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Calcium deprivation increases the palatability of calcium solutions in rats
Physiology & Behavior 84 (2005) 335 – 342
Calcium deprivation increases the palatability of calcium solutions in rats Stuart A. McCaughey*, Catherine A. Forestell, Michael G. Tordoff Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104-3308, United States Received 27 September 2004; received in revised form 5 December 2004; accepted 15 December 2004
Abstract Calcium-deprived rats have elevated intakes of CaCl2, other calcium salts, and some non-calcium compounds. We used taste reactivity to examine the effects of calcium deprivation on the palatability of CaCl2 and other solutions. Nine male Sprague–Dawley rats were calciumdeprived by maintenance on a low-calcium diet, and eight replete rats were used as controls. All rats were videotaped during intraoral infusion of the following solutions: 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, 1 mM quinined HCl, 2.5 mM sodium saccharin, and deionized water. We counted individual orofacial and somatic movements elicited by the infusions and used them to calculate total ingestive and aversive scores. Relative to controls, calcium-deprived rats gave a significantly larger number of tongue protrusions and had higher total ingestive scores for CaCl2, calcium lactate, NaCl, and MgCl2. Our results suggest that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats when they are calcium-deprived than replete, and this may be responsible for the increased intake of these solutions following calcium deprivation. D 2005 Elsevier Inc. All rights reserved. Keywords: Taste reactivity; Palatability; Calcium appetite; Mineral appetite
1. Introduction Calcium-deprived rats attempt to counteract their deficiency by seeking out and consuming substances that contain calcium [31]. They generalize this appetite to some non-calcium compounds, but not others. For example, relative to replete controls, calcium-deprived rats have elevated intakes of NaCl [14,33], but their intakes of citric acid and quinined HCl are unaffected [14,15,33] and their intakes of sweet substances are reduced [14,32]. There is evidence that calcium appetite has an unlearned component and can take place even when postingestive consequences are minimized. Calcium-deprived pups and adult rats increase intake of CaCl2 relative to replete controls in brief-access tests, which preclude postingestive effects [15,20]. Moreover, adult rats ingest unusually large amounts of CaCl2 when sham-drinking, even though they are unable to retain the calcium and thus benefit from it [23].
* Corresponding author. Tel.: +1 215 898 3770; fax: +1 215 898 2084. E-mail address: [email protected] (S.A. McCaughey). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.12.010
These results suggest that an oral factor, such as the taste of CaCl2, is sufficient to drive calcium appetite. Evidence that the taste of CaCl2 is altered by deficiency in rats comes from electrophysiological recordings of gustatory-responsive neurons in the chorda tympani nerve and nucleus of the solitary tract (NST; 18,24). In the NST, calcium deprivation results in larger taste-evoked responses to oral CaCl2, and this difference is limited to the subgroup of neurons that are most sugar-responsive. These neurons may be especially important for determining the hedonic valence of taste stimuli, and when CaCl2 activates them to a greater extent, it may cause an increase in its palatability. However, these results do not provide a direct measure of palatability per se. Insight into the hedonic properties of a substance can be gained by measuring taste reactivity. With this technique, observations are made of the orofacial and somatic reactions that accompany intraoral delivery of a solution; these individual behaviors can be classified as either ingestive or aversive and are associated with palatable or unpalatable stimuli, respectively [5,17]. Furthermore, shifts in taste reactivity have accompanied manipulations that are thought to affect palatability. For instance, when rats are sodium-
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deprived, they exhibit a larger number of ingestive behaviors and fewer aversive ones during intraoral infusion of 0.5 M NaCl [9]. Taste reactivity has also been useful for distinguishing between hedonics (blikingQ) and motivation (bwantingQ ; see [4] for a review). This distinction has been demonstrated in several studies in which changes in voluntary consumption were not accompanied by changes in taste reactivity [16,28,29,34]. The primary goal of the present study was to investigate whether calcium deprivation is accompanied by an increase in the palatability of calcium solutions. We were also interested in the specificity of calcium deprivation’s effects on taste reactivity, and so we used a broad stimulus array that included solutions that are ingested to a greater extent in calcium-deprived rats (CaCl2, calcium lactate, NaCl, and MgCl2), ones that are consumed equally (quinined HCl and H2O), and one that is ingested to a lesser degree following deprivation (saccharin).
2. Methods 2.1. Subjects Subjects were 17 male Sprague–Dawley rats that were purchased from Charles River Laboratories (Stone Ridge, NY). They were 3 weeks old when they entered the laboratory, and they were housed individually in hanging stainless steel cages (19.517.524.5 cm) in a room maintained at 23 8C and on a 12:12 h light–dark cycle. All subjects had ad libitum access to powdered food (see below) and deionized water.
underlying tissue. The animal was then placed on its back, its mouth propped open, and a 19-g needle was inserted into the mouth next to the first maxillary molar; attached to the blunt end of the needle was a length of PE-100 tubing with a flared end and a Teflon washer. Half of the rats in each group were cannulated on the left side and half on the right. The needle was pushed through the mouth and out near the top of the skull, and then pulled through the incision created earlier and out between the shoulders until the flared end of the tubing and the washer were flush against the first molar. The needle was replaced with a piece of 19-g metal tubing, which was secured by tying it to a square of polypropylene mesh that was pressed flat to the rat’s back and sutured to the overlying skin. Approximately 1 cm of the metal tubing was left sticking out between the shoulders after the incision was sutured, in order to attach the PE tubing to give infusions. For three rats in the replete group, a similar technique was used for cannulation except that both sides of the mouth were cannulated and the tubing exited the skin near the top of the head and was secured to the skull using an implant made of dental acrylic. Only one of the cannulae was used for infusions. We began the experiment with this method, but discontinued it because of concerns that we would have difficulty securing implants to the thin skulls of calciumdeprived rats. Cannulation procedure had no effect on total ingestive or aversive scores within the Replete group. Following the surgery, rats were placed under a heat lamp to recover for 1–2 h. They were given injections of the analgesic butorphanol (0.5 mg/kg at 0 and 12 h after surgery, with additional doses as needed, sc) and the antibiotic gentamicin sulfate (2.5 mg at 0, 24, and 72 h after surgery, im).
