Selective TAAR1 agonists induce conditioned taste aversion.

RATIONALE: Trace amine-associated receptor 1 (TAAR1) is the best-studied receptor of trace amines, a group of biogenic amines expressed at a relatively low level in the mammalian brain. Growing evidence suggests that TAAR1 plays a critical role in various neuropsychiatric disorders. Given that selective TAAR1 agonists were shown to produce pro-cognition and antipsychotic-like effects as well as to suppress drug use and relapse, they have been proposed to be novel treatments for mental disorders such as schizophrenia and addiction. However, the aversive effects of selective TAAR1 agonists remain largely unknown. OBJECTIVES: Here, we evaluated whether the selective TAAR1 full agonist RO5166017 and partial agonist RO5263397 could induce conditioned taste aversion (CTA). RESULTS: We found that RO5166017 and RO5263397 produced significant aversions to both saccharin and NaCl taste novelty. Furthermore, RO5166017 produced CTA to saccharin in TAAR1 heterozygous knockout (taar1±) and wild-type rats but not in TAAR1 homozygous knockout rats (taar1-/-), suggesting that TAAR1 was sufficient for the taste aversive stimulus property of RO5166017. CONCLUSIONS: Taken together, our data indicate that selective TAAR1 agonists could produce strong CTA. Our study urges careful evaluations of the aversive effects of TAAR1 agonists before translating them to clinical use for the treatment of mental disorders.

Disrupted population coding in the prefrontal cortex underlies pain aversion.

The prefrontal cortex (PFC) regulates a wide range of sensory experiences. Chronic pain is known to impair normal neural response, leading to enhanced aversion. However, it remains unknown how nociceptive responses in the cortex are processed at the population level and whether such processes are disrupted by chronic pain. Using in vivo endoscopic calcium imaging, we identify increased population activity in response to noxious stimuli and stable patterns of functional connectivity among neurons in the prelimbic (PL) PFC from freely behaving rats. Inflammatory pain disrupts functional connectivity of PFC neurons and reduces the overall nociceptive response. Interestingly, ketamine, a well-known neuromodulator, restores the functional connectivity among PL-PFC neurons in the inflammatory pain model to produce anti-aversive effects. These results suggest a dynamic resource allocation mechanism in the prefrontal representations of pain and indicate that population activity in the PFC critically regulates pain and serves as an important therapeutic target.

Effects of Bitter Substances on GI Function, Energy Intake and Glycaemia-Do Preclinical Findings Translate to Outcomes in Humans?

Bitter substances are contained in many plants, are often toxic and can be present in spoiled food. Thus, the capacity to detect bitter taste has classically been viewed to have evolved primarily to signal the presence of toxins and thereby avoid their consumption. The recognition, based on preclinical studies (i.e., studies in cell cultures or experimental animals), that bitter substances may have potent effects to stimulate the secretion of gastrointestinal (GI) hormones and modulate gut motility, via activation of bitter taste receptors located in the GI tract, reduce food intake and lower postprandial blood glucose, has sparked considerable interest in their potential use in the management or prevention of obesity and/or type 2 diabetes. However, it remains to be established whether findings from preclinical studies can be translated to health outcomes, including weight loss and improved long-term glycaemic control. This review examines information relating to the effects of bitter substances on the secretion of key gut hormones, gastric motility, food intake and blood glucose in preclinical studies, as well as the evidence from clinical studies, as to whether findings from animal studies translate to humans. Finally, the evidence that bitter substances have the capacity to reduce body weight and/or improve glycaemic control in obesity and/or type 2 diabetes, and potentially represent a novel strategy for the management, or prevention, of obesity and type 2 diabetes, is explored.

Bitter taste in silico: A review on virtual ligand screening and characterization methods for TAS2R-bitterant interactions.

The growing pharmaceutical interest in the human bitter taste receptors (hTAS2Rs) has two dimensions; i) evaluation of the bitterness of active pharmaceutical compounds, in order to develop strategies for improving patients' adherence to medication, and ii) application of ligands for extra-cellular hTAS2Rs for potential preventive therapeutic achievements. The result is an increasing demand on robust tools for bitterness assessment and screening the receptor-ligand affinity. In silico tools are useful for aiding experimental-screening, as well as to elucide ligand-receptor interactions. In this review, the ligand-based and structure-based approaches are described as the two main in silico tools for bitter taste analysis. The strengths and weaknesses of each approach are discussed. Both approaches provide key tools for understanding and exploiting bitter taste for human health applications.

