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Changes in salt intake after abdominal vagotomy: Evidence for hepatic sodium receptors
Physiology & Behavior, Vol. 26, pp. 575-582, 1981. Pergamon Press and Brain Research Publ. Printed in the U.S.A.
Changes in Salt Intake After Abdominal Vagotomy: Evidence for Hepatic Sodium Receptors R O B E R T J. C O N T R E R A S 1 A N D T H E R E S E K O S T E N
Yale University, Department o f Psychology, Box I1A Yale Station, New Haven, CT 06520 R e c e i v e d 13 August 1980 CONTRERAS, R. J. AND T. KOSTEN. Changes in salt intake after abdominal vagotomy: Evidence for hepatic sodium receptors. PHYSIOL. BEHAV. 26(4) 575-582, 1981.--Several reports have suggested that the mammalian liver contains neural receptors, innervated by the vagus nerve, that monitor the sodium concentration and osmolarity of the portal circulation. These reports have been concerned primarily with either the neurophysiological identification of these receptors or their role in the short term control of urine output. Inasmuch as relatively little is known about the role of these receptors to consummatory behavior, we investigated the effects of hepatic vagotomy in rats on sodium intake as well as on sodium output. Hepatic vagotomized (HV) rats drank less NaCI solution (0.03, 0.1, 0.3M) in 24 hr during a two-bottle test with water than sham operated rats. Comparable differences in the intakes of either water, KCI or glucose solutions were not found. The two groups of rats did not differ in their intakes of water or 0.3 M NaCI after an injection of either an osmotic load (IP, 2 M NaCI, 1% BW), deoxycorticosterone acetate (SC, 5 nag) along with furosemide (SC, 10 mg), or after 10 days of sodium deprivation. Urinary sodium output was reduced in HV rats during sodium deprivation but not when the rats had adequate levels of sodium in their diet. Because circadian patterns of water and food intake as well as body weight growth of hepatic vagotomized rats were similar to those of control rats, general malaise due to surgery and generalized deficits in motivation were ruled out as explanations for the depressed daily drinking of NaCI solutions. These findings support the existence of hepatic sodium receptors and their possible involvement to the control of sodium regulation.
Sodium receptors Liver Cellular dehydration
Sodium intake
Sodiumappetite
THE mammalian liver is believed to contain neural receptors that are sensitive to changes in the osmolarity and sodium composition of the portal circulation [22]. Support for the presence of these receptors comes from neurophysiological studies of the hepatic branch of the vagus nerve. Multi-fiber activity of afferents in the hepatic branch of the vagus respond to changes in the osmotic pressure in rat [1] and to changes in sodium concentration in rabbit [2] of the perfusate in the portal circulation. Presumably, these receptors play a role in the maintenance of steady state levels of fluid and electrolytes by activating physiological and behavioral mechanisms that act to correct for deviations from homeostasis. In rats, changes in the osmolarity of the portal, but not systemic, circulation led to rapid, dramatic changes in urine volume [10]. After simultaneous infusions of water into the hepatic portal vein and hypertonic saline into the vena cava, urine volume increased; urine volume decreased when the pattern of simultaneous infusions were reversed. The implication was that osmoregulatory mechanisms of the liver may support and perhaps precede the activation of osmoregulatory mechanisms of the central nervous system.
Hepatic vagotomy
Urine output
The notion that the liver plays a role in osmoregulation was also supported by infusion studies in dogs [15, 16, 17] and in humans [10,11] despite some studies to the contrary [9,24]. Reasonably good evidence also exists for a physiological role of hepatic sodium receptors. Increases in the sodium concentration of the portal vein were followed by increases in urinary sodium loss [7,8]. Activation of hepatic receptors has also been shown to influence consumption. Blake and Lin [3] reported that infusions of hypertonic saline into the portal vein suppressed the short-term drinking of 0.15 M sodium chloride solution, but not of water, in water deprived rats. Comparable infusions into the vena cava were without significant effect. Hepatic information is most likely transmitted by the vagus to the central nervous system for integration of drinking behavior, inasmuch as the suppression of saline drinking by portal infusions was blocked by right cervical vagotomy. The data suggest that osmo- and sodium receptors of the liver may play a role in the initiation or modification of physiological and possibly behavioral regulatory responses. With the exception of Blake and Lin's report, relatively little is known about the contribution of hepatic signals to water and
1Send reprint requests to Dr. Robert J. Contreras, Department of Psychology, Yale University, Box 11A Yale Station, New Haven, CT
06520.
Copyright © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/040575-08502.00/0
576
C()NTRERAS AND K()S:IF.N
sodium intake. The present study was carried out to ascertain the long-term effects of resection of the hepatic branch of the vagus nerve on sodium balance, with an emphasis on sodium intake coupled to analyses of sodium output. METHOD
Thirty-six male albino rats of the Sprague-Dawley strain (Charles River) weighing 320-340 g at the start of the experiment were used. These animals were housed in individual cages in a colony room maintained on a 12:12 hr light/dark cycle. The animals were given Purina pellets and deionized water to consume ad libitum unless otherwise noted. To adapt the rats to their new surroundings and to the experimenters, all the animals were handled daily for a week prior to the start o f the experiment. The 36 rats were examined in sets of twelve at three different time periods. In each case six of the twelve rats received hepatic vagotomies and six received sham operations. All 36 animals were weighed daily for three days prior to and up to two months after surgery.
Sargerv Eighteen rats received hepatic vagotomies (HV) and the other 18 rats received sham operations (SC) under sodium pentobarbital anesthesia (Pentosol, IP, 50 mg/kg body weight). F o r each animal, a midline incision was made on the animal's abdominal surface immediately posterior to the xiphisternum. This longitudinal incision exposed the median and left lateral lobes of the liver that covered the esophagus and stomach. The liver was deflected to the surgeon's left with a cotton swab. The stomach was rotated clockwise to the animal's midline and held gently in place with retraction. This straightened and slightly stretched the esophagus facilitating the exposure of the hepatic branch of the vagus nerve. The hepatic branch of the vagus was identified with the aid of a Zeiss operating microscope. A section of the hepatic branch, about 5 mm long, was removed. After sectioning, the stomach was placed in its normal position and the muscles and skin were sutured. The animal was placed on a heating pad until it recovered from the operation. Sham operations consisted of similar manipulations of the liver and stomach but leaving the hepatic branch intact.
Food and Water Intakes To assess whether hepatic vagotomy had more general effects other than on sodium balance, day-time (7 a.m.-7 p.m.) and night-time food and water intakes were measured. For measurements of food intake each rat received a 50 g portion, for each 12 hr period, of Purina pellets on the floor of its cage. A paper towel was placed under each cage to collect food spillage. At the end of each 12 hr period the uneaten food pellets with spillage were weighed. These weights were subtracted from 50 to determine the amount of food consumed. A fresh supply of pellets and a clean paper towel were provided for the next set of measurements. The intakes of deionized water in 100 ml calibrated drinking cylinders were also measured every 12 hr.
Preference Testing Twenty-four rats, 12 in each group, were given preference tests beginning approximately two weeks after surgery. Two 100 ml graduated drinking cylinders, fitted with rubber stoppets and metal drinking spouts, were attached to the front of each animal's cage. One cylinder contained deionized water
and the other a test solution. Test stimuli were presented ~n an ascending order of concentration which consisted of va~ious molar concentrations of NaCI (0.03, 0.1, 0.3), glucose (0.1, 0.3) and KC1 (0.1, 0.3) solutions made from reagenl grade chemicals in deionized water. The positions of the tw¢ drinking cylinders were interchanged daily on a random schedule and the amount of solution consumed from each cylinder was measured every 24 hr. Each test solution was presented for two consecutive days, one day on the left and one day on the right side of the cage, to control for position habits. Every third day before the presentation of another test solution, both bottles were filled with deionized water. This was done to minimize possible influences of gustatory adaptation and osmotic stress on intake from the effects of successive testing. During preference testing all animals had free access to Purina pellets.
