Figure 1. Schematic drawings of olfactory sensory neuron (OSN) function. A, Electrical activity in canonical mammalian OSNs constitutes a generator potential (a graded membrane depolarization caused by the activity of the primary signal transduction cascade, as shown in part B) and action potentials that propagate along the olfactory axons toward the olfactory bulb. B, Schematic drawing of the primary olfactory signal transduction cascade localized in the OSN cilia. Binding of an odor molecule to an odor receptor (OR) triggers activation of adenylyl cyclase type 3 (Adcy3 [ACIII in the schematic]) via the heterotrimeric G protein Golf (α subunit encoded by Gnal). This process results in the formation of cyclic adenosine monophosphate (cAMP), which in turn activates a calcium ion (Ca2+)–permeable, cyclic nucleotide–gated (CNG) cation channel (primary subunit encoded by Cnga2). Entry of Ca2+ through this channel triggers a Ca2+-activated chloride (Cl−) channel, encoded by Ano2. In mice, targeted disruption of Gnal, Adcy3, or Cnga2 leads to general anosmia. Disruption of Ano2 is dispensable for olfaction. C, Schematic view showing the anatomical organization of the olfactory system from the OSNs in the olfactory epithelium to the first synapse in the glomeruli of the olfactory bulb. The OSN axons form cranial nerve I. Mitral cell axons form the lateral olfactory tract, which transmits information from the olfactory bulb to cortical areas. Axons from OSNs expressing the same OR terminate in the same glomerulus (indicated by color coding). ATP indicates adenosine triphosphate; Na+, sodium ion.
Figure 2. Schematic model (based on original results published by Weiss et al21) depicting the presynaptic localization of voltage-gated sodium channel Nav1.7 at the first synapse in the olfactory neural pathway and its critical role in transmitter release. Loss of Nav1.7 function in olfactory sensory neurons leads to a loss of postsynaptic currents in mitral cells, indicating a lack of transmitter release at this synapse. The molecular identity of voltage-gated calcium ion channels (Cav) underlying synaptic release is not yet known. NS indicates nerve stimulation; cNav1.7−/−, conditional homozygous deletion of Nav1.7; and cNav1.7+/−, conditional heterozygous deletion of Nav1.7.
Zufall F, Pyrski M, Weiss J, Leinders-Zufall T. Link Between Pain and Olfaction in an Inherited Sodium Channelopathy. Arch Neurol. 2012;69(9):1119-1123. doi:10.1001/archneurol.2012.21
SECTION EDITOR: HASSAN M. FATHALLAH-SHAYKH, MD, PhD
Author Affiliations: Department of Physiology, University of Saarland School of Medicine, Homburg, Germany.
In a major breakthrough in our understanding of human olfaction, a recent study showed that loss-of-function mutations in the voltage-gated sodium channel Nav1.7, encoded by the gene SCN9A, cause a loss of the sense of smell (congenital general anosmia) in mice and humans. These findings are of special clinical relevance because Nav1.7 was previously known for its essential role in the perception of pain; therefore, this channel is being explored as a promising target in the search for novel analgesics. This advance offers a functional understanding of a monogenic human disorder that is characterized by a loss of 2 major senses—nociception and smell—thus providing an unexpected mechanistic link between these 2 sensory modalities.
Studies of mendelian heritable disorders and their genotype-phenotype relationships have provided major insights into complex functions of our sensory systems under normal and pathological states. These investigations led to rapid advances in our understanding of blindness, deafness, and pain disorders. However, progress in understanding the genetic basis of the human sense of smell has been slow. The complete inability to sense odors is known as general anosmia ; individuals born with this phenotype have congenital anosmia. With the exception of some syndromic cases, such as Kallmann syndrome, no causative genes for human congenital general anosmia had been identified until recently.1 In mice, genetic deletion of any of the primary olfactory signal transduction molecules (Figure 1 and the “Activation of Olfactory Sensory Neurons and Odor Perception” section) causes general anosmia.2 However, somewhat unexpectedly, humans with loss-of-function mutations in these signal transduction genes have not yet been found,1 and as a result, we do not know whether the human nose uses the same molecules for olfactory signal transduction as the mouse.
