John D. Boughter

Taste transduction: appetizing times in gustation.

&NA; Taste receptors cells sample the chemical composition of ingested material in order to provide the initial sensory information to facilitate decisions regarding its eventual acceptance or rejection. Ion channels, ionotropic and metabotropic receptors have been implicated in the initial events of transduction but until recently their identification has proven difficult. Recent advances in the identification and functional characterization of mammalian taste receptors has greatly increased our understanding of the pathways for the transduction of taste stimuli. This basic information will be critical to answer longstanding questions regarding the coding of taste information and may help elucidate the role of the taste system in the control of food intake.

A low-cost solution to measure mouse licking in an electrophysiological setup with a standard analog-to-digital converter.

Licking behavior in rodents is widely used to determine fluid consumption in various behavioral contexts and is a typical example of rhythmic movement controlled by internal pattern-generating mechanisms. The measurement of licking behavior by commercially available instruments is based on either tongue protrusion interrupting a light beam or on an electrical signal generated by the tongue touching a metal spout. We report here that licking behavior can be measured with high temporal precision by simply connecting a metal sipper tube to the input of a standard analog/digital (A/D) converter and connecting the animal to ground (via a metal cage floor). The signal produced by a single lick consists of a 100-800 mV dc voltage step, which reflects the metal-to-water junction potential and persists for the duration of the tongue-spout contact. This method does not produce any significant electrical artifacts and can be combined with electrophysiological measurements of single unit activity from neurons involved in the control of the licking behavior.

Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli

BackgroundCommon inbred mouse strains are genotypically diverse, but it is still poorly understood how this diversity relates to specific differences in behavior. To identify quantitative trait genes that influence taste behavior differences, it is critical to utilize assays that exclusively measure the contribution of orosensory cues. With a few exceptions, previous characterizations of behavioral taste sensitivity in inbred mouse strains have generally measured consumption, which can be confounded by post-ingestive effects. Here, we used a taste-salient brief-access procedure to measure taste sensitivity to eight stimuli characterized as bitter or aversive in C57BL/6J (B6) and DBA/2J (D2) mice.ResultsB6 mice were more sensitive than D2 mice to a subset of bitter stimuli, including quinine hydrochloride (QHCl), 6-n-propylthiouracil (PROP), and MgCl2. D2 mice were more sensitive than B6 mice to the bitter stimulus raffinose undecaacetate (RUA). These strains did not differ in sensitivity to cycloheximide (CYX), denatonium benzoate (DB), KCl or HCl.ConclusionB6-D2 taste sensitivity differences indicate that differences in consumption of QHCl, PROP, MgCl2 and RUA are based on immediate orosensory cues, not post-ingestive effects. The absence of a strain difference for CYX suggests that polymorphisms in a T2R-type taste receptor shown to be differentially sensitive to CYX in vitro are unlikely to differentially contribute to the CYX behavioral response in vivo. The results of these studies point to the utility of these common mouse strains and their associated resources for investigation into the genetic mechanisms of taste.

Behavioral genetics and taste

This review focuses on behavioral genetic studies of sweet, umami, bitter and salt taste responses in mammals. Studies involving mouse inbred strain comparisons and genetic analyses, and their impact on elucidation of taste receptors and transduction mechanisms are discussed. Finally, the effect of genetic variation in taste responsiveness on complex traits such as drug intake is considered. Recent advances in development of genomic resources make behavioral genetics a powerful approach for understanding mechanisms of taste.

C57BL/6J and DBA/2J mice vary in lick rate and ingestive microstructure

Fluid licking in mice is an example of a rhythmic behavior thought to be under the control of a central pattern generator. Inbred strains of mice have been shown to differ in mean or modal interlick interval (ILI) duration, suggesting a genetic‐based variation. We investigated water licking in the commonly used inbred strains C57BL/6J (B6) and DBA/2J (D2), using a commercially available contact lickometer. Results from 20‐min test sessions indicated that D2 mice lick at a faster rate than B6 mice (10.6 licks/s vs. 8.5 licks/s), based on analysis of the distribution of short‐duration ILIs (50–160 ms). This strain difference was independent of sex, extent of water deprivation or total number of licks. D2 mice also displayed a faster lick rate when the strains were tested with a series of brief (5 s) trials. However, when ingestion over the entire 20‐min session was analyzed, it was evident that D2 mice had an overall slower rate of ingestion than B6 mice. This was because of the tendency for D2 mice to have more very long pauses (>30 s) between sequences of licking bursts. Overall, it appeared that D2 mice licked more efficiently, ingesting more rapidly during excursions to the spout that were fewer and farther between.