Urinao' Electrolytes Twelve rats, six in each group, were placed in metabolism cages (Acme) for 10 days and given a powdered, sodium-free diet (Nutritional Biochemicals) to consume ad lib. For the first five days, deionized water was also always available. Beginning on Day 5, the animals were adapted to a 23 hr schedule of water deprivation with food freely available and water available for l hr. F o r this 10 day period urine specimens were collected at 9:00 a.m. daily and stored in a freezer for future analyses of sodium and potassium concentrations by flame photometry. Water intake, urine volume, and body weight measures were also recorded daily. To further assess the effects of hepatic vagotomy on sodium output, another set of twelve rats were placed in metabolism cages for eight days. In this case the animals were maintained for three days on the sodium-free diet to which 1 g of sodium chloride crystals was added to 99 g of powdered diet. This 1% NaCI mixture is the concentration of salt normally found in Purina rat pellets. This was followed by two days of a low salt diet (0.12% NaC1 to the powered sodium-free diet). F o r the last three days the animals were given the sodium-free diet without NaCl supplementation. Body weight, water intake, and urine volume measures were recorded daily, and urine specimens were saved for analyses of electrolyte concentrations.
Sodium Appetite Sodium appetite was induced in one of three different ways. The first set of animals were fed a powdered sodiumfree diet in food cups in their home cages for 10 days. This period of sodium deprivation is sufficient to induce sodium appetite [5]. Beginning on Day 5 of sodium deprivation the animals were put on a 23 hr schedule of water deprivation with food available ad lib and water available for 1 hr. Following the 1 hr drinking period all food and water were removed from each animal's cage for the ensuing hour. Then the animals were presented with distilled water in calibrated drinking tubes for 10 min. This procedure was adopted to minimize the influence of (1) food ingestion on the contents and concentration of salivary electrolytes and (2) water deprivation on taste preference. Beginning on Day 10 of salt deprivation the rats were presented with a sapid solution or distilled water during the 10 min drinking period. The solutions used were 0.1 M glucose, 0.1 M NaCI, 0.1 M KC1 and 0.3 M NaCl. This procedure has been used successfully in the measurement of salt taste thresholds [4]. The second set of animals were anesthetized with ether
HEPATIC VAGOTOMY AND SODIUM I N T A K E and received an injection of the mineralocorticoid hormone, deoxycorticosterone acetate (s.c., 5 mg in 1 ml of oil) and an injection of the natriuretic drug, furosemide (s.c., 10 mg in 1 ml aqueous solution) [6] at 9:00 a.m. The animals were returned quickly to their home cages and given a two-bottle preference test between demineralized water and 0.3 M NaC1 solution with food pellets ad lib. The 24 hr intakes of these solutions were measured for two consecutive days. Similar measurements of intake were recorded for the third set of animals after a combination of three days of dietary sodium deprivation coupled with injections of furosemide (10 mg in 1 ml aqueous solution).
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Drinking after Cellular Dehydration One set of animals was tested for drinking after cellular dehydration produced by injections of 2 M NaCI solution (IP, 1% of body weight) [12]. On the day of injections the rats were weighed and food was removed from each animal's cage. After the injections the animals were returned to their cages and presented with deionized water in 100 ml graduated drinking cylinders. Latency to drink and water intakes 30, 60, 90 and 120 min after drinking began, as well as 24 hr intakes were recorded.
Verification of Hepatic Vagotomy At the conclusion of the study the animals were sacrificed for autopsy with an injection of sodium pentobarbital. Autopsies were performed without knowledge of the animal's surgical treatment. The anterior trunk of the vagus was located with the aid of the operating microscope at the thoracic level and traced caudally to the point where the anterior trunk separates into hepatic and anterior gastric branches. For each rat records were kept describing the appearance of the esophagus, stomach and liver, and the interrupted or uninterrupted course of the hepatic branch to its target organ. We used the criterion established by Kraly [12] to judge the completeness of our vagal transections. T h a t is, whenever there was a case where any ambiguity concerning the continuity of the hepatic vagal branch, then that transection was classified as incomplete.
RESULTS
Verification of Hepatic Vagotomy With microscopic inspection hepatic innervation of the liver was found to be normal in every sham operated rat. Experimental rats could easily be identified by the general appearance of the liver, stomach and esophagus. In these cases the liver was frequently attached to either the esophagus or stomach by a new growth of tough connective tissue. This made it extremely difficult to visualize the portion of the hepatic branch remaining two months after surgery and determine if it was innervating the liver. On the basis of the criterion [12] we used to judge the completeness of the transections, three rats that received hepatic vagotomies were judged to have partial transections. Two of these animals were from the first set, one was from the second set, but none of the last set were classified as partial hepatic vagotomies. Statistical analyses were performed with the data from only those rats that were determined to be completely vagotomized.
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FIG. 1. Hepatic vagotomized (HV) and sham control (SC) animals were weighed routinely for 30 days following experimental and sham operations. The body weights plotted at five day intervals indicates that there were no differences between groups. Specifically, at the end of this 30 day period the change in body weight from preoperation weights were similar for both groups (t = 1.49, df= 17, p >0.20).
Body Weight and Food and Water Intakes Figure 1 represents the body weights of hepatic vagotomized (HV) and sham control (SC) rats for the first 30 days after surgery. At 30 days SC rats increased their body weights an average (+_S.E.) of 66.89_+7.09 g over their preoperative weights, while HV rats increased by 80.1___5.3 g. Nevertheless, these two groups of subjects did not differ in their relative increase in body weight at the end of this period (t= 1.49, df= 17, p>0.20). When measurements of food and water intake were recorded for the first eight days after surgery in one set of animals, 24 hr intakes were similar between groups. However, the HV rats tended to consume more food (8.43___0.64 g) and water (13.25-+1.95 ml) during the daytime as compared to the food (7.13___0.59 g) and water intakes of SC rats. These differences in food (t= 1.45, df=9, p<0.20) and water (t = 1.15, df=9, p<0.20) intake were not, however, significant. Figure 2 represents the ratio of diurnal water consumption to total intake. The largest differences in group means occurs in the t'n-st few days after surgery; by the end of the test period no differences are obvious. In another set of animals when daytime and nighttime intakes of food and water were measured two months after surgery no differences between groups were found.
Preference Testing The results of the two-bottle preference tests are given in Fig. 3. Analyses of the saline, glucose and KCI intakes, excluding the partial hepatic vagntomized rats, showed that the HV animals drank less of all the NaCI solutions, F(1,18) = 4.96, p <0.05, with significant differences occurring at 0.1 M NaCI (t=2.56, df=18, p<0.02) and at 0.3 M NaC1
578
CONTRERAS AND KOSTEN
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DAYS FIG. 2. For the first 8 days following the surgery of the second set of animals, water and food intakes were measured during the daytime (7 a.m.-7 p.m.) and during the nighttime. With fluid intake values expressed in ml/100 g of body weight, there was a small tendency for hepatic vagotomized (HV) animals to consume more water during the day (4.08___0.56 ml/100 g) than the controls (3.46_+0.50 ml/100 g). The total daily water intakes were more similar with the HV animals' consumption averaging 14.06_+0.54 ml/100 g and the controls drinking an average of 13.89+-0.70 ml/100 g. The food intake pattern parallelled the water intake somewhat but the differences were smaller. The water-food intake ratio was the same for both groups at all times.
(t=2.19, df=18, p<0.05). Both HV and SC rats showed a marked preference for the 0.1 M solution, modest intakes of the 0.03 M solution and very little consumption o f the 0.3 M NaCl, F(2,36)=138.4, p<0.01. Altered intakes associated with hepatic vagotomy were restricted to NaCl solutions, as the animals from both groups showed similar preferences tor glucose and relative aversions for KCI solutions. Throughout the two-bottle tests there were no differences in water consumption either during the test days or on the intervening days when deionized water was only available.
