Drinking spout orifice size affects licking behavior in inbred mice

Physiology & Behavior 85 (2005) 655 – 661

Drinking spout orifice size affects licking behavior in inbred mice Cedrick D. Dotson, Alan C. Spector * Department of Psychology and Center for Smell and Taste, University of Florida, PO Box 112250, Gainesville, FL, 32611-2250, United States Received 16 January 2005; received in revised form 8 June 2005; accepted 21 June 2005

Abstract Using a lickometer, we assessed the effect of drinking spout orifice size on the licking behavior of inbred mice [C57BL/6J, SWR/J, 129P3/J and DBA/2J]. Animals licked from drinking spout sipper tubes that had what were defined as either a large (2.7 mm) or a small (1.5 mm) orifice. Mice took approximately twice as many licks from a stationary single small orifice drinking spout than when licking from a spout with a large orifice during separate 30-min sessions. However, their total intake volume was approximately the same. We calculated that mice received a mean of 0.55 AL per lick from the drinking tubes with a small orifice and a mean of 1.15 AL per lick from the drinking tubes with a large orifice. Thus, the animals appear to have regulated their fluid intake by proportionally adjusting their licking as a function of the lick volume. On average, this regulation occurred through modulation of the size of licking bursts and not their frequency. However, strain differences in compensation strategy were observed. When licking was restricted to a series of 5-s trials in a 30-min brief access test session, the smaller orifice size increased the range of responsiveness that was expressed. Mice increased their average licks per trial by 20% and took 60% more trials when licking from a spout with a small orifice. Interestingly, when the orifice size was quasi-randomly varied within a brief access session, licking was greater from large orifice drinking spouts, suggesting that water delivered from the two orifice sizes differs in its reinforcement efficacy. These findings demonstrate that drinking spout orifice size can significantly influence experimental outcomes in licking tests involving mice and care should be taken in controlling this variable in testing the effects of taste or other factors on ingestive behavior. D 2005 Elsevier Inc. All rights reserved. Keywords: Brief access test; Fluid intake; Lickometer; Taste; Ingestive behavior; Drinking; Lick microstructure

1. Introduction The most common measure used to assess drinking in rodent models is the total volume of fluid ingested. Although convenient and interpretively meaningful, measures of total intake represent the outcome of behavior rather than the behavior itself. Alternatively, the recording and analysis of spout licking provide a more direct link to the neural mechanisms controlling ingestive behavior (e.g., [1,2]). The microstructural analysis of licking during short-term tests has been useful in the study of the effects of neural, genetic, pharmacological, and physiological manipulations on feed-

* Corresponding author. Tel.: +1 352 392 0601x288; fax: +1 352 392 7985. E-mail address: [email protected] (A.C. Spector). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.06.010

ing and drinking in rodents. Similarly, the measurement of unconditioned licking responses to various chemical stimuli presented during a series of very short trials – a procedure referred to as the brief access taste test – has enjoyed growing popularity in the assessment of gustatory function in a variety of experimental contexts (e.g., [3 –21]). Licking in the rat is influenced by environmental factors, such as the position of the animal relative to the drinking tube or the topography of access to the spout (e.g., [2,22 – 25]). The size of the drinking spout orifice can also affect the behavior of rodents when ingesting fluid from drinking spouts. For example, Freed and Mendelson [26] used female Sprague – Dawley rats to characterize schedule-induced and water-deprivation-induced drinking. They reported that rats compensated for the narrowing of the drinking spout orifices from 2.6 mm T 0.1 to 1.0 mm T 0.1 by increasing their time spent drinking, thus maintaining their intake

