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NEUROPHARMACOLOGY
Department of Anesthesiology, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania
Received June 28, 2004; accepted July 27, 2004.
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Abstract |
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- and C-fiber afferents are connected to small DRG neurons and transmit primarily nociceptive information. Opioids have a distinct effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms of this differential effect are not fully known. One of the important analgesic mechanisms of µ opioids is through inhibition of synaptic transmission by acting on voltage-gated Ca2+ channels present on the central terminals of DRG neurons (Rusin and Moises, 1995
; Kohno et al., 1999). It has been demonstrated that µ opioids inhibit voltage-gated Ca2+ channels in dissociated DRG neurons from 10 to 90% (Schroeder and McCleskey, 1993; Moises et al., 1994). It is likely that the large difference of the opioid effect on Ca2+ channels is due to the heterogeneity of DRG neurons studied. The opioid sensitivity and voltage-gated Ca2+ channels in different classes of DRG neurons remain unclear.
A novel approach to the classification of DRG neurons is derived from the requirements of subsets of neurons for specific neurotrophins because DRG neurons subserving specific sensory modalities are supported by different neurotrophins (Snider and McMahon, 1998). Based on the expression of receptors for neurotrophic factors that regulate them in adulthood, two broad classes of small DRG neurons have been classified (McMahon et al., 1994; Averill et al., 1995; Molliver et al., 1995). One class of small DRG neurons expresses the TrkA receptor for nerve growth factor (NGF) and contain neuropeptides such as substance P and calcitonin gene-related peptide. Another class of small DRG neurons possesses cell surface glycol-conjugates that can be identified by the binding of the Griffonia simplicifolia isolectin B4 (IB4) (Wang et al., 1994; Bennett et al., 1998). The IB4-positive cells express receptors for glial cell line-derived neurotrophic factor (GDNF) and are relatively "peptide poor" but express predominantly P2X3 and capsaicin receptors (TRPV1) (Bradbury et al., 1998; Guo et al., 1999). Furthermore, the central terminals of IB4-positive DRG neurons are specifically terminated in inner lamina II of the spinal cord (Pan et al., 2003). The above-mentioned neurochemical and anatomical differences have led to a proposal that IB4-positive and IB4-negative small DRG neurons may be functionally distinct groups of nociceptors (Snider and McMahon, 1998; Stucky and Lewin, 1999). However, little is known about the preferential effect of µ opioids on voltage-gated Ca2+ channels in IB4-positive and -negative DRG neurons. In the present study, we specifically determined the potential difference of the effect of the µ opioid agonist on voltage-gated Ca2+ channel currents between these two subsets of neurons.
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Materials and Methods |
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). Approximately equal number of IB4-positive and IB4-negative neurons was studied in one rat. Recordings were made within 10 h after dissociation to keep the experiment as similar to in vivo as possible and to minimize the space clamp error since DRG neurons produce neurites that are difficult to clamp after prolonged neuronal culture.
Electrophysiological Recordings. Patch electrodes with a resistance of 
2 M
were pulled from GC150TF-10 glass capillaries (i.d. 1.17 mm, o.d. 1.5 mm; Harvard Apparatus Inc., Holliston, MA) using a micropipette puller (P-97; Sutter Instrument Company, Novato, CA) and fire-polished (DMF1000; WPI, Sarasota, FL). Immediately before recording, neurons were treated with IB4-Alexa 594 (3 µg/ml; Molecular Probes, Eugene, OR) in Tyrode's solution for 10 min and then rinsed for at least 3 min. Neurons were visualized using a combination of epifluorescence illumination and differential interference contrast (20–40x) optics on an inverted microscope (Olympus Optical, Tokyo, Japan). Images of cells were taken with a charge-coupled device camera and displayed on a video monitor. The diameter of most IB4-positive rat DRG neurons was in the range of 15 to 30 µm. Thus, the similar size of IB4-negative cells was recorded for comparison. Neurons were recorded in the whole-cell configuration using an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany). Seals (1–10 G) between the electrode and cell were established in a modified Tyrode's solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4 adjusted with NaOH, osmolarity 320 mOsM). After whole-cell configuration was established, the cell membrane capacitance and series resistance were electronically compensated, as described previously (Wu and Pan, 2004). Leakage and capacitative currents were subtracted on-line using the P/4 subtraction protocol. All experiments were performed at room temperature (25°C). Signals were filtered at 1 kHz, digitized at least 10 kHz, and acquired using Pulse program (HEKA). In some control experiments, recordings were performed on neurons before they were labeled with IB4-Alexa 594.
