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Redundant vagal mediation of the synergistic satiety effect of pancreatic glucagon and cholecystokinin in sham feeding rats
Journal of the Autonomic Nervous System, 30 (1990) 13-22 Elsevier
13
JANS 01036
Redundant vagal mediation of the synergistic satiety effect of pancreatic glucagon and cholecystokinin in sham feeding rats Joseph Le Sauter and Nori Geary Department of Psychology, Columbia University, New York, NY, U.S.A.
(Received 31 July!989 ) (Revision received 19 October 1989) (Accepted 6 December 1989)
Key words: P o s t p r a n d i a l satiety; T o t a l a b d o m i n a l v a g o t o m y ; H e p a t i c v a g o t o m y ; G a s t r i c v a g o t o m y ; Celiac v a g o t o m y ; V a g o t o m y v e r i f i c a t i o n
Abstract Simultaneous intraperitoneal injection of pancreatic glucagon (PG) and cholecystokinin (CCK) results in a functionally synergistic satiety effect in non-deprived sham feeding rats ("PG-CCK satiety"). That is, PG and CCK together inhibit feeding significantly more than the sum of their individual effects. Because the individual satiety effect of each peptide on normal feeding is dependent on the abdominal vagus nerve, we tested the vagal mediation of this synergistic effect of PG plus CCK. Vagotomies were verified anatomically and, in one experiment, histologically. Total abdominal vagotomy blocked PG-CCK satiety. Neither selective vagotomies of the hepatic, the gastric, the celiac, the gastric and celiac, nor the gastric and hepatic branches, however, affected PG-CCK satiety. This indicates that the vagal contribution to the synergistic satiety effect of PG plus CCK on sham feeding is redundant. Although some vagal fibers are necessary for PG-CCK satiety, no individual branch is required for the effect, and at least two branches, the hepatic and celiac, are each sufficient for mediating it.
Introduction Both p a n c r e a t i c glucagon ( P G ) a n d cholecyst o k i n i n ( C C K ) are h y p o t h e s i z e d to signal postp r a n d i a l satiety in rats. W e recently r e p o r t e d that c o m b i n a t i o n s of P G a n d C C K result in a functionally synergistic satiety effect in n o n - d e p r i v e d rats sham feeding with o p e n gastric c a n n u l a s ( " P G - C C K satiety") [12]. T h a t is, s h a m feeding was i n h i b i t e d significantly m o r e b y s i m u l t a n e o u s
Correspondence: N. Geary, Department of Psychology, Box 28, Schermerhorn Hall, Columbia University, New York, NY 10027, U.S.A.
i n t r a p e r i t o n e a l injections o f P G a n d C C K t h a n b y the sum of the two p e p t i d e s ' i n d i v i d u a l effects. T h e m e c h a n i s m m e d i a t i n g this synergy is unknown. T h e satiety effects of P G a n d of C C K are each b l o c k e d b y total a b d o m i n a l v a g o t o m y [7,10,13,20,22,37]. Therefore, we tested the vagal d e p e n d e n c y of the synergistic P G - C C K satiety effect on s h a m feeding in n o n - d e p r i v e d rats. T o t a l a b d o m i n a l v a g o t o m y b l o c k e d the effect of P G C C K c o m b i n a t i o n s ( a n d also b l o c k e d the effect of C C K alone), b u t no selective v a g o t o m i e s did. Thus, this synergistic p e p t i d e satiety signal d e p e n d s o n a r e d u n d a n t vagal m e c h a n i s m which does n o t require the fibers o f a n y p a r t i c u l a r i n d i v i d u a l branch.
0165-1838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14 Methods
Subjects Five groups of male Sprague-Dawley rats (Charles River, Wilmington, MA), 500-700 g, were tested in succession. Rats were individually housed in solid-bottom metal cages (37 × 35 x 20 cm) with wood chip bedding (Northeastern Products, Warrensburg, NY). Pelleted rat chow (Purina 5012, St Louis, MO) was presented in hoppers hung inside the cages except during the test sessions. Tap water was available ad libitum in bottles clipped onto the cages. The room was maintained on a 12 : 12 LD schedule (lights on 07 : 30) at 20 + 2°C.
Sham feeding Rats were anesthetized with Chloropent (Fort Dodge Industries, Ft Dodge, IA; 2.5 m l / k g ) after overnight food deprivation and equipped with chronic gastric cannulae as described previously [2,6,191. Three weeks after surgery the rats were adapted to the sham feeding procedure. At 09:00, food hoppers were removed, cannulas were opened, and stomachs were flushed repeatedly with 8 - 9 ml of 0.9% NaC1 at about 30 ° C until the drainage was clear. The lavage was repeated about 30 min later to ensure that no food was left in the stomach. 1 m l / k g 0.9% NaC1 was then intraperitoneally injected, and drainage tubes (Silastic, Dow Coming, Midland, MI; length 20 cm, inner diameter 3.4 ram, outer diameter 4.6 mm, sheathed with a steel spring) were screwed into the cannulae. Rats were put in transparent plastic cages (20 × 24 × 20 cm) with slotted wire-mesh floors. The distal ends of the drainage tubes were passed through the slots to allow ingested liquid food to drain into a collection pan under the cage. Evaporated milk (Pet Industries, St. Louis, MO) and water were preser, ted in graduated tubes (Wahman, Timonium, MD). Milk intake was measured every 10 min for 40 rain. Drainage tubes were then removed, cannulae closed, and rats were returned to their home cages.
Vagotomy Groups 1 - 3 were adapted to sham feeding for 15 sessions before being reanesthesized for total or
selective vagotomy [13,33]. In groups 4 and 5 gastric cannulation and vagotomy were done in a single operation. For control operations, the abdominal vagal branches were exposed but not cut. Six total abdominal-vagotomized (TAVx) and nine control-operated rats were tested in group 1. For TAVx, about 3 m m each of the anterior (right) and posterior (left) esophageal vagal trunk was ligated and removed rostral to the bifurcation points of the hepatic and celiac branches. In group 2, eight hepatic-vagotomized (HVx) and eight control-operated rats were compared. For HVx, about 2 m m of the hepatic branch was ligated and removed. Group 3 included seven rats with gastric vagotomy (GVx) and seven control-operated rats. For GVx, 2 m m sections were tigated and removed from the anterior and the posterior gastric branches caudal to the hepatic and the celiac branches, respectively. Group 4 included eight rats with combined gastric and celiac vagotomy (GCVx), three rats with celiac vagotomy (CVx), and eight control-operated rats. For GCVx, 2-mm sections of the anterior gastric branch caudal to the hepatic branch and of the posterior trunk rostral to the celiac branches were ligated and removed. The accessory celiac branch was also cut. CVx was performed by removing sections of the celiac and accessory celiac branches. Finally, group 5 included six rats with combined gastric and hepatic vagotomy (GHVx), for which the HVx and GVx procedures were combined; three rats with separate transections of each of the abdominal vagal branches (GHCVx); two TAVx rats, and six control-operated rats. Vagotomized rats tended to lose more weight post-surgically than control-operated rats, but by 2 weeks after surgery, all groups were regaining weight and continued to do so throughout the test period. For example, in group 1 TAVx rats gained 35 + 6 g and control rats gained 50 + 3 g during 4 weeks of testing.
Procedure Rats were adapted to the sham feeding procedure for 15 sessions beginning 1 week after vagotomy. Tests were then begun when rats met the baseline criterion that the SD of each rat's mean
15 30 min intake for 3 consecutive days was less than 30% of that mean [9]. Tests were done Tuesday to Friday. Monday was an adaptation day because rats sham feed more on the first test after an interruption of the daily routine. Each rat received six tests in random order: 400 /zg/kg PG, 0.30/~g/kg CCK, simultaneous injection of 400/~g/kg PG plus 0 . 3 0 / t g / k g CCK, and three control injections. These doses were chosen because they synergistically inhibited sham feeding in previous work [12]. However, in groups 1 and 4, 0.30 /~g/kg CCK alone inhibited sham feeding in control-operated rats more than previously [12], and no synergistic effect of the PG-CCK combination was obtained. When this occurred, tests of 0.15 # g / k g CCK were done. In group 1, the original 6-day protocol was extended by six additional tests: 0.15 /~g/kg CCK, two tests of 0.15/~g/kg CCK plus 400/~g/kg PG, one test of 0.30 ffg/kg CCK plus 400 /~g/kg PG, and two control tests. In group 4, the first 6-day protocol was simply repeated in a separate experiment using 0.15/~g/kg CCK. PG was prepared by diluting 1 mg PG hydrochloride (Eli Lilly, Indianapolis, IN) mixed with lactose (49 mg) in 0.625 ml Lilly diluting fluid (1.6% glycerin, 0.2% phenol) and 0.625 ml 0.9% NaC1. Lyophillized synthetic CCK octapeptide (Squibb, Princeton, N J) was dissolved in 0.9% NaC1 with 39.2 mg lactose/ml. This vehicle was also used for control injections. For single peptide tests, 1-ml plastic syringes (Becton, Dickinson, Rutherford, N J) were filled with 0.5 m l / k g body weight of the appropriate stock solution and 0.5 m l / k g of vehicle solution. For combination treatments, syringes were filled with 0.5 m l / k g of each peptide. All injections were 1 m l / k g solution. Real feeding tests were done after the sham feeding tests in two groups whose selective vagotomies have been reported to block PG or CCK satiety effect [7,34]. PG was tested in group 2 (HVx), and CCK was tested in group 3 (GVx). The procedure was similar, except the gastric cannulae were closed and the stomachs were not lavaged.
