Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.      

Encoding olfactory signals via multiple chemosensory systems

Most animals have evolved multiple olfactory systems to detect general odors as well as social cues. The sophistication and interaction of these systems permit precise detection of food, danger, and mates, all crucial elements for survival. In most mammals, the nose contains two well described chemosensory apparatuses (the main olfactory epithelium and the vomeronasal organ), each of which comprises several subtypes of sensory neurons expressing distinct receptors and signal transduction machineries. In many species (e.g., rodents), the nasal cavity also includes two spatially segregated clusters of neurons forming the septal organ of Masera and the Grueneberg ganglion. Results of recent studies suggest that these chemosensory systems perceive diverse but overlapping olfactory cues and that some neurons may even detect the pressure changes carried by the airflow. This review provides an update on how chemosensory neurons transduce chemical (and possibly mechanical) stimuli into electrical signals, and what information each system brings into the brain. Future investigation will focus on the specific ligands that each system detects with a behavioral context and the processing networks that each system involves in the brain. Such studies will lead to a better understanding of how the multiple olfactory systems, acting in concert, offer a complete representation of the chemical world.     

Encoding Olfactory Signals via Multiple Chemosensory Systems

ABSTRACT

Most animals have evolved multiple olfactory systems to detect general odors as well as social cues. The sophistication and interaction of these systems permit precise detection of food, danger, and mates, all crucial elements for survival. In most mammals, the nose contains two well described chemosensory apparatuses (the main olfactory epithelium and the vomeronasal organ), each of which comprises several subtypes of sensory neurons expressing distinct receptors and signal transduction machineries. In many species (e.g., rodents), the nasal cavity also includes two spatially segregated clusters of neurons forming the septal organ of Masera and the Grueneberg ganglion. Results of recent studies suggest that these chemosensory systems perceive diverse but overlapping olfactory cues and that some neurons may even detect the pressure changes carried by the airflow. This review provides an update on how chemosensory neurons transduce chemical (and possibly mechanical) stimuli into electrical signals, and what information each system brings into the brain. Future investigation will focus on the specific ligands that each system detects with a behavioral context and the processing networks that each system involves in the brain. Such studies will lead to a better understanding of how the multiple olfactory systems, acting in concert, offer a complete representation of the chemical world.     

Optogenetic Patterning of Whisker-Barrel Cortical System in Transgenic Rat Expressing Channelrhodopsin-2

The rodent whisker-barrel system has been an ideal model for studying somatosensory representations in the cortex. However, it remains a challenge to experimentally stimulate whiskers with a given pattern under spatiotemporal precision. Recently the optogenetic manipulation of neuronal activity has made possible the analysis of the neuronal network with precise spatiotemporal resolution. Here we identified the selective expression of channelrhodopsin-2 (ChR2), an algal light-driven cation channel, in the large mechanoreceptive neurons in the trigeminal ganglion (TG) as well as their peripheral nerve endings innervating the whisker follicles of a transgenic rat. The spatiotemporal pattern of whisker irradiation thus produced a barrel-cortical response with a specific spatiotemporal pattern as evidenced by electrophysiological and functional MRI (fMRI) studies. Our methods of generating an optogenetic tactile pattern (OTP) can be expected to facilitate studies on how the spatiotemporal pattern of touch is represented in the somatosensory cortex, as Hubel and Wiesel did in the visual cortex.     

fMRI activation in response to odorants orally delivered in aqueous solutions

During food intake flavor perception results from simultaneous stimulation of the gustatory, olfactory and trigeminal systems. Olfactory stimulation occurs mainly through the retronasal pathway and the resulting perception is often interpreted as a taste perception, thus leading to the well-known sensory confusion between taste and olfaction. The present experiment was designed to study, with functional magnetic resonance imaging (fMRI), the cortical representation of olfactory perception in humans in response to retronasal stimulation by odorants delivered in aqueous solution. Psychophysical evaluation confirmed that the stimuli acted as pure olfactory stimuli through the retronasal pathway and did not present any taste component. Results showed activation in all brain regions previously described with neuroimaging techniques using olfactory stimulation with an odorized air flow. Piriform and orbitofrontal cortex were found activated as well as the hippocampal region, the amygdala, the insular lobe, the cingulate gyrus and the cerebellum. These results demonstrate the feasibility of efficiently stimulating the olfactory system in an fMRI scanner through the retronasal pathway with liquids delivered to the oral cavity. The presentation of olfactory stimuli in liquids to the mouth is a realistic model for the study of food-related flavor perception. This stimulation protocol furthermore allows presenting taste and olfactory stimuli separately or combined, thus allowing for direct comparisons between single modality representation, taste or olfaction, and representation of multi-modality mixtures.     

