Sensory Neurons from Nf1 Haploinsufficient Mice Exhibit Increased Excitability
Yue Wang1, G. D. Nicol1, D. Wade Clapp2,3 and Cynthia M. Hingtgen4,1
1: Department of Pharmacology and Toxicology
2: Department of Pediatrics
3: Department of Microbiology and Immunology
4: Department of Neurology
All at Indiana University School of Medicine, Indianapolis, IN
Running head: Nf1+/- Sensory Neurons Exhibit Increased Excitability
Cynthia M. Hingtgen, MD, PhD
Stark Neurosciences Research Institute
Indiana University School of Medicine
450 West Walnut Street, R2-466
Indianapolis, IN 46202
Articles in PresS. J Neurophysiol (August 10, 2005). doi:10.1152/jn.00489.2005
Copyright © 2005 by the American Physiological Society.
Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by tumor
formation. People with NF1 also can experience more intense painful responses to
stimuli, such as minor trauma, than normal. NF1 results from a heterozygous mutation
of the NF1 gene, leading to decreased levels of neurofibromin, the protein product of the
NF1 gene. Neurofibromin is a guanosine triphosphatase activating protein (GAP) for
Ras and accelerates the conversion of active Ras-GTP to inactive Ras-GDP; therefore,
mutation of the NF1 gene frequently results in an increase in activity of the Ras
transduction cascade. Using patch-clamp electrophysiological techniques, we examined
the excitability of capsaicin-sensitive sensory neurons isolated from the dorsal root
ganglia of adult mice with a heterozygous mutation of the Nf1 gene (Nf1+/-), analogous
to the human mutation, in comparison to wildtype sensory neurons. Sensory neurons
from adult Nf1+/- mice generated a more than two-fold higher number of action
potentials in response to a ramp of depolarizing current as wildtype neurons.
Consistent with the greater number of action potentials, Nf1+/- neurons had lower firing
thresholds, lower rheobase currents, and shorter firing latencies than wildtype neurons.
Interestingly, nerve growth factor augmented the excitability of wildtype neurons in a
concentration-related manner, but did not further alter the excitability of the Nf1+/-
sensory neurons. These data clearly suggest that GAPs, such as neurofibromin, can
play a key role in the excitability of nociceptive sensory neurons. This increased
excitability may explain the painful conditions experienced by people with NF1.
Keywords: dorsal root ganglia, neurofibromin, nerve growth factor, nociceptors, Ras
Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease with
an incidence of 1 in 3,500 people (Lakkis and Tennekoon 2000). 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 (Riccardi and Eichner 1992;
Creange et al. 1999; 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; Largaespada et al. 1996; Zhang et al.
1998; Cichowski and Jacks 2001). 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; Martin et al. 1990; Wallace et al.
1990; Li 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 (Ganju et al. 1998; Bron et al. 2003; 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
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 wildtype 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
Materials and Methods:
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 (NIH Publications No. 80-23) revised 1996.
Horse serum, F-12 medium, L-glutamine, and penicillin/streptomycin were
purchased from Invitrogen (Carlsbad, CA). Nerve growth factor (NGF) was purchased
from Harlan Bioproducts for Science, Inc. (Indianapolis, IN). Papain was purchased
from Worthington Biochemical Corporation 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 1-2 month 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 10-15 min incubation at 37 °C in the second set of enzymes, the tissue sample
was centrifuged for 30 seconds 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 hours
after isolation. NGF was added to the F-12 media at the time of plating, where indicated.
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
Ringers 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 MΩ 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 one second ramp of depolarizing current that had a
final amplitude of 1000 pA. The sampling frequency was 2 KHz. Second, a series of
current steps that were 200 ms in duration and of variable incremental amplitudes were
used to determine the rheobase. The sampling frequency was 1.25 KHz. At the end of
each recording, the neuron was superfused with a Ringers 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).