Behavioral observations Behavioral observations were done for groups
1-4. During the 40-min sham feeding tests, an observer rated each rat's behavior once per rain during a 0.6-s interval signalled by a low intensity tone [5]. Behaviors were classified using 24 operationally defined categories, including feeding (milk ingestion or licking mouth), grooming (ticking, biting or scratching the head, body, tail, or paws), exploring (locomotion, sniffing, rearing, or licking cage), resting (reclining in a stationary position with the abdomen supported by the cage floor and without display of any other behavior), other normal behaviors (standing, urinating, defecating, or eating feces), and anomalous behaviors (unusual postures, trembling, drooling, etc.).
Verification of vagotomy In groups 1-4 vagotomies were verified anatomically [33]. Rats were overdosed with Chloropent, and the surgical sites were examined with an operating microscope (6-40 x ). If any ambiguous fiber connection was located in the area between the ligatures, the vagotomy was judged to be incomplete, and data from such rats were discarded. According to this criterion, all the vagotomies in groups 1-3 were complete. In group 4, one GCVx rat's data were discarded because an ambiguous, possibly vagal, fiber was detected in the lesioned area. Group 5 rats were verified histologically. A tissue sample consisting of the caudal thoracic and the abdominal esophagus, the diaphragmatic hilus, the fiver surrounding the hepatic vagus, the proximal lesser gastric curvature, and adherent fat and connective tissue, was removed. A straight wire (OD 2.5 ram) was placed into the lumen of the esophagus, and a light weight (4.6 g) was attached to the stomach. The sample was suspended by the rostral end of the esophagus during fixation and processing [26], embedded in paraffin, and cut transversely at 12 /~m. Sections were stained in hematoxilin and eosin [17] and mounted. Serial sections of the esophagus from the diaphragmatic hilus to the stomach were microscopically examined (10-200 x ) by an experimenter who was blind to the behavioral data. The anterior and posterior vagal trunks were located and the surgical sites were identified by the sutures. Vagotomies were rated complete if no fiber that may
16
Fig. 1. A. Esophagus of sham-operated rat rostral to the bifurcation of the gastric and celiac branches. Note the anterior (a) and posterior (p) vagal trunks, and blood vessels (b). B. Esophagus of GHVx rat. Note remnants of ligature marking caudal end of lesion (k) and the celiac branches (c), which bifurcated from the posterior vagal trunk rostral to the lesion. C. Esophagus of TAVx rat just rostral to lesion site. Note swelling of vagal trunks. D. Lesion site of the same TAVx rat. Rostral-caudal level is similar to A. Note absence of vagal trunks.
have been a neural connection was detected in the lesion site. A l l six G H V x , b o t h T A V x , a n d two of the three G H C V x rats satisfied this criterion (Fig. 1). One G H C V x rat was j u d g e d to have received an i n c o m p l e t e v a g o t o m y b e c a u s e a c o n t i n u o u s fiber c o n n e c t i o n was d e t e c t e d all along the esophagus.
Data analysis The criteria for successful s h a m feeding tests were that ingestion b e g a n within 2 rain of milk p r e s e n t a t i o n a n d that the volume of d r a i n a g e collected was equal to or exceeded the total milk a n d water i n t a k e d u r i n g the test. D a t a f r o m unsuccessful tests were n o t included in the analysis, a n d if
three or m o r e sessions were rejected for a n y rat, n o n e of its d a t a were included. Finally, in o r d e r to increase the sensitivity of the test, rats were not i n c l u d e d if they d i d n o t continue to meet the b a s e l i n e criterion d u r i n g the three c o n t r o l tests. These criteria e l i m i n a t e d two c o n t r o l - o p e r a t e d a n d one T A V x rat in g r o u p 1, one c o n t r o l - o p e r a t e d in g r o u p 3, a n d one c o n t r o l - o p e r a t e d rat in group 4 d u r i n g the series of tests with 0 . 1 5 / ~ g / k g C C K . D u r i n g c o n t r o l tests, rats s h a m fed milk continuously a n d vigorously for 3 0 - 4 0 min, ingesting 4 0 - 5 0 m l / 3 0 rain. R a t s that s t o p p e d s h a m feeding g r o o m e d a n d rested. S o m e of these animals b e g a n a second b o u t of s h a m feeding j u s t b e f o r e the end of the test. Because the p e p t i d e s ' effects on sham
17 i n t a k e w e r e c l e a r e s t d u r i n g t h e initial b o u t o f s h a m feeding, w e a n a l y s e d the p e p t i d e s ' effects o n i n t a k e d u r i n g the first 30 m i n o f s h a m f e e d i n g as p e r c e n t i n h i b i t i o n o f the m e a n i n t a k e o n t h e c o n t r o l days, i.e.: ~ (100 ( c o n t r o l i n t a k e - t e s t i n t a k e ) / c o n t r o l i n t a k e ) / n . T h e results o f t h e rep e a t e d p e p t i d e tests in g r o u p 1 w e r e s i m i l a r a n d w e r e c o m b i n e d for analysis. T h e d a t a w e r e analysed with 2-way (control x vagotomized) A N O V A for r e p e a t e d m e a s u r e m e n t s , f o l l o w e d b y p o s t h o c tests o f d i f f e r e n c e s b e t w e e n t h e m e a n s w i t h T u k e y ' s h s d m e t h o d [8]. M i n i m u m signific a n c e level w a s 0.05. D a t a are r e p o r t e d as m e a n s + SEM. B e h a v i o r a l o b s e r v a t i o n s w e r e d o n e to determine whether peptide treatments that inhibited s h a m f e e d i n g e l i c i t e d the n o r m a l s e q u e n c e of p o s t prandial behavior. Frequencies of each behavior c a t e g o r y w e r e a n a l y s e d w i t h F r i e d m a n ' s tests foll o w e d b y p o s t - h o c tests for c o m p a r i s o n w i t h a c o n t r o l [32].
r: INHIBITION 100 :30 60 40 20 0 400 PG
0.30
T h e results w e r e clear. I n c o n t r o l - o p e r a t e d rats, injection either of CCK alone or of PG plus CCK (but not of PG alone) inhibited sham feeding, with the effect o f P G p l u s C C K u s u a l l y s i g n i f i c a n t l y l a r g e r t h a n t h e s u m o f t h e effects o f P G a l o n e a n d C C K alone. C o m p l e t e d i s c o n n e c t i o n o f t h e ab-
PG+CCK
100 80
60 40 20 0
PG+CCK 400 PG 8.15 CCK PEF'TIDE DOSE (pG/KG)
• CONTROL Results
CCK
[ ] TAUX
Fig. 2. Blockade of the inhibitory effect of simultaneous injection of PG and CCK on sham feeding in TAVx rats (group 1). Upper panel shows the results of tests with 0.30/.tg/kg CCK; lower panel shows results of tests with 0.15 /tg/kg CCK. For clarity, PG data are shown in each panel. Sham intake after vehicle injection was 43.4+ 6.4 ml in seven control-operated rats and 46.5 + 5.7 ml in five TAVx rats. * P < 0.05, * * P < 0.01 vs. vehicle; + P < 0.01 vs. control operated. Tukey's test after significant ANOVA, F (4,10) = 6.1.
TABLE I Total abdominal oagotomies block PG-CCK satiety
Data are % inhibition compared to intakes after vehicle injection, individual (R numbers) and M + SEM for group 5 with total abdominal vagotomies done by lesioning the vagal trunks rostral to their subdivisions (TAVx) or by separately lesioning each of the three branches (GHCVx). PG dose was 400 /~g/kg; CCK, 0.30 ~g/kg. * P < 0.05, * * P < 0.01 vs. vehicle, Tukey's test after significant ANOVA. PG Control (n = 6) TAVx
GHCVx
Mean + SEM
CCK
-4.3 + 11.9
R 14
34.8 ± 5.3 *
37.5
- 45.8
3.2
-6.3
R 18
-
Mean + SEM R 16 R 02 Mean + SEM
17.5 + 20.4 9.9 - 46.5 -18.3 + 28.2
-26.1 + 19.8 - 9.0 23.6 7.3 + 16.3
PG + C C K 54.6 + 11.9 * * - 54.2 17.5 -18.4 + 35.9 3.6 - 14.6 -5.5 + 9.1
18
%
% INHIBITION
INHIBITION
100 r-
100
80
80
:f.
