Chemical Senses

The social and reproductive behaviors of most mammals are modulated by pheromones, which are perceived by the vomeronasal organ (VNO). Vomeronasal transduction in vertebrates is activated through G-protein–coupled receptors, which in turn leads to the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by the activity of phospholipase C. DAG has been shown to gate the transient receptor potential channel 2, whereas IP3 may play a role in stimulating the release of calcium from the endoplasmic reticulum store. To investigate the role of the alpha subunits of Gq/11 in the transduction process, microvillar membranes from female mice VNO were preincubated with a selective C-terminal peptide antibody against Gαq/11 and then stimulated with adult male urine. Incubation of VNO membranes with antibodies against Gαq/11 blocked the production of IP3 in a dose-dependent manner. We were also able to impair the production of IP3 when we stimulated with 2-heptanone or 2,5-dimethylpyrazine in the presence of antibodies against the alpha subunit of Gq/11. 2-Heptanone is a known pheromone that has been linked to VIR receptors. Thus, our observations indicate that the alpha subunits of Gq/11 play a role in pheromonal signaling in the VNO.

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents and putative second messenger-activated currents in receptor neurons in the vomeronasal sensory epithelium of female rats. The resting membrane potential and input resistance were -45.5 +/- 2.5 mV (mean +/- SEM, n = 39) and 1.5 +/- 0.2 G omega (mean +/- SEM, n = 37). Current injection of 1-3 pA induced overshooting action potentials. The firing frequency increased with increasing current injections linearly from 1 to 10 pA and reached a plateau at 30 pA, suggesting that rat vomeronasal receptor neurons sensitively elicit action potentials in response to a small receptor potential. Under voltage clamp, voltage-dependent Na+ inward current, inward Ca2+ current, sustained outward K+ current and Ca-(2+)-activated K(+)-current were identified. Dialysis of D-inositol-1,4,5-trisphosphate (D-IP3) induced inward currents with an increase in membrane conductance in approximately 54% of the cells and inward current fluctuations in 15% of the cell. L-IP3 also induced inward currents and current fluctuations in 53 and 13% of the cells respectively. The mean amplitude of inward currents induced by 100 microM D-IP3 and L-IP3 were 84.6 +/- 14.0 pA (SEM, n = 82) and 66.1 +/- 9.4 pA (SEM, n = 100) respectively. The IP3-induced responses were blocked by elimination of Na+ and Ca2+ in the external solution or application of 10 microM ruthenium red. The present study suggested that IP3-mediated transduction pathways exist in rat vomeronasal receptor neurons.

D-glucono-1,5-lactone differs from D-glucopyranose only in that it has a C = O group instead of CHOH group at carbon atom number one. The molecule therefore possesses an intact 3,4 alpha-glycol group and is sweet. However, it autohydrolyses in water solution at room temperature, forming D-gluconic acid and D-glucono-1,4-lactone. As the solution pH falls it becomes sweet-sour and eventually almost completely sour as the generated hydronium ions dominate both the solution properties and the taste perceptions elicited. It is shown that the ratio of generated hydronium ions to unchanged lactone accords with anticipated taste quality during the first 28 min of autohydrolysis. Changes in both apparent specific volume and apparent isentropic compressibility illustrate increasing solute-solvent interaction and increasing disturbance of water structure during the course of autohydrolysis. These changes are consistent with the concurrent sweet to sour change, but do not explain the weak bitterness which also accompanies them.

The present study was undertaken to investigate the pharmacokinetics of 1,8-cineol in human subjects during prolonged inhalation. The results showed that 1,8-cineol is well absorbed from breathing air, with a peak plasma concentration after approximately 18 min. The elimination of this fragrance compound from the blood is biphasic, with a mean distribution half-life of 6.7 min and an elimination half-life of 104.6 min.

Amiloride, a sodium channel blocker, is known to suppress NaCl responses of the chorda tympani (CT) nerve in various mammalian species. In mice, the NaCl suppressing effect of amiloride is reported to differ among strains. In C57BL mice, amiloride inhibits NaCl responses to about 50% of control, whereas no such clear suppression was evident in prior studies with 129 mice. However, evidence from behavioral studies is not entirely consistent with this. Recently, it has been found that genetic backgrounds of 129 mice differ within substrains. 129X1/SvJ (formerly 129/SvJ) mice differ from the 129P3/J (formerly 129/J) strain by 25% of sequence length polymorphisms. Therefore, we examined possible substrain difference between 129P3/J and 129X1/SvJ mice in the amiloride sensitivity of electrophysiologically recorded NaCl responses. Amiloride significantly suppressed CT responses to NaCl without affecting responses to KCl both in 129P3/J and 129X1/SvJ mice. However, the magnitude of the amiloride inhibition was significantly larger (approximately 50% of control in response to 0.01-1.0 M NaCl by 100 microM amiloride) in 129X1/SvJ than in 129P3/J mice (approximately 20% of control in response to 0.03-0.3 M NaCl by 100 microM amiloride). Threshold amiloride concentration for suppression of responses to 0.3 M NaCl was 30 microM in 129P3/J mice, which was higher than that in 129X1/SvJ mice (10 microM). In 129X1/SvJ mice, the threshold amiloride concentration eliciting inhibition of NaCl responses and the magnitude of the inhibition were comparable with those in C57BL/6 mice. These results suggest that amiloride sensitivity of NaCl responses differs even among the 129 substrains, 129P3/J and 129 X1/SvJ, and the substrain difference of 129 mice in amiloride sensitivity is as large as that between two inbred strains (129P3/J and C57BL/6).