2.2. Diets 2.4. Testing chamber The diets for both groups were based on a calcium-free version of the AIN-76A diet [1], to which enough calcium carbonate was added to generate the appropriate calcium content. Eight rats (Replete group) received a diet with 150 mmol Ca++/kg diet (Dyets, Bethlehem, PA, cat. no. 113060), which exceeds the 40–68 mmol Ca++/kg required for maximal growth [2,12]. Nine rats (Ca-deprived group) were given a diet with 25 mmol Ca++/kg diet (Dyets, cat. no. 113059). Prior work has shown that 3–4 weeks maintenance on this diet effectively induces a calcium appetite and reduces plasma calcium concentrations without greatly compromising growth or survival rates [14,15,25]. 2.3. Surgery To implant intraoral cannulae, rats were anesthetized with a mixture of ketamine, xylazine, and acepromazine (100 mg/kg, 2.5 mg/kg, and 0.7 mg/kg, respectively, im). For the majority of the rats, an incision was made in the skin between the shoulder blades, a hemostat was inserted, and the skin leading to the animals’ ear was freed from the
The apparatus used for taste reactivity testing was a clear Plexiglas cylinder that measured 25 cm in diameter and 25 cm in height. A mirror was placed below the clear Plexiglas floor at a 458 angle to allow videotaping of the rat’s ventral side. The cylinder had a lid with holes cut out to allow PE100 tubing to pass freely from the infusion pump to the animal’s cannula. 2.5. Procedure Rats were handled for 7 consecutive days beginning 2–3 weeks after they arrived, after which they underwent surgery to implant an intraoral cannula (see above). They were then left undisturbed for 2 days. On the third day after surgery, the cannula was flushed out by placing the animal in the testing chamber (see above), attaching PE-100 tubing to the cannula, and infusing water through the cannula until the resulting passive drip was clear. On the next 3 days, the rat was acclimated to the infusion procedure by placing it in the chamber for 10 min, two of which were spent receiving
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an infusion of 2 ml of deionzed H2O. On the sixth day after surgery, the rat was also weighed and a 40-Al blood sample was taken from the tail in order to measure plasma ionized Ca++ concentration (Ciba-Corning Ca++/pH analyzer #634). Starting on the seventh day after surgery, subjects received infusions of one of nine different test solutions: 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, 1 mM quinined HCl, 2.5 mM sodium saccharin, and deionized H2O. All compounds were purchased from Sigma Chemical (St. Louis, MO) and were dissolved in distilled water. The stimulus array included both palatable and unpalatable compounds, and ones that are ingested to greater, lesser, and equal extents by calciumdeprived rats relative to replete ones (see Introduction). CaCl2 and NaCl were included at low and high concentrations that are thought to taste moderately and highly intense to rats, although in all cases they are consumed more following calcium deprivation [14,33]. Water was included as a control stimulus to test for generalized effects of calcium deprivation on taste reactivity behaviors, since it does not normally have a strong hedonic component and is consumed equally by replete and calcium-deprived rats in 24-h tests [14]. In short-term tests, rats consume less water when calcium-deprived than when replete [15]. We therefore also included quinined HCl as a second control stimulus that was unlikely to change in palatability following calcium deprivation, since it is consumed equally by replete and calcium-deprived rats in both short-and long-term tests [14,15] and it evokes similar gustatory responses in the rat NST regardless of calcium status [24]. Prior to the infusion, the cannula was flushed with deionized water until the passive drip was clear. Rats were videotaped during the infusions, which were at a constant rate of 1 ml/min and lasted 2 min. All subjects received all nine solutions, given once per day, and the order of delivery was counterbalanced across subjects to control for order effects. Immediately following the final infusion, each rat was weighed and another 40-Al blood sample was taken to measure plasma ionized Ca++. On the next day, each rat was given a 2-bottle preference test with deionized water and 300 mM CaCl2, in order to confirm that the low-calcium diet was effective at inducing a calcium appetite. The intakes of water and CaCl2 were determined at 24 h and expressed relative to body weights, and they were also used to calculate a percent preference for CaCl2, defined as 100%(volume of CaCl2 consumed/volume of total fluid consumed). 2.6. Taste reactivity scoring The videotapes were converted to computer files with the same temporal resolution (30 frames/s) and then were scored frame-by-frame by two experimenters who were blind as to the rat’s group. The following ingestive measures were scored: mouth movements, tongue protrusions, lateral
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tongue protrusions, and paw licks [8,17]. The following behaviors were considered aversive and scored: head shakes, paw flicks, chin rubs, paw treads, gapes, and face washes [17]. The individual behaviors listed above were added to derive total ingestive and aversive scores. Scorers also kept track of the number of frames for which the ventral surface of the rat’s face was off-screen. These frames were subtracted from the total, and the ingestive and aversive scores were expressed as the number of reactions that occurred per minute that the rat was on-screen. The number of drops of solution that fell out of the mouth was counted as passive drip. This measure was considered to be neutral, and it was not expressed relative to on-screen time, because passive drips could still be seen clearly when the rats were off-screen. 2.7. Data analysis The effects of calcium deprivation on plasma ionized Ca++ concentrations and body weights were assessed using a two-way mixed ANOVA, with group as a betweensubjects factor and day as a within-subjects factor. Post hoc t-tests were conducted to determine on which days the rats differed. Total ingestive and aversive scores, the number of passive drips, and scores for individual behaviors were each compared using a two-way mixed ANOVA, with group and solution type as factors. If there was a significant main effect of group or groupsolution interaction, then post hoc t-tests were performed to determine which solutions were responsible. The preferences of the groups for CaCl2 in 24-h tests were compared using a t-test. The distributions for CaCl2 and water intakes in 24-h tests were positively skewed in both groups, so these variables were compared between groups using Mann–Whitney U-tests. For all analyses, pb0.05 was considered significant. For some post hoc tests, we found p values between 0.05 and 0.10. We mention all such cases, describe them as btrendsQ or as bapproaching significance,Q and list the exact p value.
3. Results 3.1. Body weights The mean (FS.E.M.) weight of the Ca-deprived group was 261F25 g on the day before taste reactivity testing and 314F24 g 9 days later, after the final test. Rats in the replete group weighed 298F8 and 354F9 g before and after testing, respectively. The body weights of the two groups did not differ but both gained weight significantly across the course of the experiment (effect of day, F[1,13]=417.4, pb0.001). 3.2. Plasma Ca++ The Ca-deprived group had significantly lower plasma Ca++ concentrations than did the replete group both before
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3.3. Two-bottle test During the 24-h test conducted at the end of the experiment, the Ca-deprived group had significantly higher preferences for 300 mM CaCl2 than did the replete group (t[15]=4.7, pb0.001; Table 2). This occurred because animals in the Ca-deprived group consumed significantly more CaCl2 than did ones in the replete group (Mann– Whitney U=9, p=0.009), but the two groups had similar water intakes (Table 2). 3.4. Taste reactivity measures One hundred and thirty-three of the 153 sessions were scored by one experimenter and 39 by the other. Nineteen sessions were scored by both, which allowed for a measure of reliability. The correlations between scorers for total ingestive and aversive scores were +0.99 and +0.95, respectively, indicating a high degree of reliability. Over all tests combined, rats in the Ca-deprived group exhibited significantly more ingestive reactions than did ones in the replete group (effect of group, F[1,15]=23.2, pb0.001), and this effect was larger for some solutions than others (group solution interaction, F [8,120]=9.0, pb0.001). Post hoc tests showed that deprived rats exhibited more ingestive reactivity to 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, MgCl2, and quinined HCl (t[15]z2.7, pb0.02 in all cases; Fig. 1A). The differences for saccharin and water were not significant, but there was a trend ( p=0.06 and 0.07, respectively) in the same direction. The groups did not differ in their total aversive scores (Fig. 1B). There was a tendency toward deprived rats showing more ingestive reactivity in general. In order to address this issue more thoroughly, we compared the groups on each of the ingestive behaviors that had been scored (see Table 3). Table 1 Mean (FS.E.M.) plasma ionized Ca++ concentrations (mmol/l) in the Cadeprived and replete groups before and after infusions for taste reactivity testing Group
Before infusions
After infusions
Ca-deprived Replete
1.06F0.04* 1.36F0.04
1.03F0.06* 1.37F0.02
Samples were taken on the day prior to the start of taste reactivity tests or immediately following the last test. * pb0.05 compared with replete group.
Table 2 Mean (FS.E.M.) intakes of 300 mM CaCl2 and H2O and preferences for CaCl2 during 24-h two-bottle testing in the Ca-deprived and replete groups Group
CaCl2 intake (ml/kg)
H2O intake (ml/kg)
CaCl2 preference (%)
Ca-deprived Replete
78F9* 33F21
122F19 111F29
41F4* 16F3
* pb0.05 compared with replete group.
Rats in the Ca-deprived group showed significantly more mouth movements to all of the stimuli except 100 mM NaCl and saccharin (effect of group, F[1,15]=17.5, pb0.001; group solution interaction, F[8, 120]=2.1, p=0.04; t[15]z2.2, pb0.04 in post hoc tests), and in the former case there was a nonsignificant trend ( p=0.053). Deprived animals also gave more tongue protrusions than did replete ones (effect of group, F[1,15]=13.2, p=0.002; group solution interaction, F[8, 120]=6.1, pb0.001), and post hoc tests showed that this was due to differences for 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and MgCl2 (t[15]z2.2, pb0.05 in all cases). There was a trend ( p=0.08) toward more tongue protrusions to saccharin in the deprived rats, but the differences for quinined HCl and water did not approach significance. The groups did not 350 Mean ingestive reactions per min
and after the taste reactivity tests (effect of group, F[1,15]=29.7, pb0.001; t[15]=5.0, pb0.001 in both post hoc tests; Table 1). There was no difference between the first and second samples (effect of day, n.s.). This confirms that the low-calcium diet induced calcium deficiency effectively and that the calcium-deprived rats did not become replete based on the small amount of calcium that they received from the infusions.
