia and allodynia after CCD. However, the underlying ionic mechanisms of the increased somal excitability after CCD are not well understood.l4!1h, 百拇医药
One type of current that might contribute to the presence and frequency of spontaneous activity after CCD is Ih. Ih current, carried by hyperpolarization-activated, cyclic nucleotide-gated channels (HCN), is a nonselective cation current activated by a membrane hyperpolarization occurring, for example, at the end of an action potential. The activation of Ih produces an inward current that slowly depolarizes the membrane (Pape, 1996). As a pacemaker current, Ih contributes to the rate of spontaneous rhythmic oscillations in the heart and in the brain (DiFrancesco, 1993; Luthi and McCormick, 1998). Ih is also present in DRG neurons (Mayer and Westbrook, 1983; Scroggs et al., 1994; Villiere and McLachlan, 1996; Yagi and Sumino, 1998; Cardenas et al., 1999) and is implicated in increasing discharge rate to excitatory stimuli by limiting membrane hyperpolarization and facilitating depolarization (Yagi and Sumino, 1998). Stimuli that modulate Ih can alter neuronal excitability. For example, certain inflammatory mediators, such as serotonin, increase intracellular cAMP, which, in turn, increases Ih current by binding to the HCN channel and shifting the voltage dependence of activation (Raes et al., 1997; Cardenas et al., 1999).
In the present study, we examined the biophysical properties of Ih current in cutaneous, medium-sized somata acutely dissociated from DRGs of CCD and unoperated control rats. Our hypothesis was that CCD induces an increase in the magnitude and/or an alteration in the kinetics of activation-deactivation of Ih.gk\!, 百拇医药
Materials and Methodsgk\!, 百拇医药
Labeling of cutaneous neurons and CCD surgery. Twenty-two female Sprague Dawley rats (140-160 gm) were anesthetized with pentobarbital (40 mg/kg, i.p.). Cutaneous afferent neurons were retrogradely labeled by injecting Fluorogold solution in saline (1%, 0.05 ml; Molecular Probes, Eugene, OR) subcutaneously into the right lateral plantar region (Oyelese and Kocsis, 1996). To determine whether there was dye leakage from the subcutaneous space to the neighboring muscle, True Blue (1.5%, 0.05 ml; Molecular Probes) was injected into the exposed gastrocnemius and soleus muscles (Liu et al., 2002). Immediately after the injections, in CCD rats (n = 11), the ipsilateral, right transverse process and intervertebral foramina of L4 and L5 were exposed as described previously (Hu and Xing, 1998; Song et al., 1999). A stainless steel L-shaped rod (0.63 mm in diameter and 4 mm in length) was inserted into each foramen, one at L4 and the other at the L5 ganglion. The remaining 11 rats received no additional surgery and were used as controls.
Cell preparation. Five to 7 d after surgery, the rats were deeply anesthetized with pentobarbital (40 mg/kg, i.p.), and the L4 and L5 lumbar DRGs were exposed. In CCD rats, the correct placement of each implanted rod was confirmed. Only one DRG was discarded because of incorrect placement of the rod. DRGs were removed from control or CCD rats and placed in complete saline solution (CSS) for cleaning and mincing. The CSS contained the following (in mM): 137 NaCl, 5.3 KCl, 1 MgCl2, 3 CaCl2, 25 sorbitol, and 10 HEPES. The DRGs were then digested for 15 min with collagenase A (1 mg/ml; Boehringer Mannheim, Mannheim, Germany) and for another 15 min with collagenase D (1 mg/ml; Boehringer Mannheim) and papain (30 U/ml; Worthington, Lakewood, NJ) in CSS containing 0.5 mM EDTA and 2 µg of cysteine at 37°C as described previously (Rizzo et al., 1995). The cells were dissociated by trituration in culture medium containing 1 mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor (Boehringer Mannheim) and plated on glass coverslips coated with polyornithine and laminin. The culture medium contained equal amounts of DMEM (Invitrogen, San Diego, CA) and F-12 (Invitrogen) with 10% FCS (HyClone, Logan, UT) and 1% penicillin and streptomycin (Invitrogen). The cells were incubated at 37°C (5% CO2 balance air) for 1 hr, after which culture medium without the inhibitor was added.
