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Psychophysical Evaluation of Sweet Taste Perception in Mice


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PSYCHOPHYSICAL EVALUATION OF SW EET TASTE PERCEPTION IN MICE By CEDRICK DOTSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Cedrick Dotson

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iii ACKNOWLEDGMENTS I would like to thank my mentor, Alan Spector, Ph.D., for his support and supervision. I would also lik e to thank our senior laborat ory technician, Angela Newth, for her invaluable assistance in completing th is project. This research was supported by National Institute on Health Predoctoral National Research Service Award, # F31DC007358, granted to Cedrick D. Dotson and National Institute on Deafness and Other Communication Disorders Grant, # R01DC04574, awarded to Alan C. Spector.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Psychophysical Examination of Taste Quality in Non-Human Mammals...................1 Amino Acid Taste Perception.......................................................................................2 Sweetener Transduction............................................................................................4 Summary.....................................................................................................................10 2 EXPERIMENT 1: THE RELATIVE A FFECTIVE POTENCY OF GLYCINE, LSERINE AND SUCROSE AS ASSE SSED BY A BRIEF-ACCESS TASTE TEST IN INBRED STRAINS OF MICE...................................................................14 Background.................................................................................................................14 Methodological Details...............................................................................................17 Subjects................................................................................................................17 Taste Stimuli........................................................................................................17 Procedure.............................................................................................................18 Data Analysis.......................................................................................................19 Results........................................................................................................................ .20 Standardization Data...........................................................................................20 Sucrose................................................................................................................21 L-serine................................................................................................................22 Glycine................................................................................................................23 Discussion...................................................................................................................24 3 EXPERIMENT 2: TASTE DISCRIMINABILITY OF L-SERINE and Various SUGARS BY MICE...................................................................................................38 Background.................................................................................................................38

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v Methodological Details...............................................................................................39 Subjects................................................................................................................39 Taste Stimuli........................................................................................................39 Apparatus.............................................................................................................41 Experimental Design...........................................................................................42 Training (see Table 3-3)...............................................................................42 Testing (see Table 3-3).................................................................................44 Data Analysis.......................................................................................................45 Results........................................................................................................................ .45 Discrimination Testing........................................................................................46 Sucrose vs. L-serine.............................................................................................46 Serine group..............................................................................................46 Sucrose group...........................................................................................46 Sucrose/L-serine vs. Glucose..............................................................................48 Serine group.................................................................................................48 Sucrose group...............................................................................................49 Sucrose/L-serine vs. Maltose...............................................................................50 Serine group.................................................................................................50 Sucrose group...............................................................................................50 Sucrose vs. L-serine II.........................................................................................51 Serine group.................................................................................................51 Sucrose group...............................................................................................52 Sucrose/L-serine vs. Fructose..............................................................................53 Serine group.................................................................................................53 Sucrose group...............................................................................................54 Stimulus Control Sessions...................................................................................55 Discussion...................................................................................................................56 Monogeusia.........................................................................................................56 Sugars vs. L-serine..............................................................................................61 4 EXPERIMENT 3: PERCEIVED SIMI LARITY BETWEEN L-SERINE, LTHREONINE AND CHEMICAL COMPOU NDS REPRESENTATIVE OF THE FOUR BASIC TASTE QUALITIES..........................................................................84 Background.................................................................................................................84 Methodological Details...............................................................................................85 Subjects................................................................................................................85 Taste Stimuli........................................................................................................86 Apparatus.............................................................................................................87 Experimental Design...........................................................................................88 Davis rig training..........................................................................................88 Conditioning phase (see Table 4-2).............................................................89 Davis rig testing phase.................................................................................90 Data Analysis.......................................................................................................90 Results........................................................................................................................ .91 Conditioning Phase..............................................................................................91 Brief-Access Testing Phase.................................................................................92

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vi Sucrose CS group.........................................................................................92 L-serine CS group........................................................................................92 L-threonine CS group...................................................................................93 Discussion...................................................................................................................93 5 GENERAL DISCUSSION.......................................................................................103 LIST OF REFERENCES.................................................................................................108 BIOGRAPHICAL SKETCH...........................................................................................116

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vii LIST OF TABLES Table page 1-1 Consequences of the knockout of va rious T1R receptors on neural and tasterelated behavioral responses.....................................................................................12 1-2 % of taste buds and % TBCs in papillae..................................................................13 2-1 Mean number of licks to water SEM ta ken by the four strains when tested with sucrose, L-serine, or glycine when mice were water deprived................................32 2-2 Mean of the inter-lick interval (ILI) distribution (ms) SEM observed in the four strains of mice trained to lick e ither sucrose, L-se rine, or glycine...................33 2-3 Strains listed in order of mean Tastan t/Water Lick ratio, for sucrose, L-serine, and glycine when mice were water deprived...........................................................34 2-4 Strain listed in order of mean Standard ized Lick ratio, for sucrose, L-serine, and glycine when mice were non-deprived.....................................................................35 3-1 Stimulus concentrations...........................................................................................65 3-2 Order of stimulus discrimination pairings................................................................66 3-3 Representative training and testing pa rameters for the 2 discrimination groups.....67 3-4 Overall percentage correct during the last week of testing for the stimulus discrimination pairings.............................................................................................68 3-5 Number of stimulus-control sessions required to criterion performance.................69 4-1 Test Stimulus Arrays for the Three Conditioning Groups.......................................97 4-2 Conditioning phase schedule....................................................................................98

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viii LIST OF FIGURES Figure page 2-1 Mean ( SE) Tastant/Water Lick Ratio as a function of sucrose, L-serine, and glycine concentration for four di fferent inbred strains of mice................................36 2-2: Mean ( SE) Standardized Lick Ratio as a function of sucros e, L-serine, and glycine concentration for four di fferent inbred strains of mice................................37 3-1 Trial structure...........................................................................................................70 3-2 Individual animal (symbols) and group mean ( SEM; grey bars) data for mice trained to discriminate either su crose or L-serine from NaCl..................................71 3-3 Individual animal (symbols) and gro up mean ( SEM; grey bars) data are plotted across all test phases for mice ini tially trained to discriminate L-serine from NaCl.................................................................................................................72 3-4 Individual animal (symbols) and gro up mean ( SEM; grey bars) data are plotted across all test phases for mice in itially trained to discriminate sucrose from NaCl.................................................................................................................73 3-5 Mean ( SD) data for mice attempting to discriminate L-serine from sucrose........74 3-6 Mean ( SD) data for mice attempting to discriminate L-serine from glucose.......75 3-7 Mean ( SD) data for mice attempting to discriminate sucrose from glucose.........76 3-8 Mean ( SD) data for mice attempting to discriminate L-serine from maltose.......77 3-9 Mean ( SD) data for mice attempting to discriminate sucrose from maltose.........78 3-10 Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a second time (these mice were initially trained to discrimi nate L-serine from NaCl)........................................................................................................................79 3-11 Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a second time (these mice were initially trained to discriminate sucrose from NaCl)........................................................................................................................80 3-12 Mean ( SD) data for mice attempting to discriminate L-serine from fructose.......81

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ix 3-13 Mean ( SD) data for mice attempting to discriminate sucrose from fructose........82 3-14 Mean ( SEM) data for both groups of mice are plotted across all test phases.......83 4-1 Mean ( SEM) of the CS Suppre ssion Ratio for each conditioned stimulus (CS)..........................................................................................................................99 4-2 Mean ( SEM) Tastant/Water Lick Ratios fo r the sucrose CS group for all test stimuli.................................................................................................................100 4-3 Mean ( SEM) Tastant/Water Lick Ratios for the L-serine CS group for all test stimuli.................................................................................................................101 4-4: Mean ( SEM) Tastant/Water Lick Ratios fo r the L-threonine CS group for all test stimuli..............................................................................................................102

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PSYCHOPHYSICAL EVALUATION OF SW EET TASTE PERCEPTION IN MICE By Cedrick Dotson August 2006 Chair: Alan Spector Major Department: Psychology Despite differences in their molecular st ructure, many sugars, a subset of amino acids, and some synthetic compounds are s weet-tasting to huma ns and appear to possess a sucrose-like taste quality to non-human mammals. It has been proposed that in taste bud cells (TBCs), a family of r eceptors called the T1Rs mediates signal transduction of all of these sweeteners. However, in a brief-access test with nondeprived mice, licking responses to sucrose we re discernibly different from the responses to the amino acids tested. Experiments detailed here were designed to test the ability of mice to distinguish between L-serine and various sugars in opera nt taste discriminati on tasks. Mice were able to discriminate NaCl from sucrose (n=6 ) and L-serine (n=6) which served as training stimuli. Mice had difficulty discriminating su crose from L-serine, maltose, fructose and glucose to varying degrees depending on th e stimulus and the training history. For example, when concentration effects are ta ken into consideration, mice were unable to

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xi discriminate sucrose from glucose or fructo se suggesting that these sugars generate a unitary percept. However, these animals were able to discriminate sucrose from L-serine. Mice were also able to discriminate L-seri ne from glucose, fruc tose, and maltose, but only moderately so. Data gathered using a conditioned taste aversi on assay suggest that L-serine generates a complex taste that incl udes a sucrose-like component and that this complexity may be at the source of the limite d discriminability between L-serine and the sugars. To my knowledge, the qualitative complexity elicited by L-serine, which includes both a sweet and a b itter taste, has never been previously demonstrated before in rodents. L-serines ability to e voke multiple taste percepts would explain much of the results observed in this dissertation incl uding its lack of aff ective potency, its poor efficacy as a standard stimulus, and its discriminability from sugars. In summary, these data suggest that all of these compounds share some qualitative similarities. Therefore, it is likely that so me taste input arising from TBCs that express different T1R receptors converges somewher e along the gustatory neuraxis. However, the results of these experiments also imply that sweet-tasting L-amino acids, such as Lserine, also possess distinguish able taste characteristics.

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1 CHAPTER 1 INTRODUCTION Psychophysical Examination of Tast e Quality in Non-Human Mammals It is generally accepted that taste per ception is comprised of only a few basic qualities (e.g., sweet, salty, sour, bitter; se e Bartoshuk, 1988). However, the nature of these perceptions is difficult to study becau se these experiences cannot be directly measured and must be inferred from behavior. In animals, the systematic study of these perceptions is accomplished by the use of beha vioral procedures that are designed to characterize the relationship between physical stimuli and sensation (Blough and Blough, 1977; Berkley and Stebbins, 1990; Spector, 2003). Thes e psychophysical techniques allow for, among other things, the assessment of an animals capacity to discriminate or generalize between two chemical compounds. If an animal treats a test stimulus similarly to a trained or conditioned stimulus (i.e ., generalization), then the compounds are assumed to share some perceptual features. If an animal can be trained to discriminate between two compounds, then the stimuli involved must generate distinguishable neural signals in both the periphery and the brain. Identifying co mpounds that are behaviorally indiscriminable allows for them to be categor ized into perceptual classes. Indeed, a group of stimuli that are mutually indiscriminabl e constitute the definition of a perceptual class. Below I propose the use of psychophysical procedur es specifically designed to answer these questions. As argued above, the use of psychophysical methodology is the only way to rigorously and objectively exam ine the taste perception of animals.

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2 Amino Acid Taste Perception Explicit taste discrimination experiments using amino acids as stimuli are rare. Instead, researchers have tried to perceptually ca tegorize amino acids in rodents by using the conditioned taste aversion (C TA) generalization paradigm to quantify the degree to which these compounds are similar to prototypic al chemical stimuli thought to represent basic taste qualities (e.g., sucrose, NaCl, citr ic acid, quinine). In this procedure, an animal samples a novel taste stimulus followe d by the injection of an agent that causes visceral malaise (usually lithium chloride; LiCl). After which, researchers measure whether the subsequent avoidance conditioned to the novel tastant generalizes to other test compounds. The results of such experi ments suggest that a variety of amino acids appear to possess some degree of a qualitative similarity with the taste of sucrose. Taken together, without regard to strain and species differences, results from CTA experiments demonstrate that a subset of Damino acids and a subset of L-amino acids including L-alanine, L-prolin e, L-serine, and glycine (whi ch does not have a chiral carbon) are all treated as possessing some degree of qualitative similarity with the taste of sucrose and are thus consider ed sweet. In addition, wh en mixed with the epithelial sodium channel blocker, amiloride, aver sions conditioned to monosodium glutamate (MSG) and L-aspartic acid have also been shown to generalize to sucrose in CTA tests (e.g., Yamamoto et al. 1991; Chaudhari et al. 1996; Stapleton et al. 1999; Heyer et al. 2003). Other amino acids tested fail to fall into this category (i ncluding L-arginine, Lisoleucine, L-methionine, L-phenylalanine, Ltryptophan, D-alanine, and D-serine tested in various strain of mice; see Ninomiya et al. 1984b; Kasahara et al. 1987; Ninomiya et al. 1992; and L-arginine, L-asparagine, L-aspa rtic acid, L-glutamic acid, L-glutamine,

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3 and L-histidine tested in the golden hamster; see Nowlis et al. 1980; Yamamoto et al. 1988). A large body of psychophysical literature also suggests that these L-amino acids give rise, to some degree, to a sweet taste perception in humans (e.g., Solms et al. 1965; Schiffman and Dackis, 1975; Schi ffman and Clark, 1980; Schiffman et al. 1981; Schiffman et al. 1982; Haefeli and Glaser, 1990; Shal lenberger, 1993). These data also suggest that other L-amino acids, such as L-threonine, may also share perceptual characteristics with sucrose. Indeed, th e notion that rodents may also perceive Lthreonine as sucrose-like (i.e., sweet) is supported by the fact that L-threonine was shown to be preferred by Sprague-Daw ley rats and by ddy mice, at certain concentrations, as assessed us ing a two-bottle preference test (Pritchard and Scott, 1982a; Iwasaki et al. 1985). However, as detailed below, this fact, in and of itself, does not allow for a confident categoriz ation of a taste compound into the perceptual class humans label sweet. In general, rats unconditionally prefer t hose amino acids that have been shown to generalize with sucrose in conditioned tast e aversion tests. Using the two bottle preference test, a variety of researchers have shown that these same L-amino acids seem to be favored, to varying degrees, by different strains of mice (Iwasaki et al. 1985; Lush et al. 1995; Bachmanov et al. 2001b). Indeed, these measures are commonly used to determine whether a particular species or anim al perceives a stimulus as sweet and/or as a measure of the relative inte nsity of that percept (e.g., Bachmanov et al. 2001b). However, the use of preference measures to determine the relative qualitative similarity of a putative sweet-tasting stimulus to that of a prototypical sugar (i.e., sucrose) implies that if that compound shares a qu alitative resemblance to a partic ular sugar, then it will be

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4 unconditionally preferred. Additionally, it suggests that if a compound is not unconditionally preferred, it will not be perceived as sweet. I recently conducted a set of experiment s assessing the affective potency of sucrose, L-serine, and glycine using a brief-acces s taste test in a gustometer, the details of which are covered in Chapter 2 of this disse rtation. Suffice it to say, these data do not support the supposition that L-serine (and to a lesser extent, glycine) is unconditionally preferred by non-deprived mi ce on the basis of taste. Thus, if the aforementioned contention regarding the correspondence betwee n sweet taste quality and preference is correct, then, despite suggestions from the CTA literature, L-serine could not be considered sweet. That said, it should be noted here that the two-bottle preference test and the briefaccess taste test only assess the motivational properties of a taste stimulus, not its qualitative characteristics per se For example, rats prefer lo w concentrations of NaCl in two-bottle tests and avoid high concentrations, bu t this does not mean that the former are sweet and the latter ar e bitter. Qualitative perception is best inferred from tasks in which taste serves as a cue for some other ev ent (e.g., reinforcement) that will generate a trained directed response rega rdless of the hedonic characteri stics of the ta ste stimulus. Sweetener Transduction An understanding of the neural basis of sweetener and amino acid taste perception has been propelled by remarkable discove ries regarding the molecular biology of transduction processes in the mammalian peri pheral gustatory system. Specifically, a gene family has been identified which en codes for three 7-transmembrane spanning Gprotein coupled receptors (T1R1, T1R2, and T1R3) that bind with sugars, synthetic sweeteners, amino acids, and in some species sweet proteins (e.g., Hoon et al. 1999;

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5 Bachmanov et al. 2001a; Kitagawa et al. 2001; Max et al. 2001; Montmayeur et al. 2001; Nelson et al. 2001; Sainz et al. 2001; Li et al. 2002). Initial calcium imaging studies of receptor-ligand in teractions in a heterologous expression system, suggested that individual T1Rs are not functional, but that they, sim ilar to other class C G-protein coupled receptors, combine into heterodi meric receptor complexes. Dimerization, however, has yet to be explicitly demonstrated in this sub-family of receptors. The T1R3 receptor has been shown to combine with T1R1 or T1R2 to form functional heteromers. The T1R2+3 complex was shown, in vitro to be activated by a variety of both natural and synthetic sweeteners as well as sweet-tasting D-amino acids (Nelson et al. 2001; Zhao et al. 2003). A similar study revealed that the combination of mouse T1R1 and T1R3 gives rise to a heteromeric receptor complex that interacts with most of the twenty common L-amino acids (Nelson et al. 2002). A more recent in vitro study suggested that T1R3 may also function indepe ndently as a low affinity receptor, binding with high concentrations of natura l but not synthetic sweeteners (Zhao et al. 2003). Results from experiments on the neural and be havioral consequences of the deletion (i.e., knock-out) of one or more of the genes en coding for the T1R receptors in mice are summarized in Table 1-1. Coll ectively, these data confirm, in vivo that sugars and Lamino acids bind, selectively, with different T1R receptor complexes. These heteromeric receptors were first pur ported to be expressed, principally, in non-overlapping sets of taste bud cells. The receptor, T1R1, was reported to be mainly expressed in fungiform papillae (anterior t ongue) and in the palate (in ~20-30% of the taste bud cells (TBCs) in 100% of the buds in these receptor fields; Hoon et al. 1999). In the posterior tongue, it was purported to be rarely found in th e taste buds of the

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6 circumvallate papillae (in less than 5% of T BCs in the less than 10% of circumvallate taste buds that express T1R1; Hoon et al. 1999) and only modestly in foliate papillae (in ~10% of TBCs in the ~30% of folia te taste buds that express T1R1; Hoon et al. 1999). In contrast, T1R2 was reported to be expressed mainly in the circumvallate and foliate papillae (in ~20-30% of the TBCs in 100% of the buds in these papillae; Hoon et al. 1999). It is almost non existent in the tast e buds of the fungiform papillae (in less than 1% of fungiform taste buds; Hoon et al. 1999; but see below) and only modestly expressed in the palate (in le ss than ~5% of TBCs in the ~2 0% of palatal taste buds that express T1R2; Hoon et al. 1999). The receptor T1R3, however, was purportedly expressed in ~30% of TBCs in all th ree taste bud contai ning papillae (Nelson et al. 2001; see Table 1-2 for a summary of the result s from all of the heretofore mentioned expression pattern studies). Double-label in situ hybridization studi es showed that virtually all T1R3-expressing cells in circum vallate and foliate papillae express T1R2 (Max et al. 2001; Montmayeur et al. 2001; Nelson et al. 2001). Conversely, all T1R2 expressing cells in circumvallate and foliate pa pillae, as well as in the palate, were also reported to express T1R3 (Montmayeur et al. 2001; Nelson et al. 2001). Moreover, most of the taste cells in fungiform papillae that express T1R3 also express T1R1 (Nelson et al. 2001). These experiments, however, also implied that there ex ists a population of cells in fungiform papillae and in the palate that express T1R3 without T1R1 or T1R2 (Nelson et al. 2001). These expression data imply that the ne rves innervating the circumvallate and fungiform receptor fields (i.e., the fields of the chorda tympani and glossopharyngeal nerves, respectively) would be differentially sensitive to sugars and amino acids. For

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7 example, the chorda tympani nerve, which innervates fungiform papillae, which preferentially expresses the T1R1+3 receptor complex, should be relatively more responsive to L-amino acids than to sugars or D-amino acids. On the other hand, the glossopharyngeal nerve, which innervates th e circumvallate and foliate papillae, which preferentially expresses the T1R2+3 receptor complex, should be relatively more responsive to sugars and D-amino acids than to L-amino acids. However, the response properties of the mouse whole chorda tympani and glossopharyngeal nerves do not correspond to these predicted patterns (e.g., Ninomiya and Funakoshi, 1989; Ninomiya et al. 1993; Ninomiya et al. 2000; Danilova and Hellekant, 2003). This may suggest the existence of other receptors that are responsiv e to these ligands or it may suggest that the aforementioned expression data are inaccurate or incomplete. Indeed, more recent data do conflict with the previous studies on the expression pattern of these receptors. Contrary to these reports, th e expression of the receptor, T1R1, was shown to be greater (i.e., in a larger number of cells) than that of T1R2 and T1R3 in circumvallate papillae, albeit with a lower signal intensit y than the other two receptors (Kim et al. 2003). Using double-label in situ hybridization, these resear chers found that, in the circumvallate, almost all cells that express T1 R2 and the majority of cells that express T1R3 also express T1R1 (Kim et al. 2003). They also reported that, in fungiform papillae, half of the T1R3 expressing cells also express T1R2. As mentioned above, prior work suggested that the expression of the T1 R2 receptor in fungiform papillae is rare. Moreover, expression of T1R1 and T1R2 overl apped in fungiform papillae. Since every T1R2 positive cell ubiquitously expressed T1 R3, then T1R1 + T1R2 double positive cells must also express T1R3. Therefore, in bot h receptor fields, TBCs that co-express all

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8 three T1R receptors can be found (i.e., T1R1, T1R2, and T1R3; Kim et al. 2003). Interestingly, in the circumvallate, 66% of the T2R receptor expressing cells1 also expressed the receptor T1R1. The co-expressi on of the other two receptors with the various T2R receptors was not assessed. The fact that sugars and L-amino acids bind, selectively, with different T1R receptor complexes suggest that theses compounds may be perceptually distinct and as a result, behaviorally discriminable. Indee d, the early reports on the pattern of T1R receptor expression provide a degree of recepto r complex segregation that could support this discriminability. However, depending on the degree of receptor expression overlap on single TBCs, the data reported by Kim et al. predict that mice w ould have at least some difficulty discriminating between sugars and L-amino acids. Indeed, these data, reported by Kim et al. portray a more balanced recep tor expression pattern, at least across the circumvallate and fungiform pap illae, and correspond more closely to the response properties of gustatory nerves th an do data from the previous studies. As an important caveat, it should be not ed that peripheral nerve responses only index the nature of the signal arising from the initial processing of taste input. The central gustatory system can amp lify, attenuate, or alter feat ures of the peripheral signal (e.g., convergence). Moreover, knowledge re garding a neural response, whether of peripheral or central origin, does not in and of itself, necessarily reveal how those signals are translated into behavior (and th e associated inferred perceptions). The complexities of thes e interpretive issues are exemplified by the opposing postulations regarding bitter taste percepti on made by researchers who study receptor 1 Mix probes of T2R5, T2R8, T2R18, and T2R19 were used for analysis of T2Rs expression

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9 expression and those who study the response prop erties of TBCs. A family of G-proteincoupled taste receptors (T2Rs) were shown to bind with a structurally diverse class of bitter-tasting compounds (Adler et al. 2000; Chandrashekar et al. 2000; Matsunami et al. 2000). It was suggested that these receptors evolved to help animals avoid ingesting toxic or otherwise harmful substances. Although each one of the receptors in this family is thought to be relatively sp ecific for its ligand, many appe ar to be co-expressed in subsets of TBCs. This latter finding led re searchers to hypothesi ze that mammals could not discriminate between bitter-tasting st imuli, because a given TBC could potentially be stimulated by a wide variety of compounds (Adler et al. 2000; Chandrashekar et al. 2000). Caicedo and Roper (2001) suggested, ho wever, that TBCs are more narrowly tuned than predicted from the receptor co-exp ression. This conclusion was based on their assessment of the intracellula r calcium responses in TBCs, in situ Each bitterresponsive TBC assayed responded to only one or at most only a few of the five compounds tested (Caicedo and Roper, 2001). These researchers suggested that rats could likely discriminate between the bitte r compounds tested, based on the apparent specificity of TBCs. Spector and Kopka (2002) tested these predic tions and demonstrated that rats could not behaviorally discriminate between denat onium benzoate and qui nine hydrochloride. Data from this study strongly suggested that these two ligands produce a unitary taste sensation. These results app ear to support the molecular fi ndings, which indicted that numerous T2Rs are co-expresse d in subsets of TBCs. Neve rtheless, the specificity of TBCs observed in the periphery (based on Ca++ imaging measurements) might indeed exist. However, any segregation may be negated by a convergence of the signals

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10 generated by the TBCs into a single ne ural channel somewher e along the gustatory neuraxis. Summary Conclusions regarding the taste quality of amino acids based on data from CTA experiments suggest that a subset of amino aci ds are perceptually similar to sucrose and some other sugars and results from two-bottl e intake experiments show that these amino acids are preferred by mice. However, although L-amino acids are thought to bind exclusively with the T1R1+3 receptor comple x, strain preference behavior measured in the two-bottle intake test seems to de pend on an anomaly in the T1R2+3 complex, leaving open the possibility that other factors were influencing the relative preference for L-amino acids in this test. Moreover, data ga thered in our laboratory question whether or not these stimuli are actually preferred by mice at all on the basis of taste and as a result question the very nature of the taste quality evoked by these compounds. Indeed, the molecular biology of sweetener transduction appears to provide a neurobiological basis for behavioral discriminability (i.e., perceptual distinction) Thus, my objective was to determine the degree to which receptor specificity predicts the relative discriminability of various sweeteners, by testing whether C 57BL/6J (B6) mice can discriminate between sucrose and L-serine, as well as a variety of other sugars and putative sweeteners using an operate discrimination paradigm. To my know ledge, explicit discrimination experiments in rodents with these ligands have never b een conducted. These tasks were designed to assess whether pairs of putative sweeteners w ould be treated by animals as perceptually identical. If compounds are distinguishable, however, these discrimination tasks provide little information on the basis of the discri minability. Moreover, even if animals can discriminate between two compounds, they may also find them to be similar, relative to

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11 other taste qualities (Spector, 2003). Therefore, to determine the relative similarity of these sweeteners, as well as to provide info rmation to aide in the interpretation of the discrimination data, CTA generalization tests where also conducted with a theoretically relevant subset of the stimuli. The identification of compounds th at share perceptual features and that are behaviorally indiscriminable allows for a confident categorization of a taste compound into a perceptual class (e.g., sweetness). Moreov er, such data can be related back to the molecular biology of sweet taste in s earch of clues regarding the functional organization of the normal murine gustatory system.

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12 Table1-1: Consequences of the knockout of various T1R receptors on neural and taste-related behavioral responses Chorda Tympani Nerve Record ing Brief-Access Taste Test# STIMULUS T1R1 KO T1R2 KO T1R3 KO T1R2+3 KO T1R1 KO T1R2 KO T1R3 KO T1R2+3 KO L-serine ? ? ? ? I Normal 0 ? L-serine + 30 mM IMP* 0 Normal 0 ? ? ? ? ? L-alanine ? ? ? ? I Normal 0 ? L-alanine + 30 mM IMP* 0 Normal 0 ? ? ? ? ? D-tryptophan Normal 0 0 0 Normal 0 0 ? Sucrose Normal I I 0 Normal I I# 0 Glucose Normal I I ** (Normal) 0 Normal I I 0 I = Impaired responsiveness; 0 = No responsiveness; ? = Not te sted; IMP alone produced no nerve response; ** All results are from Zhao et al. 2003 except for those in parentheses, which are from Damak et al. 2003. # Behavioral data, not gathered in a briefaccess test, from two separate studies (Damak et al. 2003 and Delay et al. 2006) suggest that mice, lacking the recepto r T1R3 can detect the presence of sucrose.

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13 Table 1-2: % of taste buds and % TBCs in papillae Circumvallate Foliate Fungiform Palate Receptor TB TBCs refs TB TBCs refs TB TBCs refs TB TBCs refs T1R1 < 10% 5% 1 ~30%* ~10%* 1 100% ~2030% 1 100% ~2030% 1 T1R2 100% ~2030% 1 100% ~2030% 1 < 1% N/A 1 ~20% < 5% 1 T1R3 100% ~30% 2 100% ~30% 2 100% ~30% 2 100% ~30% 2 Data derived from published reports (see reference below and in te xt) of the percentages of tast e buds expressi ng a given taste receptor and the percentages of TBCs within those taste buds (see text for more details). 1All data from Hoon et al. 1999 are derived mouse and rat tongues. *These cells were reported to have a much we aker signal relative to T1R2 positive cells in the foliat e, or T1R1 cells in fungifo rm papillae or in the palate. 2 All data Nelson et al ., 20011 are derived from adult mouse tongues

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14 CHAPTER 2 EXPERIMENT 1: THE RELATIVE A FFECTIVE POTENCY OF GLYCINE, LSERINE AND SUCROSE AS ASSESSED BY A BRIEF-ACCESS TASTE TEST IN INBRED STRAINS OF MICE2 Background The molecular biology pertaining to the tr ansduction of both sugars and synthetic sweeteners as well as sweet-tasting D-amino acids, reviewed in detail in Chapter 1, is consistent with the electrophysiological and behavioral phenotypes expressed by different inbred strains of mice, but such a correspondence regarding sweet-tasting L-amino acids (and glycine) is less st raightforward. It has been known for many years that mouse strains can be differentiated according to th eir intake of and nerve responsiveness to natural and synthetic sweeteners. In gene ral, taster mice have lower preference thresholds for sweeteners in two-bottle tests and their chorda tympani nerves (CT) are more responsive to sucrose, saccharin, and various sweet-tasting D-amino acids (especially D-phenylalanine) when compared with non-taster3 mice (Capretta, 1970; Pelz et al. 1973; Fuller, 1974; Ninomiya et al. 1984; Lush, 1989; Capeless and Whitney, 1995; Bachmanov et al. 1996; Frank and Blizard, 1999; Inoue et al. 2001; Nelson et al. 2001). These taster/non-taster phenotypes in mice were genetically linked to a single chromosomal locus referred to as Sac that was later discovered to encode for the T1R3 2 A version of this Chapter has been published previously in Chemical Senses, Vol. 29 No. 6 Published by Oxford University Press. All rights reserved. 3 The phenotypic descriptors taster and non-taster may at first glance seem to denote ageusic vs. nonageusic strains, however this nomenclature is commonl y used in the literature to categorize mouse strains with varying degrees of sensitivity (i.e., low behavioral threshold vs. high behavioral threshold) to compounds such as sucrose and/or sodium saccharin.

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15 receptor (e.g., Fuller, 1974; Ramirez and Fuller, 1976; Lush, 1989; e.g., Capeless and Whitney, 1995; Lush et al. 1995). Taster and n on-taster mouse strains have different alleles of the Tas1r3 gene that give rise to receptors with slightly different amino acid sequences (e.g., Bachmanov et al. 2001a; Kitagawa et al. 2001; Max et al. 2001; Montmayeur et al. 2001; Sainz et al. 2001). Interestingly, th e taster and non-taster allele of Tas1r3 generates receptors that are functi onally similar when combined with T1R1, but the non-taster form of the T1R3 receptor displays impaired binding when combined with T1R2 (Nelson et al. 2002; Damak et al. 2003). Thus non-taster mouse strains possess a dysfunctional T1R2+3, but an apparently normal T1R1+3 receptor complex. Indeed, there is evidence that L-amino acids, which bind with the T1R1+3 receptor, stimulate the CT comparably in both taster and non-taster mice, with the possible exception of L-proline (Ninomiya et al. 1984; Inoue et al. 2001). Yet, twobottle preference for some sweet-tasting Lamino acids and glycine appears to depend on the taster status of the mouse strain based on testing w ith sugars (Lush, 1989; Capeless and Whitney, 1995; Lush et al. 1995; Bachmanov et al. 2001b). These behavioral findings are curious consideri ng that L-amino acids are believed to bind primarily with the T1R1+3 receptor which, as noted above, is thought to display similar binding properties in both tast er and non-taster mice (Nelson et al. 2002). In light of the apparent tension between the predicted behavior of mouse strains based on the molecular biology of amino aci d taste transduction and the observed behavior seen in the two-bottl e preference test, we examined the relative effectiveness of sucrose, glycine and L-serine to stimulat e licking in C57BL/6J (B6), SWR/J (SWR), DBA/2J (D2) and 129P3/J (129) mice in a briefaccess taste test. As noted above, inbred

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16 mice vary in their preference for all three of these compounds as assessed in two-bottle intake tests, and there is evidence that these compounds possess some common perceptual properties with respect to taste quality (i.e., sweet) in at least some rodents. If glycine and L-serine generate concentra tionresponse functions th at emulate sucrose, then it would suggest that these compounds are similar in their affective potency. In addition, we sought to examine the generality of the response profiles generated by these compounds by includi ng taster (B6 and SWR) a nd non-taster (129 and D2) mouse strains in the e xperimental design allowing us to make inferences regarding the effect of the non-taster form of the Tas1r3 allele on taste-guide d behavior (Capretta, 1970; Pelz et al. 1973; Fuller, 1974; Lush, 1989; Capeless and Whitney, 1995; Bachmanov et al. 1996; Max et al. 2001; Nelson et al. 2001). With some notable exceptions (Glendinning et al. 2002; Zhang et al. 2003; Zhao et al. 2003), most of the work conducted to date involving strain comparisons of unconditioned behavioral responsiveness to these com pounds has been based on two-bottle intake tests (water versus taste compound). Although taste certainl y influences the beha vior in that test paradigm, postingestive events can also influe nce intake. The brief-access taste test involves the measurement of licking during ve ry short trials with a sapid solution increasing the confidence that the responses ar e based on the oral sensory features of the stimulus. Many trials of various concentrati ons of the taste stimulus are presented during a session and concentrationres ponse functions are derived. The taste solutions are delivered in randomized blocks to minimize sy stematic carry-over effects and to mitigate the influence of postingestive factors on the response to a gi ven stimulus in the set.