*
A
* CaDep Replete
300 250 200
*
150 100
* *
*
50
*
0 45 Mean aversive reactions per min
338
B
40 35 30 25 20 15 10 5 0 Ca1
Ca2
Lac
Na1
Na2
Mg
Sac
Q
H2O
Fig. 1. Mean (FS.E.M.) total ingestive (A) and aversive (B) scores in the Ca-deprived (filled bars) and replete (open bars) groups. Scores are expressed as the number of reactions per minute that the rats were onscreen. Ca1, 30 mM CaCl2; Ca2, 300 mM CaCl2; Lac, 30 mM calcium lactate; Na1, 100 mM NaCl; Na2, 600 mM NaCl; Mg, 30 mM MgCl2; Sac, 2.5 mM sodium saccharin; Q, 1 mM quinined HCl; H2O, deionized water. *pb0.05 vs. replete group.
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Table 3 The mean (FS.E.M.) number of individual ingestive behaviors (mouth movements, tongue protrusions, lateral tongue protrusions, and paw licks) per minute of on-screen time in the Ca-deprived (CaDep) and replete groups Taste stimulus
30 mM CaCl2 300 mM CaCl2 30 mM CaLa 100 mM NaCl 600 mM NaCl 30 mM MgCl2 2.5 mM saccharin 1 mM QHCl H2O
Mouth movements
Tongue protrusions
Lateral tongue protrusions
Paw licks
CaDep
Replete
CaDep
Replete
CaDep
Replete
CaDep
Replete
66F14* 43F9* 76F15* 113F15 149F22* 48F9* 105F19 31F6* 54F12*
19F6 11F2 21F7 53F25 48F15 20F7 65F16 10F3 20F7
21F8* 3F1* 38F13* 147F28* 139F30* 8F2* 55F21 2F1 28F15
2F1 1F1 4F3 34F16 29F18 2F1 13F5 0F0 7F3
0F0 0F0 1F0 1F0 1F0 0F0 0F0 0F0 0F0
0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0
0F0 0F0 0F0 25F22 14F12 0F0 6F3 0F0 0F0
0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0
* pb0.05 compared with replete group.
differ on the number of paw licks, and although there was a significant main effect for calcium-deprived rats giving more lateral tongue protrusions relative to replete rats ( F[1,15]=4.6, pb0.05), we did not find group differences in these measures for any individual solutions. We also compared the groups on their individual aversive behaviors, in order to see whether there were differences that had been obscured by looking only at total aversive scores. Table 4 shows mean (FS.E.M.) head shakes, paw flicks, gapes, and face washes in the two groups. Data are not shown for chin rubs and paw treads because of the infrequency with which they occurred in both groups (they were observed in only 4.1% and 1.7% of the sessions that were scored, respectively). The groups did not differ on face washes, paw flicks, chin rubs, or paw treads (effect of group and groupsolution interaction, n. s.). Rats in the Cadeprived group gave significantly more head shakes to 30 mM CaCl2 and MgCl2 (groupsolution interaction, F[8, 120]=2.6, p=0.01; t[15]=2.2, pb0.05 in both post hoc tests). Although there was a significant groupsolution interaction for gapes ( F[8, 120]=2.5, p=0.01), none of the post hoc tests were significant. The number of passive drips is shown in Fig. 2. A smaller number was counted for animals in the Ca-deprived group than in the replete group (effect of group, F[1,15]=13.5, p=0.002; group solution interaction,
F[8,120]=2.6, p=0.01), and this was due to less dripping of 30 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and quinined HCl (t[15]z2.8, pb0.02 in post hoc tests). There was also a trend ( p=0.08) toward less dripping of 300 mM CaCl2 in the Ca-deprived group.
4. Discussion Calcium-deprived rats exhibited more total ingestive reactivity than did replete rats when they received infusions of 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, and quinined HCl, but total aversive scores did not differ between the groups. By themselves, these results suggest that CaCl2, calcium lactate, NaCl, MgCl2, and quinined HCl taste more palatable to calcium-deprived than replete rats. However, matters are complicated by the fact that the Ca-deprived group also showed a non-significant trend (0.05bpb0.10) toward more ingestive reactivity to saccharin and water. The lack of statistical significance for saccharin and water indicates that any difference between the groups in palatability of these solutions was marginal if it existed at all. Nevertheless, one explanation is that calcium deprivation caused both a diffuse, generalized increase in ingestive reactivity and a separate, stimulus-
Table 4 The mean (FS.E.M.) number of individual aversive behaviors (head shakes, paw flicks, gapes, and face washes) per minute of on-screen time in the Cadeprived (CaDep) and replete groups Taste stimulus
Head shakes CaDep
Replete
CaDep
Replete
CaDep
Replete
CaDep
Replete
30 mM CaCl2 300 mM CaCl2 30 mM CaLa 100 mM NaCl 600 mM NaCl 30 mM MgCl2 2.5 mM saccharin 1 mM QHCl H2O
3F1* 2F1 3F1 1F0 1F0 3F1* 3F1 2F1 4F1
2F1 1F1 2F0 2F0 2F1 1F1 2F1 1F0 2F1
7F3 4F1 4F1 3F2 3F1 7F2 6F3 4F1 6F2
3F2 0F0 2F1 4F2 2F1 3F2 2F1 1F1 3F2
2F0 10F2 0F0 0F0 1F0 3F1 0F0 8F2 0F0
3F1 6F1 1F1 0F0 2F1 3F1 1F1 5F1 1F1
21F6 16F4 9F4 5F2 4F2 18F6 10F5 20F8 22F8
12F8 5F1 4F1 18F7 7F4 13F8 9F3 7F1 8F6
* pb0.05 compared with replete group.
Paw flicks
Gapes
Face washes
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specific increase in ingestive responses that was related to an increase in palatability. We looked at individual ingestive behaviors to clarify this issue, and we found evidence for both generalized and specific effects.
effect on the ability of the Ca-deprived group to perform mouth movements normally, and thus they tended to perform more of them regardless of which solution they received. Given this possibility, we decided not to draw conclusions about the palatability of stimuli based on our mouth movement data. This decision is reinforced by the fact that mouth movements are performed frequently for solutions that are not consumed avidly [7,11] and some researchers exclude them from the total ingestive score [5,21]. A generalized effect of deprivation on mouth movements may have arisen due to hypocalcemia. Calcium plays an important role in muscle contraction, and severe calcium deprivation is known to cause tremors (i.e., tetany). However, any effect of deprivation on muscle contraction must have been limited. We did not observe any tremor in the deprived rats’ normal body movements, and the calcium deprivation was not severe enough to result in group differences in body weights. Furthermore, any generalized effects must have been restricted to mouth movements and not the other individual behaviors that we scored, based on which solutions were found to differ between the groups.
