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CCK-octapeptide injected in CSF and changes in feed intake and rumen motility
Physiology & Behavior, Vol. 24, pp. 943-950. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.
CCK-Octapeptide Injected in CSF and Changes in Feed Intake and Rumen Motility MARY ANNE DELLA-FERA
A N D C L I F T O N A. B A I L E
Department o f Clinical Studies at N e w Bolton Center, School o f Veterinary Medicine University o f Pennsylvania, Kennett Square, P A 19348 R e c e i v e d 20 D e c e m b e r 1979 DELLA-FERA, M. A. AND C. A. BAILE. CCK-octapeptide injected in CSF and changes in feed intake and rumen motility. PHYSIOL. BEHAV. 24(5) 943-950, 1980.--Cholecystokinin-octapeptide (CCK-OP) is a potent and specific suppressor of feeding in sheep. Continuous injections into the lateral cerebral ventricles in 2-hr fasted sheep of as little as 0.0l pmoles/min significantly decreased feed intake for up to 60 min, and doses of 0.638 and 2.55 pmoles/min completely suppressed feeding during the entire 3-hr injection. Since continuous intravenous injection of 2.55 pmoles/min did not affect feed intake, CCK-OP's effect on feeding is apparently mediated by the CNS. Several factors can limit feed intake independently of a primary effect on satiety, but CCK-OP-induced suppression of feed intake was due neither to decreased water intake in water-deprived sheep nor to elevated body temperature. The CNS can affect rumen function which in turn can alter feeding behavior. We also found that CCK-OP elicited changes in contraction amplitude, even in sheep without feed, similar to those which occurred with feeding during sCSF control injections. We hypothesize that CCK-OP is released in the CNS by meal-related signals from the periphery, and that it then acts in the brain to produce satiety and possibly to mediate other changes that occur during a meal. Cholecystokinin-octapeptide Sheep Water intake Rectal temperature
Lateral cerebral ventricular injections Rumen motility
N E U R O P E F F I D E involvement in the control of behaviors is currently the focus of a great deal of research. A number of peptides which have been isolated from the CNS and/or the periphery cause specific behavioral changes when applied centrally. Some of these include ACTH and vasopressin, which affect learning and memory processes [8]; enkephalins and endorphins, which affect pain perception [33]; angiotensin II, which affects drinking behavior [31]; and Factor S, which affects sleep [13]. Although several peptides can change feeding behavior, many of them do not appear to have a major role in the complex system involved in the control of feed intake. One peptide, however, has been in the forefront of research in the control of feed intake. Cholecystokinin, a 33 amino acid polypeptide originally isolated from duodenal extracts, was first shown by Gibbs, Young and Smith [17] to suppress feed intake in rats when administered peripherally. Other reports have subsequently appeared describing this effect in rats as well as in several other species [15, 16, 18, 37]. CCK and two other peptides having the biologically active part of the CCK molecule (CCK-octapeptide, CCK-OP; and caerulein, a decapeptide) have been shown to decrease feed intake without affecting water intake [17,26], and to elicit normal satiety behaviors in rats when administered systemically [1]. The structurally unrelated gastrointestinal (GI) peptides, secretin and pancreatic glucagon, had no effect; and gastrin, structurally similar to CCK but lacking the part of the molecule
CSF
Feed intake
which confers cholecystokinetic activity, was effective only at higher doses [32]. These early studies showed that CCK satisfied several criteria for a satiety agent, but a possible site of action was not apparent. Stem, Cudillo and Kruper [34], investigating the contributions of the ventromedial (VMH) and lateral (LH) hypothalamus to the suppression of feeding by CCK peptides, presented evidence that these peptides could act on the VMH to produce satiety, and Maddison [22] also showed that operant responding for food could be suppressed by lateral cerebral ventricular injections of CCK in rats. That CCK has been shown to evoke electrical changes in the VMH and L H [6] could also support a CNS site of action for CCK. Recently CCK-OP has been shown to be present in cerebrospinal fluid (CSF) [30] and in brain of a number of species [9, 10, 19, 30, 35, 36]. Because of the high concentrations [30] and the differential localization in the CNS [19] this peptide has been suggested to have specific functional roles in the brain, perhaps as a neurotransmitter or neurohumoral agent. I-n this paper we present the results of our investigation into the role of CCK-OP in the CNS control of feed intake. When administered as a continuous cerebral ventricular injection, pmole amounts of this peptide suppressed feeding in fasted sheep without increasing body temperature or decreasing water intake, and in addition, elicited changes in rumen motility consistent with changes that normally occur
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/050943-08502.00/0
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D E L L A - F E R A AND BAILE
during feeding. Thus CCK-OP is a potent and specific suppressor of feed intake and possibly may act to coordinate other changes that occur during feeding. Portions of these experiments have been published in a preliminary report [7]. METHOD
Surgical Preparation and Injection Procedure Lateral cerebral ventricular injections. Groups of four 30-kg castrated male sheep (wethers) were chronically implanted bilaterally with stainless steel cannula guides directed toward, but not entering, the lateral ventricles [28]. They were housed in individual pens in a room with constant light and temperature, with feed (Pelleted concentrate ration: medium ground corn, 44%; mixed ground hay, 40%; 0.44% protein soybean meal, 7%; dried molasses, 8%; trace minerals and vitamins, 0.5%.) and water ad lib. On treatment days the sheep were given fresh feed in order to trigger a meal, and were permitted to eat for one hr to assure satiation. The feed was then removed for 2, 4, 8, or 24 hrs. During this time sterile injection inserts were placed in the cannulas and adjusted so that the tips were in the ventricles. The inserts were attached by sterile tubing filled with the injection solution to a syringe. To determine ventricular depth the tubing was lowered below the sheep's head and the insert was adjusted until CSF flowed out. The sheep were injected unilaterally by means of syringe pumps beginning 15 rain before feed was returned. Rate of injection was 0.1 ml/min for 3 hrs. All solutions were filtered through millipore filters (Millipore Corp., Bedford, MA) prior to injection. Feed intakes were measured at intervals during and after injection and any abnormal behaviors were recorded. There were at least 24 hrs between treatment days. Intravenous injections. Thirty-kg wethers were prepared with silastic jugular catheters. They were housed and fed as above. On treatment days the sheep were given fresh feed and were permitted to eat for one hr. The feed was then removed for 2 hrs. Injections were begun 15 rain before feed was returned and lasted 3 hrs. Rate of injection was 0.1 ml/min. Feed intakes were measured at intervals during and after injection and any abnormal behaviors were recorded. There were at least 24 hrs between treatment days.
10.2 pmoles/min (N=4). Analysis of variance and Duncan's multiple range tests were used to determine differences between means. CCK-OP was also injected intravenously (IV) in 2-hr fasted sheep (N=8). The treatments were randomly assigned according to a replicated cross-over design and included sCSF and 2.55 pmoles/min CCK-OP dissolved in sCSF. Paired-t tests were used to determine differences between treatment means.
Lateral Cerebral Ventricular Injections ~1 CCK-OP and Water Intake ~f 2-hr Water-Deprived Sheep Treatments were randomly assigned to six 30-kg wethers with lateral ventricular guides according to a cross-over design and included sCSF and 0.638 pmoles/min CCK-OP. On experimental days the sheep were first given additional feed and water. One hr after presentation of the feed, the water was removed for 2 hrs. Fifteen rain before the water was returned, sterile injection inserts were placed in the cannulas and the injections were begun. Feed and water intakes were measured at intervals during and after injection. Experiments were at least 48 hrs apart. Palred-t tests were used to determine significant differences between treatment means.
Lateral Cerebral Ventricular Injections ~1 CCK-OP and Rectal Temperatures in Sheep Treatments were randomly assigned to four 30-kg wethers with lateral ventricular guides according to a replicated 2 × 2 factorial design with main effects of feed or no feed present during injection, and sCSF or 0.638 pmoles/min CCK-OP injections. On experimental days the sheep were first given additional feed for 1 hr, then the feed was removed. The injection procedure was similar to that in previous experiments. In this experiment injections were begun 105 rain after feed had been removed and lasted 75 min. Fifteen min after injections were started, feed was returned to those sheep to be fed. Rectal temperatures were measured at - 6 0 , 0, +75, and +135 rain from the start of injection, and feed intakes of sheep with feed present were measured at intervals during and after injection. Data were subjected to factorial analysis of variance.