Electrophysiological recording and drug application. Coverslips were transferred to a recording chamber that was mounted on the stage of an upright microscope (BX50-WI; Optical, Tokyo, Japan). The chamber was filled with bath solution containing the following (in mM): 125 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 10 TEA-Cl, 3 4-AP, 2 MnCl2, 1 BaCl2, and 0.001-0.005 TTX. The pH was adjusted to 7.4 with NaOH, and osmolarity was adjusted with sucrose to 290 mOsm. Neurons selected for recording were 35-45 µm in diameter and were positive for Fluorogold fluorescence and negative for True Blue fluorescence. Electrodes were fabricated from borosilicate glass Sarasota, FL) and pulled on a Flaming/Brown micropipette puller (Sutter P-97; Sutter Instrument, Novato, CA). The pipette solution contained the following (in mM): 120 K-aspartate, 18 KCl, 1 CaCl2, 2 MgCl2, 5 EGTA, 10 HEPES, 5 Na2-ATP, and 0.4 Na-GTP. The pH was adjusted to 7.2 with KOH, and osmolarity was 295 mOsm (pipette resistance, 1-2 M).
Neurons were recorded at room temperature in the whole-cell mode (Multiclamp 700A; , Union City, CA) using pClamp 8.01 software . Data were filtered at 3 kHz and digitized at 20 kHz. The voltage drop across the access resistance was compensated at 40-80%, resulting in a voltage error of <8 mV. Cesium and ZD7288 were applied locally through a perfusion pipette (100 µm in diameter) using a drug application system (ALA Scientific Instruments, Long Island, NY). All of the chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated.0mg3%, 百拇医药
Data analysis. Data were fit using pClampfit 8.1 and Origin 6.0 (Microcal Software, Northampton, MA). The goodness of fit was determined either by the "model comparison" method in pClampfit 8.1 or the logarithm of the error ratio criterion (Horn, 1987). Data are expressed as mean ± SD unless otherwise indicated. Tests for statistical differences between means for CCD and control neurons were determined with either Student's t tests or repeated-measures ANOVA (RMANOVA), followed by post hoc pairwise comparisons [Tukey's honestly significant difference (HSD) test]. The criterion for statistical significance was a value of p <0.05.
Results'@u#ds, 百拇医药
CCD increases the density of Ih current'@u#ds, 百拇医药
A total of 254 DRG neurons were recorded, 120 from 11 control and 134 from 11 CCD rats. There was no difference in cell size for the two groups. The whole-cell capacitance was 78.7 ± 3.0 pF (n = 51) for control and 74.8 ± 3.6 pF (n = 34) for CCD neurons (Student's t test; p = 0.41). To activate Ih, hyperpolarizing potentials of 60 to 120 mV were delivered in increments of 10 mV from a holding potential of 50 mV ( inset). Slowly activating, inward currents (top row) were blocked by the addition of cesium (1 mM) to the bath solution middle row), consistent with an Ih current. Cesium blocked 95.5 ± 2.4% of the inward current in control neurons (n = 12) and 95.9 ± 3.1% in CCD neurons (n = 12). In other experiments, ZD7288 (15 µM), a specific Ih channel blocker (BoSmith et al., 1993; Gasparini and DiFrancesco, 1997; Satoh and Yamada, 2000), showed a similar inhibitory effect, blocking 91.0 ± 5.2% of the inward current in CCD neurons (n = 12) and 96.5 ± 3.3% in control neurons (n = 12). The inhibition by cesium but not by ZD7288 was reversed during 30 min of washout (bottom row). The sensitivity of Ih to these inhibitors is consistent with that described previously for Ih (Yagi and Sumino, 1998; Cardenas et al., 1999). The magnitude of Ih current increased with increasing hyperpolarization (top row). At 60 mV, the mean magnitude of Ih was 22.9 ± 5.7 pA for control neurons and 53.6 ± 10.9 pA for CCD neurons. At 120 mV, Ih was 698.6 ± 85.2 pA (n = 34) for control and 1156.7 ± 115.1 pA (n = 51) for CCD neurons. When normalized to cell capacitance, CCD neurons had a significant increase in Ih current density by 66.6-61.5% compared with control neurons over the voltage range of 90 to 120 mV (control, n = 34; CCD, n = 51; two-way RMANOVA, p < 0.05) . For example, at 100 mV, the current density was 7.4 ± 0.9 pA/pF for control and 12.4 ± 1.3 pA/pF for CCD neurons, a 67% increase in the latter group
