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Over 21,000 future California residentialwildfire risk scenarios were developed on amonthly 1/8° grid, using statisticalwildfire models.We explore interactions between two global emissions scenarios, three climate models, six spatially explicit population growth scenarios derived from two growth models, and a range of parameters defining properties' vulnerability to loss. Scenarios are evaluated over two future time periods relative to historic baselines.We also explore effects of spatial resolutions for calculating household exposure to wildfire on changes in estimated future property losses. Our goal was not to produce one authoritative set of future risk scenarios but rather to understand what parameters are important for robustly characterizing effects of climate and growth on future residential property risks. By end of century, variation across development scenarios accounts for far more variability in statewide residential wildfire risks than does variation across climate scenarios. However, the most extreme increases in residential fire risks result from combining high-growth/high-sprawl scenarios with the most extreme climates considered here. Case studies for the Bay Area and the Sierra foothills demonstrate that, while land use decisions profoundly influence future residential wildfire risks, effects of diverse growth and land use strategies vary greatly around the state.

Harvesting floodwaters to recharge depleted groundwater aquifers can simultaneously reduce flood and drought risks and enhance groundwater sustainability. However, deployment of this multibeneficial adaptation option is fundamentally constrained by how much water is available for recharge (WAFR) at present and under future climate change. Here, we develop a climate-informed and policy-relevant framework to quantify WAFR, its uncertainty, and associated policy actions. Despite robust and widespread increases in future projected WAFR in our case study of California (for 56/80% of subbasins in 2070-2099 under RCP4.5/RCP8.5), strong nonlinear interactions between diversion infrastructure and policy uncertainties constrain how much WAFR can be captured. To tap future elevated recharge potential through infrastructure expansion under deep uncertainties, we outline a novel robustness-based policy typology to identify priority areas of investment needs. Our WAFR analysis can inform effective investment decisions to adapt to future climate-fueled drought and flood risk over depleted aquifers, in California and beyond., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The magnitude and pace of global change demand rapid assessment of nature and its contributions to people. We present a fine-scale global modeling of current status and future scenarios for several contributions: water quality regulation, coastal risk reduction, and crop pollination. We find that where people's needs for nature are now greatest, nature's ability to meet those needs is declining. Up to 5 billion people face higher water pollution and insufficient pollination for nutrition under future scenarios of land use and climate change, particularly in Africa and South Asia. Hundreds of millions of people face heightened coastal risk across Africa, Eurasia, and the Americas. Continued loss of nature poses severe threats, yet these can be reduced 3- to 10-fold under a sustainable development scenario., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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 (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., (Copyright © 2017 the American Physiological Society.)

We analyze the impacts of investments in sustainable land use practices on ecosystem services in the Upper Tana basin, Kenya. This work supports implementation of the Upper Tana-Nairobi Water Fund, a public-private partnership to safeguard ecosystem service provision and food security. We apply an integrated modelling framework, building on local knowledge and previous field- and model-based studies, to link biophysical landscape changes at high temporal and spatial resolution to economic benefits for key actors in the basin. The primary contribution of this study is that it a) presents a comprehensive analysis for targeting interventions that takes into account stakeholder preferences, local environmental and socio-economic conditions, b) relies on detailed, process-based, biophysical models to demonstrate the biophysical return on those investments for a practical, decision-driven case, and c) in close collaboration with downstream water users, links those biophysical outputs to monetary metrics, including: reduced water treatment costs, increased hydropower production, and crop yield benefits for agricultural producers in the conservation area. This study highlights the benefits and trade-offs that come with conducting participatory research as part of a stakeholder engagement process: while results are more likely to be decision-relevant within the local context, navigating stakeholder expectations and data limitations present ongoing challenges., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Changing climatic conditions are influencing large wildfire frequency, a globally widespread disturbance that affects both human and natural systems. Understanding how climate change, population growth, and development patterns will affect the area burned by and emissions from wildfires and how populations will in turn be exposed to emissions is critical for climate change adaptation and mitigation planning. We quantified the effects of a range of population growth and development patterns in California on emission projections from large wildfires under six future climate scenarios. Here we show that end-of-century wildfire emissions are projected to increase by 19-101% (median increase 56%) above the baseline period (1961-1990) in California for a medium-high temperature scenario, with the largest emissions increases concentrated in northern California. In contrast to other measures of wildfire impacts previously studied (e.g., structural loss), projected population growth and development patterns are unlikely to substantially influence the amount of projected statewide wildfire emissions. However, increases in wildfire emissions due to climate change may have detrimental impacts on air quality and, combined with a growing population, may result in increased population exposure to unhealthy air pollutants.