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.

In long-term two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more monosodium L-glutamate (MSG) and inosine-5'-monophosphate (IMP) compared with mice from the 129P3/J (129) strain. The goal of this study was to assess the role of afferent gustatory input in these strain differences. We measured integrated responses of the mouse chorda tympani and glossopharyngeal nerves to lingual application of compounds that evoke umami taste in humans: MSG, monoammonium L-glutamate (NH(4) glutamate), IMP and guanosine-5'-monophosphate (GMP) and also to other taste stimuli. Chorda tympani responses to MSG and NH(4) glutamate were similar in B6 and 129 mice. Chorda tympani responses to IMP and GMP were lower in B6 than in 129 mice. Responses to umami stimuli in the glossopharyngeal nerve did not differ between the B6 and 129 strains. Responses to MSG, IMP and GMP were not affected by sodium present in these compounds because B6 and 129 mice had similar neural taste responses to NaCl. This study has demonstrated that the increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli. Lack of support for the role of the chorda tympani or glossopharyngeal nerves in the enhanced consumption of MSG and IMP by B6 mice suggests that it is due to some other factors. Although results of our previous study suggest that postingestive effects of MSG can affect its intake, contribution of other gustatory components (e.g. greater superficial petrosal nerve or central gustatory processing) to the strain differences in consumption of umami compounds also cannot be excluded. Strain differences in gustatory neural responses to nucleotides but not glutamate suggest that these compounds may activate distinct taste transduction mechanisms.

To examine whether age influences taste solution preferences, we measured taste preferences of C57BL/6J and 129X1/SvJ mice given a series of 48-h 2-bottle tests with a choice between water and one of the following taste solutions: 2 mM saccharin, 5 mM citric acid, 30 microM quinine hydrochloride, 75 mM sodium chloride (NaCl), 10 mM inosine monophosphate (IMP), 50 mM calcium chloride (CaCl(2)), and 10% ethanol. We tested separate groups of male mice fed Teklad 8604 chow at ages 4, 6, 9, 12, 15, 20, 25, 30, 40, and 50 weeks and retested some of these mice at 54, 75, and 100 weeks and again at 125 weeks. Female mice fed chow were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, 100, and 125 weeks. Male mice fed AIN-93G semisynthetic diet were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, and 100 weeks. Concentration-response functions for each taste solution were collected from male and female mice fed chow aged 8 or 125 weeks. In general, the results showed that age had little effect on taste preferences. Exceptions included 1) a small increase in quinine hydrochloride preference between 54 and 125 weeks in mice of both strains and sexes, 2) a marked increase in NaCl preference between 4 and 12 weeks in female B6 mice, 3) a gradual decrease in IMP preference between 4 and 125 weeks in male and female 129 mice, 4) a marked decrease in CaCl(2) preference between 54 and 125 weeks in male and female 129 mice, and 5) a marked reduction in ethanol preference between 4 and 12 weeks in male B6 mice fed AIN-93G diet but not chow. These results show that over a wide range and with the exceptions noted, age contributes little to the variation in taste preferences observed in C57BL/6J and 129X1/SvJ mice.

Molecular mechanisms of salty taste in mammals are not completely understood. We use genetic approaches to study these mechanisms. Previously, we developed a high-throughput procedure to measure NaCl taste thresholds, which involves conditioning mice to avoid LiCl and then examining avoidance of NaCl solutions presented in 48-h 2-bottle preference tests. Using this procedure, we measured NaCl taste thresholds of mice from 13 genealogically divergent inbred stains: 129P3/J, A/J, BALB/cByJ, C3H/HeJ, C57BL/6ByJ, C57BL/6J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/BlNJ, PWK/PhJ, and SJL/J. We found substantial strain variation in NaCl taste thresholds: mice from the A/J and 129P3/J strains had high thresholds (were less sensitive), whereas mice from the BALB/cByJ, C57BL/6J, C57BL/6ByJ, CE/J, DBA/2J, NZB/BINJ, and SJL/J had low thresholds (were more sensitive). NaCl taste thresholds measured in this study did not significantly correlate with NaCl preferences or amiloride sensitivity of chorda tympani nerve responses to NaCl determined in the same strains in other studies. To examine whether strain differences in NaCl taste thresholds could have been affected by variation in learning ability or sensitivity to toxic effects of LiCl, we used the same method to measure citric acid taste thresholds in 4 inbred strains with large differences in NaCl taste thresholds but similar acid sensitivity in preference tests (129P3/J, A/J, C57BL/6J, and DBA/2J). Citric acid taste thresholds were similar in these 4 strains. This suggests that our technique measures taste quality-specific thresholds that are likely to represent differences in peripheral taste responsiveness. The strain differences in NaCl taste sensitivity found in this study provide a basis for genetic analysis of this phenotype.

We examined the effects of 5alpha-androst-16-en-3alpha-ol (3alpha-androstenol) on pulsatile luteinizing hormone (LH) secretion in human females. The frequency of the LH pulse in the follicular phase was decreased by exposing the women to 3alpha-androstenol.