Deciphering molecular mechanics in the taste masking ability of Maltodextrin: Developing pediatric formulation of Oseltamivir for viral pandemia.

Present research work was aimed at masking the bitter taste of anti- viral drug Oseltamivir phosphate (Ost) by complexing it with pea starch maltodextrin- Kleptose Linecaps® (Mld). The Ost groups involved in triggering the bitter sensation were identified by computationally assessing its interaction with human bitter taste receptor hTAS2R 38. A series of exhaustive molecular dynamics (MD) simulation was run using Schrodinger® suite to understand the type of interaction of Ost with Mld. Experimentally, complexes of Ost with Mld were realized by solution method. The complexes were characterized using differential scanning colorimetry (DSC), fourier transform-infrared spectroscopy (FT-IR), powder X-ray diffraction (PXRD), hot stage microscopy (HSM), scanning electron microscopy (SEM), proton NMR (1H-NMR) and Carbon-13 nuclear magnetic resonance (13C-NMR). Ost-oral dispersible mini tablets (ODMT) were prepared by direct compression and optimised using mixture designs. Finally, bitter taste perception of Ost-ODMT was evaluated in healthy human volunteers of either sex. Computational assessment, involving interaction of Ost with bitter receptor, predicted the involvement of free amino group of Ost in triggering the bitter response whereas, MD simulation predicted the formation of stable complex between Ost and double helical confirmation of Mld. Different characterization techniques confirmed the findings of MD simulation. Results from the taste assessment in human volunteers revealed a significant reduction in bitter taste of prepared Ost-ODMT.

The bitter tastant denatonium benzoate has no influence on the number of transient lower esophageal sphincter relaxations in health.

BACKGROUND: Administration of a bitter compound can alter the intragastric pressure (IGP) after a meal. Additionally, a negative correlation between IGP and the number of transient lower esophageal sphincter relaxations (TLESRs) has been demonstrated. However, the effect of a bitter tastant on the number of TLESRs and subsequent reflux episodes has never been investigated and it is unclear whether bitter food items should be avoided in gastro-esophageal reflux disease. We hypothesize that bitter administration in healthy volunteers (HVs) will lead to an increase in the number of TLESRs. METHODS: After an overnight fast, 20 female HVs (36 years [21-63]) underwent a high-resolution impedance manometry (HRiM) measurement. After placement of the HRiM probe, 0.1 ml/kg of a 10 mM denatonium benzoate solution (bitter) or an identical volume of water (placebo) was administered directly into the stomach. The number of TLESRs and reflux episodes was quantified 30 min before and 2 h after consumption of a high caloric meal. KEY RESULTS: There was no significant difference in the number of TLESRs or reflux episodes between the bitter and placebo condition. Additionally, no differences were observed in the nature (gas or liquid) and extent of reflux events. Lower esophageal sphincter pressures dropped significantly in the first postprandial hour to start recovering slowly back to baseline values during the second postprandial hour (p < 0.0001), without any difference between both conditions. CONCLUSIONS & INTERFERENCES: Administration of the bitter tastant denatonium benzoate has no influence on the number of TLESRs or reflux episodes.

Bitter taste receptor agonists regulate epithelial two-pore potassium channels via cAMP signaling.

BACKGROUND: Epithelial solitary chemosensory cell (tuft cell) bitter taste signal transduction occurs through G protein coupled receptors and calcium-dependent signaling pathways. Type II taste cells, which utilize the same bitter taste signal transduction pathways, may also utilize cyclic adenosine monophosphate (cAMP) as an independent signaling messenger in addition to calcium. METHODS: In this work we utilized specific pharmacologic inhibitors to interrogate the short circuit current (Isc) of polarized nasal epithelial cells mounted in Ussing chambers to assess the electrophysiologic changes associated with bitter agonist (denatonium) treatment. We also assessed release of human ß-defensin-2 from polarized nasal epithelial cultures following treatment with denatonium benzoate and/or potassium channel inhibitors. RESULTS: We demonstrate that the bitter taste receptor agonist, denatonium, decreases human respiratory epithelial two-pore potassium (K2P) current in polarized nasal epithelial cells mounted in Ussing chambers. Our data further suggest that this occurs via a cAMP-dependent signaling pathway. We also demonstrate that this decrease in potassium current lowers the threshold for denatonium to stimulate human ß-defensin-2 release. CONCLUSIONS: These data thus demonstrate that, in addition to taste transducing calcium-dependent signaling, bitter taste receptor agonists can also activate cAMP-dependent respiratory epithelial signaling pathways to modulate K2P currents. Bitter-agonist regulation of potassium currents may therefore serve as a means of rapid regional epithelial signaling, and further study of these pathways may provide new insights into regulation of mucosal ionic composition and innate mechanisms of epithelial defense.

