Salt intake by normotensive and spontaneously hypertensive rats: two-bottle and lick rate analyses

Physiology & Behavior 78 (2003) 689 – 696

Salt intake by normotensive and spontaneously hypertensive rats: two-bottle and lick rate analyses Francis W. Flynna,b,*, Bruce Culverb,c, Stephen V. Newtona,b a

Department of Zoology and Physiology, University of Wyoming, Box 3166, University Station, Laramie, WY 82071, USA b Graduate Neuroscience Program, University of Wyoming, Laramie, WY 82071, USA c School of Pharmacy, University of Wyoming, Laramie, WY 82071, USA Received 16 September 2002; received in revised form 6 January 2003; accepted 7 February 2003

Abstract Spontaneously hypertensive rats (SHR) overconsume NaCl compared to the normotensive Wistar Kyoto rat (WKY) rat. In the present experiment, two-bottle preference for NaCl (0.01, 0.03, 0.1, 0.3, 0.5, 1.0, 3.0 M) and lick rate analyses were used to identify the possible mechanisms that underlie the intake of NaCl by male SHR. Two-bottle preference and absolute NaCl intake by SHR were greater than that of WKY rats. When NaCl intake was calculated on the basis of body weight, SHR consumed more NaCl per 100 g body weight than did WKY. Also, during the one-bottle test, SHR consumed more 0.1 and 0.3 M NaCl per 100 g body weight than did WKY. The increased intake of NaCl by SHR was most evident for 0.3 M NaCl. Intake is determined by the initial rate of licking and the decline in lick rate over time. Nonlinear regression analysis of lick rate showed that the initial lick rates (licks/min) were similar for male WKY and SHR. Lick rate declined more rapidly when WKY rats drank 0.3 M than when they drank 0.1 M NaCl, a result consistent with the role of negative feedback in controlling the decay in lick rate. This concentration-dependent change in lick rate was not seen in SHR. Also, SHR lick rate for 0.1 and 0.3 M NaCl decelerated more slowly than that of WKY rats. The increased intake of hypertonic NaCl by SHR was due to a decrease in the decline in lick rate, suggesting that SHR are less responsive to ingestion – contingent negative feedback. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Sodium; Taste; Visceral feedback; Preference

1. Introduction Salt intake is linked to a number of human health challenges, including hypertension. In the 1960s, an animal model, the spontaneously hypertensive rat (SHR), was bred and introduced [32] as a model of human essential hypertension. Compared to the normotensive Wistar Kyoto rat (WKY), the SHR has a higher blood pressure (BP) and exhibits a greater preference for sodium and potassium salts [13,28]. The exaggerated preference for NaCl in the SHR adult is present even if it had been cross-fostered to normotensive rats immediately after birth [10]. The mechanism for the elevated salt intake is not known but several possibilities exist. First, alterations in brain neurochemical systems, including angiotensin II, atrial * Corresponding author. Department of Zoology and Physiology, University of Wyoming, Box 3166, University Station, Laramie, WY 82071, USA. Tel.: +1-307-766-6446; fax: +1-307-766-5625. E-mail address: [email protected] (F.W. Flynn).

natriuretic peptide, vasopressin, and tachykinin peptides may play a role in the expression of the SHR phenotype [4,24,33 – 35]. Each of these peptide systems is implicated in the control of salt intake and electrolyte balance [2,14 – 16,26], and blockade of brain angiotensin production reduces salt intake in SHR [11]. Second, altered arterial chemoreceptor activity in SHR appears to contribute to the enhanced salt intake by these rats [3]. In addition, the SHR genotype may affect the sensory controls of salt intake. For normal rats, salt intake is influenced by the oral sensory properties of the taste and visceral feedback signals. Moreover, behavioral differences in salt intake are often accompanied by altered peripheral taste nerve sensitivity and alterations in the responsiveness of higher-order gustatory neurons [5]. Indeed, one group reported an alteration in Na + ion transport across lingual epithelia in SHR compared to WKY rats [29]. The difference in sodium ion transport may be reflected in a decreased neural response of the SHR to NaCl, and this may contribute to the elevated ingestion of NaCl. Formaker and Hill [18]

