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)     

Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model

Although the trigeminal nerve innervates the meninges and participates in the genesis of migraine headaches, triggering mechanisms remain controversial and poorly understood. Here we establish a link between migraine aura and headache by demonstrating that cortical spreading depression, implicated in migraine visual aura, activates trigeminovascular afferents and evokes a series of cortical meningeal and brainstem events consistent with the development of headache. Cortical spreading depression caused long-lasting blood-flow enhancement selectively within the middle meningeal artery dependent upon trigeminal and parasympathetic activation, and plasma protein leakage within the dura mater in part by a neurokinin-1-receptor mechanism. Our findings provide a neural mechanism by which extracerebral cephalic blood flow couples to brain events; this mechanism explains vasodilation during headache and links intense neurometabolic brain activity with the transmission of headache pain by the trigeminal nerve.     

Physical Variables in the Olfactory Stimulation Process

Electrical recording from small twigs of nerve in a tortoise showed that olfactory, vomeronasal, and trigeminal receptors in the nose are responsive to various odorants. No one kind of receptor was most sensitive to all odorants. For controlled stimulation, odorant was caused to appear in a stream of gas already flowing through the nose. Of the parameters definable at the naris, temperature, relative humidity, and nature of inert gas had little effect on olfactory responses to amyl acetate, whereas odorant species, odorant concentration, and volume flow rate effectively determined the responses of all nasal chemoreceptors. An intrinsic variable of accessibility to the receptors, particularly olfactory, was demonstrated. Flow dependence of chemoreceptor responses is thought to reflect the necessity for delivery of odorant molecules to receptor sites. Since the olfactory receptors are relatively exposed, plateauing of the response with flow rate for slightly soluble odorants suggests an approach to concentration equilibrium in the overlying mucus with that in the air entering the naris. Accordingly, data for responses to amyl acetate were fitted with Beidler's (1954) taste equation for two kinds of sites being active. The requirement for finite aqueous solubility, if true, suggests substitution of aqueous solutions for gaseous solutions. A suitable medium was found and results conformed to expectations. Olfactory receptors were insensitive to variation of ionic strength, pH, and osmotic pressure.     

Sensitivity to odours in the embryo of the domestic fowl

Sensitivity to odours in the embryo of the domestic fowl was investigated on the day before hatching. Embryos were tested with four odorants: dichloroethane, cineole, amyl acetate and formic acid. Three odorants (dichloroethane, formic acid and cineole) produced an increase in the heart rate and a rise in the rates of beak-clapping and the first two increased the amount of head-shaking. Odorants had little effect on other types of activity. The response to amyl acetate varied between experiments. Blocking the nostrils with wax abolished the response. Some implications of these results are discussed briefly.     

Self- and cross-adaptation to chemical stimulation of the nasal trigeminal nerve in the rat

Electrophysiological, multi-unit responses from the ethmoidbranch of the trigeminal nerve to chemical stimuli (amyl acetate,d-carvone, l-carvone, l-menthol and toluene) were examined,using self- and cross-adaptation paradigms, to address the questionof whether different chemical stimuli may stimulate trigeminalnerve fibers using different ‘receptive pathways’and thus to suggest whether qualitative distinctions betweendifferent compounds may be made by trigeminal chemoreceptors.No adaptation occurred between l-menthol and toluene, suggestingthat these two compounds activate different receptive pathwaysin the trigeminal nerve which may be capable of making qualitativediscriminations between these two compounds. Symmetrical adaptationoccurred between amyl acetate and d-carvone, amyl acetate andl-carvone, amyl acetate and toluene, and l-carvone and d-carvonesuggesting that these compounds may activate the same receptivepathways in the trigeminal nerve which may not be capable ofmaking qualitative discriminations between these compounds.Asymmetrical adaptation occurred between amyl acetate and l-menthol,d-carvone and l-menthol, l-carvone and l-menthol, d-carvoneand toluene, and l-carvone and toluene. This implies that theprocessing of these stimuli by trigeminal nerve fibers may bemore complex than anticipated previously.     

