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1Departments of Pharmacology and Toxicology, 2Pediatrics, 3Microbiology and Immunology, and 4Neurology, Indiana University School of Medicine, Indianapolis, Indiana
Submitted 11 May 2005; accepted in final form 22 July 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). It is characterized by formation of neurofibromas (complex tumors composed of axonal processes, Schwann cells, fibroblasts and mast cells) as well as malignant tumors such as neurofibrosarcomas, malignant astrocytomas, and myeloid leukemias. In addition to tumor formation, some people with NF1 also experience a more intense painful response to stimuli, such as minor injuries, than normal (Creange et al. 1999; Riccardi and Eichner 1992; Wolkenstein et al. 2001). Although the mechanism by which the NF1 mutation causes these symptoms has not been elucidated, it is likely that the abnormal painful states involve the increased sensitivity of small-diameter nociceptive sensory neurons; cells that are known to mediate the transmission of pain.
In NF1 there is a mutation of one allele of the NF1 gene (NF1+/–). This results in reduced expression of the protein product of the NF1 gene, neurofibromin, in many cells, including neurons (Bollag and McCormick 1991; Cichowski and Jacks 2001; Largaespada et al. 1996; Zhang et al. 1998). Neurofibromin is a guanosine-triphosphatase-activating protein (GAP) that accelerates the conversion of the active form of the small G protein, Ras (Ras-GTP), to its inactive form (Ras-GDP) (Li et al. 1992; Martin et al. 1990; Wallace et al. 1990). In many cell types, mutation of the NF1 gene or its mouse correlate (Nf1), frequently results in increased basal and cytokine-stimulated Ras-GTP and enhanced activity of the downstream effectors of the Ras transduction cascade. For example, investigators have shown that the level of Ras-GTP is elevated in human NF1 neurogenic tumors (Guha et al. 1996), in mast cells from mice with a heterozygous mutation of the Nf1 gene (Nf1+/–) (Ingram et al. 2001), and in Schwann cells from embryonic mice with a homozygous mutation of the Nf1 gene (Nf1–/–) (Sherman et al. 2000). In addition, the sensory neurons from embryonic Nf1–/– mice demonstrate increased Ras activity (Klesse and Parada 1998; Vogel et al. 2000).
Among the many growth factors that activate the Ras transduction cascade, nerve growth factor (NGF) has been explored extensively for its role in pain signaling. NGF plays a critical role in the development and maintenance of sensory neurons; however, a growing body of evidence has demonstrated that NGF is an important mediator of the enhanced pain sensation (hyperalgesia) that occurs with inflammation. The content of NGF is elevated in inflamed skin (Weskamp and Otten 1987) and peripheral tissue (Aloe et al. 1992a,b). Mendell and coworkers demonstrated that NGF produces both thermal and mechanical hyperalgesia (Lewin and Mendell 1993; Lewin et al. 1993). In addition, the hyperalgesia associated with inflammation is diminished by an anti-NGF antibody (Woolf et al. 1994). By using a skin-nerve preparation, Rueff and Mendell (1996) demonstrated that NGF can increase the firing frequency of isolated saphenous nerve in response to heat stimulation. NGF also enhanced the excitability of isolated sensory neurons in culture by increasing a TTX-resistant sodium current and by suppressing a delayed-rectifier potassium current (Zhang et al. 2002). Although it is clear that NGF can sensitize sensory neurons to noxious stimuli, the intracellular cascades by which NGF exerts its effects remain poorly understood. The stimulation of either the TrkA or p75 receptor by NGF can lead to the activation of Ras transduction cascade (Blochl et al. 2004; Corbett and Alber 2001; Huang and Reichardt 2003; Susen et al. 1999). In addition, recent studies have suggested that NGF can activate downstream effectors of the Ras transduction cascade to affect changes in adult sensory neurons (Bron et al. 2003; Ganju et al. 1998; Zhuang et al. 2004). For example, Bron and colleagues have shown that NGF-induced increases in phosphorylated extracellular signal-regulated kinase (pERK) and phosphorylated Akt (pAkt), two downstream effectors of Ras activation, are associated with increases in the expression of the heat- and capsaicin-activated receptor, TRPV1, in DRG neurons and that constitutively active Ras mimics the action of NGF to increase TRPV1 expression in isolated sensory neurons (Bron et al. 2003). Based on the hypothesis that NGF-induced alteration in peripheral pain signaling may, in part, be related to activation of the Ras transduction cascade, the enhanced painful sensations experienced by people with NF1 could result from altered control of the Ras cascade because of decreased neurofibromin levels.