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volume relative to the amount consumed from the larger orifice tube. Although not explicitly measured, it is likely that the increased time spent drinking reflected an increase in the number of licks, mediated by the reduction in the volume of fluid received per lick in the ‘‘small’’ orifice condition. Indeed, research has shown that the volume of fluid received per lick significantly affects drinking behavior. Kaplan et al. [27,28] systematically varied the volume of glucose and maltodextrin obtained per lick by male Sprague – Dawley rats and demonstrated that these animals proportionally altered their licking so as to defend total intake volume. Commercial drinking spouts can be purchased with various sizes of orifices, which can then be further customized by investigators. Given that drinking spout orifice size and lick volume can have such striking effects on the number of licks emitted by rats, we sought to examine the effects of drinking spout orifice size on the microstructural parameters characterizing licking of waterdeprived mice during a 30-min one-bottle test. In addition, because use of the brief access taste test as a behavioral assay of gustatory function is increasing (see Refs. [20,29 – 31]), we tested whether drinking spout orifice size would influence licking in this task. The four inbred strains of mice we chose to study are commonly employed in taste research and some of these strains have also been used as the genetic background for gene deletion experiments [31 – 33]. It is known that the mean of the distribution characterizing interlick intervals (ILIs; defined as the time between the onset of one lick to the onset of the subsequent lick; see Ref. [1] for a review) can vary among strains of mice (e.g., [29,30]). Accordingly, we thought it was important to test the generality of any observed effects of orifice size on licking behavior by including several inbred strains of mice in the design.

2. Methods 2.1. Subjects Five male mice (Jackson Laboratories, Bar Harbor, Maine) from four different inbred strains, C57BL/6J (B6), SWR/J (SWR), 129P3/J (129), and DBA/2J (D2), served as subjects. The mice used in this study were previously used in a brief access experiment that made use of large orifice spouts (see below) and, as a result, were familiar with the apparatus and testing procedures. The mice were housed individually in polycarbonate tub cages with bedding on the

floor, in a colony room where the temperature and lighting were controlled automatically (12 h:12 h). Testing and training took place during the lights-on phase. Mice were approximately 10 weeks of age at the start of testing. Throughout testing, food (LabDiet 5001; PMI Nutrition International Inc., Brentwood, MO) was available ad libitum. Animals were placed on a water-restriction schedule in which their access to water was limited to the daily testing sessions. Although rarely necessary, mice that dropped below 80% of their free-feeding weight received 1 mL supplemental water 2 h after the end of the testing session. We tested 20 animals over 6 days and provided supplemental water on 10 out of 120 possible occasions. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. 2.2. Apparatus Testing took place in a lickometer commonly referred to as the Davis rig (Davis MS-160, DiLog Instruments, Tallahassee, FL; see Ref. [34]). This device allows a mouse access to a single tube containing a stimulus. Animals can be restricted to licking in brief trials by offering different tubes via a motorized table and access via a motorized shutter. Lick responses are recorded by computer for later analysis. 2.3. Procedure We used procedures similar to those described in Dotson and Spector [35]. All sessions were 30 min in duration. The animals were tested under a 23.5-h water-restriction schedule, over 6 days (see Table 1). On the first 2 days, the animals received access to purified water (Elix 10; Millipore, Billerica, MA) in 5-s trials delivered in stainless steel drinking spouts with vinyl stoppers, attached to glass test tubes which contained the fluid. A given trial started after the first lick. After a trial was completed, there was a 7.5-s inter-presentation interval so that a different tube could be offered. During the 30-min session, mice could initiate as many trials as possible. Three of the drinking spouts had what was defined as a large orifice (LO; OT-100, Ancare, Bellmore, NY), whereas the other three had what was defined as a small orifice (SO; OT100SP, Ancare, Bellmore, NY). Orifice diameter was determined by inserting a standard taper pin (48:1) into each sipper tube and measuring the length of the protrusion (Hole Size = (Diameter of large end) - (1/48 * Length of protrusion)). The large orifice sipper tubes were chosen, at random, from a group with a mean orifice diameter of 2.7

Table 1 Experimental procedures Procedure

Number of days tested

Presentation

Number of tubes

Orifice

Brief access with orifice size varied Stationary spout Brief access with orifice size uniform

2 2 2

5-s trials 30-min session 5-s trials

6 1 3

3 small and 3 large 1 small or 1 large 3 small or 3 large

C.D. Dotson, A.C. Spector / Physiology & Behavior 85 (2005) 655 – 661 4000 Small Orifice Large Orifice

Licks

3000

657

All of these data were analyzed with two-way strain  orifice size analyses of variance (ANOVAs). When an interaction was significant, one-way ANOVAs were conducted to test for simple effects. The conventional p  0.05 was applied as the statistical rejection criterion.

