Nav1.7 is the predominant sodium channel in rodent olfactory sensory neurons
© Ahn et al; licensee BioMed Central Ltd. 2011
Received: 21 March 2011
Accepted: 10 May 2011
Published: 10 May 2011
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© Ahn et al; licensee BioMed Central Ltd. 2011
Received: 21 March 2011
Accepted: 10 May 2011
Published: 10 May 2011
Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons).
We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs.
We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system.
Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.
Olfactory sensory neurons (OSN; also referred to as olfactory receptor neurons) are bipolar neurons adapted for peripheral odorant signal transduction and transmission centrally to the olfactory bulb. OSN peripheral terminals house a rich array of odorant receptors and a molecular amplification system which boosts receptor potentials produced by short-lived ligand-receptor binding, triggering action potentials that are transmitted centrally along unmyelinated axons which synapse on dendrites of mitral neurons in the well-organized glomeruli in the olfactory bulb within the CNS [1, 2]. The voltage-dependent sodium channels that support the initiation and propagation of action potentials in OSN are known to be tetrodotoxin-sensitive (TTX-S) . However, the molecular identity of the TTX-S channels that are expressed in OSN is not known.
Sodium channel Nav1.7 has recently emerged as a major target in pain research . This channel is preferentially expressed in peripheral neurons [5–7], and produces a fast-activating and -inactivating, slow-repriming, TTX-S current , with slow closed-state inactivation which permits a substantial inward current in response to small, slow depolarizations (ramp current) [9, 10]. Nav1.7 channels are present in most small unmyelinated fibers within the sciatic nerve , and within free nerve endings in the skin  close to the predicted peripheral trigger zone. Recently, we have shown that ERK1/2 phosphorylation of the channel hyperpolarizes activation and fast-inactivation of Nav1.7 but without changing its current density . The gating properties and subcellular localization suggest that Nav1.7 acts as a pre-synaptic threshold channel for firing action potentials which amplifies weak stimuli, for example generator and receptor potentials .
Although Nav1.7 is being explored as a therapeutic target for pain, recent data support the involvement of Nav1.7 in olfactory signaling. Human studies have shown that homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) [15–17], which is accompanied by anosmia [17–19]. Additionally, global knock-out of Nav1.7 in mice is neonatal lethal, reportedly due to lack of feeding , consistent with inability of newborn mice to smell mother's milk. We hypothesized that Nav1.7 plays a critical role in signal transmission along the olfactory sensory axis from the peripheral olfactory epithelia to the olfactory bulb . We present here molecular and cellular evidence which support the conclusion that Nav1.7 is the dominant sodium channel in rodent OSN. Early results of this study have been presented in an abstract form at the 40th annual meeting of the Society for Neuroscience, 2010, program# 848.18.
Figure 1B (Lane 1) shows amplification products (bands "a", and "b") from mouse olfactory epithelium which are consistent with the presence of a potential mixture of Nav1.1 (558 bp), Nav1.2 (561 bp) and Nav1.3 (561 bp) (band a), Nav1.6 (510 bp), Nav1.7 (501 bp), Nav1.8 (480 bp), Nav1.9 (471 bp) and Nax (501 bp) (band b); note small difference in length of amplicons for Nav1.6 and Nav1.9 due to an additional amino acid residue in this region of the mouse channels compared to their rat counterpart. Restriction enzyme analysis of the amplicons demonstrates that transcripts of Nav1.7 are the predominant subtype, similar to rat olfactory epithelium (A), with the presence of low levels of transcripts for Nav1.3, Nav1.6 and Nax; transcripts for Nav1.1, Nav1.2, Nav1.4, Nav1.5, Nav1.8 and Nav1.9 are not detected.
Nav1.7-specific primers used to amplify cDNA from mouse OSN and DRG templates.