No oral-cavity-only discrimination of purely olfactory odorants

The purely olfactory odorants coumarin, octanoic acid, phenylethyl alcohol, and vanillin had been found to be consistently identified when presented retronasally but could not be identified when presented oral-cavity only (OCO). However, OCO discrimination of these odorants was not tested. Consequently, it remained possible that the oral cavity trigeminal system might provide sufficient information to differentiate these purely olfactory odorants. To evaluate this, 20 participants attempted to discriminate vapor-phase coumarin, octanoic acid, phenylethyl alcohol, and vanillin and, as a control, the trigeminal stimulus peppermint extract, from their glycerin solvent, all presented OCO. None of the purely olfactory odorants could be discriminated OCO, but, as expected, peppermint extract was consistently discriminated. This inability to discriminate clarifies and expands the previous report of lack of OCO identification of purely olfactory odorants. Taken together with prior data, these results suggest that the oral cavity trigeminal system is fully unresponsive to these odorants in vapor phase and that coumarin, octanoic acid, phenylethyl alcohol, and vanillin are indeed purely olfactory stimuli. The OCO discrimination of peppermint extract demonstrated that the absence of discrimination for the purely olfactory odorants was odorant dependent and confirmed that the oral cavity trigeminal system will provide differential response information to some vapor-phase stimuli.     

Nociceptive inputs to C3, a motoneuron of the tentacle withdrawal reflex in Helix aspersa

The tentacle withdrawal reflex of snails is perhaps the fastest, most sensitive reflex in the animals' repertoire. We have investigated the sensory inputs to a major motoneuron (C3) mediating the reflex. The cell C3 is sensitive to both chemical and mechanical stimulation, but there is little or no discrimination of quality in chemical stimuli. Small increments in the concentration of chemical stimuli produce large changes in neuronal responses. When chemicals are applied to the afferent nerve, the effects are comparable to those caused by applications to the olfactory epithelium, suggesting that the transducing elements are unspecialized. The afferent pathway is independent of the procerebrum, which is the primary olfactory lobe. Two excitatory synaptic inputs are identified, both of which originate in the tentacle, propagate centrally and synapse directly onto C3. A small, low threshold input is assigned to dendritic sites distant from the soma. A larger, higher threshold input is assigned to proximal dendritic sites. The latter input is largely responsible for the strong activation of C3 following noxious stimulation of the tentacle. The sensory inputs to C3 have properties similar to those of fibres in the nasal branch of the vertebrate trigeminal nerve.     

Herpes Simplex Virus 1 Targets the Murine Olfactory Neuroepithelium for Host Entry

Herpes simplex virus 1 (HSV-1) is a ubiquitous and important human pathogen. It is known to persist in trigeminal ganglia (TG), but how it reaches this site has been difficult to determine, as viral transmission is sporadic, pathogenesis is complicated, and early infection is largely asymptomatic. We used mice to compare the most likely natural HSV-1 host entry routes: oral and nasal. Intranasal infection was 100-fold more efficient than oral and targeted predominantly the olfactory neuroepithelium. Live imaging of HSV-1-expressed luciferase showed infection progressing from the nose to the TG and then reemerging in the facial skin. The brain remained largely luciferase negative throughout. Infected cell tagging by viral Cre recombinase expression in floxed reporter gene mice showed nasal virus routinely reaching the TG and only rarely reaching the olfactory bulbs. Thus, HSV-1 spread from the olfactory neuroepithelium to the TG and reemerged peripherally without causing significant neurological disease. This recapitulation of typical clinical infection suggests that HSV-1 might sometimes also enter humans via the respiratory tract.     

Transient Receptor Potential Channels Encode Volatile Chemicals Sensed by Rat Trigeminal Ganglion Neurons

Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual’s physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants), environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants). In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In Xenopus laevis oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia.     