The purpose of the study was to describe the range of relative backpack weights of one group of elementary-aged children and the extent to which they exceeded recommended levels. A second purpose was to explore whether gender and age help predict the relative weight of children's backpacks. Ninety-five 8- to 12-year-old elementary school students (56% girls; 88% car or bus riders) participated. Their school backpacks were weighed, and their age, gender, and mode of transportation to school were recorded. Only 40% of the sample carried backpacks that were less than 10% of their body weights. Five percent of the students' backpacks exceeded 20% of their body weights. Neither age group nor gender significantly predicted relative backpack weight or relative weight levels. Recommendations are made for ways to reduce the weight these young children carry.

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.

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.

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

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.

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.

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.

Unlabelled: We investigated the effects of a total extract from Zanthoxylum piperitum fruit, of the volatile components of extract, and of a non-volatile fraction containing the major alkylamides of Zanthoxylum (NVA) on food intake in rats. In Experiment 1, three groups (A, B, C) of mildly food deprived rats were given one hour long feeding trials during which they were offered choices between an oat-bran wafer paired with vegetable oil and a wafer paired with a Zanthoxylum preparation in vegetable oil. Trials were divided into three blocks of two tests each. During Blocks 1 and 3, two wafers coated with vegetable oil were offered to establish baseline consumption. During Block 2, Group A was given choices between wafers coated with oil and wafers coated with total extract. Group B was offered oil coated wafers and wafers coated with the NVA fraction. Group C received two oil coated wafers. One was served on top of a screened dish containing a sample of total extract. In this manner, the rats were exposed to volatile compounds emanating from the extract but could not contact the extract. The second wafer was served on top of a screened sample of oil., Results: In Blocks 1 and 3 trials, the rats fed indiscriminately from both wafers. During Block 2, total extract, the NVA fraction and the volatile compounds from extract all significantly reduced food consumption. In Experiment 2, habituation to the NVA fraction and to the volatile constituents of extract was examined in two additional groups (D, E), using the methods described above. Baseline consumption was tested in Blocks 1 and 3 by offering two oil coated wafers. During Block 2, group D was given 10 trials with oil coated and NVA-fraction coated wafers. Group E was given 10 trials with oil coated wafers paired with the volatile constituents of total extract and with those of oil., Results: Both groups failed to habituate to the Zanthoxylum stimuli. Wafers treated with the NVA fraction and wafers paired with the volatile constituents of extract were avoided throughout the 10 test days.

We investigated the effects of zinc deficiency on carbonic anhydrase (CA) activity in the tongue epithelium and submandibular gland in rats. Male 4-week-old SD rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low-zinc), or 33.7 (zinc-sufficient) mg zinc/kg diet for 6 weeks. Rats in the fourth group (receiving 33.7 mg zinc/kg) were pair-fed against the zinc-deficient rats. Biochemical analysis at the end of the experimental period indicated that zinc deficiency significantly reduced CA activity in the tongue epithelium and submandibular gland, and the CA activity levels in these tissues seemed to parallel the dietary zinc levels. By enzyme histochemistry, an intensely positive reaction for CA was observed in the middle and basal regions of the taste buds in the circumvallate papilla in the zinc-sufficient and pair-fed (control) rats. The cells in von Ebner's glands also showed a strong positive reaction in control rats, although only a weak reaction product was found in zinc-deficient rats. These results suggest that CA activity is affected by the dietary content of zinc, which is considered to be an indispensable factor for the maintenance of normal taste sensation.