To test the hypothesis that the NF1 mutation results in increased sensory neuron excitability, we used a mouse model of NF1. These mice have a heterozygous mutation of the Nf1 gene (Nf1+/–), similar to that seen in the human disorder (Jacks et al. 1994). In this report, we demonstrate that capsaicin-sensitive sensory neurons from Nf1+/– mice exhibit enhanced excitability. Treatment of wild-type neurons with NGF mimics the increased excitability of Nf1+/– neurons. These results suggest that decreased GAP levels correlate with enhanced neuronal excitability and are consistent with the idea that GAP-regulated signaling pathways are important in the modulation of sensory neuron sensitivity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Mice heterozygous for the Nf1 mutation on a background of C57BL/6J were originally developed by Dr. Tyler Jacks (Jacks et al. 1994). All animals were housed and bred in the Indiana University Laboratory Animal Research Center and used in accordance with National Institute of Health Guide for Care and Use of Laboratory Animals (National Institutes of Health Publications No. 80–23) revised 1996.
Reagents
Horse serum, F-12 medium, L-glutamine, and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA). NGF was purchased from Harlan Bioproducts for Science (Indianapolis, IN). Papain was purchased from Worthington Biochemical, and dispase was obtained from Roche (Indianapolis, IN). Collagenase, poly-D-lysine, laminin, 5'-fluoro-2'-deoxyuridine, uridine, and standard laboratory chemicals were from Sigma (St Louis, MO).
Isolation of sensory neurons from Nf1+/– or Nf1+/– adult mice
The isolation of sensory neurons from 1- to 2-mo-old mice was accomplished using a modification of a method developed by Lindsay (1988). Briefly, the dorsal root ganglia were removed and transferred into a culture dish filled with sterilized Ca2+- and Mg2+-free Hank's balanced salts solution (HBSS) consisting of (in mM) 171 NaCl, 6.7 KCl, 1.6 Na2PO4, 0.5 KH2PO4, 6 D-glucose, and 0.01% phenol red, pH 7.3. The ganglia were incubated for 10–15 min at 37°C in HBSS containing papain (10 ng/ml) and then transferred into F-12 media containing 1 mg/ml collagenase 1A and 2.5 mg/ml dispase. After a 10- to 15-min incubation at 37°C in the second set of enzymes, the tissue sample was centrifuged for 30 s before the enzyme-containing supernatant was removed. The pellet was resuspended in F-12 media and mechanically dissociated with a fire-polished pipette until all large pieces of tissue were gone. The isolated cell suspension was plated onto plastic cover slips that were coated with 0.5% poly-D-lysine and laminin (100 µg/ml). The sensory neurons were maintained in F-12 media supplemented with 10% horse serum, 2 mM glutamine, 100 µg/ml normocin, 50 µg/ml penicillin and streptomycin, 50 µM 5-fluoro-2'-deoxyuridine, 150 µM uridine at 37°C and 3% CO2. These cells were used for electrophysiological recordings within 5–12 h after isolation. NGF was added to the F-12 media at the time of plating where indicated.
Electrophysiology
Recordings were made using the whole cell patch-clamp technique as previously described (Evans et al. 1999; Zhang et al. 2002). Briefly, a cover slip with the sensory neurons was placed in a recording chamber where the neurons were bathed in normal Ringer solution of the following composition (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.4 with NaOH. Recording pipettes were pulled from borosilicate glass tubing and typically had resistances of 2–5 M
when filled with the following solution (mM): 140 KCl, 5 MgCl2, 4 ATP, 0.3 GTP, 2.5 CaCl2, 5 EGTA (calculated free Ca2+ concentration of 
100 nM), and 10 HEPES, adjusted pH at 7.3 with KOH. Whole cell voltages were recorded with an Axopatch 200B amplifier (Axon Instruments). The data were acquired and analyzed using pCLAMP 9.0 (Axon Instruments). Only neurons that maintain resting membrane potentials more hyperpolarized than –45 mV were used in this study. In the current-clamp experiments, two protocols were used to assess the excitability. First, neurons were held at their resting potentials and injected with a 1-s ramp of depolarizing current that had a final amplitude of 1,000 pA. The sampling frequency was 2 kHz. Second, a series of current steps that were 200 ms in duration and of variable incremental amplitudes was used to determine the rheobase. The sampling frequency was 1.25 kHz. At the end of each recording, the neuron was superfused with a Ringer solution containing 100 nM capsaicin, as sensitivity to this agent is believed to be an indicator of nociceptive sensory neurons (Holzer 1991). The results presented were obtained from capsaicin-sensitive neurons only. All experiments were performed at room temperature (23°C).