Barium currents (IBa) flowing through Ca2+ channels (Moises et al., 1994; Wu and Pan, 2004) were recorded using extracellular solution consisting of 140 mM tetraethylammonium, 2 mM MgCl2, 3 mM BaCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4 adjusted with tetraethylammonium-OH, osmolarity 320 mOsM). The pipette solution contained 120 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na-GTP (pH 7.2 adjusted with CsOH, osmolarity 300 mOsM). The whole-cell voltage-gated Ca2+ channel current, carried by barium (IBa), was activated by a series of command potentials from –70 to 55 mV (150 ms, 5-mV increments in 5-s intervals) from a holding potential of –90 mV. To minimize the "run-down" associated with the whole-cell recording, GTP and ATP were included in the pipette solution. The run-down of voltage-gated Ca2+ currents typically occurs in 10 to 15 min after the whole-cell configuration was established in DRG neurons. In our experiments, the protocols for both IB4-positive and -negative cells were always completed within 10 min after rupturing the cell membrane. Furthermore, the washout data were always obtained if the drug effect (i.e., DAMGO, nimodipine, and agotoxin IVA) is reversible so that the run-down can be assessed and controlled for.
Drug Application. Drugs were dissolved in distilled water at 1000 times the final concentration and kept frozen in aliquots. Nimodipine was prepared as a stock solution dissolved in dimethyl sulfoxide. The stock solutions were diluted in extracellular solution just before use and held in a series of independent syringes connected to corresponding fused silica columns (i.d. 200 µm). The end of the parallel columns was connected to a common silica column. The distance from the column mouth to the cell examined was about 100 µm. Cells in the recording chamber were continuously bathed in extracellular 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 (WPI). Drugs and chemicals were purchased from Sigma-Aldrich except 
-conotoxin GVIA, -agatoxin IVA, -conotoxin MVIIC (Alomone Labs, Jerusalem, Israel), D-Phe-Cys-Thr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; Tocris Cookson Inc., Billerica, MO), and morphine (Astra Pharmaceutical Inc., Westborough, MA).
Double Fluorescence Labeling of IB4 and µ Opioid Receptors in DRG Cells. Under deep anesthesia with sodium pentobarbital (60 mg/ml i.p.), three rats were intracardially perfused with 250 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) and 200 ml of 10% sucrose in 0.1 M PBS (pH 7.4). The DRGs were removed quickly and postfixed in the same fixative solution and cryoprotected in 30% sucrose in PBS for 48 h at 4°C. The tissues were cut 35 µm in thickness and collected free-floating in 0.1 M PBS. The µ opioid receptor immunofluorescence labeling was performed as we described previously (Chen and Pan, 2003). Briefly, the sections were rinsed in 0.1 M PBS and blocked in 4% normal goat serum in PBS for 1 h. Sections were incubated with the primary antibody (rabbit anti-µ opioid receptor, dilution 1:1000; Neuromics, Northfield, MN) diluted in PBS containing 2% normal goat serum for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed in PBS and incubated with the secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, dilution, 5 µg/ml; Molecular Probes, Eugene, OR). The sections then were rinsed in PBS for 30 min and incubated with Alexa Fluor 594 conjugated to IB4 (dilution 2 µg/ml; Molecular Probes) diluted in PBS containing 1 mM Ca2+ for 2 h at room temperature. Finally, the sections were rinsed and mounted on slides, dried, and coverslipped. The sections were examined on a confocal microscope (Leica, Wetzlar, Germany), and areas of interest were photodocumented. Confocal laser scanning microscopy was used for accurate colocalization of fluorescent markers, because the thin (–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 colocalization was indicated by the color change and represents colocalization. To quantify the immunoreactivity of µ opioid receptors present in IB4-positive and -negative cells, the optical density of the immunoreactivity in individual DRG neurons was measured by a computer-based imaging analysis program (AIS; Imaging Research, St. Catharines, ON, Canada), as we described previously (Chen and Pan, 2003). The mean intensity of fluorescence of IB4-positive and -negative cells with a diameter of 15 to 30 µm in the same section was determined. This was analyzed by drawing regional boundaries to outline the edge of the entire section of individual IB4-positive and -negative neurons. The background mean fluorescence density was measured in the area between cells and subtracted. Measurements (relative optical density) were taken from 10 to 12 DRG sections from each animal.