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17 Methodological Details Subjects A total of 120 male naive mice (Jackson Laboratories, Bar Harbor, Maine) from four different strains, C57BL/6J (B6), SW R/J (SWR), 129P3/J (129) and DBA/2J (D2), served as subjects (n=30/strain). Within each strain, animals were randomly assigned to one of 3 stimulus groups (n=10/group). The mice were housed individually in polycarbonate shoebox cages in a colony r oom where the lighting was controlled automatically (12h: 12h). Testing and tr aining took place during the lights-on phase. Mice were habituated to the laboratory envi ronment for seven days before testing and were ~ 8 weeks of age at the start of tes ting. During this time, food and purified water (Elix 10; Millipore, Billerica, MA) were available ad libitum During periods when the animals were placed on a water-restriction schedule, mice that dropped below 80% of their free-feeding weight received 1 ml s upplemental water 2 hours after the end of the testing session. All procedures were approved by the Univer sity of Florid a Institutional Animal Care and Use Committee. Taste Stimuli All solutions were prepared daily with pur ified water and reagent grade chemicals, and were presented at room temperature. Te st stimuli consisted of 5 concentrations of sucrose (0.0625, 0.125, 0.25, 0.5, and 1.0 M; Fisher Scientific, Atlanta, GA), L-serine (0.25, 0.5, 0.75, 1.0, and 1.5 M; Sigma-Aldrich, St Louis, MO), glycine (0.25, 0.5, 0.75, 1.0, and 1.5 M; Sigma-Aldrich, St Louis, MO) and purified water. Sucrose was chosen because 1) it is a prototypical natural sweetener that is commonly used in taste experiments, 2) it has been used to differen tiate taster (B6 and SW R) from non-taster (D2 and 129) mice in two-bottle pref erence tests, and 3) binds wi th the T1R2+3, but not the

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18 T1R1+3, receptor complex. L-serine and gl ycine were chosen because 1) there is evidence that at least in so me rodents these compounds shar e a perceptual quality with sucrose, 2) they are preferred by some rodent s at mid-range concentr ations in two-bottle preference tests, and 3) appear to bind primarily with the T1R1+3, but only poorly, if at all, with the T1R2+3 receptor complex. Procedure We used a brief-access procedure sim ilar to that described by Glendinning et al (2002). Testing took place in a lickometer refe rred to as the Davi s rig (Davis MS-160, DiLog Instruments, Tallahassee, FL; see Smith, 2001). This device allowed the mouse access to a single tube containing a taste stimulus for a brief period of time (5 s) and then after a 7.5-s inter-present ation interval, a different tube was offered. The stimulus array for each compound tested included the five di fferent concentrations detailed above and purified water contained in separate bottles. A given trial started after the first lick. Presentation order was randomized without replacement in blocks so that every concentration of a stimulus and water was pr esented exactly once before the initiation of the subsequent block. Unconditioned licking res ponses were recorded for later analysis. Sessions were 30 min in duration during whic h mice could initiate as many trials as possible. The animals were first trained to lick a stationary tube of water for 30 min in the Davis rig after being placed on ~23.5-h re stricted water access schedule. Animals then received 2 days of testing with five st imulus concentrations and purified water while maintained on the water-restriction schedule. This was done to familiarize the animals with the stimulus array. The water bottles were then replaced on the home cages and the mice were tested for three days non-deprived.

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19 Data Analysis For data gathered when animals were wa ter-deprived, a Tastant/Water Lick Ratio was calculated. This ratio was derived by taki ng the average number of licks per trial for each concentration and dividing it by the average licks per trial when water was delivered. This ratio controls for individual di fferences in lick rates and for differences in motivational state. The Tastant/Water Lick Ratio is useful for analyzing responses of animals highly motivated to lick due to th e restricted water access schedule. When animals were non-deprived, the average number of licks per trial fo r each concentration was collapsed across test sessions and divide d by that animals maximum potential lick rate per trial based on the mean of the inte r-lick interval (ILI) distribution measured during training (only inter-lick in tervals greater than 50 and le ss than 200 ms were used), yielding a Standardized Li ck Ratio (see Glendinning et al. 2002). Standardizing the licking response in this fashion controls for in dividual differences in maximal lick rates. The ratio scores were analyzed with twoway strain x concentration analyses of variance (ANOVAs). When a strain main eff ect or a strain x concentration interaction was significant, 1-way ANOVAs we re conducted to test for simple effects. Differences between strains at each concen tration were evaluated using Tukeys honestly significant difference test. Differences between Standard ized Lick Ratio scores in response to a given concentration and those measured for wate r were tested with matched-t-tests. The conventional P .05 was applied as the statistical re jection criterion. Only mice that had at least 1 trial at every concentration were in cluded in the analysis of a given stimulus.

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20 Results Standardization Data Because there can be within-strain and be tween-strain differences in the local lick rate as well as in the motiv ational response to the water restriction schedule, it is important to account for these factors in any li cking measure of taste responsiveness. As recommended by Glendinning et al. (2002), the Tastant/Water Lick Ratio was calculated for animals tested when under the water-rest riction schedule and the Standardized Lick Ratio was calculated for animals tested when non-deprived to stat istically control for non-taste influences in licking. Table 2-1 c ontains the means valu es representing licks during water trials used in the calculation of the Tastant/Water Lick Ratio for the various strains and compounds. A tw o-way ANOVA on water licks re vealed a significant main effect of strain [ F (3,107) = 40.7, P < .001] and test solution [ F (2,107) = 9.15, P < .001] as well as a significant interaction [ F (6,107) = 5.31, P < .001]. One-way ANOVAs were conducted within each taste compound to test for strain differences in water licks. There was a significant main effect of strain on the mean number of licks to water when mice were tested with sucrose [ F (3,36) = 15.6, P < .001], L-serine [ F (3,36) = 15.4, P < .001] and glycine [ F (3,36) = 19.6, P < .001] in the water restriction condition. Interestingly, when mice were water deprived, one-way ANOVAs conducted within each strain to test for differences in water licks to the stimuli re vealed that the non-taster strains increased licks to water when tested with L-serine re lative to licks taken in the other stimulus conditions ( P s .001). The taster strains did not signi ficantly differ in their responses to water across stimulus conditions.

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21 Table 2-2 contains the means of the indi vidual values representing the ILI observed when water-restricted animals were licking water from a stationary spout. These means exclude the mice that were not included in the analysis of resp onses under non-deprived conditions (n = 93). The reciprocal of these values were multiplied by 5000 to derive the estimated maximum possible licks during a 5-s trial and used in the calculation of the Standardized Lick Ratio for various stra ins and compounds. As expected, a two-way ANOVA revealed a significan t effect of strain [ F (3,81) = 52.1, P < .001] but no significant stimulus effect [ F (2,81) = 0.1, P = .909] or interaction [ F (6,81) = 0.7, P = .657]. Collectively, the results from these analyses confirm the necessity for standardizing the licking data across animals and strains. Sucrose When animals were water deprived, a tw o-way ANOVA of the Tastant/Water Lick Ratios revealed a significant main effect of strain [ F (3,36) = 18.1, P < .001), a significant main effect of concentration [ F (4,144) = 5.9, P < .001] and a significant interaction [ F (12,144) = 10.4, P < .001]. Strain differences at each concentration are delineated in Table 2-3. Confirming what is apparent in Figure 2-1, separate one-way ANOVAs for each strain revealed that only the 129 mice showed a significant monotonically increasing concentration-response function [ F (4,36) = 11.9, P < .001]. Although we did not expect to find meaningful results in the water-restriction conditi on considering that mice will usually lick water at a maximal rate maki ng it difficult to ascertain a response to appetitive stimuli and we did not expect to see an aversive response profile elicited by these sweet-tasting com pounds, it appears that the 129 mice did suppress licking to water relative to the other st rains (see table 2-1) and, as a result, in creased their Tastant/Water Lick Ratio to the stimulus [ F (3,36) = 15.6, P < .001]. There were some

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22 significant concentration-depende nt effects on the Tastant/Water Lick Ratio for the other 3 strains (All F s >3.0, all P s < .05), but it is obvious that th ese functions were relatively flat and generally equal to or below a valu e of 1.0. The 129 mice had significantly higher ratios at all five concentrations compared w ith the B6 and D2 mice and at the four highest concentrations compared with the SWR mice (all P values < .05); the la tter three strains did not differ. When non-deprived, all strains clearly show ed a concentration dependent increase in licking to sucrose (see Figure 2-2; F (5,170)=531.9, P < .001), but their concentrationresponse functions significantly differed (strain x concentrat ion interaction: F (15,170) = 10.9, P < .001). Strain differences at each concen tration are delineated in Table 2-4. The SWR mice were significantly more responsive to lower sucrose concentrations compared with D2 and 129 mice. At the lowest concen tration tested (0.0625 M), the Standardized Lick Ratio was significantly greater than that for water in the SWR and B6 (both t s > 2.2, P s < .05), but not the D2 and 129, strains (both t s < -0.2, P s > .7). As the sucrose concentration was raised, however, D2 and 129 mice steeply increased their responsiveness to sucrose and eventually equa led or surpassed the licking in SWR mice. B6 mice had a concentration response profile somewhat in between the SWR and the 129 and D2 mice. At the lower concentration, B6 mice were statistica lly indistinguishable from all of the mice including the SWR, but at the two highest concen trations they were significantly less responsive compared with the other three strains. L-serine When mice were water deprived, ther e was a significant strain effect ( F (3,36) = 9.8, P < .001) on the Tastant/Water Lick Ratio, a nd a significant strain x concentration interaction (see Figure 2-1; F (12,144) = 6.6, P < .001). Surprisingly, the D2 mice

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23 actually decreased their lick rate as the L-se rine concentration was raised (Figure 2-1; F (4,36) = 15.3, P < .001), whereas the other strains disp layed relatively flat functions. Strain differences at each concentr ation are shown in Table 2-3. When animals were non-deprived, ther e was no significant difference in the Standardized Lick Ratio between the strains ( F (3,21) = 0.1, P = .9), but there was a significant effect of concentration ( F (5,105) = 4.2, P = .002), though the increase was relatively minor; there was no significant strain x concentration interaction (see Figure 22). Glycine When animals were water deprived, ther e was a significant strain effect ( F (3, 35) = 10.6, P < .001) on the Tastant/Water Lick Ratio, a nd a significant strain x concentration interaction ( F (12,140) = 5.7, P < .001). Strain differences at each concentration are delineated in Table 2-3. As was the case with sucrose, separate one-way ANOVAs indicated that only the 129 mi ce increased their Taste/Water Lick Ratio monotonically as a function of concentration ( F (4,36) = 7.1, P < .001); see Figure 2-1). This increase in licking was first significan tly greater than 1.0 at the 0.75 M concentration ( P = .022). There were some significant concentration-de pendent effects on the Tastant/Water Lick Ratio for B6 and SWR mice (All F s > 4.6, all P s < .01), but it is obvious that the functions for these strains as well as for the D2 mice were relatively flat and generally below a value of 1.0. When mice were non-deprived, there wa s a significant effect of strain ( F (3, 26) = 5.8, P = .004) on the Standardized Lick Ratio, an d a significant strain x concentration interaction ( F (15,130) = 2.9, P = .001). Strain differences at each concentration are delineated in Table 2-4. Sepa rate one-way ANOVAs of the St andardized Lick Ratios for

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24 each strain revealed that 129 ( F (5,20) = 8.1, P < .001), B6 ( F (5,40) = 3.3, P < .05), and D2 ( F (5,40) = 2.5, P < .05) mice changed their lick rate as a function of concentration, but the modest increases were apparently li mited to higher concentr ations (see Figure 22). For example, matched t-tests indicated th at the 129 strain did not display significantly elevated licking relative to water until the glycine concentration reached 1.5 M ( P < .05). For B6 and D2 mice, no concentration signifi cantly differed from water. The SWR mice did not significantly change their lick ing as a function of concentration ( F (5,30) = 0.6, P = .678). Discussion Overall, as assessed by the brief-access ta ste test, the amino acids, L-serine and glycine, paled in comparison to sucrose in th eir ability to generate licking in the mouse strains examined. Collapsed across strain, non-deprived animals licked L-serine and glycine at a mean rate of only 15.4% and 21.4%, respectively, of the maximum possible in the 5-s trial at the highest concentrati on tested (1.5 M). In striking contrast, 1.0 M sucrose (the highest concentr ation tested) elicited an av erage licking rate, collapsed across strain, that was more than 5 times highe r than that seen for L-serine and nearly 4 times higher than that seen for glycine. The relatively broad concentration range used in this study weakens the possibility that the de sign failed to capture the dynamic range of responsiveness. Thus, the results presented he re suggest that the ta ste-related affective potency of sucrose is far superior to that of glycine or L-serine. Although neither amino acid was remarkably effective at stimulating licking in non-deprived mice relative to sucrose, gl ycine generated conc entration-dependent increases in licking in water -restricted 129 mice, whereas L-serine did not. For the D2 non-taster mice, we actually observed a concentration-dependent decrease in the

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25 Tastant/Water Lick Ratio in response to L-se rine when these mice were water-deprived. Given that L-serine is thought to possess a sucrose-like taste quality, this finding was unexpected and suggests that Lserine may also bind with ot her receptors that lead to aversive responses (e.g. T2Rs), at least in the D2 strain. Ot her researchers have reported higher levels of L-serine licking relative to water by B6, 129X1/SvJ and CB6 (BALB/c x B6 hybrids) mice in a brief-access test (Zhang et al. 2003; Zhao et al. 2003). These discrepancies are likely the result of me thodological differences between the studies. More specifically, in the prior work, both food and water intake was limited in a controlled fashion, based on proce dures described by Glendinning et al. 2002, to achieve a motivational state that woul d promote stimulus sampling but would not lead to the asymptotic lick rates generally observed unde r 24-h water deprivation regimens. Based on the present results, it appears that without the additional ef fects of nutrient restriction, the gustatory properties of L-serine and glycin e alone stimulate only s light, if any, licking behavior in the mouse strains tested here. The profile of strain differences in re sponsiveness to the compounds tested here was more complex than previously reported. When non-deprive mice were tested with sucrose, in general the taster strains (B6 and SWR) were modestly more responsive at lower concentrations compared with the non-taster mice (129 and D2), but even this difference failed to reach significance for th e B6 strain. As the concentration was progressively raised, the res ponsiveness of SWR and B6 tast er mice converged with that seen in the D2 non-taster mice. Notably, the 129 non-taster mice licked the two highest concentrations of sucrose significantly more th an did all three of the other strains. In

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26 general, these results are consistent with findings obtained by ot her researchers (i.e., Glendinning et al. 2005b). When mice were water deprived, sucrose, as expected, produced licking rates comparable to water in all strains except fo r the 129 mice. The 129 mice, in fact, nearly doubled their rate of stimulus responsiveness relative to water at 1.0 M. This same pattern was seen with glycine, with th e 129 mice responding to the compound at nearly 1.5 times the rate of water at 1.5 M. Inte restingly, the D2 non-taster mice displayed concentration-dependent decrea ses in their L-serine tastant/water lick ratio when water deprived, whereas the other strains had relativel y flat curves. It appears when mice were water-deprived the non-taster strains were less motivated to lick L-serine relative to sucrose and glycine, whereas all three stimuli we re treated similarly by the taster strains. Collectively, these findings suggest that th e phenotypic descriptors taster and nontaster do not necessarily apply to the respons iveness seen at higher concentrations of putative sweeteners, at least in the brief-access test. The taster and non-taster classification is based on the preference behavior of various mouse strains to low concentrations of sweeteners in long-te rm two-bottle intake tests. The brief-access taste test differs from the two-bottle intake test in interpretively important ways. In the brief-access test, imme diate responses to small volumes of stimuli are measured raising the confidence that th e behavior is driven by taste (see Spector, 2003). Indeed, Spector et al. (1996) demonstrated that when rats are deprived of gustatory input from the 7th and 9th cranial ne rves innervating the or al cavity, they show essentially flat concentrati onresponse curves for sucrose when tested using a briefaccess paradigm providing further evidence that behavior measured using a brief-access

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27 procedure is taste-guided. In contrast, in th e two-bottle test, intake is usually measured 24 h after stimulus presentation allowing fo r postingestive factors to influence the outcome. Moreover, differences in stimulus preference at high concentrations are difficult to detect with the two-bottle pref erence test because of ceiling effects. Typically, preference ratios approach an asymptotic value of 1.0 at very low concentrations for normally preferred stimu li, after which differences are difficult to discern. Other researches have used a shorter-term one-bo ttle intake test (e.g., 6 hr.) where ceiling effects and position preferences are avoided or at least minimized (e.g., Blizard et al. 1999). But while the results obtain ed using the one-bottle test are consistent with those seen when using the twobottle intake procedure, neither test avoids the confounding effects of viscerosensory input On the other hand, the brief-access taste test does not appear to be as sensitive to changes in behavi or at low concentrations, at least when several higher con centrations are available dur ing the session. Thus, these various procedures have different dynamic ranges of sensitivity. Accordingly, it would appear that, behaviorally sp eaking, the taster/non-taster di stinction is limited to low concentrations of sweeteners. This is c onsistent with sucrose and glucose detection thresholds measured with an operant proced ure in which the hedonic value of the taste stimulus is rendered irrelevant (Eylam and Spector, 2004). Interestingly, in the Eylam and Spector study, the threshold values for glycine measured with the same procedure in the same mice did not distinguish taster and non-taster strains in as straightforward a manner. That is, non-taster 129 mice had signifi cantly higher glycine thresholds relative to B6 mice. However, the glycine threshol ds for non-taster D2 mice did not differ from those for the taster B6 and SWR mice. In stark contrast, in our study, at the higher

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28 concentrations, the 129 mice were the most respons ive strain tested in this report. These findings further highlight the difference be tween suprathreshold responsiveness and threshold sensitivity (cf., Bachmanov et al. 1997). If the T1R family of receptors mediates behavioral attraction, as postulated by some (Zhao et al. 2003), then activation of eith er receptor complex should elicit appetitive behavior. However, compounds that bind with the T1R2+3 complex are apparently much more effectiv e, at least as measured by the assay used in our study. Sucrose, which was shown to stimulate the T1R2+3 complex in a heterologous system (HEK 293), generated licking at rates at leas t four times higher than any other compound tested. Partial support for this dissociation co mes from the fact that glycine, which was also shown to stimulate the T1R2+3 complex, but to a lesser extent, in general elicited slight increases in licking at high concentrations resembling its modest ability to bind with the receptor (see Nelson et al. 2002), at least in those mi ce that sampled all of the concentrations. We found no evidence that L-serine, a compound that binds with the T1R1+3 heteromer, but not with the T1R2+3, is an effective behavior al stimulus in the brief-access test in non-deprived mice. As noted above, there is evidence that L-amino acids can stimulate significant degrees of lic king in mice that have restricted food and water access. Thus, it would appear that the affective value of stimuli that bind with the T1R1+3 receptor depends upon the nutritiona l/physiological status of the animal, whereas stimuli that bind with the T1R2+3 receptor do act like ge neral attractants. The behavioral results presented here do not relate to the electrophysiological response properties of the CT nerve in an obvious way (Frank and Blizard, 1999; Inoue et al. 2001). While all three stimuli used in our study reportedly evoke very clear

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29 concentration-related increases in CT responsiveness in B6 and 129 mice, the concentration-response functions for glycine and L-serine in non-deprived mice from these strains in the brief access test had ve ry shallow slopes. Moreover, while the magnitude of CT responses to sucrose is grea ter in B6 compared with 129 mice even at high concentrations, the 129 mice displayed mo re vigorous sucrose licking than the B6 mice at the 0.5 and 1.0 M concentrations in the br ief-access test. It is conceivable that a subclass of CT fibers might display a bette r correspondence with th e hedonic value of these stimuli and this relationship might be obscured in whole-nerve analyses (cf., Frank and Pfaffmann, 1969). However, it is likely that the affective potency of these stimuli is based on more than just input from the CT. Input from ot her peripheral nerves and the central neural circuits that tr anslate those signals into behavi or must be considered. Thus, while non-taster strains might have an impa ired peripheral signal for certain sweeteners that stimulate the T1R2+3 receptor complex, the way that input is processed by the brain can also differ from taster strains in a manne r that could augment behavior. Likewise, a robust peripheral signal for gl ycine or L-serine or any ta ste stimulus does not guarantee that a given behavioral response will be generated. In summary, we found that sucrose was the most effective compound tested, followed by glycine, and lastly L-serine in gene rating licking in the brief-access taste test. The order of affective potency se ems to be related to the ability of the stimulus to activate the T1R2+3 receptor complex. Furthermore, st rain differences in responsiveness to these compounds suggest that the current understand ing of sweet-tasting ligand transduction is insufficient in entirely explaining the observe d response profiles. For example, the fact that the 129 mice licked at rates greater th an the D2, B6 and SWR mice to the higher

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30 concentrations of sucrose would not have been predicted by the current molecular biological findings or CT nerve recordings. Apparently, the taster/ non-taster distinction which has been shown to be dependent on the polymorphism of the Tas1r3 gene encoding for the T1R3 receptor is limited to low concentrations of sucrose, whereas responsiveness to higher concentr ations of the sugar is relate d, at least in part, to other genes that might affect stimulus processing anywhere along the gustatory neuraxis. It would be instructive to repeat the behavioral tests conducted here in congenic, transgenic and/or knock-out mice in which the Tas1r3 gene has been manipulated keeping the genetic background constant to examine the explicit role of the T1R3 variants in behavioral responsiveness to mid-range and high concentrations of sugars, synthetic compounds and amino acids. The results of our study also call into question the very nature of the perceptual quality elicited by these amino acids. As noted above, there is evidence from conditioned taste aversion gene ralization experiments that rodents treat glycine and L-serine as possessing a sucrose-like taste quality (Nowlis et al. 1980; Pritchard and Scott, 1982b; Kasahara et al. 1987). Yet, in the brief-access test with the non-deprived mice tested here, the responses to sucrose were discer nibly different than those to the amino acids. Thus, it would appear that while the perception evoked by glycine and L-serine might share some qualita tive characteristic with sucrose, these amino acids might also generate additional qua lities that impact upon their affective value at least in certain species a nd strains. For example, sacch arin is both sweet and bitter tasting to humans depending on con centration (Bartoshuk, 1979; Schiffman et al. 1979). Experiments designed explicitly to test the ab ility of these mice to distinguish between sucrose, glycine, L-serine, and other Lamino acids and sugars in operant taste

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31 discrimination tasks, in addition to a more comprehensive examination of conditioned taste aversion generalization profiles should help refine the characteri zation of the qualitative similarities and differences of these taste stimuli. Such behavioral experiments can provide a functional context to guide the interpretation of findings from more molecular levels of analysis.

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32 Table 2-1: Mean number of licks to water SEM taken by the four strains when tested with sucrose, Lserine, or glycine when mice were water deprived. Sucrose L-serine Glycine B6 32.2 1.4 30.6 2.3 32.3 2.2 SWR 41.8 2.2 44.1 1.9 48.3 1.5 129 22.9 1.7 34.0 1.9* 24.8 2.3 D2 34.8 2.5 47.1 1.9* 32.8 2.9 Asterisk indicates that the 129 and D2 non-taster mice took significantly higher numbers of licks to water when tested with L-serine relative to sucrose or glycine.

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33 Table 2-2: Mean of the inter-lick interval (ILI) distri bution (ms) SEM observed in the four strains of mice trained to lick either sucrose, L-serine, or glycine. Sucrose L-serine Glycine B6 121.3 1.1 122.0 1.5 121.3 1.9 SWR 98.0 1.2 98.6 1.4 94.4 1.0 129 108.3 1.1 109.8 0.8 108.6 1.7 D2 104.4 2.3 105.9 1.8 104.7 2.8 For a given strain, the observed ILI value did not significantly differ whether tested with sucrose, L-serine, or glycine.

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34 Table 2-3: Strains listed in order of mean Tastant/Water Lick ratio, for sucrose, L-serine, and glycine when mice were water de prived. 1At concentrations at which the ANOVA detected a significant st rain effect, strains falling under the same line did not signific antly differ in Tukeys HSD posthoc comparisons (P<0.05). 0.25 M: 129=B6=SWR=D2 0.5 M: SWR129=B6=D2 0.75 M: 129SWR=B6=D2 1.0 M: 129=B6=SWR>D2 1.5 M: SWR=129=B6>D2 L-serine 1 0.25 M: 129 = B6 = SWR = D2 0.5 M: 129SWR = B6 = D2 0.75 M: 129 > B6 = SWR = D2 1.0 M: 129 > D2 = B6 = SWR 1.5 M: 129 > D2 = SWR = B6 Glycine 1 0.0625 M: 129SWR = D2 = B6 0.125 M: 129 > SWR = D2 = B6 0.25 M: 129 > SWR = D2 = B6 0.5 M: 129 > SWR = D2 = B6 1.0 M: 129 > SWR = D2 = B6 Sucrose 1

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35 Table 2-4: Strain listed in order of mean Standardized Lick ratio, for sucrose, L-serine, and glycine when mice were non-depriv ed. 1For sucrose and glycine, at concentrations at which the ANOVA detected a significant strain effect, strains falling under the same line did not significantly differ in Tukeys HSD posthoc comparisons (P<0.05). 2There were no significant st rain effects for L-serine. Water: SWR=B6=D2=129 0.0625 M: SWRB6=D2=129 0.125 M: SWRB6=129=D2 0.25 M: SWR129B6>D2 0.5 M: 129>SWR=D2=B6 1.0 M: 129>D2=SWR=B6 Sucrose 1 Water: B6 = SWR = D2 = 129 0.25 M: 129 = B6 = D2 = SWR 0.5 M: D2 = SWR = B6 = 129 0.75 M: B6 = 129 = D2 = SWR 1.0 M: B6 = 129 = SWR = D2 1.5 M: B6 = 129 = SWR = D2 L-serine 2 Water: SWR=B6=D2=129 0.25 M: B6=D2=129=SWR 0.5 M: B6=129=D2=SWR 0.75 M: B6=129=SWR=D2 1.0 M: 129B6=D2=SWR 1.5 M: 129B6=D2=SWR Glycine 1

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36 SucroseConcentration (M) 0.0650.20.30.40.50.65 0.11 Tastant/Water Lick Ratio 0.0 0.5 1.0 1.5 2.0 L-serine 0.250.40.50.60.751.5 1 Glycine 0.250.40.50.60.751.5 1 B6 SWR 129 D2 Figure 2-1: Mean ( SE) Tastant/Water Lick Ratio as a function of sucrose, L-serine and glycine concentration for four different inbred strains o f mice (n=10/stimulus/strain). The Tastant/Water Lick Ratio was calculated by dividing an animals average licks to a given taste sti mulus across trials by the average licks to water. The dashed line on the graph repres ents a Tastant/Water Lick Ratio of 1.0, which indicates licking to the taste stimulus was equivalent to licking to water. This ratio controls for differences in oral motor competence and physiological state. The se animals were tested in 3 consecutive sessions while on a 23.5 h water restriction schedule.

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37 SucroseConcentration (M) 0.0650.20.30.40.50.65 0.11 0.0 0.2 0.4 0.6 0.8 1.0 L-serine 0.250.40.50.60.751.5 1 Glycine 0.250.40.50.60.751.5 1 B6 SWR 129 D2 Standardized Lick Ratio Figure 2-2: Mean ( SE) Standardized Lick Ratio as a function of sucrose, L-serine and glycine concentration for four different inbred strains of mice. The Standardized Lick Ratio was calculated by dividing an animals average licks to a given taste stimulus across trials by the max imum potential licks in a 5-s trial, derived from that animals previously measured inter-lick interval distribution. This score is used for normal ly preferred stimuli and controls for differences in characteristic local lick rates. A score of 1.0 reflects licking to the taste stimulus that was at the maximum possible rate. These animals were tested non-deprived in 3 consecutive sessions. Only mice that had at least 1 trial at every concentration w ere included in the analysis of a given stimulus (sucrose: B6 [n=10], SWR [n=8], 129 [n=10], D2 [n=10]; L-serine: B6 [n=9], SWR [n=4], 129 [n=7], D 2 [n=5]; and glycine: B6 [n=9], SWR [n=7], 129 [n=5], D2 [n=9]).

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38 CHAPTER 3 EXPERIMENT 2: TASTE DISCRIMINAB ILITY OF L-SERINE AND VARIOUS SUGARS BY MICE Background Conclusions regarding the taste quality of amino acids based on data from CTA experiments suggest that a subset of amino aci ds is perceptually similar to sucrose and some other sugars. Consistent with this vi ew, results from two-bottle intake experiments show that these amino acids are preferred by mice. However, although L-amino acids are thought to bind exclusively with the T1R1+3 receptor complex, preference behavior measured in the two-bottle intake test s eems to depend on an anomaly in the T1R2+3 complex across strains. Moreover, data gath ered in our laboratory question whether or not these compounds are actually preferred by mice at all on th e basis of taste and as a result question the very nature of the tast e quality evoked by these compounds. Indeed, the molecular biology of sweetener transd uction appears to provi de a neurobiological basis for behavioral discriminability (i.e., perceptual distinction). Thus, the goal of the current studies was to determine the degree to which the reported receptor specificity predicts the relative discriminability of various sweeteners, by testing whether C57BL/6J (B6) mice can discriminate between sucrose and L-serine, as well as a variety of other sugars using an ope rate discrimination paradigm. To my knowledge, explicit discrimination experiments in rodents with these ligands have never been conducted.

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39 Methodological Details Subjects Adult C57BL/6J (B6) male and female mice (n=12; Jackson Laboratories, Bar Harbor, Maine), ~ 8 weeks of age on arrival, served as subjects. The B6 strain was chosen because 1) it is the most common mouse strain used in taste research, 2) has been previously characterized as a taste r strain (e.g., Capretta, 1970; Pelz et al. 1973; Fuller, 1974; Lush, 1989; Capeless and Whitney, 1995; Bachmanov et al. 1996), and 3) serves as a background strain in many knock-out, conge nic, and transgenic manipulations (e.g., Damak et al. 2003; Zhao et al. 2003). The mice were housed individually in polycarbonate cages in a colony room where the lighting was controlled automatically (12:12). Testing and training took place during the lights-on phase. After arrival in the facility, subjects had free access to pellets of laborat ory chow (Purina 5001, PMI Nutrition International Inc ., Brentwood, MO) and purified wa ter (Elix 10; Millipore, Billerica, MA). Seven days after arrival, mice were put on a restricted water-access schedule with fluid available Monday Friday during testing only. Purified water was freely available on the home cage from Fr iday afternoon through Sunday afternoon every week. Mice that dropped below 85% of hydrated weight while on water-restriction schedule, received 1 ml of supplemental wa ter ~2 hours after the end of the testing session. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Taste Stimuli All solutions were prepared daily with purified water and reagent grade chemicals and were presented at room temperature. Comparison stimuli consisted of various concentrations of sucrose, glucose, maltose, fructose, (Fisher Scien tific, Atlanta, GA) and

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40 L-serine. In addition, sodium chloride (NaCl; Fisher Scientific, Atlanta, GA) was used as a comparison stimulus to provide a contrast that could be easily discriminated from a standard stimulus during training. Sucrose, fructose, and gluc ose were chosen because 1) they are prototypical sweeteners that are commonly used in ta ste experiments, 2) with the exception of fructose, they have been used to differentiate taster (e.g., B6) from nontaster mice in two-bottle pr eference and intake tests (e .g., Stockton and Whitney, 1974; Ramirez and Fuller, 1976; Lush, 1989; Bachmanov et al. 1996; Bachmanov et al. 1997; Bachmanov et al. 2001b), as well as in an operant discrimination task (Eylam and Spector, 2004), and 3) they ar e thought to exclusively bind with the T1R2+3, but not the T1R1+3, receptor complex (Nelson et al. 2001; Nelson et al. 2002; Zhao et al. 2003). L-serine was chosen because 1) there is ev idence that at least in some rodents this compound shares a perceptual quality with sucrose (Kasahara et al. 1987), 2) it is preferred by some strains of mice at mid-range concentrations in two-bottle preference tests (Iwasaki et al. 1985), and 3) appears to bind pr imarily with the T1R1+3, but only poorly, if at all, with the T1 R2+3 receptor complex (Nelson et al. 2002; Zhao et al. 2003). Concentrations of each stimulus tested are listed in Table 3-1. These were chosen on the basis of the available behavioral data and with the intent of representing the dynamic range of responsiveness for B6 mice. Th e concentrations of sucrose and fructose selected encompass the dynamic range of be havioral responsiveness for B6 mice, as measured in a brief-access taste test (Dotson et al. 2005; Glendinning et al. 2005a; 2005b; also see Chapter 2, Figure 2-2). Glucose concentrations were chosen based on the diminished neural and behavioral sensitivity of B6 mice to glucose relative to sucrose

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41 (Ninomiya et al. 1984; Eylam and Spector, 2004). The maltose concentrations encompassed the range of behavioral pref erence for C57BL/6ByJ mice, as measured using a two-bottle intake taste test (Bachmanov et al. 2001b). Very little data has been gathered on the behavioral responsiveness of mi ce to L-amino acids. Thus, the choice of concentrations for L-serine was based on the available neural data. The whole CT nerve of ddy mice responses monotonically to an incr easing concentrations series of L-serine (0.01 M 1 M, neural thres hold = 0.003 0.01 M; Iwasaki et al. 1985). Thus, those concentrations chosen for L-serine were assumed to be within the dynamic range of responsiveness for mice. NaCl concentrati ons represented the range of behavioral responsiveness for C57BL/6ByJ mice, as meas ured in a brief-access taste test (Dotson et al. 2005) and were also thought to be with in the dynamic range of responsiveness for the substrain of B6 mice used here. It is important to stress that the broa d range of concentrations chosen helps guarantee that there will be overlapping intens ities and viscosities across the stimuli. This is important because of the need to render intensity an irrelevant cue while promoting quality as the cons istent discriminable signal. Apparatus Animals were trained and tested in a specially designed computer-controlled gustometer modified for use with mice (Spector et al. 1990; Eylam and Spector, 2002, 2003, 2004, 2005). This apparatus consisted of a modified operant chamber housed in a sound-attenuating cubicle, which had a reinforcement spout positioned on either side of a centrally positioned slot through which a mouse gained access to a sample spout. Background noise inside the sound-attenuating cubicle helped to minimize extraneous auditory cues. All stimuli and reinforcement fluids were placed in pressurized reservoirs

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42 outside the chamber. Compute r-operated solenoid valves re gulated fluid delivery from these reservoirs by controlling the amount of fl uid dispensed from the sample spout. This required that the sample shaft first be f illed with the stimulus and then, with each subsequent lick, ~ 2.0 L were deposited into the shaft. The volume per lick received from the reinforcement spouts was also ~ 2.0 L. At the end of each trial, the sample spout rotated over a funnel, was rinsed w ith purified water, and evacuated with pressurized air. Two cue lights positioned on the ceiling of the test cage above the reinforcement spouts and a house light were tu rned on or off during each trial according to the programmed trial schedule to signal the beginning and/or ending of the various phases of a trial. Experimental Design Two groups of mice were ini tially tested on their ability to discriminate either sucrose or L-serine (standard stimuli) from s odium chloride (comparison stimulus). After successful completion of this discrimination test, the comparison stimulus was changed from NaCl to one of the other compounds liste d in Table 3-2. When this discrimination was completed, the comparison stimulus was changed for a second time. This process was continued until all comparison stimuli had been paired with a given standard stimulus (see Table 3-2). Interposed between test discriminations was a series of sessions during which the animals were rete sted on the NaCl vs. standard stimulus discrimination to measure and maintain stimul us control. These were referred to as stimulus-control sessions. Training (see Table 3-3) a.) Spout training. Mice were trained to lick fro m the different spouts for fluid delivery in the gustometer by pr esenting the animals with onl y one spout each day. Water

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43 was delivered on all 3 days of this traini ng phase and was freely available ad libitum throughout a session. b.) Side training. Next, mice were trained to lic k from a specific reinforcement spout in response to the presentation of a mid-range concentration of one of the two compounds delivered through the sample spout by providing access only to the corresponding reinforcement spout. The access sl ot to the other reinforcement spout was covered. The sample solution and the matc hing reinforcement spout were alternated between days. In this phase, mice were allowed up to 180 s to respond after sampling (limited hold); no time-out contingency was in effect. c.) Alternation phase. During the alternation phase, both stimuli were presented and both reinforcement spouts were avai lable for responses. The limited hold was shortened to 15 s and a criterion number of correct responses (non-consecutively) was required for a change in the sample stimulus. The criterion, which started at four correct responses, was reduced after two sessions to two, and finally, after tw o more sessions to one. The time-out contingency was introduced in this phase as a punishment for incorrect responses. Initially, the time-out was set at 10 s. When the criterion reached 2, it was increased to 20 s, and finally increased to 30 s when the criterion reached 1. d.) Discrimination training. Mice were trained to discriminate stimuli presented in randomized blocks (discrimination traini ng). The time-out was set at 30 s. Then the two other concentrations of each stimulus we re added (discrimination testing). Mice were moved from discrimination traini ng to discrimination testing when group performance reached 85% correct responses a nd at least 75% for each animal. At the start of discrimination testing, th e limited hold was reduced to 10 s.