0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00062-3

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compared peripheral taste nerve responses of SHR and WKY rats. They found that when the chorda tympani nerve response was expressed relative to a control (0.5 M NH4Cl), there were no strain differences in response magnitudes to NaCl, with the exception of 0.05 M. They go on to conclude that the chorda tympani responsiveness and behavioral preference for NaCl may not be directly related in these strains of rats. The chorda tympani is only one of the peripheral nerves carrying taste information to the brain. The lingual branch of the glossopharyngeal nerve innervates taste receptors in the posterior portion of the tongue and NaCl elicits neural responses in this nerve [19]. However, as in the case of chorda tympani nerve responses, there are no apparent differences in SHR and WKY glossopharyngeal responses to NaCl [17]. Taken together, these studies suggest that peripheral gustatory function is not sufficiently different to account for the increased NaCl preference by SHR. While peripheral nerve responsiveness appears similar in SHR and WKY, it is not known if the processing of gustatory information at higher levels is similar in the two strains of rats. In addition to oral factors determining intake, several lines of evidence show that visceral feedback inhibits NaCl intake [25,38]. When visceral feedback is reduced or minimized, as in the sham drinking condition, sodium-deficient rats drink considerably more NaCl than when drinking normally [16,37]. The SHR genotype could cause the rat to be less sensitive to NaCl-contingent feedback and thereby facilitate NaCl intake. Behavioral tests, such as lick rate analysis [6 –9,16], have been developed whose parameters (initial lick rate, rate of decline in lick rate) are influenced by the oral stimulating property of the taste and the magnitude of the negative ingestion-contingent feedback. We used lick rate analysis and curve fitting procedures to evaluate the alternative mechanisms that result in the SHR phenotypic elevated preference for NaCl.

2. Methods 2.1. Subjects All rats were born and raised in the University of Wyoming vivarium. The original SHR and WKY breeding

stocks were purchased from Charles River Laboratory. Male SHR (n = 7) and WKY (n = 8) rats were housed in individual, suspended wire mesh cages in a temperature- and lighting-controlled room with lights on at 0800 and off at 2000 h. BP was determined using tail cuff plethysmography at the conclusion of the experiment. Mean BP of male SHR (181 ± 5 mmHg) was higher than that of the WKY rats (134 ± 6 mmHg, P < .001). SHR and WKY rats were matched for age and age-matched WKY rats weighed significantly more than did the SHR, P < .01 (Table 1) (see also Refs. [20,35]). 2.2. Two-bottle preference tests Forty-eight hour, two-bottle preference tests were conducted between distilled, deionized water and six concentrations of NaCl (0.01, 0.03, 0.1, 0.3, 1.0, 3.0 M). During the tests, rats had ad libitum access to Purina chow. The solutions were presented in glass, calibrated bottles that were attached to the fronts of the cages. NaCl was presented in an ascending order of concentrations. The starting position of the water and NaCl bottles was randomized and their positions were reversed after 24 h. After 48 h, intake of water and NaCl were recorded (nearest 1 ml). Preference ratios were computed as total NaCl intake/total NaCl + water intake  100. After each 48-h preference test, rats had access to food and tap water for 24 h, after which rats were given access to the next concentration of NaCl for 48 h. 2.3. Lick rate analyses A near-isotonic (0.1 M) and hypertonic (0.3 M) concentration of NaCl were selected for lick rate analysis using a drinking monitor (Columbus Instruments, Columbus, OH). Rats were first familiarized with the test procedure. Rats (n = 5 WKY; n = 4 SHR) were water-deprived overnight and the following morning given access to a single bottle with 0.1 M NaCl for 30 min. Food was not present during the 30min NaCl test. All of the glass bottles (100-ml graduated cylinders) were filled with approximately 80 ml of the solution. The same type of drinking tube was attached to each of the bottles and the spouts were all positioned flush with the outer wall of the test chamber. Rats had to extend their tongue approximately 3 mm to reach the tube. The test began with the first lick contact. When the tongue made

Table 1 SHR and WKY rat ingestive responses for 0.1 and 0.3 M NaCl during the 30-min intake test

WKY SHR

Mean body weight (g)

0.1 M NaCl

0.3 M NaCl

ml

ml/100 g body weight

ml/lick

ml

ml/100 g body weight

ml/lick

250 ± 9 212 ± 6 *

20.0 ± 1.3 19.6 ± 1.2

7.0 ± 0.1 9.1 ± 0.6 *

5.1 ± 0.1 4.3 ± 0.2

7.8 ± 0.3 13.8 ± 1.9 * *

2.9 ± 0.1 6.4 ± 0.9 * *

4.5 ± 0.4 3.8 ± 0.4

Data are presented as the mean ± S.E.M. * P < .05 indicates significant differences comparing SHR and WKY rats. ** P < .01 indicates significant differences comparing SHR and WKY rats.