Alkylamides isolated from the fruit of Xanthoxylum, Szechuan pepper, produce a strong tingling sensation in the mouth. In order to determine the peripheral basis of this sensation, extracellular nerve recordings were obtained from the lingual nerve of rats. The primary pungent compound, hydroxy-alpha-sanshool (HO-alpha-S), altered the levels of spontaneous activity in cool-sensitive fibers as well as inducing activity in tactile fibers, cold nociceptors and silent fibers that were insensitive to innocuous thermal or tactile stimuli. Moreover, tactile or thermal sensitivity was induced in fibers that were initially insensitive to touch or cooling. The neuronal distribution of sensitivities to capsaicin and to HO-alpha-S indicate that this compound affects neurons mediating innocuous sensations. HO-alpha-S may be useful as a model stimulus for studies of paresthesia.

Single-fiber preparations of the rat chorda tympani (CT) nerve were used to study the mechanism of action of capsaicin on salt-taste transduction. Capsaicin selectively suppressed the responses to NaCl of the CT nerve fibers (N-fibers) that are sodium-specific (insensitive or poorly sensitive to potassium). Among the more broadly responsive, cation-sensitive fibers (E-fibers) there are two subtypes, both of which responded to capsaicin but in different ways ('enhanced' type and 'suppressed' type). In both N- and E-fibers, 5% ethanol (the vehicle for capsaicin) slightly reduced the response to 100 mM NaCl. The suppressive effect of capsaicin on the response of the N-type fibers to 100 mM NaCl was significantly stronger than the effect of 5% ethanol. The suppression lasted for at least 20 s after the simultaneous application of 100 p.p.m. capsaicin-100 nM NaCl. These results indicate that 100 p.p.m. capsaicin can modify the response of CT fibers to NaCl. The observed effect of capsaicin on gustatory fibers could be the net result of opposite suppressive and enhancing processes in the taste buds cells and excited intra- or extragemmal trigeminal nerve endings.

Repellent chemicals are presumed to activate trigeminal neurons, including polymodal nociceptors, but few data are available that bear on this notion. In the present experiment, we assessed multi-unit and single-unit responses of neurons in the rat lingual trigeminal nerve to 13 candidate repellents and a thermal stimulus. All of the chemicals evoked trigeminal responses, and neural activity was predictable from available behavioral data. These results are consistent with the view that repellents are irritants. The results also suggest that electrophysiological methods may represent a useful method for screening candidate repellent compounds.

The irritating, pungent compound, capsaicin (10-20 microM), induces the formation of non-selective ion channels with a wide variety of conductances in protein-free lipid bilayers form from a mixture of zwitterionic phospholipids. The channel-forming activity of capsaicin and four of its analogs followed the sequence: resiniferatoxin > capsaicin = pelargonic acid vanillylamide > methylcapsaicin >> veratrylamine. The ability to form channels correlated with the biological activity of these compounds observed in other studies that measured 45Ca uptake into rat dorsal root ganglion cells. The correlation obtained suggests that an interaction with the lipid bilayer may be an important component of the biological activity of capsaicin.

A model of analogue-receptor binding is developed for the L-alanine receptor in the channel catfish using the AM1-SM2 and ab initio SCRF computational methods. Besides interactions involving the zwitterionic moiety of the amino acid analogue and complementary subsites on the receptor, the model suggests the presence of a hydrophobic pocket with dispersion interactions between the receptor and the residue on the amino acid analogue. Conformational analysis suggests not only a small compact active site on the receptor, but also that the analogues with the highest affinity occupy nearly identical regions of space. Although the binding interaction is dominated by the ionic terms, AM1-SM2 calculations indicate that free energy terms associated with cavity formation, solvent reorganization, and dispersion interactions can be correlated to activation and neural response. From a consideration of this model, molecular features of the analogues that are important for binding and neural response were deduced and other analogues or ligands were developed and tested.