4.1. Generalized effects
4.2. Effects of calcium deprivation on palatability
Although taste reactivity reflects the palatability of substances, it is also a set of motor responses which can be influenced by manipulations that affect muscle contraction or brain areas involved in movement. In prior studies, two methods have been used to preclude effects on motor systems as an explanation for changes in reactivity. One has been to verify that ingestive and aversive scores did not change in the same direction [5], and the other has been to test multiple taste stimuli and show differential effects across them [9]. Our results met the first criterion, since we observed significant differences between the replete and Ca-deprived groups in total ingestive, but not aversive, scores. Our support for the second criterion was less clear. We had included water and quinined HCl as control stimuli (see Methods), with the expectation that reactivity would be the same in the replete and Ca-deprived groups when they received these solutions. This was the case for nearly all of the individual behaviors that we measured. The only exception was mouth movements, which were significantly higher in calcium-deprived than replete rats for seven of the nine solutions (30 and 300 mM CaCl2, calcium lactate, 600 mM NaCl, MgCl2, quinined HCl, and water) and there was also a trend in the same direction ( p=0.053) for 100 mM NaCl. One explanation is that calcium deprivation causes an increase in palatability for all of these solutions; for CaCl2, calcium lactate, NaCl, and MgCl2, an increase in voluntary consumption accompanies this hedonic shift, but for water and quinined HCl it does not. However, a simpler explanation is that deprivation had an
The replete and Ca-deprived groups did not differ on the number of tongue protrusions, lateral tongue protrusions, or paw licks when they received our control stimuli, quinined HCl and water. We therefore conclude that calcium deprivation did not have a generalized effect on the ability of rats to perform these behaviors and that they provide a good reflection of the palatability of the stimuli that were infused. The two groups did not differ on the number of lateral tongue protrusions or paw licks for any of the solutions. However, calcium-deprived rats showed more tongue protrusions than did replete rats during infusion of 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and MgCl2. This difference provides evidence that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats following calcium deprivation, and it explains why these compounds are consumed to a greater extent by calcium-deprived rats relative to replete ones [14,15]. An increase in the palatability of calcium-containing solutions is clearly beneficial for calcium-deprived rats. It should cause them to start replenishing their calcium levels as soon as they sample an appropriate solution, without having to depend on postingestive consequences. In fact, this outcome has been found in prior work; deprived rats show avid licking of CaCl2 within the first minute of access [15]. They also consume more CaCl2 than do replete rats when sham-drinking, even though they are not able to absorb it and derive any benefit [23]. The increases in the palatability of NaCl and MgCl2 that we observed, in contrast, are not helpful and are difficult to
35
CaDep Replete
Mean drips per 2 min
30 25 20 15
* *
*
10 5
*
*
0 Ca1
Ca2
Lac
Na1
Na2
Mg
Sac
Q
H 2O
Fig. 2. Mean (FS.E.M.) number of passive drips in the Ca-deprived (filled bars) and replete (open bars) groups. Abbreviations as in Fig. 1. *pb0.05 vs. replete group.
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explain as adaptive. Moreover, ingesting large amounts of NaCl causes a further decrease in calcium status in deprived rats, because sodium and calcium excretion are linked [10,27]. In the case of MgCl2, it is possible that rats confuse it with CaCl2 because they taste similar. The two compounds evoke similar across-neuron patterns of responding, which are thought to be reflective of taste quality perceptions [24,30]. However, there is also evidence that CaCl2 and MgCl2 are distinguishable to rats [19,26], and this explanation does not work for NaCl, since there are many indications that CaCl2 and NaCl are clearly discriminable to rats [13,19,22]. We did not find any significant differences between the groups for reactivity to saccharin, and so there was no evidence that a decrease in palatability is responsible for the decrease in saccharin preference that is associated with calcium deprivation [32]. 4.3. Aversive scores Our effects were limited primarily to ingestive scores. The groups did not differ on their total aversive scores, and when we looked at the individual aversive behaviors, we found group differences only for head shakes to 30 mM CaCl2 and MgCl2. We consider these differences minor, since they were not observed for other aversive behaviors or when total aversive scores were considered. Although one might expect aversive responses to calcium solutions to be higher in the replete group relative to the Ca-deprived group, there is some precedence for negative and positive aspects of a flavor’s palatability being separate rather than integrated [6]. This was observed in a previous study in which 48 hours food deprivation increased ingestive taste reactivity responses, while aversive taste reactivity responses remained unchanged [3].
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4.5. Comparison with prior work The hedonics of CaCl2 have been examined previously in rats that were pregnant or lactating [21], and these conditions have been reported to generate a calcium appetite in some experiments [31]. The prior taste reactivity results did not support the hypothesis that calcium tastes more palatable to rats when in these states [21]. Although these results might appear to contradict ours, intake tests conducted in the earlier experiment showed that pregnant rats did not have a calcium appetite, and lactating rats had one that was significant but modest in size. Thus, our results may differ from theirs for a number of reasons, which include the size of the appetites that were generated, as well as the method of induction. 4.6. Summary Measurements of taste reactivity provided evidence that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats when they are calcium-deprived. Rats in the Ca-deprived group had significantly larger total ingestive scores and gave more tongue protrusions relative to rats in the replete group when they received these compounds. We also found that calcium-deprived rats had larger total ingestive scores for quinined HCl than did replete rats. However, this effect arose solely from a difference in the number of mouth movements, which showed evidence of a generalized effect, and so it may not have reflected a palatability shift. Overall, our results suggest that increases in palatability contribute to the increases in consumption that have been reported previously in calcium-deprived rats.
Acknowledgements 4.4. Passive drip Although palatability and ingestion are closely related, the two measures can be affected independently [16,29,34]. All infusions that we gave were 2 ml in volume, and so we can infer whether there were differences in the amount of solution ingested from the amount of passive drip. We observed significantly less passive drip to infusions of 30 mM CaCl2, calcium lactate, and 100 and 600 mM NaCl in the Ca-deprived group. Thus, calcium-deprived rats ingest more of these solutions when they are delivered by an intraoral cannula, and not just when consumed voluntarily. The groups did not differ on passive drip of MgCl2 and 300 mM CaCl2, even though deprived rats ingest more of these solutions in two-bottle tests, and they did differ on passive drip of quinined HCl, even though it is consumed equally by replete and deprived rats [14,15]. These discrepancies may be due to differences in the motivation of rats to consume these solutions based on whether they initiate the solution delivery themselves.
The authors thank Marena La Fontaine, Kazaure, Samantha Doman, and Diane Pilchak excellent technical assistance. Dr. Harvey Grill valuable help with the surgical technique for cannulation.
Hadeeza for their provided intraoral
References [1] American Institute of Nutrition. Second report of the ad hoc committee on standards for nutritional studies. J Nutr 1980;110:1726. [2] Bernhardt FW, Savini S, Tomarelli RM. Calcium and phosphorus requirements for maximum growth and mineralization of the rat. J Nutr 1969;98:443 – 8. [3] Berridge KC. Modulation of taste affect by hunger, caloric satiety, and sensory specific satiety in the rat. Appetite 1991;16:103 – 20. [4] Berridge KC. Food reward: brain substrates of wanting and liking. Neurosci Biobehav Rev 1996;20:1 – 25. [5] Berridge KC. Measuring hedonic impact in animals and infants: microstructure of affective taste reactivity patterns. Neurosci Biobehav Rev 2000;24:173 – 98.
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[6] Berridge KC, Grill HJ. Isohedonic tastes support a two-dimensional hypothesis of palatability. Appetite 1984;5:221 – 31. [7] Berridge KC, Fentress JC. Trigeminal–taste interaction in palatability processing. Science 1985;228:747 – 50. [8] Berridge KC, Grill HJ, Norgren R. Relation of consummatory responses and preabsorptive insulin release to palatability and learned aversions. J Comp Physiol Psychol 1981;95:363 – 82. [9] Berridge KC, Flynn FW, Schulkin J, Grill HJ. Sodium depletion enhances salt palatability in rats. Behav Neurosci 1984;98:652 – 60. [10] Breslau NA, McGuire JL, Zerwekh JE, Pak CY. The role of dietary sodium on renal excretion and intestinal absorption of calcium and vitamin D metabolism. J Clin Endocrinol Metab 1982;55:369 – 73. [11] Breslin PA, Spector AC, Grill HJ. A quantitative comparison of taste reactivity behaviors to sucrose before and after lithium chloride pairings: a unidimensional account of palatability. Behav Neurosci 1992;106:820 – 36. [12] Chandler PT, Cragle RG. Investigation of calcium, phosphorus and vitamin D3 relationships in rats by multiple regression techniques. J Nutr 1962;78:28 – 36. [13] Coldwell SE, Tordoff MG. Latent learning about calcium and sodium. Am J Physiol 1993;265:R1480–4. [14] Coldwell SE, Tordoff MG. Acceptance of minerals and other compounds by calcium-deprived rats: 24-h tests. Am J Physiol 1996;271:R1–10. [15] Coldwell SE, Tordoff MG. Immediate acceptance of minerals and HCl by calcium-deprived rats: brief exposure tests. Am J Physiol 1996;271:R11–7. [16] Forestell CA, LoLordo VM. Palatability shifts in taste and flavour preference conditioning. Q J Exp Psychol, B 2003;56:140 – 60. [17] Grill HJ, Norgren R. The taste reactivity test: I. Mimetic responses to gustatory stimuli in neurologically normal rats. Brain Res 1978;143: 263 – 79. [18] Inoue M, Tordoff MG. Calcium deficiency alters chorda tympani nerve responses to oral calcium chloride. Physiol Behav 1997;62: 297 – 303. [19] Leshem M, del Canho S, Schulkin J. Calcium hunger in the parathyroidectomized rat is specific. Physiol Behav 1999;67:555 – 9.