Summarized data is presented as the mean ± standard error of the mean
(S.E.M.). Statistical significance between groups was determined using a t-test or a one
way analysis of variance (ANOVA) followed by Dunnett's posthoc analysis, as
appropriate and is specified in the text. Values of p<0.05 were judged to be statistically
Sensory neurons from Nf1+/- mice have higher excitability than neurons from wildtype
To determine whether nociceptive sensory neurons with reduced levels of
neurofibromin have altered excitability, sensory neurons were isolated from Nf1+/- and
wildtype 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+/+ (panel A) and Nf1+/- (panel B) neurons,
respectively. As can be easily appreciated, identical ramps of current elicited 5 APs
from the wildtype neuron, but 14 APs from the Nf1+/- neuron. Figure 2A summarizes the
responses of 8 neurons in the wildtype group and 11 neurons in the Nf1+/- group.
Sensory neurons from Nf1+/- mice exhibited a more than 2-fold increase in the number
of APs compared to wild-type neurons for identical stimulation (6.0 ± 1.6 APs 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
Two additional parameters indicative of the level of neuronal excitability are the
firing threshold and firing latency. These measurements were determined in the same
neurons for which the number of APs evoked by the current ramp was assessed, above.
The firing threshold is the membrane voltage at which the AP is generated and was
determined as described in the legend. As summarized in Figure 2B, neurons isolated
from Nf1+/- mice had a significantly lower firing threshold compared to that of Nf1+/+
neurons (-31.7 ± 1.6 vs. -25.7 ± 1.3 mV for Nf1+/- and wildtype neurons, respectively,
p<0.05 using a t-test). A lower firing threshold suggests that the Nf1+/- neurons are
capable of generating APs at more hyperpolarized membrane potentials. Similarly, the
firing latency, or the time from the onset of the current injection to the initiation of the first
AP, was significantly shorter in the Nf1+/- sensory neurons (Figure 2C; 361 ± 55 ms and
216 ± 15 ms for Nf1+/+ and Nf1+/- neurons, respectively, p<0.05 using a t-test).
However, there was no difference in the average resting membrane potentials between
these genotypes (Figure 2D; -62.7 ± 1.8 mV and -60 ± 2.0 mV for Nf1+/+ and Nf1+/-
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 wildtype and Nf1+/- neuron are shown
in Figures 3A and 3B, respectively. As summarized in Figure 3C, Nf1+/- neurons had an
almost 3-fold lower rheobase compared to wildtype neurons (56 ± 9 pA 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
MΩ for Nf1+/+ neurons and 795 ± 79 MΩ 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 to
capsaicin-sensitive sensory neurons from wildtype mice and that this enhanced
excitability is consistent across multiple electrophysiological parameters.
Treatment with NGF enhances the excitability of wildtype sensory neurons and mimicks
the effects of the Nf1 mutation
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 wildtype and Nf1+/- sensory neurons. During the
5-12 hours 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 above, the resting membrane potential,
AP number, firing threshold, firing latency, and rheobase were measured under these
different conditions. As shown in Figure 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 wildtype mice. The number of evoked
APs was significantly higher after treatment with NGF compared to 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 to 5.9 ± 1.5 (n = 8 neurons) in the
absence of NGF for wildtype neurons. Surprisingly, there was no difference in the
number of APs elicited from Nf1+/- sensory neurons treated with NGF compared to
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.
A similar concentration-related alteration of firing latency and rheobase were
observed in the wildtype neurons treated with NGF (Figure 4C and 4D). These
parameters were significantly lower in wildtype sensory neurons treated with 30 or 100
ng/ml NGF compared to neurons not exposed to NGF. Again, there was no effect of
NGF on the firing latency or rheobase in Nf1+/- sensory neurons. Although the decrease
in firing threshold in wildtype neurons treated with the higher concentrations of NGF was
not statistically different than neurons that were not treated with NGF (Figure 4B), the
small decrease observed was sufficient to abolish the differences between genotypes
seen in the absence of NGF or in the presence of 1 ng/ml NGF. These data
demonstrate that NGF dramatically alters the excitability of capsaicin-sensitive sensory
neurons from wildtype mice, but has no effect on capsaicin-sensitive neurons
heterozygous for the Nf1 mutation. Consequently, the increased excitability that is
inherent in the Nf1+/- sensory neurons is mimicked in wildtype neurons by treatment with
In this report, we demonstrate that the small diameter, capsaicin-sensitive
sensory neurons with a heterozygous mutation of the Nf1 gene have augmented
excitability compared to wildtype neurons. These data suggest that the activation of the
Ras transduction cascade, as a consequence of mutation of the Nf1 gene, alters the
state of modulation for ion channels that regulate the capacity of sensory neurons to fire
APs as indicated by the increased number of evoked APs, a more hyperpolarized firing
threshold, and a decreased rheobase. However, those channels that maintain the
resting membrane potential and the resistance appear to be unaffected by the
heterozygous mutation of the Nf1 gene and the consequences its modifications of
downstream transduction cascade(s).