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contact with the spout approximately 100 nA current passed through the rat for a lick to be registered. Lick contacts with the spout were fed through an interface with a computer and collected in 1-min bins for 30 min. Total intake of NaCl (nearest ml) was also measured during the one-bottle test. Rats then had access to chow and water for 24 h before the start of another overnight deprivation, and had access to 0.1 M NaCl the following day. Lick data were collected during the second experience with NaCl. By the second experience, all of the water-deprived rats immediately began drinking when given access to NaCl. Rats then had ad libitum access to water and food. The procedure was then repeated with 0.3 M NaCl as the solution. Intake and the lick rate were then computed [16]. 2.4. Curve fitting

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osmolarity [Precision Systems Osmometer]) were compared in SHR and WKY. Age-matched male SHR (n = 5) and WKY (n = 5) rats were removed from their home cages and placed into individual metabolism cages. Lights in the room came on at 0800 and went off at 2000 h. Rats had access to tap water and Purina chow unless indicated. Urine output was collected into calibrated plastic centrifuge tubes. On Day 1, the calibrated bottles containing tap water were filled and returned to the cages at 1500 h and collecting tubes were attached to the funnels beneath the cages to collect urine. At 0900 the following morning water intake was measured and urine was collected. Food and water remained on the cages until that afternoon. At 1500 h water bottles were removed from the cages and urine-collecting tubes were put into position. The following morning (0900 h), after the overnight water deprivation, urine was collected and the rats were returned to their home cages.

The lick rate (licks/min) during the 30-min test for each group was quantified by fitting the lick functions to the Weibull distribution ( y = a exp[ (bt)c]) by the least squares method using Systat 8.0 [8,9]. Davis has shown that this distribution fits lick rate data during intake tests. The Weibull function provides estimates of the y-intercept, beta (b), which is an estimate of the slope, and a ‘‘shape’’ parameter c that indicates how the lick rate function deviates from an exponential function. A value of c>1 indicates that the initial rate of decline is less rapid than it is for an exponential decay function and a c < 1 indicates that the rate of decline in lick rate is greater than that for an exponential decay function [9]. The differences between lick rate curves of male SHR and WKY drinking 0.1 and 0.3 M NaCl were quantified by fitting the data to the Weibull function and examining the parameters. The curves shown in the lick rate figures (Figs. 2 and 3) are least square fits by the Weibull function to the data. A nonlinear regression analysis using group as an indicator variable was applied to the lick rate data [16,30]. For this regression, the lick rate of two groups were compared. This technique fits the lick data ( y) to a quadratic function in time (t) but the advantage of this technique is that it provides statistical tests that compare the regression coefficients of two groups. The general model is y = a + b0g + b1t + b2t2 + b3s + b4s2, where g = 0.1 (group identifier), s = g * t, s2 = g * t2 (t = time). The coefficient b3 provides an indication of whether the slopes of the two regressions are different, b4 compares the change in the overall curve of the two regressions, and b0 compares the y-intercept at time t0. 2.5. Urinary measures Both SHR and WKY rats were water-deprived during the drinking tests. It was important to determine if the water deprivation had similar effects on urine output and water/ sodium balance in the two strains of rats at the time of the drinking test. Therefore, the effects of water deprivation on urine (volume, K +, Na + [AVL Electrolyte Analyzer] and

Fig. 1. NaCl preference ratios (upper panel) and NaCl intake/100 g body weight during the preference tests (lower panel) of WKY and SHR. Data are based on the amount consumed over 48 h. The main effect of strain showed that overall, SHR preference for NaCl was greater than that of WKY rats.

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3. Results 3.1. Two-bottle intake and preference Intakes (ml) of water and NaCl were expressed per 100 g body weight and preference ratios were computed based on these values. Preference ratios, and water and NaCl intake/ 100 g body weight of WKY and SHR were analyzed using separate ANOVAs. As shown in Fig. 1, preference increased as concentration was increased to 0.1 M NaCl, and then fell off with further increases in concentration for both SHR and WKY rats [ F(6,13) = 114, P < .0001]. The significant main effect of strain revealed that overall, SHR preference ratios were greater than that of WKY rats [ F(1,13) = 8.3, P < .01]. There was no Strain  Taste interaction ( P>.15). NaCl intake was expressed per 100 g body weight and is shown in Fig. 1. NaCl intake showed a concentration-dependent effect [ F(6,13) = 38.6, P < .0001]. The main effect of strain reflected that overall SHR ingested more NaCl per 100 g body weight than did WKY rats [ F(1,13) = 18.1, P < .001]. The ANOVA comparing the water intake/100 g body weight revealed that water intake associated with hypertonic NaCl access was greater than that when isotonic or hypotonic NaCl were available to drink [ F(6,13) = 45.9, P < .0001]. There was no significant main effect of strain or Strain  Taste interaction, which indicates that the water intake during the preference tests by SHR and WKY rats was similar ( P’s>.3; data not shown). 3.2. One-bottle intake and lick rate 3.2.1. 30 min NaCl intake Absolute intake of 0.1 M NaCl was similar for SHR and WKY rats. However, when intake was expressed as a