Urinary Electrolytes Variations in the amount of sodium excreted in urine over l0 days of sodium deprivation for the second set of HV and SC rats are presented in Fig. 4. The patterns of sodium excretion were the same for both groups of rats throughout the 10 day period. The amount o f sodium eliminated in urine presumably decreased substantially on the first day of sodium deprivation from an average o f approximately 2.5 mEq/day as reported by Contreras and Hatton [5] to a value of 0.6 in the present study. Sodium output decreased even more on Day 2 from an average (-+S.E.) o f 0.64___0.11 mEq/day to 0.15-+0.02 in SC rats (t=5.00, df=5, p<0.01),
and from an average of 0.59__-0.08 mEq/day to 0.05--.0.01 in HV rats (t=6.46, df=4, p<0.01). This low level of sodium output was maintained for the remaining days of sodium deprivation, except on Day 5, when a 23 hr schedule of water deprivation was superimposed on sodium deprivation. Although sodium output in SC rats increased on Day 5 from an average ( _ S . E . ) of 0.08__-0.03 mEq on Day 4 to 0.21-+0.05 (t=3.1, dr=5, p<0.05), in HV rats the increase from 0.03_+0.01 mEq on Day 4 to 0.15_+0.07 (t= 1.84, dr=4, p>0.10) was not significant. On a daily basis the HV group always excreted less sodium than the SC group, although these differences on any one day were nonsignificant. Total sodium output for the entire 10 day period of dietary sodium deprivation was, however, less for the HV animals, F(1,9)=5.76, p<0.04. The two groups did not differ in urine volume or the amount of potassium excreted during this 10-day period. In the third set of animals metabolic testing was performed while they were on a gradual dietary reduction of sodium. With this schedule no differences in sodium loss were detected. Table 1 shows that there were no differences in total sodium loss while the animals were either on a diet containing 1% NaCl (t=0.71, df=10, p>0.10), on a 0.12% NaC1 diet (t =2.09, dr= 10, p>0.05), or on a sodium-free diet
HEPATIC VAGOTOMY AND SODIUM INTAKE
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FIG. 3. The two groups were given two-bottle preference tests between deionized water and molar concentrations of NaCI (0.03, 0.1, 0.3), glucose (0.1, 0.3) and KCI (0.1, 0.3) solutions. Each test solution was presented for two days and 24 hr intakes were recorded. Hepatic vagotomized (HV) rats ingested significantly less NaCl solutions (ml/100g/48 hr) than controls, F(1,18)=4.96, p<0.05. There were no differences in glucose, KCI or water intake.
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TABLE 1 MEAN (± S.E.M.) TOTAL URINARY SODIUM EXCRETED (mEq) IN HEPATIC VAGOTOMIZED AND SHAM CONTROLS WHILE ON SODIUM-REPLETE AND SODIUM-FREE DIET Heaptic Vagotomized N =6
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FIG. 4. The second set of animals were fed a sodium-free diet for 10 days. Beginning on Day 5, the animals of both groups were adapted to a 23 hr schedule of water deprivation. Urine specimens were collected and the volume, and sodium and potassium content of the urine were measured (Atomic absorption spectrophotometry). Hepatic vagotomized (HV) rats excreted significantly less sodium over the 10 day deprivation period, F(1,9)=5.76, p<0.04. Comparable differences in potassium output or urine volume were not found.
1% NaCl Diet (E Days 1-3)
7.40 _+ 0.31
6.83 -+ 0.73
0.12% NaC1 Diet (~ Day 4--5)
1.07 -+ 0.18
1.59 -+ 0.17
N a + free diet (~ Days 6-8)
0.22 +- 0.09
0.21 -- 0.02
Total (~ Days 1-8)
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8.67 +_ 0.36
Comparisons between groups were analyzed using t-tests for independent means. All t values indicated no differences between groups. Analyses of sodium and potassium concentrations were done by flame photometry.
580
CONTRERAS AN D KOSI [,iN TABLE 2 MEAN {_+ S.E.M.) INCREASE IN INTAKE OF 0.3 M NaCI RELATIVE TO BASELINE AFTERINDUCINGSODIUMAPPETITEBY VARIOUSMETHODSIN HEPATIC VAGOTOMIZEDAND SHAM-CONTROLRATS Hepatic Vagotomized
Sham Control
4.33 ± 0.88* N =6
6.33 ± 1.31" N=6
Sodium deprivation (10 min, ml)
DOCA + furosemide (24 hr; ml/100 g body weight) Sodium deprivation + furosemide (24 hr; ml/100 g body weight)
11.60 ± 2.11 N =5
17.17 ± 4.47 N=6
6.17 ± 3.03 N =6
8.00 ± 2.22 N =6
*These values are relative to a baseline water intake. All other values are relative to 0.3 M NaC1 intake prior to the sodium appetite challenge. Statistical comparisons between groups were analyzed using t-tests for independent means. All the t values indicated that there were no differences between the two groups of rats.
TABLE 3 MEAN (-+ S.E.M.) CUMULATIVEWATERINTAKES(ml PER 100 g OF BODY WEIGHT)AFTER CELLULAR DEHYDRATIONAT VARIOUS TIME PERIODS AFTER INITIATIONOF DRINKINGIN HEPATIC VAGOTOMIZEDAND SHAM CONTROLRATS
(t=0.37, df=10, p>0.10). The total amount of sodium excreted over this eight day period was the sam~ for both groups of rats (t =0.10, df = 10, p >0.10).
above water baselines respectively, showing no difference in their response to this sodium appetite challenge (t=1.27, dr= 10, p >0.10). Solutions of 0.1 M glucose and 0.1 M KCI were also presented during these 10 min sessions and revealed a similar drinking pattern of preference for glucose and aversion of KCI for both groups. A second set of animals were injected with a combination of DOCA and furosemide and their 24 hr intakes of 0.3 M NaCI were recorded. HV animals consumed 11.60±2.11 (ml/100 g body wt) more of this solution relative to their intake of this same solution prior to inducing sodium appetite. SC rats also increased their consumption to an average of 17.17+4.47 (ml/100 g body wt) which was found to be of similar magnitude as that of the HV rats (t=l.13, df=9, p>0.10). A combination of 3 days of dietary sodium deprivation and an injection of furosemide were given to a third set of animals. As in the above methods, both HV and SC rats showed a similar response of increased saline drinking of 0.3 M NaC1 as compared to their intakes of this solution before the sodium appetite challenge was presented. The increase in intake was 6.17+3.03 (ml/100 g body wt) for the HV rats and 8.00__-2.22 (ml/100 g body wt) for the SC rats but this difference was nonsignificant (t =0.49, dr= 10, p >0.10).
Sodium Appetite
Drinking after Cellular Dehydration
Similar increases in saline drinking occurred after inducing sodium appetite in each of the three methods as shown in Table 2. With the first method the first set of animals were maintained on a sodium-free diet for 15 days and were adapted to a 23 hr water deprivation schedule starting on the 5th day. They were given 10 min drinking tests one hour after their daily exposure to water. After 10 days of dietary sodium deprivation, 0.1 M NaCI was presented during the 10 min test period. HV animals increased their intake to 7.5±0.89 ml (mean± S.E.M.) above their water baseline; SC rats consumed 7.83-+2.46 ml more than their baseline water intake (t =0.13, df= 10, p>0.10). Sodium-free diet continued to be available for 4 more days at which time 10 min intakes of 0.3 M NaCI were recorded. Again, HV and SC rats increased their consumption to 4.33±0.88 ml and 6 . 3 3+- 1.31 ml
Latency to initiate drinking in response to injections of 2 M NaC1 were 23.5__+9.0 min for HV rats and 23.9±9,0 min for SC rats. All animals had a severe reaction to the injection and needed several minutes to recover from it. Once they recovered and began drinking, the total amount they consumed after each 30 min period did not differ between groups as shown in Table 3. When intakes during the 30 min periods were examined, HV rats drank somewhat less between the 60 and 90 minute period (0.76±0.25 ml/100 g body wt) than the SC rats (1.44±0.46 ml/100 g body wt) (t=l.30, dr=9, p>0.10). Consumption during all other periods and the cumulative intakes at the end of all the time blocks were similar for both groups of animals. Similar to the tests of sodium appetite, HV animals generally consumed slightly less than the SC rats.
Hepatic Vagotomized N=6 30 min 60 min 90 min 120 rain 24 hr
1.13 +- 0.31 2.04 - 0.48 2.80 ± 0.36 3.25 ± 0.33 7.20 -_+0.83
Sham Control N=6 1.56 _ 2.50 ± 3.85 ± 4.41 ± 8.36 ±
0.59 0.76 0.48 0.55 1.65
Statistical comparisons between groups were analyzed using t-tests for independent means. All the t values indicated that there were no differencds between the two groups of rats.