Cerebellar cortical output encodes temporal aspects of rhythmic licking movements and is necessary for normal licking frequency.

Rodents consume water by performing stereotypic, rhythmic licking movements that are believed to be controlled by brainstem pattern‐generating circuits. Previous work has shown that synchronized population activity of inferior olive neurons was phase‐locked to the licking rhythm in rats, suggesting a cerebellar involvement in temporal aspects of licking behavior. However, what role the cerebellum has in licking behavior and whether licking is represented in the high‐frequency simple spike output of Purkinje cells remains unknown. We recorded Purkinje cell simple and complex spike activity in awake mice during licking, and determined the behavioral consequences of loss of cerebellar function. Mouse cerebellar cortex contained a multifaceted representation of licking behavior encoded in the simple spike activities of Purkinje cells distributed across Crus I, Crus II and lobus simplex of the right cerebellar hemisphere. Lick‐related Purkinje cell simple spike activity was modulated rhythmically, phase‐locked to the lick rhythm, or non‐rhythmically. A subpopulation of lick‐related Purkinje cells differentially represented lick interval duration in their simple spike activity. Surgical removal of the cerebellum or temporary pharmacological inactivation of the cerebellar nuclei significantly slowed the licking frequency. Fluid licking was also less efficient in mice with impaired cerebellar function, indicated by a significant decline in the volume per lick fluid intake. The gross licking movement appeared unaffected. Our results suggest a cerebellar role in modulating the frequency of the central pattern‐generating circuits controlling fluid licking and in the fine coordination of licking, while contributing little to the coordination of the gross licking movement.

Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice

BackgroundThe detection of bitter-tasting compounds by the gustatory system is thought to alert animals to the presence of potentially toxic food. Some, if not all, bitter stimuli activate specific taste receptors, the T2Rs, which are expressed in subsets of taste receptor cells on the tongue and palate. However, there is evidence for both receptor-dependent and -independent transduction mechanisms for a number of bitter stimuli, including quinine hydrochloride (QHCl) and denatonium benzoate (DB).ResultsWe used brief-access behavioral taste testing of BXD/Ty recombinant inbred (RI) mouse strains to map the major quantitative trait locus (QTL) for taste sensitivity to QHCl. This QTL is restricted to a ~5 Mb interval on chromosome 6 that includes 24 genes encoding T2Rs (Tas2rs). Tas2rs at this locus display in total 307 coding region single nucleotide polymorphisms (SNPs) between the two BXD/Ty RI parental strains, C57BL/6J (quinine-sensitive) and DBA/2J (quinine insensitive); approximately 50% of these mutations are silent. Individual RI lines contain exclusively either C57BL/6J or DBA/2J Tas2r alleles at this locus, and RI lines containing C57BL/6J Tas2r alleles are more sensitive to QHCl than are lines containing DBA/2J alleles. Thus, the entire Tas2r cluster comprises a large haplotype that correlates with quinine taster status.ConclusionThese studies, the first using a taste-salient assay to map the major QTL for quinine taste, indicate that a T2R-dependent transduction cascade is responsible for the majority of strain variance in quinine taste sensitivity. Furthermore, the large number of polymorphisms within coding exons of the Tas2r cluster, coupled with evidence that inbred strains exhibit largely similar bitter taste phenotypes, suggest that T2R receptors are quite tolerant to variation.