60
60
40
40
20
20 0
0
400 PG
0.30 CCK
PG+CCK
F'EPTIDE DOSE (pG/KG)
• CONTROL
[] HUX
Fig. 3. Inhibition of sham feeding by simultaneous injection of PG and CCK in HVx rats (group 2). Sham intake after vehicle injection was 50.8 _+7.5 ml in eight control-operated and 48.5 _+ 6.6 ml in eight HVx rats. * P < 0.01 vs. vehicle; ~ P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,14) = 34.6.
400 PG
0.30 CCK
PG + CCK
O 15 CCK
PG + CCK
100 80 60 40 20 0
d o m i n a l v a g u s b l o c k e d o r severely a t t e n u a t e d b o t h the i n h i b i t o r y e f f e c t of C C K a l o n e a n d the synergistic effect of P G p l u s C C K . But n o selective v a g o t o m y a f f e c t e d e i t h e r i n h i b i t o r y effect. T h e b l o c k a d e o f P G - C C K satiety in g r o u p 1 T A V x rats is s h o w n in Fig. 2. I n c o n t r o l - o p e r a t e d rats, i n j e c t i o n of 0.15 t t g / k g C C K a l o n e i n h i b i t e d
% IHHIBITInN
100
400 PG
PEPTIDE DOSE (pg/kg) •
CONTROL
~
GCVX
Fig. 5. Inhibition of sham feeding by simultaneous injection of PG and CCK in GCVx rats (group 4). Sham intakes after vehicle injection were 48.9 + 4.6 ml (upper panel) and 43.9 + 4.3 ml (lower panel) in seven control-operated rats and 48.8 + 9.2 ml (upper panel) and 44.6+3.7 ml (lower panel) in seven GCVx rats. * P < 0.01 vs. vehicle; = P < 0.05, =~ P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,13)=16.9 (upper panel) and 23.2 (lower panel).
,90 60 40 20 0 400
PG
0...'30 CCK
PEPTIDE
PG+0CK
BOSE ( p G , " K G )
• CONTROL
[] GUX
Fig. 4. Inhibition of sham feeding by simultaneous injection of PG and CCK in GVx rats (group 3). Sham intake after vehicle injection was 40.6 _+4.8 ml in six control-operated and 49.0 ± 9.9 ml in seven GVx rats. * P < 0.01 vs. vehicle; # P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,11) = 10.6.
s h a m f e e d i n g b y 31.9 +_ 8.2%, P G a l o n e d i d n o t h a v e a s i g n i f i c a n t effect, a n d s i m u l t a n e o u s inject i o n of 400 / ~ g / k g P G p l u s 0.15, / ~ g / k g C C K i n h i b i t e d s h a m f e e d i n g by 64.6 _+ 13.1%, signific a n t l y m o r e t h a n the s u m of the effects of P G a n d C C K alone. I n T A V x rats, n o n e o f these treatm e n t s a f f e c t e d s h a m feeding. T h e l a r g e r d o s e of 0.30 / l g / k g C C K i n h i b i t e d s h a m f e e d i n g in c o n t r o l - o p e r a t e d rats by 57.5 +_ 9.9%, b u t d i d n o t affect T A V x rats. A l t h o u g h c o m b i n a t i o n of 400 /~g/kg PG plus 0.30/~g/kg CCK did inhibit sham f e e d i n g b y 28.6_+ 12.9% in T A V x rats, this was s i g n i f i c a n t l y less t h a n the 73.1 + 11.6% i n h i b i t i o n in c o n t r o l - o p e r a t e d rats. P G p l u s 0 . 3 0 / ~ g / k g C C K
19
~. INHIBITION 100 r80 60
20 0
400 Pg.
0.30 CCK
PG+CCK
PEPTIDE BOSE (pG/KG) •
CONTROL
[] GNUX
Fig. 6. I n h i b i t i o n of s h a m feeding b y s i m u l t a n e o u s injection of P G a n d C C K after G H V x (group 5). S h a m i n t a k e after vehicle injection was 49.0 5:8.0 m l in six control o p e r a t e d a n d 47.9 + 9.2 m l in five G H V x rats. * P < 0.05, * * P < 0.01 vs. vehicle. T u k e y ' s test after significant A N O V A , F (2,9) = 17.0.
also failed to inhibit sham feeding in the two TAVx rats and the two GHCVx rats in group 5 (Table I). In contrast to the TAVx results, neither HVx, GVx, CVx, GCVx, nor GHVx produced any change in the inhibitory effects of CCK or of PG plus CCK on sham feeding (Figs. 3-6 and Table II).
The inhibitory effect of 0.60 /~g/kg CCK on real feeding was similar in seven control-operated (48.6_+9.1%) and six GVx rats (56.9_+ 8.6%). However, HVx blocked the effect of PG on real feeding: 4 0 0 / t g / k g P G inhibited real feeding by 18.1 _+ 4.8%, P < 0.05, in seven control-operated rats vs. - 1.5 +_ 6.2%, n.s., in six HVx rats. The behavioral observations indicated that neither peptide injection nor vagotomy disrupted the normal sequence of postprandial activity. Peptide treatments that inhibited sham feeding decreased the frequency of feeding and increased the frequency of resting but did not alter the frequency or the temporal pattern of postprandial grooming and exploration and did not elicit any anomalous behaviors. This was true for both control-operated and selective-vagotomized rats. Behaviors of peptide-injected TAVx rats were similar to those of vehicle-injected control-operated rats. Because we have previously described this behavioral profile in detail [12], we do not present it here.
Discussion
When CCK doses that elicit moderate inhibitions of sham feeding in non-deprived rats are
T A B L E II
Celiac vagotomy does not block PG-CCK satiety D a t a are % i n h i b i t i o n c o m p a r e d to i n t a k e s after vehicle injection. P G dose was 4 0 0 / t g / k g . P G + C C K , T u k e y ' s test after significant A N O V A . PG
* * P < 0.01 vs. vehicle, + P < 0.01 vs.
CCK
PG + CCK
0.30 ~ g / k g C C K Control operated
(n = 8) CVx
0.15/~g/kg CCK Control operated (n = 6) CVx
M e a n 5: S E M
2.9 + 7.2
46.0 + 11.0 * *
59.7 5 : 7 . 0
**
R 02 R 04 R 20 M e a n 5: S E M
24.9 18.9 17.8 20.5 + 2.2
76.5 24.9 64.3 55.2 + 15.6 * *
85.9 39.9 78.6 68.1 5:14.3 * *
Mean + SEM
- 11.0 + 6.9
15.1 + 7.4
45.7 5:2.9 * * +
R 04 R 20 Mean + SEM
9.6 1.1 5.4 5:4.3
34.6 81.7 58.2 5:23.6 * *
71.2 92.7 82.0 5:10.8 * * +
20 intraperitoneally injected together with PG, which alone fails to inhibit sham feeding [6], sham feeding is inhibited significantly more than the sum of the peptides' individual effects [12]. The inhibition appears behaviorally specific. The normal behavioral sequence of postprandial satiety occurs when sham feeding ends [12], and neither sham water drinking [12] nor activity in running wheels (unpublished data) is inhibited by PG-CCK combinations that inhibited sham feeding by 50% or more. Thus, simultaneous injection of PG and CCK appears to elicit a functionally synergistic satiety effect in the sham feeding rat (" PG-CCK satiety"). We demonstrate here that this PG-CCK satiety effect depends on the contribution of abdominal vagal fibers. After TAVx, PG-CCK satiety is blocked or greatly attenuated. This does not appear to be the result of any debilitating effect of TAVx because TAVx and control rats both gained weight during the testing period and sham fed similar amounts (see figure legends). Despite the vagal dependency of PG-CCK satiety, we could not identify a necessary contribution of any individual subdiaphragmatic vagal branch. Selective lesions of the hepatic, gastric, and celiac branches were each without effect. Thus, none of the three major divisions of the abdominal vagus is necessary for PG-CCK satiety. Further, the lack of effect of gastric plus celiac and gastric plus hepatic vagotomy indicates that the hepatic and the celiac branches are each alone sufficient for PG-CCK satiety (gastric branch sufficiency has not been tested by observing hepatic plus celiac vagotomy). In short, the vagal mechanism mediating PG-CCK satiety is redundant. This is the clearest demonstration we know of redundancy in a vagally mediated behavioral mechanism. Our data do not disclose whether the involvement of the vagal nerves in PG-CCK satiety is in afferent or efferent limbs, or mixed. Because there is evidence that vagal afferents provide the necessary contribution to the individual satiety effects of PG and CCK on real feeding, our hypothesis is that the critical TAVx lesion blocking the synergistic satiety effect of PG and CCK is also afferent. In real feeding, blocking peripheral postganglionic muscarinic receptors with atropine methylnitrate