Data Analysis. Data were analyzed using PulseFit software program (HEKA). Whole-cell current-voltage curves for individual neurons were generated by calculating the mean peak inward current at each testing potential and normalized for cell capacitance. The percentage of inhibition of total IBa and subtypes of Ca2+ channel currents was defined as the ratio of DAMGO- or morphine-sensitive currents to total IBa. Statistical data are presented as means ± S.E.M. All comparisons between means were tested for significance using Student's unpaired t test unless otherwise indicated. P < 0.05 was considered to be statistically significant.
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Results |
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DAMGO Inhibition on Ca2+ Channel Currents in IB4-Positive and -Negative Neurons. For high voltage-activated Ca2+ channel currents, the inward IBa was typically activated at –35 mV and reached its maximum at –10 mV in most DRG neurons. DAMGO demonstrated a voltage- and concentration-dependent inhibition on IBa in all small sized cells without T-type currents (Figs. 2 and 3). DAMGO (1 µM) inhibited IBa in IB4-positive and -negative cells at the testing potentials ranging from –20 to 0 mV but did not shift the current-voltage relationship along the voltage axis (Fig. 3). The inhibition was most evident at the peak IBa (–10 mV) in both IB4-positive and -negative neurons, and became less effective at more depolarized potentials. DAMGO-sensitive IBa in IB4-negative neurons was significantly larger than that in IB4-positive cells. DAMGO inhibited 44.6 ± 4.4% (n = 14) of IBa in IB4-negative neurons and 31.4 ± 5.7% (n = 13) of IBa in IB4-positive neurons at –10 mV (P < 0.05; Fig. 3).
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The concentration-response relationship for the effect of DAMGO on IBa was obtained by perfusion of a series of concentrations of DAMGO in IB4-positive and -negative cells (Fig. 2). We applied different concentrations of DAMGO (1 nM–10 µM) in a random order. These cells were depolarized to –10 mV for 150 ms from a holding potential of –90 mV. In both IB4-positive and -negative neurons, DAMGO reached the maximal inhibition of IBa at about 2 µM (Fig. 2). At concentrations of 1, 2, and 10 µM, DAMGO inhibited 24.3 ± 2.6% (n = 21), 33.4 ± 3.5% (n = 16), and 35.8 ± 4.4% (n = 14) of IBa, respectively, in IB4-positive neurons. In IB4-negative cells, DAMGO-produced inhibitions of IBa was 35.2 ± 4.5% (n = 12), 47.8 ± 4.1% (n = 10), and 49.8 ± 3.5% (n = 9) at the concentration of 1, 2, and 10 µM, respectively. The inhibitory effect of 1 to 10 µM DAMGO on IBa in IB4-negative cells was significantly different from that in IB4-positive cells (Fig. 2; P < 0.05).
To further illustrate the difference of the magnitude of IBa inhibited by DAMGO between IB4-positive and -negative neurons, a distribution histogram was constructed to show the percentage of inhibition of IBa by DAMGO in these two groups of cells. IB4-positive and -negative cells were grouped according to the inhibition of their total IBa by 1 µM DAMGO at –10 mV. In both groups, DAMGO suppressed IBa in widely varying degrees (Fig. 3C). In 54.4% (31/57) IB4-negative cells, 1 µM DAMGO reduced the IBa greater than 40%. However, in most (84.4%, 65/77) of IB4-positive cells, inhibition of IBa by DAMGO was less than 40% (Fig. 3C).
To further examine the differential effect of µ opioids on IBa in IB4-positive and -negative cells, we tested the effect of another µ opioid agonist, morphine. Morphine is the most commonly used clinical drug acting on µ opioid receptors. In IB4-positive cells (n = 10), morphine inhibited 15.0 ± 3.0 and 23.7 ± 4.2% of IBa at 1 and 10 µM, respectively (Fig. 4). In comparison, in IB4-negative cells (n = 11), morphine inhibited 27.9 ± 3.2 and 37.1 ± 3.4% of IBa at 1 and 10 µM, respectively. At both concentrations, morphine produced a significantly larger inhibition of IBa in IB4-negative than IB4-positive cells (Fig. 4).
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To verify the role of µ opioid receptors in the effect of DAMGO on IBa, CTOP, a potent µ opioid receptor antagonist (Hawkins et al., 1989), was used. CTOP (10 µM) itself did not produce any effect on IBa in IB4-positive (n = 5) and -negative (n = 4) cells (Fig. 5A). DAMGO (1 µM) alone inhibited 29.2 ± 1.2 and 38.9 ± 9.8% of IBa in IB4-positive and -negative cells, respectively. In the presence of CTOP, DAMGO failed to inhibit IBa significantly in both IB4-positive and -negative neurons (Fig. 5).