TRPV1 receptors and nasal trigeminal chemesthesis

The trigeminal nerve responds to a wide variety of irritants. Trigeminal nerve fibers express several receptors that respond to chemicals, including TRPV1 (vanilloid) receptors, acid-sensing ion channels, P2X (purinergic) receptors, and nicotinic acetylcholine receptors. In order to assess whether TRPV1 plays a role in responses to a broad array of substances, TRPV1 (along with green fluorescent protein) was expressed in human embyonic kidney cells (HEK) 293t cells which were then stimulated with diverse trigeminal irritants. Calcium imaging was used to measure responses to capsaicin, amyl acetate, cyclohexanone, acetic acid, toluene, benzaldehyde, (-)-nicotine, (R)-(+)-limonene, (R)-(-)-carvone, and (S)-(+)-carvone. Three irritants (acetic acid and the 2 carvones) stimulated nontransfected controls. Two irritants (capsaicin and cyclohexanone) stimulated only transfected cells. The response could be eliminated with capsazepine, a TRPV1 blocker. The 5 remaining irritants were nonstimulatory in both nontransfected and transfected cells. Because all the compounds tested on HEK cells elicited neural responses from the ethmoid branch of the trigeminal nerve in rats, the 5 nonstimulatory compounds must do so by a non-TRPV1 receptor. These results suggest that TRPV1 serves as a receptor for both cyclohexanone and capsaicin in trigeminal nerve endings.     

Tyrosinase inactivation in organic solvents.

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.     

Effect of hydrotropes on solubility and mass transfer coefficient of amyl acetate

This paper presents a comprehensive study on the effect of citric acid, sodium benzoate, sodium salicylate and urea (hydrotropes) on the solubility and mass transfer coefficient for the extraction of amyl acetate in water. The influence of a wide range of hydrotrope concentration (0–3.0?mol/l) and different temperatures (303–333?K) on the solubility of amyl acetate has been studied. The influence of different hydrotrope concentrations on the mass transfer coefficients for amyl acetate–water system has been ascertained. Setschenow constant, K_s, a measure of the effectiveness of hydrotrope has been determined for each case. The solubility of amyl acetate increases with increase in hydrotrope concentration and also with system temperature. Consequent to the increase in the solubility of amyl acetate, the mass transfer coefficient was also found to increase with increase in hydrotrope concentration. A Minimum Hydrotropic Concentration (MHC) was found essential to show a significant increase in the solubility and mass transfer coefficient for amyl acetate–water system. The enhancement factor, which is the ratio of value in presence and absence of a hydrotrope is reported for both solubility and mass transfer coefficients.     

Effects of air flow on rat electroolfactogram

The electroolfactogram (EOG) previously has been used to demonstrate the regional distribution of rat olfactory epithelial odorant responses. Here, we evaluated the effects of airflow parameters on EOGs in two preparations: one where odorants were directly applied to the epithelium (opened preparation) and one where odorants were drawn through the nasal passages by an artificial sniff (closed preparation). EOG rise times served as one measure of odorant access. For isoamyl acetate (but not for limonene), rise times were slower in the lateral recesses of the closed (but not the opened) preparation. Polar odorants (amyl acetate, carvone and benzaldehyde) evoked smaller responses in the closed preparation than in the opened preparation, and these responses were particularly depressed in the lateral regions of the closed preparation. Responses to nonpolar hydrocarbon odorants (limonene and benzene) were equal in the lateral regions of both preparations, but were somewhat depressed in the medial region of the closed preparation. The responses to some polar odorants in the closed preparation were sensitive to changes in airflow parameters. These data suggest that the sorptive properties of the nose contribute substantially to determining the response of the epithelium and act to increase differences produced by inherent receptor mechanisms.     

Effects of capsaicin and nitric oxide synthase inhibitor on increase in cerebral blood flow induced by sensory and parasympathetic nerve stimulation in the rat.