In the pain system, many of the heritable monogenic pain disorders have been mapped to mutations in genes encoding ion channels, leading to a growing list of channelopathy-associated human pain syndromes.3- 5 One such ion channel that has been the focus of much recent attention is the voltage-gated and tetrodotoxin-sensitive sodium channel Nav1.7, encoded by the gene SCN9A (OMIM 603415).6 Several clinical pain syndromes have been linked to different mutations in SCN9A. In particular, loss-of-function mutations in SCN9A that cause a congenital inability to experience pain in humans are of interest.7- 10 This syndrome, previously known as congenital indifference to pain (OMIM 24300, autosomal recessive), has more recently been referred to as channelopathy-associated insensitivity to pain.7 Based on several previous findings, we hypothesized that the same Nav1.7 channel that is critical for human pain perception could also be essential for odor perception. In this report, we briefly summarize our work and that of others leading to the identification of Nav1.7 as a central ion channel for olfaction of mice and humans. Taken together, these studies identified one of the first causative genes for human congenital general anosmia and provided mechanistic insight into the critical role of this channel in axonal and synaptic signaling of olfactory sensory neurons (OSNs). These advances offer a functional understanding of a monogenic human disorder that is characterized by a loss of 2 major senses—nociception and smell—thus providing an unexpected mechanistic link between these 2 sensory modalities.
The OSNs are the chemoreceptive cells within the sensory epithelium of the nasal cavity (Figure 1). The initial steps underlying odor perception begin when odor stimuli are detected by these cells, leading to the conversion of information contained in odor molecules into electrical membrane signals.2 The OSNs are bipolar neurons in which the apical dendritic process extends to the luminal surface of the nasal cavity, where it ends in a swelling known as the dendritic knob (Figure 1A). Extending from each knob are approximately 1 dozen cilia that distribute within the mucus at the surface of the epithelium. Odor detection starts when odorants bind to specific receptor proteins in the olfactory cilia. This initiates a G protein–coupled second messenger cascade causing rapid formation of cyclic adenosine monophosphate followed by the opening of a cyclic adenosine monophosphate–gated cation channel (Figure 1B). This primary signal transduction process underlies the formation of a graded receptor potential that in turn causes the generation of action potentials. The action potentials travel along thin unmyelinated OSN axons (which form the olfactory nerve, also known as cranial nerve I) and reach the olfactory bulb, the first relay station of the olfactory forebrain (Figure 1C). The OSN axons terminate in the olfactory bulb in a delineated sphere of neuropil known as olfactory glomerulus, forming synapses from the axon terminals on juxtaglomerular interneurons and mitral/tufted cell projection neurons (Figure 1C). Twenty-five years of intense research have provided detailed information on the mechanisms underlying primary signal transduction in mammalian canonical OSNs, but, somewhat surprisingly, the search for genes required for action potential generation and conduction in these neurons did not attract a great deal of attention until recently.
Voltage-gated sodium channels underlie the generation and propagation of action potentials in electrically excitable cells, such as neurons and muscle cells. These channels form a multigene family consisting of 9 distinct genes coding for sodium channel α subunits in mice and humans.6 The Nav1.7 channel (synonyms include neuroendocrine sodium channel [NENa, NE-Na] and peripheral nerve type 1 [PN1]) was first cloned in 1995 from a human neuroendocrine cell line and was initially called the human neuroendocrine sodium channel.11 Subsequently, several other groups found the channel in peripheral nerve cells, including dorsal root and sympathetic ganglia, and it was dubbed peripheral nerve type 1.12,13
The expression of Nav1.7 in nociceptive neurons sparked an intense interest in its functional role in the pain system. Because of space limitations, we can only highlight herein a few important steps in this development and refer to several excellent reviews that summarize this line of research.3- 5,14 The first congenital pain syndrome in humans mapped to SCN9A was published in 2004,15 indicating an association between the Nav1.7 channel and primary erythermalgia, a dominant human disease associated with recurrent episodes of pain. However, nociceptor-specific deletion of Nav1.7 in mice did not produce the same phenotype,16 suggesting that the role of Nav1.7 in pain was more complex than initially thought. Today, we know that multiple mutations exist in SCN9A that lead to strikingly different pain syndromes.3- 5,14 In the context of our own work on a potential role of Nav1.7 in the olfactory system, we became interested at first in a syndrome, now known as channelopathy-associated insensitivity to pain, which is based on loss-of-function mutations in SCN9A that cause a congenital inability to experience pain in humans.7 The complete absence of pain in otherwise seemingly healthy individuals stimulated an intense search for analgesics that selectively target this sodium channel.
The finding that mutations in SCN9A are responsible for multiple human pain syndromes somewhat overshadowed other parallel developments, suggesting that Nav1.7 could have additional important roles in the nervous system. For instance, Morinville et al17 examined the distribution of Nav1.7 in the rat nervous system and concluded that the sodium channel is involved in endocrine and autonomic systems in addition to the function of pain systems. They also detected the channel in pituitary and adrenal glands. Hence, a more detailed analysis of Nav1.7 expression in discrete neuronal populations was warranted when we began our work on the role of Nav1.7 in olfaction.