fig.ommtted?*dul2, 百拇医药
The effects of CCD on the density of Ih current. A, Current responses evoked by hyperpolarizing voltage steps from a holding potential of 50 mV in a control (left) and a CCD (right) DRG neuron. The voltage protocol is shown at the bottom. Ih current amplitude was calculated from the difference between the steady-state and the instantaneous currents (i.e., the difference between the two arrows shown as an example at the last trace of the left top traces). In both panels, families of current responses on the top were recorded in the control bath solution. Middle traces, Cs+ at 1 mM. Bottom traces, After 2 min of washout. Voltage protocol is shown as an inset at bottom. B, Ih current density versus membrane potential, Vm. Squares and circles represent the data summarized from 34 control and 51 CCD neurons, respectively. Error bars indicate SEM. * Indicates that means at the same membrane voltage are significantly different.?*dul2, 百拇医药
CCD increases the conductance but not the reversal potential of Ih current
The reversal potential and the maximal Ih conductance were measured by first applying a prepulse to 100 mV to fully activate Ih and then examining the tail currents after repolarization to test potentials from 90 to 50 mV ( inset). The time point for measurement of the peak tail current is shown in(arrows). For each test voltage, the peak tail current, after subtraction of the estimated leak current, was averaged for 31 control and 31 CCD neurons, and each was fitted with a linear regression equation, as follows: Itail = Gh.max(Vm Vrev), where Itail is the peak tail current, Vm is the membrane potential of the test pulse, Gh.max is the maximal conductance, and Vrev is the reversal potential of Ih current . This fitting yielded estimates of Gh.max, Vrev of 0.15 nS/pF, 21 mV for the control neuron and 0.23 nS/pF, 23 mV for the CCD neuron. The mean Vrev values of 21.3 ± 1.4 mV (n = 31) for control neurons and 22.3 ± 1.0 mV (n = 31) for CCD neurons were not significantly different (Student's t test; p = 0.55). As expected from the data in, CCD neurons exhibited a significant increase (76%) in maximal Ih conductance, from 0.13 ± 0.01 nS/pF (n = 31) in control neurons to 0.23 ± 0.02 nS/pF (n = 31) in CCD neurons (Student's t test; p < 0.01).
fig.ommtted$., 百拇医药
Effects of CCD on the conductance and reversal potential of Ih current. A, In a control (top) and a CCD (bottom) neuron, Ih was fully activated by a hyperpolarizing prepulse to 100 mV for 1 sec, followed by a depolarization to test potentials of 90 to 50 mV (inset). Tail currents were masured at the start of the test potentials (arrows). B, Leak-subtracted tail current versus test potential. Squares and circles represent control and CCD neurons, respectively. The solid lines are the linear regressions fitted to the data points for each group. The slopes of the linear regressions were 0.15 and 0.23 nS/pF for control and CCD, respectively. The reversal potentials were 21 and 23 mV for control and CCD, respectively.$., 百拇医药
CCD does not change the voltage dependence of Ih activation$., 百拇医药
The activation curve of Ih current was estimated by measuring the tail current at 120 mV after application of prepulse potentials between 40 and 120 mV . Tail currents were normalized to the maximal current (obtained at a prepulse of - 120 mV). The activation curve for each neuron was fitted with a Boltzmann equation of the following form: Itail/Itail(max) = (1 + e((Vm V0.5)/k))1, where Vm is the membrane potential of the prepulse, V0.5 is the membrane potential at which Ih conductance is half-activated, k is a slope factor, Itail is the peak amplitude of the tail current recorded immediately after the prepulse, and Itail(max) is the tail current recorded after the maximal prepulse of 120 mV. V0.5 was measured from the activation curve , and values were not significantly different for control (74.2 ± 4.6 mV; n = 34) vs CCD (73.5 ± 4.6 mV; n = 51; Student's t test; p = 0.52) neurons. The slope factors for each group were not significantly different (6.2 ± 1.8 mV for control and 6.6 ± 1.4 mV for CCD neurons; Student's t test; p = 0.19).
