Spilanthol Enhances Sensitivity to Sodium in Mouse Taste Bud Cells.

Overconsumption of NaCl has been linked to increased hypertension-related morbidity. Compounds that can enhance NaCl responses in taste cells could help reduce human NaCl consumption without sacrificing perceived saltiness. Spilanthol is an unsaturated alkylamide isolated from the Jambu plant (Acmella oleracea) that can induce tingling, pungency, and numbing in the mouth. Structurally similar fatty acid amides, such as sanshool, elicit numbing and tingling sensations by inhibiting 2-pore-domain potassium leak channels on trigeminal sensory neurons. Even when insufficient to induce action potential firing, leak current inhibition causes depolarization and increased membrane resistance, which combine to make cells more sensitive to subsequent depolarizing stimuli, such as NaCl. Using calcium imaging, we tested whether spilanthol alters sensitivity to NaCl in isolated circumvallate taste bud cells and trigeminal sensory neurons of mice (Mus musculus). Micromolar spilanthol elicited little to no response in taste bud cells or trigeminal neurons. These same perithreshold concentrations of spilanthol significantly enhanced responses to NaCl (140 and 200 mM) in taste bud cells. Trigeminal neurons, however, exhibited response enhancement only at the highest concentrations of NaCl and spilanthol tested. Using a combination of potassium depolarization, immunohistochemistry, and Trpm5-GFP and Tas1r3-GFP mice to characterize taste bud cells by type, we found spilanthol enhancement of NaCl responses most prevalent in NaCl-responsive type III cells, and commonly observed in NaCl-responsive type II cells. Our results indicate that spilanthol enhances NaCl responses in taste bud cells and point to a family of compounds that may have utility as salty taste enhancers.

Neural basis of trigeminal chemo- and thermonociception in brown treesnakes, Boiga irregularis (Squamata: Colubridae).

To elucidate the nociceptive system of the brown treesnake, Boiga irregularis, we exposed isolated brown treesnake trigeminal neurons to thermal and chemical stimulation. We measured responses as changes in intracellular calcium using ratiometric fluorescent calcium imaging. Responses to aversive thermal and chemical identified several classes of putative nociceptors. Compounds that were aversive excited many trigeminal neurons, putative chemonociceptors. Identification as nociceptors was further supported by lack of activation by compounds that were not aversive. Brown treesnake neurons had thermal thresholds ranging from 32 to 49 °C. The distribution was discontinuous, with a population of thresholds from 32 to 45 °C and a population with thresholds > 48 °C. Thermal stimulation of 48 °C has been shown to be strongly aversive to brown treesnakes, is lethal, and suggests the presence of thermonociceptors. Thermal sensitivity of brown treesnake trigeminal neurons greatly overlaps with chemical sensitivity; only 1.1% of neurons were sensitive to only thermal stimulation. 50% of brown treesnake trigeminal neurons tested with both > 48 °C and cinnamaldehyde responded to both stimuli, identifying putative polymodal nociceptors. Although a previous study found brown treesnakes insensitive to capsicum extract containing capsaicin, brown treesnake trigeminal neurons responded to capsaicin. These findings are of evolutionary interest as well as providing potential insights into managing this significant pest species.

Fetal alcohol exposure reduces responsiveness of taste nerves and trigeminal chemosensory neurons to ethanol and its flavor components.

Fetal alcohol exposure (FAE) leads to increased intake of ethanol in adolescent rats and humans. We asked whether these behavioral changes may be mediated in part by changes in responsiveness of the peripheral taste and oral trigeminal systems. We exposed the experimental rats to ethanol in utero by administering ethanol to dams through a liquid diet; we exposed the control rats to an isocaloric and isonutritive liquid diet. To assess taste responsiveness, we recorded responses of the chorda tympani (CT) and glossopharyngeal (GL) nerves to lingual stimulation with ethanol, quinine, sucrose, and NaCl. To assess trigeminal responsiveness, we measured changes in calcium levels of isolated trigeminal ganglion (TG) neurons during stimulation with ethanol, capsaicin, mustard oil, and KCl. Compared with adolescent control rats, the adolescent experimental rats exhibited diminished CT nerve responses to ethanol, quinine, and sucrose and GL nerve responses to quinine and sucrose. The reductions in taste responsiveness persisted into adulthood for quinine but not for any of the other stimuli. Adolescent experimental rats also exhibited reduced TG neuron responses to ethanol, capsaicin, and mustard oil. The lack of change in responsiveness of the taste nerves to NaCl and the TG neurons to KCl indicates that FAE altered only a subset of the response pathways within each chemosensory system. We propose that FAE reprograms development of the peripheral taste and trigeminal systems in ways that reduce their responsiveness to ethanol and surrogates for its pleasant (i.e., sweet) and unpleasant (i.e., bitterness, oral burning) flavor attributes.NEW & NOTEWORTHY Pregnant mothers are advised to avoid alcohol. This is because even small amounts of alcohol can alter fetal brain development and increase the risk of adolescent alcohol abuse. We asked how fetal alcohol exposure (FAE) produces the latter effect in adolescent rats by measuring responsiveness of taste nerves and trigeminal chemosensory neurons. We found that FAE substantially reduced taste and trigeminal responsiveness to ethanol and its flavor components.