Coordinates (accession # BC172147 )
To examine the voltage-dependence of steady-state fast-inactivation, OSNs were held at -100 mV and the sodium currents were induced by a double pulse protocol of 500 ms prepulses from -160 mV to -20 mV in 10 mV increments, followed by a 40 ms depolarizing pulse to -20 mV to measure the fraction of available channels. The fast-inactivation curve was obtained from a Boltzmann fit to the normalized current (Figure 8D). The voltage midpoint (V1/2) and slope factor (k) of steady-state fast-inactivation were -96.4 ± 2.1 mV and 8.9 ± 0.5 mV (n = 17). Similar to activation properties, the steady-state fast-inactivation for sodium currents in OSNs is also more hyperpolarized than that (V1/2 range: -71 to -83 mV) for Nav1.7 currents in HEK293 cells [9, 30–40].
We show here that Nav1.7 is the predominant transcript in adult rat and mouse olfactory epithelium, with low abundance of Nav1.6 transcripts. Immunostaining of olfactory epithelium and the olfactory nerve show that Nav1.7 is the main sodium channel which accumulates in the thin unmyelinated fibers, with undetectable Nav1.6 immunolabeling. Co-immunostaining with the synaptic marker synaptophysin reveals a complementary distribution of Nav1.7 and Nav1.6 channels, with accumulation of Nav1.7 in presynaptic axons, and Nav1.6 in processes of mitral and granule neurons within glomeruli of the olfactory bulb. The limited Nav1.7 immunoreactivity at the presynaptic axon termini within the glomeruli may possibly reflect the dispersion of axons and their small diameter, and the robust Nav1.6 immunostaining within the glomeruli reflects the abundant expression of this channel within processes of mitral and granule neurons. Weiss et al.  recently reported the presence of Nav1.7 within mouse and human OSN, and observed Nav1.7 immunoreactivity that extended from the cell bodies of mouse OSN to their axons within olfactory glomeruli. Their results, like ours, indicate that, while Nav1.7 is the major sodium channel within OSN, it is not detectable in the mitral and granule neurons that receive synaptic inputs from the OSN. Consistent with this conclusion, they report that there is no synaptic transmission of the electric impulse from OSN to the post-synaptic neurons in mice where Nav1.7 is knocked-out in mature OSN that express the olfactory marker protein. In the aggregate, these data support the conclusion that Nav1.7 is the predominant sodium channel responsible for peripheral odorant signaling to the olfactory bulb.
Transient inward currents in rat and mouse OSNs are completely blocked by 100 nM TTX or by substitution of choline for external sodium, and action potentials are blocked by 100 nM TTX ([3, 41, 42] and this study), indicating that OSN excitability is dependent upon a TTX-S sodium channel. Consistent with the electrophysiological data, our molecular and immunostaining data show that Nav1.7, a TTX-S channel, is the predominant sodium channel in OSNs and their unmyelinated axons. Low levels of other TTX-S sodium transcripts could be amplified from olfactory epithelial cDNA templates (Figure 1A and 1B), but weak or no immunostaining for these channels was detectable in OSN and in axons within the olfactory nerve (Figure 3). While the cellular origin of sodium channel transcripts other than Nav1.7 is difficult to identify, one possibility is that low expression levels of these channels occurs in OSN, but the level of the channel protein is below the detection of our immunostaining assays. Alternatively, other cell types within the olfactory epithelium may express sodium channels other than Nav1.7. Irrespective of the source of these weakly-expressed channels, the vast abundance of Nav1.7 in the OSN points to a critical role of this channel in olfactory signal transmission, and that the presence of other sodium channels does not appear to be sufficient to rescue olfaction in humans and mice which lack Nav1.7.
The ability of Nav1.7 to boost subthreshold stimuli, for example odorant-induced receptor potential depolarization of OSN membrane, is consistent with its role as a threshold channel for firing action potentials in neurons . Single openings of the metabotropic odorant receptors have been shown to be sufficient to generate action potentials , although a recent study  has estimated that a single odorant binding event results in ~0.034 pA current while the threshold for action potential is ~1.2 pA. An elaborate Ca2+- and Cl--based signaling amplification system in the cilia has been reported to boost the odorant receptor potential for successful initiation of action potentials [1, 2]. However, the abundant expression of Nav1.7 with its demonstrable ability to boost weak depolarizations, and weak expression of other sodium channels in rodent and human OSN ( and this study), supports the conclusion that Nav1.7 plays a central role in action potential transmission along the peripheral olfactory nerve axis.