Lateral Cerebral Ventricular Injections of CCK-OP and Feed Intake in Fasted Sheep
Lateral Ventrieular Injections ~f CCK-OP and Rumen Motility in Sheep
CCK-OP (Sincalide, E. R. Squibb and Sons, Inc., Batch No. UTA-860-H/TJ-5, molecular weight, 1143; approximately 80 IDU/3500 pmoles) was injected into the lateral ventricles of 2, 4, 8, and 24-hr fasted sheep. In a preliminary experiment, treatments of no handling, sham injection, and synthetic CSF (sCSF, [28]), injection were randomly assigned to determine whether lateral ventricular injections affected feed intake. Since there was no difference between treatments, sCSF injection was used as control treatment for all experiments. Treatments were randomly assigned according to a 4×4 Latin square design, and consisted of sCSF and three concentrations of CCK-OP dissolved in sCSF. There were two series of tests with CCK-OP in 2-hr fasted sheep. In the first series the doses were 0.159, 0.638, and 2.55 pmoles/min CCK-OP injected into the lateral ventricles (N=4). The second series, which was replicated with a second group of sheep, consisted of 0.01,0.04 and 0.159 pmoles/ min CCK-OP (N=8). The doses of CCK-OP tested in 4 and 24-hr fasted sheep were 0.159, 0.638 and 2.55 pmoles/min (N=4). The doses in 8-hr fasted sheep were 0.638, 2.55 and
Four 30-kg wethers were prepared with lateral ventricular guides and tureen cannulas, and were housed and fed as in the previous experiments. Small air-filled balloons connected to pressure transducers were inserted into the rumens. Rumen contraction rate and force were measured by means of a polygraph; only primary contractions were evaluated. Treatments were randomly assigned according to a replicated 2 × 2 factorial design with main effects of feed or no feed during injection, and sCSF or 0.638 pmoles/min CCK-OP injections. On experimental days the sheep were first given additional feed for 1 hr, then the feed was removed. The injection procedure was similar to that in the previous experiments. In this experiment injections were begun 105 min after feed had been removed and lasted 75 min. Fifteen rain after injections were started, feed was returned to those sheep to be fed. Rumen motility was measured continuously from 45 min before the start of injection to 60 min after the end of injection. Mean contraction rates and amplitudes were calculated for 15 rain intervals. To more clearly show the change from the basal condition (nonfed,
CCK-OCTAPEPTIDE I N J E C T E D IN C S F A N D SATIETY
945 i n j e c t i o n of C C K - O P i n t o t h e L a t e r a l V e n t r i c l e s of 2 h F a s t e d S h e e p
preinjection), cumulative contraction rates and amplitudes were expressed as percent of baseline ( - 6 0 to - 1 5 min time period). Feed intakes of sheep with feed present during injection were measured at intervals during and after injection. Data were subjected to factorial analysis of variance.
160140-
RESULTS
Lateral Cerebral Ventricular Injections of CCK-OP and Feed Intakes of Fasted Sheep CCK-OP injected into the lateral ventricles of 2-hr fasted sheep decreased feed intake in a dose-related manner over all doses tested, for at least one time period (Figs. 1 and 2). Feed intake was decreased 56% (0<0.05) and 55% (0<0.05) after 45 min of injection of 0.01 and 0.04 pmoles/min CCKOP, respectively: these were total doses of only 0.45 and 1.8 pmoles (Fig. 1). With injection of 0.638 and 2.55 pmoles/min feed intake was essentially zero during the entire injection period (Fig. 2), and in another study, higher doses also completely suppressed feeding. Thus by the end of injection at least 5 hrs had passed since the sheep had eaten their last meal, while a normal intermeal interval for sheep fed this diet is 2 hrs [2]. Seventy-five min after the end of injection feed intake was still reduced in sheep receiving doses of 0.638 and 2.55 pmoles/min, but no dose affected 24-hr feed intake. Intravenous injection of 2.55 pmoles/min CCK-OP in 2-hr fasted sheep did not affect feed intake at any time during or after injection. This dose was greater than 200 times the lowest effective lateral ventricular dose and caused a 100% reduction in feed intake during the entire 3-hr injection period when administered in the lateral ventricle. Increasing the length of fast before lateral ventricular injection resulted in increased rate of eating in the control treated sheep (Figs. 3, 4, 5). In 4-hr fasted sheep 0.159, 0.638 and 2.55 pmoles/min CCK-OP reduced feed intake for the first 2 hrs and 2.55 pmoles/min for a total of 3 hrs, Fig. 3. In 8-hr fasted sheep 10.2 pmoles/min reduced feed intake during the entire injection period and there was a trend for a decrease with 2.55 pmoles/min (0<0.10), Fig. 4. Sheep fasted 24 hrs before injection did not decrease feed intake significantly with any dose of CCK-OP but there was a slight trend for a decrease especially evident after 3 hrs of injection with 0.638 and 2.55 pmoles/min, Fig. 5. No dose of CCK-OP caused abnormal behaviors in the sheep during or after injection. Sheep usually stood quietly or remained recumbant but alert during the experiment.
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Three-hr continuous lateral ventricular injections of 0.638 pmoles/min CCK-OP had no effect on water intake in 2-hr water-deprived sheep, although there was a slight trend for increased water intake in the first hr of injection (Fig. 6). Feed intake was reduced by CCK-OP injections for the 3 hrs after injection; consequently, the feed/water ratio was significantly reduced by CCK-OP. The low feed intakes by control sheep and the apparent lack of effect of CCK-OP on feeding during the initial periods are the result of feed not being restricted before injection as in previous experiments with CCK-OP.
Lateral Cerebral Ventricular Injections of CCK-OP and Rectal Temperature in Sheep Intraventricular injections of 0.638 pmoles/min CCK-OP
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FIG. I. Sheep were fasted for 2 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=8. TsE, treatment mean standard error.
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FIG. 2. Sheep were fasted for 2 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=4. TsE, treatment mean standard error.
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FIG. 3. Sheep were fasted for 4 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=8. T~., treatment mean standard error.
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FIG. 5. Sheep were fasted for 24 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. N=4. T~, treatment mean standard error. Feed intake
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FIG. 4. Sheep were fasted for 8 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ---p<0.01. N=4. Ts,:, treatment mean standard error.
FIG. 6. Feed intake (g), water intake (ml), and feed/water ratio (mean -+ SEM) of 2-hr water-deprived sheep during 3 hr continuous injections of sCSF (open bars) and 0.638 pmoles/min CCK-OP (hatched bars) into the lateral cerebral ventricles. Injections were begun 15 min before water was returned. Feed was available at all times before and during injection. *Different from sCSF, p<0.05. h a d n o effect o n rectal t e m p e r a t u r e o f s h e e p d u r i n g a 75-min i n j e c t i o n p e r i o d (Fig. 7). A l s o rectal t e m p e r a t u r e d u r i n g feeding was n o t different f r o m t h a t for fasting c o n d i t i o n s for e i t h e r s C S F or C C K - O P injection. F e e d i n t a k e was d e c r e a s e d by C C K - O P for all time periods. A f t e r 15 min, feed i n t a k e s of s h e e p i n j e c t e d with C C K - O P w e r e only 40% (p =0.001) o f c o n t r o l feed i n t a k e s ; a n d r e m a i n e d similarly d e c r e a s e d e v e n for o n e h r a f t e r the injection was t e r m i n a t e d (p =0.05).
CCK-OCTAPEPTIDE INJECTED IN CSF AND SATIETY
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Lateral Ventricular Injections of CCK-OP and Rumen Motility in Sheep Rate of contraction. Feeding significantly increased rumen contraction rate during the injections irrespective of treatment (Fig. 8). Although contraction rate increased to a greater degree with sCSF injection, there were no significant differences between sCSF and CCK-OP treatments for either feed present or feed absent during injection. The greatest increases occurred in the first 15 rain after feed was returned (sCSF, fed vs fasted, p=0.009; CCK-OP, fed vs fasted, p=0.04). With sCSF injections, contraction rate remained increased throughout the injection period (fed vs fasted, 30 min, p=0.01; 60 rain, p=0.04); but with CCK-OP injection, contraction rate was increased for only 30 rain (p =0.05). Force of contraction. With control (sCSF) injections feeding decreased rumen contraction force 20% (p =0.01) 15 rain and 19% (p =0.007) 30 rain after feed was returned (Fig. 9). Although there appeared to be an earlier decrease, the difference was not significant. The apparent increase in contraction force above baseline in the nonfed sheep during control injections was most likely an anticipatory response of the sheep to visual cues of feeding, since the sheep receiving feed were in the same room as the sheep not receiving feed. There was no difference in contraction force between feed present and feed absent during CCK-OP injections. In the non-fed sheep CCK-OP significantly decreased rumen contraction force. Compared to control CCK-OP decreased contraction force 13% (p=0.005) 15 rain, 24% (p=0.003) 30 min, 24% (p =0.001) 45 rain, and 20% (p =0.006) 75 min after injection was begun. In the fed sheep, contraction force was not significantly affected by CCK-OP injection compared to sCSF injection. The greatest amount of feed was eaten in the first 30 rain after presentation, most likely accounting for the greater effects on rumen motility occurring during that time interval; with CCK-OP injections, however, feed intake was significantly decreased over all time periods (Fig. 10).