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44 Testing (see Table 3-3) Mice were trained, as described above, to associate the taste of one stimulus with one reinforcement spout on one side of the samp le spout and the taste of another with the other reinforcement spout on the other side of the sample spout (counterbalanced between animals). Session length was 25 minutes. During this time, each mouse was allowed to complete as many trials as possible. Each tr ial was comprised of 4 phases: (1) the sample phase, (2) the decision phase, (3) the reinfor cement phase, and (4) the inter-trial interval (see Figure 3-1). The sample phase began when the mouse licked from the sample spout available in front of the slot. When initiati ng a trial, the mouse was required to complete an attending response by licking the dry samp le spout twice within 250 ms to trigger stimulus delivery. The mouse was allowed 5 licks or 2 s of stimulus access, whichever came first, before the sample spout was rotate d away from the slot. During this phase, the house lights were on. When the sample spout rotated away from the slot, the decision phase would begin: the house lights would be turned off and the cue lights turned on. During this phase, the mouse was required to decide which reinforcement spout to lick from. The reinforcement phase began as soon as contact was made with one of the side spouts. If a correct choice was made, the house lights were illuminated and the mouse was allowed 15 licks or 4 s of access to water reinforcement. If an incorrect choice was made or no response was initiated within 10 s (limited hold), the mouse received 30 s of time-out during which all lights were extingu ished and no fluid was delivered. When 15 licks were taken, 4 s passed, or when a time-out was completed, the sample spout rotated over a funnel and was rinsed with purified wate r and dried with pressu rized air, and then rotated back into position in front of the slot. This phase, the inter-trial interval, lasted 6 s (all lights were turned off during this phase). As mentioned above, mice were tested with

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45 a range of stimulus concentrations. Duri ng each session, three of the reservoirs were filled with different concentrations of the standard stimulus and three others with different concentrations of th e comparison stimulus (see Table 3-3). The two reservoirs connected to the reinforcement spouts were f illed with purified wate r. Presentation order was randomized without replacement in blocks so that every concentr ation of the stimuli was presented exactly once before the initiation of the s ubsequent block. Data Analysis Discriminability was evaluated using the overall proportion of correct responses as the primary dependent measure. Overall performance was assessed by collapsing all trials across both stimuli and concentrations. Concentration effects were analyzed within each stimulus. These effects, as well as ove rall performance, were tested against chance using one-sample t tests. Performance acro ss weeks was statistically analyzed using analyses of variance (ANOVAs). Only trials in which a response was made were used in the analyses. When technical problems with the gustometer occurred during an animals session, the data were discarded from the an alyses. Overall discriminability at 50% correct equals chance perfor mance. Performance approach ing this value indicated a failure to discriminate. Results As shown in Figure 3-2, both groups of mice, which were trained to discriminate a standard stimulus (e.g., sucrose or L-serine) from NaCl, lear ned the task. This initial phase of discrimination testing la sted for five weeks. During the last two weeks of this phase, both groups performed at ~85% accuracy on average. Overall performance ranged from 76 to 94% correct responses during the last week of testing for the group of mice

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46 trained to discriminate L-serine from NaCl and 76 to 92% correct responses for animals trained to discriminate sucrose from NaCl. Discrimination Testing Table 3-4 lists the perfor mance of the two groups dur ing the various phases of discrimination testing. Group performance on each of the stimulus discrimination pairings is described in greater detail below. Sucrose vs. L-serine Serine group During the first week of the second phase of discrimination testing, both groups of mice were tested for their ability to discriminate sucrose from L-serine. Overall performance dropped to levels significantly be low chance for the group of mice trained to discriminate L-serine from Na Cl [serine group; Figure 3-3; t (5) = -4.9; P < .01; null hypothesis; probability of correct response = .5]. Although the departure from chance was relatively slight (0.44), it i ndicates that there was a slight tendency for animals in this group to treat the novel stimulus as if it we re the standard, alth ough this propensity did not reach statistical significance. During the second week of testing, the performance of the serine group did not significantly differ from chance [Figure 3-3; t (5) = -0.78; P = .472; null hypothesis; probability = .5] nor did it significantly diffe r from performance levels measured during week one [ F (1,5) = 2.8, P = .156]. Sucrose group For the group of mice trained to discrimi nate sucrose from NaCl (sucrose group), overall performance also dropped precipitously during the first week of testing. These data demonstrate that changing one of the ta ste compounds in this discrimination task has

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47 the potential to substantially disrupt perfor mance in both groups. However, these mice did perform at levels above chan ce, albeit poorly [Figure 3-4; t (5) = 5.2; P < .01; null hypothesis; probability = .5]. When looking at performance across all th e individual concentrations of the two stimuli, mice were able to discriminate 0.4 and 0.6 M sucrose at levels above chance [Figure 3-5; both t -values 3.0; P s < .05; null hypothesis; probability = .5]. However, these mice did not respond to 0.2 M sucrose, as well as all three concentrations of Lserine, at levels above chance. During the second week of testing, perfor mance did significantly improve relative to that measured during th e first week [Figure 3-4; F (1,5) = 11.9, P < .05]. Overall performance appeared to improve relative to week one, because mice learned to discriminate L-serine from su crose. That is, they discriminated all concentrations of L-serine at leve ls above chance [Figure 3-5; all t -values 5.2; P s < .01; null hypothesis; probability = .5]. Although th ese mice were able to discriminate 0.6 M sucrose from L-serine [Figure 3-4; t (5) = 2.7; P < .05; null hypothesis; probability = .5], they were unable to discriminate 0.2 and 0.4 M sucrose. However, their performance towards 0.4 M sucrose did approach significance ( P = .058). Overall group performance for the sucros e group was significantly above chance, which would appear to indicate that these s timuli evoke, to some degree, discriminable neural signals. However, mice in the serine group had substantially greater difficulty executing the task. Collectively, these data s uggest that while potenti ally discriminable, the stimuli are, to some degree, qualitatively similar.

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48 Sucrose/L-serine vs. Glucose Serine group During week one of L-serine vs. glucose testing, overall performance dropped to levels significantly below chance for the serine group [Figure 3-3; t (5) = -6.4; p < .01; null hypothesis; probability = .5], again indicat ing that when animals in this group had difficulty discriminating, there was a slight, bu t statistically significant tendency for them to respond to the novel stimulus as if it was the standard [i.e., L-serine; t (5) = -5.1; P < .01; null hypothesis; probability = .5). Their performance, however, did signifi cantly improve during week two [Figure 33; F (1,5) = 50.5, P < .01]. This improvement, however, did not yield performance that was significantly greater than chance [ t (5) = 2.5; P = .052; null hypothesis; probability = .5]. These mice were tested for an additional week to see if group performance would improve. Overall performance during week three differed significantly from that measured during week one [ F (1,5) = 25.2, P < .01] but not from levels observed during week two. After three week s of testing with glucose (and two earlier weeks with sucrose), performance finally reached levels si gnificantly higher than chance [Figure 3-3; t (5) = 3.0; P < .05; null hypothesis; probability = .5]. However, individual performance during this week was highly variable. Indee d, three out of the six mice in this group appeared to have substantial difficulty pe rforming the discrimination (Figure 3-3). Overall performance ranged from 51 to 67% correct responses during week three. Mean performance appeared to improve relative to week one, because mice stopped responding to glucose as if it were L-serine. During week one, mice responded to all concentrations of gluc ose at levels significantly below chance [Figure 3-6; all t -

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49 values -2.6; P s < .05; null hypothesis; probability = .5]. That is to say, mice were responding disproportionately on the L-serine spout. During week three, mice corrected this bias and responded to all three concentrations of glucose at chance levels. During the last week of testing with gl ucose, mean overall group performance for the serine group was significantly above chan ce, which would appear to indicate that these stimuli evoke, to some degree, discrimina ble neural signals. However, as detailed above, group performance was at best medioc re and individual pe rformance was highly variable (see Figure 3-3). As with sucrose vs L-serine testing, th ese data suggest that while potentially discriminable, L-serine and glucose are, to some degree, qualitatively similar. Sucrose group As expected, mice in the sucrose group ha d great difficulty discriminating sucrose from glucose. Their overall performance never differed significantly from chance [week one or week two; Figure 3-4; both t -values 1.6; P s .179; null hypothesis; probability = .5]. In addition, performance levels obs erved during week two did not significantly differ from that measured during week one [ F (1,5) = 0.05, P = .832]. We did not run these mice for a third week for fear of losing st imulus control. Thes e data are consistent with the notion that sucrose and glucose activate the same transduction pathways (e.g., Zhao et al. 2003; see discussion below for elaboration). Mice tended to respond best to sucrose at its highest concentration (0.6 M) and best to glucose at its lowest concentration (0.5 M), although this pattern did not reach statistical significance (see Figure 3-7). In a ny event, this response strategy did not lead to overall performance levels that were grea ter than chance (however, see Sucrose vs. Fructose below).

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50 Sucrose/L-serine vs. Maltose Serine group During the first week of L-serine vs. maltose testing, overall performance was significantly higher than chance [Figure 3-3; t (5) = 3.5; P < .05; null hypothesis; probability = .5]. Mice were able to discrimi nate 0.4 and 0.6 M L-serine at levels above chance [Figure 3-8; both t -values 4.8; P s < .01; null hypothesis; probability = .5]. However, these mice did not respond to 1.0 M L-serine, as well as all three concentrations of maltose, at above chance levels. Oddly, overall performance for these mi ce, although above chance during week one, was not maintained at these levels during the last two weeks of testing. This despite the fact that mean performance increased each week during this phase of testing (see Figure 3-3). During the last two weeks of testing, performance did not significantly differ from chance [Figure 3-3; both t -values 2.4; P s .06; null hypothesis; probability = .5], nor did the values signi ficantly differ from performance levels measured during week one. As with L-serine vs. glucose testing, i ndividual performance was highly variable. Two mice substantially affected the amount of variability observed during L-serine vs. maltose testing, considerably pulling down gr oup performance (Figure 3-3). These same mice performed at ~chance levels during L-se rine vs. glucose testing (Figure 3-3). Overall performance ranged from 46 to 75% correct responses during week three. Sucrose group Unlike the failure of these mice to discri minate sucrose from glucose, they did display some reliable discrimination between sucrose and maltose. During the first week of sucrose vs. maltose testing, overall pe rformance was significantly above chance

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51 [Figure 3-4; t (5) = 4.2; P < .01; null hypothesis; probabi lity = .5]. During week one, mice were able to discriminate 0.2 M sucr ose at levels above chance [Figure 3-9; t (5) = 3.2; p < .05; null hypothesis; probability = .5]. However, these mice did not respond to 0.4 and 0.6 M sucrose, as well as all three c oncentrations of maltose, at above chance levels. Performance did not significantly im prove during weeks two or three. However, by week three, mice were able to discrimina te 0.2 M maltose and all concentrations of sucrose at levels above chance [Figure 3-9; all t -values 2.6; P s < .05; null hypothesis; probability = .5]. The ability of mice to discriminate sucr ose from maltose, detailed above, suggests that maltose is qualitatively distinctive from sucrose and the pattern of responsiveness across concentrations suggest that 0.2 M maltose is more distinguishable from sucrose relative to the higher maltose concentrations tested (i.e., 0.4 and 0.6 M maltose). Sucrose vs. L-serine II Serine group We conducted a second phase of sucrose vs. L-serine testing (Suc rose vs. L-serine II) to ascertain whether the performance of the serine group would improve on this discrimination as a function of experience. Du ring the first week of testing, performance did not significantly differ from chance [Figure 3-3; t (5) = 2.3; P = .07; null hypothesis; probability = .5]. During the next week, however, performance did reach levels significantly higher than chance [Figure 3-3; t (5) = 2.9; P < .05; null hypothesis; proba bility = .5]. Yet these levels were not significantly different from those measured during week one. Mice were then tested for an additional week to see if group performance would improve. During this final week of testi ng, overall performance did differ significantly

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52 from levels measured during week one [ F (1,5) = 17.0, P < .01] but not from that observed during week two. Mice were able to di scriminate all concentrations of L-serine at levels above chance during all three weeks of testing with these stimuli [Figure 3-10; all t -values 2.7; P s < .05; null hypothesis; probability = .5]. However, during this time, they were unable to discriminate any of the sucrose concentrations at above chance levels [Figure 3-10; all t -values 2.1; P s .09; null hypothesis; probability = .5]. This response pattern is consistent with what was s een in the last week of L-serine vs. glucose testing. That is, mice in the serine group di d not respond to any concentration of sucrose or glucose at levels above chance. As with L-serine vs. glucose and maltose testing, individu al performance was highly variable. The two mice that performe d at levels substantially below the other animals during glucose and maltose testing cont inued to do so during this phase of testing (Figure 3-3). Overall performance during week three ranged from 46 to 75% correct responses. The data presented above suggest that mice in the serine group can indeed discriminate both sucrose and glucose from L-serine. That said, this discrimination appeared to be exceedingly difficult for these animals. Sucrose group As in the first sucrose vs. L-serine discrimination task, the sucrose group did perform at levels abov e chance [Figure 3-4; t (5) = 9.7; P < .001; null hypothesis; probability = .5]. The performance during the first week of sucrose vs. L-serine II testing did not significantly differ from the last week of the first phase of sucrose vs. L-serine testing [69 and 72 % group perf ormance, respectively].

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53 Mice were able to discriminate 0.4 and 0.6 M sucrose and all three concentrations of L-serine at levels a bove chance [Figure 3-11; all t -values 4.1; P s < .01; null hypothesis; probability = .5]. As in the first pha se of sucrose vs. serine testing, mice did not respond to 0.2 M sucrose at above chance levels. Overall performance significantly improve d during week two when compared to that seen during week one [Figure 3-3; F (3,36) = 23.7, P < .01]. During this week, mice were able to discriminate all concentrations of both stimuli at levels above chance [Figure 3-11; all t -values 4.7; P s < .01; null hypothesis; probability = .5]. Performance appeared to reach asymptot ical levels during week two. Overall performance levels, by session, did not signifi cantly improve during week two [sessions 6-10: 0.79, 0.83, 0.77, 0.77, 0.75, respectively; F (4,8 = 0.336, P = .85]. As a result, mice were not tested for a third week. Sucrose/L-serine vs. Fructose Serine group During week one, the overall performance of the serine group did not significantly differ from chance [Figure 3-3; t (5) = 1.8; P = .144; null hypothesis ; probability = .5]. During the next week, however, performance did reach levels significantly higher than chance [Figure 3-3; t (5) = 4.7; P < .01; null hypothesis; proba bility = .5]. Yet these levels were not significantly different from these measured during week one. During week two, mice were able to discriminate 1.0 M L-serine, as well as 0.6 and 1.0 M fructose, at levels abov e chance [Figure 3-12; all t -values 3.1; P s < .05; null hypothesis; probability = .5]. Mice were then tested for an additiona l week to see if performance would improve. During this week, overall performance was again significantly above chance

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54 [Figure 3-3; t (5) = 4.6; P < .05; null hypothesis; probability = .5]. Performance did not differ significantly from that measured during week one or two. During this last week, mice were able to discriminate 0.6 and 1.0 M Lserine and 1.0 M fructose at levels above chance [Figure 3-12; all t -values 3.2; P s < .05; null hypothesis; probability = .5]. During this phases of discrimination test ing, individual performance variability decreased somewhat. The two mice that depressed performan ce throughout testing appeared to finally improve ever so slight ly, relative to their behavior measured in preceding phases (see Figure 3-3). Overall performance during week three ranged from 57 to 76% correct responses. Mice in the se rine group, for the most part, demonstrated that, after some experience, they can discri minate all of the sugars, including fructose from their standard stimulus. Sucrose group During the first week of sucrose vs. fr uctose testing, overall performance was significantly greater than chance [Figure 3-4; t (5) = 4.4; P < .01; null hypothesis; probability = .5]. Mice were able to discrimi nate the high concentra tions of sucrose (0.4 and 0.6 M) at levels above chance [Figure 3-13; t (5) = 3.2; p < .05; null hypothesis; probability = .5]. These mice, however, did not respond 0.3 and 0.6 M fructose at above chance levels. Interestingly, mice responded to 1.0 M fructose at levels significantly below chance [Figure 3-13; t (5) =-23.0; P < .001;null hypothesis; probability = .5]. During week two, overall performance was ag ain significantly greater than chance [Figure 3-4; t (5) = 6.9; P < .01; null hypothesis; probability = .5], but not significantly different than performance levels measured during week one. Mice were tested for an additional w eek to see if group performance would improve relative to that measured during week s one and two. During this final week of

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55 testing, performance again was significan tly higher than chance [Figure 3-4; t (5) = 2.8; P < .05; null hypothesis; probability = .5]. However, performance did not differ significantly from that observe d during week one or two. Although no significant changes in the overall percentage of correct responses were observed over the three week s of testing, the pattern of responsiveness towards the various concentrations of the two stimuli did diffe r slightly from week one to week three. By week three, the mice only responded to th e highest concentration of sucrose and the lowest concentration of fructose at above chan ce levels. This pattern of responsiveness is consistent with animals using intensity cu es to guide the discrimination (Figure 3-13; see discussion below). In summary, when mice in the sucros e group were tested on the ability to discriminate sucrose from fructose, these anim als showed that they could do so at levels significantly greater than chance. However, as detailed above, this ability was very limited (~55% correct duri ng the last week of testing) a nd appeared to result from mice responding on the basis of intensity, and not to taste quality, per se. Stimulus Control Sessions Figure 3-14 shows the performance of both groups on the first and last day of a given stimulus-control session, as well data fo r the subsequent discrimination task. The number of stimulus-control sessions require d to reach criterion performance (i.e., 85% group performance for one week or for two cons ecutive sessions at th e end of a week and individual performance 75%) is listed in Table 3-5. As can be seen, after stimuluscontrol I, the number of sessions require d to regain criterion performance dropped dramatically. Collectively, these data demons trate that stimulus control can be quickly

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56 reestablished in experienced mice even afte r a substantial period in which animals are presented with difficult discrimination tasks. Discussion Mice had difficulty, depending on the s timulus and the training history, discriminating sucrose from L-serine, maltose fructose and glucose. Indeed, when concentration effects are taken into consid eration, it appears that mice are unable to discriminate sucrose from glucose or fructose suggesting that these three sugars generate a unitary percept. Monogeusia, or the in discriminability of a class of compounds, has been demonstrated in humans with natural sweeteners (e.g., Breslin et al. 1996) and in rats with bitter tastants (Spector and Kopka, 2002). To my knowledge, monogeusia for natural sweeteners has never been demonstrated in rodents. Monogeusia All mice trained to discriminate sucros e from NaCl (i.e., sucrose group) were entirely unable to discriminate between sucr ose and glucose as assessed by their overall performance during both weeks of testing. Duri ng the first week of testing, mice did not respond to any concentration of either stimulus at levels above chance. Indeed, they responded to 2.0 M glucose at levels significantl y below chance. This indicates that the mice tended to treat the highest concentration of glucose as if it were the standard (i.e., sucrose). Although not significant, this tenden cy persisted in week two. Indeed, mice appeared to be responding on the basis of s timulus intensity. A broad concentration range was used for all compounds so that inte nsities and viscosities would overlap across stimuli. This manipulation was important so that non-qualitative cues would be rendered less redundant, causing quality to be the only c onsistent discriminable signal. However, if the concentration ranges did not sufficiently overlap, then stimulus intensity could be

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57 used as a discriminable cue, albeit not very successfully. Theoretically, if these mice were responding to two qualita tively similar stimuli on the basis of perceived intensity, then they would be expected to respond di sproportionately to a ll weak stimuli on one reinforcement spout and all strong stimuli on the other. Accordingly, as the concentration of the stimulus perc eived to be of higher intensity increases performance levels would also increase This is because, as the concentration of the stronger stimulus increases, it would become more dist inctive relative to the perceived intensity levels of the weaker stimulus. However, as the concentration of the lower intensity stimulus increases performance levels would decrease This is because, as the concentration of weaker stimulus increases, it overlaps, to a grea ter degree, with the perceived intensity levels of the stronger stimulus. It is this opposing concentration dependency that is the telltale sign of a stimulus intensity based taste discrimination. With a response strategy such as this, an imals could successfully discriminate two qualitatively identical stimuli, the concentr ation ranges of which did not sufficiently overlap, at levels greater than chance. Mice appeared to try this very response strategy when attempting to discriminate sucrose from glucose. As can be seen in Figure 3-7, mice responded to sucrose as if it were the stronger of the two stimuli being discriminated (i.e., as the concentration increased performance levels also increased) and to glucose as if it were the weak er stimulus (i.e., as the concentration increased performance levels decreased ). Indeed, by week two, mice were able to discriminate the lowest glucose concentration at levels above ch ance. However, this feat, in and of itself, did not lead to overall performance leve ls that were greater than chance.

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58 When mice were tested for the ability to discriminate sucrose from fructose, these animals showed that they could do so at le vels above chance. However, this ability appeared to result from the mice using intens ity cues. During the first week of testing with fructose, mice did not respond to any conc entration of the stimulus at above chance levels. Indeed, they responded to 1.0 M fructo se at levels signifi cantly below chance. This indicates that there was a tendency for an imals to treat the highest concentration of fructose as if it were the sta ndard (i.e., as the concentration increased performance levels decreased) This response profile is analogous to that observed during the first week of testing with glucose, referred to above. By week three, mice only responded to the highest concentration of sucrose and the lowest concentration of fructose at levels above chance. Moreover, animals tended to respond to the highest concentration of fructose and the lowest concentration of sucros e as if they were the other stimulus, but these tendencies did not meet sta tistical significance standards. As can be seen in Figure 3-13, throughout testing with these stimuli, the opposi ng concentration dependency referred to above is plainly apparent. This response pattern is ex actly what would be predicted if these animals were responding on th e basis of perceived in tensity. Thus, it is likely that the concentrations of fructose a nd sucrose chosen did not sufficiently overlap in intensity. Nevertheless, these results strongly s uggest that B6 mice cannot distinguish perceptually between the tastes of fructose, glucose, or sucrose. Because these are negative findings, we cannot conclu sively rule out that some di scriminative ability exists on the basis of quality. However, if B6 mi ce can distinguish between these compounds, they do so only poorly at best. Thus, it appears clear that in B6 mice, as in humans, all of

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59 these sugars possess a similar taste quality that humans have described as sweet (Breslin et al. 1996). The phenomenon of a class of compounds generating a unitary qualitative percept has been termed monogeusia (Breslin et al. 1996). Monogeusia for sugars likely results from an indiscriminable neural signal that originates from the stimulation of a common receptor(s). As mentioned in Chapter 1, sucrose and fructose were both shown, in vitro to stimulate the T1R2+3 receptor complex. In a separate experiment, again, al luded to in Chapter 1, it was reported that T1R2+3 knock-out mice show no neural or be havioral responsiveness to sucrose or glucose. Thus, the aforementioned behavioral data reported here support the contention that all of these sugars activate the same r eceptor, and based on the molecular biology of sweet ligand transduction, th e likely candidate is the T1R2+3 receptor complex. It should be mentioned, however, that the only index used to assess the neural responsiveness of T1R2+3 knock-out mi ce was whole nerve electrophysiological recording from the CT. Yet, it is likely that detection of these stimuli is based on more than just input from the CT. Moreover, brie f-access testing, which was used to evaluate the behavioral responsiveness of these knoc k-out mice, only assesses the motivational properties of a tastant, not the relative detectability of that s timulus, per se. As mentioned in Chapter 1, taste stimulus detection is best assessed from tasks in which taste serves as a cue for some other event (e.g., reinforcement or punishment) that will generate a trained directed response regardless of the hedonic characteristics of the taste stimulus. Indeed, recent data, gathered using such procedures, call into question the notion that the T1R2+3 receptor complex is exclusiv ely responsible for the activation of TBCs responsible for the detection of sugars. Thes e results suggest that at least one sugar can

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60 be transduced independently of at least one of the members of the T1R2+3 receptor complex. It was reported that mutant mice, lacking the receptor T1 R3, show absolutely no deficit in their ability to dete ct the presence of sucrose (Delay et al. 2006). It should be mentioned, however, that these data are so mewhat controversial and have yet to be replicated. That said, it does appear possible for the T1R2 receptor to bind with taste ligands in the absence of T1R3 (e.g., Nie et al. 2005; Temussi, 2006), although this remains to be demonstrated in vivo Maltose, however, appears to generate a dis tinctive taste quality relative to sucrose, depending on concentration. Although mi ce could only discriminate the lowest concentration of maltose at levels above chance, they were able to discriminate all three concentrations of sucrose successfully during the last week of te sting. This response pattern is quite different from that obs erved when these mice were attempting to discriminate sucrose from either glucose or fr uctose. Therefore, these data suggest that, although qualitatively similar, sucrose and malto se must generate a discriminable neural signal at the periphery. Surpri singly, however, it is the lowe st concentration of maltose that appears to be the most distinctive, suggesting that as the concentration increases, maltose becomes qualitatively more similar to sucrose. The discriminability of maltose and sucrose has been demonstrated, in r odents and humans, by a variety of other researchers (e.g., Ninomiya et al. 1984; Spector and Gr ill, 1988; Breslin et al. 1996; Spector et al. 1997). In addition to sucrose and glucose, T1R2 +3 knock-out mice also show no neural or behavioral responsiveness to maltose. If thes e data are correct, and the T1R2+3 receptor complex mediates the transduction of both su crose and maltose, then some other factor

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61 must be influencing the nature of the signa l arising from the pe riphery. Possibilities include ligand binding/TBC activation characteris tics that lead to differential signaling (e.g., rise & decay). It is also possible that other undiscovered members of the T1R family of receptors or other mechanisms of ligand transduction ex ist that generate a discriminable neural signal. It would be informative to see if T1R2+3 knock-out mice could detect the presence of maltose in a task explicitly designed to assess taste thresholds (e.g., an operate detection task). If so, data such as these would definitively prove that maltose could be transduced i ndependently of the receptors T1R2 and/or T1R3. Sugars vs. L-serine All mice in the sucrose group learned to di scriminate sucrose and L-serine. After many weeks of experience with sucrose as a st andard, they were able to discriminate sucrose from L-serine ~80% of the time during th e last week of testing with these stimuli. As a group, these mice responded to all concentr ations of both stimuli at levels above chance. Mice in the serine group were able to discriminate all the concentrations of Lserine from sucrose. These mice were also ab le to discriminate L-serine from glucose, fructose, and maltose at levels above chance. The data presented above suggest that sucr ose and L-serine are distinguishable. On the other hand, the data also s uggest that the two stimuli shar e some qualitative features. A comparison of the performance of animal s when they were discriminating their respective standard stimulus from NaCl during the stimulus -control sessions (i.e., ~85 90% correct responses) to that observed when they were attempting to discriminate sucrose from L-serine indicates that there wa s some degree of per ceptual confusion (see Figures 3-3 and 3-4).

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62 In addition to maintaining and providi ng a measure of stimulus control, the stimulus-control sessions also provided a pe rformance standard based on a discrimination task that was easy for the animals to accomplish. Because these stimuli (i.e., sucrose/L-serine vs. NaCl) are thought to be independently coded at the periphery (i.e., independently transduced; e.g., Zhang et al. 2003), it was postulat ed that mice could perform this discrimination task without a grea t deal of difficulty. It was assumed that deviation from 100% correct responding dur ing these sessions was the result of nonsensory factors (e.g., motivational state, task difficulty, etc). T hus, during the various discrimination testing phases, it was belie ved that deviation from the performance standards observed during the stimulus-control sessions indi cated increasing perceptual confusion. This uncertainty likely result ed from comparison stimuli evoking neural signals that are more similar to one anothe r relative to those ge nerated by sucrose/Lserine and NaCl. Indeed, despite five weeks of testing with these stimuli, mice trained to discriminate L-serine from Na Cl (i.e., serine group) never responded to any concentration of sucrose at levels above chance. During th e first phase of sucrose vs. L-serine testing, animals in the sucrose group were unable to discriminate 0.2 M sucros e from L-serine. It took until the second week of the second phase before they were able to learn the discrimination. These findings imply that 0.2 M sucrose tastes more similar to L-serine than do the higher concentrations of sucrose. Accordingly, L-serine, across the range of concentrations tested, likely evokes a mild sweetness relative to that elicited by 0.4 and 0.6 M sucrose. This fact might contribute to the lack of appetitive behavior displayed towards this stimulus detailed in Chapter 1.

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63 Collectively, these data suggest that at least some of the signals generated by the receptor(s) responsible for the transduction of these stimuli converge somewhere alone the gustatory neuraxis. Might this converg ence be at the initial site of stimulus transduction (e.g., the TBCs themselves)? As mentioned in Chapter 1, Kim et al. 2003 reported, contrary to previous studies, that there exists a population of TBCs that coexpress all three T1R receptors. Perhaps these cells mediate the signals that are responsible for the qualitative similarity betw een these stimuli. It should be said, however, that even if they do exist, it remains unclear what the response properties of such a cell would be. Anothe r possibility is that sweet -tasting amino acids achieve their qualitative similarity with sucrose by binding with the same receptor(s) (e.g., T1R2+3). Indeed, as mentioned in Chapte r 2, the putative sweet-tasting amino acid glycine appears able to bind with the T1R2 +3 and the T1R1+3 receptor complex. This hypothesis is consistent with the fact, as mentioned in Chapter 2, that two-bottle preference for sweet-tasting L-amino aci ds and glycine appears to depend on the taster status of the mouse strain based on testing with sugars. Perhaps L-serine, like glycine, has the ability to activate transduc tion pathways also activated by sucrose. However, it should be mentioned that in an operant discrimination ta sk designed to assess taste stimulus thresholds, values for glycin e in mice did not distinguish taster and nontaster strains in straightforward a manner (E ylam and Spector, 2004). That said, glycine detectability may be unaffected by the pol ymorphism in the T1R3 receptor. As mentioned in Chapter 2, this polymorphism se lectively affects the functionality of the T1R2+3 receptor complex. Thus, taste inpu t from remaining func tional receptors (e.g., T1R1+3) could be sufficient to maintain task performance. However, the loss of taste

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64 input from the T1R2+3 receptor complex, which mediates, at least in part, the affective potency of highly appetitive nature sweetener s such as sucrose, would likely impact upon the relative affective valence of glyc ine, if not upon its threshold levels. Training history had a huge impact on the abil ity of mice to discriminate all of the stimuli, particularly L-serine from sucrose. Mice, in the serine group, appeared to have more difficulty discriminating L-serine from sucrose relative to animals in the sucrose group. Indeed, two mice in the serine group we re unable to discrimi nate L-serine from all of the sugars. This training history as ymmetry may have resulted from a relative difference in the efficacy of sucrose and Lserine as standards (i.e., compounds to which all comparison stimuli are discriminate d against). This difference in efficacy likely results from the degree of qualitative purity elicit ed by sucrose and L-serine, which is discussed in detail in the following chapters.