Predominant Qualities Evoked by Quinine, Sucrose, and Capsaicin Associate With PROP Bitterness, but not TAS2R38 Genotype.

Genetic variability in the ability to taste thiourea compounds has been studied for 80+ years. Over the last 3 decades, many studies have reported perceived intensity of concentrated propylthiouracil (PROP) associates with greater intensity from a broad range of stimuli, including nonbitter tastants, irritants, and retronasally delivered odorants. Thus, PROP phenotype has become a common measure of individual differences in orosensation. Much, but not all, of the phenotypic variation in PROP bitterness is explained by TAS2R38 polymorphisms. While differences in PROP bitterness are clearly due to genetic variation, mechanistically it is challenging to envision how this receptor (narrowly tuned to the N-C=S moiety) relates to overall orosensory response. Here, we report data for 200+ individuals who had been genotyped for TAS2R38 and phenotyped for PROP in a laboratory setting. Participants also reported the intensity of quinine, capsaicin, and sucrose on a general Labeled Magnitude Scale. Our data recapitulate earlier reports associating PROP bitterness with the intensity of the predominant qualities of sucrose, quinine, and capsaicin; however, we also find correlations between the intensities of sucrose, quinine, and capsaicin were much stronger with each other than with PROP. As expected, TAS2R38 diplotype did not associate with the intensity of sucrose, quinine, or capsaicin. The strength of PROP-capsaicin and PROP-sucrose relationships increased after grouping participants by TAS2R38 diplotype, with the greatest increases in association observed within homozygotes. Collectively, this suggests the suprathreshold intensity of PROP is a confounded phenotype that captures both genetic variation specific to N-C=S compounds and overall orosensation.

Conditioned taste avoidance induced by the combination of heroin and cocaine: Implications for the use of speedball.

Speedball (heroin + cocaine) is a prevalent drug combination among intravenous drug users. Although its use is generally discussed to be a function of changes in the rewarding effects of either or both drugs, changes in the aversive effects of either drug may also be impacted (weakened) by the combination. To address this latter possibility and its potential role in the use of speedball, the present studies examined the interaction of cocaine and heroin in taste avoidance conditioning. In Experiment 1, male Sprague-Dawley rats were given access to a novel saccharin solution and then injected with either vehicle or heroin (3.2 mg/kg, IP) followed immediately by various doses of cocaine (10, 18 or 32 mg/kg, SC). At the two lowest doses of cocaine, only animals injected with the drug combination (H + C) displayed a taste avoidance relative to control subjects (taste avoidance was induced with both the combination and the high dose of cocaine). At no dose did animals injected with the combination of heroin and cocaine drink more than animals injected with cocaine alone. In Experiment 2, male Sprague-Dawley rats were similarly treated but injected with vehicle or cocaine (10 mg/kg) followed by injections of various doses of heroin (1.8, 3.2, 5.6 or 10 mg/kg). At the three highest doses of heroin, only animals injected with the drug combination (C + H) displayed significant avoidance relative to control subjects (no avoidance was evident with the combination of cocaine and the low dose of heroin). At no dose did animals injected with the combination of cocaine and heroin drink more than animals injected with heroin alone. Together, these results suggest that the aversive effects of heroin and cocaine are not attenuated by co-administration by cocaine and heroin, respectively. The importance of this for the use of speedball was discussed.

Combinatorial Pharyngeal Taste Coding for Feeding Avoidance in Adult Drosophila.

Taste drives appropriate food preference and intake. In Drosophila, taste neurons are housed in both external and internal organs, but the latter have been relatively underexplored. Here, we report that Poxn mutants with a minimal taste system of pharyngeal neurons can avoid many aversive tastants, including bitter compounds, acid, and salt, suggesting that pharyngeal taste is sufficient for rejecting intake of aversive compounds. Optogenetic activation of selected pharyngeal bitter neurons during feeding events elicits changes in feeding parameters that can suppress intake. Functional dissection experiments indicate that multiple classes of pharyngeal neurons are involved in achieving behavioral avoidance, by virtue of being inhibited or activated by aversive tastants. Tracing second-order pharyngeal circuits reveals two main relay centers for processing pharyngeal taste inputs. Together, our results suggest that the pharynx can control the ingestion of harmful compounds by integrating taste input from different classes of pharyngeal neurons.