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FIG. 8. Rumen contraction rate (expressed as percent of baseline--mean rate during the -60 to - 15 min time period) of 2-hr fasted sheep with feed present (solid line) or feed absent (dashed line) during 75 rain injections of sCSF (circles) or 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned to those sheep to be fed (zero time). *Different from feed absent, same treatment, p<0.05. Ts~:, treatment standard error (%).
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FIG. 9. Rumen contraction amplitude (expressed as percent of baseline--meanamplitude during the -60 to - 15 rain time period) of 2-hr fasted sheep with feed present (solid line) or feed absent (dashed line; during 75 min injections of sCSF (circles) or 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned to those sheep to be fed (zero time). *Different from feed absent, same treatment, p<0.05. Different from sCSF, feed absent, p<0.05. TSE, treatment standard error (%).
948
D E L L A - F E R A AND BAILE
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FIG. 10. Feed intake (mean _+ SEM) of 2-hr fasted sheep during 75 min injections of sCSF (circles) of 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned (zero time). Rumen motility was measured concurrently. *Different from sCSF, p <0.05. **Different from sCSF. p<0.01.
DISCUSSION
Although recent studies have demonstrated the presence of CCK, primarily CCK-OP, in brain tissue of several species (e.g., [10, 19, 30]), as is true of a number of recently isolated brain peptides, the function of CCK-OP in the CNS is not yet known. The regional localization of CCK-OP in rat brain, as reported by Innis et al. [19], suggests specific functions for this peptide. The periaqueductal gray and medial hypothalamus, both of which are in close proximity to the ventricular system, had the highest concentration of CCKOP-containing cells; and the medial hypothalamus, of course, is known to be involved in the control of feed intake. The results of our studies are strong support for a role for brain CCK-OP in satiety. When injected continuously into the lateral ventricles over 45 rain, as little as 0.45 pmoles CCK-OP (0.01 pmoles/min) reduced feed intake 56% in 2-hr fasted sheep. CCK activity in human CSF has been reported to range from 0.004 to 0.06 pmoles/ml [30], and we have measured similar concentrations of CCK in sheep CSF (0.002 to 0.07 pmoles/ml, range; 0.013 _+ 0.003 pmoles/ml for satiated sheep) (Della F e r a , M. A., C. A. Baile, B. Schneider, manuscript in preparation). The concentration in the injection solution of the above dose of CCK-OP was only 0.1 pmoles/ml and if 0.2 ml of C S F were formed per min by the sheep [28] then in the steady-state condition the 0.1 mt injection per min would result in 0.05 pmoles/ml increase in concentration. Thus, we are approaching physiological levels with an effective dose of CCK-OP. Higher doses of CCK-OP, 0.638 and 2.55 pmoles/min, completely suppressed feeding of 2-hr fasted sheep during the entire 3-hr injection period. In effect this doubled their normal intermeal interval, since sheep under these conditions of housing and feeding have been shown to initiate a meal every 2 to 3 hrs [2]. The CCK-OP induced suppression of feeding also appears to interact in a consistent manner with the deprivation state of the sheep, since greater amounts of CCK-OP were required to decrease feeding with increased hunger drives. For example,
compared to the 0.01 pmoles/min dose effective in 2-hr fasted sheep, 0.059 pmoles/min was the lowest dose that decreased feed intake in 4-hr fasted sheep by a similar percent, and 10.2 pmoles/min was the only dose that decreased feed intake significantly in 8-hr fasted sheep. The effect of intraventricular injections of CCK peptides has also been investigated in the rat. Stern. Cudillo and Kruper [34] reported that 0. l0 and 0.25 ~g caerulein, 80 and 190 pmoles, respectively, decreased feed intake when injected as a bolus into the lateral ventricles of rats. In addition, Maddison [22] found that operant responding for food was depressed in rats after bolus intraventricular injections of CCK-33 (0.05, 0.10 and 0.20 I.D.U.: approximately 5, 10 and 20 pmoles, respectively). However, Nemeroffet al. [27] reported that as much as 3 ~g CCK-OP (2625 pmoles or 60 I.D.U.) injected in the lateral ventricles was required to inhibit stress-induced eating in rats. We have found that rats under various lighting and feeding schedules also do not respond to CCK-OP administered as a continuous lateral ventricular injection in doses comparable to those that were effective in sheep [7]; thus under these conditions, rats appear to be less sensitive to CCK-OP than are sheep. Because a number of factors have been shown to limit feed intake in ruminants, e.g., heat load [3], dehydration [38], gastric or intestinal distention [4], nutritional deficiencies [11], and illness [20], we investigated whether the CCK-OP induced decrease in feed intake was a secondary effect. Under the conditions of our experiments, factors that might become important in this regard would include heat load induced by a rise in body temperature, and dehydration, either of which might be caused by CCK-OP. Gengler et a/. [14] showed that cows whose body temperatures were increased by means of heating coils in the tureens, decreased both feed and water intakes. Bhattacharya and Warner [3] also reported a decrease in feed intake when body temperature was elevated by infusing warm water into the rumens of cows, thus the reduced feed intake reported by Gengler et al. [14] was apparently not secondary to the reduced water intake. In our experiment although rectal temperatures increased slightly (less than 0.5°C) during injections there was no difference between the two treatments. This increase in temperature was due in part to activity that occurs with feeding, and is similar to that previously reported to occur during the normal circadian cycle at the time of day the experiments were done [25]. Dehydration, resulting in an increased body fluid osmolarity, is known to inhibit feeding of animals, including ruminants. Utley et al. [38] showed that restricting water intake to 60% of ad lib levels reduced voluntary feed intake of steers. English [12] restricted water intake of sheep and also observed a decreased voluntary feed intake. Thus if CCK-OP acted primarily as an inhibitor of water intake, a reduction in feed intake would also occur. In our experiment, water intake of 2-hr water-deprived sheep was not affected by lateral ventricular injections of CCK-OP. Feed intakes were not significantly reduced until "after 3 hrs of injection, most likely due to the fact that they had not been feed-deprived before injection; however, the reduced feed intake after 3 hrs of injection of CCK-OP in conjunction with a water intake similar to that during control injections resulted in an apparent increase in the amount of water consumed per unit of feed. Normal water intake during injection of CCK-OP also indicates that the sheep were not depressed and that their activity was not impaired. The effect of CCK-OP on tureen motility adds an interest-
CCK-OCTAPEPTIDE INJECTED IN CSF AND SATIETY ing aspect to this peptide's activity in the CNS, since the CNS has been shown to affect various aspects of gastrointestinal function [29], which in turn could alter feeding behavior [24]. In our experiment under control conditions the amplitude of rumen contractions was decreased in the first 30 min of a meal following a 2-hr fast. As is shown in Fig. 9, CCK-OP injected continuously into the lateral ventricles caused a similar decrease in the amplitude of contractions. That this action on the rumen is independent of the effect of feeding is shown by the fact that even in nonfed sheep, injections of CCK-OP resulted in decreased amplitude of contractions. Also under control conditions, feeding increased contraction rate, as previously shown [5]. CCK-OP did not affect rumen contraction rate, independent of its effect on feeding. The smaller increase in contraction rate during the meal with CCK-OP injections compared to that with control injections is most likely due to the fact that feed intake during CCK-OP injections was less than half of that during control injections. Leek and Harding [21] have identified in each of the two gastric centers of the medulla two distinct circuits responsible for the control of rumen motility. One neuronal network is concerned with the rate of primary cycle movements and another is concerned with their form and amplitude. Because we have found a selective effect of CCK-OP on amplitude but not rate, we propose that this
949 peptide has an inhibitory effect on the neurons of the "form and amplitude circuit" but does not affect the "rate circuit" neurons. Alternatively, CCK-OP could affect higher centers which influence activity of the "form and amplitude circuit." Based on the results presented here, we propose that one function of CCK-OP in the brain is to produce satiety. We hypothesize that signals from the periphery, that are related to feeding activity, cause release of CCK-OP in the brain where it then acts in specific areas to cause termination of the meal and possibly other satiety-related changes, as indicated by its effect on rumen function. We also suggest that the CSF plays an active role in transporting CCK-OP to its site(s) of action, although CCK-OP might be released directly at its sites of action and the role of the CSF may only be passive removal of the peptide. However the proposed sites of action are likely to be periventricular, since suppression of feeding occurred as early as 15 min after beginning injection. ACKNOWLEDGEMENTS This work was supported in part by NIH Training Grant 0205110, NIH Grant AM-21413, and NSF Grant PCM 75-23128. We are grateful to Dr. S. J. Lucania of E. R. Squibb and Sons, Inc. for supplying the CCK-OP, and also to Debby Keim for technical assistance.