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65 Table 3-1: Stimulus concentrations Concentrations Sucrose 0.2, 0.4, 0.6 M Glucose 0.5, 1.0, 2.0 M Maltose 0.2, 0.4, 0.6 M Fructose 0.3, 0.6, 1.0 M L-serine 0.4, 0.6, 1.0 M NaCl 0.2, 0.4, 0.6 M

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66 Table 3-2: Order of stimulus discrimination pairings Group Standard Stimulus Comparison Stimuli 1 Sucrose NaCl L-serine Glucose Maltose L-serine Fructose 2 L-serine NaCl Sucrose Glucose Maltose Sucrose Fructose

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67 Table 3-3: Representative trai ning and testing parameters for the 2 discrimination groups Phase Stimuli Limited Hold Sample Licks/time(s) Reinf. Licks/time(s) Timeou t(s) Presentation Schedule Spout Training H2O None None Constant Side Training Middle Conc. of Comparison Stimulus 1 and Middle Conc. of Standard Stimulus 180 5/2 15/30 0 Constant Alternation Middle Conc. of Comparison Stimulus 1 and Middle Conc. of Standard Stimulus 15 5/2 15/30 10, 20, or 30 Criterion (4-1) Discrimination Training Middle Conc. of Comparison Stimulus 1 and Middle Conc. of Standard Stimulus 15 5/2 15/4 30 Randomized blocks Discrimination Testing All Conc. Comparison Stimulus 1 and All Conc. Standard Stimulus 10 5/2 15/4 30 Randomized blocks Discrimination Testing All Conc. Comparison Stimulus 2 and All Conc. Standard Stimulus 10 5/2 15/4 30 Randomized blocks Discrimination Testing All Conc. Comparison Stimulus 3 and All Conc. Standard Stimulus 10 5/2 15/4 30 Randomized blocks Discrimination Testing All Conc. Comparison Stimulus 1 and All Conc. Standard Stimulus 10 5/2 15/4 30 Randomized blocks Discrimination Testing All Conc. Comparison Stimulus 4 and All Conc. Standard Stimulus 10 5/2 15/4 30 Randomized blocks

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68 Table 3-4: Overall percentage correct during the last week of testing for the stimulus discrimination pairings. Standard Stimulus Comparison Stimuli Sucrose NaCl L-serine SCI Glucose SCII Maltose SCIII L-serine SCIV Fructose Overall % Correct 85% 69% 85% 52% 82% 63% 84% 79% 89% 56% L-serine NaCl Sucrose SCI Glucose SCII Maltose SCIII Sucrose SCIV Fructose Overall % Correct 85% 48% 86% 58% 83% 60% 85% 65% 83% 65% SC = stimulus-control sessions

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69 Table 3-5: Number of stimuluscontrol sessions required to criterion performance. Stimulus Control Sessions SCI SCII SCIII SCIV Sucrose 15 5 5 5 L-serine 20 5 7 5

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70 Figure 3-1: Trial structure (see text for more details). or No response in 10 sec Time Out No fluid 30 sec Incorrect Sampling Phase 5 licks or 2 sec Decision Phase 10 s Reinforcement H2O 15 licks or 4 s Correct Intertrial Interval 6 sec Trial Structure

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71 Discrimination Testing 45 OVERALL PROPORTION CORRECT 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose vs. NaCl L-serine vs. NaCl Weeks 45 0.5 0.6 0.7 0.8 0.9 1.0 Figure 3-2: Individual animal (symbols) and group mean ( SEM; grey bars) data for mice trained to discriminate either sucrose or L-serine from NaCl Performance on all trials with a lever press is depicted collapsed across all stimuli during a week. Chance performance equaled 0.5.

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72 SerVSuc 12 OVERALL PROPORTION CORRECT 0.4 0.5 0.6 0.7 0.8 0.9 1.0 SerVGlu 123 SerVMalWeeks 123 SerVSucII 123 SerVFru* 123 "Serine" Group Figure 3-3: Individual animal (symbols) and group mean ( SEM; grey bars) data are plotted across all test phases for mice init ially trained to discriminate Lserine from NaCl. Performance on all trials with a lever press is depicted collapsed across all stimuli during a week. Chance p erformance equaled 0.5. (*) One mouse was removed from L-serine vs. fructose testing because of illness.

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73 SucVSer 12 OVERALL PROPORTION CORRECT 0.4 0.5 0.6 0.7 0.8 0.9 1.0 "Sucrose" Group SucVGlu 12 SucVMalWeeks 123 SucVSerII 12 SucVFru 123 Figure 3-4: Individual animal (symbols) and group mean ( SEM; grey bars) data are plotted across all test phases for mice init ially trained to discriminate sucrose from NaCl. Performance on all trials with a lever pres s is depicted collapsed across all stimuli during a week. Chance performance equaled 0.5.

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74 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.40.61 L-serine Sucrose vs. L-serine "Sucrose" Group 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.40.61 Week 1 Figure 3-5: Mean ( SD) data for mice attempting to discriminate L-serine from sucrose. These mice were initially trained to discriminate sucrose from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed ac ross a week. Chance pe rformance equaled 0.5.

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75 Week 2 Concentration (M) 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.512 Week 1 L-serine vs. Glucose 0.40.61 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 L-serine 0.512 Week 3 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.512 Glucose Figure 3-6: Mean ( SD) data for mice attempting to discriminate L-serine from glucose. These mice were initially trained to di scriminate L-serine from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.

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76 Week 1 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.512 Glucose Sucrose vs. Glucose 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.512 Figure 3-7: Mean ( SD) data for mice attempting to discriminate sucrose from glucose. These mice were initially trained to discriminate sucrose from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed ac ross a week. Chance pe rformance equaled 0.5.

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77 Week 2 Concentration (M) 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.20.40.6 Week 3 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.20.40.6 Maltose Week 1 L-serine vs. Maltose 0.40.61 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 L-serine 0.20.40.6 Figure 3-8: Mean ( SD) data for mice attempting to discriminate L-serine from maltose. These mice were initially trained to di scriminate L-serine from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.

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78 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.20.40.6 Week 3 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.20.40.6 Maltose Week 1 Sucrose vs. Maltose 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.20.40.6 Figure 3-9: Mean ( SD) data for mice attempting to discriminate sucrose from maltose. These mice were initially trained to dis criminate sucrose from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.

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79 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.40.61 Week 1 Sucrose vs. L-serine II "Serine" Group 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.40.61 Week 3 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.40.61 L-serine Figure 3-10: Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a s econd time. These mice were init ially trained to discriminate Lserine from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance p erformance equaled 0.5.

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80 Week 1 Sucrose vs. L-serine II "Sucrose" Group 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.40.61 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.40.61 L-serine Figure 3-11: Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a second time. These mice were initially trained to discriminate sucrose from NaCl. Performance, by concentration, on all trials with a lever pre ss is depicted collapsed across a week. Chance performance equaled 0.5.

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81 Week 1 L-serine vs. Fructose 0.40.61 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 L-serine 0.30.61 Week 2 Concentrartion (M) 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.30.61 Week 3 0.40.61 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.30.61 Fructose Figure 3-12: Mean ( SD) data for mice attempting to discriminate L-serine from fructose. These mice were initially trained to discriminate L-serine from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.

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82 Week 1 Sucrose vs. Fructose 0.20.40.6 Performance 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Sucrose 0.30.61 Week 2 Concentration (M) 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.30.61 Week 3 0.20.40.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.30.61 Fructose Figure 3-13: Mean ( SD) data for mice attempting to discriminate sucrose from fructose. These mice were initially trained to d iscriminate sucrose from NaCl. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.

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83 SVS FL OVERALL PROPORTION CORRECT 0.5 0.6 0.7 0.8 0.9 1.0 "Sucrose" Group "Serine" Group SCII FL SVM FL SCIII FL SS2 FL SCI FL SVG FL SCIV FL Discrmination Testing Phases SVF FL Figure 3-14: Mean ( SEM) data for both groups of mice are plo tted across all test phases. Perfor mance on all trials with a lev er press is depicted averaged across all stimuli in a session. Only the first (F) and last (L) day of each phase are shown. The s ucrose group was initially traine d to discriminate sucrose from NaCl. The serine group was initially trained to discriminate L-serine from NaCl. Chance pe rformance equaled 0.5 (SVS = S ucrose vs. Lserine; SVG = Sucrose/L-serine vs. Glucose; SVM = Sucrose/L-seri ne vs. Maltose; SS2 = Sucrose vs. L-serine II; SVF = Sucrose/Lserine vs. Fructose; SC = stimulus-control sessions).

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84 CHAPTER 4 EXPERIMENT 3: PERCEIVED SIMI LARITY BETWEEN L-SERINE, LTHREONINE AND CHEMICAL COMPOUNDS REPRESENTATIVE OF THE FOUR BASIC TASTE QUALITIES Background The results presented in Chapter 3, as well as those from CTA generalization studies, suggest that a subset of L-amino aci ds, including L-serine, share a qualitative similarity with the taste of sucrose. Howeve r, data detailed in Chapter 2 show that Lserine appears to be unlike sucrose in its ab ility to generate licking in mice. Thus, while it would appear that L-serine might share some qualitative characteristics with sucrose, this amino acid does not share the affectiv e potency of this sugar. One possible explanation for this finding is that L-serine generates additional qua lities that impact upon its affective value. For example, sacchar in is both sweet and bitter tasting to humans depending on concentratio n (e.g., Bartoshuk, 1979; Schiffman et al. 1979). Although, saccharin was initiall y thought to only activate th e T1R2+3 receptor complex, researchers has recently demonstrated, using an in vitro preparation, that it also activates the human bitter receptors hTAS2R43 and hT AS2R44 at concentrations known to elicit a bitter-taste perception in humans (Kuhn et al. 2004). It is quite pos sible that at least some L-amino acids also activate separate tr ansduction pathways that lead to different taste perceptions. Although all 20 common L-amino acids, including L-serine, were shown to interact with the mouse T1R1+3 receptor complex (Nelson et al. 2002), behavioral studies in rodent s and humans suggest that th e taste qualities evoked by Lamino acids are varied (e.g., Ninomiya et al. 1984; Shallenberger, 1993). These data

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85 suggest that L-amino acids may activate other taste transduction pathways independent of the T1R1+3 receptor complex. To my knowledge, the only CTA generaliza tion study that directly addressed the taste quality(ies) evoked by L-serine in mice was conducted by Kasahara and colleagues (1987). These researchers showed that when conditioned to avoid 0.2 M sucrose, ddy mice generalized the aversion to 0.2 M L-serine However, these re searchers did not use L-serine as a conditioning stimulus (only as a test stimulus). If also used as a conditioning stimulus, then perhaps mice c onditioned to avoid L-serine would have generalized an aversion to one or more of the other tested compounds thought to represent other basic tastes. Thus, to inve stigate the taste quali ties evoked by L-serine, CTA generalization tests were conducted in the current investigation. In addition, the similarity of the L-amino acid, threonine, to compounds representative of the four basic tastes was also investigated. I am unawar e of any prior CTA generalization experiments that have explicitly examined the qualitative similarity of L-threonine and compounds representative of the basic taste qualities. Methodological Details Subjects Adult C57BL/6J (B6) male mice (n=44; J ackson Laboratories, Bar Harbor, Maine), ~ 10 weeks of age on arrival, served as s ubjects. The use of B6 mice allowed for a comparison of results with those obtained in Experiment 2. The mice were housed individually in polycarbonate cages in a colony room where the lighting was controlled automatically (12:12). Testing and training took place during the lights-on phase. After arrival in the facility, subjects had free access to pellets of laboratory chow (Purina 5001, PMI Nutrition International Inc., Brentwood, MO ) and purified water (Elix 10; Millipore,

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86 Billerica, MA) for six days before training and testing took place. All procedures, described below, were approved by the Inst itutional Animal Care and Use Committee at the University of Florida. Taste Stimuli All solutions were prepared daily with purified water and reagent grade chemicals and presented at room temperature. The co mpounds that served as conditioned stimuli (CSs) were sucrose, L-serine, and L-threoni ne. The logic of choosing sucrose and Lserine as CSs was the same as that used to justify their inclusion in the discrimination experiment. That logic was described in deta il in Chapter 3. The choice of L-threonine as a CS was based on the fact that it reportedly gives rise to a sweet taste in humans (e.g., Shallenberger, 1993) and is pref erred, at certain concentratio ns, by rodents (Pritchard and Scott, 1982a; Iwasaki et al. 1985). The panel of test s timuli (TSs) was composed of various concentrations of sucrose, L-seri ne, L-threonine, NaCl, citric acid (Fisher Scientific, Atlanta, GA), a nd quinine hydrochloride (QHCl; Sigma-Aldrich, St Louis, MO). Sucrose, NaCl, citric acid, and QHCl were chosen as TSs because they are frequently used as prototypical representa tives of compounds that elicit a sweet, salty, sour, and bitter taste, respectively (S chiffman and Erickson, 1980). L-serine, Lthreonine, and sucrose were also used as TSs to allow for the assessment of any generalization asymmetries. Asymmetrical relationships can aris e when the conditioning and test stimuli, albeit similar, are not qual itatively identical. Yet, generalization, or the lack thereof, can also occur based on stimul us characteristics othe r than quality (i.e., stimulus intensity; see Nowlis, 1974). As a result, if the conditioning and test stimuli evoke a qualitatively identical percept, but the TS is of a low intens ity relative to the CS, then expression of the learned aversion may be weak or non-existent. Thus, the use of

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87 more than one concentration increases ones confidence that a learned aversion towards a CS will generalize to at least one of the con centrations of a TS (Spector and Grill, 1988; Spector, 2003). In view of this, two con centrations of each compound were included in the test stimulus arrays. Thes e concentrations are listed in Table 4-1. The concentrations chosen for L-serine, L-threonine, and sucrose were th e same as those used in the discrimination experiment described in Chapte r 3. For QHCl, citric acid, and NaCl, an attempt was made to choose concentrations that would produce comparable sensation magnitudes. Stimulus concentrations th at produced ~equivalent degrees of lick avoidance (Tastant/Water Lick Ratio: taste s timulus licks/water licks) were chosen from the dynamic range of behavioral responsivene ss for C57BL/By6J mice, as measured in a brief-access taste test (Dotson et al. 2005). For the high concentration, stimuli that produced a ~50% decrease in the lick rate of animals, relative to water, were chosen. For the low concentration, those th at produced a ~25% decrease were chosen. These values were chosen in an attempt to elicit, in non-conditioned mice, lick rates to these normally avoided stimuli that were sufficiently high enough to allow any differences between conditioned and non-conditioned mice to be fully discernible. Apparatus Lickometer training and te sting took place in an appa ratus commonly referred to as the Davis rig (Davis MS-160, DiLog Inst ruments, Tallahassee, FL; see Smith, 2001). This device allows a mouse access to a singl e sipper tube containing a stimulus. Animals can also be restricted to licking in brief trials (5 s) by offering access to the different tubes via a motorized table and shutter. An 8-s inter-presentation in terval was interposed between stimulus presentations. The test ar ray for each mouse included the two different concentrations of each test stimulus detail ed above, the CS, and purified water all of

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88 which were contained in separa te bottles (i.e., fourteen stimul us sipper tubes; in addition to the stimulus tubes, a non-s timulus rinse sipper tube wa s also included in the array see details below). A given trial started upon the first lick. Each lick on a sipper tube was registered by a contact circuit. These re sponses were recorded by computer for later analysis. During the conditioning phase, intake test s were conducted in the home cages. Fluids were presented in 25 ml graduated pipe ttes fitted with stainless steel sipper tubes on one end. Pipettes were secured to the shel f above with cable clip s to reduce spillage. Intake was measured to the nearest 0.1 ml during this phase. Experimental Design Davis rig training The mice were trained under a restricted water-access schedule. Water bottles were removed from the home cages the day before the start of the training phase. The mice were first trained for 2 daily consecutive 30-min sessions in the Davis rig with a stationary sipper tube contai ning purified water positioned in front of the access slot. The mice were allowed to take as many licks as possible within a 30min session. Next, the mice were trained for 3 days in the Davis rig with a brief-access paradigm, in which access to water was available in 5-s trials from fourteen different sipper tubes. A water rinse (5 lick maximum) presentation was inte rposed between all trials. This was done so that stimulus presentation was consistent with that during the Davis rig test session (see below). Presentation order was randomized without replacement in blocks. The mice were allowed to complete as many tria ls as possible with in the 25-min session.

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89 Conditioning phase (see Table 4-2) Water bottles were removed the day before the start of the conditioning phase. During this phase, starting at 0900, each mouse received water from a drinking spout in its home cage for 15 min at the same time each day (the start of the trial for animals was staggered by 5 min to allow time for intake measurements and injections). Approximately 5 h after the start of each animals morn ing session, the mice were given access to purified water for 1 h to allow for rehydrati on. After three days of one-bottle water testing, the animals were divided into six gr oups (n = 8 mice per gr oup) according to the CS (L-serine, L-threonine, or sucrose) a nd unconditioned stimulus (US; LiCl or NaCl) they would receive. Mice were assigned to groups on the basis of their body weight, mean water intake during the first three days of the conditi oning phase, mean licks/trial, and mean number of trials dur ing the last three da ys of Davis rig trai ning. There were no significant differences between the groups regarding these parameters. Subsequently, five conditioning trials follo wed in which mice were presented with the appropriate CS for 15 min, immediately fo llowed by an intraperitoneal (i.p.) injection (3.0 mEq/kg body wt) of either 0.15 M LiCl1 or 0.15 M NaCl. The purpose of the LiCl injection was to induce visceral malaise. Mice that drank less than 0.1 mL of their respective CS, had ~0.1 mL infused in their or al cavity with a syringe before receiving the US injection. Water bottles were re turned to the home cages ~5 hours after a conditioning trial. The following day, wate r bottles were removed from the cages, starting at 0915, on the same staggered sche dule used on the previous conditioning phase 1 The 1st US injections for all three experimental groups were carried out with LiCl dissolved in 0.15 M NaCl. All subsequent injections were completed with LiCl dissolved in purified water. Also, four mice died after the 1st injection for unknown reasons. As a result, n = 6 8 mice per group.

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90 day. Approximately 5 h after the removal of each animals water bottle, the mice were again given access to purified water for 1 h to re-establish the 18-h schedule of water restriction. The next conditioning trial was se parated by one more day of the restricted water-access schedule. After the fifth condi tioning trial, water bottles were replaced on the home cages for one day. Davis rig testing phase Water bottles were removed ~23.5 h befo re each animals brief-access testing phase began. On the first day of testing, th e mice were presented with 5-s water trials from fourteen sipper tubes, as described above for brief-access training. This water testing was intended to reacquaint the mice to the task in the Davis rig and to increase their motivation for licking on the following te st day. On the next day, the mice were presented with 5-s trials of their specific test stimulus array. A water rinse (5-lick maximum) presentation was interposed between the test trials for all stimuli to help control for potential carryover effects (see St John et al. 1994; Boughter et al. 2002). Presentation order was randomized without repl acement in blocks so that every stimulus and water was presented exactly once before th e initiation of the subsequent block. The mice were allowed to complete as many tria ls as possible within the 25-min session. Data Analysis A CS-suppression ratio was de rived by dividing the CS intake before the first conditioning trial by the CS intake before the fifth (final) trial for each mouse. A ratio of 1.0 signifies equal intake between the first and last conditioning trials, while a ratio less than or greater than 1.0 signifies decreased or increased intake, resp ectively, in the last conditioning trial relative to the first trial.

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91 In the brief-access test, Tastant/Water Lick Ratios were calculated for each mouse representing the mean number of test stimul us licks per trial divi ded by the mean number of water stimulus licks per trial (water rinse tr ials were not included in the analysis). All of these data were analyzed with analys es of variance (ANOVAs) and t-tests. The statistical rejection criteri on was set at the conventiona l value of .05. Bonferroni adjustments were also performed to control for the use of multiple comparison on the same data set. This extremely conservative standard is reported for the benefit of the reader (see Figures 4-2, 4-3, and 4-4). However, the desi gn was based on testing the response of the control and experimental mice to each stimulus. Thus, we chose to base our interpretation on the unadjusted values which are also detailed. Results Conditioning Phase All of the LiCl-injected groups demonstr ated convincing evidence of an aversion acquired to their respective CS during th e conditioning phase. T-tests revealed a significant difference from a value of 1.0 in th e CS suppression ratio for all three LiCl injected groups [Figure 4-1; all t -values -8.4, P < .001]. This indicates that there was a significant decrease in CS intake between the first and fifth conditioning trial for these groups. There was also a significa nt decrease in CS intake fo r the saline injected serine group on the fifth conditioning da y relative to the first [ t (6) = 3.0, P < .05]. However, this decrease was slight and, as a result, in terpretively insignifi cant. Indeed, the CS suppression ratio for each Li Cl-injected group was significantly lower than its corresponding control group [Figure 4-1; all t -values 5.0, P < .001]. Collectively, these data confirm the effectiveness of the conditioning procedures. However, it is well known that, relative to rats, mice have a greate r resistance to CTA acquisition procedures

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92 (Ninomiya et al. 1984; Kasahara et al. 1987; Welzl et al. 2001). Thus, of mice in the three LiCl conditioning groups, only those that consumed less than 0.5 ml during the fifth and final conditioning session were included in the subsequent Brief-Access Testing Phase. Only two mice (out of 8 in the su crose conditioning group) consumed more than 0.5 ml and thus were discarded from all analyses reported he re. The fact that sucrose conditioned mice were the most resistant to the procedures is consiste nt with at least one published report on the use of sugars as CSs, albeit in the rat (Smith, 1971). Brief-Access Testing Phase Sucrose CS group T-tests indicted that, relative to contro ls, mice conditioned to avoid 0.4 M sucrose showed strong suppression to all three con centrations of sucrose [Figure 4-2; all t -values 3.6, P s < .01]. A one-way ANOVA indicated th at the Tastant/Water Lick Ratios of these concentrations did not si gnificantly differ. Relative to controls, sucrose conditioned mice did not avoid any other stimul us in the test stimulus array. L-serine CS group T-tests revealed that, relative to contro ls, mice conditioned to avoid 0.6 M L-serine also showed strong suppression to all three concentrations of L-serine [Fi gure 4-3; all t values 2.3, P s < .05]. A one-way ANOVA indicated th at the Tastant/Water Lick Ratios of these concentrations did not significantly differ. Relative to controls, these mice also avoided both concentrations of sucrose [Figure 4-3; both t -values 3.4, P s < .01] and Lthreonine [Figure 4-3; both t -values 2.4, P s < .05]. The Tastant/Water Lick Ratios of the two sucrose concentrations did not signifi cantly differ. However, a paired t-test revealed that the conditioned mice avoided 0.7 M Lthreonine sign ificantly more than 0.175 M L-threonine [ t (7) = 4.0, P < .01]. Surprisingly, in addition to sucrose and L-

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93 threonine, L-serine conditioned mice also avoided 0.625 mM QHCl to a significantly greater degree than did control mice [Figure 4-3; t (13) = 2.5, P < .05]. These animals did not avoid any other stimulus in the test stimulus array. L-threonine CS group Relative to animals in the control group, mice conditioned to avoid 0.35 M Lthreonine, similar to the other two conditio ned groups, showed strong suppression to all three concentrations of th eir CS [Figure 4-4; all t -values 2.5, P s < .05]. A one-way ANOVA indicated that the Tastant/Water Lick Ratios of these con centrations did not significantly differ. Relative to controls, these mice also avoided 0.6 M sucrose [Figure 4-4; t (11) = 2.5, P < .05] and 1.0 M L-serine [Figure 4-4; t (11) = 3.2, P < .01]. These animals did not avoid any other stim ulus in the test stimulus array. Discussion The results from Experiment 3 demonstrat ed that animals trained to avoid 0.6 M Lserine subsequently avoided both concentrations of sucrose. These data suggest that Lserine possesses a sucrose-like taste quality to B6 mice. This finding is consistent with the only CTA experiment on the taste of L-se rine in rodents, as well as the large body of human psychophysical studies and the data reported in the preceding chapter. In addition, L-threonine also appears to possess a sucrose-like taste quality to these animals. This has never been explicitly demonstrat ed in rodents. Collectively, data from Experiments 2 and 3 suggest that at least some of the signals genera ted by the receptor(s) responsible for the transduc tion of these stimuli (i.e., L-amino acid sweeteners and sucrose) converge somewhere in the gustato ry system. The mechanisms potentially underlying this qualitative similarity were discussed in detail in Chapter 3.

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94 Interestingly, the generalization seen to wards sucrose by mice trained to avoid Lserine was not reversible. That is, mice trained to avoid 0.4 M sucrose did not avoid either of the L-serine concentrations tested or, for that matter, either concentration of Lthreonine. Yet, L-serine did reversibly ge neralize with L-threonine These data, as well as those gathered in Experiment 2, suggest that sweet-tasting L-amino acids, while more similar to sucrose than to other prot otypical representative s of the basic taste qualities, possess discriminable ta ste characteristics. The source of the discriminable cue may lie in the pattern of receptor expression. TBCs that express receptor(s) which respond to L-serine (e.g., T1R1+3) may genera te discrete neural signals that remain distinguishable as the info rmation ascends through the nervous system. However, another possibility is suggested by the genera lization pattern observed in Experiment 3. The results from this experiment suggest that to B6 mice, L-serine may be perceived as both sweet and bitter. At least on e published study has reported that humans perceive the taste of L-serine crystals as sweet, with a bitte r/sour aftertaste (Haefeli and Glaser, 1990). Humans also state that the ar tificial sweetener, saccharin, gives rise to a similar taste perception (i.e., sweet, with a bitter aftertaste; e.g., Bartoshuk, 1979; Schiffman et al. 1979). The putative bitte r-taste evoked by this stimulus would appear to provide a plausible explanat ion for the difference in the affective potency of L-serine and sucrose. Because of the inherent floor effect involved with measuring conditioned avoidance to naturally aversi ve compounds in a CTA design, th e fact that animals trained to avoid L-serine significantly suppressed their licking of quini ne relative to control mice raises our confidence that this amino acid po ssesses a quinine-like taste component. The

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95 response of control animals to a bitter TS su ch as quinine is naturally suppressed, making further avoidance difficult to discern (see Smith and Theodore, 1984 for an example). Nevertheless, mice conditioned to avoid L-se rine suppressed their lick responses to quinine above and beyond these factors. That said, it would be in structive to repeat Experiment 3 using a taste assessment procedur e in which the affective characteristic of the stimuli are rendered irre levant (e.g., Grobe and Specto r, 2006). Such a procedure would not be plagued by the problems menti oned above and as a result, would likely be more sensitive to any bitter-taste potent ially elicited by the CSs (e.g., L-threonine). It would also be informative to repeat Experiment 3 with mice lacking one or more of the T1R receptors. This would reduce or eliminate the neural input from TBCs that express the family of receptors that are likely responsible for the relative similarity of L-serine and sucrose. These manipulations would allow for a more focused evaluation of the bitter taste-evoking potenti al of L-serine. I would predic t that one or more of these groups would generalize an aver sion learned to L-serine more strongly to quinine than would wild-type mice. Based on responses of T1R knock-out mice reported in Zhao et al. 2003, in at least one group of these animal s (e.g., T1R1 and/or T1R3 knock-out mice; see Table 1-1 or Zhao et al. 2003), L-serine would not e voke T1R peripheral neural input, and as a result, it would poten tially taste more purely bitter. Interestingly, as detailed in Chapter 1, 66% of the T2R-expressing cells in the circumvallate papilla also e xpressed T1R1, which, in combination with T1R3, is thought to bind with L-amino acids, including L-serine It has been postulated by some, that rodents will reject amino acids in proportion to their toxicity (Pritchard and Scott, 1982a). However, it is not known whether taste input ha s a role in this reje ction. The discovery

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96 of a bitter receptor responsible for Lamino acid rejection would allow for a conformation that this behavior is based, to a certain extent, in peripheral taste physiology. As covered earlier in this chapte r, two bitter receptors were recently shown to be responsible for mediating the bitter afte rtaste elicited by saccharin. As detailed in Chapter 2, water-restricted D2 mice avoide d L-serine in a concentration dependent manner. This is consistent w ith the fact that L-serine ap pears to evoke a bitter taste perception. However, in this same experiment B6 mice did not avoid the stimulus. It is well known that these two strain s differ in their sensitivity to various bitter tasting compounds (e.g., B6 mice are more sensitive to quinine and D2 mice are more sensitive to raffinose undecaacetate; Boughter et al. 2005; Nelson et al. 2005). These behavioral phenotypes can be exploited to help identify any potential polymor phisms (e.g., in genes encoding for T2R receptors) that contribute to the observed behavioral divergence (i.e., L-serine avoidance).

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97Table 4-1: Test Stimulus Arrays for the Three Conditioning Groups Test Stimuli Sucrose L-Threonine L-Serine QHCl Citric Acid NaCl CS# High Low CS# High Low CS# High Low High Low High Low High Low Group 1 0.4 M 0.6 M 0.2 M 0.7 M 0.175 M 1.0 M 0.4 M 0.625 mM 0.325 mM 13.25 mM 6.25 mM 0.375 M 0.175 M Group 2 0.6 M 0.2 M 0.7 M 0.175 M 0.6 M 1.0 M 0.4 M 0.625 mM 0.325 mM 13.25 mM 6.25 mM 0.375 M 0.175 M Group 3 0.6 M 0.2 M 0.35 M 0.7 M 0.175 M 1.0 M 0.4 M 0.625 mM 0.325 mM 13.25 mM 6.25 mM 0.375 M 0.175 M *High = ~50% of the lick rate relative to water; low = ~75% of the lick rate relative to water. See text for more details. #These were concentrations used in Experiment 2

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98 Table 4-2: Conditioning phase schedule Conditioning phase AM 15-m water access 15-m CS access US injection Water bottles removed 15-m water access 15-m CS access US injection Water bottles removed 15-m water access 15-m CS access US injection Water bottles removed 15-m water access 15-m CS access US injection Water bottles removed 15-m water access 15-m CS access US injection PM 1-h water access Water bottles returned 5-h after injection 1-h water access 1-h water access Water bottles returned 5-h after injection 1-h water access 1-h water access Water bottles returned 5-h after injection 1-h water access 1-h water access Water bottles returned 5-h after injection 1-h water access 1-h water access Water bottles returned 5-h after injection 3 days 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day

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99 CS Suppression Ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ***### L-serine --> NaCl L-threonine --> NaCl Sucrose --> NaCl L-serine --> LiCl L-threonine --> LiCl Sucrose --> LiCl Figure 4-1: Mean ( SEM) of the CS Suppression Ra tio for each conditioned stimulus (CS). A ratio of 1.0 signifies equal intake between the first and last conditioning trials, while a ratio less than or greater than 1.0 signifies decreased or increase d intake, respectively, in the last conditioning trial relative to the first trial. An asterisk (* ) denotes a significant difference from a ratio value of 1.0 while a pound (#) indicates a CS Suppression Ratio for each LiCl-injected group that was significantly lower than its corresponding control group.

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100 Sucrose CALCAHNLNHQLQHSLSHSucL'CS'SucHTLTH Tastant/Water Lick Ratio 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 ***### Saline LiCl Figure 4-2: Mean ( SEM) Tastant/Water Lick Ratios for the sucrose CS group for a ll test stimuli. The Tastant/Water Lick Ratio was calculated by d ividing an animals average licks to a given taste stimulus across trials by the average licks to water. The dashed line on the graph repr esents a Tastant/Water Lick Ratio of 1.0, which indicates licking to the taste stimulus was equivalent to licking to water. An asterisk (*) denotes a significant difference between the Tastant/Water Lick Ratios of the control and the co nditioned groups. A pound (#) denotes a significant difference t hat survives the Bonferroni alpha adjustment. (CAL = 6.25 mM citric acid, CAH = 13.25 mM citric acid; NL = 0.175 M NaCl, NH = 0.375 M Na Cl; QL = 0.325 mM QHCl, QH = 0.625 mM QHCl; SL = 0.4 M L-serine, SH = 1.0 M L-serine; SucL = 0.2 M sucrose, SucH = 0.6 M Sucro se; TL = .0.175 M L-threonine, TH = 0.7 M Lthreonine; CS = 0.4 M sucrose).

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101 L-serine CALCAHNLNHQLQHSL'CS'SHSucLSucHTLTH Tastant/Water Lick Ratio 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 ** ** **# # # Saline LiCl Figure 4-3: Mean ( SEM) Tastant/Water Lick Ratios for the L-serine CS group for a ll test stimuli. The Tastant/Water Lick Ratio was calculated by dividing an animals average licks to a given taste stimulus across trials by the average licks to water. The dashed line on the graph repr esents a Tastant/Water Lick Ratio of 1.0, which indicates licking to the taste stimulus was equivalent to licking to water. An asterisk (*) denotes a significant difference between the Tastant/Water Lick Ratios of the control and the co nditioned groups. A pound (#) denotes a significant difference t hat survives the Bonferroni alpha adjustment. (CAL = 6.25 mM citric acid, CAH = 13.25 mM citric acid; NL = 0.175 M NaCl, NH = 0.375 M Na Cl; QL = 0.325 mM QHCl, QH = 0.625 mM QHCl; SL = 0.4 M L-serine, SH = 1.0 M L-serine; SucL = 0.2 M sucrose, SucH = 0.6 M Sucro se; TL = .0.175 M L-threonine, TH = 0.7 M Lthreonine; CS = 0.6 M L-serine).