fig.ommttedlk;v\y$, http://www.100md.com
Effects of CCD on the voltage dependence of Ih activation. A, Ih was activated from a holding potential of 50 mV to prepulse potentials ranging from 40 to 120 mV, followed by a step to a test potential of 120 mV (voltage protocol as shown at bottom). The magnitude of the tail current at the start of the test potential was used as an index of Ih activation (arrows in insets a and b). The tail currents of a control (top) and a CCD (middle) neuron are shown. B, Activation curves obtained by fitting the data of a control (squares) and a CCD (circles) neuron (as shown in A, top and middle, respectively) with single Boltzmann functions. The fitting yielded midpoint potentials, V0.5, and slope factors, k, of 76.2 and 5.3 mV for control and 77.4 and 6.5 mV for CCD neurons, respectively.lk;v\y$, http://www.100md.com
CCD increases the rate of activation of Ih currentlk;v\y$, http://www.100md.com
Ih was activated by a series of hyperpolarizing test potentials of 60 to 120 mV from a holding potential of 50 mV ( inset). Test potentials of shorter duration were used at more hyperpolarized potentials to avoid damaging the cell membrane. The time course of activation of Ih was fitted to first- and second-order exponential functions. On the basis of the criterion of Horn (1987), the current traces were not well described by a single exponential function but rather by the sum of two exponential functions. The equation was of the following form: Ih(t) = Af e(t/f) + Ase(t/s), where Ih(t) is the amplitude of the current at time t, and Af and As are the amplitude coefficients of the fast (f) and slow (s) time constants, respectively. The relative proportion of the total current in the slow and fast components did not differ significantly across activation potential or between control and CCD neurons (two-way RMANOVA, p > 0.05). In general, Ih current activated faster at more hyperpolarized potentials . For example, from 80 to 120 mV, the mean f decreased from 302 to 68 msec in control neurons and from 175 to 52 msec in CCD neurons . f was significantly faster in CCD than in control neurons for voltages of 70 to 90 mV (n = 17 for control; n = 21 for CCD; two-way RMANOVA; Tukey's HSD; p < 0.05) . From 80 to 120 mV, the mean s decreased from 1134 to 636 msec in control neurons and from 925 to 445 msec in CCD neurons. s was significantly faster in CCD than in control neurons for voltages of 90 to 110 mV (n = 17 for control; n = 21 for CCD; two-way RMANOVA; Tukey's HSD; p < 0.05) .
fig.ommtted)62t.ne, http://www.100md.com
Effect of CCD on the activation kinetics of Ih current. A, Current responses of a control (top) and a CCD (bottom) neuron each evoked by a series of hyperpolarizing voltage steps of 60 to 120 mV from a holding potential of 50 mV. Step duration was varied from 2150 to 1150 msec (inset at bottom right). Each solid line is a fitting of the sum of two exponentials to the data points (open circle) obtained at each hyperpolarizing voltage. Note the faster activation of Ih currents at each voltage in the CCD neuron. B, The mean ratio of Af to Af + As versus membrane potential for control (squares) and CCD (circles) neurons. C, D, The mean time constants f (C) and s (D) versus membrane potential for control (squares) and CCD (circles). * Indicates that means are significantly different.)62t.ne, http://www.100md.com
The deactivation kinetics were estimated using an envelope technique described by Mayer and Westbrook (1983). Neurons were subjected to a prepulse potential of 110 mV to fully activate Ih, followed by a repolarization (deactivation) to a test potential of varying duration, followed, in turn, by a return to 90 mV. The magnitude of deactivation was calculated by measuring the amount of Ih remaining after the test potential. The measurements were obtained at test potentials of 60, 50, 40, and 30 mV .