did not affect either PG [7] or CCK satiety [34]. In contrast, selective transection of the dorsal, but not the ventral, medullary vagal rootlets, blocked CCK satiety [35]; surgical lesions of the portion of the nucleus of the solitary tract receiving hepatic vagal afferents blocked PG satiety [38]; and chemical lesion of small diameter sensory neurons by intraperitoneal capsaicin injection attenuated both CCK and PG satiety [29,30]. Both PG and CCK appear to act peripherally to signal satiety [7,22,34]. Thus, their synergistic interaction might occur either peripherally, if one of the peptides potentiates some effect of the other in the abdomen, or centrally, if the peptides elicit independent afferent signals that converge in the CNS. The present results, taken together with previous studies of the vagal mediation of the peptides' satiety effects on real feeding, suggest that the most likely alternative is that PG potentiates CCK's peripheral action. Hepatic afferents appear to be necessary and sufficient for mediating the contribution of the abdominal vagus to PG satiety during real feeding ([7,20,39], but see also [3,39]). Yet neither hepatic nor hepatic-gastric vagotomy attenuated PG-CCK's sham feeding satiety effect. Indeed, selective hepatic vagotomy blocked the satiety effect of PG on real feeding in the same rats in which it failed to block the effect of PG plus CCK on sham feeding. Thus, CCK apparently did not potentiate a hepatic vagal satiety signal to inhibit sham feeding. Further, since the hepatic vagus alone was unnecessary for PG-CCK satiety, this effect obviously did not depend on the central convergence of a hepatic vagal afferent signal caused by PG with an independent signal related to CCK. It is unclear whether the functional redundancy in PG-CCK satiety occurs because the satiety signal arises at a single site innervated by different vagal branches or because the signal can be generated in multiple abdominal sites. Either mechanism is plausible. For example, the gastric, celiac and hepatic vagal branches all appear to control pancreatic insulin secretion: the dorsal motor nucleus of the vagus appears to project to the pancreas via each branch [28], and the insulin response to electric stimulation of the cervical vagus can be decreased by selective vagotomy of
21
each of them [4]. Alternatively, redundancy may be mediated by central convergence of different branches. Such convergence appears to occur even in the primary sensory nuclei of visceral afferents. Abdominal vagal afferents terminate most heavily in the dorsal medial nucleus of the solitary tract and in the commissural nucleus, with the terminal regions of the gastric, celiac, and hepatic branches overlapping each other to some extent [1,14,24,25, 31]. Further, inputs projecting via different abdominal vagal branches have been shown to converge on individual neurons of the nucleus of the solitary tract [11]. CCK and PG may each signal satiety by directly stimulating vagal afferents. It is interesting, therefore, that CCK binding sites are present in each of the branches of the abdominal vagus [21]. Further, at least in the hepatic branch, some afferents can be stimulated by either CCK or PG [23]. Unfortunately, the effect of PG-CCK combinations on neural activity has not been tested yet. The effect of CCK alone on sham feeding, like that of PG-CCK combinations, was blocked by total abdominal vagotomy but not by any selective vagotomy. This extends previous reports that neither gastric nor gastric-celiac vagotomy blocks peripheral CCK's satiety effect [10,13]. Thus, a redundant vagal mechanism may mediate the satiety effect of CCK alone as well as P G - C C K satiety. Similarly, a satiety effect of bombesin may be mediated by redundant vagal and spinal visceral afferents. Intraperitoneal bombesin injections decreased meal size in rats with total abdominal vagotomies and in rats with T6 spinal cord transections and T 3 - T 6 dorsal rhizotomies, but not in rats with both lesions [36]. The failures to detect an effect of selective abdominal vagotomy on the satiety effect of CCK on either real feeding or sham feeding [10,13] differ from Smith et al.'s [34] report that selective gastric vagotomy was sufficient to block CCK's satiety effect on real feeding. Although our results do not resolve this issue, our histological verification procedure convinces us that our failure to block PG-CCK or CCK satiety with gastric vagotomies did not result from incomplete lesions. Many methods have been used to verify vagotomies [16,27,33], but histological examination of the
esophagus has not previously been described. Esophageal histology appears to present several advantages. For example, it includes afferent fibers in the verification, which other functional [16] and histological [27] tests do not. Gross anatomical examination also includes afferents, but it is sometimes impossible to identify and track neural fibers using gross examination when connective tissue adheres to the lesion site. In conclusion, a redundant vagal mechanism appears to mediate the synergistic satiety effect of simultaneous intraperitoneal injection of PG and CCK in sham feeding rats. Difficult electrophysiological and behavioral tests will probably be required to understand the nature of this redundancy. Given the clarity of the effect and the apparent importance of synergy and redundancy in peripheral neuroendocrine controls of feeding [4,9,11,12,36], this effort seems worthwhile.
Acknowledgements We thank Debra Rosenzweig, Jennifer Fudge, and Zsolt Stockinger for help with the experiments. This research was supported by National Institutes of Health Grant DK-32448 to N. Geary. Preliminary reports of these data were given at the IXth International Conference on the Physiology of Food and Fluid Intake, Seattle, WA, July 1986, and at the 17th Annual Meeting of the Society for Neuroscience, New Orleans, LA, November 1987 (Soc. Neurosci. Abst., 13 (1987) 587).
References 1 Adachi, A., Projection of the hepatic vagal nerve in the medulla oblongata, J. Auton. Nerv. Syst., 10 (1984) 287-293. 2 Antin, J., Gibbs, J., Holt, J., Young, R.C. and Smith, G.P., Cholecystokinin elicits the complete behavioral sequence of satiety in rats, J. Comp. Physiol. Psychol., 89 (1975) 784790. 3 Bellinger, L.L. and Williams, F.E., Glucagon and epinephrine suppression of food intake in liver-denervated rats, Am. J. Physiol., 251 (1986) R349-R358. 4 Berthoud, H.R., Niijima, A., Sauter, J.-F. and Jeanrenaud, B., Evidence for a role of the gastric, coeliac, and hepatic branches in vagally stimulated insulin secretion in the rat, J. Auton. Nerv. Syst., 7 (1983) 97-110.