REFERENCES 1. Antin, J., J. Gibbs, J. Holt, R. C. Young and G. P. Smith. CCK elicits the complete behavioral sequence of satiety in rats. J. comp. physiol. Psychol. 89: 784-790, 1975. 2. Baile, C. A. Control of feed intake in ruminants. In: Digestion and Metabolism in the Ruminant, edited by I. W. McDonald and A. C. I. Warner. Armidale N.S.W., Australia: University of New England Publishing Unit, 1975, pp. 333-350. 3. Bhattacharya, A. N. and R. G. Warner. Influence of varying rumen temperature on central cooling or warming and on regulation of voluntary feed intake in dairy cattle. J. Dairy Sci. 51: 1481-1489, 1968. 4. Campling, R. C., M. Freer and C. C. Balch. Factors affecting the voluntary intake of food by cows. 2. The relationship between the voluntary intake of roughages, the amount of digesta in the reticulorumen, and the rate of disappearance of digesta from the alimentary tract. Br. J. Nutr. 15: 531-540, 1961. 5. Church, D. C. In: Digestive Physiology and Nutrition o f Ruminants. Vol. l, 2nd edition. Corvallis, Oregon: O and B Books, 1979, pp. 6%98. 6. Dafny, N. and E. D. Jacobson. CCK and central nervous regulation of appetite. In: Gastrointestinal Hormones, edited by J. C. Thompson. Austin: Univ. of Texas Press, 1975, pp. 643-649. 7. Della-Fera, M. A. and C. A. Baile. Cholecystokinin-octapeptide: continuous picomole injections into the cerebral ventricles suppress feeding. Science 206: 471-473, 1979. 8. deWeid, D. and W. H. Gispen. Behavioral effects of peptides. In: Peptides in Neurobiology, edited by H. Gainer. New York: Plenum Press, 1977, pp. 397-447. 9. Dockray, G. J. Immunochemical evidence of CCK-like peptides in brain. Nature 264: 568-570, 1976. 10. Dockray, G. J., R. A. Gregory, J. B. Huchinson, J. Ieuan Harris and M. J. Runswick. Isolation, structure and biological activity of two CCK-octapeptides from sheep brain. Nature 274: 711713, 1978. 11. Elliot, R. C. Voluntary intake of low protein diets by ruminants. II. Intake of food by sheep. J. agric. Sci. Cambridge 69: 383390, 1967. 12. English, P. B. Water and electrolyte balance in sheep. I. External balances of water, sodium, potassium, and chloride. Res. vet. Sci. 7: 233-239, 1966.
13. Fencl, V., G. Kosky and J. R. Pappenheimer. Factors in cerebrospinal fluid from goats that affect sleep and activity in rats. J. Physiol., Lond. 216: 565-589, 1971. 14. Gengler, W. R., F. A. Martz, H. D. Johnson, G. F. Kraus and L. Hahn. Effect of temperature on food and water intake and rumen fermentation. J. Dairy Sci. 53: 434-437, 1970. 15. Gibbs, J., J. P. Falasco and P. F. McHugh. Cholecystokinindecreased feed intake in rhesus monkeys. Am. J. Physiol. 230: 15-18, 1976. 16. Gibbs, J. and G. P. Smith. Cholecystokinin and satiety in rats and rhesus monkeys. Am. J. clin. Nutr. 30: 758-761, 1977. 17. Gibbs, J., R. C. Young and G. P. Smith. Cholecystokinindecreased food intake in rats. J. comp. physiol. Psychol. 84: 488-495, 1973. 18. Houpt, T. R., S. M. Anika and N. C. Wolff. Satiety effects of cholecystokinin and caerulein in rabbits. Am. J. Physiol. 235: R23-R28, 1978. 19. Innis, R. B., F. M. A. Correa, G. R. Uhl, B. Schneider and S. H. Snyder. Cholecystokinin octapeptide-like immunoreactivity: Histochemical localization in rat brain. Proc. natn. Acad. Sci. U.S.A. 76: 521-525, 1979. 20. Kronfeld, D. S. Ketone body metabolism, its control, and its implications in pregnancy toxemia, acetonaemia, and feeding standards. In: Physiology o f Digestion and Metabolism in the Ruminant, Proc. int. Symp., 3rd, Cambridge, England, 1969; edited by A. T. Phillipson. Newcastle, England: Oriel Press, 1970, pp. 566-583. 21. Leek, B. F. and R. H. Harding. Sensory nervous receptors in the ruminant stomach and the reflex control of reticulo-ruminal motility. In: Digestion and Metabolism in the Ruminant, edited by I. W. McDonald and A. C. I. Warner, Proc. IV int. Symp. Ruminant Physiology, Armidale, N.S.W., Australia: Univ. New England Publishing Unit, 1975, pp. 60-76. 22. Maddison, S. Intraperitoneal and intracranial cholecystokinin depress operant responding for food. Physiol. Behav. 19: 81% 824, 1977. 23. Martin, F. H., J. R. Seoane and C. A. Baile. Feeding in satiated sheep elicited by intraventricular injections of CSF from fasted sheep. Life Sci. 13: 177-184, 1973.
950
24. McHugh, P. R. and T. H. Moran. The gastro-enteric coordination active in short-term (meal time) satiety of Rhesus monkeys. East. Psychol. Ass. Ann. meet., Philadelphia, PA, April 18-21. 1979, p. 17. 25. Mendel, V. E. and G. V. Raghavan. A study of diurnal temperature patterns in sheep. J. Physiol. 174: 206-216, 1964. 26. Mueller, K. and S. Hsiao. Specificity of CCK satiety effect: reduction of food but not water intake. Pharmac. Biochem. Behav. 6: 643-646, 1977. 27. Nemeroff, C. B., A. J. Osbahr III, G. Bissett, G. Jahnke, M. A. Lipton and A. J. Prange, Jr. Cholecystokinin inhibits tail pinchinduced eating in rats. Science 200: 793-794, 1978. 28. Pappenheimer, J. R., S. H. Heisey, E. F. Jordan and J. de C. Downer. Perfusion of the cerebral ventricular system in unanesthetized goats. A m . J. Physiol. 203: 763-774, 1962. 29. Ralph, T. L. and P. E. Sawchenko. Differential effects of lateral and ventromedial hypothalamic lesions on gastrointestinal transit in the rat. Brain Res. Bull. 3:11-14, 1978. 30. Rehfeld, J. F. and C. Kruse-Larsen. Gastrin and CCK in human CSF. Brain Res. 155: 1%26, 1978. 31. Simpson, J. B., M. L. Mangiapane and H. D. Dellman. Central receptor sites for angiotensin-induced drinking. Fedn Proc. 37: 2676-2682, 1978.