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Effect of IB4 Labeling on the Effect of DAMGO on IBa. We then determined whether the smaller effect of DAMGO on IBa in IB4-positive neurons was caused by IB4 dye binding to the membrane of these cells. In seven additional neurons, we first recorded the IBa elicited by depolarizing the neuron from –90 to –10 mV without prior IB4 labeling. Under this condition, IBa was inhibited 29.7 ± 2.9% by 1 µM DAMGO. These neurons were then verified to be IB4-positive cells by subsequent perfusion of the IB4-Alexa 594 (3 µg/ml) solution for 3 min and washed for at least another 3 min using normal external solution (neurons not labeled by IB4 were excluded from analysis). The inhibitory effect of 1 µM DAMGO was then reexamined in the same cell. DAMGO inhibited 29.1 ± 3.3% of IBa in the same neurons after IB4 labeling, which was not significantly different from the initial effect of DAMGO on IBa before IB4 labeling (Fig. 6; P > 0.05).
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µ Opioid Receptor Immunoreactivity in IB4-Positive and -Negative DRG Neurons. To determine the µ opioid receptor distribution in IB4-positive and -negative DRG neurons, double fluorescence labeling of µ opioid receptors and IB4 was performed. The omission of the primary antibody resulted in negative labeling in the DRG sections. Most of the IB4-positive cells had a diameter between 15 and 30 µm. Among small-diameter (15–30-µm) cells, about 65% of DRG neurons were IB4-positive (Fig. 7). The µ opioid receptor immunoreactivity was present in different sizes of DRG neurons. Furthermore, the µ opioid receptor immunoreactivity was significantly higher in IB4-negative than IB4-positive cells with a diameter of 15 to 30 µm. The relative optical density of µ opioid receptor-immunoreactivity in 73 IB4-negative and 88 IB4-positive cells was 2.35 ± 0.25 and 1.99 ± 0.26 (P < 0.05), respectively.
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Subtypes of Voltage-Gated Ca2+ Currents Inhibited by DAMGO. To determine which high voltage-gated calcium currents subtypes were inhibited by DAMGO in IB4-positive and -negative neurons, the selective Ca2+ channel blockers nimodipine (5 µM; L-type), -conotoxin GVIA (2 µM; N-type), -agatoxin IVA (100 nM; P/Q-type), and -conotoxin MVIIC (500 nM; N-and P/Q-type) were combined appropriately to define L-, N-, P/Q-, and R-type Ca2+ channels (Figs. 8 and 9) (Randall and Tsien, 1995; McDonough et al., 1996). Because 100 nM -agatoxin IVA is not sufficient to block the Q-type Ca2+ channel (Randall and Tsien, 1995), -conotoxin MVIIC was coapplied with -agatoxin IVA to isolate L-and R-type currents. In this protocol, the cells were depolarized from –90 to –10 mV for 150 ms. The effect of DAMGO (1 µM) was initially tested without blockers to determine its inhibition on total IBa. After the initial effect of DAMGO was washed out, a series of grouped blockers were used to isolate a defined subtype of Ca2+ channel currents before the effect of DAMGO (1 µM) was reexamined (Figs. 8 and 9).
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DAMGO had a significantly greater inhibition of total IBa in IB4-negative (43.9 ± 2.9%; n = 29) than that in IB4-positive cells (30.0 ± 1.7%; n = 40) (P < 0.05). Nimodipine and -conotoxin GVIA were then applied together to isolate the P/Q- and R-type IBa. The drug-resistant fraction of IBa was considered as the R-type Ca2+ current. Notably, this subtype was almost negligible in small DRG neurons (Figs. 8 and 10). Furthermore, in both IB4-positive and -negative neurons, DAMGO had no significant effect on the R-type IBa (Figs. 8 and 10). In the presence of both nimodipine and -conotoxin GVIA, DAMGO produced a greater inhibition on IBa in IB4-negative (8.5 ± 1.4%; n = 16) cells than that in IB4-positive cells (4.5 ± 0.4%; n = 21) (Figs. 8 and 10; P < 0.05). To further determine the effect of DAMGO on R-type IBa, -conotoxin MVIIC and -agatoxin IVA were coapplied with nimodipine and -conotoxin GVIA. DAMGO failed to inhibit the remaining IBa. Cd2+ (300 µM) completely blocked the remaining IBa insensitive to all the blockers used in IB4-positive and -negative neurons (Fig. 8).