[20] Leshem M, del Canho S, Schulkin J. Ontogeny of calcium preference in the parathyroidectomized rat. Dev Psychobiol 1999;34:293 – 301. [21] Leshem M, Levin T, Schulkin J. Intake and hedonics of calcium and sodium during pregnancy and lactation in the rat. Physiol Behav 2002;75:313 – 22. [22] Lewis M. Discrimination between drives for sodium chloride and calcium. J Comp Physiol Psychol 1968;65:208 – 12. [23] McCaughey SA, Tordoff MG. Calcium-deprived rats sham-drink CaCl2 and NaCl. Appetite 2000;34:305 – 11. [24] McCaughey SA, Tordoff MG. Calcium deprivation alters gustatoryevoked activity in the rat nucleus of the solitary tract. Am J Physiol 2001;281:R971–8. [25] McCaughey SA, Fitts DA, Tordoff MG. Lesions of the subfornical organ decrease the calcium appetite of calcium-deprived rats. Physiol Behav 2003;79:605 – 12. [26] Morrison GR. Behavioural response patterns to salt stimuli in the rat. Can J Psychol 1967;21:141 – 52. [27] Nakamura TS, Ichikawa T, Sakamaki T, Sako K, Fujie M, Kurashina T, et al. Effect of saline infusion on urinary calcium excretion in essential hypertension. Am J Hypertens 1991;4:113 – 8. [28] Parker LA. Rewarding drugs produce taste avoidance but not taste aversion. Neurosci Biobehav Rev 1995;19:143 – 51. [29] Pelchat ML, Grill HJ, Rozin P, Jacobs J. Quality of acquired responses to tastes by Rattus norvegicus depends on type of associated discomfort. J Comp Psychol 1983;97:140 – 53. [30] Scott TR, Mark GP. The taste system encodes stimulus toxicity. Brain Res 1987;414:197 – 203. [31] Tordoff MG. Calcium: taste, intake, and appetite. Physiol Rev 2001;81:1567 – 97. [32] Tordoff MG, Rabusa SH. Calcium-deprived rats avoid sweet compounds. J Nutr 1998;128:1232 – 8. [33] Tordoff MG, Ulrich PM, Schulkin J. Calcium deprivation increases salt intake. Am J Physiol 1990;259:R411–9. [34] Wyvell CL, Berridge KC. Intra-accumbens amphetamine increases the conditioned incentive salience of sucrose reward: enhancement of reward bwantingQ without enhanced blikingQ or response reinforcement. J Neurosci 2000;20:8122 – 30.
Calcium deprivation increases the palatability of calcium solutions in rats Stuart A. McCaughey*, Catherine A. Forestell, Michael G. Tordoff Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104-3308, United States Received 27 September 2004; received in revised form 5 December 2004; accepted 15 December 2004
Abstract Calcium-deprived rats have elevated intakes of CaCl2, other calcium salts, and some non-calcium compounds. We used taste reactivity to examine the effects of calcium deprivation on the palatability of CaCl2 and other solutions. Nine male Sprague–Dawley rats were calciumdeprived by maintenance on a low-calcium diet, and eight replete rats were used as controls. All rats were videotaped during intraoral infusion of the following solutions: 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, 1 mM quinined HCl, 2.5 mM sodium saccharin, and deionized water. We counted individual orofacial and somatic movements elicited by the infusions and used them to calculate total ingestive and aversive scores. Relative to controls, calcium-deprived rats gave a significantly larger number of tongue protrusions and had higher total ingestive scores for CaCl2, calcium lactate, NaCl, and MgCl2. Our results suggest that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats when they are calcium-deprived than replete, and this may be responsible for the increased intake of these solutions following calcium deprivation. D 2005 Elsevier Inc. All rights reserved. Keywords: Taste reactivity; Palatability; Calcium appetite; Mineral appetite
1. Introduction Calcium-deprived rats attempt to counteract their deficiency by seeking out and consuming substances that contain calcium [31]. They generalize this appetite to some non-calcium compounds, but not others. For example, relative to replete controls, calcium-deprived rats have elevated intakes of NaCl [14,33], but their intakes of citric acid and quinined HCl are unaffected [14,15,33] and their intakes of sweet substances are reduced [14,32]. There is evidence that calcium appetite has an unlearned component and can take place even when postingestive consequences are minimized. Calcium-deprived pups and adult rats increase intake of CaCl2 relative to replete controls in brief-access tests, which preclude postingestive effects [15,20]. Moreover, adult rats ingest unusually large amounts of CaCl2 when sham-drinking, even though they are unable to retain the calcium and thus benefit from it [23].
* Corresponding author. Tel.: +1 215 898 3770; fax: +1 215 898 2084. E-mail address: [email protected] (S.A. McCaughey). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.12.010
These results suggest that an oral factor, such as the taste of CaCl2, is sufficient to drive calcium appetite. Evidence that the taste of CaCl2 is altered by deficiency in rats comes from electrophysiological recordings of gustatory-responsive neurons in the chorda tympani nerve and nucleus of the solitary tract (NST; 18,24). In the NST, calcium deprivation results in larger taste-evoked responses to oral CaCl2, and this difference is limited to the subgroup of neurons that are most sugar-responsive. These neurons may be especially important for determining the hedonic valence of taste stimuli, and when CaCl2 activates them to a greater extent, it may cause an increase in its palatability. However, these results do not provide a direct measure of palatability per se. Insight into the hedonic properties of a substance can be gained by measuring taste reactivity. With this technique, observations are made of the orofacial and somatic reactions that accompany intraoral delivery of a solution; these individual behaviors can be classified as either ingestive or aversive and are associated with palatable or unpalatable stimuli, respectively [5,17]. Furthermore, shifts in taste reactivity have accompanied manipulations that are thought to affect palatability. For instance, when rats are sodium-
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deprived, they exhibit a larger number of ingestive behaviors and fewer aversive ones during intraoral infusion of 0.5 M NaCl [9]. Taste reactivity has also been useful for distinguishing between hedonics (blikingQ) and motivation (bwantingQ ; see [4] for a review). This distinction has been demonstrated in several studies in which changes in voluntary consumption were not accompanied by changes in taste reactivity [16,28,29,34]. The primary goal of the present study was to investigate whether calcium deprivation is accompanied by an increase in the palatability of calcium solutions. We were also interested in the specificity of calcium deprivation’s effects on taste reactivity, and so we used a broad stimulus array that included solutions that are ingested to a greater extent in calcium-deprived rats (CaCl2, calcium lactate, NaCl, and MgCl2), ones that are consumed equally (quinined HCl and H2O), and one that is ingested to a lesser degree following deprivation (saccharin).
2. Methods 2.1. Subjects Subjects were 17 male Sprague–Dawley rats that were purchased from Charles River Laboratories (Stone Ridge, NY). They were 3 weeks old when they entered the laboratory, and they were housed individually in hanging stainless steel cages (19.517.524.5 cm) in a room maintained at 23 8C and on a 12:12 h light–dark cycle. All subjects had ad libitum access to powdered food (see below) and deionized water.
underlying tissue. The animal was then placed on its back, its mouth propped open, and a 19-g needle was inserted into the mouth next to the first maxillary molar; attached to the blunt end of the needle was a length of PE-100 tubing with a flared end and a Teflon washer. Half of the rats in each group were cannulated on the left side and half on the right. The needle was pushed through the mouth and out near the top of the skull, and then pulled through the incision created earlier and out between the shoulders until the flared end of the tubing and the washer were flush against the first molar. The needle was replaced with a piece of 19-g metal tubing, which was secured by tying it to a square of polypropylene mesh that was pressed flat to the rat’s back and sutured to the overlying skin. Approximately 1 cm of the metal tubing was left sticking out between the shoulders after the incision was sutured, in order to attach the PE tubing to give infusions. For three rats in the replete group, a similar technique was used for cannulation except that both sides of the mouth were cannulated and the tubing exited the skin near the top of the head and was secured to the skull using an implant made of dental acrylic. Only one of the cannulae was used for infusions. We began the experiment with this method, but discontinued it because of concerns that we would have difficulty securing implants to the thin skulls of calciumdeprived rats. Cannulation procedure had no effect on total ingestive or aversive scores within the Replete group. Following the surgery, rats were placed under a heat lamp to recover for 1–2 h. They were given injections of the analgesic butorphanol (0.5 mg/kg at 0 and 12 h after surgery, with additional doses as needed, sc) and the antibiotic gentamicin sulfate (2.5 mg at 0, 24, and 72 h after surgery, im).