To explore the possibility that compounds which were identified as pheromones in experimental animals mediate human menstrual synchrony, we examined the relationship between menstrual synchrony and the ability to smell putative pheromones, 5alpha-androst-16-en-3alpha-ol (3alpha-androstenol) and 5alpha-androst-16-en-3-one (5alpha-androstenone). When we examined menstrual synchrony among 64 women living together in a college dormitory, we found that 24 (38%) of them became synchronized with room-mates in 3 months. Afterwards, dilution series of 3alpha-androstenol and 5alpha-androstenone and the control odorant (pyridine) were presented to the 64 women and sensitivity to the odors was compared between synchronized and non-synchronized women. No difference was found between the two groups of women in the detection threshold for pyridine, indicating that general olfactory ability did not differ between them. The detection threshold for 3alpha-androstenol of synchronized women was significantly lower than that of non-synchronized women, but no difference in the threshold for 5alpha-androstenone was found between synchronized and non-synchronized women. These results indicate that the women who showed menstrual synchrony had a higher sensitivity to 3alpha-androstenol but not necessarily to 5alpha-androstenone.

Myriapods represent an arthropod lineage, that originating from a marine arthropod ancestor most likely conquered land independently from hexapods and crustaceans. Establishing aerial olfaction during a transition from the ocean to land requires molecules to be detected in gas phase instead of in water solution. Considering that the olfactory sense of myriapods has evolved independently from that in hexapods and crustaceans, the question arises if and how myriapods have solved the tasks of odor detection and odor information processing in air. Comparative studies between arthropod taxa that independently have established a terrestrial life style provide a powerful means of investigating the evolution of chemosensory adaptations in this environment and to understand how the arthropod nervous system evolved in response to new environmental and ecological challenges. In general, the neuroethology of myriapods and the architecture of their central nervous systems are insufficiently understood. In a set of experiments with the centipede Scutigera coleoptrata, we analyzed the central olfactory pathway with serial semi-thin sectioning combined with 3-dimensional reconstruction, antennal backfilling with neuronal tracers, and immunofluorescence combined with confocal laser-scanning microscopy. Furthermore, we conducted behavioral experiments to find out if these animals react to airborne stimuli. Our results show that the primary olfactory and mechanosensory centers are well developed in these organisms but that the shape of the olfactory neuropils in S. coleoptrata is strikingly different when compared with those of hexapods and malacostracan crustaceans. Nevertheless, the presence of distinct neuropils for chemosensory and mechanosensory qualities in S. coleoptrata, malacostracan Crustacea, and Hexapoda could indicate a common architectural principle within the Mandibulata. Furthermore, behavioral experiments indicate that S. coleoptrata is able to perceive airborne stimuli, both from live prey and from a chemical extract of the prey. These results are in line with the morphological findings concerning the well-developed olfactory centers in the deutocerebrum of this species.

The olfactory-mediated responses to the sex hormone 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) were studied in spermiated and regressed male crucian carp (Carassius carassius L.). The position and spontaneous locomotor activity of single male crucian carp were continuously recorded in an artificial stream. 17 alpha,20 beta-P (final concentration 10(-11) M) was supplied to one half and its ethanol carrier to the other half of the test area. Milt volume and gonadotropin (GtH-II) concentration in the plasma were also measured. The smell of 17 alpha,20 beta-P significantly increased both the GtH-II concentration in the plasma and the volume of strippable milt in spermiated crucian carp. Behaviorally, the side of the test area scented with 17 alpha,20 beta-P was significantly avoided by spermiated males. None of the described effects of 17 alpha,20 beta-P on spermiated males were observed for the regressed crucian carp. In view of the lack of response from regressed crucian carp we suggest that the observed avoidance behavior of 17 alpha,20 beta-P by spermiated males is a relevant reaction for spawning male crucian carp. The results are well in accordance with responses obtained in the closely related goldfish and gives strong support that the wild male crucian carp use the 17 alpha,20 beta-P signal from the females to prepare for the coming spawning.

Linoleic, oleic, and stearic fatty acids, presented vapor-phase retronasally, were discriminable from blanks and each other, but the same concentrations, oral-cavity-only (OCO), were not discriminable from blanks. It remained possible that higher concentrations might be discriminable OCO. To evaluate this, participants attempted to discriminate undiluted linoleic, oleic, or stearic acids, vapor-phase OCO, from blanks. For each fatty acid, participants received 5 stimulus delivery containers (SDCs) in 2 trials; 4 SDC held blanks, the fifth, a fatty acid. As a “positive control” in 2 trials, participants received vapor-phase OCO peppermint extract and blanks. For all trials, the task was to select the 1 different SDC. It was found that the 1 different SDC was selected in 24% of stearic, 32% of linoleic, 47% of oleic acid, and in 92% of peppermint trials; discriminations (the 1 different SDC selected in both trials) occurred in 0%, 16%, 26%, and 84% of pairs, respectively. Correct selections for oleic acid differed from chance, P = 0.0004, but not for linoleic acid, P = 0.125, or stearic acid, P = 0.345, Bonferroni corrected. Vapor-phase oleic acid can be an oral cavity trigeminal stimulus, linoleic acid might be (uncorrected P = 0.0384), but vapor-phase stearic acid cannot be.

