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Department of Anesthesiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania
Submitted 9 December 2004; accepted in final form 6 January 2005
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and C-fiber afferents are critical for detecting noxious stimuli and initiating pain sensation (Harper and Lawson 1985
; Slugg et al. 2000). These DRG neurons are functionally diverse and have different phenotypes. The roles of different classes of nociceptors in acute and chronic pain conditions are not clear. A new approach to the classification of DRG neurons is based on the requirement of subsets of neurons for specific neurotrophins (Molliver et al. 1995; Snider and McMahon 1998). Two broad classes of small DRG neurons have been classified: one class expresses the high-affinity nerve growth factor (NGF) receptor TrkA and responds to NGF (Averill et al. 1995; McMahon et al. 1994; Molliver et al. 1995) and contains neuropeptides such as calcitonin gene-related peptide and substance P. Another class of small DRG neurons expresses the glial-derived neurotrophic factor (GDNF) receptor complex and responds to GDNF in vitro and in vivo (Bennett et al. 1998; Kashiba et al. 2001; Wang et al. 1994). Although these GDNF-dependent neurons are "peptide-poor", they contain cell surface glycoconjugates that bind to the Griffonia simplicifolia isolectin B4 (IB4) (Kashiba et al. 2001; Plenderleith and Snow 1993; Silverman and Kruger 1990). These neurochemical differences suggest that IB4-binding and IB4-negative small DRG neurons are functionally distinct (Snider and McMahon 1998; Stucky and Lewin 1999). In this regard, IB4-positive DRG neurons have a higher density of tetrodotoxin-resistant (TTX-R) Na+ currents and exhibit a longer action potential duration and a higher threshold of firing than IB4-negative neurons of mice (Stucky and Lewin 1999). Also, the density of N-type Ca2+ channel currents is significantly higher in IB4-positive than that in IB4-negative rat DRG neurons (Wu and Pan 2004; Wu et al. 2004). However, the potential differences in other ion channels between IB4-positive and -negative DRG neurons have not been determined previously.
Voltage-gated K+ channels (Kv) are important in the control of electrical properties and excitability of neurons. The native Kv currents in sensory neurons include 2 major types: sustained delayed (IK) and A-type. The A-type Kv currents can be further divided into at least 2 subtypes, fast-inactivating (IA) and slow-inactivating (ID), based on their inactivation kinetics and sensitivities to tetraethylammonium (TEA) and 4-aminopyridine (4-AP) (Everill et al. 1998; Gold et al. 1996; Liu and Simon 2003; McFarlane and Cooper 1991; Safronov et al. 1996). Axotomy causes a profound reduction in Kv1 family subunits, especially the Kv1.4 that is associated with A-type Kv currents in small-sized DRG cells (Everill and Kocsis 1999; Kim et al. 2002; Rasband et al. 2001). Therefore information about the different types of Kv currents and their functional significance in IB4-positive and -negative DRG neurons is important to define the role of subsets of nociceptive neurons in chronic neuropathic pain. In this study, we determined the possible difference in A-type Kv currents between IB4-positive and -negative DRG neurons. We also studied the contribution of A-type Kv currents to the firing properties of these 2 groups of DRG neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All the procedures used conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine. Male Sprague–Dawley rats (4- to 6-wk-old, Harlan, Indianapolis, IN) were anesthetized with halothane and then rapidly decapitated. The thoracic and lumbar segments of vertebral column were surgically removed. The DRGs and the nerve roots were quickly dissected out and transferred immediately onto Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA) on ice. Then the DRGs were dissected free of attached connective tissues under a microscope and minced with fine spring scissors. The minced ganglion fragments were placed in a flask containing 5 ml of DMEM in which trypsin (type III, 0.5 mg/ml, Sigma, St. Louis, MO) and collagenase (type I, 1 mg/ml, Sigma) had been dissolved. After incubation at 34°C in a shaking water bath for 30 min, soybean trypsin inhibitor (type II-s, 1.25 mg/ml, Sigma) was added to terminate tryptic digestion. The resulting cell suspension was centrifuged (500 rpm, 6 min) to precipitate the dissociated DRG neurons. The supernatant was removed, and the cells were replenished with DMEM. Cells were subsequently plated onto a 35-mm culture dish containing poly-L-lysine (50 µg/ml) precoated coverslips and incubated in 5% CO2 at 37°C for 
1 h.