2.2. Diets 2.4. Testing chamber The diets for both groups were based on a calcium-free version of the AIN-76A diet [1], to which enough calcium carbonate was added to generate the appropriate calcium content. Eight rats (Replete group) received a diet with 150 mmol Ca++/kg diet (Dyets, Bethlehem, PA, cat. no. 113060), which exceeds the 40–68 mmol Ca++/kg required for maximal growth [2,12]. Nine rats (Ca-deprived group) were given a diet with 25 mmol Ca++/kg diet (Dyets, cat. no. 113059). Prior work has shown that 3–4 weeks maintenance on this diet effectively induces a calcium appetite and reduces plasma calcium concentrations without greatly compromising growth or survival rates [14,15,25]. 2.3. Surgery To implant intraoral cannulae, rats were anesthetized with a mixture of ketamine, xylazine, and acepromazine (100 mg/kg, 2.5 mg/kg, and 0.7 mg/kg, respectively, im). For the majority of the rats, an incision was made in the skin between the shoulder blades, a hemostat was inserted, and the skin leading to the animals’ ear was freed from the
The apparatus used for taste reactivity testing was a clear Plexiglas cylinder that measured 25 cm in diameter and 25 cm in height. A mirror was placed below the clear Plexiglas floor at a 458 angle to allow videotaping of the rat’s ventral side. The cylinder had a lid with holes cut out to allow PE100 tubing to pass freely from the infusion pump to the animal’s cannula. 2.5. Procedure Rats were handled for 7 consecutive days beginning 2–3 weeks after they arrived, after which they underwent surgery to implant an intraoral cannula (see above). They were then left undisturbed for 2 days. On the third day after surgery, the cannula was flushed out by placing the animal in the testing chamber (see above), attaching PE-100 tubing to the cannula, and infusing water through the cannula until the resulting passive drip was clear. On the next 3 days, the rat was acclimated to the infusion procedure by placing it in the chamber for 10 min, two of which were spent receiving
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an infusion of 2 ml of deionzed H2O. On the sixth day after surgery, the rat was also weighed and a 40-Al blood sample was taken from the tail in order to measure plasma ionized Ca++ concentration (Ciba-Corning Ca++/pH analyzer #634). Starting on the seventh day after surgery, subjects received infusions of one of nine different test solutions: 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, 1 mM quinined HCl, 2.5 mM sodium saccharin, and deionized H2O. All compounds were purchased from Sigma Chemical (St. Louis, MO) and were dissolved in distilled water. The stimulus array included both palatable and unpalatable compounds, and ones that are ingested to greater, lesser, and equal extents by calciumdeprived rats relative to replete ones (see Introduction). CaCl2 and NaCl were included at low and high concentrations that are thought to taste moderately and highly intense to rats, although in all cases they are consumed more following calcium deprivation [14,33]. Water was included as a control stimulus to test for generalized effects of calcium deprivation on taste reactivity behaviors, since it does not normally have a strong hedonic component and is consumed equally by replete and calcium-deprived rats in 24-h tests [14]. In short-term tests, rats consume less water when calcium-deprived than when replete [15]. We therefore also included quinined HCl as a second control stimulus that was unlikely to change in palatability following calcium deprivation, since it is consumed equally by replete and calcium-deprived rats in both short-and long-term tests [14,15] and it evokes similar gustatory responses in the rat NST regardless of calcium status [24]. Prior to the infusion, the cannula was flushed with deionized water until the passive drip was clear. Rats were videotaped during the infusions, which were at a constant rate of 1 ml/min and lasted 2 min. All subjects received all nine solutions, given once per day, and the order of delivery was counterbalanced across subjects to control for order effects. Immediately following the final infusion, each rat was weighed and another 40-Al blood sample was taken to measure plasma ionized Ca++. On the next day, each rat was given a 2-bottle preference test with deionized water and 300 mM CaCl2, in order to confirm that the low-calcium diet was effective at inducing a calcium appetite. The intakes of water and CaCl2 were determined at 24 h and expressed relative to body weights, and they were also used to calculate a percent preference for CaCl2, defined as 100%(volume of CaCl2 consumed/volume of total fluid consumed). 2.6. Taste reactivity scoring The videotapes were converted to computer files with the same temporal resolution (30 frames/s) and then were scored frame-by-frame by two experimenters who were blind as to the rat’s group. The following ingestive measures were scored: mouth movements, tongue protrusions, lateral
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tongue protrusions, and paw licks [8,17]. The following behaviors were considered aversive and scored: head shakes, paw flicks, chin rubs, paw treads, gapes, and face washes [17]. The individual behaviors listed above were added to derive total ingestive and aversive scores. Scorers also kept track of the number of frames for which the ventral surface of the rat’s face was off-screen. These frames were subtracted from the total, and the ingestive and aversive scores were expressed as the number of reactions that occurred per minute that the rat was on-screen. The number of drops of solution that fell out of the mouth was counted as passive drip. This measure was considered to be neutral, and it was not expressed relative to on-screen time, because passive drips could still be seen clearly when the rats were off-screen. 2.7. Data analysis The effects of calcium deprivation on plasma ionized Ca++ concentrations and body weights were assessed using a two-way mixed ANOVA, with group as a betweensubjects factor and day as a within-subjects factor. Post hoc t-tests were conducted to determine on which days the rats differed. Total ingestive and aversive scores, the number of passive drips, and scores for individual behaviors were each compared using a two-way mixed ANOVA, with group and solution type as factors. If there was a significant main effect of group or groupsolution interaction, then post hoc t-tests were performed to determine which solutions were responsible. The preferences of the groups for CaCl2 in 24-h tests were compared using a t-test. The distributions for CaCl2 and water intakes in 24-h tests were positively skewed in both groups, so these variables were compared between groups using Mann–Whitney U-tests. For all analyses, pb0.05 was considered significant. For some post hoc tests, we found p values between 0.05 and 0.10. We mention all such cases, describe them as btrendsQ or as bapproaching significance,Q and list the exact p value.
3. Results 3.1. Body weights The mean (FS.E.M.) weight of the Ca-deprived group was 261F25 g on the day before taste reactivity testing and 314F24 g 9 days later, after the final test. Rats in the replete group weighed 298F8 and 354F9 g before and after testing, respectively. The body weights of the two groups did not differ but both gained weight significantly across the course of the experiment (effect of day, F[1,13]=417.4, pb0.001). 3.2. Plasma Ca++ The Ca-deprived group had significantly lower plasma Ca++ concentrations than did the replete group both before
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3.3. Two-bottle test During the 24-h test conducted at the end of the experiment, the Ca-deprived group had significantly higher preferences for 300 mM CaCl2 than did the replete group (t[15]=4.7, pb0.001; Table 2). This occurred because animals in the Ca-deprived group consumed significantly more CaCl2 than did ones in the replete group (Mann– Whitney U=9, p=0.009), but the two groups had similar water intakes (Table 2). 3.4. Taste reactivity measures One hundred and thirty-three of the 153 sessions were scored by one experimenter and 39 by the other. Nineteen sessions were scored by both, which allowed for a measure of reliability. The correlations between scorers for total ingestive and aversive scores were +0.99 and +0.95, respectively, indicating a high degree of reliability. Over all tests combined, rats in the Ca-deprived group exhibited significantly more ingestive reactions than did ones in the replete group (effect of group, F[1,15]=23.2, pb0.001), and this effect was larger for some solutions than others (group solution interaction, F [8,120]=9.0, pb0.001). Post hoc tests showed that deprived rats exhibited more ingestive reactivity to 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, MgCl2, and quinined HCl (t[15]z2.7, pb0.02 in all cases; Fig. 1A). The differences for saccharin and water were not significant, but there was a trend ( p=0.06 and 0.07, respectively) in the same direction. The groups did not differ in their total aversive scores (Fig. 1B). There was a tendency toward deprived rats showing more ingestive reactivity in general. In order to address this issue more thoroughly, we compared the groups on each of the ingestive behaviors that had been scored (see Table 3). Table 1 Mean (FS.E.M.) plasma ionized Ca++ concentrations (mmol/l) in the Cadeprived and replete groups before and after infusions for taste reactivity testing Group
Before infusions
After infusions
Ca-deprived Replete
1.06F0.04* 1.36F0.04
1.03F0.06* 1.37F0.02
Samples were taken on the day prior to the start of taste reactivity tests or immediately following the last test. * pb0.05 compared with replete group.