While the molecular and immunostaining data show dominant expression of Nav1.7 channels in rodent and human OSN ( and this study), whole-cell patch-clamp recordings of mouse OSN show a TTX-S current with hyperpolarized activation and inactivation; threshold for activation was near -70 mV with a peak around -25 mV and V1/2 of -41 mV (Figure 8). Data presented in Figure 3B by Weiss et al  are in agreement with hyperpolarized voltage dependence for activation (threshold and peak) in OSN. These data are consistent with a published report of V1/2 of -48 mV from isolated P5-P15 rat OSN . The voltage-dependence of activation for sodium current in OSNs is more hyperpolarized than those for human Nav1.7 currents (V1/2 range: -16 to -29 mV) in HEK293 cells [9, 30–40] or DRG neurons [10, 46], or of TTX-S currents in native DRG neurons (-23 to -28 mV) [47–49]. Similarly, a wide range of V1/2 for steady-state inactivation of sodium current has been reported for rat OSNs: -96 mV for adult mouse (this study), -87 mV for P5-P15 rat , and -110 mV , -107 mV , and -105 mV  for adult rat OSNs. The wide range of reported V1/2 may arise from the use of neurons from neonatal, juvenile or adult rats, different recording buffers, time in culture and other technical issues. However, the V1/2 of -96 mV that we obtained is hyperpolarized compared to those (V1/2 range: -71 to -83 mV) reported for Nav1.7 current in HEK cells [9, 30–40] or DRG neurons [10, 46, 53] or the TTX-S currents (-66 to -72 mV) in native DRG [47–49]. Since we report in this study that the sequence of the Nav1.7 cDNA is identical in mouse OSN and DRG templates, modulation of the Nav1.7 channels by post-translational modification and possible interaction with cell-specific channel partners, rather than a different Nav1.7 splicing isoform, is likely responsible for the altered gating properties of this channel in OSN versus DRG neuronal backgrounds.
Using RT-PCR, we also observed (Figure 1A) Nav1.6 mRNA at low levels in mouse and rat olfactory epithelium (the only detectable sodium channel transcript other than Nav1.7 in mouse tissue), but found no detectable immunostaining signal in rat OSN or olfactory nerve axons, which suggest a limited contribution of this channel to peripheral olfactory signal transmission. Studies on Scn8a medtg mice which lack Nav1.6 channels support our view that Nav1.7 is essential for olfaction in mice. While global knock-out of Nav1.7 in mice is neonatal lethal , total loss of Nav1.6 in mice is juvenile lethal although these mice are indistinguishable from WT or heterozygote littermates in terms of feeding and open field behavior for the first 10-14 days after birth [54–56]. Neonatal lethality of Nav1.7 knock-out mouse has been linked to lack of feeding , while death of Nav1.6 knock-out mouse is linked to muscle degeneration [54–56]. These data are consistent with a minor role for Nav1.6 in OSN excitability and olfactory signal transmission, at least within the first two weeks after birth.
Recent data have shown that Nav1.7, which is normally considered a threshold sodium channel , is critical to nerve signal transduction and transmission in two sensory neuronal pathways: nociception and olfaction. The predominant expression of Nav1.7 in OSN ( and this study), compared to other channels, provides a reasonable explanation for anosmia in human subjects [15, 17–19] and mice  when this channel is not functional. Thus, Nav1.7 appears to be critically important for olfactory signaling by OSN. In contrast to OSN where Nav1.7 expression predominates, Nav1.7 is co-expressed with several other channels within DRG neurons which signal pain , and these channels are distributed to the peripheral free endings of the axons in the epidermis . These observations, together with the profound loss of pain sensibility in CIP, point to a dominant role of Nav1.7 in pain-signaling, although the exact mechanism is not well understood. Intriguingly, Nav1.7 is present in sympathetic neurons and gain-of-function mutations that depolarize resting membrane potential cause hypoexcitability of these neurons . However, Nav1.7-related CIP patients do not report significant sympathetic dysfunction [15–18], thus it appears that Nav1.7 does not play an equally central role in signal transduction/transmission in sympathetic neurons. These data show that the contribution of Nav1.7 channels to neuronal activity appears to be neuronal-type dependent.