DRG cells labeling and electrophysiological recordings
The neurons selected for recordings were 15–30 µm because IB4 labels small-diameter DRG neurons of rats (Wu and Pan 2004; Wu et al. 2004). A distinct feature of IB4-conjugated fluorescent dyes is that they can bind and label living DRG neurons (Stucky and Lewin 1999; Wu and Pan 2004; Wu et al. 2004). Immediately before recording, neurons were treated with IB4-Alexa Fluor 594 (3 µg/ml, Molecular Probes, Eugene, OR) in Tyrode solution (in mM: 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose; pH 7.4 adjusted with NaOH, osmolarity 320 mOsm) for 5 min and then rinsed for 3 min. In some control experiments, recordings were performed on neurons before they were labeled with IB4-Alexa Fluor 594. Then, IB4-Alexa Fluor 594 solution was perfused for 2 min on these patched neurons, which were subsequently washed for 3–4 min and the Kv currents were recorded again to assess the effect of the dye on the Kv currents. An approximately equal number of IB4-positive and -negative neurons was studied in any given rat. Recordings were made within 10 h after dissociation to keep the experiment as similar to in vivo conditions as possible and to minimize the space-clamp error (Wu and Pan 2004; Wu et al. 2004). All neurons selected for recordings had overshooting action potentials and resting membrane potentials more negative than –45 mV.
Patch electrodes with a resistance of 2–4 M
were pulled from GC150TF-10 glass capillaries (ID 1.17 mm, OD 1.5 mm, Harvard Apparatus, Holliston, MA) using a micropipette puller (P-97 Sutter Instruments, Novato, CA) and fire-polished. Neurons were visualized using a combination of epifluorescence illumination and differential interference contrast (20–40x) optics on an inverted microscope (Olympus, Tokyo, Japan). Images of cells were taken with a CCD camera and displayed on a video monitor. Neurons were patched in the whole cell configuration and recorded at a holding potential of –80 or –120 mV using an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany). Seals (1–10 G) between the electrode and the cell were established. After whole cell configuration was established, the cell membrane capacitance and series resistance were electronically compensated. Leak currents were subtracted using the on-line P/4 protocol. All experiments were performed at room temperature (about 25°C). Signals were filtered at 1 kHz, digitized at 10 kHz, and acquired using Pulse program (HEKA).
To selectively record K+ currents and minimize the contribution from Ca2+ and Na+ currents, the extracellular solution contained (in mM): 150 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 1 CdCl2, and 10 D-glucose (pH 7.4 adjusted with KOH, osmolarity 330 mOsm) (Liu and Simon 2003). The extracellular solution also contained 15 µM ZD7288 to block hyperpolarization-activated currents of the DRG neurons (Yao et al. 2003). Electrodes were filled with solution containing (in mM): 120 potassium gluconate, 20 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5 Na2-ATP, and 1 CaCl2 (pH 7.2 adjusted with KOH, osmolarity 300 mOsm). The outward currents recorded under these conditions were completely blocked by inclusion of Cs+ in the pipette solution. The protocol used to measure Kv current activation was performed at a holding potential of –80 mV and consisted of 100-ms depolarization pulses from –70 to 60 mV in 10-mV increments with a 2-s interval. In some DRG neurons, cells were held at –120 mV and depolarized by a series of pulses from –80 to 60 mV for 100 ms in 10-mV steps (Yang et al. 2004). Action potentials in DRG neurons were elicited in Tyrode solution by injection of a series of depolarizing currents (from 0 to 250 pA in 30- to 50-pA increments, 400-ms duration) and recorded using the whole cell current-clamp method. The intracellular solution contained (in mM): 124 KCl, 2 MgCl2, 1 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP (pH 7.2 adjusted with KOH, osmolarity 290 mOsm).