Table 2 Mean (FS.E.M.) intakes of 300 mM CaCl2 and H2O and preferences for CaCl2 during 24-h two-bottle testing in the Ca-deprived and replete groups Group
CaCl2 intake (ml/kg)
H2O intake (ml/kg)
CaCl2 preference (%)
Ca-deprived Replete
78F9* 33F21
122F19 111F29
41F4* 16F3
* pb0.05 compared with replete group.
Rats in the Ca-deprived group showed significantly more mouth movements to all of the stimuli except 100 mM NaCl and saccharin (effect of group, F[1,15]=17.5, pb0.001; group solution interaction, F[8, 120]=2.1, p=0.04; t[15]z2.2, pb0.04 in post hoc tests), and in the former case there was a nonsignificant trend ( p=0.053). Deprived animals also gave more tongue protrusions than did replete ones (effect of group, F[1,15]=13.2, p=0.002; group solution interaction, F[8, 120]=6.1, pb0.001), and post hoc tests showed that this was due to differences for 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and MgCl2 (t[15]z2.2, pb0.05 in all cases). There was a trend ( p=0.08) toward more tongue protrusions to saccharin in the deprived rats, but the differences for quinined HCl and water did not approach significance. The groups did not 350 Mean ingestive reactions per min
and after the taste reactivity tests (effect of group, F[1,15]=29.7, pb0.001; t[15]=5.0, pb0.001 in both post hoc tests; Table 1). There was no difference between the first and second samples (effect of day, n.s.). This confirms that the low-calcium diet induced calcium deficiency effectively and that the calcium-deprived rats did not become replete based on the small amount of calcium that they received from the infusions.
*
A
* CaDep Replete
300 250 200
*
150 100
* *
*
50
*
0 45 Mean aversive reactions per min
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B
40 35 30 25 20 15 10 5 0 Ca1
Ca2
Lac
Na1
Na2
Mg
Sac
Q
H2O
Fig. 1. Mean (FS.E.M.) total ingestive (A) and aversive (B) scores in the Ca-deprived (filled bars) and replete (open bars) groups. Scores are expressed as the number of reactions per minute that the rats were onscreen. Ca1, 30 mM CaCl2; Ca2, 300 mM CaCl2; Lac, 30 mM calcium lactate; Na1, 100 mM NaCl; Na2, 600 mM NaCl; Mg, 30 mM MgCl2; Sac, 2.5 mM sodium saccharin; Q, 1 mM quinined HCl; H2O, deionized water. *pb0.05 vs. replete group.
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Table 3 The mean (FS.E.M.) number of individual ingestive behaviors (mouth movements, tongue protrusions, lateral tongue protrusions, and paw licks) per minute of on-screen time in the Ca-deprived (CaDep) and replete groups Taste stimulus
30 mM CaCl2 300 mM CaCl2 30 mM CaLa 100 mM NaCl 600 mM NaCl 30 mM MgCl2 2.5 mM saccharin 1 mM QHCl H2O
Mouth movements
Tongue protrusions
Lateral tongue protrusions
Paw licks
CaDep
Replete
CaDep
Replete
CaDep
Replete
CaDep
Replete
66F14* 43F9* 76F15* 113F15 149F22* 48F9* 105F19 31F6* 54F12*
19F6 11F2 21F7 53F25 48F15 20F7 65F16 10F3 20F7
21F8* 3F1* 38F13* 147F28* 139F30* 8F2* 55F21 2F1 28F15
2F1 1F1 4F3 34F16 29F18 2F1 13F5 0F0 7F3
0F0 0F0 1F0 1F0 1F0 0F0 0F0 0F0 0F0
0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0
0F0 0F0 0F0 25F22 14F12 0F0 6F3 0F0 0F0
0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0 0F0
* pb0.05 compared with replete group.
differ on the number of paw licks, and although there was a significant main effect for calcium-deprived rats giving more lateral tongue protrusions relative to replete rats ( F[1,15]=4.6, pb0.05), we did not find group differences in these measures for any individual solutions. We also compared the groups on their individual aversive behaviors, in order to see whether there were differences that had been obscured by looking only at total aversive scores. Table 4 shows mean (FS.E.M.) head shakes, paw flicks, gapes, and face washes in the two groups. Data are not shown for chin rubs and paw treads because of the infrequency with which they occurred in both groups (they were observed in only 4.1% and 1.7% of the sessions that were scored, respectively). The groups did not differ on face washes, paw flicks, chin rubs, or paw treads (effect of group and groupsolution interaction, n. s.). Rats in the Cadeprived group gave significantly more head shakes to 30 mM CaCl2 and MgCl2 (groupsolution interaction, F[8, 120]=2.6, p=0.01; t[15]=2.2, pb0.05 in both post hoc tests). Although there was a significant groupsolution interaction for gapes ( F[8, 120]=2.5, p=0.01), none of the post hoc tests were significant. The number of passive drips is shown in Fig. 2. A smaller number was counted for animals in the Ca-deprived group than in the replete group (effect of group, F[1,15]=13.5, p=0.002; group solution interaction,
F[8,120]=2.6, p=0.01), and this was due to less dripping of 30 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and quinined HCl (t[15]z2.8, pb0.02 in post hoc tests). There was also a trend ( p=0.08) toward less dripping of 300 mM CaCl2 in the Ca-deprived group.
4. Discussion Calcium-deprived rats exhibited more total ingestive reactivity than did replete rats when they received infusions of 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, and quinined HCl, but total aversive scores did not differ between the groups. By themselves, these results suggest that CaCl2, calcium lactate, NaCl, MgCl2, and quinined HCl taste more palatable to calcium-deprived than replete rats. However, matters are complicated by the fact that the Ca-deprived group also showed a non-significant trend (0.05bpb0.10) toward more ingestive reactivity to saccharin and water. The lack of statistical significance for saccharin and water indicates that any difference between the groups in palatability of these solutions was marginal if it existed at all. Nevertheless, one explanation is that calcium deprivation caused both a diffuse, generalized increase in ingestive reactivity and a separate, stimulus-
Table 4 The mean (FS.E.M.) number of individual aversive behaviors (head shakes, paw flicks, gapes, and face washes) per minute of on-screen time in the Cadeprived (CaDep) and replete groups Taste stimulus
Head shakes CaDep
Replete
CaDep
Replete
CaDep
Replete
CaDep
Replete
30 mM CaCl2 300 mM CaCl2 30 mM CaLa 100 mM NaCl 600 mM NaCl 30 mM MgCl2 2.5 mM saccharin 1 mM QHCl H2O
3F1* 2F1 3F1 1F0 1F0 3F1* 3F1 2F1 4F1
2F1 1F1 2F0 2F0 2F1 1F1 2F1 1F0 2F1
7F3 4F1 4F1 3F2 3F1 7F2 6F3 4F1 6F2
3F2 0F0 2F1 4F2 2F1 3F2 2F1 1F1 3F2
2F0 10F2 0F0 0F0 1F0 3F1 0F0 8F2 0F0
3F1 6F1 1F1 0F0 2F1 3F1 1F1 5F1 1F1
21F6 16F4 9F4 5F2 4F2 18F6 10F5 20F8 22F8
12F8 5F1 4F1 18F7 7F4 13F8 9F3 7F1 8F6
* pb0.05 compared with replete group.