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102 L-threonine CALCAHNLNHQLQHSLSHSucLSucHTL'CS'TH Tastant/Water Lick Ratio 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 * * *# # Saline LiCl Figure 4-4: Mean ( SEM) Tastant/Water Lick Ratios for the L-threonine CS group fo r all test stimuli. The Tastant/Water Lick Ratio was calculated by dividing an animals average li cks to a given taste stimulus across trials by th e average licks to water. The dashed line on th e graph represents a Tastant/Water Lick Ratio of 1.0, which indicates licking to the taste stimulus was equivalent to licking to water. An asterisk (*) denotes a significant difference between the Tastant/Water Lick Ratios of the control and the conditioned groups. A pound (#) denotes a s ignificant difference that survives the Bonferroni alpha adjustment. (CAL = 6.25 mM citric acid, CAH = 13.25 mM c itric acid; NL = 0.175 M NaCl, NH = 0.375 M NaCl; QL = 0.325 mM QHCl, QH = 0.625 mM QHCl; SL = 0.4 M L-serine, SH = 1.0 M L-serine; SucL = 0.2 M sucrose, SucH = 0.6 M Sucrose; TL = .0.175 M L-threonine, TH = 0.7 M Lthreonine; CS = 0.35 M Lthreonine).

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103 CHAPTER 5 GENERAL DISCUSSION The first experiment presented is this di ssertation was designed to assess the tasterelated affective potency of glyc ine, L-serine, and sucrose. As a result of the complete lack of appetitive behavior observed during te sting with the putative sweetener L-serine, we explicitly tested the ability of mice to di stinguish between L-serine and various sugars in an operant taste discrimination task. Additionally, we examined the ability of mice to distinguish between an assortment of natu ral sweeteners. Finally, CTA generalization tests were conducted to make inferences re garding the taste quali ty(ies) evoked by some putative sweet-tasting L-amino acids. The resu lts of these studies were consistent with the notion that all of these compounds share some qualitati ve similarities. Mice had difficulty, depending on the stimulus and th e training history, discriminating sucrose from L-serine, maltose, fructose and glucose. However, despite th e apparent qualitative similarity of L-serine and the natural sweeteners tested in the current report, mice appeared to possess a limited ability to distinguish between them. The results of CTA generalization experiments imply that L-seri ne evokes a complex taste perception that includes a sucrose-like taste quali ty. Indeed, this complexit y, like the taste of saccharin to humans, appears to result from, at least in part, the generation of both a sweet and bitter taste, and may provide the cue that distinguishes L-serine from sucrose, as well as from other sugars. Along with the rela tively weak sweet-taste evoked by L-serine postulated in Chapter 3, its ability to generate a bitter-taste would appear to provide an

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104 additional plausible explanation for the differenc e in the affective pote ncy of L-serine and sucrose. It should be pointed out that some of the concentrations of the amino acids tested, although within the range of th at commonly used to assess the behavioral responsiveness of rodents in other experiments (e.g., Pritchard and Scott, 1982a; Iwasaki et al. 1985; Zhang et al. 2003; Zhao et al ., 2003), were relatively hi gh. Since stimuli at these concentrations would not likely be encountered in nature, the response of the gustatory system to these stimuli is of questionable biological relevance. That said, based on electrophysiological data, both th e chorda tympani (Pritchard and Scott, 1982a; Iwasaki et al. 1985; Inoue, et al. 2001; Danilova and Hellekant, 2003) and glossopharyngeal nerves (Danilova and Hellekant, 2003) disp lay monotonically increasing responses across a high concentration range of L-amino acids including L-serine and L-threonine. These results suggest that investiga ting the responses of rodents to a broad concentration array of various amino acids may yet provide ecologi cally relevant information. In any event, studying ligands that robustly active the gusta tory system could prove useful as we attempt to uncover the principles governing the functional organiza tion of taste in the periphery. During Experiment 2, training history had a huge impact on the ability of mice to discriminate all of the stimuli, particularly L-serine from sucrose. Mice, trained to discriminate L-serine from Na Cl (i.e., serine group), appear ed to have more difficulty discriminating L-serine from sucrose relativ e to animals in the sucrose group. Indeed, two mice in the serine group were unable to discriminating L-serine from all of the sugars. Although there are other possible explanations for this training history

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105 asymmetry, one likely explanation arises from the apparent qualitative complexity of Lserine. In Experiment 3, mice did not genera lize an aversion learned to sucrose to any other stimulus in the TS array. Thus, sucrose appears to be more unita ry in its perceptual quality. As a result, mice trained with the more complex stimulus array (i.e., L-serine) may have had a greater expectation of the qualita tive variability of their standard stimulus (e.g., pure sweetness at low c oncentrations and sweetness and bitterness at higher concentrations) than did animals in the su crose group (pure sweet ness across the range of concentrations). If so, mice in the sucrose group would have an easier time discriminating a complex comparison stimulus from their qualitativ ely pure standard stimulus. Mice in the serine group, on th e other hand, would have more difficulty discriminating a pure comparison stimul us, which, depending on concentration, appears to evoke qualities si milar to those elicited by thei r complex standard, again depending on concentration. This was exemp lified by the fact that during the first week of L-serine vs. sucrose or Lserine vs. glucose testing, mice had a tendency to respond to the comparison stimulus as if it were the standard. Mice in th e sucrose group never responded in such a way. To test this hypothesis, two groups of mice could be trained, as in Experiment 2, to discriminate a standard stimulus from an easily discriminable comparison stimulus (NaCl). One group would be given a qualitati vely pure standard (sucrose), while the other would be given an artifi cially complex standard (e.g., a sucrose/quinine mixture). As in Experiment 2, once the animals were tr ained in the task, the comparison stimulus (NaCl) could be replaced with the standard stimulus from th e other group, so that both sets of animals would be discriminating sucr ose from a sucrose/quinine mixture. Based

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106 on the hypothesis mentioned above, I would predic t that B6 mice trained to discriminate an artificially complex standard from NaCl would have more difficultly with this discrimination task. In summary, the findings here suggest that all of the putative sweet tasting L amino acids and sugars tested in the current report share some qualitative features. However, the results of these experiments al so imply that sweet-tasting L-amino acids, such as L-serine, also possess distinguishable taste characteristics. To my knowledge, the qualitative complexity elicited by L-serine, wh ich includes a bitter taste, has never been previously demonstrated before in rodents. Yet, the ability of L-se rine to evoke multiple taste percepts would explain mu ch of the behavioral results observed in th is dissertation (i.e., the efficacy of L-serine as a standard /training stimulus, its lack of affective potency, and its discriminability from sugars). In addition, monogeusia for sucrose, gluc ose, fructose, and to a lesser extent, maltose has been demonstrated here for the fi rst time in rodents. However, we cannot conclusively rule out the exis tence of some discriminative competency if these animals were tested under different e xperimental procedures. That said, if B6 mice can discriminate between these sugars, our results at least imply an extremely limited ability. Thus, these data suggest that the periphera l input evoked by these sugars is channeled into a single neural pathway as it ascends th rough the nervous system. This convergence likely occurs in the TBCs themselves (i.e ., stimulation of a co mmon receptor(s) (e.g., T1R2+3 receptor complex.). Lastly, these data provide a sorely need functional context in which to interpret the neurobiology of sweet taste. This beha vioral framework will allow researchers to

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107 evaluate the taste-related cons equences of manipulation of the gustatory system, assumed to affect sweet-taste percep tion (e.g., taste receptor knockouts), with the knowledge of how normal mice react to these stimuli. Pr eviously experiments have been beset by unconfirmed assumptions regarding the taste qua lity(ies) elicited by these stimuli and the subsequent behavioral responses evoke by th eir ingestion (e.g., if rodents generalize a sucrose aversion to a TS in a CTA experiment, then that TS will be unconditionally preferred). These findings also generate ma ny questions to answer. For example, which T1R receptors, if any, contribute to the qualitati ve similarity of sucrose and L-serine? As mentioned in Chapter 4, the pattern of beha vioral generalization would be expected to vary greatly between various T1R knock-out mice. By providing a better u nderstanding of the function of chemosensory systems, studies such as these can lead to improveme nts in the diagnosis and treatment of smell and taste disorders and contri bute to our knowledge of how sensory systems operate and how their signals are integrat ed in the brain, as well as add to our understanding of the controls of food se lection and intake.

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108 LIST OF REFERENCES Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J., and Zuker, C. S. (2000) A novel family of mammalian taste receptors. Cell, 100, 693-702. Bachmanov, A. A., Li, X., Reed, D. R., Ohme n, J. D., Li, S., Chen, Z., Tordoff, M. G., de Jong, P. J., Wu, C., West, D. B., Chatterjee, A., Ross, D. A., and Beauchamp, G. K. (2001a) Positional cloning of the m ouse saccharin preference (Sac) locus. Chem. Senses, 26, 925-933. Bachmanov, A. A., Reed, D. R., Ninomiya, Y., Inoue, M., Tordoff, M. G., Price, R. A., and Beauchamp, G. K. (1997) Sucrose consumption in mice: major influence of two genetic loci affecting peripheral sensory responses. Mamm. Genome, 8, 545-548. Bachmanov, A. A., Reed, D. R., Tordoff, M. G., Price, R. A., and Beauchamp, G. K. (1996) Intake of ethanol, sodi um chloride, sucrose, citric acid, and quinine hydrochloride solutions by mice: a genetic analysis. Behav. Genet., 26, 563-573. Bachmanov, A. A., Tordoff, M. G., and Beauchamp, G. K. (2001b) Sweetener preference of C57BL/6ByJ and 129P3/J mice. Chem. Senses, 26, 905-913. Bartoshuk, L. M. (1979) Bitter taste of saccharin related to the genetic ability to taste the bitter substance 6-n-propylthiouracil. Science, 205, 934-935. Bartoshuk, L. M. (1988) Taste In Stevens, S. S., and Atkinson, R. C. (eds), Stevens' handbook of experimental psycho logy. Wiley, New York, pp. 461-499. Berkley, M. A., and Stebbins, W. C. (1990). Comparative perception Wiley, New York. Blizard, D. A., Kotlus, B., and Frank, M. E. (1999) Quantitative trait loci associated with short-term intake of sucrose, sacc harin and quinine solutions in laboratory mice. Chem. Senses, 24, 373-385. Blough, D., and Blough, P. (1977) Animal psychophysics In Honig, W. K., and Staddon, J. E. R. (eds), Handbook of opera nt behavior. Prentice-Hall, Englewood Cliffs, N.J., pp. 514-539. Boughter, J. D., Jr., Raghow, S., Nelson, T. M., and Munger, S. D. (2005) Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli. BMC Genet., 6, 36.

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115 Temussi, P. (2006) The history of sweet taste: not exactly a piece of cake. J. Mol. Recognit., 19, 188-199. Welzl, H., D'Adamo, P., and Lipp, H. P. (2001) Conditioned taste aversion as a learning and memory paradigm. Behav. Brain res., 125, 205-213. Yamamoto, T., Matsuo, R., Fujimoto, Y ., Fukunaga, I., Miyasak a, A., and Imoto, T. (1991) Electrophysiological and behavioral -studies on the taste of umami substances in the rat. Physiol. Behav., 49, 919-925. Yamamoto, T., Matsuo, R., Kiyomitsu, Y., and Kitamura, R. (1988) Taste effects of 'umami' substances in hamsters as studied by electr ophysiological and conditioned taste aver sion techniques. Brain res., 451, 147-162. Zhang, Y., Hoon, M. A., Chandrashekar, J., Mueller, K. L., Cook, B., Wu, D., Zuker, C. S., and Ryba, N. J. (2003) Coding of sweet, bitte r, and umami tastes: different receptor cells shar ing similar signaling pathways. Cell, 112, 293-301. Zhao, G. Q., Zhang, Y., Hoon, M. A., Chandrashekar, J., Erlenbach, I., Ryba, N. J., and Zuker, C. S. (2003) The receptors for mammalian sweet and umami taste. Cell, 115, 255-266.

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116 BIOGRAPHICAL SKETCH Cedrick Dotson obtained a B achelor of Arts degree in psychology from the University of West Florida in Pensacola, Florida, in 1998. He received a Master of Science degree in clinical psychology from the University of Florida in 2001. In 2002, he began his doctoral training in the behavioral neuroscience di vision in the Department of Psychology at the University of Florida. In August 2006 he earned his Ph.D. in psychology with a specialization in behavioral neuroscience. Hi s research interests are in the neural basis of gustation.


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Title: Psychophysical Evaluation of Sweet Taste Perception in Mice
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PSYCHOPHYSICAL EVALUATION OF SWEET TASTE PERCEPTION IN MICE


By

CEDRICK DOTSON













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Cedrick Dotson
















ACKNOWLEDGMENTS

I would like to thank my mentor, Alan Spector, Ph.D., for his support and

supervision. I would also like to thank our senior laboratory technician, Angela Newth,

for her invaluable assistance in completing this project. This research was supported by

National Institute on Health Predoctoral National Research Service Award, # F31-

DC007358, granted to Cedrick D. Dotson and National Institute on Deafness and Other

Communication Disorders Grant, # R01-DC04574, awarded to Alan C. Spector.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES .................... ............... ................... .......... .. vii

LIST OF FIGURES ............... .......................... .... .... .... .. ........ .. viii

A B ST R A C T ................. .......................................................................................... x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Psychophysical Examination of Taste Quality in Non-Human Mammals.................. 1
A m ino A cid T aste Perception .................................................................. ...............2
"Sw eetener" Transduction ................................................................................ 4
S u m m ary ................................0.............................

2 EXPERIMENT 1: THE RELATIVE AFFECTIVE POTENCY OF GLYCINE, L-
SERINE AND SUCROSE AS ASSESSED BY A BRIEF-ACCESS TASTE
TEST IN INBRED STRAINS OF M ICE .........1........ .. .............. ...............14

Background .................... ...... ...................... 14
M ethodological D etails............ ... ......................................................... .. .... .. .. .... 17
Subjects ............... ........ .......................17
Taste Stimuli ............... ......... ........ .......... 17
P ro c e d u re ................................................... ...................................1 8
Data Analysis ......................................................................... ......... ................... 19
Results ............... ....... ............ ................. ......... 20
Stan d ardization D ata ..................................................................................... 2 0
S u c ro se ................................................................2 1
L -serin e ...............22................. .........................
G ly c in e ................................................................2 3
Discussion ............... ......... .........................24

3 EXPERIMENT 2: TASTE DISCRIMINABILITY OF L-SERINE and Various
SUGARS BY MICE ................. ......... ..................38

B a c k g ro u n d .................................................................................................... 3 8










Methodological Details................ .. .................... .........39
Subjects ............... .................................39
Taste Stimuli ............... ......... .............. ................... 39
Apparatus ................................................41
E x p erim ental D esign ..................................................................................... 42
Training (see Table 3-3) .................................... ............... 42
Testing (see Table 3-3).................................... .................. 44
D ata A n a ly sis ................................................................................................. 4 5
Results .......................................................... ...............45
D iscrim nation Testing ............................................................. 46
S u cro se v s. L -serin e ....................................................................................... 4 6
"Serine" group ............................... ............... 46
S u cro se" g ro u p ..................................................................................... 4 6
Sucrose/L-serine vs. Glucose ................................. ............... 48
S e rin e g ro u p ........................................................................................... 4 8
Sucrose group ............................................. ......... .........49
Sucrose/L-serine vs. M altose............................................... 50
S e rin e g ro u p ........................................................................................... 5 0
Sucrose group ................................. .......................... .... ...... 50
Sucrose vs. L -serine II................................................ ........ 51
S e rin e g ro u p ........................................................................................... 5 1
Sucrose group ................................. .......................... .... ...... 52
Sucrose/L-serine vs. Fructose................................ ................... 53
S e rin e g ro u p ........................................................................................... 5 3
Sucrose group ................................. .......................... .... ...... 54
Stimulus Control Sessions ..................................................... 55
D isc u ssio n ............................................................................................................. 5 6
M o n o g e u sia ................................................................................................... 5 6
Sugars v s. L -serine ............................................................6 1

4 EXPERIMENT 3: PERCEIVED SIMILARITY BETWEEN L-SERINE, L-
THREONINE AND CHEMICAL COMPOUNDS REPRESENTATIVE OF THE
FOUR BASIC TASTE QUALITIES .............................................. 84

Background .................... ...... ...................... 84
M eth o d olog ical D etails......................................................................................... 8 5
Subjects ............... ........ ......................85
Taste Stim uli ............... .................................. ..................... 86
A p p aratu s ................................87............................
E x p erim ental D esig n ..................................................................................... 8 8
Davis rig training................. .............. ......... 88
Conditioning phase (see Table 4-2) ..................................... ......89
D avis rig testing phase ....................................................... 90
D ata A n a ly sis ................................................................................................. 9 0
R e su lts .................................. ......................................................9 1
C conditioning P hase.....................................................9 1
Brief-Access Testing Phase .................................................. 92


v









Sucrose CS group ........... .. ......... .... ............ ... .... .......92
L -serine C S group ................................ .............. .................. ..............92
L-threonine CS group ......... ............... ................... 93
D iscu ssio n ......... .................................... ............................9 3

5 GENERAL DISCU SSION ............. .................. .............. .............................. 103

LIST OF REFEREN CE S ......... .................................. ........................ ............... 108

B IO G R A PH ICA L SK ETCH ......... ................. ...................................... .....................116
















LIST OF TABLES


Table p

1-1 Consequences of the "knockout" of various T1R receptors on neural and taste-
related behavioral responses.......................................................... ............... 12

1-2 % of taste buds and % TBCs in papillae .......................... ....... ............... 13

2-1 Mean number of licks to water SEM taken by the four strains when tested with
sucrose, L-serine, or glycine when mice were water deprived. ............................32

2-2 Mean of the inter-lick interval (ILI) distribution (ms) SEM observed in the
four strains of mice trained to lick either sucrose, L-serine, or glycine .............. 33

2-3 Strains listed in order of mean Tastant/Water Lick ratio, for sucrose, L-serine,
and glycine when mice were water deprived. .................................. .................34

2-4 Strain listed in order of mean Standardized Lick ratio, for sucrose, L-serine, and
glycine when mice were non-deprived...................... ... ........................ 35

3-1 Stim ulu s concentration s ........................................ .............................................65

3-2 Order of stimulus discrimination pairings............... ............................................. 66

3-3 Representative training and testing parameters for the 2 discrimination groups .....67

3-4 Overall percentage correct during the last week of testing for the stimulus
discrim nation pairing ......... ................. ................. .................... ............... 68

3-5 Number of stimulus-control sessions required to criterion performance ...............69

4-1 Test Stimulus Arrays for the Three Conditioning Groups....................................97

4-2 C conditioning phase schedule......................................................... .....................98















LIST OF FIGURES


Figure page

2-1 Mean ( SE) Tastant/Water Lick Ratio as a function of sucrose, L-serine, and
glycine concentration for four different inbred strains of mice.............................36

2-2: Mean ( SE) Standardized Lick Ratio as a function of sucrose, L-serine, and
glycine concentration for four different inbred strains of mice ............................37

3 -1 T rial stru ctu re ...................................................................... 7 0

3-2 Individual animal (symbols) and group mean ( SEM; grey bars) data for mice
trained to discriminate either sucrose or L-serine from NaCl.................................71

3-3 Individual animal (symbols) and group mean ( SEM; grey bars) data are
plotted across all test phases for mice initially trained to discriminate L-serine
from N aC l ...................................... ................................................ 72

3-4 Individual animal (symbols) and group mean ( SEM; grey bars) data are
plotted across all test phases for mice initially trained to discriminate sucrose
from N aC l ...................................... ................................................ 7 3

3-5 Mean (+ SD) data for mice attempting to discriminate L-serine from sucrose........74

3-6 Mean (+ SD) data for mice attempting to discriminate L-serine from glucose .......75

3-7 Mean (+ SD) data for mice attempting to discriminate sucrose from glucose.........76

3-8 Mean (+ SD) data for mice attempting to discriminate L-serine from maltose.......77

3-9 Mean (+ SD) data for mice attempting to discriminate sucrose from maltose.........78

3-10 Mean (+ SD) data for mice attempting to discriminate L-serine from sucrose for
a second time (these mice were initially trained to discriminate L-serine from
N aC 1) ...............................................................................7 9

3-11 Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for
a second time (these mice were initially trained to discriminate sucrose from
N aC 1) ...............................................................................8 0

3-12 Mean ( SD) data for mice attempting to discriminate L-serine from fructose.......81









3-13 Mean (+ SD) data for mice attempting to discriminate sucrose from fructose ........82

3-14 Mean (+ SEM) data for both groups of mice are plotted across all test phases......83

4-1 Mean (+ SEM) of the CS Suppression Ratio for each conditioned stimulus
(C S )... .................................................................................... 9 9

4-2 Mean ( SEM) Tastant/Water Lick Ratios for the sucrose CS group for all test
stim u li ................................................. ........................................10 0

4-3 Mean ( SEM) Tastant/Water Lick Ratios for the L-serine CS group for all test
stim u li ................................................. ........................................10 1

4-4: Mean ( SEM) Tastant/Water Lick Ratios for the L-threonine CS group for all
te st stim u li ..............................................................................................................1 0 2

















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PSYCHOPHYSICAL EVALUATION OF SWEET TASTE PERCEPTION IN MICE

By

Cedrick Dotson

August 2006

Chair: Alan Spector
Major Department: Psychology

Despite differences in their molecular structure, many sugars, a subset of amino

acids, and some synthetic compounds are "sweet-tasting" to humans and appear to

possess a "sucrose-like" taste quality to non-human mammals. It has been proposed that

in taste bud cells (TBCs), a family of receptors called the T1Rs mediates signal

transduction of all of these "sweeteners." However, in a brief-access test with non-

deprived mice, licking responses to sucrose were discernibly different from the responses

to the amino acids tested.

Experiments detailed here were designed to test the ability of mice to distinguish

between L-serine and various sugars in operant taste discrimination tasks. Mice were

able to discriminate NaCl from sucrose (n=6) and L-serine (n=6) which served as training

stimuli. Mice had difficulty discriminating sucrose from L-serine, maltose, fructose and

glucose to varying degrees depending on the stimulus and the training history. For

example, when concentration effects are taken into consideration, mice were unable to









discriminate sucrose from glucose or fructose suggesting that these sugars generate a

unitary percept. However, these animals were able to discriminate sucrose from L-serine.

Mice were also able to discriminate L-serine from glucose, fructose, and maltose, but

only moderately so. Data gathered using a conditioned taste aversion assay suggest that

L-serine generates a complex taste that includes a sucrose-like component and that this

complexity may be at the source of the limited discriminability between L-serine and the

sugars. To my knowledge, the qualitative complexity elicited by L-serine, which

includes both a "sweet" and a "bitter" taste, has never been previously demonstrated

before in rodents. L-serine's ability to evoke multiple taste percepts would explain much

of the results observed in this dissertation including its lack of affective potency, its poor

efficacy as a "standard" stimulus, and its discriminability from sugars.

In summary, these data suggest that all of these compounds share some qualitative

similarities. Therefore, it is likely that some taste input arising from TBCs that express

different T1R receptors converges somewhere along the gustatory neuraxis. However,

the results of these experiments also imply that "sweet-tasting" L-amino acids, such as L-

serine, also possess distinguishable taste characteristics.














CHAPTER 1
INTRODUCTION

Psychophysical Examination of Taste Quality in Non-Human Mammals

It is generally accepted that taste perception is comprised of only a few basic

qualities (e.g., sweet, salty, sour, bitter; see Bartoshuk, 1988). However, the nature of

these perceptions is difficult to study because these experiences cannot be directly

measured and must be inferred from behavior. In animals, the systematic study of these

perceptions is accomplished by the use of behavioral procedures that are designed to

characterize the relationship between physical stimuli and sensation (Blough and Blough,

1977; Berkley and Stebbins, 1990; Spector, 2003). These psychophysical techniques

allow for, among other things, the assessment of an animal's capacity to discriminate or

generalize between two chemical compounds. If an animal treats a test stimulus similarly

to a trained or conditioned stimulus (i.e., generalization), then the compounds are

assumed to share some perceptual features. If an animal can be trained to discriminate

between two compounds, then the stimuli involved must generate distinguishable neural

signals in both the periphery and the brain. Identifying compounds that are behaviorally

indiscriminable allows for them to be categorized into perceptual classes. Indeed, a

group of stimuli that are mutually indiscriminable constitute the definition of a perceptual

class. Below I propose the use of psychophysical procedures specifically designed to

answer these questions. As argued above, the use of psychophysical methodology is the

only way to rigorously and objectively examine the taste perception of animals.










Amino Acid Taste Perception

Explicit taste discrimination experiments using amino acids as stimuli are rare.

Instead, researchers have tried to perceptually categorize amino acids in rodents by using

the conditioned taste aversion (CTA) generalization paradigm to quantify the degree to

which these compounds are similar to prototypical chemical stimuli thought to represent

basic taste qualities (e.g., sucrose, NaC1, citric acid, quinine). In this procedure, an

animal samples a novel taste stimulus followed by the injection of an agent that causes

visceral malaise (usually lithium chloride; LiC1). After which, researchers measure

whether the subsequent avoidance conditioned to the novel tastant generalizes to other

test compounds. The results of such experiments suggest that a variety of amino acids

appear to possess some degree of a qualitative similarity with the taste of sucrose.

Taken together, without regard to strain and species differences, results from CTA

experiments demonstrate that a subset of D-amino acids and a subset of L-amino acids

including L-alanine, L-proline, L-serine, and glycine (which does not have a chiral

carbon) are all treated as possessing some degree of qualitative similarity with the taste of

sucrose and are thus considered "sweet." In addition, when mixed with the epithelial

sodium channel blocker, amiloride, aversions conditioned to monosodium glutamate

(MSG) and L-aspartic acid have also been shown to generalize to sucrose in CTA tests

(e.g., Yamamoto et al., 1991; Chaudhari et al., 1996; Stapleton et al., 1999; Heyer et al.,

2003). Other amino acids tested fail to fall into this category (including L-arginine, L-

isoleucine, L-methionine, L-phenylalanine, L-tryptophan, D-alanine, and D-serine tested

in various strain of mice; see Ninomiya et al., 1984b; Kasahara et al., 1987; Ninomiya et

al., 1992; and L-arginine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine,









and L-histidine tested in the golden hamster; see Nowlis et al., 1980; Yamamoto et al.,

1988). A large body of psychophysical literature also suggests that these L-amino acids

give rise, to some degree, to a sweet taste perception in humans (e.g., Solms et al., 1965;

Schiffman and Dackis, 1975; Schiffman and Clark, 1980; Schiffman et al., 1981;

Schiffman et al., 1982; Haefeli and Glaser, 1990; Shallenberger, 1993). These data also

suggest that other L-amino acids, such as L-threonine, may also share perceptual

characteristics with sucrose. Indeed, the notion that rodents may also perceive L-

threonine as "sucrose-like" (i.e., "sweet") is supported by the fact that L-threonine was

shown to be preferred by Sprague-Dawley rats and by ddy mice, at certain

concentrations, as assessed using a two-bottle preference test (Pritchard and Scott, 1982a;

Iwasaki et al., 1985). However, as detailed below, this fact, in and of itself, does not

allow for a confident categorization of a taste compound into the perceptual class humans

label sweet.

In general, rats unconditionally prefer those amino acids that have been shown to

generalize with sucrose in conditioned taste aversion tests. Using the two bottle

preference test, a variety of researchers have shown that these same L-amino acids seem

to be favored, to varying degrees, by different strains of mice (Iwasaki et al., 1985; Lush

et al., 1995; Bachmanov et al., 2001b). Indeed, these measures are commonly used to

determine whether a particular species or animal perceives a stimulus as "sweet" and/or

as a measure of the relative intensity of that percept (e.g., Bachmanov et al., 2001b).

However, the use of preference measures to determine the relative qualitative similarity

of a putative "sweet-tasting" stimulus to that of a prototypical sugar (i.e., sucrose) implies

that if that compound shares a qualitative resemblance to a particular sugar, then it will be









unconditionally preferred. Additionally, it suggests that if a compound is not

unconditionally preferred, it will not be perceived as "sweet."

I recently conducted a set of experiments assessing the affective potency of

sucrose, L-serine, and glycine using a brief-access taste test in a gustometer, the details of

which are covered in Chapter 2 of this dissertation. Suffice it to say, these data do not

support the supposition that L-serine (and to a lesser extent, glycine) is unconditionally

preferred by non-deprived mice on the basis of taste. Thus, if the aforementioned

contention regarding the correspondence between "sweet" taste quality and preference is

correct, then, despite suggestions from the CTA literature, L-serine could not be

considered "sweet."

That said, it should be noted here that the two-bottle preference test and the brief-

access taste test only assess the motivational properties of a taste stimulus, not its

qualitative characteristics per se. For example, rats prefer low concentrations of NaCl in

two-bottle tests and avoid high concentrations, but this does not mean that the former are

"sweet" and the latter are "bitter." Qualitative perception is best inferred from tasks in

which taste serves as a cue for some other event (e.g., reinforcement) that will generate a

trained directed response regardless of the hedonic characteristics of the taste stimulus.

"Sweetener" Transduction

An understanding of the neural basis of sweetener and amino acid taste perception

has been propelled by remarkable discoveries regarding the molecular biology of

transduction processes in the mammalian peripheral gustatory system. Specifically, a

gene family has been identified which encodes for three 7-transmembrane spanning G-

protein coupled receptors (T1R1, T1R2, and T1R3) that bind with sugars, synthetic

sweeteners, amino acids, and in some species "sweet" proteins (e.g., Hoon et al., 1999;









Bachmanov et al., 2001 a; Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al.,

2001; Nelson et al., 2001; Sainz et al., 2001; Li et al., 2002). Initial calcium imaging

studies of "receptor-ligand" interactions in a heterologous expression system, suggested

that individual T1Rs are not functional, but that they, similar to other class C G-protein

coupled receptors, combine into heterodimeric receptor complexes. Dimerization,

however, has yet to be explicitly demonstrated in this sub-family of receptors. The T1R3

receptor has been shown to combine with T1R1 or T1R2 to form functional

"heteromers." The T1R2+3 complex was shown, in vitro, to be activated by a variety of

both natural and synthetic sweeteners as well as "sweet-tasting" D-amino acids (Nelson

et al., 2001; Zhao et al., 2003). A similar study revealed that the combination of mouse

T1R1 and T1R3 gives rise to a "heteromeric" receptor complex that interacts with most

of the twenty common L-amino acids (Nelson et al., 2002). A more recent in vitro study

suggested that T1R3 may also function independently as a low affinity receptor, binding

with high concentrations of natural but not synthetic sweeteners (Zhao et al., 2003).

Results from experiments on the neural and behavioral consequences of the deletion (i.e.,

knock-out) of one or more of the genes encoding for the T1R receptors in mice are

summarized in Table 1-1. Collectively, these data confirm, in vivo, that sugars and L-

amino acids bind, selectively, with different T1R receptor complexes.

These heteromeric receptors were first purported to be expressed, principally, in

non-overlapping sets of taste bud cells. The receptor, T1R1, was reported to be mainly

expressed in fungiform papillae (anterior tongue) and in the palate (in -20-30% of the

taste bud cells (TBCs) in 100% of the buds in these receptor fields; Hoon et al., 1999). In

the posterior tongue, it was purported to be rarely found in the taste buds of the









circumvallate papillae (in less than 5% of TBCs in the less than 10% of circumvallate

taste buds that express T1R1; Hoon et al., 1999) and only modestly in foliate papillae (in

-10% of TBCs in the -30% of foliate taste buds that express T1R1; Hoon et al., 1999).

In contrast, T1R2 was reported to be expressed mainly in the circumvallate and foliate

papillae (in -20-30% of the TBCs in 100% of the buds in these papillae; Hoon et al.,

1999). It is almost non existent in the taste buds of the fungiform papillae (in less than

1% of fungiform taste buds; Hoon et al., 1999; but see below) and only modestly

expressed in the palate (in less than -5% of TBCs in the -20% of palatal taste buds that

express T1R2; Hoon et al., 1999). The receptor T1R3, however, was purportedly

expressed in -30% of TBCs in all three taste bud containing papillae (Nelson et al., 2001;

see Table 1-2 for a summary of the results from all of the heretofore mentioned

expression pattern studies). Double-label in situ hybridization studies showed that

virtually all T1R3-expressing cells in circumvallate and foliate papillae express T1R2

(Max et al., 2001; Montmayeur et al., 2001; Nelson et al., 2001). Conversely, all T1R2

expressing cells in circumvallate and foliate papillae, as well as in the palate, were also

reported to express T1R3 (Montmayeur et al., 2001; Nelson et al., 2001). Moreover,

most of the taste cells in fungiform papillae that express T1R3 also express T1R1 (Nelson

et al., 2001). These experiments, however, also implied that there exists a population of

cells in fungiform papillae and in the palate that express T1R3 without T1R1 or T1R2

(Nelson et al., 2001).

These expression data imply that the nerves innervating the circumvallate and

fungiform receptor fields (i.e., the fields of the chorda tympani and glossopharyngeal

nerves, respectively) would be differentially sensitive to sugars and amino acids. For









example, the chorda tympani nerve, which innervates fungiform papillae, which

preferentially expresses the T1R1+3 receptor complex, should be relatively more

responsive to L-amino acids than to sugars or D-amino acids. On the other hand, the

glossopharyngeal nerve, which innervates the circumvallate and foliate papillae, which

preferentially expresses the T1R2+3 receptor complex, should be relatively more

responsive to sugars and D-amino acids than to L-amino acids. However, the response

properties of the mouse whole chorda tympani and glossopharyngeal nerves do not

correspond to these predicted patterns (e.g., Ninomiya and Funakoshi, 1989; Ninomiya et

al., 1993; Ninomiya et al., 2000; Danilova and Hellekant, 2003). This may suggest the

existence of other receptors that are responsive to these ligands or it may suggest that the

aforementioned expression data are inaccurate or incomplete. Indeed, more recent data

do conflict with the previous studies on the expression pattern of these receptors.