2000

3. Results 1000

3.1. Stationary spout (30-min presentation) 0 B6

SWR

129

D2

Fig. 1. Average number of licks (Tse) taken per session during the stationary spout condition for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

mm T 0.8%, whereas the small orifice sipper tubes were chosen randomly from a group with a mean diameter of 1.52 mm T 1.6%. The presentation order of the stimuli was randomized without replacement in blocks of six. On Day 3, half of the mice were tested with a single stationary SO spout for 30 min (i.e., no trials) and the other half received water from a LO spout. These test conditions were then switched on Day 4. Intake was determined on those days by weighing the glass tubes and drinking spouts before and after each session. We did not observe any spillage during the experiment. In addition, spillage tests with an empty apparatus confirmed the absence of any detectable loss of fluid from the tubes. On Day 5, mice were tested again using the brief access procedure (5-s trials), but this time, only a single spout orifice size was used in the session. Half of the mice were tested with water delivered from three spouts that had SOs and the other half received water from three spouts with LOs. The orifice size condition was reversed for all animals on Day 6. Presentation order among the three spouts was randomized without replacement in blocks.

When allowed access to a single tube for 30 min, there was a significant effect of orifice size on total number of licks per session ( F(1,16) = 32.3, p < 0.01) and a significant effect of strain ( F(3,16) = 3.7, p < 0.05), but the interaction was not significant ( F(3,16) = 0.2, p = 0.92). Mice licked from SO drinking spouts substantially more than they did from the LO spouts (Fig. 1). As expected, there was a significant main effect of orifice size on lick volume ( F(1,16) = 28.3, p < 0.01). However, there was no strain effect ( F(3,16) = 1.3, p = 0.3) and no interaction ( F(3,16) = 0.5, p = 0.66) (Fig. 2). Overall, we calculated that animals received a mean of 0.55 AL per lick from the SO tubes and a mean of 1.15 AL per lick from the LO tubes (Fig. 2). As a result, animals, while licking far more from the SO tubes, did not differ in the amount of water they ingested. There was no effect of orifice size ( F(1,16) = 2.1, p = 0.17) or strain ( F(3,16) = 2.4, p = 0.11) on the total intake per session and no significant interaction ( F(3,16) = 0.3, p = 0.83; see Fig. 3). A two-way ANOVA of the ILI revealed a significant main effect of orifice size ( F(1,16) = 8.9, p < 0.01), a significant main effect of strain ( F(3,16) = 23.6, p < 0.01) and a significant interaction ( F(3,16) = 4.2, p < 0.05). Oneway ANOVAs revealed that although 129, B6, and D2 mice licked slightly faster from the SO spouts, this difference only reached significance for the 129 strain ( F(1,4) = 11.5, p < 0.05). Nevertheless, as can be seen in Fig. 4, the magnitude of this effect was relatively minor. SWR mice

2.4. Data analysis 2.5 Small Orifice Large Orifice

2.0 Volume per Lick (µL)

In addition to measuring total licks and volume ingested, the interlick interval (ILI), burst number, and burst size were quantified during the stationary spout condition. The ILI was defined as the time between the onset of one lick to the onset of the subsequent lick. Only ILIs between 50 ms and 200 ms were included in the ILI analysis (see Refs. [2,24,36,37]). A burst of licks was operationally defined as a series of consecutive licks ending with a pause  1000 ms [see Ref. 36]. For the brief access tests, number of licks taken per trial and number of trials initiated were measured. Animals licked from multiple representatives of a particular orifice size during brief access testing (i.e., either 3 SO and/ or 3 LO within a brief access test session). The results from the individual same-size tubes did not differ and were therefore averaged together.