Drug preparations and perfusion
Drugs were dissolved in distilled water at 1,000 times the final concentration and kept frozen in aliquots. The stock solutions were diluted in the appropriate external solution just before use and held in a series of independent syringes connected to corresponding fused silica columns (ID 200 µm). The end of the parallel columns was connected to a common silica column. The distance from the column mouth to the cell studied was about 100 µm. Cells in the recording chamber were continuously bathed in external solution. Each drug solution was delivered to the recording chamber by gravity, and rapid solution exchange (about 200 ms) was achieved by controlling the corresponding valve switch (World Precision Instruments). Both TEA and 4-AP were purchased from Sigma.
Immunofluorescence labeling of Kv1.1, Kv1.2, and Kv1.4 subunits
To study the possible types of Kv subunits associated with A-type Kv currents in IB4-positive DRG neurons, we double-labeled DRG neurons with IB4 and Kv1.1, Kv1.2, or Kv1.4 subunit. We chose Kv1.1, Kv1.2, and Kv1.4 because these subunits can form A-type Kv channels and are expressed in small- and medium-sized DRG neurons of rats (Rasband et al. 2001). Under deep anesthesia with sodium pentobarbital (60 mg/kg, administered intraperitoneally), rats were perfused intracardially with 250 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and 4% sucrose (pH 7.4). The DRGs were removed quickly and postfixed for 2 h in the same fixative solution and cryoprotected in 10% sucrose in PBS for 48 h at 4°C. The sections were cut to 30 µm in thickness and collected free floating in 0.1 M PBS. For Kv1.1, Kv1.2, and Kv1.4 immunofluorescence staining, the sections were rinsed in 0.02 M PBS and blocked in 4% normal goat serum in PBS for 1 h. Then, the sections were incubated with the respective primary antibody diluted in PBS containing 2% normal goat serum and 0.3% TX-100 for 2 h at room temperature and overnight at 4°C. The primary antibodies used include rabbit anti-Kv1.1 (1:50, Chemicon, Temecula, CA), mouse anti-Kv1.2 (1:200, Upstate, Lake Placid, NY), and rabbit anti-Kv1.4 (1:100, Chemicon; and Alomone Labs, Jerusalem, Israel) (Ishikawa et al. 2003). Subsequently, sections were rinsed in PBS and incubated with the secondary antibody (goat anti-rabbit or anti-mouse IgG-Alexa Fluor 488, Molecular Probes, dilution: 5 µg/ml). The sections then were rinsed and incubated with IB4-Alexa Fluor 594 diluted in 0.1 M Tris buffer containing 1 mM Ca2+ for 2 h at room temperature (Wu et al. 2004). Finally, the sections were rinsed and mounted on slides, dried, and coverslipped. The sections were examined on a confocal microscope (Leica, Wetzlar, Germany) and the areas of interest were photodocumented. Confocal laser scanning microscopy was used for accurate co-localization of fluorescent markers, because the thin (about 0.3 µm) optical sectioning generated by the confocal microscope eliminates the confounding effect of out-of-focus fluorescence. In the higher magnification images, the co-localization is indicated by the color change (yellow) and represents co-localization (Wu et al. 2004). Negative controls were performed by omitting the primary antibody or replacing the primary antibody with nonimmune serum from the same species. The specificity of the above antibodies was also evaluated by preabsorption with the corresponding immunogen for 1 h at 30°C.