Research on the neurobiological substrates of taste-related learning and memory is a broad and rapidly evolving mul- tidisciplinary field. Historically, research on taste aversion learning has contributed critically both to the behavioral and the neurobiological assessment of learning systems. Since the discovery by Garcia et al. (1955) of conditioned taste aversion and its peculiar features, such as one-trial and long-delay learning, taste aversion learning represented a challenge for learning theorists and contributed decisively to the development of modern learning theories. Also, the search for the brain circuit responsible of taste-illness asso- ciation has revealed a complex interaction among a variety of areas located at different brain levels from brain stem to cortex. During the ninth European Congress of Psychology, a symposium was held where 5 speakers discussed the recent developments in the brain mechanisms of taste memory and neural plasticity. Their talks reflected how the research field has widened both in the types of processes and brain areas studied as well as levels of analysis aimed and technical approaches applied. First, an increased interest in the study of the neurobiological mechanisms not only of taste aversion but also of the safe taste memory was evident because the effect of nonrewarded taste exposure on habit- uation of neophobia and latent inhibition were issues of interest for the speakers. This is consistent with wider theo- retical approaches to taste memory, such as that proposed by Bermudez-Rattoni (2004). Also, from the speakers' talks, it became evident that the study of brain areas additional to those involved in taste and visceral processing may be nec- essary for a complete understanding of the neural plasticity required for taste memory. Second, research aimed not only at the system but also at the cellular and molecular levels of analysis was presented. Third, the range of approaches covered by the speakers included data from lesion, elec- trophysiological recording, immunoreactivity mapping, neurochemical stimulation, and blockade, as well as devel- opmental approaches. Federico Bermudez-Rattoni opened the symposium offer- ing a comprehensive review of the brain mechanisms under- lying the neural representation of safe and aversive taste memory. He presented data supporting a role of cortical glu- tamatergic transmission and metabotropic glutamate recep- tors on taste aversion, taste neophobia, and attenuation of neophobia. His data suggest that the formation of the safe taste memory needs the temporal inactivation of the gluta- matergic system during the first period of time after taste consumption. Ilene Bernstein reviewed studies assessing gene expression in a brain-distributed system underlying taste, visceral, and associativeprocessing.Differentialgeneexpressionasafunc- tion of taste novelty throughout the circuit subserving con- ditioned taste aversion was discussed. Based on her studies that examined the induction of fos-like immunoreactivity in response to taste preexposure, Ilene Bernstein proposed the synthesis and subsequent degradation of an immediate early gene such as c-fos as a potential molecular substrate for changes in conditioning strength over a range of intervals and pointed to central amygdala as a potential site for taste-visceral association relevant for the formation of con- ditioned taste aversions. The talk by Takashi Yamamoto emphasized the separated amygdala substrates underlying the sensory and hedonic changes induced by taste aversion learning. The data pre- sented suggest that the enhanced activation induced by the aversive taste originates in the central amygdala and the he- donic shift from appetitive to aversive originates in the baso- lateral amygdala. By reviewing electrophysiological and recentc-fosimmunohistochemicalstudies,hepresentedadis- tributed neural system underlying conditioned taste aversion expression that includes the parabrachial nucleus, amygdala, insular cortex, supramammillary nucleus, nucleus accum- bens, and ventral pallidum. Milagros Gallo added a developmental approach to the study of taste memory. After reviewing data showing the

Gastrin-releasing-peptide(1-27) (GRP) has been implicated in the regulation of satiety and appetite in numerous paradigms. However, the specific site and mode of action of this gut-brain peptide has not been elucidated. The following experiment examined the effects of GRP on taste responses to sucrose in the behaving rat. A brief-exposure, multi-bottle gustometer was used to provide rats with momentary access to six different concentrations of sucrose in a single test session. This procedure has been used previously, resulting in monotonically increased licking behavior as concentrations of sucrose increase. Differing injection procedures were employed such that rats were tested immediately after i.p. injection or 5 min after i.p. injection of 5 nmol/kg body wt of GRP. Results indicate that GRP does reduce the oral reinforcing properties of sucrose, but the effect is transient, diminishing significantly within 5 min after injection.

The glucocorticoid RU 28362 was employed to identify glucocorticoid receptors in the olfactory mucosa of the guinea-pig. Results demonstrate significant binding of RU 28362 and suggest that the olfactory mucosa is a target site for glucocorticoid action. Chem. Senses 22: 313–319, 1997.

The epithelial sodium channel (ENaC) blocker amiloride has been shown to increase the behaviorally measured NaCl detection threshold in mice. In this study, a conditioned taste aversion (CTA) paradigm was used to examine whether 100 microM amiloride has a perceptible taste that could contribute to this observed decrease in behavioral responsiveness. Eighty-four C57BL/6J (B6) and 64 DBA/2J (D2) mice were divided into eight groups (n=8-12 per group), in which half received an injection of 0.15 M LiCl (2 mEq/kg) and the other half an equivalent saline injection, in three conditioning trials. The four conditioned stimuli were 100 microM amiloride hydrochloride, water, 0.1 and 0.3 M NaCl. Neither strain demonstrated acquisition of a CTA to amiloride in a brief-access (BA) taste test (5 s trials in the gustometer). Although 0.3 M NaCl is inherently aversive, its pairing with LiCl led to significantly further decreases in licking during the BA test on salt trials in both strains. The D2 strain clearly avoided 0.1 M NaCl, whereas avoidance of this stimulus was more equivocal in B6 mice. The inefficacy of amiloride to serve as a conditioned stimulus in taste aversion learning involving three LiCl pairings suggests that the effects of this ENaC blocker on taste-related behavioral responses to NaCl are likely due to its pharmacological interference with sodium taste transduction.