fig.ommtted]o9l|, 百拇医药
Effects of CCD on the deactivation of Ih current. A, Current response (top) of a control neuron evoked by a triple-pulse voltage protocol (bottom). A hyperpolarizing potential of 110 mV was followed by different durations of a depolarization to 30 mV to deactivate Ih current and a hyperpolarization to 90 mV to reactivate Ih (the number of traces was reduced for clarity). The amplitude of Ih current (shown as an example in the right trace) activated by the third pulse was used to calculate the deactivation time constant. B, Current responses of a control cell (same cell as in A) to the triple protocol but with deactivating potentials of 30, 40, 50, and 60 mV (a-d, respectively). Current traces were truncated at the end of b-d to keep the same time scale as that in a. The number of data points in each current trace in both A and B were reduced to . C, The effect of potential on the time course of Ih current deactivation. Data points are fit by a single exponential. D, Mean time constant of deactivation of Ih current versus membrane potential for control and CCD neurons (squares and circles, respectively). The single exponential functions fitted to the data points for each group are not significantly different for CCD and control neurons.
Because the data were not equally spaced in this experiment, the Levenberg-Marquardt method (Clampfit; ) was used for curve fitting. A single exponential equation fitted the data best as determined using a model comparison procedure (pClampfit) (. The deactivation of Ih became faster when membrane potentials were more depolarized. For example, in a control neuron, the deactivation time constants were 512.3, 342.6, 228.2, and 141.4 msec at potentials of 60, 50, 40, and 30 mV, respectively . The deactivation time constants of Ih were plotted as a function of test potential for control and CCD neurons. The functions were well described by (msec) = 15.8 + 9.9 exp(Vm/17) for control and (msec) = 15.6 + 12.1 exp(Vm/18) for CCD neurons. The deactivation time constants of Ih from both groups showed a clear voltage dependence, with an e-fold decrease of 17 mV (control) or 18 mV (CCD) depolarization . However, at each depolarizing voltage, the deactivation time constants were not significantly different between the two groups (n = 19 for control and n = 23 for CCD neurons; two-way RMANOVA; p > 0.05) . For example, mean time constants were 73.4 ± 7.0 (control) and 77.0 ± 6.2 (CCD) msec at 30 mV.
Ih does not contribute to the resting membrane potentials of control or CCD neurons998e%|{, 百拇医药
To determine whether Ih contributes to resting membrane potential, neurons were recorded in the current-clamp mode with no holding current. A periodic hyperpolarizing pulse (0.3 to 0.7 nA, 200 msec) was applied every 3 sec for the measurement of membrane resistance and to demonstrate the presence of "depolarization sag," the latter an index of Ih current. Under control conditions, there was no significant difference between the mean resting potentials of control and CCD neurons (58 ± 4 mV (n = 24) and 57 ± 6 mV (n = 17), respectively; Student's t test; p = 0.42). Application of Ih channel blockers Cs+ (1 mM) and ZD7288 (15 µM) eliminated the depolarization sag ( bottom) but did not change resting membrane potential (top).998e%|{, 百拇医药
fig.ommtted998e%|{, 百拇医药
The effects of Ih on the resting membrane potential. Current-clamp recordings of the resting membrane potential (Vrest) of a control (A) and a CCD (B) neuron. The respective initial values of Vrest for the control and CCD neurons were 53 and 56 mV. Current pulses of 0.6 nA, 200 msec, were injected into each cell every 3 sec to elicit a hyperpolarization and sag in voltage response. The horizontal bars indicate the durations of application of the Ih blockers Cs+ (1 mM) and ZD7288 (15 µM). A, B, Top trace, Voltage responses to the current injection; bottom trace, enlarged voltage traces to show the reduction in sag produced by the blocker. Note that, in both control and CCD neurons, both 1 mM Cs+ and 15 µM ZD7288 did not alter resting potential but had similar effects in abolishing the sag.