Gustatory, trigeminal, and olfactory aspects of nicotine intake in three mouse strains.

Studies of nicotine consumption in rodents often intend to investigate nicotine's post-absorptive effects, yet little is known about the pre-absorptive sensory experience of nicotine drinking, including gustatory, trigeminal, and olfactory influences. We conditioned taste aversion (CTA) to nicotine in males of 3 inbred mouse strains: C57BL/6J, DBA/2J, and 129X1/SvJ by repeatedly pairing 150 µg/ml nicotine drinking with lithium chloride injections. Generalization to a variety of bitter, sour, sweet, salty, and irritant solutions and to nicotine odor was then examined. Nicotine CTA generalized to the bitter stimulus quinine hydrochloride and the chemosensory irritant spilanthol in all strains. It also showed strain specificity, generalizing to hydrogen peroxide (an activator of TRPA1) in C57BL/6J mice and to the olfactory cue of nicotine in DBA/2J mice. These behavioral assays demonstrate that the sensory properties of nicotine are complex and include multiple gustatory, irritant, and olfactory components. How these qualities combine at the level of perception remains to be assessed, but sensory factors clearly exert an important influence on nicotine ingestion and their contribution to net intake of nicotine should not be neglected in animal or human studies.

Multiple cation channels mediate increases in intracellular calcium induced by the volatile irritant, trans-2-pentenal in rat trigeminal neurons.

Trans-2-Pentenal (pentenal), an alpha,beta-unsaturated aldehyde, induces increases in [Ca(2+)](i) in cultured neonatal rat trigeminal ganglion (TG) neurons. Since all pentenal-sensitive neurons responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC) and neurons from TRPA1 knockouts failed to respond to pentenal, TRPA1 appears to be sole initial transduction site for pentenal-evoked trigeminal response, as reported for the structurally related irritant, acrolein. Furthermore, because the neuronal sensitivity to pentenal is strictly dependent upon the presence of extracellular Na(+)/Ca(2+), as we showed previously, we investigated which types of voltage-gated sodium/calcium channels (VGSCs/VGCCs) are involved in pentenal-induced [Ca(2+)](i) increases as a downstream mechanisms. The application of tetrodotoxin (TTX) significantly suppressed the pentenal-induced increase in [Ca(2+)](i) in a portion of TG neurons, suggesting that TTX-sensitive (TTXs) VGSCs contribute to the pentenal response in those neurons. Diltiazem and omega-agatoxin IVA, antagonists of L- and P/Q-type VGCCs, respectively, both caused significant reductions of the pentenal-induced responses. omega-Conotoxin GVIA, on the other hand, caused only a small decrease in the size of pentenal-induced [Ca(2+)](i) rise. These indicate that both L- and P/Q-type VGCCs are involved in the increase in [Ca(2+)](i) produced by pentenal, while N-type calcium channels play only a minor role. This study demonstrates that TTXs VGSCs, L- and P/Q-type VGCCs play a significant role in the pentenal-induced trigeminal neuronal responses as downstream mechanisms following TRPA1 activation.

Multiple types of sensory neurons respond to irritating volatile organic compounds (VOCs): calcium fluorimetry of trigeminal ganglion neurons.

Many volatile organic compounds (VOCs) are significant environmental irritants that stimulate somatosensory nerve endings to produce pain and irritation. We measured intracellular calcium in cultured trigeminal ganglion neurons to characterize the cellular mechanisms and chemical structural determinants underlying sensitivity to VOCs. Trigeminal neurons responded to homologous series of alcohols (C4-C7) as well as saturated and unsaturated aldehydes in a concentration dependent manner. Ranked in terms of threshold to recruit neurons by compounds of the same carbon chain length, enaldehyde

Zanthoxylum piperitum (DC), a potential feeding deterrent for mammals: studies with Microtus ochrogaster (Wagner).