Data analysis and curve fitting
Data were analyzed using the PulseFit software program (HEKA). Whole cell current-voltage (I–V) curves for individual neurons were generated by calculating the mean peak outward current at each testing potential and normalized for cell capacitance. The amplitude of the Kv current was measured at the peak. 4-AP and TEA-sensitive Kv currents were obtained by subtracting the 4-AP and TEA-resistant Kv current, respectively, from the total Kv current. Data were fit to the Boltzmann equation
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is the slope of the function, C is a constant (= 0 in the IV relation), Imax is the maximal Kv current density, and Vm is the membrane potential. The action potential characteristics were analyzed using a peak detection program (MiniAnalysis, Synaptosoft, Leonia, NJ). The action potential threshold was defined as the lowest current injected that elicited an action potential with an overshoot. The frequency was calculated as the maximum number of action potentials elicited at the activation threshold. The action potential overshoot was determined from 0 mV to the peak of an action potential. The duration of the action potential was measured at 50 and 75% of the peak amplitude from the resting membrane potential, because the 50% amplitude was close to the base of the action potential and the 75% amplitude was close to the inflection on the falling phase of the action potential (Stucky and Lewin 1999). Statistical data are presented as means ± SE. Comparisons between means were tested for significance using paired and unpaired Student's t-test or ANOVA test. P < 0.05 was considered to be statistically significant. |
RESULTS |
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) and -negative (566.7 ± 46.5 M) neurons. To determine the difference in the total whole cell Kv currents between IB4-positive and -negative DRG neurons, K+ currents were elicited by depolarizing pulses from –70 to 60 mV for 150 ms with 10-mV increments in 2-s intervals. The outward A-type (IA and ID) and delayed (IK) Kv currents were delineated using 25 mM TEA and 5 mM 4-AP, blockers for native IK and A-type Kv currents in DRG neurons, respectively (Everill et al. 1998; Liu and Simon 2003; Safronov et al. 1996). In the preliminary study, a series of TEA concentrations (25, 50, and 75 mM) were applied to examine its effects on A-type currents in DRG neurons. Because 50 and 75 mM TEA exhibited partial inhibition of the peak A-type current, 25 mM TEA was the concentration used in our study. Different types of Kv currents in IB4-positive and -negative neurons
Different types of native Kv currents in IB4-positive and -negative DRG neurons were separated using 25 mM TEA and 5 mM 4-AP (cells were depolarized from –70 to 60 mV in 10-mV steps from a holding potential of –80 mV). The 15 IB4-positive neurons could be further separated into 2 populations with different components of 4-AP–sensitive A-type and TEA-sensitive IK currents (Fig. 1, A and B). In 10 of 15 IB4-positive cells (66%), there was a predominant 4-AP–sensitive IA component (Fig. 1A). In the remaining 5 (34%) IB4-positive cells, the 4-AP–sensitive Kv currents displayed both fast (IA)- and slow (ID)-inactivating components, and the total Kv current showed little inactivation (Fig. 1B). By contrast, in 17 IB4-negative neurons examined, there were 3 subpopulations of cells with different rise and decay kinetics of Kv currents (Fig. 1, C, D, and E). All of the 17 IB4-negative cells exhibited a major proportion of TEA-sensitive IK and small 4-AP–sensitive A-type (i.e., IA and ID) Kv currents, compared with those in IB4-positive neurons. In general, IB4-positive cells had a higher total Kv current density and a greater contribution of 4-AP–sensitive A-type currents to the total Kv current (Fig. 2A). Conversely, there was a larger contribution of TEA-sensitive IK currents to the total Kv current in IB4-negative than in IB4-positive cells (Fig. 2A).
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). In both IB4-positive and -negative neurons tested, the Kv currents in most cells exhibited an initial fast decay followed by a delayed slow-inactivating component (Fig. 3, A and C). In a small proportion of neurons in both groups, the total Kv currents displayed only a slow-inactivation component (Fig. 3, B and D). Further treatment with 25 mM TEA and 5 mM 4-AP revealed a similar difference in IA, ID, and IA subtypes between IB4-positive and -negative neurons (Fig. 2B), as described above. Using this pulse protocol, the IA current was significantly larger in both IB4-positive (19.6% increase at 60 mV) and -negative (20.4% increase at 60 mV) DRG cells. In the presence of both 25 mM TEA and 5 mM 4-AP, the remaining peak current was very small in both IB4-positive (6.4%) and -negative (5.8%) neurons (Fig. 3).