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. In order to elucidate the specific
channels that are modulated by the Nf1 mutation, additional voltage-clamp studies are
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 (Martin et al. 1990; Wallace et al. 1990; Li 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; Klesse and Parada 1998; Sherman et al. 2000; Vogel et al.
2000; Ingram et al. 2001). 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 a week (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 wildtype 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 (Susen et al. 1999;
Blochl et al. 2004). In addition, Hida and colleagues demonstrated that ceramide can
activate Ras in cultured oligodendrocytes (Hida et al. 1998). 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
The authors would like to thank Shannon L. Roy for technical assistance and Michael R.
Vasko for valuable discussions. This research was supported by a Young Investigator
Award from the National Neurofibromatosis Foundation and the Department of Defense
Neurofibromatosis Research Program New Investigator Award (DAMD 17-03-1-0227) to
C.M.H., an APRC Supplement (5R01CA074177-07) to D.W.C. and, in part, from NIH
R01NS046084 to G.D.N.
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Figure 1. Nf1+/- sensory neurons generate more action potentials (APs) in
response to a given stimulus than wildtype neurons. Tracings from representative
sensory neurons isolated from either a wildtype (panel A) or a Nf1+/- mouse (panel B)
illustrate the APs evoked by the ramp of current. The neurons were held at their resting
membrane potential and APs were evoked by injecting a one second ramp of
depolarizing current that had a final amplitude of 1000 pA. The dV/dt trace represents
the differentiation of the membrane voltage. The baseline dV/dt is calculated as the
average of all the values between the injection of the ramp current and just prior to the
initiation of the AP. The firing threshold is the membrane voltage corresponding to the
point at which dV/dt exceeds the baseline value by 20-fold. The firing latency is
calculated as the time between the onset of the current ramp and the time at which the
firing threshold is attained.
Figure 2. Nf1+/- sensory neurons exhibit increased excitability compared to
wildtype neurons. Open circles indicate values from individual wildtype neurons
(Nf1+/+) and filled circles indicate values from individual Nf1+/- neurons. To the right of
each series of individual values is the mean ± SEM for the specific genotype (The error
for the firing latency of Nf1+/- neurons in panel F is small enough to be obscured by the
filled circle). The values in each panel are from the same 8 wildtype and 11 Nf1+/-
neurons shown in Figure 1. The asterisk indicates a statistical difference between
genotypes using a t-test (p<0.05).
Figure 3. Sensory neurons from Nf1+/- mice show a lower rheobase compared to
wildtype neurons. Sensory neurons were held at their resting membrane potential, and
injected with current steps 200 ms in duration that began with a -20 pA step and
increased by 20 pA increments until an AP was elicited. The rheobase is taken as the
minimum current required to elicit an AP. Panels C and D show a summary of the
rheobase and input resistance values, respectively, for 8 wildtype and 11 Nf1+/-
neurons. The mean ± SEM is given to the right of each group (The errors for the
rheobase and input resistance of Nf1+/- neurons is small enough to be obscured by the
filled circles). The asterisk indicates a statistical difference between genotypes using a t-
Figure 4. NGF treatment of wildtype sensory neurons mimics the increased
excitability of Nf1+/- sensory neurons. Sensory neurons isolated from adult mice
were maintained in growth media for 5-12 hours in the absence or presence of NGF.
The columns represent the mean ± S.E.M. (5-12 neurons per group). A depolarizing
current ramp (1000 pA) was used to elicit APs. Asterisks indicate a statistically
significant difference between genotypes at a given concentration of NGF using a t-test
(p<0.05). The @ indicates a statistically significant difference between NGF treatments
within a given genotype using a one-way ANOVA followed by a Dunnet’ s posthoc