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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.
Fluid licking is a highly stereotyped behavior in rats, mice and many other mammals that involves the rhythmic co-ordination of muscle groups involved in tongue protrusion and retraction, jaw opening and closing and swallowing. The co-ordination of these oromotor movements is thought to be under the control of central pattern generators (CPGs), motor ‘programs’ extant among premotor neuron networks that send rhythmic inputs to motor neuron pools in cranial nerve nuclei V, VII and XII (for reviews see Nakamura & Katakura 1995; Travers et al. 1997). Current evidence suggests a substrate for rhythmic licking organized among premotor neurons in the medullary reticular formation (RF). Premotor neurons associated with intrinsic and extrinsic tongue muscles are located in a number of medullary and pontine RF cell groups (Travers & Rinaman 2002; Travers et al. 2005). Neurons rhythmically active during licking are found in both the parvocellular and intermediate zones of the RF (e.g. Travers et al. 1997), and reversible lesion studies in awake rat preparations suggest a necessary role for the rostrolateral medullary RF (Chen & Travers 2003). However, the specific identity and physiological properties of the neurons and networks that underlie the CPG for licking are unknown.
The species of choice for investigating the physiological and anatomical substrates of licking has been the rat (Weijnen 1998) but the study of strains of mice with different lick or ingestion rates holds substantial promise for genetic approaches to the study of oromotor CPGs (e.g. Okayasu et al. 2003; Tomiyama et al. 2004). Horowitz et al. (1977) examined ad lib fluid licking over a series of 20-h periods in undeprived C57BL/6 (B6) and DBA/2 (D2) mice, and their F1 progeny, using an infrared-beam lickometer. Local lick rate, as defined by interlick intervals (ILIs) <390 ms differed substantially between strains; B6 mice exhibited a slower lick rate (mean ILI ≈ 130 ms) than D2 mice (mean ILI ≈ 97 ms), and F1 mice expressed an intermediate rate (mean ILI ≈ 109 ms). These data indicated robust and genetically influenced differences in lick rate, and that the strain difference was stable over time and in response to different stimuli.
Other strain differences in licking have been reported: Smith et al. (2001) showed a significant strain lick-rate difference between water-deprived inbred mice, with SWR/J mice possessing a shorter modal ILI (faster lick rate; 109 ms) than AKR/J mice (129 ms) in a short (30 s) trial. More recently, Dotson & Spector (2005) assessed lick rate in four strains of mice (B6, D2, 129P3 and SWR) in a commercially available lickometer (Davis MS160). These strains differed significantly in terms of mean ILI when the analysis was limited to ILIs 50–200 ms, with D2 and SWR mice licking significantly faster than the other strains.
In order to provide a broader characterization of inbred strain differences in lick rate and the robustness of their generalizability, we examined licking in water-restricted B6 and D2 mice across several temporal and situational contexts. We utilized a licking microstructure analysis to evaluate how licking in these strains was organized across various time frames, ranging locally from one lick cycle to the next, to more broadly across bursts of licking as well as over an entire ingestion bout. Treatments that influence meal size tend more often to influence the size and number of bursts of licking rather than the rate of licking intrinsic to bursts (Davis 1996; Spector et al. 1998). Therefore, understanding principal strain differences in the organization of licking in both local lick rate and at the level of bursts and pauses will provide a better platform to genetically evaluate the operating characteristics of food intake mechanisms in a variety of genetic models of obesity.
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By several different measures (ILI distribution, lick rate within bursts and lick rate in short trials), D2 mice possessed a significantly faster lick rate than B6 mice. This effect was independent of sex. Importantly, the strain difference in lick rate was robust over a 4-day period, and therefore independent of prolonged water deprivation, or total licks (which increased in both strains across the 4-day period). B6 mice appeared to have at least a subtle increase in lick rate resulting from experience, with significantly shorter MPI values on day 2, or days 2–4 in the extended-testing group. No such modulation was observed in D2 mice, although D2 mice licked considerably faster overall. These results confirm and extend previously published reports of lick rates, or differences in lick rate, between these strains (Dotson & Spector 2005; Glendinning et al. 2002; Horowitz et al. 1977).