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Because only IB4-positive neurons bind to IB4-Alexa Fluor 594, we determined whether the observed differences in the total and 4-AP–sensitive A-type Kv current density between IB4-positive and -negative neurons were a result of IB4-Alexa Fluor 594 binding to the cell membrane. In 8 separate DRG neurons, we measured the total Kv and A-type current (in the presence of 25 mM TEA) and then perfused the cell with IB4-Alexa Fluor 594 for 1–2 min followed by a wash period of 3–4 min. We then recorded both total Kv and A-type current from the same cell. Both the total Kv and 4-AP–sensitive A-type current densities were not significantly altered by application of IB4-Alexa Fluor 594 in these 8 IB4-positive neurons examined (Fig. 3C).
Influence of A-type Kv currents on action potentials in DRG neurons
It has been shown that IB4-positive and -negative DRG neurons of mice have distinct firing properties (Stucky and Lewin 1999). We next determined whether the difference in the density of 4-AP–sensitive A-type current between IB4-positive and -negative rat DRG neurons contributes to distinct firing properties of these 2 groups of neurons. All 24 of the IB4-positive neurons fired multiple action potentials upon current injection (Fig. 4A). Although 13 of 22 (70%) IB4-negative neurons displayed repetitive firing, about 30% (9/22) of IB4-negative neurons showed only one action potential (Fig. 4B). In a separate group of IB4-positive (n = 10) and -negative (n = 8) neurons, the action potentials were elicited by depolarizing current injection after initial hyperpolarizing current (–300 pA) injection. The firing properties of action potential of these neurons were not different from those evoked by depolarizing current injection only.
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Kv1.1, Kv1.2, and Kv1.4 immunoreactivities in DRG neurons
For Kv1.1, Kv1.2, and Kv1.4 subunits, negative controls (omitting the primary antibody and replacing the primary antibody with nonimmune serum from the same species) resulted in no detectable immunostaining of DRG neurons. Furthermore, there was no evident labeling after preabsorption of the primary antibody with the corresponding immunogen. Although both the Kv1.1 and Kv1.2 immunoreactivities were predominantly present in DRG neurons with a diameter of >30 µm, it was also distributed in some cells with a soma size between 15 and 30 µm (Fig. 6, A and B). Notably, double fluorescence labeling showed that none of the IB4-positive DRG neurons exhibited the Kv1.1 or Kv1.2 immunoreactivity (Fig. 6, A and B). In contrast, the Kv1.4 immunoreactivity was distributed in a large number of small-diameter (15–30 µm) DRG neurons (Fig. 6C). Two different Kv1.4 primary antibodies produced a similar labeling profile in the DRG. Furthermore, double fluorescence labeling revealed that all IB4-positive DRG neurons contained the Kv1.4 immunoreactivity (Fig. 6C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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IB4-positive and -negative DRG neurons are the 2 broad classes of small-diameter nociceptive neurons (Snider and McMahon 1998). Whereas IB4-positive DRG neurons express receptors for GDNF (Bennett et al. 1998), most IB4-negative DRG neurons express TrkA receptors for NGF (Snider and McMahon 1998). Both classes of DRG neurons are involved in physiological detection of nociceptive stimuli. For instance, rats and mice deprived of NGF during embryonic development by antibodies or gene targeting are unable to respond to painful stimuli (Crowley et al. 1994; Smeyne et al. 1994). Recent studies have also documented that humans with mutations of TrkA receptors or NGF beta gene cannot detect painful stimuli (Einarsdottir et al. 2004; Mardy et al. 2001). On the other hand, it has been shown that depletion of IB4-positive DRG neurons with a cytotoxin targeting of IB4 in adult rats leads to decreased sensitivities to noxious stimuli (Vulchanova et al. 2001). The different physiological functions of IB4-positive and -negative DRG neurons in acute and chronic pain states remain to be established. The difference in the firing properties of IB4-positive and -negative small DRG neurons has been largely attributed to the high density of TTX-R Na+ channels in IB4-positive neurons (Stucky and Lewin 1999). In addition to Na+ channels, A-type Kv channels are also involved in regulation of neuronal firing. In the present study, we observed that the density of whole cell Kv currents was significantly larger in IB4-positive than in IB4-negative DRG neurons of rats. Furthermore, the fraction of 4-AP–sensitive A-type currents was much larger in IB4-positive than in IB4-negative neurons. In contrast, the density of TEA-sensitive IK currents was significantly higher in IB4-negative than in IB4-positive cells. The possibility that IB4 labeling affected the Kv channels is unlikely because labeling with IB4-Alexa Fluor 594 did not significantly alter the magnitude of both A-type and total Kv currents. Thus these data strongly suggest that IB4-positive DRG neurons are endowed with a high density of 4-AP–sensitive A-type Kv currents.