Regional Variation of Bitter Taste and Aftertaste in Humans.

Despite widespread and persistent myths of a tongue map, all 5 prototypical taste qualities are sensed over the entire tongue. However, modern psychophysical data also suggest there may be more nuanced differences in suprathreshold intensity across oral loci, especially for bitterness. Here, we test whether bitter stimuli matched for whole-mouth intensity differ in perceived intensity across regions of the oral cavity in 2 experiments. Experiment 1 consisted of a whole-mouth sip and spit approach and Experiment 2 consisted of a spatial taste test using cotton swabs. In Experiment 1, participants (n = 63) rated overall intensity of 3 bitter solutions at 5 different loci (front, middle, back of tongue; roof of mouth; and lip). Temporal effects were explored using in-mouth and aftertaste ratings. In Experiment 2, participants (n = 48) rated the intensity of quinine and Tetralone solutions after solutions were painted on fungiform, circumvallate, and foliate papillae with a swab. After the spatial taste test, participants completed a questionnaire on self-reported beer intake. Analysis of variance results of both experiments show a significant locus by stimulus interaction, suggesting different bitterants were perceived differently across the various loci. This result was apparently driven by low-intensity ratings for Tetralone on the anterior tongue. Aftertaste ratings in Experiment 1 also revealed significant temporal effects: ratings on the anterior tongue decreased for all bitterants and ratings for quinine decreased at all loci. Reasons for these effects are not known but may suggest differential expression of bitter taste receptors or differences in bitter agonist-receptor binding affinity across tongue regions.

Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers.

BACKGROUND AND PURPOSE: Like chili peppers, gingers produce pungent stimuli by a group of vanilloid compounds that activate the nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel. How these compounds interact with TRPV1 remains unclear. EXPERIMENTAL APPROACH: We used computational structural modelling, functional tests (electrophysiology and calcium imaging), and mutagenesis to investigate the structural mechanisms underlying ligand-channel interactions. KEY RESULTS: The potency of three principal pungent compounds from ginger -shogaol, gingerol, and zingerone-depends on the same two residues in the TRPV1 channel that form a hydrogen bond with the chili pepper pungent compound, capsaicin. Computational modelling revealed binding poses of these ginger compounds similar to those of capsaicin, including a "head-down tail-up" orientation, two specific hydrogen bonds, and important contributions of van der Waals interactions by the aliphatic tail. Our study also identified a novel horizontal binding pose of zingerone that allows it to directly interact with the channel pore when bound inside the ligand-binding pocket. These observations offer a molecular level explanation for how unique structures in the ginger compounds affect their channel activation potency. CONCLUSIONS AND IMPLICATIONS: Mechanistic insights into the interactions of ginger compounds and the TRPV1 cation channel should help guide drug discovery efforts to modulate nociception.

Conditioned taste aversion versus avoidance: A re-examination of the separate processes hypothesis.

Rats not only avoid ingesting a substance associated with LiCl toxicosis, but they display rejection reflexes (e.g., gapes) to its taste; this latter response is thought to reflect disgust or taste aversion. Prior work has shown that rats also avoid consuming foods/fluids associated with other adverse gastrointestinal (GI) effects like lactose indigestion but without the concomitant change in oromotor responses (taste reactivity; TR) indicative of aversion. Because of interpretive limitations of the methods used in those studies, we revisited the taste aversion-avoidance distinction with a design that minimized non-treatment differences among groups. Effects on intake and preference (Experiments 1a, 1b, and 2), as well as consummatory (TR, Experiment 1a and 1b) and appetitive (Progressive Ratio, Experiment 2) behaviors to the taste stimulus were assessed after training. In both experiments, rats were trained to associate 0.2% saccharin (CS) with intraduodenal infusions of LiCl, Lactose, or NaCl control. Rats trained with 18% lactose, 0.3 and 1.5 mEq/kg dose of LiCl subsequently avoided the taste CS in post-training single-bottle intake tests and two-bottle choice tests. However, only those trained with 1.5 mEq/kg LiCl displayed post-conditioning increases in taste CS-elicited aversive TR (Experiment 1a and 1b). This dose of LiCl also led to reductions in breakpoint for saccharin. The fact that conditioned avoidance is not always accompanied by changes in other common appetitive and/or consummatory indices of ingestive motivation further supports a functional dissociation between these processes, and highlights the intricacies of visceral influences on taste-guided ingestive motivation.