We present here molecular and immunolabeling data that demonstrate that Nav1.7 is the predominant sodium channel in OSN and along olfactory nerve fibers. Gain-of-function mutations of Nav1.7 cause hyperexcitability of DRG neurons, underlying pain symptoms in inherited erythromelalgia and PEPD; however, it has not been reported that patients with these disorders also manifest hyperosmia. In contrast, patients with Nav1.7-related CIP report anosmia, and the data presented in this study provide a molecular basis for anosmia in these patients. Nav1.7-specific blockers are being pursued as a highly targeted approach for the treatment of pain. Our data suggest that hyposmia or anosmia are potential side effects that need to be taken into consideration in the clinical application of these therapeutics.
Sprague-Dawley male rats (adult, 225-250 gm, Harlan, Indianapolis, IN) and C57BL/6 mice (adult, 25-30 gm, Harlan) were housed under a 12 hr light/dark cycle in a pathogen-free area with ad libitum access to water and food. The experimental procedures were approved by the VA Connecticut Healthcare System Institutional Animal Care and Use Committee, in accordance with NIH guidelines and conform to the guidelines of the Committee for Research and Ethical Issues of the IASP.
Rats and mice were deeply anaesthetized with CO2, decapitated, and olfactory epithelium was quickly removed and immediately frozen in liquid nitrogen. Total RNA was extracted using RNeasy mini kit (Qiagen, Valancia, CA) and RNA was eluted in 30-50 μl of H2O. First strand cDNA was reverse transcribed in a 20 μl reaction volume including 7 μl total RNA, 200 ng random primers and 200 U SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA), and 40 U RNase inhibitor (Roche Biosciences, Indianapolis, IN). The buffer consisted of: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM DTT and 0.5 mM dNTP. The reaction proceeded at 25°C for 5 min, 50°C for 90 min and then terminated by heating to 70°C for 15 min. A parallel reaction was performed as a negative control by substituting sterile water for the reverse transcriptase enzyme (data not shown).
A multiplex PCR was used to amplify Nav channel transcripts (Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, and Nax) which may be present in the cDNA pool as previously described [21–23]. Primers were designed against highly conserved sequences in domain I of sodium channel α subunits. Sequences of the four forward and three reverse primers (F1-F4 and R1-R3) are as follows: F1 5'-AATCCCTGGAATTGGTTGGA-3', F2 5'-GACCCRTGGAACTGGCTGGA-3', F3 5'-GACCCGTGGAACTGGTTAGA-3', F4 5'-GATCTTTGGAACTGGCTTGA-3'; R1 5'-CAAGAAGGCCCAGCTGAAGGTGTC-3', R2 5'-GAGGAATGCCCACGCAAAGGAATC-3', R3 5'-AAGAAGGGACCAGCCAAAGTTGTC-3'. Amplification was performed in a 60 μl reaction volume using 4 μl first-strand cDNA, 1-3 μM of each primer and 5 U of Expand Long Template DNA polymerase enzyme mixtures (Roche). The PCR reaction buffer contained 2.75 mM MgCl2 and detergents. Amplification was carried out in two stages using a programmable thermal cycler (PTC-200, MJ Research, Cambridge, MA). First, a denaturation step at 94°C for 2 min, an annealing step at 57°C for 2 min and an elongation step at 68°C for 2 min. Second, a denaturation step at 94°C for 30 sec, an annealing step at 57°C for 45 sec and an elongation step at 68°C for 45 sec. The second stage was repeated 39 times for a total of 40 cycles, with the elongation step in the last cycle extended to 10 min. Control PCR reactions in which the template was substituted by H2O produced no amplification products (data not shown).