Total extract from the fruit of Szechuan pepper (Zanthoxylum piperitum DC), the volatile components of the extract and a non-volatile fraction containing alkylamides (NVA fraction) are feeding deterrents for rats. The present study investigated the effectiveness of these natural repellents in prairie voles (Microtus ochrogaster Wagner). Two-choice feeding trials were conducted during which food-deprived voles were offered choices between oat-bran wafers. In Experiment 1, 10 voles were given three sets of feeding trials, each 2 h long. Baseline consumption was established during the first set of two trials by offering a choice between two oat-bran wafers dipped in ethanol, the control solvent. During the second set of two trials the voles were given a choice between an oat-bran wafer dipped in ethanol and a wafer dipped in Zanthoxylum extract. During the third set the voles were given a choice between a wafer served on top of a screened dish containing a sample of ethanol and a wafer served on top of a dish containing a sample of extract. In this manner the voles were exposed to volatile compounds emanating from the extract but could not contact it. Wafers dipped in extract were almost completely avoided. The volatile components of extract also significantly reduced food intake. In Experiment 2, habituation to the volatile constituents of extract was examined in 16 Zanthoxylum-naïve voles. Baseline consumption was established by offering two wafers served on top of screened dishes containing ethanol. This was followed by twelve tests during which a choice between a wafer served above a sample of ethanol and a wafer served above a sample of extract was given. The voles failed to habituate to the volatile components of extract, consistently consuming less of the wafers served above extract. In Experiment 3 a dose-response curve to Zanthoxylum extract was established, using 12 stimulus-naive voles. After baseline consumption was established, the animals were given two tests each, presenting a choice between a control wafer and a wafer dipped in a dilution of extract (0.001-100 g liter(-1)). Only concentrations of 10 and 100 g liter(-1) reduced food intake. In Experiment 4 the effects of the non-volatile fraction of extract were compared to those of whole extract. Vegetable oil was used as solvent. Eight stimulus-naïve voles were given two tests with a choice between an oil-dipped and an extract-dipped wafer. A second group of eight voles received two tests with a choice between an oil-dipped and NVA-dipped wafer. Extract-dipped wafers were avoided, but the NVA fraction had no effect on food consumption.

Age related decreases in neural sensitivity to NaCl in SHR-SP.

To determine whether neurophysiological taste responses of young and old rats are different, recordings were made from the whole chorda tympani nerve which innervates taste buds on the anterior tongue. SHR-SP (Stroke-Prone Spontaneously Hypertensive Rats) in two age groups were studied. Chemical stimuli included single concentrations of 250 mM NH(4)Cl, 100 mM NaCl, 100 mM KCl, 500 mM sucrose, 20 mM quinine-hydrochloride, 10 mM HCl, 10 mM monosodium glutamate (MSG), 10 mM L- glutamic acid (L-Glu) and an NaCl concentration series. The magnitude of the neural response (response ratio) was calculated by dividing the amplitude of the integrated response by the amplitude of the spontaneous activity that preceded it. Substantial neural responses to all chemicals were obtained at both ages. The responses to KCl, sucrose, quinine-hydrochloride, HCl, monosodium glutamate (MSG) and glutamic acid (Glu) did not change with age, but the response to NaCl did decrease significantly. The profile of the response/concentration function for NaCl differed with age. In particular, the responses to solutions more concentrated than 100 mM NaCl were significantly weaker in aged than in young SHR-SPs. We also observed that recovery from amiloride treatment on the tongue of SHR-SPs was faster in aged rats than in young ones, suggesting that there is some functional difference in the sodium-specific channels on the taste cell. These results suggest that aged SHR-SP may be less able than young SHR-SPs to discriminate among higher concentrations of NaCl solutions.

Calcium responses of chicken trigeminal ganglion neurons to methyl anthranilate and capsaicin.

Using digital fluorescence imaging, we determined the effects of methyl anthranilate (MA), an avian irritant, and capsaicin (CAP), a mammalian irritant, on intracellular calcium ([Ca(2+)](i)) in chicken trigeminal neurons. Concentration-response functions indicated that the threshold for inducing increases in [Ca(2+)](i) was higher for CAP (30 micromol l(-1)) than for MA (10 micromol l(-1)). The maximum magnitudes of [Ca(2+)](i) in response to MA and CAP were compared after normalization to 40 mmol l(-1) KCl. At equal concentrations (300 micro mol l(-1)), trigeminal neurons responded with a greater change in [Ca(2+)](i) to MA (78% of KCl) than to CAP (43% of KCl). Furthermore, at 300 micromol l(-1), 48% of neurons responded to MA whereas only 16% responded to CAP. The increases in [Ca(2+)](i) induced by both MA and CAP were dependent upon extracellular calcium. While the calcium responses to MA were also dependent on extracellular sodium, responses to CAP were not. There were separate but overlapping populations of neurons sensitive to MA and CAP. Taken together, the higher threshold concentration of CAP, the higher response magnitude to MA than CAP and the greater number of neurons sensitive to MA than CAP provide a rationale for the observed behavioral differences of birds to these two compounds. Finally, the findings that the calcium responses to MA and CAP have different ion dependencies and that there are separate populations sensitive to these compounds suggest different transduction mechanisms mediating chicken trigeminal responses to MA and CAP.