22 5 Geary, N. and Smith, G.P., Pancreatic glucagon and postprandial satiety in the rat, Physiol. Behav., 28 (1982) 313322. 6 Geary, N. and Smith, G.P., Pancreatic glucagon fails to inhibit sham feeding in the rat, Peptides, 3 (1982) 163-166. 7 Geary, N. and Smith, G.P. Selective hepatic vagotomy blocks pancreatic glucagon's satiety effect. Physiol. Behav., 31 (1983) 391-394. 8 Hays, W.L. Statistics (3rd edn.), Holt, Rinehart and Winston, New York, 1981. 9 Hinton, V., Rosofsky, M., Granger, J. and Geary, N. Combined injection potentiates the satiety effects of pancreatic glucagon, cholecystokinin, and bombesin. Brain Res. Bull., 17 (1986) 615-619. 10 Kraly, F.S. Vagotomy does not alter cholecystokinin's inhibition of sham feeding. Am. J. Physiol., 246 (1984) R829-R831. 11 Laughton, W.B., Campfield, A. and Nelson, D.O. Hepatic portal and gastric afferent processing in nucleus of the solitary tract of the rat. Soc. Neurosci. Abst., 13 (1987) 385. 12 Le Sauter, J. and Geary, N. Pancreatic glucagon and cholecystokinin synergistically inhibit sham feeding in rats. Am. J. Physiol., 253 (1987) R719-R725. 13 Le Sauter, J., Goldberg, B. and Geary, N. CCK inhibits real and sham feeding in gastric vagotomized rats. Physiol. Behav., 44 (1988) 527-534. 14 Leslie, R.A., Gwyn, D.G. and Hopkins, D.A. The central distribution of the cervical vagus and gastric afferent and efferent projections in the rat. Brain Res. Bull., 8 (1982) 37-43. 15 Lorenz, D.A. and Goldman, S.A. Vagal mediation of the cholecystokinin satiety effect in rats. Physiol. Behav., 29 (1982) 599-604. 16 Louis-Sylvestre, J. Validation of tests of completeness of vagotomy in rats, In J.G. Kral, T.L. Powley and C.McC. Brooks (Eds), Vagal Nerve Function: Behavioral and Methodological Considerations. Elsevier, Amsterdam, 1983, p. 345. 17 Luna, L.G. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (3rd Edn.), McGrawHill, New York, 1968. 18 MacIsaac, L. and Geary, N. Partial liver denervations dissociate the inhibitory effects of pancreatic glucagon and epinephrine on feeding. Physiol. Behav., 35 (1985) 233-237. 19 Martin, C.F. and Gibbs, J. Bombesin elicits satiety in sham feeding rats. Peptides, 1 (1980) 131-134. 20 Martin, J.R., Novin, D. and VanderWeele, D.A. Loss of glucagon suppression of feeding after vagotomy in rats. Am. J. Physiol., 234 (1978) E314-E318. 21 Moran, T.H., Smith, G.P., Hostetler, A.M. and McHugh, P.R. Transport of cholecystokinin (CCK) binding sites in subdiaphragmatic vagal branches. Brain Res., 415 (1987) 149-152. 22 Morley, J.R., Levine, A.S., Kniep, J. and Grace, M. The
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effect of vagotomy on the satiety effects of neuropeptides and naloxone. Life Sci., 30 (1982) 1943-1947. Niijima, A. Glucose-sensitive afferent nerve fibers in the liver and their role in food intake and blood glucose regulation. J. Auton. Nerv. Syst., 9 (1983) 207-220. Norgren, R. Afferent interactions of cranial nerves involved in ingestion. J. Auton. Nerv. Syst., 9 (1983) 67-77. Norgren, R. and Smith, G.P. Central distribution of subdiaphragmatic vagal branches in the rat. J. Comp. Neurol., 273 (1988) 207-223. Prechtl, J.C. and Powley, T.L. Organization and distribution of the rat subdiaphragmatic vagus and associated paraganglia. J. Comp. Neurol., 235 (1985) 182-195. Powley, T.L., Fox, E.A. and Berthoud, H.R. Retrograde tracer technique for assessment of selective and total subdiaphragmatic vagotomies. Am. J. Pl~vsiol., 253 (1987) R361R370. Rinaman, L. and Miselis, R. The organization of vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J. Auton. Nerv. Syst., 21 (1987) 109125. Ritter, R.C. and Ladenheim, E.E. Capsaicin pretreatment attenuates suppression of food intake by cholecystokinin. Am. ~L Physiol., 248 (1985) R501-R504. Ritter, S. and Weatherford, S.C. Capsaicin pretreatment blocks glucagon-induced suppression of food intake. Appetite, 7 (1986) 291. Rogers, R.C. and Hermann, G.E. Central connections of the hepatic branch of the vagus nerve: A horseradish peroxidase histochemical study. J. Auton. Nerv. Svst., 7 (1983) 165-174. Siegel, S. Nonparametric Statisti~:s" for the Behavioral Sciences (2nd edn.), McGraw Hill, New York, 1988. Smith, G.P. and Jerome, C. Effects of total and selective abdominal vagotomies on water intake in rats. J. Auton. Nerv. Syst., 9 (1983) 259-271. Smith, G.P., Jerome, C., Cushin, B., Eterno, R. and Simansky, K.J. Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science, 213 (1981) 1036-1037. Smith, G.P., Jerome, C. and Norgren, R. Afferent axons in abdominal vagus mediate satiety effect of cholecystokinin in rats. Am. J. Physiol., 249 (1985) R638-R641. Stuckey, J.A., Gibbs, J. and Smith, G.P. Neural disconnection of gut from brain blocks bombesin-induced satiety. Peptides, 6 (1985) 1249-1252. VanderWeele, D.A., Geiselman. P.J. and Novin, D. Pancreatic glucagon, food deprivation and feeding in intact and vagotomized rabbits. PhysioL Behav., 23 (1979) 155-158. Weatherford, S.C. and Ritter, S. Lesion of vagal afferent terminals impairs glucagon-induced suppression of food intake. Physiol. Behav., 43 (1988) 645-650. Weatherford, S.C. and Ritter, S. Glucagon satiety: Diurnal variation after hepatic branch vagotomy or intraportal alloxan. Brain Res. Bull., 17 (1986) 545-549.
13
JANS 01036
Redundant vagal mediation of the synergistic satiety effect of pancreatic glucagon and cholecystokinin in sham feeding rats Joseph Le Sauter and Nori Geary Department of Psychology, Columbia University, New York, NY, U.S.A.
(Received 31 July!989 ) (Revision received 19 October 1989) (Accepted 6 December 1989)
Key words: P o s t p r a n d i a l satiety; T o t a l a b d o m i n a l v a g o t o m y ; H e p a t i c v a g o t o m y ; G a s t r i c v a g o t o m y ; Celiac v a g o t o m y ; V a g o t o m y v e r i f i c a t i o n
Abstract Simultaneous intraperitoneal injection of pancreatic glucagon (PG) and cholecystokinin (CCK) results in a functionally synergistic satiety effect in non-deprived sham feeding rats ("PG-CCK satiety"). That is, PG and CCK together inhibit feeding significantly more than the sum of their individual effects. Because the individual satiety effect of each peptide on normal feeding is dependent on the abdominal vagus nerve, we tested the vagal mediation of this synergistic effect of PG plus CCK. Vagotomies were verified anatomically and, in one experiment, histologically. Total abdominal vagotomy blocked PG-CCK satiety. Neither selective vagotomies of the hepatic, the gastric, the celiac, the gastric and celiac, nor the gastric and hepatic branches, however, affected PG-CCK satiety. This indicates that the vagal contribution to the synergistic satiety effect of PG plus CCK on sham feeding is redundant. Although some vagal fibers are necessary for PG-CCK satiety, no individual branch is required for the effect, and at least two branches, the hepatic and celiac, are each sufficient for mediating it.
Introduction Both p a n c r e a t i c glucagon ( P G ) a n d cholecyst o k i n i n ( C C K ) are h y p o t h e s i z e d to signal postp r a n d i a l satiety in rats. W e recently r e p o r t e d that c o m b i n a t i o n s of P G a n d C C K result in a functionally synergistic satiety effect in n o n - d e p r i v e d rats sham feeding with o p e n gastric c a n n u l a s ( " P G - C C K satiety") [12]. T h a t is, s h a m feeding was i n h i b i t e d significantly m o r e b y s i m u l t a n e o u s
Correspondence: N. Geary, Department of Psychology, Box 28, Schermerhorn Hall, Columbia University, New York, NY 10027, U.S.A.
i n t r a p e r i t o n e a l injections o f P G a n d C C K t h a n b y the sum of the two p e p t i d e s ' i n d i v i d u a l effects. T h e m e c h a n i s m m e d i a t i n g this synergy is unknown. T h e satiety effects of P G a n d of C C K are each b l o c k e d b y total a b d o m i n a l v a g o t o m y [7,10,13,20,22,37]. Therefore, we tested the vagal d e p e n d e n c y of the synergistic P G - C C K satiety effect on s h a m feeding in n o n - d e p r i v e d rats. T o t a l a b d o m i n a l v a g o t o m y b l o c k e d the effect of P G C C K c o m b i n a t i o n s ( a n d also b l o c k e d the effect of C C K alone), b u t no selective v a g o t o m i e s did. Thus, this synergistic p e p t i d e satiety signal d e p e n d s o n a r e d u n d a n t vagal m e c h a n i s m which does n o t require the fibers o f a n y p a r t i c u l a r i n d i v i d u a l branch.
0165-1838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14 Methods
Subjects Five groups of male Sprague-Dawley rats (Charles River, Wilmington, MA), 500-700 g, were tested in succession. Rats were individually housed in solid-bottom metal cages (37 × 35 x 20 cm) with wood chip bedding (Northeastern Products, Warrensburg, NY). Pelleted rat chow (Purina 5012, St Louis, MO) was presented in hoppers hung inside the cages except during the test sessions. Tap water was available ad libitum in bottles clipped onto the cages. The room was maintained on a 12 : 12 LD schedule (lights on 07 : 30) at 20 + 2°C.
Sham feeding Rats were anesthetized with Chloropent (Fort Dodge Industries, Ft Dodge, IA; 2.5 m l / k g ) after overnight food deprivation and equipped with chronic gastric cannulae as described previously [2,6,191. Three weeks after surgery the rats were adapted to the sham feeding procedure. At 09:00, food hoppers were removed, cannulas were opened, and stomachs were flushed repeatedly with 8 - 9 ml of 0.9% NaC1 at about 30 ° C until the drainage was clear. The lavage was repeated about 30 min later to ensure that no food was left in the stomach. 1 m l / k g 0.9% NaC1 was then intraperitoneally injected, and drainage tubes (Silastic, Dow Coming, Midland, MI; length 20 cm, inner diameter 3.4 ram, outer diameter 4.6 mm, sheathed with a steel spring) were screwed into the cannulae. Rats were put in transparent plastic cages (20 × 24 × 20 cm) with slotted wire-mesh floors. The distal ends of the drainage tubes were passed through the slots to allow ingested liquid food to drain into a collection pan under the cage. Evaporated milk (Pet Industries, St. Louis, MO) and water were preser, ted in graduated tubes (Wahman, Timonium, MD). Milk intake was measured every 10 min for 40 rain. Drainage tubes were then removed, cannulae closed, and rats were returned to their home cages.