Sodium taste transduction is thought to occur via an amiloride-sensitive, sodium-selective pathway and an amiloride-insensitive, cation nonselective, anion-dependent pathway(s). It has been shown by others that amiloride, an epithelial sodium channel (ENaC) blocker, significantly reduces the chorda tympani nerve response to lingually applied NaCl in C57BL/6 (B6) mice but not in DBA/2 (D2) mice, suggesting that the latter strain might not possess functional ENaCs in taste receptor cells. We psychophysically measured and compared taste detection thresholds of NaCl and sodium gluconate (NaGlu) prepared with and without 100 microM amiloride in these two strains (eight/strain). Mice were trained and tested in a two-response operant signal detection procedure conducted in a gustometer. Surprisingly, no strain effect was found for the detection thresholds of both salts (approximately 0.05-0.06 M). Moreover, these thresholds were increased by almost an order of magnitude by amiloride adulteration of the solutions. This marked effect of amiloride on sodium detection thresholds suggests that ENaCs are necessary for normal sensitivity to sodium salts in both strains. In addition, because NaGlu is thought to stimulate primarily the amiloride-sensitive pathway, especially at low concentrations, the similarity of NaCl and NaGlu thresholds (r > 0.81 both strains) suggests that ENaCs are also sufficient to support the detection of sodium in weak solutions by B6 and D2 mice.

Odorant-binding proteins (OBPs) are thought to be responsible for the transport of semiochemicals across hydrophobic interfaces to olfactory receptors. In insects, a second class of OBPs with four conserved cysteines has been variously named as sensory appendage proteins, olfactory segment-D proteins, and chemosensory proteins (CSPs). The physiological functions of these proteins have remained elusive. Here we report a comprehensive survey of both genome and expressed sequence tags (EST) databases. This showed that CSPs are apparently only present in the phylum, Arthropoda, and in two subphyla, Crustacea and Uniramia. This is the first report of a putative CSP in Crustacea and suggests that the origin of these genes predates the divergence of Uniramia and Crustacea. For the Uniramia, we identified 74 new genes encoding putative CSPs of insect species from 10 different orders. Using tissue-specific EST libraries, we have examined the relative expression of putative CSP genes in many tissues from 22 insect species suggesting that the genes are expressed widely. One Drosophila CSPs is expressed sixfold higher in head than other CSPs. One Bombyx mori CSPs was found at a very high level in pheromone gland, and for the first time, six CSPs were identified in B. mori compound eyes. The different frequencies of CSP transcripts were observed between solitary and gregarious EST libraries of Locusta migratoria.

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any prefererence for NaCl at any concentration in two-bottle preference tests. Because 100 μM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068–0.273 M) in F-344 rats with and without 100 μM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.

Performance on olfactory tests can be influenced by a number of stimulus characteristics including chemical structure, concentration, perceptual similarity, and previous experience with the test odorants. Few of these parameters have been extensively characterized in the Fischer 344 rat strain. To investigate how odor quality affects perception in this rat strain, we measured how graded perceptual similarity, created by varying carbon chain length across a series of homologous alcohol pairs, influenced odor discrimination using a liquid-motivated go/no-go task. We employed an automated, liquid-dilution olfactometer to train Fischer 344 rats (N = 8) on a 2-odor discrimination task. Six odorants (1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, and 1-octanol) were arranged to produce 15 novel odorant pairs differing between 1 and 5 carbon atoms; testing sessions included presentation of only 1 pseudorandomly assigned pair daily (200 trials). Results show that although rats can learn to discriminate between any 2 odorant pairs, performance declines systematically as the pairs become more structurally similar and, therefore, more perceptually confusing. As such, the easier discrimination pairs produced reliable ceiling effects across all rats, whereas performance for the difficult discrimination pairs was consistently worse, even after repeated testing. These data emphasize the importance of considering odorant stimulus dimensions in experimental designs employing olfactory stimuli. Moreover, establishing baseline olfactory performance in Fischer 344 rats may be particularly useful for predicting age-related cognitive decline in this model.

Adult Fischer 344 (F344) rats fail to display any preference for NaCl solutions at concentrations typically preferred by other rat strains. To determine whether this behavior is due to a strain difference in NaCl detection threshold, a conditioned taste aversion (CTA) was first established to a suprathreshold concentration of NaCl (0.1 M). Then, a series of dilute NaCl solutions, ranging from 0.0 to 0.011 M NaCl, were presented to F344 (n = 16) and Wistar (n = 16) rats. The lowest concentration at which there was a reliable difference in the preference scores of conditioned and control rats was defined as the detection threshold. Results indicate that the detection threshold for NaCl lies between 0.001 and 0.002 M NaCl for both F344 and Wistar rats. The addition of the sodium channel blocker amiloride to the NaCl solutions raised the detection threshold 10-fold to 0.03-0.04 M NaCl for both strains of rats. These results suggest that the NaCl detection thresholds of F344 and Wistar rats are similar and that these strains do not differ in the degree to which amiloride raises this threshold.