HEPATIC VAGOTOMY AND SODIUM INTAKE
DISCUSSION The results of this study demonstrated that rats that had the hepatic branch of the vagus nerve resected consumed significantly less of most NaC1 solutions while they were maintained on an ad libitum feeding schedule. However, the relative intakes of the various molar concentrations of NaCI solutions were similar for the two groups of rats. That is the animals consumed increasingly greater amounts with increasing concentration to a maximum at 0.1 M. Then at 0.3 M, N~/CI solution intake declined substantially. The two group~, of rats differed only in their absolute intakes, with the NaC1 intakes of hepatic vagotomized (HV) rats being somewhat depressed. Hepatic vagotomy did not affect the animals' intakes of water, KCI or glucose solutions. Thus, the lowered salt intakes of HV rats were not the result of general malaise due to the surgery. These data are consistent with the notion that sodium receptors are present in the portal circulation of the mammalian liver which may be important for sodium regulation. By detecting changes in the sodium concentration of plasma, these receptors may influence physiological and behavioral responses for maintaining steady state levels of sodium. Along with the reduced NaCI intake, measurements of hrinary output revealed a subtle change in the amount of 'sodium, but not of potassium, excreted after 10 days of sodium deprivation. Hepatic vagotomized rats excreted less sodium than sham control rats. From a glance this result was unexpected because it suggested that severing the hepatic receptors better equipped the animals to respond to salt deprivation. Although it was fortunate in this case, a change in sodium excretion compared to control levels represents a deviation from normality. Besides on Day 5 when water deprivation was superimposed on sodium deprivation control animals increased their sodium output. It was presumably maladaptive for HV rats not to increase their sodium output to compensate for a reduction in their water to food intake ratio. We considered that the modest deviation in total sodium output was not peculiar to sodium deprivation but might also be apparent while the animals were maintained on a diet that contained adequate sodium levels. When the diet contained 1% NaCl (the concentration in Purina Rat Pellets), a concentration that is approximately" 10 times the amount needed [26], or 0.12% NaCl, a low but adequate sodium level [14], no significant trends in urinary sodium output were observed. A single measurement of urinary sodium output taken at the end of a 24 hr period is perhaps insensitive to the dynamic variations that occur much earlier, although on an a priori basis, the pattern of sodium excretion during sodium deprivation does not necessarily have to be the same as that during conditions where the animals' diet contains normal sodium levels. According to an homeostatic viewpoint, subtle excursions from sodium balance during free-feeding conditions underlies the cumulative intakes of saline and water over the course of a day. Sodium deprivation represents one end of this continuum where the excursion from homeostasis is more abrupt and severe. The specificity of the effect of hepatic vagotomy on changes in consummatory behavior and metabolic response to sodium supports the existence of sodium receptors rather than osmoreceptors. Inasmuch as hepatic vagotomy presumably disrupted the normal function of postulated osmoreceptors, then in addition to NaC1 intake, KCI, water, and glucose intakes should have also been altered. As was
58! selectively reviewed in the introduction, hepatic sodium receptors have been indicated by electrophysiological [2] and infusion [7, 8, 19, 25] studies although the controversy of sodium versus osmoreceptors still persists. It is possible that both hepatic sodium and osmoreceptors exist and contribute to both sodium- and osmoregulation, but the conditions of the present study favored the contribution of sodium receptors. With the loss of these sodium receptors in the liver through denervation of the hepatic branch of the vagus, there were altered responses to the presence of sodium rather than a complete loss of all sodium regulatory mechanisms. The decreased saline drinking and sodium excretion supported this assessment as did the smaller, although nonsignificant, intakes of hypertonic saline after inducing sodium appetite. In regard to sodium appetite the system may have been driven to the extent that the operation of other sodium regulatory mechanisms overrode the lost contribution of the hepatic sodium receptors. Since latency was not measured in these tests, it is not known whether the HV animals were sluggish in their initiation to drink. It is also not known whether the severed afferent fibers of the hepatic vagus transmitted a false reading of the amount of sodium in plasma. The afferent signal concerning the sodium status of the portal circulation may be important to the cessation of NaC1 intake. A signal indicating a reduced sodium need may account for the decreased saline drinking both during ad libitum conditions and during sodium deprivation. In fact, when sodium appetite was induced by sodium deprivation, the reduced urinary sodium loss of HV rat may have lessened the need and appetite for sodium as compared to sham control rats, resulting in reduced intake. The data from the present investigation were consistent with those of Kraly, Gibbs and Smith [13] and Martin, Geiselman and Novin [18] indicating that transection of the hepatic branch of the vagus did not disrupt osmotically induced drinking. Moreover, hepatic vagotomy did not alter daily food and water intake, findings in agreement with those previously reported [1]. We also did not observe any changes in body weight in apparent contrast to that of earlier findings [23]. In sum, our data suggest that the hepatic branch of the vagus nerve is not crucial for the control of body weight, and food and water intake. Although the effects of hepatic vagotomy were modest, the specific changes in NaC1 intake are important for understanding the contribution of hepatic receptors to the maintenance of sodium homeostasis. The neural fibers that compose the hepatic branch are but a small proportion of the total number of fibers that constitute the abdominal vagus. We were interested in producing a selective lesion of the abdominal vagus and then look for both subtle as well as more general behavioral and physiological consequences. That selective hepatic vagutomy produced subtle rather than general consequences supports the proposed existence of hepatic sodium receptors and their possible involvement to the control of sodium regulation. Transection of the hepatic branch affected the daily drinking of NaC1 solutions across the concentrations tested, even though preference-aversion functions were unaffected. The drinking of water, KCI and glucose solutions were unaffected. Moreover, circadian patterns of intake of water and food were maintained as well as normal body weight growth after hepatic vagotomy. Ruled out as possible explanations are general malaise due to surgery, and generalized deficits in motivation. It is not known whether the observed changes in NaC1 intake were
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due to afferent or efferent vagal function. There is precedent, h o w e v e r , for the suggestion that afferents o f the abdominal vagus mediate changes in drinking [13]. Precisely where and through what m e c h a n i s m the central n e r v o u s system integrates peripheral sensory information to modify drinking behavior is unknown. N e v e r t h e l e s s , there is good e v i d e n c e that hepatic sodium-and o s m o r e c e p t o r s activate neurons in the regions of the brain, possibly i n v o l v e d in the control of
drinking b e h a v i o r [20,21]. The present data support the contribution o f hepatic sodium receptors to behavioral regulation. ACKNOWLEDGEMENTS This research was supported by NIH Biomedical Research Support Grant 5-507-RR-07015 and by a grant from NIH, NHLBI Grant HL-24732 to R. J. Contreras.
REFERENCES 1. Adachi, A., A. Niijima and H. L. Jacobs. A hepatic osmoreceptor mechanism in the rat: electrophysioiogical and behavioral studies. Am. J. Physiol. 231: 1043-1049, 1976. 2. Andrews, W. H. H. and J. Orbach. Sodium receptors activating some nerves of perfused rabbit livers. Am. J. Physiol. 227: 1273-1275, 1974. 3. Blake, W. D. and K. K. Lin. Hepatic portal vein infusion of glucose and sodium solutions on the control of saline drinking in the rat. J. Physiol., Lond. 274: 12%139, 1978. 4. Contreras, R. J. and F. A. Catalanotto. Sodium deprivation in rats: Salt thresholds are related to salivary sodium concentrations. Behav. Neural Biol. 29: 303-314, 1980. 5. Contreras, R. J. and G. I. Hatton. Gustatory adaptation as an explanation for dietary-induced sodium appetite. Physiol. Behav. 15: 56%576, 1975. 6. Cruz, C., I. Perelle and G. Wolf. Methodologic aspects of sodium appetite: An addendum. Behav. Biol. 20: 96-103, 1977. 7. Daly, J. J., J. W. Roe and P. Horrocks. A comparison of sodium excretion following the infusion of saline into the systemic and portal veins in the dog: Evidence for a hepatic role in the control of sodium excretion. Clin. Sci. 33: 481-487, 1%7. 8. DeWardener, H. E., I. H. Mills, W. F. Clapham and C. J. Hayter. Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin. Sci. 21: 24%258, 1961. 9. Glasby, M. A. and D. J. Ramsay. Hepatic osmoreceptors? J. Physiol., Lond. 243: 765-776, 1974. 10. Haberich, F. J. Osmoreception in the portal circulation. Fedn Proe. 27: 1137-1141, 1%8. 11. Kiil, J. and D. Andersen. Evidence of a gastro-hepatic osmoregulation in humans and the influcence of vagotomy on its activity. Scand. J. Gastroenterol. 7: 573-581, 1972. 12. Kraly, F. S. Abdominal vagotomy inhibits osmotically induced drinking in the rat. J. comp. physiol. Psychol. 92: 999-1013, 1978. 13. Kraly, F. S., J. Gibbs and G. P. Smith. Disordered drinking after abdominal vagotomy in rats. Nature 258: 226-228, 1975. 14. Kriksey, A. and R. Pike. Some effects of high and low sodium intakes during pregnancy in the rat. I. Food consumption, weight gain, reproductive performance, electrolyte balances, plama total protein and protein fraction in normal pregnancy. J. Nutr. 77: 33-42, 1962.