Vagotomy Groups 1 - 3 were adapted to sham feeding for 15 sessions before being reanesthesized for total or
selective vagotomy [13,33]. In groups 4 and 5 gastric cannulation and vagotomy were done in a single operation. For control operations, the abdominal vagal branches were exposed but not cut. Six total abdominal-vagotomized (TAVx) and nine control-operated rats were tested in group 1. For TAVx, about 3 m m each of the anterior (right) and posterior (left) esophageal vagal trunk was ligated and removed rostral to the bifurcation points of the hepatic and celiac branches. In group 2, eight hepatic-vagotomized (HVx) and eight control-operated rats were compared. For HVx, about 2 m m of the hepatic branch was ligated and removed. Group 3 included seven rats with gastric vagotomy (GVx) and seven control-operated rats. For GVx, 2 m m sections were tigated and removed from the anterior and the posterior gastric branches caudal to the hepatic and the celiac branches, respectively. Group 4 included eight rats with combined gastric and celiac vagotomy (GCVx), three rats with celiac vagotomy (CVx), and eight control-operated rats. For GCVx, 2-mm sections of the anterior gastric branch caudal to the hepatic branch and of the posterior trunk rostral to the celiac branches were ligated and removed. The accessory celiac branch was also cut. CVx was performed by removing sections of the celiac and accessory celiac branches. Finally, group 5 included six rats with combined gastric and hepatic vagotomy (GHVx), for which the HVx and GVx procedures were combined; three rats with separate transections of each of the abdominal vagal branches (GHCVx); two TAVx rats, and six control-operated rats. Vagotomized rats tended to lose more weight post-surgically than control-operated rats, but by 2 weeks after surgery, all groups were regaining weight and continued to do so throughout the test period. For example, in group 1 TAVx rats gained 35 + 6 g and control rats gained 50 + 3 g during 4 weeks of testing.
Procedure Rats were adapted to the sham feeding procedure for 15 sessions beginning 1 week after vagotomy. Tests were then begun when rats met the baseline criterion that the SD of each rat's mean
15 30 min intake for 3 consecutive days was less than 30% of that mean [9]. Tests were done Tuesday to Friday. Monday was an adaptation day because rats sham feed more on the first test after an interruption of the daily routine. Each rat received six tests in random order: 400 /zg/kg PG, 0.30/~g/kg CCK, simultaneous injection of 400/~g/kg PG plus 0 . 3 0 / t g / k g CCK, and three control injections. These doses were chosen because they synergistically inhibited sham feeding in previous work [12]. However, in groups 1 and 4, 0.30 /~g/kg CCK alone inhibited sham feeding in control-operated rats more than previously [12], and no synergistic effect of the PG-CCK combination was obtained. When this occurred, tests of 0.15 # g / k g CCK were done. In group 1, the original 6-day protocol was extended by six additional tests: 0.15 /~g/kg CCK, two tests of 0.15/~g/kg CCK plus 400/~g/kg PG, one test of 0.30 ffg/kg CCK plus 400 /~g/kg PG, and two control tests. In group 4, the first 6-day protocol was simply repeated in a separate experiment using 0.15/~g/kg CCK. PG was prepared by diluting 1 mg PG hydrochloride (Eli Lilly, Indianapolis, IN) mixed with lactose (49 mg) in 0.625 ml Lilly diluting fluid (1.6% glycerin, 0.2% phenol) and 0.625 ml 0.9% NaC1. Lyophillized synthetic CCK octapeptide (Squibb, Princeton, N J) was dissolved in 0.9% NaC1 with 39.2 mg lactose/ml. This vehicle was also used for control injections. For single peptide tests, 1-ml plastic syringes (Becton, Dickinson, Rutherford, N J) were filled with 0.5 m l / k g body weight of the appropriate stock solution and 0.5 m l / k g of vehicle solution. For combination treatments, syringes were filled with 0.5 m l / k g of each peptide. All injections were 1 m l / k g solution. Real feeding tests were done after the sham feeding tests in two groups whose selective vagotomies have been reported to block PG or CCK satiety effect [7,34]. PG was tested in group 2 (HVx), and CCK was tested in group 3 (GVx). The procedure was similar, except the gastric cannulae were closed and the stomachs were not lavaged.
Behavioral observations Behavioral observations were done for groups
1-4. During the 40-min sham feeding tests, an observer rated each rat's behavior once per rain during a 0.6-s interval signalled by a low intensity tone [5]. Behaviors were classified using 24 operationally defined categories, including feeding (milk ingestion or licking mouth), grooming (ticking, biting or scratching the head, body, tail, or paws), exploring (locomotion, sniffing, rearing, or licking cage), resting (reclining in a stationary position with the abdomen supported by the cage floor and without display of any other behavior), other normal behaviors (standing, urinating, defecating, or eating feces), and anomalous behaviors (unusual postures, trembling, drooling, etc.).
Verification of vagotomy In groups 1-4 vagotomies were verified anatomically [33]. Rats were overdosed with Chloropent, and the surgical sites were examined with an operating microscope (6-40 x ). If any ambiguous fiber connection was located in the area between the ligatures, the vagotomy was judged to be incomplete, and data from such rats were discarded. According to this criterion, all the vagotomies in groups 1-3 were complete. In group 4, one GCVx rat's data were discarded because an ambiguous, possibly vagal, fiber was detected in the lesioned area. Group 5 rats were verified histologically. A tissue sample consisting of the caudal thoracic and the abdominal esophagus, the diaphragmatic hilus, the fiver surrounding the hepatic vagus, the proximal lesser gastric curvature, and adherent fat and connective tissue, was removed. A straight wire (OD 2.5 ram) was placed into the lumen of the esophagus, and a light weight (4.6 g) was attached to the stomach. The sample was suspended by the rostral end of the esophagus during fixation and processing [26], embedded in paraffin, and cut transversely at 12 /~m. Sections were stained in hematoxilin and eosin [17] and mounted. Serial sections of the esophagus from the diaphragmatic hilus to the stomach were microscopically examined (10-200 x ) by an experimenter who was blind to the behavioral data. The anterior and posterior vagal trunks were located and the surgical sites were identified by the sutures. Vagotomies were rated complete if no fiber that may
16
Fig. 1. A. Esophagus of sham-operated rat rostral to the bifurcation of the gastric and celiac branches. Note the anterior (a) and posterior (p) vagal trunks, and blood vessels (b). B. Esophagus of GHVx rat. Note remnants of ligature marking caudal end of lesion (k) and the celiac branches (c), which bifurcated from the posterior vagal trunk rostral to the lesion. C. Esophagus of TAVx rat just rostral to lesion site. Note swelling of vagal trunks. D. Lesion site of the same TAVx rat. Rostral-caudal level is similar to A. Note absence of vagal trunks.
have been a neural connection was detected in the lesion site. A l l six G H V x , b o t h T A V x , a n d two of the three G H C V x rats satisfied this criterion (Fig. 1). One G H C V x rat was j u d g e d to have received an i n c o m p l e t e v a g o t o m y b e c a u s e a c o n t i n u o u s fiber c o n n e c t i o n was d e t e c t e d all along the esophagus.
Data analysis The criteria for successful s h a m feeding tests were that ingestion b e g a n within 2 rain of milk p r e s e n t a t i o n a n d that the volume of d r a i n a g e collected was equal to or exceeded the total milk a n d water i n t a k e d u r i n g the test. D a t a f r o m unsuccessful tests were n o t included in the analysis, a n d if
three or m o r e sessions were rejected for a n y rat, n o n e of its d a t a were included. Finally, in o r d e r to increase the sensitivity of the test, rats were not i n c l u d e d if they d i d n o t continue to meet the b a s e l i n e criterion d u r i n g the three c o n t r o l tests. These criteria e l i m i n a t e d two c o n t r o l - o p e r a t e d a n d one T A V x rat in g r o u p 1, one c o n t r o l - o p e r a t e d in g r o u p 3, a n d one c o n t r o l - o p e r a t e d rat in group 4 d u r i n g the series of tests with 0 . 1 5 / ~ g / k g C C K . D u r i n g c o n t r o l tests, rats s h a m fed milk continuously a n d vigorously for 3 0 - 4 0 min, ingesting 4 0 - 5 0 m l / 3 0 rain. R a t s that s t o p p e d s h a m feeding g r o o m e d a n d rested. S o m e of these animals b e g a n a second b o u t of s h a m feeding j u s t b e f o r e the end of the test. Because the p e p t i d e s ' effects on sham
17 i n t a k e w e r e c l e a r e s t d u r i n g t h e initial b o u t o f s h a m feeding, w e a n a l y s e d the p e p t i d e s ' effects o n i n t a k e d u r i n g the first 30 m i n o f s h a m f e e d i n g as p e r c e n t i n h i b i t i o n o f the m e a n i n t a k e o n t h e c o n t r o l days, i.e.: ~ (100 ( c o n t r o l i n t a k e - t e s t i n t a k e ) / c o n t r o l i n t a k e ) / n . T h e results o f t h e rep e a t e d p e p t i d e tests in g r o u p 1 w e r e s i m i l a r a n d w e r e c o m b i n e d for analysis. T h e d a t a w e r e analysed with 2-way (control x vagotomized) A N O V A for r e p e a t e d m e a s u r e m e n t s , f o l l o w e d b y p o s t h o c tests o f d i f f e r e n c e s b e t w e e n t h e m e a n s w i t h T u k e y ' s h s d m e t h o d [8]. M i n i m u m signific a n c e level w a s 0.05. D a t a are r e p o r t e d as m e a n s + SEM. B e h a v i o r a l o b s e r v a t i o n s w e r e d o n e to determine whether peptide treatments that inhibited s h a m f e e d i n g e l i c i t e d the n o r m a l s e q u e n c e of p o s t prandial behavior. Frequencies of each behavior c a t e g o r y w e r e a n a l y s e d w i t h F r i e d m a n ' s tests foll o w e d b y p o s t - h o c tests for c o m p a r i s o n w i t h a c o n t r o l [32].