Contrary to these reports, the expression of the receptor, T1R1, was shown to be greater

(i.e., in a larger number of cells) than that of T1R2 and T1R3 in circumvallate papillae,

albeit with a lower signal intensity than the other two receptors (Kim et al., 2003).

Using double-label in situ hybridization, these researchers found that, in the

circumvallate, almost all cells that express T1R2 and the majority of cells that express

T1R3 also express T1R1 (Kim et al., 2003). They also reported that, in fungiform

papillae, half of the T1R3 expressing cells also express T1R2. As mentioned above, prior

work suggested that the expression of the T1R2 receptor in fungiform papillae is rare.

Moreover, expression of T1R1 and T1R2 overlapped in fungiform papillae. Since every

T1R2 positive cell ubiquitously expressed T1R3, then T1R1 + T1R2 double positive cells

must also express T1R3. Therefore, in both receptor fields, TBCs that co-express all









three T1R receptors can be found (i.e., T1R1, T1R2, and T1R3; Kim et al., 2003).

Interestingly, in the circumvallate, 66% of the T2R receptor expressing cells1 also

expressed the receptor T1R1. The co-expression of the other two receptors with the

various T2R receptors was not assessed.

The fact that sugars and L-amino acids bind, selectively, with different T1R

receptor complexes suggest that theses compounds may be perceptually distinct and as a

result, behaviorally discriminable. Indeed, the early reports on the pattern of T1R

receptor expression provide a degree of receptor complex segregation that could support

this discriminability. However, depending on the degree of receptor expression overlap

on single TBCs, the data reported by Kim et al. predict that mice would have at least

some difficulty discriminating between sugars and L-amino acids. Indeed, these data,

reported by Kim et al., portray a more balanced receptor expression pattern, at least

across the circumvallate and fungiform papillae, and correspond more closely to the

response properties of gustatory nerves than do data from the previous studies.

As an important caveat, it should be noted that peripheral nerve responses only

index the nature of the signal arising from the initial processing of taste input. The

central gustatory system can amplify, attenuate, or alter features of the peripheral signal

(e.g., convergence). Moreover, knowledge regarding a neural response, whether of

peripheral or central origin, does not in and of itself, necessarily reveal how those signals

are translated into behavior (and the associated inferred perceptions).

The complexities of these interpretive issues are exemplified by the opposing

postulations regarding "bitter" taste perception made by researchers who study receptor


1 Mix probes of T2R5, T2R8, T2R18, and T2R19 were used for analysis of T2Rs expression









expression and those who study the response properties of TBCs. A family of G-protein-

coupled taste receptors (T2Rs) were shown to bind with a structurally diverse class of

"bitter-tasting" compounds (Adler et al., 2000; Chandrashekar et al., 2000; Matsunami et

al., 2000). It was suggested that these receptors evolved to help animals avoid ingesting

toxic or otherwise harmful substances. Although each one of the receptors in this family

is thought to be relatively specific for its ligand, many appear to be co-expressed in

subsets of TBCs. This latter finding led researchers to hypothesize that mammals could

not discriminate between "bitter-tasting" stimuli, because a given TBC could potentially

be stimulated by a wide variety of compounds (Adler et al., 2000; Chandrashekar et al.,

2000). Caicedo and Roper (2001) suggested, however, that TBCs are more narrowly

tuned than predicted from the receptor co-expression. This conclusion was based on their

assessment of the intracellular calcium responses in TBCs, in situ. Each "bitter-

responsive" TBC assayed responded to only one, or at most only a few of the five

compounds tested (Caicedo and Roper, 2001). These researchers suggested that rats

could likely discriminate between the "bitter" compounds tested, based on the apparent

specificity of TBCs.

Spector and Kopka (2002) tested these predictions and demonstrated that rats could

not behaviorally discriminate between denatonium benzoate and quinine hydrochloride.

Data from this study strongly suggested that these two ligands produce a unitary taste

sensation. These results appear to support the molecular findings, which indicted that

numerous T2Rs are co-expressed in subsets of TBCs. Nevertheless, the specificity of

TBCs observed in the periphery (based on Ca" imaging measurements) might indeed

exist. However, any segregation may be negated by a convergence of the signals









generated by the TBCs into a single neural channel somewhere along the gustatory

neuraxis.

Summary

Conclusions regarding the taste quality of amino acids based on data from CTA

experiments suggest that a subset of amino acids are perceptually similar to sucrose and

some other sugars and results from two-bottle intake experiments show that these amino

acids are preferred by mice. However, although L-amino acids are thought to bind

exclusively with the T1R1+3 receptor complex, strain preference behavior measured in

the two-bottle intake test seems to depend on an anomaly in the T1R2+3 complex,

leaving open the possibility that other factors were influencing the relative preference for

L-amino acids in this test. Moreover, data gathered in our laboratory question whether or

not these stimuli are actually preferred by mice at all on the basis of taste and as a result

question the very nature of the taste quality evoked by these compounds. Indeed, the

molecular biology of "sweetener" transduction appears to provide a neurobiological basis

for behavioral discriminability (i.e., perceptual distinction). Thus, my objective was to

determine the degree to which receptor specificity predicts the relative discriminability of

various "sweeteners," by testing whether C57BL/6J (B6) mice can discriminate between

sucrose and L-serine, as well as a variety of other sugars and putative sweeteners using an

operate discrimination paradigm. To my knowledge, explicit discrimination experiments

in rodents with these ligands have never been conducted. These tasks were designed to

assess whether pairs of putative sweeteners would be treated by animals as perceptually

identical. If compounds are distinguishable, however, these discrimination tasks provide

little information on the basis of the discriminability. Moreover, even if animals can

discriminate between two compounds, they may also find them to be similar, relative to









other taste qualities (Spector, 2003). Therefore, to determine the relative similarity of

these "sweeteners," as well as to provide information to aide in the interpretation of the

discrimination data, CTA generalization tests where also conducted with a theoretically

relevant subset of the stimuli.

The identification of compounds that share perceptual features and that are

behaviorally indiscriminable allows for a confident categorization of a taste compound

into a perceptual class (e.g., sweetness). Moreover, such data can be related back to the

molecular biology of "sweet" taste in search of clues regarding the functional

organization of the "normal" murine gustatory system.







12


Tablel-1: Consequences of the "knockout" of various T1R receptors on neural and taste-related behavioral
responses
Chorda Tympani Nerve Recording Brief-Access Taste Test
STIMULUS
T1R2+3 T1R2+3
T1R1 KO T1R2 KO T1R3 KO T1R1 KO T1R2 KO T1R3 KO
KO KO

L-serine ? ? ? ? I Normal 0 ?

L-srine +30n 0 Normal 0 ? ? ? ? ?
IMP*

L-alanine ? ? ? ? I Normal 0 ?

L-alanine + 30 mM
L-alanine M 0 Normal 0 ? ? ? ? ?
IMP*

D-tryptophan Normal 0 0 0 Normal 0 0 ?

Sucrose Normal I I 0 Normal I I# 0

Glucose Normal 0 Normal I I 0
1 (Normal)
I = Impaired responsiveness; 0 = No responsiveness; ? = Not tested; IMP alone produced no nerve response; ** All results are from Zhao
et al., 2003 except for those in parentheses, which are from Damak et al., 2003.

# Behavioral data, not gathered in a brief-access test, from two separate studies (Damak et al., 2003 and Delay et al., 2006) suggest that
mice, lacking the receptor T1R3 can detect the presence of sucrose.














Table 1-2: % of taste buds and % TBCs in papillae
Circumvallate Foliate Fungiform Palate
Receptor TB TBCs refs TB TBCs refs TB TBCs refs TB TBCs refs

-20- -20-
T1R1 < 10% 5% 1 -30%* -10%* 1 100% 0 1 100% 0 1
30% 30%

-20- -20-
T1R2 100% 300 1 100% 30 1 < 1% N/A 1 -20% < 5% 1
30% 30%


T1R3 100% -30% 2 100% -30% 2 100% -30% 2 100% -30% 2

Data derived from published reports (see reference below and in text) of the percentages of taste buds expressing a given taste receptor
and the percentages of TBCs within those taste buds (see text for more details).

1All data from Hoon et al., 1999 are derived mouse and rat tongues.

*These cells were reported to have a much weaker signal relative to T1R2 positive cells in the foliate, or T1R1 cells in fungiform
papillae or in the palate.

2All data Nelson et al., 2001 are derived from adult mouse tongues















CHAPTER 2
EXPERIMENT 1: THE RELATIVE AFFECTIVE POTENCY OF GLYCINE, L-
SERINE AND SUCROSE AS ASSESSED BY A BRIEF-ACCESS TASTE TEST IN
INBRED STRAINS OF MICE2

Background

The molecular biology pertaining to the transduction of both sugars and synthetic

sweeteners as well as "sweet-tasting" D-amino acids, reviewed in detail in Chapter 1, is

consistent with the electrophysiological and behavioral phenotypes expressed by different

inbred strains of mice, but such a correspondence regarding "sweet-tasting" L-amino

acids (and glycine) is less straightforward. It has been known for many years that mouse

strains can be differentiated according to their intake of and nerve responsiveness to

natural and synthetic sweeteners. In general, "taster" mice have lower preference

thresholds for sweeteners in two-bottle tests and their chorda tympani nerves (CT) are

more responsive to sucrose, saccharin, and various "sweet-tasting" D-amino acids

(especially D-phenylalanine) when compared with "non-taster"3 mice (Capretta, 1970;

Pelz et al., 1973; Fuller, 1974; Ninomiya et al., 1984; Lush, 1989; Capeless and Whitney,

1995; Bachmanov et al., 1996; Frank and Blizard, 1999; Inoue et al., 2001; Nelson et al.,

2001). These taster/non-taster phenotypes in mice were genetically linked to a single

chromosomal locus referred to as Sac that was later discovered to encode for the T1R3


2 A version of this Chapter has been published previously in Chemical Senses, Vol. 29 No. 6 C Published
by Oxford University Press. All rights reserved.

3 The phenotypic descriptors "taster" and "non-taster" may at first glance seem to denote ageusic vs. non-
ageusic strains, however this nomenclature is commonly used in the literature to categorize mouse strains
with varying degrees of sensitivity (i.e., low behavioral threshold vs. high behavioral threshold) to
compounds such as sucrose and/or sodium saccharin.









receptor (e.g., Fuller, 1974; Ramirez and Fuller, 1976; Lush, 1989; e.g., Capeless and

Whitney, 1995; Lush et al., 1995). Taster and non-taster mouse strains have different

alleles of the Taslr3 gene that give rise to receptors with slightly different amino acid

sequences (e.g., Bachmanov et al., 2001a; Kitagawa et al., 2001; Max et al., 2001;

Montmayeur et al., 2001; Sainz et al., 2001). Interestingly, the taster and non-taster

allele of Taslr3 generates receptors that are functionally similar when combined with

T1R1, but the non-taster form of the T1R3 receptor displays impaired binding when

combined with T1R2 (Nelson et al., 2002; Damak et al., 2003). Thus non-taster mouse

strains possess a dysfunctional T1R2+3, but an apparently normal T1R1+3 receptor

complex. Indeed, there is evidence that L-amino acids, which bind with the T1R1+3

receptor, stimulate the CT comparably in both taster and non-taster mice, with the

possible exception of L-proline (Ninomiya et al., 1984; Inoue et al., 2001). Yet, two-

bottle preference for some "sweet-tasting" L-amino acids and glycine appears to depend

on the "taster" status of the mouse strain based on testing with sugars (Lush, 1989;

Capeless and Whitney, 1995; Lush et al., 1995; Bachmanov et al., 2001b). These

behavioral findings are curious considering that L-amino acids are believed to bind

primarily with the T1R1+3 receptor which, as noted above, is thought to display similar

binding properties in both taster and non-taster mice (Nelson et al., 2002).

In light of the apparent tension between the predicted behavior of mouse strains

based on the molecular biology of amino acid taste transduction and the observed

behavior seen in the two-bottle preference test, we examined the relative effectiveness of

sucrose, glycine and L-serine to stimulate licking in C57BL/6J (B6), SWR/J (SWR),

DBA/2J (D2) and 129P3/J (129) mice in a brief-access taste test. As noted above, inbred









mice vary in their preference for all three of these compounds as assessed in two-bottle

intake tests, and there is evidence that these compounds possess some common

perceptual properties with respect to taste quality (i.e., "sweet") in at least some rodents.

If glycine and L-serine generate concentration-response functions that emulate sucrose,

then it would suggest that these compounds are similar in their affective potency.

In addition, we sought to examine the generality of the response profiles generated

by these compounds by including taster (B6 and SWR) and non-taster (129 and D2)

mouse strains in the experimental design allowing us to make inferences regarding the

effect of the non-taster form of the Taslr3 allele on taste-guided behavior (Capretta,

1970; Pelz et al., 1973; Fuller, 1974; Lush, 1989; Capeless and Whitney, 1995;

Bachmanov et al., 1996; Max et al., 2001; Nelson et al., 2001). With some notable

exceptions (Glendinning et al., 2002; Zhang et al., 2003; Zhao et al., 2003), most of the

work conducted to date involving strain comparisons of unconditioned behavioral

responsiveness to these compounds has been based on two-bottle intake tests (water

versus taste compound). Although taste certainly influences the behavior in that test

paradigm, postingestive events can also influence intake. The brief-access taste test

involves the measurement of licking during very short trials with a sapid solution

increasing the confidence that the responses are based on the oral sensory features of the

stimulus. Many trials of various concentrations of the taste stimulus are presented during

a session and concentration-response functions are derived. The taste solutions are

delivered in randomized blocks to minimize systematic carry-over effects and to mitigate

the influence of postingestive factors on the response to a given stimulus in the set.









Methodological Details

Subjects

A total of 120 male naive mice (Jackson Laboratories, Bar Harbor, Maine) from

four different strains, C57BL/6J (B6), SWR/J (SWR), 129P3/J (129), and DBA/2J (D2),

served as subjects (n=30/strain). Within each strain, animals were randomly assigned to

one of 3 stimulus groups (n=10/group). The mice were housed individually in

polycarbonate shoebox cages in a colony room where the lighting was controlled

automatically (12h: 12h). Testing and training took place during the lights-on phase.

Mice were habituated to the laboratory environment for seven days before testing and

were 8 weeks of age at the start of testing. During this time, food and purified water

(Elix 10; Millipore, Billerica, MA) were available ad libitum. During periods when the

animals were placed on a water-restriction schedule, mice that dropped below 80% of

their free-feeding weight received 1 ml supplemental water 2 hours after the end of the

testing session. All procedures were approved by the University of Florida Institutional

Animal Care and Use Committee.

Taste Stimuli

All solutions were prepared daily with purified water and reagent grade chemicals,

and were presented at room temperature. Test stimuli consisted of 5 concentrations of

sucrose (0.0625, 0.125, 0.25, 0.5, and 1.0 M; Fisher Scientific, Atlanta, GA), L-serine

(0.25, 0.5, 0.75, 1.0, and 1.5 M; Sigma-Aldrich, St Louis, MO), glycine (0.25, 0.5, 0.75,

1.0, and 1.5 M; Sigma-Aldrich, St Louis, MO) and purified water. Sucrose was chosen

because 1) it is a prototypical natural sweetener that is commonly used in taste

experiments, 2) it has been used to differentiate taster (B6 and SWR) from non-taster (D2

and 129) mice in two-bottle preference tests, and 3) binds with the T1R2+3, but not the









T1R1+3, receptor complex. L-serine and glycine were chosen because 1) there is

evidence that at least in some rodents these compounds share a perceptual quality with

sucrose, 2) they are preferred by some rodents at mid-range concentrations in two-bottle

preference tests, and 3) appear to bind primarily with the T1R1+3, but only poorly, if at

all, with the T1R2+3 receptor complex.

Procedure

We used a brief-access procedure similar to that described by Glendinning et al.

(2002). Testing took place in a lickometer referred to as the Davis rig (Davis MS-160,

DiLog Instruments, Tallahassee, FL; see Smith, 2001). This device allowed the mouse

access to a single tube containing a taste stimulus for a brief period of time (5 s) and then

after a 7.5-s inter-presentation interval, a different tube was offered. The stimulus array

for each compound tested included the five different concentrations detailed above and

purified water contained in separate bottles. A given trial started after the first lick.

Presentation order was randomized without replacement in blocks so that every

concentration of a stimulus and water was presented exactly once before the initiation of

the subsequent block. Unconditioned licking responses were recorded for later analysis.

Sessions were 30 min in duration during which mice could initiate as many trials as

possible. The animals were first trained to lick a stationary tube of water for 30 min in

the Davis rig after being placed on -23.5-h restricted water access schedule. Animals

then received 2 days of testing with five stimulus concentrations and purified water while

maintained on the water-restriction schedule. This was done to familiarize the animals

with the stimulus array. The water bottles were then replaced on the home cages and the

mice were tested for three days non-deprived.









Data Analysis

For data gathered when animals were water-deprived, a Tastant/Water Lick Ratio

was calculated. This ratio was derived by taking the average number of licks per trial for

each concentration and dividing it by the average licks per trial when water was

delivered. This ratio controls for individual differences in lick rates and for differences in

motivational state. The Tastant/Water Lick Ratio is useful for analyzing responses of

animals highly motivated to lick due to the restricted water access schedule. When

animals were non-deprived, the average number of licks per trial for each concentration

was collapsed across test sessions and divided by that animal's maximum potential lick

rate per trial based on the mean of the inter-lick interval (ILI) distribution measured

during training (only inter-lick intervals greater than 50 and less than 200 ms were used),

yielding a Standardized Lick Ratio (see Glendinning et al. 2002). Standardizing the

licking response in this fashion controls for individual differences in maximal lick rates.

The ratio scores were analyzed with two-way strain x concentration analyses of

variance (ANOVAs). When a strain main effect or a strain x concentration interaction

was significant, 1-way ANOVAs were conducted to test for simple effects. Differences

between strains at each concentration were evaluated using Tukey's honestly significant

difference test. Differences between Standardized Lick Ratio scores in response to a

given concentration and those measured for water were tested with matched-t-tests. The

conventional P < .05 was applied as the statistical rejection criterion. Only mice that had

at least 1 trial at every concentration were included in the analysis of a given stimulus.









Results

Standardization Data

Because there can be within-strain and between-strain differences in the local lick

rate as well as in the motivational response to the water restriction schedule, it is

important to account for these factors in any licking measure of taste responsiveness. As

recommended by Glendinning et al. (2002), the Tastant/Water Lick Ratio was calculated

for animals tested when under the water-restriction schedule and the Standardized Lick

Ratio was calculated for animals tested when non-deprived to statistically control for

non-taste influences in licking. Table 2-1 contains the means values representing licks

during water trials used in the calculation of the Tastant/Water Lick Ratio for the various

strains and compounds. A two-way ANOVA on water licks revealed a significant main

effect of strain [F(3,107) = 40.7, P < .001] and test solution [F(2,107) = 9.15, P < .001]

as well as a significant interaction [F(6,107) = 5.31, P < .001]. One-way ANOVAs were

conducted within each taste compound to test for strain differences in water licks. There

was a significant main effect of strain on the mean number of licks to water when mice

were tested with sucrose [F(3,36) = 15.6, P < .001], L-serine [F(3,36) = 15.4, P < .001]

and glycine [F(3,36) = 19.6, P < .001] in the water restriction condition. Interestingly,

when mice were water deprived, one-way ANOVAs conducted within each strain to test

for differences in water licks to the stimuli revealed that the non-taster strains increased

licks to water when tested with L-serine relative to licks taken in the other stimulus

conditions (Ps < .001). The taster strains did not significantly differ in their responses to

water across stimulus conditions.









Table 2-2 contains the means of the individual values representing the ILI observed

when water-restricted animals were licking water from a stationary spout. These means

exclude the mice that were not included in the analysis of responses under non-deprived

conditions (n = 93). The reciprocal of these values were multiplied by 5000 to derive the

estimated maximum possible licks during a 5-s trial and used in the calculation of the

Standardized Lick Ratio for various strains and compounds. As expected, a two-way

ANOVA revealed a significant effect of strain [F(3,81) = 52.1, P < .001] but no

significant stimulus effect [F(2,81) = 0.1, P = .909] or interaction [F(6,81) = 0.7, P =

.657]. Collectively, the results from these analyses confirm the necessity for

standardizing the licking data across animals and strains.

Sucrose

When animals were water deprived, a two-way ANOVA of the Tastant/Water Lick

Ratios revealed a significant main effect of strain [F(3,36) = 18.1, P < .001), a significant

main effect of concentration [F(4,144) = 5.9, P < .001] and a significant interaction

[F(12,144) = 10.4, P < .001]. Strain differences at each concentration are delineated in

Table 2-3. Confirming what is apparent in Figure 2-1, separate one-way ANOVAs for

each strain revealed that only the 129 mice showed a significant monotonically increasing

concentration-response function [F(4,36) = 11.9, P < .001]. Although we did not expect

to find meaningful results in the water-restriction condition considering that mice will

usually lick water at a maximal rate making it difficult to ascertain a response to

appetitive stimuli and we did not expect to see an aversive response profile elicited by

these "sweet-tasting" compounds, it appears that the 129 mice did suppress licking to

water relative to the other strains (see table 2-1) and, as a result, increased their

Tastant/Water Lick Ratio to the stimulus [F(3,36) = 15.6, P < .001]. There were some









significant concentration-dependent effects on the Tastant/Water Lick Ratio for the other

3 strains (All Fs >3.0, all Ps < .05), but it is obvious that these functions were relatively

flat and generally equal to or below a value of 1.0. The 129 mice had significantly higher

ratios at all five concentrations compared with the B6 and D2 mice and at the four highest

concentrations compared with the SWR mice (all P values < .05); the latter three strains

did not differ.

When non-deprived, all strains clearly showed a concentration dependent increase

in licking to sucrose (see Figure 2-2; F(5,170)=531.9, P < .001), but their concentration-

response functions significantly differed (strain x concentration interaction: F(15,170) =

10.9, P < .001). Strain differences at each concentration are delineated in Table 2-4. The

SWR mice were significantly more responsive to lower sucrose concentrations compared

with D2 and 129 mice. At the lowest concentration tested (0.0625 M), the Standardized

Lick Ratio was significantly greater than that for water in the SWR and B6 (both ts > 2.2,

Ps < .05), but not the D2 and 129, strains (both ts < -0.2, Ps > .7). As the sucrose

concentration was raised, however, D2 and 129 mice steeply increased their

responsiveness to sucrose and eventually equaled or surpassed the licking in SWR mice.

B6 mice had a concentration response profile somewhat in between the SWR and the 129

and D2 mice. At the lower concentration, B6 mice were statistically indistinguishable

from all of the mice including the SWR, but at the two highest concentrations they were

significantly less responsive compared with the other three strains.

L-serine

When mice were water deprived, there was a significant strain effect (F(3,36) =

9.8, P < .001) on the Tastant/Water Lick Ratio, and a significant strain x concentration

interaction (see Figure 2-1; F(12,144) = 6.6, P < .001). Surprisingly, the D2 mice









actually decreased their lick rate as the L-serine concentration was raised (Figure 2-1;

F(4,36) = 15.3, P < .001), whereas the other strains displayed relatively flat functions.

Strain differences at each concentration are shown in Table 2-3.

When animals were non-deprived, there was no significant difference in the

Standardized Lick Ratio between the strains (F(3,21) = 0.1, P = .9), but there was a

significant effect of concentration (F(5,105) = 4.2, P = .002), though the increase was

relatively minor; there was no significant strain x concentration interaction (see Figure 2-

2).

Glycine

When animals were water deprived, there was a significant strain effect (F(3, 35) =

10.6, P < .001) on the Tastant/Water Lick Ratio, and a significant strain x concentration

interaction (F(12,140) = 5.7, P < .001). Strain differences at each concentration are

delineated in Table 2-3. As was the case with sucrose, separate one-way ANOVAs

indicated that only the 129 mice increased their Taste/Water Lick Ratio monotonically as

a function of concentration (F(4,36) = 7.1, P < .001); see Figure 2-1). This increase in

licking was first significantly greater than 1.0 at the 0.75 M concentration (P = .022).

There were some significant concentration-dependent effects on the Tastant/Water Lick

Ratio for B6 and SWR mice (All Fs > 4.6, all Ps < .01), but it is obvious that the

functions for these strains as well as for the D2 mice were relatively flat and generally

below a value of 1.0.

When mice were non-deprived, there was a significant effect of strain (F(3, 26) =

5.8, P = .004) on the Standardized Lick Ratio, and a significant strain x concentration

interaction (F(15,130) = 2.9, P = .001). Strain differences at each concentration are

delineated in Table 2-4. Separate one-way ANOVAs of the Standardized Lick Ratios for









each strain revealed that 129 (F(5,20) = 8.1, P < .001), B6 (F(5,40) = 3.3, P < .05), and

D2 (F(5,40) = 2.5, P < .05) mice changed their lick rate as a function of concentration,

but the modest increases were apparently limited to higher concentrations (see Figure 2-

2). For example, matched t-tests indicated that the 129 strain did not display significantly

elevated licking relative to water until the glycine concentration reached 1.5 M (P < .05).

For B6 and D2 mice, no concentration significantly differed from water. The SWR mice

did not significantly change their licking as a function of concentration (F(5,30) = 0.6, P

=.678).

Discussion

Overall, as assessed by the brief-access taste test, the amino acids, L-serine and

glycine, paled in comparison to sucrose in their ability to generate licking in the mouse

strains examined. Collapsed across strain, non-deprived animals licked L-serine and

glycine at a mean rate of only 15.4% and 21.4%, respectively, of the maximum possible

in the 5-s trial at the highest concentration tested (1.5 M). In striking contrast, 1.0 M

sucrose (the highest concentration tested) elicited an average licking rate, collapsed

across strain, that was more than 5 times higher than that seen for L-serine and nearly 4

times higher than that seen for glycine. The relatively broad concentration range used in

this study weakens the possibility that the design failed to capture the dynamic range of

responsiveness. Thus, the results presented here suggest that the taste-related affective

potency of sucrose is far superior to that of glycine or L-serine.

Although neither amino acid was remarkably effective at stimulating licking in

non-deprived mice relative to sucrose, glycine generated concentration-dependent

increases in licking in water-restricted 129 mice, whereas L-serine did not. For the D2

non-taster mice, we actually observed a concentration-dependent decrease in the









Tastant/Water Lick Ratio in response to L-serine when these mice were water-deprived.

Given that L-serine is thought to possess a sucrose-like taste quality, this finding was

unexpected and suggests that L-serine may also bind with other receptors that lead to

aversive responses (e.g. T2Rs), at least in the D2 strain. Other researchers have reported

higher levels of L-serine licking relative to water by B6, 129X1/SvJ and CB6 (BALB/c x

B6 hybrids) mice in a brief-access test (Zhang et al., 2003; Zhao et al., 2003). These

discrepancies are likely the result of methodological differences between the studies.

More specifically, in the prior work, both food and water intake was limited in a

controlled fashion, based on procedures described by Glendinning et al. 2002, to achieve

a motivational state that would promote stimulus sampling but would not lead to the

asymptotic lick rates generally observed under 24-h water deprivation regimens. Based

on the present results, it appears that without the additional effects of nutrient restriction,

the gustatory properties of L-serine and glycine alone stimulate only slight, if any, licking

behavior in the mouse strains tested here.

The profile of strain differences in responsiveness to the compounds tested here

was more complex than previously reported. When non-deprive mice were tested with

sucrose, in general the "taster" strains (B6 and SWR) were modestly more responsive at

lower concentrations compared with the "non-taster" mice (129 and D2), but even this

difference failed to reach significance for the B6 strain. As the concentration was

progressively raised, the responsiveness of SWR and B6 taster mice converged with that

seen in the D2 non-taster mice. Notably, the 129 non-taster mice licked the two highest

concentrations of sucrose significantly more than did all three of the other strains. In









general, these results are consistent with findings obtained by other researchers (i.e.,

Glendinning et al., 2005b).

When mice were water deprived, sucrose, as expected, produced licking rates

comparable to water in all strains except for the 129 mice. The 129 mice, in fact, nearly

doubled their rate of stimulus responsiveness relative to water at 1.0 M. This same

pattern was seen with glycine, with the 129 mice responding to the compound at nearly

1.5 times the rate of water at 1.5 M. Interestingly, the D2 non-taster mice displayed

concentration-dependent decreases in their L-serine tastant/water lick ratio when water

deprived, whereas the other strains had relatively flat curves. It appears when mice were

water-deprived the non-taster strains were less motivated to lick L-serine relative to

sucrose and glycine, whereas all three stimuli were treated similarly by the taster strains.

Collectively, these findings suggest that the phenotypic descriptors "taster" and "non-

taster" do not necessarily apply to the responsiveness seen at higher concentrations of

putative sweeteners, at least in the brief-access test.

The taster and non-taster classification is based on the preference behavior of

various mouse strains to low concentrations of sweeteners in long-term two-bottle intake

tests. The brief-access taste test differs from the two-bottle intake test in interpretively

important ways. In the brief-access test, immediate responses to small volumes of stimuli

are measured raising the confidence that the behavior is driven by taste (see Spector,

2003). Indeed, Spector et al. (1996) demonstrated that when rats are deprived of

gustatory input from the 7th and 9th cranial nerves innervating the oral cavity, they show

essentially flat concentration-response curves for sucrose when tested using a brief-

access paradigm providing further evidence that behavior measured using a brief-access









procedure is taste-guided. In contrast, in the two-bottle test, intake is usually measured

24 h after stimulus presentation allowing for postingestive factors to influence the

outcome. Moreover, differences in stimulus preference at high concentrations are

difficult to detect with the two-bottle preference test because of ceiling effects.

Typically, preference ratios approach an asymptotic value of 1.0 at very low

concentrations for normally preferred stimuli, after which differences are difficult to

discern. Other researches have used a shorter-term one-bottle intake test (e.g., 6 hr.)

where ceiling effects and position preferences are avoided or at least minimized (e.g.,

Blizard et al., 1999). But while the results obtained using the one-bottle test are

consistent with those seen when using the two-bottle intake procedure, neither test avoids

the confounding effects of viscerosensory input. On the other hand, the brief-access taste

test does not appear to be as sensitive to changes in behavior at low concentrations, at

least when several higher concentrations are available during the session. Thus, these

various procedures have different dynamic ranges of sensitivity. Accordingly, it would

appear that, behaviorally speaking, the taster/non-taster distinction is limited to low

concentrations of sweeteners. This is consistent with sucrose and glucose detection

thresholds measured with an operant procedure in which the hedonic value of the taste

stimulus is rendered irrelevant (Eylam and Spector, 2004). Interestingly, in the Eylam

and Spector study, the threshold values for glycine measured with the same procedure in

the same mice did not distinguish taster and non-taster strains in as straightforward a

manner. That is, non-taster 129 mice had significantly higher glycine thresholds relative

to B6 mice. However, the glycine thresholds for non-taster D2 mice did not differ from

those for the taster B6 and SWR mice. In stark contrast, in our study, at the higher









concentrations, the 129 mice were the most responsive strain tested in this report. These

findings further highlight the difference between suprathreshold responsiveness and

threshold sensitivity (cf, Bachmanov et al., 1997).

If the T1R family of receptors mediates "behavioral attraction," as postulated by

some (Zhao et al., 2003), then activation of either receptor complex should elicit

appetitive behavior. However, compounds that bind with the T1R2+3 complex are

apparently much more effective, at least as measured by the assay used in our study.

Sucrose, which was shown to stimulate the T1R2+3 complex in a heterologous system

(HEK 293), generated licking at rates at least four times higher than any other compound

tested. Partial support for this dissociation comes from the fact that glycine, which was

also shown to stimulate the T1R2+3 complex, but to a lesser extent, in general elicited

slight increases in licking at high concentrations resembling its modest ability to bind

with the receptor (see Nelson et al., 2002), at least in those mice that sampled all of the

concentrations. We found no evidence that L-serine, a compound that binds with the

T1R1+3 heteromer, but not with the T1R2+3, is an effective behavioral stimulus in the

brief-access test in non-deprived mice. As noted above, there is evidence that L-amino

acids can stimulate significant degrees of licking in mice that have restricted food and

water access. Thus, it would appear that the affective value of stimuli that bind with the

T1R1+3 receptor depends upon the nutritional/physiological status of the animal,

whereas stimuli that bind with the T1R2+3 receptor do act like general "attractants."

The behavioral results presented here do not relate to the electrophysiological

response properties of the CT nerve in an obvious way (Frank and Blizard, 1999; Inoue et

al., 2001). While all three stimuli used in our study reportedly evoke very clear









concentration-related increases in CT responsiveness in B6 and 129 mice, the

concentration-response functions for glycine and L-serine in non-deprived mice from

these strains in the brief access test had very shallow slopes. Moreover, while the

magnitude of CT responses to sucrose is greater in B6 compared with 129 mice even at

high concentrations, the 129 mice displayed more vigorous sucrose licking than the B6

mice at the 0.5 and 1.0 M concentrations in the brief-access test. It is conceivable that a

subclass of CT fibers might display a better correspondence with the hedonic value of

these stimuli and this relationship might be obscured in whole-nerve analyses (cf., Frank

and Pfaffmann, 1969). However, it is likely that the affective potency of these stimuli is

based on more than just input from the CT. Input from other peripheral nerves and the

central neural circuits that translate those signals into behavior must be considered. Thus,

while non-taster strains might have an impaired peripheral signal for certain sweeteners

that stimulate the T1R2+3 receptor complex, the way that input is processed by the brain

can also differ from taster strains in a manner that could augment behavior. Likewise, a

robust peripheral signal for glycine or L-serine or any taste stimulus does not guarantee

that a given behavioral response will be generated.