Discussion48c2v|, 百拇医药
The major finding of this study is that CCD increased the density of Ih current in DRG somata because of an increase in Ih conductance and also increased the rate of activation of Ih current.48c2v|, 百拇医药
Characterization of Ih in DRG neurons48c2v|, 百拇医药
The kinetics of activation of Ih observed in this study are generally consistent with that described previously by other laboratories. We found that the activation kinetics of Ih are voltage dependent and are best described by a double exponential in rat DRG neurons. The f and s were similar to values reported for neonatal rat DRG neurons (Wang et al., 1997). The complicated kinetics of activation may reflect the presence of multiple isoforms of the Ih channel. Four members of a gene family encoding mammalian HCN channels (HCN1-HCN4) have been cloned in recent years. When expressed in HEK 293 cells, recombinant HCN1-HCN4 channels demonstrate unique activation kinetics, i.e., with the fastest time constant for HCN1 and gradually slower time constants for HCN2-HCN4 (Moosmang et al., 2001). The two distinct time constants obtained in our preparation may indicate that two kinds of homomeric channels exist in DRG neurons, as has been proposed for sinoatrial node cells (Moosmang et al., 2001). This is consistent with the observation of Chaplan et al. (2001) that both HCN1 and HCN3 isoforms are present in rat DRG.
The parameters of steady-state activation of Ih observed in our recordings are consistent with those reported previously for DRG neurons in rodents. We obtained a half-activation voltage of 74 mV, consistent with the value (73 mV) obtained previously for medium-sized rat DRG neurons (Cardenas et al., 1999). Our V0.5 value is more positive than those of neonatal rat DRG neurons (Wang et al., 1997), possibly because of the absence of ATP in their recording pipette. ATP has been shown to cause a depolarizing shift of V0.5 in mouse DRG neurons (Raes et al., 1997). The Ih conductance of 0.13 nS/pF at 100 mV is within range of values obtained in a variety of neuronal types (Pape, 1996), and our reversal potential value of 21 mV is virtually identical to those reported previously for DRG neurons in rodents (Raes et al., 1997; Wang et al., 1997).3, 百拇医药
Effect of CCD on Ih3, 百拇医药
A major finding of our study is that the magnitude of Ih increased after CCD. The immediate cause of the increase in Ih may be complicated, because this model is presumably associated with ischemia-hypoxia and inflammation. A local inflammation and acidosis could recruit mast cells and macrophages that release inflammatory mediators, such as 5-HT, prostaglandin E2, ATP, NGF, and cytokines (Millan, 1999. These mediators can activate cellular mechanisms that alter the expression of HCN channels, change the intrinsic properties of HCN channels, and/or increase the amount of cAMP binding to the Ih channel or enhancing the phosphorylation processes. Results using other models of neuropathic pain have had inconsistent effects on Ih. After transection of the sciatic nerve, Ih was found to decrease in one study (Abdulla and Smith, 2001), but, in another study, transection of the spinal nerve caused Ih to increase (Chaplan et al., 2001). A reconciliation among these models and observations is not immediately obvious.
There are several possible explanations for the increase in magnitude of Ih observed in this study. (1) There may be a change in HCN isoform, as occurs for sodium currents after peripheral axotomy (Dib-Hajj et al., 1999; Ishikawa et al., 1999; Kim et al., 2001). However, we believe this to be unlikely, because CCD did not alter the ratio of Af/(Af + As). Thus, if there are two homomers of the HCN channel, neither of them became dominant after CCD. (2) A "beta " -subunit for the HCN channel family may exist (Yu et al., 2001), and changes in its expression could modify the Ih current properties, as has been observed in Na+, Ca2+, and K+ channels (Isom et al., 1994). (3) Intracellular messengers, particularly cAMP, may have modified Ih. HCN channels have a cyclic binding domain located in the C terminal (Wainger et al., 2001) and PKA consensus phosphorylation sites (Santoro et al., 1998; Gauss and Seifert, 2000; Vargas and Lucero, 2002). Electrophysiological studies have shown that cAMP enhances Ih by altering the voltage dependence of activation (Ingram and Williams, 1996; Cardenas et al., 1999). However, we did not observe a change in activation properties after CCD in our experiments. These possibilities provide several possible paths for the pursuit of additional experiments.