function of body weight, 0.1 M NaCl intake/100 g body weight was significantly greater in SHR as compared with WKY ( P < .03). SHR consumed more 0.3 M NaCl compared to WKY both in terms of absolute intake and intake/ 100 g body weight ( P’s < .01) (Table 1). WKY rats consumed significantly less 0.3 than 0.1 M NaCl ( P < .001). In contrast, there was no significant difference in the amount of 0.1 and 0.3 M NaCl ingested by SHR. The average lick volume (total amount of NaCl consumed/total licks) was determined and compared using a Strain  Taste ANOVA (Table 1). Although not reaching statistical significance, SHR tended to take a smaller volume per lick than did WKY [ F(1,7) = 4.4, P < .07]. In addition, the Strain  Taste interaction was not significant ( P=.7). The significant main effect of taste [ F(1,7) = 6.9, P < .03], reflected that the lick volume for hypertonic 0.3 M NaCl was significantly less than that for 0.1 M NaCl. 3.2.2. 0.1 M NaCl cumulative licks and lick rate The cumulative number of licks were compared using a Strain  Time repeated-measures ANOVA (Fig. 2). The total number of licks increased significantly during the 30-min test [ F(29,203) = 473.5, P < .0001]. There was a significant Strain  Time interaction [ F(29,203) = 7.9, P < .0001]. Additional post hoc comparisons showed that the cumulative licks increased similarly for SHR and WKY during the first 10 min of the test. After that time, cumulative licks by SHR significantly outpaced that of the WKY rats ( P’s < .05). Despite the higher number of licks, the absolute intake of 0.1 M NaCl was similar in SHR and WKY. This can be accounted for by the slightly smaller volume that was ingested per lick by the SHR.

Fig. 2. Cumulative licks (left panel) and lick rate (right panel) for 0.1 M NaCl during the 30-min intake test. The lines (solid = WKY, dashed = SHR) are least squares fits of the lick rate at 1-min intervals to the Weibull function. Cumulative licks increased significantly during the test. SHR licked significantly more than did the WKY rats and SHR lick rate declined more slowly than WKY, * P’s < .05.

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The nonlinear regression analysis showed that there was no significant difference in the initial lick rate of SHR and WKY rats for 0.1 M NaCl (Fig. 2). As ingestion continued, however, there were significant differences in the slope ( P < .04) and the rate of deceleration in the lick rate ( P < .02). Overall, the initial rate of decline of lick rate of SHR was less rapid than in WKY rats (c = 1.1 ± 0.3, c = 0.6 ± 0.1, respectively). This result suggests that NaCl ingestion-contingent feedback had less of an inhibitory effect on ingestion by SHR than WKY rats. 3.2.3. 0.3 M NaCl cumulative licks and lick rate There were significant strain differences in cumulative licks for hypertonic NaCl [ F(3,15) = 20.5, P < .0001] (Fig. 3). The strain difference in licking appeared by the third minute of the test ( P < .01), and continued for the remainder of the test period ( P’s < .01). Fig. 3 shows the lick rate of WKY and SHR drinking 0.3 M NaCl. There was no significant difference in the initial lick rates of SHR and WKY rats. The rate of decline in lick rate was significantly less in SHR than in WKY rats ( P < .02). Lick rate of SHR declined slower than an exponential decay (c = 1.5 ± 0.1) while WKY rat lick rate for hypertonic NaCl declined faster than an exponential rate (c = 0.8 ± 0.09). 3.2.4. NaCl concentration comparisons Within-group comparisons indicated that the cumulative licks of WKY rats for 0.3 M NaCl was significantly less than that for 0.1 M NaCl [ F(1,4) = 64.8, P < .001]. Licking for 0.3 M was less than that for 0.1 M NaCl by the second minute of the test ( P < .01). In contrast, there was no main effect of taste on licking by SHR ( P>.2). The significant interaction of Taste  Time indicated that SHR licking for

Fig. 4. Mean ( ± S.E.M.) water intake and overnight urinary output on successive nights by SHR and WKY when water was present and when water had been removed from the cages. Asterisk indicates a significant group difference, P < .01.