DELI~A-FERA AND BAILE
32. Smith, G. P., J. Gibbs and R. C. Young. Cholecystokinin and intestinal satiety in the rat. Fedn Proc. 33: 1146-1149, lC)74. 33. Snyder, S. H. Peptide neurotransmitter candidates in the brain: Focus on Enkephalin, Angiotensin II, and Neurotensin. In: Th~ Hypothalamus, edited by S. Reichlin, R. J. Baldessarini and .!. B. Martin. New York: Raven Press, 1978, pp. 233-244. 34. Stern, J. J., C. A. Cudillo and J. Kruper. Ventromedial hypothalamus and short-term suppression of feeding by caerulein in male rats..I, comp. physical, P~y~hol. 90: 484-490. 1976, 35. Straus, E., J. E. Muller, H. Schoi, F. Paronetto and R. S. Yalow. lmmunohistochemical localization in rabbit brain of a peptide resembling the COOH terminal octapeptide of CCK. Pro~'. mJtn. A('ad. Sci. U.S.A. 74: 3033-3034. t974 36. Straus, E. and R. S. Yalow. CCK in br.,dns uf ubese and nonobese mice. Science 203: 68--70, 1979. 37. Sturdevant, A. L. and H. Goetz. Cholecystokinin both stimulates and inhibits human food intake. Nature 261: 714-715, 1976. 38. Utley, P. F., N. W. Bradley and J. A. Boling. Effect of restricted water intake on feed intake, nutrient digestability and nitrogen metabolism in steers..I. A~tim. S~i. 31: 130-135, 1970,
CCK-Octapeptide Injected in CSF and Changes in Feed Intake and Rumen Motility MARY ANNE DELLA-FERA
A N D C L I F T O N A. B A I L E
Department o f Clinical Studies at N e w Bolton Center, School o f Veterinary Medicine University o f Pennsylvania, Kennett Square, P A 19348 R e c e i v e d 20 D e c e m b e r 1979 DELLA-FERA, M. A. AND C. A. BAILE. CCK-octapeptide injected in CSF and changes in feed intake and rumen motility. PHYSIOL. BEHAV. 24(5) 943-950, 1980.--Cholecystokinin-octapeptide (CCK-OP) is a potent and specific suppressor of feeding in sheep. Continuous injections into the lateral cerebral ventricles in 2-hr fasted sheep of as little as 0.0l pmoles/min significantly decreased feed intake for up to 60 min, and doses of 0.638 and 2.55 pmoles/min completely suppressed feeding during the entire 3-hr injection. Since continuous intravenous injection of 2.55 pmoles/min did not affect feed intake, CCK-OP's effect on feeding is apparently mediated by the CNS. Several factors can limit feed intake independently of a primary effect on satiety, but CCK-OP-induced suppression of feed intake was due neither to decreased water intake in water-deprived sheep nor to elevated body temperature. The CNS can affect rumen function which in turn can alter feeding behavior. We also found that CCK-OP elicited changes in contraction amplitude, even in sheep without feed, similar to those which occurred with feeding during sCSF control injections. We hypothesize that CCK-OP is released in the CNS by meal-related signals from the periphery, and that it then acts in the brain to produce satiety and possibly to mediate other changes that occur during a meal. Cholecystokinin-octapeptide Sheep Water intake Rectal temperature
Lateral cerebral ventricular injections Rumen motility
N E U R O P E F F I D E involvement in the control of behaviors is currently the focus of a great deal of research. A number of peptides which have been isolated from the CNS and/or the periphery cause specific behavioral changes when applied centrally. Some of these include ACTH and vasopressin, which affect learning and memory processes [8]; enkephalins and endorphins, which affect pain perception [33]; angiotensin II, which affects drinking behavior [31]; and Factor S, which affects sleep [13]. Although several peptides can change feeding behavior, many of them do not appear to have a major role in the complex system involved in the control of feed intake. One peptide, however, has been in the forefront of research in the control of feed intake. Cholecystokinin, a 33 amino acid polypeptide originally isolated from duodenal extracts, was first shown by Gibbs, Young and Smith [17] to suppress feed intake in rats when administered peripherally. Other reports have subsequently appeared describing this effect in rats as well as in several other species [15, 16, 18, 37]. CCK and two other peptides having the biologically active part of the CCK molecule (CCK-octapeptide, CCK-OP; and caerulein, a decapeptide) have been shown to decrease feed intake without affecting water intake [17,26], and to elicit normal satiety behaviors in rats when administered systemically [1]. The structurally unrelated gastrointestinal (GI) peptides, secretin and pancreatic glucagon, had no effect; and gastrin, structurally similar to CCK but lacking the part of the molecule
CSF
Feed intake
which confers cholecystokinetic activity, was effective only at higher doses [32]. These early studies showed that CCK satisfied several criteria for a satiety agent, but a possible site of action was not apparent. Stem, Cudillo and Kruper [34], investigating the contributions of the ventromedial (VMH) and lateral (LH) hypothalamus to the suppression of feeding by CCK peptides, presented evidence that these peptides could act on the VMH to produce satiety, and Maddison [22] also showed that operant responding for food could be suppressed by lateral cerebral ventricular injections of CCK in rats. That CCK has been shown to evoke electrical changes in the VMH and L H [6] could also support a CNS site of action for CCK. Recently CCK-OP has been shown to be present in cerebrospinal fluid (CSF) [30] and in brain of a number of species [9, 10, 19, 30, 35, 36]. Because of the high concentrations [30] and the differential localization in the CNS [19] this peptide has been suggested to have specific functional roles in the brain, perhaps as a neurotransmitter or neurohumoral agent. I-n this paper we present the results of our investigation into the role of CCK-OP in the CNS control of feed intake. When administered as a continuous cerebral ventricular injection, pmole amounts of this peptide suppressed feeding in fasted sheep without increasing body temperature or decreasing water intake, and in addition, elicited changes in rumen motility consistent with changes that normally occur
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/050943-08502.00/0
944
D E L L A - F E R A AND BAILE
during feeding. Thus CCK-OP is a potent and specific suppressor of feed intake and possibly may act to coordinate other changes that occur during feeding. Portions of these experiments have been published in a preliminary report [7]. METHOD
Surgical Preparation and Injection Procedure Lateral cerebral ventricular injections. Groups of four 30-kg castrated male sheep (wethers) were chronically implanted bilaterally with stainless steel cannula guides directed toward, but not entering, the lateral ventricles [28]. They were housed in individual pens in a room with constant light and temperature, with feed (Pelleted concentrate ration: medium ground corn, 44%; mixed ground hay, 40%; 0.44% protein soybean meal, 7%; dried molasses, 8%; trace minerals and vitamins, 0.5%.) and water ad lib. On treatment days the sheep were given fresh feed in order to trigger a meal, and were permitted to eat for one hr to assure satiation. The feed was then removed for 2, 4, 8, or 24 hrs. During this time sterile injection inserts were placed in the cannulas and adjusted so that the tips were in the ventricles. The inserts were attached by sterile tubing filled with the injection solution to a syringe. To determine ventricular depth the tubing was lowered below the sheep's head and the insert was adjusted until CSF flowed out. The sheep were injected unilaterally by means of syringe pumps beginning 15 rain before feed was returned. Rate of injection was 0.1 ml/min for 3 hrs. All solutions were filtered through millipore filters (Millipore Corp., Bedford, MA) prior to injection. Feed intakes were measured at intervals during and after injection and any abnormal behaviors were recorded. There were at least 24 hrs between treatment days. Intravenous injections. Thirty-kg wethers were prepared with silastic jugular catheters. They were housed and fed as above. On treatment days the sheep were given fresh feed and were permitted to eat for one hr. The feed was then removed for 2 hrs. Injections were begun 15 rain before feed was returned and lasted 3 hrs. Rate of injection was 0.1 ml/min. Feed intakes were measured at intervals during and after injection and any abnormal behaviors were recorded. There were at least 24 hrs between treatment days.
10.2 pmoles/min (N=4). Analysis of variance and Duncan's multiple range tests were used to determine differences between means. CCK-OP was also injected intravenously (IV) in 2-hr fasted sheep (N=8). The treatments were randomly assigned according to a replicated cross-over design and included sCSF and 2.55 pmoles/min CCK-OP dissolved in sCSF. Paired-t tests were used to determine differences between treatment means.
Lateral Cerebral Ventricular Injections ~1 CCK-OP and Water Intake ~f 2-hr Water-Deprived Sheep Treatments were randomly assigned to six 30-kg wethers with lateral ventricular guides according to a cross-over design and included sCSF and 0.638 pmoles/min CCK-OP. On experimental days the sheep were first given additional feed and water. One hr after presentation of the feed, the water was removed for 2 hrs. Fifteen rain before the water was returned, sterile injection inserts were placed in the cannulas and the injections were begun. Feed and water intakes were measured at intervals during and after injection. Experiments were at least 48 hrs apart. Palred-t tests were used to determine significant differences between treatment means.
Lateral Cerebral Ventricular Injections ~1 CCK-OP and Rectal Temperatures in Sheep Treatments were randomly assigned to four 30-kg wethers with lateral ventricular guides according to a replicated 2 × 2 factorial design with main effects of feed or no feed present during injection, and sCSF or 0.638 pmoles/min CCK-OP injections. On experimental days the sheep were first given additional feed for 1 hr, then the feed was removed. The injection procedure was similar to that in previous experiments. In this experiment injections were begun 105 rain after feed had been removed and lasted 75 min. Fifteen min after injections were started, feed was returned to those sheep to be fed. Rectal temperatures were measured at - 6 0 , 0, +75, and +135 rain from the start of injection, and feed intakes of sheep with feed present were measured at intervals during and after injection. Data were subjected to factorial analysis of variance.