Response times provide essential subthreshold perceptual data that extend beyond accuracy alone. Behavioral reaction times (RTs) were used to characterize rats' ability to detect individual odorants in a series of complimentary binary odorant mixture ratios. We employed an automated, liquid-dilution olfactometer to train Fischer 344 rats (N = 8) on an odor identification task using nonreinforced probe trials. Binary mixture ratios composed of aliphatic odorants (citral and octanol) were arranged such that relative contributions of the 2 components varied systematically by a factor of 1% (v/v). Odorant concentrations for the target (S+), control (S-), and mixture (S+:S-) odorants were presented relative to threshold for each rat. Rats were initially trained to respond by licking at a spout to obtain liquid reward for either citral or octanol as the reinforced target (S+) odorant. After achieving 100% accuracy, rats were transferred to variable ratio (VR 2) reinforcement for correct responding. Nonreinforced probe trials (2 per block of 22 trials) were tested for each mixture ratio and recorded as either S+ (rats lick-responded in the presence of the mixture) or S- (rats refrained from licking), thereby indicating detection of the trained, S+ odorant. To determine the perceived salience for each ratio, RTs (latency from odorant onset to lick response) were recorded for each trial. Consistent with previous studies, RTs for both odorants were shortest (~150-200ms) when the probe trials consisted of a single, monomolecular component. Binary mixtures that contained as little as 1% of the S-, nontarget odorant, however, were sufficiently different perceptually to increase behavioral RTs (i.e., rats hesitated longer before responding); RTs changed systematically as a function of the binary ratio. Interestingly, the rate of RT change was dependent on which odorant served as the S+, suggesting an asymmetric interaction between the 2 odorants. The data demonstrate the value of behavioral RT as a sensitive measure of suprathreshold perceptual responding. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

One hundred and ninety three odor detection thresholds, ODTs, obtained by Nagata using the Japanese triangular bag method can be correlated as log (1/ODT) by a linear equation with R2 = 0.748 and a standard deviation, SD, of 0.830 log units; the latter may be compared with our estimate of 0.66 log units for the self-consistency of Nagata's data. Aldehydes, acids, unsaturated esters, and mercaptans were included in the equation through indicator variables that took into account the higher potency of these compounds. The ODTs obtained by Cometto-Muñiz and Cain, by Cometto-Muñiz and Abraham, and by Hellman and Small could be put on the same scale as those of Nagata to yield a linear equation for 353 ODTs with R2 = 0.759 and SD = 0.819 log units. The compound descriptors are available for several thousand compounds, and can be calculated from structure, so that further ODT values on the Nagata scale can be predicted for a host of volatile or semivolatile compounds. See full article at: http://escholarship.org/uc/item/9b72q2v9

The marine dinoflagellate Gambierdiscus toxicus produces highly lipophilic, polycyclic ether toxins that cause a seafood poisoning called ciguatera. Ciguatoxins (CTXs) and gambierol represent the two major causative agents of ciguatera intoxication, which include taste alterations (dysgeusiae). However, information on the mode of action of ciguatera toxins in taste cells is scarce. Here, we have studied the effect of synthetic CTX3C (a CTX congener) on mouse taste cells. By using the patch-clamp technique to monitor membrane ion currents, we found that CTX3C markedly affected the operation of voltage-gated Na+ channels but was ineffective on voltage-gated K+ channels. This result was the exact opposite of what we obtained earlier with gambierol, which inhibits K+ channels but not Na+ channels. Thus, CTXs and gambierol affect with high potency the operation of separate classes of voltage-gated ion channels in taste cells. Our data suggest that taste disturbances reported in ciguatera poisoning might be due to the ability of ciguatera toxins to interfere with ion channels in taste buds.

Individual differences in sensitivity to the putative human pheromone androstadienone were investigated in three experiments. In experiment 1, the absolute detection threshold for androstadienone was determined to be 211 micro M using the method of constant stimuli. Detection for the related compound estratetraenol was also investigated but a threshold could not be determined. In experiment 2, using an adaptive threshold test on 100 participants, the sensitivity distribution for androstadienone, but not for the reference odor phenylethyl alcohol, was bimodal, with a smaller group of individuals with a high sensitivity to androstadienone (supersmellers). A lack of correlation between thresholds for androstadienone and phenylethyl alcohol further suggested that the bimodality for androstadienone was not due to individuals with a high general olfactory sensitivity. In line with an earlier observation, there was a statistical tendency for women to be more sensitive to androstadienone than men. Results of experiment 3 preclude the possibility that the bimodal sensitivity distribution for androstadienone would depend on individual differences in trigeminal activation. Altogether, the current study suggests that olfactory sensitivity to androstadienone is bimodally distributed in the population with a subgroup consisting of highly sensitive people.

The absolute configuration of 2-sec-butyl-4,5-dihydrothiazole (DHT) in urine of adult male mice was determined through chiral trifluoroacetyl derivative capillary chromatography by comparing the retention time with synthetic standards. (S)-DHT was extracted from fresh urine, while neither (R)-DHT nor the racemization of (S)-DHT were detected. We can conclude that DHT in urine possesses the S configuration, although we cannot exclude a minor component in the R configuration. (S)-DHT was then characterized for binding to the complex of major urinary proteins of male mouse urine (MUP) and for a behavioral response, the competitive scent marking behavior (countermarking). The binding constant of (S)-DHT to MUP (determined by competitive displacement) was 8.2 +/- 0.6 microM (mean +/- SD) and was 10.5 +/- 0.6 microM for R-DHT, thus excluding a relevant difference in binding. (S)-DHT modified countermarking in a peculiar way. Male mice were slow in countermarking urinary spots streaked 2 days earlier and on top of which (S)-DHT was added shortly before the test. This response was not seen when adding (S)-DHT to freshly streaked urinary spots or to clean paper. Unlike (S)-DHT, (R)-DHT prompted countermarking rather than delaying it. We can further conclude that (S)-DHT in male mouse urine is an aversive chemosignal for countermarking.