Are all other sensory modalities fully preserved in patients with channelopathy-associated insensitivity to pain? This question remained unclear until very recently, but a literature search uncovered early indications pointing to an association between congenital indifference to pain and sense of smell deficits in several case reports. Thrush summarized the previous literature and defined congenital insensitivity to pain using the criterion that “all other sensory modalities should be intact or only minimally impaired.”18(p369) At the same time, he noted that “no convincing evidence of olfactory sensation was found”18(p380) in his patients with congenital insensitivity to pain.18 Losa et al described 3 women with autosomal recessive congenital analgesia and complete absence of labor pain; they concluded that “all affected individuals had anosmia but no deficits of autonomic nervous system functions.”19(p1303) Hirsch et al described 2 brothers with congenital indifference to pain and stated in their abstract that “all other sensory modes are intact”20(p851) in these patients. Later in the report, however, they noted that in one of the patients, “neurologic examination revealed that the patient was anosmic, but had normal taste.”20(p852) All these reports had in common a lack of primary data concerning the smell tests. Likewise, no information described what odors were tested and how the results were analyzed, making an objective assessment of the stated conclusions difficult.
In a study by Goldberg et al,8 patients with confirmed Nav1.7 loss-of-function mutations underwent testing for their ability to sense odors and were found, in most cases, to be anosmic. No actual data of the smell tests were provided and no methods for these tests were described.8 Therefore, it remained unclear what was tested and how the tests were performed. Furthermore, Goldberg et al8 stated in the abstract of their study that “patients have severely impaired pain perception, but are otherwise essentially normal.” Thus, despite the fact that all these investigations pointed, in one way or another, to the possibility that the sense of smell could be affected in individuals with congenital analgesia, it remained uncertain whether this was actually the case.
In our own work,21 we documented a complete absence of odor detection in 3 patients with SCN9A loss-of-function mutations and concluded that Nav1.7 is an essential requirement for human olfaction. We proposed that such odor-sensing defects are caused by a critical role of Nav1.7 in the primary OSNs and, consistent with this hypothesis, verified that Nav1.7 is normally expressed in human OSNs.21
Subsequently, our experimental strategy was to move to a mouse model, examine the expression of Nav1.7 in the mouse olfactory system, develop a conditional gene deletion approach, and investigate the effects of the gene deletion at the cellular and systems level. Real-time quantitative reverse transcription–polymerase chain reaction analyses identified Nav1.7 as the most abundant sodium channel in mouse OSNs. Remarkably, immunohistochemical analysis revealed the most striking Nav1.7 staining not in the olfactory epithelium but in individual glomeruli of the olfactory bulb. Such glomeruli constitute a complex neuropil that includes the presynaptic OSN boutons (Figure 1). Colocalization studies verified that Nav1.7 occupies a critical presynaptic location at the first synapse in the olfactory system,21 and these results were essentially confirmed by others.22
We then used the Cre- LoxP system to delete the channel in OSNs in a tissue-specific and time-dependent manner. This genetic approach revealed offspring that were viable but showed a clear deficit in suckling behavior,21 which is typical for anosmic mice.
On the basis of its biophysical properties, Nav1.7 has been proposed to play a more general role in the transformation of a graded receptor potential into an action potential sequence in sensory neurons.14 However, we were unable to confirm such a role for Nav1.7 in OSNs. Instead, we found clear evidence that the presence of Nav1.7 in the sections of OSN axons within the olfactory glomerulus is an essential and nonredundant requirement for synaptic transfer at the first synapse in the olfactory pathway. In OSNs lacking Nav1.7, presynaptic electrical stimulation failed to elicit postsynaptic responses in olfactory bulb mitral cells, consistent with a loss of glutamate release in OSNs (Figure 2).21 Therefore, we concluded that Nav1.7 is critical for a voltage-dependent influx of calcium ions in presynaptic OSN terminals, although this conclusion has not yet been shown directly. The deficit in synaptic signaling was not due to a loss of synapse formation during development.21
Taken together, these findings revealed an unexpected function of Nav1.7 in the control of presynaptic transmitter release of canonical OSNs (Figure 2). This result is fully consistent with early predictions suggesting that one function of Nav1.7 could be to support the voltage-dependent influx of calcium ions, in turn triggering the release of hormones in some neuroendocrine cells, including adrenal chromaffin cells.11 In light of our findings, this idea now requires further investigation.
Finally, we analyzed several odor-guided behaviors in the mutant mice and found that the presence of Nav1.7 in OSNs is essential for the display of innate attraction to species-specific social odors and food odors. We also observed that the mutant mice no longer display several other vital behaviors, including predator odor avoidance, short-term odor learning, and maternal pup retrieval.21 Therefore, our results not only indicated that conditional deletion of Nav1.7 from OSNs causes a congenital general anosmia phenotype, linking the results in mice with those in humans, but they also provided a mechanistic basis for the observed loss of odor perception. Finally, they provided an unexpected mechanistic link between 2 different sensory modalities, that is, nociception and smell.