15. Liang, C. C. The influence of hepatic portal circulation on urine flow. J. Physiol., Lond. 214: 571-581, 1971. 16. Lytin, H. Untersuchungen tiber den einfluss intravenrs intraportal und oral zugefiihrter hypotoner kochsalzlrsungen anf die diurese des hundes. Z. ges. exp. Med. 149: 193-210, 1%9. 17. Lytin, H. Untersuchungen am Menschen fiber die wirkung von oral und intravenfs zugefiihrter kochsalzrsungen auf die harnausscheidung. Z. ges. exp. Med. 149: 211-225, 1%9. 18. Martin, J. R., P. J. Geiselman and D. Novin. Drinking to intracellular dehydration following vagotomy in rats. Physiol. Behav. 23: 527-537, 1979. 19. Passo, S. S., J. R. Thomborough and A. B. Rothballer. Hepatic receptors in control of sodium excretion in anesthetized cats. Am. J. Physiol. 224: 373-375, 1973. 20. Rogers, R. C., D. Novin and L. L. Butcher. Hepatic sodium and osmoreceptors activate neurons in the ventrobasal thalamus. Brain Res. 168: 398--403, 1979. 21. Rogers, R. C., D. Novin and L. L. Butcher. Electrophysiological and neuroanatomical studies of hepatic portal osmo- and sodium-receptive afferent projections within the brain. J. auton. herr. syst. 1: 183-202, 1980. 22. Sawchenko, P. E. and M. I. Friedman. Sensory functions of the liver. Am. J. Physiol. 236: R5-R20, 1979. 23. Sawchenko, P. E., M. I. Friedman and R. M. Gold. Hepatic vagotomy selectively alters the diurnal distribution of food intake in female rats. Presentation to the East. Psychol. Assoc., Philadelphia, 1979. 24. Schneider, E. G., J. O. Davis, C. A. Robb, J. S. Baumber, J. A. Johnson and F. S. Wright. Lack of evidence for a hepatic osmoreceptor mechanism in conscious dogs. Am. J. Physiol. 218: 42--45, 1970. 25. Strandoy, J. W. and H. E. Williamson. Evidence for an hepatic role in the control of Na excretion. Proc. Soc. Exp. Biol. (NY) 133: 419-422, 1970. 26. Warner, R. G. und L. H. Breur. Nutrient requirements of the laboratory rat. In: Nutrient Requirements o f Laboratory Animals. Washington, D.C.: National Academy of Sciences, 1972, pp. 56--93.
Changes in Salt Intake After Abdominal Vagotomy: Evidence for Hepatic Sodium Receptors R O B E R T J. C O N T R E R A S 1 A N D T H E R E S E K O S T E N
Yale University, Department o f Psychology, Box I1A Yale Station, New Haven, CT 06520 R e c e i v e d 13 August 1980 CONTRERAS, R. J. AND T. KOSTEN. Changes in salt intake after abdominal vagotomy: Evidence for hepatic sodium receptors. PHYSIOL. BEHAV. 26(4) 575-582, 1981.--Several reports have suggested that the mammalian liver contains neural receptors, innervated by the vagus nerve, that monitor the sodium concentration and osmolarity of the portal circulation. These reports have been concerned primarily with either the neurophysiological identification of these receptors or their role in the short term control of urine output. Inasmuch as relatively little is known about the role of these receptors to consummatory behavior, we investigated the effects of hepatic vagotomy in rats on sodium intake as well as on sodium output. Hepatic vagotomized (HV) rats drank less NaCI solution (0.03, 0.1, 0.3M) in 24 hr during a two-bottle test with water than sham operated rats. Comparable differences in the intakes of either water, KCI or glucose solutions were not found. The two groups of rats did not differ in their intakes of water or 0.3 M NaCI after an injection of either an osmotic load (IP, 2 M NaCI, 1% BW), deoxycorticosterone acetate (SC, 5 nag) along with furosemide (SC, 10 mg), or after 10 days of sodium deprivation. Urinary sodium output was reduced in HV rats during sodium deprivation but not when the rats had adequate levels of sodium in their diet. Because circadian patterns of water and food intake as well as body weight growth of hepatic vagotomized rats were similar to those of control rats, general malaise due to surgery and generalized deficits in motivation were ruled out as explanations for the depressed daily drinking of NaCI solutions. These findings support the existence of hepatic sodium receptors and their possible involvement to the control of sodium regulation.
Sodium receptors Liver Cellular dehydration
Sodium intake
Sodiumappetite
THE mammalian liver is believed to contain neural receptors that are sensitive to changes in the osmolarity and sodium composition of the portal circulation [22]. Support for the presence of these receptors comes from neurophysiological studies of the hepatic branch of the vagus nerve. Multi-fiber activity of afferents in the hepatic branch of the vagus respond to changes in the osmotic pressure in rat [1] and to changes in sodium concentration in rabbit [2] of the perfusate in the portal circulation. Presumably, these receptors play a role in the maintenance of steady state levels of fluid and electrolytes by activating physiological and behavioral mechanisms that act to correct for deviations from homeostasis. In rats, changes in the osmolarity of the portal, but not systemic, circulation led to rapid, dramatic changes in urine volume [10]. After simultaneous infusions of water into the hepatic portal vein and hypertonic saline into the vena cava, urine volume increased; urine volume decreased when the pattern of simultaneous infusions were reversed. The implication was that osmoregulatory mechanisms of the liver may support and perhaps precede the activation of osmoregulatory mechanisms of the central nervous system.
Hepatic vagotomy
Urine output
The notion that the liver plays a role in osmoregulation was also supported by infusion studies in dogs [15, 16, 17] and in humans [10,11] despite some studies to the contrary [9,24]. Reasonably good evidence also exists for a physiological role of hepatic sodium receptors. Increases in the sodium concentration of the portal vein were followed by increases in urinary sodium loss [7,8]. Activation of hepatic receptors has also been shown to influence consumption. Blake and Lin [3] reported that infusions of hypertonic saline into the portal vein suppressed the short-term drinking of 0.15 M sodium chloride solution, but not of water, in water deprived rats. Comparable infusions into the vena cava were without significant effect. Hepatic information is most likely transmitted by the vagus to the central nervous system for integration of drinking behavior, inasmuch as the suppression of saline drinking by portal infusions was blocked by right cervical vagotomy. The data suggest that osmo- and sodium receptors of the liver may play a role in the initiation or modification of physiological and possibly behavioral regulatory responses. With the exception of Blake and Lin's report, relatively little is known about the contribution of hepatic signals to water and
1Send reprint requests to Dr. Robert J. Contreras, Department of Psychology, Yale University, Box 11A Yale Station, New Haven, CT
06520.
Copyright © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/040575-08502.00/0
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sodium intake. The present study was carried out to ascertain the long-term effects of resection of the hepatic branch of the vagus nerve on sodium balance, with an emphasis on sodium intake coupled to analyses of sodium output. METHOD
Thirty-six male albino rats of the Sprague-Dawley strain (Charles River) weighing 320-340 g at the start of the experiment were used. These animals were housed in individual cages in a colony room maintained on a 12:12 hr light/dark cycle. The animals were given Purina pellets and deionized water to consume ad libitum unless otherwise noted. To adapt the rats to their new surroundings and to the experimenters, all the animals were handled daily for a week prior to the start o f the experiment. The 36 rats were examined in sets of twelve at three different time periods. In each case six of the twelve rats received hepatic vagotomies and six received sham operations. All 36 animals were weighed daily for three days prior to and up to two months after surgery.