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.     

Receptor guanylyl cyclases in mammalian olfactory function

The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system.     

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.     

The human operculo-insular cortex is pain-preferentially but not pain-exclusively activated by trigeminal and olfactory stimuli

Increasing evidence about the central nervous representation of pain in the brain suggests that the operculo-insular cortex is a crucial part of the pain matrix. The pain-specificity of a brain region may be tested by administering nociceptive stimuli while controlling for unspecific activations by administering non-nociceptive stimuli. We applied this paradigm to nasal chemosensation, delivering trigeminal or olfactory stimuli, to verify the pain-specificity of the operculo-insular cortex. In detail, brain activations due to intranasal stimulation induced by non-nociceptive olfactory stimuli of hydrogen sulfide (5 ppm) or vanillin (0.8 ppm) were used to mask brain activations due to somatosensory, clearly nociceptive trigeminal stimulations with gaseous carbon dioxide (75% v/v). Functional magnetic resonance (fMRI) images were recorded from 12 healthy volunteers in a 3T head scanner during stimulus administration using an event-related design. We found that significantly more activations following nociceptive than non-nociceptive stimuli were localized bilaterally in two restricted clusters in the brain containing the primary and secondary somatosensory areas and the insular cortices consistent with the operculo-insular cortex. However, these activations completely disappeared when eliminating activations associated with the administration of olfactory stimuli, which were small but measurable. While the present experiments verify that the operculo-insular cortex plays a role in the processing of nociceptive input, they also show that it is not a pain-exclusive brain region and allow, in the experimental context, for the interpretation that the operculo-insular cortex splay a major role in the detection of and responding to salient events, whether or not these events are nociceptive or painful.     

The nasal cavity is a route for prion infection in hamsters

Animals that naturally acquire the prion diseases have a well-developed olfactory sense that they utilize for a variety of basic behaviors. To assess the potential for the nasal cavity to serve as a point of entry for prion diseases, a small amount of prion-infected brain homogenate was placed inferior to the nostrils of hamsters, where it was immediately sniffed into the nasal cavity. Hamsters extra-nasally inoculated with the HY strain of transmissible mink encephalopathy (TME) agent had an incubation period that was not significantly different from per os inoculation of the same dose of the HY TME agent. However, the efficiency of the nasal route of inoculation was determined to be 10 to 100 times greater based on endpoint dilution analysis. Immunohistochemistry on tissues from hamsters killed at 2-week intervals after inoculation was used to identify the disease-associated form of the prion protein (PrP(d)) to determine the route of prion neuroinvasion. Nasal mucosa-associated lymphoid tissue and submandibular lymph nodes initially accumulated PrP(d) as early as 4 weeks postinfection. PrP(d) was first identified in cervical lymph nodes at 8 weeks, in the mesenteric lymph nodes, spleen, and Peyer's patches at 14 weeks, and in the tongue 20 weeks after inoculation. Surprisingly, there was no evidence of PrP(d) in olfactory epithelium or olfactory nerve fascicles at any time after inoculation. Therefore, the HY TME agent did not enter the central nervous system via the olfactory nerve; instead, PrP(d) accumulated in elements of the cranial lymphoreticular system prior to neuroinvasion.     

Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice

In rodents, the nasal cavity contains two separate chemosensory epithelia, the main olfactory epithelium, located in the posterior dorsal aspect of the nasal cavity, and the vomeronasal/accessory olfactory epithelium, located in a capsule in the anterior aspect of the ventral floor of the nasal cavity. Both the main and accessory olfactory systems play a role in detection of biologically relevant odors. The accessory olfactory system has been implicated in response to pheromones, while the main olfactory system is thought to be a general molecular analyzer capable of detecting subtle differences in molecular structure of volatile odorants. However, the role of the two systems in detection of biologically relevant chemical signals appears to be partially overlapping. Thus, while it is clear that the accessory olfactory system is responsive to putative pheromones, the main olfactory system can also respond to some pheromones. Conversely, while the main olfactory system can mediate recognition of differences in genetic makeup by smell, the vomeronasal organ (VNO) also appears to participate in recognition of chemosensory differences between genetically distinct individuals. The most salient feature of our review of the literature is that there are no general rules that allow classification of the accessory olfactory system as a pheromone detector and the main olfactory system as a detector of general odorants. Instead, each behavior must be considered within a specific behavioral context to determine the role of these two chemosensory systems. In each case, one system or the other (or both) participates in a specific behavioral or hormonal response.     