A-type Kv currents have been implicated in the delay of the spike onset and the decrease in the firing frequency of central neurons (Pongs 1999). We further determined whether the difference in the density of 4-AP–sensitive A-type Kv currents between IB4-positive and -negative neurons contributes to the distinct firing properties of these 2 groups of cells. We observed that the action potential duration was significantly longer in IB4-positive than in IB4-negative rat DRG neurons. However, inhibition of A-type Kv currents with 4-AP did not have a significant effect on action potential duration and the resting membrane potential in either group of neurons. These data are consistent with the finding that the TTX-R Na+ and high-voltage–activated Ca2+ channels are the most important determinants for these 2 parameters of action potentials in the DRG neurons (Blair and Bean 2002). Nevertheless, we found that IB4-positive neurons had a longer latency of action potential generation in response to current injection compared with that of IB4-negative DRG neurons of rats. We observed that some IB4-negative DRG neurons fired only a single action potential. This is probably explained by the presence of large-conductance Ca2+-activated K+ currents or M (KCNQ) currents in these cells (Passmore et al. 2003; Zhang et al. 2003). Importantly, 4-AP significantly reduced the latency of action potential generation and increased the firing frequency of IB4-positive but not IB4-negative neurons. Furthermore, 4-AP significantly reduced the amount of current injection required to evoke action potentials in IB4-positive but not in IB4-negative neurons. These data suggest that the high density of 4-AP–sensitive A-type Kv currents contributes importantly to the higher basal firing threshold and electrogenesis of IB4-positive DRG neurons. The A-type Kv channels probably function to suppress neuronal excitability by clamping the membrane potential near the resting level to prevent the membrane potential from reaching the threshold for action potential generation in these IB4-positive neurons.
The 4-AP–sensitive A-type Kv channels in many central neurons consist of Kv1.1 and/or Kv1.2 coassembled with Kv1.4 (Cooper et al. 1998; Rhodes et al. 1995; Sheng et al. 1992). The native A-type Kv channels in the DRG are formed by heteromeric subunits, especially those of the Shaker gene (Kv1) subfamily (Rasband et al. 2001). In the DRG, Kv1.4 has been identified to be the subunit primarily expressed in small-diameter cells (Rasband et al. 2001). In this study, we compared the difference in the Kv1.1, Kv1.2, and Kv1.4 immunoreactivities between IB4-positive and -negative DRG neurons. Similar to the previous study (Rasband et al. 2001), we observed that the Kv1.1 and Kv1.2 immunoreactivity was mainly present in large- and medium-sized DRG neurons. On the other hand, the Kv1.4 immunoreactivity was distributed to most small-diameter DRG neurons. An important finding of the present study is that all the IB4-positive neurons had Kv1.4 subunit immunoreactivity. Interestingly, no immunoreactivity for the Kv1.1 and Kv1.2 subunits was found in the IB4-positive neurons. These results suggest that Kv1.4 probably is an important subunit to form 4-AP–sensitive A-type Kv currents in IB4-positive neurons, and Kv1.1 and Kv1.2 subunits may constitute the TEA-sensitive IK current in IB4-negative neurons. It should be noted that other subunits in the Kv1 and Kv4 subfamilies also could be important in forming functional A-type Kv currents in DRG neurons. A complete identification of different Kv subunits in IB4-positive and -negative DRG neurons is beyond the scope of this study and warrants further investigation. There is a profound decrease in the Kv1.1–Kv1.4 subunits encoding A-type Kv currents in the DRG after peripheral nerve injury in rats (Kim et al. 2002; Rasband et al. 2001). Because A-type Kv currents preferentially regulate the excitability of IB4-positive DRG neurons, it is possible that reduction of Kv1 family subunits would primarily cause hyperactivity of this group of nociceptors after peripheral nerve injury.