Effects of electrical stimulation of the nerve bundles including sensory and parasympathetic nerves innervating cerebral arteries on cerebral blood flow (CBF) and mean arterial blood pressure (MABP) were investigated with a laser-Doppler flowmeter and a blood pressure monitoring system in anesthetized rats pretreated with and without capsaicin. The electrode was hooked on the nerve bundles including the distal nasociliary nerve from trigeminal nerve and parasympathetic nerve fibers from sphenopalatine ganglion. In control rats, the nerve stimulation for 30 s increased CBF in the ipsilateral side and MABP. Hexamethonium attenuated the increase in CBF and abolished that in MABP. Under treatment with hexamethonium, N(G)-nitro-L-arginine (L-NNA, 1 mg/kg) significantly attenuated the stimulation-induced increase in CBF, which was restored by the addition of L-arginine. Although the dose of L-NNA was raised up to 10 mg/kg, the stimulation-induced increase in CBF was not further inhibited and was never abolished. In capsaicin-pretreated rats, magnitudes of the stimulation-induced increases in CBF and MABP were lower than those in control rats. Hexamethonium attenuated the increase in CBF and abolished that in MABP. Under treatment with hexamethonium, L-NNA abolished the stimulation-induced increase in CBF in capsaicin-pretreated rats. In conclusion, nitric oxide released from parasympathetic nerves and neuropeptide(s) released antidromically from sensory nerves may be responsible for the increase in CBF in the rat. The afferent impulses by nerve stimulation may stimulate the trigeminal nerve and lead to the rapid increase in MABP, which partly contributes to the increase in CBF.     

Odor sensitivity mediated by the trigeminal nerve in the pigeon

Absolute behavioral thresholds of three pigeons to amyl acetatein air were elevated at least 2.6 log units by radical bilateralolfactory nerve resection and remained relatively unchangedover many sessions of threshold testing. No evidence of postoperativeability to discriminate qualitatively between amyl acetate andbutyl acetate was observed. These results contrast with previousreports that olfactory nerve resection did not prevent discriminationbetween amyl acetate and butyl acetate and that sensitivityto amyl acetate increased with continued postoperative testing.We suggest that these earlier results may have been due, atleast in part, to reconstituted olfactory nerves.     

Effects of antibodies against odorant binding proteins on electrophysiological responses to odorants

Monoclonal antibodies against two olfactory mucosal proteins, one with affinity for anisole-like and the other for benzaldehyde-like compounds, were applied to mouse olfactory epithelium. Responses to three odorants (anisole, benzaldehyde and amyl acetate) were measured. Of 26 antibodies, three (12%) inhibited responses only to the odorant with affinity for the antigen, nine (35%) inhibited responses to all three odorants, and 14 (54%) were without effect. None reduced responses by as much as 50%. The data support the hypothesis that there is a class of related proteins in olfactory neuronal cell membranes that function as receptor molecules and that other mechanisms also mediate odorant stimulation.     

A genome-wide study on the perception of the odorants androstenone and galaxolide

Twin pairs and their siblings rated the intensity of the odorants amyl acetate, androstenone, eugenol, Galaxolide, mercaptans, and rose (N = 1573). Heritability was established for ratings of androstenone (h (2) = 0.30) and Galaxolide (h(2) = 0.34) but not for the other odorants. Genome-wide association analysis using 2.3 million single nucleotide polymorphisms indicated that the most significant association was between androstenone and a region without known olfactory receptor genes (rs10966900, P = 1.2 × 10(-7)). A previously reported association between the olfactory receptor OR7D4 and the androstenone was not detected until we specifically typed this gene (P = 1.1 × 10(-4)). We also tested these 2 associations in a second independent sample of subjects and replicated the results either fully (OR7D4, P = 0.00002) or partially (rs10966900, P = 0.010; N = 266). These findings suggest that 1) the perceived intensity of some but not all odorants is a heritable trait, 2) use of a current genome-wide marker panel did not detect a known olfactory genotype-phenotype association, and 3) person-to-person differences in androstenone perception are influenced by OR7D4 genotype and perhaps by variants of other genes.     

Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis strain JHM

The present study assessed the functional consequences of viral infection with a neurotropic coronavirus, designated MHV OBLV, that specifically targets central olfactory structures. Using standard operant techniques and a 'go, no-go' successive discrimination paradigm, six BALB/c mice were trained to discriminate between the presentation of an air or odor stimulus (three mice for each of the odorants propanol and propyl acetate). Two additional BALB/c mice were trained to discriminate between the presentation of air and the presentation of either vanillin or propionic acid. Following criterion performance, each mouse received an additional 2000 trials of overtraining. At completion of overtraining one mouse from the propanol and propyl acetate groups were allocated as untreated. The remaining six mice were inoculated with 300 microl of the OBLV stock per nostril for a total of 1.5 x 10(6) p.f.u. in 600 microl. Following a 1 month rest, untreated and inoculated animals were again tested on their respective air versus odor discrimination task. Untreated animals immediately performed at criterion levels. In contrast, inoculated animals varied in their capacity to discriminate between air and odorant. Five of the six inoculated mice showed massive disruption of the olfactory bulb, including death of mitral cells; the other was more modestly affected. In addition, the density of innervation of the olfactory mucosa by substance P-containing trigeminal fibers is also affected by inoculation. Those mice that remained anosmic to the training odorants had the most severe reduction in mitral cell number and substance P fiber density among the inoculated animals.     