The role of endogenous pituitary adenylyl cyclase activating polypeptide (PACAP) in nicotine self-administration, reward and aversion.

Pituitary adenylyl cyclase activating polypeptide (PACAP) and its receptors (PAC1, VPAC1, and VPAC2) are localized in brain regions implicated in stress response, reward seeking and aversive responses, raising the possibility that PACAP may be involved in motivational effects of nicotine. To test this hypothesis, we used two-bottle choice (TBC) and place conditioning paradigms and assessed if nicotine preference or conditioned place preference (CPP) or aversion (CPA) induced by nicotine would be altered in mice lacking PACAP compared to their wild-type controls. In the TBC paradigm, mice had access to two water bottles during the first week and then one of the water bottles was switched to nicotine solution (20, 40 and then 80 µg/mL). The volume of water and nicotine consumed was measured every day. In the place conditioning paradigm, mice were tested for baseline place preference on day 1, received conditioning with saline versus a low (0.25) or high (1 mg/kg) dose nicotine and, respectively, tested for CPP or CPA 24 h following the last conditioning. We discovered that mice lacking PACAP compared to their wild-type controls exhibited more preference for nicotine over water in the TBC paradigm, particularly at the two higher concentrations of nicotine. While the rewarding action of the low dose nicotine was not altered in mice lacking PACAP, the aversive effect of the high dose nicotine was blunted in these mice compared to their wild-type controls. The present results suggest that endogenous PACAP may play a functional role in nicotine preference and its aversive effect.

Tastant-Evoked Arc Expression in the Nucleus of the Solitary Tract and Nodose/Petrosal Ganglion of the Mouse Is Specific for Bitter Compounds.

Despite long and intense research, some fundamental questions regarding representation of taste information in the brain still remain unanswered. This might in part be due to shortcomings of the established methods that limit the researcher either to thorough characterization of few elements or to analyze the response of the entirety of neurons to only one stimulus. To overcome these restrictions, we evaluate the use of the immediate early gene Arc as a neuronal activity marker in the early neural structures of the taste pathway, the nodose/petrosal ganglion (NPG) and the nucleus of the solitary tract (NTS). Responses of NPG and NTS neurons were limited to substances that taste bitter to humans and are avoided by mice. Arc-expressing cells were concentrated in the rostromedial part of the dorsal NTS suggesting a role in gustatory processing. The use of Arc as a neuronal activity marker has several advantages, primarily the possibility to analyze the response of large numbers of neurons while using more than one stimulus makes Arc an interesting new tool for research in the early stages of taste processing.

The Utility of the Artificial Taste Sensor in Evaluating the Bitterness of Drugs: Correlation with Responses of Human TASTE2 Receptors (hTAS2Rs).

The purpose of this study was to examine the ability of the artificial taste sensor to evaluate the bitterness of drugs by comparing the responses of the taste sensor with documented responses of human TASTE2 receptors (hTAS2Rs). For this purpose 22 bitter compounds, used as ingredients of pharmaceutical medicines in Japan and known ligands of hTAS2Rs, were selected for testing. Their solutions (0.01, 0.03, 0.1 mM) were evaluated by five different taste sensors (AC0, AN0, BT0, C00, AE1). Correlations between physicochemical parameters of the compounds and the responses of the taste sensors and hTAS2Rs were evaluated. From taste sensor measurements, diphenidol, haloperidol, diphenhydramine, dextromethorphan and papaverine, all ligands of hTAS2R 10 and/or hTAS2R14, were predicted to express strong bitterness, surpassing that of quinine. Responses of taste sensors BT0 were found to be significantly correlated with responses of hTAS2R14. High log P values (≧2.73) and responses of hTAS2R14 were also significantly correlated (** p<0.01, chi-square test). In conclusion, taste sensor BT0 is highly sensitive to bitterness and correlates significantly with hTAS2R14, making it useful for evaluating the bitterness of hydrophobic compounds which respond to hTAS2R14 and their inhibitors.