The rat olfactory epithelium contains a specific water-soluble 45-kDa protein. This protein is recognized by anti-peptide antibodies which react with α-subunits of the known G-proteins. The 45-kDa protein has been isolated using DEAE-chromatography and gel-exclusion chromatography. The content of 45-kDa protein is about 2% of the total soluble proteins of the olfactory mucosa and it is located at the mucociliary surface. According to photo-affinity labeling, the 45-kDa protein possesses a high affinity to GTP and exhibits low GTP hydrolytic activity. The functions of the 45-kDa protein are discussed. Chem, Senses 21: 181–188, 1996.

Few studies have investigated long-term odor recognition memory, although some early observations suggested that the forgetting rate of olfactory representations is slower than for other sensory modalities. This study investigated recognition memory across 64 days for high and low familiar odors and faces. Memory was assessed in 83 young participants at 4 occasions; immediate, 4, 16, and 64 days after encoding. The results indicated significant forgetting for odors and faces across the 64 days. The forgetting functions for the 2 modalities were not fundamentally different. Moreover, high familiar odors and faces were better remembered than low familiar ones, indicating an important role of semantic knowledge on recognition proficiency for both modalities. Although odor recognition was significantly better than chance at the 64 days testing, memory for the low familiar odors was relatively poor. Also, the results indicated that odor identification consistency across sessions, irrespective of accuracy, was positively related to successful recognition. © The Author 2015. Published by Oxford University Press.

Advantame is a new ultrahigh-intensity noncaloric sweetener derived from aspartame and approved for human use. Rats and mice are not attracted to the taste of aspartame and this study determined their preference for advantame. In 24-h choice tests with water, C57BL/6J mice and Sprague-Dawley rats were indifferent to advantame at concentrations of 0.01, 0.03, and 0.1mM but significantly preferred 0.3 and 1mM advantame to water. Both species also preferred 1mM advantame to 1mM saccharin in direct choice tests, but preferred 10mM saccharin to 1mM advantame, which is near the solubility limit for this sweetener. Mice also preferred 1mM advantame to 1mM sucralose or acesulfame K, but preferred both sweeteners at 10mM to 1mM advantame. In addition, mice preferred 1mM advantame to 1 and 10mM aspartame. Thus, advantame is a potent sweetener for rodents but, because of limited solubility, is not an effective alternative to saccharin, sucralose, or acesulfame K at higher concentrations. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evidence in the literature shows that in rodents, some taste-responsive neurons respond to both quinine and acid stimuli. Also, under certain circumstances, rodents display some degree of difficulty in discriminating quinine and acid stimuli. Here, C57BL/6J mice were trained and tested in a 2-response operant discrimination task. Mice had severe difficulty discriminating citric acid from quinine and 6-n-propylthiouracil (PROP) with performance slightly, but significantly, above chance. In contrast, mice were able to competently discriminate sucrose from citric acid, NaCl, quinine, and PROP. In another experiment, mice that were conditioned to avoid quinine by pairings with LiCl injections subsequently suppressed licking responses to quinine and citric acid but not to NaCl or sucrose in a brief-access test, relative to NaCl-injected control animals. However, mice that were conditioned to avoid citric acid did not display cross-generalization to quinine. These mice significantly suppressed licking only to citric acid, and to a much lesser extent NaCl, compared with controls. Collectively, the findings from these experiments suggest that in mice, citric acid and quinine share chemosensory features making discrimination difficult but are not perceptually identical.

Little is known about how specific genes influence taste function in mammals. One of the most promising ways to fill this void is to screen the progeny of chemically mutagenized (or genetically altered) mice for aberrant taste phenotypes and then identify the mutated gene(s) that is associated with each taste anomaly. To exploit this approach, a high-throughput and robust screening procedure is needed. We have attempted to meet this demand by developing an automated procedure that assesses taste responsiveness of individual mice to palatable and unpalatable taste stimuli. We focused on three taste stimuli (quinine hydrochloride, QHCl; sodium chloride, NaCl; and sucrose) and one mouse strain (C57BL/6). We used a commercially available gustometer system that both monitors the licking responses of mice and controls the presentation of each taste stimulus during successive 5 s trials. We describe a screening procedure that (after 2 days of simple training) can generate a concentration-response curve for NaCl or sucrose during a single 30 min test session, and for QHCl over three 30 min test sessions. A normative database based on the responses of 98 mice subjected to our screening procedure is also presented. We envision that investigators could use this normative database to assess taste function in the progeny of mutagenized (or genetically altered) mice. Any mouse that deviates significantly-e.g. three standard deviations (SD)-from the mean of the normative database would be flagged as having a potentially interesting mutation. We also developed an additional second screen for identifying mice with oromotor abnormalities. This latter screen is necessary because oromotor problems could lead to false positives or negatives in the screen for taste function, but is also useful for researchers interested in genes influencing oromotor circuitry. Throughout the development of the screening protocol, we sought to balance two conflicting demands: the need to maximize the screen's sensitivity and minimize its duration. This screen represents a significant improvement over the common two-bottle preference test because it assesses taste function more specifically and in a fraction of the time.

Numerous animals use chemical cues within their environments to execute various behaviors. One of these behaviors is orientation to an odor source. Crayfish, in particular, can orient to food sources under a number of different conditions. It has not been determined, however, what kind of search strategy these animals employ to successfully locate a food source. To determine the role of antennae and antennules in this behavior and to investigate different modes of orientation behavior, the orientation patterns of crayfish with complete and partial antennal lesions were examined. Detailed analysis of orientation paths confirmed that crayfish could not locate odor sources with either bilateral or unilateral lesions. This suggests that crayfish are using the spatial information obtained from these appendages to successfully orient. Animals using information from the bilaterally paired appendages in the control group exhibited increased walking speed, increased speed to source and decreased heading angles towards the source compared to these measurements taken from lesioned groups. There was no significant difference in any parameters between animals with unilateral or bilateral lesions. This strongly suggests that these animals are reliant on the spatial comparison of differences between bilaterally paired olfactory appendages for successful orientation.