0.3 M NaCl was less than that for 0.1 M only during the very last 2 min of the test ( P’s < .05). Additional regression analyses were computed that compared the lick rates of each group when drinking 0.1 and 0.3 M NaCl. SHRs were unresponsive to the difference in NaCl concentration because the lick rate of SHR rats drinking 0.1 and 0.3 M NaCl was not significantly different. In contrast, analysis of WKY rats revealed differential responses to the 0.1- and 0.3-M NaCl. In particular, the rate of decline of lick rate (slope) was greater when WKY were drinking 0.3 M (b = 0.2 ± 0.1) compared to when drinking 0.1 M NaCl (b = 0.06 ± 0.1) ( P < .06).

Fig. 3. Cumulative licks (left panel) for 0.3 M NaCl during the 30-min intake test. Cumulative licks increased significantly during the test. SHR licked significantly more than did the WKY rats ( * * P < .0001). The lines (solid = WKY, dashed = SHR) are least squares fits of the lick rate at 1-min intervals (right panel) to the Weibull function. Asterisk indicates that the rate of decay in lick rate was significantly less for SHR than it was for WKY rats, P < .02.

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Fig. 5. SHR and WKY urine osmolarity and electrolyte concentrations (mean ± S.E.M.) when water was present and when rats were water-deprived. Water deprivation affected urine osmolarity, Na + , and K + concentrations equally in SHR and WKY.

3.2.5. Urine measurements WKY rats drank significantly more water overnight than did SHR rats [t(8) = 4.2, P < .01] (Fig. 4). Overall, urine output by the two strains was not significantly different (Fig. 4) and the ratio of urine output to water intake was similar for both WKY (0.21 ± 0.5 ml) and SHR (0.20 ± 0.3 ml). Urine sodium and potassium concentrations and urine osmolarity were not significantly different in SHR and WKY rats (Fig. 5). During the overnight water deprivation, urine output was far less than when rats had water access and the urine volume was similar for both WKY and SHR (  1 ml). Overnight water deprivation led to a similar increase in the urinary sodium and potassium excretion for WKY and SHR. Also, urine osmolarity was similar in waterdeprived WKY and SHR (Fig. 5).

4. Discussion A number of papers report that SHR consume more NaCl than do normotensive WKY rats [10 – 12,20]. Examination of the present preference and intake data shows a consistent pattern of the SHR having greater preference for, and higher intakes of, NaCl solutions compared to WKY rats. The greatest difference between SHR and WKY appeared when the rats were given access to 0.3 M NaCl both during the preference test and the one-bottle test. Interestingly, examination of the preference data from Ferrell and Gray [12] also show that in adult rats, the most striking difference between SHR and WKY occurred when rats had access to 0.3 M NaCl. The amount that an animal ingests is determined by the initial rate of ingestion and the decay in the rate of ingestion. The initial lick rate reflects the animal’s responsiveness to the oral stimulating properties of the taste while the rate of the decay in lick rate reflects the responsiveness to the postingestional stimulation [7,8]. As discussed earlier, the effect of the SHR genotype on salt intake could reflect an

enhanced excitatory influence of salt taste, a decrease in the effectiveness of salt ingestion-contingent negative feedback to reduce intake, or a combination of the two processes. Initial lick rate was similar in SHR and WKY rats drinking NaCl. This pattern indicates that SHR do not respond differently than do WKY rats to the oral sensory properties of NaCl; an interpretation that is consistent with the gustatory electrophysiological results comparing SHR and WKY rats [17,18]. Since the initial lick rate was similar for SHR and WKY rats, factors other than orosensory stimulation must have led to the increased intake of NaCl by SHR. Several lines of evidence show that visceral feedback inhibits NaCl intake by rats [16,37,38]. Also, the greater the negative feedback, the more rapid the lick rate decays. The negative feedback results directly from the accumulation of the ingested substance in the gut and also through learned associations of the taste with postingestional stimulation [8,9,16]. In the present study, lick rate by WKY rats declined more rapidly when drinking 0.3 M than when drinking 0.1 M NaCl, a result in keeping with the greater postingestional stimulation resulting from the ingestion of a hypertonic salt solution. This concentration-dependent change in the decline in lick rate was not seen in SHR. Also, the slope of the decay in lick rate for both 0.1 and 0.3 M NaCl was less in SHR than in WKY rats, and this pattern indicates that the SHR genotype was less responsive to NaCl ingestion-contingent negative feedback. The effect of the slower decline in lick rate on intake was most evident when comparing the total intake of hypertonic NaCl by SHR and WKY. Exactly what ingestion-contingent cue(s) the SHR was less responsive to is not specified by this study. Lick rate for 0.3 M NaCl declined rapidly in the WKY. Within-group comparison showed that the WKY lick rate for 0.1 and 0.3 M NaCl was different within 3 min. Based on changes in lick rate during normal and sham drinking tests, Davis et al. [8,9] concluded that conditioned negative feedback signals operate during the first several minutes of