Central neuronal pathways of the cardioinhibitory reflex in the isopod crustacean Bathynomus doederleini

We have already identified central neurons for cardioinhibition and cardioacceleration in Bathynomus, an isopod crustacean. The 1st thoracic ganglion (TG1) has cardioinhibitory neurons, which we call CIs, while the 2nd and 3rd thoracic ganglia (TG2 and TG3) have cardioacceleratory neurons, which we call CA1s and CA2s. We examined neuronal pathways for cardioinhibitory reflexes in whole animal preparations, using intracellular and extracellular recording methods. Cardiac inhibition in response to a variety of external stimuli was mediated by activation of CIs and inhibition of both CAs. When preparations had the ventral nerve cord intact, CIs were activated by excitatory postsynaptic potentials and CAs were inhibited by inhibitory postsynaptic potentials in response to tactile stimuli applied to sensilla setae on appendages and afferent stimuli applied to ganglionic roots of the thoracic ganglia. However, stimulation of ganglionic nerve roots of TG2 and TG3, or tactile stimulation of the body surface, failed to evoke inhibition of CAs in preparations in which both the cerebral ganglion and TG1 had been excised. These results suggest that TG1 is an indispensable central region for the excitation of CI and for inhibition of CA neurons, induced by tactile stimuli and by stimuli applied to nerve roots of TG2 and TG3.     

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca²⁺. Two family members, ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose. We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.     

Grueneberg ganglion neurons are activated by a defined set of odorants

Based on a variety of recent findings, the Grueneberg ganglion (GG) in the vestibule of the nasal cavity is considered as an olfactory compartment. However, defined chemical substances that activate GG neurons have not been identified. In this study, the responsiveness of murine GG cells to odorants was examined by monitoring the expression of the activity-dependent gene c-Fos. Testing a number of odorous compounds, cells in the GG were found to respond to dimethylpyrazine (DMP) and a few related substances. These responses were dose-dependent and restricted to early postnatal stages. The DMP-responsive GG cells belonged to the subset of GG neurons that coexpress the signaling elements V2r83, GC-G, and CNGA3. These cells have been previously reported to respond to cool ambient temperatures as well. In fact, cool temperatures enhanced DMP-evoked responses of GG cells. These findings support the concept that the GG of neonatal mice operates as a dual sensory organ that is stimulated by both the odorous compound DMP and cool ambient temperatures.     

A comparison of the discriminatory ability and sensitivity of the trigeminal and olfactory systems to chemical stimuli in the tiger salamander

Summary Trigeminal receptors can respond to a wide variety of chemical stimuli, but it is unknown whether these receptors mediate discrimination between chemical stimuli matched for equal perceptual intensity. The present electrophysiological and behavioral experiments address this issue using tiger salamanders, Ambystoma tigrinum, and four compounds (amyl acetate, cyclohexanone, butanol, and d-limonene). In addition, the relative sensitivities of the trigeminaland olfactory systems to these compounds are compared. In electrophysiological cross-adaptation experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), there was complete cross adaptation such that only concentrations above the background (crossa-dapting) stimulus concentration elicited responses, suggesting that chemical stimuli may stimulate trigeminal receptors nonspecifically. In behavioral experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), only animals with intact olfactory nerves could discriminate between perceptually equivalent concentrations, that is concentrations that elicited the same level of responding. Both electrophysiologically and behaviorally, the trigeminal system exhibited higher thresholds than the olfactory system. We conclude that trigeminal chemoreceptors, at least in salamanders, are unable to discriminate between these two pairs of compounds when matched for equal perceptual intensity, and that trigeminal chemoreceptors are less sensitive than olfactory receptors.Abbreviations AA amyl acetate - CH cyclohexanone - LI d-limonene - BU butanol - EOG electro-olfactogram - ISI interstim-ulus interval - ONX olfactory nerve cut - ppm parts per million (1 l of compound in vapor phase/1l of air=1 ppm)