Oral sensitivity to acids mediates a range of irritation, from desirable sensations of pungency in various foods and beverages to the pain associated with harmful levels of acidity and inflammation. To characterize the role of the epithelial and vascular processes in neural sensitivity to acid applied to the lingual mucosa, we measured lingual trigeminal nerve responses in rats to acidic stimuli of varying physicochemical properties. Stimulatory efficacy was strongly correlated with lipophilicity (octanol-H2O partition coefficient) for the series of straight-chain fatty acids from one to six carbons. Neither acid dissociation constant nor the pH of stimulus solutions correlated with stimulatory efficacy for the fatty acids and six other organic acids. The hypothesis that weakly acidic stimuli gain access to trigeminal nerve endings primarily through the lipid phase of the epithelium was supported by these findings. Further support for this hypothesis is the result that LaCl3, an inhibitor of epithelial tight junctions, had no effect on the neural response to n-pentanoic acid. The potential relevance of buffering-clearance in modulating responses to acid was indicated by the finding that the application of 1 mM capsaicin to the tongue reversibly inhibited the neural response to acid, as well as causing plasma extravasation in lingual epithelium.

Single fibers of the rat chorda tympani nerve were used to study the mechanism of action of the antibiotic novobiocin on salt taste transduction. In the rat, novobiocin selectively enhanced the responses of sodium-specific and amiloride-sensitive chorda tympani nerve fibers (N type) without affecting more broadly responsive cation-sensitive and amiloride-insensitive fibers (E type). In the presence of amiloride, novobiocin was ineffective at enhancing the response of N-type fibers toward sodium chloride. Novobiocin also increased the conductance of bilayers formed from neutral lipids by forming nonrectifying ion channels with low conductance (approximately 7 pS in 110 mM NaCl), long open times (several seconds and longer), and high cation selectivity. Amiloride did not alter either the conductance or kinetics of these novobiocin channels. These observations suggest that even though novobiocin is able to form cation channels in lipid bilayers, and possibly in cell membranes as well, its action on the salt-taste response is through modulation of existing amiloride-sensitive sodium channels.

The goal of this study was to examine the role of the enzyme, carbonic anhydrase, in oral trigeminal chemoreception with particular regard to the reception of CO2. Using both single and multiunit recordings of trigeminal neurons in the lingual nerve of rat, we measured responses to cool (24 degrees C), noxiously hot (55 degrees C) and cold (8 degrees C) H2O, NH4Cl and supersaturated solutions of CO2 (24 degrees C and 33 degrees C). The importance of peripheral carbonic anhydrase was tested by inhibiting enzyme activity with acetazolamide (15 mg/kg b.w.). Single unit responses to CO2 and HCl suggest that neural sensitivity to CO2 is not simply a function of extraepithelial pH. Responses to CO2 were significantly inhibited by acetazolamide while the responses to thermal stimuli and NH4Cl were not. The results support a role for carbonic anhydrase in trigeminal responses to CO2. Furthermore, the results suggest that intraepithelial acidification mediated by carbonic anhydrase may be the basis for sensitivity to CO2.

L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[(3)H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,β-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC50s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of theα-carboxylic acid and theα-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[(3)H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.

In order to understand the molecular determinants of amino acid taste receptor binding and activation, structure/activity studies were performed using analogs of L-alanine in a competitive ligand binding assay and a taste neurophysiological preparation. The presence of both the amine and carboxylic acid in a charged and unhindered form is a primary requisite for a ligand to both bind and activate L-alanine receptors. Although a number of carboxylic acid derivatives are moderately good stimuli, their neural activity derives from action at receptors different from L-alanine receptors. Of the molecular parameters examined, chirality and molecular volume of the side chain are the most important factors in determining the binding and stimulatory efficacy of L-amino acid taste stimuli. Electronegativity of the side chain did not correlate with receptor site binding. Heterologous ligand-induced enhancement of the binding of L-[3H]alanine by a purified taste membrane preparation is described. Neurophysiological experiments support the hypothesis that this phenomenon may be a basis of peripheral sensory interactions.