Lateral Cerebral Ventricular Injections of CCK-OP and Feed Intake in Fasted Sheep
Lateral Ventrieular Injections ~f CCK-OP and Rumen Motility in Sheep
CCK-OP (Sincalide, E. R. Squibb and Sons, Inc., Batch No. UTA-860-H/TJ-5, molecular weight, 1143; approximately 80 IDU/3500 pmoles) was injected into the lateral ventricles of 2, 4, 8, and 24-hr fasted sheep. In a preliminary experiment, treatments of no handling, sham injection, and synthetic CSF (sCSF, [28]), injection were randomly assigned to determine whether lateral ventricular injections affected feed intake. Since there was no difference between treatments, sCSF injection was used as control treatment for all experiments. Treatments were randomly assigned according to a 4×4 Latin square design, and consisted of sCSF and three concentrations of CCK-OP dissolved in sCSF. There were two series of tests with CCK-OP in 2-hr fasted sheep. In the first series the doses were 0.159, 0.638, and 2.55 pmoles/min CCK-OP injected into the lateral ventricles (N=4). The second series, which was replicated with a second group of sheep, consisted of 0.01,0.04 and 0.159 pmoles/ min CCK-OP (N=8). The doses of CCK-OP tested in 4 and 24-hr fasted sheep were 0.159, 0.638 and 2.55 pmoles/min (N=4). The doses in 8-hr fasted sheep were 0.638, 2.55 and
Four 30-kg wethers were prepared with lateral ventricular guides and tureen cannulas, and were housed and fed as in the previous experiments. Small air-filled balloons connected to pressure transducers were inserted into the rumens. Rumen contraction rate and force were measured by means of a polygraph; only primary contractions were evaluated. Treatments were randomly assigned according to a replicated 2 × 2 factorial design with main effects of feed or no feed during injection, and sCSF or 0.638 pmoles/min CCK-OP injections. On experimental days the sheep were first given additional feed for 1 hr, then the feed was removed. The injection procedure was similar to that in the previous experiments. In this experiment injections were begun 105 min after feed had been removed and lasted 75 min. Fifteen rain after injections were started, feed was returned to those sheep to be fed. Rumen motility was measured continuously from 45 min before the start of injection to 60 min after the end of injection. Mean contraction rates and amplitudes were calculated for 15 rain intervals. To more clearly show the change from the basal condition (nonfed,
CCK-OCTAPEPTIDE I N J E C T E D IN C S F A N D SATIETY
945 i n j e c t i o n of C C K - O P i n t o t h e L a t e r a l V e n t r i c l e s of 2 h F a s t e d S h e e p
preinjection), cumulative contraction rates and amplitudes were expressed as percent of baseline ( - 6 0 to - 1 5 min time period). Feed intakes of sheep with feed present during injection were measured at intervals during and after injection. Data were subjected to factorial analysis of variance.
160140-
RESULTS
Lateral Cerebral Ventricular Injections of CCK-OP and Feed Intakes of Fasted Sheep CCK-OP injected into the lateral ventricles of 2-hr fasted sheep decreased feed intake in a dose-related manner over all doses tested, for at least one time period (Figs. 1 and 2). Feed intake was decreased 56% (0<0.05) and 55% (0<0.05) after 45 min of injection of 0.01 and 0.04 pmoles/min CCKOP, respectively: these were total doses of only 0.45 and 1.8 pmoles (Fig. 1). With injection of 0.638 and 2.55 pmoles/min feed intake was essentially zero during the entire injection period (Fig. 2), and in another study, higher doses also completely suppressed feeding. Thus by the end of injection at least 5 hrs had passed since the sheep had eaten their last meal, while a normal intermeal interval for sheep fed this diet is 2 hrs [2]. Seventy-five min after the end of injection feed intake was still reduced in sheep receiving doses of 0.638 and 2.55 pmoles/min, but no dose affected 24-hr feed intake. Intravenous injection of 2.55 pmoles/min CCK-OP in 2-hr fasted sheep did not affect feed intake at any time during or after injection. This dose was greater than 200 times the lowest effective lateral ventricular dose and caused a 100% reduction in feed intake during the entire 3-hr injection period when administered in the lateral ventricle. Increasing the length of fast before lateral ventricular injection resulted in increased rate of eating in the control treated sheep (Figs. 3, 4, 5). In 4-hr fasted sheep 0.159, 0.638 and 2.55 pmoles/min CCK-OP reduced feed intake for the first 2 hrs and 2.55 pmoles/min for a total of 3 hrs, Fig. 3. In 8-hr fasted sheep 10.2 pmoles/min reduced feed intake during the entire injection period and there was a trend for a decrease with 2.55 pmoles/min (0<0.10), Fig. 4. Sheep fasted 24 hrs before injection did not decrease feed intake significantly with any dose of CCK-OP but there was a slight trend for a decrease especially evident after 3 hrs of injection with 0.638 and 2.55 pmoles/min, Fig. 5. No dose of CCK-OP caused abnormal behaviors in the sheep during or after injection. Sheep usually stood quietly or remained recumbant but alert during the experiment.
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Three-hr continuous lateral ventricular injections of 0.638 pmoles/min CCK-OP had no effect on water intake in 2-hr water-deprived sheep, although there was a slight trend for increased water intake in the first hr of injection (Fig. 6). Feed intake was reduced by CCK-OP injections for the 3 hrs after injection; consequently, the feed/water ratio was significantly reduced by CCK-OP. The low feed intakes by control sheep and the apparent lack of effect of CCK-OP on feeding during the initial periods are the result of feed not being restricted before injection as in previous experiments with CCK-OP.
Lateral Cerebral Ventricular Injections of CCK-OP and Rectal Temperature in Sheep Intraventricular injections of 0.638 pmoles/min CCK-OP
120 17
180 19
240 28
FIG. I. Sheep were fasted for 2 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=8. TsE, treatment mean standard error.
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_y
li
60 19
120 16
180 ,6
2,~0 29
~ a m n
rain Ts~
15 30 19 18
FIG. 2. Sheep were fasted for 2 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=4. TsE, treatment mean standard error.
946
DELLA-FERA / Injection of C C K - O P into t h e Lateral Ventricles of 4 h Fasted S h e e p
2804
/
520-
AND BAILE
I n j e c t i o n of C C K - O P into t h e L a t e r a l V e n t r i c l e s of 2 4 h F a s t e d S h e e p ,.
-----sCSF
--~--.159 Pm.oles C C K . O P
2401
--o--.638
480-
mm
--e-- 2.55
-"
440' 400"
ff/S~
i60] ea'~/
360"
U.
~ 320"
--.--sCSF
40
min TsE
"
"
A -
A -
15 22 23
240"
A A ]
A t--
do
--1
'
'
120 30
27
240 38
min
1
3
TeE 46 48
FIG. 3. Sheep were fasted for 4 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ--p<0.01. N=8. T~., treatment mean standard error.
--~--.159 ~ - ~ C C K - O P --o-- 638 man --'--'2.55
200.
'
180 32
ff _~r/ ~"
6
'o
56
• ,,~-RF --o--.838 ~-:-~----3-C C K - O P ._e.. 2.55 rain --,--10.2
32&
1 8' 0
53
66
2 4'0
67
FIG. 5. Sheep were fasted for 24 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. N=4. T~, treatment mean standard error. Feed intake
Injection of C C K - O P into the Lateral Ventricles of 8 h Fasted Sheep ~ 360'
1 2' 0
Water Intake
(g}
Feed/Water
(el)
Iglml)
40. 40
.8"
30
.6"
Am / /
~
/
30"
280' *- 240'
* 20
.ao .
200"
~ 160"
/?
10
10"
e
15 60 180
I~ 120"
i
.4-
!
.2 °
15 60 180 Time
15 60 180
(min)
80" 40"
I--,%
,*
i
rain
15 ~0
Tst 46 49
l
,I-
|
dO
50
120
46
180 48
240 ! 56
FIG. 4. Sheep were fasted for 8 hrs before injection and feed was returned 15 min after beginning injection (zero time). They were injected for 3 hrs at a rate of 0.1 ml/min. Treatment means without a common letter are different, AB--p<0.05, YZ---p<0.01. N=4. Ts,:, treatment mean standard error.
FIG. 6. Feed intake (g), water intake (ml), and feed/water ratio (mean -+ SEM) of 2-hr water-deprived sheep during 3 hr continuous injections of sCSF (open bars) and 0.638 pmoles/min CCK-OP (hatched bars) into the lateral cerebral ventricles. Injections were begun 15 min before water was returned. Feed was available at all times before and during injection. *Different from sCSF, p<0.05. h a d n o effect o n rectal t e m p e r a t u r e o f s h e e p d u r i n g a 75-min i n j e c t i o n p e r i o d (Fig. 7). A l s o rectal t e m p e r a t u r e d u r i n g feeding was n o t different f r o m t h a t for fasting c o n d i t i o n s for e i t h e r s C S F or C C K - O P injection. F e e d i n t a k e was d e c r e a s e d by C C K - O P for all time periods. A f t e r 15 min, feed i n t a k e s of s h e e p i n j e c t e d with C C K - O P w e r e only 40% (p =0.001) o f c o n t r o l feed i n t a k e s ; a n d r e m a i n e d similarly d e c r e a s e d e v e n for o n e h r a f t e r the injection was t e r m i n a t e d (p =0.05).