Data analysis
Summarized data are presented as the means +/– SE. Statistical significance between groups was determined using a t-test or a one-way ANOVA followed by Dunnett's post hoc analysis, as appropriate and is specified in the text. Values of P < 0.05 were judged to be statistically significant.
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RESULTS |
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To determine whether nociceptive sensory neurons with reduced levels of neurofibromin have altered excitability, sensory neurons were isolated from Nf1+/– and wild-type mice. One measure of neuronal excitability is the number of APs elicited by a given amount of depolarizing current. Figure 1 shows representative recordings in response to the current ramp from single Nf1+/+ (A) and Nf1+/– (B) neurons, respectively. As can be easily appreciated, identical ramps of current elicited 5 APs from the wild-type neuron, but 14 APs from the Nf1+/– neuron. Figure 2A summarizes the responses of 8 neurons in the wild-type group and 11 neurons in the Nf1+/– group. Sensory neurons from Nf1+/– mice exhibited a more than twofold increase in the number of APs compared with wild-type neurons for identical stimulation (6.0 +/– 1.6 and 14.8 +/– 2.2 APs for Nf1+/+ and Nf1+/– neurons, respectively, P < 0.05 using a t-test). All of the neurons of both genotypes were capsaicin-sensitive as determined at the end of the protocol.
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These results demonstrate that the firing threshold was reduced in neurons isolated from the Nf1+/– mice. Consistent with this observation was our finding that the rheobase (the minimum amount of current required to evoke an AP) also was reduced in Nf1+/– neurons. Representative tracings from a wild-type and Nf1+/– neuron are shown in Fig. 3, A and B, respectively. As summarized in Fig. 3C, Nf1+/– neurons had an almost threefold lower rheobase compared with wild-type neurons (56 +/– 9 and 154 +/– 36 pA for 11 Nf1+/– and 8 Nf1+/+ neurons, respectively, using a t-test). As shown in Fig. 3D, the input resistance was not significantly different between the two genotypes (712 +/– 191 M for Nf1+/+ neurons and 795 +/– 79 M for Nf1+/– neurons). Taken together, these data clearly demonstrate that capsaicin-sensitive sensory neurons isolated from mice that are heterozygous for the Nf1 mutation exhibit enhanced excitability compared with capsaicin-sensitive sensory neurons from wild-type mice and that this enhanced excitability is consistent across multiple electrophysiological parameters.
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Because NGF is a growth factor known to alter the excitability of nociceptive sensory neurons and is an activator of the Ras transduction cascade, we examined the actions of NGF on excitability in both wild-type and Nf1+/– sensory neurons. During the 5–12 h wherein the neurons were maintained in culture and prior to obtaining these recordings, the neurons were maintained in media containing either no added NGF or different concentrations of NGF. As described in the preceding text, the resting membrane potential, AP number, firing threshold, firing latency, and rheobase were measured under these different conditions. As shown in Fig. 4A, NGF caused a concentration-related increase in the number of APs elicited by a standard ramp of depolarizing current in capsaicin-sensitive sensory neurons isolated from wild-type mice. The number of evoked APs was significantly higher after treatment with NGF compared with that obtained in its absence. For example, after exposure to 100 ng/ml NGF, the number of evoked APs increased to 14.8 +/– 2.8 (n = 6 neurons) compared with 5.9 +/– 1.5 (n = 8 neurons) in the absence of NGF for wild-type neurons. Surprisingly, there was no difference in the number of APs elicited from Nf1+/– sensory neurons treated with NGF compared with those maintained in the absence of NGF. As a consequence, the difference in AP number observed between the genotypes was abolished after treatments with the higher concentrations of NGF.
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DISCUSSION |
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There is evidence to support the modulation of ion channels by activation of the Ras transduction cascade. For example, Fitzgerald and Dolphin (1997) demonstrated that the microinjection of an activated K-Ras isoform enhances the voltage-gated calcium current in dorsal root ganglia (DRG) neurons from neonatal rats. Similarly, blocking Ras activation with a peptide that inhibits the interaction of Ras with the TrkA-Src complex or inhibiting Ras signaling with a neutralizing antibody, reduces these calcium currents. In addition, co-expression of constitutively active Ras and an inward-rectifier potassium channel in HEK cells causes a decrease in the inward-rectifying potassium current. This reduction in current is blocked by the mitogen-activated protein kinase/ERK kinase (Mek) inhibitor PD98059 (Giovannardi et al. 2002). These data are consistent with the ability of increased Ras activation to enhance neuronal excitability as observed in the isolated Nf1+/– sensory neurons. To elucidate the specific channels that are modulated by the Nf1 mutation, additional voltage-clamp studies are necessary.