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.

1. Taste receptors for L-alanine and L-arginine in the channel catfish, Ictalurus punctatus, are differentially reactive to N-ethylmaleimide (NEM) and p-chloromercuribenzenesulphonic acid (pCMBS). 2. The binding of L-[3H]alanine by a sedimentable membrane fraction (Fraction P2) isolated from taste epithelium was inhibited by both NEM and pCMBS while the binding of L[3H]arginine was unaffected. 3. Inhibition of the binding of L-[3H]alanine by pCMBS was reversible with dithiothreitol (DTT). 4. NEM (10(-3) M) inhibited multi-unit neural responses to both 10(-4) M L-alanine and 10(-4) M L-arginine, while pCMBS had little effect on neural responses. 5. Pretreatment of intact taste epithelium before the preparation of Fraction P2 with NEM caused strong inhibition of L-[3H]alanine binding, while pretreatment with pCMBS caused weak inhibition. 6. The presence of L-alanine during the reaction of pCMBS or NEM with taste plasma membranes did not substantially protect against the inhibition of L-[3H]alanine binding.

Diet manipulation, an habituation test, and chemical analysis of urinary free amino acids were used to demonstrate that bullhead catfish (Ictalurus nebulosus) naturally detect the body odors of conspecifics and respond to them in a predictable fashion. These signals are used in dominance and territorial relationships and lead to increased aggression toward chemical "strangers." The results support the general notion that nonspecific metabolites, as well as specific pheromones, are important in chemical mediation of social behavior.

Specific binding of amino acid taste stimuli is known to occur to a sedimentable fraction (P2) from catfish (Ictalurus punctatus) taste epithelium or to purified plasma membranes from that fraction. L-Alanine, a potent taste stimulus for the catfish, binds in a reversible and saturable manner to these preparations. The extent to which the enantiomeric stimuli, L- and D-alanine, interact with the same or different receptor/transduction processes is investigated here both electrophysiologically and biochemically. With an electrophysiological assay, L-alanine was the more potent stimulus across a concentration range of 10(-9)-10(-3) M, yet both enantiomers displayed approximately the same threshold. The concentration-electrophysiological response functions for each enantiomer were different. That of L-alanine was approximately linear across the (log) concentration range while that of D-alanine was non-linear, with small but definitely observable responses being noted from 10(-9)-10(-5) M D-alanine, and larger incremental responses thereafter. With most of the nerve bundle preparations studies, L- and D-alanine cross-adapted one another, but this cross-adaptation was not always complete. Experiments in which both L- and D-alanine were present in a 1:1 mixture of equally stimulatory concentrations suggested the existence of receptor or transduction processes unique to each enantiomer. Biochemically binding studies demonstrated high affinity binding sites for both enantiomers with values of Kd-app for L-alanine of 1.5 microM and for D-alanine of 25 microM. For both enantiomers, additional lower-affinity binding sites were observable. The capacity of the lower-affinity sites was particularly great for D-alanine. The enantiomers competed one with the other for binding, with L-alanine showing greater competitive ability than D-alanine at low concentrations. For the high affinity sites, double-reciprocal plots of the data suggested a competitive mechanism. The lower affinity sites for D-alanine were less accessible to L-alanine compared with the high affinity sites of D-alanine. Both the biochemical and electrophysiological results indicate that while a portion of the responses to L- and D-alanine occurs through a common receptor/transduction process, there exist independent receptor/transduction processes for the enantiomers, L- and D-alanine.

We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.