CCK-OCTAPEPTIDE INJECTED IN CSF AND SATIETY
947
..--2".. ;::: :':-.7 "cs'
4o.5.1 0 v
=s
40.0' _
Feed p r e s e n t
~ CCK-OP
Feed a b s e n t
" 0.638 pmoleslmin
180.160.
sCSF /
a ~ a
~CCK-OP
/
. 0.638pmoles/mm
~ O 140"
d z 120"
£ 39.0-
38.5
-co-go-
100Injection
80-
40 ~
•
60
6
o 6 2'o go 6'o go lO'O Time
(min)
FIG. 7. Rectal temperature (C; mean -+ SEM) of sheep during 75 min injections of sCSF or 0.638 pmoles/min CCK-OP into the lateral cerebral ventricles.
Lateral Ventricular Injections of CCK-OP and Rumen Motility in Sheep Rate of contraction. Feeding significantly increased rumen contraction rate during the injections irrespective of treatment (Fig. 8). Although contraction rate increased to a greater degree with sCSF injection, there were no significant differences between sCSF and CCK-OP treatments for either feed present or feed absent during injection. The greatest increases occurred in the first 15 rain after feed was returned (sCSF, fed vs fasted, p=0.009; CCK-OP, fed vs fasted, p=0.04). With sCSF injections, contraction rate remained increased throughout the injection period (fed vs fasted, 30 min, p=0.01; 60 rain, p=0.04); but with CCK-OP injection, contraction rate was increased for only 30 rain (p =0.05). Force of contraction. With control (sCSF) injections feeding decreased rumen contraction force 20% (p =0.01) 15 rain and 19% (p =0.007) 30 rain after feed was returned (Fig. 9). Although there appeared to be an earlier decrease, the difference was not significant. The apparent increase in contraction force above baseline in the nonfed sheep during control injections was most likely an anticipatory response of the sheep to visual cues of feeding, since the sheep receiving feed were in the same room as the sheep not receiving feed. There was no difference in contraction force between feed present and feed absent during CCK-OP injections. In the non-fed sheep CCK-OP significantly decreased rumen contraction force. Compared to control CCK-OP decreased contraction force 13% (p=0.005) 15 rain, 24% (p=0.003) 30 min, 24% (p =0.001) 45 rain, and 20% (p =0.006) 75 min after injection was begun. In the fed sheep, contraction force was not significantly affected by CCK-OP injection compared to sCSF injection. The greatest amount of feed was eaten in the first 30 rain after presentation, most likely accounting for the greater effects on rumen motility occurring during that time interval; with CCK-OP injections, however, feed intake was significantly decreased over all time periods (Fig. 10).
%~'---e.
TSE
0.3
12.6
~~~___..~
3'o
e'o
7.1
Time (rain) 3,4
9'o
1 ,o 2,0
FIG. 8. Rumen contraction rate (expressed as percent of baseline--mean rate during the -60 to - 15 min time period) of 2-hr fasted sheep with feed present (solid line) or feed absent (dashed line) during 75 rain injections of sCSF (circles) or 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned to those sheep to be fed (zero time). *Different from feed absent, same treatment, p<0.05. Ts~:, treatment standard error (%).
L
120.
~ 'E
,^
A,--.--A
Feed p r e s e n t Feed a b s e n t
} CCK-OP ) 0,638 pmeles/min
11o-
g 1oo90.
~ . I1
70
<
60 N %e
6 0,4
a'o 1.1
1.1
6'o Time ( min ) 0.8
9'o 1.2
FIG. 9. Rumen contraction amplitude (expressed as percent of baseline--meanamplitude during the -60 to - 15 rain time period) of 2-hr fasted sheep with feed present (solid line) or feed absent (dashed line; during 75 min injections of sCSF (circles) or 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned to those sheep to be fed (zero time). *Different from feed absent, same treatment, p<0.05. Different from sCSF, feed absent, p<0.05. TSE, treatment standard error (%).
948
D E L L A - F E R A AND BAILE
210-
t
=
sCSF
1"
C C K - O P 0.638 pmoles/min
~m~
|
180" 150-
IT
120"
l
90"
T,
-'F'i
J
60" 30"
( -15
Injection
6 15
L
6b
3'0 Time
9'0
150
(min)
FIG. 10. Feed intake (mean _+ SEM) of 2-hr fasted sheep during 75 min injections of sCSF (circles) of 0.638 pmoles/min CCK-OP (triangles) into the lateral cerebral ventricles. Injections were begun 15 min before feed was returned (zero time). Rumen motility was measured concurrently. *Different from sCSF, p <0.05. **Different from sCSF. p<0.01.
DISCUSSION
Although recent studies have demonstrated the presence of CCK, primarily CCK-OP, in brain tissue of several species (e.g., [10, 19, 30]), as is true of a number of recently isolated brain peptides, the function of CCK-OP in the CNS is not yet known. The regional localization of CCK-OP in rat brain, as reported by Innis et al. [19], suggests specific functions for this peptide. The periaqueductal gray and medial hypothalamus, both of which are in close proximity to the ventricular system, had the highest concentration of CCKOP-containing cells; and the medial hypothalamus, of course, is known to be involved in the control of feed intake. The results of our studies are strong support for a role for brain CCK-OP in satiety. When injected continuously into the lateral ventricles over 45 rain, as little as 0.45 pmoles CCK-OP (0.01 pmoles/min) reduced feed intake 56% in 2-hr fasted sheep. CCK activity in human CSF has been reported to range from 0.004 to 0.06 pmoles/ml [30], and we have measured similar concentrations of CCK in sheep CSF (0.002 to 0.07 pmoles/ml, range; 0.013 _+ 0.003 pmoles/ml for satiated sheep) (Della F e r a , M. A., C. A. Baile, B. Schneider, manuscript in preparation). The concentration in the injection solution of the above dose of CCK-OP was only 0.1 pmoles/ml and if 0.2 ml of C S F were formed per min by the sheep [28] then in the steady-state condition the 0.1 mt injection per min would result in 0.05 pmoles/ml increase in concentration. Thus, we are approaching physiological levels with an effective dose of CCK-OP. Higher doses of CCK-OP, 0.638 and 2.55 pmoles/min, completely suppressed feeding of 2-hr fasted sheep during the entire 3-hr injection period. In effect this doubled their normal intermeal interval, since sheep under these conditions of housing and feeding have been shown to initiate a meal every 2 to 3 hrs [2]. The CCK-OP induced suppression of feeding also appears to interact in a consistent manner with the deprivation state of the sheep, since greater amounts of CCK-OP were required to decrease feeding with increased hunger drives. For example,
compared to the 0.01 pmoles/min dose effective in 2-hr fasted sheep, 0.059 pmoles/min was the lowest dose that decreased feed intake in 4-hr fasted sheep by a similar percent, and 10.2 pmoles/min was the only dose that decreased feed intake significantly in 8-hr fasted sheep. The effect of intraventricular injections of CCK peptides has also been investigated in the rat. Stern. Cudillo and Kruper [34] reported that 0. l0 and 0.25 ~g caerulein, 80 and 190 pmoles, respectively, decreased feed intake when injected as a bolus into the lateral ventricles of rats. In addition, Maddison [22] found that operant responding for food was depressed in rats after bolus intraventricular injections of CCK-33 (0.05, 0.10 and 0.20 I.D.U.: approximately 5, 10 and 20 pmoles, respectively). However, Nemeroffet al. [27] reported that as much as 3 ~g CCK-OP (2625 pmoles or 60 I.D.U.) injected in the lateral ventricles was required to inhibit stress-induced eating in rats. We have found that rats under various lighting and feeding schedules also do not respond to CCK-OP administered as a continuous lateral ventricular injection in doses comparable to those that were effective in sheep [7]; thus under these conditions, rats appear to be less sensitive to CCK-OP than are sheep. Because a number of factors have been shown to limit feed intake in ruminants, e.g., heat load [3], dehydration [38], gastric or intestinal distention [4], nutritional deficiencies [11], and illness [20], we investigated whether the CCK-OP induced decrease in feed intake was a secondary effect. Under the conditions of our experiments, factors that might become important in this regard would include heat load induced by a rise in body temperature, and dehydration, either of which might be caused by CCK-OP. Gengler et a/. [14] showed that cows whose body temperatures were increased by means of heating coils in the tureens, decreased both feed and water intakes. Bhattacharya and Warner [3] also reported a decrease in feed intake when body temperature was elevated by infusing warm water into the rumens of cows, thus the reduced feed intake reported by Gengler et al. [14] was apparently not secondary to the reduced water intake. In our experiment although rectal temperatures increased slightly (less than 0.5°C) during injections there was no difference between the two treatments. This increase in temperature was due in part to activity that occurs with feeding, and is similar to that previously reported to occur during the normal circadian cycle at the time of day the experiments were done [25]. Dehydration, resulting in an increased body fluid osmolarity, is known to inhibit feeding of animals, including ruminants. Utley et al. [38] showed that restricting water intake to 60% of ad lib levels reduced voluntary feed intake of steers. English [12] restricted water intake of sheep and also observed a decreased voluntary feed intake. Thus if CCK-OP acted primarily as an inhibitor of water intake, a reduction in feed intake would also occur. In our experiment, water intake of 2-hr water-deprived sheep was not affected by lateral ventricular injections of CCK-OP. Feed intakes were not significantly reduced until "after 3 hrs of injection, most likely due to the fact that they had not been feed-deprived before injection; however, the reduced feed intake after 3 hrs of injection of CCK-OP in conjunction with a water intake similar to that during control injections resulted in an apparent increase in the amount of water consumed per unit of feed. Normal water intake during injection of CCK-OP also indicates that the sheep were not depressed and that their activity was not impaired. The effect of CCK-OP on tureen motility adds an interest-