1.5

1.0

0.5

0.0 B6

SWR

129

D2

Fig. 2. Mean volume (Tse) taken per lick when mice were tested during the stationary spout condition for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

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A

3.0 Small Orifice Large Orifice

2.5

500 Small Orifice 400

Large Orifice

Burst Licks

Intake (mL)

2.0 1.5 1.0 0.5

200

100

0.0 SWR

129

Fig. 3. Mean total intake (T se) per session when mice were tested during the stationary spout condition for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

actually licked slightly faster from the LO spouts, but this difference failed to reach significance. There was no effect of orifice size on the number of bursts initiated in a session, although there was a significant main effect of strain ( F(3,16) = 12.2, p < 0.01; Fig. 5B). The strain  orifice interaction ( F(3,16) = 2.0, p = 0.15) was not significant. In contrast, there was a significant main effect of orifice size on the number of licks taken within a burst ( F(1,16) = 11.1, p < 0.01); there was also a significant main effect of strain ( F(3,16) = 9.2, p < 0.01) and a significant strain  orifice size interaction ( F(3,16) =3.7, p < 0.05) (Fig. 5A). However, for each strain, none of the separate one-way ANOVAs on orifice size reached statistical significance. Specific identification of the origin of the strain  orifice size interaction in burst licks was likely obscured by the small sample size of the groups. An interesting strain difference emerged in the drinking pattern. B6 mice initiated many bursts of few licks, whereas SWR mice initiated less bursts of many licks. The D2 and 129 strains fell in between these two extremes. A one-way ANOVA conducted on burst licks from the SO spout indicated a significant strain effect ( F(3,16) = 7.3, p < 0.01); 160 Small Orifice Large Orifice

140 120 100 80 60 40 20 0 B6

SWR

129

0

D2

D2

Fig. 4. The mean of the interlick interval distributions (Tse) for the mice during the stationary spout condition for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

B

B6

SWR

129

D2

B6

SWR

129

D2

80

60 Burst Number

B6

Interlick Interval (msec)

300

40

20

0

Fig. 5. (A) Average number of licks (T se) taken within a burst and (B) average number of bursts (T se) taken in a session during the stationary spout condition for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

Tukey post hoc comparisons revealed that SWR mice licked significantly more within a burst compared to the B6, 129, and D2 mice. These latter three strains did not differ from one another. An analysis of burst licks in the LO condition also revealed a significant strain effect ( F(3,16) = 8.8, p < 0.01). Tukey post hoc comparisons revealed that SWR mice licked significantly more than did B6, 129, and D2 strains. Again, these latter three strains did not differ from one another. A one-way ANOVA conducted on the number of bursts initiated from the SO spout indicated a significant strain effect ( F(3,16) = 8.6, p < 0.01); Tukey post hoc comparisons revealed that the B6 mice took significantly more bursts compared with SWR and D2 mice; 129 mice did not significantly differ from B6, SWR, or D2 mice. An analysis of burst number in the LO condition revealed a significant strain effect F(3,16) = 6.5, p < 0.01). Tukey post hoc comparisons revealed that B6 mice took significantly more bursts from the LO spouts compared with SWR and 129 mice; D2 mice did not significantly differ from B6, SWR, or 129 mice. There was no effect of orifice size on the amount of water consumed per burst ( F(1,16) = 0.03, p = 0.85), although there was a significant main effect of strain ( F(3,16) = 9.8, p < 0.01). There was no significant strain  orifice interaction ( F(3,16) = 2.4, p = 0.11). Averaged across strain, mice

C.D. Dotson, A.C. Spector / Physiology & Behavior 85 (2005) 655 – 661 70 Small Orifice Large Orifice

60 50

Trials

received a mean of 0.082 T 0.022 mL per burst from the SO drinking spouts and a mean of 0.08 T 0.012 mL per burst from the LO tubes. Collapsed across orifice size the mean (Tse) burst volume values were B6 = 0.029 T 0.004 mL, SWR = 0.175 T 0.038 mL, 129 = 0.073 T 0.017 mL, and D2 = 0.047 T 0.006 mL.