In summary, this study demonstrates a clear difference in the density of 4-AP–sensitive A-type Kv currents between the IB4-positive and -negative small DRG neurons. Furthermore, our data suggest that the A-type Kv currents act to suppress the initiation of action potentials in IB4-positive neurons. This study provides complementary new information to our understanding of the distinct functions of IB4-positive and -negative DRG neurons and their potential roles in nociception. It is reasonable to expect that development of selective A-type Kv channel openers would be effective in the treatment of chronic neuropathic pain caused by damage to IB4-positive primary afferent neurons. Further studies are required to establish the role of subsets of sensory neurons in various acute and chronic pain states so that more effective treatment strategies can be designed.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H.-L. Pan, Department of Anesthesiology, H187, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (E-mail: hpan{at}psu.edu)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, and Priestley JV. A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 18: 3059–3072, 1998.
Blair NT and Bean BP. Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J Neurosci 22: 10277–10290, 2002.
Cooper EC, Milroy A, Jan YN, Jan LY, and Lowenstein DH. Presynaptic localization of Kv1.4-containing A-type potassium channels near excitatory synapses in the hippocampus. J Neurosci 18: 965–974, 1998.
Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, MacMahon SB, Shelton DL, Levinson AD, and Phillips HS. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76: 1001–1011, 1994.[CrossRef][ISI][Medline]
Einarsdottir E, Carlsson A, Minde J, Toolanen G, Svensson O, Solders G, Holmgren G, Holmberg D, and Holmberg M. A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Hum Mol Genet 13: 799–805, 2004.
Everill B and Kocsis JD. Reduction in potassium currents in identified cutaneous afferent dorsal root ganglion neurons after axotomy. J Neurophysiol 82: 700–708, 1999.
Everill B, Rizzo MA, and Kocsis JD. Morphologically identified cutaneous afferent DRG neurons express three different potassium currents in varying proportions. J Neurophysiol 79: 1814–1824, 1998.
Gold MS, Shuster MJ, and Levine JD. Characterization of six voltage-gated K+ currents in adult rat sensory neurons. J Neurophysiol 75: 2629–2646, 1996.
Harper AA and Lawson SN. Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones. J Physiol 359: 31–46, 1985.
Ishikawa T, Nakamura Y, Saitoh N, Li WB, Iwasaki S, and Takahashi T. Distinct roles of Kv1 and Kv3 potassium channels at the calyx of Held presynaptic terminal. J Neurosci 23: 10445–10453, 2003.
Kashiba H, Uchida Y, and Senba E. Difference in binding by isolectin B4 to trkA and c-ret mRNA-expressing neurons in rat sensory ganglia. Brain Res Mol Brain Res 95: 18–26, 2001.[Medline]
Kim DS, Choi JO, Rim HD, and Cho HJ. Downregulation of voltage-gated potassium channel alpha gene expression in dorsal root ganglia following chronic constriction injury of the rat sciatic nerve. Brain Res Mol Brain Res 105: 146–152, 2002.[Medline]
Liu L and Simon SA. Modulation of IA currents by capsaicin in rat trigeminal ganglion neurons. J Neurophysiol 89: 1387–1401, 2003.
Mardy S, Miura Y, Endo F, Matsuda I, and Indo Y. Congenital insensitivity to pain with anhidrosis (CIPA): effect of TRKA (NTRK1) missense mutations on autophosphorylation of the receptor tyrosine kinase for nerve growth factor. Hum Mol Genet 10: 179–188, 2001.
McFarlane S and Cooper E. Kinetics and voltage dependence of A-type currents on neonatal rat sensory neurons. J Neurophysiol 66: 1380–1391, 1991.