CCK-OCTAPEPTIDE INJECTED IN CSF AND SATIETY ing aspect to this peptide's activity in the CNS, since the CNS has been shown to affect various aspects of gastrointestinal function [29], which in turn could alter feeding behavior [24]. In our experiment under control conditions the amplitude of rumen contractions was decreased in the first 30 min of a meal following a 2-hr fast. As is shown in Fig. 9, CCK-OP injected continuously into the lateral ventricles caused a similar decrease in the amplitude of contractions. That this action on the rumen is independent of the effect of feeding is shown by the fact that even in nonfed sheep, injections of CCK-OP resulted in decreased amplitude of contractions. Also under control conditions, feeding increased contraction rate, as previously shown [5]. CCK-OP did not affect rumen contraction rate, independent of its effect on feeding. The smaller increase in contraction rate during the meal with CCK-OP injections compared to that with control injections is most likely due to the fact that feed intake during CCK-OP injections was less than half of that during control injections. Leek and Harding [21] have identified in each of the two gastric centers of the medulla two distinct circuits responsible for the control of rumen motility. One neuronal network is concerned with the rate of primary cycle movements and another is concerned with their form and amplitude. Because we have found a selective effect of CCK-OP on amplitude but not rate, we propose that this
949 peptide has an inhibitory effect on the neurons of the "form and amplitude circuit" but does not affect the "rate circuit" neurons. Alternatively, CCK-OP could affect higher centers which influence activity of the "form and amplitude circuit." Based on the results presented here, we propose that one function of CCK-OP in the brain is to produce satiety. We hypothesize that signals from the periphery, that are related to feeding activity, cause release of CCK-OP in the brain where it then acts in specific areas to cause termination of the meal and possibly other satiety-related changes, as indicated by its effect on rumen function. We also suggest that the CSF plays an active role in transporting CCK-OP to its site(s) of action, although CCK-OP might be released directly at its sites of action and the role of the CSF may only be passive removal of the peptide. However the proposed sites of action are likely to be periventricular, since suppression of feeding occurred as early as 15 min after beginning injection. ACKNOWLEDGEMENTS This work was supported in part by NIH Training Grant 0205110, NIH Grant AM-21413, and NSF Grant PCM 75-23128. We are grateful to Dr. S. J. Lucania of E. R. Squibb and Sons, Inc. for supplying the CCK-OP, and also to Debby Keim for technical assistance.
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13. Fencl, V., G. Kosky and J. R. Pappenheimer. Factors in cerebrospinal fluid from goats that affect sleep and activity in rats. J. Physiol., Lond. 216: 565-589, 1971. 14. Gengler, W. R., F. A. Martz, H. D. Johnson, G. F. Kraus and L. Hahn. Effect of temperature on food and water intake and rumen fermentation. J. Dairy Sci. 53: 434-437, 1970. 15. Gibbs, J., J. P. Falasco and P. F. McHugh. Cholecystokinindecreased feed intake in rhesus monkeys. Am. J. Physiol. 230: 15-18, 1976. 16. Gibbs, J. and G. P. Smith. Cholecystokinin and satiety in rats and rhesus monkeys. Am. J. clin. Nutr. 30: 758-761, 1977. 17. Gibbs, J., R. C. Young and G. P. Smith. Cholecystokinindecreased food intake in rats. J. comp. physiol. Psychol. 84: 488-495, 1973. 18. Houpt, T. R., S. M. Anika and N. C. Wolff. Satiety effects of cholecystokinin and caerulein in rabbits. Am. J. Physiol. 235: R23-R28, 1978. 19. Innis, R. B., F. M. A. Correa, G. R. Uhl, B. Schneider and S. H. Snyder. Cholecystokinin octapeptide-like immunoreactivity: Histochemical localization in rat brain. Proc. natn. Acad. Sci. U.S.A. 76: 521-525, 1979. 20. Kronfeld, D. S. Ketone body metabolism, its control, and its implications in pregnancy toxemia, acetonaemia, and feeding standards. In: Physiology o f Digestion and Metabolism in the Ruminant, Proc. int. Symp., 3rd, Cambridge, England, 1969; edited by A. T. Phillipson. Newcastle, England: Oriel Press, 1970, pp. 566-583. 21. Leek, B. F. and R. H. Harding. Sensory nervous receptors in the ruminant stomach and the reflex control of reticulo-ruminal motility. In: Digestion and Metabolism in the Ruminant, edited by I. W. McDonald and A. C. I. Warner, Proc. IV int. Symp. Ruminant Physiology, Armidale, N.S.W., Australia: Univ. New England Publishing Unit, 1975, pp. 60-76. 22. Maddison, S. Intraperitoneal and intracranial cholecystokinin depress operant responding for food. Physiol. Behav. 19: 81% 824, 1977. 23. Martin, F. H., J. R. Seoane and C. A. Baile. Feeding in satiated sheep elicited by intraventricular injections of CSF from fasted sheep. Life Sci. 13: 177-184, 1973.
950
24. McHugh, P. R. and T. H. Moran. The gastro-enteric coordination active in short-term (meal time) satiety of Rhesus monkeys. East. Psychol. Ass. Ann. meet., Philadelphia, PA, April 18-21. 1979, p. 17. 25. Mendel, V. E. and G. V. Raghavan. A study of diurnal temperature patterns in sheep. J. Physiol. 174: 206-216, 1964. 26. Mueller, K. and S. Hsiao. Specificity of CCK satiety effect: reduction of food but not water intake. Pharmac. Biochem. Behav. 6: 643-646, 1977. 27. Nemeroff, C. B., A. J. Osbahr III, G. Bissett, G. Jahnke, M. A. Lipton and A. J. Prange, Jr. Cholecystokinin inhibits tail pinchinduced eating in rats. Science 200: 793-794, 1978. 28. Pappenheimer, J. R., S. H. Heisey, E. F. Jordan and J. de C. Downer. Perfusion of the cerebral ventricular system in unanesthetized goats. A m . J. Physiol. 203: 763-774, 1962. 29. Ralph, T. L. and P. E. Sawchenko. Differential effects of lateral and ventromedial hypothalamic lesions on gastrointestinal transit in the rat. Brain Res. Bull. 3:11-14, 1978. 30. Rehfeld, J. F. and C. Kruse-Larsen. Gastrin and CCK in human CSF. Brain Res. 155: 1%26, 1978. 31. Simpson, J. B., M. L. Mangiapane and H. D. Dellman. Central receptor sites for angiotensin-induced drinking. Fedn Proc. 37: 2676-2682, 1978.
DELI~A-FERA AND BAILE
32. Smith, G. P., J. Gibbs and R. C. Young. Cholecystokinin and intestinal satiety in the rat. Fedn Proc. 33: 1146-1149, lC)74. 33. Snyder, S. H. Peptide neurotransmitter candidates in the brain: Focus on Enkephalin, Angiotensin II, and Neurotensin. In: Th~ Hypothalamus, edited by S. Reichlin, R. J. Baldessarini and .!. B. Martin. New York: Raven Press, 1978, pp. 233-244. 34. Stern, J. J., C. A. Cudillo and J. Kruper. Ventromedial hypothalamus and short-term suppression of feeding by caerulein in male rats..I, comp. physical, P~y~hol. 90: 484-490. 1976, 35. Straus, E., J. E. Muller, H. Schoi, F. Paronetto and R. S. Yalow. lmmunohistochemical localization in rabbit brain of a peptide resembling the COOH terminal octapeptide of CCK. Pro~'. mJtn. A('ad. Sci. U.S.A. 74: 3033-3034. t974 36. Straus, E. and R. S. Yalow. CCK in br.,dns uf ubese and nonobese mice. Science 203: 68--70, 1979. 37. Sturdevant, A. L. and H. Goetz. Cholecystokinin both stimulates and inhibits human food intake. Nature 261: 714-715, 1976. 38. Utley, P. F., N. W. Bradley and J. A. Boling. Effect of restricted water intake on feed intake, nutrient digestability and nitrogen metabolism in steers..I. A~tim. S~i. 31: 130-135, 1970,