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.     

Direct behavioral evidence for retronasal olfaction in rats

The neuroscience of flavor perception is becoming increasingly important to understand abnormal feeding behaviors and associated chronic diseases such as obesity. Yet, flavor research has mainly depended on human subjects due to the lack of an animal model. A crucial step towards establishing an animal model of flavor research is to determine whether the animal uses the retronasal mode of olfaction, an essential element of flavor perception. We designed a go- no go behavioral task to test the rat's ability to detect and discriminate retronasal odorants. In this paradigm, tasteless aqueous solutions of odorants were licked by water-restricted head-fixed rats from a lick spout. Orthonasal contamination was avoided by employing a combination of a vacuum around the lick-spout and blowing clean air toward the nose. Flow models support the effectiveness of both approaches. The licked odorants were successfully discriminated by rats. Moreover, the tasteless odorant amyl acetate was reliably discriminated against pure distilled water in a concentration-dependent manner. The results from this retronasal odor discrimination task suggest that rats are capable of smelling retronasally. This direct behavioral evidence establishes the rat as a useful animal model for flavor research.     

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.     

Biofiltration of ethyl acetate and amyl acetate using a composite bead biofilter

Biodegradation kinetic behaviors of ethyl acetate and amyl acetate in a composite bead biofilter were investigated. The composite bead was the spherical PVA/peat/KNO(3)/GAC composite bead which was prepared in our previous works. Both microbial growth rate and biochemical reaction rate were inhibited at higher inlet concentration. For the microbial growth process, the microbial growth rate of ethyl acetate was greater than that of amyl acetate in the inlet concentration range of 100-400ppm. The degree of inhibitive effect was almost the same for ethyl acetate and amyl acetate in this concentration range. The half-saturation constant K(s) values of ethyl acetate and amyl acetate were 16.26 and 12.65ppm, respectively. The maximum reaction rate V(m) values of ethyl acetate and amyl acetate were 4.08 and 3.53gCh(-1)kg(-1) packed material, respectively. Zero-order kinetic with the diffusion limitation could be regarded as the most adequate biochemical reaction model. For the biochemical reaction process, the biochemical reaction rate of ethyl acetate was greater than that of amyl acetate in the inlet concentration range of 100-400ppm. The inhibitive effect for ethyl acetate was more pronounced than that for AA in this concentration range. The maximum elimination capacity of ethyl acetate and amyl acetate were 82.3 and 37.93gCh(-1)m(-3) bed volume, respectively. Ethyl acetate degraded by microbial was easier than amyl acetate did.     

Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.     

Response patterns of single neurons in the tortoise olfactory epithelium and olfactory bulb

The responses to odor stimulation of 40 single units in the olfactory mucosa and of 18 units in the olfactory bulb of the tortoise (Gopherus polyphemus) were recorded with indium-filled, Pt-black-tipped microelectrodes. The test battery consisted of 27 odorants which were proved effective by recording from small bundles of olfactory nerve. Two concentrations of each odorant were employed. These values were adjusted for response magnitudes equal to those for amyl acetate at –2.5 and –3.5 log concentration in olfactory twig recording. Varying concentrations were generated by an injection-type olfactometer. The mucosal responses were exclusively facilitory with a peak frequency of 16 impulses/sec. 19 mucosal units responded to at least one odorant and each unit was sensitive to a limited number of odorants (1–15). The sensitivity pattern of each unit was highly individual, with no clear-cut types, either chemical or qualitative, emerging. Of the 18 olfactory bulb units sampled, all responded to at least one odorant. The maximum frequency observed during a response was 39 impulses/sec. The bulbar neurons can be classified into two types. There are neurons that respond exclusively with facilitation and others that respond with facilitation to some odorants and with inhibition to others. Qualitatively or chemically similar odorants did not generate similar patterns across bulbar units.