Sargerv Eighteen rats received hepatic vagotomies (HV) and the other 18 rats received sham operations (SC) under sodium pentobarbital anesthesia (Pentosol, IP, 50 mg/kg body weight). F o r each animal, a midline incision was made on the animal's abdominal surface immediately posterior to the xiphisternum. This longitudinal incision exposed the median and left lateral lobes of the liver that covered the esophagus and stomach. The liver was deflected to the surgeon's left with a cotton swab. The stomach was rotated clockwise to the animal's midline and held gently in place with retraction. This straightened and slightly stretched the esophagus facilitating the exposure of the hepatic branch of the vagus nerve. The hepatic branch of the vagus was identified with the aid of a Zeiss operating microscope. A section of the hepatic branch, about 5 mm long, was removed. After sectioning, the stomach was placed in its normal position and the muscles and skin were sutured. The animal was placed on a heating pad until it recovered from the operation. Sham operations consisted of similar manipulations of the liver and stomach but leaving the hepatic branch intact.
Food and Water Intakes To assess whether hepatic vagotomy had more general effects other than on sodium balance, day-time (7 a.m.-7 p.m.) and night-time food and water intakes were measured. For measurements of food intake each rat received a 50 g portion, for each 12 hr period, of Purina pellets on the floor of its cage. A paper towel was placed under each cage to collect food spillage. At the end of each 12 hr period the uneaten food pellets with spillage were weighed. These weights were subtracted from 50 to determine the amount of food consumed. A fresh supply of pellets and a clean paper towel were provided for the next set of measurements. The intakes of deionized water in 100 ml calibrated drinking cylinders were also measured every 12 hr.
Preference Testing Twenty-four rats, 12 in each group, were given preference tests beginning approximately two weeks after surgery. Two 100 ml graduated drinking cylinders, fitted with rubber stoppets and metal drinking spouts, were attached to the front of each animal's cage. One cylinder contained deionized water
and the other a test solution. Test stimuli were presented ~n an ascending order of concentration which consisted of va~ious molar concentrations of NaCI (0.03, 0.1, 0.3), glucose (0.1, 0.3) and KC1 (0.1, 0.3) solutions made from reagenl grade chemicals in deionized water. The positions of the tw¢ drinking cylinders were interchanged daily on a random schedule and the amount of solution consumed from each cylinder was measured every 24 hr. Each test solution was presented for two consecutive days, one day on the left and one day on the right side of the cage, to control for position habits. Every third day before the presentation of another test solution, both bottles were filled with deionized water. This was done to minimize possible influences of gustatory adaptation and osmotic stress on intake from the effects of successive testing. During preference testing all animals had free access to Purina pellets.
Urinao' Electrolytes Twelve rats, six in each group, were placed in metabolism cages (Acme) for 10 days and given a powdered, sodium-free diet (Nutritional Biochemicals) to consume ad lib. For the first five days, deionized water was also always available. Beginning on Day 5, the animals were adapted to a 23 hr schedule of water deprivation with food freely available and water available for l hr. F o r this 10 day period urine specimens were collected at 9:00 a.m. daily and stored in a freezer for future analyses of sodium and potassium concentrations by flame photometry. Water intake, urine volume, and body weight measures were also recorded daily. To further assess the effects of hepatic vagotomy on sodium output, another set of twelve rats were placed in metabolism cages for eight days. In this case the animals were maintained for three days on the sodium-free diet to which 1 g of sodium chloride crystals was added to 99 g of powdered diet. This 1% NaCI mixture is the concentration of salt normally found in Purina rat pellets. This was followed by two days of a low salt diet (0.12% NaC1 to the powered sodium-free diet). F o r the last three days the animals were given the sodium-free diet without NaCl supplementation. Body weight, water intake, and urine volume measures were recorded daily, and urine specimens were saved for analyses of electrolyte concentrations.
Sodium Appetite Sodium appetite was induced in one of three different ways. The first set of animals were fed a powdered sodiumfree diet in food cups in their home cages for 10 days. This period of sodium deprivation is sufficient to induce sodium appetite [5]. Beginning on Day 5 of sodium deprivation the animals were put on a 23 hr schedule of water deprivation with food available ad lib and water available for 1 hr. Following the 1 hr drinking period all food and water were removed from each animal's cage for the ensuing hour. Then the animals were presented with distilled water in calibrated drinking tubes for 10 min. This procedure was adopted to minimize the influence of (1) food ingestion on the contents and concentration of salivary electrolytes and (2) water deprivation on taste preference. Beginning on Day 10 of salt deprivation the rats were presented with a sapid solution or distilled water during the 10 min drinking period. The solutions used were 0.1 M glucose, 0.1 M NaCI, 0.1 M KC1 and 0.3 M NaCl. This procedure has been used successfully in the measurement of salt taste thresholds [4]. The second set of animals were anesthetized with ether
HEPATIC VAGOTOMY AND SODIUM I N T A K E and received an injection of the mineralocorticoid hormone, deoxycorticosterone acetate (s.c., 5 mg in 1 ml of oil) and an injection of the natriuretic drug, furosemide (s.c., 10 mg in 1 ml aqueous solution) [6] at 9:00 a.m. The animals were returned quickly to their home cages and given a two-bottle preference test between demineralized water and 0.3 M NaC1 solution with food pellets ad lib. The 24 hr intakes of these solutions were measured for two consecutive days. Similar measurements of intake were recorded for the third set of animals after a combination of three days of dietary sodium deprivation coupled with injections of furosemide (10 mg in 1 ml aqueous solution).
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Verification of Hepatic Vagotomy At the conclusion of the study the animals were sacrificed for autopsy with an injection of sodium pentobarbital. Autopsies were performed without knowledge of the animal's surgical treatment. The anterior trunk of the vagus was located with the aid of the operating microscope at the thoracic level and traced caudally to the point where the anterior trunk separates into hepatic and anterior gastric branches. For each rat records were kept describing the appearance of the esophagus, stomach and liver, and the interrupted or uninterrupted course of the hepatic branch to its target organ. We used the criterion established by Kraly [12] to judge the completeness of our vagal transections. T h a t is, whenever there was a case where any ambiguity concerning the continuity of the hepatic vagal branch, then that transection was classified as incomplete.
RESULTS
Verification of Hepatic Vagotomy With microscopic inspection hepatic innervation of the liver was found to be normal in every sham operated rat. Experimental rats could easily be identified by the general appearance of the liver, stomach and esophagus. In these cases the liver was frequently attached to either the esophagus or stomach by a new growth of tough connective tissue. This made it extremely difficult to visualize the portion of the hepatic branch remaining two months after surgery and determine if it was innervating the liver. On the basis of the criterion [12] we used to judge the completeness of the transections, three rats that received hepatic vagotomies were judged to have partial transections. Two of these animals were from the first set, one was from the second set, but none of the last set were classified as partial hepatic vagotomies. Statistical analyses were performed with the data from only those rats that were determined to be completely vagotomized.
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FIG. 1. Hepatic vagotomized (HV) and sham control (SC) animals were weighed routinely for 30 days following experimental and sham operations. The body weights plotted at five day intervals indicates that there were no differences between groups. Specifically, at the end of this 30 day period the change in body weight from preoperation weights were similar for both groups (t = 1.49, df= 17, p >0.20).
Body Weight and Food and Water Intakes Figure 1 represents the body weights of hepatic vagotomized (HV) and sham control (SC) rats for the first 30 days after surgery. At 30 days SC rats increased their body weights an average (+_S.E.) of 66.89_+7.09 g over their preoperative weights, while HV rats increased by 80.1___5.3 g. Nevertheless, these two groups of subjects did not differ in their relative increase in body weight at the end of this period (t= 1.49, df= 17, p>0.20). When measurements of food and water intake were recorded for the first eight days after surgery in one set of animals, 24 hr intakes were similar between groups. However, the HV rats tended to consume more food (8.43___0.64 g) and water (13.25-+1.95 ml) during the daytime as compared to the food (7.13___0.59 g) and water intakes of SC rats. These differences in food (t= 1.45, df=9, p<0.20) and water (t = 1.15, df=9, p<0.20) intake were not, however, significant. Figure 2 represents the ratio of diurnal water consumption to total intake. The largest differences in group means occurs in the t'n-st few days after surgery; by the end of the test period no differences are obvious. In another set of animals when daytime and nighttime intakes of food and water were measured two months after surgery no differences between groups were found.