What is the basis of the lick-rate difference? We hypothesize that there is a difference in the organizational properties of the oromotor CPG, although the possibility certainly exists that other factors, such as anatomical differences, play a major role. Age-matched adult B6 mice have a larger overall mandible size than D2 mice, which could in theory correspond to slower jaw movements and lick rate (Carvalho & Gerstner 2004; Lovell et al. 1984). However, B6 × D2 F1 mice were found to possess a larger mandible size than either parent strain, despite possessing an intermediate lick rate (Horowitz et al. 1977; Lovell et al. 1984; unpublished data from our laboratory). We also measured tongue length, width and weight (anterior tongue portion) in 10 B6 and 10 D2 mice (both sexes) and did not find a significant correlation between any of these variables and MPI score. However, studies of skeletal muscle indicate that a subset of fast- and slow-twitch hind limb muscles are heavier in B6 than D2 mice (Lionikas et al. 2003, 2005). This is a polygenic trait, and the authors observed significant effects of sex. It is possible that one or more genes influencing the weight of specific muscles such as extrinsic tongue muscles, as opposed to the tongue itself, could contribute to strain differences in lick rate.
An extremely important consideration attending all of these studies is the time frame of analysis used to evaluate ILIs. The current study shows that different analysis time frames (i.e. analyses of ILIs limited to different upper range cutoffs – 160, 500 or 1000 ms) can produce different, even opposing, conclusions about treatment/strain effects on the rate of licking. For example, as the time frame of analysis was expanded, the faster lick rate of D2 was progressively reduced until they were shown to lick slower than B6 mice when averaged over the entire test period. The aforementioned studies used different criteria for ILI analysis, calling into question whether the various conditions affected properties of the CPG controlling lick rate in the primary ILI distribution (those less than 160 ms), or ILIs of longer durations reflecting an influence on mechanisms that engage or disengage the CPG. Our findings indicate that the effects of deprivation or experience on lick rates reflecting CPG output are of considerably less influence than previously reported.
The net effect of the two phenotype differences in primary lick rate and licking microstructure is that D2 mice appear to lick in a more efficient manner overall. These differences perhaps reflect a difference in ingestion or meal-taking strategies between the two strains. This difference is clarified by analysis of the relative distributions of bursts and pauses within the ingestion period. Traditionally, the mean length of bursts is considered to be reflective of gustatory influences of the tastant as burst size increases linearly with increases in the concentrations of palatable solutions and it decreases with naturally or conditioned aversive tastants (Baird et al. 2005; Spector & St. John 1998; Spector et al. 1998). Although D2 mice expressed a faster intrinsic lick rate, the mean burst size/duration was not significantly different suggesting comparable taste reactivity across strains, although only water was used as a taste stimulus.
The principal strain differences between B6 and D2 mice in terms of burst/pause distribution were in the number of bursts expressed and the duration of intervals (pauses) between those bursts. Overall, D2 mice took significantly fewer bursts at the spout, and they expressed, on balance, proportionally more long pauses (>30 s) and proportionally fewer short pauses (<30 s) between bursts, resulting in an average pause length almost threefold longer than that for B6 mice among males (Fig. 5). This difference resulted in the overall slower rate of ingestion for D2 mice over the course the entire drinking period. Treatments affecting satiety-related processes have prototypical effects on the distributions of bursts and pauses. As satiety increases toward the end of meals, pauses on average tend to grow longer in duration (Davis 1996). In addition, treatments that reduce or enhance meal size tend to, respectively, decrease or increase the number of bursts in the meal (Davis & Levine 1977; Davis et al. 1994, 1995, 1997, 1998; Eisen et al. 2001; Schwartz et al. 1999). Varying the size of the sipper tube orifice also increases burst length, but not number of bursts, in mice. Either B6 or D2 mice took nearly twice as many licks in a 30-min session when presented with a 1.5 mm tube orifice than with a 2.7 mm orifice, although neither the total amount consumed nor the mean ILI were changed (Dotson & Spector 2005).
Overall, it is clear that B6 and D2 mice exhibit different and complex profiles of licking microstructure. It would be worthwhile to perform a videographic analysis to determine the nature of the other behaviors expressed by D2 mice during the longer intervals between bursts (e.g. grooming, sleep, stereotypy) to further characterize the differential portfolios of behavioral expression exhibited by these two strains. In any case, licking and ingestive phenotypes are ideal candidates for genetic analysis using derivative populations of the parent inbred strains. Moreover, it will be important and feasible in future research to determine how patterns of ingestion vary or systematically breakdown with controlled, specified mutations. Indeed, recent studies in mice and other species further support the utility of genetic approaches for dissecting the organization of CPGs and locomotor networks (Kiehn & Kullander 2004; Kullander 2005).