Functional implications of increased Ih in CCD neurons&*, http://www.100md.com
CCD increases the excitability of DRG neurons, as indicated by an increased incidence of spontaneous activity and subthreshold oscillations in membrane potential and lowered current and action potential thresholds (Hu and Xing, 1998; Song et al., 1999; Zhang et al., 1999; Xing et al., 2001). The increased Ih current conductance we observed may account for the higher incidence of spontaneous activity and contribute to neuropathic pain. The latter is supported by the observation that ZD7288 blocked neuropathic pain caused by spinal nerve ligation (Chaplan et al., 2001).&*, http://www.100md.com
In CCD neurons, a repolarization of an action potential will produce a greater depolarization because of increased Ih and thus have a greater chance of evoking another action potential, possibly enhancing the occurrence of repetitive discharge either autonomously or in response to excitatory stimuli. For example, in rat DRG neurons, an increase of Ih by 5-HT increased the number of anode break discharges. A reduction of Ih by clonidine decreased the rate of evoked multispike intervals (Yagi and Sumino, 1998; Cardenas et al., 1999). In hippocampal neurons, inhibition of Ih by its blocker ZD7288 reduced the frequency of spontaneous activity and blocked the evoked multispike discharge (Maccaferri and McBain, 1996; Gasparini and DiFrancesco, 1997). An increase of Ih mediated through the -adrenergic receptor by the -agonist isoproterenol (ISP) increased the frequency of spontaneous spikes recorded from rat cerebellar basket cells, whereas ZD7288 decreased the spike frequency and abolished the ISP-induced increase in spike discharges (Saitow and Konishi, 2000).
In conclusion, the increase in Ih current density that we observed after CCD could contribute to an increased incidence of spontaneous activity and subthreshold oscillations in membrane potential and thus to the genesis of neuropathic pain.ay9@[, 百拇医药
Referencesay9@[, 百拇医药
Abdulla FA, Smith PA (2001) Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons. J Neurophysiol 85:644-658 .ay9@[, 百拇医药
BoSmith RE, Briggs I, Sturgess NC (1993) Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br J Pharmacol 110:343-349 .ay9@[, 百拇医药
Cardenas CG, Mar LP, Vysokanov AV, Arnold PB, Cardenas LM, Surmeier DJ, Scroggs RS (1999) Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J Physiol (Lond) 518:507-523 .ay9@[, 百拇医药
Chaplan SR, Guo HQ, Lee DH, Butler MP, Velumian A, Dubin AE (2001) Markedly increased DRG Ih and HCN mRNA accompany neuropathic pain in the rat. Soc Neurosci Abstr 27:54.11 .
Dib-Hajj SD, Fjell J, Cummins TR, Zheng Z, Fried K, LaMotte R, Black JA, Waxman SG (1999) Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic pain. Pain 83:591-600 .4q, http://www.100md.com
DiFrancesco D (1993) Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55:455-472 .4q, http://www.100md.com
Gasparini S, DiFrancesco D (1997) Action of the hyperpolarization-activated current (Ih) blocker ZD 7288 in hippocampal CA1 neurons. Pflügers Arch 435:99-106 .4q, http://www.100md.com
Gauss R, Seifert R (2000) Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels. Chronobiol Int 17:453-469 .4q, http://www.100md.com
Horn R (1987) Statistical methods for model discrimination: applications to gating kinetics and permeation of the acetylcholine receptor channel. Biophys J 51:255-263 .4q, http://www.100md.com
Hu SJ, Xing JL (1998) An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain 77:15-23 .4q, http://www.100md.com
Ingram SL, Williams JT (1996) Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons. J Physiol (Lond) 492:97-106 .