r: INHIBITION 100 :30 60 40 20 0 400 PG
0.30
T h e results w e r e clear. I n c o n t r o l - o p e r a t e d rats, injection either of CCK alone or of PG plus CCK (but not of PG alone) inhibited sham feeding, with the effect o f P G p l u s C C K u s u a l l y s i g n i f i c a n t l y l a r g e r t h a n t h e s u m o f t h e effects o f P G a l o n e a n d C C K alone. C o m p l e t e d i s c o n n e c t i o n o f t h e ab-
PG+CCK
100 80
60 40 20 0
PG+CCK 400 PG 8.15 CCK PEF'TIDE DOSE (pG/KG)
• CONTROL Results
CCK
[ ] TAUX
Fig. 2. Blockade of the inhibitory effect of simultaneous injection of PG and CCK on sham feeding in TAVx rats (group 1). Upper panel shows the results of tests with 0.30/.tg/kg CCK; lower panel shows results of tests with 0.15 /tg/kg CCK. For clarity, PG data are shown in each panel. Sham intake after vehicle injection was 43.4+ 6.4 ml in seven control-operated rats and 46.5 + 5.7 ml in five TAVx rats. * P < 0.05, * * P < 0.01 vs. vehicle; + P < 0.01 vs. control operated. Tukey's test after significant ANOVA, F (4,10) = 6.1.
TABLE I Total abdominal oagotomies block PG-CCK satiety
Data are % inhibition compared to intakes after vehicle injection, individual (R numbers) and M + SEM for group 5 with total abdominal vagotomies done by lesioning the vagal trunks rostral to their subdivisions (TAVx) or by separately lesioning each of the three branches (GHCVx). PG dose was 400 /~g/kg; CCK, 0.30 ~g/kg. * P < 0.05, * * P < 0.01 vs. vehicle, Tukey's test after significant ANOVA. PG Control (n = 6) TAVx
GHCVx
Mean + SEM
CCK
-4.3 + 11.9
R 14
34.8 ± 5.3 *
37.5
- 45.8
3.2
-6.3
R 18
-
Mean + SEM R 16 R 02 Mean + SEM
17.5 + 20.4 9.9 - 46.5 -18.3 + 28.2
-26.1 + 19.8 - 9.0 23.6 7.3 + 16.3
PG + C C K 54.6 + 11.9 * * - 54.2 17.5 -18.4 + 35.9 3.6 - 14.6 -5.5 + 9.1
18
%
% INHIBITION
INHIBITION
100 r-
100
80
80
:f.
60
60
40
40
20
20 0
0
400 PG
0.30 CCK
PG+CCK
F'EPTIDE DOSE (pG/KG)
• CONTROL
[] HUX
Fig. 3. Inhibition of sham feeding by simultaneous injection of PG and CCK in HVx rats (group 2). Sham intake after vehicle injection was 50.8 _+7.5 ml in eight control-operated and 48.5 _+ 6.6 ml in eight HVx rats. * P < 0.01 vs. vehicle; ~ P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,14) = 34.6.
400 PG
0.30 CCK
PG + CCK
O 15 CCK
PG + CCK
100 80 60 40 20 0
d o m i n a l v a g u s b l o c k e d o r severely a t t e n u a t e d b o t h the i n h i b i t o r y e f f e c t of C C K a l o n e a n d the synergistic effect of P G p l u s C C K . But n o selective v a g o t o m y a f f e c t e d e i t h e r i n h i b i t o r y effect. T h e b l o c k a d e o f P G - C C K satiety in g r o u p 1 T A V x rats is s h o w n in Fig. 2. I n c o n t r o l - o p e r a t e d rats, i n j e c t i o n of 0.15 t t g / k g C C K a l o n e i n h i b i t e d
% IHHIBITInN
100
400 PG
PEPTIDE DOSE (pg/kg) •
CONTROL
~
GCVX
Fig. 5. Inhibition of sham feeding by simultaneous injection of PG and CCK in GCVx rats (group 4). Sham intakes after vehicle injection were 48.9 + 4.6 ml (upper panel) and 43.9 + 4.3 ml (lower panel) in seven control-operated rats and 48.8 + 9.2 ml (upper panel) and 44.6+3.7 ml (lower panel) in seven GCVx rats. * P < 0.01 vs. vehicle; = P < 0.05, =~ P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,13)=16.9 (upper panel) and 23.2 (lower panel).
,90 60 40 20 0 400
PG
0...'30 CCK
PEPTIDE
PG+0CK
BOSE ( p G , " K G )
• CONTROL
[] GUX
Fig. 4. Inhibition of sham feeding by simultaneous injection of PG and CCK in GVx rats (group 3). Sham intake after vehicle injection was 40.6 _+4.8 ml in six control-operated and 49.0 ± 9.9 ml in seven GVx rats. * P < 0.01 vs. vehicle; # P < 0.01 vs. sum of individual effects of PG and of CCK. Tukey's test after significant ANOVA, F (2,11) = 10.6.
s h a m f e e d i n g b y 31.9 +_ 8.2%, P G a l o n e d i d n o t h a v e a s i g n i f i c a n t effect, a n d s i m u l t a n e o u s inject i o n of 400 / ~ g / k g P G p l u s 0.15, / ~ g / k g C C K i n h i b i t e d s h a m f e e d i n g by 64.6 _+ 13.1%, signific a n t l y m o r e t h a n the s u m of the effects of P G a n d C C K alone. I n T A V x rats, n o n e o f these treatm e n t s a f f e c t e d s h a m feeding. T h e l a r g e r d o s e of 0.30 / l g / k g C C K i n h i b i t e d s h a m f e e d i n g in c o n t r o l - o p e r a t e d rats by 57.5 +_ 9.9%, b u t d i d n o t affect T A V x rats. A l t h o u g h c o m b i n a t i o n of 400 /~g/kg PG plus 0.30/~g/kg CCK did inhibit sham f e e d i n g b y 28.6_+ 12.9% in T A V x rats, this was s i g n i f i c a n t l y less t h a n the 73.1 + 11.6% i n h i b i t i o n in c o n t r o l - o p e r a t e d rats. P G p l u s 0 . 3 0 / ~ g / k g C C K
19
~. INHIBITION 100 r80 60
20 0
400 Pg.
0.30 CCK
PG+CCK
PEPTIDE BOSE (pG/KG) •
CONTROL
[] GNUX
Fig. 6. I n h i b i t i o n of s h a m feeding b y s i m u l t a n e o u s injection of P G a n d C C K after G H V x (group 5). S h a m i n t a k e after vehicle injection was 49.0 5:8.0 m l in six control o p e r a t e d a n d 47.9 + 9.2 m l in five G H V x rats. * P < 0.05, * * P < 0.01 vs. vehicle. T u k e y ' s test after significant A N O V A , F (2,9) = 17.0.
also failed to inhibit sham feeding in the two TAVx rats and the two GHCVx rats in group 5 (Table I). In contrast to the TAVx results, neither HVx, GVx, CVx, GCVx, nor GHVx produced any change in the inhibitory effects of CCK or of PG plus CCK on sham feeding (Figs. 3-6 and Table II).
The inhibitory effect of 0.60 /~g/kg CCK on real feeding was similar in seven control-operated (48.6_+9.1%) and six GVx rats (56.9_+ 8.6%). However, HVx blocked the effect of PG on real feeding: 4 0 0 / t g / k g P G inhibited real feeding by 18.1 _+ 4.8%, P < 0.05, in seven control-operated rats vs. - 1.5 +_ 6.2%, n.s., in six HVx rats. The behavioral observations indicated that neither peptide injection nor vagotomy disrupted the normal sequence of postprandial activity. Peptide treatments that inhibited sham feeding decreased the frequency of feeding and increased the frequency of resting but did not alter the frequency or the temporal pattern of postprandial grooming and exploration and did not elicit any anomalous behaviors. This was true for both control-operated and selective-vagotomized rats. Behaviors of peptide-injected TAVx rats were similar to those of vehicle-injected control-operated rats. Because we have previously described this behavioral profile in detail [12], we do not present it here.