Having established an essential role for Nav1.7 function in olfaction, the question arises as to whether this channel also plays a critical role in other chemosensory systems and whether it might even play a more general role in the control of neurotransmitter or hormone release. One system that comes to mind is the taste system. Although a function of sodium channels in taste cells is not well established, evidence suggests that Nav1.7 is expressed in a subset of taste cells of the mouse tongue.23 Because these cells lack olfactory marker protein expression, Cre-mediated Nav1.7 deletion did not occur in these cells in our conditional Nav1.7-null mice and, therefore, we did not test the mice for taste deficits. However, in humans with congenital analgesia, 2 case reports pointed out specifically that “he was . . . anosmic, but taste recognition was correct though delayed”18(p373) and “that the patient was anosmic, but had normal taste.”20(p852) We believe that these findings are not yet fully conclusive and will require further investigations.
Several other chemosensory systems besides the main olfactory system exist in the mouse nose. These systems include an accessory (vomeronasal) olfactory system, a Grueneberg ganglion, the organ of Masera, and the guanylyl cyclase type D system.2 Each of these olfactory subsystems express specific receptors, downstream molecules for second messenger generation, and ion channels for signal detection and transduction.2 It is currently unclear whether these subsystems use the same sodium channels for action potential generation and conduction as canonical OSNs or whether sensory neurons in each subsystem have evolved distinct sets of sodium channels for these functions.
The identification of a sodium channel subunit as a causative gene for an inherited form of general anosmia has significantly advanced our understanding of the genetic basis of the human sense of smell. On the basis of these findings, we propose that the first synapse of the olfactory system should be a rewarding target in the search for additional anosmia-related gene defects. Very little is still known about the contribution of other voltage-gated channels, such as potassium and calcium channels, to action potential propagation and transmitter release at this synapse. Likewise, many of the molecular components involved in the presynaptic release machinery at this synapse remain to be identified. Like the cellular and behavioral phenotypes described in our work,21 any mutation causing disruption of synaptic release and signaling at the first olfactory synapse will cause massive olfactory dysfunction. Loss-of-function mutations in SCN9A are unlikely to be the only mutations causing a general anosmia phenotype. In this context, a variety of gain-of-function mutations for SCN9A have been identified.3- 5,14 An interesting investigation would be to determine whether such gain-of-function mutations alter odor perception in humans.
With respect to sodium channel function in OSNs, which channels underlie the conversion of odor-stimulated graded potentials into action potential sequences remain unclear. We identified Nav1.3 as an additional sodium channel expressed in OSNs,21 and future gene deletion studies will be required to determine its function in olfaction. No human hereditary diseases are yet linked to Nav1.3 (SCN3A) dysfunction in the Online Mendelian Inheritance in Man catalog.
At present, researchers are interested in the mechanisms that underlie neural map formation in the mouse olfactory system, especially with respect to OSN axonal targeting of olfactory bulb glomeruli. Some of the available evidence indicates that neural activity plays a role in this process. Future experiments should test whether Nav1.7 or other sodium channel subunits, such as Nav1.3, are involved in this function.
Finally, we must remember that the Nav1.7 channel is currently being explored as a promising target for the pharmacotherapy of pain in humans. We wait with interest to see whether pharmacological blockade of this channel in vivo will also affect our sense of smell.
Correspondence: Frank Zufall, PhD, Department of Physiology, University of Saarland School of Medicine, Kirrbergerstrasse 1, Bldg 58, D-66421 Homburg, Germany (email@example.com).
Accepted for Publication: January 9, 2012.
Published Online: June 25, 2012. doi:10.1001/archneurol.2012.21
Author Contributions:Study concept and design: Zufall, Pyrski, Weiss, and Leinders-Zufall. Acquisition of data: Pyrski and Weiss. Analysis and interpretation of data: Zufall, Pyrski, and Leinders-Zufall. Drafting of the manuscript: Zufall. Critical revision of the manuscript for important intellectual content: Zufall, Pyrski, Weiss, and Leinders-Zufall. Obtained funding: Zufall and Leinders-Zufall. Administrative, technical, and material support: Zufall and Leinders-Zufall. Study supervision: Zufall and Leinders-Zufall.
Financial Disclosure: None reported.
Funding/Support: This study was supported by grants SFB 530 and SFB 894 from the Deutsche Forschungsgemeinschaft (Drs Zufall and Leinders-Zufall) and by the Volkswagen Foundation (Dr Leinders-Zufall).
Additional Contributions: We thank our colleagues who participated in the work summarized herein and apologize to those whose work we could not cite owing to space limitations. Gabriele Moerschbaecher provided administrative help.