Preference Testing The results of the two-bottle preference tests are given in Fig. 3. Analyses of the saline, glucose and KCI intakes, excluding the partial hepatic vagntomized rats, showed that the HV animals drank less of all the NaCI solutions, F(1,18) = 4.96, p <0.05, with significant differences occurring at 0.1 M NaCI (t=2.56, df=18, p<0.02) and at 0.3 M NaC1
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(t=2.19, df=18, p<0.05). Both HV and SC rats showed a marked preference for the 0.1 M solution, modest intakes of the 0.03 M solution and very little consumption o f the 0.3 M NaCl, F(2,36)=138.4, p<0.01. Altered intakes associated with hepatic vagotomy were restricted to NaCl solutions, as the animals from both groups showed similar preferences tor glucose and relative aversions for KCI solutions. Throughout the two-bottle tests there were no differences in water consumption either during the test days or on the intervening days when deionized water was only available.
Urinary Electrolytes Variations in the amount of sodium excreted in urine over l0 days of sodium deprivation for the second set of HV and SC rats are presented in Fig. 4. The patterns of sodium excretion were the same for both groups of rats throughout the 10 day period. The amount o f sodium eliminated in urine presumably decreased substantially on the first day of sodium deprivation from an average o f approximately 2.5 mEq/day as reported by Contreras and Hatton [5] to a value of 0.6 in the present study. Sodium output decreased even more on Day 2 from an average (-+S.E.) o f 0.64___0.11 mEq/day to 0.15-+0.02 in SC rats (t=5.00, df=5, p<0.01),
and from an average of 0.59__-0.08 mEq/day to 0.05--.0.01 in HV rats (t=6.46, df=4, p<0.01). This low level of sodium output was maintained for the remaining days of sodium deprivation, except on Day 5, when a 23 hr schedule of water deprivation was superimposed on sodium deprivation. Although sodium output in SC rats increased on Day 5 from an average ( _ S . E . ) of 0.08__-0.03 mEq on Day 4 to 0.21-+0.05 (t=3.1, dr=5, p<0.05), in HV rats the increase from 0.03_+0.01 mEq on Day 4 to 0.15_+0.07 (t= 1.84, dr=4, p>0.10) was not significant. On a daily basis the HV group always excreted less sodium than the SC group, although these differences on any one day were nonsignificant. Total sodium output for the entire 10 day period of dietary sodium deprivation was, however, less for the HV animals, F(1,9)=5.76, p<0.04. The two groups did not differ in urine volume or the amount of potassium excreted during this 10-day period. In the third set of animals metabolic testing was performed while they were on a gradual dietary reduction of sodium. With this schedule no differences in sodium loss were detected. Table 1 shows that there were no differences in total sodium loss while the animals were either on a diet containing 1% NaCl (t=0.71, df=10, p>0.10), on a 0.12% NaC1 diet (t =2.09, dr= 10, p>0.05), or on a sodium-free diet
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•
Control
TABLE 1 MEAN (± S.E.M.) TOTAL URINARY SODIUM EXCRETED (mEq) IN HEPATIC VAGOTOMIZED AND SHAM CONTROLS WHILE ON SODIUM-REPLETE AND SODIUM-FREE DIET Heaptic Vagotomized N =6
.4O
Sham Control N =6 i
o .2-
DAYS
FIG. 4. The second set of animals were fed a sodium-free diet for 10 days. Beginning on Day 5, the animals of both groups were adapted to a 23 hr schedule of water deprivation. Urine specimens were collected and the volume, and sodium and potassium content of the urine were measured (Atomic absorption spectrophotometry). Hepatic vagotomized (HV) rats excreted significantly less sodium over the 10 day deprivation period, F(1,9)=5.76, p<0.04. Comparable differences in potassium output or urine volume were not found.
1% NaCl Diet (E Days 1-3)
7.40 _+ 0.31
6.83 -+ 0.73
0.12% NaC1 Diet (~ Day 4--5)
1.07 -+ 0.18
1.59 -+ 0.17
N a + free diet (~ Days 6-8)
0.22 +- 0.09
0.21 -- 0.02
Total (~ Days 1-8)
8.60 _+ 0.59
8.67 +_ 0.36
Comparisons between groups were analyzed using t-tests for independent means. All t values indicated no differences between groups. Analyses of sodium and potassium concentrations were done by flame photometry.
580
CONTRERAS AN D KOSI [,iN TABLE 2 MEAN {_+ S.E.M.) INCREASE IN INTAKE OF 0.3 M NaCI RELATIVE TO BASELINE AFTERINDUCINGSODIUMAPPETITEBY VARIOUSMETHODSIN HEPATIC VAGOTOMIZEDAND SHAM-CONTROLRATS Hepatic Vagotomized
Sham Control
4.33 ± 0.88* N =6
6.33 ± 1.31" N=6
Sodium deprivation (10 min, ml)
DOCA + furosemide (24 hr; ml/100 g body weight) Sodium deprivation + furosemide (24 hr; ml/100 g body weight)
11.60 ± 2.11 N =5
17.17 ± 4.47 N=6
6.17 ± 3.03 N =6
8.00 ± 2.22 N =6
*These values are relative to a baseline water intake. All other values are relative to 0.3 M NaC1 intake prior to the sodium appetite challenge. Statistical comparisons between groups were analyzed using t-tests for independent means. All the t values indicated that there were no differences between the two groups of rats.
TABLE 3 MEAN (-+ S.E.M.) CUMULATIVEWATERINTAKES(ml PER 100 g OF BODY WEIGHT)AFTER CELLULAR DEHYDRATIONAT VARIOUS TIME PERIODS AFTER INITIATIONOF DRINKINGIN HEPATIC VAGOTOMIZEDAND SHAM CONTROLRATS
(t=0.37, df=10, p>0.10). The total amount of sodium excreted over this eight day period was the sam~ for both groups of rats (t =0.10, df = 10, p >0.10).
above water baselines respectively, showing no difference in their response to this sodium appetite challenge (t=1.27, dr= 10, p >0.10). Solutions of 0.1 M glucose and 0.1 M KCI were also presented during these 10 min sessions and revealed a similar drinking pattern of preference for glucose and aversion of KCI for both groups. A second set of animals were injected with a combination of DOCA and furosemide and their 24 hr intakes of 0.3 M NaCI were recorded. HV animals consumed 11.60±2.11 (ml/100 g body wt) more of this solution relative to their intake of this same solution prior to inducing sodium appetite. SC rats also increased their consumption to an average of 17.17+4.47 (ml/100 g body wt) which was found to be of similar magnitude as that of the HV rats (t=l.13, df=9, p>0.10). A combination of 3 days of dietary sodium deprivation and an injection of furosemide were given to a third set of animals. As in the above methods, both HV and SC rats showed a similar response of increased saline drinking of 0.3 M NaC1 as compared to their intakes of this solution before the sodium appetite challenge was presented. The increase in intake was 6.17+3.03 (ml/100 g body wt) for the HV rats and 8.00__-2.22 (ml/100 g body wt) for the SC rats but this difference was nonsignificant (t =0.49, dr= 10, p >0.10).