The identity of the α-subunits expressed in rat or mouse olfactory epithelium were determined by a combination of length polymorphism and restriction endonuclease analysis of the PCR products. Typically 1/20th of the PCR products were digested for 1 hr at the recommended temperature and the products resolved by electrophoresis in a 2% agarose gel. Fragment sizes were determined by comparison to a standard 100-bp ladder molecular weight marker (Invitrogen). DNA was visualized by ethidium bromide fluorescence and the gel image was digitized by a Kodak Image Station 440 CF (Kodak, Rochester, NY).
The Nav1.7 cDNA from mouse OSN and DRG templates were amplified using 6 primer pairs which amplified overlapping fragments (Table 1). Amplicons were purified using spin columns (Qiagen), and cloned into pGEM-Teasy vectors (Promega Inc.). The identity of the fragments was determined by sequencing of both strands at the W. Keck core facility of Yale School of Medicine. Sequence analysis was done using Lasergene and BLAST software.
Rats were deeply anesthetized with ketamine/xylazine (80/5 mg/kg, i.p.) and transcardially perfused with PBS and then ice-cold fixative solution containing 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4. Olfactory epithelium was removed and fixed for an additional 2-4 hr in the fixative solution and then transferred to a 4% paraformaldehyde solution containing 30% sucrose overnight at 4°C. Twelve micron cryosections were cut and tissue processed for non-radioactive in situ hybridization detection of Nav1.7 mRNA as previously described . Briefly, sections were deproteinized with proteinase K (10 μg/ml), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, and incubated in pre-hybridization buffer (50% formamide, 5 × SSC, 5 × Denhardt's solution, 100 μg/ml salmon sperm DNA; Sigma, St. Louis, MO) for 1 hr at room temperature followed by hybridization buffer (50% formamide, 10% dextran sulfate, 5 × SSC, 1 × Denhardt's solution, 100 μg/ml salmon sperm DNA, Sigma) containing digoxigenin (DIG)-UTP-labeled Nav1.7 (1.0 ng/μl) riboprobes overnight at 58°C. The slides were then sequentially incubated in: (1) 4 × SSC, 5 min; (2) 2 × SSC, 2 × 10 min each; (3) RNase A solution (20 μg/ml; Sigma) in 10 mM Tris/500 mM NaCl/1 mM EDTA, pH 8.0, for 45 min at 37°C; (4) 2 × SSC, 2 × 10 min each; (5) 0.2 × SSC, 3 × 20 min each at 58°C; (6) 100 mM Tris/150 mM NaCl, pH 7.5, 1 min; (7) blocking solution, containing 100 mM Tris/150 mM NaCl/2% normal sheep serum/1% BSA, 30 min, (8) alkaline phosphatase-labeled anti-DIG antibody (1:500 in blocking solution; Roche) overnight at 4°C; (9) 100 mM Tris/150 mM NaCl, pH 7.5, 4 × 5 min each; (10) 100 mM Tris/100 mM NaCl/50 mM MgCl2, pH 9.5, 4 × 5 min each; (11) NBT/X-phos solution [384 μg/ml ρ-nitro-blue tetrazolium chloride (NBT) and 188 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (X-phos) in 100 mM Tris/100 mM NaCl/50 mM MgCl2, pH 9.5]. The reaction was stopped by rinsing in 10 mM Tris/1 mM EDTA, pH 8.0. Sections were incubated with 300 nM 4', 6-diamidino-2-phenylindole (DAPI) to label olfactory sensory nuclei.