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Similarly, the N-type IBa was differentiated by coapplication of nimodipine and -agatoxin GVIA in separate group of neurons. Since the R-type current was very small and DAMGO had no effect on the R-type current, it is reasonable to assume that the fraction of IBa inhibited by DAMGO was N-type calcium channels (Figs. 9 and 10). DAMGO-produced inhibition of N-type IBa was significantly larger in IB4-negative cells (36.6 ± 5.2%; n = 13) than IB4-positive neurons (25.6 ± 2.1%; n = 19) (P < 0.05). Since 100 nM -agatoxin IVA does not completely block the Q-type Ca2+ channel in rat cerebellar granule neurons (Randall and Tsien, 1995), it is possible that a small component of IBa in the presence of nimodipine and -agatoxin GVIA may be the P/Q-type. Furthermore, to assess the effect of DAMGO on the L-type Ca2+ current, the effect of DAMGO on IBa was tested in the presence of -conotoxin GVIA, -conotoxin MVIIC, and -agatoxin IVA. DAMGO had no significant effect on the isolated L-type IBa in IB4-positive (0.1 ± 0.3%; n = 12) and -negative (0.6 ± 0.7%; n = 9) neurons (Figs. 9 and 10).
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Discussion |
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DRG neurons with thinly myelinated A fibers are rich in low voltage-activated (T-type) Ca2+ channels (Scroggs and Fox, 1992a,b). In our study, such T-type currents were only present in a few small-sized IB4-positive and -negative cells. Consistent with the previous study (Schroeder and McCleskey, 1993; Moises et al., 1994), we found that DAMGO had no significant effect on T-type Ca2+ currents in both IB4-positive and -negative DRG neurons. T-type Ca2+ channels are pharmacologically and physiologically heterogeneous (Cribbs et al., 1998; Perez-Reyes et al., 1998). Of the three subtypes (
1G, 1H, and 1I), only 1H is expressed predominantly in the rat DRG neurons (Talley et al., 1999). The Ca2+ channel inhibition produced by G protein-coupled receptor agonists is mediated primarily by the 
subunit (Ikeda, 1996). It has been demonstrated that T-type 1H (CaV3.2) is only inhibited by the 22 subunit (Wolfe et al., 2003). Thus, lack of inhibition of DAMGO on T-type Ca2+ channel currents suggests that µ opioid receptors are not coupled to G proteins with the 22 dimer in both IB4-positive and -negative DRG neurons.
We observed that the high voltage-activated Ca2+ currents in DRG neurons without T-type currents were profoundly inhibited by DAMGO and morphine. Furthermore, the fraction of IBa inhibited by DAMGO and morphine was significantly greater in IB4-negative than IB4-positive cells. The possibility that IB4 labeling attenuated the effect of DAMGO on IBa is unlikely, since the fraction of IBa inhibited by DAMGO before IB4 labeling was not significantly different from that after IB4 labeling. Because CTOP, a potent selective µ opioid receptor antagonist (Hawkins et al., 1989), blocked the inhibitory effect of DAMGO on IBa, it strongly suggests that DAMGO inhibits high voltage-gated Ca2+ channels through µ opioid receptors. The high voltage-gated Ca2+ channels are important for nociceptive neurotransmission (Jun et al., 1999; Vanegas and Schaible, 2000; Saegusa et al., 2001). In this regard, mice lacking N-type Ca2+ channels display reduced responses to a painful stimulus and attenuated neuropathic pain symptoms (Kim et al., 2001; Saegusa et al., 2001). The µ opioids can reduce Ca2+-dependent neurotransmitter release by inhibition of high voltagegated Ca2+ channels (Schroeder and McCleskey, 1993). Thus, the differential modulation of Ca2+ channels by µ opioids in IB4-positive and -negative neurons may have a different effect on neurotransmitter releases from the central terminals of IB4-positive and -negative DRG neurons.