Aversive learning of odor-heat associations in ants.

Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general.

Heterogeneity in the Drosophila gustatory receptor complexes that detect aversive compounds.

Animals must detect aversive compounds to survive. Bitter taste neurons express heterogeneous combinations of bitter receptors that diversify their response profiles, but this remains poorly understood. Here we describe groups of taste neurons in Drosophila that detect the same bitter compounds using unique combinations of gustatory receptors (GRs). These distinct complexes also confer responsiveness to non-overlapping sets of additional compounds. While either GR32a/GR59c/GR66a or GR22e/GR32a/GR66a heteromultimers are sufficient for lobeline, berberine, and denatonium detection, only GR22e/GR32a/GR66a responds to strychnine. Thus, despite minimal sequence-similarity, Gr22e and Gr59c show considerable but incomplete functional overlap. Since the gain- or loss-of-function of Gr22e or Gr59c alters bitter taste response profiles, we conclude a taste neuron's specific combination of Grs determines its response profile. We suspect the heterogeneity of Gr expression in Drosophila taste neurons diversifies bitter compound detection, improving animal fitness under changing environmental conditions that present a variety of aversive compounds.

Immobility responses are induced by photoactivation of single glomerular species responsive to fox odour TMT.

Fox odour 2,4,5-trimethyl thiazoline (TMT) is known to activate multiple glomeruli in the mouse olfactory bulb (OB) and elicits strong fear responses. In this study, we screened TMT-reactive odourant receptors and identified Olfr1019 with high ligand reactivity and selectivity, whose glomeruli are located in the posterodorsal OB. In the channelrhodopsin knock-in mice for Olfr1019, TMT-responsive olfactory-cortical regions were activated by photostimulation, leading to the induction of immobility, but not aversive behaviour. Distribution of photoactivation signals was overlapped with that of TMT-induced signals, but restricted to the narrower regions. In the knockout mice, immobility responses were reduced, but not entirely abolished likely due to the compensatory function of other TMT-responsive glomeruli. Our results demonstrate that the activation of a single glomerular species in the posterodorsal OB is sufficient to elicit immobility responses and that TMT-induced fear may be separated into at least two different components of immobility and aversion.

Long-Term Alcohol Drinking Reduces the Efficacy of Forced Abstinence and Conditioned Taste Aversion in Crossed High-Alcohol-Preferring Mice.

BACKGROUND: Negative outcomes of alcoholism are progressively more severe as the duration of problem of alcohol use increases. Additionally, alcoholics demonstrate tendencies to neglect negative consequences associated with drinking and/or to choose to drink in the immediate presence of warning factors against drinking. The recently derived crossed high-alcohol-preferring (cHAP) mice, which volitionally drink to heavier intoxication (as assessed by blood ethanol [EtOH] concentration) than other alcohol-preferring populations, as well as spontaneously escalating their intake, may be a candidate to explore mechanisms underlying long-term excessive drinking. Here, we hypothesized that an extended drinking history would reduce the ability of 2 manipulations (forced abstinence [FA] and conditioned taste aversion [CTA]) to attenuate drinking. METHODS: Experiment 1 examined differences between groups drinking for either 14 or 35 days, half of each subjected to 7 days of FA and half not, to characterize the potential changes in postabstinence drinking resulting from an extended drinking history. Experiment 2 used a CTA procedure to assess stimulus specificity of the ability of an aversive flavorant to decrease alcohol consumption. Experiment 3 used this taste aversion procedure to assess differences among groups drinking for 1, 14, or 35 days in their propensity to overcome this aversion when the flavorant was mixed with either EtOH or water. RESULTS: Experiment 1 demonstrated that although FA decreased alcohol consumption in mice with a 14-day drinking history, it failed to do so in mice drinking alcohol for 35 days. Experiment 2 showed that the addition of a flavorant only suppressed alcohol drinking if an aversion to the flavorant was previously established. Experiment 3 demonstrated that an extended drinking history expedited extinction of suppressed alcohol intake caused by a conditioned aversive flavor. CONCLUSIONS: These data show that a history of long-term drinking in cHAP mice attenuates the efficacy of interventions that normally reduce drinking. Analogous to alcoholics who may encounter difficulties in limiting their intake, cHAP mice with long drinking histories are relatively insensitive to both abstinence and signals of harmful consequences. We propose that the cHAP line may be a valid model for adaptations that occur following the extended heavy alcohol drinking.