Rats were deeply anesthetized with ketamine/xylazine (80/5 mg/kg, i.p.) and transcardially perfused with 0.01 M PBS (pH 7.4) followed by ice-cold 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer (pH 7.4). The olfactory epithelium was removed, immersion-fixed for an additional 20 min (total fixation time 30 min) and cryoprotected with 30% (w/v) sucrose in PBS overnight at 4°C. Ten-μm thick cryosections were mounted on slides (Fisher, Pittsburgh, PA) and processed for detection of Nav1.7 protein as described previously . In brief, sections were incubated in the following (1) blocking solution (PBS containing 5% cold water fish skin gelatin, 3% normal donkey serum, 2% BSA, 0.1% Triton X-100, and 0.02% sodium azide) for 15 min at room temperature; (2) primary antibody(ies) singly or in combination [mouse anti-Nav1.1 (1:100, Antibodies, Inc., Davis, CA); mouse anti-Nav1.2 (1:100, Antibodies, Inc.); rabbit anti-Nav1.6 (1:100, Sigma); rabbit anti-Nav1.7 (1:250, Y083 ); goat anti-olfactory mature protein (OMP) (1:200 Wako, Richmond, VA); mouse anti-peripherin (1:1000, Abcam), and mouse anti-synaptophysin (1:50, GeneTex, Irvine, CA)] in blocking solution overnight at 4°C; (3) PBS, 6 × 5 min each; (4) appropriate secondary antibodies in blocking solution for 6-8 hr at room temperature; (5) PBS, 6 × 5 min each.
OSN cultures from adult C57BL/6 mice were done according to report by Sosnowski et al  with some modifications. In brief, mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.), decapitated, and olfactory tissue was dissected and immediately placed in ice-cold Ca2+/Mg2+-free HBSS. After freeing olfactory epithelium from other tissues, olfactory epithelium was rinsed in ice-cold Ca2+/Mg2+-free HBSS and minced to 1 mm pieces. Trypsin (0.125% in Ca2+/Mg2+-free HBSS) treatment was used to dissociate epithelial tissue at 35°C for 30 min with gentle agitation. Trypsin was inactivated by OSN medium (MEM containing 10% fetal bovine serum and antibiotics). Dispersed cells were mixed gently and centrifuged at 1200 × g for 2 min. The pellet was resuspended in 1 ml OSN medium with a fire-polished glass pipette and filtered through a 40-μm mesh. Approximately 50 μl of cell suspension was plated on a poly-D-lysine/laminin coated coverslip in 24-well plate (BD Biosciences). Cultures were placed in a humidified 35°C incubator receiving 5% CO2. One hour later, cells were fed with 450 μl of OSN medium with fresh NGF (50 ng/ml). Half the medium was replaced daily and supplied with NGF. Cultures were used for patch-clamp recording on the same day of culture.
The whole-cell voltage-clamp recording was conducted at room temperature (21-23°C) using an Axopatch 200B amplifier (Molecular Devices, Union City, CA). The bath solution contained (in mM): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 glucose and 20 HEPES, pH 7.3 with NaOH (adjusted to 320 mOsm with sucrose), and the pipette solution contained (in mM): 140 CsF, 1 EGTA, 10 NaCl, 10 mM HEPES, pH 7.3 with CsOH (adjusted to 310 mOsm with sucrose). 20 mM TEA-Cl was included in the bath solution to block endogenous potassium current. Fire-polished electrodes were fabricated from capillary glass (PG10165-4, World Precision Instruments, Sarasota, FL) using a P-97 puller (Sutter Instrument Co., Novato, CA). The resistance of recording pipettes in the bath solution was 2-5 MΩ. Whole-cell capacitive currents were compensated with analog compensation and 60-80% series resistance compensation was applied to minimize voltage errors. The voltages were not corrected for liquid junction potential. The currents were filtered at 5 kHz, acquired at 100 kHz, and then digitized using pClamp 10 software and Digidata 1440A (Molecular Devices). The Origin 8.1 software (OriginLab Corporation, Northampton, MA) was used for data analysis. Data are presented as means ± S.E.
paroxysmal extreme pain disorder
congenital insensitivity to pain
olfactory sensory neuron
olfactory mature protein
olfactory nerve layer
microtubule associated protein 2
dorsal root ganglion
superior cervical ganglion
We thank Dr. Charles Greer for helpful discussions and Bart Toftness for excellent technical assistance. This work was supported by the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University.
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