659

40 30

3.2. Brief access procedure with orifice size uniform 20

When the mice were presented with either 3 LO or 3 SO drinking spouts within a brief access test session, there was a significant main effect of orifice size on the number of licks taken per trial ( F(1,16) = 15.3, p < 0.01) and a significant effect of strain ( F(3,16) = 7.3, p < 0.01; Fig. 6). There was no significant strain  orifice interaction ( F(3,16) = 0.4, p = 0.76). Additionally, all strains took more trials per session when tested with SO drinking spouts (Fig. 7) as indicated by a significant main effect of orifice on the number of trials taken per session ( F(1,16) = 19.3, p < 0.01), with no effect of strain ( F(3,16) = 1.9, p = 0.18) and no strain  orifice interaction ( F(3,16) = 0.3, p = 0.82). We did not attempt to measure total intake because we were concerned about the accuracy of doing so, given the number of drinking tubes and the small volumes involved. However, assuming that the average lick volume for each orifice size calculated in the stationary spout condition is relatively constant, we used these two values to estimate intake. Consistent with the results when only a single stationary spout was available, the mice ingested similar amounts of water on the brief access test regardless of which orifice size was presented (LO = 1.073 mL; SO = 1.038 mL collapsed across strain). A strain  orifice size two-way ANOVA indicated a significant main effect of strain ( F(3,16) =11.7, p < 0.01), but orifice size and the interaction were not significant. 3.3. Brief access procedure with orifice size varied When tested using the brief access procedure with both SO and LO tubes available within the same session, there Brief Access with Orifice Size Uniform

10 0 B6

SWR

129

D2

Fig. 7. Average number of trials (Tse) taken in a session when mice were tested with only one spout orifice size within a session brief access test session for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

was a significant main effect of orifice size on the number of licks taken per trial ( F(1,16) = 36.5, p < 0.01) and a significant effect of strain ( F(3,16) = 52.3, p < 0.01), but the interaction was not significant ( F(3,16) = 0.7, p = 0.56). Surprisingly, mice licked more from LO drinking spouts, than from SO spouts (Fig. 6). When we estimated volume consumed from each orifice size, a two-way ANOVA indicated significant effects of strain ( F(3,16) = 4.0, p < 0.05) and orifice size ( F(3,16) = 40.3, p < 0.01), but no interaction. The difference in intake between the two orifice sizes (LO : 0.709 mL; SO : 0.199 mL) was not unexpected given the difference in licks and lick volume. We also compared the estimated total intake when the orifice size was uniform and when the orifice size was varied in the brief access test. The intake from the SO tube in the uniform condition did not significantly differ from the total intake in the varied condition (0.908 mL; F(1,16) = 1.0, p = 0.34). The intake from the LO tube in the uniform condition did significantly differ from the total intake in the varied condition ( F(1,16) = 5.5, p < 0.05), but the volumes were nonetheless quite similar. Overall, it Brief Access with Orifice Size Varied

60 Small Orifice Large Orifice

Mean Licks per Trial

50 40 30 20 10 0 B6

SWR

129

D2

B6

SWR

129

D2

Fig. 6. Left panel: Average number of licks (Tse) taken per trial when mice were tested with only one spout orifice size within a brief access test session. Right panel: Average number of licks (Tse) taken per trial when mice were tested with both spout orifice sizes within a brief access test session for each spout orifice size and each strain. B6 = C57BL/6J; SWR = SWR/J; 129 = 129P3/J; D2 = DBA/2J.

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appears that total intake during the brief access test, while a bit lower than that observed under the stationary spout condition, was regulated.