Discussion
When CCK doses that elicit moderate inhibitions of sham feeding in non-deprived rats are
T A B L E II
Celiac vagotomy does not block PG-CCK satiety D a t a are % i n h i b i t i o n c o m p a r e d to i n t a k e s after vehicle injection. P G dose was 4 0 0 / t g / k g . P G + C C K , T u k e y ' s test after significant A N O V A . PG
* * P < 0.01 vs. vehicle, + P < 0.01 vs.
CCK
PG + CCK
0.30 ~ g / k g C C K Control operated
(n = 8) CVx
0.15/~g/kg CCK Control operated (n = 6) CVx
M e a n 5: S E M
2.9 + 7.2
46.0 + 11.0 * *
59.7 5 : 7 . 0
**
R 02 R 04 R 20 M e a n 5: S E M
24.9 18.9 17.8 20.5 + 2.2
76.5 24.9 64.3 55.2 + 15.6 * *
85.9 39.9 78.6 68.1 5:14.3 * *
Mean + SEM
- 11.0 + 6.9
15.1 + 7.4
45.7 5:2.9 * * +
R 04 R 20 Mean + SEM
9.6 1.1 5.4 5:4.3
34.6 81.7 58.2 5:23.6 * *
71.2 92.7 82.0 5:10.8 * * +
20 intraperitoneally injected together with PG, which alone fails to inhibit sham feeding [6], sham feeding is inhibited significantly more than the sum of the peptides' individual effects [12]. The inhibition appears behaviorally specific. The normal behavioral sequence of postprandial satiety occurs when sham feeding ends [12], and neither sham water drinking [12] nor activity in running wheels (unpublished data) is inhibited by PG-CCK combinations that inhibited sham feeding by 50% or more. Thus, simultaneous injection of PG and CCK appears to elicit a functionally synergistic satiety effect in the sham feeding rat (" PG-CCK satiety"). We demonstrate here that this PG-CCK satiety effect depends on the contribution of abdominal vagal fibers. After TAVx, PG-CCK satiety is blocked or greatly attenuated. This does not appear to be the result of any debilitating effect of TAVx because TAVx and control rats both gained weight during the testing period and sham fed similar amounts (see figure legends). Despite the vagal dependency of PG-CCK satiety, we could not identify a necessary contribution of any individual subdiaphragmatic vagal branch. Selective lesions of the hepatic, gastric, and celiac branches were each without effect. Thus, none of the three major divisions of the abdominal vagus is necessary for PG-CCK satiety. Further, the lack of effect of gastric plus celiac and gastric plus hepatic vagotomy indicates that the hepatic and the celiac branches are each alone sufficient for PG-CCK satiety (gastric branch sufficiency has not been tested by observing hepatic plus celiac vagotomy). In short, the vagal mechanism mediating PG-CCK satiety is redundant. This is the clearest demonstration we know of redundancy in a vagally mediated behavioral mechanism. Our data do not disclose whether the involvement of the vagal nerves in PG-CCK satiety is in afferent or efferent limbs, or mixed. Because there is evidence that vagal afferents provide the necessary contribution to the individual satiety effects of PG and CCK on real feeding, our hypothesis is that the critical TAVx lesion blocking the synergistic satiety effect of PG and CCK is also afferent. In real feeding, blocking peripheral postganglionic muscarinic receptors with atropine methylnitrate
did not affect either PG [7] or CCK satiety [34]. In contrast, selective transection of the dorsal, but not the ventral, medullary vagal rootlets, blocked CCK satiety [35]; surgical lesions of the portion of the nucleus of the solitary tract receiving hepatic vagal afferents blocked PG satiety [38]; and chemical lesion of small diameter sensory neurons by intraperitoneal capsaicin injection attenuated both CCK and PG satiety [29,30]. Both PG and CCK appear to act peripherally to signal satiety [7,22,34]. Thus, their synergistic interaction might occur either peripherally, if one of the peptides potentiates some effect of the other in the abdomen, or centrally, if the peptides elicit independent afferent signals that converge in the CNS. The present results, taken together with previous studies of the vagal mediation of the peptides' satiety effects on real feeding, suggest that the most likely alternative is that PG potentiates CCK's peripheral action. Hepatic afferents appear to be necessary and sufficient for mediating the contribution of the abdominal vagus to PG satiety during real feeding ([7,20,39], but see also [3,39]). Yet neither hepatic nor hepatic-gastric vagotomy attenuated PG-CCK's sham feeding satiety effect. Indeed, selective hepatic vagotomy blocked the satiety effect of PG on real feeding in the same rats in which it failed to block the effect of PG plus CCK on sham feeding. Thus, CCK apparently did not potentiate a hepatic vagal satiety signal to inhibit sham feeding. Further, since the hepatic vagus alone was unnecessary for PG-CCK satiety, this effect obviously did not depend on the central convergence of a hepatic vagal afferent signal caused by PG with an independent signal related to CCK. It is unclear whether the functional redundancy in PG-CCK satiety occurs because the satiety signal arises at a single site innervated by different vagal branches or because the signal can be generated in multiple abdominal sites. Either mechanism is plausible. For example, the gastric, celiac and hepatic vagal branches all appear to control pancreatic insulin secretion: the dorsal motor nucleus of the vagus appears to project to the pancreas via each branch [28], and the insulin response to electric stimulation of the cervical vagus can be decreased by selective vagotomy of
21
each of them [4]. Alternatively, redundancy may be mediated by central convergence of different branches. Such convergence appears to occur even in the primary sensory nuclei of visceral afferents. Abdominal vagal afferents terminate most heavily in the dorsal medial nucleus of the solitary tract and in the commissural nucleus, with the terminal regions of the gastric, celiac, and hepatic branches overlapping each other to some extent [1,14,24,25, 31]. Further, inputs projecting via different abdominal vagal branches have been shown to converge on individual neurons of the nucleus of the solitary tract [11]. CCK and PG may each signal satiety by directly stimulating vagal afferents. It is interesting, therefore, that CCK binding sites are present in each of the branches of the abdominal vagus [21]. Further, at least in the hepatic branch, some afferents can be stimulated by either CCK or PG [23]. Unfortunately, the effect of PG-CCK combinations on neural activity has not been tested yet. The effect of CCK alone on sham feeding, like that of PG-CCK combinations, was blocked by total abdominal vagotomy but not by any selective vagotomy. This extends previous reports that neither gastric nor gastric-celiac vagotomy blocks peripheral CCK's satiety effect [10,13]. Thus, a redundant vagal mechanism may mediate the satiety effect of CCK alone as well as P G - C C K satiety. Similarly, a satiety effect of bombesin may be mediated by redundant vagal and spinal visceral afferents. Intraperitoneal bombesin injections decreased meal size in rats with total abdominal vagotomies and in rats with T6 spinal cord transections and T 3 - T 6 dorsal rhizotomies, but not in rats with both lesions [36]. The failures to detect an effect of selective abdominal vagotomy on the satiety effect of CCK on either real feeding or sham feeding [10,13] differ from Smith et al.'s [34] report that selective gastric vagotomy was sufficient to block CCK's satiety effect on real feeding. Although our results do not resolve this issue, our histological verification procedure convinces us that our failure to block PG-CCK or CCK satiety with gastric vagotomies did not result from incomplete lesions. Many methods have been used to verify vagotomies [16,27,33], but histological examination of the
esophagus has not previously been described. Esophageal histology appears to present several advantages. For example, it includes afferent fibers in the verification, which other functional [16] and histological [27] tests do not. Gross anatomical examination also includes afferents, but it is sometimes impossible to identify and track neural fibers using gross examination when connective tissue adheres to the lesion site. In conclusion, a redundant vagal mechanism appears to mediate the synergistic satiety effect of simultaneous intraperitoneal injection of PG and CCK in sham feeding rats. Difficult electrophysiological and behavioral tests will probably be required to understand the nature of this redundancy. Given the clarity of the effect and the apparent importance of synergy and redundancy in peripheral neuroendocrine controls of feeding [4,9,11,12,36], this effort seems worthwhile.
Acknowledgements We thank Debra Rosenzweig, Jennifer Fudge, and Zsolt Stockinger for help with the experiments. This research was supported by National Institutes of Health Grant DK-32448 to N. Geary. Preliminary reports of these data were given at the IXth International Conference on the Physiology of Food and Fluid Intake, Seattle, WA, July 1986, and at the 17th Annual Meeting of the Society for Neuroscience, New Orleans, LA, November 1987 (Soc. Neurosci. Abst., 13 (1987) 587).
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