Sodium Appetite
Drinking after Cellular Dehydration
Similar increases in saline drinking occurred after inducing sodium appetite in each of the three methods as shown in Table 2. With the first method the first set of animals were maintained on a sodium-free diet for 15 days and were adapted to a 23 hr water deprivation schedule starting on the 5th day. They were given 10 min drinking tests one hour after their daily exposure to water. After 10 days of dietary sodium deprivation, 0.1 M NaCI was presented during the 10 min test period. HV animals increased their intake to 7.5±0.89 ml (mean± S.E.M.) above their water baseline; SC rats consumed 7.83-+2.46 ml more than their baseline water intake (t =0.13, df= 10, p>0.10). Sodium-free diet continued to be available for 4 more days at which time 10 min intakes of 0.3 M NaCI were recorded. Again, HV and SC rats increased their consumption to 4.33±0.88 ml and 6 . 3 3+- 1.31 ml
Latency to initiate drinking in response to injections of 2 M NaC1 were 23.5__+9.0 min for HV rats and 23.9±9,0 min for SC rats. All animals had a severe reaction to the injection and needed several minutes to recover from it. Once they recovered and began drinking, the total amount they consumed after each 30 min period did not differ between groups as shown in Table 3. When intakes during the 30 min periods were examined, HV rats drank somewhat less between the 60 and 90 minute period (0.76±0.25 ml/100 g body wt) than the SC rats (1.44±0.46 ml/100 g body wt) (t=l.30, dr=9, p>0.10). Consumption during all other periods and the cumulative intakes at the end of all the time blocks were similar for both groups of animals. Similar to the tests of sodium appetite, HV animals generally consumed slightly less than the SC rats.
Hepatic Vagotomized N=6 30 min 60 min 90 min 120 rain 24 hr
1.13 +- 0.31 2.04 - 0.48 2.80 ± 0.36 3.25 ± 0.33 7.20 -_+0.83
Sham Control N=6 1.56 _ 2.50 ± 3.85 ± 4.41 ± 8.36 ±
0.59 0.76 0.48 0.55 1.65
Statistical comparisons between groups were analyzed using t-tests for independent means. All the t values indicated that there were no differencds between the two groups of rats.
HEPATIC VAGOTOMY AND SODIUM INTAKE
DISCUSSION The results of this study demonstrated that rats that had the hepatic branch of the vagus nerve resected consumed significantly less of most NaC1 solutions while they were maintained on an ad libitum feeding schedule. However, the relative intakes of the various molar concentrations of NaCI solutions were similar for the two groups of rats. That is the animals consumed increasingly greater amounts with increasing concentration to a maximum at 0.1 M. Then at 0.3 M, N~/CI solution intake declined substantially. The two group~, of rats differed only in their absolute intakes, with the NaC1 intakes of hepatic vagotomized (HV) rats being somewhat depressed. Hepatic vagotomy did not affect the animals' intakes of water, KCI or glucose solutions. Thus, the lowered salt intakes of HV rats were not the result of general malaise due to the surgery. These data are consistent with the notion that sodium receptors are present in the portal circulation of the mammalian liver which may be important for sodium regulation. By detecting changes in the sodium concentration of plasma, these receptors may influence physiological and behavioral responses for maintaining steady state levels of sodium. Along with the reduced NaCI intake, measurements of hrinary output revealed a subtle change in the amount of 'sodium, but not of potassium, excreted after 10 days of sodium deprivation. Hepatic vagotomized rats excreted less sodium than sham control rats. From a glance this result was unexpected because it suggested that severing the hepatic receptors better equipped the animals to respond to salt deprivation. Although it was fortunate in this case, a change in sodium excretion compared to control levels represents a deviation from normality. Besides on Day 5 when water deprivation was superimposed on sodium deprivation control animals increased their sodium output. It was presumably maladaptive for HV rats not to increase their sodium output to compensate for a reduction in their water to food intake ratio. We considered that the modest deviation in total sodium output was not peculiar to sodium deprivation but might also be apparent while the animals were maintained on a diet that contained adequate sodium levels. When the diet contained 1% NaCl (the concentration in Purina Rat Pellets), a concentration that is approximately" 10 times the amount needed [26], or 0.12% NaCl, a low but adequate sodium level [14], no significant trends in urinary sodium output were observed. A single measurement of urinary sodium output taken at the end of a 24 hr period is perhaps insensitive to the dynamic variations that occur much earlier, although on an a priori basis, the pattern of sodium excretion during sodium deprivation does not necessarily have to be the same as that during conditions where the animals' diet contains normal sodium levels. According to an homeostatic viewpoint, subtle excursions from sodium balance during free-feeding conditions underlies the cumulative intakes of saline and water over the course of a day. Sodium deprivation represents one end of this continuum where the excursion from homeostasis is more abrupt and severe. The specificity of the effect of hepatic vagotomy on changes in consummatory behavior and metabolic response to sodium supports the existence of sodium receptors rather than osmoreceptors. Inasmuch as hepatic vagotomy presumably disrupted the normal function of postulated osmoreceptors, then in addition to NaC1 intake, KCI, water, and glucose intakes should have also been altered. As was
58! selectively reviewed in the introduction, hepatic sodium receptors have been indicated by electrophysiological [2] and infusion [7, 8, 19, 25] studies although the controversy of sodium versus osmoreceptors still persists. It is possible that both hepatic sodium and osmoreceptors exist and contribute to both sodium- and osmoregulation, but the conditions of the present study favored the contribution of sodium receptors. With the loss of these sodium receptors in the liver through denervation of the hepatic branch of the vagus, there were altered responses to the presence of sodium rather than a complete loss of all sodium regulatory mechanisms. The decreased saline drinking and sodium excretion supported this assessment as did the smaller, although nonsignificant, intakes of hypertonic saline after inducing sodium appetite. In regard to sodium appetite the system may have been driven to the extent that the operation of other sodium regulatory mechanisms overrode the lost contribution of the hepatic sodium receptors. Since latency was not measured in these tests, it is not known whether the HV animals were sluggish in their initiation to drink. It is also not known whether the severed afferent fibers of the hepatic vagus transmitted a false reading of the amount of sodium in plasma. The afferent signal concerning the sodium status of the portal circulation may be important to the cessation of NaC1 intake. A signal indicating a reduced sodium need may account for the decreased saline drinking both during ad libitum conditions and during sodium deprivation. In fact, when sodium appetite was induced by sodium deprivation, the reduced urinary sodium loss of HV rat may have lessened the need and appetite for sodium as compared to sham control rats, resulting in reduced intake. The data from the present investigation were consistent with those of Kraly, Gibbs and Smith [13] and Martin, Geiselman and Novin [18] indicating that transection of the hepatic branch of the vagus did not disrupt osmotically induced drinking. Moreover, hepatic vagotomy did not alter daily food and water intake, findings in agreement with those previously reported [1]. We also did not observe any changes in body weight in apparent contrast to that of earlier findings [23]. In sum, our data suggest that the hepatic branch of the vagus nerve is not crucial for the control of body weight, and food and water intake. Although the effects of hepatic vagotomy were modest, the specific changes in NaC1 intake are important for understanding the contribution of hepatic receptors to the maintenance of sodium homeostasis. The neural fibers that compose the hepatic branch are but a small proportion of the total number of fibers that constitute the abdominal vagus. We were interested in producing a selective lesion of the abdominal vagus and then look for both subtle as well as more general behavioral and physiological consequences. That selective hepatic vagutomy produced subtle rather than general consequences supports the proposed existence of hepatic sodium receptors and their possible involvement to the control of sodium regulation. Transection of the hepatic branch affected the daily drinking of NaC1 solutions across the concentrations tested, even though preference-aversion functions were unaffected. The drinking of water, KCI and glucose solutions were unaffected. Moreover, circadian patterns of intake of water and food were maintained as well as normal body weight growth after hepatic vagotomy. Ruled out as possible explanations are general malaise due to surgery, and generalized deficits in motivation. It is not known whether the observed changes in NaC1 intake were
582
CONTRERAS AND KOSTEN
due to afferent or efferent vagal function. There is precedent, h o w e v e r , for the suggestion that afferents o f the abdominal vagus mediate changes in drinking [13]. Precisely where and through what m e c h a n i s m the central n e r v o u s system integrates peripheral sensory information to modify drinking behavior is unknown. N e v e r t h e l e s s , there is good e v i d e n c e that hepatic sodium-and o s m o r e c e p t o r s activate neurons in the regions of the brain, possibly i n v o l v e d in the control of
drinking b e h a v i o r [20,21]. The present data support the contribution o f hepatic sodium receptors to behavioral regulation. ACKNOWLEDGEMENTS This research was supported by NIH Biomedical Research Support Grant 5-507-RR-07015 and by a grant from NIH, NHLBI Grant HL-24732 to R. J. Contreras.
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