Variation in Nicotine Consumption in Inbred Mice Is Not Linked to Orosensory Ability

Genetic studies of nicotine addiction in mice have utilized the oral self-administration model. However, it is unclear if strain differences in nicotine consumption are influenced by variation in bitter taste sensitivity. We measured both nicotine consumption and nicotine brief-access licking behavior in several commonly used inbred strains of mice that were previously shown to differ in nicotine consumption. A/J (A), C57BL/6J (B6), and DBA/2J (D2) mice were given a 2-bottle choice test with a single concentration of nicotine (75 microg/ml; nicotine vs. water). Mice of these strains were also tested with a range of nicotine concentrations (5-400 microg/ml) using a brief-access test, which measures orosensory response and minimizes postingestive effects. Although B6 mice consumed more 75-microg/ml nicotine than A or D2 mice in the 2-bottle test, these strains did not differ in level of aversion to nicotine when tested with the brief-access procedure. Strain differences in orosensory response to nicotine were not found; yet, differences emerged during the 2-bottle tests. This study provides evidence that variation in intake level of nicotine is likely not due to differences in taste or trigeminal sensitivity but likely due to postingestive factors.

Subnuclear organization of parabrachial efferents to the thalamus, amygdala and lateral hypothalamus in C57BL/6J mice: a quantitative retrograde double labeling study

The present study investigated the subnuclear organization of collateralized efferent projection patterns from the mouse parabrachial nucleus (PbN), the second taste relay in rodents, to higher gustatory centers, including the ventroposteromedial nucleus of the thalamus (VPMpc), central nucleus of the amygdala (CeA) and lateral hypothalamus (LH). We made injections of the retrograde tracer red and green latex microspheres into the VMPpc and CeA (VPMpc-CeA group), VMPpc and LH (VPMpc-LH group) or CeA and LH (CeA-LH group, n=6 for each group). Injections into these areas preferentially resulted in retrograde labeling in the ipsilateral PbN in all groups. Cells projecting to the VPMpc, CeA, and LH were generally found in all subnuclei, but were differentially distributed. VPMpc-projecting cells predominated in gustatory-related subnuclei, CeA-projecting neurons predominated in the external lateral (el) subnucleus, and concentrated labeling was observed in the dorsal lateral subnucleus (dl) following LH injection. Double-labeled neurons were found for all groups, almost entirely ipsilaterally and primarily in the medial (m), waist area (wa), ventral lateral (vl) and el subnuclei. These results suggest that PbN neurons in different subdivisions have different projection and collateralization patterns to the VPMpc, CeA and LH. Functional implications of these projections are discussed with an emphasis on their roles in taste.

Bitter taste stimuli induce differential neural codes in mouse brain

A growing literature suggests taste stimuli commonly classified as “bitter” induce heterogeneous neural and perceptual responses. Here, the central processing of bitter stimuli was studied in mice with genetically controlled bitter taste profiles. Using these mice removed genetic heterogeneity as a factor influencing gustatory neural codes for bitter stimuli. Electrophysiological activity (spikes) was recorded from single neurons in the nucleus tractus solitarius during oral delivery of taste solutions (26 total), including concentration series of the bitter tastants quinine, denatonium benzoate, cycloheximide, and sucrose octaacetate (SOA), presented to the whole mouth for 5 s. Seventy-nine neurons were sampled; in many cases multiple cells (2 to 5) were recorded from a mouse. Results showed bitter stimuli induced variable gustatory activity. For example, although some neurons responded robustly to quinine and cycloheximide, others displayed concentration-dependent activity (p<0.05) to quinine but not cycloheximide. Differential activity to bitter stimuli was observed across multiple neurons recorded from one animal in several mice. Across all cells, quinine and denatonium induced correlated spatial responses that differed (p<0.05) from those to cycloheximide and SOA. Modeling spatiotemporal neural ensemble activity revealed responses to quinine/denatonium and cycloheximide/SOA diverged during only an early, at least 1 s wide period of the taste response. Our findings highlight how temporal features of sensory processing contribute differences among bitter taste codes and build on data suggesting heterogeneity among “bitter” stimuli, data that challenge a strict monoguesia model for the bitter quality.