Paw flicks
Gapes
Face washes
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specific increase in ingestive responses that was related to an increase in palatability. We looked at individual ingestive behaviors to clarify this issue, and we found evidence for both generalized and specific effects.
effect on the ability of the Ca-deprived group to perform mouth movements normally, and thus they tended to perform more of them regardless of which solution they received. Given this possibility, we decided not to draw conclusions about the palatability of stimuli based on our mouth movement data. This decision is reinforced by the fact that mouth movements are performed frequently for solutions that are not consumed avidly [7,11] and some researchers exclude them from the total ingestive score [5,21]. A generalized effect of deprivation on mouth movements may have arisen due to hypocalcemia. Calcium plays an important role in muscle contraction, and severe calcium deprivation is known to cause tremors (i.e., tetany). However, any effect of deprivation on muscle contraction must have been limited. We did not observe any tremor in the deprived rats’ normal body movements, and the calcium deprivation was not severe enough to result in group differences in body weights. Furthermore, any generalized effects must have been restricted to mouth movements and not the other individual behaviors that we scored, based on which solutions were found to differ between the groups.
4.1. Generalized effects
4.2. Effects of calcium deprivation on palatability
Although taste reactivity reflects the palatability of substances, it is also a set of motor responses which can be influenced by manipulations that affect muscle contraction or brain areas involved in movement. In prior studies, two methods have been used to preclude effects on motor systems as an explanation for changes in reactivity. One has been to verify that ingestive and aversive scores did not change in the same direction [5], and the other has been to test multiple taste stimuli and show differential effects across them [9]. Our results met the first criterion, since we observed significant differences between the replete and Ca-deprived groups in total ingestive, but not aversive, scores. Our support for the second criterion was less clear. We had included water and quinined HCl as control stimuli (see Methods), with the expectation that reactivity would be the same in the replete and Ca-deprived groups when they received these solutions. This was the case for nearly all of the individual behaviors that we measured. The only exception was mouth movements, which were significantly higher in calcium-deprived than replete rats for seven of the nine solutions (30 and 300 mM CaCl2, calcium lactate, 600 mM NaCl, MgCl2, quinined HCl, and water) and there was also a trend in the same direction ( p=0.053) for 100 mM NaCl. One explanation is that calcium deprivation causes an increase in palatability for all of these solutions; for CaCl2, calcium lactate, NaCl, and MgCl2, an increase in voluntary consumption accompanies this hedonic shift, but for water and quinined HCl it does not. However, a simpler explanation is that deprivation had an
The replete and Ca-deprived groups did not differ on the number of tongue protrusions, lateral tongue protrusions, or paw licks when they received our control stimuli, quinined HCl and water. We therefore conclude that calcium deprivation did not have a generalized effect on the ability of rats to perform these behaviors and that they provide a good reflection of the palatability of the stimuli that were infused. The two groups did not differ on the number of lateral tongue protrusions or paw licks for any of the solutions. However, calcium-deprived rats showed more tongue protrusions than did replete rats during infusion of 30 and 300 mM CaCl2, calcium lactate, 100 and 600 mM NaCl, and MgCl2. This difference provides evidence that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats following calcium deprivation, and it explains why these compounds are consumed to a greater extent by calcium-deprived rats relative to replete ones [14,15]. An increase in the palatability of calcium-containing solutions is clearly beneficial for calcium-deprived rats. It should cause them to start replenishing their calcium levels as soon as they sample an appropriate solution, without having to depend on postingestive consequences. In fact, this outcome has been found in prior work; deprived rats show avid licking of CaCl2 within the first minute of access [15]. They also consume more CaCl2 than do replete rats when sham-drinking, even though they are not able to absorb it and derive any benefit [23]. The increases in the palatability of NaCl and MgCl2 that we observed, in contrast, are not helpful and are difficult to
35
CaDep Replete
Mean drips per 2 min
30 25 20 15
* *
*
10 5
*
*
0 Ca1
Ca2
Lac
Na1
Na2
Mg
Sac
Q
H 2O
Fig. 2. Mean (FS.E.M.) number of passive drips in the Ca-deprived (filled bars) and replete (open bars) groups. Abbreviations as in Fig. 1. *pb0.05 vs. replete group.
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explain as adaptive. Moreover, ingesting large amounts of NaCl causes a further decrease in calcium status in deprived rats, because sodium and calcium excretion are linked [10,27]. In the case of MgCl2, it is possible that rats confuse it with CaCl2 because they taste similar. The two compounds evoke similar across-neuron patterns of responding, which are thought to be reflective of taste quality perceptions [24,30]. However, there is also evidence that CaCl2 and MgCl2 are distinguishable to rats [19,26], and this explanation does not work for NaCl, since there are many indications that CaCl2 and NaCl are clearly discriminable to rats [13,19,22]. We did not find any significant differences between the groups for reactivity to saccharin, and so there was no evidence that a decrease in palatability is responsible for the decrease in saccharin preference that is associated with calcium deprivation [32]. 4.3. Aversive scores Our effects were limited primarily to ingestive scores. The groups did not differ on their total aversive scores, and when we looked at the individual aversive behaviors, we found group differences only for head shakes to 30 mM CaCl2 and MgCl2. We consider these differences minor, since they were not observed for other aversive behaviors or when total aversive scores were considered. Although one might expect aversive responses to calcium solutions to be higher in the replete group relative to the Ca-deprived group, there is some precedence for negative and positive aspects of a flavor’s palatability being separate rather than integrated [6]. This was observed in a previous study in which 48 hours food deprivation increased ingestive taste reactivity responses, while aversive taste reactivity responses remained unchanged [3].
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4.5. Comparison with prior work The hedonics of CaCl2 have been examined previously in rats that were pregnant or lactating [21], and these conditions have been reported to generate a calcium appetite in some experiments [31]. The prior taste reactivity results did not support the hypothesis that calcium tastes more palatable to rats when in these states [21]. Although these results might appear to contradict ours, intake tests conducted in the earlier experiment showed that pregnant rats did not have a calcium appetite, and lactating rats had one that was significant but modest in size. Thus, our results may differ from theirs for a number of reasons, which include the size of the appetites that were generated, as well as the method of induction. 4.6. Summary Measurements of taste reactivity provided evidence that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats when they are calcium-deprived. Rats in the Ca-deprived group had significantly larger total ingestive scores and gave more tongue protrusions relative to rats in the replete group when they received these compounds. We also found that calcium-deprived rats had larger total ingestive scores for quinined HCl than did replete rats. However, this effect arose solely from a difference in the number of mouth movements, which showed evidence of a generalized effect, and so it may not have reflected a palatability shift. Overall, our results suggest that increases in palatability contribute to the increases in consumption that have been reported previously in calcium-deprived rats.
Acknowledgements 4.4. Passive drip Although palatability and ingestion are closely related, the two measures can be affected independently [16,29,34]. All infusions that we gave were 2 ml in volume, and so we can infer whether there were differences in the amount of solution ingested from the amount of passive drip. We observed significantly less passive drip to infusions of 30 mM CaCl2, calcium lactate, and 100 and 600 mM NaCl in the Ca-deprived group. Thus, calcium-deprived rats ingest more of these solutions when they are delivered by an intraoral cannula, and not just when consumed voluntarily. The groups did not differ on passive drip of MgCl2 and 300 mM CaCl2, even though deprived rats ingest more of these solutions in two-bottle tests, and they did differ on passive drip of quinined HCl, even though it is consumed equally by replete and deprived rats [14,15]. These discrepancies may be due to differences in the motivation of rats to consume these solutions based on whether they initiate the solution delivery themselves.
The authors thank Marena La Fontaine, Kazaure, Samantha Doman, and Diane Pilchak excellent technical assistance. Dr. Harvey Grill valuable help with the surgical technique for cannulation.
Hadeeza for their provided intraoral
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