Ishikawa K, Tanaka M, Black JA, Waxman SG (1999) Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy. Muscle Nerve 22:502-507 .qx, 百拇医药
Isom LL, De Jongh KS, Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12:1183-1194 .qx, 百拇医药
Kim DS, Yoon CH, Lee SJ, Park SY, Yoo HJ, Cho HJ (2001) Changes in voltage-gated calcium channel alpha(1) gene expression in rat dorsal root ganglia following peripheral nerve injury. Brain Res Mol Brain Res 96:151-156 .qx, 百拇医药
Liu CN, Devor M, Waxman SG, Kocsis JD (2002) Subthreshold oscillations induced by spinal nerve injury in dissociated muscle and cutaneous afferents of mouse DRG. J Neurophysiol 87:2009-2017 .qx, 百拇医药
Luthi A, McCormick DA (1998) H-current: properties of a neuronal and network pacemaker. Neuron 21:9-12 .qx, 百拇医药
Maccaferri G, McBain CJ (1996) The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J Physiol (Lond) 497:119-130 .
Mayer ML, Westbrook GL (1983) A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones. J Physiol (Lond) 340:19-45 .y*), 百拇医药
Millan MJ (1999) The induction of pain: an integrative review. Prog Neurobiol 57:1-164 .y*), 百拇医药
Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A (2001) Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem 268:1646-1652 .y*), 百拇医药
Oyelese AA, Kocsis JD (1996) GABAA-receptor-mediated conductance and action potential waveform in cutaneous and muscle afferent neurons of the adult rat: differential expression and response to nerve injury. J Neurophysiol 76:2383-2392 .y*), 百拇医药
Pape HC (1996) Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58:299-327 .y*), 百拇医药
Raes A, Wang Z, van den Berg RJ, Goethals M, Van de Vijver G, van Bogaert PP (1997) Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons. Pflügers Arch 434:543-550 .
Rizzo MA, Kocsis JD, Waxman SG (1995) Selective loss of slow and enhancement of fast Na+ currents in cutaneous afferent dorsal root ganglion neurones following axotomy. Neurobiol Dis 2:87-96 .:?, 百拇医药
Saitow F, Konishi S (2000) Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells. J Neurophysiol 84:2026-2034 .:?, 百拇医药
Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR (1998) Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717-729 .:?, 百拇医药
Satoh TO, Yamada M (2000) A bradycardiac agent ZD7288 blocks the hyperpolarization-activated current (I(h)) in retinal rod photoreceptors. Neuropharmacology 39:1284-1291 .:?, 百拇医药
Scroggs RS, Todorovic SM, Anderson EG, Fox AP (1994) Variation in IH, IIR, and ILEAK between acutely isolated adult rat dorsal root ganglion neurons of different size. J Neurophysiol 71:271-279 .:?, 百拇医药
Song XJ, Hu SJ, Greenquist KW, Zhang JM, LaMotte RH (1999) Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia. J Neurophysiol 82:3347-3358 .
Vargas G, Lucero MT (2002) Modulation by PKA of the hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons. J Membr Biol 188:115-125 .9, 百拇医药
Villiere V, McLachlan EM (1996) Electrophysiological properties of neurons in intact rat dorsal root ganglia classified by conduction velocity and action potential duration. J Neurophysiol 76:1924-1941 .9, 百拇医药
Wainger BJ, DeGennaro M, Santoro B, Siegelbaum SA, Tibbs GR (2001) Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature 411:805-810 .9, 百拇医药
Wang Z, Van Den Berg RJ, Ypey DL (1997) Hyperpolarization-activated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture. J Neurophysiol 78:177-186 .9, 百拇医药
Xing JL, Hu SJ, Long KP (2001) Subthreshold membrane potential oscillations of type A neurons in injured DRG. Brain Res 901:128-136 .9, 百拇医药
Yagi J, Sumino R (1998) Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons. J Neurophysiol 80:1094-1104 .9, 百拇医药
Yu H, Wu J, Potapova I, Wymore RT, Holmes B, Zuckerman J, Pan Z, Wang H, Shi W, Robinson RB, El-Maghrabi MR, Benjamin W, Dixon J, McKinnon D, Cohen IS, Wymore R (2001) MinK-related peptide 1: a beta subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ Res 88:E84-E87 .9, 百拇医药
Zhang JM, Song XJ, LaMotte RH (1999) Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion. J Neurophysiol 82:3359-3366 .(Hang Yao David F. Donnelly Chao Ma and Robert H. LaMotte)