Once Ras-GTP recruits the kinase, Raf, to the cell membrane, a cascade of downstream effectors is activated. The role of GAPs, such as neurofibromin, is to catalyze the hydrolysis of active Ras-GTP to inactive Ras-GDP and, thereby, halt the activation of downstream cascades (Li et al. 1992; Martin et al. 1990; Wallace et al. 1990). As a result of the Nf1 mutation, there is an increase in cellular levels of Ras-GTP, pERK, and pAkt, both at rest and when cells are exposed to growth factors that activate these cascades (Guha et al. 1996; Ingram et al. 2001; Klesse and Parada 1998; Sherman et al. 2000; Vogel et al. 2000). NGF is one such growth factor that can activate the Ras transduction cascade. Recently, several investigations demonstrated that the Ras transduction cascade and the downstream effectors of this pathway may play a key role in the actions of NGF to sensitize sensory neurons. For example, Zhuang et al. (2005) reported that in the spinal nerve ligation model of neuropathic pain, pERK levels were increased in spinal cord neurons and the DRG. NGF injected into the rat hindpaw also increased p-ERK labeling in TrkA-containing neurons in the DRG (Averill et al. 2001). In addition, peripheral inflammation increased the levels of phosphorylated p38 (p-p38), another member of the mitogen-activated kinase family, in nociceptive sensory neurons (Ji et al. 2002). This increase in p-p38 was correlated with an increase in expression of TRPV1 and thermal hyperalgesia, and all three of these responses were blocked by treatment with NGF antisera prior to the initiation of the inflammation. Treatment with constitutively active Ras mimicked the action of NGF to increase TRPV1 expression in isolated sensory neurons and increased neuronal levels of pERK and pAkt (Bron et al. 2003). In addition, the Mek inhibitor, PD98059, reduced the capsaicin sensitivity of neurons that were treated with NGF for 1 wk (Ganju et al. 1998). Although most of these investigations have focused on the role of components of the Ras transduction cascade in NGF-mediated changes of TRPV1 expression and capsaicin responses, they do not exclude the possibility of other Ras-mediated changes in the excitability of nociceptive sensory neurons. In general, these observations are consistent with our findings that NGF increased the excitability of wild-type mouse sensory neurons and mimicked the enhanced excitability that was intrinsic to the Nf1+/– sensory neurons.
It is possible that a further enhancement of excitability in the Nf1+/– neurons treated with NGF was not observed because the mutated neurons had already attained their maximum ability to fire APs when stimulated by depolarizing currents. In this case, the Nf1+/– neurons would be unable to fire more APs when treated with NGF. Alternatively, there may be compensatory mechanisms modulating NGF-induced sensitization of the neurons with reduced neurofibromin. Potential compensatory mechanisms could include downregulation of the TrkA or p75 receptors or modulation of other downstream components of these cascades. Interestingly, Zhang et al. (2002) reported that NGF-induced sensitization of rat sensory neurons is mediated by ceramide, a product of the p75 receptor-activated transduction cascade (Dobrowsky et al. 1994). However, it is possible that there is overlap or cross-talk between the cascades activated by Trk A and those activated by p75. There is evidence that NGF can stimulate the Ras transduction cascade through activation of the p75 receptor in expression systems and neuronal cells that do not express TrkA (Blochl et al. 2004; Susen et al. 1999). In addition, Hida and colleagues (1998) demonstrated that ceramide can activate Ras in cultured oligodendrocytes. Therefore, if NGF enhances neuronal excitability via Ras-dependent pathways mediated through TrkA or p75 or both, it is not surprising that this effect of NGF is lost in cells with constitutive activation of the Ras cascade, like those from Nf1+/– mice. It would be very informative to examine the role of both of these receptors in the sensory neuronal excitability induced by activation of the Ras pathway.
These data clearly suggest that GAPs, such as neurofibromin, can play a key role in modulating the excitability of nociceptive sensory neurons. A clearer understanding of the mechanisms underlying the enhanced neuronal excitability in sensory neurons with the Nf1 mutation, similar to the human disorder NF1, and how this sensitization may be modified in injury could lead to better therapies for the painful conditions associated with NF1 or chronic painful conditions that arise from other ailments.
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GRANTS |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. M. Hingtgen, Stark Neurosciences Research Institute, Indiana University School of Medicine, 450 W. Walnut St., R2-466, Indianapolis, IN 46202 (E-mail: chingtge{at}iupui.edu)
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REFERENCES |
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, values from individual wild-type neurons (Nf1+/+); 
, values from individual Nf1+/– neurons. To the right of each series of individual values is the mean +/– SE for the specific genotype (The error for the firing latency of Nf1+/– neurons in F is small enough to be obscured by 