In summary, we found that sucrose was the most effective compound tested,

followed by glycine, and lastly L-serine in generating licking in the brief-access taste test.

The order of affective potency seems to be related to the ability of the stimulus to activate

the T1R2+3 receptor complex. Furthermore, strain differences in responsiveness to these

compounds suggest that the current understanding of "sweet-tasting" ligand transduction

is insufficient in entirely explaining the observed response profiles. For example, the fact

that the 129 mice licked at rates greater than the D2, B6 and SWR mice to the higher









concentrations of sucrose would not have been predicted by the current molecular

biological findings or CT nerve recordings. Apparently, the taster/non-taster distinction

which has been shown to be dependent on the polymorphism of the Taslr3 gene

encoding for the T1R3 receptor is limited to low concentrations of sucrose, whereas

responsiveness to higher concentrations of the sugar is related, at least in part, to other

genes that might affect stimulus processing anywhere along the gustatory neuraxis. It

would be instructive to repeat the behavioral tests conducted here in congenic, transgenic

and/or knock-out mice in which the Taslr3 gene has been manipulated keeping the

genetic background constant to examine the explicit role of the T1R3 variants in

behavioral responsiveness to mid-range and high concentrations of sugars, synthetic

compounds and amino acids. The results of our study also call into question the very

nature of the perceptual quality elicited by these amino acids. As noted above, there is

evidence from conditioned taste aversion generalization experiments that rodents treat

glycine and L-serine as possessing a sucrose-like taste quality (Nowlis et al., 1980;

Pritchard and Scott, 1982b; Kasahara et al., 1987). Yet, in the brief-access test with the

non-deprived mice tested here, the responses to sucrose were discernibly different than

those to the amino acids. Thus, it would appear that while the perception evoked by

glycine and L-serine might share some qualitative characteristic with sucrose, these

amino acids might also generate additional qualities that impact upon their affective value

at least in certain species and strains. For example, saccharin is both sweet and bitter

tasting to humans depending on concentration (Bartoshuk, 1979; Schiffman et al., 1979).

Experiments designed explicitly to test the ability of these mice to distinguish between

sucrose, glycine, L-serine, and other L-amino acids and sugars in operant taste






31


discrimination tasks, in addition to a more comprehensive examination of conditioned

taste aversion generalization profiles should help refine the characterization of the

qualitative similarities and differences of these taste stimuli. Such behavioral

experiments can provide a functional context to guide the interpretation of findings from

more molecular levels of analysis.







32





Table 2-1: Mean number of licks to water SEM taken by the four strains when tested with sucrose, L-
serine. or glvcine when mice were water deprived.


Sucrose L-serine Glycine
B6 32.2 + 1.4 30.6 + 2.3 32.3 + 2.2
SWR 41.8 2.2 44.1 1.9 48.3 1.5
129 22.9 + 1.7 34.0 + 1.9* 24.8 + 2.3
D2 34.8 + 2.5 47.1 1.9* 32.8 + 2.9


Asterisk indicates that the 129 and D2 "non-taster" mice took significantly higher numbers of licks to water
when tested with L-serine relative to sucrose or glycine.


'











Table 2-2: Mean of the inter-lick interval (ILI) distribution (ms) + SEM observed in the four strains of mice
trained to lick either sucrose, L-serine, or glycine.
Sucrose L-serine Glycine
B6 121.3 + 1.1 122.0 + 1.5 121.3 + 1.9
SWR 98.0 + 1.2 98.6 + 1.4 94.4 + 1.0
129 108.3 + 1.1 109.8 + 0.8 108.6 + 1.7
D2 104.4 + 2.3 105.9 + 1.8 104.7 + 2.8
For a given strain, the observed ILI value did not significantly differ whether tested with sucrose, L-serine,
or glycine.
















Table 2-3: Strains listed in order of mean Tastant/Water Lick ratio, for sucrose, L-serine, and glycine when mice were water deprived.


Sucrose'


0.0625 M 129 > SWR = D2 = B6

0.125 M 129 > SWR = D2 = B6

0.25 M 129 > SWR = D2 = B6

0.5 M 129 > SWR = D2 = B6

1.0 M 129 > SWR = D2 = B6


Lserinel

0.25 M 129 = B6 = SWR = D2


0.5 M SWR > 129 = B6 = D2


0.75 M 129 > SWR = B6 = D2

1.0 M 129 = B6 = SWR > D2

1.5 M SWR = 129 = B6 > D2


Qycine

0.25 M 129 = B5 = SWR = D2


0.5 M 129 > SWR = B = D2

0.75 M 129 > B5 = SWR = D2

1.0 M 129 > D2 = B = SWR

1.5 M 129 > D2 = SWR = B6


1At concentrations at which the ANOVA detected a significant strain effect, strains falling under the same line did not significantly differ in Tukey's HSD
posthoc comparisons (P<0.05).


















Table 2-4: Strain listed in order of mean Standardized Lick ratio, for sucrose, L-serine, and glycine when mice were non-deprived.


Sucrose'

Water: SWR = B6 = D2 = 129


0.0625 M: SWR > B6 = D2 = 129


0.125 M: SWR > B6 = 129 = D2

0.25 M: SWR > 129 > B6 > D2

0.5 M: 129 > SWR = D2 = B6

1.0 M: 129 > D2 = SWR = B6


Lserine2

Water: B6 = SWR = D2 = 129

0.25 M 129 = B6 = D2 = SWR

0.5 M D2 = SWR = B6 = 129

0.75 M B6 = 129 = D2 = SWR

1.0 M B6 = 129 = SWR = D2

1.5 M B6 = 129 = SWR = D2


Glycine

Water: SWR = B6 = D2 = 129

0.25 M: B6 = D2 = 129 = SWR

0.5 M: B6 = 129 = D2 = SWR

0.75 M: B6 = 129 = SWR = D2


1.0 M: 129 > B6 = D2 = SWR


1.5 M: 129 > B6 = D2 = SWR


1For sucrose and glycine, at concentrations at which the ANOVA detected a significant strain effect, strains falling under the same line did not significantly
differ in Tukey's HSD posthoc comparisons (P<0.05).

2There were no significant strain effects for L-serine.





















20 B6 Sucrose L-serine Glycine

2 -- 129
D2
15-



10 -



05



nn


02 03 04 05 065


1 025 04 05 06 075 1


15 025 04 05 06 075 1


Concentration (M)


Figure 2-1: Mean (+ SE) Tastant/Water Lick Ratio as a function of sucrose, L-serine, and glycine concentration for four different inbred strains of mice
(n=10/stimulus/strain). The Tastant/Water Lick Ratio was calculated by dividing an animal's average licks to a given taste stimulus across trials by
the average licks to water. The dashed line on the graph represents a Tastant/Water Lick Ratio of 1.0, which indicates licking to the taste stimulus
was equivalent to licking to water. This ratio controls for differences in oral motor competence and physiological state. These animals were tested
in 3 consecutive sessions while on a 23.5 h water restriction schedule.


0065 01




















S08 -0- SR
129
vD2
S06








00
0065 01 02 03 04 05 065 1 025 04 05 06 075 1 15 025 04 05 06 075 1 15

Concentration (M)


Figure 2-2: Mean (+ SE) Standardized Lick Ratio as a function of sucrose, L-serine, and glycine concentration for four different inbred strains of mice. The
Standardized Lick Ratio was calculated by dividing an animal's average licks to a given taste stimulus across trials by the maximum potential licks
in a 5-s trial, derived from that animal's previously measured inter-lick interval distribution. This score is used for normally preferred stimuli and
controls for differences in characteristic local lick rates. A score of 1.0 reflects licking to the taste stimulus that was at the maximum possible rate.
These animals were tested non-deprived in 3 consecutive sessions. Only mice that had at least 1 trial at every concentration were included in the
analysis of a given stimulus (sucrose: B6 [n=10], SWR [n=8], 129 [n=10], D2 [n=10]; L-serine: B6 [n=9], SWR [n=4], 129 [n=7], D2 [n=5]; and
glycine: B6 [n=9], SWR [n=7], 129 [n=5], D2 [n=9]).














CHAPTER 3
EXPERIMENT 2: TASTE DISCRIMINABILITY OF L-SERINE AND VARIOUS
SUGARS BY MICE

Background

Conclusions regarding the taste quality of amino acids based on data from CTA

experiments suggest that a subset of amino acids is perceptually similar to sucrose and

some other sugars. Consistent with this view, results from two-bottle intake experiments

show that these amino acids are preferred by mice. However, although L-amino acids are

thought to bind exclusively with the T1R1+3 receptor complex, preference behavior

measured in the two-bottle intake test seems to depend on an anomaly in the T1R2+3

complex across strains. Moreover, data gathered in our laboratory question whether or

not these compounds are actually preferred by mice at all on the basis of taste and as a

result question the very nature of the taste quality evoked by these compounds. Indeed,

the molecular biology of "sweetener" transduction appears to provide a neurobiological

basis for behavioral discriminability (i.e., perceptual distinction). Thus, the goal of the

current studies was to determine the degree to which the reported receptor specificity

predicts the relative discriminability of various "sweeteners," by testing whether

C57BL/6J (B6) mice can discriminate between sucrose and L-serine, as well as a variety

of other sugars using an operate discrimination paradigm. To my knowledge, explicit

discrimination experiments in rodents with these ligands have never been conducted.









Methodological Details

Subjects

Adult C57BL/6J (B6) male and female mice (n=12; Jackson Laboratories, Bar

Harbor, Maine), 8 weeks of age on arrival, served as subjects. The B6 strain was

chosen because 1) it is the most common mouse strain used in taste research, 2) has been

previously characterized as a "taster" strain (e.g., Capretta, 1970; Pelz et al., 1973; Fuller,

1974; Lush, 1989; Capeless and Whitney, 1995; Bachmanov et al., 1996), and 3) serves

as a background strain in many knock-out, congenic, and transgenic manipulations (e.g.,

Damak et al., 2003; Zhao et al., 2003). The mice were housed individually in

polycarbonate cages in a colony room where the lighting was controlled automatically

(12:12). Testing and training took place during the lights-on phase. After arrival in the

facility, subjects had free access to pellets of laboratory chow (Purina 5001, PMI

Nutrition International Inc., Brentwood, MO) and purified water (Elix 10; Millipore,

Billerica, MA). Seven days after arrival, mice were put on a restricted water-access

schedule with fluid available Monday Friday during testing only. Purified water was

freely available on the home cage from Friday afternoon through Sunday afternoon every

week. Mice that dropped below 85% of hydrated weight while on water-restriction

schedule, received 1 ml of supplemental water -2 hours after the end of the testing

session. All procedures were approved by the Institutional Animal Care and Use

Committee at the University of Florida.

Taste Stimuli

All solutions were prepared daily with purified water and reagent grade chemicals

and were presented at room temperature. "Comparison stimuli" consisted of various

concentrations of sucrose, glucose, maltose, fructose, (Fisher Scientific, Atlanta, GA) and









L-serine. In addition, sodium chloride (NaC1; Fisher Scientific, Atlanta, GA) was used as

a comparison stimulus to provide a contrast that could be easily discriminated from a

standard stimulus during training. Sucrose, fructose, and glucose were chosen because 1)

they are prototypical sweeteners that are commonly used in taste experiments, 2) with the

exception of fructose, they have been used to differentiate taster (e.g., B6) from non-

taster mice in two-bottle preference and intake tests (e.g., Stockton and Whitney, 1974;

Ramirez and Fuller, 1976; Lush, 1989; Bachmanov et al., 1996; Bachmanov et al., 1997;

Bachmanov et al., 2001b), as well as in an operant discrimination task (Eylam and

Spector, 2004), and 3) they are thought to exclusively bind with the T1R2+3, but not the

T1R1+3, receptor complex (Nelson et al., 2001; Nelson etal., 2002; Zhao etal., 2003).

L-serine was chosen because 1) there is evidence that at least in some rodents this

compound shares a perceptual quality with sucrose (Kasahara et al., 1987), 2) it is

preferred by some strains of mice at mid-range concentrations in two-bottle preference

tests (Iwasaki et al., 1985), and 3) appears to bind primarily with the T1R1+3, but only

poorly, if at all, with the T1R2+3 receptor complex (Nelson et al., 2002; Zhao et al.,

2003).

Concentrations of each stimulus tested are listed in Table 3-1. These were chosen

on the basis of the available behavioral data and with the intent of representing the

dynamic range of responsiveness for B6 mice. The concentrations of sucrose and fructose

selected encompass the dynamic range of behavioral responsiveness for B6 mice, as

measured in a brief-access taste test (Dotson et al., 2005; Glendinning et al., 2005a;

2005b; also see Chapter 2, Figure 2-2). Glucose concentrations were chosen based on the

diminished neural and behavioral sensitivity of B6 mice to glucose relative to sucrose









(Ninomiya et al., 1984; Eylam and Spector, 2004). The maltose concentrations

encompassed the range of behavioral preference for C57BL/6ByJ mice, as measured

using a two-bottle intake taste test (Bachmanov et al., 2001b). Very little data has been

gathered on the behavioral responsiveness of mice to L-amino acids. Thus, the choice of

concentrations for L-serine was based on the available neural data. The whole CT nerve

of ddy mice responses monotonically to an increasing concentrations series of L-serine

(0.01 M 1 M, neural threshold = 0.003 0.01 M; Iwasaki etal., 1985). Thus, those

concentrations chosen for L-serine were assumed to be within the dynamic range of

responsiveness for mice. NaCl concentrations represented the range of behavioral

responsiveness for C57BL/6ByJ mice, as measured in a brief-access taste test (Dotson et

al., 2005) and were also thought to be within the dynamic range of responsiveness for

the substrain of B6 mice used here.

It is important to stress that the broad range of concentrations chosen helps

guarantee that there will be overlapping intensities and viscosities across the stimuli.

This is important because of the need to render intensity an irrelevant cue while

promoting quality as the consistent discriminable signal.

Apparatus

Animals were trained and tested in a specially designed computer-controlled

gustometer modified for use with mice (Spector et al., 1990; Eylam and Spector, 2002,

2003, 2004, 2005). This apparatus consisted of a modified operant chamber housed in a

sound-attenuating cubicle, which had a reinforcement spout positioned on either side of a

centrally positioned slot through which a mouse gained access to a sample spout.

Background noise inside the sound-attenuating cubicle helped to minimize extraneous

auditory cues. All stimuli and reinforcement fluids were placed in pressurized reservoirs









outside the chamber. Computer-operated solenoid valves regulated fluid delivery from

these reservoirs by controlling the amount of fluid dispensed from the sample spout. This

required that the sample shaft first be filled with the stimulus and then, with each

subsequent lick, 2.0 [iL were deposited into the shaft. The volume per lick received

from the reinforcement spouts was also 2.0 [iL. At the end of each trial, the sample

spout rotated over a funnel, was rinsed with purified water, and evacuated with

pressurized air. Two cue lights positioned on the ceiling of the test cage above the

reinforcement spouts and a house light were turned on or off during each trial according

to the programmed trial schedule to signal the beginning and/or ending of the various

phases of a trial.

Experimental Design

Two groups of mice were initially tested on their ability to discriminate either

sucrose or L-serine (standard stimuli) from sodium chloride (comparison stimulus). After

successful completion of this discrimination test, the comparison stimulus was changed

from NaCl to one of the other compounds listed in Table 3-2. When this discrimination

was completed, the comparison stimulus was changed for a second time. This process

was continued until all comparison stimuli had been paired with a given standard

stimulus (see Table 3-2). Interposed between "test" discrimination was a series of

sessions during which the animals were retested on the NaCl vs. standard stimulus

discrimination to measure and maintain stimulus control. These were referred to as

"stimulus-control" sessions.

Training (see Table 3-3)

a.) Spout training. Mice were trained to lick from the different spouts for fluid

delivery in the gustometer by presenting the animals with only one spout each day. Water









was delivered on all 3 days of this training phase and was freely available ad libitum

throughout a session.

b.) Side training. Next, mice were trained to lick from a specific reinforcement

spout in response to the presentation of a mid-range concentration of one of the two

compounds delivered through the sample spout by providing access only to the

corresponding reinforcement spout. The access slot to the other reinforcement spout was

covered. The sample solution and the matching reinforcement spout were alternated

between days. In this phase, mice were allowed up to 180 s to respond after sampling

(limited hold); no time-out contingency was in effect.

c.) Alternation phase. During the alternation phase, both stimuli were presented

and both reinforcement spouts were available for responses. The limited hold was

shortened to 15 s and a criterion number of correct responses (non-consecutively) was

required for a change in the sample stimulus. The criterion, which started at four correct

responses, was reduced after two sessions to two, and finally, after two more sessions to

one. The time-out contingency was introduced in this phase as a punishment for incorrect

responses. Initially, the time-out was set at 10 s. When the criterion reached 2, it was

increased to 20 s, and finally increased to 30 s when the criterion reached 1.

d.) Discrimination training. Mice were trained to discriminate stimuli presented

in randomized blocks (discrimination training). The time-out was set at 30 s. Then the

two other concentrations of each stimulus were added (discrimination testing). Mice

were moved from discrimination training to discrimination testing when group

performance reached 85% correct responses and at least 75% for each animal. At the

start of discrimination testing, the limited hold was reduced to 10 s.









Testing (see Table 3-3)

Mice were trained, as described above, to associate the taste of one stimulus with

one reinforcement spout on one side of the sample spout and the taste of another with the

other reinforcement spout on the other side of the sample spout (counterbalanced between

animals). Session length was 25 minutes. During this time, each mouse was allowed to

complete as many trials as possible. Each trial was comprised of 4 phases: (1) the sample

phase, (2) the decision phase, (3) the reinforcement phase, and (4) the inter-trial interval

(see Figure 3-1). The sample phase began when the mouse licked from the sample spout

available in front of the slot. When initiating a trial, the mouse was required to complete

an "attending response" by licking the dry sample spout twice within 250 ms to trigger

stimulus delivery. The mouse was allowed 5 licks or 2 s of stimulus access, whichever

came first, before the sample spout was rotated away from the slot. During this phase,

the house lights were on. When the sample spout rotated away from the slot, the decision

phase would begin: the house lights would be turned off and the cue lights turned on.

During this phase, the mouse was required to decide which reinforcement spout to lick

from. The reinforcement phase began as soon as contact was made with one of the side

spouts. If a correct choice was made, the house lights were illuminated and the mouse

was allowed 15 licks or 4 s of access to water reinforcement. If an incorrect choice was

made or no response was initiated within 10 s (limited hold), the mouse received 30 s of

time-out during which all lights were extinguished and no fluid was delivered. When 15

licks were taken, 4 s passed, or when a time-out was completed, the sample spout rotated

over a funnel and was rinsed with purified water and dried with pressurized air, and then

rotated back into position in front of the slot. This phase, the inter-trial interval, lasted 6 s

(all lights were turned off during this phase). As mentioned above, mice were tested with









a range of stimulus concentrations. During each session, three of the reservoirs were

filled with different concentrations of the standard stimulus and three others with

different concentrations of the comparison stimulus (see Table 3-3). The two reservoirs

connected to the reinforcement spouts were filled with purified water. Presentation order

was randomized without replacement in blocks so that every concentration of the stimuli

was presented exactly once before the initiation of the subsequent block.

Data Analysis

Discriminability was evaluated using the overall proportion of correct responses as

the primary dependent measure. Overall performance was assessed by collapsing all

trials across both stimuli and concentrations. Concentration effects were analyzed within

each stimulus. These effects, as well as overall performance, were tested against chance

using one-sample t tests. Performance across weeks was statistically analyzed using

analyses of variance (ANOVAs). Only trials in which a response was made were used in

the analyses. When technical problems with the gustometer occurred during an animal's

session, the data were discarded from the analyses. Overall discriminability at 50%

correct equals chance performance. Performance approaching this value indicated a

failure to discriminate.

Results

As shown in Figure 3-2, both groups of mice, which were trained to discriminate a

standard stimulus (e.g., sucrose or L-serine) from NaC1, learned the task. This initial

phase of discrimination testing lasted for five weeks. During the last two weeks of this

phase, both groups performed at -85% accuracy on average. Overall performance ranged

from 76 to 94% correct responses during the last week of testing for the group of mice









trained to discriminate L-serine from NaC1 and 76 to 92% correct responses for animals

trained to discriminate sucrose from NaC1.

Discrimination Testing

Table 3-4 lists the performance of the two groups during the various phases of

discrimination testing. Group performance on each of the stimulus discrimination

pairings is described in greater detail below.

Sucrose vs. L-serine

"Serine" group

During the first week of the second phase of discrimination testing, both groups of

mice were tested for their ability to discriminate sucrose from L-serine. Overall

performance dropped to levels significantly below chance for the group of mice trained to

discriminate L-serine from NaCl serinene" group; Figure 3-3; t(5) = -4.9; P < .01; null

hypothesis; probability of correct response = .5]. Although the departure from chance

was relatively slight (0.44), it indicates that there was a slight tendency for animals in this

group to treat the novel stimulus as if it were the standard, although this propensity did

not reach statistical significance.

During the second week of testing, the performance of the serine group did not

significantly differ from chance [Figure 3-3; t(5) = -0.78; P = .472; null hypothesis;

probability = .5] nor did it significantly differ from performance levels measured during

week one [F(1,5) = 2.8, P = .156].

"Sucrose" group

For the group of mice trained to discriminate sucrose from NaCl (sucrose group),

overall performance also dropped precipitously during the first week of testing. These

data demonstrate that changing one of the taste compounds in this discrimination task has









the potential to substantially disrupt performance in both groups. However, these mice

did perform at levels above chance, albeit poorly [Figure 3-4; t(5) = 5.2; P < .01; null

hypothesis; probability = .5].

When looking at performance across all the individual concentrations of the two

stimuli, mice were able to discriminate 0.4 and 0.6 M sucrose at levels above chance

[Figure 3-5; both t-values > 3.0; Ps < .05; null hypothesis; probability = .5]. However,

these mice did not respond to 0.2 M sucrose, as well as all three concentrations of L-

serine, at levels above chance.

During the second week of testing, performance did significantly improve relative

to that measured during the first week [Figure 3-4; F(1,5) = 11.9, P < .05].

Overall performance appeared to improve, relative to week one, because mice

learned to discriminate L-serine from sucrose. That is, they discriminated all

concentrations of L-serine at levels above chance [Figure 3-5; all t-values > 5.2; Ps < .01;

null hypothesis; probability = .5]. Although these mice were able to discriminate 0.6 M

sucrose from L-serine [Figure 3-4; t(5) = 2.7; P < .05; null hypothesis; probability = .5],

they were unable to discriminate 0.2 and 0.4 M sucrose. However, their performance

towards 0.4 M sucrose did approach significance (P = .058).

Overall group performance for the sucrose group was significantly above chance,

which would appear to indicate that these stimuli evoke, to some degree, discriminable

neural signals. However, mice in the serine group had substantially greater difficulty

executing the task. Collectively, these data suggest that while potentially discriminable,

the stimuli are, to some degree, qualitatively similar.









Sucrose/L-serine vs. Glucose

Serine group

During week one of L-serine vs. glucose testing, overall performance dropped to

levels significantly below chance for the serine group [Figure 3-3; t(5) = -6.4; p < .01;

null hypothesis; probability = .5], again indicating that when animals in this group had

difficulty discriminating, there was a slight, but statistically significant tendency for them

to respond to the novel stimulus as if it was the standard [i.e., L-serine; t(5) = -5.1; P <

.01; null hypothesis; probability = .5).

Their performance, however, did significantly improve during week two [Figure 3-

3; F(1,5) = 50.5, P < .01]. This improvement, however, did not yield performance that

was significantly greater than chance [t(5) = 2.5; P = .052; null hypothesis; probability =

.5].

These mice were tested for an additional week to see if group performance would

improve. Overall performance during week three differed significantly from that

measured during week one [F(1,5) = 25.2, P < .01] but not from levels observed during

week two. After three weeks of testing with glucose (and two earlier weeks with

sucrose), performance finally reached levels significantly higher than chance [Figure 3-3;

t(5) = 3.0; P < .05; null hypothesis; probability = .5]. However, individual performance

during this week was highly variable. Indeed, three out of the six mice in this group

appeared to have substantial difficulty performing the discrimination (Figure 3-3).

Overall performance ranged from 51 to 67% correct responses during week three.

Mean performance appeared to improve, relative to week one, because mice

stopped responding to glucose as if it were L-serine. During week one, mice responded

to all concentrations of glucose at levels significantly below chance [Figure 3-6; all t-









values < -2.6; Ps < .05; null hypothesis; probability = .5]. That is to say, mice were

responding disproportionately on the "L-serine" spout. During week three, mice

corrected this bias and responded to all three concentrations of glucose at chance levels.

During the last week of testing with glucose, mean overall group performance for

the serine group was significantly above chance, which would appear to indicate that

these stimuli evoke, to some degree, discriminable neural signals. However, as detailed

above, group performance was at best mediocre and individual performance was highly

variable (see Figure 3-3). As with sucrose vs. L-serine testing, these data suggest that

while potentially discriminable, L-serine and glucose are, to some degree, qualitatively

similar.

Sucrose group

As expected, mice in the sucrose group had great difficulty discriminating sucrose

from glucose. Their overall performance never differed significantly from chance [week

one or week two; Figure 3-4; both t-values < 1.6; Ps > .179; null hypothesis; probability

= .5]. In addition, performance levels observed during week two did not significantly

differ from that measured during week one [F(1,5) = 0.05, P = .832]. We did not run

these mice for a third week for fear of losing stimulus control. These data are consistent

with the notion that sucrose and glucose activate the same transduction pathways (e.g.,

Zhao et al., 2003; see discussion below for elaboration).

Mice tended to respond best to sucrose at its highest concentration (0.6 M) and best

to glucose at its lowest concentration (0.5 M), although this pattern did not reach

statistical significance (see Figure 3-7). In any event, this response strategy did not lead

to overall performance levels that were greater than chance (however, see Sucrose vs.

Fructose below).









Sucrose/L-serine vs. Maltose

Serine group

During the first week of L-serine vs. maltose testing, overall performance was

significantly higher than chance [Figure 3-3; t(5) = 3.5; P < .05; null hypothesis;

probability = .5]. Mice were able to discriminate 0.4 and 0.6 M L-serine at levels above

chance [Figure 3-8; both t-values > 4.8; Ps < .01; null hypothesis; probability = .5].

However, these mice did not respond to 1.0 M L-serine, as well as all three

concentrations of maltose, at above chance levels.

Oddly, overall performance for these mice, although above chance during week

one, was not maintained at these levels during the last two weeks of testing. This despite

the fact that mean performance increased each week during this phase of testing (see

Figure 3-3). During the last two weeks of testing, performance did not significantly

differ from chance [Figure 3-3; both t-values < 2.4; Ps > .06; null hypothesis; probability

= .5], nor did the values significantly differ from performance levels measured during

week one.

As with L-serine vs. glucose testing, individual performance was highly variable.

Two mice substantially affected the amount of variability observed during L-serine vs.

maltose testing, considerably pulling down group performance (Figure 3-3). These same

mice performed at -chance levels during L-serine vs. glucose testing (Figure 3-3).

Overall performance ranged from 46 to 75% correct responses during week three.

Sucrose group

Unlike the failure of these mice to discriminate sucrose from glucose, they did

display some reliable discrimination between sucrose and maltose. During the first week

of sucrose vs. maltose testing, overall performance was significantly above chance









[Figure 3-4; t(5) = 4.2; P < .01; null hypothesis; probability = .5]. During week one,

mice were able to discriminate 0.2 M sucrose at levels above chance [Figure 3-9; t(5) =

3.2; p < .05; null hypothesis; probability = .5]. However, these mice did not respond to

0.4 and 0.6 M sucrose, as well as all three concentrations of maltose, at above chance

levels. Performance did not significantly improve during weeks two or three. However,

by week three, mice were able to discriminate 0.2 M maltose and all concentrations of

sucrose at levels above chance [Figure 3-9; all t-values < 2.6; Ps < .05; null hypothesis;

probability = .5].

The ability of mice to discriminate sucrose from maltose, detailed above, suggests

that maltose is qualitatively distinctive from sucrose and the pattern of responsiveness

across concentrations suggest that 0.2 M maltose is more distinguishable from sucrose

relative to the higher maltose concentrations tested (i.e., 0.4 and 0.6 M maltose).

Sucrose vs. L-serine II

Serine group

We conducted a second phase of sucrose vs. L-serine testing (Sucrose vs. L-serine

II) to ascertain whether the performance of the serine group would improve on this

discrimination as a function of experience. During the first week of testing, performance

did not significantly differ from chance [Figure 3-3; t(5) = 2.3; P = .07; null hypothesis;

probability = .5].

During the next week, however, performance did reach levels significantly higher

than chance [Figure 3-3; t(5) = 2.9; P < .05; null hypothesis; probability = .5]. Yet these

levels were not significantly different from those measured during week one.

Mice were then tested for an additional week to see if group performance would

improve. During this final week of testing, overall performance did differ significantly









from levels measured during week one [F(1,5) = 17.0, P < .01] but not from that

observed during week two. Mice were able to discriminate all concentrations of L-serine

at levels above chance during all three weeks of testing with these stimuli [Figure 3-10;

all t-values > 2.7; Ps < .05; null hypothesis; probability = .5]. However, during this time,

they were unable to discriminate any of the sucrose concentrations at above chance levels

[Figure 3-10; all t-values < 2.1; Ps > .09; null hypothesis; probability = .5]. This

response pattern is consistent with what was seen in the last week of L-serine vs. glucose

testing. That is, mice in the serine group did not respond to any concentration of sucrose

or glucose at levels above chance.

As with L-serine vs. glucose and maltose testing, individual performance was

highly variable. The two mice that performed at levels substantially below the other

animals during glucose and maltose testing continued to do so during this phase of testing

(Figure 3-3). Overall performance during week three ranged from 46 to 75% correct

responses.

The data presented above suggest that mice in the serine group can indeed

discriminate both sucrose and glucose from L-serine. That said, this discrimination

appeared to be exceedingly difficult for these animals.

Sucrose group

As in the first sucrose vs. L-serine discrimination task, the sucrose group did

perform at levels above chance [Figure 3-4; t(5) = 9.7; P < .001; null hypothesis;

probability = .5]. The performance during the first week of sucrose vs. L-serine II testing

did not significantly differ from the last week of the first phase of sucrose vs. L-serine

testing [69 and 72 % group performance, respectively].









Mice were able to discriminate 0.4 and 0.6 M sucrose and all three concentrations

of L-serine at levels above chance [Figure 3-11; all t-values > 4.1; Ps < .01; null

hypothesis; probability = .5]. As in the first phase of sucrose vs. serine testing, mice did

not respond to 0.2 M sucrose at above chance levels.

Overall performance significantly improved during week two when compared to

that seen during week one [Figure 3-3; F(3,36) = 23.7, P < .01]. During this week, mice

were able to discriminate all concentrations of both stimuli at levels above chance [Figure

3-11; all t-values > 4.7; Ps < .01; null hypothesis; probability = .5].

Performance appeared to reach asymptotical levels during week two. Overall

performance levels, by session, did not significantly improve during week two [sessions

6-10: 0.79, 0.83, 0.77, 0.77, 0.75, respectively; F(4,8 = 0.336, P = .85]. As a result, mice

were not tested for a third week.

Sucrose/L-serine vs. Fructose

Serine group

During week one, the overall performance of the serine group did not significantly

differ from chance [Figure 3-3; t(5) = 1.8; P = .144; null hypothesis; probability = .5].

During the next week, however, performance did reach levels significantly higher than

chance [Figure 3-3; t(5) = 4.7; P < .01; null hypothesis; probability = .5]. Yet these

levels were not significantly different from these measured during week one. During

week two, mice were able to discriminate 1.0 M L-serine, as well as 0.6 and 1.0 M

fructose, at levels above chance [Figure 3-12; all t-values > 3.1; Ps < .05; null hypothesis;

probability = .5].

Mice were then tested for an additional week to see if performance would

improve. During this week, overall performance was again significantly above chance









[Figure 3-3; t(5) = 4.6; P < .05; null hypothesis; probability = .5]. Performance did not

differ significantly from that measured during week one or two. During this last week,

mice were able to discriminate 0.6 and 1.0 M L-serine and 1.0 M fructose at levels above

chance [Figure 3-12; all t-values > 3.2; Ps < .05; null hypothesis; probability = .5].

During this phases of discrimination testing, individual performance variability

decreased somewhat. The two mice that depressed performance throughout testing

appeared to finally improve ever so slightly, relative to their behavior measured in

preceding phases (see Figure 3-3). Overall performance during week three ranged from

57 to 76% correct responses. Mice in the serine group, for the most part, demonstrated

that, after some experience, they can discriminate all of the sugars, including fructose

from their standard stimulus.

Sucrose group

During the first week of sucrose vs. fructose testing, overall performance was

significantly greater than chance [Figure 3-4; t(5) = 4.4; P < .01; null hypothesis;

probability = .5]. Mice were able to discriminate the high concentrations of sucrose (0.4

and 0.6 M) at levels above chance [Figure 3-13; t(5) = 3.2; p < .05; null hypothesis;

probability = .5]. These mice, however, did not respond 0.3 and 0.6 M fructose at above

chance levels. Interestingly, mice responded to 1.0 M fructose at levels significantly

below chance [Figure 3-13; t(5) =-23.0; P < .001;null hypothesis; probability = .5].

During week two, overall performance was again significantly greater than chance

[Figure 3-4; t(5) = 6.9; P < .01; null hypothesis; probability = .5], but not significantly

different than performance levels measured during week one.

Mice were tested for an additional week to see if group performance would

improve relative to that measured during weeks one and two. During this final week of









testing, performance again was significantly higher than chance [Figure 3-4; t(5) = 2.8; P

< .05; null hypothesis; probability = .5]. However, performance did not differ

significantly from that observed during week one or two.