It is important to learn about changes in both taste and odor perceptions with increasing age, because the taste of foods we encounter in our daily life is strongly affected by their smell. This study discusses the difference in qualitative taste and odor discrimination between the elderly and the young. Tastants and odorants used in this study were presented not as single stimuli but as a taste mixture (sucrose and tartaric acid) and an odor mixture (β-phenylethyl alcohol and γ-undecalactone). The results showed that quality discrimination abilities of the elderly subjects for both taste and odor were significantly lower than those of the young subjects, indicating a decline in quality discrimination abilities related to age. Also, a moderate but significant correlation was observed between the taste discrimination ability and the odor discrimination ability. We measured thresholds for single-taste and odor components in mixtures and compared them between the elderly and the young to investigate the cause for these findings.

Drosophila larvae and adults exhibit a naturally occurring genetically based behavioural polymorphism in locomotor activity while foraging. Larvae of the rover morph exhibit longer foraging trails than sitters and forage between food patches, while sitters have shorter foraging trails and forage within patches. This behaviour is influenced by levels of cGMP-dependent protein kinase (PGK) encoded by the foraging (for) gene. Rover larvae have higher expression levels and higher PGK activities than do sitters. Here we discuss the importance of the for gene for studies of the mechanistic and evolutionary significance of individual differences in behaviour. We also show how structure-function analysis can be used to investigate a role for mushroom bodies in larval behaviour both in the presence and in the absence of food. Hydroxyurea fed to newly hatched larvae prevents the development of all post-embryonically derived mushroom body (MB) neuropil. This method was used to ablate MBs in rover and sitter genetic variants of foraging to test whether these structures mediate expression of the foraging behavioural polymorphism. We found that locomotor activity levels during foraging of both the rover and sitter larval morphs were not significantly influenced by MB ablation. Alternative hypotheses that may explain how variation in foraging behaviour is generated are discussed.

Chronic suppurative otitis media (CSOM), a severe form of middle ear infection, affects most Australian Aboriginal children with up to 50% in some communities suffering hearing loss as a consequence. To date, there is no information on whether repeated exposure to the pathogens that characterize CSOM and that are present in the upper respiratory airway affect olfactory function. Accordingly, this study aimed to determine whether 1) there was a high prevalence of olfactory loss in Aboriginal children and 2) hearing loss is a predictor of olfactory loss. Two hundred and sixty one 9- to 12-year-old Aboriginal children from 16 rural communities reported to have high prevalences of CSOM and hearing loss were assessed for olfactory loss using a 16-odor identification test and hearing loss. One child was found to be anosmic, 4 were slightly hyposmic, and 42 had hearing loss. No relationship was found between olfactory loss and hearing loss. The test-retest reliability of the 16-odor identification test was 0.98. It was concluded that CSOM does not appear to affect olfactory function in the long term and that hearing loss in Aboriginal children is not a predictor of olfactory loss.

Twenty-two experienced panelists rated odor intensity of aqueous solutions of citral, octen-1-ol-3, and hexanal. The panel assessed unmixed components and mixtures (9 binary and 4 ternary). In sensory sessions dedicated to mixtures (n = 6), evaluation was focused on one target odor, presented at a fixed concentration. All components had lower odor intensity on mixed presentations. In many cases, information obtained from simpler systems was not extended to complex mixtures. In a mixture, the competition between odorant molecules on qualitative aspects (dominance/suppression) imbalanced components contribution, anticipated from the quantitative distribution. Hexanal appeared to be the potentially weaker odorant in paired combinations, whereas octen-1-ol-3 had a lower relative impact on ternary systems. Suppression of the odor of octen-1-ol-3 and a concomitant increase in the odor of hexanal was common to all ternary mixtures. Reciprocal inhibition of octen-1-ol-3 and citral odors through perceptual interactions was suspected. Mutual suppression is suspected to have eased the perception of hexanal intensity.

Memory for sweet taste intensities in different media during 125 h was investigated using three concentrations (w/w) of sucrose: 4.21% (0.125 M), 8.28% (0.25 M) and 16.06% (0.5 M). Sucrose was dissolved in four media [water with no tastant and water with 0.73% (0.125 M) sodium chloride, 0.04% (0.002 M) citric acid and 0.04% (0.002 M) caffeinel as standard stimuli. Subjects (n = 39) were assigned into four groups, each group performing the memory task in one medium only. After tasting each standard the subjects reproduced the subjective taste intensity immediately and after 1 2 mm and 1, 5, 25 and 125 h by mixing portions of low (0%) and high (29.75%; 1 M) concentrations (w/w) of sucrose and by tasting and retasting (ad libitum procedure). The produced sucrose concentrations increased significantly from the first session to the 125 h time interval. There was a significant difference between immediately reproduced standard concentrations and concentrations produced after all time intervals. Relative differences from standard (δi/i) differed only between concentrations produced immediately and after 125 h time interval. The low (4.21%) concentration showed larger differences from standard than the high (16.06%) concentration. The added tastant had no effect on the results.