4. Discussion Consistent with the data on rat licking behavior, in the stationary spout condition, mice, as a group, took about twice as many licks from the SO drinking spout than from the LO drinking spout, but their total intake volume was approximately the same. Thus, the mice appear to have regulated their fluid intake by proportionally adjusting their licking as a function of lick volume. We measured a number of variables that have been shown to affect intake to see how the mice actually adjusted their behavior. Although there was a significant effect of orifice size on the ILI in the 129 strain, the magnitude of the difference was slight, suggesting that, in mice, ILI is relatively immune to changes in lick volume, at least within the range examined here. In contrast to its lack of effect on ILI, manipulation of the drinking spout orifice size had striking effects on burst licks. Collapsed across strain, average burst licks from SO drinking spouts were approximately two times that of LO drinking spouts. Because orifice size had no significant effect on the number of bursts initiated and because lick volume from SO drinking spouts was approximately half that from LO drinking spouts, intake regulation occurred by increasing the size of drinking bursts, not by increasing their frequency. Although the effects of orifice size on licking behavior were relatively consistent, given the low statistical power resulting from the relatively small sample size, some group differences may have been obscured. Be that as it may, as can be seen in Fig. 6, the SWR mice more than doubled the number of licks taken in a burst in the SO condition when compared to the number of licks taken in the LO condition, while actually decreasing the numbers of bursts within a session by approximately 25%. B6 mice, on the other hand, increased the number of bursts in a session by 48% when drinking from the SO spouts relative to the LO spouts, while only averaging a 22.5% increase in burst licks, an increase far lower than the 120% value displayed by the SWR strain. The way that mice adjust to alterations in lick volume may depend on their natural pattern of licking in short-term tests. B6 mice tended to drink in many small bursts, whereas SWR mice tended to initiate less bursts but with many licks and larger volume. The 129 and D2 mice had patterns somewhat in between these two extremes. These findings are consistent with those found by Eylam and Spector [38] who assessed the licking patterns of B6, 129, and SWR mice to various concentrations of sucrose under water-restricted and non-restricted conditions. These researchers found that B6 mice, when licking to water in the water-restricted condition, took more burst in a session compared with SWR mice, which took more licks within a burst. The licking pattern of

129 mice appeared somewhat similar to the SWR strain. The basis for these strain differences in licking patterns remains to be clarified, but to the extent that burst number is more of a reflection of appetitive processes (i.e., behaviors that involve orientation and approach to food) and that burst size is more of a reflection of consummatory processes (i.e., licking, chewing, etc.—see [39] for a review of appetitive versus consummatory responses), this phenotypic variation could potentially be exploited to learn more about the factors influencing the organization of ingestive behavior. Despite these interpretive issues, it is clear that mice, like rats [27,28], can also display behavioral flexibility in the maintenance of total intake when the volume received per lick is manipulated by altering the spout orifice size. Moreover, if we assume that the average lick volume measured in the stationary spout condition for each orifice size was relatively constant across experiments, then the estimated volumes of water ingested suggest that total intake was also regulated during the brief access test sessions. When the orifice size was not varied during a brief access test session, the smaller orifice size increased the range of responsiveness that was expressed. When the SO drinking tubes were available, the mice increased their average licks per trial by 20% relative to that seen when only the LO drinking spouts were available, although the actual change in licking varied as a function of strain. More impressively, however, was that, as a whole, mice took 60% more trials with the SO compared with the LO drinking spouts. Thus, use of SO drinking spouts in brief access tests increases the amount of behavior generated. When the orifice size was quasi-randomly varied within a brief access session, licking from LO drinking spouts was greater than from SO drinking spouts. These findings suggest that water delivered from the two orifice sizes differs in its reinforcement efficacy. This is presumably the result of the increased volume of fluid received per lick. This interpretation is consistent with findings by Snyder and Hulse [40], who demonstrated that rats will progressively increase their rate of licking as a function of the volume of fluid received per lick up to 0.005 cm3. At lick volumes above 0.005 cm3, a decrease in average lick rate was observed. This decrease was purportedly mediated by more frequent pausing between bursts of licks for these larger volumes. Our data show that testing mice using the brief access procedure without controlling for orifice size could introduce random variation in behavioral responses as a result of uncontrolled differences in the reinforcement efficacy of the test stimuli. It should be mentioned here that although we believe that the behavioral effects seen in this experiment when manipulating orifice size are mediated by the volume of fluid received per lick, it is possible that other factors, also presumably influenced by orifice size, are mediating or contributing to the observed responses. For example, surface area of tongue in contact with droplet or adverse somatosensory contact with the orifice rim, etc.

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Collectively, these findings extend the body of research on the environmental effects on licking behavior in rodents (e.g., [2,22 –25,27,28,40]). Moreover, based on the results from the brief access test procedures presented here, experimenters should take care to use a relatively uniform spout orifice size when applying this assay because variations in this parameter can have striking effects on the behavior observed.

Acknowledgments We would like to thank Ross Henderson for his technical advice and also for providing the drinking spout sipper tubes used in this experiment. Supported by NIDCD grant R01-DC04574.

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