Although no significant changes in the overall percentage of correct responses were

observed over the three weeks of testing, the pattern of responsiveness towards the

various concentrations of the two stimuli did differ slightly from week one to week three.

By week three, the mice only responded to the highest concentration of sucrose and the

lowest concentration of fructose at above chance levels. This pattern of responsiveness is

consistent with animals using "intensity" cues to guide the discrimination (Figure 3-13;

see discussion below).

In summary, when mice in the sucrose group were tested on the ability to

discriminate sucrose from fructose, these animals showed that they could do so at levels

significantly greater than chance. However, as detailed above, this ability was very

limited (-55% correct during the last week of testing) and appeared to result from mice

responding on the basis of intensity, and not to taste quality, per se.

Stimulus Control Sessions

Figure 3-14 shows the performance of both groups on the first and last day of a

given stimulus-control session, as well data for the subsequent discrimination task. The

number of stimulus-control sessions required to reach criterion performance (i.e., 85%

group performance for one week or for two consecutive sessions at the end of a week and

individual performance > 75%) is listed in Table 3-5. As can be seen, after stimulus-

control I, the number of sessions required to regain criterion performance dropped

dramatically. Collectively, these data demonstrate that stimulus control can be quickly









reestablished in experienced mice even after a substantial period in which animals are

presented with difficult discrimination tasks.

Discussion

Mice had difficulty, depending on the stimulus and the training history,

discriminating sucrose from L-serine, maltose, fructose and glucose. Indeed, when

concentration effects are taken into consideration, it appears that mice are unable to

discriminate sucrose from glucose or fructose, suggesting that these three sugars generate

a unitary percept. "Monogeusia," or the indiscriminability of a "class" of compounds,

has been demonstrated in humans with natural sweeteners (e.g., Breslin et al., 1996) and

in rats with "bitter" tastants (Spector and Kopka, 2002). To my knowledge, monogeusia

for natural sweeteners has never been demonstrated in rodents.

Monogeusia

All mice trained to discriminate sucrose from NaCl (i.e., sucrose group) were

entirely unable to discriminate between sucrose and glucose as assessed by their overall

performance during both weeks of testing. During the first week of testing, mice did not

respond to any concentration of either stimulus at levels above chance. Indeed, they

responded to 2.0 M glucose at levels significantly below chance. This indicates that the

mice tended to treat the highest concentration of glucose as if it were the standard (i.e.,

sucrose). Although not significant, this tendency persisted in week two. Indeed, mice

appeared to be responding on the basis of stimulus intensity. A broad concentration

range was used for all compounds so that intensities and viscosities would overlap across

stimuli. This manipulation was important so that non-qualitative cues would be rendered

less redundant, causing quality to be the only consistent discriminable signal. However,

if the concentration ranges did not sufficiently overlap, then stimulus intensity could be









used as a discriminable cue, albeit not very successfully. Theoretically, if these mice

were responding to two qualitatively similar stimuli on the basis of perceived intensity,

then they would be expected to respond disproportionately to all "weak" stimuli on one

reinforcement spout and all "strong" stimuli on the other. Accordingly, as the

concentration of the stimulus perceived to be of higher intensity increases, performance

levels would also increase. This is because, as the concentration of the "stronger"

stimulus increases, it would become more distinctive relative to the perceived intensity

levels of the "weaker" stimulus. However, as the concentration of the lower intensity

stimulus increases, performance levels would decrease. This is because, as the

concentration of "weaker" stimulus increases, it overlaps, to a greater degree, with the

perceived intensity levels of the "stronger" stimulus. It is this opposing concentration

dependency that is the telltale sign of a stimulus intensity based taste discrimination.

With a response strategy such as this, animals could successfully discriminate two

qualitatively identical stimuli, the concentration ranges of which did not sufficiently

overlap, at levels greater than chance. Mice appeared to try this very response strategy

when attempting to discriminate sucrose from glucose. As can be seen in Figure 3-7,

mice responded to sucrose as if it were the "stronger" of the two stimuli being

discriminated (i.e., as the concentration increased, performance levels also increased)

and to glucose as if it were the "weaker" stimulus (i.e., as the concentration increased,

performance levels decreased). Indeed, by week two, mice were able to discriminate the

lowest glucose concentration at levels above chance. However, this feat, in and of itself,

did not lead to overall performance levels that were greater than chance.









When mice were tested for the ability to discriminate sucrose from fructose, these

animals showed that they could do so at levels above chance. However, this ability

appeared to result from the mice using intensity cues. During the first week of testing

with fructose, mice did not respond to any concentration of the stimulus at above chance

levels. Indeed, they responded to 1.0 M fructose at levels significantly below chance.

This indicates that there was a tendency for animals to treat the highest concentration of

fructose as if it were the standard (i.e., as the concentration increased, performance

levels decreased). This response profile is analogous to that observed during the first

week of testing with glucose, referred to above. By week three, mice only responded to

the highest concentration of sucrose and the lowest concentration of fructose at levels

above chance. Moreover, animals tended to respond to the highest concentration of

fructose and the lowest concentration of sucrose as if they were the "other" stimulus, but

these tendencies did not meet statistical significance standards. As can be seen in Figure

3-13, throughout testing with these stimuli, the opposing concentration dependency

referred to above is plainly apparent. This response pattern is exactly what would be

predicted if these animals were responding on the basis of perceived intensity. Thus, it is

likely that the concentrations of fructose and sucrose chosen did not sufficiently overlap

in intensity.

Nevertheless, these results strongly suggest that B6 mice cannot distinguish

perceptually between the tastes of fructose, glucose, or sucrose. Because these are

negative findings, we cannot conclusively rule out that some discriminative ability exists

on the basis of quality. However, if B6 mice can distinguish between these compounds,

they do so only poorly at best. Thus, it appears clear that in B6 mice, as in humans, all of









these sugars possess a similar taste quality that humans have described as "sweet"

(Breslin et al., 1996). The phenomenon of a class of compounds generating a unitary

qualitative percept has been termed "monogeusia" (Breslin et al., 1996).

Monogeusia for sugars likely results from an indiscriminable neural signal that

originates from the stimulation of a common receptor(s). As mentioned in Chapter 1,

sucrose and fructose were both shown, in vitro, to stimulate the T1R2+3 receptor

complex. In a separate experiment, again, alluded to in Chapter 1, it was reported that

T1R2+3 knock-out mice show no neural or behavioral responsiveness to sucrose or

glucose. Thus, the aforementioned behavioral data reported here support the contention

that all of these sugars activate the same receptor, and based on the molecular biology of

"sweet" ligand transduction, the likely candidate is the T1R2+3 receptor complex.

It should be mentioned, however, that the only index used to assess the neural

responsiveness of T1R2+3 knock-out mice was whole nerve electrophysiological

recording from the CT. Yet, it is likely that detection of these stimuli is based on more

than just input from the CT. Moreover, brief-access testing, which was used to evaluate

the behavioral responsiveness of these knock-out mice, only assesses the motivational

properties of a tastant, not the relative detectability of that stimulus, per se. As mentioned

in Chapter 1, taste stimulus detection is best assessed from tasks in which taste serves as

a cue for some other event (e.g., reinforcement or punishment) that will generate a trained

directed response regardless of the hedonic characteristics of the taste stimulus.

Indeed, recent data, gathered using such procedures, call into question the notion

that the T1R2+3 receptor complex is exclusively responsible for the activation of TBCs

responsible for the detection of sugars. These results suggest that at least one sugar can









be transduced independently of at least one of the members of the T1R2+3 receptor

complex. It was reported that mutant mice, lacking the receptor T1R3, show absolutely

no deficit in their ability to detect the presence of sucrose (Delay et al., 2006). It should

be mentioned, however, that these data are somewhat controversial and have yet to be

replicated. That said, it does appear possible for the T1R2 receptor to bind with taste

ligands in the absence of T1R3 (e.g., Nie et al., 2005; Temussi, 2006), although this

remains to be demonstrated in vivo.

Maltose, however, appears to generate a distinctive taste quality relative to sucrose,

depending on concentration. Although mice could only discriminate the lowest

concentration of maltose at levels above chance, they were able to discriminate all three

concentrations of sucrose successfully during the last week of testing. This response

pattern is quite different from that observed when these mice were attempting to

discriminate sucrose from either glucose or fructose. Therefore, these data suggest that,

although qualitatively similar, sucrose and maltose must generate a discriminable neural

signal at the periphery. Surprisingly, however, it is the lowest concentration of maltose

that appears to be the most distinctive, suggesting that as the concentration increases,

maltose becomes qualitatively more similar to sucrose. The discriminability of maltose

and sucrose has been demonstrated, in rodents and humans, by a variety of other

researchers (e.g., Ninomiya et al., 1984; Spector and Grill, 1988; Breslin et al., 1996;

Spector et al., 1997).

In addition to sucrose and glucose, T1R2+3 knock-out mice also show no neural or

behavioral responsiveness to maltose. If these data are correct, and the T1R2+3 receptor

complex mediates the transduction of both sucrose and maltose, then some other factor









must be influencing the nature of the signal arising from the periphery. Possibilities

include ligand binding/TBC activation characteristics that lead to differential signaling

(e.g., rise & decay). It is also possible that other undiscovered members of the T1R

family of receptors or other mechanisms of ligand transduction exist that generate a

discriminable neural signal. It would be informative to see if T1R2+3 knock-out mice

could detect the presence of maltose in a task explicitly designed to assess taste

thresholds (e.g., an operate detection task). If so, data such as these would definitively

prove that maltose could be transduced independently of the receptors T1R2 and/or

T1R3.

Sugars vs. L-serine

All mice in the sucrose group learned to discriminate sucrose and L-serine. After

many weeks of experience with sucrose as a standard, they were able to discriminate

sucrose from L-serine -80% of the time during the last week of testing with these stimuli.

As a group, these mice responded to all concentrations of both stimuli at levels above

chance. Mice in the serine group were able to discriminate all the concentrations of L-

serine from sucrose. These mice were also able to discriminate L-serine from glucose,

fructose, and maltose at levels above chance.

The data presented above suggest that sucrose and L-serine are distinguishable. On

the other hand, the data also suggest that the two stimuli share some qualitative features.

A comparison of the performance of animals when they were discriminating their

respective standard stimulus from NaCl during the stimulus-control sessions (i.e., -85 -

90% correct responses) to that observed when they were attempting to discriminate

sucrose from L-serine indicates that there was some degree of perceptual confusion (see

Figures 3-3 and 3-4).









In addition to maintaining and providing a measure of stimulus control, the

stimulus-control sessions also provided a performance standard based on a discrimination

task that was "easy" for the animals to accomplish. Because these stimuli (i.e.,

sucrose/L-serine vs. NaC1) are thought to be independently coded at the periphery (i.e.,

independently transduced; e.g., Zhang et al., 2003), it was postulated that mice could

perform this discrimination task without a great deal of difficulty. It was assumed that

deviation from 100% correct responding during these sessions was the result of non-

sensory factors (e.g., motivational state, task difficulty, etc). Thus, during the various

discrimination testing phases, it was believed that deviation from the performance

standards observed during the stimulus-control sessions indicated increasing perceptual

confusion. This "uncertainty" likely resulted from comparison stimuli evoking neural

signals that are more similar to one another relative to those generated by sucrose/L-

serine and NaC1.

Indeed, despite five weeks of testing with these stimuli, mice trained to

discriminate L-serine from NaCl (i.e., serine group) never responded to any concentration

of sucrose at levels above chance. During the first phase of sucrose vs. L-serine testing,

animals in the sucrose group were unable to discriminate 0.2 M sucrose from L-serine. It

took until the second week of the second phase before they were able to learn the

discrimination. These findings imply that 0.2 M sucrose tastes more similar to L-serine

than do the higher concentrations of sucrose. Accordingly, L-serine, across the range of

concentrations tested, likely evokes a mild "sweetness" relative to that elicited by 0.4 and

0.6 M sucrose. This fact might contribute to the lack of appetitive behavior displayed

towards this stimulus detailed in Chapter 1.









Collectively, these data suggest that at least some of the signals generated by the

receptor(s) responsible for the transduction of these stimuli converge somewhere alone

the gustatory neuraxis. Might this convergence be at the initial site of stimulus

transduction (e.g., the TBCs themselves)? As mentioned in Chapter 1, Kim et al., 2003

reported, contrary to previous studies, that there exists a population of TBCs that co-

express all three T1R receptors. Perhaps these cells mediate the signals that are

responsible for the qualitative similarity between these stimuli. It should be said,

however, that even if they do exist, it remains unclear what the response properties of

such a cell would be. Another possibility is that "sweet-tasting" amino acids achieve

their qualitative similarity with sucrose by binding with the same receptor(s) (e.g.,

T1R2+3). Indeed, as mentioned in Chapter 2, the putative "sweet-tasting" amino acid

glycine appears able to bind with the T1R2+3 and the T1R1+3 receptor complex. This

hypothesis is consistent with the fact, as mentioned in Chapter 2, that two-bottle

preference for "sweet-tasting" L-amino acids and glycine appears to depend on the

"taster" status of the mouse strain based on testing with sugars. Perhaps L-serine, like

glycine, has the ability to activate transduction pathways also activated by sucrose.

However, it should be mentioned that in an operant discrimination task designed to assess

taste stimulus thresholds, values for glycine in mice did not distinguish taster and non-

taster strains in straightforward a manner (Eylam and Spector, 2004). That said, glycine

detectability may be unaffected by the polymorphism in the T1R3 receptor. As

mentioned in Chapter 2, this polymorphism selectively affects the functionality of the

T1R2+3 receptor complex. Thus, taste input from remaining functional receptors (e.g.,

T1R1+3) could be sufficient to maintain task performance. However, the loss of taste









input from the T1R2+3 receptor complex, which mediates, at least in part, the affective

potency of highly appetitive nature sweeteners such as sucrose, would likely impact upon

the relative affective valence of glycine, if not upon its threshold levels.

Training history had a huge impact on the ability of mice to discriminate all of the

stimuli, particularly L-serine from sucrose. Mice, in the serine group, appeared to have

more difficulty discriminating L-serine from sucrose relative to animals in the sucrose

group. Indeed, two mice in the serine group were unable to discriminate L-serine from

all of the sugars. This training history asymmetry may have resulted from a relative

difference in the efficacy of sucrose and L-serine as "standards" (i.e., compounds to

which all comparison stimuli are discriminated against). This difference in efficacy

likely results from the degree of qualitative "purity" elicited by sucrose and L-serine,

which is discussed in detail in the following chapters.










Table 3-1: Stimulus concentrations
Concentrations
Sucrose 0.2, 0.4, 0.6 M
Glucose 0.5, 1.0, 2.0 M
Maltose 0.2, 0.4, 0.6 M
Fructose 0.3, 0.6, 1.0 M
L-serine 0.4, 0.6, 1.0 M
NaCl 0.2, 0.4, 0.6 M






66



Table 3-2: Order of stimulus discrimination pairing
Standard
Group Standad Comparison Stimuli
Stimulus
1 Sucrose NaCl L-serine Glucose Maltose L-serine Fructose
2 L-serine NaCl Sucrose Glucose Maltose Sucrose Fructose











Table 3-3: Representative training and testing parameters for the 2 discrimination groups


SLimited Sample Reinf. Timeou Presentation
Phase Stimuli
Hold Licks/time(s) Licks/time(s) t(s) Schedule

Spout Training HzO None None Constant
Middle Conc. of
Comparison Stimulus
Side Training 180 5/2 15/30 0 Constant
and
Middle Conc. of
Standard Stimulus
Middle Conc. of
Comparison Stimulus
1 10, 20, Criterion
Alternation 15 5/2 15/3020,
and or 30 (4-1)
Middle Conc. of
Standard Stimulus
Middle Conc. of
Comparison Stimulus
Discrimination 1 Randomized
15 5/2 15/4 30
Training and blocks
Middle Conc. of
Standard Stimulus
All Conc. Comparison
Discrimination Stimulus 1 and Randomized
10 5/2 15/4 30
Testing All Conc. Standard blocks
Stimulus
All Conc. Comparison
Stimulus 2
Discrimination Randomized
Te g and 10 5/2 15/4 30 b
Testing blocks
All Conc. Standard
Stimulus
All Conc. Comparison
Stimulus 3
Discrimination Randomized
and 10 5/2 15/4 30 o
Testing blocks
S All Conc. Standard
Stimulus
All Conc. Comparison
> Stimulus 1
Discrimination St s Randomized
Te g and 10 5/2 15/4 30 b
Testing blocks
All Conc. Standard
Stimulus
All Conc. Comparison
Stimulus 4
Discrimination Randomized
Te g and 10 5/2 15/4 30 b
Testing blocks
All Conc. Standard
Stimulus







68



Table 3-4: Overall percentage correct during the last week of testing for the stimulus discrimination pairings.


NaCl


Comparison Stimuli


L-serine


Glucose


SCII


Maltose SCIII


L-serine


SCIV


Fructose


85% 69% 85% 52% 82% 63% 84% 79% 89% 56%

serine NaCi Sucrose SCI Glucose SCII Maltose SCIII Sucrose SCIV Fructose

85% 48% 86% 58% 83% 60% 85% 65% 83% 65%

SC = stimulus-control sessions


Standard
Stimulus


Overall
%
Correct

Overall
%
Correct






69



Table 3-5: Number of stimulus-control sessions required to
criterion performance.
Stimulus Control Sessions
SCI SCII SCIII SCIV
Sucrose 15 5 5 5
L-serine 20 5 7 5



























Figure 3-1: Trial structure (see text for more details).







71


Discrimination Testing


Sucrose vs. NaC1


o



V
V V



-*

*


1.0



0.9



0.8



0.7



0.6



0.5


L-serine vs. NaC1


Weeks


Figure 3-2: Individual animal (symbols) and group mean (+ SEM; grey bars) data for mice trained to
discriminate either sucrose or L-serine from NaC1. Performance on all trials with a lever press is
depicted collapsed across all stimuli during a week. Chance performance equaled 0.5.


1.0



0.9



0.8



0.7



0.6



0.5


V
v
0
v



r











"Serine" Group


1 2 1 2 3 1 2 3 1 2 3 1 2 3


Weeks

Figure 3-3: Individual animal (symbols) and group mean (+ SEM; grey bars) data are plotted across all test phases for mice initially trained to discriminate L-
serine from NaC1. Performance on all trials with a lever press is depicted collapsed across all stimuli during a week. Chance performance equaled 0.5.
(*) One mouse was removed from L-serine vs. fructose testing because of illness.












"Sucrose" Group


SucVSer


0

~r


1 2
1 2


SucVGlu


1 2


SucVMal


I I I


SucVSerII


1 2


SucVFru









0
0


1 I I
123


Weeks

Figure 3-4: Individual animal (symbols) and group mean (+ SEM; grey bars) data are plotted across all test phases for mice initially trained to discriminate
sucrose from NaC1. Performance on all trials with a lever press is depicted collapsed across all stimuli during a week. Chance performance equaled
0.5.


1.0


0.9


0.8


0.7


0.6


0.5










Sucrose vs. L-serine
"Sucrose" Group


0.2 0.4 0.6 0.4 0.6 1
0.2 0.4 0.6 0.4 0.6 1


0.2 0.4 0.6 0.4 0.6 1
0.2 0.4 0.6 0.4 0.6 1


Concentration (M)


Figure 3-5: Mean (+ SD) data for mice attempting to discriminate L-serine from sucrose. These mice were
initially trained to discriminate sucrose from NaC1. Performance, by concentration, on all trials
with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


Week 1


Week 2


0.2

01












L-serine vs. Glucose


Week 2


0.4 0.6 1 0.5 1 2


0.4 0.6 1 0.5 1 2


0.4 0.6 1 0.5 1 2


Concentration (M)

Figure 3-6: Mean ( SD) data for mice attempting to discriminate L-serine from glucose. These mice were initially trained to discriminate L-serine from NaC1.
Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


Week 1


Week 3










Sucrose vs. Glucose


0.3

0.2

0.1

0.0


Week 1


Week 2


0.2 0.4 0.6 0.5 1 2 0.2 0.4 0.6 0.5 1 2


Concentration (M)


Figure 3-7: Mean (+ SD) data for mice attempting to discriminate sucrose from glucose. These mice were
initially trained to discriminate sucrose from NaC1. Performance, by concentration, on all trials
with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.












L-serine vs. Maltose


Week 2


0.4 0.6 1 0.2 0.4 0.6


0.4 0.6 1 0.2 0.4 0.6


0.4 0.6 1 0.2 0.4 0.6


Concentration (M)

Figure 3-8: Mean ( SD) data for mice attempting to discriminate L-serine from maltose. These mice were initially trained to discriminate L-serine from NaC1.
Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


Week 1


Week 3


0.3

0.2

0.1












Sucrose vs. Maltose


Week 2


0.2 0.4 0.6 0.2 0.4 0.6


0.2 0.4 0.6 0.2 0.4 0.6


0.2 0.4 0.6 0.2 0.4 0.6


Concentration (M)

Figure 3-9: Mean (+ SD) data for mice attempting to discriminate sucrose from maltose. These mice were initially trained to discriminate sucrose from NaC1.
Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


Week 1


Week 3


0.3

0.2

0.1













Sucrose vs. L-serine II
"Serine" Group


Week 1


1.0

0.9 -

0.8

0.7

0.6 -

0.5 -

0.4 -

0.3 -

0.2 -

0.1


Week 2


0.2 0.4 0.6 0.4 0.6 1


Week 3


0.2 0.4 0.6 0.4 0.6


Concentration (M)


Figure 3-10: Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a second time. These mice were initially trained to discriminate L-
serine from NaC1. Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled
0.5.


Sucrose

0.2 0.4 0.6 0.4 0.6 1






80


Sucrose vs. L-serine II
"Sucrose" Group


Week 1


0.2 0.4 0.6 0.4


1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1


0.6 1


Week 2


0 L-serine
I I I 0 0
0.2 0.4 0.6 0.4 0.6 1


Concentration (M)

Figure 3-11: Mean ( SD) data for mice attempting to discriminate L-serine from sucrose for a second
time. These mice were initially trained to discriminate sucrose from NaC1. Performance, by
concentration, on all trials with a lever press is depicted collapsed across a week. Chance
performance equaled 0.5.


0.3

0.2

0.1

















Week 1


0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1


L-serine vs. Fructose



Week 2


0.4 0.6 1 0.3 0.6 1


Week 3


0.4 0.6 1 0.3 0.6 1


Concentrartion (M)


Figure 3-12: Mean ( SD) data for mice attempting to discriminate L-serine from fructose. These mice were initially trained to discriminate L-serine from NaC1.
Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


O L-serine 0.1
I I I I 0.0
0.4 0.6 1 0.3 0.6 1













Sucrose vs. Fructose


Week 2


0.2 0.4 0.6 0.3 0.6 1


0.2 0.4 0.6 0.3 0.6 1


0.2 0.4 0.6 0.3 0.6 1


Concentration (M)

Figure 3-13: Mean ( SD) data for mice attempting to discriminate sucrose from fructose. These mice were initially trained to discriminate sucrose from NaC1.
Performance, by concentration, on all trials with a lever press is depicted collapsed across a week. Chance performance equaled 0.5.


Week 3














Discrimination Testing Phases


SCI SCII SCIII SCIV
1.0
0 SVS SVG SVM SS2 SVF


0.9


O 0.8 -


S0.7 i
0


S0.6 r
(0.5 I -I
5 ----- -- -- -
0 o,__, ,_L--O-- ,0 .., ...........
FL FL FL FL FL FL FL FL FL

o "Sucrose" Group
"Serine" Group


Figure 3-14: Mean (+ SEM) data for both groups of mice are plotted across all test phases. Performance on all trials with a lever press is depicted averaged across
all stimuli in a session. Only the first (F) and last (L) day of each phase are shown. The "sucrose" group was initially trained to discriminate sucrose
from NaC1. The serinee" group was initially trained to discriminate L-serine from NaC1. Chance performance equaled 0.5 (SVS = Sucrose vs. L-
serine; SVG = Sucrose/L-serine vs. Glucose; SVM = Sucrose/L-serine vs. Maltose; SS2 = Sucrose vs. L-serine II; SVF = Sucrose/L-serine vs.
Fructose; SC = stimulus-control sessions).













CHAPTER 4
EXPERIMENT 3: PERCEIVED SIMILARITY BETWEEN L-SERINE, L-
THREONINE AND CHEMICAL COMPOUNDS REPRESENTATIVE OF THE FOUR
BASIC TASTE QUALITIES

Background

The results presented in Chapter 3, as well as those from CTA generalization

studies, suggest that a subset of L-amino acids, including L-serine, share a qualitative

similarity with the taste of sucrose. However, data detailed in Chapter 2 show that L-

serine appears to be unlike sucrose in its ability to generate licking in mice. Thus, while

it would appear that L-serine might share some qualitative characteristics with sucrose,

this amino acid does not share the affective potency of this sugar. One possible

explanation for this finding is that L-serine generates additional qualities that impact

upon its affective value. For example, saccharin is both "sweet" and "bitter" tasting to

humans depending on concentration (e.g., Bartoshuk, 1979; Schiffman et al., 1979).

Although, saccharin was initially thought to only activate the T1R2+3 receptor complex,

researchers has recently demonstrated, using an in vitro preparation, that it also activates

the human "bitter" receptors hTAS2R43 and hTAS2R44 at concentrations known to elicit

a "bitter-taste" perception in humans (Kuhn et al., 2004). It is quite possible that at least

some L-amino acids also activate separate transduction pathways that lead to different

taste perceptions. Although all 20 common L-amino acids, including L-serine, were

shown to interact with the mouse T1R1+3 receptor complex (Nelson et al., 2002),

behavioral studies in rodents and humans suggest that the taste qualities evoked by L-

amino acids are varied (e.g., Ninomiya et al., 1984; Shallenberger, 1993). These data









suggest that L-amino acids may activate other taste transduction pathways independent of

the T1R1+3 receptor complex.

To my knowledge, the only CTA generalization study that directly addressed the

taste quality(ies) evoked by L-serine in mice was conducted by Kasahara and colleagues

(1987). These researchers showed that when conditioned to avoid 0.2 M sucrose, ddy

mice generalized the aversion to 0.2 M L-serine. However, these researchers did not use

L-serine as a conditioning stimulus (only as a test stimulus). If also used as a

conditioning stimulus, then perhaps mice conditioned to avoid L-serine would have

generalized an aversion to one or more of the other tested compounds thought to

represent other basic tastes. Thus, to investigate the taste qualities evoked by L-serine,

CTA generalization tests were conducted in the current investigation. In addition, the

similarity of the L-amino acid, threonine, to compounds representative of the four basic

tastes was also investigated. I am unaware of any prior CTA generalization experiments

that have explicitly examined the qualitative similarity of L-threonine and compounds

representative of the basic taste qualities.

Methodological Details

Subjects

Adult C57BL/6J (B6) male mice (n=44; Jackson Laboratories, Bar Harbor, Maine),

-10 weeks of age on arrival, served as subjects. The use of B6 mice allowed for a

comparison of results with those obtained in Experiment 2. The mice were housed

individually in polycarbonate cages in a colony room where the lighting was controlled

automatically (12:12). Testing and training took place during the lights-on phase. After

arrival in the facility, subjects had free access to pellets of laboratory chow (Purina 5001,

PMI Nutrition International Inc., Brentwood, MO) and purified water (Elix 10; Millipore,









Billerica, MA) for six days before training and testing took place. All procedures,

described below, were approved by the Institutional Animal Care and Use Committee at

the University of Florida.

Taste Stimuli

All solutions were prepared daily with purified water and reagent grade chemicals

and presented at room temperature. The compounds that served as conditioned stimuli

(CSs) were sucrose, L-serine, and L-threonine. The logic of choosing sucrose and L-

serine as CSs was the same as that used to justify their inclusion in the discrimination

experiment. That logic was described in detail in Chapter 3. The choice of L-threonine

as a CS was based on the fact that it reportedly gives rise to a sweet taste in humans (e.g.,

Shallenberger, 1993) and is preferred, at certain concentrations, by rodents (Pritchard and

Scott, 1982a; Iwasaki et al., 1985). The panel of test stimuli (TSs) was composed of

various concentrations of sucrose, L-serine, L-threonine, NaC1, citric acid (Fisher

Scientific, Atlanta, GA), and quinine hydrochloride (QHC1; Sigma-Aldrich, St Louis,

MO). Sucrose, NaC1, citric acid, and QHC1 were chosen as TSs because they are

frequently used as prototypical representatives of compounds that elicit a sweet, salty,

sour, and bitter taste, respectively (Schiffman and Erickson, 1980). L-serine, L-

threonine, and sucrose were also used as TSs to allow for the assessment of any

generalization asymmetries. Asymmetrical relationships can arise when the conditioning

and test stimuli, albeit similar, are not qualitatively identical. Yet, generalization, or the

lack thereof, can also occur based on stimulus characteristics other than quality (i.e.,

stimulus intensity; see Nowlis, 1974). As a result, if the conditioning and test stimuli

evoke a qualitatively identical percept, but the TS is of a low intensity relative to the CS,

then expression of the learned aversion may be weak or non-existent. Thus, the use of









more than one concentration increases ones confidence that a learned aversion towards a

CS will generalize to at least one of the concentrations of a TS (Spector and Grill, 1988;

Spector, 2003). In view of this, two concentrations of each compound were included in

the test stimulus arrays. These concentrations are listed in Table 4-1. The concentrations

chosen for L-serine, L-threonine, and sucrose were the same as those used in the

discrimination experiment described in Chapter 3. For QHC1, citric acid, and NaC1, an

attempt was made to choose concentrations that would produce comparable sensation

magnitudes. Stimulus concentrations that produced -equivalent degrees of lick

avoidance (Tastant/Water Lick Ratio: taste stimulus licks/water licks) were chosen from

the dynamic range of behavioral responsiveness for C57BL/By6J mice, as measured in a

brief-access taste test (Dotson et al., 2005). For the high concentration, stimuli that

produced a -50% decrease in the lick rate of animals, relative to water, were chosen. For

the low concentration, those that produced a -25% decrease were chosen. These values

were chosen in an attempt to elicit, in non-conditioned mice, lick rates to these normally

avoided stimuli that were sufficiently high enough to allow any differences between

conditioned and non-conditioned mice to be fully discernible.

Apparatus

Lickometer training and testing took place in an apparatus commonly referred to

as the Davis rig (Davis MS-160, DiLog Instruments, Tallahassee, FL; see Smith, 2001).

This device allows a mouse access to a single sipper tube containing a stimulus. Animals

can also be restricted to licking in brief trials (5 s) by offering access to the different

tubes via a motorized table and shutter. An 8-s inter-presentation interval was interposed

between stimulus presentations. The test array for each mouse included the two different

concentrations of each test stimulus detailed above, the CS, and purified water all of









which were contained in separate bottles (i.e., fourteen stimulus sipper tubes; in addition

to the stimulus tubes, a non-stimulus "rinse" sipper tube was also included in the array -

see details below). A given trial started upon the first lick. Each lick on a sipper tube

was registered by a contact circuit. These responses were recorded by computer for later

analysis.

During the conditioning phase, intake tests were conducted in the home cages.

Fluids were presented in 25 ml graduated pipettes fitted with stainless steel sipper tubes

on one end. Pipettes were secured to the shelf above with cable clips to reduce spillage.

Intake was measured to the nearest 0.1 ml during this phase.

Experimental Design

Davis rig training

The mice were trained under a restricted water-access schedule. Water bottles were

removed from the home cages the day before the start of the training phase. The mice

were first trained for 2 daily consecutive 30-min sessions in the Davis rig with a

stationary sipper tube containing purified water positioned in front of the access slot. The

mice were allowed to take as many licks as possible within a 30-min session. Next, the

mice were trained for 3 days in the Davis rig with a brief-access paradigm, in which

access to water was available in 5-s trials from fourteen different sipper tubes. A water

rinse (5 lick maximum) presentation was interposed between all trials. This was done so

that "stimulus" presentation was consistent with that during the Davis rig test session (see

below). Presentation order was randomized without replacement in blocks. The mice

were allowed to complete as many trials as possible within the 25-min session.









Conditioning phase (see Table 4-2)

Water bottles were removed the day before the start of the conditioning phase.

During this phase, starting at 0900, each mouse received water from a drinking spout in

its home cage for 15 min at the same time each day (the start of the trial for animals was

staggered by 5 min to allow time for intake measurements and injections). Approximately

5 h after the start of each animal's morning session, the mice were given access to

purified water for 1 h to allow for rehydration. After three days of one-bottle water

testing, the animals were divided into six groups (n = 8 mice per group) according to the

CS (L-serine, L-threonine, or sucrose) and unconditioned stimulus (US; LiCl or NaC1)

they would receive. Mice were assigned to groups on the basis of their body weight,

mean water intake during the first three days of the conditioning phase, mean licks/trial,

and mean number of trials during the last three days of Davis rig training. There were no

significant differences between the groups regarding these parameters.

Subsequently, five conditioning trials followed in which mice were presented with

the appropriate CS for 15 min, immediately followed by an intraperitoneal (i.p.) injection

(3.0 mEq/kg body wt) of either 0.15 M LiC11 or 0.15 M NaC1. The purpose of the LiCl

injection was to induce visceral malaise. Mice that drank less than 0.1 mL of their

respective CS, had -0.1 mL infused in their oral cavity with a syringe before receiving

the US injection. Water bottles were returned to the home cages -5 hours after a

conditioning trial. The following day, water bottles were removed from the cages,

starting at 0915, on the same staggered schedule used on the previous conditioning phase



1 The 1st US injections for all three experimental groups were carried out with LiCl dissolved in 0.15 M
NaC1. All subsequent injections were completed with LiCl dissolved in purified water. Also, four mice
died after the 1st injection for unknown reasons. As a result, n = 6 8 mice per group.