McMahon SB, Armanini MP, Ling LH, and Phillips HS. Expression and coexpression of Trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets. Neuron 12: 1161–1171, 1994.[CrossRef][ISI][Medline]
Molliver DC, Radeke MJ, Feinstein SC, and Snider WD. Presence or absence of TrkA protein distinguishes subsets of small sensory neurons with unique cytochemical characteristics and dorsal horn projections. J Comp Neurol 361: 404–416, 1995.[CrossRef][ISI][Medline]
Passmore GM, Selyanko AA, Mistry M, Al-Qatari M, Marsh SJ, Matthews EA, Dickenson AH, Brown TA, Burbidge SA, Main M, and Brown DA. KCNQ/M currents in sensory neurons: significance for pain therapy. J Neurosci 23: 7227–7236, 2003.
Plenderleith MB and Snow PJ. The plant lectin Bandeiraea simplicifolia I-B4 identifies a subpopulation of small diameter primary sensory neurones which innervate the skin in the rat. Neurosci Lett 159: 17–20, 1993.[CrossRef][ISI][Medline]
Pongs O. Voltage-gated potassium channels: from hyperexcitability to excitement. FEBS Lett 452: 31–35, 1999.[CrossRef][ISI][Medline]
Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, and Trimmer JS. Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci USA 98: 13373–13378, 2001.
Rhodes KJ, Keilbaugh SA, Barrezueta NX, Lopez KL, and Trimmer JS. Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain. J Neurosci 15: 5360–5371, 1995.[Abstract]
Safronov BV, Bischoff U, and Vogel W. Single voltage-gated K+ channels and their functions in small dorsal root ganglion neurones of rat. J Physiol 493: 393–408, 1996.[ISI]
Sheng M, Tsaur ML, Jan YN, and Jan LY. Subcellular segregation of two A-type K+ channel proteins in rat central neurons. Neuron 9: 271–284, 1992.[CrossRef][ISI][Medline]
Silverman JD and Kruger L. Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers. J Neurocytol 19: 789–801, 1990.[CrossRef][ISI][Medline]
Slugg RM, Meyer RA, and Campbell JN. Response of cutaneous A- and C-fiber nociceptors in the monkey to controlled-force stimuli. J Neurophysiol 83: 2179–2191, 2000.
Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA, and Barbacid M. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368: 246–249, 1994.[CrossRef][Medline]
Snider WD and McMahon SB. Tackling pain at the source: new ideas about nociceptors. Neuron 20: 629–632, 1998.[CrossRef][ISI][Medline]
Stucky CL and Lewin GR. Isolectin B(4)-positive and -negative nociceptors are functionally distinct. J Neurosci 19: 6497–6505, 1999.
Vulchanova L, Olson TH, Stone LS, Riedl MS, Elde R, and Honda CN. Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neuroscience 108: 143–155, 2001.[CrossRef][ISI][Medline]
Wang H, Rivero-Melian C, Robertson B, and Grant G. Transganglionic transport and binding of the isolectin B4 from Griffonia simplicifolia I in rat primary sensory neurons. Neuroscience 62: 539–551, 1994.[CrossRef][ISI][Medline]
Wu ZZ, Chen SR, and Pan HL. Differential sensitivity of N- and P/Q-type Ca2+ channel currents to a mu opioid in isolectin B4-positive and -negative dorsal root ganglion neurons. J Pharmacol Exp Ther 311: 939–947, 2004.
Wu ZZ and Pan HL. High voltage-activated Ca2+ channel currents in isolectin B4-positive and -negative small dorsal root ganglion neurons of rats. Neurosci Lett 368: 96–101, 2004.[CrossRef][ISI][Medline]
Yang EK, Takimoto K, Hayashi Y, de Groat WC, and Yoshimura N. Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy. Neuroscience 123: 867–874, 2004.[CrossRef][ISI][Medline]
Yao H, Donnelly DF, Ma C, and LaMotte RH. Upregulation of the hyperpolarization-activated cation current after chronic compression of the dorsal root ganglion. J Neurosci 23: 2069–2074, 2003.
Zhang XF, Gopalakrishnan M, and Shieh CC. Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons. Neuroscience 122: 1003–1011, 2003.[CrossRef][ISI][Medline]
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