The greater sensitivity of N-and P/Q-type Ca2+ currents to opioids in IB4-negative than IB4-positive neurons is an unexpected but intriguing finding, since IB4-positive (possessing TRPV1) neurons are generally considered to play a more important role in nociception (Guo et al., 1999; Vulchanova et al., 2001). The µ opioid receptors are negatively coupled to N- and P/Q-type Ca2+ channels (Moises et al., 1994; Rusin and Moises, 1995). We found both N- and P/Q-type Ca2+ channels were inhibited by DAMGO to a larger degree in IB4-negative than in IB4-positive cells. This could be due to a higher density of N- and P/Q-type Ca2+ channels or µ opioid receptors present in IB4-negative neurons. However, we have found that there is no significant difference in P/Q-type Ca2+ channel currents between IB4-positive and -negative cells (Wu and Pan, 2004). In fact, the current density of N-type Ca2+ channels is significantly higher in IB4-positive than -negative DRG neurons (Wu and Pan, 2004). Thus, the differential modulation of IBa by opioids is probably due to different densities of µ opioid receptors in these two subgroups of cells. This possibility is supported by our immunocytochemistry data showing that the µ opioid receptor immunoreactivity was approximately 18% higher in IB4-negative than -positive small DRG neurons. A recent study also has shown that the level of µ opioid receptor mRNA is proportional to the degree of inhibition of Ca2+ currents by DAMGO (Silbert et al., 2003). Thus, a higher density of µ opioid receptors in IB4-negative cells could result in a greater inhibition of IBa. The expression of µ opioid receptors in subsets of DRG cells may be regulated by different neurotrophic factors. For example, in NGF overexpressed mice, there is an increase in the mRNA of µ opioid receptor in DRG neurons. By contrast, the mRNA of µ opioid receptor is decreased in DRG neurons of mice overexpressed with GDNF (Zwick et al., 2003). Furthermore, it has been shown that intrathecal NGF restores opioid effectiveness in an animal model of neuropathic pain (Cahill et al., 2003). Alternatively, it has been shown that protein kinase C is preferentially expressed in IB4-positive DRG neurons (Molliver et al., 1995), and protein kinase C can affect the µ opioid receptor coupling to N-type Ca2+ channels (King et al., 1999). Thus, lesser inhibition of voltage-gated Ca2+ channels by µ opioids in IB4-positive cells may be due to the attenuated coupling between µ opioid receptors and Ca2+ channels by protein kinase C. Other possibilities, including the differential influence of neurotrophins and the phosphorylation and/or dephosphorylation state of voltage-activated Ca2+ channels, may account for the different sensitivity of N- and P/Q-type Ca2+ currents to µ opioids in IB4-positive and -negative DRG cells.
The precise roles of IB4-positive (expressing GDNF receptors) and IB4-negative (expressing NGF receptors) DRG neurons in nociception remain less clear, although some studies suggest that both groups are important for normal pain perception (Johnson et al., 1980; Crowley et al., 1994; Smeyne et al., 1994; Vulchanova et al., 2001). For example, 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). On the other hand, rats and mice deprived of NGF during embryonic development by antibodies or gene targeting are unable to respond to painful stimuli (Johnson et al., 1980; Crowley et al., 1994; Smeyne et al., 1994). Furthermore, recent studies have documented that humans with mutations of TrkA receptors or NGF gene are unable to detect pain (Mardy et al., 2001; Einarsdottir et al., 2004). A higher density of µ opioid receptors and preferential inhibition of high voltage-gated Ca2+ channels in IB4-negative cells (expressing TrkA receptors for NGF) suggest that nociception mediated by this subset of DRG neurons may be more effectively controlled by the µ opioids. In this regard, we have recently observed that the analgesic effect of intrathecal morphine was profoundly potentiated in rats treated with resiniferatoxin (unpublished data), which primarily depletes IB4-positive DRG neurons (Pan et al., 2003). Thus, this observation is consistent with the notion that µ opioids may be particularly efficacious in inflammatory persistent pain involving NGF-responsive and IB4-negative nociceptive afferent neurons (Zwick et al., 2003).
In summary, this study demonstrates a clear difference in the sensitivity of N- and P/Q-type Ca2+ channel currents to the µ opioid agonist between IB4-positive and -negative DRG neurons. The findings from this present study may help to explain the wide variation in neuron-to-neuron sensitivity to opioids reported in previous studies. This new information is important for a better understanding of the analgesic mechanisms of µ opioids and their effects on subpopulations of putative nociceptors.
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Footnotes |
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ABBREVIATIONS: DRG, dorsal root ganglion; NGF, nerve growth factor; IB4, Griffonia simplicifolia isolectin B4; GDNF, glial cell line-derived neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin; CTOP, D-Phe-Cys-Thr-D-Trp-Orn-Thr-Pen-Thr-NH2.
Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology, H187, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033-0850. E-mail: hpan{at}psu.edu
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