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ingesting hypertonic solutions (NaCl and sucrose). The conditioned feedback results from the association of the taste and any number of its postingestional effects. After this time, the direct postingestional stimulation from the accumulation of fluid in the gastrointestional tract operates to suppress lick rate [8,9]. In the present experiment, rats were familiarized with NaCl prior to the lick test. This prior experience with NaCl would have allowed for an association of the taste and its postingestional stimulation, and may explain the observed rapid decline in WKY rat lick rate for hypertonic NaCl. Lick volume (3– 6 ml) was similar to previous reports [1,22,23,36,39]. Other reports show that the volume removed by each lick is not constant but is affected by the composition and concentration of the taste solution [1,23]. For example, the lick volume for 0.4% saccharin is about two thirds of that for 0.1% saccharin [1] and lick volume increases with increases in sucrose concentration [21]. Similarly, in the present experiment, the comparison of the lick volumes for the two concentrations of NaCl revealed subtle microbehavioral adjustments. Increasing the NaCl concentration from 0.1 to 0.3 M led to a significant decrease in lick volume by both SHR and WKY rats. The concentration-dependent decrease in lick volume further shows that SHR are responsive to the orosensory properties of NaCl. SHR tended to ingest less 0.1 and 0.3 M NaCl per lick than did the WKY. While not statistically significant, this smaller volume would explain why the absolute intake of 0.1 M NaCl was not different for the two groups in spite of the SHR having a higher number of cumulative licks and lick rate decaying more slowly than in WKY rats. The SHR genotype affected ingestive behaviors (lick rate and lick volume) in ways that were not revealed by the measurement of absolute intake (ml). In regards to 0.3 M, the cumulative licks of SHR were considerably greater than that of the WKY, and this high cumulative frequency offsets the slightly smaller lick volume to result in a greater total intake of hypertonic NaCl. One possible explanation for the smaller lick volume relates to the overall behavior of SHR. SHR are characterized as having higher general levels of activity and eat more rapidly than do WKY rats [27,31]. One speculation is that the enhanced behavioral reactivity of the SHR affected the duration that the tongue made contact with the spout, and the amount of fluid ingested per lick. SHR and WKY rats were water-deprived prior to the lick rate tests. In order to determine whether SHR and WKY were in a similar water/sodium balance following the deprivation and at the time of testing, urine output was collected and analyzed. Measurement of water intake and urinary output suggest that SHR and WKY rats were equivalent in water/sodium balance at the time of testing. Urine analyses indicated that SHR and WKY responded similarly to water deprivation by reducing and concentrating their urinary output. As such, the differences in lick responses cannot be attributed to a differential effect of

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the water deprivation on the fluid and electrolyte balance in the two strains. In conclusion, these results show that initial rate of ingestion of NaCl is similar for SHR and WKY rats. Lick rate of WKY drinking NaCl declined more rapidly than it did in SHR. There was no difference in the absolute intake of 0.1 M because SHR tended to consume less per lick than did WKY. Lick rate declined more rapidly when WKY were drinking hypertonic NaCl compared to when drinking NaCl, but this concentration-dependent change was absent in SHR. The slow decline in lick rate of SHR drinking hypertonic NaCl resulted in their overconsumption of hypertonic NaCl relative to WKY. The difference in the intake of hypertonic NaCl was not due to differences in the initial rate of ingestion but instead reflected differences in the SHR responsiveness to negative feedback cues controlling lick rate.

Acknowledgements This research was funded by NIH grants DK 50586 and NCRR award P20 RR15640 to F.W.F.

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